WO2002018922A2

WO2002018922A2 - High-throughput method for determining the enantiomeric purity of chiral compounds

Info

Publication number: WO2002018922A2
Application number: PCT/EP2001/009833
Authority: WO
Inventors: Manfred Theodor Reetz; Detlev Belder; Klaus M. KÜHLING; Heike Hinrichs; Alfred Deege
Original assignee: Studiengesellschaft Kohle Mbh
Priority date: 2000-08-29
Filing date: 2001-08-25
Publication date: 2002-03-07
Also published as: DE10042451A1; WO2002018922A3

Abstract

The invention relates to a high-throughput method for determining the enantiomeric purity of chiral compounds, by using electrophoretic separation, with the aid of parallel CE capillaries or microfluidic systems. According to said method, chiral sectors are added to the electrolyte, or the enantiomers to be determined are converted into electrophoretically discernable diastereomers, prior to the electrophoretic separation.

Description

High-throughput method for the determination of the enantiomeric purity of chiral compounds

The present invention relates to the high-throughput determination of the enantiomeric purity of chiral compounds by electrophoretic separation using parallel CE capillaries or using microfluidic systems.

The development of effective processes for the generation of extensive libraries of chiral chemical catalysts by methods of combinatorial chemistry [Reviews: B. Jandeleit, DJ Schaefer, TS Powers, HW Turner, WH Weinberg, Angew. Chem. 1999, 111, 2648-2689; Angew. Chem., Int. Ed. 1999, 38, 2494-2532; b) PP Pescarmona, JC van der Waal, IE Maxwell, T. Maschmeyer, Catal. Lett. 1999, 63, 1-11] or for the production of libraries of enantioselective biocatalysts by in vitro evolution [a) MT 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; b) MT Reetz, K.-E. Jaeger, Chem. — Eur. J. 2000, 6, 407-412] is the subject of current research. The decisive factor for the success of these new technologies is the availability of effective, rapid methods for searching and finding the most enantioselective catalysts or biocatalysts from the respective catalyst libraries. While there are many effective methods available in combinatorial drug chemistry to search large libraries of biologically active compounds [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], there are only a few methods for high-throughput screening of enantioselective catalysts, biocatalysts or optically active agents. The determination of the enantiomeric excess (ee) of the products of stereoselective substance conversions is normally carried out conventionally with the aid of gas or liquid chromatography on chiral stationary phases [WA König, Gas Chromatography Enantiomer Separation with Modified Cyclodextrins, Hüthig, Heidelberg, 1992; Chiral Separation by HPLC (Ed .: AM Krstulovic), Ellis Horwood, Chichester, 1989]. Although precise ee values can be determined here, such conventional methods have the disadvantage that only a limited number of samples can be examined per unit time, typically less than a few dozen ee determinations per day. In combinatorial catalysis, however, thousands of samples are produced every day.

The first assays were recently developed to solve analytical problems of this kind. So z. B. in the context of an investigation into the in vitro evolution of enantioselective lipases developed a relatively rough test method, according to which the course of enantioselective hydrolysis of chiral carboxylic acid esters can be determined [MT 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]. The hydrolyses of (R) - and (S) -configured carboxylic acid p-nitrophenol esters catalyzed by lipase mutants on microtiter plates are tracked spectrophotometrically over time, whereby the most enantioselective mutants can be identified quickly. Apart from the fact that no exact ee values are accessible, this process is limited to the class of chiral carboxylic acids. The same applies to one related test method [LE Janes, RJ Kazlauskas, J. Org. Chem. 1997, 62, 4560-4561]. This restriction also applies to related 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 completely different approach to identifying chiral catalysts relates to IR thermography [MT Reetz, MH Becker, KM Kühling, A. Holzwarth, Angew. Chem. 1998, 110, 2792-2795; Angew. Chem., Int. Ed. 1998, 37, 2547-2650]. However, it has not yet been possible to quantify the enantioselectivity or enantiomeric purity of a chiral product. Another analytical method relates to the mass spectrometry of isotope-labeled pseudo-enantiomeric and pseudo-prochiral substrates [MT Reetz, MH Becker, H.-W. Klein, D. Stöckigt, Angew. Chem. 1999, 111, 1872; Angew. Chem., Int. Ed. 1999, 38, 1758-1761]. This means that around 1000 ee determinations can be carried out per day for the first time. However, the method is limited to the analysis of kinetic resolution and reactions of prochiral substrates with enantiotopic groups.

