U.S. patent application number 09/824279 was filed with the patent office on 2001-08-30 for antibody catalysis of enantio- and diastereo-selective aldol reactions.
Invention is credited to Barbas, Carlos F., Lerner, Richard A., Zhong, Guofu.
Application Number | 20010018201 09/824279 |
Document ID | / |
Family ID | 27022979 |
Filed Date | 2001-08-30 |
United States Patent
Application |
20010018201 |
Kind Code |
A1 |
Barbas, Carlos F. ; et
al. |
August 30, 2001 |
Antibody catalysis of enantio- and diastereo-selective aldol
reactions
Abstract
Nine efficient aldolase antibodies were generated using hapten
2. This hapten combines, in a single molecule, structural
components employed for reactive immunization with structural
components employed for forming a transition state analog of the
aldol reaction. Characterization of two of these antibodies reveals
that they are highly proficient (up to 1000-fold better than any
other antibody catalyst) and enantioselective catalysts for aldol
and retro-aldol reactions and exhibit enantio- and
diastereo-selectivities opposite that of antibody 38C2.
Inventors: |
Barbas, Carlos F.; (Del Mar,
CA) ; Lerner, Richard A.; (La Jolla, CA) ;
Zhong, Guofu; (San Diego, CA) |
Correspondence
Address: |
THE SCRIPPS RESEARCH INSTITUTE
OFFICE OF PATENT COUNSEL, TPC-8
10550 NORTH TORREY PINES ROAD
LA JOLLA
CA
92037
US
|
Family ID: |
27022979 |
Appl. No.: |
09/824279 |
Filed: |
April 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09824279 |
Apr 2, 2001 |
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09458367 |
Dec 9, 1999 |
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6210938 |
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09458367 |
Dec 9, 1999 |
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09415453 |
Oct 8, 1999 |
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Current U.S.
Class: |
435/188.5 ;
435/280; 435/326; 530/400; 530/408; 562/430 |
Current CPC
Class: |
C07D 277/36 20130101;
C12P 7/26 20130101; C07B 2200/07 20130101; C07K 16/40 20130101;
C07D 417/06 20130101; C07D 493/04 20130101; C07D 277/24 20130101;
C07D 277/34 20130101; C12P 17/16 20130101; C12P 41/002 20130101;
A61P 35/00 20180101; C12P 17/18 20130101; C12N 9/0002 20130101 |
Class at
Publication: |
435/188.5 ;
435/326; 435/280; 530/400; 530/408; 562/430 |
International
Class: |
C12N 009/00; C12N
005/16; C12P 021/08; C07K 016/00; C07C 317/00 |
Goverment Interests
[0002] This invention was made with government support under the
National Cancer Institute grant No. CA 27489. The U.S. government
has certain rights in the invention.
Claims
What is claimed is:
1. A hapten represented by the following structure: 11where n is
greater than or equal to 2 and less than or equal to 8.
2. A hapten according to claim 1 that mimics the transition state
of an aldol reaction as found in Class I aldolases.
3. A hapten according to claim 1 wherein n is greater than or equal
to 4 and less than or less than or equal to 6.
4. A hapten according to claim 1 wherein n is five.
5. A transition state immunoconjugate represented by the following
structure: 12where n is greater than or equal to 2 and less than or
equal to 8.
6. A transition state immunoconjugate according to claim 5 wherein
n is greater than or equal to 4 and less than or equal to 6.
7. A transition state immunoconjugate according to claim 5 wherein
n is five.
8. A transition state immunoconjugate according to claim 5 wherein
the carrier protein is keyhole limpet hemocyanin (KLH).
9. A process for producing a catalytic monoclonal antibody for
catalyzing an aldol reaction, the process comprising the following
steps: Step A: eliciting an immune response within an immune
responsive subject by injecting a sterile solution of a
hapten-carrier protein, said hapten-carrier protein including a
sulfone .beta.-diketone hapten; then Step B: isolating and cloning
an antibody producing cell from the immune responsive subject of
said Step A which expresses a catalytic antibody for catalyzing the
aldol reaction; and then Step C: isolating the aldolase catalytic
antibody expressed by the antibody producing cell isolated and
cloned in said Step B.
10. A process for producing a catalytic monoclonal antibody
according to claim 9 wherein the hapten-carrier protein is
represented by the following structure: 13where n is greater than
or equal to 2 and less than or equal to 5.
11. A process for producing a catalytic monoclonal antibody
according to claim 9 wherein n is 5.
