Polyclonal antibody-catalysed hydrolysis of a β-lactam

Article abstract of DOI:10.1039/b110135h

We report the first example of antibody-catalysed hydrolysis of a β-lactam where the antibodies were generated by a simple transition-state analogue; in this example the antibodies are polyclonal.

Full text of DOI:10.1039/b110135h

Polyclonal antibody-catalysed hydrolysis of a b-lactam
a
b
a
c
d
Elizabeth L. Ostler, Marina Resmini, Guillaume Boucher, Nickolas Romanov, Keith Brocklehurst
a
and Gerard Gallacher*
a
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb,
Brighton, UK BN2 4GJ. E-mail: g.gallacher@bton.ac.uk
Department of Chemistry, Queen Mary, University of London, Mile End Rd, London, UK E1 4NS
Erevanskkaya 14-a, K25 03087. Kiev-87, Ukraine
Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary,
University of London, Mile End Rd, London, UK E1 4NS
b
c
d
Received (in Cambridge, UK) 6th November 2001, Accepted 13th December 2001
First published as an Advance Article on the web 14th January 2002
We report the first example of antibody-catalysed hydrolysis
of a b-lactam where the antibodies were generated by a
simple transition-state analogue; in this example the anti-
bodies are polyclonal.
product inhibition is unlikely because the ring-opened product
is very different from both the substrate and the phosphate
transition state analogue, and lastly (vi) it is intermediate in
reactivity between a nitrophenyl ester and a nitroanilide. Given
these advantages it is surprising that this structure has not been
the subject of catalytic antibody investigations until now.
Since the first demonstration1,2 of catalytic antibodies, the field
has grown rapidly with large numbers of catalytic antibodies
being described3 for a range of chemical reactions. However,
although there is a plethora of antibodies that catalyse the
,4
5
–11
hydrolysis of esters and carbonates, only a few
have been
generated that catalyse the hydrolysis of amides. Consequently
the production of catalytic antibodies for amide hydrolysis is the
focus of considerable current effort3
,4,12
and success promises a
wealth of applications in medicine and biotechnology.
We here report that hydrolysis of the b-lactam 3 is
accelerated by the anti-nitrophenylphosphate antibodies at pH
values between 8 and 10. This rate acceleration is completely
inhibited by the phosphate transition state analogue 1b,
demonstrating that the observed activity is due to a binding site
specific for the transition state analogue 1b (preliminary
Here we report that antibodies that were designed to catalyse
the hydrolysis of a carbonate have now been found to catalyse
also the hydrolysis of a structurally related amide, a b-lactam.
This is the first indication that it is possible to obtain catalytic
antibodies with b-lactamase activity by immunisation with a
simple transition-state analogue. We have shown previously13
that polyclonal antibodies generated by the transition state
analogue 1a catalyse the hydrolysis of the carbonate 2, and these
catalytic antibodies have been the subject of extensive in-
experiments give a value for K of 1.5mM). For the antibody
i
preparation 270-26,13 (2 mM IgG, pH 9, 37 °C) a plot of initial
rate for the catalysed reaction against concentration of substrate
3
M
is shown in Fig. 1. At this pH the catalysed reaction has K =
0 mM and kcat = 1.3 3 10
vestigation.1
4,15
We work with total IgG purified from sheep
13
2
5 21
3
s , on the assumption that the
antisera (via salt fractionation, and chromatography over
protein G then Sephadex), and our most recent report16
established that a polyclonal catalytic antibody preparation
contained at most 8% catalytic antibodies, and probably less
than 1%.
concentration of catalytic sites = 2 3 [IgG]. If the proportion of
16
catalytic antibodies is assumed to be 8% then kcat = 1.6 3
2
4
21
1
0
s . This antibody activity is sustained for multiple
turnovers (the catalytic reaction was followed for more than 6
turnovers).
Further evidence that the catalysed hydrolysis of the b-lactam
is indeed antibody-mediated was provided by investigations of
substrate specificity. The hydrolyses of N-(2-nitrophenyl)azeti-
17
din-2-one 5 (the 2-substituted analogue) and 4-nitrophe-
nylformanilide 6 were not catalysed by the catalytic antibody
preparation 270-26. It is of particular interest that the for-
manilide 6 is more labile towards hydrolysis than the substrate
b-lactam 3. One possible explanation for this observation might
Current work involves studies with a variety of 4-ni-
troanilides and 4-nitrophenyl esters and carbonates, with the
aim of determining the minimum recognition features required
for catalysis by these antibody preparations. One such com-
pound is N-(4-nitrophenyl)azetidin-2-one 3.17 This b-lactam
has the following advantages as a substrate for catalytic
antibody investigations: (i) it is a reactive amide whose
hydrolysis is activated by both ring strain and electronic effects,
Fig. 1 Plot of initial rate against concentration of substrate 3. The points
(
ii) the product of hydrolysis, the amino acid carboxylate 4,
represent values obtained in initial rate experiments corrected for back-
contains a nitroaniline chromophore which facilitates kinetic
26 21
ground hydrolysis (knon-cat = 6.4 3 10
s ), and the curve represents best
measurements, (iii) base promoted hydrolysis, in the absence of
211
21
fit values of Vmax (5.11 ± 0.78 3 10
M s ) and K
M
(30 ± 10mM)
catalyst, is well characterised,1
2,18
(iv) the nitrophenyl group
obtained by fitting the data to the Michaelis–Menten equation by non-linear
provides recognition and binding in antibody studies, (v)
regression using an unweighted least squares analysis (r2 = 0.974).
2
26
CHEM. COMMUN., 2002, 226–227
This journal is © The Royal Society of Chemistry 2002

