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1. ee 1 21 3 How can I access Linux files from a PC running MS Windows 4 21 4 Can Irun NMRanalyst on an Apple Macintosh ccccccsssscccccecceeeceeeeeeeeeeeneens 4 21 5 How to display new MS Windows applications as foreground windows 4 21 6 Any UNIX editor available from the Windows UNIX Shell 5 21 7 How to copy and paste text in Windows NMRanalyst cc cccccccceeeeeeeeeeeneees 5 21 8 What should I do about the Windows XP NMRanalyst security aert 6 21 9 How can I get NMRanalyst printing to work wcccciscvsnsssnerssannebacnecenssaveneveessarensceonadic 6 21 10 How can I capture NMRgraph and NMR plot displays 0 0 0 ccccccececeeeeeeeseees 7 21 11 Why can t I switch NMRanalyst to a different spectrum type cccceceeeeeees 8 21 12 Why are the EEN 8 21 13 How to fix incorrect NMRplot or NMRgraph renderng 9 21 14 How to increase the paging space On Lmusx 10 21 15 How to specify correlations for the Assemblelt structure generation 10 CHAPTER 22 January 11 2010 User Manual CHAPTER 1 Overview NMRanalyst automates the analysis of one to three dimensional NMR spectra It reduces experimental multidimensional NMR data to a list of detected spin systems eliminating the tedious manual interpretation of raw NMR data NMRanalyst s AssembleIt workwindow con tains the Findit VerifyIt and AssembleIt components The FindIt component identifies the best matching structures for
2. 2 3 7 5 DQF COSY prednisone s sseesseseieseiisseieeseriesssierssrirsssieesrrerssrren 2 3 1 2 S ACTONYM MEANING 2 eeeeseeseeseesessessesessessessesseeseeees ee 12 coupling constant mapping rang 15 6 Se Bee i deriving carbon carbon correlation 17 2 17 3 January 11 2010 22 3 NMRanalyst Manual Index geminal coupling ceeeeeeeseeeseeeseeseeeeeeeeeeees 17 3 maximum number of points 14 5 long range coupling 17 3 output Die 14 3 Mapping parameters oe eee eee 15 7 15 8 pDurmose of 14 1 optimization detection parameters 15 8 transform coefficients oo 14 2 schematic ee E EE E GE 14 4 spectral diagonal sesser 1 3 Vicinal couplmg 17 3 FID weighting functions 4 14 DVD Content WEE OT 12 8 file selection noe 11 1 E BEE eege 11 1 11 2 emulator find files MMA EE GEES 9 3 Virtual P N 21 4 Findlt siii 17 6 error adding structures to database 9 15 ambiguous signal assignment 0 c cece 16 1 fitting area EE 9 4 NMRanalyst analysis cc cecscscecsseseseeseseseeeseen 1 3 incorrect display permission teg 9 5 FRED multiple NMRanalyst running eseeseee1ee111se 12 8 see Ge 1 1 NMRanalyst program not found 9 3 ee phase miematch 15 10 16 1 16 8 signal area overlan 16 1 G Start CLLOLS cceecsscscsessscsececessesessersesececsvsteeceveveeeeeeee 9 9 a too many signals Found 13 6 goodness of fit criterion EE 9 5 multiple AANA esc cascscasesctaxescstancasoetseanics
3. F2 Mapping Ponts 15 6 J Mapping Point 0 15 6 Numerics 1D Analysis workwindow 1110111011102 13 1 Id_analysis program effect of signal splitting 0 0c eee 13 5 maximum number of points 13 5 nucleus observe frequency u 13 4 SE 13 1 peak overlap criterion s 13 6 peak oickimg eseseseseses estes 13 5 positive negative gignals cesses 13 6 spectral width erte 13 4 spectrum identification test 13 4 Start Of gpectrum eects tseseeeeteeeeees 13 5 subtracting signals from data 13 8 supported data types sees 13 2 zero TIOE gedet 13 5 3D SPECTRUM example appleatpon 7 5 for all 3D spectrum types ccccceeeeeeeeeeeeeee 7 5 loading procpar Die 7 6 optimization detection parameters 15 9 sensitive analysis 7 7 spectral aimulapton eset eeeees 15 2 A ADEQUATE ACTONYM MEANING oo eee es eee esesseseesesesteeseeeeeees 1 2 advantage of F1 DQ fDeouencies 17 3 comparison with HMBC ue eee 17 3 deriving carbon carbon correlation 17 2 Mapping parameters c c cece estes 15 7 optimization detection parameters 15 9 SQ or DQ frequencies in E 17 3 ambiguous signal ass1gmnment 15 4 analysis speed relation to phase functions eee 15 12 analyze program causes for correlation rejection eee 15 10 correlation summary line eee 15 10 coupling constant mapping points 15 6 detection parameter optimization 0 0 0 15
4. kg Add User Structures to FindIt Database Progress 100 Adding GriitestyClLlIHl1iBros mol SUCCESS structure ID 1 Adding G ytesty ClLLIHl Nzo3_ mol Error This structure if identical to the FindIt database structure of ID 5323896 FAILED to add this structure to the FindIt database Adding G ytestyCl2HleNz03_ mol SUCCESS structure ID Zi Added Z af the 3 input structures in amp s Task completed Done Continue Abort Help When adding removing or extracting structures the shown popup with progress information is displayed If a structure is already con tained in the FindIt data base its ID is displayed and it won t be added again During the adding process the Abort button allows stopping the process When the adding removing or extracting process 1s com pleted the Done and Continue buttons are enabled Done closes the popup Continue brings up the FindIt Data base Manager popup for further structure processing The complete progress messages are saved in the file findit 1log in the current NMRDATA directory After upgrading to a new version of NMRanalyst the user added structures in the FindIt data base will be out of date To upgrade the user structures follow these steps First extract all the user structures to an empty directory Next delete all the user structures from the Findit database Finally re add the saved user struc
5. Automated Analysis of 1D Through 3D NMR Spectra Structure Dereplication and Structure Elucidation Version 3 6 Patents U S 5 218 299 and 5 572 125 British 0 577 770 German 692 31 690 6 08 Copyright NMRanalyst Software This copyright notice applies to all parts of the software and documentation except for the 1 Enscript and 2 GNU Ghostscript programs the 3 Java Runtime 4 JavaHelp 5 GL2PS 6 Mesa 7 Visualization Toolkit VTK and 8 Freely Distributable LIBM libraries and the 9 PC UNIX toolset See the corresponding manual chapters for specific copyright information for the third party software Copyright C 2010 by ScienceSoft LLC All Rights Reserved It is illegal to copy modify or otherwise distribute this software without the prior specific written consent of ScienceSoft LLC Reinhard Dunkel Ph D ScienceSoft LLC 9934 Pinehurst Dr Sandy UT 84092 http www ScienceSoft net dunkel ScienceSoft net Phone and FAX 801 816 0163 SCIENCESOFT LLC AND ITS AGENTS DISCLAIM ANY AND ALL WARRANTIES WITH REGARD TO THE SOFTWARE AND DOCUMENTATION WHETHER EXPRESSED OR IMPLIED AND INCLUDING ANY WARRANTIES OF MERCHANTABILITY OR FITNESS FOR PARTICULAR PURPOSE IN NO EVENT SHALL SCIENCESOFT LLC OR ANY OF ITS AGENTS BE LIABLE FOR ANY DAMAGES WHETHER GENERAL SPECIAL INDIRECT OR CONSEQUENTIAL DAMAGES RESULTING FROM LOSS OF USE DATA OR PROFITS WHETHER IN ANY ACTION UNDER CONTRACT NEGLIGENCE OR OTH
6. Key FnADEQ por cosy por cosy report Leg To include DQF COSY correla tions in the carbon skeleton determination specify the location of its Report workwindow report log file A bond is only observable by DQF COSY if its carbons are protonated The proton frequencies of a DQF COSY correlation are mapped to the directly bonded car bons Geminal proton proton couplings are reliably identified since both proton resonances are bonded to the same carbon Vicinal proton proton couplings are the desired ones Proton proton couplings over more than three bonds are undesired Their coupling constants are gen erally below 3 Hz NMRanalyst marks such correlations as potential long range correlations and does not save them as atom atom correlation for the molecular structure generation Key FnCOSY HMBC Pmmc report Leg E wel To include HMBC correlations in the carbon skeleton determination specify the location of its Report workwindow report log file To observe a carbon carbon bond using HMBC data at least one of the bonded carbons has to be protonated AssemblelIt can recover unobserved bonds if 3 bond HMBC correlations involving the unobserved bond are observed HMBC correlations correspond to 2 bond 3 bond and longer range proton carbon couplings Longer range couplings are compute intensive to consider during the structure elucidation HMBC correlations stronger than the specified Weak threshold are assumed to be either 2 bond or 3
7. Tutorial I Using NMRanalyst The Report Workwindow mined parameter values and the corresponding error marginal standard deviation values are saved INADEQUATE 2D Analysis Extract Bond Patterns From 2D Spectrum File Edit Spectrum Type Workwindow Process Tools Help THFUT FILES F 1D Analysis Output File eschen out al AHALYSIS PARAMETERS Analyze Correlation Index He Thresholds Integral Precision Ww Report Multiple Minima Hap F2 Frequencies Hz F2 Mapping Points Hap Coupling Constant Hz J Mapping Points Estimated Coupling Constant Hz Mapping _ 2 Integrals _ F1 Phase _ F1 Relaxation mes _ 4 Integrals _ F Phase _ F Relaxation Unused Detection WwW Integrals _ F1 Phase _ F1 Relaxation Unused _ 4 Integrals _ F Phase _ F Relaxation hm Fi Phase Is 64650 lo o113505 ppm F Phase ilo 63785 a 0032270 ppm AD Analysis 2D FFT 20 analysis 3 7 The Report Workwindow Generating a report is the final step of the numerical analysis of a multidimensional NMR spectrum Information from the previous workwindows are summarized and presented as a list of identified correlations here carbon carbon bonds Display the shown Report work January 11 2010 3 9 Tutorial I Using NMRanalyst The Report Workwindow window by clicking the Report tab Click Start and the generation of this report completes nearly instantan
8. chmod u w SNMRDATA INADEQUATE January 11 2010 9 9 Using the NMRanalyst Window The Speed Button Panel In case that the Loo file and its backup file 1log bck have no write per missions and NMRanalyst fails to obtain write access on these files the following popup window is shown Cancel removes this popup window Change the file permissions and try again Workwindovw File Permissions x Mo write access foar tile INADEQUATE d_analysis log Please correct the file permissions or choose another WMRDATA directory Cancel When Start is clicked and no permission problems exist the workwindow input screen changes to the output screen to show the output of the computation The computation can be termi nated using the Stop button This Start Stop button is set as the default button so that typing the Enter key is equivalent to clicking the button Help Directory Editor This button starts a popup from which the directories used by the NMRanalyst software can be changed See CHAPTER 10 Using the Directory Editor for details Clicking the UNIX Shell button opens a UNIX shell window for command line input Clicking this button exits from NMRanalyst NMRanalyst xj If no computations are in progress the shown popup is displayed Exit terminates NMRanalyst Cancel returns to the NMRanalyst application window Ok to exit HHMRanalyst Exit Cance
9. 13 2 Output Files OUTPUT FILES This input field allows Numerical Description of Signals eschen out DU specifying the name of the generated output file con taining the numerical description of identified 1D resonances Information about all detected resonances in the spectrum is stored in this file chemical shift integral transverse relaxation January 11 2010 13 3 1D Analysis Workwindow Spectral Description time and phase for each resonance per line The detected resonances are numbered m the order of decreasing chemical shift This file is read and used by the FFT nD Analysis Report and AssembleIt workwindows If the name of the output file is changed in this input field it needs to be changed in related workwindows as well Key FnOut Plot Data of Spectrum eene piot DI Tfa file name is specified the experimental spectrum individual simulated resonances the sum of all simulated reso nances simulated spectrum and the difference between experimental and simulated spec tra residual spectrum are stored These spectra can be plotted using the Graphic workwindow Key FnSpcA Plot Data of Parameter Values Ir If a file name is specified a plot file containing the determined phase baseline and error values is created The file also contains the derived 1D baseline and phase correction functions Key FnParm 13 3 Spectral Description SPECTRAL DESCRIPTION In this input field an iden Iden
10. 2010 2 1 NMRanalyst Installation Contents of the NMRanalyst Distribution 2 2 Contents of the NMRanalyst Distribution NMRanalyst contains a database of over 15 9 million unique chemical structures It 1s shipped on one DVD The DVD contains a self extracting installation program for Linux or for MS Windows and the structure database It also contains additional NMR datasets and most of their analyses are described in the tutorials After the software installation consider copying the example datasets to the installation directory to allow other users easy access to them The NMRanalyst distribution consists of the following top level directories and files ANALYST LINUX bin Self extracting installer of the Red Hat Linux Enterprise NMRanalyst software Its data subdirectory contains NMR FIDs for the prednisone structure elucidation for test and practise purposes 980 MBytes ANALYST WIN s xe Self extracting installer of the Microsoft Windows NMRanalyst software It contains a UNIX shell with UNIX user commands Its data subdirectory contains NMR FIDs for the prednisone structure elucidation for test and practise purposes 970 MBytes Manual pdf The NMRanalyst manual is contained in the JavaHelp online documentation This PDF version 1s optimized for printing It can be displayed and printed by the Adobe Acrobat reader free download from http www adobe com products acro bat readstep htm1 3 MBytes SETUCCOO ER tee SErPUC
11. FFT and nD Analysis workwindows for the selected multidimensional spectrum type The analyze program controlled by the nD Analysis workwindow exam ines one fitting area at a time and does not attempt to assemble the analysis results from differ ent fitting areas The program report summarizes this information and provides warning messages in case of ambiguous signal assignments a phase mismatch of a reported correlation with the overall phase functions or overlapping correlation areas The AssembleIt workwindow can combine the Report workwindow results from different spectrum types Its FindIt component identifies the best matching molecular structures for the observed NMR data Its AssembleIt component derives the most likely skeletons from this often incomplete and inconsistent correlation information see CHAPTER 17 Assemblelt Workwindow for details January 11 2010 16 1 Report Workwindow Input File 16 1 Input File INPUT FILE The label of the input field 2 Analysis Output File inadequate corz D reflects the dimensionality of the selected spectrum type Enter in this input field the file name specified in the nD Analysis workwindow Determined D Correlations field The file contains the numerical description of identified patterns in the analyzed multidimensional spectrum Key Fn2D 16 2 Output Files OUTPUT FILES The function of the Report Molecular Structure structure plot al workwindow 1S to summa rize
12. For the visual NMR data analysis phase sensitive spectra are phase corrected This improves the resolution of the displayed spectrum by removing dispersive signal components Absorp tion resonances have a higher amplitude and are easier to distinguish visually from noise NMRanalyst does not phase correct data for the numerical analysis as it analyzes all phase components two for 1D four for 2D and eight for 3D phase sensitive spectra Phasing merely redistributes information between the analyzed phase components and frequency dependent phasing distorts line shapes But NMRanalyst automatically determines phase functions They are used to display experimental data phase corrected They are also used so that NMRanalyst does not have to redetermine the phase of every spin system in each dimen sion This improves analysis speed and sensitivity January 11 2010 4 8 Tutorial Il Setting Analysis Parameters Determining Phase Functions di T NMRanalyst determines HH F1 phases the phase of every 2D of 5 6465 0 01135 f INADEQUATE correla s 7eaeo craa o oooooer2 tion for both spectral aa dimensions Determined phases can be plotted against their spectral fre quencies Interpolation of these phases yields the F1 and F2 phase functions NMRanalyst determined them during the last Report workwindow run Switch to the Graphic workwindow input screen click on the File With Plot Data folder button on the right double click on the fi
13. Inc Binary Code License Agreement READ THE TERMS OF THIS AGREEMENT AND ANY PROVIDED SUPPLEMENTAL LICENSE TERMS COLLEC TIVELY AGREEMENT CAREFULLY BEFORE OPENING THE SOFTWARE MEDIA PACKAGE BY OPENING THE SOFTWARE MEDIA PACKAGE YOU AGREE TO THE TERMS OF THIS AGREEMENT IF YOU ARE ACCESS ING THE SOFTWARE ELECTRONICALLY INDICATE YOUR ACCEPTANCE OF THESE TERMS BY SELECTING THE ACCEPT BUTTON AT THE END OF THIS AGREEMENT IF YOU DO NOT AGREE TO ALL THESE TERMS PROMPTLY RETURN THE UNUSED SOFTWARE TO YOUR PLACE OF PURCHASE FOR A REFUND OR IF THE SOFTWARE IS ACCESSED ELECTRONICALLY SELECT THE DECLINE BUTTON AT THE END OF THIS AGREEMENT 1 LICENSE TO USE Sun grants you a non exclusive and non transferable license for the internal use only of the accompa nying software and documentation and any error corrections provided by Sun collectively Software by the number of users and the class of computer hardware for which the corresponding fee has been paid 2 RESTRICTIONS Software is confidential and copyrighted Title to Software and all associated intellectual property rights is retained by Sun and or its licensors Except as specifically authorized in any Supplemental License Terms you may not make copies of Software other than a single copy of Software for archival purposes Unless enforcement is prohibited by applicable law you may not modify decompile or reverse engineer Software You acknowledge that Software is not designed licen
14. Key FnSpec 14 3 Spectral Description SPECTRAL DESCRIPTION For the identification of Identification Text the multidimensional spectrum an arbitrary text string can be specified in this input field The first 200 characters of the entered string are stored with the spectrum The software neither requires an identification string nor assigns a meaning to the specified string The string is displayed in further analysis steps for spectrum identification Key TxSpec Observe Frequency E NMRanalyst analyzes up to three dimensional NMR spectra Two dimensional spectra have F1 and F2 spectral axes and 3D spectra have an additional F3 axis An Observe Frequency field is provided for each dimension If the observe fre quency is the same for different dimensions some of these fields might not be needed and are dimmed Key HzPpmF1 HzPpmF1U HzPpmF3 HzPpmF3U Spectral wiath_ x2 Specify in the provided F1 through F2 F1 through F3 for 3D input fields the spectral width along the corresponding spectral dimension with unit used For January 11 2010 14 3 FFT Workwindow Spectral Description a 2D INADEQUATE spectrum the F1 spectral width cannot be smaller than the F2 spectral width Otherwise signal assignment ambiguities would result through multiple aliasing of signals Keeping both spectral widths the same allows the most effective use of spectrometer time For a heteronuclear 2D spectrum acquired on a Varian spe
15. Place Heteroatoms x of Af ml St asf SI as sal wl ar Madd Likely C C Double Bonds Ww Order Structures by 7C Shift Agreement 2 82 The shown structure results gs Ze A8 TS Sida 6 167 li a apo 123 74 21141783 87 40 of A we et ae KS 58 81 S34 419 66 08 383 aha 185 03 Sra di A 18 73 The structure can be repositioned to a more common orientation Click on the 66 08 50 8 Hz bond to select it The bond line aq changes to a red color upon selection 15 49 211 41 Select Structures Rotate on ff ee Bond ube vertical seruc 210 19 50 45 w tures Flip Horizontal and 18 73 ago e fe Structures Pilip Vertical 55 05 ar an 48 75 g To deselect the bond click in an empt 7 a r a T e f pty 126 99 419i 35 56 area This gives the shown prednisone sda ada 8 Structure 185 03 T 18 33 13 i za a 123 74 31 52 January 11 2010 3 13 Tutorial I Using NMRanalyst Exiting the Software 3 9 Exiting the Software To terminate NMRanalyst click the Exit button in the NMRanalyst window A popup appears asking for confirmation before exiting NMRanalyst and its started windows January 11 2010 3 14 CHAPTER 4 Tutorial IT Setting Analysis Parameters In CHAPTER 3 Tutorial I Using NMRanalyst input parameters for the analysis of the prednisone dataset are provided For new datasets NMRanalyst contains default settings for the supported spectrum types A load function is p
16. SECTION 7 4 Analyz ing 3D Spectra incorporating NMRanalyst programs into shell scripts macros and other software systems SECTION 7 5 Incorporating NMRanalyst Programs in Other Software and the online help system SECTION 7 6 The On line Help System In NMRanalyst select Edit Preferences set Mode to Full NMRanalyst and select the Show All Input Fields switch as this tutorial describes less commonly used features of the software Click the OK button 7 1 Using an HSQC FID for the findit Structure Identification SECTION 3 1 findit Script for Molecular Structure Identification bases the structure identi fication on a 1D carbon FID findit can substitute a missing proton or carbon FID by the corre sponding F1 and or F2 projection of an HSQC or HMQC spectrum SECTION 6 1 ID Carbon 1D Proton Edited HSQC and HMBC Analyses shows the strychnine HSQC spec trum and its F1 and F2 projections The 2D spectrum phasing does not matter for absolute value projections findit converts both projections to NMRanalyst format It then determines best matching structures January 11 2010 7 1 Tutorial V Additional NMRanalyst Features Using an HSQC FID for the findit Structure Copy the strychnine directory from the Tutorial IV to a temporary location Delete its proton and carbon FIDs to keep findit from using them From the NMRanalyst UNIX Shell window execute the following commands to run findit o cp r E
17. Unob served Bonds to7 4 Bond HMBC Correlations to 1 and start this workwindow One potential gibberellic acid structure is generated in 5 minutes on a 3 06 GHz PC and it is the correct one Extracting carbon list information from the 2D spectra requires more user interaction than analyzing a 1D carbon spectrum But it is faster than acquiring a 1D carbon spectrum It potentially allows using smaller sample quantities or shorter total acquisition times As described in SECTION 2 2 Contents of the NMRanalyst Distribution the NMRanalyst dis tribution contains the ca 200 micro gram dihydrotestosterone FIDs For under one milligram of compound the acquisition of a 1D carbon spectrum is practically impossible But its struc ture elucidation succeeds with the techniques of this chapter See http www science soft net dihydrotestosterone index html for details A limitation is the required separation among carbons to be distinguished Deriving quater nary carbons is done from HMBC Due to the possible F1 skew of HMBC resonances car bons need to be separated 0 12 ppm this is also the default value from other carbons for reliable distinction Protonated carbons can be determined from the phase sensitive HSQC Here the HSQC F1 resolution is important Carbons down to a fifth of this resolution are dis tinguishable by the software which means down to thirteen Hz for the gibberellic acid HSQC Incompletely resolved multiplets in F2 or closel
18. and NMR SPEC shell variables OK commits the current input values and exits from the editor Cancel exits from this window and leaves input values unchanged While computations are in progress the OK button is disabled dimmed When clicking on a history button NMRDIR NMRUSER NMRDATA NMRSPEC a list of previous shell variable entries 1s displayed The folder buttons at the right side of the text fields open a Filebox for locating the desired directory See CHAPTER 11 Using the Filebox Popup for details January 11 2010 10 1 Using the Directory Editor NMRDIR NMRUSER NMRDATA and NMR 10 1 NMRDIR NURUSER NMRDATA and NMRSPEC Shell Variables gege Wixi The NMRDIR NMRUSER NMRDATA and Items Evfstrichnines Selection Evfconotoxin MOES Evfdihydrotestosterones Evfgibberellic_ Evoligamer_ 3D SPECTRUM Evforednisone_noiD13scy Evfstrichnine_ Evsucrose_ INADEQUATE EJANALYS T WiMdata NMRSPEC buttons in the Directory Editor show a his tory list of the corresponding shell variables Up to 50 previous entries for each shell variable are saved in the SHOME analyst setup file For example the shown history list is started by the NMRDATA button and allows setting the NMRDATA shell variable acids Tm These shell variables have the following functions EvANALYST WIN datas Cancel Help HIRD IR HERDATA Specifies the NMRanalyst installation d
19. and Number of Points is 320 Run the 1D Analysis workwindow and the generic L N line list consisting of 78 resonances is generated January 11 2010 6 5 Tutorial IV Advanced Structure Elucidation 15N HMBC Spectrum Analysis oi VN HMBC Spectrum Analysis 40 GO an 100 120 140 H pn With selected N15 HMBC spectrum type switch to the 2D Analysis work window Set Fl 1D Analysis Output File to nitrogen out Due to the use of the generic ION Tine list set Map F1 Frequencies to 58 Hz to cover the 1D Analysis 115 Hz line spacing Set Thresholds Integral to 1 5 In the Report workwindow set Redetermined F1 Resonance List 1 EE Ee gen out Run the 2D Analysis and it runs the Report workwindow From the Graphic workwindow display the n15 hmbc spec file with analyzed correlation locations n15 hmbc_ areas plot as shown Determined nitrogen frequen cies are saved in the specified nni trogen out so an exhaustive search of the nitrogen axis 1s no longer needed N 37 842 48 26 63 aa ia i DN 59 89 Sg A N LEO 14227 47 99 F ada 42 28 In the 2D Analysis workwindow set the F1 1D Analysis Output File to nnitrogen out and delete the Map Fl Frequencies entry In the Report workwindow delete the Redetermined F1 Reso nance List entry to not overwrite the newly created line list Now run the 2D Analysis again and it auto runs the Report workwindow Switch to the Assemblelt workwindow Change t
20. pling constant of 7 Hz vicinal coupling for unfixed conformation and for the analysis of HMBC and N15 HMBC spectra a long range CH coupling constant of 8 Hz typical HMBC pulse sequence optimization Mapping of the coupling constant can be used to overcome inaccuracies in the specified or the default initial coupling constants For COUPLED HSQC a oy value of 150 Hz is assumed Alkyne protons have a 240 250 Hz coupling constant and are not detected by this value Increase this value to 245 Hz for their detection Key USrdcc UsrJdccU Max Ridge Correlation This input field is displayed for the ADEQUATE HMBC N15 HMBC HSQC COUPLED HSQC and N15 HSQC spectrum types These spectra can con tain ridges in F1 or sinc wiggles for underdigitized signals This threshold for ridge volume vs correlation volume is used to avoid the misinterpretation of such ridges as correlations The default value for HMBC and N15 HMBC is 5 5 for COUPLED HSQC and N15 HSQC 5 and for other spectrum types 10 To increase the effectiveness of this ridge suppression increase the specified Map F1 Frequencies mapping range Key Ridge DQF COSY Mapping _ Integrals WFI Phase __imused _ need _ 4 Integrals WF Phase _ Relaxation _ Unused ADEQUATE HETCOR HMBC N15 HMBC HSQC N15 HSQC Mapping WF1 Phase vr Phase Fa Relaxation _ F2 Relaxation 2 Wray V Prog Nucl Magn Reson Spectrosc 1979 13 177 Janua
21. strychnine C tmp cd C tmp strychnine rm E Proton fid carbon tid findit oO o2 o PERR dd aE ae a ae eA HEE A AEE A HE EAE EAE a AEE aa aaa aaa aaa dg dg dg dg dg dd EREEREER EHH d findit Formula Constraints C 2010 ScienceSoft LLC Best matching of 14 5 million molecular structures for 1D H amp C FIDs it uncompressed JCAMP dx files proton amp carbon line lists HSQC or Formula Constraints Use NMRanalyst to inspect intermediate steps f tH HH HH HH HE EE HH HE HE EH HH OE EEE td dr OE EOE EEE SEE EEE EE tu C tmp strychnine Varian HSOC Cl3 ghsqe fid procpar Transforming HSQC projection s Determining most likely FindIt structures Li 0 964760 5304 SCH 04942377 E ZT31L17 of 02929914 16345008 ae O 9Z29067 110313816 of Us926625 10045330 6 0 925083 17556896 Te 10 924929 2165 790 03 0 943604 1160301437 9 U 922883 11489638 LOt Ws 972923 7400761 The top structure PubChem CID 5304 is the correct strychnine structure Replacing the pro ton and carbon spectra by HSQC projections leads to some compromises Integrals of a proton spectrum identify likely CH and OCH3 groups For the proton projection only the signal heights are known but not their line shapes and derived integrals With regard to the carbon projection only carbons with attached protons are detected by HSQC So findit sets in the AssemblelIt workwindow the F1 projection in the 1D DEPT rather than in the 1D
22. the screen number of zero The DISPLAY variable can also be set manually To display the current value of the DIS PLAY variable use echo SDISPLAY on the client side where the analyst program should be run This shell variable setting can be changed for the Bourne and Korn shells with TCP IP stands for Transfer Control Protocol Internet Protocol January 11 2010 9 4 Using the NMRanalyst Window Error Incorrectly Set DISPLAY Variable DISPLAY host 0 export DISPLAY where host is the network node name such as hydrogen company com or the IP network address such as 123 456 789 01 of the computer on which you want to dis play the windows Since standard Linux workstations have only one display per CPU the display number will always be 0 The colon in the example specifies a TCP IP connection When the client and server are running on the same computer the special DISPLAY value 0 or localhost 0 or unix 0 can be used Note that a DISPLAY value specified inside NMRanalyst is only valid for the current NMRanalyst session A DISPLAY value specified from the Linux command line is active only for the programs started from that shell 2 The analyst program does not have the permission to display the application window on the X Window server identified by the DISPLAY variable The X Window server must grant permission to other computers to display windows on it So the owner of the X Win dow server process the
23. 3 EE 14 2 e VA tcc E 14 3 e y ca rer ec seeSesteecc ste eat tees 17 5 HzPpmF3U RE 14 3 BIC OS tbe 17 3 IntLimit E 15 3 EE ee 15 2 IS 17 9 EE egenen 17 2 Eege 15 4 PGE UD scxinedcsspeo steer vnecnstnsarteucantebestbamebesinnsleasestisas 13 2 LAMDIG occ cseee eee eseeeeeceeseeetesesnssessetsseeeeneees 16 8 Pa KE 14 1 LAmbigutes esse cesesessesseseseeeeeneees 17 5 Enbnd 17 6 LAssemblelt c ccccscsecsscssccessssssesesessesestsesevseees 17 8 ICC ET 17 3 11818 oy E Mee ee eee eee eer nen 17 5 January 11 2010 22 6 NMRanalyst Manual Index Eeer 17 6 HS E 18 5 LFindItPos c ceceeceececescseeeseesessesesesesseseseseseeeee 17 7 MUMPTG oes ccecceesceeseeeessessssessetseestssesesesseateees 17 8 LFindItPosMB sosser 17 7 NUMMatch 6 rr 17 6 MU TE I A TE 13 8 GUTS CPUC osa iencisaleseriisitses iasaid ceacoteset 17 9 Te Gb rears perches sieaece stdin tne 13 8 Oi a icttecce eget sctstetesstt civ t teare een eens 14 3 Pe lOO is eeserteecrereseceren ce enrennerdcuedietarenest 13 8 ASS ER 18 6 LHeter0H E 17 4 PA ee 13 6 15 4 LHidel egend 18 3 lee ee 18 5 IER E 13 8 P E 13 8 IERT 13 8 In EE 13 8 Eege 13 8 ee 13 7 IR E 13 8 EE asta seep erase ew xed cesses 18 2 LOvrlp sc taieatinadstisnesisnapisinvednssulbesdeebamsteshigaeliabensssien 16 9 RangeF 1 oiececccecccsccsseeessssessesssstseesseetsessenees 15 5 EPS E 16 8 Range A RER 15 5 LShiftRules oie cess cesesesesesseseseseseeeee 17 4 RETT S 15 5 Lomp RE 16 9 Ranger U saro 15 5 Dy 0 EE 17 7 Range
24. 3D spectra an isosurface plot are created This type of plots provides the greatest detail available on each correlation The plot contains the phase corrected experimental simulated and residual spec tral data for one spectral region All phase components of the hypercomplex data are saved and the desired phase component can be selected when displaying the data Key Cor rPlot 15 5 Automatically Running Report After nD Analysis San REPORT WORKWINDOW AFTER SUCCESSFUL analysts Selecting this RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS automatically starts the Report workwindow after the nD Anal ysis workwindow runs to completion This auto run functionality is a feature of the NMRana lyst user interface and hence does not apply to a backgrounded analyze computing program Key ARunReport January 11 2010 15 13 CHAPTER 16 Report Workwindow This chapter describes the Report workwindow summarizing the automated two and three dimensional spectral analysis results Its input fields and resulting program output are explained To display all the input fields available in this workwindow select from the Edit menu Preferences SetMode Full NMRanalyst Show All Input Fields switches and click OK See CHAPTER 12 Using the Workwindows for a general description of the function and use of a workwindow Program report controlled by the Report workwindow assembles the information obtained from the 1D Analysis
25. Detection deselected F1 Phase 1 32221 7 0 0020997 ppm and F2 Phase 0 38455 0 0548430 ppm Run the 2D Analy sis and it runs the Report workwindow In the Graphic workwindow specify the same phase functions as above and display the shown hsqc spec file January 11 2010 6 3 Tutorial IV Advanced Structure Elucidation 1D Carbon 1D Proton Edited HSQC and 408 411 3 05 3 09 AN 7 Z iz 4 02 44i BA 200 A0 2E E AE 0003 EN g o Ae 407 410 3 04 307 3 68 2 31 g 5 2 70 557 52 48 306 2 67 1 41 623 26 63 426 1 38 42g EA 3 64 2 28 7 20 04 999 116 01 e7 4 02 i 4 23 a72 7 E 128 38 255 7 17 yE 122 347 39i 7 13 3 3 1 3 TE 303 340 5 26 ot 12 7 35 d The resulting shown struc 8 ture plot file illustrates the 2e2 ee sois ar2ei detected correlations in graphi dos cal form 7 11 622 08 710 Elie e TU ben GE Select spectrum type HMBC Switch to the FFT workwindow and load file CLS ghmbc fid procpar Increase F1 Start of Spectrum to 2313 039 and run the FFT Switch to the 2D Analysis workwindow The strongest HMBC correlations result from 2 bond and 3 bond couplings Often the 2D Analysis workwindow Integral threshold can be set so that a sufficient number of 2 bond and 3 bond correlations are detected without detection of longer range correlations But longer range HMBC correlations are indistinguishable from 2 bond and 3 bond correlations
26. Min Integral Precision uu 13 6 Min Signal Distance occ 13 5 N nD Analysis workwindow s 15 1 NMRanalyst analysis Steps siccsteanerepaninnveninactstiisuienicoasteoisecneys 12 1 Assembleht 17 1 avoiding overlap errors 16 9 background computations 0 0cccceeeeeeeeees 9 11 changing spectrum Iepe 21 8 changing UNIX file permissions ss 11s0e11110 9 9 COPY and paste a ec ccs cssesstssetsee testes 21 5 correlation areas searched formula 15 3 CUSTOMIZING windows sssi 10 3 directory editor Dmceton cece eeeeeeeeeeees 9 1 disk space redguitemente eee 2 2 distribution E 2 1 distribution tape contents 0 cece eee 2 2 excluding weak aignals 16 3 January 11 2010 22 8 NMRanalyst Manual Index file locking by backgrounded process 12 8 meaning bond line style 19 11 filebox description 11 1 molecular display 19 5 filebox 11 1 MOUSE FUNCTIONS sssseiiseeeiieseeireeereireeerrreeeerrn 19 12 input fields seeiieeeieeeiireeeieerirresrrrrerrrrsrrereeren 4 1 MOVING iteMS 19 12 installation directorg 2 4 number free atom valences cee eee 19 10 installation instructions 9 3 OUtpUt Summary 0 eee cesses eeeeeeeseeeseeeeees 3 12 manually terminating computations 9 10 PostScript output cece esesteeeeeeeees 19 3 new name for FRED 21 1 undo redo change 19 3 NOE build up Curves sessies 16 10 USC Ol A ete 3 12 phase funct
27. OF LIABILITY ARISING OUT OF OR RELATED TO THE USE OF OR INABILITY TO USE SOFTWARE EVEN IF SUN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES In no event will Sun s liability to you whether in contract tort including negligence or otherwise exceed the amount paid by you for Software under this Agreement The foregoing limitations will apply even if the above stated warranty fails of its essential purpose 6 Termination This Agreement is effective until terminated You may terminate this Agreement at any time by destroying all copies of Software This Agreement will terminate immediately without notice from Sun if you fail to comply with any provi sion of this Agreement Upon Termination you must destroy all copies of Software 7 Export Regulations All Software and technical data delivered under this Agreement are subject to US export control laws and may be subject to export or import regulations in other countries You agree to comply strictly with all such laws and reg ulations and acknowledge that you have the responsibility to obtain such licenses to export re export or import as may be required after delivery to you 8 U S Government Restricted Rights If Software is being acquired by or on behalf of the U S Government or by a U S Government prime contractor or subcontractor at any tier then the Government s rights in Software and accompanying doc umentation will be only as set forth in this Agreement this 1s in accorda
28. Param Workwindow Workwindow Workwin Folder button Sash button eter Tabs each shows input screen dow output starts Filebox adjusts rela input a clock while contains all screen con for selection tive size of fields computation isin parameter set tains program of directories input and out progress tings output and files put screens spectrum type as the selected spectrum type The following three sections SECTION 9 2 Error Message analyst not found SECTION 9 3 Error Incorrectly Set DISPLAY Vari able and SECTION 9 4 NMRanalyst Cannot Find Its Installation Directory provide information on correcting problems that may be encountered during the start of NMRanalyst Should no problems be encountered at this stage skip to SECTION 9 5 Structure and Func tion of the Application Window January 11 2010 9 2 Using the NMRanalyst Window Error Message analyst not found 9 2 Error Message analyst not found This error message indicates your shell cannot locate the analyst program This is caused by an incorrectly set PATH variable or incorrectly installed software Try to find program analyst in default locations for system wide installation usr local bin bin usr bin and for local installations SHOME bin and other users bin directory On MS Windows also search the various top level directories of disk drives If the analyst pro gram cannot be found in program directories try to locate
29. Set the Integral threshold high enough to exclude noise January 11 2010 6 4 Tutorial IV Advanced Structure Elucidation Generation of a Generic IDN Resonance List and spectral distortions from detection Set this field now to 1 5 run the 2D Analysis work window and it auto runs the Report workwindow From the Graphic workwindow display the shown hmbc spec 6 2 Generation of a Generic DN Resonance List A quality 1D proton spectrum can be acquired in seconds But for nuclei such as carbon or nitrogen acquiring the 1D spectrum tends to be slower than acquiring HSQC and HMBC spectra For strychnine the LN HMBC spectrum was acquired without a corresponding 1D nitrogen spectrum Similar to SECTION 5 5 Structure Identification Without 1D Carbon Spectrum a generic line list needs to be generated by NMRanalyst to describe the nitrogen frequencies Select spectrum type N15 HMBC and switch to its FFT workwindow Load file N15 ghmbc fid procpar Run the LN HMBC spectrum transform To generate the generic resonance list for this nitrogen dimension switch to the 1D Analysis workwindow Set the Input File Format menu to Generate Generic List which hides input sections that do not apply Set Use Relaxation Time s to0 015 Copy the four values from the FFT workwindow F1 dimension to the 1D Analysis workwin dow fields of the same name Observe Frequency is 50 655 MHz Spectral Width is 9117 154 Hz Start of Spectrum is 261 032 Hz
30. Start of Spectrum is set to 1754 059 inthe 1D Analysis workwindow The 2D INADEQUATE spectrum solvent is specified as CDCL But the spectrum shows no CDCl but DMSO d resonances So also set F2 Start of Spectrumto1754 059 Run the workwindow 4 5 Displaying All Workwindow Input Fields For the routine use of the software many input fields are not needed and are not displayed Select from NMRanalyst the Edit menu Preferences popup Select the Show January 11 2010 4 7 Tutorial Il Setting Analysis Parameters Determining Phase Functions All Input Fields switch and click OK Now all settings of the software are dis played mm REPORT wm aerer successruL were Only the completed 1D Analysis and FFT workwindows OO require additional acquisition specific information to be loaded Switch to the 2D Analysis workwindow At the bottom of its input screen the RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS switch is shown This switch is selected by default whether it is displayed or not Run the workwindow As soon as the spectral analysis completes NMRanalyst switches to the Report workwindow and runs it to summarize the analysis results With the 2D analysis default settings all but one prednisone carbon carbon bond are detected The 49 78 to 50 45 ppm carbon bond is highly second order and is not detected well by the used pulse sequence The following refinement will detect this bond 4 6 Determining Phase Functions
31. a copy of the provided oligomer 3D SPECTRUM dataset Start the Directory Editor specify the oligomer 3D SPECTRUM directory in the NMRDATA field delete the NMRSPEC entry and click OK 1 Courtesy of Paul A Keifer Ph D Varian Inc January 11 2010 7 5 Tutorial V Additional NMRanalyst Features Analyzing 3D Spectra The 1D line lists for the 3D analysis are normally generated generically as described in SEC TION 5 5 Structure Identification Without 1D Carbon Spectrum and SECTION 6 2 Gen eration of a Generic N Resonance List Other generation methods described for 2D spectra can be used as well For this heteronuclear 3D dataset the three line lists files C lines N lines H lines are provided Select the FFT workwindow and load HNCO fid procpar A 3D Varian procpar file does not indicate which decoupler fre quency belongs to which spectral dimension So follow NMRanalyst s mapping convention for the data acquisition as implemented in shell script varian2txt or modify this shell script to fit your 3D acquisition preferences When the Transform Order menu lists the pulse sequence used such as Varian hnco in this case select the corresponding item to request a native FFT If the used pulse sequence is not listed select the Coefficient File item and the 40 transform coeffi cients are read from the file specified in the 3D FFT Coefficient File field The coef coefficient file for the current dataset 1s su
32. allow the PC or several PCs direct access to specified Linux file systems For example acquired NMR data from the Linux side can be accessed directly from the PC without having to copy these files or directo ries to the PC and NMRanalyst results can be written directly to the Linux file system Another useful Samba feature is printing from the PC to printers on the Linux side 21 4 Can Irun NMRanalyst on an Apple Macintosh Dr Dimitris Argyropoulos Varian Germany tested Windows NMRanalyst on an Apple Pow erBook G4 running Mac OS 10 2 2 and Virtual PC with Windows 2000 Professional Virtual PC involves the emulation of an Intel processor So Windows NMRanalyst becomes several times slower than running natively on an Intel processor of similar speed But it functions as expected 21 5 How to display new MS Windows applications as foreground windows Microsoft enabled a Windows feature by default years ago Applications were not allowed to display foreground windows or new foreground windows were not recognized as belonging to the current foreground application When starting a new NMRanalyst UNIX Shell Help NMRplot or NMRgraph window on such Windows versions the new window was shown January 11 2010 21 4 Frequently Asked Questions Any UNIX editor available from the Windows below other displayed windows The application would then flash in the taskbar 3 times by default indicating this window was denied the privilege to
33. apa EN 11 6 8 Identifying the Molecular Structure Through Shift Prediction ccccccceceeees 12 CHAPTER7 Tutorial V Additional NMRanalyst Features 7 1 7 1 Using an HSQC FID for the findit Structure Identification cccceeeeeeeeeeeeees l 7 2 Displaying Experimental Simulated and Residual NMR Snpecta 3 CS UE E A o E 5 E i EEN J 7 5 Incorporating NMRanalyst Programs in Other Software 8 DE Thenie seo E 9 Reference Manual CHAPTER 8 Using the Help System c e 8 1 S1 A E Mee so caiseienssontrnacin sor unsnwtnncedinndeansbaleeateneen iene ANENE EE Eiana iea 1 Sa Navigating Through the Help Pe EE 2 s9 Kampe Ui EE E cecil ee ees 2 E SR er iscsi eens 4 January 11 2010 5 NMRanalyst Manual Contents CHAPTER 9 Using the NMRanalyst Window 6 ccccccceeeeeeeeees 9 NN WEE ere Ones See nent Sr eT Seb EEN l 9 2 Error Message analyst not found sisiicecisasssscensssvacnanavessadesrasiarievnsanedssennenntatasensaees 3 Poo Error heorectiy Set DISPLAY Variable dicen cine netic rete 4 9 4 NMRanalyst Cannot Find Its Installation Directory ennnesseseesssossssnsserreneee 6 H Structure and Function of the Application Wumdow 6 Ae Me ve EE 7 Ne Pe Pon E 8 98 The Application Window Menus a cssasins ecco nsonictninniancanadeneniansananiorar tenanmiatpeioannes 11 oa Tie Pe a E 17 9 10 Copyright Enscript Propra eereeirereneiieeini ceneni ia iii 18 9 11 Copyright GNU Ghostscript Program s ccces
34. be improved but NMRanalyst already effectively supports using NMR as a structure elucidation tool 1 2 Identification of Best Matching Molecular Structures The most powerful NMR structure elucidation is based on 2D INADEQUATE spectra but they are insensitive The next best approach are indirect detection methods involving DQF COSY HSQC or HMQC and HMBC spectra Both approaches are covered in this manual PubChem http pubchem ncbi nim nih gov has published over 15 9 million distinct small molecular structures Identifying the best matching structures from this collec tion for available NMR data is faster and can be more reliably automated than a full structure elucidation FindIt VerifyIt and the derived findit script are described in the manual and we have recently published the approach 1 Reinhard Dunkel Xinzi Wu Identification of organic molecules from a structure database using proton and carbon NMR analysis results Journal of Magnetic Resonance 188 2007 97 110 January 11 2010 1 4 Overview Using this Manual 1 35 Using this Manual This manual consists of three parts Part one is the NMRanalyst User Manual It begins with CHAPTER 1 Overview It proceeds to the software installation CHAPTER 2 NMRana lyst Installation Then five recipe style tutorials CHAPTER 3 Tutorial I Using NMRan alyst CHAPTER 4 Tutorial II Setting Analysis Parameters CHAPTER 5 Tutorial III Combining A
35. bond correlations HMBC correlations at or below the Weak threshold can involve any number of bonds Key FnHMBC THHMBC H15_HMBC az meest Log Ca _weax To place nitrogens in derived molecular skeletons N HMBC and or HSQC spectra can be used Specify the location of the N HMBC Report workwindow report log file in this field To observe a nitrogen carbon bond using this data the bonded carbon has to be protonated But AssemblelIt can recover unobserved bonds if 3 bond HMBC correlations involving the unobserved bond are January 11 2010 17 3 Assemblelt Workwindow Combine NMR Analysis Results observed See the input field above for the use of the Weak threshold Key FnNHMBC ThHNHMBC woe _fis0c report 1og EU Information about direct carbon proton bond information is required for running the Assemblelt task Inverse detected hetero nuclear correlation experiments like HSQC and HMQC are supported Multiplicity editing of these spectra is recommended The detected phase difference between methyls and methines vs methylene groups is used to set the number of free valences for each carbon Key FnH SQC TADEQURTE _ INADEQUATE report log a To include 2D INADEQUATE correlations in the carbon skeleton determination specify the location of its Report workwin dow report log file Its correlations are a direct way to determine carbon carbon bond information In contrast to indirect detection spectra no protonation of in