12. Antibody molecules or molecules containing antibody combining
site portions that catalyze an aldol addition reaction produced
according to the following method: Step A: eliciting an aldolase
immune response within an immune response subject by vaccination
with a sterile solution containing the appropriate concentration of
an aldol transition state immunoconjugate, said aldol transition
state immunoconjugate including a sulfone .beta.-diketone hapten;
then Step B: isolating and cloning an antibody producing cell from
the immune responsive subject of said Step A which expresses a
catalytic aldolase antibody; and then Step C: isolating the
catalytic aldolase antibody expressed by the antibody producing
cell isolated and cloned in said Step B.
13. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the aldol transition
state immunoconjugate is represented by the following structure:
14where n is greater than or equal to 2 and less than or equal to
8.
14. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein n of the aldol
transition state immunoconjugate is 5.
15. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 85A2, having ATCC accession number
HB ______.
16. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 85C7, having ATCC accession number
HB ______.
17. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 92F9, having ATCC accession number
HB ______.
18. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 93F3, having ATCC accession number
HB ______.
19. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 84G3, having ATCC accession number
HB ______.
20. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 84G11, having ATCC accession
number HB ______.
21. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 84H9, having ATCC accession number
HB ______.
22. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 85H6, having ATCC accession number
HB ______.
23. Antibody molecules or molecules containing antibody combining
site portions according to claim 12 wherein the antibody producing
cell of said Step B is hybridoma 90G8, having ATCC accession number
HB ______.
24. An antibody producing cell which secretes antibody molecules or
molecules containing antibody combining site portions that catalyze
an aldol addition reaction, the antibody producing cell being
produced according to the following method: Step A: eliciting an
aldolase immune response within an immune response subject by
vaccination with a sterile solution containing the appropriate
concentration of the aldol transition state immunoconjugate, said
aldol transition state immunoconjugate including a sulfone
.beta.-diketone hapten; and then Step B: isolating and cloning the
antibody producing cell from the immune responsive subject of said
Step A which expresses a catalytic aldolase antibody.
25. An antibody producing cell according to claim 24 wherein the
aldol transition state immunoconjugate is represented by the
following structure: 15where n is greater than or equal to 2 and
less than or equal to 8
26. The antibody producing cell of claim 24 that is a
hybridoma.
27. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 85A2.
28. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 85C7.
29. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 92F9.
30. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 93F3.
31. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 84G3.
32. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 84G11.
33. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 84H9.
34. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 85H6.
35. A hybridoma according to claim 24 having ATCC accession number
HB ______ which is capable of expressing antibody 90G8.
36. An improved process for catalyzing an aldol reaction between
aldehyde and a ketone reactants, wherein the improvement comprises
contacting said aldehyde and ketone reactants with antibody
molecules or molecules containing antibody combining site portions
that catalyze an aldol addition reaction produced according to the
following method: Step A: eliciting an aldolase immune response
within an immune response subject by vaccination with a sterile
solution containing the appropriate concentration of an aldol
transition state immunoconjugate, said aldol transition state
immunoconjugate including a sulfone .beta.-diketone hapten; then
Step B: isolating and cloning an antibody producing cell from the
immune responsive subject of said Step A which expresses a
catalytic aldolase antibody; and then Step C: isolating the
catalytic aldolase antibody expressed by the antibody producing
cell isolated and cloned in said Step B.
37. An improved process for catalyzing an aldol reaction according
to claim 36 wherein the aldol transition state immunoconjugate of
said Step A is represented by the following structure: 16where n is
greater than or equal to 2 and less than or equal to 8.
38. An improved process for catalyzing an aldol reaction according
to claim 36 wherein the ketone is selected from a group consisting
of members represented by the following structures: 17
39. An improved process for catalyzing, an aldol reaction according
to claim 36 wherein the aldehyde is selected from a group
consisting of members represented by the following structures:
18
40. An improved process for catalyzing an aldol reaction between
aldehyde and ketone reactants, wherein the improvement comprises
contacting said aldehyde and ketone reactants with antibody
molecules selected from a group consisting of 85A2, 85C7, 92F9,
93F3, 84G3, 84G11, 84H9, 85H6 and 90G8.
41. An improved process for catalyzing an aldol reaction according
to claim 40 wherein the ketone is selected from a group consisting
of members represented by the following structures: 19
42. An improved process for catalyzing an aldol reaction according
to claim 40 wherein the aldehyde is selected from a group
consisting of members represented by the following structures:
20
43. An improved kinetic resolution of .beta.-hydroxyketones from a
racemic mixture by means of a retro-aldol reaction, wherein the
improvement comprises catalyzing said retro-aldol reaction with
antibody molecules or molecules containing antibody combining site
portions that catalyze an aldol addition reaction produced
according to the following method: Step A: eliciting an aldolase
immune response within an immune response subject by vaccination
with a sterile solution containing the appropriate concentration of
an aldol transition state immunoconjugate, said aldol transition
state immunoconjugate including a sulfone .beta.-diketone hapten;
then Step B: isolating and cloning an antibody producing cell from
the immune responsive subject of said Step A which expresses a
catalytic aldolase antibody; and then Step C: isolating the
catalytic aldolase antibody expressed by the antibody producing
cell isolated and cloned in said Step B.
