EP1481258A1 - Procede de selection a haut rendement pour la determination de l'enantioselectivite de catalyseurs, de biocatalyseurs et d'agents - Google Patents

Procede de selection a haut rendement pour la determination de l'enantioselectivite de catalyseurs, de biocatalyseurs et d'agents

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Publication number
EP1481258A1
EP1481258A1 EP03708123A EP03708123A EP1481258A1 EP 1481258 A1 EP1481258 A1 EP 1481258A1 EP 03708123 A EP03708123 A EP 03708123A EP 03708123 A EP03708123 A EP 03708123A EP 1481258 A1 EP1481258 A1 EP 1481258A1
Authority
EP
European Patent Office
Prior art keywords
nmr
chiral
throughput
isotope
agents
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03708123A
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German (de)
English (en)
Inventor
Manfred T. Reetz
Patrick Tielmann
Andreas Eipper
Richard Mynott
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Studiengesellschaft Kohle gGmbH
Original Assignee
Studiengesellschaft Kohle gGmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Studiengesellschaft Kohle gGmbH filed Critical Studiengesellschaft Kohle gGmbH
Publication of EP1481258A1 publication Critical patent/EP1481258A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy

Definitions

  • the present invention relates to a method for determining the enantioselectivity of kinetic resolution and of asymmetric reactions of prochiral compounds by using isotope-labeled substrates or with the aid of chiral auxiliary reagents, a high-throughput NMR spectrometer being used as the detection system in an automated measurement process.
  • the invention thus enables simple high-throughput screening of enantioselective catalysts, biocatalysts or agents.
  • a photometer assay enables the tracking of enantioselective hydrolysis of lipase variants in microtiter plates.
  • the disadvantage is that no exact ee values are accessible and this method is restricted to the class of chiral carboxylic acids. Similar restrictions apply to a related test method [LE Janes, RJ Kazlauskas, J. Org. Chem. 1997, 62, 45460-45461]. Furthermore, this restriction applies to processes that are based on color changes of pH indicators during ester hydrolysis [LE Janes, AC Löwendahl, RJ Kazlauskas, Chem.-Eur. J. 1998, 4, 2324-2331].
  • a method for using DNA microarrays to determine enantiomeric excesses enables a high sample throughput, but the assay contains four steps and is therefore cumbersome, and the method is also not generally applicable [GA Korbel, G. Lalic, MD Shair, J. At the. Chem. Soc. 2001, 123, 361-362].
  • the recently presented use of coupled enzyme reactions for the determination of enantiomeric excesses (EMDee) has an excessively high error range of +/- 10% ee and can only be used to a limited extent [P. Abato, CT Seto, J. Am. Chem. Soc. 2001, 123, 9206-9207].
  • NMR spectroscopy as the detection system in an automated measurement process in the process for high-throughput determination of the enantioselectivity of reactions caused by chiral catalysts, biocatalysts or chiral agents is used.
  • isotope-labeled substrates are used which can be detected by NMR spectroscopy.
  • the present invention can also be used to conveniently track those enantioselective substance conversions in which a prochiral compound without enantiotopic groups changes into a chiral product.
  • the enantiomeric excess can be determined by quantifying the NMR signals of the isotope-labeled substrates.
  • the chiral products and / or starting materials of the reactions to be investigated are treated with enantiomerically pure agents for derivatization and the NMR signals of the resulting diastereomers are quantitatively evaluated for ee determination.
  • the ee determination can also be carried out by using chiral solvents or chiral shift reagents. In both embodiments of the invention, a throughput of 1000 and more samples per day is possible.
  • Figure 1 a) Asymmetric transformations of pseudo-enantiomers (a and b), pseudo-meso (c) and / «eu-io-prochiral (d) compounds.
  • FG represents the functional group, FG 'or FG "symbolize the functional groups formed by the implementation; the isotope marking is identified by an asterisk (*).
  • Figure 2 Derivatization of enantiomeric mixtures with chiral auxiliary reagents for quantification by means of NMR analysis.
  • Figure 3 Experimental setup of a high-throughput screening system for enantioselectivity using NMR with isotope-labeled substrates.
  • Figure 4 Experimental setup of a high-throughput screening system for enantioselectivity using NMR and chiral auxiliary reagents or chiral agents or solvents.
  • Figure 5 Kinetic resolution of 1-phenylethyl acetate: comparison of the ee determination between chiral GC and high-throughput NMR.
  • Figure 6 Methyl signal of the diacetate in the NMR-NMR spectrum with natural 13 C satellites at a measurement frequency of 300 MHz.
  • Figure 8 Diastereomeric splitting in the 1H-NMR spectrum of the CH group of the ester of racemic phenylethanol with MTPA at a measuring frequency of 300 MHz.
  • the detection systems used in the present invention are nuclear magnetic resonance spectrometers, in particular those with a flow cell, for high-throughput operation