be a different binding mode in the case of the formanilide such
that catalysis does not occur. These data demonstrate that the
catalysis is both specific for the 4-nitro substituent and is
extremely limited in tolerance for alternative reaction centres,
characteristics consistent with antibody catalysis.
Notes and references
1
2
3
4
5
6
A. Tramontano, K. D. Janda and R. A. Lerner, Science, 1986, 234,
566–1570.
S. J. Pollack, J. W. Jacobs and P. G. Schultz, Science, 1986, 234,
570–1573.
R. A. Lerner, S. J. Benkovic and P. G. Schultz, Science, 1991, 252,
59–667.
J. D. Stevenson and N. R. Thomas, Nat. Prod. Rep., 2000, 17,
35–577.
K. D. Janda, D. Schloeder, S. J. Benkovic and R. A. Lerner, Science,
988, 241, 1188–1191.
1
1
6
5
1
M. T. Martin, T. S. Angeles, R. Sugasawara, N. I. Aman, A. D. Napper,
M. J. Darsley, R. I. Sanchez, P. Booth and R. C. Titmas, J. Am. Chem.
Soc., 1994, 116, 6508–6512.
We have demonstrated antibody catalysis of the hydrolysis of
an N-aryl-b-lactam by antibodies raised to an immunogen that
was not designed for this purpose. There have been only two
other reports of antibody-catalysed hydrolysis of b-lactams.
7 F. Benedetti, F. Berti, A. Colombatti, C. Ebert, P. Linda and F. Tonizzo,
Chem. Commun., 1996, 1417–1418.
8 C. S. Gao, B. J. Lavey, C. H. L. Lo, A. Datta, P. Wentworth and K. D.
Janda, J. Am. Chem. Soc., 1998, 120, 2211–2217.
10
One of these antibodies was produced by an antiidiotypic
approach, and the other11 via reactive immunisation. Therefore,
9 B. Gong, P. G. Schultz and S. S. Yoon, J. Korean Chem. Soc., 1998, 42,
4
62–466.
0 B. Avalle, D. Thomas and A. Friboulet, FASEB J., 1998, 12,
055–1060.
1 F. Tanaka, H. Almer, R. A. Lerner and C. F. Barbas, Tetrahedron Lett.,
999, 40, 8063–8066.
the example described above is the first report of antibodies
with b-lactamase activity generated via immunisation with a
simple transition-state analogue. The use of immunogens more
closely related to the substrate structure (e.g. cyclic phospho-
nates) would be expected to produce antibodies with improved
b-lactamase activity. The combination of stability (t0.5 ~ 2
weeks at pH 8) and susceptibility to antibody-catalysed
hydrolysis makes N-aryl-b-lactams attractive targets for the
development of prodrugs that can be activated by catalytic
antibodies. For example, we are now investigating the b-lactam
1
1
1
1
12 G. M. Blackburn and S. X. Deng, Biochem. Soc. Trans., 1993, 21,
1102–1107; O. Ersoy, R. Fleck, M. J. Blanco and S. Masamune, Bioorg.
Med. Chem., 1999, 7, 279; V. M. Yomtova, S. D. Kyurkchiev, N. N.
Slavcheva and I. P. Ivanov, Biocatal. Biotransform., 1998, 16, 307.
1
3 G. Gallacher, C. S. Jackson, M. Searcey, G. T. Badman, R. Goel, C. M.
Topham, G. W. Mellor and K. Brocklehurst, Biochem. J., 1991, 279,
8
71–881.
7
as a potential nitrogen mustard prodrug for use in Antibody-
1
4 G. Gallacher, C. S. Jackson, M. Searcey, R. Goel, G. W. Mellor, C. Z.
Smith and K. Brocklehurst, Eur. J. Biochem., 1993, 214, 197–207.
5 M. Resmini, R. Vigna, C. Simms, N. J. Barber, E. P. HagiPavli, A. B.
Watts, C. Verma, G. Gallacher and K. Brocklehurst, Biochem. J., 1997,
19
Directed Abzyme Prodrug Therapy (ADAPT).
1
3
26, 279–287.
1
1
6 M. Resmini, S. Gul, S. Carter, S. Sonkaria, C. M. Topham, G. Gallacher
and K. Brocklehurst, Biochem. J., 2000, 346, 117–125.
7 Synthesised according to the method described in Hiroki Takahata,
Yoshinori Ohnishi and Takao Yamazaki, Heterocycles, 1980, 14(4),
4
67–469.
1
8 G. M. Blackburn and J. D. Plackett, J. Chem. Soc., Perkin Trans. 2,
1
3
973, 1366–1371; T. J. Broxton and L. W. Deady, J. Org. Chem., 1974,
9, 2767; A. R. Butler, K. A. Freeman and D. E. Wright, J. Chem. Soc.,
We thank The Wellcome Trust for financial support (grant
reference 060295). Guillaume Boucher was supported by a
TMR award (ERBFMRXCT 980 193) from the European
Commission.
Perkin Trans. 2, 1977, 765–9.
19 P. Wentworth, A. Datta, D. Blakey, T. Boyle, L. J. Partridge and G. M.
Blackburn, Proc. Natl. Acad. Sci., 1996, 93, 799–803.
CHEM. COMMUN., 2002, 226–227
227