44. An improved kinetic resolution of .beta.-hydroxyketone
according to claim 43 wherein the aldol transition state
immunoconjugate of said Step A is represented by the following
structure: 21where n is greater than or equal to 2 and less than or
equal to 8 wherein the acemic mixture is treated with a catalytic
amount of the antibody in an aqueous buffer to give a simple
ketone, an aldehyde containing a phenyl ring and an
enantiomerically enriched .beta.-hydroxyketone.
45. An improved kinetic resolution of .beta.-hydroxyketones from a
racemic mixture by means of a retro-aldol reaction, wherein the
improvement comprises catalyzing said retro-aldol reaction with
antibody molecules selected from a group consisting of 85A2, 85C7,
92F9, 93F3, 84G3, 84G11, 84H9, 85H6 and 90G8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of application Ser. No.
09/415,453, filed Oct. 8, 1999, whose disclosure is incorporated
herein by reference.
FIELD OF INVENTION
[0003] 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
[0004] 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). Alaternatively, 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.
[0005] To address the problem of the de novo generation of aldolase
enzymes, a strategy of reactive immunization using .beta.-dike tone
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 .beta.-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.
Bjornestedt, 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.
[0006] 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
[0007] 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.
[0008] Illustrated in FIG. 1 is a novel sulfone .beta.-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
.beta.-diketone hapten 2 also includes the .beta.-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).
[0009] 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.
[0010] 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: 1
[0011] 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.
[0012] Another aspect of the invention is directed to a transition
state immunoconjugate represented by the following structure: 2
[0013] 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).
[0014] 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 .beta.-diketone hapten. In a
preferred mode, the hapten-carrier protein is represented by the
following structure: 3
[0015] 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.
[0016] 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 molecules or
molecules containing antibody combining site portions are produced
by eliciting an aldolase immune response within an immune response
subject by vaccination with a sterile solution containing the
appropriate concentration of an aldol transition state
immunoconjugate. The aldol transition state immunoconjugate is of
the type which includes a sulfone .beta.-diketone hapten. In a
preferred mode, the hapten-carrier protein is represented by the
following structure: 4
[0017] 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. Preferred antibody
molecules or molecules containing antibody combining site portions
include hybridoma 85A2, having ATCC accession number HB ______;
.hybridoma 85C7, having ATCC accession number HB ______; hybridoma
92F9, having ATCC accession number HB ______; hybridoma 93F3,
having ATCC accession number HB ______; hybridoma 84G3, having ATCC
accession number HB ______; hybridoma 84G11, having ATCC accession
number HB ______; hybridoma 84H9, having ATCC accession number HB
______; hybridoma 85H6, having ATCC accession number HB ______;
hybridoma 90G8, having ATCC accession number HB ______.
[0018] Another aspect of the invention is directed to an antibody
producing cell which secretes antibody molecules or molecules
containing antibody combining site portions that catalyze an aldol
addition reaction. The antibody producing cell is produced by
eliciting an aldolase immune response within an immune response
subject by vaccination with a sterile solution containing the
appropriate concentration of the aldol transition state
immunoconjugate, The aldol transition state immunoconjugate
includes a sulfone .beta.-diketone hapten. In a preferred mode, the
aldol transition state immunoconjugate is represented by the
following structure: 5
[0019] 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 converted to a hybridoma.
Preferred hybridomas include hybridoma 85A2, having ATCC accession
number HB ______; hybridoma 85C7, having ATCC accession number HB
______; hybridoma 92F9, having ATCC accession number HB ______;
hybridoma 93F3, having ATCC accession number HB ______; hybridoma
84G3, having ATCC accession number HB ______; hybridoma 84G11,
having ATCC accession number HB ______; hybridoma 84H9, having ATCC
accession number HB ______; hybridoma 85H6, having ATCC accession
number HB ______; hybridoma 90G8, having ATCC accession number HB
______.
[0020] Another aspect of the invention is directed to an improved
kinetic resolution of .beta.-hydroxyketones from a racemic mixture
by means of a retro-aldo reaction using the catalytic monoclonal
antibodies for catalyzing aodol reactions described herein.