  • Suitable nuclei for this are ⁇ , 19 F, 31 P and 13 C, whereby the method can be extended to other types of nuclei (e.g. U B, l5 N, 29 Si).
  • the method can be used to find or optimize chiral catalysts, biocatalysts or chiral agents for asymmetric reactions.
  • chiral catalysts, chiral agents or biocatalysts such as enzymes, antibodies, ribozymes or phages for the kinetic resolution of compounds such as alcohols, carboxylic acids, carboxylic esters, amines, amides, olefins, alkynes, phosphines, phosphonites, phosphites, phosphates, halides , Oxiranes, thiols, sulfides, sulfones, sulfoxides, sulfonamides and their derivatives and combinations; b) chiral catalysts, chiral agents or biocatalysts for the stereoselective conversion of prochiral compounds, with or without enantiotopic groups, the substrates belonging to the substance classes of carboxylic acids, carboxylic acid esters,
  • the first embodiment of the invention is based on the use of isotope-labeled substrates in the form of seudo-enantiomeric or pseudo-prochiral compounds (FIG. 1), C-labeled substrates being used in particular.
  • the second embodiment makes use of chiral auxiliary reagents (FIG. 2).
  • substrates 1 and 2 or 1 and 7 which differ in the absolute configuration and in the isotope labeling in the functional group FG or in the radical R 2, are prepared in enantiomerically pure form and in a ratio of 1: 1 mixed so that a racemate is simulated ( Figure la or b).
  • an enantioselective conversion in which the chemical reaction takes place on the functional group (ideally a kinetic resolution up to a conversion of 50%)
  • real enantiomers 3 and 4 are formed in addition to unlabelled and labeled achiral by-products 5 and 6, or it
  • the psewd ⁇ enantiomers 3 and 8 are formed. If the desymmetrization of prochiral compounds is carried out (FIG. 1c or d), pseudo-enantiomers also arise.
  • isotope labeling is dispensed with. Rather, the enantiomeric mixtures of asymmetrically proceeding reactions are implemented with enantiomerically pure chiral derivatization agents, NMR shift agents or solvents to form diastereomeric compounds or complexes, which are then investigated by high-throughput NMR spectroscopy (FIG. 4).
  • Figure 2 can be used as chiral auxiliary reagents such.
  • B mandelic acid, mandelic acid chloride, O-methylmandelic acid (MPA), O-methylmandelic acid chloride, atrolactic acid, atrolactic acid chloride, ⁇ -methoxy- ⁇ -trif uormethyl-phenylacetic acid (MTPA, Mosher's acid), -methoxy- ⁇ -trifluoromethyl-phenylacetic acid chloride
  • the assay for the high-throughput screening of an asymmetric reaction by means of NMR is designed in such a way that in the case of a kinetic resolution, a pseudo-R & cem & t is first produced from enantiomerically pure isotope-labeled and unlabeled substrate. Then the racemate cleavage, e.g. B. in 96-well microtiter plates, with the addition of the catalyst. Finally, the samples are introduced into the flow cell of the NMR device using a pipetting and sample application robot (FIG. 3).
  • Example 1 Kinetic resolution of 1-phenylethyl acetate The kinetic resolution of 1-phenylethyl acetate by hydrolysis, catalyzed by e.g. B. enzymes such as lipases (wild type or variants) is monitored according to Figure 3 in a high-throughput assay, d. H. enantioselectivity and conversion are determined.
  • the solvent is removed on a rotary evaporator and the crude product is columnared over silica gel with dichloromethane. After removing the solvent in vacuo and briefly drying in a high vacuum, 1.24 g (92%) of the desired product are obtained as a clear liquid.
  • Table 1 Mixtures from 35 ⁇ l to 700 ⁇ l CDC1 3 .
  • the measurement method can be reduced to a cycle time of approximately one minute. This does not affect the accuracy of the analysis, the backmixing with the previous sample remains less than 1%. Typical results are summarized in Table 2.
  • Table 2 Mixtures of 1.3 to 1.7 mg on 1 ml CDC1 3 in the high-throughput NMR method (approx. 1 min per cycle).
  • Table 3 Mixtures from 10 ⁇ l to 700 ⁇ l CDC1 3.
  • Example 3 Enantioselective hydrolysis of mesol, 4-diacetoxy-2-cyclopentene This example relates to the reaction of a pseudo-proc i tal compound which carries enantiotopic groups (here acetoxy groups).
  • the alcohol was first derivatized with Mosher's acid chloride to produce the corresponding diastereomeric esters.
  • the samples were then in tested with a high-throughput NMR device and the ee values calculated by automatic integration of the CH signals of the diastereomers in the ⁇ -NMR spectrum.
  • the enantiomeric purity of the same samples was determined by gas chromatography.
  • the ee values determined by means of high-throughput NMR and GC are compared in Table 5.
  • the alcohol was derivatized analogously to Example 4 with Mosher's acid chloride in order to prepare the corresponding diastereomeric esters.
  • the samples were then tested in a high-throughput NMR device and the ee values were calculated by automatically integrating the CH signals of the diastereomers in the 1H-NMR spectrum.
  • the enantiomeric purity of the same samples was determined by gas chromatography.
  • the ee values determined using a high-throughput NMR device and GC are compared in Table 6.
  • the ratios of the CH signals of the diastereomers were automatically evaluated using the AMLX @ software from Bruker.