We have now found that a general solution to the analytical problem described is possible if, for high-throughput determination of the enantiomeric purity of chiral compounds, electrophoretic separation using parallel CE capillaries (variant 1) or channels in microfluidic systems (variant 2) is used, chiral selectors being added to the electrolyte or the enantiomers to be determined being converted into electrophoretically distinguishable diastereomers prior to electrophoretic separation. The method according to the invention allows the ee determination of thousands of samples per day, typically 7,000 to 50,000. The basis of the invention is conventional capillary electrophoresis (CE), with which electrophoretic separations of enantiomers are possible by adding suitable chiral selectors to the electrolyte [B. Chankvetadze, Capillary Electrophoresis in Chiral Analysis, Wiley, Chichester, 1997]. However, the previously described approaches in sequential mode of operation do not allow high throughput screening due to the relatively long analysis times. As chiral selectors, it is known that preferred cyclodextrins (CD) are used as so-called pseudostationary phases in the CE. Derivatives of cyclodextrins, other carbohydrates, chiral crown ethers, peptides, chiral calixerenes or chiral detergents also serve as selectors.

An important area of application for capillary electrophoresis is DNA analysis and sequencing. Different technical implementations have been implemented, particularly for use in the human genome project, in order to significantly increase the sample throughput in the CE. This includes capillary array electrophoresis (CAE), in which a large number of capillaries are operated in parallel in order to enable automated DNA separations and sequencing with high throughput [a) XC Huang, MA Quesada, RA Mathies, Anal. Chem. 1992, 64, 2149-2154; b) H. Kambahara, S. Takahashi, Nature 1993, 361, 565-566; c) NJ Dovichi, Electrophoresis 1997, 18, 2393-2399; d) G. Xue, H. Pang, ES Yeung, Anal. Chem. 1999, 71, 2642-2649; e) S. Behr, M. Mätzig, A. Levin, H. Eickhoff, C. Heller, Electrophoresis 1999, 20, 1492-1507]. Special devices of this type tailored to DNA analysis are now commercially available. B. the MegaBACE ^® system [the MegaBACE ^® device is from Amersham Pharmacia Biotech (Freiburg, Germany) commercially available], consisting of 6 bundles of 16 capillaries each for processing standard 96-well microtiter plates.

Variant 1 of the invention for the high-throughput screening of enantiomeric purity of chiral compounds makes use of this CAE technology.

Variant 2 makes use of microchip technology.

To illustrate the first variant of the invention, the super-high-throughput ee determination of chiral amines is described, although the method is in no way restricted to this class of substance. Type 3 chiral amines represent important intermediates in the synthesis of pharmaceuticals and crop protection agents [a) Chirality in Industry: The Commercial Manufacture and Applications of Optically Active Compounds (ed .: AN Collins, GN Sheldrake, J. Crosby), Wiley, Chichester , 1992; b) Chirality in Industry II: Developments in the Commercial Manufacture and Applications of Optically Active Compounds (ed .: AN Collins, GN Sheldrake, J. Crosby), Wiley, Chichester, 1997; c) RA Sheldon, Chirotechnology: Industrial Synthesis of Optically Active Compounds, Dekker, New York, 1993, d) F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, /. Prakt. Chem./Chem.-Ztg. 1997, 339, 381-384]. They can be prepared, inter alia, by catalytic reductive amination of ketones 1, Markovnikov addition of NH ₃ to olefins 2 or hydrolytic enzyme-catalyzed kinetic resolution of amides 4 (or back-reaction).

