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    Overview of Custom Peptide Synthesis


    Proteins are present in every living cell and possess a variety of biochemical activities. They appear as enzymes, hormones, antibiotics, and receptors. They compose a major portion of muscle, hair, and skin. Consequently, scientists have been very interested in synthesizing them in the laboratory. This interest has developed into a major synthetic field known as Peptide Synthesis. The major objectives in this field are four-fold:

    1. To verify the structure of naturally occurring peptides as determined by degradation techniques.
    2. To study the relationship between structure and activity of biologically active protein and peptides and establish their molecular mechanisms.
    3. To synthesize peptides that are of medical importance such as hormones and vaccines.
    4. To develop new peptide-based immunogens.

    Solid Phase Peptide Synthesis (SPPS)

    The fundamental premise of this technique involves the incorporation of N-a-amino acids into a peptide of any desired sequence with one end of the sequence remaining attached to a solid support matrix. While the peptide is being synthesized usually by stepwise methods, all soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away at the end of each coupling step. After the desired sequence of amino acids has been obtained, the peptide can be removed from the polymeric support.

    The general scheme for solid phase peptide synthesis is outlined in Figure 1. The solid support is a synthetic polymer that bears reactive groups such as -OH. These groups are made so that they can react easily with the carboxyl group of an N-a-protected amino acid, thereby covalently binding it to the polymer. The amino protecting group (X) can then be removed and a second N-a-protected amino acid can be coupled to the attached amino acid. These steps are repeated until the desired sequence is obtained. At the end of the synthesis, a different reagent is applied to cleave the bond between the C-terminal amino acid and the polymer support; the peptide then goes into solution and can be obtained from the solution.


    Fmoc Strategy in SPPS

    The crucial link in any polypeptide chain is the amide bond, which is formed by the condensation of an amine group of one amino acid and a carboxyl group of another. Generally, an amino acid consists of a central carbon atom (called the a-carbon) that is attached to four other groups: a hydrogen, an amino group, a carboxyl group, and a side chain group. The side chain group, designated R, defines the different structures of amino acids. Certain side chains contain functional groups that can interfere with the formation of the amide bond. Therefore, it is important to mask the functional groups of the amino acid side chain.

    The general scheme which outlines the strategy of Fmoc synthesis is shown in Figure 2. Initially, the first Fmoc amino acid is attached to an insoluble support resin via an acid labile linker. Deprotection of Fmoc, is accomplished by treatment of the amino acid with a base, usually piperidine. The second Fmoc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via TFA cleavage.

    Fmoc Cleavage

    The removal of peptides in solid phase peptide synthesis is primarily done by acidolysis. The Fmoc chemistry employs the use of weak acids such as TFA or TMSBr. Various scavengers are included to protect the peptide from carbocations generated during cleavage which can lead to side reactions. These additives usually include thiol compounds, phenol, and water.

    The following protecting groups are compatible with TFA and TMSBr cleavage:

    Arg(Boc)2 Cys(Acm) Lys(Boc)
    Arg(Mtr) Cys(Trt) Lys(Fmoc)
    Arg(Pbf) Gln(Tmob) Lys(Mtt)
    Arg(Pmc) Gln(Trt) Ser(tBu)
    Asn(Tmob) Glu(OtBu) Thr(tBu)
    Asn(Trt) His(Boc) Tyr(tBu)
    Asp(OtBu) His(Trt)

    Depending on the type of protecting groups present, certain combinations of scavengers must be used. For instance, when either Boc and t-Butyl groups are present, their carbocation counterparts (t-butyl cations and t-butyltrifluoroacetate) can react with Trp, Tyr, and Met to form their t-butyl derivatives. While EDT is a very efficient scavenger for t-butyl trifluoroacetate, it does not protect Trp from t-butylation. Therefore, water must be added in order to suppress alkylation. The indole ring of Trp and the hydroxyl group of Tyr are especially susceptible to the reactivity of the cleaved Pmc group. Again, water has been shown to be effective in suppressing this reaction. Similar occurrences can happen with the Trt and Mtr groups. Therefore, scavengers in the appropriate combination will greatly reduce the amount of side reactions.

    Boc Strategy in SPPS

    The general scheme which outlines the strategy of Boc synthesis is shown in Figure 3. Initially, the first Boc amino acid is attached to an insoluble support resin via a HF cleavable linker. Deprotection of Boc, is accomplished by treatment of the amino acid with TFA. The second Boc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via HF cleavage.

    Boc Cleavage

    The Boc chemistry employs the use of strong acids such as HF, TFMSOTf, or TMSOTf. Various additives, usually thiol compounds are added to protect the peptide from the carbocations generated during cleavage.

