Peptide User Guide
Contents
1. Glycopeptides N and O Glycosylations of proteins are important post translational modifications as well Glycosylated pep tides may act as a stimulator in the immune system The demand for such compounds is constantly increasing even though their synthesis still represents a very chal lenging task The synthetic problems are due to a variety of reasons such as the limited choice of protecting groups for the glycoside moiety and the high lability of the O glycosidic bond Bachem is one of the few suppliers of Fmoc Ser GalNAc Ac o D OH Fmoc Thr GalNAc Ac o D OH which are employed for obtaining glycopeptides Lipopeptides Substantial difficulties have to be expected during the purification of lipopeptides due to their increased hydro phobicity Before starting the synthesis of the lipopeptide the sequence to be lipidated is studied carefully to de termine the most suitable position for the introduction of the lipid moiety A considerable number of palmitoylated peptides has been successfully synthesized at Bachem The lipophilic cysteine derivatives Fmoc Pam Cys OH and Pam Cys OH are available from stock These compounds which resemble the N terminus of the lipoprotein from the outer membranes of F coli have been used at Bachem for synthesizing immunogenic conjugates such as peptide mitogens or vaccines Radiolabeled Peptides Even though it proceeds smoothly the labeling of tyrosine with 25 requires2. 80 is equiva lent to 1 25 g gross weight 1g net 0 8 1 25 g gross weight Which purity is requested Please refer to the table on p 5 What does immunograde mean Immunograde peptides are peptides purified up to 60 70 Their main use is in the production of polyclonal antibodies At Bachem the crude peptide obtained after cleavage from the resin is NEVER sold as Immunograde peptide even if it is sufficiently pure It is ALWAYS sub jected to preparative HPLC to remove the contaminants remaining from the cleavage and subsequent work up What are the remaining impurities Not all impurities can be removed by a single HPLC run Usually a few peptidic contaminants remain mostly dele tion sequences peptides lacking one or more amino ac ids of the target sequence Such by products differ only slightly in polarity hence their removal may fail Truncation sequences which may be generated deliber ately by capping steps to avoid the formation of dele tion peptides incompletely deprotected peptides and by products generated during the synthesis or during the final cleavage may also be found among the impurities What kind of MS analyses are provided by Bachem Electrospray ionization ESI Bruker micrOTOF Mass range 50 to 3000 amu accuracy 5 ppm resolution gt 10 000 FWHM Additionally MALDI TOF and Q TOF are routinely used How to perform an amino acid analysis AAA Amino acid analysis is performed b
3. During solid phase peptide synthesis a peptide which is anchored by its C terminus to an insoluble polymer is assembled by the successive addition of protected amino acids constituting its primary structure Hence the peptide is elongated in the C to N direction Peptides are synthesized from the C terminus to the N terminus of the sequence A synthetic cycle consists of e Cleavage of the o amino protecting group Washing steps to remove the cleavage reagent Coupling of the protected amino acid Washing steps to remove excessive material As the growing chain is linked to an insoluble sup port excesses of reagents and by products can be re moved by repetitive washings with appropriate solvents Only solvents which swell the peptide resin properly can be used for deprotection and coupling whereas the washing protocol may include shrinking steps After completion of the synthesis the desired pep tide is cleaved from the resin Usually this cleavage step is performed with acids of varying strength The Solid Support Polystyrene crosslinked with 1 divinylbenzene is still the most popular carrier resin in SPPS It is chemically inert under the conditions of SPPS and it is readily de rivatized allowing the introduction of a large variety of anchoring groups The resulting resin swells sufficiently in solvents suitable for SPPS The choice of the anchoring moiety is determined by the chosen synthetic strategy and by the
4. interchim BACHEM Peptide User Guide A brief introduction into synthesis methods handling and design of peptides Abstract Almost 40 years of experience in peptide synthesis and the world s largest group of peptide chemists in the industry make Bachem your ideal partner for the custom synthesis of peptides and complex organic molecules Bachem offers a full range of technologies which are available at four company owned production sites two subsidiaries in the USA and two in Europe We produce research grade peptides as well as GMP grade material from simple peptides to the most complex peptidomi metics or synthetic proteins Our experts will support you in the design of your peptides and peptide derivatives The aim of this monograph is to present a general survey of the methods of peptide production and to provide answers to the most frequently asked questions of the end user This publication is focused on peptides used for research purpose milligram to gram scale Copyright by Bachem AG 4416 Bubendorf Switzerland Reproduction forbidden without permission Table of Contents Peptide Synthesis The Principle of Solid Phase Peptide Synthesis SPPS Solid Support Protecting Groups Fmoc tBu or Boc Bzl Strategy Long Peptides Peptide Purification Quality Control of Peptides Definition of Purity Definition of Net Peptide Content Impurities Batch to batch Variability of Peptides Recommended Purit
5. Usually an additional step is required to generate another salt form which will add to the price Hence we will quote for the TFA salt if you do not explic request a different counterion such as the acetate or the hydrochloride Due to deviating purification protocols certain peptides e g acidic peptides will be delivered as ammonium salts Do have to order a minimal amount What is the maximum quantity feasible at Bachem The minimal quantity to be ordered depends on the re quested purity and is 5 mg for Immunograde peptides and 2 mg for the other grades of purity There is no upper limit at Bachem for the amount of re search and GMP peptides Our facilities enable us to produce peptides even up to ton scale Peptide User Guide 15 16 Peptide User Guide Questions Related to the Synthesis How do you synthesize peptides Peptides are synthesized by chemical methods either by solid phase synthesis or by classical solution phase methods The peptide is elongated starting from the C terminus to the N terminus of the sequence What does SPPS mean Solid Phase Peptide Synthesis SPPS can be defined as the process in which a peptide is constructed by succes sive addition of the protected amino acids constituting its sequence anchored via its C terminus to an insoluble polymer For more information please ask for your brochure The Bachem Practice of SPPS What do Boc and Fmoc strategy mean T
6. is stabilized by an N terminal Pyr a very common feature in bioactive peptides Substance P is a perfect model for N terminal degrada tion by diketopiperazine formation during storage This side reaction may occur if a Pro follows the N terminal amino acid and especially if the amino acids adjacent to this residue are unhindered e g Gly 8 Sheet Formation Even though formation cannot be categorized as a side reaction it has to be mentioned in this context as it is the cause of many problems occurring during synthe sis and handling of a peptide Incomplete solvation of the growing peptide chains due to B sheet formation during SPPS leads to formation of deletion sequences and other by products As demonstrated by structural analysis of model peptides forming B hairpin structures in aqueous solution a range of amino acids shows a propensity to be incorporated in B sheets Gln lle Leu Phe Trp Thr Tyr and Val rank highest among them Hence peptides containing a large proportion or clusterings of these amino acids may show the tendency to aggregate Naturally the sequence will influence the extent of B sheet formation as well and the solubility of the peptide The conservative replacement of Gln by Asn can help Thr may be substituted by Ser A slightly altered choice of the partial sequence of a protein to be synthesized may result in reduced aggregation As Pro residues are the most efficient means to disru
7. Receptor ligand interaction studies quantitative 29575 6 Blocking and competition assays quantitative ELISA and RIA quantitative n vivo in vitro studies Western blotting studies non quantitative gt 80 Enzyme substrate studies non quantitative Phosphorylation studies e Production of polyclonal antibodies Immuno e Determination of the titer of antibodies in grade standard ELISA Peptide Analysis Bachem s custom peptides are depending on the re quested purity grade accompanied by the analytical data obtained by 2 3 methods HPLC The purity of the peptide is determined by RP HPLC The chromatogram additionally indicates the number and relative amount of by products MS The molecular mass of the peptide is determined by mass spectrometry to confirm that the correct product will be delivered Moreover the mass spectrum displays the masses of the main impurities Bachem routinely performs ESLMS electrospray ionization and MALDI TOF MS ma trix assisted laser desorption ionization time of flight At Bachem all custom peptides from Immuno grade to gt 97 are purified by RP HPLC Peptide User Guide 5 6 Peptide User Guide NPC The net peptide content is assessed by amino acid analysis AAA and or by elemental analysis as it cor responds to the nitrogen content of the peptide Addition ally AAA allows to verify the amino acid composition of the peptide Especially for sho
