Amino acids solid phase peptide synthesis

The actual process of solid-phase peptide synthesis, outlined in Figure 27.15, begins with the attachment of the C-terminal amino acid to the chloromethylated polymer in step 1. Nucleophilic substitution by the carboxylate anion of an A-Boc-protected C-terminal... 

Menifield successfully automated all the steps in solid-phase peptide synthesis, and computer-controlled equipment is now commercially available to perform this synthesis. Using an early version of his peptide synthesizer, in collaboration with coworker Bemd Gutte, Menifield reported the synthesis of the enzyme ribonuclease in 1969. It took them only six weeks to perform the 369 reactions and 11,391 steps necessary to assemble the sequence of 124 amino acids of ribonuclease. 

Solid-phase peptide synthesis does not solve all purification problems, however. Even if every coupling step in the ribonuclease synthesis proceeded in 99% yield, the product would be contfflninated with many different peptides containing 123 amino acids, 122 amino acids, and so on. Thus, Menifield and Gutte s six weeks of synthesis was followed by four months spent in purifying the final product. The technique has since been refined to the point that yields at the 99% level and greater are achieved with cunent instrumentation, and thousands of peptides and peptide analogs have been prepared by the solid-phase method. 

Solid-phase peptide synthesis (Section 27.18) Method for peptide synthesis in which the C-terminal amino acid is covalently attached to an inert solid support and successive amino acids are attached via peptide bond formation. At the completion of the synthesis the polypeptide is removed from the support. 

Polymer-supported esters are widely used in solid-phase peptide synthesis, and extensive information on this specialized protection is reported annually. Some activated esters that have been used as macrolide precursors and some that have been used in peptide synthesis are also described in this chapter the many activated esters that are used in peptide synthesis are discussed elsewhere. A useful list, with references, of many protected amino acids (e.g., -NH2, COOH, and side-chain-protected compounds) has been compiled/ Some general methods for the preparation of esters are provided at the beginning of this chapter conditions that are unique to a protective group are described with that group/ Some esters that have been used as protective groups are included in Reactivity Chart 6. 

Chapter 26, Biomolecules Amino Acids. Peptides, and Proteins—The chapter has been updated, particularly in its coverage of solid-phase peptide synthesis. 

The novel concept of synthesizing a molecule while attached to a swollen cross-linked resin bead was introduced and demonstrated by R. B. Merrifield with the solid-phase peptide synthesis method about 20 years ago (1,2). The procedure involves the covalent attachment of an amino-acid residue to the polymer bead followed by the addition of subsequent amino-acid units in a stepwise manner under conditions that do not disrupt the attachment to the support. At the completion of the assembly of the peptide, the product is cleaved from the resin and recovered. The macro-scopically insoluble support provides convenient containment of the desired product so that isolation and purification from soluble co-products in the synthesis can be achieved by simple... 

A recent development in this context is the Liberty system introduced by CEM in 2004 (see Fig. 3.25). This instrument is an automated microwave peptide synthesizer, equipped with special vessels, applicable for the unattended synthesis of up to 12 peptides employing 25 different amino acids. This tool offers the first commercially available dedicated reaction vessels for carrying out microwave-assisted solid-phase peptide synthesis. At the time of writing, no published work accomplished with this instrument was available. 

Fig. 3 Important 19F-labelled amino acids, (a) Compounds that are wo-steric to native amino acids can be incorporated into proteins biosynthetically, but they possess too many degrees of torsional freedom to be useful for ssNMR structure analysis, (b) In these artificial amino acids the 19F-reporter group is rigidly attached to the peptide backbone. They can be incorporated by solid-phase peptide synthesis, but some problems can arise due to racemisation (4F-Phg, 4CF3-Phg), steric hindrance of coupling (F3-Aib) or HF elimination (fluoro-Ala, F3-Ala). 4F-Phg is additionally problematic due to an ambiguity of the side-chain rotamer. The preferred 19F-labels for ssNMR structure analysis are CF3-Bpg and CF3-Phg (as suitable substitutes for Leu, lie, Met, Val and Ala), as well as F3-Aib and CF3-MePro...

Fields GB, Noble RL (1990) Solid-phase peptide-synthesis utilizing 9-fluorenylmethoxycar-bonyl amino-acids. Int J Pept Prot Res 35 161-214... 

GE Reid, RJ Simpson. Automated solid-phase peptide synthesis use of 2-(17f-ben-zotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids. Anal Biochem 200, 301, 1992. 