    The following protecting groups are compatible with HF cleavage:

    Arg(Mts) Cys(4-MeOBzl) His(Z)
    Arg(Tos) Glu(OBzl) Lys(Cl-Z)
    Asp(OBzl) Glu(OcHex) Ser(Bzl)
    Asp(OcHex) His(Bom) Thr(Bzl)
    Cys(Acm) His(Dnp) Trp(CHO)
    Cys(4-MeBzl) His(Tos) Tyr(Br-Z)
    Asp(OtBu) His(Trt)

    The following protecting groups are compatible with TFMSOTf cleavage:

    Arg(Mts) His(Bom) Met(O)
    Asp(OBzl) His(Dnp)) Ser(Bzl)
    Cys(Acm) His(Tos) Thr(Bzl)
    Cys(4-MeBzl) His(Z) Trp(CHO)
    Glu(OBzl) Lys(Cl-Z) Tyr(Br-Z)

    The following protecting groups are compatible with TMSOTf cleavage:

    Arg(Mts) Glu(OcHex) Trp(CHO)
    Arg(Mbs) His(Bom) Trp(Mts)
    Asp(OBzl) Lys(Cl-Z) Tyr(Br-Z)
    Asp(OcHex) Met(O) Tyr(Bzl)
    Cys(Acm) Ser(Bzl) Tyr(Cl-Bzl)
    His(Bom) Thr(Bzl)


    General Coupling Methods in SPPS

    Coupling reactions in SPPS require the acylation reactions to be highly efficient to yield high-purity peptides.

    Coupling Methods in Fmoc SPPS

    The most widely used coupling method in Fmoc SPPS is the activated ester method either pre-formed (pre-activated species) or in situ (without pre-activation). Initially, the p-nitrophenyl and N-hydroxysuccinimide (ONSu) activated esters were the predominantly used forms (1-2). However, even in the presence of HOBt, the coupling reactions tended to be slow. In addition, ONSu esters of Fmoc amino acids were prone to the formation of the side product succinimido-carbonyl-b-alanine-N-hydroxysuccinimide ester (3-4). The most commonly used activated esters presently are the pentafluorophenyl (OPfp) ester and the 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester (5-7). In the presence of HOBt, the rate of reaction is very rapid and the reaction is efficient with minimal side product formation. On the other hand, many coupling reactions can be done in situ using activating reagents such as DCC, HBTU, TBTU, BOP, or BOP-Cl. The direct addition of carbodiimide is considered to be the best choice (8-13). HBTU and TBTU would rank second, followed by BOP and finally BOP-Cl. With regards to ester coupling, the following order was found: BOP/HOBt > carbodiimide/HOBt ~ carbodiimide/ODhbT > DCC/OPfp (14-15).

    More recently, 1-hydroxy-7-azabenzotriazole (HOAt) and its corresponding uronium salt analog O-(7-azabenzotrizol-1-yl)-1,1,3,3, tetra-methyluronium hexafluorophosphate (HATU) have been developed and found to have a greater catalytic activity than their HOBt and HBTU counterparts. The use of HOAt and HATU enhances coupling yields, shortens coupling times, and reduces racemization. Consequently, these reagents are suitable for the coupling of sterically hindered amino acids, thereby ensuring greater success in the synthesis of difficult peptides (16-17).

    Coupling Methods in Boc SPPS

    The carbodiimides, primarily DCC, were the coupling reagents of choice for many years (18). The major drawbacks encountered were the precipitation of dicyclohexylurea during the activation and acylation processes and the numerous side reactions associated with its usage. Several carbodiimides which produced soluble ureas were developed, such as diisopropylcarbodiimide (DIC), t-butyl methyl- and t-butylethyl-carbodiimides (19-22), but these did not resolve the problem of side reactions. Consequently, new types of activating agents were developed. The first of these was BOP (23), PyBroP (24-25) PyBOP (26), HBTU (27), TBTU (28), and HATU (29). All of these reagents require bases for activation.

    All of the DCC and DCC-related derivatives discussed previously work by the formation of the symmetrical anhydride. The symmetrical anhydrides are usually very reactive and have been used extensively in SPPS, especially in Boc synthesis (30-33). Attempts at incorporating symmetrical anhydrides to Fmoc amino acids were met with some difficulties (34-36). For instance, symmetrical anhydrides prepared from N-(3-dimethylamino propyl)-N'-ethyl-carbodiimide•HCl, upon formation of the 2-alkoxy-5(4H)-oxazolone intermediate, rearranged in the presence of carbodiimides and tertiary amines (37). Also, not all of the Fmoc symmetrical anhydrides are soluble in DCM or remain insoluble regardless of the solvent used (38).

    An alternative to the symmetrical anhydride is the mixed anhydride which is a carboxylic-carbonate or carboxylic-phosphinic mixed anhydride. Typically, these anhydrides are prepared by reacting either isobutyl- or isopropyl-chloroformate and substituted phosphinic chlorides with the N-a-protected amino acid (39-42). The reaction is typically rapid with little or no side reactions (43-46).

    A type of mixed anhydride, N-carboxyanhydrides (NCA's), also known as Leuchs' anhydride have been widely used for the preparation of polyamino acids (47). This class of compounds combines N-a-protection with carboxyl group activation. Once reacted with another amino acid or peptide residue, the NCA releases carbon dioxide as its only by-product. NCA derivatives are easily prepared by treating a-amino acids with phosgene (48-51). The resulting NCA derivatives usually crystallize out and are ready for use under strictly defined conditions. These conditions require the pH to be carefully controlled during synthesis. At pH < 10, the peptide-carbamate (produced by the reaction between the NCA and the peptide or amino acid residue) tends to lose carbon dioxide with the generation of a free a-amino end group with resulting polymerization. At pH 10.5, hydrolytic decomposition of the NCA occurs. Therefore, the reaction is performed at pH 10.2. Another required condition is that the reaction proceeds for 2 minutes at 0C with vigorous stirring. The resulting product is free of racemization and bears a free a-amino group that can be extended by addition of another anhydride.