36. changes 2 F rst A Pretsch E Robien W Comprehensive parameter set for the prediction of the 13C_NMR chemical shifts of sp hybridized carbon atoms in organic compounds Anal Chim Acta 1990 233 213 222 Pretsch E Forst A Robien W Parameter set for the prediction of the 13C NMR chemical shifts of ep and sp hybrid ized carbon atoms in organic compounds Anal Chim Acta 1991 248 415 428 Schaller R B Arnold C Pretsch E New parameters for predicting IH NMR chemical shifts of protons attached to carbon atoms Anal Chim Acta 1995 312 95 105 Matter U E Pascual C Pretsch E Pross A Simon W Sternhell S Estimation of the chemical shifts of olefinic protons using additive increments II the compilation of additive increments for 43 functional groups Tetrahedron 1969 25 691 697 Beeby J Sternhell S Hoffmann Ostenhof T Pretsch E Simon W Estimation of the chemical shifts of aromatic protons using additive increments Anal Chem 1973 45 1571 1573 Pretsch E Buhlmann P Affolter C Structure Determination of Organic Compounds Tables of Spectral Data 3rd ed Springer Berlin 2000 January 11 2010 19 4 NMRgraph Molecular Correlation Editor Pull Down Menus View The View menu contains items to alter the display of all RES ei SUE Structures Zoom R354 EES oles NMRgraph s structure layout is applied at the start of Hide Correlation Labels Ctrl B 75 Hi
37. data to Sun or its service provider about those specific processes to help Sun understand and optimize them Sun does not associate the data with personally identifiable information You can find more information about the data Sun collects at http java com data For inquiries please contact Sun Microsystems Inc 4150 Network Circle Santa Clara California 95054 U S A LFI 143333 Form ID 011801 January 11 2010 9 21 Using the NMRanalyst Window Copyright PC Cygwin UNIX Toolset 9 13 Copyright PC Cygwin UNIX Toolset Most of the provided UNIX tools are covered by the GNU General Public License GPL though some are public domain and others have a Berkeley style copyright See http cygwin com for the source code a verbatim copy of the GPL license and the available documentation about this tool set The Cygwin software is not an integral part of NMRanalyst nor does ScienceSoft support it It is provided AS IS in the hope that its UNIX like commands will be found helpful January 11 2010 9 22 CHAPTER 10 Using the Dir eclory Editor Directory Editor ell The Directory Editor can be started from the Directory Editor H H oOo al button in the NMRanalyst applica HMRUSER E ANALYST WIN UserData al f wenn EE al tion window as described in CHAP 7 TER 9 Using the NMRanalyst HMRSPEC rue QUATE E e Window The editor window shows Cancel the current values of the NMRDIR NMRUSER NMRDATA
38. identified 2D correlations bond signals to the pre viously determined 1D resonance frequencies SECTION 3 4 The ID Analysis Workwindow CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 RESONANCE 4 Shift Shift J Index Shift J Index Shift J Index Shift J Index ppm ppm Hz ppm Hz ppm Hz ppm Hz 211 41 87 40 46 29 7166 08 40 71 8 210 19 58 81 37 95 28 49 78 37 80 30 135 031127 0 50 81 ES e 43 167 18 123 7 63 86 60 41 91 36 15 67 31 52 38 67 71 155 05 127 0 62 66 75 41 91 40 53 83 126 99 185 0 50 81 42 155 0 62 66 75 123 74 165 0 53 56 43 167 2 63 86 60 87 396 211 4 46 29 7 50 45 37 17 122 33 56 37 41 127 66 077 211 4 40 71 8 58812121072 37 95 28 41 91 33 74 148 35 56 28 82 149 50 448 87 40 37 17 122 49 78 30 297 156 48 75 31 89 157 15 49 36 34 165 49 777 210 2 37 80 30 50245 30 27 156 48 749 50 45 31 89 157135 56 36 22 176 22 82 33 16 180 41 913 167 2 36 15 67 155 0 40 53 83 58 81 33 74 148 18 73 32 83 188 35 555 58 81 28 82 149 48 75 36 22 176 33 13 34 82 191 33 562 87 40 37 41 127 22 82 32 76 198 335130 35 56 34 82 191131452 31 41 201 S1 2518 167 2 38 67 WINS e E 31 41 201 22 2824 48 75 33 16 180 33 56 32 76 198 18373041291 32 88 188 15 494 50 45 36 34 165 2 The integral precision is the number of marginal standard deviations that the determined integral value of the bond li
39. is granted under this Agreement Additional restrictions for developers and or publishers licenses are set forth in the Supplemental License Terms 4 LIMITED WARRANTY Sun warrants to you that for a period of ninety 90 days from the date of purchase as evidenced by acopy of the receipt the media on which Software is furnished if any will be free of defects in materials and workman ship under normal use Except for the foregoing Software is provided AS IS Your exclusive remedy and Sun s entire lia bility under this limited warranty will be at Sun s option to replace Software media or refund the fee paid for Software Any implied warranties on the Software are limited to 90 days Some states do not allow limitations on duration of an implied warranty so the above may not apply to you This limited warranty gives you specific legal rights You may have others which vary from state to state 5 DISCLAIMER OF WARRANTY UNLESS SPECIFIED IN THIS AGREEMENT ALL EXPRESS OR IMPLIED CONDITIONS REPRESENTATIONS AND WARRANTIES INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT ARE DIS CLAIMED EXCEPT TO THE EXTENT THAT THESE DISCLAIMERS ARE HELD TO BE LEGALLY INVALID 6 LIMITATION OF LIABILITY TO THE EXTENT NOT PROHIBITED BY LAW IN NO EVENT WILL SUN OR ITS LICENSORS BE LIABLE FOR ANY LOST REVENUE PROFIT OR DATA OR FOR SPECIAL INDIRECT CONSE QUENTIAL INCIDENTAL OR PUNITIVE DAMAGES HOWEVER
40. list in this popup shows from top the oldest value to bottom the newest value previous values of the text field Any previous value can be selected and the value is copied back to the input field when OK 1s clicked The Cancel button removes the History popup without changing the text field content For input fields in the input screen like the one m the previous example the standard editing facilities are implemented An input field can be edited by moving the mouse pointer to the desired character position and clicking the first left mouse button to give the input field the keyboard focus Text entered from the keyboard is inserted at the current cursor position The text cursor can be moved using the keyboard left and right arrow keys The Backspace key can be used to erase the character before the text cursor The following table describes the implemented additional control characters sup ported by workwindow input fields Ctrl Insert ont EST Delete Shift Insert Ctrl b Ctrl f Cee d Ctrl k Home End Down Arrow copy clipboard cut clipboard paste clipboard backward one character forward one character kill next character kill characters from cursor position to end of line move cursor position to beginning of line move cursor position to end of line show next history entry for the input field January 11 2010 12 4 Using the Workwindows The Workwindow Input Scree
41. plant hormone Its natural occurrence controls the plant development In gardening GA is used for seed germination growth promoter early flowering and increasing the yield of greenhouse tomatoes January 11 2010 5 1 Tutorial Ill Combining Analysis Results Analysis of 1D Spectra 5 1 Analysis of 1D Spectra NMR based structure elucidations start with the analysis of 1D survey spectra The proton spectrum is fast to acquire and is regularly used Carbon and nitrogen 1D spectra are less sen sitive to acquire NMRanalyst supports deriving their shifts indirectly as described later 1n this chapter From the NMRanalyst application window click the Directory Editor button Set NMRDATA to the copied gibberellic acid directory set NURSPEC to HMBC and click OK Switch to the 1D Analysis workwindow Load from directory carbon fid the spec trum specific parameter file procpar Run the workwindow From the Graphic work experimental Window display the cre IL ated and shown carbon plot file All resonances are detected as shown by their black color The strong reso nances around 40 ppm belong to the DMSO d solvent In the 1D Analy sis workwindow load from directory HMBC the file ld ave Sie HOG This procedure loads the reference corrected carbon resonances in the input screen and selects the Remove switches for the seven DMSO d resonances Rerun the workwindow 195 180 160 140 120 100 ao Bo AC 20 0 5 Fre
42. s Output file nD Analysis al analyze txt workwindow gt analyze log Plot file report log EE sl xe workwindow p report log Plot files Ett bog Graphic Graphic Ae workwindow graphic log Spectrum Graphical Presentation Result Summary Simulation January 11 2010 12 9 cuaptert3 1D Analysis Workwindow This chapter describes the details of the 1D Analysis workwindow All its input fields are explained along with their default settings for the analysis of carbon spectra To display all the input fields select from the Edit menu Preferences Select the Full NMRanalyst and the Show All Input Fields switches and click OK Depending on the selected spectrum type different default settings are provided For a general description of function and use of a workwindow see CHAPTER 12 Using the Workwin dows The 1D Analysis workwindow provides the user interface to the 1d analysis program Its primary use is to read a 1D FID or Bruker or Varian software transformed spectrum and to analyze it Its output is used as input for the multidimensional spectral analysis and the Findit VerifyIt and AssemblelIt generation of molecular structures When no acquired 1D spectral data 1s available similar information can be generated A line list of shifts can be converted to NMRanalyst format A two dimensional spectrum can be projected on its axes and peak picked Three dimensional projections need to be generated by the Varian VNMR or othe
43. settings of the input screen objects the default settings The NMRanalyst software comes with a complete set of default txt files for each sup ported spectrum type These default files are used whenever the current SNMRDATA SNMR SPEC directory does not contain an appropriate txt file Upon the initial display of a workwindow or a NMRDATA directory or a spectrum type change the corresponding txt file is loaded automatically The following popup should not be encountered if the NMRana lyst software is installed as described in CHAPTER 2 NMRanalyst Installation However a user can supply modified txt default files and require the workwindow to use them by setting the NMRUSER shell variable appropriately If txt files are missing from a modified spectrum specific NMRUSER directory the following popup results Click Cancel or Exit whichever button is dis played to abort the load process Check the Directory Edi tor NMRUSER and NMRSPEC variables Either change the variables or copy the missing txt file into the SNMRUSER SNMRSPEC directory Workwindow Load Warning q x Could not load default file 1d_analysis tt f from the EJAMNACYS TWI Midata NADE GLATE nor the FNMRUSERINADEQJATE directory Cancel Help Workwindow History Information x The workwindow input screen keeps an unlimited history of previous input values Should this information exceed 200 KBytes the shown popup i
44. soon as the user changes input screen values The following are the input value files corresponding to the six workwin dows of the NMRanalyst application 1D Analysis workwindow 1d analysis txt FFT workwindow PT Ose nD Analysis workwindow analyze txt Report workwindow report CRE Assemblelt workwindow assembleit txt Graphic workwindow graphic txt An input value file contains the input screen values as a set of key value pairs The txt file name ending stems from the fact that these are plain text files The following examples explain the kinds of interface objects used in the input screens 1D FID Spectrum or Line List la eiaveia al The above example contains three related interface objects widgets The button on the left describes the function of the input line and shows a History popup described below when clicked The text field in the middle is used to enter one piece of information such as a number or file name described below The folder button at the right side of the example starts a File box allowing the interactive location of directories and files as described in CHAPTER 11 Using the Filebox Popup January 11 2010 12 3 Using the Workwindows The Workwindow Input Screen History 1D FID Spectrum or Line List wl Items Selection agang OK Cancel Help The button with the text label in the previous figure dis plays the shown History popup when clicked The his tory
45. specifying initial values for the graphic program When started the graphic program translates the input values into the nmr graph or nmrplot command and starts the corresponding plotting application All plotting applications are run as background processes It may take minutes for the plot rendering pro gram to show the requested plot January 11 2010 18 6 CHAPTER 19 NMkeraph Molecular Correlation Editor NMRanalyst creates a numerical description of spin systems identified in multidimensional NMR spectra An intuitive presentation of this information is the NMRgraph plot of identified molecular correlations For example the 2D INADEQUATE analysis results are displayed by NMRgraph as the identified molecular carbon skeleton The AssembleIt module combines structural information from several spectrum types Resulting structural information 1s dis played by NMRgraph Molecular structures in Molfile or SDfile format can be displayed Car bon and proton chemical shifts can be predicted and used to sort molecular structures by the agreement between observed and predicted shifts NMRgraph is a structure editor and its interactive features are described in this chapter Carbon atoms are represented by their chemical shifts in parts per million ppm Heteroatoms show the element symbol potentially followed by the chemical shift Correlations between atoms are represented as lines or arrows The correlation labels show for DQF COSY HMBC and
46. supported spectrum types VERSION Current version and possibly patch level of the NMRanalyst software ban Directory containing programs libraries shell scripts FindIt structures and chemical shift information cygwin Directory in Windows NMRanalyst to emulate the UNIX Shell and the UNIX commands available from this shell data January 11 2010 2 4 NMRanalyst Installation Preparation for the Linux Installation Small dataset 1D carbon and 2D INADEQUATE of prednisone discussed in the tutori als enscript Program enscript and supporting files for converting text to PostScript gs Ghostscript program and supporting files for printing PostScript files on non PostScript printers help NMRanalyst Manual in JavaHelp format for online display Jre Sun Java Runtime Environment JRE Standard Edition Version 5 0 2 53 Preparation for the Linux Installation The Linux system wide NMRanalyst installation requires root privileges An individual user can install the NMRanalyst software for private use without root privileges l For a system wide installation obtain super user privileges now by typing O su root s Password and by supplying the password Normally the user command prompt changes to for the root account 2 Insert the NMRanalyst installation DVD in a local drive and the shown DVD logo appears on the desktop Right click on this logo and select Mount and mount the
47. the UNIX Shell and issue the command rm r Garbon With selected HMBC spectrum type display the 1D Analysis workwindow Set Input File Format to Generate Generic List to generate an initial carbon reso nance list Copy the FFT workwindow F1 settings into the corresponding 1D Analysis input fields Observe Frequency of 125 704 MHz Spectral Width of 22522 523 Hz Start of Spectrumof 879 678 Hz and Number of Points of 1024 Click Start The resulting 254 carbon resonance frequencies are 88 0646 Hz apart covering the whole HMBC F1 carbon range They are saved in the specified file carbon out Now analyze the HSQC spectrum with carbon out Switch to the HSQC spectrum type Its 1D Analysis and FFT workwindows are unaffected Switch to the 2D Analysis workwin dow The F1 1D Analysis Output File uses the carbon out generated list To cover its resonance spacing set Map F1 Frequencies to 45 Hz half the resonance spacing Increase Thresholds Integral to 70 as the extensive mapping creates slightly larger values than before Select the F1 Phase and F2 Phase switches in the Mapping and Detection sections Remove both phase functions Fl Phase and F2 Phase fields as they were determined with the help of the 1D carbon spectrum In the Report workwindow specify the Redetermined Fl Resonance List as carbon hsqc out Run the 2D Analysis workwindow and it auto runs the Report work window Twelve carbon resonances are stored in carbo
48. the NMRanalyst installation direc tory called ANALYST WIN or ANALYST LINUX depending on platform Default installation locations are usr local HOME opt export home usr home and the top level directories of PC drives If the installation directory can be found start ana lyst from its bin subdirectory If the installation directory cannot be found either then determine if the analyst program is accessible anywhere on the computer using the Bourne and Korn shell command O find type f name analyst print 2 gt dev null If this step fails NMRanalyst is not installed or not accessible from the current user account In either case consult your system administrator If the analyst program can be found the problem with locating the analyst program is caused by an incompletely set PATH vari able A UNIX shell only automatically searches for a program in the directories which are listed in the shell s PATH variable The current value of the PATH variable with directories separated by colons can be displayed by echo SPATH Add the directory in which you found the analyst program say usr local bin to the PATH variable for the Bourne and Korn shells with PATH SPATH usr local bin export PATH To avoid having to retype these settings after every login the above PATH extension com mand should be added to the user s profile file for the Bourne and Korn shells January 11 2010 9 3 Using the NMRanalyst Win
49. the determined molecular structural information The graph of determined correlations is saved when a file name is provided in this input field The graph can be displayed using the Graphic workwin dow See CHAPTER 18 Graphic Workwindow For 2D INADEQUATE spectra the resulting graph shows the determined carbon skeleton For heteronuclear spectra the displayed fragments consist of one carbon atom with correlated proton frequencies For NOESY ROESY and TOCSY spectra each displayed fragment con tains a proton surrounded by correlated proton frequencies The Assemblelt workwindow allows combining the information from different spectrum types and deriving the possible car bon skeleton s from it Key FnSk1t Phase Functions Jr esegier a Specify a name for the plot file of determined phase values and phase functions This plot shows the precision with which the report program determines the spectral Phase Functions The plot file is created when a file name is specified in this field Use the Graphic workwindow to display it Key FnPse Correlation Locations inadequate_areas p1ot ss The Report workwindow produces a connectivity table and warnings This output file contains a plot of areas of 2D or January 11 2010 16 2 Report Workwindow Output Files volumes of 3D spectrum showing the location of determined correlation signals The plot file is created when a file name is specified in this input field Use the Graphic wor
50. the printer name has the format computer name printer name NMRana lyst changes Windows backslashes to regular slashes Make sure this computer_name can be reached over the network and printer name corresponds to a shared printer On Linux printer names from the Print Manager can be used directly whether they are locally or remotely attached If the current workwindow output screen contains text print it by clicking the Print but ton in the NMRanalyst Print Settings popup Otherwise specify any text file in the Print File field and print it If the resulting page is printed but contains formatting instructions or graphic symbols select a more appropriate printer driver The printer settings are saved in file analyst and are used for future NMRanalyst printing until explicitly changed You may reset the PC default printer setting above if it was changed In case of further printing problems save PostScript files to disk and find a way to print them 21 10 How can I capture NMReraph and NMRplot displays On MS Windows display the desired plot and copy the whole screen contents onto the clip board by pressing the Print Screen keyboard key Then start a drawing program such as Start Programs Accessories Paint paste the clipboard content in the drawing area crop the image as desired and save the image into a file On Linux capture the screen image with the X Window dump utility and select the window to be captured by cl
51. this workwindow Key F1Pse0 F1Pse1 F1Pse2 through F3Pse0 F3Psel F3Pse2 By default the plot type for 2D spectra is Contour The Sur face option shows the surface of the 2D spectrum carpet plot The Surface plots are similar to the whitewash mode of display ing 2D spectra but now the spectra are true 3D objects shown with depth perception perspective and can be rotated Key plotType 2D Plot Type A phase insensitive 2D spectrum consists of two a phase sensitive 2D spectrum of four and a phase sensitive 3D spectrum of eight phase components NMRanalyst spectra contain these phase components The phase component to be plotted can be selected from this menu In the menu label R stands for the real part containing the absorption mode signals after phasing Label I stands for the imaginary part containing January 11 2010 18 5 Graphic Workwindow Function of the Graphic Programs the dispersion mode signals The first character of the label corresponds to the F1 dimension the second one to the F2 dimension etc The default phase component is RR RRR Key phase NMRplot application supports axis labeling in ppm Hertz Hz and point num bers NP NMRanalyst spectrum files contain the spectral referencing and can be displayed in all the three units without requiring additional information Key unit 18 4 Function of the Graphic Programs The Graphic workwindow provides an interface for
52. ware acceleration slider to None Click OK to apply the settings On Linux black lines from the edges of moved atoms or correlation lines may remain on an NMRegraph display window To remove such artifacts click the right mouse button over an empty space on the desktop From the displayed menu select Refresh Desktop January 11 2010 21 9 Frequently Asked Questions How to increase the paging space on Linux 21 14 How to increase the paging space on Linux To overcome limitations of available physical memory Linux uses virtual memory hard disk space often called paging space or swap space The following table describes adding an additional 100 MBytes of paging space on Red Hat Linux Operating System Task and UNIX Command Red Hat Linux List all paging spaces sbin swapon s Create new paging file bin dd if dev zero of swapfile bs 1024 count 102400 sbin mkswap swapfile Activate paging file sbin swapon swapfile Modifying the system paging spaces requires root privileges To permanently make this modification edit etc fstab to include swapfile swap swap defaults 9 i0 21 15 How to specify correlations for the Assemblelt structure generation AssemblelIt generates possible molecular structures from correlations carbon carbon nitro gen carbon etc contained in a specified plot file These correlations are typically cre ated by combining NMRanalyst spectral analysis results But c
53. 010 Tutorial Ill Combining Analysis Results Assemblelt HSQC amp HMBC Derived Gibberellic 5 4 Assemblelt HSQC amp HMBC Derived Gibberellic Acid Structure Hetero nuclear 2D NMR spectra can be acquired of samples an order of magnitude smaller than for the direct carbon skeleton determination through 2D INADEQUATE Assemblelt is our approach to solving the resulting puzzle of deriving the molecular carbon skeleton The detected hetero nuclear information can be incomplete or ambiguous NMRanalyst allows up to a specified number of experimental correlations to be 4 bond or even to be incorrect assign ments and the best possible structures can still be derived Display the AssembleIt workwindow It first combines results from analyzed spectrum types Specified non existing input files in the COMBINE NMR ANALYSIS RESULTS section cause a warning and are ignored From the row of switches select Consider Chemi cal Shift Rules to identify likely double bonds and the location of carbonyl groups Also select Ambiguities to consider correlations with assignment ambiguities Result ing carbon carbon correlations are stored in the file specified in the Output Atom Atom Correlations field which is set to CC_corrs plot by default The input field is dis played when the Edit Preferences Show All Input Fields switch is selected Assemblelt derives the most likely molecular skeleton s from the above derived correlations It generat
54. 010 11 2 CHAPTER 12 Using the Workwindows The NMRanalyst analysis of multidimensional spectra can be divided into four separate com puting tasks the analysis of one or more 1D spectra the Fast Fourier Transform FFT of the multidimensional FID or import of a Fourier transformed spectrum the spin system identifi cation in the multidimensional spectrum and the summary of obtained analysis results Two further tasks are supported AssemblelIt provides the identification of best matching from mil lions of candidate structures dereplication verification of a specified structure and combi nation of analysis results from several spectra and derivation of possible molecular skeletons The other task is the graphic display of spectral plots and molecular structures For all the tasks the user can 1 specify input values for the task 2 start and control the progress pause continue background foreground and stop the task and 3 examine the program out put The workwindow provides the input output functionality using a consistent user inter face The NMRanalyst application provides the process control features for all the workwindows A workwindow can be displayed by clicking on the 1D Analysis 2D FFT 2D Analysis Report AssembleIt and Graphic tabs in the NMRanalyst window Each workwindow controls a separate program which can be run independently of other programs and even independently of the user interface 1 Bu
55. 10 estimated coupling Constant 15 6 estimated variation of 1D frequencies 15 5 22 1 January 11 2010 NMRanalyst Manual Index estimating coupling Constants 15 6 example 3D analysis c ceccccecsscseseesesesteseeteseeeee 7 6 excluding weak signals seeiiseeeiseeeireeeerseeen 16 3 function OF sexes dasscateiscdacstebeicesinneasdustandasseortaestonsnes 12 2 general features occ esses 15 1 Mapping parameters oo eee esses 15 8 number of mapping points 15 6 ST EE 3 8 Se E 15 2 saving fitting volume pilote 7 7 searching specific spectral regions 15 3 specifying spectrum fupe 15 2 anti phase correlation DOF COSY n 1 3 INADEQUATE AA Le Assemblelt specifying correlations 21 10 assembleit program 3 membered rings eeceeeeseseeseeeseeetseeseseeseeeeeees 17 9 A membered ring 0 cscscseeecssesseseesessessseseeseeeee 17 9 adding additional bonde 17 8 allowing correlation violations 0001100011 17 9 ambiguity in heteronuclear data 17 9 Assemblelt component ccccccccceseeseeeeeeees 17 1 correlation circle d agram 17 5 correlation not detected c ceceeeeseeeeeeeeees 17 8 deriving unobserved correlation 17 3 17 4 17 8 PUG TASK E 17 1 Meaning atom color 19 10 meaning correlation line style 0001100111000 19 11 missing COTTE AtIONS eects ese eeseeeeeeeeeeees 17 1 number free atom valences cceeeeeeee 19 10 proton and
56. 11 2010 20 4 NMRplot Plotting Multidimensional Spectra Copyright Mesa The Help menu Nmrplot displays the current manual chap GER ter Tutorial shows Tutorial II with application examples The EE NMRplot application is started by the Graphic workwindow Files dis nD Analysis Workwindow played by NMRplot can be created by the 1D Analysis nD Analysis and EE Report workwindows The menu items 1D Analysis Workwin dow nD Analysis Workwindow Report Work window and Graphic Workwindow display the manual chapters on the 1D Analysis nD Analysis Report and Graphic workwindows respectively About NMRplot shows program version information About MiiRplot 20 4 Copyright Mesa Copyright C 1999 2005 Brian Paul All Rights Reserved Permission is hereby granted free of charge to any person obtaining a copy of this software and associated documentation files the Software to deal in the Software without restriction including without limitation the rights to use copy modify merge publish distribute sublicense and or sell copies of the Software and to permit persons to whom the Software is fur nished to do so subject to the following conditions The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software THE SOFTWARE IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EXPRESS OR IMPLIED INCLUDING BUT NOT
57. 1416 ChiSq 101814574E 01 The information about a correlation labeled 6 1s shown The first line 6 C12 C13 COR RELATION Fa 30 038 Fb 22 013 J 31 97 1s the summary line discussed below The following lines from top to bottom contain the parameters for both spins of the reported correlation Tal 2 Both integrals for spin A Ib1 2 Both integrals for spin B Fa bb F2 transition frequency of spins A and B Pa Pb F2 phase of spins A and B Ta Tb Effective relaxation time of spins A and B ierg F2 acquisition time and coupling constant Fd Pd _ FI transition frequency and phase Tr Td Fl relaxation and acquisition time January 11 2010 16 4 Report Workwindow Select Items to Be Reported The six columns of numbers in this table specify from left to right the initial estimate the final value and the error value for the first parameter and the initial final and error value for the second parameter A negative error value indicates that the corresponding initial parameter estimate was not optimized during the regression analysis EXAMPLE In the above parameter list the estimated coupling constant J for this bond in a 2D INADEQUATE spectrum is 35 0000 Hz and the best fit determined coupling constant is 31 9676 Hz with a marginal standard deviation uncertainty of 0 2669 Hz The last line in the above parameter list gives some statistical information about the reported pattern Of particular interest is the agreement valu
58. 1d fid and then file procpar 2 In the FFT workwindow Load Filebox select directory 2d fid and then file proc par For Varian format FID data the directory name always ends on fid and the file with acquisition parameters is always called procpar 3 The sucrose sample does not contain a referencing compound and its D20 solvent contains no carbons So no solvent resonances need to be removed in the 1D Analysis workwindow January 11 2010 4 13 Tutorial Il Setting Analysis Parameters Analysis of the Sucrose VNMR Created Spec 61 8032 104 05 467 76 87 We 74 41 s9 1 73 42 62 73 The determined sucrose carbon skeleton is shown A 2D INADEQUATE spectrum does not detect carbon oxygen bonds and two carbon fragments result To reproduce stated shifts and coupling constants make sure the report Log file is loaded in the 2D Analysis workwindow to set and lock the F1 and F2 phase functions 60 55 ee d I A1 mm Edem GE ee l eg Am EE 4 11 Analysis of the Sucrose VNMR Created Spectrum Besides transforming FIDs NMRanalyst can import and analyze phase sensitive spectra cre ated by Varian eg VNMR and Bruker eg XWIN NMR software Incentives to analyze externally created spectra include e The data was acquired on a spectrometer of an unsupported vendor or is in an unsupported format As long as the Varian or Bruker software can transform this data NMRanalyst can analyze the resulting spectrum e The pulse sequence use
59. 2 12 NMRanalyst Manual Index UNIX Shell button 9 10 user manual Content 1 5 v RTA e T 17 7 VNMR color cell usage 21 8 W weighting Tunctpon 13 2 Kee 2 1 problem displaying foreground window 21 4 supported computer platforms 0 cceee eee 2 1 TweakUI application eects 21 5 Windows NMRanalyst accessing UNIX Dies 21 4 changing drive Jetter 21 3 characters in directory amp file names 2 7 21 2 SE KE nee ro 21 5 SE 21 1 device characterg 2 7 EE E 2 6 Notepad uge 21 5 NTFS access Demmiseiong eee 21 2 printing on UNIX printers eee 21 4 problem displaying foreground window 21 5 process killed on Jogout 21 2 Samba software 21 4 specific Characteristics c ccceceeseeseseeeeeeeees 21 1 specifying drive letter oo eee 21 3 TweakUI application oo 21 5 UNC path names esses eseseeseseeseeeee 21 3 tM Sta At EE 2 8 EE 21 5 Windows 2000 2 1 Windows Vista 2 1 Windows XP 2 1 WordPad use 21 5 workwindow backgrounding Computattons 9 1 check symbol use 12 6 clock C10 5 21 8 editing using emacs Commandes 12 4 graphic workwindow features 18 1 history WINdOW USE ou eeeeeseeseseesee este eeeeeeeeeees 12 4 Input screen 9 8 12 3 12 5 input screen default 12 6 loading input values tees 12 3 nmrgraph EE 19 2 numerical input cesses tees eeeeeeeeees 12 5 numerical unit selecton eee 12 5 output log Ble cess setseseseeteneees 12 7 OUtPUt SCLEEN ooo ecc
60. 20 1 ChemDraw eXchange output 19 2 NOESY display of cortelattons 19 1 1 2 ACTONYM MEANING oo eeeeseeseeseeseesessesessessessesseeseeees keyboard key Dmcttons 19 13 SSC e optimization detection parameters 15 9 MDL Molfile output 19 2 MDL SDfile om 19 2 Number of Ponts 14 4 Meaning atom color 19 10 meaning atom label s 19 1 January 11 2010 22 9 NMRanalyst Manual Index O increasing plot resolution 0 eee eeees 18 2 integral distribution oo eee eeeeeeeee tees 16 3 Observe Freguency 13 4 14 3 15 5 NOE build up Curve seess 16 11 oligomer overlay pilote 20 3 AGAGAAAG Sequence ovna 7 5 phase correction oo eeeeeeceeeseseeseseseesteesteeseeeeeees 18 5 demo dataset 2 3 phase baseline error values unn UE HNCO spectrum ccccsccsccesssssssssssesssessssessessssesevnese 7 5 plot title essesiseseisseiessseesssriessresssrresssreessreessrres 18 2 plotting phase Duncpons cece 16 2 output time requirement c ceeseseseee ese teeeeeeeees 18 6 meet 12 9 SS reaqnisone OUT UL SCTE N eege ee 12 2 12 7 P demo dataset cccccccccsscecsssssssssessesessessessssesseeeess 2 2 Output Screen button oo esses 9 8 preference P non default background color 9 13 printing paging swapping space bjl0e format eee eee neon 9 17 Increasing ON Linus 21 10 bj200 format 9 17 BEE 21 10 COO O Format E 9 17 ee oe 9 14 bjc800 EE 9 17 WW SE 9 17 peak picking EEN 9 17 comparison to NMRanalyst un Le c
61. 3 4 Analysis Parameters Peak Picking Threshold The peak picking step identifies resonances in the 1D spec trum worth further examination Only resonances above the specified threshold times the noise level average distance between adjacent spectral points are selected for further investi gation The higher the value entered in this input field the smaller the number of identified resonances When this threshold 1s set to a negative value the single resonance analysis 1s per formed using its absolute value The normally subsequent resonance cluster optimization 1s then not performed Key HLimit Min Signal Distance xz This parameter specifies how much resonance splitting can occur before each additional splitting is considered a separate resonance The default value for proton spectra is one and for other spectrum types 1 7 Hertz or the digital resolution whichever is higher Key WidthL WidthLU Correction FID Points Most NMR baseline distortions result from intensity distor tions of the first few FID points Rather than backward linear prediction 1d_ analysis cor rects for distorted FID points in the frequency domain Dunkel R United States Patent 5 218 299 Specify in this input field the number of FID points of which intensities need to be corrected Proton spectra typically require one and carbon spectra correction of three FID points Bruker spectra might show super smilies at both spectral edges T
62. 8 NMRgraph Molecular Correlation Editor Pull Down Menus structures a user can choose whether to display carbon shift prediction details for all struc tures or only structures with selected atoms or bonds Un Shift Agreements H Shift Details c Shift Agreements and Uc Shift Details display shift prediction related text information in a popup Selected text in this popup can be copied to the clipboard or printed by clicking the right mouse button over the Print All Copy Selection Print Selection text and choosing Copy Selection or Print Selection To select all text in this popup choose Select A11 To print all text independent of selection state choose PEINE ALL NMRgraph Place Heteroatoms E wll Selecting Place Heteroatoms from the Prediction menu brings up the shown popup Max Max Max H Max eff WI mol KI Select possible heteroatom s to be placed into the dis played structure s to minimize the difference between si Ip s Jet SS SS SI al predicted and observed carbon chemical shifts For each selected heteroatom type the maximum number Madd Likely cc Double Bonds of heteroatoms to be added can be specified Select the Moraer structures by shift agreement heteroatom to enable its input field f_o Cancel wep If the Add Likely C C Double Bonds switch is selected a single bond between two sp car bons is changed to a double bond if ea
63. 860 Se Se gt Recommended Threshold Integral I max 371 826 I 11 223 I max 353 832 I 13 844 January 11 2010 Tutorial Ill Combining Analysis Results Analysis of Gradient HMBC Spectrum 1 72 S 3 L642 2 Pt 1 2 AE 1 70 5 L116 11 313 2 49 50 90 24 A7 K ACC KC E EE Ae mt Vie mm E EN eg AC wen EH E Dim CA e Ae HU 141 al al LA a 9 38 73 15 1 1 30 307 2501 2A 1845 50 Lem mm E 2 16 372 22 659 Sr 14 L63 2132 16 50 44 12 106 2 14 44 Set the 2D Analysis Thresholds Integral field to 56 505 as suggested Rerun the 2D Analysis which auto runs the Report workwindow Display the shown structure plot file of HSQC derived molecular structural results The multiplicity editing of the HSQC inverts CH relative to CH and CH resonances Identi fied CH carbons are shown in green CH ones in blue and CH ones and proton resonance frequencies in orange 5 3 Analysis of Gradient HMBC Spectrum Switch to the HMBC spectrum type and display the FFT workwindow Load from directory ghmbc 12Hz fidthe procpar file The 1D carbon spectrum was referenced by 93 722 Hz so update this Fl Start of Spectrum to 879 678 Hz Run the FFT workwin dow Switch to the 2D Analysis workwindow Run the 2D Analysis which auto runs the Report workwindow For this absolute value HMBC only two of four 2D phase components were acquired Pro ton pr
64. Analysis Parameters The History Mechanism for Input Values 4 9 The History Mechanism for Input Values History File With Plot Data Jj wll Every input field in NMRanalyst keeps a history of pre vious entries Display the Graphic workwindow input screen and click on the File With Plot Data but ton The displayed popup is shown This capability can be used as an undo facility Furthermore selecting previ ous values from the history list is more convenient than typing them in again The current value is the bottom entry in this list and the default value is the first entry in the list For an empty input field the history list entry is empty Double click now on structure plot Fazi1 42_Fhe 40_J46 3 plot Selection siucturepiot to copy it in the input field The background color of this input field changes to the default color OK Cancel Help Workwindow To make current input screen settings the default values click the Output Screen Op Workwindow pull down menu and select Make Default This mere Mi mechanism is helpful to declare the current settings as the starting point eer before further alterations which are then easily visible as non default 1D Analysis cet values 2D FFT Ctrl T _2D Analysis Ctrl M Report Clap _Assemblel Ctri A4 Graphic Ctrl G 4 10 Directory Editor and the Sucrose FID Analysis The 8 2 mg sucrose MF C12H22011 D20 solvent 2D INADEQUATE dataset was
65. CAUSED REGARDLESS OF THE THEORY OF LIABILITY ARISING OUT OF OR RELATED TO THE USE OF OR INABILITY TO USE SOFTWARE EVEN IF SUN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES In no event will Sun s liability to you whether in contract tort including negligence or otherwise exceed the amount paid by you for Software under this Agreement The January 11 2010 9 19 Using the NMRanalyst Window Copyright Java Runtime Environment foregoing limitations will apply even if the above stated warranty fails of its essential purpose Some states do not allow the exclusion of incidental or consequential damages so some of the terms above may not be applicable to you 7 TERMINATION This Agreement is effective until terminated You may terminate this Agreement at any time by destroying all copies of Software This Agreement will terminate immediately without notice from Sun if you fail to comply with any provision of this Agreement Either party may terminate this Agreement immediately should any Software become or in either party s opinion be likely to become the subject of a claim of infringement of any intellectual property right Upon Termination you must destroy all copies of Software 8 EXPORT REGULATIONS All Software and technical data delivered under this Agreement are subject to US export con trol laws and may be subject to export or import regulations in other countries You agree to comply strictly with all such laws and regul
66. Car bon input field HMQC spectra give worse F1 line shapes than HSQC projections But the HSQC or HMQC spectra remain faster to acquire than a 1D carbon spectrum Overall proton and carbon 1D FIDs are preferable for structure identification over 2D spectrum projections January 11 2010 7 2 Tutorial V Additional NMRanalyst Features Displaying Experimental Simulated and Resid 7 2 Displaying Experimental Simulated and Residual NMR Spectra For spectral analyses the Fourier transformed spectrum is created Phase functions are deter mined and locked when feasible The Report workwindow saves the location of identified cor relations Combining this information in a phased experimental spectrum with detected correlation locations and 1D spectral projections is the default way to visualize these results SECTION 4 7 Prednisone Spectrum With 1D Carbon amp Correlation Locations gives an example For the detailed visual inspection of a correlation area the experimental simulated and residual spectral areas can be created and displayed as described in SECTION 4 8 Experimental Simulated and Residual Spectrum of One Correlation This section describes creating such plots for the complete spectrum Switch NMRanalyst to the strychnine directory created in the previous tutorial To create a simulated HSQC spectrum switch to the HSQC spectrum type and the Report workwindow output screen In the middle of the report program output 1
67. Copyright C 2005 The International Union of Pure and Applied Chemistry IUPAC International Chemical Identifier InChI contact secretariat iupac org The InChI program is free software you can redistribute it and or modify it under the terms of the GNU Lesser General Pub lic License as published by the Free Software Foundation http www opensource org licenses lgpl license php 19 7 Copyright NIH PubChem ftp ftp ncbi nlm nih gov pubchem README Fair Use Disclaimer Databases of molecular data on the NCBI FTP site include such examples as nucleotide sequences GenBank protein sequences macromolecular structures molecular variation gene expression and mapping data They are designed to provide and encourage access within the scientific community to sources of current and comprehensive information Therefore NCBI itself places no restrictions on the use or distribution of the data contained therein However some submitters of the original data may claim patent copyright or other intellectual property rights in all or a portion of the data they have submitted NCBI is not in a position to assess the validity of such claims and therefore cannot provide comment or unrestricted permission concerning the use copying or distribution of the information contained in the molecular databases January 11 2010 19 13 cuapter20 NMRplot Plotting Multidimensional Spectra NMRanalyst automates the analysis of multidimen
68. DVD January 11 2010 2 5 NMRanalyst Installation Installing NMRanalyst mkdir media NMRanalyst bin mount dev hdc media NMRanalyst mount block device dev hdc is write protected mounting read only To permanently enable user mounting of this DVD drive update the etc fstab file Replace owner by users in the mount line and the mounting line should be similar to dev cdrom mnt cdrom udf iso9660 noauto users exec kudzu ro 0 O0 2 4 Installing NMRanalyst The installation consists of a simple series of installation windows allowing modification of default installation parameters With default settings it takes about ten minutes to install NMRanalyst To install NMRanalyst on Windows log in as Administrator or use an account with sys tem privileges Microsoft Windows does not allow overwriting files which are in use Stop running user programs or reboot the PC Insert the NMRanalyst DVD double click the desk ee top POSS icon and double click the CD DVD file system icon 3 dows Vista right click the NMRanalyst installer the file ANALYST WIN exe and choose Properties Click the Compatibility tab Select the check box Run this pro gram in compatibility mode for and select Windows XP Service Pack 2 Also select the check box Run this program as an administrator and click the OK button Now Double click the NMRanalyst installer If installing under Win To install NMRanalyst on Linux follow th
69. E profile for the Bourne and Korn shells For example a Bourne or Korn shell user can install customized workwindow input screen files by 1 copying the USerData directory from the installation directory to the SHOME directory 2 modifying the txt files in one of its subdirectories and 3 setting NMRUSER in the profile setup file to use the modified directory with NMRUSER SHOME UserData export NMRUSER January 11 2010 10 3 Using the Directory Editor NMRDIR NMRUSER NMRDATA and NMR It is still possible to change NMRUSER to another directory from the Directory Editor or to load input screen value files on an individual basis from another directory using the NMRana lyst Load button When clicking on the Directory Editor OK button several dialog win dows might appear nee oe all If the specified NMRDATA directory does not exist the shown popup is displayed to query whether a new directory should be created Create creates the specified directory and Can cel removes the popup window A new NMRSPEC directory 1s always created without questions The specified WMRDATA directory Evtcholesterol does not exist Create this directory Create Cancel Help Directory Shell Variable Changes xj In case of permission problems or a non existent NMRDIR or NMRUSER directory the following popup is displayed Cancel removes the popup win dow and Reset restores the last va
70. E E EAA EA 2 e ne E 4 loa Generate NOE Build up i yee sirinin naie einean A a aaa 10 CHAPTER 17 Assemblelt Workwindow ccccccccseccccccesneeesceees 17 1 Ee Combne NMR Analysis Restis esasen AEEA EERTE 2 17 2 Findit Identify Database Structures Best Matching NMR Data 6 17 3 VerifyIt Rate Specified Structure by Agreement With NMR Data f January 11 2010 7 NMRanalyst Manual Contents 17 4 Assemblelt Elucidate Molecular Structure From NMR Data 8 CHAPTER 18 Graphic Workwindow ccccccccceeseececcecseeeeeeeseees 18 1 ee NEE l Ioe NAR e E 2 18 3 Multidimensional Spectrum Specific Settings nccicccscscccessesaxtanavesvarcscasnsanceetereaavetents 3 184 Fuerz ihe Graphic Programmis 5c isiccariserviservcarveionvieeicientounrieianionimieeeimmen 6 CHAPTER 19 NMRgraph Molecular Correlation Editor 19 1 KS We POTON TEE ee ET ea 10 Wo The er Propeny EE 11 E e K KW NR EE 13 De C e 1 EE 13 197 Copright NIH Publ eege ENKER 13 CHAPTER 20 NMRplot Plotting Multidimensional Spectra 20 1 ALL SPor E l 1 er 1 W Ee ME EE 2 SEKR Aeren 5 20 5 Copyright OpenGL to PostScript Printing Library OT 27 5 20 6 Copyright Visualization Toolkit VTK sisisisidisisisntitnaiemiiouionneiimamaimanaen 6 Appendix CHAPTER 24 Frequently Asked Oueeiioue 21 1 January 11 2010 8 NMRanalyst Manual Contents 21 1 How are the CCBond FRED and NMRanalyst programs related l 21 2 Any differences between MS Windows and LINUX NMRanalyst
