J. Am. Chem. Soc. 1997, 119, 10251-10252
10251
Reactive Immunization Strategy Generates
Antibodies with High Catalytic Proficiencies
differing enantioselectivities would be generated and that during
screening, antibodies with the required (+)-(S)-selectivity could
4
a,e
be highlighted.
Following synthesis and coupling of hapten 3 to carrier
proteins, 129 Gix mice were immunized with a keyhole limpet
Chih-Hung L. Lo, Paul Wentworth, Jr., Kyung Woon Jung,
Juyoung Yoon, Jon A. Ashley, and Kim D. Janda*
9
+
haemocyanin (KLH)-3 conjugate. Monoclonal antibodies were
10
produced by standard techniques and purified from hybridoma
Department of Chemistry
The Scripps Research Institute and
The Skaggs Institute for Chemical Biology
1
1
supernatants as described previously.
Of 20 monoclonal
antibodies that bound to a bovine serum albumin (BSA)-3
conjugate, 12 catalyzed the hydrolysis of the substrate rac-2.12
The kinetic parameters and enantioselectivities of the five most
active antibody catalysts were studied in detail (Table 1). In
all cases the catalysis was competitively inhibited by both
1
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La Jolla, California 92037
ReceiVed May 5, 1997
1
3
phosphonates 4 and 5.
This cross-reactivity supports the
notion that catalysis occurs in the antibody combining site and
that the RI strategy involves antibody recognition of structural
components along the hydrolysis coordinate of the phosphodi-
ester hapten. A defining precept of RI is the installation of
chemical reactivity within the antibody binding-site to the
hapten. This was determined kinetically for antibody 15G12,
Acyl-transfer processes are ubiquitous in both biochemical
metabolism and organic chemistry and as such have remained
a major focus for antibody catalysis since the first reports of
1
these programmable proteins appeared in the mid-1980s. The
numerous antigens utilized to elicit catalysts for these reactions
can be broadly categorized as being either transition-state
analogues (TSA) or bait and switch haptens.2 To expand the
scope and improve the catalytic abilities of antibodies we are
directing efforts toward new strategies for immunogen design.
Recently we reported a method termed reactive immunization
which catalyzes the hydrolysis of the hapten analogue 4 (K )
m
-3
-1
2
32 µM, kcat ) 2.1 × 10 min ). Interestingly, no phospho-
nylation of 15G12 was detected during this process, and in
3
contrast to our previous report, no acyl-antibody intermediate
was detected, suggesting either that acylation is rate-limiting
or that the chemical reactivity engendered by RI, in this case,
is not covalent in origin.
(RI), which utilizes a labile phosphonate diester hapten which
can either hydrolyze at physiological pH or trap a nucleophile
at the B-cell level of the immune response.3 We have reasoned
that the combination of these events, which incorporate chemical
reactivity and transition state stabilization into antibody selec-
tion, may encompass a better overall strategy for catalyst
The most active members of the panel have enhancement
5
ratios, kcat/kuncat, of greater than 10 , highlighting the catalytic
power imparted by RI. Antibodies 5A9 and 6C7 have high
substrate specificity, which coupled with their high turnover
numbers, kcat, results in reaction specificity constant values, kcat/
3
generation. In the present study we combine the known
4
stereoselectivity of antibodies with the new RI approach to
5
-1
-1
14a
generate catalysts for a kinetic resolution of a racemic mixture
Km > 10 M min . Wolfenden has defined the catalytic
proficiency of a biocatalyst by division of its second-order
specificity constant with the background rate of substrate
hydrolysis. By this rationale, the three most active monoclonals,
5A9, 6C7, and 15G12 have proficiency constants of 4.66, 2.75,
5
of p-methylsulfonylphenyl esters of naproxen 1 (Scheme 1).
6
Chemical synthesis of 1 leads to a racemic mixture and
consequently there is a considerable effort being devoted to
improve the methods for its asymmetric synthesis and resolu-
tion.7
9
-1
14b
and 2.20 × 10 M , respectively.
These values are among
the highest achieved by catalytic antibodies for any acyl transfer
process, whether elicited by transition state analogue, bait and
switch, or heterologous immunization approaches,15 and are
comparable with a number of lipases and esterases (10 -10
M ), including A-esterase (EC 3.1.1.2).
The hapten chosen for immunization, phosphonate diester 3,
hydrolyzes under physiological conditions with expulsion of a
p-methylsulfonylphenol leaving group (pKa 7.8).8 By immuniz-
9
12
ing with a racemate, it was expected that antibody catalysts with
-
1
16a
16b
(
1) (a) Tramontano, A.; Janda, K. D.; Lerner, R. A. Science 1986, 234,
At present, industrial production of (S)-naproxen 1 involves
1
566-1570. (b) Pollack, S. J.; Jacobs, J. W.; Schultz, P. G. Science 1986,
diastereomeric crystallization of a racemic acid mixture.6
2
34, 1570-1573.