[0021] Another aspect of the invention is directed to an improved
process for catalyzing an aldol reaction between aldehyde and
ketone reactants. In the improved process, the aldehyde and ketone
reactants are contacted with antibody molecules or molecules
containing antibody combining site portions that catalyze an aldol
addition reaction. The antibody molecules or molecules containing
antibody combining site portions are produced by eliciting an
aldolase immune response within an immune response subject by
vaccination with a sterile solution containing the appropriate
concentration of an aldol transition state immunoconjugate. The
aldol transition state immunoconjugate includes a sulfone
.beta.-diketone hapten. In a preferred mode, the hapten-carrier
protein is represented by the following structure: 6
[0022] 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. Preferred antibody
molecules or molecules containing antibody combining site portions
include hybridoma 85A2, having ATCC accession number HB ______;
.hybridoma 85C7, having ATCC accession number HB ______; hybridoma
92F9, having ATCC accession number HB ______; hybridoma 93F3,
having ATCC accession number HB ______hybridoma 84G3, having ATCC
accession number HB ______; hybridoma 84G11, having ATCC accession
number HB ______; hybridoma 84H9, having ATCC accession number HB
______; hybridoma 85H6, having ATCC accession number HB ______;
hybridoma 90G8, having ATCC accession number HB ______. Preferred
ketones include the following compounds: 7
[0023] Preferred aldehydes include the following compounds: 8
[0024] In a preferred mode, the antibody molecules are selected
from a group consisting of 85A2. 85C7, 92F9, 93F3, 84G3, 84G11,
84H9, 85H6 and 90G8.
BRIEF DESCRIPTION OF FIGURES
[0025] FIG. 1 illustrates both the prior art hapten (compound 1)
and the claimed hapten (compound 2). Both haptens are employable
for the generation of aldolase antibodies.
[0026] FIG. 2 illustrates the mechanism of antibody catalyzed aldol
reaction and reactive immunization with hapten 2 for the generation
of new aldolase antibodies. The transition state formed during the
aldol reaction and the transition state analog formed during
reactive immunization are juxtaposed so as to illustrate their
structural similarity.
[0027] FIG. 3 illustrates an antibody 93F3 catalyzed aldol
reaction. Antibody 93F3 was formed by reactive immunization using
hapten 2.
[0028] FIG. 4 is a table illustrating antibody 93F3 and 84G3
catalyzed kinetic resolutions by retro-aldolization. [A] Antibody
93F3 was used. Absolute configurations assigned by comparing aldol
products with those obtained from ab38C2 catalyzed reactions. [B]
Antibody 84G3 was used.
[0029] FIG. 5 is a table illustrating antibody 93F3 catalyzed aldol
reactions. [A] Absolute configurations assigned by asymmetric
synthesis of the aldols (I. Paterson, et al., Tetrahedron 1990, 46,
4663-4684). [B] Antibody 93F3 was used in all reactions.
[0030] FIG. 6 illustrates a table illustrating kinetic parameters
for additional antibody catalyzed aldol and retro-aldol reactions.
[A] Conditions: All data was determined in phosphate buffered
saline (PBS) at pH 7.4. [b] Per antibody active site. k.sub.cat and
K.sub.m were obtained by fitting experimental data to non-linear
regression analysis using Grafit software. [c] Aldol reactions with
a unit [M]. [d] Retro-aldol reactions with a unit [M.sup.-1]. [e]
B. List, et al., Proc. Natl. Acad. Sci. USA 1998, 95, 15351-15355.
[f] G. Zhong, et al., Angew. Chem. Int. Ed. Engl. 1998, 37,
2481-2484. [g] J. Wagner, et al., Science 1995, 270, 1797-1880.
DETAILED DESCRIPTION
[0031] Mice were immunized with the sulfone .beta.-diketone hapten
2 coupled to the carrier protein keyhole limpet hemocyanin (KLH)
and 17 monoclonal antibodies were prepared and purified as
described. All antibodies were first screened for their ability to
covalently react with 2,4-pentanedione to form a stable enaminone
(WV at .lambda..sub.max 316 nm) (J. Wagner, et al., Science 1995,
270, 1797-1880). Nine antibodies, 85A2, 85C7, 92F9, 93F3, 84G3,
84G11, 84H9, 85H6 and 90G8, showed the characteristic enaminone
absorption maximum at 316 nm after incubation with
2,4-pentanedione. All antibodies were then assayed with fluorescent
and UV active retro-aldol substrates (.+-.)-3 and (.+-.)-4,
respectively (B. List, et al., Proc. Natl. Acad. Sci. USA 1998, 95,
15351-15355; and G. Zhong, et al., Angew. Chem. Int. Ed. Engl.
1998, 37, 2481-2484). Catalysis was observed only with antibodies
that had demonstrated enaminone formation with 2,4-pentanedione.