8. a licence and the protective equipment for handling radioactive compounds 5l labeled pep tides should be used up rapidly 60 days due to the rather short halflife of the radioisotope Bachem holds the required licenses for handling radioactive material and owns protective equip ment for the production of l radiolabeled peptides Peptides Labeled with Stable Isotopes The production of peptides labeled with stable isotopes such as 5N or 2H is limited only by the commercial availability of the correspondingly labeled amino acids Isotope labels are especially useful in NMR studies of peptides A range of protected N labeled amino acids is available from stock at Bachem Further labeled amino acids will be acquired if they are commercially avail able for the synthesis of suitably protected derivatives The price of the peptide will have to be increased in ac cordance with our expenses Fluorophores and Chromophores A broad range of derivatives of fluorophores and chro mophores suitable for the labeling of peptides is avail able at Bachem to meet our customers requirements In most cases the dyes are introduced either N terminally or C terminally The synthesis of a C terminally labeled peptide usually is more complex than obtaining an N terminal modification The incorporation of a label at the N terminus merely means an additional step in the SPPS protocol even though more elaborate coupling proce dures
9. aggregation impeded by repetitive TFA treatment Acid sensitive peptides and derivatives e g O glycosylated or sulfated peptides Long Peptides up to 100 Amino Acids Our customers request for long peptides up to 80 100 amino acids is increasing Such large molecules could be successfully synthesized at Bachem by stepwise SPPS following the described strategies However with increas ing peptide length this standard approach may fail To Peptide Purification The properties of individual peptide depend on the composition and sequence of amino acids Acidolytic cleavage following SPPS yields a crude prod uct containing the desired peptide and impurities such as deletion peptides truncated peptides incompletely de protected peptides modified peptides scavengers and by products derived from the cleaved protecting groups All these contaminants have to be removed Purification of synthetic peptides is routinely carried out by reversed phase high performance liquid chromatography RP HPLC using C moditied silica as the stationary phase and UV peak detection The target peptide and impuri ties are retained by the stationary phase depending on their hydrophobicity Very polar contaminants will elute fulfill our customers requirements nevertheless the Native Chemical Ligation NCL technology was established at Bachem NCL was developed by Kent as a viable alterna tive to stepwise SPPS for synthesizing very l
10. comparison and evaluation of the chromatograms The conversion should take 45 min to 1 hr at room tempera acetic acid as to lower the pH to pH 4 and thus stop the reaction For removal of by products and excessive DTT apply the solution to a desalting column e g Sepha dex G 10 G 25 or BioGel P 4 P 10 and elute with diluted acetic acid use an ACOH concentration in which the peptide is readily soluble if lyophilization is intended To avoid reoxidation the reduced peptides should be used rapidly Please keep in mind that DTT is readily oxidized it should be handled and stored in a dry and inert atmosphere DTT solutions should be freshly prepared Disulfide bridges can be cleaved even at low pH applying TCEP Tris 2 carboxyethyl phosphine Lit J Wu amp J T Watson Protein Sci 6 391 398 1997 How long will delivery take The delivery time for custom peptides varies from 2 to weeks It depends on the requested purity grade and the complexity of the synthesis The synthesis and purifica tion of highly complex molecules may take considerably longer Note The requested peptide could already be listed in our catalog comprising more than 9000 products If this is the case it can be delivered overnight Bachem owns four production sites situated in Switzer land and the USA so the production of your compound can be transferred according to your requirements We are your reliable partner producing a simple rese
11. depends on the method of disulfide bond formation The consecutive for mation of two to three disulfide bridges has to be consid ered as routine a larger number requires sophisticated Cys protection schemes The number of S S bonds which can be generated simultaneously is not limited either the BACHEN The Bachem practice of SPPS Tips and tricks from the experts at Bachem compiled by M Mergler amp J P Durieux Bachem AG peptide will fold into the required conformation in solu tion or the method does not work Thus peptides con taining up to 5 disulfide bonds have been produced at Bachem How many phosphorylated residues can be incorporated in a peptide Two to three phosphorylated residues per peptide are a reasonable limit What would be referred to as a standard modification Standard modifications affect the final price of the pep tide only slightly as they can be easily integrated into a routine synthetic protocol This includes acetylation ami dation biotinylation mono phosphorylation FITC label ing and similar modifications What is the effect of N terminal acetylation and C termi nal amidation Acetylation and amidation reduce the overall charge of a peptide thus the solubility may decrease These modifica tions could increase the metabolic stability of the peptide as they prevent enzymatic degradation by exopeptidas es If the peptide comprises a partial sequence of a pro tein correspondin
12. may be required the sterically hindered dye de rivatives will couple sluggishly Additionally dyes can be linked to the peptide by selective reaction with a cysteine thiol moiety or the less hindered group of lysine The insertion of a spacer moiety between the dye and the peptide helps to avoid interactions between the label and the peptide which will help retain conformation and biological activity Additional effects may be attained by varying the length the flexibility and the hydrophilicity of the spacer The flexible non polar w amino carboxylic acids e g acid and the hydrophilic 2 2 amino ethoxy ethoxy acetic acid AEEAc are readily coupled to the N terminus or to the Lys side chain When devising FRET substrates the F rster distance i e the distance between fluorophore and quencher allowing an efficiency of energy transfer of 50 usually 20 90 has to be achieved at the minimum to obtain a good quenching effect It depends on the type of fluorophore quencher pair Only a limited number of amino acids can be inserted between dye and quencher moiety otherwise the background fluorescence may reach unacceptable levels The incorporation of a flexible spacer may disturb the energy transfer For more detailed information please visit our website or ask for our Technical Note and Product Monograph mentioned below BAacHEen Technical Notes Chromophors Fluorophors
13. parameters small change of one of these param eters may turn a barely resolved shoulder into a closely eluting peak which can be integrated and thus quanti fied This is demonstrated by the HPLC profiles above For scrutinizing synthetic peptides by RP HPLC and other methods the expert knowledge and the know how of the analyst are of utmost importance Bachem s analytical de partment profits from decades of experience in analyzing peptides combined with cutting edge HPLC equipment Lyophilizates of peptides contain varying amounts of non covalently bound water Normally the peptide is de livered as the TFA salt which results from the RP HPLC pu rification The side chain functionalities of Arg Lys and His and the free N terminus will form trifluoroacetates small amounts of TFA may adhere to the peptide These contaminants cannot be detected by analytical HPLC Other salt forms of your peptide e g acetate hy drochloride will be produced upon special request Absolute Quantity Purity x NPC quantity of of x peptide lyophilizate 10 000 Definition of the Net Peptide Content NPC If not requested otherwise peptides are isolated and provided as trifluoroacetates containing residual water In accordance with the number of basic functionalities Absorbance 210 220 nm Impurity not visible AC Retention Time On request Bachem will analyze your peptide in two or more dif
