Carbohydrate Protein Conjugates (CPC): The Design of New Materials to Stabilize Enzymes
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Carbohydrate Protein Conjugates (CPC): The Design of New Materials to Stabilize Enzymes Tara G. Hill,t Peng Wang,t,§ Lynn M. Oehlert,§ Michael E. Huston,t Charles A. Wartchow,t M. Bradley Smith,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. Intense efforts have been directed at the stabilization of proteins because of their potential uses in organic synthesis, diagnostics, and the pharmaceutical industry. These efforts have resulted in a number of methods to stabilize enzymes including adsorbtion on inert supports or ion exchange resins, entrapment within a gel (with or without crosslinking of the gel or protein), covalent attachment to beads or polymeric supports, inclusion in micelles, chemical derivatization of the protein and mutagenesis. 1,2 However, these methods do not provide a general approach to solving the problem of protein stability. We believed that the multi-site attachment of a carbohydrate-based macromolecule to the surface of a protein would provide structural stability and a water-like microenvironment for the protein under harsh reaction conditions. As part of our program for the preparation and study of biological macromolecules, we have discovered a series of carbohydrate-based materials that stabilize proteins. We report here the synthesis of a series of carbohydrate-based polymers, the coupling of proteases to these materials through their e-lysine residues, and their stability at elevated temperatures in distilled water and buffered solutions. The following paper demonstrates the use of these carbohydrate protein conjugates of proteases (CPC-proteases) for the catalytic formation of peptide bonds in organic media. We prepared a series of aminoglucose-based monomers, la-3a, by reaction of the appropriate amine with methacryloyl chloride in methanol. 3 Treatment of la-3a with ammonium persulfate in water at temperatures from 50C - 70'C gave the carbohydrate-based macromolecules lb-3b in yields of >80%.4,5 These water-soluble materials contain a high -1tThe Ohio State University, Department of Chemistry, Columbus, OH 43210 *University 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. 01991 Materials Research Society
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density of masked aldehyde functionality and have absolute molecular weights of >4 x 106 with polydispersities -OH
(j'>-OH
NaBH 3 CN
I••O C -IOH /-H --NH
1-CPC(ENZYME) - 3-CPCWNZYME)
lb -- 3b
la-3a
O
ENZYME-Chymotrypsln (CT)
Trypsin (Try) SubtilIsin BPN' (BPN')
C.OH
0
=
S.Obo
HO O pH
o OH
HO O?*H
OH 2
OH)-OH OH 3
respective carbohydrate protein conjugates.8 The kcat and Km values of the native enzymes
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