In the context of the invention, the electrophoretic conditions for the enantiomer separations of the chiral amines 3 are first varied using a conventional single-capillary CE system. In order to enable parallel detection in capillary array systems by means of laser-induced fluorescence detection (LIF) (see below), the amines 3 are reacted with fluorescein isothiocyanate (FITC) 4 to form the derivatives 5. Sufficient antipode separations of the substrates can be achieved by using different commercially available cyclodextrin derivatives (CD) as chiral selectors.

The parameters obtained in such preliminary tests in conventional single-capillary CE systems are now transferred to a suitable capillary array electrophoresis system. Such multi-capillary systems are commercially available, for. B. the MegaBACE® device from Amersham Pharmacia Biotech (Freiburg, Germany), CAE instruments are also available from Beckmann Coulter (Fullerton, CA, USA) and PE Apilied Biosystems (Foster City, CA, USA). The correct adaptation of the experimental parameters is crucial for success. Due to the special configuration of such devices tailored to DNA analysis (only electrokinetic injection and detection only possible at the anode), the electrolyte system must be adapted. Electrolytes, with which very good results have been obtained in single-capillary CE systems, only deliver very unstable electrophoretic runs with electrical current which fluctuates greatly for individual capillaries. The results, which are not very reproducible, can be partly explained by the fact that an electrokinetic injection of the anionic analytes is problematic even when the cathodic EOF is only weakly present, as in the capillaries coated with polyacrylamide. In the context of the invention, however, reproducible results of high quality are obtained when using a viscous electrolyte, e.g. B. after the addition of linear polyacrylamide (LPA) or one Cellulose derivative or another polymer is worked so that the electroosmotic flow is suppressed. With the CAE device thus adapted, (+) / (-) mixtures of amines 5 can be analyzed efficiently. The electropherograms obtained in parallel are automatically evaluated and thus deliver ee values. If the CAE-determined ee values are checked with those of the corresponding non-derivatized amines (+) / (-) - 3, which are determined by gas chromatography using a chiral stationary phase, the agreement is very good. This proves the reliability of the CAE method.

With this analysis method, 7,000 to 50,000 ee determinations can be carried out per day. Such a super high throughput screening of enantioselectivity is not possible with any other system currently available [B. Jandeleit, D. J. Schaefer, T. S. Powers, H. W. Turner, W. H. Weinberg, Angew. Chem. 1999, 111, 2648-2689; Angew. Chem. Int. Ed. 1999, 38, 2494-2532]. Given that there are many benefits associated with the CAE, e.g. B. smallest sample amounts, almost no solvent consumption, absence of high pressure pumps and valves, very variable use of different, relatively inexpensive chiral phases, the assay described here is particularly attractive industrially.

Variant 2 of the invention for drastically increasing the analysis throughput when using capillary electrophoresis to determine the enantiomeric purity (% ee) makes use of the microchip technology [DJ Harrison, K. Fluri, K. Seiler, Z. Fan, CS Effenhauser, A. Manz, Science (Washington, D.C., 1883-) 1993, 261, 895-897; SC Jacobson, R. Hergenroder, LB Koutny, RJ Warmack, JM Ramsey, Anal. Chem. 1994, 66, 1107-1113; L. D. Hutt, DP Glavin, JL Bada, RA Mathies, Anal. Chem. 1999, 71, 4000-4006; I. Rodriguez, LJ Jin, SFY Li, Electrophoresis 2000, 21, 211-219]. Photolithographic techniques for producing one or more capillaries on microchips are preferably used here. Such CE microchips made of plastic or glass have hitherto been used for the analysis of oligonucleotides, DNA sequence fragments, amino acids, DNA restriction fragments and PCR products. In this highly miniaturized system, extremely fast separations can be obtained within seconds to minutes. While both plastic and glass chips can be used to separate biomolecules in aqueous electrolytes, plastic microchips are not stable enough to separate conventional organic compounds in electrolytes that contain organic solvents. Since suitable devices for operating CE chips are not yet commercially available, an instrument is produced within the scope of the invention, equipped with 2 high-voltage devices, high-voltage switches and with LIF detectors (argon ion laser). Chiral separations of the FITC-derivatized amines 5 can thus be carried out using CE glass chips from Micralyne (Edmonton, Canada). For this purpose, similar electrolyte systems are used as in the first version, but without the addition of LPA. This allows separations of enantiomers, as in the CAE described above, to be achieved, but with significantly shorter analysis times per separation channel. By using higher field strengths, which requires the use of special high-voltage changeover switches, separations of enantiomers are possible in a few seconds. Since microfluidic structures can easily be parallelized in microchips, there is an even higher throughput of analyzes, with parallel instead of sequential Working method, easy to implement. Automated instruments that make this possible are currently being developed and will then enable a sample throughput of> 50,000 ee determinations per day using our method.

