0
–1
–2
–3
–4
–5
the shape of the transition state, yet elicits efficient catalysts, it
is likely to generate the necessary charge complementarity in
the active site. Thus, haptenic tetrahedral geometry is a
desirable feature but not a prerequisite to generating hydrolytic
antibodies. This study, together with the information reported
about a cyclopropenone-containing protease inhibitor, high-
lights the potential applications of the rarely used cycloprop-
enone functionality in the future design of both haptens and
enzyme inhibitors.
We are grateful to Dr S. C. Sinha for early contributions to
this work and to D. M. Kubitz for antibody preparation. We
thank Pharmore Therapeutics and The Skaggs Institute for
Chemical Biology for financial support. F. G. thanks the Lady
Davis foundation for a postdoctoral fellowship.
y = 0.567x –8.041 r2 = 0.988
y = 0.487x –4.523 r2 = 0.982
Catalyzed reaction
k
Background reaction
Notes and References
† E-mail: keinan@scripps.edu
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
pH
1 G. Fisher, Enzyme Mechanisms, ed. M. I. Page and A. Williams, The
Royal Society of Chemistry, London, 1987, p. 227.
Fig. 1 pH–rate profile of 12G2-catalyzed hydrolysis of 6b
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.
Table 1 Kinetic parameters 1025 for the 12G2 catalyzed hydrolysis of the
studied substrates
kun/1025
kcat/1022
min21
Substrate
min21
Km/mm
kcat/kun
6b
7b
8
3.6
7.0
2.7
100
480
90
3.5
1.4
0.3
970
200
110
by HPLC. The background (uncatalyzed) reaction rate constants
(kun) were determined in the presence of 12G2 and 1 (0.001 ml,
EtOH solution, equimolar to the substrate).11 The catalyzed
reaction rate constants (kcat) were obtained from Lineweaver–
Burk plots of the kinetic data.
The pH rate profiles (log k vs. pH) of the catalyzed and
uncatalyzed hydrolysis of 6b between pH 5.5 and 8 (Fig. 1)
show that both reactions have the same linear dependence on
pH. The fact that both lines in Fig. 1 exhibit the same slope
suggests that catalysis does not involve any strain/rehybridiza-
tion effect and both reactions proceed along the same mecha-
nism, namely, general base catalysis.
A comparative study of the substrate range was carried out at
pH 6.5. The kinetic parameters for three different substrates,
derived from the corresponding Lineweaver–Burk plots are
given in Table 1. While rate enhancement of 2–3 orders of
magnitude is achieved with substrates 6b, 7b and 8, no catalysis
is observed with 6a and 7a, a fact that highlights the importance
of the nitro group as a substrate recognition element. Compar-
ison of the kinetic data of 6b and 7b indicates that the presence
of an acetamido group in the substrate decreases both substrate
binding and catalytic efficiency. This observation supports the
above-mentioned conclusion that the antibody cannot distort the
substrate to fit the binding pocket of 12G2. Ester 8 binds to
12G2 as well as 6b does but its catalyzed hydrolysis is one order
of magnitude slower.
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. dH(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 2 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-phenylcyclo-
propenone (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. dH(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+).
Interestingly, very little is known about natural products
containing a cyclopropenone fragment.12 A papain inhibitor
possessing a cyclopropenone moiety exhibits a Ki in the
submicromolar level. The mechanism for the action of this
unique protease inhibitor still remains unclear.13
Catalytic antibodies offer unique opportunities to examine
mechanistic hypotheses and the relative importance of indivi-
dual design elements in catalysis. We have shown here that
antibodies elicited against hapten 1 catalyze ester hydrolysis
with a 1000-fold rate enhancement. As hapten 1 does not mimic
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.
Received in Cambridge, UK, 9th January 1998; 8/00274F
866
Chem. Commun., 1998