71. E directory Other pre ferred locations are opt export home usr and home The installation program also creates a logical link from the chosen installation directory to the directory specified in the Choose Link Folder window during the installation To start the installed software on Microsoft Windows from the KN menu click gt AlPrograms Click NwRanalyst3s and then click the Wi nmranayst3z6 item If a different location is specified in the Choose Shortcut Folder window during the installation start NMRanalyst from the chosen location Alternatively double clicking the analyst exe file inside the installation directory also starts NMRanalyst See CHAPTER 3 Tutorial I Using NMRanalyst for a description of the use of the software The Windows NMRanalyst includes a UNIX shell started from the UNIX Shell button in the NMRanalyst application window It contains the standard UNIX file utilities including ftp zcat tar zip unzip etc to help copying datasets See SECTION 4 1 The UNIX Shell Window for details On Linux start the installed NMRanalyst from a shell window by typing O analyst January 11 2010 2 7 NMRanalyst Installation De installing NMRanalyst If no logical links were created during the installation start the program directly from the installation directory ANALYST LINUX bin analyst Except for a small dataset inside the ANALYST WIN and ANALYST LINUX directories which is used for t
72. ER TORTIOUS ACTION ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE Trademarks Assemblelt FindIt NMRanalyst NMRgraph NMRplot and VerifyIt are trademarks of ScienceSoft LLC ChemDraw and ChemOffice are trademarks of CambridgeSoft Corporation InChI is a registered trademark of the International Union of Pure and Applied Chemistry IUPAC Intel and Pentium are registered trademarks of Intel Corporation Linux is a registered trademark of Linus Torvalds MDL is a registered trademark of MDL Information Systems Inc Microsoft Windows Vista Microsoft Windows XP and Microsoft Windows 2000 are trademarks of Microsoft Corporation OpenGL is a trademark of Silicon Graphics Inc PostScript is a registered trademark Acrobat and PDF trademarks of Adobe Systems Inc Red Hat is a registered trademark of Red Hat Inc Java and JavaHelp are trademarks of Sun Microsystems Inc UNIX is a registered trademark licensed exclusively by X Open Company Ltd VNMR VnmrJ VXR UNITY and UNITYplus are trademarks of Varian Inc XWIN NMR and CryoProbe are trademarks of Bruker BioSpin Corporation Other brand or product names are the trademarks or registered trademarks of their respective holders CHAPTER 0 CHAPTER 1 CHAPTER 2 January 11 2010 User Manual OV CIV CW eene E E N E E EE l I LI TheAunomaed EE sucina araa iii 1 2 Identification of Best Matching Molecular Structures ccceccc
73. GCEZS These 26 st ruct files contain the FindIt structure database The NMRanalyst installer copies these files into the proper location during the installation 2 32 GBytes in total dihydrotestosterone Ca 200 micro gram AP 600 MHz proton gHSQC gHMBC and gDQF COSY datasets for practising the dihydrotestosterone structure elucidation using Bruker format data 28 MBytes gilbDereLllic acid January 11 2010 2 2 NMRanalyst Installation Contents of the NMRanalyst Distribution Varian 500 MHz ID proton and carbon multiplicity edited gHSQC and gHMBC gibber ellic acid datasets CHAPTER 5 Tutorial III Combining Analysis Results describes the carbon skeleton determination from this heteronuclear data 6 MBytes Ci bperellicC acid DEE Varian 500 MHz 1D proton multiplicity edited gHSQC and gHMBC gibberellic acid datasets CHAPTER 5 Tutorial II Combining Analysis Results describes the carbon skeleton determination from this heteronuclear data without using a 1D carbon spectrum 6 MBytes oligomer sD SPECTRUM The analysis of this 500 MHz Varian 3D HNCO dataset is described in CHAPTER 7 Tutorial V Additional NMRanalyst Features For larger 3D datasets see the following human ubiquitin datasets 3 MBytes prednisone Varian INOVA 500 3 6 mg ml proton 34 s carbon 4 h 51 m gHMBC 1 h 37 m and gHSQC 48 m 2 s prednisone in DMSO d for practising the structure elucidation 8 MBytes strychnine Varian 500 MH
74. HORS HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGE THE AUTHORS AND DISTRIBUTORS SPECIFICALLY DISCLAIM ANY WARRANTIES INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NON INFRINGEMENT THIS SOFTWARE IS PROVIDED ON AN AS IS BASIS AND THE AUTHORS AND DISTRIBUTORS HAVE NO OBLIGATION TO PROVIDE MAINTENANCE SUPPORT UPDATES ENHANCE MENTS OR MODIFICATIONS January 11 2010 20 6 Appendix CHAPTER 21 Frequently Asked Questions 21 1 How are the CCBond FRED and NMRanalyst programs related Our research program of the automated 2D INADEQUATE spectral analysis was called CCBond Carbon Carbon BOND Varian Inc marketed it under their trade name FRED Full Reduction of Entire Datasets After extension to the analysis of standard 2D and 3D NMR spectra we renamed the software to NMRanalyst 21 2 Any differences between MS Windows and LINUX NMRanalyst The following subsections describe the minor differences 1 Dunkel R Ph D Thesis University of Utah 1990 January 11 2010 21 1 Frequently Asked Questions Any differences between MS Windows and 21 2 1 Backgrounded Computations Killed on Logout NMRanalyst allows backgrounding computations On Linux backgrounded computations run to completion even if the user logs out Microsoft MS Windows kills user processes on logout 21 2 2 Directory and File Names not Case Sensitive On Linux
75. INADEQUATE spectra the determined coupling constant in Hertz For 3D spectra the correlation label shows the chemical shift in the third dimension and for all the other spec trum types the correlation label shows the signal integral of the detected spin system NMRgraph not only displays determined structures or atom correlations but also allows mod ifying displayed structures The AssembleIt workwindow allows specifying additional chemi cal knowledge for intermediate structures using the editing capabilities of NMRgraph See CHAPTER 17 AssembleIt Workwindow for details January 11 2010 19 1 NMRgraph Molecular Correlation Editor Pull Down Menus 19 1 Pull Down Menus pe NMRgraph has pull down menus for accessing its structural display and Sle ctr shit O editing functionality Insert Save List e SE Open allows opening a new file for display Save Molfile Save SDfile Insert inserts structures read from an input file at the bottom Save ChemDraw a of the current NMRgraph display Print Ctrl P Exit Ctrl X Save saves changes to the current NMRgraph display in its original file format and name Save Structure saves the current NMRgraph display in an NMRanalyst structure file The filename can be specified through a Filebox and a plot ending is appended if no filename ending is specified The saved file can be redisplayed to continue the NMRgraph editing Save Molfile saves the dis
76. LIMITED TO THE WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT IN NO EVENT SHALL BRIAN PAUL BE LIABLE FOR ANY CLAIM DAM AGES OR OTHER LIABILITY WHETHER IN AN ACTION OF CONTRACT TORT OR OTHERWISE ARISING FROM OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFT WARE 20 5 Copyright OpenGL to PostScript Printing Library GL2PS GL2PS an OpenGL to PostScript Printing Library Copyright C 1999 2002 Christophe Geuzaine E mail geuz geuz org URL http www geuz org gl2ps January 11 2010 20 5 NMRplot Plotting Multidimensional Spectra Copyright Visualization Toolkit VTK This library is free software you can redistribute it and or modify it under the terms of the GNU Library General Public License as published by the Free Software Foundation either version 2 of the License or at your option any later version This library is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU Library General Public License for more details You should have received a copy of the GNU Library General Public License along with this library if not write to the Free Software Foundation Inc 675 Mass Ave Cambridge MA 02139 USA For a verbatim copy of the GNU LIBRARY GENERAL PUBLIC LICENSE please see file SNMRDIR enscript LGPL The source code
77. NMRanalyst findit Script for Molecular Structure Identifica minutes on a 3 06 GHz PC Close the NMRgraph display by selecting Exit from the File pull down menu DC NMRgraph Findit matches File Edit View Structures Prediction January 11 2010 3 5 Tutorial l Using NMRanalyst Selecting Full NMRanalyst Mode 3 2 Selecting Full NMRanalyst Mode zi For the structure elucidation of the prednisone datasets select the full NMRanalyst function ality From the NMRanalyst Edit pull down menu select the Prefer ences item This operation is also referred to as selecting Edit Prefer ences From the started popup set the Mode to Full NMRanalyst and deselect the Show ALL Input Fields switch as shown Click the OK button or press the keyboard Enter key Mode Full WMRanalyst d _ Show 411 Input Fields Defaults OR Cancel Help 3 3 Selecting 2D INADEQUATE Spectrum Type Spectrum Type The window title of NMRanalyst displays the selected spectrum type The win _ 14 ADEQUATE dow displayed in SECTION 3 1 findit Script for Molecular Structure Identifi deeg cation is set to INADEQUATE If your window is set to another spectrum HETCOR ieee type select the NMRanalyst Spectrum Type INADEQUATE item as _ N415_HMBC shown HSQC _ COUPLED_HS0C Ni Hor INADEQUATE _ NOESY _ ROESY TOCSY _ 3D_SPECTRUM 3 4 The ID Analysis Workwindow At the bo
78. NMRgraph Molecular Correlation Editor Pull Down Menus Invert Selection 1s activated when at least one atom or correlation is selected It deselects all the selected items and selects all the previously unselected items For example to delete all the displayed structures except one select the structure of interest Select Invert Selection andthen Delete Selection Atom Properties 1s active when a single atom is selected It starts the Atom Prop erty Editor Double clicking on an atom with left mouse button also starts its property editor See SECTION 19 2 The Atom Property Editor for details Correlation Properties becomes active when a single correlation bond is selected It starts the Correlation Property Editor described in SECTION 19 3 The Correla tion Property Editor Double clicking on a bond also starts its property editor eee xj Selecting Preferences starts the shown popup Selected atom and bond label sizes affect display and print Atom Label Font Size 11 pt oe 2 j Sizes Carbon and proton shift prediction can be based on rules Bond Label Font Size 10 pt 3 DEE or on up to six sphere HOSE codes The order of applying HOSE Code Before Rule Based these two methods can be selected Clicking OK saves the current settings and the settings are effective for the current and ox canca Hew future NMRgraph applications Cancel closes the popup without applying
79. NOESY and ROESY spectrum types The shown switch selects the generation of NOE build up curves NMRanalyst specializes on the automated analysis of experimental multidimen sional NMR spectra This NOE build up curve extension provides a simple but powerful way to combine information from a series of NMR spectra The derived build up curve slopes are more reliable for the determination of the 3D structure of molecules than the individual signal volumes Key BuildUp Mixing Time m Up to five NOE spectra acquired with different mixing times can be incor Tine 1 porated in the build up curves The mixing times entered in these input fields should be specified in milli seconds and the software accepts val Se ues between zero and 60 000 ms one minute The spectrum file name input fields described below and the corresponding mixing time need to be specified in the same line of the table The order in which the spectra are listed in the table does not matter Key MixT1 MixT5 2 Analysis Output File For NOE Spectrum The build up curve generation starts with the real tr EI analysis results of the corresponding compo File i al nent spectra It is recommended to carefully analyze the spectrum of the highest mixing time since this spectrum will have the highest signal to noise S N ratio Only the identified correlations need then be analyzed in the spectra acquired with shorter mixing times Finally all nD Analysis workwindow output f
80. Patents 5 218 299 and 5 572 125 British Patent 0 577 770 German Patent 692 31 690 6 08 January 11 2010 4 9 Tutorial Il Setting Analysis Parameters Prednisone Spectrum With 1D Carbon amp Corre tion Fl Phase and F2 Phase switches are deselected as shown Run the 2D Analysis which auto runs the Report workwindow 4 7 Prednisone Spectrum With 1D Carbon amp Correlation Locations Switch to the Graphic workwindow input screen In the File With Plot Data input field specify inadequate spec the spectrum cre ated by the FFT workwindow Run the workwin dow to display the shown spectrum plot It shows the phased experimental prednisone 2D INADE QUATE spectrum with the 1D carbon spectrum The location of NMRanalyst identified correla tions are indicated by rectangular bounding boxes The spectral Fl sweep width vertical axis was acquired with equal width as the F2 axis Had this spectrum been acquired with a larger F1 spectral range the green bounding boxes of aliased resonances would have been detected outside the shown spectrum The unaliased correlations are indicated by black bounding boxes F2 200 80 160 4140 120 100s a DU 40 ppm 4 8 Experimental Simulated and Residual Spectrum of One Correlation Detailed plots are only created when requested To identify the weakest detected prednisone correlation switch to the Report workwindow and click Output Screen Scroll up to the Correlation Detection Pro
81. PubChem collection is shipped with NMRanalyst Select FindIt Database Manager from the NMRanalyst Tools menu to add further structures see SECTION 9 8 The Application Window Menus for details Key LFindIt Input Molecular Formula Constraint Specify the molecular for mula constraint to be considered when matching the database structures Atoms can be speci fied in any order and multiple entries of the same atom type are combined A number or number range can be specified following each element If no number is specified one such element is assumed If only is specified zero to a hundred such atoms are considered The range 3 5 would mean three to five preceding elements are considered If an element should not be present specify the element with the number zero If no constraint is specified all data base structures are considered the default setting Specify a trailing if further unspeci fied atom types can appear in matching structures So NO N letter zero star means any molecule without nitrogen matches C H O means molecules containing only any number of carbon hydrogen and oxygen match Key mFormula Molecular Weight Range Tel When the molecular formula is unknown the weight of the unknown might be known from a mass spectrum Specify the minimum weight in Dalton g mol in the left field and the maximum weight in the right field By default these input fields are empty a
82. QC the default value is 30 Hz Key RangeJ RangeJU J Mapping Points Only the INADEQUATE HMBC N15 HMBC COUPLED HSQC and DQF COSY spectral analyses support the determination of scalar coupling constants and display this input field Specify in this input field how many mapping points should be used to examine the Map Coupling Constant range A maximum number of 100 mapping points are accepted by NMRanalyst For a detailed discussion of this input field see the description for the F2 Mapping Points input field Key MapJ Estimated Cowling Constant 1 z Only the INADEQUATE HMBC N15 HMBC COUPLED HSQC and DQF COSY spectral analyses support the determination of scalar coupling constants and display this input field For the regression analysis of INADEQUATE January 11 2010 15 6 nD Analysis Workwindow Analysis Parameters spectral regions a one bond carbon carbon coupling constant Clee needs to be estimated The default estimate for Jcc is chosen on the basis of possible hybridization of the carbons involved as follows epp 35 Hz sp sp 45 Hz sp sp 60 Hz The INADEQUATE pulse sequence contains a coupling constant parameter often called JCC for which the data acquisition provides the optimal detection This JCC value or any other value assumed to give a good initial Joc estimate can be specified in this input field By default the regression analysis of DQF COSY spectral regions uses a proton proton cou
83. The Ctr1 S crx displayed at the right hand side is the keyboard short cut for this menu item Print sends the color PostScript file of the displayed plot to the printer specified in the NMRanalyst Print Settings popup Exit exits the displayed NMR plot application January 11 2010 20 2 NMRplot Plotting Multidimensional Spectra Pull Down Menus View Reset returns to the display with which NMRplot Reset ctrl R started Previous Zoom restores the previous rubber band Previous zoom Ctri z zooming level Black Background and White Back White Background Cito Eege ground switch the background color for displayed and saved ground Ctri B l l l ae Se plots Parallel Proj ection and Perspective Pro Horizontal F2 Ort jection switch perspective for the plot off and on respectively gen Surface renders the displayed surface or iso surface plot as Reset op filled surfaces Wireframe shows such plot in wireframe repre White Background amw sentation For 2D contour plots Horizontal F1 and Hor E es seen Be i zontal F2 display F1 and F2 respectively along the horizontal Parallel Projection ma 1 e X axis For 2D surface plots F1 Side and F2 Side 7 TT EH TTT reorient the spectrum to show the corresponding side of the spec w SUMACE Ctri U f HANED coup trum For 3D spectral displays F3 F2 Si de J F3 F1 Ee cum Side and F2 F1 Side show the corresponding spectrum F2 Sid
84. The best structure found so far is saved in temporary files during the generation process When the generation is paused or completes the temporary structure files are replaced by the Best Structures number of most likely structures Up to 1000 best structures can be requested Key numSt ruct January 11 2010 17 9 CHAPTER 18 Graphic Workwindow The Graphic workwindow displays 1D Analysis nD Analysis Report and AssemblelIt work window plot files experimental simulated and residual 2D and 3D NMR spectra as well as molecular structures saved in supported file formats The following plot types are supported by the Graphic workwindow 1 plots of determined 1D and 2D phase and baseline parame ters with error values and interpolation functions 2 1D spectral plots 3 surface carpet plot of 2D fitting areas 4 isosurface plot of 3D fitting volumes 5 location plot of detected correlations 6 plot of determined parameter precisions and integrals 7 NOESY and ROESY NOE build up curves 8 2D and 3D spectral plots from NMRanalyst spectrum files 9 graph of determined molecular correlations and 10 molecular structures in NMRanalyst plot file Molfile and SDfile formats See CHAPTER 12 Using the Workwindows for a general description of the function and use of a workwindow The Graphic workwindow input screen is shown in CHAPTER 3 Tutorial I Using NMRanalyst Examples of its use are described in CHAPTER 4 Tutori
85. Title 18 2 NMRplot Specific Settings The following input fields affect all the plots displayed by the NMRplot application HMRPLOT SPECIFIC SETTINGS The default axis titles can X Incis Title be overwritten from these reis tite input fields Key xTi zis titie tle yTitle zTitle Plot Window Size A plot window size depending on plot types is supplied by default This size can be overwritten by this entry The relative scaling of the plot axes inside the plot window is determined by the Relative Size input fields described below Key size Text size The size of plot labels and of the axial numbering is chosen relative to the plot size determined by the Size field above The default Text Size value is 1 Key tSize January 11 2010 18 2 Graphic Workwindow Multidimensional Spectrum Specific Settings A Franes The AA in this label stands for anti aliasing Displayed text and NMR spec tra are approximated by the used screen resolution NMRplot has no inherent plot resolution limits and increasing the plot resolution sufficiently resolves rendering problems But given a fixed screen resolution for displaying plots anti aliasing can be used to increase the rendering quality Using anti aliasing the requested number of frames is rendered using a displacement of less than one pixel and all frames are averaged and displayed By default a value of 2 is used for some anti alias rendering improvement For maximum re
86. Toolset cnnan 0 2 9 22 E EEN 15 9 15 10 r eee nee ne meee ere a ae 0 2 9 18 digital filtering EE 4 14 13 2 Freely Distributable LIN 0 2 13 9 g GL2PS brary rrn pe SE Sp GNU f Ghost e 0 2 E E Java runtime envitronment 0 2 9 18 directory editor JavaHe lp u ecceccescsscsssessesssessessssessssseseesteseenees 0 2 8 4 Directory Editor button 9 10 Mesn 0 2 NMRDATA cccccccccsseceseseeseseseseees 10 2 10 3 12 6 INR le ER 0 2 NME DIR eenia T 10 2 Open Source software 0 2 NMR SPE Ceroctis ce rr reer tert 10 1 10 3 Visualization Toolkit NIK 0 2 20 6 NKMERUISER ereeschen 10 2 12 6 E Pi Pe A oe 13 5 setting SUMMATY s escscesenesenenenenenenenenenenn 10 2 variable settings u 10 1 COUPLED HSQC l disk space requirements Mapping parameters oo eee esses 15 8 Pe 22 2 3 optimization detection parameters 15 9 REENEN ee FindIt structure database o 0 0 ccceeceeeeeeeeeeeees 2 2 gibberellic HEES 2 3 D Linux NMbanalvst 2 2 oligomer 3D SPECTRUNM ccc cece 2 3 S NN 14 2 EEGEN 7 3 data low 12 9 sucrose INADEOUATE 2 3 data format ubiquitin HNCACB SEENEN eer ed SEENEN NEEN 2 4 EE EE 4 14 ubiquitin HNCO DE 2 4 Bruker AMX amp DMX 13 2 14 2 Windows NMRanalvst 2 2 Varian VNMR ossssssaiiiiiiiieren 4 12 13 2 14 2 distribution media 2 2 datasets documentation dihydrotestosterone 0 esses eesteeeeseeeeeeee 2 2 SE 8 1 gibberellic acid 2 2 2 3 PDF tie Aa ee eet 8 1 oligomer 3D SPECTRUNM
87. a and experience in analyzing such spec tra will yield insights on how to set this Weak threshold Set the AssembleIt workwindow HMBC Weak field to 2 2 declaring the bottom one sixth of these correlations to potentially contain longer range correlations A similar Weak threshold could be set for N15 HMBC As only two nitrogens were detected the obtainable speed improvement is limited and consider ing all of these correlations to be potential longer range correlations is fine Now select the AssembleIt ELUCIDATE MOLECULAR STRUCTURE FROM NMR DATA section Typically about 20 of expected 2 bond HMBC correlations are not observed due to unfavorable ICH coupling constants Set Evaluate Unobserved Bonds to 10 for Assemblelt to try to derive up to this number of unobserved bonds from longer range cor relations For aromatic systems 3 bond correlations are typically stronger than 2 bond ones Set the Evaluate option menu to Bonds Over E to more likely recover such bonds Finally set 4 Bond HMBC Correlations to 4 and Long Range HMBC Correlations to 1 to have Assemblelt consider up to this number of longer range correlations below the Weak threshold Start the workwindow and in about 8 minutes on a January 11 2010 6 7 Tutorial IV Advanced Structure Elucidation Using DQF COSY 3 06 GHz PC Assemblelt reports 6 structures consistent with the specified strychnine data They are saved in the file CC corrs plot 1 Without the AssemblelIt ex
88. a fitting volume several phase components might need to be examined The NMRplot window allows chang ing the display among the phase components through the Component menu January 11 2010 7 7 Tutorial V Additional NMRanalyst Features Incorporating NMRanalyst Programs in Other HNCO and HNCACB datasets of human ubiquitin are provided SECTION 2 2 Contents of the NMRanalyst Distribution for practising the 3D analysis of a larger protein See http www sciencesoft net 3DAnalysis html for further instructions 7 5 Incorporating NMRanalyst Programs in Other Software NMR software typically includes a peak picking program for the simplistic computerized spectral analysis NMRanalyst s programs were written to be included in other software sys tems as super peak picker The NMRanalyst user interface is an example of how to inte grate the analysis code into software packages The names of the computing programs are ld analysis fft analyze report assembleit and graphic the programs have a exe postfix in the MS Windows version The computing programs read input from the corresponding txt file 1d analysis txt fft txt analyze txt report txt assembleit txt and graphic txt and write output and error mes sages to the screen The txt files are normal text files and contain one key value pair each line The key is the name of a variable to be set Supported variable names are described in the corresponding wor
89. ach workwindow provides the user interface for one analysis step See CHAPTER 12 Using the Workwindows for details The desired workwindow can be selected by clicking on the corresponding tab Each workwindow tab has a keyboard mnemonic that is typing Alt or Ctr1 and the underlined character e g T for the FFT workwindow simultaneously is equivalent to clicking the tab Only one workwindow can be displayed at a time but NMRanalyst allows all workwindows to perform computations simultaneously 1D Analysis 2D F rT 2D Analysis Report Aesembieh 9 6 The Workwindow Tabs The workwindows provide access to the analysis software and appear as a stack of folders in the application window Clicking on a folder tab displays the corresponding workwindow SECTION 9 5 Structure and Function of the Application Window shows the row of folder tabs for the analysis of 2D spectra For the analysis of 3D spectra the tabs appear as shown in SECTION 9 1 Starting NMRanalyst The position of the tabs and associated workwindows from left to right corresponds to the order in which a typical analysis is performed When a workwindow is selected the corresponding tab is displayed in front of the others When the computation of the workwindow is started the tab displays a clock icon to the left of the workwindow name e g 2 anaysis The clock icon disappears as soon as the computa tion is completed Right clicking on a workwin
90. acquired overnight on a Varian UNITYplus 500 spectrometer using a ee Nanoprobe Start NMRana lyst This sucrose INADEQUATE dataset is provided with the NMRanalyst software 2 Courtesy of Paul A Keifer Ph D Varian Inc January 11 2010 4 12 Tutorial Il Setting Analysis Parameters Directory Editor and the Sucrose FID Analysis Assuming it was installed in the UNIX system directory usr local and should be copied to the login directory start the UNIX Shell and issue the commands cp r usr local sucrose INADEQUATE HOME cd SHOME sucrose INADEQUATE Otherwise load it from the original NMRanalyst distribution medium e g a DVD Rename the contained INADEQUATE directory so the provided analysis parameters are not found still from the UNIX Shell ZS mv INADEQUATE INADEQUATE org gt lt Directory Editor xj Click the Directory Editor SIRDIR pusr oca sanaysay O e button at the top of the NMRanalyst EIERE window to display the shown popup wepan none xcceona sucrose_mmavequarcA e All NMRanalyst directories can be set NMRSPEC TNADEQUATE EI from this popup Set the current working directory NMRDATA to __Gancel SHOME sucrose INADEQUATE Set its spectrum type NMRSPEC to INADEQUATE and click OK Use the procedure described for prednisone Only small adjustments are needed for this sucrose dataset 1 In the 1D Analysis workwindow Load Filebox select for this Varian format dataset directory
91. al II Setting Analysis Parameters For the printer setup used by the graphic programs see the Print Popup settings described in CHAPTER 9 Using the NMRanalyst Window This chap ter describes the function of all Graphic workwindow input fields grouped into several input screen sections To display all the input fields select from the Edit menu Prefer ences Selectthe Show All Input Fields switch and click OK 18 1 Settings for All Plots The entries in this input section apply to all the supported plot formats January 11 2010 18 1 Graphic Workwindow NMRplot Specific Settings SETTINGS FOR ALL PLOTS The only entry required File With Plot Data structure plot for plotting pre calculated data or 2D and 3D NMR spectra is the file name of the plot data possibly with a path name Program graphic deter mines from the format of the specified file the graphics program to use for rendering the data If a Report workwindow molecular correlations output file a Molfile or a SDfile is specified the NMRgraph application is called If a non existing file is specified the NMRegraph applica tion is started with an empty window and a warning message For the other NMRanalyst plots the NMRplot application is invoked Key FnPlot Piot Title J All plots have a default title derived from the kind of plot requested or from the file name of the plot data This input field can be used to overwrite the default title Key p
92. analysis results It contains over 15 9 million common molecular structures and further ones can be added The VerifyIt component rates a specified structure by its agreement with NMR results It can compare the rating with all the FindIt structures for further confirmation of the specified structure The AssembleIt component performs the struc ture elucidation It combines the analysis results of several spectra and derives the most likely molecular skeletons from this often incomplete and ambiguous information The NMRplot program displays experimental simulated and residual spectra as contour sur face and isosurface plots The NMRgraph program displays and allows editing molecular structures It predicts proton and carbon chemical shifts It completes determined molecular skeletons by adding likely bond multiplicities and NMR unobserved heteroatoms The NMRanalyst spectral analysis software is based on a mathematical spin system model NMRanalyst analyzes all acquired phase components simultaneously for maximum sensitiv ity This provides maximum sensitivity for various types of multidimensional spectra This approach contrasts with other computerized analysis strategies such as peak picking which January 11 2010 1 1 Overview The Automated Spectral Analysis ignore valuable spectral information By using this novel mathematical approach NMRana lyst often excels compared to even an experienced spectroscopist in the sensitivity relia
93. ancel discards all changes 19 3 The Correlation Property Editor een ell When one correlation is selected in NMRgraph the Correla correlation Labe1 FET tion Properties kel menu item becomes active and starts the shown Correlation Property Editor when clicked The Corre eee eae EE lation Label text field at the top of the editor allows editing Steg 7 the label Bond Type Single Double Triple Up Down Line Style Solid Dotted Dashed Dot Dashed and Arrow None Left Right Both for the correlation can be chosen from the option menus The current correlation settings are previewed in the text field at the bottom OK in this editor applies the current settings and Cancel discards all changes Correlations are drawn in four line styles solid dotted dashed and dot dashed When corre lations have directions thy are shown as arrows AssemblelIt represents 2D INADEQUATE or DQF COSY correlations as solid lines as they represent bonds An ADEQUATE correlation is represented as a solid arrow as it represents a bond with an observed direction A HSQC HMBC derived correlation can be a bond or longer range correlation and it has a direc tion Without assignment ambiguity the correlation is shown as a dotted otherwise as a dashed line For final molecular structures the Report workwindow reports detected bonds as solid lines Bonds for which an ambiguity phase mismatch or correlation overlap warning was iss
94. and F Relaxation switches select the optimization of non linear parameters which are time consuming to optimize Key Map1 Map8 Not accessible from interface MapF1A MapF2A MapF1F DQF COSY Detection W Integrals WF1 Phase MW Couplings _WUnused _ 4 Integrals WF Phase _ Relaxation l January 11 2010 15 8 nD Analysis Workwindow Analysis Parameters ADEQUATE HETCOR HMBC N15 HMBC Detection MF1 Phase WF Phase _ F1 Relaxation _ F Relaxation HSQC N15 HSQC Detection WF1 Phase WF Phase _ Unused wi Integral _ F1 Relaxation _ F2 Relaxation _Umsed _ Unused COUPLED HSQC Detection W 2 Integrals WF1 Phase _ F1 Relaxation wi Integral _ Unused WF Phase _ F Relaxation _ Unused INADEQUATE Detection W Integrals WF1 Phase _ F1 Relaxation ms _ 4 Integrals WF Phase _ F Relaxation Unused NOESY ROESY TOCSY Detection 2 Integrals WF1 Phase WF Phase _ Relaxation 3D SPECTRUM Detection WF1 Phase WF Phase WF Phase _ Integral _ F1 Relaxation _ F2 Relaxation _ F3 Relaxation _ ined The mapping step described above is optimized to rapidly identify possible correlation sig nals The detection step described here further optimizes the spin system descriptions These final parameter values are reported in the analyze program output and are used for spectral simulations The switches indicate which com
95. and 1D Residual Spec trum menu items are shown for displaying hiding the selected 1D spectra For phase function plots F1 F2 and F3 for 3D spectra only phase components can be dis played or hidden by toggling F1 Phases F2 Phases and F3 Phases respec tively Component menu applies to 1D spectrum plots 2D contour plots 2D surface plots surface plots of 2D fitting areas and iso surface plots of 3D fitting volumes For the 1D plots the selected item determines whether the absorption or the dispersion mode of a 1D spectrum is displayed For the 2D and 3D plots the selected item determines the displayed phase component A phase component is specified in the order F1 F2 F3 and R stands for real and I for imaginary After phase correction R contains the absorption mode and I the dispersion mode resonances For 2D F1 phase insensitive spec tra the IR and II items in this menu are deactivated Component vw Absorption RR Dispersion Hl Unit menu applies to 2D contour 2D surface 3D isosurface 2D and 3D ae ee correlation location 1D spectrum and 1D parameter plots The selected item PointNumber crer determines the unit displayed along the spectral axes Build up Curves Build up Curves menu is only shown for plots of build up Previous Plot Cin Page UR curves Previous Plot and Next Plot menu items can be Hot Plot Ctrl Page Down selected to scroll through all build up curves January
96. antage of locking phase functions is that during the mapping step fewer patterns are considered to be potential correlations During the detection step locked phase functions provide a powerful filter to increase the dis tinction between correlation and noise patterns For step by step instructions on how to deter mine and use phase functions see CHAPTER 4 Tutorial Il Setting Analysis Parameters Key F1Pse0 FlPsel F2Pse0 F2Psel F3Pse0 F3Psel1 15 4 Select Plot Data to be Saved The plotting switches in this section should be unselected when analyzing whole datasets Plot generation slows down the analysis and consumes disk space Created plots can be displayed by the Graphic workwindow SELECT PLOT DATA TO BE SAVED These two switches allow Simulated Spectrum _ Add Correlations _ Ada Diagonal Resonances creating a simulated spec trum When the Add Correlations and Add Diagonal Resonances switches are selected the simu lated best fit patterns of each analyzed correlation is added to the simulated spectrum The January 11 2010 15 12 nD Analysis Workwindow Automatically Running Report After nD Analy Add Diagonal Resonances switch can be selected to add such signals to the simu lated spectrum Key Add2PF DiagPF Each Correlation _ Combined Experimental Simulated Residual Spectrum This switch allows saving the analyzed spectral region For 2D fitting areas a surface carpet plot and for
97. ants youa non exclusive non transferable limited license without fees to reproduce internally and use internally the Software complete and unmodified for the purpose of designing developing and testing your Programs B License to Distribute Software Subject to the terms and conditions of this Agreement and restrictions and exceptions set forth in the Software README file including but not limited to the Java Technology Restrictions of these Supplemental Terms Sun grants youa non exclusive non transferable limited license without fees to reproduce and distribute the Software provided that 1 you distribute the Software complete and unmodified and only bundled as part of and for the sole purpose of running your Programs 11 the Programs add significant and primary functionality to the Software 111 you do not distribute additional software intended to replace any component s of the Software iv you do not remove or alter any proprietary legends or notices contained in the Software v you only distribute the Software subject to a license agreement that protects Sun s interests consistent with the terms contained in this Agreement and vi you agree to defend and indemnify Sun and its licensors from and against any damages costs liabilities settlement amounts and or expenses including attor neys fees incurred in connection with any claim lawsuit or action by any third party that arises or results from the use or dis tribution of an
98. ar skele ton s from specified carbon nitrogen and heteroatom bonded proton correlations The challenge is that the specified atom atom correlations do not necessarily correspond to direct bonds Longer range or incorrect correlation assignments are expected as described below This workwindow section is displayed when this switch is selected Key LAssembleIt Input Aren Aten Correlations C_corrs plot EU The result of combined spectral analyses is a structure file containing a circle diagram of known atom atom correla tions This file can be displayed and edited from the Graphic workwindow to enter further information For example carbon atoms belonging to solvent or impurity resonances can be removed If a carbon chemical shift indicates the presence of bonded heteroatoms or unsatura tion in the molecule the number of free valences of the affected carbons can be reduced Additional known correlations can be added Specify in this field the structure file with the final set of atom atom correlations for the structure determination Key FnPlot Evaluate Fragment a A molecular fragment consists of the atoms connected directly or indi rectly by specified correlations Only one fragment is evaluated at a time and the Fragment is 1 by default corresponding to the largest fragment The maximum allowed Fragment is 50 Key numF rag Unobserved Bonds 0 The 2D INADEQUATE spectrum type can detect carbon carbon bonds even between unp
99. arbons symmetrically dis posed about the diagonal as shown Because the double quantum frequency in F1 direction is the sum of the chemical shifts of the two coupled carbons an initial analysis of a proton decoupled 1D carbon spectrum can be used to identify the regions of the 2D INADEQUATE spectrum which may contain correlation signals For each pair of resonances identified in the 1D spectrum two small regions of the spectrum are defined by the small rectangles shown in the figure above In case of a bond between the two carbons under consideration the charac teristic pattern is contained within this fitting area Here the AB and BC fitting areas con tain the required patterns but the AC fitting area does not consistent with the bonding of the three carbon fragment shown The objective of NMRanalyst is to examine the fitting area appropriate for each pair of carbon resonances and to determine whether or not it contains a correlation bond signal SNH cu This analysis strategy extends to other spectrum types by using the appropriate spin system model Shown in this sche matic is the DQF COSY spin system The spectrum contains an active coupling of two protons Each correlation coupling signal is composed of 16 anti phase transi tions The spectral diagonal is normally overly crowded and NMRanalyst only examines the off diagonal transitions in Hz the two shown fitting areas What can be improved by using this autom
100. ated spectra In the Graphic workwindow set File With Plot Data tohsqe spec Subtract Spectrum to hsqc spec sim and run the workwindow To adjust the size of a plot in these windows press the right mouse button in a plot window and move January 11 2010 7 4 Tutorial V Additional NMRanalyst Features Surface Plot of a 2D Spectrum 7 3 Surface Plot of a 2D Spectrum As in the last section switch to the strychnine dataset and set the spectrum type to HSQC Scroll to the bottom of the Graphic workwin dow input screen set 2D Plot Type to Surface to render the surface rather than the default spec tral contour plot To display the experimental strychnine spectrum Set File With Plot Data to hsgce spec and delete the Sub tract Spectrum Relative Size Y and Contour Lines Max entries NMR resonances tend to be small compared to the screen resolution Increase Plot Window Size to 600 Start the workwindow to obtain the shown plot It can be interactively positioned rotated and zoomed as described in CHAPTER 20 NMR plot Plotting Multidimensional Spectra 7 4 Analyzing 3D Spectra HNCO The oligomer 3D SPECTRUM dataset contains the 3D HNCO FID of Resii i Rest i R 0 5 mg of the AGAGAAAG oligomer containing a sequence of amino ma acids Alanin and Glycin NMRanalyst s 3D SPECTRUM spectrum type x a amp wa analyzes all phase sensitive 3D spectra as though they were heteronuclear spectra Create
101. ated spectral analysis 1 A major limitation of NMR is its lack of sensitivity A phase sensitive 2D dataset is acquired with four phase components and each phase sensitive 3D dataset with eight Half of the phase components are acquired sequentially and hence their noise content is uncorre lated NMRanalyst evaluates all phase components simultaneously by nonlinear regression analysis making full use of the acquired data January 11 2010 1 3 Overview Identification of Best Matching Molecular 2 The visual spectrum analysis can be described as pattern recognition What sticks out of the noise level and looks like an expected signal Can this signal be assigned to other sig nals in the same or other spectra or 1s it perhaps a spectral artifact NMRanalyst looks for the whole spin system potentially consisting of several transitions NMRanalyst starts with 1D resonance information and only searches the areas in the multi dimensional spectrum which could contain spin systems The automated analysis is more selective than the visual pattern recognition It is also more sensitive as it can reliably detect signals even for signal to noise challenged spectra where resonances are not visible 3 The molecular structure determination using NMR remains labor intensive Why not auto mate the steps which don t require the creative capabilities of a spectroscopist Spectros copist In A Box is our goal for NMRanalyst Things remain to
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103. ation s 17 2 default locations on Linux 2 7 MAPPIN PALAMEteTS en get Je default locations on Windows eeseeeiese 2 7 optimization detection parameters 15 9 from CD ROM HNCO preparation on Lime 2 5 oligomer analysis E 1 1 integral precision definition e EE 13 6 HSQC Integral Precision Threshold 00 00 15 3 ACTONYM MEANING oo eeeeeeesceeseeeseseesesseseseeseesesseeees 1 2 distinguishing methyl methylene methine 17 4 Mapping parameters s 15 7 J multiplicity editing oo esses 17 4 Ivi optimization detection parameters 15 9 Sopot E A oo 9 18 hypercomplex data JavaHelp online bein 8 1 de itO ee 15 13 provided runtime envitronment cee 2 5 I K Identification Text 13 4 key name IN ADEQU ATE PLA PIAUIIES sos sdececslesascinsnceceecnsensbeseaoeseucdatdsosannsouadsscns 18 3 1 2 EE 15 13 ACTONYM MEANING oo eecceeeceeseesesseseesessesseseesessesseeees RAIS ON soe srastece cass feotentenetenscesteosinancerren 17 8 anti phase doublet 1 2 EE EE pea 17 4 ARunbenort 15 13 eege 13 6 January 11 2010 22 5 NMRanalyst Manual Index Eeer 15 11 FS OG ee 17 4 Dud 15 11 FHINAD u ceccccccccscsecececececsescecsesecececeesrevsnsesececeeeneee 17 4 EE 15 11 teen 16 3 BAPI E 15 11 PaP ee eeg 16 3 BndF2A ooeeecccccccsccecesecesececsesesececsesvsvsuseseceeseavevaee 15 11 Ent ocapon cccccececesssssescseseseceesesesesesesesecseeveee 18 4 BondNo oue ececeecesessesececsesvsecese
104. ations and acknowledge that you have the responsibility to obtain such licenses to export re export or import as may be required after delivery to you 9 TRADEMARKS AND LOGOS You acknowledge and agree as between you and Sun that Sun owns the SUN SOLARIS JAVA JINI FORTE and iPLANET trademarks and all SUN SOLARIS JAVA JINI FORTE and iPLANET related trade marks service marks logos and other brand designations Sun Marks and you agree to comply with the Sun Trademark and Logo Usage Requirements currently located at http www sun com policies trademarks Any use you make of the Sun Marks inures to Sun s benefit 10 U S GOVERNMENT RESTRICTED RIGHTS If Software is being acquired by or on behalf of the U S Government or by a U S Government prime contractor or subcontractor at any tier then the Government s rights in Software and accom panying documentation will be only as set forth in this Agreement this is in accordance with 48 CFR 227 7201 through 227 7202 4 for Department of Defense DOD acquisitions and with 48 CFR 2 101 and 12 212 for non DOD acquisi tions 11 GOVERNING LAW Any action related to this Agreement will be governed by California law and controlling U S fed eral law No choice of law rules of any jurisdiction will apply 12 SEVERABILITY If any provision of this Agreement is held to be unenforceable this Agreement will remain in effect with the provision omitted unless omission would frustrate the in
105. babilities table The weakest bond is at the bot tom of this table January 11 2010 4 10 Tutorial Il Setting Analysis Parameters Experimental Simulated and Residual Spec E 24147 099999127 CORRELATION 1 8 211 416 674402 46 29 Switch to the 2D Analysis workwindow In the input field Analyze Correlation Index s specify the index number 7 Scroll to the bottom of the input screen and select the Combined Experimental Simulated Residual Spectrum check button deselect the RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS check button and run the workwindow The creation of file Fa211 42 Fb87 40 J46 3 plot is reported Experimental Switch to the input screen Delete the Analyze Correlation Index s entry again Deselect the Combined Experimental Simu lated Residual Spectrum switch and Simulated SUCCESSFUL ANALYSIS switch again Change to the Graphic workwindow set File With Eesen 7 v i Fa211 42 Fb87 40 J46 3 plot and click Start The 87 40 ppm anti phase doublet on anti phase part of this bond on the left side requires this software s three times higher sensitivity over select the RUN REPORT WORKWINDOW AFTER Een Data to Residual the right side is observable But the 211 42 ppm the visual analysis for detection Spry Display file structure plot from the Graphic workwindow The complete prednisone carbon skeleton is now determined January 11 2010 4 11 Tutorial Il Setting