    Solution Phase Synthesis

    Stepwise condensation is based on the repetitive addition of single N-a-protected amino acids to a growing amino component, generally starting from the C-terminal amino acid of the chain to be synthesized. The process of coupling individual amino acids can be accomplished through employment of the carbodiimide (52-53), the mixed carbonic anhydride (54-55), or the N-carboxyanhydride methods (56-57). The carbodiimide method involves coupling N- and C- protected amino acids by using DCC as the coupling reagent. Essentially, this coupling reagent promotes dehydration between the free carboxyl group of an N-protected amino acid and the free amino group of the C-protected amino acid, resulting in the formation of an amide bond with precipitation of the by- product, N,N'-dicylcohexylurea. This method, however, is hampered by side reactions which can result in racemization (58-59) or in the presence of a strong base, the formation of 5(4H)-oxazolones (60) and N-acylureas (61). Fortunately, these side reactions can be minimized, if not altogether eliminated, by adding a coupling catalyst such as N-hydroxysuccinimide (HOSu) or 1-hydroxybenzotriazole (HOBt). In addition, this method can be employed to synthesize the active ester derivatives of N-protected amino acids (62). In turn, the resulting activated ester will react spontaneously with any other C-protected amino acid or peptide to form a new peptide.

    In cases where separation of the activated ester from the by-product dicyclohexylurea proves to be difficult, the mixed carbonic anhydride method can be employed. This method consists of two stages: the first stage involves activating the carboxyl group of an N-a-protected amino acid with an appropriate alkyl chlorocarbonate, such as ethylchlorocarbonate (63), or preferably isobutylchlorocarbonate (64). Activation occurs in an organic solvent in the presence of a tertiary base. The second stage involves reacting the carbonic anhydride with a free amine component of an amino acid or a peptide unit. The carbonic anhydride is usually added at a 14-fold excess over the amine component.

    The mixed carbonic anhydride method is noted for being highly effective at low temperatures, resulting in high yields and pure products. However, it does have its short-comings. For instance, there is a tendency for the anhydride derivative to undergo racemization as a result of the strong activation of the carbonyl group. This problem does not occur when N-a-urethane protecting groups, such as Cbz or t-Boc, are employed (65-66). Furthermore, as a result of their high reactivity, mixed carbonic anhydrides are prone to the formation of 5(4H)-oxazolones (67), urethanes (68-69), diacyimides (70-71), esters (72), and are subject to disproportion (73-74). Conditions which prompt such side reactions to occur are high temperatures, prolonged activation times (the time interval between the addition to the alkylchlorocarbonate and the amine component after the mixed anhydride is formed), steric bulk of the amine component, and incomplete formation of the mixed anhydride. Fortunately, most of these side reactions, except for oxazalone and urethane formation, can be substantially reduced by performing the reaction at low temperature (~ -15C) and allowing for shorter activation times (~ 1-2 min). To minimize the formation of oxazolone and urethane derivatives, the following conditions must be implemented: 1) dried organic solvents such as ethyl acetate, tetrahydrofuran, t-butanol, or acetonitrile must be used (75); 2) the tertiary base, N-methylmorpholine, should be used (76); and 3) Cbz- or Boc-N-a-protected amino acids must be utilized (77).

    Although isobutyl- and ethylchlorocarbonate are typically used to form carbonic anhydrides, other coupling reagents do exist. For example, N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline (EEDQ) (78) and N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ) (79) were developed to react with the carboxyl component to form the ethyl- or isobutylcarbonate derivative. Unlike the classical anhydride procedure, EEDQ and IIDQ do not require base nor low reaction temperatures. Typically, the procedure involves reacting equimolar amounts of the carboxyl and amine components in an organic solvent (a wide variety of solvents can be used) (80) at 0.1 M to 0.4 M concentrations. Then EEDQ or IIDQ is added in 5-10% excess and the mixture is allowed to stir for 15-24 hours at room temperature. After removal of the solvent, in vacuo, the residue is dissolved in ethyl acetate and washed with 1N NaHCO3, 10% citric acid, and salt water, then dried with Na2SO4 (anhydrous), and evaporated. The product can then be recrystallized or purified by chromatography. While this method circumvents the use of base, it is still subject to racemization and urethane side product formation at levels comparable to those found in the classical anhydride approach. Consequently, its only advantage may be that it is easy and convenient to use. It should be noted that a detailed comparison of the two methods has not been carried out to this date.

    HPLC Analysis and Purification (81-84)

    Analytical HPLC utilizes columns and pumping systems that can withstand and deliver very high pressures enabling the use of very fine particles (3-10 microns) as packing material. Consequently, peptides can be resolved with a high degree of resolution in a short time interval (i.e., minutes).

    Two common HPLC purification methods are, ion exchange and reverse phase. Ion exchange HPLC is based on direct charge interactions between the peptide and the stationary phase. The column support is derivatized with an ionic species that maintains a particular charge over a certain pH range, while the peptide or peptide mixture exhibits an opposite charge which is dependent on its amino acid composition. Separation is dependent on charge interactions. The peptide is eluted by changing the pH, the ionic strength, or both. Typically, a solution of low ionic strength is used; the ionic strength of the solution is then gradually or step-wise increased until the peptide is eluted from the column. One example of ion exchange separation incorporates the use of strong cation exchange columns such as sulfoethylaspartimide which separates on the basis of positive charge at an acidic pH.

