Use of antibodies to dissect the components of a catalytic event. The cyclopropenone hapten

Abstract

Antibodies elicited against the planar cyclopropenone hapten 1 efficiently catalyze ester hydrolysis, highlighting the importance of charge rather than shape complementarity as a design element of hydrolytic antibodies.

References

1. G. Fisher, Enzyme Mechanisms, ed. M. I. Page and A. Williams, The Royal Society of Chemistry, London, 1987, p. 227.
2. P. G. Schultz and R. A. Lerner, Science, 1995, 269, 1835; J. R. Jacobsen and P. G. Schultz, Curr. Opin. Struct. Biol., 1995, 5, 818; G. MacBeath and D. Hilvert, Chem. Biol., 1996, 3, 433; E. Keinan and R. A. Lerner, Israel J. Chem., 1996, 36, 113; N. R. Thomas, Nat. Prod. Rep., 1996, 479.
3. K. D. Janda, M. I. Weinhouse, D. M. Schloeder, R. A. Lerner and S. J. Benkovic, J. Am. Chem. Soc., 1990, 112, 1274; A. I. Khalaf, G. R. Proctor, C. J. Suckling, L. H. Bence, J. I. Irvine and W. H. Stimson, J. Chem. Soc., Perkin Trans. 1, 1992, 1475.
4. For the use of haptens with trimethylammonium and phosphate groups on two vicinal carbon atoms, see: H. Suga, O. Ersoy, T. Tsumuraya, J. Lee, A. J. Sinskey and S. Masamune, J. Am. Chem. Soc., 1994, 116, 487.
5. For the use of two differently charged haptens in heterologous immunization to generate two catalytic residues in the antibody's active site, see: H. Suga, O. Ersoy, S. F. Williams, T. Tsumuraya, M. N. Margolies, A. J. Sinskey and S. Masamune, J. Am. Chem. Soc., 1994, 116, 6025; T. Tsumuraya, H. Suga, S. Meguro, A. Tsunakawa and S. Masamune, J. Am. Chem. Soc., 1995, 117, 11 390.
6. K. T. Potts and J. S. Baum, Chem. Rev., 1974, 74, 189.
7. H. Tsukada, H. Shimanouchi and Y. Sasada, Tettrahedron Lett., 1973, 27, 2455; H. Tsukada, H. Shimanouchi and Y. Sasada, Chem. Lett., 1974, 639.
8. C. W. Bird and A. F. Hamer, Org. Prep. Proced. Int., 1970, 2, 79.
9. Preparation of 5∶4(0.55 g, 2.5 mmol) was dissolved in dry THF (25 ml). Glutaric anhydride (1.42 g, 12.5 mmol) was added and the mixture was stirred at room temp. for 12 h. Work-up and purification by column chromatography (silica gel, EtOAc–hexane 4∶1) afforded 5(0.21 g, 25%) in the form of a colorless solid. δ_H(CDCl₃ with 2 drops CD₃OD) 8.05 (s 1 H), 7.90 (s br, 2 H), 7.84 (s br, 1 H), 7.59 (d, J 9.72, 1 H), 7.50 (s, 3 H), 7.41 (t, J 7.48, 1 H), 2.35 (t, J 7.68, 2 H), 2.31 (t, J 6.76, 2 H), 1.91 (t, J 7.80, 2 H); m/z(ESI) 334 (M – H⁺).
10. Preparation of 1: sodium nitrate (0.025 g, 0.298 mmol) was added to a stirred solution of 2-[3-(4-carboxybutanamido)phenyl]-3-phenylcyclopropenone (0.1 g, 0.298 mmol) in conc. sulfuric acid (1 ml). The mixture was stirred for 1 h at room temp., then heated to 100 °C for 1 h and poured into ice–water. The precipitate was washed with water, dissolved in EtOAc and passed through a silica gel bed using EtOAc to give 1(0.053 g, 31%) in the form of a colorless solid. δ_H(CD₃OD) 8.87 (s, 1 H), 8.52 (d br, 1 H, J 8.28), 8.50 (s, 1 H), 8.44 (d br, 1 H, J 7.04), 7.95 (t, 1 H, J 7.72), 7.82 (d, 1 H, J 8.04), 7.76 (d, 1 H, J 7.44), 7.66 (s, 1 H), 7.61 (t, 1 H, J 7.80); m/z(LSI) 381 (MH⁺).
11. No change in the rate of the 12G2-catalyzed hydrolysis of 6b could be detected in the presence of ethanol (0.001 ml). Partial inhibition of the reaction was observed in the presence of compounds 2, 3 and 5(1 equiv. with respect to the substrate).
12. T. Okuda, K. Yokose, T. Furumai and H. Maruyama, J. Antibiot., 1984, 37, 718; H. Tokuyama, M. Isaka, E. Nakamura, R. Ando and Y. Morinaka, J. Antibiot., 1992, 45, 1148.
13. R. Ando, Y. Morinaka, H. Tokuyama, M. Isaka and E. Nakamura, J. Am. Chem. Soc., 1992, 115, 1174.