14. the aggregation and solubilization of amyloid B protein fragments For the solubilization of amyloid B protein 1 42 product number 1368 Bachem recommends the use of a 0 1 M aqueous ammonia solution 1 mg ml pH gt 9 We advise the use of NH ions to increase the pH value without disturbing the peptide structure J Busdglio et al dissolved the lyophilized amyloid B protein 1 40 1194 to a stock concentration of 1 0 mg ml in either deionized water 35 acetonitrile 0 1 TFA or 100 96 DMSO and then diluted it 1 100 directly into tissue culture medium Lyophilized amyloid protein 1 40 was poorly soluble in salt containing buffers e g PBS but could be introduced into saline containing solutions affer being initially dissolved in one of the three vehicles described above HPLC Purification A low yield or complete loss of the peptide on the HPLC column may occur during the purification of hydrophobic peptides Considerable difficulties have to be expected during the production of very hydrophobic long peptides and very short hydrophilic peptides The production of acidic peptides containing free cyste ines is one of the more precarious tasks as during purifi cation and handling in neutral or basic solution extreme caution is required to prevent the oligomerization of the peptide by disulfide bond formation Even if the production of a peptide seems rather straight forward unexpected problem
15. Spectral Properties and Characteristics Bachem offers a series of peptide based enzyme substrates linked to chromophors or fluorophors The advantage of these chromogenic and fluorogenic substrates is the facile spectrometrical detection and analysis of the reaction products The follow ing guide contains some useful information about the various kinds of substrates Chromophors The characteristic feature of chromophors to absorb light of UV and visible wavelengths from 200 nm to 400 nm and from 400 nm to 800 nm respectively can be used to determine their concentration by absorption photometry The method measures the decrease in light intensity when light passes through a colored solution The distance which the light has to pass through a solution is called the path length With a line arly rising concentration of the chromophor solution the intensity of the emergent beam of light falls off exponentially The absorbance A is defined as follows log 1 log 1 T intensity of transmitted light intensity of incident light l l transmittance Since the absorbance also called extinction or optical density O D is linear to the concen tration and the path length the Lambert Beer s law can be applied the law is limited since it is only valid for highly diluted solutions Lambert Beer s law A excxd A absorbance dimensionless concentration mol l M d path length cm molar decadic absorption coeffici
16. acids Most of them are available as No protected de rivatives from stock so they can be inserted into your peptide without delay Standard Amino Acids D Amino acids Modifications N Methylamino acids Citrulline 6 Hydroxylysine B Alanine Unusual Amino Acids 4 Fluoro phenylalanine Norleucine acid Unnatural Amino Acids and Building Blocks Disulfide Bridges The synthesis of peptides containing three or more disul tide bridges still poses a great challenge for the chem ist Complex orthogonal protection schemes have to be devised if the disulfide bridges have to be formed con secutively If the dissolved peptide does not fold sponta neously into a conformation allowing simultaneous oxida tion of the cysteines yielding the correct bridging i e the secondary structure of the natural bioactive compound there is no alternative to the more time consuming and expensive approach involving selective S S bond forma tion On the other hand unnatural folding patterns as well as designed conformations can be obtained only by consecutive bridge formation The outcome of such complex syntheses is difficult to predict several orthogo nal protection schemes and orders of bridge formation may have to be evaluated Bachem may have to ask for generous time limits and milestone payments when fulfill ing such orders Peptide User Guide 9 10 Peptide User Guide Cyclization via Amide Bond Besi
17. al Methods What is an ADS ADS stands for Analytical Data Sheet It contains the fol lowing data Lot number Type of product catalog or custom synthesized Product number e Product description name and sequence Molecular formula Relative molecular mass BACHEM Analytical Data Sheet Lot number 1003828 not for drug use 1 1465 4012873 Suc Ala Ala Pro Phe AMC Cu Hs NO 661 71 Appearance white powder Appearance of solution clear colorless solution 10 mg ml in MeOH Identification ESI MS 661 73u average mass Identification TLC complies with authentic material Purity HPLC 99 0 TFA 99 0 NaCIO Purity TLC gt 98 TLC conditions chloroform methanol acelic acid 77 5 15 7 5 chloroform methanol acelic acid H O 70 42 0 5 10 plate silicagel 60 Fps detected by UV ninhydrin chlorine tolidine Assay elemental analysis 95 8 Nth 10 58 96 Nid 10 14 95 Tests performed depending on the type of product and the purity requested e g o Appearance o Molecular mass obtained by mass spectrometry o Amino acid analysis o Purity determined by HPLC o N content determined by elemental analysis o Solubility Where are the ADS available The ADS can be downloaded from the Bachem website www bachem com Please click on Analytical Data Sheet in the left side menu displayed on our homepage You have to enter the product number and the lot number then clic
18. arch peptide in mg scale as well as the most complex peptido mimetic in kg to ton scale Additionally we can produce your active compound under cGMP conditions which in cludes the preparation of the required documentation Bachem guarantees for best quality at the most competi tive price Bachem is the Number One in Peptides Peptide User Guide 19 20 Peptide User Guide Abbreviations Amino Acid Analysis Aad o Aminoadipyl Abu o Aminobutyryl ADS Analytical Data Sheet AEEAc 2 2 Amino ethoxy ethoxyacetic acid 7Amido 4 methylcoumarin Asu o Aminosuberyl Asx Asn or Asp Boc HButyloxycarbonyl BSA Bovine Serum Albumin Bzl Benzyl cGMP Current Good Manufacturing Practice Dab o Y Diaminobutyryl Dap o B Diaminopropionyl DMF Dimethyltormamide DMSO Dimethylsulfoxide DOTA 1 4 7 10 Tetraazacyclododecane 1 4 7 1O tetraacetic acid Diethylenetriaminepentaacetic acid DTT 1 A4 Dithio DL threitol FITC Fluorescein isothiocyanate Fmoc 9 Fluorenylmethyloxycarbonyl FRET Gal GC Glx GMP HPLC KLH MS Nle NPC Orn OVA Pam PBS pNA Pyr RP HPLC SPPS tBu TFA Fluorescence Resonance Energy Transfer Galactosyl Gas Chromatography Gln or Glu or Pyr AAA Good Manufacturing Practice High Performance Liquid Chromatography Keyhole Limpet Hemocyanin Mass Spectrometry Norleucyl Net Peptide Content Ornithyl Ovalbumin Palmitoyl Phosphate Buffered Saline 4 Nitroanil
19. asic Arg R His H Lys K Nonpolar hydrophobic Ala A lle I Leu L Met M Phe F Pro P Trp W Val V Polar uncharged Asn Cys C Gly G Gln Q Ser S Thr T Tyr Y Bl Acidic Asp D Glu E Bachem issues a lot specific analytical data sheet ADS with each peptide describing the solubility of the product in a specific solvent If appropriate for the customers application we recommend using the solvent indicated on the ADS Otherwise we suggest testing the solubility with a smal amount of the sample following the guidelines below The solubility properties of a peptide are primarily dependent on its amino acid composition We recommend you to check the amino acid sequence of a peptide before choosing the appropriate solvent Basic peptides number of basic amino acids gt number of acidic amino acids should be dissolved in a small amount of an acidic solvent such as acetic acid or trifluoroacetic acid and then diluted to the desired concentration For stock solutions which are highly diluted for their application use 8096 acetic acid for reconstitution of the peptide Otherwise use 2096 acetic acid to reduce the putative negative effect of acetate on the assay system Acidic peptides number of acidic amino acids gt number of basic amino acids should be reconstituted a small amount of a basic solvent such as 0 1 NH OH and then diluted with water to the desired concentrat
20. des disulfide bridging cyclization of a peptide can be achived by other methods Head to tail cyclization an amide bond formation between the N and the C ter minus of the peptide can be achieved following SPPS of the side chain protected peptide Stabilization of a desired conformation is gained by the side chain cycliza tion of amino and groups which requires an additional level of orthogonality Such lactam bridges are formed between Asp Glu Aad Asu and Dap Dab Orn Lys i e ring size flexibility and the direction of bond formation can be varied Additionally these amide bonds are more stable than disulfide bridges If required other modes of cyclization e g formation of thioethers metathesis can be performed Phosphopeptides O Phosphorylation is a very common post translational modification of proteins Hence phosphorylation is a popular modification of peptides though the introduction of phosphorylated sites is limited to two to three residues per molecule For synthesizing the moditied peptides O phosphorylated derivatives of Tyr Ser and Thr are employed These derivatives especially the Ser and Thr derivative are sterically demanding compounds Thus subsequently sluggish couplings have to be expected We recommend introducing the phosphorylated sites close to the N terminus Bachem has gained extensive know how in syn thesizing peptides containing two to three phos phorylated residues