106. be displayed as a foreground win dow Newer versions of Windows no longer interfere with the operation of NMRanalyst and its sub windows On MS Windows systems still showing this problem download the TweakUI application from Microsofts web site http www microsoft com win dowsxp downloads powertoys xppowertoys mspx Follow the instructions for installation Start TweakUI from the Control Panel select the General tab and deselect the Prevent apps from stealing focus checkbox 21 6 Any UNIX editor available from the Windows UNIX Shell The Notepad started with Start Programs Accessories Notepad and WordPad are the native MS Windows editors The NMRanalyst UNIX Shell offers an enhanced vi editor for UNIX style editing 21 7 How to copy and paste text in Windows NMRanalyst To copy text select the text with pressed left mouse button NMRanalyst s Edit Copy menu item copies the selected text into the CLIPBOARD To paste the CLIPBOARD contents into a MS Windows program an NMRanalyst input field or the UNIX Shell simultaneously press the keyboard Shift and Insert keys In the UNIX Shell holding Shift and clicking the left mouse button works as well The only known limitation is that the UNIX Shell may hang when text containing UNIX line endings e g multi line Windows NMRana lyst output screen text is pasted into it January 11 2010 21 5 Frequently Asked Questions What should do about th
107. ber under which further information can be found con 7 94 _ 105 214 163 10 49 i 103 22 161 12 8 15 Es 84 42 wg 53 84 cerning the corresponding correlation signal Display the P I distribution file preci sions plot for this dataset NMRanalyst is significantly more sensitive than required for the analysis of this spectrum As for 2D the software is very beneficial for analyzing low S N 3D datasets SECTION 4 8 Experimental Simulated and Residual Spectrum of One Correla Experimental na AM gg a REN Con describes the creation of experimen TT TS E WW tal simulated and residual surface plots of an analyzed 2D spectral fitting window For 3D spectra the equivalent plot shows the experimental simulated and residual 3D iso surfaces In the 3D Analysis work window set the Analyze Correla tion Index s field to 49 the second weakest correlation select the Combined Experimental Simulated Resid ual Spectrum switch and run the analysis of this correlation From the Graphic work window display the created Fal62 92 Fb90 91 Fc7 89 plot file Like other NMRplot displays this plot can be resized rotated zoomed and moved The sinc character of 3D signals might cause them to not be equally visible in all eight fitting volume phase compo nents This does not compromise the computerized spectral analysis as all eight phase compo nents are examined simultaneously But to get the best display of a signal in
108. bility accuracy and speed of the data analysis The software supports the analysis of the following and equivalent spectrum types e Adequate sensitivity DoublE QUAnTum spEctroscopy ADEQUATE e Double Quantum Filtered COrrelation SpectroscopY DQF COSY e HETeronuclear CORrelation HETCOR e Heteronuclear Multi Bond Connectivity HMBC e Heteronuclear Single Quantum Coherence HSQC HMQC e Incredible Natural Abundance DoublE QUAntum Transfer Experiment INADEQUATE e Nuclear Overhauser Effect SpectroscopY NOESY e Rotating frame Overhauser Effect SpectroscopY ROESY e TOtal Correlation SpectroscopY TOCSY and e 3D spectra 1 1 The Automated Spectral Analysis RS The first application of NMRanalyst was EH CH CH for 2D INADEQUATE spectra The sche matic explains this application for a gt CH CH CH molecular fragment Its 1D pro ton decoupled S spectrum is shown at the back of the schematic In the 2D spec trum a pair of bonded carbons e g CH CH gives rise to a pair of anti phase doublets centered at the chemical shifts of the two carbons v and vp and split approximately by the carbon carbon cou OH CH7 CH pling constant J Each of these AB spec January 11 2010 1 2 Overview The Automated Spectral Analysis tral patterns is displaced along the F1 axis by the sum of the two relevant chemical shifts V4tVp This spin system results in a bond pattern for each pair of bonded c
109. bination of parameter values is optimized during the Detection stage for the selected spectrum type Every spin system identified during the Mapping stage is further refined based on the selected Detection parameters before deciding if the found pattern is a valid correlation signal In addition to the selected switches the chemical shifts in F1 through F3 are always refined if appropriate and for DQF COSY HMBC N15 HMBC COUPLED HSQC and INADEQUATE spectra the coupling constant is refined as well January 11 2010 15 9 nD Analysis Workwindow Analysis Parameters The 2 Couplings switch for DQF COSY spectra determines an independent F1 and F2 coupling constant The DQF COSY active coupling spin system is symmetrical and can be described by a single coupling constant But when an overlapping passive coupling is present a single coupling constant could be determined as large as the sum of the active and the pas sive coupling Selecting the 2 Couplings switch safeguards against this misinterpreta tion The HSQC COUPLED HSQC and 3D SPECTRUM spectral types can encode further spin system information by creating signals in positive and negative absorption Select the Integral switch if positive and negative integrals are expected in the spectral absorption mode When phases are locked spin systems with negative integrals are only recognized as correlations 1f this switch is selected The analyze program optimizes the selected Det
110. ble for download over the Internet and the commercial versions are shipped on DVD For supported hardware and oper ating system configurations see SECTION 2 1 Supported Computer Platforms The instal lation DVD contents and the function of supplied top level directories are described in SECTION 2 2 Contents of the NMRanalyst Distribution A wizard style installation SECTION 2 4 Installing NMRanalyst and deinstallation SECTION 2 5 De installing NMRanalyst procedure is used for the supported platforms SECTION 2 6 NMRanalyst Software Support provides pointers for harder to resolve instal lation and usage challenges 2 1 Supported Computer Platforms NMRanalyst is supported on Intel compatible PCs running Red Hat Linux Enterprise 5 amp binary compatibles e g CentOS or Microsoft Windows 2000 XP amp Vista with modified installation NMRanalyst is fully functional on Windows Vista But its installer built by the InstallAnywhere software needs to be executed on Vista in the XP compatibility mode After installation NMRanalyst runs under Windows Vista without using the compatibility mode The disk space requirements for the NMRanalyst installation are described in SECTION 2 2 Contents of the NMRanalyst Distribution Analyzing spectra with NMRanalyst requires about 120 MBytes of virtual memory The memory size required by the NMRplot spectral dis plays scales with the size of the data to be displayed January 11
111. bute the Software subject to a license agreement that protects Sun s interests consistent with the terms contained in this Agreement and v agree to defend and indemnify Sun and its licensors from and against any damages costs liabilities settlement amounts and or expenses including attorneys fees incurred in connection with any claim lawsuit or action by any third party that arises or results from the use or distribution of any and all Programs and or Software 3 License to Distribute Redistributables In addition to the license granted in Section 1 Software Internal Use and Develop ment License Grant of these Supplemental Terms subject to the terms and conditions of this Agreement including but not limited to Section 3 Java Technology Restrictions of these Supplemental Terms Sun grants you a non exclusive non trans ferable limited license to reproduce and distribute those files specifically identified as redistributable in the Software README file Redistributables provided that 1 you distribute the Redistributables complete and unmodified unless otherwise specified in the applicable README file and only bundled as part of your Programs 11 you do not distribute additional software intended to supersede any component s of the Redistributables 111 you do not remove or alter any pro prietary legends or notices contained in or on the Redistributables iv you only distribute the Redistributables pursuant to a license agre
112. can be downloaded from http www geuz org gl2ps and can be ordered from ScienceSoft LLC for the cost of distribution 20 6 Copyright Visualization Toolkit VTK Copyright c 1993 1998 Ken Martin Will Schroeder Bill Lorensen This software is copyrighted by Ken Martin Will Schroeder and Bill Lorensen The following terms apply to all files associ ated with the software unless explicitly disclaimed in individual files This copyright specifically does not apply to the related textbook The Visualization Toolkit ISBN 013199837 4 published by Prentice Hall which is covered by its own copyright The authors hereby grant permission to use copy and distribute this software and its documentation for any purpose pro vided that existing copyright notices are retained in all copies and that this notice is included verbatim in any distributions Additionally the authors grant permission to modify this software and its documentation for any purpose provided that such modifications are not distributed without the explicit consent of the authors and that existing copyright notices are retained in all copies Some of the algorithms implemented by this software are patented observe all applicable patent law IN NO EVENT SHALL THE AUTHORS OR DISTRIBUTORS BE LIABLE TO ANY PARTY FOR DIRECT INDIRECT SPECIAL INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OF THIS SOFTWARE ITS DOCUMENTATION OR ANY DERIVATIVES THEREOF EVEN IF THE AUT
113. carbon buts 17 2 proton bonded to heteroatom s sss11001110 17 4 PULP OSC ipiius 17 1 reducing free valences ccc cceseeseeneeeeeeees 17 8 removing solvent resonances 17 8 specified false correlation ccceceeeee eee 17 9 specifying fragment number 17 8 structures sorted by likelihood 0 0 0010000000000 17 9 structures violating Consttraimnts 17 9 supported spectrum epes 17 2 unpaired sp2 Carbong eee 17 9 Verifylt component cece eee teeeeeeeeees 17 1 WAYS tO Store StrUCtUTES oo eects 17 9 wrong input Cortclatnons cceeeeeeeeeeees 17 1 Assemblelt workwimdow 17 1 automation Mining repor xsceresceenstaccsesiscansarsaacdeameoaicen 15 13 B brackets meaning Of ops pesspschenstosinemsesininsileorti resi sasacesaveatunsnients 1 6 breaches Of contract 7 9 Bruker acquisition formats oo eee ese eeeeeeeee estes 14 2 AMX DMX support 13 2 14 2 analyzing spectrum files 13 2 14 2 data formats ebe 14 2 loading parameter files 00 0 0 9 8 13 3 14 2 A WIN NM R erene E 14 2 build up curve corr2plot script 16 11 M X Ng PANTS Sis eege 16 10 Negative slope 16 11 spectral artifact sess essences 16 11 C CCBond program 21 1 computer platforms January 11 2010 22 2 NMRanalyst Manual Index SUP US aicese erener ene aa e 2 1 ubiquitin HNCACB osese 2 3 Reg ubiquitin HNCO sisser 2 4 See shell script detection step copyright TUPI CHOU Ro GE 15 9 Cygwin
114. cceeeeesssseteeeeeees Lo Uen ior Mama BETEN 1 4 Typographic and Keyboard Conventions cccccccccccccccccceeeeeeesesssssssssesssseeeeaeeeees NMRanalyst Installation cccccccc cect teeeecceee eee 2 1 3 E a Fa TO EEN 2 2 Contents ofthe NMRanalyst Distribution sents ee ses eecssccenercanernerenenerioioeenereetereenaest 2 3 Preparation for the Linux a EN Ge noallie NMR EE 2 5 De installing NMRamalyst ccc ccccccccccceeeeeeseseeessesessnssseeeeeeeeeeeeeceeeeseseeeeseeeeeas Sekt INURE cece eres Table of Contents NMRanalyst Manual NMRanalyst Manual Contents CHAPTER 3 CHAPTER 4 CHAPTER 5 January 11 2010 See ee ee ee ee ee ee ee ee e ee ee ee ee ee e Tutorial I Using NMRanalyst 3 1 findit Script for Molecular Structure Identification 000000ee0eeeeneneeenne 3 2 Selecting Full NMRanalyst Mode 3 0 Selecting 2D INADEQUATE Spectrum Type asciccesccistecescccccsiersesuensncce 3 4 The 1D Analysis Workwindow 0 cccccccccccceesesesssecccceeeeeeeeeeeeeeeeesseseeeeeas do Terr E EE 36 ThenD Analysis WorkwindOW ageet innein aai aia Se See vr See Deernmmed Eerst 2a e E Tutorial IT Setting Analysis Parameters SE e Stel NEE dee 4 2 1D Analysis Workwindow Settings for Prednisone cccccccccccceeeeeeeeees A3 VerifyIt Molecular Structure Verification and Shift Assignments 4 4 FFT Workwindow Settings for Prednisone nnnennnnneennenesse
115. ch color selection Click OK to set the current selec tions in both the General and Color tabs as the preferences The pref erences are used by NMRanalyst until they are altered again Defaults restores the preferences to the initial values Cancel closes the popup without applying any change x Background Color for Hon default Value __ De ooo 7 mma eee DERRRETEENEA ZU CIERSSRRRSISICIEIEISIRRRSCT CIE eee Pee ERR EECE EES Preview Sample Text Sample Text Defaults OK Cancel January 11 2010 9 13 Using the NMRanalyst Window The Application Window Menus Spectrum Type The Spectrum Type menu displays the 2D spectrum types for which 1H 1 ADEQUATE specialized analysis settings are available Other 2D spectrum types can likely EE be analyzed by one of the provided ones All phase sensitive 3D spectra can SE be analyzed with the provided heteronuclear 3D spectrum type by selecting _N15_HMBC the 3D SPECTRUM menu item HSQC _ COUPLED_HSOC _N15_HSQC INADEQUATE NOESY _IROESY TOCSY _ 3D_SPECTRUM Workwindow Make Default erases all history information of the selected work EH cm window input screen Hence the current values of all input screen items SE in the selected workwindow become the default values and all input Restore To Default fields and buttons return to their default background color Clear 4D Analysis oo History deletes the o
116. ch of the two carbons has at least one free valence A single bond representing a 2D INADEQUATE correlation with a coupling constant between 55 and 150 Hz is also turned into a double bond Select Order Structures by tc Shift Agreement to sort structures by average difference between predicted and observed carbon shifts If all heteroatoms are deselected in the popup clicking OK clears previously added heteroatoms from the displayed structure s If the Add Likely C C Double Bonds switch is deselected clicking OK restores the original single bonds Cancel closes the popup without applying changes January 11 2010 19 9 NMRgraph Molecular Correlation Editor The Atom Property Editor NMRgraph Atom Limit i ell NMRgraph can display up to 7000 atoms If this limit is exceeded during heteroatom placement the shown popup ang heteroatoms The heteroatom placement Appears Clicking OK acknowledges the error and failed ta complete Please delete some atoms closes the popup and try placing heteroatoms again Heip NMRgraph displays the current chapter Tutorial dis NMRgraph plays Tutorial II with application examples NMRgraph is started by the See Graphic workwindow Files to be displayed by NMRgraph can be created assemblet Workwindow OY the Report or the Assemblelt workwindows Report Workwin Graphic Workwindow dow AssembleIt Workwindow and Graphic About NMRgraph Workwindow
117. character of DQF COSY and 2D INADE QUATE spectral spin systems has already been incorporated in the analysis model and hence all determined integral parameters should be positive When the phase functions are not locked a sign difference between I1 and I2 and between I3 and I4 can also be used to reject a pattern Whenever a negative sign of an integral value is encountered the summary line of the fitting area shows the I neg indicator as in 89 C 6 C14 No correlation detected I neg dIl1 dI2 dI3 To assign for example a 2D correlation signal to a pair of 1D resonances the range given in the Map F2 Frequencies input field described above is used to decide whether or not the pattern under consideration can result from the two 1D resonances corresponding to the current fitting area The indicators Fa and Fb are used in summary lines to indicate that an identified pattern does not meet this requirement 65 C GC 9 No correlation detected Fa Fb Finally when the optimization of a relaxation time 1s requested in the Detection section a determined negative time rejects the corresponding pattern This condition is shown in the summary line by the T2 neg indicator as in 52 C 14 C 17 No correlation detected T2 neg Initial parameter values should be optimized only if the corresponding values are clearly determined by the data Key Bnd1 Bnd8 Not accessible from interface BndF1A BndF2A BndF1F Fi Phase pm Ifthe phase swi
118. cribed earlier in the Output Files section and the plot can be inspected using the Graphic workwindow Even ifthe Correlation Table s With Warnings on switch is not selected selecting the Improper Phase switch displays such correlations in a different linestyle in the NMRgraph correlation editor See CHAPTER 19 NMRgraph Molecular Correlation Editor for details Key LPseMm 2 NMRanalyst uses an improved version of the algorithm given in Dunkel R U S Patent 5 218 299 for resolving the 2 7 ambiguities in 1D and 7 ambiguities in determined multidimensional phase values January 11 2010 16 8 Report Workwindow Select Items to Be Reported _ Fitting area overlay When two correlation patterns identified in different fitting areas are not identical which would indicate an ambiguity but both fitting areas partly overlap the report program issues an overlap warning if the Fitting Area Overlap switch is selected Overlap windows 105 288 with transitions A A B B NMRanalyst was designed to distinguish expected spin systems from noise Overlapping cor relation signals can neither be described as isolated signals nor as random noise and a severe overlap of two or more correlation patterns can possibly lead to a false positive correlation test The best approach is to acquire the spectrum with sufficient resolution to avoid such problems If this is not feasible the overlap warnings allow quickly identifying the
119. ctrometer the Fl Spectral Width input field should be set according to the sw1 and the F2 input field according to the sw VNMR parameter Key swF1 swF1U swF3 SwF3U Start of Setzer ERD Specify in the provided F1 through F2 F1 through F3 for 3D input fields the low frequency limit of the corresponding spectral axis and set the unit used The 1D and the multidimensional spectra should be acquired under the same experimental conditions e g same sweep width and transmitter offset If that is the case the Start of Spectrum values of both spectra are identical For a heteronuclear 2D spectrum acquired on a Varian spectrometer the corresponding Start of Spectrum values can be calculated as Pie rfpl rfl Fripp yfl If the sweep width sw or the transmitter offset tof is different between the 1D and multi dimensional spectral axes then the reference peak position has to be calculated as EDD reif sw P swl P 2 rof tof P Not every spectrometer can reproduce the exact acquisition parameters Despite this correc tion additional frequency mapping might be necessary during the nD Analysis workwindow run If the 2D spectrum contains a visible signal such as a solvent line the 2D data can also be referenced using the spectrometer software before the FFT and nD Analysis workwindows are run Key spF1 spF1U spF3 spF3U Munber of Points The F1 through F3 Number of Points input fields allow specification of th
120. d inthe Enter path or folder name field Every directory contains a pointer to the parent directory represented in the list by two dots It allows traversing January 11 2010 11 1 Using the Filebox Popup Interactive Item Specification the directory tree upwards To change all displayed lists to the parent directory of the currently displayed directory position the Folders list using the vertical scroll bar until the parent link shows double click it using the left mouse button To traverse the directory tree in the downward direction double click the subdirectory name from the Folders list If the specification of a file name is requested locate the directory containing the file Side by side with the Folders list is a Files list containing the files currently in the directory specified inthe Enter path or folder name input field Select a file name by click ing on the name once Then click OK to return the selected path file name to the calling application To specify a non existing file name select the directory where the file should reside as described above and then type the new file name inthe Enter file name input field The Enter path or folder name input field can be edited like the Enter file name input field This allows changing the directory and file names displayed in the lists Clicking on the Update button at the bottom of the window updates the lists according to the specified string January 11 2
121. d requires an unsupported FID transform order digital filtering or weighting functions From the previous section the sucrose NMRanalyst parameters are configured to analyze FIDs The Varian VNMR software stores transformed spectra with all its phase components in file vnmrl vnmrsys exp datdir data the question mark represents a number between and 9 The data files are provided in the sucrose INADEQUATE subdirecto ries 1d fidand 2d fid Change the 1D Analysis workwindow 1D FID Spectrum or Line List field to 1d fid data and click Start Change the FFT workwin dow 2D FID or Spectrum field to 2d fid data and click Start Run the 2D Analysis workwindow which auto runs the Report workwindow From the Graphic workwin dow display the carbon skeleton It 1s the same as the one from the previous section except for minor coupling constant deviations January 11 2010 4 14 Tutorial Il Setting Analysis Parameters Analysis of the Sucrose VNMR Created Spec o As in SECTION 4 3 VerifyIt Molecular Structure Verification and va mm Shift Assignments create the shown carbon assignments for the ren T 1115 mol sucrose structure VerifyIt only assigns the 1D carbon spec 10405 2 trum results to this structure It agrees with the 2D INADEQUATE ai detected carbon carbon skeleton D aA TLH oO 72 36 d o Gei os Q Q January 11 2010 4 15 CHAPTERS Tutorial III Combining Analysis Results Hx CHAPTER 3 Tutorial I U
122. de Solid Correlations op creo NMRgraph when the input file does not contain atom Hide Dotted Correlations 110 og coordinates or when selecting the Redo Layout deel Val menu item Molecular structures are displayed by the lay Hide Dot Dashed Correlations 700 ith f ll ft t e h t d t t b tt R EC HES GES out a gorithm from left to right and top to bottom Reap Show H Shift Differences Ctrl D Restore plying the layout algorithm to the currently displayed Hide Predicted C Shifts Show C Shift Differences Ctrl R structures 1s particularly useful after adding or removing structures atoms or correlations Selecting a percentage from the Zoom submenu enlarges or reduces the distance between atoms in the displayed structures accordingly The size of the displayed atom and correlation labels remain unchanged The label size can be changed through Preferences as described above Excessive zooming out can lead to overlap of displayed items Switching the correlation labels off with the Hide Correlation Labels menu item can reduce the yen Selecting Restore from this submenu resets the display to the initial zoom level Hide Atom Labels Show Atom Labels toggles the display of atom labels on or off Hide Correlation Labels Show Correlation Labels toggles the dis play of correlation labels on or off For DQF COSY HMBC and INADEQUATE a correla tion label shows the determined coupling constant in Hertz For 3D sp
123. display Copy 1s similar to Cut except that it does not remove the structures copied Paste adds the structures on the clipboard to the display These menu items can be used to transfer structures from one NMRgraph application to another Sort by H Shift Agreement Sart by C Shift Agreement Molecular Formula and Weight International Chemical Identifier If no atoms and correlations are selected Flip Horizon tal flips all displayed structures horizontally If any atom or correlation is selected only the structures containing at least one selected atom or correlation are flipped horizontally Flip Vertical is similar except that it flips structures vertically Rotate 90 rotates structures clockwise by 90 degrees When only one correlation is selected Rotate Bond to Horizontal and Rotate Bond to Vertical are enabled They rotate the structure with the selected correlation so that at the end of the rotation the selected correlation is horizontal or vertical NMRgraph s layout algorithm attempts to obtain a natural two dimensional representation of the molecular structure But not every structure can be represented without crossing bonds or overlapping atoms To optimize layout speed NMRgraph does not explore all possible arrangements It starts with one atom and positions additional atoms around it To explore fur ther layouts select one atom in the structure and the Layout Structure From Selected Atom item b
124. display the manual chapters on these workwindows About NMRgraph shows program version information 19 2 The Atom Property Editor NMRgraph Atom Property Editor wl When one atom is selected in NMRegraph the Atom oars ele Properties menu item becomes active and starts atm Laver e the shown Atom Property Editor when clicked The Atom aaae mane Type and Atom Charge None Positive Negative can EE eege be chosen from the option menus The Atom Label text Screen Coordinates 295 195 field allows editing the atom label The Number of Free Correlation 37 2 to atom 88 26 Atom Valences option menu only applies to and hence is ie eat ee visible for carbon or nitrogen atoms For carbon and nitrogen Correlation 36 3 to atom 16 36 atoms the atom label color indicates the number of free valences and the number of bonded hydrogens The screen ea GEES ies eal coordinates of an atom is also displayed in the editor The list at the bottom shows the label of each correlation and the label of each atom correlated to this atom This connectivity information cannot be edited in this list Use the Correlation Property Editor described below to edit the correlation labels and the functions described in SECTION 19 4 Mouse Operations to change the atom con January 11 2010 19 10 NMRgraph Molecular Correlation Editor The Correlation Property Editor nectivities OK in this editor applies the current settings and C
125. djcolor format 9 17 E 13 5 cdjmono fonmat oennnnnnnnnn 9 17 phase function deskjet 6 00 01 ae 9 17 2D phase function plot 4 9 determining Linux printer mames 21 6 correcting spectral data 18 5 determining Windows printer names 21 6 KS djet500 format 9 17 phase sensitive spectrum epson format 9 17 distinction from absolute value 0 c cccce 14 2 CpSONe FOrMAL oes esse ess esseteeseseeeeeseees 9 17 plotting and graphics formatting with ghostscript ccc 9 17 IR RE 4 3 4 4 E E 2 5 3D fitting volume plot 7 7 increasing plot resolution 00 0 0 eeeeeeeee 18 2 accuracy OF plots sa ciatosesecie sieenascdesrnaccsnsccateocenassesn 18 3 laserjet format u 9 17 BEEN 18 3 lbp8 format 9 17 available graphics summarg 18 1 ljet3 format ceccscccscescssecssesessesseeecsseessessseeesseeeee 9 17 displaying overlapped Ines 16 9 ljet4 Format 9 17 expansion for more detail 4 5 ljetplus format 9 17 general input parameters 00 00 00 18 1 locally attached printet eee 21 7 January 11 2010 22 10 NMRanalyst Manual Index Network printer eee eseseseeseseseeeseeseeeee 21 7 prednisone output oes eects 3 10 pjx1300 format 9 17 redetermining 1D resonance uu 16 9 PostScript format 0 ccceeeee eee eee 9 17 21 7 removing ambiguous signals 0 0 cccceeeeeee 16 9 Print Manager 21 6 selecting reported information 16 4 16 7 T4081 Format 9 17 specifying 2D mmm 16 2 resolving problems esses
126. dow The workwindow area consists of an input screen at the top and an output screen at the bottom of the window When the area of the workwindow input and output screens exceeds the work window display area the horizontal and vertical scroll bars can be used to scroll to the area of interest Between the input and output screens is a sash a separator line with a small rectangu lar button at the right side To change the relative visible area of the input and output screens drag the sash vertically as desired When entering input values in the workwindow the sash is normally at the bottom of the window so only the input screen is visible When computa tions are in progress the sash is at the top of the window and the output screen is displayed Besides using the sash the Input Screen Output Screen button at the top of 2 Press the first left mouse button over the sash and move the mouse with the button pressed January 11 2010 12 2 Using the Workwindows The Workwindow Input Screen the NMRanalyst window or the corresponding menu item can be used to switch between the two screens See CHAPTER 9 Using the NMRanalyst Window for details 12 1 The Workwindow Input Screen The input screen allows the user to modify the input values for the computing task supported by the workwindow The input values in the input screen are initially loaded from a corre sponding input value file The workwindow updates this file as
127. dow Error Incorrectly Set DISPLAY Variable 953 Error Incorrectly Set DISPLAY Variable On Linux the NMRanalyst application needs to establish a display connection to the X Win dow display server The Windows version simply uses the local display in case of connection problems All NMRanalyst versions show a message indicating a DISPLAY error if no con nection to the screen can be established The following list provides reasons and corrective actions k The most common problem is an unset or incorrectly set DISPLAY shell variable The DISPLAY variable identifies the display server the display number the type of network connection and optionally the screen number to be used Whenever program analyst detects problems with opening the display connection the following message is displayed O analyst Starting NMRanalyst 3 6 from usr local ANALYST LINUX Exception in thread main java lang InternalError Can t connect to XLI window server using localhosti 0 0 as the value of the DISPLAY variable On a properly configured network a specified computer name gets automatically trans lated into the corresponding network IP address Should this name resolution fail either specify the network IP address or have the system administrator add the mapping between the name of your display computer and its IP address in the file etc hosts Program analyst assumes by default a TCP IP connection with both the display and
128. dow Menus Functions available through the speed buttons and the workwindow tabs are also available as menus They are located at the top of the application window The NMRanalyst functions are available through pull down menus File Edit SpectrumType Workwindow Pro cess Tools and Help pe Load reads a file into the selected workwindow input screen Load oi Print starts the Print popup described in SECTION 9 9 The Print a Popup Exit exits from the software The Load and January 11 2010 9 11 Using the NMRanalyst Window The Application Window Menus Exit items can also be found as speed buttons in the application window The Load item has an accelerator that is typing Ctrl L is identical to opening the File menu and selecting the Load menu item Similarly typing Ctrl I is equivalent to selecting the Exit menuitem An underlined letter in a menu item indi cates the keyboard mnemonic When a menu is opened typing the underlined letter is equiva lent to selecting the corresponding menu item Edit Cut cuts and Copy copies the selected text to the CLIPBOARD Cut ctx The CLIPBOARD content is accessible to other applications Paste at ne copies the CLIPBOARD content to the text field with the input focus Find in Output Screen Find Next F3 Preferences To search text in the output screen click Find in ma ee Output Screen from the Edit menu The sh
129. dow tab shows the popup menu DECH which allows reposi tioning the stack of workwindow tabs toward the top of the application window After the workwindow tabs are moved to the top right clicking on a tab again brings up the popup menu EISE which allows moving the workwindow tabs back to the bottom of the application window January 11 2010 9 7 Using the NMRanalyst Window The Speed Button Panel 9 7 The Speed Button Panel Output Screen f koaa ee f start ee f unnesnen EEN The pull down menus described in SECTION 9 8 The Application Win dow Menus provide access to all NMRanalyst functions The upper section of the application window contains speed buttons for convenient access to frequently used functions The underlined letter in a button label indi cates the keyboard mnemonic that is typing Alt or Ctr1 and the underlined letter simultaneously is equivalent to clicking the button This button toggles the workwindow display area between the Input Screen sy and the Output Screen by moving the separating sash Starting a computation with the Start button toggles the display area to the output screen Load Clicking this button starts a Filebox popup for loading a txt 1log olot Varian procpar or Bruker acqu file The txt files are the native parameter files of NMRanalyst The other file formats are converted to txt format by this load procedure by calling in this order the varian2txt bruker2tx
130. e Agree It specifies which fraction of the signal power in the experimental correlation area can be explained by the reported pattern A perfect fit no noise would give an agreement value of one The reported agreement value of 0 1416 14 indicates a strong correlation signal given the detection limit of NMRana lyst When the Summary Lines option is selected from the Report About Correla tions option menu the following type of list 1s generated CORRELATION Summary Lines if 6 C 12 C 13 CORRELATION Fa 30 038 Fb 22 013 J leo it T6 C 3 C 23 CORRELATION Fa 48 792 Fb 22 016 J 33 62 WO SAE 3 CORREGLATTON ee 94 045 Fo 48 790 J 32 66 it 31 C 3 C 4 CORRELATION Fa 48 794 Fb 45 381 J 29 13 it 32 C 4 C 14 CORRELATION Fa 45 383 Fb 19 644 J 37 37 it 34 C A C 157 CORRELATION Fas 45 364 Fb 19 374 J 36 93 Each line describes one identified correlation signal and lists the correlation number the num ber of each of the correlated 1D resonances both chemical shifts and for DQF COSY HMBC N15 HMBC COUPLED HSQC and INADEQUATE spectra the coupling constant For other spectrum types the average integral instead of the coupling constant is listed 1 The numbers are if not modified by the user the position of the 1D resonances in the decreasing frequency sorted 1D spectral line list s January 11 2010 16 5 Report Workwindow Select Items to Be Reported For the Nothing menu item neither the s
131. e Ctrl 2 surface View Reset Ctr R w White Background Cito Black Background Ctri B Parallel Projection Ctri A w Perspective Projection Ctr E Surface Ctri U Wiretrame Litt F3 F2 Side Lil F3 F1 Side Ctri 2 F2 F1 Side Ctri 3 Overlay menu applies to 2D contour 3D isosurface 1D spec Ge SE Se Se trum and phase function plots The items in this menu selectively vn BQalve slghals r 7 en ous Set men cme Switch the visibility of displayed components on and off For 2D v1D Experimental Spectum outen contour and 3D isosurface plots nD Positive Signals v 1D Simulated Spectrum ol nD Negative Signals and nD Correlation AUD ieee Dalai A Locations if specified display hide the positive signals neg 10 Component Resonances Ctrl C i ative signals and correlation locations respectively F1 Phases Ctrl 4 Fo Phases Ctrl 2 FS Phases Girl January 11 2010 20 3 NMRplot Plotting Multidimensional Spectra Pull Down Menus For 1D spectrum plots 1D Experimental Spectrum 1D Simulated Spec trum and 1D Residual Spectrum plot the selected spectra The residual spectrum is the difference between experimental and simulated spectra 1D Component Reso nances plots the individual overlapping signals of which the sum is equal to the simulated Spectrum If a 1D spectrum is added to the F1 or F2 dimension of a 2D contour plot 1D Experi mental Spectrum 1D Simulated Spectrum
132. e DMSO d solvent center reso nance around 40 3 while it should be at 39 5 ppm Display the 1D Analysis workwindow Click the Output Screen button to display the analysis results again At the bottom of the output screen NMRanalyst identifies the DMSO d solvent resonances and the spectral referencing is 109 960 Hz off from the expected referencing Experimental Simulated AU A 40 2 40 39 75 Frequency ppr DMSO Resonances 16 17 18 19 20 21 22 Start of Spectrum 109 960 1753 281 Hz To load the determined reso 15 41 9133 1 899 0 52328 0 616 E 16 40 0009 TIE p 15521 0 596 nance descriptions with refer 17 39 8337 3 091 0 12240 Lo 594 ence correction in the 1D E o Lo K M e mer ee Analysis input screen load 20 39 3329 7 254 o 11044 0 588 g from directory INADEQUATE 21 9 1660 2 668 0 10872 Lo 587 w thefileld analysis log 22 38 9982 186 2394 0 585 wi click Load and select 23 5 5553 665 0 22188 Lo 524 EE tINApDE QUATE 1d analysis lo g Scroll to the bottom of the 1D Analysis input screen in which the numerical description of January 11 2010 4 5 Tutorial Il Setting Analysis Parameters Verifylt Molecular Structure Verification and the determined 29 carbon resonances is loaded The removal of the seven solvent resonances is already requested by selected Remove check buttons for each solvent resonance as shown Rerun the 1D spectral analysis Display the r
133. e SECTION 2 3 Preparation for the Linux Instal lation preparation steps Start the installation program by executing sh MOUNT POINT ANALYST LINUX bin where MOUNT POINT is the DVD mount directory likely media NMRanalyst Some Linux shells might fail to run this installer so specify sh in this command line to execute it January 11 2010 2 6 NMRanalyst Installation Installing NMRanalyst After starting the NMRanalyst installer follow the on screen instructions Under Linux a user cannot eject a DVD if its mount directory is currently being accessed So change to a directory other than the DVD mount directory e g cd tmp before trying to eject the DVD On Microsoft Windows the default installation directory for the software is ANALYST WIN located on the drive containing the operating system When specifying a different directory leave ANALYST WIN as the installation directory name use a backslash rather than a Linux slash to separate directories in the path name and avoid pathnames containing special characters such as a plus sign comma semicolon equal sign or square bracket which can cause problems for a Linux shell Changing to a different device character works fine as long as the new device exists and has sufficient disk space On Linux the recommended location for a system wide installation is usr local The recommended location for a single user installation is the user s SHOM
134. e Windows XP 21 8 What should I do about the Windows XP NMRanalyst security alert Windows Security Alert EA Starting NMRanalyst under Windows XP may 9 To help protect your computer Windows Firewall has blocked Cause the shown security alert A similar Security E alert may result from starting the FindIt Database Manager of the NMRanalyst application NMRanalyst communicates with NMRgraph and NMRplot through sockets to handle printing plots displaying help information and aborting adding user structures to the FindIt database No network connection is ever attempted Click Keep Blocking NMRanalyst continues to function properly and this popup won t show Do you want to keep blocking this program Yi Name Launch nywhere GUI Publisher Zero G Keep Blocking Unblock Ask Me Later Windows Firewall has blocked this program from accepting connections from the Internet or a network IF you recognize the program or trust the publisher you can unblock it When should unblock a program again 21 9 How can I get NMRanalyst printing to work On Red Hat Linux from the desktop Main Menu click System Settings and then Printing to display the name and state of known printers On MS Windows select the Start menu Settings item Printers item The dis played Print Manager window shows an icon for each known printer If none is displayed see the Windows help system on how to add one If severa
135. e graph is queued to the printer specified in the NMRanalyst Print Settings popup See SECTION 21 9 How can I get NMRanalyst printing to work for a description on how to print this PostScript file on a non PostScript printer Exit exits from NMRgraph NMRgraph windows started from NMRanalyst are exited when NMRanalyst exits Edit The Edit menu items allow editing displayed structures Undo Change Ctri z2 Clear ShitttDelete The Undo Change reverses the last performed modification of the displayed structures Then Redo Change 1s displayed to reverse the Undo Change modification Remove Atom Labels Remove Protons Select All Ctrl A Delete Selection Delete Invert Selection Clear clears the current display A shortcut is to use the key board Shift Delete key combination Atom Properties Ctri h Correlation Properties 9 Ctri h Preferences Remove Atom Labels resets all the atom labels to the corre sponding atom types Remove Protons deletes all the protons and their bonds from the NMRgraph display Select Al11 selects all the displayed structures See SECTION 19 4 Mouse Opera tions for possible manipulations of selected structures Delete Selection is activated when at least one atom or correlation is selected It deletes the selected items When deleting an atom its correlations bonds are also removed A shortcut is to use the keyboard Delete key January 11 2010 19 3
136. e is loaded from the NMRSPEC subdirectory of the NMRUSER directory If NMRUSER is unset upon program startup this variable defaults to SNMRDIR UserData Directory NMRUSER contains files used by NMRanalyst which a user might want to modify such as default workwindow input screen values To modify these files create a copy of the whole directory normally called UserData modify the files as desired and set the NNRUSER variable to the modified directory User added structures to the FindIt database are also stored in the NNRUSER direc tory In order to add structures to the FindIt database a user must have write permis sion to the NMRUSER directory In case that a user does not have write access to the default NMRUSER directory SNMRDIR UserData a copy of the directory can be copied to a new location and the NMRUSER variable can be set accordingly The NMRSPEC shell variable specifies the spectrum type to be analyzed The value must be one of the supported types otherwise an error popup window is displayed Changing variable NMRSPEC in the Directory Editor and applying the change is equivalent to changing the spectrum type from the NMRanalyst menu See CHAP TER 9 Using the NMRanalyst Window for details Note that NURDIR NMRUSER NMRDATA and NMRSPEC are regular UNIX Linux shell variables User specified values always overwrite program defaults To permanently set these shell variables assign them values from the shell setup file SHOM
137. e is saved by appending bck backup to the old file name before writing the new 10g file Changing between workwindows or exiting and restarting NMRanalyst does not alter the content of any workwindow input or output screen January 11 2010 12 7 Using the Workwindows The Workwindow File Locking Workwindow Output Screen File Size H wl The shown popup is displayed if there is insuffi cient computer memory to load a Log file into File E ANALYSTAiNidatalnaAbe UATE We port log e e k A hi fil A S is too large to load into the Report workwindow Output Screen Its corresponding Wor window T IS Tle 1S auto matically renamed by appending bck to the original file name OK removes the popup Please reduce the size of this file _ok Help When a text region is selected in the output screen clicking the right mouse but ton above the output screen brings up the shown popup menu Selected text can be copied to the clipboard or printed by choosing Copy Selection or Print Selection To select all text in the output screen choose Select A11 To print all text independent of selection state choose Print A11 Print All Copy Selection Print Selection 12 3 The Workwindow File Locking The shown popup is displayed if one copy of NMRan alyst tries to access a txt file already in use by another NMRanalyst program Clicking Cancel or Exit aborts the action which led to this txt fi
138. e points to be used in each spectral dimension Typically these input fields are left blank indicating that all acquired points in the FID should be used for the data analy sis By specifying the number of points in these input fields the data in the corresponding dimension can be truncated or zero filled The maximum number of points for complex data January 11 2010 14 4 FFT Workwindow Spectral Description in each dimension is 2 65536 A specified value represents the number of points used by the Fourier transform without zero filling to the next power of two Key npF1 npF3 January 11 2010 14 5 CHAPTER 15 nD Analysis Workwindow This chapter describes the nD Analysis workwindow The individual input fields are explained along with standard settings for the analysis of routine spectra To display all the input fields available in this workwindow select from the Edit menu Prefer ences Set Mode Full NMRanalyst Show All Input Fields switches and click OK The analysis performed by this workwindow is the heart of the NMRanalyst software The corresponding analyze program determines the correlation information from two and three dimensional spectra Using the output of the 1D Analysis and FFT workwindows the analyze program searches all multidimensional signal regions to find spin systems of interest See CHAPTER 12 Using the Workwindows for a general description of the function and use of a workwi
139. ecomes active Click it and the structure is rearranged using the selected start atom hoort by In Shift Agreement and Sort by Shite Agreement sort the structures by the average difference between predicted and observed proton or carbon shifts respectively The structures are repositioned from left to right and then from top to bot tom The structure with best agreement is positioned at the top left corner of the display area January 11 2010 19 7 NMRgraph Molecular Correlation Editor Pull Down Menus If no atoms and correlations are selected Molecular Formula and Weight displays in a popup the molecular formula and weight for each structure If any atom or corre lation 1s selected molecular formula and weight are displayed only for the structures contain ing at least one selected atom or correlation International Chemical Identifier displays in a popup the InChI IUPAC International Chemical Identifier for each structure if no atoms and correlations are selected or only for the structures containing at least one selected atom or correlation if any atom or correlation is currently selected Prediction H Shifts predicts shifts for protons connected to carbon atoms in H Shifts On the currently displayed structures The predicted proton shifts are shown i Se PEN in square brackets beside each carbon atom label H Shift SSC Se Agreements displays in a popup the average difference between C Shift A
140. ect assignments These correlations are dis played by NMRgraph as correlation circle diagram See CHAPTER 18 Graphic Workwin dow for details The displayed correlations can be edited see CHAPTER 19 NMRgraph Molecular Correlation Editor Atoms are labeled by their chemical shift in ppm INADE QUATE derived bonds are shown as solid lines between atom labels with the determined car bon carbon coupling constant in Hertz as bond label ADEQUATE derived correlations are shown as solid arrows Their direction corresponds to the direction in which they were detected and the correlation label gives one involved proton frequency in ppm DQF COSY HMBC and N15 HMBC derived correlations are ambiguous and are shown as dotted arrows with a label indicating one of the involved proton frequencies in ppm Correlations with ambiguous assignment or of a strength below a specified Weak threshold are shown as dashed lines Key FnCorr January 11 2010 17 5 Assemblelt Workwindow Findit Identify Database Structures Best 17 2 Findit Identify Database Structures Best Matching NMR Data MFinalt IDENTIFY DATABASE STRUCTURES BEST MATCHING HMR DATA When this switch is selected this workwindow section is displayed The FindIt task compares experimentally observed carbon and or proton chemical shifts with predicted ones for known structures in its database A database with over 14 5 million structures from the National Institutes of Health NIH