Study of all antibodies for their ability to catalyze the aldol
addition of acetone to the aldehydes,
3-(4'-acetamidophenyl)propanal (12) and 4-isobutyramidobenzaldehyde
(13), identified the same catalysts. All antibody catalyzed aldol
and retro-aldol reactions followed Michaelis-Menten kinetics and
were inhibited by addition of a stoichiometric amount of
2,4-pentanedione. These results are consistent with the programming
of a reactive amine in covalent catalytic mechanism of these
antibodies. The output of catalysts prepared using this hapten, 9
of 17, is significantly greater than previous studies with hapten 1
where 2 of 20 antibodies were catalysts.
[0032] Deposit of Hybridomas:
[0033] Deposits for hybridoma 84G3, having ATCC accession number HB
______, for hybridoma 85H6, having ATCC accession number HB ______,
for hybridoma 93F3, having ATCC accession number HB ______, for
hybridoma 85A2, having ATCC accession number HB ______, for
hybridoma 85C7, having ATCC accession number HB ______, for
hybridoma 92F9, having ATCC accession number HB ______, for
hybridoma 84G1, having ATCC accession number HB ______, for
hybridoma 84H9, having ATCC accession number HB ______, and for
hybridoma 90G8, having ATCC accession number HB ______, were made
in compliance with the Budapest Treaty requirements that the
duration of the deposits should be for 30 years from the date of
deposit at the depository or for the enforceable life of a U.S.
patent that matures from this application, whichever is longer. The
hybridoma cell lines will be replenished should any of them become
non-viable at the depository, under the terms of the Budapest
Treaty, which assures permanent and unrestricted availability of
the progeny of the hybridomas to the public upon issuance of the
pertinent U.S. patent or upon laying open to the public of any U.S.
or foreign patent application, whichever comes first, and assures
availability of the progeny to one determined by the U.S.
Commissioner of Patents and Trademarks to be entitled thereto
according to 35 U.S.C. .sctn.122 and the Commissioner's rules
pursuant thereto (including 37 CFR .sctn. 1.14 with particular
reference to 886 OG 638). The assignee of the present application
has agreed that if the hybridoma deposit should die or be lost or
destroyed when cultivated under suitable conditions, it will be
promptly replaced on notification with a viable specimen of the
same hybridoma. Availability of the deposit is not to be construed
as a license to practice the invention in contravention of the
rights granted under the authority of any government in accordance
with its patent laws.
[0034] In order to compare these antibodies with the commercially
available aldolase antibody 38C2, several aldol and retro-aldol
reactions were chosen for study. In these studies, antibodies 93F3
and 84G3 were characterized in detail.
[0035] Scope and Synthetic Utility:
[0036] To begin to probe the synthetic scope and enantioselectivity
of these antibodies, their utility for kinetic resolutions of
.beta.-hydroxyketones was characterized. Racemic aldols 3-7 were
treated with (0.2-0.4 Mol %) ab93F3 (or ab84G3) in aqueous buffer
as previously described for ab38C2 (G. Zhong, et al., Angew. Chem.
Int. Ed. Engl. 1998, 37, 2481-2484). In each case high-performance
liquid chromatography (HPLC) indicated that the retro-aldolization
reactions halted at .about.50% conversion showing that the antibody
was highly enantioselective. The unconverted aldols were recovered
and studied using chiral-phase HPLC. Comparison with
enantiomerically-enriched standards, according to the method of I.
Paterson, et al., Tetrahedron 1990, 46, 4663-4684, indicated that
the catalyst was highly enantioselective and provided the unreacted
S-aldols with ee's typically greater than 96% (FIG. 4). Antibody
38C2 provides the corresponding R-aldols by kinetic resolution,
thus ab93F3 is its antipodal complement. Study of ab84G3 revealed
an enantioselectivity similar to ab93F3 and identified two
catalysts with enantioselectivities similar to ab38C2.
[0037] Catalysis of the synthetic reaction of acetone was then
characterized with four different aldehydes, 12, 13,
4-nitrobenzaldehyde (14) and 4-nitrocinnamaldehyde (15), to provide
aldols 5 and 8-10. Chiral-phase HPLC analysis demonstrated that the
enantioselectivities of ab93F3 and ab84G3 catalyzed aldol addition
reactions are substrate dependant. Aldols R-5, R-9 and R-10 are
provided in essentially enantiomerically pure form with either
catalyst while a moderate enantioselectivity is obtained in the
synthesis of S-8 (ee 69% with ab93F3 or 54% with ab84G3)(see FIG.
5). The ee values obtained with these catalysts are quite similar
to those obtained with ab38C2, however, the enantioselectivity is
reversed.
[0038] To examine the diastereoselectivity of ab93F3, the reaction
of 3-pentanone to provide aldol-11 was characterized. In this case
ab93F3 provided aldol syn-11 as the major product. The antibody
93F3 exhibited diastereo- and enantio-selectivities that differ
from that obtained with ab38C2. Antibody 93F3 provides 11 with a de
of 90% (syn-.alpha.-isomer) and an ee of 90% while ab38C2 provides
11 with a de of 62% (anti-isomer) and an ee of 59%.