    Reverse phase HPLC conditions are essentially the reverse of normal phase chromotography. The peptide binds on the column through hydrophobic interactions and is eluted by decreasing the ionic strength (i.e., increasing the hydrophobicity of the eluent). Generally, the column supports are composed of hydrocarbon alkane chains which are covalently attached to silica. These chains range from C4 to C18 carbon atoms in length. Since elution from the column is a function of the hydrophobicity, the longer chain hydrocarbon columns are better for small, highly charged peptides. On the other hand, large hydrophobic peptides elute better using short chain hydrocarbon supports. However, in general practice, these two types of columns can be used interchangeably with little significant differences. Other types of supports consist of aromatic hydrocarbons such as phenyl groups.

    A typical run usually consist of two buffers, 0.1% TFA-H2O and 80% acetonitrile/0.1% TFA-H2O, which are mixed using a linear gradient with a flow rate which will give a 0.5% to 1.0% change per minute. Typical columns for analytical and purification runs are 4.6 x 250 mm (3-10 microns) and 22 x 250 mm (10 microns), respectively. If radial packed columns are used, then column sizes are 8 x 100 mm (3-10 microns) and 25 x 100 mm (10 microns), respectively. A variety of other buffers can contain many different types of reagents such as 0.1% heptafluorobutyric acid, 0.1% phosphoric acid, dilute HCl, formic acid (5-60%, pH 2-4), 10-100 mM NH4HCO3, sodium/ammonium acetate, TFA/TEA, sodium or potassium phosphate, or triethylammonium phosphate (pH 4-8). In addition, water miscible eluents can also be added such as methanol, propanol, and isopropanol. Therefore, many combinations of solvents and additives for a buffer are possible. It should be noted that silica-based reverse phase column packing must not be exposed to high pH's or even slightly basic pH's for extended periods of time because the column can be destroyed at those pH levels.

    The crude peptide obtained from SPPS will contain many by-products which are a result of deletion or truncated peptides as well as side products stemming from cleaved side chains or oxidation during the cleavage and deprotection process. Earlier purification methods included ion exchange, partition, and counter current chromatography. Recent purification methods include reverse phase HPLC which is generally successful with peptides containing 60 residues or less. In conjunction, ion exchange HPLC can be used in cases where reverse phase HPLC does not work.

    Typically, analytical HPLC results are used to determine the purification conditions. For example, if a peptide elutes out at 30% (0.1% TFA) aqueous acetonitrile (determined by analytical HPLC analysis), a buffer containing a lower concentration of acetonitrile is chosen such that the peptide peak will come out 4-5 minutes after the solvent peak under isocratic conditions (e.g., 28% (0.1% TFA) aqueous acetonitrile). The purification conditions will entail using a linear gradient of 16-35% (0.1% TFA) aqueous acetonitrile over one or two hours depending on the type of column chosen. The collected fractions will then be checked by analytical HPLC, using the buffer chosen for isocractic conditions.

    Handling and Storage of Peptides

    Peptides have widely varying solubility properties. The main problem associated with the dissolution of a peptide is secondary structure formation. This formation is likely to occur with all but the shortest of peptides and is even more pronounced in peptides containing multiple hydrophobic amino acid residues. Secondary structure formation can be promoted by salts. It is recommended first to dissolve the peptide in sterile distilled or deionized water. Sonication can be applied if necessary to increase the rate of dissolution. If the peptide is still insoluble, addition of a small amount of dilute (approximately 10%) acetic acid (for basic peptides) or aqueous ammonia (for acidic peptides) can facilitate dissolution of the peptide.

    For long-term storage of peptides, lyophilization is highly recommended. Lyophilized peptides can be stored for years at temperatures of -20C or lower with little or no degradation. Peptides in solution are much less stable. Peptides are susceptible to degradation by bacteria so they should be dissolved in sterile, purified water.

    Peptides containing methionine, cysteine, or tryptophan residues can have limited storage time in solution due to oxidation. These peptides should be dissolved in oxygen-free solvents. To prevent the damage caused by repeated freezing and thawing of peptides, dissolving the amount needed for the immediate experiment and storing the remaining peptide in solid form is recommended.