141. ection parameters and decides whether or not a valid correlation signal was found according to the adjusted values When a valid cor relation signal is detected a summary line for this fitting area is shown like a IG E CTS CORRELATIONS a 22 166 FOs elek J 702 If no potential correlation signal can be found in the fitting area the summary line looks as follows 71 C 9 C 15 No correlation detected No pattern found When more than one potential correlation signal results from the optimization of the selected Detection parameters each of these signal patterns is examined further In case no detected correlations result from the potential patterns the signal pattern leading to the small est sum of square residuals is listed in the summary line with an explanation why this pattern is not a valid correlation signal The most frequently encountered reason for rejection of a pos sible correlation signal is an insufficient parameter precision If only the overall integral of a spin system is determined no Integrals switch selected the corresponding correla tion area summary line may read t aC OC 13 ONG iG6rrelataom detected dil January 11 2010 15 10 nD Analysis Workwindow Analysis Parameters When the phase functions are locked as indicated by unselected phase switches in the Detection section patterns can be rejected based on an inappropriate sign of the deter mined integral parameters Note that the anti phase
142. ectra CHAPTER 7 Tutorial V Additional NMRanalyst Features describes the analysis of 3D spectra and further software features The NMR data for the prednisone compound used in the first two tutorials were provided by Stephen H Grode Ph D MF C21H2605 14 9 mg in DMSO d Dual CryoProbe Shigemi tube Bruker 500 MHz DRX 500 spectrometer The related files are in the directory ANALYST ARCH data where ARCH is LINUX on Linux and WIN on MS Windows On MS Windows data is NMRanalyst s default working directory when the software is ger To start NMRanalyst on Windows assuming a default installation click on the FS menu button at the left bottom of the desktop click gt allPrograms Click NMRanalyst36 aNd Choose Wi NMRanalyst 3 6 1 Corticosteroid to reduce swelling It is used for many conditions such as skin diseases breathing problems artritis cer tain cancers and for hormone replacement January 11 2010 3 1 Tutorial I Using NMRanalyst findit Script for Molecular Structure Identifica On Linux create a copy of the data directory contained in the software installation direc tory The location of the installation directory is determined during the software installation Recommended locations are HOME home SUSER usr local opt export home usr and home If the installation directory cannot be found see CHAPTER 9 Using the NMRanalyst Window Make a copy of the data directory
143. ectra the correlation label shows the resonance frequency in the third dimension For all the other spectrum types a label shows the determined spin system integral 3 Bremser W Expectation ranges of 13C NMR chemical shifts Magn Reson Chem 1985 23 271 275 Steinbeck C Krause S Kuhn S NMRShiftDB constructing a free chemical information system with open source components J Chem Inf Comput Sci 2003 43 1733 1739 4 NMRgraph s layout algorithm is derived from Bley K Brandt J Dengler A Frank R Ugi I J Chem Research S 1991 261 amp J Chem Research M 1991 2601 2689 5 Item positions are stored in screen pixels When distances among atoms approach the screen resolution rounding errors might become noticeable when zooming in or out January 11 2010 19 5 NMRgraph Molecular Correlation Editor Pull Down Menus Hide Solid Correlations Show Solid Correlations Hide Dot ted Correlations Show Dotted Correlations Hide Dashed Cor relations Show Dashed Correlations and Hide Dot Dashed Correlations Show Dot Dashed Correlations toggle the display of corre lations with different line styles on or off The meanings of the different correlation line styles are described in SECTION 19 3 The Correlation Property Editor Hide Predicted H Shifts Show Predicted H Shifts is enabled after proton shift prediction is requested from the Prediction menu It toggles the display of
144. ectral resonances can lead to incorrectly derived atom atom correlations Specify the maximum number of 4 bond and longer range correlations to consider in these input fields Up to 50 4 bond and 50 longer range correlations can be evaluated by Assemblelt If more 4 bond correlations are detected than specified the excess number is considered as long range violations AssemblelIt reports molecular structures up to the specified numbers of 4 bond and long range correlations Key vio4Bond vioLongRange Unpaired sp Gros n molecular skeletons true sp carbons appear paired as both carbons con tribute an electron to the double bond The heuristic used is that CH CH and C carbons with a shift between 100 and 168 5 ppm are assumed to be sp As this is not always true the pair ing of assumed ep carbons can be switched off Key unpairedSp2 _ 3 Menbered Rings _ 4 Menbered Rings By default Assemblelt does not report structures containing 3 Membered Rings and 4 Membered Rings Due to the ambiguity of long range hetero nuclear data small ring systems are in agreement with even linear chains of carbon atoms When such features are expected in the molecule select the corresponding switches More possible structures result but reported structures are sorted in decreasing rating So only if the specified correlations support such structures will they obtain a high ranking Key ring3 ring4 Save Best structures 100
145. ednisone Click the 1D Analysis tab in the NMRanalyst window As no customized settings are available NMRanalyst provides default settings Unknown spec trum related fields such as Observe Frequency Spectral Width and Start of Spectrum remain empty Click the Load button at the top of the application window In the displayed Filebox popup double click in the Folders list on the left the direc tory named 5 and double click in the Files list on the right file acgus containing the spectral parameters This load process is also described as load from direc tory 5 file acqus or simply load 5 acqus Enter path or folder name E ANALYST WINtd atai5 Filter Mon Hidden Files d Folders Enter file name lacqus OK Update Cancel Help The spectrum specific parameters are loaded by this procedure Click Start This opera tion is also described as running the workwindow Twenty eight 1D resonances are reported The 1D Analysis workwindow saves the resulting phase corrected experimental and simulated spectra in file carbon plot Switch to the Graphic workwindow click the NMRanalyst window Graphic tab Set the File With Plot Data input field to carbon plot by either typing the new name or January 11 2010 4 3 Tutorial Il Setting Analysis Parameters 1D Analysis Workwindow Settings for Pred by clicking on the folder button at the right side and choosing this file from the started F
146. ement that protects Sun s interests consistent with the terms contained in the Agreement and v you agree to defend and indemnify Sun and its licensors from and against any damages costs liabilities settlement amounts and or expenses including attorneys fees incurred in connection with any claim lawsuit or action by any third party that arises or results from the use or distribution of any and all Programs and or Software 4 Java Technology Restrictions You may not modify the Java Platform Interface JPI identified as classes contained within the java package or any subpackages of the java package by creating additional classes within the JPI or other wise causing the addition to or modification of the classes in the JPI In the event that you create an additional class and asso ciated API s which i extends the functionality of the Java platform and 11 is exposed to third party software developers for the purpose of developing additional software which invokes such additional API you must promptly publish broadly an accurate specification for such API for free use by all developers You may not create or authorize your licensees to create additional classes interfaces or subpackages that are in any way identified as java Javax sun or similar convention as specified by Sun in any naming convention designation January 11 2010 8 6 Using the Help System Copyright JavaHelp 5 Java Runtime Availability Ref
147. enanesea 15 4 Estimated Coupling Constant 15 6 Graphic workwindow EE 18 1 l See also plotting and graphics Exu OON eera 9 10 H F hard disk fast fourier transform installation directory Size uo estes 2 2 point number specification S EE Eege ge l 3 5 paging swapping RE eegene 2 1 sch 0 fft program help DD iranis formi Ordf saicsscssseaiteeaestentietectiecnetceeve 14 2 full text search 8 4 coef file for 3D transform ieee Eise EE ee 8 3 January 11 2010 22 4 NMRanalyst Manual Index NMRanalyst online bein 2 5 coupling constant determination 15 6 online Manual directory uu ccc cece eeeeeseseeeeees 2 5 coupling constant mapping range 15 6 PDF NMRanalyst Manual 2 2 deriving carbon carbon correlation 00 4 17 2 table of contents 0 cece esecessetseseseeseseseseteeeeeees 8 3 DOF ais 1 3 HETCOR estimating coupling constants 0 c ccceeee 15 7 se EAE T sees etc reece ecco 1 2 deet 1 3 isotope shifts mm 15 4 15 5 Mapping parameters cccececcsececseseessseseeseseeeee 15 7 15 2 E E 15 9 mapping parameters e optimization detection parameters 15 9 heteronuclear spectrum prednisone sample dataset 2 5 MAPPING parameters EE Es sucrose sample dataset sees eeeeseeeees 4 12 history mechanism 10 2 12 4 12 6 INPUt screen 12 2 12 3 12 6 HMBC Input Screen button 9 8 SE E l 2 installation deriving carbon carbon correl
148. eously kg INADEQUATE Report Summarize 2D Spectral Analysis File Edit Spectrum Type Workwindow Process Tools Help SELECT ITEMS TO BE REPORTED Report About Correlations Hothing d Hon Correlations Hothing d Warnings On M mbiguous Signal Assignments Improper Phaze _ Fitting Area Overlap Redetermine _ ib Chemical Shifts The following table of correlation patterns is displayed use the vertical scroll bar to scroll to about the middle of the program output Index P I Prob Cl C2 Fa Fb J 180 10 402 1 0000000 CORRELATION 13 19 48 754 22 829 33 16 191 10 364 1 0000000 CORRELATION 15 17 35 560 33 137 34 82 176 9 134 1 0000000 CORRELATION 13 15 48 756 35 560 36 22 7 2 747 0 9999912 CORRELATION 1 8 211 416 87 402 46 29 CUTOFF 22000 Ke January 11 2010 3 10 Tutorial I Using NMRanalyst The Report Workwindow The identified bonds with determined integrals above the user specified threshold CUTOFF are listed The table shows from left to right the correlation index number the determined integral precision P T 7 the statistical confidence the bond assignment carries the location of the bonded carbons in the line list and the resonance frequencies and the coupling constant determined for this bond pattern It is more intuitive to inspect correlation information using the displayed correlation table Since 2D INADEQUATE spectra are homonuclear each cor relation appears twice This table assigns
149. equency ppm The analysis of these spectrum types 1s covered by previous tutorials Switch NMRanalyst to the copied strychnine directory Select the HMBC spectrum type Change to the 1D Analy sis workwindow Load from directory car bon fid the file proc par and run the 1D Analysis workwindow Load HMBC 1d_ analysis log to add the table of obtained reference corrected numerical signal descriptions with CDCl solvent resonances marked for removal Rerun the workwindow Display the shown carbon spectrum file carbon plot January 11 2010 6 2 Tutorial IV Advanced Structure Elucidation 1D Carbon 1D Proton Edited HSQC and 2 89 H if 6 5 4 3 a Frequency ppm at an 1 dL 100 rs S ppr E E EE EE DEE BEE ENEE F2 H H e 4 4 1 Bpm a Switch to the HSQC spec Experimental trum type and select the ou ID Analysis workwin dow for the 1D proton spectrum analysis Load file pro ton iidprocpar Run the analysis Display the created and shown Procon pLoOC spec trum Switch to the FFT workwindow Load C13 gug EE EE set Fl Start Of SpeCeErum 1025554119 taking into account the 6 052 Hz correc tion from the 1D carbon spectral analy sis and run the workwindow Switch to the 2D Analysis workwindow After optimization as described in the previ ous tutorial the final 2D Analysis parameters are TaresnoLas Integral of 66 917 Fl Phase and F2 Phase switches in Map ping and
150. er formats See http www cs wisc edu ghost doc printer htm for further information on the functionality and printer compatibility of gs supplied printer drivers Since a user may not know the correct printer driver Other Printer inthe To pull down menu is selected by default which invokes the Windows native dialog or Linux kprinter dialog for printing Specify in the To text field the printer to use By default the first available value from the last used entry stored in file analyst the LPDEST shell variable the PRINTER shell variable or the system default printer is shown Under Windows if a specified printer name does not work specify the printer s port name in this field instead See SECTION 21 9 How can I get NMRanalyst printing to work for further printer setting information The Pages Sheet menu allows printing multiple text pages on a single sheet of paper Button Print saves the printer settings and prints the specified file Apply Set tings only saves the current printer settings Cancel removes the dialog without print ing January 11 2010 9 17 Using the NMRanalyst Window Copyright Enscript Program 9 10 Copyright Enscript Program Copyright c 1995 Markku Rossi Author Markku Rossi lt mtr ik1 fi gt This file is part of GNU enscript This program is free software you can redistribute it and or modify it under the terms of the GNU General Public License as published by the Free Soft
151. er to the appropriate version of the Java Runtime Environment binary code license currently located at http www java sun com jdk index htm1 for the availability of runtime code which may be dis tributed with Java applets and applications 6 Trademarks and Logos You acknowledge and agree as between you and Sun that Sun owns the SUN SOLARIS JAVA JINI FORTE and iPLANET trademarks and all SUN SOLARIS JAVA JINI FORTE and iPLANET related trademarks service marks logos and other brand designations Sun Marks and you agree to comply with the Sun Trademark and Logo Usage Requirements currently located at http www sun com policies trademarks Any use you make of the Sun Marks inures to Sun s benefit 7 Source Code Software may contain source code that is provided solely for reference purposes pursuant to the terms of this Agreement Source code may not be redistributed unless expressly provided for in this Agreement Some source code may contain alternative license terms that apply only to that source code file 8 Termination for Infringement Either party may terminate this Agreement immediately should any Software become or in either party s opinion be likely to become the subject of a claim of infringement of any intellectual property right For inquiries please contact Sun Microsystems Inc 4150 Network Circle Santa Clara California 95054 LFI 135834 Form ID 011801 January 11 2010 8 7 CHAPTER 9 Using the NMRanaly
152. es above zero January 11 2010 3 11 Tutorial l Using NMRanalyst Determined Prednisone Structure For example the first line STT VT EE 26 29 7100 08 40 71 8 specifies that the carbon atom resonating in the 1D spectrum at 211 41 ppm is bonded to the carbons resonating at 1 87 40 ppm with J 46 29 Hz index 7 and 2 66 08 ppm with J 40 71 Hz index 8 3 8 Determined Prednisone Structure 126 39 210 19 aid Zeen Ee The correlation table can be displayed graphi cally Select the Graphic tab specify in the File With Plot Data input field the Report workwindow generated struc ture plot and click Start to obtain the shown structure display A carbon atom is labeled by the 1D carbon resonance frequency in ppm A bond is represented by a line between both bonded atoms and labeled by the determined coupling constant in Hz Com parison with the prednisone structure at the beginning of this tutorial shows that all the prednisone carbon carbon bonds are identified January 11 2010 Tutorial Il Using NMRanalyst Determined Prednisone Structure The prednisone molecular formula indicates that this structure contains oxygen atoms While NMR does not observe them observed carbon shifts reflect their pres ence Select in the NMRgraph window Predic tion Place Heteroatoms In the started popup make sure that oxygen O is selected and click the OK button NMARgraph
153. es up to the number of requested most likely structures Most important for building structures are 2 bond HMBC correlations Some are not detected For the gibberellic acid structure generation select the AssembleIt ELUCIDATE MOLECULAR STRUCTURE FROM NMR DATA section Set its Evaluate Unobserved Bonds to 7 to have Assemblelt derive up to this number of unobserved bonds from other detected correlations Run the AssembleIt workwindow The exhaustive structure generation takes under minute on a 3 06 GHz PC and one structure results January 11 2010 5 7 Tutorial Ill Combining Analysis Results Assemblelt HSQC amp HMBC Derived Gibberellic oy at re i From the Graphic workwindow display the i 1 t shown CC_corrs plot file first The col en Ge erg S za ored labels in this circle diagram represent the TE e ae Ka 19 gibberellic acid carbon shifts A red atom Zeg EE II tan COlor identifies a quaternary green a CH blue A Al ii a Ch and orange a CH group The selected Ee KS i n A Pig A E Consider Chemical CHITE o pe i ei rs P Riesi switch further reduces the number of pel i FR ais i f free valences of some carbons This is indi oh an E d Se cated by an appended atom suffix of s d t or A ee Leg S q Singlet unprotonated doublet one d r E EE EE bonded proton triplet two bonded protons BRE ye FEE OF OG or quadruplet three bonded protons repre sha ag A fst sents the number of corresponding
154. eseseeeeeeeeee 21 6 statistical information 0 0 0 0 16 5 setting default printer 0 cece 21 6 USC of 3 10 S16 Ol OF formal EN 9 17 WOLKWINdOW o oo ecesessessessesessesseseeseesesseseesessessesesee 3 11 a file bet 9 17 21 7 Report workwindow 16 1 EE 9 17 Resume menu tem 9 14 private software installation 2 5 ROESY probe ACTONYM MEANING ooo eee es secs esseeseeesestseseeeesees 1 2 EE 4 12 BER ee 15 9 procpar E 12 9 S R Sampa SONU W ALS sissien ea ceii 21 4 reference manual shell script chapters ee Stee ARE 1 5 bruker2txt 9 8 13 3 tA report program for format CONVELSIONS c cscsscsssesesseeseestesesseeees 9 9 GE 16 7 E E 9 8 ANAL EEN 3 11 PO A EAEE AEE 9 8 build up curve generation 16 10 protein ilog oe eeeceeceeeseseesessessesessssesesessvssetseeaeeeee 2 4 correlation output description 16 4 EE 9 8 correlation EE 3 11 E EE ER Ee 16 2 DISPLAY 9 4 ER 16 1 LppEST nnen 9 17 O a O DZ NMRDATA wassnnennennnn 4 13 10 1 Een 16 2 O NMRDIR 10 1 non correlation features 16 6 NMRSPEC 4 13 10 1 EE 16 5 NMRUSER 10 1 Overlapping st m 16 9 IN sic cite cease ne AEAEE OAA 9 3 parameters eegene 16 4 PRINTER 9 17 phase MaN SENNA CM copu2tca res aecconeeastantvaneedteseeeterecosa eons 16 8 plotting phase Dmnctons eects 16 2 Sign of Phased Signals eeee 13 6 precision CistribUtiON cece esses eseeteeneeees 16 3 single user mstallatnen g January 11 2010 22 11 NMRanalyst Manual Index
155. esonances can often be fitted as an overlap of Lorentzian component lines with different phase values as selected by the Random Phase option The Pos amp Neg and Random Phase settings yield identical results for clusters of one or two res onances But for three or more resonances per cluster the Random Phase setting contin ues to determine one phase parameter for each resonance in the cluster while the Pos amp Neg setting determines a linear phase function for all resonances in the cluster For the Pos amp Neg and Random Phase options it is not clear which resonances are supposed to be positive and which ones are supposed to be negative So the 1d analysis program phases the spectrum so that the majority of resonances are in posi tive absorption The numerical results of the 1D and multidimensional spectral analysis are not affected by this ambiguity but the y axis of plotted 1D data might be shown reversed from the desired result Key PseSht Analysis Region fron sem L to Low Frequency m These two input fields allow limiting the analysis of the 1D spectrum to the specified spectral range Normally both input fields are left blank and the first input field defaults to the sum of the Start of Spectrum and Spectral Width input values and the second field to the Start of Spectrum value corresponding to the analysis of the whole spectrum Notice that the low field resonance corresponding to the highe
156. esulting carbon plot file from the Graphic workwindow as before Confirm the seven DMSO d resonances are removed except for minor residuals which are identical to the ones observed before removing these resonances 4 3 VerifyIt Molecular Structure Verification and Shift Assignments gt SECTION 3 1 findit Script for Molecular Structure Identification cov Os Le ers FindIt the determination of best matching structures for NMR data ANA Aa Often the likely structure of a molecule is known and only needs to be ot validated by NMR Perhaps the molecule was synthesized by well estab lished reactions and only its identity needs to be confirmed Each NMRanalyst supplied sample dataset directory contains the Molfile of the examined molecu lar structure From the Graphic workwindow display the shown 4900 mol prednisone structure Bonded hydrogen atoms are implied and not explicitly displayed Click the AssembleIt tab Select the VerifyIt RATE SPECIFIED STRUC TURE BY AGREEMENT WITH NMR DATA switch Set its Proposed Structure to 4900 mol and Assigned Carbon Shifts to 4900 plot Run the workwindow VerifyIt shows a detailed comparison of the detected 1D carbon results with the expected molecular structure The overall prednisone rating is 0 993954 ona scale of 1 0 for perfect agreement and 0 0 for no agreement Compared to over 15 9 million FindIt structures of small organic molecules this specified structure obtains the top posit
157. et Carbon to the generated ncarbon out list Start this workwindow The best matching structure identified has the PubChem CID of 6466 and is gibberellic acid 5 6 Structure Elucidation Without 1D Carbon Spectrum With the previously generated ncarbon out the gibberellic acid structure elucidation can be completed without using the acquisition time intensive 1D carbon spectrum Repeat the HSQC and HMBC spectrum analysis using ncarbon out Select the HSQC spectrum type and its 2D Analysis workwindow Set F1 1D Analysis Output File to ncar bon out Delete the Map F1 Frequencies entry In the Report workwindow delete the Redetermined Fl Resonance List entry Run the 2D Analysis workwindow again and it runs the Report workwindow Load the determined phase functions in the Graphic and 2D Analysis workwindows Run the 2D Analysis workwindow and the Report workwin dow is auto run January 11 2010 5 10 Tutorial Ill Combining Analysis Results Structure Elucidation Without 1D Carbon Spec Switch to the HMBC spectrum type and its 2D Analysis workwindow Set Fl 1D Analy Sis Output Filetoncarbon out Thresholds Integral to 0 26 and delete the Map Fl Frequencies tand Thresholds Agreement entries In the Report workwindow remove the Redetermined F1 Resonance List entry Run the 2D Analysis and it runs the Report workwindow In the AssemblelIt workwindow set the input field Carbon to ncarbon out Set in the AssemblelIt section the Evaluate
158. fearrann n 15 6 EE 15 8 AS O E 15 6 EE 15 8 EE 16 6 lee 15 6 PRG FA WE 13 8 E EE 15 8 PR AIO EE 13 8 Man EE 15 8 tee 15 7 dE E 15 8 EE 17 9 MapP cie A 15 6 ng 17 9 gege 15 6 OM 16 6 EE 18 5 BS ee 13 3 MAX Weight 17 6 e Bc a oe nT en 13 2 7001 SOS S001 1 Fe 17 6 Seier 13 2 MAV eege 18 5 EE 18 2 mtnMWeight 17 6 SO E 13 5 14 4 EAR H 16 10 SET 13 5 14 4 leet 16 10 Ree 14 4 HS 1 E 13 6 ZE E 14 4 EE EE E 13 6 EE 13 4 14 4 MUIMiGN L eee ess essesscesesscssessessessescsesseseeseen 15 4 sw 13 4 14 4 EI E 14 5 EN 14 4 mb 14 5 S eA O 14 4 E 13 5 E aer PT 17 2 January 11 2010 22 7 NMRanalyst Manual Index fal LY LS ee 17 3 TTDI E WEE 17 4 E 18 2 EE 13 4 14 3 WEE 18 6 EEGEN Eee 17 9 Eelere 15 7 IRC KEE 15 7 EE 17 9 ViOLONGRANGE eieiei 17 9 Ke d BE 13 5 Ke REU r 13 5 EE eiert 18 3 KE 18 2 SATO nn 18 3 SEI 18 2 Ge EE 18 3 KE 18 2 keyboard COMMANAS siiin 12 4 12 5 L line list generic generation 0 ecccccsecsecseesecsecseesecseesseseens 6 5 linear prediction oo cecccccccccccseesesseeseeseeeeesees 13 2 Linux supported computer platforms 0 c ceeeeeee 2 1 virtual memory teguitements 21 10 Linux NMRanalyst HATS EE E 2 6 un installation EE 2 8 log Dies 12 7 12 9 M Mac Os WIA BE 21 4 Make Default menu tem 9 14 manual Appendix a 21 1 reference manual 1 5 TE BEE 1 5 Map Coupling Constant 1 15 6 Map F2 Frequencies 00 00 15 11 Mapping Darameterg 15 8 Min Cluster Dustance 13 6
159. file and directory names are case sensitive MS Windows file systems are case pre serving but not case sensitive A new file called Data overwrites an existing file called data in the same directory NMRanalyst default names are unique and work as expected on case insensitive file systems When choosing new names make sure they differ by more than the capitalization 21 2 3 Handling of File and Directory Access Permissions Linux provides read write and execute file permissions for the owner group members and other users MS Windows uses different kinds of file systems with different access permis sions On NTFS all files have an owner group which is not set by default and access con trol list ACL permissions Windows NMRanalyst maps the MS Windows file access permissions to the closest Linux settings 21 2 4 No Special Characters Allowed in Path or File Names Microsoft Windows allows special characters in directory and file names which can cause problems for the UNIX Shell started from NMRanalyst Avoid the use of special characters such as a plus sign comma semicolon equal sign or square bracket in file and directory names which cause problems when used unquoted in a UNIX shell 2 NTFS is the native file system for Windows 2000 XP and Vista January 11 2010 21 2 Frequently Asked Questions Any differences between MS Windows and 21 2 5 Microsoft Windows Drive Letters and UNC Path Names Linu
160. g ees Using proton excitation and EEN ADEQUATE can be several times more sensitive than 2D INADEQUATE and this spectrum type can be acquired on an indirect detection probe Switch to the 1 1 ADEQUATE spectrum type In the FFT workwindow load adequate dq fid procpar and adjust F1 Start of Spectrum to 14597 577 considering the 6 052 Hz referencing correction from the 1D carbon spectrum Run this workwindow In the 2D Analysis workwindow set Thresholds Integral to 1200 to only consider stronger resonances Run the workwindow and it auto runs the Report work window Load file ADEQUATE report log in the Graphic and 2D Analysis workwindows to set the phase functions Rerun the 2D Analysis workwindow and it auto runs the Report workwindow Display the adequate spec file with correla tion locations using the Graphic workwindow as shown Unaliased correlation areas are shown as black and F1 aliased correlations as green bound uv 4 ing boxes Like 2D INADEQUATE the 1 1 lt o 1 ADEQUATE signal to noise ratio is too limited to make spectral 1D projections useful So the 1D proton spectrum is shown along the F2 axis The 1D carbon spectrum does not have the double quantum format of the F1 axis and is not shown ve Kai 3 TTT Ff D 00 220 240 d re D F tamed 260 DH pm ee a ee F2 S T D 5 4 A 2 U ppm January 11 2010 6 10 Tutorial IV Advanced Structure Elucidation Adding Heteroatoms 42 28 Run the Assemb
161. g the File With Subtract Spectrum Plot Data input field described in Section 18 1 Settings for All Plots an experimental or simulated spectrum can be specified for plotting The spectrum specified in this field is subtracted from the File With Plot Data spec January 11 2010 18 3 Graphic Workwindow Multidimensional Spectrum Specific Settings trum before plotting A residual spectrum can be plotted as the difference between the experi mental and simulated spectra as long as both spectra have the same file format and the same number of spectral points Key FnSubtract Aaa Corr Locations inedequate_areas plot EU Locations of detected corre lations can be added to 2D contour and 3D isosurface spectral plots Specify the name of the file containing correlation locations in this input field Key FnLocation Add F1 Im Spectrum EI A 1D spectrum can be Add F 1D Spectrum Iesch zier a added to either the F1 or F2 dimension of a 2D con tour plot Specify the name of the file containing the 1D spectrum in these input fields Since these input fields only apply to 2D contour plots they are hidden when the currently selected spectrum type is 3D SPECTRUM Key Fn1DSpcF1 FniDSpcF2 contour Lines tin max steps The 2D contour and 3D iso surface plots use the specified Contour Lines or Iso Surface values as intensity contour or surface values When not specified a Max input field value is supplied based o
162. gen spectrum is 50 times worse than carbon Acquisition of a 1D carbon spectrum often takes as much time as a multiplicity edited HSQC HMBC and DQF COSY spectra combined The acquisition of a nitrogen spectrum is impractical Equivalent resonance information can be derived from HSQC and HMBC type spectra If a file name is specified the F1 resonance list is redeter mined and saved From HSQC only protonated resonances can be obtained An HMBC is often not phase sensitive in F1 So use as F1 resolution no less than the distance between the closest heteroatoms to be resolved This field is displayed for the HMBC N15 HMBC HSQC COUPLED HSQC and N15 HSQC spectrum types Key FnResF1 January 11 2010 16 3 Report Workwindow Select Items to Be Reported 16 3 Select Items to Be Reported When the Everything option is selected all informa tion determined for every correlation signal is displayed Report About Correlations 6 C 12 C 13 CORRELATION Fa 30 038 Fb 22 013 J 31 97 Tal 2 65 8241 555 824 1 000 65 8241 555 824 67 77 Ib1 2 65 8241 585 838 1 000 65 8241 585 838 65 30 Fa Fb 30 0482 30 0382 2637E 02 22 0183 29 0199 2362E 0 Pa Pb 2 21128 1 98546 1980 2 24784 2 08620 1836 Ta Tb 200000 200000 1 000 221400 221400 1 000 Ts J 233319 233319 1 000 35 0000 31 9676 2669 Fd Pd 52 0665 52 0511 1 000 149412 197273 8358E 0 Tr Td 100000 100000 1 000 109368E 01 109368E 01 1 000 iter 8 Agree
163. greements observed and predicted proton shifts for each structure It takes a carbon C Shift Details atom label as the observed shift for the proton s attached to the carbon Place Heteroatoms NMRgraph 1H Shift Details 2 x H Shift Details shows in a popup detailed derivation of predicted proton chemical shifts If any atoms or bonds are selected in dis played structures choosing this menu item brings up the shown popup A user can choose whether P cancel om to display proton shift prediction details for all structures or only structures with selected atoms or bonds Shift prediction details can be lengthy If numerous structures are currently displayed it may be more convenient to select a few interesting structures and only display their shift prediction details Show Shift Prediction Details for ALL Structures e Only Structures With Selected Atoms or Bonds Li Shifts predicts carbon shifts for displayed structures The predicted carbon shifts are shown in parentheses beside each carbon atom label 19C Shift Agreements displays in a popup the average difference between observed and predicted carbon shifts for each structure It treats a carbon atom label as the observed carbon shift 195C Shift Details displays detailed derivation of predicted carbon chemical shifts in a popup Similar to Up Shift Details if any atoms or bonds are selected in displayed January 11 2010 19
164. haustive listing of all possible structures the first found structure might be regarded as the correct one But display the CC _corrs plot 1 file of possible strychnine structures All structures have the correct number of carbons and nitrogens correct atom valences and explain the observed HSQC and HMBC correlations within the specified limits One of these structures is likely the correct carbon and nitrogen skeleton of strychnine But further information is helpful to identify which one is the correct one 6 5 Using DQF COSY The HMBC 2 bond Jou 3 bond Clow and longer range Jcn correlation ambiguity cannot be resolved experimentally A valuable source of unambiguous correlation information is an additional DQF COSY spectrum HMBC can detect a bond if one of its bonded carbons is protonated For DQF COSY bond detection both carbons need to be protonated But then their vicinal coupling identifies their carbon carbon bond January 11 2010 6 8 Tutorial IV Advanced Structure Elucidation Using DQ 1 1 ADEQUATE Switch to spectrum type DQF COSY and its FFT workwindow Load gdqf cosy fid procpar and run its transform In the 2D Analysis workwin dow set Thresholds Integral to 1 deselect the F1 Phase switch for Mapping and Detection set F1 Phase 1 56517 0 0017787 ppm and F2 Phase 0 06599 0 0905206 ppm Run the 2D Analy sis and it auto runs the Report workwin dow In the Graphic workwindow set the same phase fu
165. he 1D Nitrogen field to nnitro gen out and start the workwindow Display the created NC_corrs plot file It is a circle diagram As only a few correlations were derived click the NMRgraph View Redo Layout menu item to obtain the shown lay out January 11 2010 6 6 Tutorial IV Advanced Structure Elucidation Strychnine Structures Derived From Previous 6 4 Strychnine Structures Derived From Previous Analysis Results From the previous analysis results the possible strychnine structures can be derived Return to spectrum type HMBC not N15 HMBC and select the AssembleIt workwindow Select the COMBINE NMR ANALYSIS RESULTS section Set 1D Nitrogen to nnitro gen out Delete the 1 1 ADEQUATE DQF COSY and INADEQUATE field entries For faster structure generation the HMBC Weak field should be specified To see how to choose this value deselect the AssembleIt ELUCIDATE MOLECULAR STRUCTURE FROM NMR DATA section in this input screen and start the workwindow The middle of the output screen shows the detected 127 HMBC correlations HMBC HMBC report log 499 87 MHz F1Diff 0 155026 F2Diff 0 016914 Fl Range 9 74 F2 Range 6 50 ee 225 Ges 3 H2 35 Fl 140 032 F2 4 092 es 20 120 2 889 Cl 10 H2 34 Fls 77 391 F2 4 090 I 16 070 127 ae Cl 9 H2 10 Fl 116 005 F2 7 198 I 1 507 The strongest HMBC correlation has an integral of 20 120 and the weakest one an integral of 1 507 A consistent way to acquire HMBC spectr
166. he atom label with the left mouse button to start its Atom Property Editor From the editor the atom type atom label atom charge and number of free valences for carbon and nitrogen only can be set To add a correlation between two existing atoms hold down the Ctr1 keyboard key press the right mouse button over one of the atoms drag the mouse cursor to the other atom a red dotted line appears and release the mouse button and the Ctrl key To set the correlation type style and label double click on it with the left mouse button and set its properties from the Correlation Property Editor To move a correlation between atoms right click the correlation at the side of the atom to be disconnected drag the correlation with pushed right mouse button to the new atom a red dot ted line appears intermediately and release the mouse button The correlation is moved to the new atom January 11 2010 19 12 NMRgraph Molecular Correlation Editor Keyboard Key Operations 19 5 Keyboard Key Operations Keyboard keys can be used to scroll the NMRgraph display The following table describes supported key operations Home scroll to the top left display corner End scroll to the bottom right display corner Page Down move display down by one screen Page Up move display up by one screen Ctrl Page Down move display to right by one screen Ctrl Page Up move display to left by one screen 19 6 Copyright InChI
167. he tutorials and installation testing additional datasets are only loaded when explicitly copied 2 5 De installing NMRanalyst The de installation procedure described here permanently removes NMRanalyst from a user s computer After the de installation NMRanalyst can be installed again An older version of NMRanalyst should first be de installed before the newer version is installed To un install exit from NMRanalyst On Microsoft Windows from the menu click gt allPrograms Click NMRanalyst36 and then click the uninstaliNwiRanalyst36 item Under Windows Vista the de installation needs to be run in the XP compatibility mode Before running the de installer right click on the uninstali nmRanayst36 item and choose Properties Click the Com patibility tab Select the check box Run this program in compatibility mode for and select Windows XP Service Pack 2 Also select the check box Run this program as an administrator and click the OK button On Win dows 2000 and XP if no shortcut is created during the installation start the Control t9 Addor Panel and double click on the Remove icon The NMRanalyst installation creates the Programs NMRanalyst for Windows entry in the Add Remove list Select this item click the Change Remove button and follow the displayed instructions To un install NMRanalyst on Linux type from a shell window O s uninstall analyse If no logical links were created dur
168. hey result from digital filtering imperfections Specifying 99 or any value above 10 selects a model free baseline correc January 11 2010 13 5 1D Analysis Workwindow Analysis Parameters tion The mentioned values are used by default when no other value is specified Key BaseN Min Cluster Distance 1 x This value specifies the frequency range included around a resonance for determining its numerical description Two neighboring resonances are consid ered to overlap if their specified ranges overlap In this case both resonances are assigned to the same cluster of overlapping resonances and the parameter values of all cluster resonances are optimized simultaneously to account for the effects of their mutual overlap All resonances in a spectrum mutually overlap Fortunately effects of resonance overlap decrease rapidly with resonance distance and become negligible Set this input field to several times the expected width at half height of the widest resonance of interest Key mSigHz mSigHzU Min Integral Precision The Min Integral Precision is defined as the inte gral value of a detected resonance divided by its error value marginal standard deviation Noise patterns will typically yield integral precisions around one 1 e the integral error value is as high as the detected resonance integral When this input field value is increased the weaker resonances which are poorly defined by the data are removed first Th
169. ical Shifts switch is mutually exclusive with this Ambiguous Signal Assignment switch because it removes all but the most proba ble assignment from a collection of mutually ambiguous assignments Selecting the Ambiguous Signal Assignment switch displays ambiguous correlations in a dif ferent linestyle in the NMRgraph correlation editor See CHAPTER 19 NMRgraph Molecu lar Correlation Editor for details Key LAmbig Mimroper Phase When the Improper Phase switch is selected those correlations with phase values inconsistent with the determined phase functions are identified in the Connec tivity Table CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 Shift Shift J Index Shift J Index Shift J Index ppm ppm Hz ppm Hz ppm Hz 7 Eee 135 50 132 63 60 7 31 128 54 65 4 12 Phase mismatch index s call 12 An individual phase value determined from a 1D spectrum can be shifted by 27 360 degrees and two phase values from a single transition determined from a multidimensional spectrum can be shifted by m 180 degrees in two orthogonal directions without changing their appear ance For example for 2D INADEQUATE spectra the bond signal model has three phase val ues one F1 and two F2 phases A phase mismatch occurs when one phase is shifted an odd and the other phases an even number of times or the other way around The shifted phases and determined phase functions are saved in a plot file as des
170. icking on it O xwd gt tmp p xwd January 11 2010 21 7 Frequently Asked Questions Why can t switch NMRanalyst to a different The captured screen image can be displayed with xwud lt tmp p xwd amp This might be required for applications which cannot directly capture nmrplot OpenGL images To obtain a higher than screen resolution save the NMRplot or NMRgraph display in a PostScript file File Save PostScript To create a 200 dpi 2800x2800 pixel image from the vector graphic PostScript file use type the command as a single line gs GdNOPAUSE dBATCH sOutputFile myFile jpeg r200 9g2800x2800 SDEVICE jpeg myFile ps 21 11 Why cant I switch NMRanalyst to a different spectrum type NMRanalyst does not allow changing the spectrum type while computations are in progress Workwindows with ongoing computations are identified by the clock icon on their folder tab Either wait for the remaining computations to finish or join each active workwindow and ter minate the computation by clicking Stop A new spectrum type can be selected after the last computation terminates 21 12 Why are the NMRplot colors wrong NMRanalyst NMRegraph and NMRplot are Java applications and are displayed on Linux operating systems by its X Window system NMRplot itself uses a small number of shared color cells So it does not interfere with the operation of other running X Window applica tions But if another running a
171. ida tion from a combination of direct carbon carbon correlations 2D INADEQUATE heteronu clear correlations ADEQUATE or HMBC and proton proton correlations DQF COSY Such experimental correlations pose challenges for example expected correlations may not be observed Among the observed correlations some may correspond to long range gt 3 bond correlations and some may result from incorrect assignments AssemblelIt can still derive the carbon skeleton or molecular fragments despite these challenges January 11 2010 17 1 Assemblelt Workwindow Combine NMR Analysis Results The AssembleIt workwindow input fields are described in this chapter See CHAPTER 12 Using the Workwindows for a general description of a workwindow 17 1 Combine NMR Analysis Results COMBINE HMR ANALYSIS RESULTS The molecular skeleton determination consists of two steps This first one reads NMRanalyst analysis results For running the subsequent Assemblelt task it derives possible atom atom correlations from ADEQUATE DQF COSY HMBC N15 HMBC and or INADEQUATE results Results from an HSQC spectrum plus those from at least one of the previous spectrum types need to be specified This panel is only active when this switch is selected Key Task1 m Proton proton out DI A structure determination typically starts with the determination of proton and carbon chemical shifts All multidimen sional resonances are then assigned to these 1D reso
172. ield is displayed when the Transform Order option menu below is set to Coefficient File The specified file should contain the transform coefficients in VNMR coef file format For 3D data the first four lines in the file should contain eight coefficients each and specify how the F2 interferograms are formed The fifth row contains the eight coefficients specifying how the F1 interferogram is formed See the VNMR documentation for details Key Coefs2D or FnCoef Fil RSL Eo Ke A NMRanalyst s input data formats are Varian VNMR FIDs and spectra data file format and Bruker AMX and DMX FIDs or spectra for 2D named 2rr 2ri 2ir 2ii and for 3D named 3rrr 3rri 3rir 3rii 3irr Siri 3iir 3iii For Bruker AMX amp DMX input files use the Load button in the NMRanalyst window and select a ser or pdata 2rr or equivalent file located in the acquired data directory This procedure calls the bruker2txt script to assemble the needed spectral information from the Bruker acquisition and if present processing parameter files After calling bruker2txt do not alter the input file name as it determines 1f this file is assumed to contain a Bruker FID or preprocessed spectrum Bruker FIDs can contain any combination of TPPI States States TPPI and other acquisition formats for each spectral dimension Please report if data from a standard pulse sequence is not correctly handled so NMRanalyst can be extended accordingly In case of proble
173. ient only for accurate acquisition and referencing of the 1D and 2D spectra and in the absence of significant signal overlap in the 2D spectrum For homonuclear and heteronuclear correlation spectra the default mapping range is 10 Hz A referencing difference between the 1D and the multidimensional spectra can be compen sated for by analyzing the multidimensional spectrum with the Map F Frequencies values greater than their default values The referencing of the multidimensional spectral axes can then be corrected by the average determined frequency shift However an increased input value slows the analysis significantly and increases the number of ambiguous signal assign ments Keeping the acquisition conditions of the 1D and the multidimensional spectra identi cal and acquiring the 2D spectrum with sufficient resolution to limit signal overlap problems is preferable Key RangeF1 RangeF3 RangeF1U RangeF3U 1 Hansen P E Isotope Effects on Nuclear Shielding in Annual Reports on NMR Spectroscopy Webb G A Ed Aca demic Press New York 1983 Vol 15 pp 105 234 January 11 2010 15 5 nD Analysis Workwindow Analysis Parameters 2 Mapping Points Depending on the selected spectrum type up to three spectral dimen sions F1 through F3 can be mapped The specified frequency ranges are mapped using the number of mapping points specified in these F1 through F3 input fields The density of map ping poi
174. ile box Run the workwindow to display the shown overlaid spectra ku NMARplot CfANALYST WINfdatafcarbon plot File View Overlay Component Unit Help 1 06 0 8 0 6 DA DA 214 200 180 160 140 120 100 GU DO 40 16 Frequency pem In the displayed plot the experimental spectrum is drawn in yellow first and then the simu lated spectrum is drawn on top in black No resonance of significance should remain in yel low as this would imply it was not properly identified by the automated analysis Small differences between detected and simulated resonances result from magnet inhomogeneity imperfect shimming and spectral noise Here the spectral baseline is sufficiently flat and all resonances are identified January 11 2010 4 4 Tutorial Il Setting Analysis Parameters 1D Analysis Workwindow Settings for Pred The seven DMSO d sol vent resonances in this spectrum are around 40 ppm Place the cursor at the top left edge of the area of interest in this spectral plot drag the cur sor with pressed left mouse button and a yel low rubber band indi cates the currently selected region Release the mouse button and the selected area redraws enlarged If the area is still too large zoom in again If the area is not correct select Reset from the View pull down menu at the top of this window to undo all zooming or select Previous Zoom to undo the last one The shown enlarged spectral area shows th
175. iles of the component spectra should be entered in these input fields and the corresponding mixing times should be entered in the Mixing Times ms fields described above Key FnNOE1 FANOE 5 Save Build Up Curves in File puila up plot DU Use this input field to spec ify a name for the created plot file If no name is specified build up plot is used The two dollar signs in the default name expand to a unique process ID at run time The build up curve plot is created when the Report workwindow is run with the GENERATE NOE BUILD UP CURVES switch selected and the Mixing Time ms and2D Analysis Output File For NOE Spectrum input fields contain valid entries The numerical January 11 2010 16 10 Report Workwindow Generate NOE Build up Curves analysis results are displayed from the Report workwindow output screen and the created plot can be displayed by the Graphic workwindow Key FnBuild NMRanalyst provides the user interface for this build up curve generation The report pro gram calls the corr2plot shell script for the actual curve generation Script corr2plot eliminates curves with negative slope likely diagonal signals When three or more mixing time spectra are given it eliminates those build up curves containing only one determined volume possibly a spectral artifact Each build up curve shows all determined volumes with error values and the best linear interpolation of these weighted points The corr2plot scrip