As analytical methods for the detection of the enantiomers z. B. used: fluorescence, UV / VIS, IR, electrical conductivity, electrochemical methods, refractometry, circular dichroism or mass spectrometry.

If the laser-induced fluorescence detection is used, fluorescence labeling is often necessary. Amines can e.g. can be derivatized with the following compounds: with isothiocyanate derivatives, such as fluorescein isothiocyanate (FTTC), tetramethylrhodamine isothiocyanate (TRITC), phenyl isothiocyanate (P TC), S-1- (l-naphthyl) ethyl isothiocyanate (SNEJT), S-phenylethylisothiocyanate; Succinimide ester derivatives such as e.g. 7- (diethylamino) coumarin-3-carboxylsuccinimide ester (DCCS), with chloroformate derivatives, such as 9-fluorenylmethyl chloroformate (FMOC), 2- (9-anhryl) ethyl chloroformate (AEOC), (+) / (-) l- (9-fluorenyl) ethyl chloroformate (FLEC); Isoindole derivatives such as o-phthalaldehyde (OPA), naphthalenes-2,3-dicarboxyaldehyde (NDA), 3- (4-carboxybenzoyl) -2-quinolinecarboxaldehyde (CBQCA) and with other reagents such as fluorescamine, 4-phenylspiro [furan-2 (3H ) -l'-phthalan] -3,3'- dione, 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole.

The following reagents can be used to derivatize reducing groups such as aldehydes or keto functions in sugars: 2-aminopyridine (AP), p-aminobenzoic acid (PABA), ethyl p-aminobenzoate (EP AB), 2-aminoacridone (AC), p-aminobenzonitrile, 9-aminopyrene-l, 4-trisulfate (APTS), 5-aminonaphtyl-2-sulfonic acid (5-ANSA), R / S -1-phenylethylamine (SPEA). For the derivatization of alcohols, for example, [1- (9-fluorenyl) ethyl chloroformate (FLEC) or 9-fluorenyl methyl chloroformate (FMOC) can be used. A large number of other fluorescent markers can be used to derivatize different functionalities such as, for example, diethyldithiocarbamic acid (DDTC) for the alkyl chloride functionality; Dansyl hydrazine (DNSH), 2,4-dinitrophenyl hydrazine (DNPH), 4-methoxy-l, 2-phenyl diamine (MPD), o-phenyl diamine (OPD) for dicarbonyls; 7-aminonaphthyl-1,3-disulfonic acid (7-ANDA), 5- (aminoacetoamido) fluorescein (AAF), 1-pyrendiazomethane (PDAM) for the carbonyl functionality.

With the two versions of chiral high-throughput capillary electrophoresis according to the invention, the analysis of other chiral substance classes is also possible. These include chiral alcohols, diols, thio alcohols, amino alcohols, amino acids, cyanohydrins, halohydrins and other organic compounds with functional groups.