    1. Atherton, E. et al. J. Chem. Soc. Perkin Trans. I, 538-546 (1981).
    2. Fields, G. B. et al. PNAS USA 85, 1384-1388 (1988).
    3. Atherton, E. and Sheppard, R. C. J. Chem. Soc. Chem. Commun., 165-166 (1985).
    4. Gross, H. and Bilk, L. Tetrahedron 24, 6935-6939 (1968).
    5. Kisfaludy, L. and Schon, I. Synthesis, 325-327 (1983).
    6. Snow, J. T. Peptides: The Drugs of the Future and Derivatized Amino Acids for Synthesis, Calbiochem Corporation, Rockford, IL (1988).
    7. Konig, W. and Geiger, R. Chem. Ber. 103, 2034-2040 (1970).
    8. Merrifield, R. B. et al. Anal. Biochem. 174, 399-414 (1988).
    9. Knorr, R. et al. Tetrahedron Lett. 30, 1927-1930 (1989).
    10. Fournier, A. et al. Int. J. Peptide Protein Res. 33, 133-139 (1989).
    11. Diago-Meseguer, J. et al. Synthesis, 547-551 (1980).
    12. Knorr, R. et al. Peptides 1990, Giralt, E. and Andreu, D., Eds., The Netherlands, Escom, Leiden, 62-64 (1991).
    13. Gausepohl, H. et al. Peptides: Chemistry and Biology, Smith, J. A. and Rivier, J. E., Eds., The Netherlands, Escom, Leiden (1991).
    14. Hudson, D. J. Org. Chem. 5, 617-624 (1988).
    15. Merrifield, R. B. et al. Anal. Biochem. 174, 399-414 (1988).
    16. Carpino, L. A. J. Amer. Chem. Soc. 115, 4397 (1993).
    17. Carpino, L. A. et al. J. Chem. Soc. Commun., 201, (1994).
    18. Barany, G. and Merrifield, R. B., The Peptides, Gross,E. and Meienhofer, J. Eds., Academic Press, New York, 2, 1-284 (1979).
    19. Sarantakis, D. et al. Biochem. Biophys. Res. Commun. 73, 336-342 (1976).
    20. Hudson, D. et al. Peptide Chemistry 1985, Kiso, Y., Ed., Protein Research Foundation, Osaka, 413-418 (1986).
    21. Izdebski, J. and Pelka, J. Peptides 1984, Ragnarsson, U., Ed., Almqvist & Wiksell International, Stockholm, 113-116 (1984).
    22. Izdebski, J. et al. Int. J. Peptide Protein Res. 33, 77-81 (1989).
    23. Castro, B. et al. Peptides 1976, Loffet, A., Ed., Editions de l'Universite de Bruxelles, Belgium, 79-84 (1976).
    24. Coste, J. et al. Peptides: Synthesis, Stucture, and Function, Rivier, J. and Marshall, G. R., Eds., Escom, Leiden (1989).
    25. Castro, B. et al. The Eleventh American Peptide Symposium Abstracts, P130 (1989).
    26. Coste, J. et al. Tetrahedron Lett. 31, 205-208 (1990).
    27. Knorr, R. et al. Peptides 1988, Jung, G. and Bayer, E., Eds., Walter de Gruyter & Company, Berlin, 37-39 (1989).
    28. Gausepohl, H. et al. Peptides: Chemistry and Biology, Smith, J. A. and Rivier, J. E., Eds., The Netherlands, Escom, Leiden (1991).
    29. Carpino, L. A. J. Amer. Chem. Soc. 115, 4397 (1993).
    30. Hagenmaier, H. and Frank, H., Hoppe-Seylers Z. Physiol. Chem. 353, 1973-1976 (1972).
    31. Yamashiro, D. et al. J. Org. Chem. 38, 3561-3565 (1973).
    32. Noble, R. L. et al. J. Amer. Chem. Soc. 98, 2324-2328 (1976).
    33. Noble, R. L. et al. Int. J. Peptide Protein Res. 10, 385-393 (1977).
    34. Liu, Y. Z. and Meienhofer, J. Peptides: Structure and Function, Deber, C. M., Hruby, V. J., and Kopple, K. D., Eds., Pierce Chemical Co., Rockford, IL, 281-284 (1985).
    35. Brown, E., et al. J. Chem. Soc. Perkin Trans. I, 75-82 (1983).
    36. Atherton, E. et al. J. Chem. Soc. Commun., 537-539, 539-540 (1978).
    37. Benioton, N. L. and Chen, F. M. F. J. Chem. Soc. Chem. Commun., 1225-1227 (1981).
    38. Harrison, J. L. et al. Techniques in Protein Chemistry, Hugli, T. E., Ed., Academic Press, San Diego, 505-516 (1989).
    39. Chaturvedi, N. et al. Chemical Synthesis and Sequencing of Peptides and Proteins, Lui, T. Y., Schechter, A. N., Heinrikson, R. L., and Condliffe, P. G., Eds., Elsevier North Holland, Amsterdam, 169-177 (1981).
    40. Fuller, W. D. et al. Peptides: Synthesis, Structure, Function, Rich, D. H. and Gross, E., Eds., Pierce Chemical Company, Rockford, IL, 210-204 (1981).
    41. Chen, F. M. F. and Benoiton, N. L. Can. J. Chem. 65, 619-625 (1987).
    42. Benoiton, N. L. and Chen, F. M. F. Peptides: Chemistry and Biology, Marshall, G. R., Ed., Ecsom, Leiden, 152-156 (1988).
    43. Chaturvedi, N. et al. Chemical Synthesis and Sequencing of Peptides and Proteins, Lui, T. Y., Schechter, A. N., Heinrikson, R. L., and Condliffe, P. G., Eds., Elsevier North Holland, Amsterdam, 169-177 (1981).
    44. Fuller, W. D. et al. Peptides: Synthesis, Structure, Function, Rich, D. H. and Gross,E., Eds., Pierce Chemical Company, Rockford, IL, 210-204 (1981).
    45. Benoiton, N. L. et al. Int. J. Peptide Protein Res. 31, 577-580 (1988).
    46. Ramage, R. et al. J. Chem. Soc. Perkin Trans. I, 1617-1622 (1985).
    47. Katchalski, E. and Sela, M. Adv. Protein Chem. 8, 243-492 (1958).
    48. Hirschmann, R. et al. J. Org. Chem. 32, 3415-3425 (1967).
    49. Hirschmann, R. Intra-Sci. Chem. Rep. 5, 203-228 (1971).
    50. Katakai, R. J. Org. Chem. 40, 2697-2702 (1975).
    51. Katakai, R. and Nakayama, Y. Biopolymers 15, 747-755 (1976).
    52. Kurzer, F. and Douraghi-Zadeh, K. Chem. Rev. 67, 107-152 (1967).
    53. Finn, F. M. and Hofmann, K. Proteins, 3rd Ed. 2, 105-253 (1976).
    54. Schroder, E. and Lubke, K. The Peptides, Academic Press, New York, 1&2 (1965).
    55. Denkewalter, R. G. et al. J. Amer. Chem. Soc. 88, 3163 (1966).
    56. Hirschmann, R. et al. J. Org. Chem. 32, 3412 (1967).
    57. Anderson, G. W. and Callahan, F. M., J. Amer. Chem. Soc. 80, 2902-3000 (1958).
    58. Hofman, K et al. Amide Resin, J. Amer. Chem. Soc. 80, 1486-1489 (1958).
    59. Konig, W. and Geiger, R. Chem. Ber. 80, 103 (1970).
    60. Wunsch, E. and Dress, F. Chem, Ber. 9, 110-120 (1966).
    61. Bodanszky, M., Acta Chim. Acad. Sci. Hung 10, 335-346 (1956).
    62. Wieland, T. and Bernhard, H. Justus Liebigs Ann. Chem. 572, 190-194 (1951).
    63. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
    64. Determann, H. Peptides, Proceedings of the 8th European Peptide Symposium, Beyermann, H. C., van der Linde, A., and Massen van der Brink, W., Eds., North Holland, Amsterdam, 73-78 (1967).
    65. Weygand, F. et al. Angew. Chem, Int. Ed. Engl. 2, 183-188 (1963).
    66. Vaughan, J. R., Jr. J. Amer. Chem. Soc. 74, 6137-6139 (1952).
    67. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
    68. Battersby, A. R. and Robinson, J. C. J. Chem. Soc. 259-269 (1955).
    69. Bodanszky, M. and Tolle, J. C. Int. J. Peptide Protein Res. 10, 380-384 (1977).
    70. Merrifield, R. B. et al. J. Org. Chem. 39, 660-668 (1977).
    71. Goodman, M. and Stueben, K. C. J. Org. Chem. 27, 3409-03416 (1962).
    72. Koppel, K. D. and Renick, R. J. J. Org. Chem. 23, 1565-1567 (1958).
    73. Tarbell, D. S. Acc. Chem. Res. 2, 296-300 (1969).
    74. Albertson, N. F., Org. React. 12, 157-355 (1962).
    75. Longosz, E. J. and Tarbell, D. S. J. Org. Chem. 26, 2161-2169 (1961).
    76. Anderson, G. W. Peptides: Chemistry and Biochemistry, Weinstein, B. and Lande, S., Eds., Deker, New York, 255-266 (1970).
    77. Anderson, G. W. et al. J. Amer. Chem. Soc. 89, 5012-5017 (1967).
    78. Belleau, D. and Malek, G. J. Amer. Chem. Soc. 90, 1651-1652 (1968).
    79. Kiso, Y. et al. Chem. Pharm. Bull. 21, 2507-2510 (1973).
    80. Belleau, D. and Malek, G. J. Amer. Chem. Soc. 90, 1651-1652 (1968).
    81. Reviews in High Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation Mant, R. S. and Hodges, R. S., Eds., CRC Press Inc., Boca Raton, FL, 33432, USA (1991).
    82. River, J. et al. J. Chromatography 288, 303 (1984).
    83. Wilson, K. J. et al. Biochemistry Journal 199, 31 (1984).
    84. Lloyd-Williams, P. et al. Int. J. Peptide Protein Res. 37, 58 (1991).

