Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-12-09
2001-04-03
Patterson Jr., Charles L. (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S188500
Reexamination Certificate
active
06210938
ABSTRACT:
FIELD OF INVENTION
The invention relates to antibody catalyzed aldol reactions. More particularly the invention relates to enantio- and diastereo-selective aldol reactions and to antibodies that catalyzed such reactions.
BACKGROUND
The aldol reaction is a C—C bond forming reaction that is key to the practice of synthetic organic chemistry. For reviews of the aldol reaction, see: a) S. Masamune, et al.,
Angew. Chem. Int. Ed. Engl
. 1985, 24, 1-30; b) C. H. Heathcock,
Aldrichim. Acta
1990, 23, 99-111; c) D. A. Evans,
Science
1988, 240, 420-426; d) C. H. Heathcock, et al, in
Comprehensive Organic Synthesis
, Vol. 2 (Eds. B. M. Trost, I. Fleming, C. H. Heathcock), Pergamon, Oxford, 1991, pp. 133-319; e) C. J. Cowden, et al.,
Org. React
. 1997, 51, 1; f) A. S. Franklin, et al.,
Contemp. Org. Synth
. 1994, 1, 317. As a result of its utility, intensive effort has been applied to the development of catalytic enantioselective variants of this reaction. Catalytic enantioselective aldol reactions are typically accomplished with preformed enolates and chiral transition metal catalysts (S. G. Nelson,
Tetrahedron: Asymmetric
1998, 9, 357-389; A. Yanagisawa, et al.,
J. Am. Chem. Soc
. 1997, 119, 9319-9320; E. M. Carreira, et al.,
J. Am. Chem. Soc
. 1995, 117, 3649-3650; D. A. Evans, et al.,
J. Am. Chem. Soc
. 1997, 119, 10859-10860; and D. J. Ager, et al.,
Asymmetric Synthetic Methodology
(CRC Press, Inc.: Florida, 1996). Alternatively, catalytic enantioselective aldol reactions may be achieved with natural aldolase enzyme catalysts (C.-H. Wong, et al.,
Enzymes in Synthetic Organic Chemistry
(Pergamon, Oxford, 1994); C.-H. Wong, et al.,
Angew. Chem. Int. Ed. Engl
. 1995, 34, 412-432; and W.-D. Fessner,
Current Opinion in Chemical Biology
1998, 2, 85-89). With transition metal catalyzed aldol reactions, enantioselectivity is readily reversed by exchange of the chiral ligand that directs the stereochemical course of the reaction. With enzymes, however, a general approach to the reversal of enantioselectivity is not available.
To address the problem of the de novo generation of aldolase enzymes, a strategy of reactive immunization using &bgr;-diketone haptens to program into antibodies a chemical mechanism analogous to that used by nature's Class I aldolase enzymes was developed. The chemistry of this class of enzymes is based on a unique chemically reactive lysine residue that is essential to the covalent mechanism of these catalysts.
FIG. 1
illustrates a prior art hapten, viz., compound 1, having a &bgr;-diketone functionality employable as a reactive immunogen capable of trapping a chemically reactive lysine residue in the active site of an antibody. Covalent trapping was facilitated by intramolecular hydrogen bonding that acts to stabilize an enaminone in the active site of the antibody. The chemical mechanism leading up to the stabilized enaminone should match that of Class I aldolases over this portion of the reaction coordinate. Given the mechanistic symmetry around the C—C bond forming transition state, this approach allowed for the programming of this multi-step reaction mechanism into antibodies (C. F. Barbas III, et al.,
Science
1997, 278, 2085-2092). The efficient antibody catalysts that resulted, ab38C2 (Aldrich reagent) and ab33F12 have been shown to catalyze a broad array of enantioselective aldol and retro-aldol reactions (R. Björnestedt, et al.,
J. Am. Chem. Soc
. 1996, 118, 11720-11724; G. Zhong, et al.,
J. Am. Chem. Soc
. 1997, 119, 8131-8132; T. Hoffmann, et al.,
J. Am. Chem. Soc
. 1998, 120, 2768-2779; and S. C. Sinha, et al.,
J. Am. Chem. Soc
. 1999, submitted). For an alternative aldolase antibody strategy see J. L. Reymond,
Angew. Chem. Int. Ed. Engl
. 1995, 34, 2285-2287 or J. L. Reymond, et al.,
J. Org. Chem
. 1995, 60, 6979.
