Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2000-05-18
2001-12-04
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S188500
Reexamination Certificate
active
06326176
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. More particularly, these antibodies are shown to catalyze intermolecular ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions are also identified.
BACKGROUND
The aldol reaction is arguably one of the most important C—C bond forming reactions employed in synthetic transformations. Traditionally, the aldol reaction has been a proving ground for the development of asymmetric synthetic strategies. In the 1980's the aldol reaction experienced a renaissance with the development of numerous strategies to effect highly stereoselective aldols (For reviews of the adol reaction, see Heathcock, C. H. in
Asymmetric Synthesis,
Morrison, J. D., ed., Academic Press, New York, Vol. 3, 1984; Evans et al
Topics in Stereochemistry
1982, 12, 1; Masamune et al.
Angew. Chem. Int. Ed. Engl.
1985, 24, 1; Heathcock et al.
Aldrichim. Acta
1990, 23, 99.; Heathcock, C. H.
Science
1981, 214, 395.; Evans, D. A. ibid. 1988, 240, 420.; Masamune et al.
Angew. Chem. Int. Ed. Engl.
1985, 24, 1.; Evans, D. A.; Nelson, J. V.; Taber, T. R.
Top Stereochem.
1982, 13, 1.; Heathcock, C. H.; et al, in
Comprehensive Organic Synthesis,
Trost, B. M., Ed. (Pergamon, Oxford, 1991), Vol. 2, pp. 133-319; Peterson, I.
Pure Appl. Chem.
1992, 64, 1821).
Generally, this has been most successfully achieved through the use of stoichiometric quantities of chiral auxiliaries. In recent years the design of stereoselective catalysts of the aldol reaction has become a topic of interest. Most notable of these approaches is the Carreira aldol reaction where a chiral Ti(IV) complex (2-10 mol %) catalyzes the enantioselective addition of 2-methoxypropene to aldehydes with 66-98% ee (Yanagisawa, A; Matsumoto, Y.; Nakashima, H.; Asakawa, K.; Yamamoto, H.
J. Am. Chem. Soc.
1997, 119, 9319., and references therein; Bach, T.
Angew. Chem. Int. Ed. Engl.
1994, 33, 417., and references therein; Yamada, Y. M. A.; Yoshikawa, N.; Sasai, H.; Shibasaki, M.
Angew. Chem. Int. Ed. Engl.
1997, 36, 1871. (b) Carreira, E. M.; Lee, W.; Singer, R. A.
J. Am. Chem. Soc.
1995, 117, 3649).
As a challenge to traditional organic methodology, the application of natural aldolase enzymes as synthetic catalysts has yielded numerous efficient syntheses of stereochemically complex molecules, particularly in the area of carbohydrate synthesis . Since no asymmetric catalysts exhibits the scope of reactivity required to meet every synthetic challenge there is a need for methodologies that allow for the development of asymmetric catalysts. This is true of both transition metal based as well as enzyme based catalysts. For example, while the Carreira Ti(IV) complex is limited in scope to the use of the enolate equivalent 2-methoxypropene, fructose 1,6-diphosphate aldolase is limited to the use of dihydroxyacetone phosphate as the aldol donor substrate (Gijsen, H. J. M.; Qiao, L.; Fitz,W.; Wong, C.-H.
Chem. Rev.
1996, 96, 443. (b) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T.
Angew. Chem. Int. Ed. Engl.
1995, 34, 412-432. (b)Henderson, I.; Sharpless, K. B.; Wong, C.-H.
J. Am. Chem. Soc.
1994, 116, 558. (c) Wong, C.-H.; Whitesides, G. M.
Enzymes in Synthetic Organic Chemistry
(Pergamon, Oxford, 1994); Bednarski, M. D., in
Comprehensive Organic Synthesis,
Trost, B. M., Ed (Pergamaon, Oxford, 1991), vol. 2, 455; Gijsen, H. J. M., Wong, C.-H. ibid. 1995, 117, 2947; Wong, C.-H. et al. ibid. 1995, 117, 3333; Chen, L.; Dumas, D. P., Wong, C.-H. ibid. 1992, 114, 741).