    Tips for Peptide Synthesis

    Reconstitution and Storage of Peptides

    Peptides are usually supplied as a fluffy, freeze-dried material in serum vials. Store peptides in a freezer after they have been received. In order to reconstitute the peptide, distilled water or a buffer solution should be utilized. Some peptides have low solubility in water and must be dissolved in other solvents such as 10% acetic acid for a positively charged peptide or 10% ammonium bicarbonate solution for a negatively charged peptide. Other solvents that can be used for dissolving peptides are acetonitrile, DMSO, DMF, or isopropanol. Use the minimal amount of these non-aqueous solvents and add water or buffer to make up the desired volume. After peptides are reconstituted, they should be used as soon as possible to avoid degradation in solution. Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20C. Repeated thawing and refreezing should be avoided.

    Methods to Dissolve Peptides

    The best way to dissolve a peptide is to use water. For peptides that are not soluble in water, use the following procedure:

    1. For acidic peptides, use a small amount of base such as 10% ammonium bicarbonate to dissolve the peptide, dilute with water to the desired concentration. Do not use base for cysteine-containing peptides.
    2. For basic peptides, use a small amount of 30% acetic acid, dilute with water to the desired concentration.
    3. For a very hydrophobic peptide, try dissolving the peptide in a very small amount of DMSO, dilute with water to the desired concentration.
    4. For peptides that tend to aggregate (usually peptides containing cysteines), add 6 M urea, 6 M urea with 20% acetic acid, or 6 M guanidine•HCl to the peptide, then proceed with the necessary dilutions.

