Solid-phase peptide synthesis
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Solid-phase peptide synthesis (SPPS), pioneered by Robert Bruce Merrifield, caused a paradigm shift within the peptide synthesis community, and it is now the standard method for synthesizing peptides and proteins in the lab. SPPS allows for the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids.

Small porous beads are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus 'immobilized' on the solid-phase and can be retained during a filtration process while liquid-phase reagents and by-products of synthesis are flushed away.

The general principle of SPPS is one of repeated cycles of deprotection-wash-coupling-wash. The free N-terminal amine of a solid-phase attached peptide is coupled (see below) to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.

The overwhelmingly important consideration is to generate extremely high yield in each step. For example, if each coupling step were to have 99% yield, a 26-amino acid peptide would be synthesized in 77% final yield (assuming 100% yield in each deprotection); if each step were 95%, it would be synthesized in 25% yield. Thus each amino acid is added in major excess (2~10x) and coupling amino acids together is highly optimized by a series of well-characterized agents.

There are two majorly used forms of SPPS C Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by either of these two groups and added onto a deprotected amino acid chain.

Automated synthesizers are available for both techniques, though many research groups continue to perform SPPS manually.

SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields.

Since its introduction over 40 years ago, SPPS has been significantly optimized. First, the resins themselves have been optimized. Furthermore, the 'linkers' between the C-terminal amino acid and polystyrene resin have improved attachment and cleavage to the point of mostly quantitative yields. The evolution of side chain protecting groups has limited the frequency of unwanted side reactions. In addition, the evolution of new activating groups on the carboxyl group of the incoming amino acid have improved coupling and decreased epimerization. Finally, the process itself has been optimized. In Merrifield's initial report, the deprotection of the -amino group resulted in the formation of a peptide-resin salt, which required neutralization with base prior to coupling. The time between neutralization of the amino group and coupling of the next amino acid allowed for aggregation of peptides, primarily through the formation of secondary structures, and adversely affected coupling. The Kent group showed that concomitant neutralization of the -amino group and coupling of the next amino acid led to improved coupling. Each of these improvements has helped SPPS become the robust technique that it is today.