To address the problem of the de novo generation of protein catalysts of the aldol reaction, we recently described the development of two aldolase catalytic antibodies 38C2 and 33F12. These antibodies were raised against the &bgr;-diketone hapten 1 which served as a chemical trap to imprint the lysine-dependent class I aldolase mechanism in the active site of the antibody. The suggested mechanism for the selection process of antibodies 38C2 and 33F12 during immunization is shown in FIG.
6
. The &egr;-amino group of the lysine residue reacts with a carbonyl function of the &bgr;-diketone moiety of 1 to form a &bgr;-keto hemiaminal followed by dehydration to give a &bgr;-keto imine that finally tautomerizes into a stable enaminone 2. Consequently, the hapten is now covalently bound in the binding pocket. The mechanistic similarity between this stoichiometric reaction and the accepted enamine mechanism of class I aldolase enzymes has been discussed in detail elsewhere (Wagner, J.; Lerner, R. A.; Barbas III, C. F.
Science
1995, 270, 1797. (b) Zhong, G.; Hoffmann, T.; Lerner, R. A.; Danishefsky, S.; Barbas III, C. F.
J. Am. Chem. Soc.
1997, 119, 8131. (c) Barbas III, C. F.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Björnestedt, R.; List, B.; Anderson, J.; Stura, E. A.; Wilson, E. A.; Lerner, R. A.
Science
1997, 278, 2085).
The formation of the enaminone has been monitored by UV spectroscopy (with hapten 1: lmax=318 nm, &egr;~15000) and is complete within seconds to a few minutes, depending on whether antibodies were incubated with hapten 1, or other diketones such as 2,4-pentanedione or 3-methyl 2,4-pentanedione. Antibodies 38C2 and 33F12 have been previously shown to catalyze aldol reactions of some aliphatic ketones donors with two different aldehyde acceptors having a 4-acetanilide substituent in the &bgr;-position as well as intramolecular aldol reactions that allowed for our recent antibody catalyzed synthesis of the Wieland-Miescher ketone (Zhong et al., ibid). Moreover, both antibodies were found to catalyze the decarboxylation reactions of aromatic &bgr;-keto acids by the formation of a Schiff base between the &egr;-amino group of the lysine residue and the keto group of the substrate (Björnestedt, R.; Zhong, G.; Lerner, R. A.; Barbas III, C. F.
J. Am. Chem. Soc.
1996, 118, 11720).
What is needed is antibodies which can catalyze many aldol addition reactions with varying substrates producing desired enantiomeric outcomes and in some cases to catalyze their subsequent dehydration to yield aldol condensation products.
BRIEF SUMMARY OF THE INVENTION
The invention is directed to the use of catalytic antibodies for catalyzing aldol condensation reactions and retroaldol reactions.
One aspect of the invention is directed to a method for catalyzing an aldol condensation between an aldol donor substrate and an aldol acceptor substrate for producing a &bgr;-hydroxy ketone. A catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is admixed with sufficient amounts of the aldol donor substrate and aldol acceptor substrate in a reaction medium for producing a reaction admixture. The aldol donor substrate is of a type which includes a reactive carbonyl group and an unbranched carbon adjacent to the carbonyl group. The catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff's base intermediate with the reactive carbonyl group of the aldol donor substrate. The above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or catalytically active molecule to catalyze the aldol condensation between the aldol donor substrate and the aldol acceptor substrate for producing the &bgr;-hydroxy ketone. The aldol donor substrate is either a ketone donor substrate or an aldehyde donor substrate. The aldol acceptor substrate is either a ketone acceptor substrate or an aldehyde acceptor substrate. However, the following proviso applies: if the aldol donor substrate is the ketone donor substrate and the aldol acceptor substrate is the aldehyde acceptor substrate, then the ketone donor su
Barbas Carlos F.
Lerner Richard A.
List Benjamin
Zhong Guofu
Northrup Thomas E.
Patterson Jr. Charles L.
The Scripps Research Institute
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