[0039] To further characterize the scope of reactions catalyzed by
these antibodies, a variety of ketones were employed as aldol donor
substrates in reaction with aldehyde 14. Preliminary results
indicate that in addition to acetone and 3-pentanone, seven
ketones: 2-butanone, 3-methyl-2-butanone, 2-pentanone,
cyclopentanone, cyclohexanone, hydroxyacetone, and fluoroacetone,
are substrates. Thus these antibodies share the characteristic
broad scope observed previously with ab38C2.
[0040] Kinetic Studies:
[0041] The results of kinetic studies of three retro-aldol
reactions and one aldol addition reaction are provided (FIG. 6). In
most cases studied, the catalytic proficiency of ab93F3 and ab84G3
exceeds that of ab38C2, as determined by the method of A. R.
Radzicka, et al., Science 1995, 267, 90-93. In the aldol reaction
of acetone with aldehyde 12 that provides S-8, a 3-fold increase in
the catalytic proficiency is observed. An overall trend towards
increased efficiency is consistent with the notion that inclusion
of transition state analogy into the hapten design results in
increased catalytic efficiency. This effect is particularly evident
with substrate 7 where a 10.sup.3-fold increase in proficiency over
ab38C2 is observed. Based on the success of this substrate, analog
16 was synthesized. 9
[0042] Since in antibody based resolutions of aldols, the
unprocessed enantiomer can be inhibitory to the processing of the
enantiomer that is the substrate for the antibody (B. List, et al.
J. Am. Chem. Soc. 1999, 121, in press), R-16 was isolated using
chiral-phase HPLC. Study of the kinetics of retro-aldolization of
R-16 by ab84G3, revealed that it was processed by the antibody
extremely rapidly with a k.sub.cat of 1.4 s.sup.-1. Study of the
uncatalyzed reaction revealed that R-16 was not more chemically
reactive than the corresponding methoxy derivative 7, and that the
antibody provides a rate enhancement k.sub.cat/k.sub.un of
2.3.times.10.sup.8. The catalytic proficiency of ab84G3 for the
retro-aldolization of aldol R-16 is approximately 1000-fold higher
than that reported for any other catalytic antibody (N. R. Thomas,
Appl. Biochem. Biotechnol. 1994, 47, 345-72; and G. Zhong, et al.,
Angew. Chem. Int. Ed. Engl. 1998, 37, 2481-2484). The catalytic
efficiency of the antibody for this substrate, 3.3.times.10.sup.5
s.sup.-1M.sup.-1, compares favorably with the efficiency of
nature's muscle aldolase, 4.9.times.10.sup.4 s.sup.-1M.sup.-1, in
the retro-aldolization of its substrate fructose-1,6-bisphosphate
(A. J. Morris, et al. Biochemistry 1994, 33, 12291-12297, data for
muscle aldolase was reported at 4.degree. C.). 10
[0043] 1. 4-(4'-Iodophenylcarbamoyl)butyric Acid Methyl Ester
(101):
[0044] 4-Iodophenylamine (6.0 g, 27 mmol) was dissolved in 240 mL
of dried methylene chloride. Triethylamine (3.9 mL, 27 mmol) was
added. Methyl 4-(chloroformyl)butyrate (4.2 mL, 28 mmol) was added
dropwise. After 30 min of standing, the reaction mixture was washed
with 50 mL of aqueous HCl (0.5 M). The organic phase was dried over
magnesium sulfate. Evaporation of solvent gave 8.3 g of the ester
(101) for a yield 81%.
[0045] 2. 4-[4'-(3"-Oxobutyl)phenylcarbamoyl]butyric Acid Methyl
Ester (102):
[0046] 4-(4'-iodophenylcarbamoyl)butyric acid methyl ester (4.9 g,
14 mmol) was added to 16 mL of dried DMF, then tetrabutylammonium
chloride (3.9 g, 14 mmol), sodium bicarbonate (2.9 g, 35 mmol) and
3-buten-2-ol (21 mmol) were added. The mixture was stirred for 10
min. Then palladium chloride (0.57 g, 3.2 mmol) was added. The
reaction mixture was kept stirring at room temperature for 24 h
under nitrogen. It was diluted with 120 mL of ethyl acetate, washed
with 25 mL of 5% hydrochloric acid and 2.times.25 mL of brine and
dried over magnesium sulfate. Evaporation of solvent gave crude
product, which was purified by column chromatography on silica gel
(ethyl acetate/hexane: 70/30), 2.7 g of pure
4-[4'-(3"-oxobutyl)phenylcarbamoyl]butyric acid methyl ester (102)
was obtained for a yield of 66%.