176. ing the installation execute this command directly from the installation directory ANALYST Linux UninstallerData uninstall analyst User created files and files shared with other applications may not get un installed After un January 11 2010 2 8 NMRanalyst Installation NMRanalyst Software Support installing the entire ANALYST ARCH replacing ARCH by WIN or LINUX installation directory can be deleted to remove remaining files 2 6 NMRanalyst Software Support NMRanalyst is an ongoing medium size software project Despite best efforts on our part shortcomings might be discovered and we need to know about them to correct them For up to date support information and contact information please visit http www ScienceSoft net January 11 2010 2 9 CHAPTER3 Tutorial I Using NMRanalyst Five tutorials are provided covering FindIt VerifyIt and the full NMRanalyst features This tutorial covers determining likely structures with the findit script and through an NMRanalyst structure elucidation Input values are provided CHAPTER 4 Tutorial II Setting Analysis Parameters practises setting analysis parameters for new data sets CHAPTER 5 Tutorial II Combining Analysis Results introduces the molecular carbon skeleton determination from sensitive indirect detection spectra CHAPTER 6 Tutorial IV Advanced Structure Elu cidation adds placing nitrogen atoms in molecular skeletons and using DQF COSY and 1 1 ADEQUATE sp
177. ion for the observed NMR data January 11 2010 4 6 Tutorial Il Setting Analysis Parameters FFT Workwindow Settings for Prednisone i To derive this structure rating VerifyIt assigns observed sac aia _ carbon shifts to the specified structure This assigned struc ture is saved in the specified 4900 plot file Display it 355 05 a ee from the Graphic workwindow as shown in the top figure ii ai ai For the following shift prediction select in the NMRgraph Jm Aerm mam window Edit Preferences Inthe popup set S SE SS Shift Prediction Method to HOSE Code o Before Rule Based and click OK To compare 0 09 3 A observed with predicted shifts select Prediction e ead 19C Shifts To display the shift differences select ei as SR Su View Show 13c Shift Differences as Ng hown in the bottom figure Diff to 5 shown in the bottom figure Differences up to 5 ppm repre Aa vn o0 sent a reasonable agreement for carbons For shifts closer E e de EE than 5 ppm exchanged assignments might result 4 4 FFT Workwindow Settings for Prednisone Switch to the FFT workwindow Like the 1D Analysis workwindow before it initially only contains default INADEQUATE parameters Load the spectrum specific parameters by load ing from the directory named 7 the file acqus It is important to keep the referencing of all spectra consistent To bring the major DMSO d solvent resonance to 39 5 ppm the 1D car bon spectrum
178. ions for signal validation 15 12 workwindow Features 19 2 BETEN 9 17 nmrplot program DEE 2 5 ee 18 6 EH Ge EE l 1 color problems 21 8 raw analysis outmut eee eseeeseeeeeeeeee 19 1 contour line Colors s s s 18 4 setting DISPLAY variable 9 4 exit 20 2 EE CERN 7 5 at in EE 5 9 implementation anti aliasing 0 0 ccccceeee 18 3 software EEN GENEE 7 e interactive features oo tetas 20 1 EE MOUSE interactions ceeeeeeeeesess esses esses 20 1 EES 2 1 NO resolution Jmits eseeeeeseeeeeeeee 18 3 supported peo ee SEENEN 1 2 PC mouse operatong esse eeeeeeeeeees 20 2 system wide installation ccc cccceeeeteeeeees 2 5 aa Plot panning secre ana 20 2 termination Of Session seeeeiseeeeieeeereeerne 3 14 plot rotation s ssssssseeiesseieseeriesesiesserresssressrerssrres 20 2 use in other programs eesseseressssrsrsnsoen 7 1 7 8 plot spinning ool esse estes tsetse 20 2 nmrgraph program REESEN 20 2 3D fitting volume 7 7 plotting 2D amp 3D spectra wees 18 2 adding MCU S veri ccececsdesestoentncsrradrvrscerceoresteeedvaon 19 12 projection 3D spectrum on 2D plane 18 3 as molecular correlation editor ceeeeeee 19 1 EU E EE 20 2 Atom Property Editor 19 10 rubber band ZOOMING 0 0 eeeeeeeeeeeeeees 20 2 Bond Property Editor 0 1100011100111000 19 4 19 11 slice display of 3D spectrum oo eee 18 3 carbon skeleton display 0 0 cccecseeeeeeeeeees 16 2 spectral phasing
179. irectory If the NMRDIR variable is not set before NMRanalyst is started the variable is set automatically given that the instal lation directory is in a default location See CHAPTER 2 NMRanalyst Installation and CHAPTER 9 Using the NMRanalyst Window for details Being able to change NMRDIR allows one to switch between different installation directories However the components of different NMRanalyst versions might not be compati ble So switching between installation directories of different software versions 1s not encouraged Identifies the NMRanalyst working directory If it is unset upon program startup NMRDATA defaults on Linux to the current directory and on MS Windows to SNMRDIR data All user specified files without pathname are saved in and loaded from this directory All NMRanalyst interface files txt log and log bck are written in the NNRSPEC subdirectory of the NNRDATA directory ep SNMRDATA INADEQUATE report log To switch between analysis directories change the NMRDATA variable from the Directory Editor To change the analysis spectrum type change the NMRanalyst Spectrum Type menu as January 11 2010 10 2 Using the Directory Editor NMRDIR NMRUSER NMRDATA and NMR maen HERSPEC described in CHAPTER 9 Using the NMRanalyst Window or change the NMR SPEC variable as desired below If the NMRDATA directory does not contain an input file txt for a selected workwindow then a default fil
180. is input param eter should be specified as follows set the value to 3 and inspect the simulated and residual spectra If weaker resonances are not detected decrease this value If the program reports too many resonances such as impurities or other undesired spectral features slightly raise the specified value Note that only those resonances that have been initially identified in the peak picking step are passed through the filter If a resonance of interest 1s not identified initially then the peak picking analysis parameters Peak Picking Threshold Min Signal Distance andMin Cluster Distance need to be adjusted accordingly Key PInt i In most 1D spectra all resonances can be phased to positive absorption using a linear phase function If that is the case d this option menu should be set to Positive This set ting allows negative resonances during the spectral simula tion to be eliminated and the phases of three or more resonances in a cluster to be fitted by a linear phase function Some spectra contain both positive and negative resonances For exam ple a DEPT 135 spectrum shows CH and CH carbon resonances in positive and CH ones in Sign of Phased Signals Positive January 11 2010 13 6 1D Analysis Workwindow Analysis Parameters negative absorption Such a spectrum can be analyzed by selecting the Pos amp Neg Positive and Negative resonances setting in this option menu Non Lorentzian r
181. k overlap criterion peak picking positivenegative signals spectral width spectrum identification text start of spectrum subtracting signals fram data supported data types zero filling 30_SPECTRUM example application for all 3D spectrum types loading procpar file optimization detection parameters sensitive analysis spectral simulation The navigation subwindow on the left side of the help window allows navigating and finding topics in the help system The figure shows it with the table of con tents tab selected To expand or collapse sub topics in a chapter double click the chapter title The selected topic in the table of contents is synchronized with the text shown in the content subwindow The figure shows the navigation subwindow with the index tab selected The index 1s an alphabetical list of keywords organized as a hierarchy One keyword may contain sub levels of keywords Click on a leaf key word to view the content associated with it Alterna tively enter a phrase in the Find input field located above the index hierarchy and hit the Enter keyboard key The first index entry con taining the phrase will automatically be selected and its associated content displayed Keep typing Enter will find subsequent occurrences of the phrase in the index As for the table of contents the selected index entry and the content displayed are synchronized January 11 2010 Using the Help System Co
182. kwindow reference chapter The workwindow input screen provides a con venient way to edit the key value pairs ina txt file but any text editor can be used as well Click on the UNIX Shel1 button in the NMRanalyst window to start a UNIX shell win dow Change the current directory to a desired analysis directory such as the data directory used in CHAPTER 3 Tutorial I Using NMRanalyst For spectrum type INADEQUATE simply issue the command 6 ld analysis INADEQUATE The output from the analysis of the 1D spectrum which normally appears in the workwindow output screen is now written to the shell from which it was started The computing processes of NMRanalyst can be run from any program or script which can issue operating system com mands January 11 2010 7 8 Tutorial V Additional NMRanalyst Features The On line Help System Please note that NMRanalyst programs are protected by the software contract Even if incorporated in other software they fall under the same distribution restrictions as the original NMRanalyst software 7 6 The On line Help System The five tutorials contain the information to follow discussed examples Further questions can be answered through the NMRanalyst Manual contained in the online help CHAPTER 8 Using the Help System provides detailed information on how to use the help system In the NMRanalyst window select Manual from the Help pull down menu and the shown help window a
183. kwindow to display it Key FnArea Precision Distribution Ibrecisions plot iY Agreement Distribution faseements plot EU The normal Gaussian dis tribution of parameter precisions or agreements of non correlation patterns allows calculating correlation detection probabilities Specify a name in this input field to save a plot file of the distribution of analyzed correlation areas the threshold for detected correlations and the cal culated distribution function Use the Graphic workwindow to display it Key FnIP Integral Distribution jintewralsplot OO al The P rec 1 S 1 on D S So tribution described above allows effective distinction of signals from random noise But spectral distortions especially T1 ridges are not random and often consist of signals which match the spin system model Specify a filename in this input field to save the integral distribution and use the Graphic workwindow to display it NMRanalyst determined integrals correspond to the integrals of well phased signals Based on this integral distribution plot choose an integral value threshold above the noise and ridge signal responses To exclude the weak signals specify this threshold in the nD Analysis workwindow Thresholds Integral field as described in CHAPTER 15 nD Analysis Workwindow and rerun the analysis Key FnInt Redetermined PI Resonance List al Acquiring a 1D carbon spectrum is 5700 times less sensitive than a 1D proton spectrum A 1D nitro
184. l Help January 11 2010 9 10 Using the NMRanalyst Window The Application Window Menus NMRanalyst Exit Procedure i x The shown popup 1s displayed if Some workwindows are still computing any workwindow IS computing Terminate terminates all computations Terminate aborts the com zm aite tram met putations and exits from Cancel returns to HMRanalyst NMRanalyst Cancel Background continues all computations as Causes the exit request to be background processes using the ignored Background specified priority setting and exits from NMRanalyst causes the computations to con Priority value for background processes tinue in the background while CT slow fee rast NMRanalyst exits Backgrounding allows exitin remit cies _ Becta u from the application degen dow and on Linux even logging out without interfering with ongoing computations A background process com petes with interactive users for computer cycles Hence a slider 1s provided to set the priority of the background process created through the Background but ton The left side of the slider corresponds to the lowest possible priority Slow that the background process can be given At this lowest priority the background process should not interfere with the interactive use of the computer The right side of the slider corresponds to the highest priority Fast a user can give back ground processes 9 8 The Application Win
185. l assignments Ambiguities are reported by the report program in the Connectivity Table when the Ambiguous Signal Assignment switch is selected All ambigu ous interpretations are listed with additional messages CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 Shift Shift J Index Shift J Index Shift J Index ppm ppm Hz ppm Hz ppm Hz Lees Ost oUa 25 0 1054 Ambiguity index 105 and index s 288 163 79 50 46 35 5 226 Ambiguity index 288 and index s 105 An ambiguity is reported when the same correlation signal is found while examining two overlapping fitting areas In the 2D INADEQUATE table shown both ambiguous regions involve the resonance at 50 48 ppm which is correlated bonded to either the carbon reso nating at 16 77 ppm 105 or that at 16 79 ppm 288 The three Hz F1 shift difference between the two resonances 500 MHz spectrometer is smaller than possible isotope shifts between 1D and 2D carbon resonances causing both assignments to be indistinguishable When an ambiguity is encountered information from other sources is needed to uniquely assign the involved correlations carbon carbon bonds This ambiguity might be a disap pointing discovery to the spectroscopist However one of the major strengths of NMRanalyst is to reliably detect and report such ambiguities January 11 2010 16 7 Report Workwindow Select Items to Be Reported The Redetermine 1D Chem
186. l printers are accessible from the PC first notice which printer is currently selected as default to reset this setting later Now click on the icon of the desired printer with the right mouse button select Set as Default Printer Right click the selected printer icon again select Properties item and click Print Test Page button If the selected printer does not correctly print the test page follow the Windows help system suggestions Start NMRanalyst if not already running Select File menu Print item to start its Print Settings popup controlling all NMRanalyst print settings Set the To option menu to the appropriate print format For a Windows printer select a printer driver named closest to the printer name If none of the listed printer drivers matches the desired printer choose January 11 2010 21 6 Frequently Asked Questions How can capture NMRgraph and NMRplot dis Other Printer from the option menu to invoke the Windows native dialog for printing For Linux try the PostScript Printer setting Delete the input field entry on this line and click Apply Settings Now display the Print Settings popup again and this field contains the default printer name If on MS Windows it is LPT or COM with a number instead of the question mark it is a printer attached to the local PC If on MS Windows it is a shared printer connected to another PC on the local network or a Samba exported share from a Linux system
187. ld analysis parameter values which accumulate 2D FFT Ctrl T _J2D Analysis Ctrl h _ Report Ctrl F _Assemblelt Ctri A Graphic Ctri G whenever workwindow input screen values are changed Restore To Default resets the current values of all input screen items to their default values without erasing any history information The remaining items correspond to the functions described in SECTION 9 6 The Work window Tabs and in SECTION 9 7 The Speed Button Panel For a full description of each workwindow see CHAPTER 3 Tutorial I Using NMRanalyst and CHAPTER 7 Tutorial V Additional NMRanalyst Features Process From the Process menu Start runs the current workwindow The Stat ep Pause item is dimmed if no computation is in progress As soon as a compu tation is started this item is activated See SECTION 9 7 The Speed Button Panel for the description of its functions Tools The Directory Editor and UNIX Shell menu Directory Editor cry items start the Directory Editor and the UNIX shell window respec ee tively The functions are identical to those described in SECTION 9 7 Findit Database Manager The Speed Button Panel January 11 2010 9 14 Using the NMRanalyst Window The Application Window Menus FindIt Database Manager H b x Selecting FindIt Database Man Mada User Structures ager starts the shown popup for Structure File IT e adding extracting or remo
188. le conflict Workwindow File Locking H x e The MMROATA directory EANAL GT Wihrdatar is locked by another active HM anahet with PIC 244 For interactive computing processes associated with a workwindow and for backgrounded computing processes the corresponding P Log output file is kept locked while computations are in progress A newly started workwindow recon nects to a running background process if possible 12 4 Summary of Workwindow File Usage Each NMRanalyst workwindow is associated with three files These files are automatically created and managed and are stored in directory SNMRDIR SNMRSPEC The workwindow input screen values are stored ina txt file as described in SECTION 12 1 The Workwin January 11 2010 12 8 Using the Workwindows Summary of Workwindow File Usage dow Input Screen The workwindow output screen information is saved in a corresponding x log file as described in SECTION 12 2 The Workwindow Output Screen Whenever computations are started in a workwindow the previous log file is renamed to log bck and a new 1o0g file is created All other files created by NMRanalyst are described in the subsequent workwindow reference chapters The following figure shows the data flow of the NMRanalyst software 1D FID s nD FID Ee or spectrum or spectrum EES ld Ee EE E 1D Analysis Output file FFT Frt txt id analysis Log workwindow workwindow Et loo Output file
189. le phases plot and click Start to display the shown plot Phases are given on the vertical axis in radians to convert them to degrees multiply them by 180 r and the marginal standard deviation in phase and frequency is shown as one tilted error bar through each phase value This phasing works so well we pat ented it For comparing determined phase functions remember that a phase value only influ ences the appearance of the NMR spectrum through the sine and cosine of its value So a phase function shifted by 27 is identical to the unshifted one For higher dimensional spectra simultaneously shifting the phase in two orthogonal dimensions by x does not alter the spec tral phasing either O 6378 0 00323 f Phase radians 0 639 0 0033 f 0 00000042 5 64 ku 426 400 350 300 250 200 160 100 50 14 Frequency ppm Mapping _ 2 Integrals _ F1 Phase _ F1 Relaxation ime To use these phase func _ 4 Integrals jor Phase F2 Relaxation e tions for the correction of Detection 2 Integrals _ F1 Phase _ F1 Relaxation ime spectral plots load in the A Integrals F Phase F Relaxation _ Unused Graphic workwindow SCgeseg a lb ees xm from directory INADE Fe Phase Joes 0 0032270 mpm QUATE the file report log Also load these functions in the 2D Analysis workwindow to improve the spectral analysis Now in the 2D Analysis input screen both linear phase functions are set and the Mapping and Detec 1 U S
190. lelIt workwindow and display the cre ated CC_corrs plot Reformat the circle diagram 77 39 with the NMRgraph View Redo Layout and switch off the correlation labels with View Hide 9 Correlation Labels for the shown results 5989 E etie 3140 7663 3 Zi 0 05 ike fe 128 44 52 48 aa 132 45 26 63 S178 7 S d aps 142 055 160d ei 31 40 59 69 E ee KM ee N 150 1 140 05 47 94 a dies S 127 4 gra 165 15 KM az EEN ze P AA 4226 3 T 60 05 ks 128 44 52 48 p 132 45 26 63 51 78 To e apa afa idz gs TIR e 31 40 59 89 ger E 150 1 140 05 f i H 47 93 a pe ade E 77 39 iiia sr 6441 2 6944 Return to spectrum type HMBC and its Assemblelt workwindow Set 2D 1 1 ADEQUATE to ADE QUATE report log and run this workwindow The structure determination takes under one sec ond and two structures remain Display the shown CC corrs plot 1 Both structures are reported with four 4 Bond one Long Range and seven Unobserved Bond correlations 6 7 Adding Heteroatoms The Assemblelt derived strychnine structure represents the possible carbon and nitrogen skel etons From the edited HSQC the resonance frequency and number of bonded hydrogen for January 11 2010 6 11 Tutorial IV Advanced Structure Elucidation Identifying the Molecular Structure Through each carbon are known though they are not displayed But it is known which carbons have remaining unexplained free valences Free valences result from bonds t
191. lid shell variable entry The specified MWMRDIR directory EVANALYST HPS f does not exist or access denied Flease correctthe pathname Reset NMRDIR Cancel Help January 11 2010 10 4 chapter Using the Filebox Popup IS 1 Filebox Load 1D Analysis Paramete When NMRanalyst requires a directory name or a file name the file selection popup is displayed This popup is referred to as the Filebox and the window title indi cates which item to specify EK x Enter path or folder name E ANALYST WINtd atai5 Filter Mon Hidden Files d Folders A requested item can be typed in the lower Enter file name input field Alternatively the Enter file name item can be assembled interactively by selecting items from the Folders and Files lists The OK button exits the Filebox and returns the speci fied Enter file name entry to the calling applica tion The Update button updates the Folders and Files lists according to the specified Enter path or folder name string The Cancel button quits the Filebox Enter file name lacqus OK Update Cancel Help 11 1 Interactive Item Specification Selecting a directory or an existing file from a list is less error prone than directly typing its name inthe Enter file name input field The string inthe Enter path or folder name input field contains a directory The Filebox provides a list of subdirectories of the directory specifie
192. located inside the installation directory here assumed to be usr local ANALYST LINUX to one s working directory join this directory and start the software cp r usr local ANALYST LINUX data HOME cd SHOME data analyst AP oO o 3 1 findit Script for Molecular Structure Identification A major application of NMR is the determination of an unknown molecular structure The NMRanalyst findit script described here automates the identification of best matching struc tures based on molecular formula 1D proton FID 1D carbon FID or HSQC HMQC 1D spectral projections Ideally the best identified structure among the over 14 5 million consid ered structures is the correct one As a nearly infinite number of small molecules could be syn thesized even this huge number of known molecular structures does not guarantee to contain the correct structure Instead of the correct structure similar molecular structures may be reported Only the full structure elucidation identifies an unknown But the findit identifica tion of similar structures is automated and can be applied routinely The full structure elucida tion requires more NMR data and can be too resource intensive for routine applications January 11 2010 3 2 Tutorial I Using NMRanalyst findit Script for Molecular Structure Identifica Start the shown NMRanalyst application window as described in the previous section This application window shows the state with which
193. mined structures SECTION 6 7 Adding Heteroatoms Dr P ter Sandor Varian Deutschland GmbH graciously provided 500 MHz datasets of 20 mg strychnine MF C H 22N 0 gt acquired in CDCl using a 3 mm tube The provided 1D proton acquisition time 1 1 min 1D carbon 39 4 min multiplicity edited C gHSQC 39 min ZC gHMBC o 1 h 17 min IN gHMBC 3 h 12 min gDQF COSY 1 h 36 min and DQ 1 1 ADEQUATE 19 h 21 min spectra are used in this tutorial Copy the supplied strychnine dataset containing the experimental data Delete its ADEQUATE DQF COSY HMBC HSQC and N15 HMBC subdirectories This removes the provided NMRanalyst parameter settings for this compound In NMRanalyst start Edit Prefer Strychnine 1 Strychnine is an alkaloid extract obtained from the dried ripe seeds of Strychnos nux vomica a small tree of the East Indies In the past strychnine has been used as an antiseptic stomach tonic circulatory stimulant central nervous sys tem stimulant and medication for the relief of constipation Strychnine is still in limited use today as a bird mammal and insect control agent January 11 2010 6 1 Tutorial IV Advanced Structure Elucidation 1D Carbon 1D Proton Edited HSQC and ences setMode to Full NMRanalyst and deselect the Show All Input Fields switch 6 1 ID Carbon ID Proton Edited HSQC and HMBC Analyses Experimental Simulated 127 160 146 170 106 DO BD 4 18 Fr
194. mpty to have all correlation areas examined Specific correlation areas can be examined by listing in this input field a sequence of correla tion numbers and or ranges of correlation numbers separated by spaces commas tabs colons semicolons or any combination thereof A range of correlation numbers is specified by the first and last number of the range separated by a dash For example the input 2 10 50 3 specifies that correlation numbers 2 and 3 as well as 10 through 50 should be examined Key BondNo Thresholds Integral NMRanalyst fits a spin system model to the data By default only the Threshold Precision for DQF COSY HMBC and N15 HMBC called Agree ments value described below is used to keep noise patterns from being misinterpreted as correlations This Thresholds Integral allows specifying the minimal integral a simu lated spin system has to reach before being identified as a correlation To determine this threshold analyze the dataset and display the determined integral distribution see the Report workwindow Integral Distribution field description Choose a value above the main integral distribution as described below HMBC and DQF COSY resonances result from couplings which can be below the acquired digital F2 resolution The spin system resonances overlap and largely cancel each other due to their opposite signs anti phase For these spectrum types the absolute value integral of reso nances 1s tested against this I
195. ms using a Bruker FID try to transform the data using Bruker s software e g XWIN NMR and analyze the resulting spec trum with NMRanalyst instead To analyze spectra taken on NMR spectrometers of other vendors import the data into the Varian VNMR or Bruker software to Fourier transform the data and then analyze the resulting phase sensitive spectrum with NMRanalyst Key Dat Fmt Transform Order Coefficient File This option menu only shows for 3D FIDs and applies to cae NMRanalyst s native FFT but not to VNMR s f t3d pro ena ages gram The directly acquired dimension F3 in 3D is trans arian ghn_cach formed first The linear combination of phase components for arian ghcch tocsy arian gnoesyChsqc the F2 and F1 interferograms is determined through the selected pulse sequence The fastest transform results from January 11 2010 14 2 FFT Workwindow Output File choosing the used pulse sequence from this menu If no appropriate pulse sequence is listed use the Coefficient File item and specify VNMR s coef file containing the 40 transform coefficients for 3D phase sensitive transforms in the 3D FFT Coefficient File field above Key OrderFT 14 2 Output File OUTPUT FILE Enter the name of the Phase Sensitive Spectrum jinadequate spec multidimensional spec trum to be created through the FFT This file is read by the nD Analysis and the Report workwindows and can be dis played by NMRplot
196. n Up Arrow show previous history entry for the input field Regions of text can be selected and used in the subsequent operations Clicking on a word twice with the first left mouse button selects the word clicking on text three times selects all text in the input field and pressing the first left mouse button and moving the mouse with the button pressed dragging selects the corresponding text area A selected area can be removed using the Delete key The selected area can be copied to another input field possibly in another workwindow by first selecting the Copy item from the Edit pull down menu then clicking on the final destination with the first left mouse button and finally selecting the Paste item from the Edit pull down menu All workwindow input screen text fields are limited to one line of text and entered newline characters are ignored All text input fields are scrollable horizontally and can hold at least 200 input characters inde pendent of their displayed length Cut Clicking the right mouse button above an input field with selected text brings up the shown popup menu Cut removes the selected text and pastes it to the clipboard Copy copies the selected text to the clipboard Paste adds any text on the clipboard to this input field History brings up the history popup for this input field Tee x Most input fields request a numerical input value and ignore entered characters which cannot be pa
197. n the standard deviation of spectral intensity values The Min input field defaults to the negative Max input field value For 2D surface plots specified values define clipping planes cutting off signals exceeding the specified intensity range The Steps input field is only used for 2D contour plots Its value specifies the number of contour lines to be drawn possible values are 1000 to 1000 The default value of 2 requests one negative and one positive contour line If a value of 1 0 or 1 is specified the default value is used If the Min value is negative and the Max value positive contour lines are drawn outside this specified intensity range to avoid the noise level around zero intensity If both threshold values are either negative or positive the contour lines are drawn between the two thresholds The 2D contour lines are color coded for easy distinction For positive contours orange represents the lowest intensity and yellow the highest intensity with a linear color interpolation between these values For negative contours blue represents the lowest absolute intensity and cyan the highest absolute intensity Linear color interpolation is used for intensi ties between these values When Steps contains a negative number only one negative con tour line is drawn and the remaining lines are used for positive contours This causes positive January 11 2010 18 4 Graphic Workwindow Multidimensional Spectrum Specific Settings
198. n hsqc out 2 The carbon dimension was referenced based on the 1D spectrum So strictly the original referencing should be used But either approach works January 11 2010 5 9 Tutorial Ill Combining Analysis Results Structure Elucidation Without 1D Carbon Spec Switch to the HMBC spectrum type and its 2D Analysis workwindow The F1 1D Analy Sis Output File is set to the generic carbon out SetMap F1 Frequencies to 45 Hz to cover the resonance spacing in this generic list Set Thresholds Integral to 1 1 and Agreement to 30 In the Report workwindow set Redetermined F1 Res onance List tocarbon hmbc out Run the 2D Analysis workwindow and it auto runs the Report workwindow This exhaustive HMBC analysis takes about 15 minutes on a 3 06 GHz PC but is much faster than acquiring a carbon spectrum Fourteen identified carbon res onances are stored in carbon hmbc out Now the combined carbon line list can be gener ated From the UNIX Shell window execute O lt mergez list carbon tee cur Carbon hmoc out 0 12 gt EE EE EE This merges the both line lists keeping HSQC determined carbon resonances and adding the HMBC ones over 0 12 ppm apart from other resonances The resulting 19 resonances are saved in the file ncarbon out Select the HSQC spectrum type and switch to the AssemblelIt workwindow Select the Fin dIt IDENTIFY DATABASE STRUCTURES BEST MATCHING NMR DATA switch In the COMBINE NMR ANALYSIS RESULTS section s
199. nalysis Results CHAPTER 6 Tutorial IV Advanced Structure Elucidation and CHAPTER 7 Tutorial V Additional NMRanalyst Features cover using this software effectively It is highly recommended to work through the tutorials using the software before analyzing and interpreting research datasets Part two of this manual is the NMRanalyst Reference Manual starting with CHAPTER 8 Using the Help System It covers all the software features and should be consulted for spe cific questions Part three of this manual is the appendix It contains the FAQ list CHAPTER 21 Frequently Asked Questions To find answers also consult the detailed index 1 4 Typographic and Keyboard Conventions Words typeset in Color indicate hypertext links which might not show in the printed manual Click on them to locate related information Typewriter style indicates computer terms such as program input and output shell variables or command names Command line examples are prefixed by a letter indicating the required user privileges execute command with super user root privileges January 11 2010 1 5 Overview Typographic and Keyboard Conventions O 5 execute command with user privileges Keyboard keys and user interface buttons or switches are shown in square brackets such as Leck January 11 2010 chapter NMRanalyst Installation The NMRanalyst evaluation software with test datasets is availa
200. nances Specify the 1D Analysis work window output file for the proton spectrum or the HSQC report log file in this input field Key FnProton Carnon Jess DU Specify the 1D Analysis workwin dow output file for the carbon spectrum This can also be a generated generic line list or a car bon resonance list derived from F1 frequencies of heteronuclear spectra Key FnCarbon Mitrogen _ fpitrogen out DU Specify the 1D Analysis workwin dow output file for a nitrogen spectrum Given the NMR challenges for direct LSN observa tion this entry will likely be a N HMBC derived or generic line list Key FnNitrogen Dert faept out This input field is displayed if the Findit or VerifyIt instead of the Assemblelt task is selected Specify a DEPT spectrum in this field Key FnDEPT 2D 1 1 ADEQUATE ADEQUATE report Log E To include ADEQUATE information in the molecular skeleton determination specify the location of its Report work window report log file In contrast to HMBC ADEQUATE only shows direct bonds January 11 2010 17 2 Assemblelt Workwindow Combine NMR Analysis Results without longer range correlations AssembleIt supports ADEQUATE spectra with single quantum SQ or double quantum DQ frequencies in F1 SQ ADEQUATE spectra have the same assignment challenges as HMBC when several carbons in the HSQC spectrum have indistinguishable proton chemical shifts These challenges can be avoided by using DQ ADE QUATE
201. nce with 48 CFR 227 7201 through 227 7202 4 for Department of Defense DOD acquisitions and with 48 CFR 2 101 and 12 212 for non DOD acquisitions 9 Governing Law Any action related to this Agreement will be governed by California law and controlling U S federal law No choice of law rules of any jurisdiction will apply 10 Severability If any provision of this Agreement is held to be unenforceable this Agreement will remain in effect with the provision omitted unless omission would frustrate the intent of the parties in which case this Agreement will immediately terminate 11 Integration This Agreement is the entire agreement between you and Sun relating to its subject matter It supersedes all prior or contemporaneous oral or written communications proposals representations and warranties and prevails over any conflicting or additional terms of any quote order acknowledgment or other communication between the parties relating to its subject matter during the term of this Agreement No modification of this Agreement will be binding unless in writing and signed by an authorized representative of each party January 11 2010 8 5 Using the Help System Copyright JavaHelp JAVAHELP TM VERSION 2 0 SUPPLEMENTAL LICENSE TERMS These supplemental license terms Supplemental Terms add to or modify the terms of the Binary Code License Agreement collectively the Agreement Capitalized terms not defined in these Supplemen
202. nctions as above and display the dqf cosy spec file and the correlation locations are also dis played as shown Th oth pee an ii ep 11 mi KR P o r pa T a a Tr rrr rrr rrr rrr rrt ppm F2 ZS D 6 gt 4 A 2 1 Pm aa Run the Assemblelt workwindow From the Graphic work window display the created CC corrs plot Reformat i a the circle diagram with the NMRgraph View Redo Layout and switch off the correlation labels through View Hide Correlation Labels to obtain the shown correlations display 60 05 LISfde zst20 Ade ze1240d bdAle 2et27 i d Zbie 2ebtn1p Return to spectrum type HMBC and the AssembleIt workwindow to add this DQF COSY information Set 2D DQF COSY to DOF COSY report log Run this workwindow Two possible strychnine structures result in a few seconds 6 6 Using DO 1 1 ADEQUATE The first two tutorials cover the carbon carbon bond detection from a 2D INADEQUATE spectrum This would resolve remaining ambiguities But it is over an order of magnitude less January 11 2010 6 9 Tutorial IV Advanced Structure Elucidation Using DQ 1 1 ADEQUATE sensitive than HSQC HMBC and DQF COSY spectra The ADEQUATE acronym stands for Adequate sensitivity DoublE QUAnTum spEctroscopy The AD ALT D detects H C C moieties It is known as 1 1 ADEQUATE due to the use of June and Ie c couplings Like 2D INADEQUATE 1 1 ADEQUATE depends on both carbons involved bein
203. nd all database structures are considered Key minWeight maxWeight Output Matching Structures I finait piot im Specify the name of the out put file for saving database structures best matching the NMR data Key FnFind Huber of Structures 0 Specify the maximum number of best matching structures to be saved in the output file By default the top 10 best matches are saved Up to 1000 structure matches can be requested Key numMatch January 11 2010 17 6 Assemblelt Workwindow Verifylt Rate Specified Structure by Agreement 17 3 Verifylt Rate Specified Structure by Agreement With NMR Data MVerityIt RATE SPECIFIED STRUCTURE BY aGRERENT Em mR pp The Verifylt task rates the consistency between a proposed structure and MNR data by comparing the predicted and observed proton and or carbon chemical shifts High consistency generates a high rating with 1 0 being the perfect matching and 0 0 being no consistency VerifyIt can also assign observed shifts to the pro posed structure This workwindow section is shown when this switch is selected Key LVerifylIt Input Proposed Structure 1 EU Specify the expected struc ture in this field The structure file can be in the NMRgraph plot file format or in Molfile for mat Key FnVerify Output Assigned Proton Shitts DU Ventfvlt determines the most likely assignment of observed proton shifts for the proposed structure When a name is speci fied in this inpu
204. ndering speed this value can be set to 0 or 1 and displayed plots are only accurate up to the size of one screen pixel A maximum of 20 anti aliasing frames can be specified Key AAFrames Relative Size x x 2 If no values are specified in these input fields a X Y or X Y Z for 3D plots and 2D surface plots ratio optimal to the plot displayed will be used If a value is specified in at least one of the input fields the input fields without a speci fied value default to 1 Two dimensional plots except for 2D surface plots ignore any value specified in the Relative Size Z field For surface plots of 2D spectra a specified Z value scales the displayed intensity values Several interesting effects can be obtained by changing these aspect ratios For example a 3D spectrum can be projected onto the 2D F1 F3 plane by setting Y to 0 To display slices through 3D spectra specify a small range in the F Frequency Min and Max fields described below and set the corresponding slice rela tive size to a small or zero value Key xScale yScale zScale m e riot tegema Seyeral types of plots e g phase function plots 1D parameter plots 1D spectrum plots and build up curves have a legend area at the right side of the plot window By default the legend area is displayed Select this switch to hide the legend area Key LHideLegend 18 3 Multidimensional Spectrum Specific Settings MULTIDIMENSIOWAL SPECTRUM SPECIFIC SETTINGS Usin
205. ndow 15 1 Input Files INPUT FILES Specify in the F1 through Fi 1D Analysis Output File fe_igiinessout EN F2 F1 through F3 for 3D TE input fields the file with F3 1D Analysis Output File festaiinessout EI resonance positions along each axis of the multidi mensional spectrum For homo nuclear 2D spectra DQF COSY INADEQUATE NOESY ROESY and TOCSY only the F2 1D Analysis Output File input field is dis played since the indirectly determined F1 resonance frequencies are identical to the directly acquired ones For 2D heteronuclear spectrum types ADEQUATE HETCOR HMBC N15 HMBC HSQC COUPLED HSQC and N15 HSQC both the F1 and the F2 reso January 11 2010 15 1 nD Analysis Workwindow Output Files nances have to be specified For 3D spectra the general heteronuclear spectrum type is assumed If several axes of the multidimensional spectrum show the same possible resonance frequencies specify the same 1D line list in the corresponding input fields Key Fn1DF1 Fn1DF3 2D FFT Output File inadequate spec The label of the input field changes with the dimensionality of the selected spectrum type Specify in this input field the name of the phase sensitive multidimensional spectrum created by the FFT workwindow Key FnCube 15 2 Output Files OUTPUT FILE Specify a name for the nD Determined 2D Correlations inadequate corr al Analysis workwindow output file For each examined multidimensional spectral
206. non correlations can be displayed by selecting the Everything option Key RNBond Warnings On Ambiguous Signal Assignments sw Improper Phase _ Fitting Area Overlap The Report workwindow shows a table assigning all multidimensional spectral resonances to the 1D resonances for each spectral direction For heteronuclear spectra one table is displayed listing the correla tions to the F1 resonances one listing the correlations to the F2 resonances etc For each cor relation the respective chemical shift coupling constant or average detected integral and correlation index number are given CORRELATION to RESONANCE 1 RESONANCE 2 RESONANCE 3 January 11 2010 16 6 Report Workwindow Select Items to Be Reported Shift Shift J Index Shift J Index Shift J Index ppm ppm Hz ppm Hz 7 ppm Hz az EE Eh Ch IEN 20205 EE 32 92 32 40 9 44 This 2D INADEQUATE Connectivity Table fragment specifies that the 1D carbon resonance at 56 86 ppm is correlated to three carbons resonating in the 1D spectrum at 45 39 30 05 and 9 32 ppm The table also shows the corresponding coupling constants 31 8 28 6 and 40 9 Hz and the correlation index numbers 36 52 and 44 The index numbers allow locating further information about the correlations as described for the Report About Correlations option menu M ambiguous Signal assignments 1 WO dimensional spectra can give rise to ambiguous correlation signa
207. ns moves back and forth between visited pages A prints the current text and 5 allows cus tomizing print settings January 11 2010 8 2 Using the Help System Using the JavaHelp Navigation ol gt AMRanalhst Manual EJ User Manual gt b ET CHAPTER 1 Overview gt 1 1 The Automated Spectral Analysis gt 1 2 Identification of Best Matching Structures gt 1 3 Using this Manual gt 1 4 Typographic and Keyboard Conventions CHAPTER 2 NitRanalyst Installation p a CHAPTER 3 Tutorial Using NMRanalyst D zt fin dit Scr ipt fo f Mo lecular St ructure Identification gt 3 2 Selecting Full NMRanalyst Mode 3 3 Selecting 2D INADEQUATE Spectrum Type gt 3 4 The 10 Analysis vorkw nd O gt 3 5 The FFT Workwindow gt 3 6 The nD Analysis VWorkwindow gt 37 The Report vvorkwindow 3 8 Determined Prednisone Structure gt 3 9 Exiting the Software CHAPTER 4 Tutorial Il Setting Analysis Parameters CHAPTER 5 Tutorial IW Combining Analysis Results KAS CHAPTER amp Tutorial W Advanced Structure Elucidation a CHAPTER 7 Tutorial Additional NMRanalyst Features E Reference Manual EIN CHAPTER 8 Using the Help System CHAPTER 9 Using the WMRanalst Window Fc CHAPTER 10 Using the Directory Editor spectral width F2 Mapping Points J Mapping Points 1D Analysis workwindow F1d_analysis program effect of signal splitting maximum number of points nucleus observe frequency overview pea
208. ntegral threshold Key IntLimit Thresholds gt Precision OF Agreement A detected pattern 1s reported as a corre lation based on this threshold value For DQF COSY HMBC and N15 HMBC spectrum types this is the agreement in percent between simulated and experimental spectra For other spectrum types it is the determined absolute value integral divided by its uncertainty mar January 11 2010 15 3 nD Analysis Workwindow Analysis Parameters ginal standard deviation The higher this value the more likely the pattern is a genuine corre lation signal By default NMRanalyst chooses this value such that reported correlation signals have at least a 99 9 probability of being genuine correlations This input field allows overwriting this default threshold The Report workwindow provides a detection probability for each reported correlation After the initial analysis of the spectrum display the precisions plot or agreements plot The used threshold is shown in the distribution plot as a vertical red line Adjust its position to lie outside the Gaussian distribution of noise values Key PInt consider F1 aliasing This switch is displayed for the ADEQUATE HETCOR HMBC N15 HMBC HSQC COUPLED HSQC and N15 HSQC spectrum types Normally 2D spectra are acquired covering the full range of the corresponding atom The ADEQUATE F1 range has to cover two carbon chemical shift ranges This requires a considerable spectral res
209. nts is dF Range points where Range is twice the value specified in the previous input field and points is the value specified in this input field The smallest mapping point value is Pinin Pmiq Range 2 dF 2 and the largest mapping point value is Pax Pmig Range 2 dF 2 where P nig 1s the estimated parameter value The mapping points are opti mally positioned in the mapping range NMRanalyst currently supports a maximum of 20 specified mapping points Key MapF1 MapF3 Map Cowling Constant This field is only shown for INADEQUATE HMBC N15 HMBC COUPLED HSQC and DQF COSY spectral analyses supporting the determination of scalar coupling constants The coupling constant of a two spin system 1s dif ficult to estimate from prior knowledge See the description of the Estimated Coupling Constant input field below The mapped coupling constant range should be chosen to cover all possible deviations from the initial estimates A 2D INADEQUATE experiment can reliably detect coupling constants 10 Hz around the set coupling constant JCC used in the pulse sequence The default mapping range depends on the estimated coupling constant For an estimated coupling constant under 50 Hz the map ping range is 10 Hz For a coupling constant above 50 Hz the default mapping range 1s 15 Hz For a DQF COSY spectrum the default value is the Estimated Coupling Con stant value described below minus two Hz For COUPLED HS
