Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-25
2004-11-30
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S280000
Reexamination Certificate
active
06824982
ABSTRACT:
This application is a 371 of PCT/EP00/02121 filed on Mar. 10, 2000.
The present invention relates to a method for determining the enantioselectivity of kinetic resolution processes and of asymmetrically proceeding reactions of prochiral compounds bearing enantiotopic groups by using isotope-labeled substrates so that the reaction products can be quantitatively determined with an isotope-specific detection system, e.g., an ESI mass spectrometer. In connection with an automated sampler the method can be employed for high throughput screening. The relevant enantioselective conversions can be induced by chiral homogeneous or heterogeneous catalysts, biocatalysts or stoichiometric amounts of optically active agents.
The development of effective methods for generating extensive libraries of chiral chemical catalysts by methods of combinatorial chemistry [a) G. Liu, J. A. Ellman, J. Org. Chem. 1995, 60, 7712-7713; b) K. Burgess, H.-J. Lim, A. M. Porte, G. A. Sulikowski, Angew. Chem. 1996, 108, 192-194; Angew. Chem., Int. Ed. Engl. 1996, 35, 220-222; c) B. M. Cole, K. D. Shimizu, C. A. Krueger, J. P. A. Harrity, M. L. Snapper, A. H. Hoveyda, Angew. Chem. 1996, 108, 1776-1779; Angew. Chem., Int. Ed. Engl. 1996, 35, 1668-1671; d) C. Gennari, H. P. Nestler, U. Piarulli, B. Salom, Liebigs Ann./Recl. 1997, 637-647] or for the preparation of libraries of enantioselective biocatalysts by in-vitro evolution [M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963; Angew. Chem., Int. Ed. Engl. 1997, 36, 2830-2932] is currently under investigation. A critical aspect of the success of these novel technologies is the existence of effective and rapid methods for the screening of the enantioselective catalysts or biocatalysts from the respective catalyst libraries. While many effective methods for the screening of large libraries of biologically active compounds are available in the combinatorial chemistry of active substances [a) F. Balkenhohl, C. von dem Bussche-Hünnefeld, A. Lansky, C. Zechel, Angew. Chem. 1996, 108, 2436-2488; Angew. Chem., Int. Ed. Engl. 1996, 35, 2288-2337; b) J. S. Früchtel, G. Jung, Angew. Chem. 1996, 108, 19-46; Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42; c) Chem. Rev. 1997, 97 (2), 347-510 (special edition about combinatorial chemistry); d) S. R. Wilson, A. W. Czarnick, Combinatorial Chemistry: Synthesis and Application, Wiley, N.Y., 1997], the development of methods for the high throughput screening of enantioselective catalysts, biocatalysts or optically active agents is still at the beginning. The determination of the enantiomeric excess (ee) of the products of stereo-selective conversions is normally effected classically by means of gas or liquid chromatography on chiral stationary phases [G. Schomburg, Gaschromatographie: Grundlagen, Praxis, Kapillartechnik, 2nd Ed., VCH, Weinheim, 1987; K. K. Unger, Packings and stationary phases in chromatographic techniques, Series Chromatographic science; Vol. 47, Marcel Dekker, New York, 1990]. Although precise ee values can be determined thereby, such conventional methods have a disadvantage in that only a limited number of samples can be examined per unit time since the times required for analysis depend on the respective retention times.
The first suggestions for solving analytical problems of this kind have been made recently. Thus, for example, in the scope of a study on the in-vitro evolution of enantioselective lipases, a relatively rough test method has been developed according to which the course of enantioselective hydrolyses of chiral carboxylate esters can be determined [M. T. Reetz, A. Zonta, K. Schimossek, K. Liebeton, K.-E. Jaeger, Angew. Chem. 1997, 109, 2961-2963; Angew. Chem., Int. Ed. Engl. 1997, 36, 2830-2932]. Thus, the time course of the hydrolysis of carboxylic acid p-nitrophenol esters of (R)- and (S)-configurations catalyzed by lipase mutants is monitored spectrophotometrically on microtiter plates, whereby the most enantioselective mutants can be identified quickly. Apart from the fact that no exact ee values are possible, this method is limited to the chiral carboxylic acid class of substances. The same applies to a related test method [L. E. Janes, R. J. Kazlauskas, J. Org. Chem. 1997, 62, 4560-4561]. Also subject to this limitation are related methods which are based on the color change of pH indicators during ester hydrolysis [L. E. Janes, A. C. Löwendahl, R. J. Kazlauskas, Chem.—Eur. J. 1998, 4, 2324-2331]. A totally different approach for the identification of chiral catalysts is based on infrared thermography [M. T. Reetz, M. H. Becker, K. M. Kühling, A. Holzwarth, Angew. Chem. 1998, 110, 2792-2795; Angew. Chem., Int. Ed. 1998, 37, 2547-2650]. However, the further development of this method to enable the quantitative analysis of enantioselective reactions still remains to be done.
The present invention remedies these defects by employing partially or completely isotope-labeled substrates or substrates having an isotope distribution which deviates from the natural distribution for kinetic resolution processes or for stereoselective reactions with prochiral substrates containing enantiotopic groups. This permits the use of an isotope-specific detection system, for example, a mass-spectrometric ionization method, for the quantitative determination of the conversion or the relative proportions of the pseudo-enantiomers or of enantiomeric excess.
As compared to previous approaches, the present invention offers the following advantages:
1) Exact determination of the ee values of kinetic resolution processes and of asymmetrically proceeding conversions of prochiral compounds bearing enantiotopic groups, no limitations being made with respect to the class of substances or the type of reaction.
2) Exact determination of the conversion of the reactions mentioned under 1).
3) Rapid or high throughput testing of the data mentioned under 1) and 2), at least 1000 determinations per day being possible in particular.
The detection systems used in the present invention are mass spectrometers, especially those using electro-spray ionization (ESI) [J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Science (Washington, D.C.) 1989, 246, 64-71] or matrix-assisted laser desorption/ionization (MALDI) [a) K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, T. Yoshida, Rapid Commun. Mass Spectrom. 1988, 2, 151-153; b) M. Karas, F. Hillenkamp, Anal. Chem. 1988, 60, 2299-2301]. In connection with automated sampler (use of one or more sample charging robots and microtiter plates), optionally with the use of several spectrometers, the method according to the invention is suitable as a high throughput screening method.
The method can be used for finding or optimizing chiral catalysts or chiral agents for asymmetrically proceeding reactions. These include:
a) chiral catalysts or chiral agents for the kinetic resolution of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, and their derivatives;
b) chiral catalysts (e.g., chiral homogeneous or chirally modified heterogeneous catalysts, chiral metal complexes) or chiral agents for the stereoselective conversion of prochiral compounds whose enantiotopic groups include one or more functional groups from the classes of substances of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides, or their derivatives;
c) biocatalysts, e.g., enzymes, antibodies, proteins, hormones, phages, ribozymes, peptides or other biopolymers, for the kinetic optical resolution of alcohols, carboxylic acids, carboxylate esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides, oxiranes, thiols, sulfides, sulfone
Becker Michael Heinrich
Klein Heinz-Werner
Reetz Manfred T.
Stöckigt Detlef
Gitomer Ralph
Norris & McLaughlin & Marcus
Studiengesellschaft Kohle mbH
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