[0047] 3. 4-[4'-(3"-Hydroxybutyl)phenylcarbamoyl]butyric Acid
Methyl Ester (103):
[0048] At 0.degree. C., sodium borohydride (0.22 g, 3 mmol) was
added in portions to 4-[4'-(3"-oxobutyl)-phenylcarbamoyl]butyric
acid methyl ester (1.6 , 5.6 mmol) in 25 mL of dried methanol. The
reaction mixture was kept at 0.degree. C. for 1 h. Then it was
poured into 200 mL of ammonium chloride saturated ice-water. It was
extracted with 3.times.100 mL of ethyl acetate. The combined
organic phases were dried over sodium sulfate. Evaporation of the
solvent gave 1.5 g of 4-[4'-(3"-hydroxybutyl)-
phenylcarbamoyl]butyric acid methyl ester (103) with a yield
94%.
[0049] 4. 4-[4'-(3"-Acetylsulfanylbutyl)phenylcarbamoyl]butyric
Acid Methyl Ester (104):
[0050] 4-[4'-(3"-Hydroxybutyl)phenylcarbamoyl]butyric acid methyl
ester (200 mg, 0.68 mmol) was dissolved in 6 mL of dry methylene
chloride. Triethylamine (140 .mu.L, 1.02 mmol) was added. In a
second flask, 2-fluoro-1-methylpyridinium p-toluenesulfonate (250
mg, 0.88 mmol) was suspended in 6 mL of dry methylene chloride. The
above solution was added to
4-[4'-(3"-hydroxybutyl)phenylcarbamoyl]butyric acid methyl ester in
dry methylene chloride and stirred for 1 h. The solvent was
evaporated and the residue was dissolved in 6 mL of dry DMF.
Potassium thioacetate was added and heated to 80.degree. C. for one
and a half hours. The reaction mixture was diluted with 80 mL of
ethyl acetate and washed with 2.times.20 mL of water. The organic
phases were dried over magnesium sulfate. Evaporation of solvent
followed by column chromatography (methylene chloride/diethyl
ether: 1:3) to afford yellowish product (104) (186 mg, yield
78%).
[0051] 5. 4-[4'-(3"-Mercaptobutyl)phenylcarbamoyl]butyric Acid
Methyl Ester (105):
[0052] 4-[4'-(3"-acetylsulfanylbutyl)phenylcarbamoyl]butyric acid
methyl ester (165 mg, 0.47 mmol) was dissolved in 4 mL of methanol.
Potassium carbonate (6.5 mg, 0.047 mmol) was added. The mixture was
stirred for 3 h. The solvent was evaporated and the residue was
purified by column chromatography (methylene chloride/diethyl
ether: 1:3) to afford
4-[4'-(3"-mercaptobutyl)phenylcarbamoyl]butyric acid methyl ester
(105) (110 mg, yield 74%).
[0053] 6.
4-{4'-[3"-(2'"-Oxopropylsulfanyl)butyl]phenylcarbamoyl}butyric Acid
Methyl Ester (106):
[0054] 4-[4'-(3"-mercaptobutyl)phenylcarbamoyl]butyric acid methyl
ester (110 mg, 0.35 mmol) was dissolved in 5 mL of methylene
chloride. Triethylamine (144 .mu.L, 1.05 mmol) and chloroacetone
(138 .mu.L, 1.75 mmol) were added. The reaction was stirred
overnight. The solvent was evaporated and the residue was purified
by column chromatography (methylene chloride/diethyl ether: 1:3) to
give 4-{4%-[3"-(2'"-oxopropyls-
ulfanyl)butyl]phenyl-carbamoyl}butyric acid methyl ester (106) (92
mg, yield 72%).
[0055] 7.
4-{4'-[3"-(2"'-oxopropyl-3"'-sulfonyl)butyl]phenylcarbamoyl}buty-
ric Acid Methyl Ester (107):
[0056]
4-{4'-[3"-(2'"-Oxopropylsulfanyl)butyl]phenylcarbamoyl}butyric acid
methyl ester (128 mg, 0.25 mmol) was dissolved in 3 mL of methylene
chloride. At 0.degree. C., mCPBA (87 mg, 0.25 mmol) in 2 mL of
methylene chloride was slowly added to the above solution. After
two and half hours, the solvent was partly evaporated and the
reaction mixture was diluted with 15 mL of ethyl acetate. Then the
reaction mixture was washed with 10 mL of sodium bicarbonate (1.0
M). The organic phase was dried over magnesium sulfate. Evaporation
of solvent followed by column chromatography (methylene
chloride/diethyl ether: 1:3) to afford
4-{4'-[3"-(2"'-oxopropyl-3"'-sulfonyl)butyl]phenylcarbamoyl}butyric
acid methyl ester (107) (100 mg, yield 91%).