What is needed is a method for increasing the repertoire of catalysts for this reaction. In particular, antibodies with antipodal reactivity are needed. What is needed is a new hapten design concept for providing more efficient reaction programming.
SUMMARY
It is disclosed herein that a limitation of the design of prior art hapten 1 is that it does not address the tetrahedral geometry of the rate-determining transition state of the C—C bond forming step (J. Wagner, et al.,
Science
1995, 270, 1797-1880). For discussions of the transition state geometry of the aldol reaction, see: a) H. E. Zimmerman, et al.,
J. Am. Chem. Soc
. 1957, 79, 1920; b) S. E. Denmark, et al.,
J. Am. Chem. Soc
. 1991, 113, 2177-2194 and references therein; c) C. Gennari, et al.,
Tetrahedron
1992, 48, 4439-4458.
Illustrated in
FIG. 1
is a novel sulfone &bgr;-diketone hapten, viz., compound 2, which overcomes this limitation by containing structural features common to the transition state analog approach that has been successful for so many reactions (P. G. Schultz and R. A. Lerner,
Science
1995, 269,1835-1842; and N. R. Thomas,
Nat. Prod. Rep
. 1996, 13, 479-511). The sulfone &bgr;-diketone hapten 2 also includes the &bgr;-diketone functionality, which is key to the reactive immunization strategy. The tetrahedral geometry of the sulfone moiety in hapten 2 mimics the tetrahedral transition state of C—C bond forming step and therefore facilitates nucleophilic attack of the enaminone intermediate on the acceptor aldehyde (FIG.
2
).
It is disclosed herein that combining transition state analogy and reactive immunization design into a single hapten results in an increase with respect to both the output of catalysts from the immune system as well as their efficiency as catalysts. This strategy resulted in the characterization of the most proficient antibody catalysts prepared to date. Antibodies 93F3 and 84G3 catalyze a wide array of aldol reactions with ee's in most cases studied exceeding 95%. With acetone as the aldol donor substrate a new stereogenic center is formed by attack on the re-face of the aldehyde, providing the antipodal complement of ab38C2 in aldol reactions. Through aldol and retro-aldol reactions both aldol enantiomers may be accessed. These catalysts are shown to provide access to a wide variety of enantiomerically enriched synthons with application to natural product syntheses.
One aspect of the invention is directed to a hapten that combines a structure that mimics a transition state of an aldol reaction as found in Class I aldolases together with a structure employable in a reactive immunization. In a preferred embodiment, the hapten is represented by the following structure:
In the above structure, n is greater than or equal to 2 and less than or equal to 8. Alternatively, n may be greater than or equal to 4 and less than or less than or equal to 6. In a preferred embodiment, n is five.
Another aspect of the invention is directed to a transition state immunoconjugate represented by the following structure:
In the above structure, n is greater than or equal to 2 and less than or equal to 8; alternative, n may be greater than or equal to 4 and less than or equal to 6; or alternatively, n is five. A preferred carrier protein is keyhole limpet hemocyanin (KLH).
Another aspect of the invention is directed to a process for producing a catalytic monoclonal antibody for catalyzing an aldol reaction. In the first step of the process, an immune response is elicited within an immune responsive subject by injecting a sterile solution of a hapten-carrier protein. The hapten-carrier protein is of a type which includes a sulfone &bgr;-diketone hapten. In a preferred mode, the hapten-carrier protein is represented by the following structure:
Then, an antibody producing cell which expresses a catalytic antibody for catalyzing the aldol reaction is isolated and cloned from the immune responsive subject. And then, aldolase catalytic antibody is isolated as it is expressed by the antibody producing cell isolated and cloned in the previous step.
Another aspect of the invention is directed to antibody molecules or molecules containing antibody combining site portions that catalyze an aldol addition reaction. The antibody mol
Barbas Carlos F.
Lerner Richard A.
Zhong Guofu
Lewis Donald G.
Patterson Jr. Charles L.
The Scripps Research Institute
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