    Preparation of HBTU/HOBt Solution for the Peptide Synthesizer

    1. Preparation of 0.5 M HOBt in DMF:
      • Weigh 13.5 g anhydrous HOBt (0.1 mol, MW 135.1) [100 g, # 21003; 500 g, # 21004] into a 250 mL graduated cylinder.
      • Add DMF until the 200 mL level is reached.
    2. Preparation of 0.45 M HBTU/HOBt solution:
      • Add the solution prepared in step 1 to 37.9 g HBTU (0.1 mol, MW 379.3) [100 g, # 21001; 500 g, # 21002] contained in a beaker or an Erlenmayer flask.
    3. Stir for about 15 min with a magnetic stirring bar until HBTU is dissolved.
    4. Filter the solution through a fine pore size sintered glass funnel.
    5. Pour the filtered solution into an appropriate bottle for attachment to a peptide synthesizer.
    * This solution is stable at room temperature for at least six weeks.

    Biotinylation of Amino Group

    1. Wash 0.1 mmol resin with DMF.
    2. Dissolve 0.244 g (+)-biotin (1 mmol, MW 244.3) [1 g, # 21100; 5 g, # 21101] in 5 mL DMF-DMSO (1:1) solution. A little warming is necessary.
    3. Add 2.1 mL 0.45 M HBTU/HOBt solution and 0.3 mL DIEA to the solution prepared in step 2.
    4. Add the activated biotin solution to the resin and let stir overnight.
    5. Check resin to make sure coupling is complete as evidenced by negative ninhydrin test (colorless).
    6. Wash resin with DMF-DMSO (1:1) (2x) to remove excess (+)-biotin.
    7. Wash resin with DMF (2x) and DCM (2x).
    8. Let the resin dry before proceeding to cleavage.

    Procedure for Loading Fmoc-Amino Acid to 2-Chlorotrityl Chloride Resin

    1. Weigh 10 g 2-chlorotrityl chloride resin (15 mmol) [1 g, # 22229; 5 g, # 22230] in a reaction vessel, wash with DMF (2x), swell the resin in 50 mL DMF for 10 min, drain vessel.
    2. Weigh 10 mmol Fmoc-amino acid in a test tube, dissolve Fmoc-amino acid in 40 mL DMF, transfer the solution into the reaction vessel above, add 8.7 mL DIEA (50 mmol), swirl mixture for 30 min at room temperature.
    3. Add 5 mL methanol into the reaction vessel and swirl for 5 min.
    4. Drain and wash with DMF (5x).
    5. Check substitution.
    6. Add 50 mL 20% piperidine to remove the Fmoc group. Swirl mixture for 30 min.
    7. Wash with DMF (5x), DCM (2x), put resin on tissue paper over a foam pad and let dry at room temperature overnight under the hood. Cover the resin with another piece of tissue paper, press lightly to break aggregates.
    8. Weigh loaded resin.
    9. Pack in appropriate container.

    Procedure for Checking Substitution of Fmoc-Amino Acid Loaded Resins

    1. Weigh duplicate samples of 5 to 10 mg loaded resin in an eppendorf tube, add 1.00 mL 20% piperidine/DMF, shake for 20 min, centrifuge down the resin.
    2. Transfer 100 L of the above solution into a tube containing 10 mL DMF, mix well.
    3. Pipette 2 mL DMF into each of the two cells (reference cell and sample cell), set spectrophotometer to zero. Empty the sample cell, transfer 2 mL of the solution from step 2 into the sample cell, check absorbance.
    4. Subs = 101(A)/7.8(w)
      A = absorbance
      w = mg of resin
    5. Check absorbance three times at 301 nm, calculate average substitution.

    Manual Fmoc Synthesis (0.25 mmol)

    1. Wash resin with DMF (4x) and then drain completely.
    2. Add approximately 10 mL 20% piperidine/DMF to resin. Shake for one min and drain.
    3. Add another 10 mL 20% piperidine/DMF. Shake for 30 min.
    4. Drain reaction vessel and wash resin with DMF (4x). Make sure there is no piperidine remaining. Check beads using ninhydrin test, beads should be blue.
    5. Coupling Step - Prepare the following solution:
      1 mmol Fmoc-amino acid
      2.1 mL 0.45 M HBTU/HOBT (1mmol)
      348 L DIEA (2 mmol)
      Add above solution to the resin and shake for a minimum of 30 min. This coupling step can be longer if desired.
    6. Drain reaction vessel and wash resin with DMF (4x).
    7. Perform Ninhydrin test:
      • If negative (colorless), proceed to step 2 and continue synthesis.
      • If positive (blue), return to step 5 and re-couple the same Fmoc-amino acid. Increase the coupling time if necessary.