21. ent x mol x cm M x cm the units for found in the literature are usually M cm Knowing the molar absorption coefficient of a chromophor in solution its concentration can be calculated according to the Lambert Beer s law A zxd Please note that molar absorption coefficients may depend on the temperature pH and the ionic strength of a solution Enzyme Substrates and Inhibitors C terminal chromophores and fluorophores such as pNA and AMC are applied in substrates for the detection and quantitation of enzymatic activity A different type of C terminal residue is required to turn a substrate interact ing with the active center of the enzyme into an inhibitor binding reversibly or even irreversibly to this site The in troduction of aldehyde hydroxamate fluoromethylketone or chloromethylketone functions are amongst the most common C terminal modifications of peptides for gener ating effective inhibitors The incorporation of such highly reactive moieties requires the adaptation of the synthetic strategy to each case but our specialists can rely on their vast experience in SPPS and solution chemistry Bachem offers a vast choice of chromophores and fluorophores for the design of enzyme sub strates Our experience and chemical know how allows us to synthesize all types of inhibitors O Acylated Peptides The peptide hormone ghrelin containing an O acylated serine residue and its analogs have found wide
22. eptide decreases with increasing length Nevertheless many exceptions to this rule can be found in the literature Although quite a few examples for the synthesis of pep tides containing up to 100 residues have been published the solid phase synthesis of very long peptides still pre sents a challenging task Small Peptides 12 to 5 Amino Acids Standard Research Peptides medium sized 4 to 50 Amino acids Long Peptides 40 to 80 Amino Acids At Bachem each sequence is scrutinized by our chemists before quotation They will inform you about potential problems associated with your peptide design If unexpected difficulties occur during synthesis or purification we will inform you especially if the agreed purity cannot be attained The synthesis of short peptides consisting of less than 5 predominantly hydrophobic amino acids may pose a problem as well as such molecules are hardly soluble Hence purification is impeded The subdivision into small medium sized and long pep tides and eventually small proteins is quite arbitrary An approximate classification is summarized in the scheme below Small Proteins 70 to 120 Amino Acids eee eS aS Se 0 10 30 Polarity The solubility of a peptide in aqueous systems and con sequently the ease of purification by reversed phase HPLC are strongly dependent on the overall amino acid composition The coded amino acids can be divided into the four group
23. ethanol propanol isopropanol or in mix tures of these solvents with water Acidic peptides can normally be reconstituted in basic buffers basic peptides in acids and if provided as salts of strong acids water could be an alternative whereas the solubility behaviour of zwitterionic peptides is difficult to predict Basic peptides number of basic amino acids including the N terminal amino group gt number of acidic amino acids including the C terminal carboxyl moiety should be dissolved in a small amount of an acidic solvent such as acetic acid or trifluoroacetic acid and then diluted to the desired concentration For stock solutions which are high ly diluted for their application use 80 acetic acid for reconstitution of the peptide Otherwise use 20 acetic acid to reduce the potentially negative effect of acetic acid on the assay system If delivered as trifluoroacetates peptides containing a relatively large proportion of Arg and Lys residues tend to be soluble in water Acidic peptides number of acidic amino acids including the C terminal carboxyl group gt number of basic amino acids including the N terminal amino group should be reconstituted in a small amount of a basic solvent such as 0 1 aqueous NH and then diluted with water to the desired concentration Acidic peptides may be soluble in PBS pH 7 4 Please note that peptides containing free cysteines should be dissolved in carefully degassed acidic buffers a
24. ferent buffer systems though additional HPLC analyses increase the cost of a custom product Actually we refer to the lowest value of HPLC purity we obtain as the final puri ty not the average present in the peptide they may contain a considerable number of counterions Besides lyophilizates of such salts are rather hygroscopic Both water and counter ions reduce the net peptide content The net peptide content is defined as the percentage of peptides relative to non peptidic material mostly counter ions and moisture At Bachem peptides used for quantitative studies are always provided with their Net Peptide Content Net peptide content and purity are not equivalent as the NPC includes peptidic contaminants A low NPC has to be expected for peptides containing a large proportion of basic amino acids even if they are extremely pure Hydrophilic peptides can absorb considerable amounts of moisture The NPC can vary from batch to batch depending on the conditions of final purification and lyophilization The NPC is determined by amino acid analysis and as the non peptidic contaminants do not contain nitrogen it be determined by elemental analysis Net peptide content and purity have to be taken into consideration when preparing solutions of biologically active peptides for assays Impurities After isolation and purification impurities may still con taminate the peptide amongst them deletion sequences pep
25. g to an active site the terminal acetyla tion amidation will generate a closer mimic of the native protein Hence this simple modification may increase the biological activity of a peptide considerably not merely by prolonging its half life What do you recommend for avoiding pyroglutamate formation in case of an N terminal Gln Pyroglutamate formation can be prevented by N termi nal acetylation or especially when synthesizing partial sequences incorporation of the preceding amino acid or omission of the Gln Even the acceptance of a certain extent of Pyr formation could be an option Is a spacer required for introducing a fluorescent label Most fluorescent dyes are large aromatic molecules The incorporation of such bulky moieties may influence the biological activity of a peptide an effect which can be alleviated by interposing a flexible spacer On the other hand the introduction of a spacer cannot be recommended when performing structural studies or when devising FRET substrates In the latter case the re sponse could be modified We strongly advise to search the literature for precedents when considering the incor poration of a linker Do we have to expect batch to batch variability Lot to lot variability will increase when producing low pu rity batches However even when obtaining very pure peptides gt 95 or gt 97 the net peptide content can vary from batch to batch Questions Related to Purity and Analytic
26. ials Dry ice shipment will increase the price of your product considerably but it is available Upon request Are custom peptides supplied as gross weight or as net peptide weight Custom peptides are supplied as gross weight unless re quested otherwise Are peptides containing free Cys supplied as mono mers The purity stated for peptides containing free Cys residues signifies the monomer content at the time the analytical HPLC was recorded As air oxidation cannot be complete ly excluded in the meantime we suggest reducing the peptide before use by treatment with dithiothreitol DTT For a short description of the cystine reduction using DTT please see our Technical Note Reversal of Inadvertent Oxidation of Cys containing Peptides which can be downloaded from our homepage Could you provide special packing Special packaging and vialing are available upon re quest but will be charged additionally Conclusion Why choose Bachem for your custom synthesis Thanks to almost 40 years of experience in peptide chem istry Bachem has acquired a unique know how in this field Our team of peptide chemists will advise and sup port you in every respect when you design peptides and complex organic molecules for your research needs With the largest independent production capacity on the market Bachem offers high flexibility reliable quality at competitive prices and short delivery times Automatic and semi automatic synthesizers
27. ide Pyroglutamyl Reversed Phase High Performance Liquid Chromatography Solid Phase Peptide Synthesis Butyl Trifluoroacetic acid Xaa Yaa Unspecified o amino acid HoN H3C OH H3C Leucine lle Tr 131 18 204 23 p 113 16 186 21 C H 4 NO C Hi NO H5N mn 2 i H3C CH3 N H Isoleucine Tryptophan Ala OKs C H NO HN OH Alanine Met 149 21 12 0 Ch 5 HN Su OH H4C S Methionine e Pro 115 13 97 12 i o a Proline Copyright by Bachem AG Switzerland Reproduction forbidden without permission Cys 103 14 C H NO S HN od Cysteine Asn 132 12 114 10 C H N O H2N O HN OH Asparagine Val 11715 99 13 C H NO HN H3C OH CH Valine Periodic Chart of Amino Acids www bachem com s Gly 57 05 C H NO OH Glycine 50 Ser 87 08 C H NO 3 HN as Serine m Basic Non polar hydrophobic Polar uncharged Acidic BACHE Y T r T 181 19 y jer Jm Glin 128 13 TARO H3N OH HoN Glutamine Thr 163 17 101 10 C H NO C H NO HN HN HO OH ae Tyrosine Threonine Letter Ami 3 Letter Amino UD E Ser x 105 09 Relative Molecular 2 87 08 Mass CHNO M H20 H3N Chemical Molecular Formula Structure HO OH Serine Chemical Name Al