210. o heteroatoms and higher bond multiplicities which are not detected by the used NMR spectra Based on the obtained molecular carbon chemical shift assignments further predictions about bonded het eroatoms and higher bond orders can be derived from their expected effect on observed car bon chemical shifts To add heteroatoms to the previously displayed structures select in NMRgraph Predic tion Place Heteroatoms Inthe started popup confirm O oxygen is selected as heteroatom to consider and click OK Oxygen atoms are placed in the structure to mini mize the difference between observed and predicted carbon chemical shifts The resulting structures are sorted by shift agreements ee Select Prediction we Shift Agree e 42 53 es ments The average differences between the Gi an AA E Gau Predicted and observed carbon shifts are 0 160 SN ee or aa no and 7 992 ppm for the two structures So the E a f gt agma shown top structure is the most likely one and it A Dn a agrees with the complete strychnine structure 14 0 Ta oe wb shown at the beginning of this tutorial Dy SS d ba d 127 44 77 39 169 15 dd 42 28 64 41 6 8 Identifying the Molecular Structure Through Shift Prediction Using only HMBC and HSQC derived ambiguous carbon carbon correlations tends to become compute intensive for larger molecules where numerous candidate structures result The chemical shift represents the chemical environment of each atom S
211. o placing heteroatoms and sorting structures according to agreement of the carbon shifts with prediction can identify the most likely structure In SECTION 6 4 Strychnine Structures Derived From Previous Analysis Results six possible structures result from HMBC and HSQC correlations Placing heteroatoms and sorting these structures in carbon shift agreement order identifies the correct strychnine structure In SECTION 6 5 Using DQF COSY adding DQF COSY information January 11 2010 6 12 Tutorial IV Advanced Structure Elucidation Identifying the Molecular Structure Through decreases the possible strychnine skeletons to two Adding oxygen atoms and sorting the structures in carbon shift agreement order also identifies the correct strychnine skeleton So 1 1 ADEQUATE information often is not needed for the structure elucidation of smaller mol ecules January 11 2010 6 13 CHAPTERT Tutorial V Additional NMRanalyst Features The previous four tutorials cover the NMRanalyst analysis of 1D and 2D spectra the Findit structure identification and the Assemblelt structure elucidation This tutorial adds determin ing molecular structures from an HSQC FID SECTION 7 1 Using an HSQC FID for the findit Structure Identification generating simulated spectra SECTION 7 2 Displaying Experimental Simulated and Residual NMR Spectra and 2D surface plots SECTION 7 3 Surface Plot of a 2D Spectrum It also covers analyzing 3D data
212. oints to the help system SS When selecting On Item the mouse cursor turns into question current werkwindow Mark shape Clicking on any NMRanalyst application window interface Using Help object displays its help information NMRgraph NMRoplot If the help system is started from the NMRanalyst application window switching back and forth between the help window and the application win dow works as expected However when started from a popup window the help window blocks access to the NMRanalyst application window In order to return to inter acting with the application window close the help window On Item About NMRanabyst January 11 2010 8 1 Using the Help System Navigating Through the Help Text 6 2 Navigating Through the Help Text The NMRanalyst help system consists of a toolbar at the top a navigation subwindow on the left and a content subwindow on the right The navigation subwindow contains three tabs for switching between the table of contents index and full text search displays The NMRanalyst help text is organized in 21 chapters Each chapter in the help text has mmm navigational buttons at the beginning and the end for changing to the previous and subsequent chapters Colored and underlined phrases are hypertext links and can be followed by single clicking on the link 8 3 Using the JavaHelp Navigation The toolbar at the top of the NMRanalyst JavaHelp window contains four butto
213. olution and the acquired F1 range is often chosen smaller allowing resonances outside the acquired range to alias back into the acquired range Other spectrum types have similar F1 challenges Select this switch to have NMRanalyst consider multiple F1 aliasing Acquire aliased spectra such that aliased and non aliased resonances do not fall at the same F1 fre quencies Otherwise ambiguities in the correlation assignment may result Key LAlias M Report Multiple Minima This switch is displayed for the INADEQUATE spectrum type Overlapping correlation signals in multidimensional spectra can lead to ambiguous signal interpretations Multiple Minima in the goodness of fit criterion for a single fitting area indicate that multiple correlations were found in this fitting area This information 1s required to identify all ambiguities in the identified signal assignments For 2D INADEQUATE spectra this switch is selected by default The resonance frequency of a carbon atom in the 1D carbon spectrum and in the 2D INADEQUATE spectrum differs by up to 0 027 ppm due to isotope shifts caused by the neighboring 5C nucleus In high resolu tion 2D INADEQUATE data F2 resolution lt 1 Hz if the 1D carbon spectrum contains reso nances less than 0 03 ppm apart several bond patterns might be found in one fitting area If there are no two carbon resonances 0 03 ppm or less apart or if plotting is requested and should be limited to the strongest identified pat
214. on scripts can be called interactively from the Linux command line for example O gt yarianztxt lds lt fid procpar gt 1d analysis txt This button shows the deactivated dimmed Pause label if no computation is in progress When a computation is started this button shows the activated Pause label Clicking this button suspends pauses the computa tion and the button label changes to Resume Clicking this button again con tinues the computation from the point where it was suspended and changes the button label to Pause A suspended computation can be terminated using the Stop button To start a workwindow computation requires write permissions on the spectrum specific subdirectory under the NMRDATA directory and the corresponding x log file and its backup file log bck NMRanalyst obtains write permis sions on the directory and files when possible The following popup window appears if NMRanalyst cannot obtain write access to the spectrum specific subdi rectory Workwindow File Permissions H Ei Cancel removes the popup window f Directory permissions can only be changed No write access for directory NMRDATA DYANALYST PCidatalINADEQUATE by the directory owner or a user with system MENE EENIA privileges To manually add write permis choose another NMRDATA directory sions to a directory ep SNMR DATA INADEQUATE type the following _cance Hep command from the NMRanalyst UNIX Shell window
215. orrelations obtained from publications and other means can be saved in a plot file which can be used for the molec ular structure elucidation To create the plot file with the molecular structure editor NMRgraph display an existing plot file Delete all its atoms and correlations Enter the desired correlations Set the atom type and number of free non hydrogen valences from the Atom Property Editor Set correla January 11 2010 21 10 Frequently Asked Questions How to specify correlations for the Assemblelt tion types from the Correlation Property Editor For correlations which are known to be bonds such as derived from INADEQUATE or 1 1 ADEQUATE use the correlation settings Bond Type Single Line Style Solid Arrow Both For ambiguous correlations such as derived from HMBC or DQF COSY use Bond Type Single Line Style Dotted Arrow Both Save these correlations in a file such as new plot In the AssemblelIt workwindow deselect COMBINE NMR ANALYSIS RESULTS select AssembleIt BR LUCIDATE MOLECULAR STRUCTURE FROM NMR DATA specify in the Input Atom Atom Corrections field the created new plot file If the field is not visible select Edit menu Preferences item set Mode to Full NMRanalyst select the Show All Input Fields switch and click the OK button Set the structure gener ation options and run the workwindow January 11 2010 21 11 CHAPTER 22 Index Symbols
216. oton couplings during the HMBC pulse sequence cause F2 phases to scatter over the 27 phase range and no linear phase function can be derived For HMBC don t set phase func tions so NMRanalyst determines individual F1 and F2 phases for every correlation January 11 2010 5 5 Tutorial Ill Combining Analysis Results Analysis of Gradient HMBC Spectrum Ee ee e RE i q D E 4 th Ai 1 d Ei 20 S oe a a 8 RH m Bi ES TA a Au ao S g e 10 f 120 p wo F 10 p D Unset detection threshold DU Carrelation Area Integral a ink ofa SS ee ee a H p pm EE ef cee F2 6 5 4 3 2 1 ppm Display the shown integrals plot file of the correlation integral distribution To avoid the report of longer range cor relations set the 2D Anal ysis workwindow Tncesnoloas IN e gral to 0 26 Rerun the 2D Analysis which auto runs the Report workwin dow wkt g D KE DEE ree E E E EE ae From the Graphic workwindow display the shown HMBC spectrum set File With Plot Data to hmbc spec As for the HSQC spectrum before check if the strong resonances were detected as indicated by rectangular bounding boxes The pairs of signals close to the spectral diagonal 100 to 200 Hz apart are incompletely suppressed direct C H bond correlations Weak cor relations are likely 4 bond or longer range correlations which are not needed for the structure elucidation January 11 2
217. our software provider for a license file Se ats Rae Feature has expired 2 i EE Expire date 01 jul 2001 server in this software causes License zait DHAMALYST WIN license det License path D 4NALYST WiIMfilicense dat h l l FLESIm error 1 359 System Error 2 No such file or directory FLEXIm error 10 32 t l D ana ySIS nD ana ySIS For Further inf tion refer to the FL Elo End User M l a Assemblelt and NMRgraph programs to stop functioning when the NMRDIR license dat file is not correctly installed left figure or when the evaluation time period has expired right figure The Windows NMRanalyst is also available for perma nent installations not using this license server January 11 2010 21 3 Frequently Asked Questions How can access Linux files from a PC running 21 5 How can l access Linux files from a PC running MS Windows If the computers are not networked then write a DVD or other medium on the Linux side and load it on the PC To copy individual files over a network start the ftp program on MS Win dows with Start Run and specify ftp computer name Log in type bin for binary mode and use get filename to copy the filename file to the PC running MS Windows If files from the Linux computer are accessed frequently install the free Samba software on the Linux computer The Samba software with documentation can be downloaded from http samba org Configure Samba to
218. own popup is then displayed Enter the text to be searched in axa the Find Text input field Select the Match ma Cancel Hp Case check box for case sensitive search Click the Find button to start searching from the top of the output screen The first match if any is then high lighted To continue searching for the next match select Find Next from the Edit menu or hit the F3 short cut key Cancel closes the popup without searching Find Text in Output Screen R x January 11 2010 9 12 Using the NMRanalyst Window The Application Window Menus Selecting Preferences from the Edit menu brings up the shown popup In the Full NMRanalyst mode all the six workwindows 1D Analysis FFT nD Analy sis Report AssemblelIt and Graphic are dis played Ifthe FindIt amp VerifyIt mode is selected only the three relevant workwin dows 1D Analysis AssemblelIt and Graphic are displayed For the routine use of the soft ware many input fields are not needed and hence are hidden by default Select the Show All Input Fields check box to display all the available fields Preferences Mode Full HERanalyst _ Show ALl Input Fields Defaults E Cancel Help Clicking on the Color tab switches to the shown display From here choose the back ground color to identify interface widgets such as text fields with non default values The preview area updates with ea
219. pectrum 40 D DU 100 120 F2 D 4 3 2 1 ppm ppm In the Graphic workwindow set File With Plot Data to hsgqce spec and display this spectrum by starting this workwindow Typically 2D INADE QUATE described in CHAPTER 4 Tutorial II Setting Analysis Parame ters and 1 1 ADEQUATE spectra described in CHAPTER 6 Tutorial IV Advanced Structure Elucidation have too low a signal to noise ratio to create meaningful spectral projections But for the other spectrum types 1D projections of a 2D spectrum are shown by default Were all the correlations detected The undetected resonance around 40 ppm in Fl and 2 5 ppm in F2 arises from partly undeuterated DMSO d solvent and should not be detected The F1 43 ppm and F2 5 1 ppm resonance is too far off diagonal and should not have been detected The Report workwindow output screen suggests how to adjust the integral threshold to avoid such problems HSQC Resonances of Wrong Sign it 7121 C 18 H 42 Fl 16 579 F2 1 627 gt Recommended Threshold Integral T Mex 3593 832 I 11 673 Positive HSQC Resonances too Far From Strongest One for Same Carbon Zoe C Y He Fle sa PAS 353930 184 C 19 H 5e Fl 142569 F2 0 986 Se Se gt Recommended Threshold Integral Fi max 3 858 I 44 059 Fi max 1 061 I 56 504 HSQC Resonance Much Weaker Than Strongest One for Same Carbon 596 C 16 H 15 Fl 42 711 His 5 115 716 Cis H 39 Fl 16 532 F2 1
220. played structures in MDL Molfile format A mol ending is appended if no filename ending is specified Created mol files can be imported by programs supporting the Molfile format such as by NMRgraph itself Save SDfile saves the displayed structures in MDL SDfile format A sdf ending is appended if no filename ending is specified Created sdf files can be imported by pro grams supporting the SDfile format including NMRgraph itself Save ChemDraw saves the displayed structures in CambridgeSoft Corporation binary ChemDraw eXchange CDX format A cdx ending is appended if no filename end ing 1s specified Created cdx files from all the supported platforms can be displayed under MS Ma with molecular drawing programs such as ChemOffice and ChemDraw version 4 or later 1 Free download of MS Windows ChemOffice Net Viewer 2004 from http chemstore cambridgesoft com January 11 2010 19 2 NMRgraph Molecular Correlation Editor Pull Down Menus Save PostScript saves PostScript output of the currently displayed graph for printing or inclusion into other documents A ps ending is appended if no filename ending 1s specified All the displayed molecular structures are scaled to fit on one page The created PostScript file cannot be read back into NMRgraph for further modification If further modifi cations are desired use Save Structure above Print prints the NMRgraph display in PostScript format Th
221. ppears The navigation section at the left side allows easy navigation among help topics The Help menu at the top of the NMRanalyst window provides access to frequently used help topics SS Help System of WMRanalyst ajal aju BB aiRanalyst Manua i User Manual b aJ CHAPTER 1 Overdew Ek A CHAPTER 3 NMRanalyst ine b aJ CHAPTER 3 Tutorial Using gt J CHAPTER 4 Tutorial Il Settir a CHAPTER 5 Tutorial ill Cor p a CHAPTER 6 Tutorial IV Ades CHAPTER T Tutorial Y Addii zI Reference Manual b J CHAPTER 8 Using the Help gt aJ CHAPTER 9 Using the NMR J CHAPTER 10 Using the Dire r 7 CHAPTER 11 Using the File a CHAPTER 12 Using the Wor Ek J CHAPTER 13 10 Analysis 4 h 23 CHAPTER 14 FFT Workwind gt E CHAPTER 15 nD Analysis W b aJ CHAPTER 16 Report Work CHAPTER 17 Assemblelt W r DJ CHAPTER 18 Graphic Work b od CHAPTER 19 Hptiorapnb Wu J CHAPTER 20 NMiRplot Ploti zI Appendix J CHAPTER 721 Frequently Ast e Ce Automated Analysis of 1D Through 3D NMR Spectra January 11 2010 7 9 Reference Manual CHAPTER 8 Using the Help System The NMRanalyst help system is provided in JavaHelp and as PDF file Manual pdf This chapter explains the JavaHelp application accessible from the software 8 1 Starting the On Line Help Help The shown Help menu located at the right side of the top NMRanalyst Manual menu bar provides various task specific access p
222. pplication reserves a large number of color cells for its private use other running applications have to share whatever color cells are left To resolve NMR plot color problems exit the application reserving the available color cells If Varian VNMR or VnmrJ are displayed on your X Window system they likely cause this problem Exit them to free over 80 private color cells Then start NMRplot and it shows in its intended colors January 11 2010 21 8 Frequently Asked Questions How to fix incorrect NMRplot or NMRgraph ren 21 13 How to fix incorrect NMRplot or NMReraph rendering Problems with the NMRanalyst graphic rendering are typically caused by the window system or graphic card configuration On Microsoft Windows the color of a displayed NMRplot image may change during mouse interactions or window resizing A NMRplot display may not redraw correctly when overlap ping with other display windows Random dots may appear on a NMRgraph display Should incorrect rendering of NMRplot or NMRgraph occur decrease the number of bits of color information stored for each screen pixel and decrease the hardware optimization level From the MS Windows Control Panel double click the Display icon Select the Settings tab and reduce the number of bits used for each screen pixel from the Colors option menu e g from True Color 32 bt tO hugh Color 16 bit Click the Advanced button select the Troubleshooting tab and drag the Hard
223. pplied and VNMR can create coefficient files for standard Varian pulse sequences Choose one of these two native transforms now and per form it by clicking Start To analyze the 3D HNCO spectrum click the 3D Analysis tab and click Start The analysis of this 3D spectrum takes about one minute Display of Correlation Information 1H 1 C 1 N 1 No correlation detected dil 2 H 1 C 1 N 2 CORRELATION Fl 163 393 F2 105 190 F3 8 174 3 H 1 C 1 N 3 CORRELATION Fl 163 391 F2 105 190 F3 8 174 140 H 5 C 4N 7 No correlation detected dIl January 11 2010 7 6 Tutorial V Additional NMRanalyst Features Analyzing 3D Spectra 8 172 105 19 163 39 TO summarize the 3D analysis results run the Report workwindow Three phase functions file phases plot 3D fitting volumes file vol umes plot integral integrals plot and parameter precision dis so a ms tributions precisions plot anda structure plot file are saved 99 9 90 91 16292 CQuivalent to the 2D spectrum analyses Display the shown struc ture plot file from the Graphic workwindow This oligomer has eight amino acids and seven HNCO signals are expected and observed The carbo 7 93 0300 04 15 40 NY carbons shifts in red representing the 4 carbon valences are bonded to LN atoms shifts in green representing the 3 nitrogen valences which are bonded to protons shifts in orange representing one proton valence Bond labels show the correlation index num
224. predicted proton shifts on or off Hide H Shift Differences Show H Shift Differences is enabled after proton shift prediction is requested from the Prediction menu It toggles the display of differences between observed and predicted proton shifts on or off When this menu item 1s selected a carbon atom label is treated as the observed shift for the proton s attached to the carbon Hide Predicted c Shifts Show Predicted 17C Shifts is enabled after carbon shift prediction is requested from the Prediction menu It toggles the display of predicted carbon shifts on or off Hide tC Shift Differences Show C Shift Differences is enabled after carbon shift prediction is requested from the Prediction menu It toggles the display of differences between observed and predicted carbon shifts on or off When this menu item is selected a carbon atom label is taken as the observed carbon shift January 11 2010 19 6 NMRgraph Molecular Correlation Editor Pull Down Menus Structures The Structures menu contains items that apply to con Cut cru nected structures Copy Ctrl C Paste Ctrl EES Cut copies the structures with selected atoms and or correla Flip Vertical tions or all the displayed structures in case that no atoms and Rotate 90 Rotate Bond To Horizontal Rotate Bond To Vertical Layout Structure From Selected Atom correlations are selected to the clipboard and removes the struc tures from the
225. processing parameters The JCAMP DX FID item specifies an uncompressed JCAMP DX FID The jcamp2txt script is used for its parameter and FID conversions All FID data is included in the specified file Regular compressions are ineffective for FID data So it is assumed to be specified uncompressed The Import Line List item allows converting a table of resonances into NMRanalyst format The simplest format for the line list is one shift number per line It is interpreted as a shift in ppm This format is used to enter carbon resonances A second column can be speci fied to indicate the integrals This format is frequently used for proton information When sev eral resonances are included in one integral divide its value through the number of contained resonances for each resonance Only methyl or methoxy group resonances need to be speci fied by the ratios 3 H 1 5 1 5 H 0 75 1 5 0 75 H or multiples thereof for reliable group rec ognition Only the relative size of integrals matters not their absolute values The Generate Generic List item allows generating a generic 1D resonance list when no experimental data is available Key Dat Fmt Use Relaxation Tine s When Import Line List or Generate Generic List is selected this input field is displayed and allows specifying the effective relaxation time to be used for all resonances When not specified a one Hz linewidth 0 3183 s relaxation time is assumed Key Set T2
226. pyright JavaHelp Hitting areal Bee CHAPTER 145 nD Analysis VWworkwindow 13 CHAPTER 16 Report Workwindow 6 CHAPTER 1 Overview 2 CHAPTER 3 Tutorial Using NMRanalyst 9 CHAPTER 20 NMR plot Plotting Multidimensional Spectra 4 CHAPTER 18 Graphic Workwindow we D CHAPTER Tutorial Y Additional MMRanalyst Features we D CHAPTER 12 Using the Workwindows ee D CHAPTER 9 Using the NMRanalyst Window bei 5 CHAPTER 4 Tutorial Il Setting Analysis Parameters Gei 4 CHAPTER 19 MMRgraph Molecular Correlation Editor bei 3 CHAPTER 131D Analysis Workwindow qe 1 CHAPTER 21 Frequently Asked Questions 2 CHAPTER 8 Using the Help System 1 CHAPTER 6 Tutorial lv Advanced Structure Elucidation Gei 1 NMRanalyst Manual The NMRanalyst online help system supports full text search To use this search facility display the search tab as shown Type the phrase to be searched in the Find input field and hit Enter The titles of topics of which content contains the phrase are listed and ranked according to how well they match the search criterion The topic with the highest number of occurrences of the searched phrase is displayed at the top and is selected Its associated content is displayed with each occurrence of the phrase highlighted To view another topic in the search result list single click on its title If no occurrences of the phrase can be found no result list is displayed 68 4 Copyright JavaHelp Sun Microsystems
227. quency ppm January 11 2010 5 2 Tutorial Ill Combining Analysis Results Analysis of Multiplicity Edited HSQC Spectrum From the Spectrum Experimental TYPE menu select ee HSQC Switch to the ID Analysis workwin dow Load the spectrum specific parameters from directory proton fid file procpar Run the analysis of the 1D proton data and display the shown proton plot file 01 ki 14 6 14 12 10 H 4 2 D 12 Frequency ppm 5 2 Analysis of Multiplicity Edited HSQC Spectrum For the gibberellic acid structure elucidation switch to the FFT workwindow Set the acquisi tion specific parameters by loading from directory ghsqc fid the file procpar The 1D carbon spectrum was referenced by 93 722 Hz so update this F1 Start of Spectrum value to 954 79 Hz Run the FFT workwindow Change to the 2D Analysis workwindow Run the 2D Analysis and the Report workwindow is auto run The next step is to load and lock the phase functions to distinguish CH CH and CH groups from this multiplicity edited HSQC spectrum In the Graphic workwindow load the HSQC report log containing the determined linear phase functions so the following dis played 2D HSQC 1s phased Also load these phase functions in the 2D Analysis workwindow to improve the analysis results Run the 2D Analysis and it auto runs the Report workwindow January 11 2010 5 3 Tutorial Ill Combining Analysis Results Analysis of Multiplicity Edited HSQC S
228. r frequency value should be entered in the left input field If the 1D and 2D spectra of a dataset were acquired with different spectral widths use these input fields to limit the analysis of the 1D spectrum and hence the 2D spectrum to the com mon spectral range Another reason for specifying the 1D spectral range 1s to focus on a par ticularly challenging spectral area for the optimization of analysis parameters Key FqF rom FqFromu FqTo FqTou January 11 2010 13 7 1D Analysis Workwindow General 1D Resonance Analysis 13 5 General 1D Resonance Analysis The input fields allow specification and optionally locking of initial parameter values for up to 300 resonances Resonances with selected Remove switch are subtracted from the experi mental dataset before the data analysis This mechanism allows removing solvent and impuri ties from the spectrum The 1d analysis program optimizes the remaining specified resonances EE mr resonance Panes This switch allows displaying and activating or hiding and deacti vating the table of initial parameter estimates If selected the specified initial values are used instead of the values generated by the peak picking algorithm For the routine analysis of 1D spectra this button should be deselected Key LFit Resonance Frequency ppm Integral Relaxation s Phase rad Remove In this table every line of 1 input fields describes one spectral resonance Lines wi
229. r software to create line lists to be imported into NMRanalyst This workwindow can also generate generic 1D resonance lists This generic information allows the complete analysis of multidimensional spectra without requiring experimental 1D spectra 13 1 Input File Generate Generic List January 11 2010 13 1 1D Analysis Workwindow Input File THPUT ELE Spacing Between Generated Signals ppm d Varian Bruker AMX amp DMX JCAMP DX Import Line List IHPUT FILE 1D FID Spectrum or Line List fia eiaveia al When neither a 1D FID spectrum or line list is available set the Input File Format option menu described below to Generate Generic List The first input field shown above is displayed Specify the desired distance between resonances in agreement with the mapping information for the analysis of the multidimensional spectrum If no value is specified the default value of 4 points is used When this workwindow is run the generic 1D line list with the specified line spacing 1s created When experimental 1D information is available specify the name of the file inthe 1D FID Spectrum or Line List input field for conversion to NMRanalyst format Varian VNMR format data files can also be specified in this input field allowing to use VNMR pre processing linear prediction digital filtering weighting functions etc before the NMRana lyst data analysis Key SigDist SigDistU FnFID The 1d analy
230. region the initial best fit and error values marginal standard deviations of possible correlation patterns are written to this file Key FnBond ap Simulated Spectrum inadequate spec sim E For the optimal detection of correlations in multidimensional spectra NMRanalyst fits a simulation of the expected spin system to the experimental data The simulated spectrum containing all best fit spin systems is hence a natural way to present the analysis results When the Add Correlations or Add Diagonal Resonances switch is selected NMRanalyst writes 2D and 3D spectral simulations in its internal format If no file name is specified in this input field the file name from the 2D FFT Output File input field is used with a sim postfix The 2D and 3D spectral simulations can be displayed by the nmrplot program described in CHAPTER 4 Tutorial II Setting Analysis Parameters Key FnSim January 11 2010 15 2 nD Analysis Workwindow Analysis Parameters 15 3 Analysis Parameters AHALYSIS PARAMETERS Specify which correlation Analyze Correlation Index s _ areas the analyze pro gram should examine Up to 999 999 correlation areas can be examined For a given number of 1D resonances n the maximum number of correlation areas in 2D homonuclear spectra is n n 1 2 The number of possible multidimensional heteronuclear correlations is the product of resonances 1n all speci fied 1D input files Leave this input field e
231. resonances E cup which would be observed in a proton coupled carbon spectrum The dotted lines in this circle diagram represent the derived unambiguous correlations and the dashed lines the ambiguous ones 1444 1786s 173 0s 81 Display the resulting structure file 7 h fh CC corrs plot 1 In this case only the correct Dam agaga IT gibberellic acid carbon skeleton is consistent with all j the experimental correlations In this display bonds which cannot contain an unobserved heteroatom are d shown as solid lines The other bonds are shown ma dotted line style Bonds which are not observed but were derived from longer range correlations are iden tified by a question mark label The other bond labels show the proton frequency over which the bond was derived The direction over which a bond is observed is indicated by arrows 16 504 gng do January 11 2010 5 8 Tutorial Ill Combining Analysis Results Structure Identification Without 1D Carbon 5 5 Structure Identification Without 1D Carbon Spectrum Acquiring a 1D carbon spectrum for the gibberellic acid structure identification or elucidation sounds harmless But its 3 h 37 min acquisition time exceeds the remaining 1D proton multi plicity edited HSQC and HMBC acquisition times combined This section describes generat ing a carbon resonance list from the HSQC and HMBC spectra From the current gibberellic acid directory remove the carbon FID and related information Start
232. ressing the left mouse button Two dimensional contour 2D location plots of correlation signals precision distribution integral values phase function 1D parameter 1D spectrum and build up curve plots support zooming and moving but not rotat ing or spinning For these 2D plots pressing and dragging the left mouse button draws a rect angular box rubber band over the displayed spectrum upon release of the left mouse button the spectral area inside the rubber band 1s enlarged To abort a rubber band zooming type the Esc keyboard key before releasing the left mouse button The display of these 2D plots can be shifted to a different area Press the middle mouse button over the displayed area and drag the mouse in the desired direction The edge of the acquired spectrum is indicated by a red dashed line Upon the completion of a rubber band zoom or shift the axis labels update 20 3 Pull Down Menus Pull down menus are located at the top of the NMRplot window Besides mouse interactions the menus and menu items can be activated by typing the F10 keyboard key and navigated through the left right up and down arrow keys Not all menus and menu items apply to each plot type So only applicable menus and menu items are shown for a displayed plot Details of each menu and menu item are discussed below File Save PostScript saves the displayed plot as a color PostScript E EE Gs file It starts a Filebox popup for specifying the file name
233. rotonated carbons But from heteronuclear NMR alone a bond between two unprotonated carbons is not directly detectable Specify the maximum number of unobserved bonds to be added during the structure generation in this field Up to 50 unobserved bonds can be considered AssemblelIt can derive the presence of such bonds from observed 3 bond heter onuclear HMBC correlations involving the unobserved bond Key addBonds Ho Bonds Over 3 HMBC Correlations Bonds not directly observed can be derived by combining con nectivity information over several HMBC correlations Two HMBC correlations are combined by default to derive additional Ho Bonds Over 3 HHBC Correlations Bonds Over sp sp Bonds Over sp any Bonds Over Any 3 HMBC Correlations unobserved bonds Conjugated systems typically have 3 bond January 11 2010 17 8 Assemblelt Workwindow Assemblelt Elucidate Molecular Structure HMBC couplings stronger than 2 bond ones Often only 3 bond correlations are detected Select Bonds Over sp2 sp2 to consider bonds between ep carbons over 3 cor relations Select Bonds Over sp2 any to allow bonds involving at least one sp carbon and Bonds Over Any 3 HMBC Correlations for bonds between any car bons over 3 correlations Key L3CorrBond 4 Bond HBC Correlations Long Range BC Correlations J Indistinguishable chemical shifts 4 bond or longer range HMBC correlations and incompletely suppressed sp
234. rovided to add Bruker and Varian FID and spectra specific parameters to these generic settings So the number of parameters which may need optimization is limited The procedure to optimize them is described in this tutorial 4 1 The UNIX Shell Window If an NMRanalyst application is still running exit it to remove it with its started plot applica tions Now start NMRanalyst and change to the supplied data directory as described in the previous tutorial CHAPTER 3 Tutorial I Using NMRanalyst For the analysis of a new dataset no customized input value files are available Select from the Spectrum Type menu 1 1 ADEKQUATE January 11 2010 4 1 Tutorial Il Setting Analysis Parameters The UNIX Shell Window E Click the UNIX ANALYST WIN data P 21 Shell button at the top of the NMRanalyst win dow A shell window similar to the shown one appears It can execute UNIX commands even on Microsoft Windows Rename the INADE QUATE directory con taining current parameter values from this shell 4 window mv INADEQUATE INADEQUATE org When no spectrum specific directory is present NMRanalyst loads generic files from the SNMRUSER INADEQUATE directory Now change NMRanalyst back to the INADEQUATE spectrum type Spectrum Type menu INADEQUATE item January 11 2010 4 2 Tutorial Il Setting Analysis Parameters 1D Analysis Workwindow Settings for Pred 4 2 1D Analysis Workwindow Settings for Pr
235. rt of a valid number If the final entry is not a valid number the shown popup appears Restore resets the input field to the last valid entry The text 788 623 entered in the previous numerical input field is nota valid number Restore Help Some input fields expect a file name entry These input fields have a folder button at the right side When typing the Space key NMRanalyst auto completes the file name as long as the file name can be uniquely deter mined Otherwise a white space is inserted in the input field The workwindow input screen utilizes an option menu for choosing one item out of a fixed list of items For example the frequency unit can be chosen by clicking on the shown option button When this button is clicked with the first left mouse button the menu with the unit choices 1s displayed and the desired item can be selected January 11 2010 12 5 Using the Workwindows The Workwindow Input Screen _3 Nenbered Rings 4 Menbered Rings For toggling between two possible states of an item the work window input screen uses check boxes as shown A check box is selected when the check symbol shows All input screen objects recognize their default setting To guide the user s attention all input screen objects with modified non default values are shown in a color different from the default background color Make Default in the Workwindow pull down menu allows making the current
236. rt the NMRanalyst application window See CHAPTER 10 Using the Directory Editor for details on using the Directory Editor 9 5 Structure and Function of the Application Window The application window provides access to all NMRanalyst functions The window consists of the following parts 4 INADEQUATE 1D Analysis Obtain Numerical Description of 13C Resonances H iol xi The window title dis plays the currently selected spectrum type and Reeg and a short explanation of the function of the selected workwindow AB ot At the top right corner of the application window is the NMRanalyst logo When the currently selected workwindow is computing the logo is ani mated File Edit Spectrum Type Workwindow Process Tools Help At the top of the appli cation window is a row of pull down menus providing access to all application window func tions The menus can alternatively be accessed through the keyboard keys The F10 key makes the pull down menus accessible from the keyboard and the File menu button is highlighted The other menus can be accessed with the Left or Right cursor key The Down or Up cursor key selects items in the currently opened menu January 11 2010 9 6 Using the NMRanalyst Window The Workwindow Tabs l t Ganie At the bottom of the application window is a row of workwindow tabs The analysis of multidimensional NMR spectra is controlled by workwin dows displayed in the application window E
237. ry 11 2010 15 7 nD Analysis Workwindow Analysis Parameters COUPLED HSQC Mapping _ Integrals WF1 Phase _ F1 Relaxation _ Unused Unused WE Phase _ F Relaxation _ Unused INADEQUATE Mapping _ 2 Integrals WF1 Phase _ Fi Relaxation _ Unused _ 4 Integrals MF2 Phase _ F2 Relaxation _ Unused NOESY ROESY TOCSY Mapping _ 2 Integrals WF1 Phase WF Phase _ Relaxation 3D SPECTRUM Mapping WF1 Phase WF Phase WF3 Phase Unused _ F1 Relaxation _ F Relaxation _ F3 Relaxation _Umsed These switches indicate which parameter values are to be optimized during the Mapping stage for the selected spectrum type In addition to the selected switches the chemical shifts in Fl through F3 are mapped if appropriate and for DQF COSY HMBC N15 HMBC COUPLED HSQC and INADEQUATE spectra the coupling constant is mapped as well The 2 Integrals and 4 Integrals switches are mutually exclusive When the Detection F1 Relaxation switch described below is selected the resonance fre quency in F1 direction is also optimized to overcome possible inaccuracies in the relaxation time caused by this resonance frequency For the initial analysis of a dataset the phase switches should be selected until the phase func tions are determined Once phase functions are determined these switches can be deselected so that the response surface can be mapped rapidly in a linear fashion The F Phase
238. s Correlation Summary LOS 106 108 215 304 306 308 395 396 477 479 597 602 698 701 703 708 807 904 905 1002 1004 1021 1022 1200 1206 1207 1209 1326 1329 1413 1416 1487 1490 1498 1500 1582 1692 1697 1703 1704 Select the three lines of identified correlation index numbers Click the right mouse button and select Copy Selection Switch to the 2D Analysis workwindow Click the Analyze Correlation Index s field to select it and delete any previous entry Click the right mouse button and choose Paste Scroll to the bottom of the input screen and select the Add Correlations switch Deselect the RUN REPORT WORKWINDOW AFTER SUCCESSFUL ANALYSIS switch Run the workwindow Every best fit correlation area is added to the simulated spectrum January 11 2010 7 3 Tutorial V Additional NMRanalyst Features Displaying Experimental Simulated and Resid 120 ppm it up increase or down decrease The shown three spectra display from top to bot tom the experimental simulated and residual spectra To create the upper one delete in the Graphic workwindow the Add Corr Loca tions Add Fl 1D Spectrum and Add F2 1D Spectrum entries Set File With Plot Data tohsqce spec Relative Size Yto 0 33 Contour Lines Max to 0 03 and start the workwindow To create the simulated spectrum change File With Plot Data to hsqc spec sim and run the workwindow The residual spectrum is the difference between the experimental and simul
239. s displayed Clear History deletes all history information for this input screen and Keep History proceeds without delet ing old input values Over 200 KB of history information has e accumulated for this workwindow input screen To improve program performance clear this history information Clear History keep History Help January 11 2010 12 6 Using the Workwindows The Workwindow Output Screen Workwindow Subprocess xi If a NMRanalyst computing program is not avail f able this popup is shown OK returns to NMRan EAA E anaie exe INADEQUATE aaen alyst See CHAPTER 2 NMRanalyst Installation ue for corrective actions Check the NiiRanalyst software installation and the PATH and MMRDIR variable settings ge Help 12 2 The Workwindow Output Screen The workwindow output screen is used to display the output of the running computation Dur ing computation the text displayed in the output screen is also written to a 1og file named according to the following table 1D Analysis workwindow ld analysis log FFT workwindow EE OG nD Analysis workwindow analyze log Report workwindow report log AssemblelIt workwindow assembleit log Graphic workwindow graphic log Whenever a workwindow is selected the corresponding Loo file from the previous analy sis if any is automatically loaded into the output screen When the computation of a work window is started a previous 10g fil
240. sed or intended for use in the design construction operation or maintenance of any nuclear facility Sun dis claims any express or implied warranty of fitness for such uses No right title or interest in or to any trademark service mark logo or trade name of Sun or its licensors is granted under this Agreement January 11 2010 8 4 Using the Help System Copyright JavaHelp 3 LIMITED WARRANTY Sun warrants to you that for a period of ninety 90 days from the date of purchase as evidenced by a copy of the receipt the media on which Software is furnished if any will be free of defects in materials and workman ship under normal use Except for the foregoing Software is provided AS IS Your exclusive remedy and Sun s entire liabil ity under this limited warranty will be at Sun s option to replace Software media or refund the fee paid for Software 4 DISCLAIMER OF WARRANTY UNLESS SPECIFIED IN THIS AGREEMENT ALL EXPRESS OR IMPLIED CON DITIONS REPRESENTATIONS AND WARRANTIES INCLUDING ANY IMPLIED WARRANTY OF MERCHANT ABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT ARE DISCLAIMED EXCEPT TO THE EXTENT THAT THESE DISCLAIMERS ARE HELD TO BE LEGALLY INVALID 5 LIMITATION OF LIABILITY TO THE EXTENT NOT PROHIBITED BY LAW IN NO EVENT WILL SUN OR ITS LICENSORS BE LIABLE FOR ANY LOST REVENUE PROFIT OR DATA OR FOR SPECIAL INDIRECT CONSE QUENTIAL INCIDENTAL OR PUNITIVE DAMAGES HOWEVER CAUSED REGARDLESS OF THE THEORY
241. signals to be filled solidly which allows them to be distinguished from negative signals on black and white printouts Key minV maxV nSteps Multidimensional NMR spectra are acquired with pulse sequences containing an evolution time for each spectral dimension with the last evolution time equal to the detection time Since the evolution times are named t through t the corresponding spectral dimensions are named F1 through Fn Following the tradition of displaying the highest spectral dimension along the horizontal x axis F2 1s displayed along the x axis and F1 along the y axis for 2D spectra For 3D spectra F3 is displayed along the x axis F2 along the y axis and F1 along the z axis Another tradition in NMR 1s to reverse spectral axes for displaying NMR spectra and NMRplot application uses this convention F1 Phase pm NMRanalyst does not phase correct data for F2 Phase ll v mmm the spectral analysis since frequency depen F3 Phase ll pm dent phase functions distort the signal line shapes Experimental simulated and the corresponding residual spectra in NMRanalyst format are saved unphased Phase corrected data is only created for plotting These input fields allow specifying the phase functions to be applied to the data before the data is plotted By default these input fields are left empty and no phase correction is applied These fields can be set automatically by loading the Report workwindow output file in
242. sing NMRanalyst and CHAPTER 4 Tuto a ee rial II Setting Analysis Parameters use 2D INADEQUATE for the C H c C molecular carbon skeleton determination Indirect detection NMR can ae Bo vO improve the detection sensitivity over an order of magnitude The graphi SS 8 8 cal illustration shows a carbon proton bond is observable through an S ae m HSQC spectrum Combining this information with 2 bond and 3 bond HMBC correlations provides carbon carbon bond information The AssembleIt workwindow combines the analysis results from different spectrum types It then derives the most likely molecular skeleton s from this often incomplete and ambiguous infor mation Dr P ter Sandor Varian Deutschland GmbH graciously provided 500 MHz NMR datasets of gibberellic acid MF C19H gt 50 Using a 7 mg ml sample in DMSO d a 1D carbon acquisition time 3 h 37 min 1D proton 14 s multiplicity edited gHSQC 31 min and absolute value gHMBC 2 h 31 min were acquired Sim ilar to the CHAPTER 4 Tutorial II Setting Analysis Parameters create a copy of the supplied gibberellic acid dataset containing the experimental data FIDs Delete its HMBC and HSQC subdirectories to start with default analysis parameters In NMRanalyst start Edit Preferences setMode to Full NMRanalyst deselect the Show All Input Fields switch and click OK OD Gibberellic Acid 1 The plant growth regulator gibberellic acid GA is a potent
243. sional NMR spectra The NMRplot appli cation provides the link between the visual and NMRanalyst s automated analysis The quality of NMRanalyst results can be visualized using experimental simulated and residual 1D through 3D spectra Derived information such as phase functions correlation locations NOE build up curves and detailed area plots can also be visualized This chapter describes the NMRplot application with its interactive features 20 1 Spectral Phasing NMRplot can be started by the Graphic workwindow If the specified plot file is in NMRana lyst spectral file format the spectrum is phase corrected by the phase functions specified in the Fl Phase F2 Phase andF3 Phase input fields in the Graphic workwin dow before plotting See CHAPTER 16 Report Workwindow and SECTION 18 3 Multi dimensional Spectrum Specific Settings for details 20 2 Mouse Interactions The NMRplot application supports the following mouse interactions January 11 2010 20 1 NMRplot Plotting Multidimensional Spectra Pull Down Menus Left mouse button select rubber band area in 2D plots to redisplay as enlarged spectral plot rotate spectrum for 3D and 2D surface plots Middle mouse button move spectrum Right mouse button enlarge or reduce zoom spectrum and Ctrl left button spin 3D spectrum and 2D surface plots On a two button mouse the missing middle mouse button is emulated by holding the Shift keyboard key and p
244. sis program can read experimental data in Varian Bruker AMX amp DMX and JCAMP DX format This option menu sets the expected data for Import Line List mat Generate Generic List Input File Format Varian JCAMP DX FID Besides FIDs the Varian and Bruker AMX amp DMX settings allow reading prepro cessed spectra in Bruker called 1r and 1i and Varian VNMR called data files The Bruker and Varian software can import spectra from different spectrometer vendors and pro vide data manipulation capabilities e g linear prediction weighting filtering By analyzing preprocessed phase sensitive spectrum files these facilities can be used before the NMRana lyst data analysis For Bruker AMX amp DMX input files use the Load button in the 1 The NMRanalyst computing processes expect a list of key value pairs as input For users of the NMRanalyst graphical interface the names of the parameters keys are irrelevant However when working from a command line or integrating NMRanalyst programs in other software the key names allow setting analysis parameters For every workwindow inter face widget the corresponding key name is given in parenthesis January 11 2010 13 2 1D Analysis Workwindow Output Files NMRanalyst window and specify the fid or pdata 1r file in the acquired data directory This calls the bruker2txt script to assemble spectral information from the Bruker acquisition and if present
