The Use of Carbohydrate Protein Conjugates of Proteases [CPC(Proteases)] for the Catalytic Formation of Peptide Bonds
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The Use of Carbohydrate Protein Conjugates of Proteases [CPC(Proteases)] for the Catalytic Formation of Peptide Bonds Peng Wang,*,§ Tara G. Hill,t Mark D. Bednarski,t,§,* and Matthew R. Callstromt,§,* Contribution from the Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, the Department of Chemistry, University of California at Berkeley, Berkeley, California 94720, and the Center for Advanced Materials; Materials Sciences Division; Lawrence Berkeley Laboratory, Berkeley, California 94720. The discovery of catalysts that can selectively couple unprotected peptide fragments would revolutionize protein chemistry by allowing convergent polypeptide synthesis.1-4 Proteolytic enzymes have the capability to perform this chemistry because the protein can specifically recognize and bind to C-terminal and N-terminal peptide sequences, activate the C-terminal peptide sequence by forming an acyl-enzyme intermediate, and couple the two peptide fragments together. 5-9 However, barriers that limit the use of proteases as catalysts for convergent peptide synthesis include (i) the stability of proteolytic enzymes in organic solvent systems; 10 (ii) a simple and effective C-terminal and N-terminal protecting group strategy; and (iii) the isolation of the polypeptide product from the reaction mixture. 5 In the previous paper we reported the stabilization of enzymes by the covalent attachment of proteins through their e-lysine residues to a series of carbohydrate-based macromolecules. 11 In this paper we report the use of carbohydrate protein conjugates of proteases [CPC(proteases)] as catalysts for peptide bond synthesis and a general strategy for convergent oligopeptide synthesis. We have prepared carbohydrate protein conjugates of (t-chymotrypsin [1CPC(CT)], thermolysin [1-CPC(Th)], and subtilisin BPN' [1-CPC(BPN')] to evaluate the use of these materials as catalysts for the synthesis of peptide bonds in organic solvents (Scheme i).2.3,9-13 We choose to use acetonitrile, dioxane and tetrahydrofuran as solvents for this chemistry to demonstrate the catalytic efficiency and generality of the use of CPC-proteases in organic media and because oligopeptides are soluble in these solvents or mixtures of these solvents with water (Table I).9m, 14, 15 Entries 1, 2 and 3 of Table I show that 1-CPC(CT) is
tThe Ohio State University, Department of Chemistry, Columbus, OH 43210 fUniversity of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 §Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Mat. Res. Soc. Symp. Proc. Vol. 218. 91991 Materials Research Society
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Scheme I
OH OH HO eHO, HO tHO, 0 OH 0 OH
1
00NH2NYME E NaBH 3 CN
OH H0 -(HO 0
HNOH I
H HO. OOH
1-CPC(ENZYME) ENZYME= Chymotrypsin (CT) ThermolysIn (Th) Subtillsin BPN' (BPN')
catalytically active in these solvents and that high yields of dipeptide can be synthesized using 90% acetonitrile containing 5% water and 5% triethylamine. The Vmax for the formation of peptide bonds in acetonitrile is approximate
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