28. ides containing two or more sulfated tyrosines could be obtained Further examples of modified peptides and complex pep tidomimetic compounds synthesized at Bachem could be added to this list Feasibility studies for the synthesis of pseudopeptides containing hydroxyethylene or other isosteric bonds peptide alcohols depsipeptides or what ever you might conceive as your new chemical entity can be conducted by our experts We will be very pleased to send you a customized quotation Bachem can rapidly deliver peptide chelator conjugate but the complexation of the appropriate radionuclide has to be performed by the customer Care and Handling of Peptides Please keep in mind to wear a dust respirator when handling larger amounts of peptide lyophilizates If stored under appropriate conditions peptides are rather stable Nevertheless they should not be stored in solu tion not even in sterile oxygen free solution as they may slowly chemically degrade Frozen solutions may be kept for a few weeks For longer storage peptides should be kept as lyophili zate in a tightly closed container at lt 15 C the lower the temperature the better long term storage at 50 C or lower Especially peptides containing Asn Gln Met Cys and or Trp have limited shelf lives However they may be shipped at room temperature and for short term use they may be stored in a refrigerator at 4 C Handling of Lyophilized Peptide
29. idues require special care to avoid oxidation Oxy gen free water buffers or reducing agents such as 1 4 dithio DL threitol DTT Bachem Cat No Q 1225 have to be used Ask Bachem for our technical documentation on handling and solubilization of peptides Delivery Time The production of a custom peptide consists of four steps synthesis purification lyophilization and analysis Synthesis and purification are the most time consuming steps Synthesis Potential problems low yield incomplete deprotection and coupling e The peptide cannot be obtained in acceptable purity by standard Fmoc tBu SPPS Changing the type of car rier resin or a different synthetic strategy may lead to the desired product complete failure of synthesis Storage of Peptides in Solution e Dissolve peptides in an appropriate buffer cf Solubi lization of peptides p 13 For storage peptide solutions should be aliquoted and kept frozen below 15 C Long term storage of peptide solutions cannot be recommended especially when the peptide contains Asn Gln Cys Met or Trp BACHEM Technical Notes Care and Handling of Amyloid Peptides Bachem offers a series of amyloid peptides The potential problems with the solubility and aggregation of synthetic amyloid B proteins suggest an urgent need for information Following this we have listed a selection of references for our customers We hope you will find some informati
30. in all sizes and the ad equate equipment for purification and lyophilization are available for synthesizing your peptide in mg to ton scale Reactors of all sizes are available at Bachem for the synthesis of complex organic compounds Technical Notes Reversal of Inadvertent Oxidation of Cys containing Peptides Peptides containing free thiol functions may oxidize yielding dimers or oligomers during storage even as lyophilizates at low temperatures Peptides provided as acetates are more sensitive towards Cys oxidation than the corresponding trifluoroacetates or hydrochlo e oxidation rate is pH dependent disulfide bond formation is rapid at neutral or slightly basic pH A small amount of oxygen dissolved in water or buffers will suffice under these conditions Disulfide bridge formation is reversible the bonds can be reduced under slightly basic conditions using dithiothreitol Q 1225 pH 7 9 5 is the optimum pH range for reductions with DTT An inert atmosphere has to be maintained throughout the process Prepare an aqueous 0 1 M NaHCO solution pH 8 5 Bubble nitrogen or argon through the solution for 5 min for removing the dissolved oxygen Dissolve the peptide max 5 mg ml Add an excess of DTT 3 eq per mol peptide HPLC monitoring of the course of the reduction is optional It is recommended if the proportion of oxidized material is rather large It may be helpful to oxidize a sample intentionally for
31. ion Please note that the use of NH OH should be avoided with peptides that contain cysteine residues because at alkaline pH the formation of disulfide bonds is promoted Bachem Leading beyond peptides Can predict if a peptide is soluble in aqueous buffer Unfortunately the solubility of a peptide in water cannot be predicted just by studying the structure However a few clues can be deduced from the sequence a relatively short peptide containing lys and Arg residues will be soluble in aqueous buffers especially as all basic func tionalities will be protonated in peptides sold as acetate or trifluoroacetate salts The guanidine function of Arg is a strong base whereas the group of Lys is a moderately strong base By contrast acidic peptides containing a large proportion of Asp and Glu tend to be insoluble in water but they are readily dissolved by diluted ammonia and by basic buffers The side chain carboxy functions are rather weak acids they are con siderably less acidic than the C terminal carboxyl group How should peptides be stored For long term storage the lyophilizate of the peptide should be kept in the deep freeze at 15 C For short time storage a refrigerator 4 will suffice How does Bachem ship peptides Is dry ice required for the shipment Dry ice is not required for the shipment of peptides Express delivery due to the high stability of the lyo philizates in sealed v
32. k on continue to get your ADS as PDF file Which analytical data do you provide The kind of analytical data accompanying your product depends on the chosen purity range Grade of Purity Data Obtained by HPLC MS Net Peptide Content Determination AAA on request HPLC MS Net Peptide Content Determination AAA HPLC MS Net Peptide Content Determination AAA except for the minimum quantity 2 mg How do you purify the peptide Purification of synthetic peptides is carried out by RP HPLC and in some cases by ion exchange chromatography What does HPLC purity mean The purity determined by HPLC corresponds to the percentage of requested peptide in relation to the total amount of material absorbing at 220 nm i e the desired product peptidic by products and other impurities What does net peptide content mean The net peptide content NPC is the fraction of peptidic material i e the requested peptide and the peptidic im purities relative to counter ions and residual water The latter do not contain nitrogen allowing the determination of the net peptide content by elemental analysis Addi tionally it can be determined by AAA Peptide User Guide 17 18 Peptide User Guide What does gross weight mean The gross weight of a peptide sample comprises the weight of the peptide the salt counter ions and the re sidual water Gross weight Net weight NPC An example 1 g net of peptide NPC
33. l information is compiled to the best of our knowledge We cannot be made liable for any possible errors or misprints Furthermore the terms of sales and delivery of the current main catalog are in force Peptide User Guide 21 Visit us today and let us know what you are looking for www bachem com access to more than 9 000 amino acid derivatives peptides and related immunology products custom synthesis service for peptide synthesis peptide conjugation and custom kit formulation For more information prices and availability please contact your nearest Bachem office www interchim com Winterchim 211 bis Avenue Kennedy BP 1140 Agence Paris Normandie 03103 Montlugon France 33 0 1 41 32 34 40 33 0 4 70 03 88 55 Fax 33 0 1 47 91 23 90 Fax 33 0 4 70 03 82 60 e mail interchim paris interchim com e mail interchim interchim com Published by Bachem Holding AG Hauptstrasse 144 4416 Bubendorf Switzerland July 2009 BRCHETI1
34. ni tored at 220 nm Fractions containing sufficiently pure target peptide as determined by analytical HPLC are pooled and lyophilized If the desired compound can not be obtained sufficiently pure by RP HPLC applying the standard TFA system an appropriate combination of buffer systems will be developed If the C stationary phase is too hydrophobic e g when purifying less polar peptides other column packing materials are selected Peptide User Guide 3 4 Peptide User Guide Quality Control of Peptides Definition of Peptide Purity The purity of the lyophilized target peptide is deter mined by analytical RP HPLC followed by UV detection at 220 nm It is quantitied as area percentage as it cor Absorbance 210 220 nm Impurity visible Ak Retention Time Schematic Analytical RP HPLC Chromatograms responds to the area of the main peak in relation to the total area of all peaks i e all material including the requested peptide which absorbs at this wavelength Amide bonds and other chromophors absorb at 220 nm whereas water and residual salts are not detected UV spectrophotometrically The ability of this method to de tect and quantify impurities eluting in the proximity of the product peak i e an adequate resolution is essential The resolution of analytical HPLC can be improved by ju dicious choice of the buffer system the stationary phase the steepness of the gradient the column temperature and other
