RESEARCH INTERESTS:

The Kent research group is devoted to inventing and using new chemistries to reveal how proteins work in nature. To that end, we develop novel methods for the total synthesis of proteins that enable us to apply advanced physical methods in unprecedented ways to understand the chemical basis of protein function. Our goal is to then demonstrate that knowledge by the design and construction of protein molecules with novel properties.

Chemical Protein Synthesis
The total synthesis of natural products is arguably the most important intellectual endeavor in the area of synthetic organic chemistry - it drives methodology forward and in the process generates new molecular entities. Proteins are the most abundant, exciting, and challenging class of natural products. Proteins are the molecular machines of the living world, performing nearly all of the functions in the cell and playing vital roles in human biology and medicine. With the success of the genome sequencing projects, proteins are being discovered at an unprecedented rate – a single recent publication described several million novel protein molecules, known only as open reading frame data encoded in the genomic DNA. In addition, it has been estimated that there are more than one million venom-derived proteins, each of which has potent and specific biological activities.

The robust synthesis of protein molecules was one of the ‘grand challenges’ of organic chemistry in the twentieth century. Our laboratory invented the chemical ligation methods that met this challenge, and that have enabled the practical total synthesis of proteins. Chemical protein synthesis is generally applicable to the efficient preparation of polypeptides containing 300 or more amino acids. Total synthesis enables the versatile incorporation of non-coded amino acids into proteins, and the modification and labeling of protein molecules, without restriction as to the sites, numbers, and kinds of chemical moieties being introduced.

We prepare the long polypeptide chains of protein molecules by the chemoselective reaction (‘chemical ligation’) of unprotected peptide segments containing unique, mutually reactive functional groups. The most powerful of these ligation chemistries is thioester-mediated, amide-forming ligation, termed ‘native chemical ligation’. The resulting polypeptide chains are then folded with great efficiency to give high purity synthetic proteins. The covalent structure of the molecule is confirmed by mass spectrometry and the three dimensional folded structure of the synthetic protein is determined by Xray crystallography.

Recently, we introduced ‘kinetically controlled ligation’, a novel and highly sophisticated chemistry for the fully convergent synthesis of large protein molecules. Currently we are exploring novel insertion reactions for the creation of molecular diversity in preformed molecular scaffolds, and the use of polymer-supported ligation chemistries for the synthesis of proteins.

Chemistry of Enzyme Catalysis
We take full advantage of the flexibility provided by total protein synthesis to study the physical organic chemistry of how enzyme molecules work. Backbone engineering of the amide bonds in the polypeptide chain has been used to delete critical H-bonding interactions and to evaluate the effects on enzyme function. 13C & 15N NMR probe nuclei have been introduced at unique single atom sites to elucidate critical aspects of the chemical basis of enzyme catalysis. Single molecule fluorescence spectroscopy is being used to probe the functional properties of uniquely chemical analogues of the enzyme molecule.

Mirror Image Protein Molecules
The chemical synthesis approach enables us to make enantiomeric ‘D-protein’ molecules not found in nature. We have pioneered the use of racemic protein crystallography to determine the Xray structures of proteins that will otherwise not crystallize, and to obtain protein electron density maps of unprecedented accuracy.

The mirror image enzyme HIV-1 protease prepared by total chemical synthesis
{Image credit: Art Olson, TSRI}

We are also prototyping ‘mirror image drug discovery’, the use of mirror image protein targets to identify novel lead compounds from protein libraries. Synthesis of the enantiomer of the identified protein binders gives unique molecules, that could not have been discovered using the natural target, with the specificity and potency of antibodies and effective against the protein found in nature. The resulting D-protein therapeutic candidates are small enough to be made by existing chemical methods of production.

Selected References

1. Design and total synthesis of [GluA4(OβThrB30)]insulin (‘ester insulin’): a minimal proinsulin surrogate that can be chemically converted into human insulin. Youhei Sohma, Qing-Xin Hua, Jonathan Whittaker, Michael A. Weiss, Stephen B. H. Kent, Angewandte Chemie Int. Ed. Engl., 49, 5489 - 5493 (2010). Cover

2. An investigation into the origin of the dramatically reduced reactivity of peptide-prolyl-thioesters in native chemical ligation. Samuel B. Pollock, Stephen B.H. Kent, Chem. Commun., [Dec 21. Epub ahead of print] (2010).

3. Total chemical synthesis of human proinsulin. Samuel Luisier, Michal Avital-Shmilovici, Michael A. Weiss, Stephen B.H. Kent, Chem. Commun., 46, 8177 – 8179 (2010).

4. X-ray structure of native scorpion toxin BmBKTx1 by racemic protein crystallogaraphy using direct methods. Kalyaneswar Mandal, Brad L. Pentelute, Valentina Tereshko, Anthony A. Kossiakoff, Stephen B. H. Kent, J. Am. Chem. Soc., 131, 1362-3 (2009).

5. Role of a salt bridge in the model protein crambin explored by chemical protein synthesis: X-ray structure of a unique protein analogue, [V15A]crambin-αcarboxamide. Duhee Bang, Valentina Tereshko, Anthony A. Kossiakoff, Stephen B.H. Kent, Molecular BioSystems, 5, 750 - 756 (2009).

6. Total chemical synthesis of proteins. Chemical Society Reviews, 38, 338-51 (2009).

7. Total chemical synthesis and racemic protein crystallography used to determine the X-ray structure of plectasin by direct methods. Protein Science, 18, 1146-1154 (2009). Cover

8. Dynamics of flap structures in three HIV-1 protease inhibitor complexes probed by total chemical synthesis and pulse-EPR spectroscopy. J. Am. Chem. Soc., 131, 884-5 (2009).

9. Comparative properties of insulin-like growth factor 1 (IGF-1) and [Gly7D-Ala]IGF-1 prepared by total chemical synthesis. Angew. Chem. Int. Ed. Eng., 47, 1102-1106 (2008).

10. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. J. Am. Chem. Soc., 130, 9695-9701 (2008).

11. Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site. Bioorg. & Med. Chem. Letters, 18, 4554-7 (2008).

12. Convergent chemical synthesis and crystal structure of a 203 amino acid ‘covalent dimer’ HIV-1 protease enzyme molecule. Angew. Chem. Int. Ed. Engl., 46, 1667-1670 (2007).

13. Convergent chemical synthesis and high resolution X-ray structure of human lysozyme. Proc. Natl. Acad. Sci. USA, 104, 4846-4851 (2007).

14. Kinetically-controlled ligation for the convergent chemical synthesis of proteins. Angew. Chem. Int. Ed. Engl. 45, 3985-3988 (2006).

15. Dissecting the energetics of protein α-helix C-cap termination through chemical protein synthesis. Nature Chemical Biology, 2, 139-143 (2006).