Several reports of enzymatic resolution of naproxen alkyl esters
(
2) (a) Janda, K. D.; Shevlin, C. G.; Lo, C.-H. L. In ComprehensiVe
Supramolecular Chemistry; Yakito, M., Ed.; Pergamon: London, 1996; Vol.
4
1
, pp 43-78. (b) Lavey, B. J.; Janda, K. D. ACS. Symp. Ser. 1995, 604,
(8) The phosphonate diester 3 hydrolyzes to its monoester with a half-
life, t1/2, of 2 d in phosphate-buffered saline (PBS) (200 mM, pH 7.4) at
310 K as determined by HPLC.
(9) The synthesis and spectroscopic data for hapten 3 is reported in the
Supporting Information. Protein conjugates were prepared by the sulfo-
NHS ester method in PBS (10% DMF).
(10) (a) K o¨ hler, G.; Howe, S. C.; Milstein, C. Eur. J. Immunol. 1976, 6,
292-295. (b) K o¨ hler, G.; Milstein, C. Ibid. 1976, 6, 511-519.
(11) Janda, K. D.; Schloeder, D.; Benkovic, S. J.; Lerner, R. A. Science
1988, 241, 1188-1191.
(12) BSA an “off the shelf protein” which has been reported to accelerate
medium-sensitive reactions (Hollfelder, F.; Kirby, A. J.; Tawfik, D. S.
Nature 1996, 383, 60-63. Kikuchi, K.; Thorn, S. N.; Hilvert, D. J. Am.
Chem. Soc. 1996, 118, 8184-8185) does not catalyze the hydrolysis of
rac-2.
(13) For assay conditions and Ki data see Supporting Information.
(14) (a) Radzicka, A. R.; Wolfenden, R. A. Science 1995, 267, 90-93.
(b) Kinetic investigation of the antibodies in Table 1 revealed neither
accumulation of detectable acylated intermediate nor pronounced curving
of the progress curves during antibody-catalyzed hydrolysis of 2, both of
which lends support to this analysis [Koshland, D. E., Jr. Bio. ReV. Camb.
Philos. 1953, 28, 416-418. Wolfenden, R. A. Annu. ReV. Biophys. Bioeng.
1976, 5, 271-306].
(15) Data taken from the following key reviews: Thomas, N. R. Appl.
Biochem. Biotechnol. 1994, 47, 345-372 and references cited therein.
Stewart, J. D.; Benkovic, S. J. Nature 1995, 375, 388-391 and references
cited therein.
23-137.
(
3) Wirsching, P.; Ashley, J. A.; Lo, C.-H. L.; Janda, K. D.; Lerner, R.
A. Science 1995, 270, 1775-1782.
(
4) For leading papers on antibody stereoselectivity, see: (a) Janda, K.
D.; Benkovic, S. J.; Lerner, R. A. Science 1989, 244, 437-440. (d) Shoji,
I.; Weinhouse, W. I.; Janda, K. D.; Lerner, R. A.; Danishefsky, S. J. J. Am.
Chem. Soc. 1990, 113, 7763-7764. (e) Fujii, I.; Lerner, R. A.; Janda, K.
D. J. Am. Chem. Soc. 1991, 113, 8258-8529. (f) Pollack, S. J.; Hsuin, P.;
Schultz, P. G. J. Am. Chem. Soc. 1992, 114, 2257-2258.
(
5) Profens are a widely prescribed class of non-steroidal anti-inflam-
matory drugs (NSAIDs) for the treatment of rheumatoid arthritis [Payan,
D. G.; Shearn, M. A. In Basic and Clinical Pharmacology; Katzung, B.
G., Ed.; Appleton and Lange: London, 1989; pp 431-432]. Naproxen 1,
a prominent member of this drug class, exhibits both stereoselective activity
and disposition [(a) Wechter, W. J.; Loughhead, R. J.; Reischer, G. J.;
VanGiessen, G. J.; Kaiser, D. G. Biochem. Biophys. Res. Commun. 1974,
6
1, 833-835. (b) Roszowski, A. P.; Rooks, W. H., II; Tomolonis, A. J.;
Miller, L. M. J. Pharmacol. Exp. Ther. 1971, 179, 114-117].
6) Harrison, I. T.; Lewis, B.; Nelson, P.; Rooks, W.; Roszowski, A.;
Tomolonis, A.; Fried, J. H. J. Med. Chem. 1970, 13, 203-207.
7) (a) Gu, Q.-Ming; Chen, C.-S.; Sih, C. J. Tetrahedron Lett. 1986, 27,
(
(
1
763-1766. (b) Hern a´ iz, M. J.; S a´ nchez-Montero, J. M.; Sinisterra, J. V.
Tetrahedron Lett. 1994, 50, 10749-10760. (c) Manimaran, T.; Stahly, G.
P. Tetrahedron: Assymetry 1993, 4, 1949-1954. (d) Pirkle, W. H.; Welch,
C. J.; Lamm, B. J. Org. Chem. 1992, 5, 3854-3860. (e) Pirkle, W. H.;
Liu, Y. J. Org. Chem. 1994, 59, 6911-6916.
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