35. o acids are hydrophobic In most cases the biological activity of the peptide remains unchanged On the other hand the polarity of the pep tide is slightly increased by the oxidation of the thioether The biological activities of reduced and oxidized peptide often vary interesting effects may be generated by this readily available modification Tyr and especially Trp containing peptides should be protected from intense sunlight as both amino acids are susceptible to photo oxidation Oxidation of the lateral phenol and indole moieties by oxygen radicals is a rather common posHranslational modification of proteins Ad ditionally the indole ring is acid sensitive Asp containing peptides are susceptible to acid catalyzed aspartimide formation The ease of cyclization markedly depends on the nature of the subsequent amino acid In case of Asp Pro the peptide is cleaved The motifs Asp Gly and to a lesser degree Asp Ser are especially prone to aspartimide formation The subsequent hydrolysis of the ring yields a mixture of the B Asp peptide and the na tive sequence The concomitant racemisation of Asp ag gravates the situation Aspartimide formation is equally involved in the base catalyzed deamidation of Asn N terminal Gln shows an extreme tendency to form cyclic pyroglutamate Pyr N terminal acylation will suppress this side reaction When coupling a Pyr derivative instead of Gln a better defined product is obtained The peptide
36. on which could be helpful for your experimental work with our peptides According to the amyloid hypothesis the deposition of amyloid B protein in senile plaques is a primary event in the pathological cascade for Alzheimer s disease AD The amyloid B protein consists of 39 43 amino acid residues derived from proteolysis of a larger B amyloid precursor protein B APP With our present state of knowledge aggregation or aging of the amyloid B protein is an essential requirement for neurotoxicity of amyloid B protein During this aging process the amyloid undergoes conformational conversions from soluble monomeric random coil or a helix conformation into aggregated B sheet structures 1 2 3 Several studies demonstrated that this conformational change within the aggregation process was affected by a number of factors such as the length of the amyloid B peplide 4 solvent hydrophobicity 5 ionic strength 6 7 pH 8 9 10 peptide concentration initial aggregation state 11 12 buffer type peptide counterions e g CF COO vs CI I3 and the presence of partially oxidized or preaggregated forms seeds of the peptide 4 7 Given that it is not astonishing that even the smallest modifications could drastically change the aggregation rate respectively the solubility Solubilization and aggregation of lyophilized amyloid peptides In the following section you will find some examples from literature which describe
37. ong peptides Synthetic strategies comprising stepwise elongation of the peptide may yield a very impure crude product which cannot be purified by standard chromatographic proto cols The chemoselective coupling of unprotected peptide fragments is the essential feature of NCL thus subsequent purification is reduced to removing unreacted fragments The required segments are obtained by SPPS Even the chemical synthesis of small proteins has become feasible at least research quantities 10 20 mg could be obtained employing a combination of stepwise SPPS and chemical ligation The synthesis of proteins by this convergent ap proach is a viable alternative to standard recombinant technologies offering a plethora of additional options However due to the high costs of NCL this technology will not become a routine method for synthesizing long peptides for research purposes As the necessary know how and the required equipment for performing Boc and Fmoc synthe ses are available at Bachem the synthetic strate gy for your peptide can be optimized Bachem has already succeeded in the synthesis of very complex peptides which could not be produced elsewhere at the beginning with aqueous 0 1 TFA then the po larity of the eluent is gradually reduced by continuously increasing the proportion of the less polar modifier ace tonitrile a linear gradient is formed the concentration of TFA is kept constant The elution of material is mo
38. pt sec ondary structures pseudoproline derivatives have been introduced for facilitating SPPS These derivatives are obtained from Ser or Thr which limits their use to the synthesis of peptides containing a Ser or Thr in suitable positions They are introduced as dipeptides Fmoc Xaa wPro OH which will increase the production costs sub stantially Ser or Thr are regenerated during acidolytic cleavage The purity of the crude material will be consid erably higher but the purification will be impeded by the low solubility of the aggregating peptide Methods for obtaining solubilized peptide derivatives withstanding the cleavage from the resin are under investigation Peptide Modifications A choice of modifications of peptides available at Bachem is listed below Acetylation acylation Amidation Biotinylation Conjugation to a carrier protein Incorporation of unusual amino acids Disulfide bridges single and multiple Cyclizations head to tail side chain lactam bridges Phosphorylation Glycosylation e Lipopeptides e Radiolabeling with 5 Labeling with stable isotopes Labeling with fluorophores and chromophores Enzyme substrates and inhibitors e C terminal alcohol and ester groups e Stabilizing modifications PEGylation N methylated derivatives reduced peptide bonds etc Incorporation of a chelating agent DOTA Highly acid sensitive modifications sulfation Boc Acet
39. rt peptides e g enzyme substrates el emental analysis replaces AAA as an additional confir mation of identity The table below presents a compilation of the standard analyses provided by Bachem in relation to the required peptide amount and purity Standard Analytical Methods Available at Bachem Intermediate on request We must emphasize that standard custom peptides are not suitable for human use Peptides intended for use in humans have to be synthesized in a cGMP environment Production under cGMP conditions has to be requested explicitly Bachem is the world leader in the production of cGMP peptides including the required documentation There is no room for compromise at Bachem concerning the quality of our products Our experts from QC will be pleased to answer all your questions concerning your Analytical Data Sheet ADS How to Design Your Custom Peptide When conceiving a peptide sequence for custom synthe sis the feasibility of its synthesis has to be kept in mind A range of factors influences the outcome of a peptide synthesis and the properties of the target peptide includ ing its stability These aspects should be considered be fore definitively placing an order for a custom synthesis Our experts will support your search for an optimal but feasible sequence without additional charge Length of Peptide As the number of potential by products grows with each additional step the purity of the crude p
40. s Peptides tend to be hygroscopic therefore allow the vial to reach ambient temperature in a desiccator prior to opening and weighing out the peptide Adsorption of water reduces the overall peptide content it may de crease stability Weigh out peptides quickly and reseal vial tightly Solubilization of Peptides The reconstitution of a hydrophobic peptide may turn into an almost insurmountable obstacle in the course of your assay As the properties of peptides can vary extraordi narly we cannot offer you a standard protocol for dis solving a peptide We can only offer you a large number of tips and tricks gained from our broad experience in handling these compounds The solubility of a peptide is determined mainly by its po larity The amino acid composition will provide a first in dication before choosing a solvent the sequence should be studied and the number of acidic basic and neutral residues should be determined The side chain functional ities of acidic and basic amino acids will be charged at physiological pH The nature of the N and the C terminal functionality has to be taken into consideration The tral amino acids can be roughly divided into two catego ries non polar i e more or less hydrophobic residues and polar residues Peptides containing a large proportion of hydrophobic amino acids and or polar uncharged amino acids tend to be soluble in organic solvents such as DMSO DMF acetonitrile m
41. s may occur Hence we prefer to give you an approximate delivery time instead of a fixed term Depending on the requested purity and amount of peptide the delivery time varies from 10 days to several weeks Most Frequently Asked Questions Questions Related to the Calculation of Prices Bachem adheres to a competitive price policy based on chart prices for each range of purity For a given purity grade the price will depend on the sequence and the requested quantity What is the relationship between the production costs and the quantity and purity requested The production costs of custom made peptides depend markedly on the requested quantity and purity as de scribed below Costs Increase of purity considerably increases the production costs Purity 100 Costs Increase of quantity per order results in lower cost per unit Quantity Questions Related to Quotation Inquiries and Orders How should the peptide sequence be presented Starting from the N terminal amino acid please use the three letter code as standard An unmodified N terminus is depicted as H not NH a free C terminus as OH nof Modifications required on the lateral chain of amino acids are written in brackets following the abbre viation of the amino acid to be modified If not explicitly denoted otherwise all amino acids are introduced as L enantiomers If you require a more complex peptide or if you have problems in producing an unambig