Example 1. Capillary electrophoretic separation of the chiral amines 3

First, the amines were derivatized with fluorescein isothiocyanate (FJTC) to produce the corresponding fluorescent derivatives. 100 μL of the amine solution with a concentration of 6.7 μmol / L in acetone, with 12.7 μL of a FITC solution with a concentration of 26 μmol / L in acetone were added to 50 μL of a 20 mmol / L borate buffer at pH 9 and then 1 : 10 diluted with methanol.

Suitable methods for capillary electrophoretic antipode separation using the automated classic single-capillary CE system with UV detection (HP 3D-CE, Agilent, Waldbronn, Germany) were developed for the chiral FTTC derivatives produced in this way. For this purpose, electrolyte systems were mixed with cylodextrins (CD) as chiral additives. Through targeted variation of the following parameters: 1) type and derivative of the cyclodextrin, 2) concentration of the CD, 3) pH of the electrolyte, electrolyte systems were finally developed in which a separation of the enantiomers of the different substrates was obtained. These results are summarized in Table 1.

Adequate chiral separations of FTTC-cyclohexylethylamine. (5c) could be obtained, for example, with the following electrolyte composition: 40 mM CHES buffer at pH 9 with 6.25 mM γ-CD. FIG. 1 shows a chiral CE separation of the FTTC-labeled compounds obtained in this way, which was obtained with the following experimental parameters: voltage: 30 kV, 60 μA; Injection 40 mbar, 2 sec; Detection: UV at 200 nm; Capillary coated with polyvinyl alcohol: 40 cm eff. Length, 50 μm inside diameter.

Example 2. High-throughput method for determining the ee of R / S-cyclohexylethylamine libraries using CAE

For high-throughput screening of libraries of R / S-cyclohexylethylamine which were present in standard 96-well microtiter plates, each sample was first derivatized with FΪTC as described in Example 1 and diluted with water in a ratio of 1: 5000. The samples were then transferred with the microtite plates into the commercially available CAE system MegaBACE (Amersham Pharmacia Biotech, Freiburg, Germany) and analyzed there in parallel. The electrolyte used was the electrolyte system previously developed in the one-capillary system, to which linear polyacrylamide (LPA) was added. By adding LPA, which increases the viscosity and reduces the electroosmotic flow, a high reproducibility and robustness of the method could be obtained. For this purpose, the 5: 1 electrolyte described above was mixed with the highly viscous LPA buffer (long read matrix) from Amersham Pharmacia. FIG. 2 shows the electropherograms obtained from an analysis of a library consisting of 12 solutions of +/- mixtures of cyclohexylethylamine by means of CAE, which were obtained with the following experimental parameters: voltage: -10 kV / 8 μA, electrokinetic injection: -2 kV / 9 s, capillary array from Amersham Pharmacia Biotech. The signals received were evaluated with software support and the ee values determined. As a control, the enantiomeric purity of the corresponding non-derivatized samples was determined by gas chromatography using a chiral stationary phase (Ivadex-l / PS086 df: 0.15 μm, iD: 250 μm, 1:25 m). The ee values determined by means of CAE and GC are compared in Table 2, the numbering of the samples corresponds to that of the signals in Figure 2. Sample ee 3c (GC) ee 5c (CAE)

[%] [%]

1 98> 95

2 88 90

3 84 82

4 74 70

5 74 70

6 38 38

7 18 16

8 18 16

9 0 0

10 0 2

11 54 52

12 54 52

Table 2. Comparison of the experimental results for the determination of the enantiomeric excess of samples from (R) / (S) -3c using GC and of corresponding samples from (R) / (S) -5c using CAE.

