Building a protein can be likened to a jigsaw puzzle. Stephen Kent of the University of Chicago, US, puts the pieces together

A long-held goal of protein science has been to apply chemistry's tools to understand how a protein's structure leads to its function. Ideally this would be achieved by total chemical synthesis which, once achieved for a particular protein target, would give complete control over the protein's covalent structure. Simply modifying the synthesis would allow a chemist to incorporate site-specific labels and create a wide variety of novel chemical analogues.

Chemical ligation was used to create an active analogue of erythropoietin - a protein that stimulates red blood cell production

However, until recently, proteins have resisted total chemical synthesis because of their large size. The smallest proteins have about 50 amino acid residues in their polypeptide chain, while proteins of typical size have in the region of 300. Yet chemical peptide synthesis cannot routinely make molecules containing more than around 40 to 50 amino acid residues. This was the conundrum that - until the mid-1990s - confronted the researcher who wished to apply the full power of chemical science to study proteins.

Now, all that has changed. In the early 1990s, a new principle was proposed:  chemoselective condensation of unprotected peptide segments. This has led to the efficient total chemical synthesis of proteins. In a chemoselective condensation reaction, two unique, mutually reactive chemical functional groups are employed, one on each of the reacting peptide segments. These groups are designed to react with one another, but to NOT react with any of the other functional groups found in protein-derived peptides. When these two peptide segments are mixed in solution, an unambiguous reaction ensues to form a single product in essentially quantitative yield. The process is called chemical ligation. 

The chemical ligation principle surmounts the difficulties that limited traditional solution and solid phase synthetic approaches. Use of unprotected peptide building blocks means that the starting materials, intermediate products, and the final full length synthetic polypeptide chain can all be purified by standard methods, and characterised by high resolution techniques such as reverse phase HPLC and electrospray mass spectrometry. Reactions can be carried out at high concentrations, since the lack of protecting groups means the peptides are freely soluble in solvents such as aqueous 6M guanidine.HCl. Consequently, reactions are rapid and go to completion. The full length synthetic polypeptide chains fold with high efficiency to give the tertiary structure, including disulfide bonds, of the functional protein molecule.

Chemical ligation has enabled a wide variety of proteins to be prepared efficiently, some containing 200 or more amino acid residues. For example, native chemical ligation, which links a protein segment containing a thioester to one with an N-terminal cysteine residue, was used to make a 203 amino acid residue form of the HIV-1 protease enzyme, with full catalytic activity. The technique has also been used to create an analogue of erythropoietin (see figure), a protein that stimulates red blood cell production. The glycoprotein mimetic had a mass of over 50 kiloDaltons and showed enhanced properties including a ~3-fold extended lifetime in vivo. 

In a tribute to George W Kenner and colleagues, who thirty years ago attempted the total synthesis of the enzyme consensus lysozyme by conventional solution methods, chemical ligation has led to a fully convergent chemical synthesis of human lysozyme. Four unprotected peptide segments were connected to give a 130 amino acid polypeptide chain that was folded to give a protein with four disulfide bridges. The crystalline synthetic enzyme had full activity.

An elegant and practical solution to the grand challenge of protein synthesis, chemical ligation offers a way to make designer proteins with novel, and improved, properties. It also enables the science of chemistry to be applied, without limitation, towards unravelling the molecular basis of protein function. 

Read more in Stephen Kent's tutorial review 'Total chemical synthesis of proteins' in Chemical Society Reviews.

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