245. software ET 0 2 de installation c cccccecccsccsesesssesecessscsecesevsesesesenevees 2 8 MIDI 0 2 teen 2 6 Microsoft Windows 2000 0 2 SUD OE E 2 9 Microsoft Windows Vista 0 2 version 2 4 Microsoft Windows XP 0 2 space requirements NMRanaly EE 0 2 NN E 0 2 CDG E E EA A E 2 2 EE 0 2 Spectral Width sssrini s ELE Deere veer eter E EE 0 2 grat button 9 9 IS E ene ee OT nT OT 0 2 et eet 0 2 Start Of Spectrum 14 4 SC Sei 0 2 Stop button 9 9 SUN Attert 0 2 BEE UNI 0 2 data acquisition EE 4 12 UNITYplus GE 0 2 demo dataset ccc 2 3 END ee 0 2 AA DEE 0 2 UPEP USS VR 0 2 becoming SEENEN 2 3 NWINNMR ooooooooooooooooooooooooooooooco 0 2 supported computer platforms el Ttoral system wide installation 1111000011111000 2 5 copying sample data 3 2 tt E 12 3 12 9 T TOCSY U ACTONYM MEANING oo eeeeeeseseeeeseseeseseesesseseesteeseeeee l 2 UNC optimization detection parameters 15 9 PC uniform naming convention ccscccscsssccsece 21 3 trademark UNIX Adobe Acrobat EE 0 2 changing file permissions EE 9 9 Assemblelt 0 2 E FORMAL EN 11 1 ChemDraw cccceccsececesesesesecesesecesscecscetsceesceesesearseees 0 2 Bega deen acces cence ccc 9 3 ChemOffece 0 2 ENEE 91 4 CryoProbe biede Eege Dee ege 0 2 shell on WindOWS ooooooooooooooooooooooooocoooecc S Pe El 0 2 SU Commande 75 VANS EE 0 2 SWAP Commande 71 10 deeg 0 2 SWAPON Commande 21 10 JavaHellp o 0 2 January 11 2010 2
246. spectral regions that could cause misinterpretations of the data Selecting the Fitting Area Overlap switch displays such overlapping correlations in a different linestyle in the NMRgraph correlation editor See CHAPTER 19 NMRgraph Molecular Correlation Editor for details Key LOvr1p Redetermine _ 1D Chemical Shifts Without selection of this switch multidimensional reso nances are assigned to 1D resonances and the 1D resonance values are shown in the displayed correlation tables and related structure plots If this switch is selected new lists of 1D reso nances are determined from the multidimensional data Then the correlation tables and struc ture plots show resonance frequencies directly determined from the multidimensional spectrum Selecting this switch removes ambiguous assignments from the correlation tables except for the most likely interpretation It is recommended to not select this switch if the 1D information was determined from an actual spectrum and to select it if generic 1D information or spectral projections were used for the analysis For carbon and nitrogen HSQC and HMBC spectra this switch is not shown Chemical shifts are redetermined if a Redetermined F1 Resonance List value is specified Key LSimp1 January 11 2010 16 9 Report Workwindow Generate NOE Build up Curves 16 4 Generate NOE Build up Curves Sean noe am curves The panel for the generation of NOE build up curves is displayed for
247. sseeesees esse tssessstsseseseseeneees 12 7 setting priority value iseer 9 1 text editing occ cece eees esses eeetseseseseeseeeeteees 12 5 A X Window allowing display of windows 9 5 capturing screen Image 21 7 Color problems esses esesesesesseseeeeeseees 21 8 setting DISPLAY variable 9 4 Starting server 9 5 Xhost commande 9 5 xinit Commande 9 5 XW commande 21 7 XWUC Commande 21 7 January 11 2010 22 13 NMRanalyst Manual Index LZ aro ee 13 5 EEN 19 5 January 11 2010 22 14
248. sssseeeneseneeeeeeee 4 5 Displaying All Workwindow Input Fields cccccecceseeessccecceeeeeeeeeeeees 4 6 Determining Phase Funcnons 4 7 Prednisone Spectrum With 1D Carbon amp Correlation Locations 4 8 Experimental Simulated and Residual Spectrum of One Correlation 4 9 The History Mechanism for Input Values cccccssssssssccceceeeeeeeeeeeeeeees 4 10 Directory Editor and the Sucrose FID Analysis cccccccccccccccceeeceeeeeeeeees 4 11 Analysis of the Sucrose VNMR Created Spectrum ccc ccccccceeeeeeeeees EE E EE E E E E E E E E E Tutorial IIH Combining Analysis Results SL Anbo D e EN 5 2 Analysis of Multiplicity Edited HSQC Spectrum i cccccceeeeeeneees 5 3 Analysis of Gradient HMBC Spectum cccccccceeseesessssesssssseeeeeees 5 4 AssembleIt HSQC amp HMBC Derived Gibberellic Acid Structure 5 5 Structure Identification Without 1D Carbon Spectrum 000000000000000000000 NMRanalyst Manual Contents 5 6 Structure Elucidation Without 1D Carbon Spectrum cccccceeeesesseeseeeeeeees 10 CHAPTER 6 Tutorial IV Advanced Structure Elucidation 6 1 6 1 1D Carbon 1D Proton Edited HSQC and HMBC Analvaes ceeeeeeeeeees 2 6 2 Generation of a Generic N Resonance List 5 6 3 N HMBC EE EE 6 6 4 Strychnine Structures Derived From Previous Analysis Results ccc sesseees 7 63 Usame DOF COSY TEE 8 DO geese eee eae 9
249. st nor does ScienceSoft support it It is provided AS IS in the hope that its PostScript to Printer Control Language PCL document conversion will be found help ful 9 12 Copyright Java Runtime Environment Sun Microsystems Inc Binary Code License Agreement for the JAVA 2 PLATFORM STANDARD EDITION RUNTIME ENVIRONMENT 5 0 January 11 2010 9 18 Using the NMRanalyst Window Copyright Java Runtime Environment 1 DEFINITIONS Software means the identified above in binary form any other machine readable materials including but not limited to libraries source files header files and data files any updates or error corrections provided by Sun and any user manuals programming guides and other documentation provided to you by Sun under this Agreement General Purpose Desktop Computers and Servers means computers including desktop laptop and tablet computers or servers used for general computing functions under end user control such as but not specifically limited to email general purpose Internet browsing and office suite productivity tools The use of Software in systems and solutions that provide ded icated functionality other than as mentioned above or designed for use in embedded or function specific software applications for example but not limited to Software embedded in or bundled with industrial control systems wireless mobile telephones wireless handheld devices kiosks TV STB Blu ray Disc devices telem
250. st Window The shown NMRanalyst window provides the user interface for the automated analysis of multidimensional NMR spectra Its function is to display and control the subwindows of the software the Help system the Directory Editor the UNIX shell window the graphic win dows and the workwindows managing all computations 9 1 Starting NMRanalyst To start NMRanalyst on MS Windows from the menu click AlPrograms click J NwRanalyst36 and then click Wi NMRanalyst36 On Linux start NMRanalyst from a shell window using analyst Starting NMRanalyst 3 6 from usr local ANALYST LINUX The above command starts NMRanalyst as a background process to free the terminal for fur ther input Optionally a spectrum type can be specified as a command line parameter For example analyst HMBC starts NMRanalyst with HMBC instead of the default last used January 11 2010 9 1 Using the NMRanalyst Window Starting NMRanalyst History Speed Button Pull down Window title NMRanalyst buttons Panel fast menu applica shows spectrum logo animated recall access to fre tion window typeandfunction when selected previous quently used commandsand of selected Workwindow values commands help Workwindow computing Scroll bars Mi 30_SPECTRUM 3D Analysis Extract Correlations from Heteronuctear 30 File pa Spectrum Type Workwindow Process Tools Fres Spectrum Analyze outp Fl line lif Fz line F3 Line Jo LZ
251. t is a simple text file in Bourne shell syntax It can be customized as desired January 11 2010 16 11 CHAPTER 17 ASS emblel t Wor kwindow The Assemblelt workwindow combines NMRanalyst results from various spectral analyses and derives the likely molecular structure A full structure elucidation may require many NMR spectra to be acquired The Findit task of the AssembleIt workwindow compares avail able spectra of an unknown sample with a database of known molecular structures This matching is based on the molecular formula weight range proton protonated carbon e g DEPT 135 or HSQC carbon spectrum results or any combination thereof The supplied data base consists of over 14 5 million small organic molecular structures from the NIH PubChem collection The best matching structures reported by FindIt can be displayed by the Graphic workwindow Often the likely structure of a compound is known Perhaps the compound was synthesized using well established reactions The VerifyIt task compares a specified structure with the NMR data The expected structure can be specified in the NMRgraph native plot or Molfile format See CHAPTER 19 NMRgraph Molecular Correlation Editor for a graphical way to enter the structure VerifyIt explains the consistency between the proposed structure and the NMR data from which an overall agreement rating 1s derived The third task of this workwindow is Assemblelt It supports the molecular skeleton eluc
252. t log2txt and plot2txt shell scripts The varian2txt script extracts for the 1D Analysis and FFT workwindows the rel evant information from Varian VNMR parameter files named procpar The bruker2txt script extracts for the 1D Analysis and FFT workwindows the rel evant information from Bruker parameter files Workwindow Load 1D Analysis Parameters i x The L g2txt script provides a mechanism to transfer output from The selected file EVANALYST WihidatalNaADEQUATEManalyze tf i S cannot be loaded because it does not contain recognizable one workwindow to another work information for this 1D Analysis workwindow window input screen The script sup ports the transfer of linear phase Hel 5 cance tee functions from the Report workwin dow output screen to the nD Analysis or the Graphic workwindow input screen and the transfer of signal description from the 1D Analysis workwindow output screen to the signal table in its input screen The plot2txt script extracts linear phase function parameters from a phase function plot file This information is used by the nD Analysis and Graphic January 11 2010 9 8 Using the NMRanalyst Window The Speed Button Panel workwindows If none of the scripts generate information appropriate for the cur rent workwindow the following popup shows Cancel aborts the loading process All the format conversions are provided as shell scripts for easy customization The conversi
253. t 2D and 3D Free Induction Decays FIDs can be transformed or spec January 11 2010 3 7 Tutorial Il Using NMRanalyst The nD Analysis Workwindow tra in Bruker and Varian format can be imported Click Start and the transform of this 2D INADEQUATE FID takes only seconds INADEQUATE FFT Fast Fourier Transform 2D FID File Edit SpectrumType Workwindow Process Tools CG THPUT FILES 2 rim or ten pe EI 2D FFT Coefficients i Aly fl al FID Spectrum Format Bruker AMX amp DHX i SPECTRAL DESCRIPTION Identification Text Dr Mar 28 17 22 25 2002 EST UT 5h quest IRIS u data quest nmr mi Fi Observe PEREN HHz Spectral Width zsne2 656642 Hz Start of Spectemn Hz Humber of Points 125 F2 Observe Frequency 125 772134 Hz Spectral Width 25062 656642 Hz Start of Spectrum 1754 059 EZ Humber of Points 2048 3 6 The nD Analysis Workwindow Click the 2D Analysis tab to switch to the shown workwindow SECTION 1 1 The Automated Spectral Analysis explains this 2D INADEQUATE spectrum analysis Click Start For every examined fitting area a one line report is displayed For identified bonds the chemical shift frequencies and the determined coupling constant are shown All numerical values determined by this analysis are saved in the text file named inade quate corr For every bond region examined the initial parameter estimates the deter January 11 2010 3 8
254. t field the specified structure with assigned proton shifts is saved in NMR graph plot file format Key FnHShifts Assigned Carbon Shifts 4900 plot EU VerifyIt determines the most likely assignment of observed carbon shifts for the proposed structure When a name is specified in this input field the specified structure with assigned carbon or protonated carbon only DEPT shifts is saved in NMRgraph plot file format Key FnShifts Report Mrinart Position If this switch is selected after VerifyIt determines the rating of the expected structure it rates all the structures in the FindIt database against the observed carbon and or proton shifts It then reports the rating position of the expected structure among all the Findit database structures Key LFindItPos _MFindit Position With Molecular Formia This switch is similar to the above FindIt Position switch except that only the FindIt database structures with the same molecular formula as the expected structure are rated Key LFindIt PosMF January 11 2010 17 7 Assemblelt Workwindow Assemblelt Elucidate Molecular Structure 17 4 Assemblelt Elucidate Molecular Structure From NMR Data MassenbleTt ELUCIDATE MOLECULAR STRUCTURE FRON mR DATA Atom atom correlations derived from spectral analysis results can be edited as described in CHAPTER 19 NMRgraph Molecular Correla tion Editor The function of this Assemblelt task is to derive the most likely molecul
255. tal Terms shall have the same meanings ascribed to them in the Agreement These Supplemental Terms shall supersede any inconsistent or conflicting terms in the Agreement or in any license contained within the Software 1 Software Internal Use and Development License Grant Subject to the terms and conditions of this Agreement including but not limited to Section 4 Java TM Technology Restrictions of these Supplemental Terms Sun grants you a non exclu sive non transferable limited license to reproduce internally and use internally the binary form of the Software complete and unmodified for the sole purpose of designing developing and testing your Java applets and applications intended to run on the Java platform Programs 2 License to Distribute Software In addition to the license granted in Section Software Internal Use and Development License Grant of these Supplemental Terms subject to the terms and conditions of this Agreement including but not limited to Section 4 Java Technology Restrictions Sun grants you a non exclusive non transferable limited license to reproduce and distribute the Software in binary form only provided that you 1 distribute the Software complete and unmodified and only bundled as part of your Programs 11 do not distribute additional software intended to replace any component s of the Software 111 do not remove or alter any proprietary legends or notices contained in the Software iv only distri
256. tches inthe Detection sec F2 Phase E m tion are selected the analyze program F3 Phase Bj mm determines phase values for each identified correlation pattern The report program which is controlled by the Report workwindow can interpolate the determined phase values January 11 2010 15 11 nD Analysis Workwindow Select Plot Data to be Saved of each spectral dimension by a linear phase function which can be entered in these input fields Input fields without a specified phase value default to zero It is important to understand the determination and use of phase functions When specifying the phase function without locking the phase parameters a slight speed advantage is achieved during the analysis because better starting values for the phases are available The time deter mining step in the analysis is the mapping of the response surface for each fitting area Using default parameters this mapping is designed to detect even bad deviations of real correlation patterns from initial parameter estimates If the phase function in any direction is unknown the mapping of the response surface has to be performed in a slow nonlinear way So it 1s advantageous though not required to determine approximate phase functions and to lock the phase parameters during the mapping step in order to perform the response surface mappings in a linear way The linear analysis 1s about ten times faster than a nonlinear approach A fur ther adv
257. tent of the parties in which case this Agreement will imme diately terminate 13 INTEGRATION This Agreement is the entire agreement between you and Sun relating to its subject matter It supersedes all prior or contemporaneous oral or written communications proposals representations and warranties and prevails over any conflicting or additional terms of any quote order acknowledgment or other communication between the parties relating to its subject matter during the term of this Agreement No modification of this Agreement will be binding unless in writing and signed by an authorized representative of each party SUPPLEMENTAL LICENSE TERMS These Supplemental License Terms add to or modify the terms of the Binary Code License Agreement Capitalized terms not defined in these Supplemental Terms shall have the same meanings ascribed to them in the Binary Code License Agreement These Supplemental Terms shall supersede any inconsistent or conflicting terms in the Binary Code License Agreement or in any license contained within the Software January 11 2010 9 20 Using the NMRanalyst Window Copyright Java Runtime Environment A Software Internal Use and Development License Grant Subject to the terms and conditions of this Agreement and restric tions and exceptions set forth in the Software README file incorporated herein by reference including but not limited to the Java Technology Restrictions of these Supplemental Terms Sun gr
258. tern in a fitting area this button can be dese lected to simplify the program output Key MulMin January 11 2010 15 4 nD Analysis Workwindow Analysis Parameters Map F2 Frequencies x To find a resonance along one axis in a multidimen sional spectrum the corresponding 1D resonance frequency is used as initial estimate The 1D and nD resonance frequencies might differ somewhat A search area in the multidimensional spectrum around the 1D resonance frequency can be specified The number of starting points to use for the search inside this specified mapping range can be specified as well see below The program analyze knows the spectral resolution along each spectral axis as well as the approximate linewidth from the corresponding 1D resonance So the automatically chosen number of mapping points is normally appropriate When using generic line lists the mapping range has to cover half the chosen distance between generated signals For 2D INADEQUATE spectra the default mapping range is the 0 027 ppm maximum iso tope shift times the Observe Frequency value specified in the FFT workwindow For a 500 MHz spectrometer the default mapping range for 2D INADEQUATE spectra is 0 027ppm 125 697Hz ppm 3 395 Hz The actual value used by the program analyze is shown at the start of the program in the output line Mapping Density F Range 3 39 F Pts 1 J Range 10 00 J Pts 2 The default 2D INADEQUATE frequency range is suffic
259. th an empty Fre quency ppm field are ignored If a frequency value is specified in the Frequency ppm field the correspond ing resonance is analyzed starting with the initial values provided in the other input fields of this line The Integral field defaults to 1 the Relaxation s field to 0 31830988 corresponding to a one Hz linewidth at half height and the Phase rad field to 0 Each specified resonance can be removed from the spectrum by selecting the corresponding Remove switch Key LFregql LI1 Relaxl Psel LI1L through LFreqg300 LI300 Relax300 Pse300 LI300L tear taste mg ges tame To clear all entries in this 1D resonance table click the Clear Table button at the bottom of the workwindow The Undo Clear Table button is then enabled and can be used to restore the values in the table ed Lines to Table Initially only 25 lines are displayed in the table To add more lines click the Add Lines to Table button This table can contain up to 300 lines January 11 2010 13 8 1D Analysis Workwindow Copyright Freely Distributable LIBM 13 6 Copyright Freely Distributable LIBM Copyright C 2004 by Sun Microsystems Inc All rights reserved Permission to use copy modify and distribute this software is freely granted provided that this notice is preserved January 11 2010 13 9 cuaptenta FIT Workwindow The FFT workwindow is used to Fast Fourier Transform FFT a multidimensional Free Ind
260. the software was last exited Click on the UNIX Shell button at the top right of the application window INADEQUATE 1D Analysis Obtain Numerical Description of 13C Resonances File Edit Spectrum Type Workwindow Process Tools Help I IHPUT FILE 1b FID Spectrum or Line List tia al Input File Format Bruker AMX amp DMX d SPECTRAL DESCRIPTION Identification Text IG Mar 28 09 46 41 2002 EST UT 5h questIRIS uydata quest nur m Ubservre Erequency 125 772134 MHz r Spectral Width aner 656642 Hz r bart ob Spectrum IER Hz AHALYSIS PARAMETERS Min Integral Precision Sign of Phased Signals Positive Analysis Region from pen AV to Low Frequency l pem d USE INITIAL RESOHAHCE PARAMETERS Resonance Frequency ppm Integral Relaxation s Phase rad Remove 1 211 409 0 944 0 42208 2 540 _ 2 210 193 1 206 0 38189 La 527 DEEN go i re lt 1D Analysis The shown UNIX shell window is displayed Issue in it the findit command to analyze the car bon proton 1D FID and or HSQC HMQC spectral projections in the current directory The best matching structures from the 15 9 million known structures are listed with the placement number structure rating and PubChem Compound ID CID in parentheses Structure names and compound biological activities can be obtained from http pub chem ncbi nlm nih gov search through identified CIDs If molecular formula or Jan
261. thencccsccadennantecntevcceanceiesctsanceaueecadecsonvecee 18 9 12 Copyright Java Runtime Environment sacs ciiersrestecisensrionstsaioeseinraavereonnsenimeaeneeaseees 18 21 Comit PE e Toole nuina iE 22 CHAPTER 10 Using the Directory Editor 0 ccccccccccccec ee eeeeeeees 10 1 10 1 NMRDIR NMRUSER NMRDATA and NMRSPEC Shell Variables 2 CHAPTER 11 Using the Filebox Popup ccccccccccecccccccceeeeeeeeees 11 1 11 1 Interactive Item Specification cccccccccssssssssessscsnsceecceccceccceeeeeeeeseeessssesanseaces 1 CHAPTER 12 Using the Workwindowg 12 1 121 The norkomdow nomi TEE 3 i22 The EE 7 i23 ri File E E 8 12 4 Summary of Workwindow File Usage occa ccdiscoovsciosannonsiecasconvscennicsnsavevinbenstaseiceassies 8 CHAPTER 13 1D Analysis WorkwindoW ccccccccceccccceces eee tneeeees 13 1 January 11 2010 6 NMRanalyst Manual Contents Dea E E l SSC ET 3 Sch estate 4 ER o EN 5 I3 General ID Resonance E 8 13 6 Copyright Fre ly Distributable LIBM siicescascssveaivicasdeedsvenatacsua pberehenasacesieneseieaensvdes 9 CHAPTER 14 FFT Horkwaindon 14 1 A D E l ERT E 3 ER EE 3 CHAPTER 15 nD Analysis WorkwindowW cccccccseecccccceeeeetneeeees 15 1 SH T EE LS E er TEEN 2 15 3 Getreide EEN 3 Ds Select Pi Dan ir De reece eerie eee 12 15 5 Automatically Running Report After nD Analysis cccccceeeeessesseeeeeeeeeees 13 CHAPTER 16 Report Horkwindon 16 1 Aoa AE aE AE 2 e E e 1 gt
262. tification Text tification text for the 1D spectrum can be specified The specified text appears m the 1D Analysis workwindow output screen and the generated numerical description file The first 200 characters specified in this Identification Text field are displayed by the computing processes The text has no effect on the data anal ysis Key TxSpec Observe Frequency wz Enter the observe frequency of the nucleus under consider ation For example on a 500 MHz spectrometer the 13C observe frequency is around 125 7 MHz On a Varian instrument the observe frequency can be obtained from the VNMR param eter sfrq Key HzPpmF1 HzPpmF1U Spectral width x Provide the value of the spectral width and choose the appropriate unit using the option menu This is the parameter sw in Varian s VNMR software Key swF1 swF1U January 11 2010 13 4 1D Analysis Workwindow Analysis Parameters Start of spectrun He Specify the frequency of the right most low frequency limit of the acquired 1D spectrum If a Varian instrument was used to acquire the data this value can be obtained by subtracting the parameter rf1 from the parameter rfp Key spF1 SpF1U Munber of Points Specify the positive number of phase sensitive points up to 65536 to be Fourier transformed If no value 1s specified all acquired FID points are used If a value is specified the FID is truncated or zero filled as requested Key nPts 1
263. ttom of the NMRanalyst application window click the 1D Analysis tab The 1D analysis has already been completed by the findit script at the beginning of this tutorial January 11 2010 3 6 Tutorial I Using NMRanalyst The FFT Workwindow and the results are stored in the directory data FINDIT To manually run the analysis again click the Start button at the top of the workwindow or simply type the keyboard Enter key This FID transform and spectral analysis takes a few seconds kd INADEQUATE 1D Analysis Obtain Numerical Description of 13C Resonances File Edit Spectrum Type Workwindow Process Tools Help IHPUT FILE 1D FID Spectrum or Line List BE al Input File Format Bruker AMX amp DMX r SPECTRAL DESCRIPTION Identification Text IG Mar 26 09 46 41 2002 EST UT 5h questIRIS uydata quest nur m Ubservre EPrequency 125 772134 MHz r Spectral Width aner 656642 Hz r Start of spectrin IESSE Hz r AHALYSIS PARAMETERS Hin Integral Precision Sign of Phased Signals Positive Analysis Region from pen dE to Low Frequency pen W USE IHITIAL RESOHAHCE PARAMETERS Resonance Frequency ppm Integral Relaxation Tei Phase rad Remove 1 211 409 0 944 o 42208 2 540 2 210 193 1 206 0 38189 2 527 _ q has nas In oan In aaas 2 346 l Kee 1D Analysis 3 5 The FFT Workwindow Click the 2D FFT tab to switch to the shown FFT workwindow Bruker AMX DMX and Varian VNMR forma
264. tton labels change depending on the dimensionality of the selected spectrum type en 2D Analysis 3D Analysis January 11 2010 12 1 Using the Workwindows kg INADEQUATE 2D Analysis Extract Bond Patterns From 20 Spectrum T 2 Ip x File Edit Spectrum Type Workwindow Process Tools Help ri THPUT FILES F ID Analysis Output File eschen out al AHALYSIS PARAMETERS Analyze Correlation Index Mes Thresholds Integral Precision sw Report Multiple Hinima Hap F2 Frequencies Hz d F2 Mapping Points SE l4 Wo correlation detected JI neg dil dle 15 CORRELATION Fa 48 756 Fh 35 560 l Wo correlation detected I_neg dil dle 17 No correlation detected I_neg dil dle 16 Wo correlation detected I_neg dil dle 19 CORRELATION Fa 48 754 Fbs 22 029 ZU Wo correlation detected I_neg dil dle l No correlation detected I_neg dI Fa Fh l5 Wo correlation detected I_neg dIl dIz Fa Fb 16 Wo correlation detected dIl dle 17 No correlation detected I_neg dIl dle Fh 16 Wo correlation detected I_neg dil dle 19 Wo correlation detected I_neg dIl dIz Fa Fb ZU CORRELATION Fa 41 915 Fh 18 733 159 l4 C 21 No correlation detected I neg dIl Oz Fb 4D Analysis 2D FFT 20 analysis RIS E E SIs Sie Sie GE EIS Gre cre EIS Ge Gre EIS ors Let Ver gt Wen KE a e ein L t en VE e EE e Kleng Hl Je et fe Dt E JC D JC ee JH D 3 The figure shows the NMRanalyst window with displayed nD Analysis workwin
265. tures to the FindIt database Help The Help menu provides fast access to several important manual chapters Manual The NMRanalyst software reuses an existing Help window whenever possi Overview Tutorial Current Workwindow ble Hence it is not necessary to quit the Help window manually since this window may be reused and is removed when the NMRanalyst application Using Help exits For details see CHAPTER 8 Using the Help System About SE NMRanalyst shows program version information NMRplot On Item About NMRanabyst January 11 2010 9 16 Using the NMRanalyst Window The Print Popup 9 9 The Print Popup Print Settings sl The shown popup is started from the Print File TMADEQUATE 1Ld_ analysis lag im _ Seiection Print 3 item n the File MEU It Ap can be used to print a specified file or to To Other Printer fnitrogen 1 eto al Owen re e a a a aca eee san It ed vides the printer drivers PostScript Pages Sheet 1E Font 10 pt Paper Letter b410e b 200 betio b c800 ert Apply Settings Cancel Hep edeskiet Cdj550 cd color cdjmono deskjet djet500 epson Windows only epsonc Win dows only laserjet lbp8 Windows only 1jet3 ljet4 ljetplus pjx1300 r4081 Windows only stcolor Windows only and uniprint NMRanalyst creates print and plot output in PostScript format Program gs Ghostscript 1s used for conversion to other print
266. uary 11 2010 3 3 Tutorial I Using NMRanalyst findit Script for Molecular Structure Identifica constraints are known specify them as findit command line options as described in SEC TION 17 2 Findit Identify Database Structures Best Matching NMR Data UNIX Shell E Ioj x Lehome dunkel data Findit HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHRHHHHHHHHHHHRHHHHHHHH Findit CFormula_ConstraintsJ Co 2009 ScienceSoft LLC Eest matching of 15 9 million molecular structures For 1D Hel FIDs uncompressed JCAMP dv Files proton amp carbon line lists HSQC or Formula_Constraints Use AMRanalyst to inspect intermediate steps HHHHHHHHRHHH RH HHHHRRRRR RRR RH HHH RR RRR RRR RH HHH RR RRR RRR RR HHHRRRRRRRR RRR homedunkel data Eruker 10 C 5Sacqus Analyzing 10D carbon FID or JCAMP Det 29 resonances C referencing correction solvent removal reanalysist 21 resonances Determining most likely FindIt structures 1A Ciel Soleo 4900 et 0 973014 384596857 St O 9 7301d 24669807 dr O 970856 22615241 Br 0 965318 21528905 6 0 958169 11767664 ft 0 958169 23602176 Br 0 958039 17962854 3 0 958021 ot 91436 10 0 954752 216646350 homedunkel datal Jj The final findit step is the shown NMRgraph display of best matching structures The findit FID analysis identification of best matching structures and structure display take about 6 January 11 2010 3 4 Tutorial I Using
267. uction Decay FID or to import a pre processed Bruker or Varian spectrum To display all the input fields available in this workwindow select from the Edit menu Prefer ences Set Mode Full NMRanalyst Show All Input Fields switch and click OK See CHAPTER 12 Using the Workwindows for a general descrip tion of the function and use of a workwindow 14 1 Input Files INPUT FILES Specify the experimental 2D FID or Spectrum inadequate fid tid al multidimensional data in this input field The for mat of the specified file is specified by the option menu below Supported formats include an FID file or spectrum in Bruker or Varian format To use Varian VNMR to create phase sensi tive 3D spectra rather than using the native transform specify the location of the VNMR experiment directory containing the 3D FID in this input field Then NMRanalyst s fft program calls VNMR s ft3d program repeatedly as required to create the 8 phase compo nents of a phase sensitive 3D spectrum Key FnFID1 FFT Coefficients oo al The upper field is dis played for 2D data It aD FFT Coefficient File allows the specification of 4 F1 absolute value or 8 January 11 2010 14 1 FFT Workwindow Input Files F1 phase sensitive FFT transform coefficients If no coefficients are provided default ones are used and program fft tries to guess whether the 2D FID contains F1 phase sensitive data For 3D spectra the second f
268. ued are shown in a dotted line style In AssemblelIt derived carbon skeletons the dotted linestyle is used to indicate that the shown correlation might not be a direct bond but could contain another atom such as an oxygen forming an ether group January 11 2010 19 11 NMRgraph Molecular Correlation Editor Mouse Operations 19 4 Mouse Operations Mouse operations are provided to select move and add atoms and correlations bonds Selections are done using the left mouse button To select a single 1tem and to deselect all the currently selected items if any click on the desired item To select or deselect items from the current set of selected items press the Ctrl keyboard key while clicking on the desired items A group of items can be selected by sweeping out an area dragging with pressed left mouse button To move a single atom press the right mouse button middle and right mouse buttons have the same function on a three button mouse over the item drag the mouse with pressed mouse button to the desired location and then release the mouse button Corresponding correlations follow the moved atom To move groups of items select the items first as described above move one of the selected items and the other selected items follow accordingly To add an atom hold down the Ctr1 keyboard key and press the right mouse button over an empty screen area The new atom 1s placed at the current cursor location Double click on t
269. ummary lines nor the numerical values for detected correlations are reported Key RBond _ Hon Correlations Nothing AJ The items of this option menu correspond to the Report About Correlations option menu above Non Corre lations typically are not of interest So the Nothing option is selected by default However if an expected correla tion is not reported the corresponding summary line can be inspected to see why program analyze did not identify this pattern as a correlation Non CORRELATION Summary Lines H 1c 7C 9 No correlation detected No pattern found 2 C 6 C 10 No correlation detected dI4 Fa 3C 8C 9 No correlation detected dI4 Fa 4C 7C 8 No correlation detected dI1l1 dI4 5C 5 C11 No correlation detected dIl dI4 Fa Fb 7C 30C 14 No correlation detected dIl dI4 The best fit pattern found for fitting area 1 was not identified as a correlation because at least one of the determined resonance frequencies lies outside the fitting area The best fit pat tern found in fitting region 5 was not reported as a correlation because the determined parameter precisions for the A doublet OTI and the B doublet d1I4 are not sufficiently high and both determined transition frequencies are significantly different from the 1D reso nance frequencies Fa Fb For a more detailed description see the explanation of the Detection switches in CHAPTER 15 nD Analysis Workwindow The determined numerical values for all
270. user who started the server should issue the command xhost host where host is the name or network IP address of the computer on which the analyst application is running It is also possible to use the xhost command no host name specified which allows all computers to display windows on the display server 3 The X Window server identified by the DISPLAY variable is not running The X Window system can normally be started with SXIN In case of further problems consult the system administrator 2 This convenient approach can compromise the computer security in an unsafe network environment January 11 2010 9 5 Using the NMRanalyst Window NMRanalyst Cannot Find Its Installation Direc 9 4 NMRanalyst Cannot Find Its Installation Directory NMRanalyst only loads the currently needed programs and help files in computer memory When further information is needed it is loaded from the installation directory at run time Upon startup NMRanalyst tries to locate the installation directory by inspecting the NMRDIR shell variable and by searching standard installation locations for the installation directory If these attempts fail the Directory Editor rather than the NMRanalyst application window is shown with a popup explaining the problem encountered Set the correct installation directory in the Directory Editor possibly using the Filebox popup and click the Directory Editor OK button to sta
271. ving user RER IS structures from the FindIt database To add structures select the Add User _ Extract User Added Structures Structures switch Then specify El either a file or a directory containing the _ Remove User Added Structures structures to be added If a directory D e name is specified all files in the directory S are processed but sub directories are oK Cancel Help ignored The supported file formats include NMRgraph native structure files plot Molfiles mol and SDfiles sdf To remove a structure select the Remove User Added Structure switch and specify the structure ID to delete Only user added structures can be removed When adding a structure to the database the assigned structure ID is displayed in the progress message The ID can later be used to remove the structure To remove all the user added structures select the A11 switch To extract structures from the FindIt database select Extract User Added Structures and specify a directory to store extracted structures in Extracting structures does not remove the structures from the FindIt database Only user added structures can be extracted Each extracted structure is saved in a SDfile Click OK to start adding removing or extracting structures Cancel closes the popup without applying any changes Save in Directory January 11 2010 9 15 Using the NMRanalyst Window The Application Window Menus
272. volved carbons is necessary But its sensitivity is over an order of magnitude less than that for indirect detection spectra Key FnINAD 8 n f Consider M Chemical shift Rules Experimentally only proton carbon and nitrogen correlation infor mation is detectable by NMR for the Assemblelt molecular skeleton determination Bond multiplicities and the location of unobserved bonded heteroatoms e g oxygen sulphur halo gens except fluorine are unobservable Two rules are provided to automatically reduce the number of free carbon valences for these cases If a carbon chemical shift lies between 100 and 168 5 ppm its free valence count is reduced by one If the carbon shift is 168 5 or above it corresponds to either a C O or C S group and its free valences are reduced by two These carbon shift rules can be disabled by deselecting this Consider Chemical Shift Rules switch The number of free valences can also be manually edited as described in CHAPTER 19 NMRgraph Molecular Correlation Editor Key LShiftRules _iteteroaton Protons Protons in HMBC spectra show long range couplings to carbons indepen dently of the type of atom they are bonded to By default only the correlations of protons bonded to carbon and for N HMBC spectra bonded to nitrogen are reported Select this switch if correlations of protons bonded to heteroatoms e g oxygen should be reported and saved in the atom atom correlations file Ke
273. vsesececevsveecevevssecasavsvees 15 3 FnNHMBC ooaiaaoanoansoisoossoonsonnsinroriroriorererrrrenna 17 4 BuildUp E 16 10 FONitrogen oo ceececccccsecseseesesseseestssesesesesseseeateees 17 2 COeCSS2D oeeeecceeccsescsecececeescsescsesecececsesveveesceceeseevaee 14 2 FONOE 1 ooececcceccccccsescsecececeescecsesesececscareversesneeeeeens 16 10 EE ee 15 13 FaN ODS ees 16 10 Datbmt 13 3 14 2 rg sescentae EAN 13 4 RTE a E AAEE AE 15 13 Eege 13 4 FIPSCO eserine 15 12 18 5 FnPlot w ccccccccccccccscscsscscscsccescscseseeccacseseeeeees 17 8 18 2 F1Psel ooeeeeccscsesececeseccecesesesececsceseeseseeeees 15 12 18 5 FOProton cccscsccscsesecesececsescecsececscecsesvsvsnsesceceeeree 17 2 i A AEREE ee ere eT 18 5 Ee 16 2 PA YTE O 15 12 Fnbest ooceccececcsscsesssessecscessssecesevsesececsrseseavevsececs 16 3 Fe E 15 12 tee 17 7 F3PSECO oreesa 15 12 18 5 Engm voocccccccccccscscscscscescscsesecccacscsesscecsesesceacscseseeeeaes 15 2 F3PSC1 vececcecccssecesscsesessecessvsssesecevsreeeeveees 15 12 18 5 FnSklt ooccecccccscccsseccsscescssecessesesevsseeseseseveecevseeeeeeees 16 2 SC eegen 18 5 FES DO ee 13 4 UNE A P EEA 15 2 TE GC AEE EA 14 3 MIDP eebe EE 15 2 En ubtract 18 4 Ba SC ae pets 18 4 Ee 17 7 PAD DE E 18 4 Bee 13 7 FO2D A 16 2 FQFLOMmU 0 eececececcssesssseeeessesessesesseseesesestsesseeeees 13 7 E 17 3 EE 13 7 Ee 16 3 PL OU deeg 13 7 eu eer 15 2 EH eege 13 5 Enfuild 16 11 HZPpmF 1 oiiire 13 4 14 3 Ent arbon 17 2 HzPpmF1U oeiee 13 4 14
274. ware Foundation either version 2 or at your option any later version This program is distributed in the hope that it will be useful but WITHOUT ANY WARRANTY without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE See the GNU General Public License for more details You should have received a copy of the GNU General Public License along with this program see the file COPYING If not write to the Free Software Foundation 59 Temple Place Suite 330 Boston MA 02111 1307 USA The file COPYING can be found in directory SNMRDIR enscript and the enscript source code can be downloaded from http people ssh fi mtr genscript or can be ordered from ScienceSoft LLC for the cost of distribu tion Enscript is not an integral part of NMRanalyst nor does ScienceSoft LLC support it It is provided AS IS in the hope that its conversion of text into formatted PostScript will be found helpful 9 11 Copyright GNU Ghostscript Program GNU Ghostscript is distributed under the GNU General Public License GPL which allows free use and free copying and redistribution under certain conditions including in some cases commercial distribution See file SNMRDIR enscript COPYING orhttp www gnu org copyleft gpl html for details The source code can be downloaded from http www gnu org or can be ordered from ScienceSoft LLC for the cost of distribution The GNU Ghostscript software is not an integral part of NMRanaly
275. x systems organize files in a single tree like file system of directories Microsoft Win dows typically has several file system trees distinguished by a letter of the alphabet the so called drive letter Windows NMRanalyst specifies the drive letter e g C as part of the path name in the format C some_ pathname To change to a different drive letter from a NMRanalyst filebox type the new drive letter in the Enter path or folder name input field and click the Update button to update the Folders and Files lists To access network filesystems the MS Explorer can map network resources to drive letters Another way is to use the uniform naming convention UNC UNC paths start with two slashes and consist of server name share name directory tree and file name like in myComputer home ANALYST WIN VERSION While Linux uses slashes to separate directories in path names MS Windows uses back slashes NMRanalyst accepts both notations and converts specified back slashes in UNC paths into slashes 21 2 6 Incorporation of a License Server in Windows NMRanalyst Flexible License Manager H Ei A C offer a time limited eval NMRanalyst checkout Failed Cannot Find license file Flexible License Manager J x uation version of W indows The license Files for server network addresses attempted are NMR d t A li listed below Use LM_LICEMSE_FILE to use a different license file Ee analvst 1cense or contact y
276. y LHeteroH January 11 2010 17 4 Assemblelt Workwindow Combine NMR Analysis Results Bonds HMBC spectra often contain incompletely suppressed direct carbon proton bonds Selecting this switch will remove correlations which could result from such unsuppressed direct bonds By default this switch is unselected as valid HMBC correlations might be elim inated through it as well Key LRBondsCH Mimbiguities If this switch is unselected Assemblelt only reports correlations having a unique assignment HSQC correlations may map one proton frequency to more than one carbon or nitrogen atom When this switch is selected such correlations are listed with an ambiguous comment and are saved using a dashed line style Such correlations are used in the Assemblelt structure elucidation The HMBC and N15 HMBC analysis result input fields above have a Weak threshold input field Correlations below these thresholds are treated as ambiguous So these Weak input fields are deactivated when this switch is deselected Key LAmbigu ities Output Atom Atom Correlations C_corrs plot im Information about carbon and nitrogen correlations derived from the spectrum types above is saved in the specified plot file These correlations include direct bond information longer range correlations resulting typically from DQF COSY and HMBC and occasionally incorrect information resulting from misinterpretation of spectral features or incorr
277. y and all Programs and or Software C Java Technology Restrictions You may not create modify or change the behavior of or authorize your licensees to cre ate modify or change the behavior of classes interfaces or subpackages that are in any way identified as java javax sun or similar convention as specified by Sun in any naming convention designation D Source Code Software may contain source code that unless expressly licensed for other purposes is provided solely for reference purposes pursuant to the terms of this Agreement Source code may not be redistributed unless expressly provided for in this Agreement E Third Party Code Additional copyright notices and license terms applicable to portions of the Software are set forth in the THIRDPARTYLICENSEREADME txt file In addition to any terms and conditions ofany third party open source freeware license identified in the THIRDPARTYLICENSEREADME txt file the disclaimer of warranty and limitation of liability provisions in paragraphs 5 and 6 of the Binary Code License Agreement shall apply to all Software in this distribution F Termination for Infringement Either party may terminate this Agreement immediately should any Software become or in either party s opinion be likely to become the subject of a claim of infringement of any intellectual property right G Installation and Auto Update The Software s installation and auto update processes transmit a limited amount of
278. y spaced carbons with bonded protons at the same F2 frequency further limit this distinction For analyzing your own datasets work at least through CHAPTER 6 Tutorial IV Advanced Structure Elucidation As described in this section and CHAPTER 6 Tutorial IV Advanced Structure Elucidation the 4 Bonds HMBC Correlation and Long Range HMBC Correlation fields might need to be specified Also the AssembleIt workwindow Weak threshold should be specified to accelerate the structure generation The FindIt structure iden tification reliably finds similar structures Full AssemblelIt structure elucidations are more likely to encounter unexpected challenges making them less reliably than FindIt applications January 11 2010 5 11 CHAPTER 6 N utor ial I V Advanced Structure Elucidation The previous tutorials CHAPTER 3 Tutorial I Using NMRanalyst CHAPTER 4 Tuto rial II Setting Analysis Parameters and CHAPTER 5 Tutorial II Combining Analysis Results cover the basic NMRanalyst spectral analysis This tutorial covers generating a generic line list for nitrogen resonances SECTION 6 2 Generation of a Generic L N Reso nance List positioning nitrogen atoms in determined molecular carbon skeletons SEC TION 6 3 N HMBC Spectrum Analysis using additional spectra to reduce the number of possible molecular skeletons SECTION 6 5 Using DQF COSY and SECTION 6 6 Using DQ 1 1 ADEQUATE and placing unobserved heteroatoms in deter
279. z 1D proton and carbon multiplicity edited C gHSQC C gHMBC SN gHMBC gradient DQF COSY and DQ 1 1 ADEQUATE spectra for the strychnine alkaloid CHAPTER 6 Tutorial IV Advanced Structure Elucidation describes its struc ture elucidation 19 MBytes sucrose INADEQUATE Varian UNITYplus 500 MHz 1D carbon and 2D INADEQUATE sucrose datasets The VNMR created spectrum as well as the FIDs are provided for practising the automated analysis of VNMR pre processed spectra as described in CHAPTER 4 Tutorial II Set ting Analysis Parameters 5 MBytes pig itin HNCACB January 11 2010 2 3 NMRanalyst Installation Contents of the NMRanalyst Distribution Varian 600 MHz 3D gradient HNCACB of 0 2 mM ISN and 1 C enriched human ubiq uitin The data was acquired with two transients per increment resulting in a challenging S N 17 MBytes ubrgurtin NCO Varian 600 MHz 3D gradient HNCO of 0 2 mM LSN and 3C enriched human ubiquitin These analysis results can be combined with the ubiquitin HNCACB results to deter mine protein backbone assignments using the protein21o0g script 9 MBytes The installation directory for the supported platforms ANALYST LINUX or ANA LYST WIN contains the following files and directories after installation COPYRIGHT RELEASE NOTES UninstallerData Directory with program and supporting files for uninstalling the NMRanalyst software UserDatea Directory with default input parameter files for the

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