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Chemical Synthesis of Peptides
In contrast to the traditional biotechnological practice of obtaining peptides or proteins by isolation from cellular hosts through protein expression, advances in chemical techniques for the synthesis and ligation of peptides has allowed for the total synthesis of some peptides and proteins. Chemical synthesis of proteins is a valuable tool in chemical biology as it allows for the introduction of non-natural amino acids as well as residue specific incorporation of “posttranslational modifications” such as phosphorylation, glycosylation, acetylation, and even ubiquitination. These capabilities are valuable for chemical biologists as non-natural amino acids can be used to probe and alter the functionality of proteins, while post translational modifications are widely known to regulate the structure and activity of proteins. Although strictly biological techniques have been developed to achieve these ends, the chemical synthesis of peptides often has a lower technical and practical barrier to obtaining small amounts of the desired protein. Given the widely recognized importance of proteins as cellular catalysts and recognition elements, the ability to precisely control the composition and connectivity of polypeptides is a valued tool in the chemical biology community and is an area of active research. While chemists have been making peptides for over 100 years, the ability to efficiently and quickly synthesize short peptides came of age with the development of Bruce Merrifield’s solid phase peptide synthesis (SPPS). Prior to the development of SPPS, the concept of step-by-step polymer synthesis on an insoluble support was without chemical precedent. The use of a covalently bound insoluble polymeric support greatly simplified the process of peptide synthesis by reducing purification to a simple “filtration and wash” procedure and facilitated a boom in the field of peptide chemistry. The development and “optimization” of SPPS took peptide synthesis from the hands of the specialized peptide synthesis community and put it into the hands of the broader chemistry, biochemistry, and now chemical biology community. SPPS is still the method of choice for linear synthesis of polypeptides up to 50 residues in length and has been implemented in commercially available automated peptide synthesizers. One inherent shortcoming in any procedure that calls for repeated coupling reactions is the buildup of side products resulting from incomplete couplings and side reactions. This places the upper bound for the synthesis of linear polypeptide lengths at around 50 amino acids, while the “average” protein consists of 250 amino acids. Clearly, there was a need for development of “non-linear” methods to allow synthetic access to the average protein.

Although the shortcomings of linear SPPS were recognized not long after its inception, it took until the early 1990’s for effective methodology to be developed to ligate small peptide fragments made by SPPS, into protein sized polypeptide chains (for recent review of peptide ligation strategies see review by Dawson et al ). The oldest and best developed of these methods is termed native chemical ligation. Native chemical ligation was unveiled in a 1994 paper from the laboratory of Stephen B. H. Kent. Native chemical ligation involves the coupling of a C-terminal thioester and an N-terminal cysteine residue, ultimately resulting in formation of a “native” amide bond. Further refinements in native chemical ligation have allowed for kinetically controlled coupling of multiple peptide fragments, allowing access to moderately sized peptides such as an HIV-protease dimer and human lysozyme. Even with the successes and attractive features of native chemical ligation, there are still some drawbacks in the utilization of this technique. Some of these drawbacks include the installation and preservation of a reactive C-terminal thioester, the requirement of an N-terminal cysteine residue (which is the second least common amino acid in proteins ), and the requirement for a sterically unincumbering C-terminal residue. Other strategies that have been used for ligation of peptide fragments using the acyl transfer chemistry first introduced with native chemical ligation include expressed protein ligation, sulfurization/desulfurizationtechniques , use of removable thiol auxiliaries.

Expressed protein ligation allows for the biotechnological installation of a C-terminal thioester using intein biochemistry, thereby allowing the appendage of a synthetic N-terminal peptide to the recombinantly produced C-terminal portion. This technique allows for access to much larger proteins as only the N-terminal portion of the resulting protein has to be chemically synthesized. Both sulfurization/desulfurization techniques and the use of removable thiol auxiliaries involve the installation of a synthetic thiol moiety to carry out the standard native chemical ligation chemistry, followed by removal of the auxiliary/thiol. These techniques help to overcome the requirement of an N-terminal cysteine needed for standard native chemical ligation, although the steric requirements for the C-terminal residue are still limiting. A final category of peptide ligation strategies include those methods not based on native chemical ligation type chemistry. Methods that fall in this category include the traceless Staudinger ligation, azide-alkyne dipolar cycloadditions , and imine ligations.

Major contributors in this field today include Stephen B. H. Kent, Philip E. Dawson, and Tom W. Muir, as well as many others involved in methodology development and applications of these strategies to biological problems.