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  • Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • High Energy & Nuclear Physics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne un procédé à haut rendement, par spectroscopie RMN, pour la détermination de l'énantiosélectivité de réactions d'allure asymétrique. Les réactions peuvent être provoquées par des catalyseurs, agents ou biocatalyseurs chiraux, de manière à pouvoir évaluer leur énantiosélectivité. Dans un mode de mise en oeuvre, on utilise des pseudo-énantiomères ou des substrats pseudo-prochiraux marqués par des isotopes, de telle façon que l'intégration des signaux RMN des substrats et/ou des produits respectifs permette la détermination quantitative de l'énantiosélectivité. L'emploi d'une structure d'appareil automatisée, avec utilisation de plaques à microtitration, robots et appareils RMN à haut rendement, joue un rôle déterminant pour l'obtention d'un procédé à haut rendement. Dans une seconde forme de mise en oeuvre de l'invention, on utilise la structure d'appareil automatisée, en vue de détecter quantitativement, sous forme de diastéréomères, les produits et/ou les adduits dérivatisés avec des agents exempts d'énantiomères. Dans les deux modes de mise en oeuvre, il est possible d'effectuer au moins 1000 déterminations de valeurs ee par jour, avec une précision d'au moins ? 5 %.
EP03708123A 2002-03-01 2003-02-22 Procede de selection a haut rendement pour la determination de l'enantioselectivite de catalyseurs, de biocatalyseurs et d'agents Withdrawn EP1481258A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10209177 2002-03-01
DE10209177A DE10209177A1 (de) 2002-03-01 2002-03-01 Ein Hoch-Durchsatz-Screening-Verfahren zur Bestimmung der Enantioselektivität von Katalysatoren, Biokatalysatoren und Agenzien
PCT/EP2003/001825 WO2003075031A1 (fr) 2002-03-01 2003-02-22 Procede de selection a haut rendement pour la determination de l'enantioselectivite de catalyseurs, de biocatalyseurs et d'agents

Publications (1)

Publication Number Publication Date
EP1481258A1 true EP1481258A1 (fr) 2004-12-01

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EP03708123A Withdrawn EP1481258A1 (fr) 2002-03-01 2003-02-22 Procede de selection a haut rendement pour la determination de l'enantioselectivite de catalyseurs, de biocatalyseurs et d'agents

Country Status (7)

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US (1) US20050153358A1 (fr)
EP (1) EP1481258A1 (fr)
JP (1) JP2005526963A (fr)
AU (1) AU2003212262A1 (fr)
CA (1) CA2477476A1 (fr)
DE (1) DE10209177A1 (fr)
WO (1) WO2003075031A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2007134446A1 (fr) * 2006-05-23 2007-11-29 The University Of Western Ontario Criblage a haut debit d'exces enantiomere (ee)

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Publication number Priority date Publication date Assignee Title
US5146166A (en) * 1990-08-06 1992-09-08 Chemagnetics, Inc. Method and apparatus for enhancing sample analysis rate in magnetic resonance spectroscopy
US6063633A (en) * 1996-02-28 2000-05-16 The University Of Houston Catalyst testing process and apparatus
US6316616B1 (en) * 1998-04-02 2001-11-13 President And Fellows Of Harvard College Parallel combinatorial approach to the discovery and optimization of catalysts and uses thereof
DE19913858A1 (de) * 1999-03-26 2000-09-28 Studiengesellschaft Kohle Mbh High-Throughput-Screening-Verfahren zur Bestimmung der Enantioselektivität asymmetrisch verlaufender Reaktionen

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See references of WO03075031A1 *

Also Published As

Publication number Publication date
DE10209177A1 (de) 2003-09-18
AU2003212262A1 (en) 2003-09-16
US20050153358A1 (en) 2005-07-14
WO2003075031A1 (fr) 2003-09-12
JP2005526963A (ja) 2005-09-08
CA2477476A1 (fr) 2003-09-12

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