S-S Wang, ST Chen, KT Wang, RB Merrifield. 4-Methoxybenzyloxycarbonyl amino acids in solid phase peptide synthesis. Int J Pept Prot Res 30, 662, 1987. 

E Atherton, JL Holder, MMeldal, RC Sheppard, RM Valerio. 3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl esters of fluorenylmethoxycarbonyl amino acids as self-indicating reagents for solid phase peptide synthesis. J Chem Soc Perkin Trans 1 2887, 1988. 

E Falb, T Yechezkel, Y Salitra, C Gilon. In situ generation of Fmoc-amino acid chlorides using >/s-(trich loro methyl (carbonate and its utilization for difficult couplings in solid-phase peptide synthesis. J Pept Res 53, 507, 1998. 

Solid-phase peptide synthesis offers a fast and convenient route for many peptides when isotope-enriched compounds are not required. Classical synthesis additionally permits the use of non-natural amino acids and allows site-specific isotope labeling. Although Fmoc protected 15N-labeled amino adds are commercially available, the cost of such synthesis is usually prohibitive, and the peptides from chemical synthesis require perdeuterated detergents and unfortunately exclude investigation of internal dynamics through measurement of 15N relaxation. 

Pellarini F, Pantarotto D, Da Ros T, Giangaspero A, Tossi A, Prato M (2001) A novel [60]fullerene amino acid for use in solid-phase peptide synthesis. Org. Lett. 3 1845-1848. 

Yang J, Alemany LB, Driver J, Hartgerink JD, Barron AR (2007a) Fullerene-derivatized amino acids Synthesis, characterization, antioxidant properties, and solid-phase peptide synthesis. Chem. Eur. J. 13 2530-2545. 

Work in the Imperiali laboratory has also focused on exploring the ability of minimal peptide scaffolds to augment the rate of coenzyme-mediated transaminations [22-25]. To accomplish this, a strategy has been developed in which the core functionality of the coenzyme is incorporated as an integral constituent of an unnatural coenzyme amino acid chimera construct. Thus, non-cova-lent binding of the coenzyme to the peptide or protein scaffold is unnecessary. Both the pyridoxal and pyridoxamine analogs have been synthesized in a form competent for Fmoc-based solid phase peptide synthesis (SPPS) (Fig. 7) [23,24]. 

One advantage of the coenzyme amino acid chimera approach is that it is compatible with solid phase peptide synthesis. Consequently, the reactive functionality can be readily and selectively delivered to any site in the peptide. Additionally, both natural and unnatural residues can be incorporated throughout the peptide scaffold, and related compounds can be investigated rapidly by combinatorial synthesis techniques. 

A thiazolium amino acid (Taz) has been developed which can be utilized to mimic TDP-dependent enzyme function [52]. In this strategy, illustrated in Fig. 15, the commercially available amino acid 4-thiazolylalanine is incorporated into peptides by solid phase peptide synthesis. Prior to deprotection of the amino acid side chains and cleavage of the peptide from the resin, the thiazole amino acid is alkylated with an alkyl halide to generate the corresponding thiazolium amino acid having various N3-substituents (BzTaz = 3-benzyl-Taz, NBTaz = 3-nitrobenzyl-Taz). 

Use of Proteases in Peptide Synthesis. Typically peptides are synthesized the standard solid or liquid phase methodologies (56, 57). However, both of these techniques require harsh chemical reactions which are detrimental to certain amino acids. Furthermore, in practical terms most peptide syntheses are limited to the range of 30 to 50 amino acid residues. Hence, peptide synthesis is still somewhat problematic in many cases. In certain situations, the alternative method of peptide synthesis using proteases is an attractive choice. With this form of synthesis, one can avoid the use of the noxious and hazardous chemicals used in solid or liquid phase peptide synthesis. Since the reactions are enzyme catalyzed, racemization of the peptide bond does not occur. This technique has been used with success in the synthesis and semisynthesis of several important peptides including human insulin (55,59). 

The first version of solid-phase peptide synthesis (SPPS) to be developed used the /-Boc group as the amino-protecting group. It can be cleaved with relatively mild acidic treatment, and TFA is usually used. The original coupling reagent was dicyclohexylcar-... 

In 1962, solid-phase peptide synthesis was introduced, and described in detail in 1963.1171 This new synthetic principle was developed with the hope that it would simplify and accelerate the synthesis of peptides. The idea was to covalently anchor the C-terminal residue of a peptide to an insoluble support and then to assemble the remaining amino acids in a stepwise manner with activated amino acids while the peptide was in the insoluble solid phase, and finally to cleave the peptide from the solid support and liberate it into solution. The general methodology is shown in Scheme 6. 

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