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Carbohydrate Protein Conjugates (CPC): The Design of New Materials to Stabilize Enzymes

Published online by Cambridge University Press:  15 February 2011

Tara G. Hill ,
Peng Wang ,
Lynn M. Oehlert ,
Michael E. Huston ,
Charles A. Wartchow ,
M. Bradley Smith ,
Mark D. Bednarski  and
Matthew R. Callstromt
Tara G. Hill
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210
Peng Wang
Affiliation:
University of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Lynn M. Oehlert
Affiliation:
University of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Michael E. Huston
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210
Charles A. Wartchow
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210
M. Bradley Smith
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210
Mark D. Bednarski
Affiliation:
University of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Matthew R. Callstromt
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
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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. 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.

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 218: Symposium V – Materials Synthesis Based on Biological Processes , 1990 , 7
Copyright
Copyright © Materials Research Society 1991

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References

References and Footnotes

1. For selected reviews on enzyme immobilization methods and studies on immobilized enzymes, see: Chibata, I. Immobilized Enzymes; Halsted Press: New York; 1978. Trevan, M. D. Immobilized Enzymes; Wiley: New York; 1980. Zaborsky, O. R. Immobilized Enzymes; CRC Precs: Cleveland; 1973. Inaki, Y. In Functional Monomers and Polymers; Takemoto, K.; Inaki, Y.; Ottenbrite, R. M., Eds.; Dekker. New York; 1987; p 461. Mosbach, K. Meth. Enzymol. 1976, 44, 999. Larson, P.-O.; Mosbach, K. Meth. Enzymol. 1976, 44, 183. Chang, T. M. S. Meth. Enzymol. 1976, 44, 201. Chibata, I.; Fukui, S.; Wingard, L. B., Jr. Eds. Enzyme Engineering; Plenum: New York; 1982; Vol.6. Carr, P. W.; Bowers, L. D.; Immobilized Enzymes in Analytical and Clinical Chemistry; Wiley: New York; 1980. Katchalski, E.; Silman, I.; Goldman, R. Adv. Enzymol. 1971, 34, 445.Google Scholar
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3. Spectral data for 1a: IR (KBr): 3350, 2925, 1720, 1650, 1615, 1540, 1430, 1360, 1328, 1261, 1215, 1130, 1111, 1078, 1058, 1021, 960, 910, 860, 790, 775, 699 cm−1; 1H NMR (250 MHz, D2O) A 1:1 mixture of α and β anomers, α anomer δ 1.86 (m, 3 H), 3.42 (m, 1 H), 3.71 (m, 2 H), 3.76 (m, 1 H), 3.78 (m, 1 H), 3.89 (dd, J = 3.5 and 10.8 Hz, 1 H), 5.16 (d, J = 3.5 Hz, 1 H), 5.40 (m, 1 H), 5.63 (m, 1 H); β anomer: δ 1.86 (m, 3 H), 3.40 (m, 1 H), 3.55 (m, 1 H), 3.67 (m, 1 H), 3.69 (m, 1 H), 3.81 (m, 1 H), 4.71 (d, J = 8.4 Hz, 1 H), 5.40 (m, 1 H), 5.63 (m, 1 H); 13C NMR (62.9 MHz, D2O) A 1:1 mixture of α and β anomers, δ 20.60 (q), 20.63 (q), 57.14 (d), 59.75 (d), 63.49 (t), 63.65 (t), 72.84 (d), 73.03 (d), 73.41 (d), 74.45 (d), 76.59 (d), 78.82 (d), 93.71 (d), 97.72 (d), 123.93 (t), 124.10 (t), 141.92 (s), 142.17 (s), 175.29 (s), 175.47 (s). Anal. Calcd for C22H49NO6Si4 (α- and β-2-deoxy-2-N-methacrylamido-1,3,4,6- tetra-O-trimethylsilyl-D-glucose): C, 49.35; H, 9.16. Found: C, 49.20; H, 9.28. Spectral data for 2a: IR (KBr): 3337, 2927, 1747, 1657, 1612, 1544, 1452, 1378, 1327, 1277, 1214, 1157, 1052, 935, 872, 805 cm−1; 1H NMR: (250 MHz, D2O) δ 1.88 (s, 3 H), 3.2–4.5 (complex, 7 H), 5.17 (d, J = 3.62 Hz, 1 H), 5.40 (s, 1 H), 5.65 (s, 1 H); 13C NMR: (62.9 MHz, D2O) A mixture of α and β anomers, δ 20.36 (q), 20.74 (q), 56.87 (d), 57.28 (d), 60.24 (d), 63.46 (t), 63.66 (t), 70.67 (d), 70.83 (d), 72.57 (d), 74.15 (d), 79.98 (d), 101.64 (d), 106.71 (d), 123.94 (t), 124.01 (t), 142.18 (s, area 2), 175.74 (s), 175.93 (s). Anal. Calcd for C22H49 NO6Si4 (α- and β-3-deoxy-3-N-methacrylamido-1,2,4,6-tetra-O-trimethylsilyl-D-glucose): C, 49.35; H, 9.16. Found: C, 49.10; H, 9.23. Spectral data for 3a: IR (KBr): 3364, 2914, 1722, 1648, 1609, 1524, 1444, 1364, 1319, 1244, 1214, 1137, 1069, 1040, 924, 794, 737 cm−1; 1H NMR (250 MHz, D2O) δ 1.74 (s, 3 H), 3.0–3.6 (complex, 7 H), 4.14 (d, J = 8.22 Hz, 1 H), 5.25 (s, 1 H), 5.48 (s, 1 H); 13C NMR (75.5 MHz, D2O) δ 18.04 (q, area 2), 40.52 (t), 40.70 (t), 70.16 (d), 71.51 (d), 71.79 (d), 71.84 (d), 73.30 (d), 73.50 (d), 74.23 (d), 75.98 (d), 99.31 (d), 103.39 (d), 121.41 (t, area 2), 139.29 (s, area 2), 172.39 (s), 172.46 (s); HRMS m/z for C22H49NO6Si4 (α- and β-6-deoxy-6-N-methacrylamido-1,2,3,4-tetra-O-trimethylsilyl-D-glucose), calcd 520.2403 [(M-CH3)+], found 520.2439.Google Scholar
4. Spectral data for 1b: IR (KBr) 3371, 2926, 1747, 1631, 1547, 1301, 1201, 1146, 1033, 988, 846, 771 cm−1; 1H NMR (250 MHz, D2O) δ 0.92 (br), 1.73 (br), 3.34 (br), 3.73 (br), 4.98 (d br); 13C NMR (125.8 MNz, D2O) δ 17.51 (q), 45.49 (s), 46.21 (t), 55.33 (d), 61.54 (t), 71.23 (d, area 2), 72.41 (d), 91.06 (d), 180.08 (s). Anal. Calcd for C10H17NO6•H2O: C, 45.28; H, 7.17. Found: C, 43.90; H, 7.59. Gel permeation chromatography found Mn = 4.3 × 106 (Z = 1.3). Spectral data for 2b: IR (KBr) 3356, 2926, 1726, 1639, 1560, 1303, 1201, 1078, 869, 844 cm−1; 1H NMR (250 MHz, D2O) δ 1.1 (br), 1.8 (br), 3.0–4.2 (m), 4.8 (br), 5.1 (br); 13C NMR (62.5 MHz, D2O) δ 19.65 (q), 47.34 (s), 48.00 (t), 57.02 (d), 59.74 (t), 62.71 (d, area 2), 70.17 (d), 93.38(d), 182.27 (s). Anal. Calcd for C10H17NO6•H2O: C, 45.28; H, 7.17. Found: C, 45.59; H, 7.48. Gel permeation chromatography found Mn = 4.2 × 106 (Z = 1.4). Spectral data for 3b: IR (KBr) 3327, 2924, 1727, 1637, 1541, 1442, 1280, 1197, 1132, 1052, 883, 814 cm−1; 1H NMR (200 MHz, D2O) δ 0.90 (br), 1.73 (br), 3.19 (br), 3.41 (br), 3.63 (br), 4.03 (br), 5.17 (br). 13C NMR (62.5 MHz, D2O) δ 17.82 (q), 42.48 (t, area 2), 46.11 (s), 73.10 (d, area 2), 74.06 (d), 75.44 (d), 76.71 (d), 180.55 (s). Anal. Calcd for C10H17NO6: C, 48.58; H, 6.88. Found: C, 48.78; H, 7.10. Gel permeation chromatography found Mn = 3.8 × 107 (Z = 1.3).Google Scholar
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