CARBOHYDRATE PROTEIN CONJUGATES OF PROTEASES

点击翻译按钮获取中文摘要

In this proposal, we outline the synthesis of new materials that stabilize proteases in organic solvents for their use in the catalytic formation of peptide bonds. We detail the synthesis of these new carbohydrate based materials, the preparation of carbohydrate protein conjugates of proteases [CPC(proteases)], and explore the use of these materials for the enzyme catalyzed synthesis of oligo- and polypeptides in organic solvent systems. Our investigations on the use of CPC (proteases) should extend to other enzymes and may lead to new opportunities for the use of proteins in organic synthesis. Specifically, our goals include: * The synthesis of a series of carbohydrate-functionalized macromolecules that contain masked aldehyde functionality, with charged, hydrophobic or hydrophilic groups. We propose the preparation of carbohydrate-based macromolecules that contain functionality that can effectively control the surface properties of proteases to preserve their catalytic activity in organic solvents. * The synthesis and characterization of carbohydrate protein conjugates of proteases. We plan to study conjugates of proteases for two reasons: (1) proteases are well-characterized enzymes (X-ray crystallographic data, mechanistic detail, etc.) which may allow us to develop a fundamental understanding of the mechanism of stabilization of these enzymes conjugates in organic solvents, and (2) proteases are important for the development of convergent. methodology in polypeptide synthesis. * The development of carbohydrate protein conjugates of proteases [CPC(proteases)] for convergent polypeptide synthesis. We will study the fundamental problems associated with the enzymatic synthesis of peptide bonds: hydrolysis of activated substrates, transamidation, substrate specificity, etc. We will also use CPC(proteases) for the coupling of large peptide fragments in organic solvents (and organic solvent-water mixtures) such as acetonitrile, dimethylformamide and dimethylsulfoxide with the overall goal being the development of the convergent synthesis of polypeptides and proteins.