    Synthesis of Phosphotyrosine-Containing Peptides Using Fmoc-Phosphotyrosine

    Reagent: N-a-Fmoc-O-phosphotyrosine [1 g, # 20254; 5 g, # 20255]

    1. For 0.1 mmol or 0.25 mmol synthesis, use 0.483 g Fmoc-Tyr(PO3H2)-OH (1 mmol, MW 483.4) . For ABI synthesizers, pack Fmoc-Tyr(PO3H2)-OH in a cartridge.
    2. The cycle program for coupling Fmoc-Tyr (PO3H2)-OH is the same as for other Fmoc-amino acids except for the coupling time (see step 3). (Note: ABI synthesizers use HBTU/HOBT as the activating reagent.)
    3. The coupling time for Fmoc-Tyr(PO3H2)-OH needs to be increased. For ABI model 430A peptide synthesizer, insert several steps (i.e., vortex on, wait 990 sec, vortex off, to increase the coupling time). For ABI model 431A peptide synthesizer, add additional "I"s. Overnight coupling may be necessary for some sequences.
    4. After the coupling step for Fmoc-Tyr(PO3H2)-OH, perform ninhydrin test to ensure complete coupling. Negative (colorless) ninhydrin test indicates complete coupling, while a positive (blue) ninhydrin test indicates incomplete coupling.
    5. Increase the coupling time of the amino acid residues after the phosphotyrosine or perform double coupling. (Note: The coupling of amino acids after the phosphotyrosine can be difficult.)
    6. There is a limit on the number of amino acid residues that can be coupled after the hosphotyrosine. Since the phospho group is unprotected, side reactions are likely to ccur. (Note: Peptides have been successfully coupled with sequences containing up o ten additional amino acids following the phosphotyrosine residue.)

    Simultaneous Synthesis of Peptides Which Differ in the C-Termini Using 2-Chlorotrityl Resin and Wang Resin*

    Peptides which differ in the C-termini can be simultaneously synthesized in one reaction vessel by employing resins that possess different cleavage properties. The resins used were the weak acid labile 2-chlorotrityl resins and the TFA labile Wang resins. The success of this approach was shown by the co-synthesis of ACTH (4-10) with ACTH (4-11) and Neuropeptide Y, a C-terminal amide peptide with its corresponding C-terminal free acid analog.

    *Hong A., Le T., and Phan T. Techniques in Protein Chemistry VI, 531-562 (1995).

    Cleavage Protocol to Produce Fully Protected Peptide

    Starting Resin: Chlorotrityl resins

    Reagents for 1 g Peptide-Resin:
    1 mL acetic acid (AcOH)
    2 mL trifluoroethanol (TFE)
    7 mL dichloromethane (DCM)

    1. Prepare above mixture.
    2. Add peptide-resin to the mixture and let it stir at room temperature for 1 h.
    3. Filter and wash resin with 10 mL TFE:DCM (2:8) (2x) to ensure that all of the product is recovered.
    4. Evaporate the solvent until there is less than 5 mL of liquid.
    5. Add ether to a test tube containing about 100 mL of the above solution. Check solubility of the fully protected peptide in ether. If the product precipitates, proceed to step 6. If no precipitate is observed, proceed to step 7.
    6. Add cold ether to the residual liquid in step 4 to precipitate the fully protected peptide. Filter through a fine sintered funnel to obtain the product.
    7. Some fully protected peptides are soluble in ether. In this case, add water to precipitate them out. Filter through a fine sintered funnel to obtain the product.

    Procedure for Removing Mtt group from Fmoc-Lys(Mtt) on Solid Phase

    Fmoc-Lys(Mtt)-OH [1 g, # 20093; 5 g, #20094]

    1. Swell resin in DCM.
    2. Wash resin with 3% TFA/DCM (2x) (since the resin is swollen in DCM, this step of washing the resin quickly with 3% TFA/DCM ensures that the actual concentration of TFA is 3%).
    3. Shake the resin in 3% TFA for 10 min.
    4. Repeat step 3.
    5. Wash resin with DCM (3x), DMF (3x), isopropanol (3x), and DCM (3x).
    6. Let the resin dry in air.

    Procedure for FITC Labeling of Peptides

    FITC [1 g, # 20151]
    Fmoc-e-Ahx-OH [1 g, # 20957; 5 g, # 20958]

    1. Couple Fmoc-e-Ahx-OH to the amino terminal of the peptide-resin using standard coupling conditions.
    2. "De-Fmoc" with piperidine using the standard 20% piperidine procedure.
    3. Wash resin with DMF (3-4x).
    4. Swell resin with DCM and drain.
    5. Prepare solution of 1.1 equivalent of FITC in pyridine/DMF/DCM (12:7:5). Use just enough solution to form a slurry with the resin. Do not use too much solution since the rate of the reaction is proportionate to the concentration of the solution.
    6. Add the solution prepared in step 2 to the resin.
    7. Let mix overnight.
    8. Check the completion of the reaction using ninhydrin test.
    9. If the coupling of FITC to the amino group is not complete, ninhydrin test will give a blue color. Repeat the coupling with FITC (steps 5-7) if necessary.
    10. Wash resin with DMF (2x), isopropanol (2x), and DCM (2x).

    Procedure for Fluorescein Labeling of Peptides

    5-carboxyfluorescein (5-FAM) [0.1 g, # 24623; 0.5 g, # 24624)

    Use standard coupling method to couple 5-carboxyfluorescein to the amino group of the peptide. For cost saving purposes, use 2x excess compared to the mmol of resin, instead of the standard 4x excess used for Fmoc-amino acids. For 0.1 mmol synthesis, use 75 mg 5-carboxyfluorescein, 76 mg HBTU, and 70 mL DIEA.

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