42. s shown below basic non polar hydrophobic polar uncharged and acidic see also the Periodic Chart of Amino Acids at p 21 for a visual representation Peptides containing a large proportion of basic and acid ic amino acids are readily soluble in aqueous buffers at physiological pH pH 7 whereas a large number of basic residues facilitates the dissolution in acidic solvent systems such as 0 1 aqueous TFA used for preparative chromatography A large proportion of polar amino ac ids will improve the solubility of the peptide as well The insertion of a Pro in the sequence may break a second ary structure or disrupt aggregation Both effects increase the solubility The presence of Pro residues facilitates the SPPS of sequences which would aggregate otherwise 60 20 Bachem scientists are the leading experts for producing research peptides and small proteins Classification of Amino Acids Basic Arg His Lys Non polar hydrophobic Ala Ile Leu Met Phe Pro Trp Val Polar uncharged Asn Cys Gly Gln Ser Thr Tyr Number of Amino Acids Peptide User Guide 7 8 Peptide User Guide A close look at the sequence will allow a rough predic tion of polarity and solubility of the peptide and thus the anticipation of problems during synthesis and purifica tion Difficulties can be expected when synthesizing pep tides containing a large proportion of non polar amino acids Practically insoluble produc
43. s the thiol moieties will be rapidly oxidized at pH 2 7 to di sulfides Neutral or highly hydrophobic peptides containing a high proportion of polar uncharged amino acids and or hydrophobic amino acids should be dissolved in a small amount of an organic solvent such as DMSO DMF ace tic acid acetonitrile methanol propanol or isopropanol and then diluted with water or buffer to the desired con centration Please keep in mind that high concentrations of these solvents are incompatible with biological systems such as cells Peptides prone to aggregation such as the B amyloid fragments require a special treatment Ask Bachem for your technical brochure on han dling and solubilization of B amyloid peptides For peptides containing methionine or free cysteine resi dues the use of DMSO is discouraged as it may oxidize the side chain functionalities Denaturating agents such as urea or guanidinium hydro chloride may be used to solubilize peptides which tend to Peptide User Guide 13 14 Peptide User Guide aggregate As these additives interfere with most biologi cal systems their application is rather limited The reconstitution of a peptide may take time occasion ally up to several hours Sonication for several minutes in a water bath may be helpful to accelerate the dissolution of larger particles However excessive warming of the sample should be avoided Please note that peptides containing Trp Met or Cys res
44. spread application in obesity research Bachem gained consid erable experience in performing this modification during the synthesis of many ghrelin analogs BACHEM Product Monograph FRET Substrates uem c PRET TOSS lle Lys Thr Glu Glu lle Ser Glu Val Asn Leu Asp Ala Glu Phe Acceptor Donor whey lle Lys Thr Glu Glu lle Ser Glu Val Asn Leu OH H Asp Ala Glu Phe i gt A A v Abstract Wi Fluorescence Quenching nergy from an excited The energy transfer is influenced by from each other and the relative Bi FRET Donor and Acceptors Wi Substrates Peptide User Guide 11 12 Peptide User Guide Stabilizing Modifications A range of modifications for prolonging the half life and increasing the metabolic stability of bioactive pep tides can be performed at Bachem including selective PEGylation incorporation of N methylamino acids and generation of pseudo peptide bonds resisting enzymatic cleavage e g reduced peptide bonds CH NH Peptides Containing Chelating Groups Complexes formed between peptides bearing a chelat ing moiety such as DOTA or DTPA and radionuclides are increasingly used as imaging agents or for radionuclide delivery The derivatives of the chelators required for the coupling with the peptide are synthesized in house Acid Sensitive Modifications Bachem s chemists are able to fine tune their synthetic tactics so precisely that even highly acid sensitive pep t
45. t be used in biological assays even if the assay could be conducted employing a low purity product The material obtained after cleavage from the resin and precipitation still may contain a range of harmful non peptidic impurities e g small amounts of scavengers Fortunately peptides which were purified by standard procedures and lyophilized will contain only traces in the ppm range of cytotoxic non peptidic con taminants such as residual solvents and scavengers from cleavage Only TFA cannot be removed completely due to salt formation If residual TFA may pose a problem we recommend ordering a more biocompatible salt form of the active peptide However as an additional ion exchange step will be re quired the price of the custom peptide will have to be adjusted Recommended Peptide Purity for Varying Applications Four standard product grades are offered by Bachem but intermediate purity ranges can be provided on de mand The lower the level of purity the lower the price will be The correlation between purity and price is not linear efforts and costs for obtaining very pure peptides 97 99 may increase exponentially Applications e NMR studies Crystallography studies e Peptides used as reference in final gt 97 quantitative studies Enzyme substrate studies Receptor ligand interaction studies Blocking and competition assays Production of monoclonal antibodies e Enzyme substrate studies quantitative e
46. tides lacking at least one of the required amino acids incompletely deprotected sequences truncated peptides by products formed during peptide synthesis or under the conditions of cleavage Except for TFA all potentially cytotoxic reagents used in the course of the synthesis should have been removed by the washings preceding the final cleavage or during the purification process Traces of residual solvents can be determined by gas chromatography GC if required Batch to batch Variability of Peptides The purity of a peptide i e the proportion of desired product can vary from batch to batch When a peptide is ordered at 80 purity the quality of the product can range between 80 and 100 The lower the requested purity the broader the observed variability between two lots Hence results obtained from quantitative assays could vary unpredictably depending on the quality of the particular batch Batches of low purity contain a consid erable number of peptidic by products Proportion and structure of these contaminants will vary from batch to batch Peptidic impurities may show biological activity as well but not necessarily the activity of the target peptide In the worst case they may interfere with the assay The NPC can vary as well It is influenced by the polarity of the peptide the conditions of lyophilization the condi tions and duration of storage contact with humidity and many other parameters Crude peptides should no
47. ts may result which cannot be purified The conservation of the biological activity limits solubiliz ing modifications to a peptide But merely a minor reduc tion of length or the incorporation of charged residues at the termini may help to avoid the predicted difficulties This aspect has to be kept in mind when selecting partial sequences of a protein for custom synthesis Amino Acids Prone to Undergo Side Reactions Amino Acids Sensitive to Oxidation Met Trp and in particular free Cys are susceptible to oxidation Hence peptides containing these amino acids have to be handled with appropriate care They should be dissolved only in carefully degassed solvents The oxidation of a Cys containing peptide yields a di sulfide bridge i e a cystine peptide Met is converted into a sulfoxide Both transformations are reversible Inter and intramolecular disulfide bonds between the Cys thiol groups are formed very rapidly at pH gt 7 The bridging may be reversed by treatment with reducing agents such as dithiothreitol DTT Hence peptides containing free cysteine residues should be dissolved in buffer systems including a reductant If a cysteine is not absolutely re quired for the biological activity it can be replaced by the hydrophobic isoster Abu o aminobutyric acid or by the polar Ser The latter may participate in reactions of the native peptide Met can be replaced by the inert isosteric norleucine Nle residue Both amin