Example 3: High-throughput method for determining the ee of R / S-2-heptylamine using CAE

For the high-throughput screening of R / S-2-heptylamine solution which were present in standard 96 microtitre plates, each sample was first derivatized analogously to Example 2 with FTTC. The samples were then transferred to a CAE system with 96 capillaries working in parallel with the microtite plates and analyzed there. A 40 mM CHES buffer at pH 9 with 15% acetonitrile and 40 mM γ-CD was used as the electrolyte. With an increased separation voltage of 20 kV compared to Example 2, the 96 substrates present in a microtitre plate could be analyzed in less than 8 minutes. An exemplary electropherogram is shown in FIG. 3. In connection with an automatic computer-aided data evaluation it was possible to carry out 15,000 ee determinations per day.

Example 4 High-throughput method for determining the ee of R / S-1- (ρara-tolyl) ethylamine

For high-throughput screening of R Sl- (para-tolyl) ethylamine, each sample was first derivatized with FTTC analogously to Example 2. The samples were then transferred to a CAE system with 96 capillaries working in parallel with the microtite plates and analyzed there. A 40 mM CHES buffer with 5 mM HE-ß-CD was used as the electrolyte. When using a separation voltage of 25 kV and when using unoccupied capillaries with a dominant high electroosmotic flow, the 96 substrates present in a microtiter plate could be analyzed in less than 4 minutes. In connection with an automatic computer-aided data evaluation, it was possible to carry out 34000 ee determinations per day. Example 5: High-speed analysis for determining the ee of R / S-cyclohexylethylamine using CE microchips

Racemic cyclohexylethylamine, which was present in a concentration of 0.67 μM / L, was first reacted with FTTC according to Example 1 and diluted with water in a ratio of 1: 10000. Chiral separation could be achieved with CE microchips from Micralyne (Edmonton, Canada) using a self-made instrument in less than 2 minutes, as shown in FIG. The same electrolyte was used as for Fig. 1, a voltage of 1 kV was applied to the electrodes for sample inlet, buffer outlet and buffer inlet for sample application, while the sample outlet was at ground potential. The voltage was then switched over so that the following potentials were applied to the four electrodes: buffer inlet lkV, sample inlet 0.6 kV, sample outlet 0.6 kV, buffer outlet 0V. The detection was carried out with a modified laser-induced fluorescence detector from Picometrics (Toulouse, France) equipped with an argon ion laser. The experimental setup is shown schematically in FIG. 5.

Claims

claims

1. A method for high-throughput determination of the enantiomeric purity of chiral compounds, characterized in that the enantiomers are electrophoretically in parallel CE capillaries or in channels of microfluidic systems. are separated, chiral selectors being added to the electrolyte or the enantiomers being converted into electrophoretically distinguishable diastereomers before the separation.

2. The method according to claim 1, wherein the parallel CE capillaries are used in the form of capillary arrays.

3. The method according to claim 1, wherein chiral organic compounds are used as selectors, which interact with the enantiomers to be analyzed.

4. The method according to claim 3, wherein cyclodextrins, derivatives of cyclodextrins, further carbohydrates, chiral crown ethers, peptides, chiral calixerenes or chiral detergents serve as selectors.

5. The method according to claims 1-4, wherein an additive for increasing the viscosity is added to the electrolyte.

6. The method of claim 5, wherein the additive is a linear polyacrylamide, a cellulose derivative or another polymer.

7. The method according to claim 1, wherein the microfluidic systems are used in the form of microchips.

8. The method according to claim 1, wherein chiral fluorescent markers are used as chiral derivatization reagents for converting the enantiomers into diastereomers.

9. The method according to claim 8, wherein the chiral fluorescent marker used is one of the following compounds: R / S- [l- (9-fluorenyl) ethyl] chloroformate (FLEC), R / Sl- (l-naphthyl) ethyl isothiocyanate (S ETT) or R / S phenylethyl isothiocyanate (SAMBI)

10. The method according to claim 1, wherein for the detection of the enantiomers fluorescence, UV / VIS, IR, electrical conductivity, electrochemical methods, refractometry, circular dichroism or mass spectrometry are used.

11. The method according to claims 1-7, wherein for detection with fluorescence or UV / VIS, the enantiomers are derivatized before separation with compounds which contain fluorophoric or chromophoric groups.