Solid-phase synthesis of peptides

Traditional approaches to peptide synthesis were based on solution methods using convergent synthetic schemes, with isolation and characterization of each intermediate. This provided well-defined products with confidence in their final structures, but at the price of increased labor and losses at each synthetic step in separating product from reagents and byproducts. Until recently, the synthesis of small proteins (60 to 100 residues) has been dominated by fragment condensation in solution with maximal protection of side chains. The desired protein sequence is divided into peptide segments of about 10 residues, which is about the maximum length of peptide readily prepared by stepwise addition in solution. After each reaction, the product is isolated and fully characterized before proceeding with the next reaction. Once the set of fragments has been prepared, they are combined pairwise to generate segments of approximately 20 residues. These are then combined pairwise to generate fragments of approximately 40 residues, and this fragment condensation continues until the desired sequence is obtained. The protecting groups on side chains are then removed to give the fully unprotected peptide chain, which is allowed to fold, resulting in the desired protein. First, the 80-residue protein is divided into eight 10-residue segments that are prepared by stepwise elongation requiring nine coupling and nine deprotection steps, with isolation of each intermediate to give a completely protected fragment with both amino and carboxyl termini protected. Depending on the role of the segment, either its N- or C-terminus is deprotected and reacted with its adjacent fragment. This process is continued until the entire protein is assembled, deprotected, purified, and allowed to fold.

Figure 1. A common implementation of the BOC strategy for peptide synthesis.

In order to get selectivity in the reaction, all reactive groups except the two that are desired to react can be protected transiently with chemically stable protecting groups. This is referred to as maximal protection and would be preferred, except for incomplete removal and side reactions associated with deprotection of the fully protected peptide product containing a heterogeneous set of protecting groups. In solid-phase synthesis, assembly of the correct sequence is often quite facile, but incomplete removal of the protecting groups can lead to an intractable mixture. By careful control of reaction conditions (aqueous, pH, etc.), one can eliminate some of the side-chain protection to minimize problems associated with protecting group removal. This is referred to as minimal protection and is preferred if selective acylation of the amino terminus by the carboxyl of the N-terminal peptide can be achieved.

Solid Phase Peptide Synthesis - RealPeptide

Figure 2. The alternative FMOC/Wang support strategy which allows ready synthesis of protected peptide fragments as

Illustrative are the solid-phase protocols for the two strategies (Boc and Fmoc) commonly used for the synthesis of peptides. The Boc strategy (Fig. 1) is often combined with a 1% to 2% cross-linked polystyrene support and a benzyl ester linkage to the polymer, requiring strong acid such as hydrogen fluoride for deprotection. The procedure favored by most synthetic laboratories uses an acid-labile linkage similar to the p-methoxybenzyl ester linkage of the Wang resin and a base-labile amino protecting group, the fluorenylmethyloxycarbonyl (Fmoc), on the added amino acids (Fig. 2). One can use side-chain protection with similar acid lability to the Wang linkage to give free peptide upon cleavage, or use more stable side-chain protection to give the protected peptide for fragment condensation after purification and characterization. In the latter case, a final deprotection with strong acid such as HF is required.

Solid-phase peptide synthesis: ..

While many proteins contain cysteine residues, and some have them optimally spaced for fragment assembly, many do not, and one of the research goals in this area is the extension of these concepts to allow coupling at residues other than the ^-terminus of cysteine residues. Some success has been achieved with ligation at X-Gly and Gly-X sites (14), as well as at X-His sites (15). Another development that bodes well for the chemical synthesis of larger proteins is the adaptation of chemical ligation to solid-phase using agarose as the polymeric support. This should allow repetitive ligation of smaller fragments to generate the larger protein stepwise on the polymer.

Solid Phase Synthesis - Solid-Phase Combinatorial …

Aziridine-containing cyclic tetrapeptides possess great potential for covalent protein labeling and can be valuable in screening libraries. Chung . describe the synthesis, cyclization, and site-specific modification of these scaffolds.