48. type of C terminus of the desired peptide Protecting Groups Two categories of protecting groups are required for synthesizing peptides groups allowing temporary pro tection of the o amino group and permanent pro tecting groups blocking the side chain functionalities of the amino acids The latter groups have to withstand the conditions of the repetitive cleavages of the temporary protecting group usually they are removed only during the cleavage from the carrier resin Untimely removal of protecting groups is a common cause for the formation of by products The best strategy to avoid this risk consists of introducing temporary and permanent protecting groups which can be removed by differing chemical mecha nisms i e orthogonal protection Truly orthogonal pro tecting groups may be split off with absolute selectivity and in any order The classical Boc Bzl strategy does not fulfill this requirement as both groups are cleaved with acid However their acid lability differs sufficient ly to afford selective removal of the o amino protection The combination Boc Bzl may be called quasi orthogo nal The pairing Fmoc tBu on the other hand is truly orthogonal The temporary o amino group is deblocked with base piperidine Thus TFA dabile and simultane ously base stable groups as tBu and Boc in combination with a TFA labile anchor are the perfect choice for side chain protection Orthogonal protection schemes permit milder o
49. uous presentation of your required structure we are pleased to help you Examples N terminal acetylation A peptide amide Xaa Yaa NH A peptide carrying a biotinyl residue attached to the Lys side chain Xaa Lys biotinyl Yaa A head to tail cyclized peptide Xaa Yaa A peptide containing a D amino acid Xaa D Yaa A peptide containing an N methylated amino acid Xaa IN Me Yaa or Xaa MeYaa If an unambiguous abbreviation for a moiety is lacking please give out the full name e g chloroacetyl What do on N terminus OH on C terminus signify H signifies a free N terminus i e NH or NH Pro OH denotes a free C terminus a carboxylic acid please do not omit the hyphens The three letter code of an amino acid stands for NH CHR CO i e H Xaa OH stands for H NH CHR CO OH IF the N terminus has to be acetylated the H should be replaced by Ac When the C terminus is amidated the OH has to be re placed by NH Replacement by signifies a C terminal aldehyde What kind of information should be given Additional information concerning the purity and quan tity you require is most important Further material such as literature references would be highly welcome especially if you are requesting the syn thesis of an unusual peptide Should we specify the requested salt form IF not specified otherwise custom peptides are delivered as TFA salts
50. verall reaction conditions as well as the synthe sis of partly protected or side chain modified peptides Fmoc tBu or Boc Bzl Strategy The Boc Bzl strategy can be traced back to the begin nings of SPPS Merrifield s pioneering work This method ology requires anchoring groups which tolerate repeti tive TFA treatment HX lt Tinker P coupling of Fmoc AA PG OH o TOCO HM further coupling and deprotection steps BE 6569 09 ORCA trifluoroacetic acid TFA peptide General Scheme of Fmoc SPPS X NH AA Amino Acid PG Protecting Group TFA labile not all amino acids require side chain protection Usually the inorganic acid HF is employed for the fi nal cleavage which limits the batch size in this step and the choice of reactor Even though many remark able synthetic successes employing Boc Bzltechnol ogy are recorded in the literature the development of orthogonal protection schemes increased the flex ibility of the solid phase method The Fmoc tBu strat egy see scheme is the most popular amongst them It can be automated far more conveniently than the Boc Bzl strategy and it can be scaled as needed Additional levels of orthogonality allow the synthesis of highly com plex peptides Nevertheless depending on the sequence the Boc Bzl strategy still can remain a viable alternative Routine synthesis requires special equipment Base labile peptides difficult sequences
51. wo SPPS strategies have been developed which are now considered standard procedure the Boc Bzl and the Fmoc tBu protection scheme They differ in the types of protecting groups used for the free o amino group and the side chain functionalities Boc signifies t butyloxycar bonyl Bzl benzyl tBu tert butyl and Fmoc stands for 9 fluorenylmethyloxycarbonyl What is the maximum peptide length feasible by chemi cal synthesis Due to the continuing improvement of the methodology the maximum peptide length accessible by SPPS was steadily increased It depends on the peptide sequence Peptide comprising approximately 50 amino acids can be considered as the upper limit in routine SPPS By adapting the synthetic protocols Bachem could obtain nu merous peptides consisting of about 100 residues Such molecules can be regarded as small proteins How many free cysteines in a peptide are acceptable 4 to 5 free thiol moieties depending on the amino acid composition of the peptide is our upper limit A replace ment of Cys residues by less sensitive isosteric serines should always be considered as these stable analogs will be obtained in higher purity Peptides containing a single free thiol group may be oxidized yielding dimers cyclic peptides or oligomers may be obtained from pep tides containing several Cys residues Hence we have to lower the purity on offer How many disulfide bridges can be obtained The maximal number of disulfide bridges
52. y Grades for Varying Applications Peptide Analysis How to Design Your Custom Peptide Length of Peptide Polarity Amino Acids Prone to Undergo Side Reactions B Sheet Formation Peptide Modifications Care and Handling of Peptides Handling of Lyophilized Peptides Solubilization of Peptides Storage of Peptides in Solution Delivery Time Most Frequently Asked Questions Questions Related to the Calculation of Prices Questions Related to Quotation Inquiries and Orders Questions Related to the Synthesis Questions Related to Purity and Analytical Methods Questions Related to Handling and Storage Conclusion Abbreviations A ONNNN N Cn CO CONN N 13 13 14 14 15 13 15 16 17 18 19 20 Peptide User Guide 1 2 Peptide User Guide Peptide Synthesis Peptides can be obtained chemically by classical solu tion synthesis by solid phase peptide synthesis SPPS or by a combination of both methods which can in volve native chemical ligation Normally at Bachem the synthesis of peptides is carried out on solid phase whereas the classical approach is chosen for synthe sizing di and tripeptides and occasionally C termi nally modified peptides such as enzyme substrates In the following paragraphs we will discuss the solid phase approach in more detail as this methodology is of utmost importance for the synthesis of peptides The Principle of Solid Phase Peptide Synthesis
53. y liquid phase hy drolysis of the peptide with constant boiling hydrochloric acid 6N HCl followed by pre column derivatization of the free amino acids by AccQ Fluor The derivatized amino acids are separated by RP HPLC using a C 4 um column Integration of the individual peaks allows the determination of the amino acid composition of the pep tide hydrolysate However the indole moiety of Trp is destroyed during hydrolysis the side chain amide groups of Asn and Gln and the lactam cycle of Pyr will be hy drolyzed yielding Asp and Glu respectively Hence the AAA lists these amino acids as Asx and Glx Questions Related to Handling and Storage How to dissolve a peptide Please refer to the section Solubilization of Peptides and see our Technical Note Solubilization of Peptides 7 which can be downloaded from our website BACHE Technical Notes Solubilization of Peptides The solubility of a peptide is primarily dependent on the physical properties of its amino acids Amino acids can be classified as acidic basic polar uncharged or nonpolar hydrophobic Peptides with a high content of nonpolar hydrophobic amino acids or polar uncharged amino acids are preferentially solubilized in organic solvents such as DMF methanol propanol isopropanol or DMSO whereas overall acidic peptides can normally be reconstituted in basic solvents and overall basic peptides in acidic solvents Families of Amino Acids B
54. ylation amidation and biotinylation are most fre quently requested by our customers As these modifications only minimally increase our input they can be offered as routine operations which will not be charged additionally If you are interested in a modifi cation not mentioned in this compilation please inquire Conjugation to a Carrier Protein Three standard proteins are used at Bachem as carriers for peptides KLH BSA and OVA Usually the peptide is coupled to the protein either via its N terminus or via the SH functionality of a cysteine Conjugation via a thiol group is the preferred method as it is highly selective Thus an additional Cys is coupled either to the C termi nus or more practically after completion of the SPPS if the desired peptide lacks this amino acid The addition of an N terminal Cys allows obtaining the Cys derivative re quired for the conjugation together with the peptide lack ing the Cys for binding assays from the same batch Bachem has an excellent reputation for synthesizing peptides containing multiple disulfide bridges Bachem offers the largest range of special amino acids available for custom peptide syn thesis Do not hesitate to ask for your poster Periodic Chart of Unusual o Amino Acids Unusual Amino Acids o Amino acid bearing unusual side chain functionalities and turn mimetics are useful tools for peptide design Bachem offers an exceptional choice of unusual amino
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