[0057] 8.
4-{4'-[3"-(2"',4"'-Dioxopentane-3"'-sulfonyl)butyl]phenylcarbamo-
yl}butyric Acid Methyl Ester (108):
[0058] To the mixture of acetic acid (4.7 mg, 0.08 mmol, 1.2 eq)
and .beta.-diketone sulfone 107 (26 mg, 0.07 mmol) in 2 mL of dried
dimethylformamide was DEPC (13 mg, 0.08 mmol, 1.2 eq), followed by
addition of triethylamine (21 mg, 0.21 mmol, 3.2 eq). The reaction
mixture was stirred at 0.degree. C. for 2 h, and then at room
temperature for 20 h. After evaporation of the solvent, the residue
was dissolved in benzene-ethyl acetate (1/1) (25 mL) and washed
with 10% aq. sulfuric acid (10 mL) and 5% aq. sodium bicarbonate
(15 mL). The organic phase was dried over sodium sulfate.
Evaporation of solvent gave the crude product which was purified by
column chromatography (hexane/ethyl acetate: 4/1) to afford 18 mg
of .beta.-diketone sulfone 108 with a yield 74%.
[0059] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.81 (s, 1H),
7.34 (d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H), 3.61 (s, 3H), 2.88
(m, 1H), 2.63 (t, J=7.3 Hz, 2H), 2.51 (d, J=7.3 Hz. 2H), 2.39 (t,
J=7.1 Hz, 2H), 2.10 (pent, J=7.3 Hz, 2H), 2.05 (s, 6H), 1.80 (m,
2H), 1.30 (d, J=7.1 Hz, 3H); MS m/z: 462 (M+Na.sup.+, 82%), 440
(M+H.sup.+, 53%); C.sub.21H.sub.29O.sub.7NS (439.52).
[0060] 9.
4-{4'-[3"-(2"',4"'-Dioxopentane-3"'-sulfonyl)butyl]phenylcarbamo-
yl}butyric Acid (109):
[0061] .beta.-diketone sulfone 108 (18 mg, 0.041 mmol) was added to
2 mL of lithium hydroxide solution (30 mM). The reaction mixture
was stirred for 2 h at room temperature, then it was acidified by 1
M aqueous hydrochloric acid. .beta.-diketone sulfone hapten 109 was
isolated by extraction with ethyl acetate. There was obtained 16 mg
of .beta.-diketone sulfone hapten 109 for a yield of 92%.
[0062] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.88 (s, 1H),
7.44 (d, J=9.0 Hz, 2H), 6.99 (d, J=9.0 Hz, 2H), 2.87 (m, 1H), 2.62
(t, J=7.2 Hz, 2H), 2.50 (d, J=7.2 Hz, 2H), 2.41 (t, J=7.3 Hz, 2H),
2.10 (pent, J=7.2 Hz, 2H), 2.02 (s, 6H), 1.77 (m, 2H), 1.29 (d,
J=7.3 Hz, 3H); MS m/z: 426 (M+H.sup.+, 98%);
C.sub.20H.sub.27O.sub.7NS (425.50).
[0063] 10.
4-{4'-[3"-(2"',4"'-Dioxopentane-3"'-sulfonyl)butyl]phenylcarbam-
oyl}butyric Acid N-succinimoyl Ester (110):
[0064] .beta.-diketone sulfone hapten 109 (18 mg, 0.037 mmol), DCC
(11 mg, 0.052 mmol) and N-hydroxysuccinimide (2.5 mg, 0.052 mmol)
were added to 3 mL of 1,4-dioxane under nitrogen. The reaction
mixture was stirred at room temperature (it was a clear solution)
for overnight. Then the reaction mixture was filtered, washed with
3.times.20 mL of diethyl ether. The solvent of ethereal solution
was evaporated under vacuum. The crude product was obtained, which
was further purified by column chromatography (ethyl
acetate/hexane: 4/1) on silica gel to give 16 mg pure activated
ester 110. with a yield of 86%. .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. 7.82(s, 1H), 7.33 (d, J=9.0 Hz, 2H),7.01 (d, J=9.0 Hz, 2H),
2.88 (m, 1H), 2.80 (s. br, 4H), 2.62 (t, J=7.3 Hz, 2H), 2.50 (d,
J=7.3 Hz. 2H), 2.38 (t, J=7.2 Hz, 2H), 2.11 (pent, J=7.3 Hz, 2H),
2.04 (s, 6H), 1.82 (m, 2H), 1.31 (d,J=7.2 Hz, 3H); MS
(electrospray) n/z: pos. 531 (M+Na.sup.+, 44%), 509 (M+H.sup.+,
76%); C.sub.24H.sub.30O.sub.9NS (508.56).
* * * * *