A high-throughput screening method for asymmetric catalytic reactions
By introducing labeled groups into the substrates of asymmetric catalytic reactions and carrying out click chemistry reactions, combined with ion mobility mass spectrometry analysis, the problem of high-throughput screening of asymmetric catalytic reactions has been solved, and rapid and accurate determination of ee values has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN UNIV
- Filing Date
- 2023-09-15
- Publication Date
- 2026-06-23
Smart Images
Figure CN117330621B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of testing technology, and in particular to a high-throughput screening method for asymmetric catalytic reactions. Background Technology
[0002] Asymmetric catalysis is the primary method for synthesizing chiral compounds. However, due to the complexity of the structure and reactivity relationship of chiral catalytic systems, establishing asymmetric chemical reaction methodologies typically requires significant experimental time and effort. Changes in any factor among catalysts, substrates, additives, and reaction conditions can have a substantial impact on experimental results. Therefore, determining the optimal reaction conditions and more universally applicable combinations for the target product using conventional reaction screening methods is extremely challenging. High-throughput screening, capable of acquiring large amounts of experimental data in a very short time, has become an important method for exploring complex chemical spaces and related reaction mechanisms. Although high-throughput reaction screening techniques have been successfully applied in fields such as chemical reaction condition optimization, drug discovery, and life sciences, the bottleneck of rapid analysis of enantiomeric excess (ee) values of chiral products limits the development of high-throughput screening research and methods for asymmetric catalytic reactions.
[0003] To achieve rapid determination of ee values, various analytical techniques such as optical probes, 19 Strategies such as F-NMR and pseudoenantiomer mass labeling enable rapid chiral analysis of chiral compounds in specific chemical scenarios. However, for complex asymmetric synthetic reactions, chiral chromatography remains the most commonly used method for determining the ee value. To ensure good separation, even without considering the cumbersome selection of chiral stationary phases and sample pretreatment steps, the long time required for chromatographic separation significantly limits the efficiency of asymmetric reaction screening. Therefore, the screening and optimization of asymmetric catalytic reactions currently cannot fully consider multiple combinations of conditions as it does for other types of reactions, and it is difficult to discover the optimal reaction system in a short time.
[0004] Ion mobility mass spectrometry (IMS) enables the separation of isomers on a millisecond timescale based on the difference in migration rates of gaseous ions with the same mass-to-charge ratio (m / z) in an electric field, allowing for precise qualitative and quantitative analysis of the separated isomers. Currently, IMS is widely used for rapid isomer analysis. Enantiomers within isomers share the same ion mobility, while most products of asymmetric catalytic reactions possess only a single chiral center. This prevents the direct use of IMS to determine the ee value of asymmetric catalytic reaction products. Therefore, achieving high-throughput screening of asymmetric catalytic reactions is both challenging and significant. Summary of the Invention
[0005] In view of the above-mentioned deficiencies of the prior art, in a first aspect of the present invention, a high-throughput screening method for asymmetric catalytic reactions with high accuracy and simplicity is provided, comprising the following steps:
[0006] (1) Prefunctionalization of the substrate:
[0007] By selecting an asymmetric catalytic reaction system and introducing labeling groups into the substrate molecule structure, labeled substrates can be obtained.
[0008] (2) High-throughput experiments on asymmetric reactions:
[0009] The labeled substrate was subjected to high-throughput screening of asymmetric reactions to obtain a high-throughput reaction mixture;
[0010] (3) Diastereoisomerization of the product:
[0011] A chiral reagent is added to the high-throughput reaction mixture. The chiral reagent and the labeled substrate in the high-throughput reaction mixture undergo a derivatization reaction to convert the substrate into a diastereomer, thus obtaining a derivatization reaction solution.
[0012] (4) Rapid chiral analysis by ion mobility mass spectrometry:
[0013] The derived reaction solution was analyzed by ion mobility mass spectrometry to complete rapid chirality analysis.
[0014] Preferably, in step (1), the labeling group is a group that can be subsequently modified for derivatization, and the introduction of the labeling group does not have a substantial impact on the stereoselectivity of the selected reaction.
[0015] More preferably, in step (1), the labeling group is an alkynyl group or an azide group, and the labeling group is located at the molecular end of the labeled substrate.
[0016] Preferably, in step (3), an internal standard is added to the high-throughput reaction mixture to determine the yield of the reaction process in which the labeled group is introduced by the internal standard method.
[0017] More preferably, the internal standard contains the same terminal labeling group as the labeled substrate.
[0018] More preferably, the molar ratio of the labeled substrate to the internal standard is 0.1 to 10:1.
[0019] Preferably, in step (3), the chiral reagent has an optical purity of ≥98%, and its molecular structure contains a rigid skeleton including any one of naphthalene ring, fluorenyl, and adamantane, as well as a reactive group corresponding to the labeling group in the labeling substrate.
[0020] In this invention, the chiral reagent has sufficiently high optical purity (≥98%), meaning it does not include the R configuration. This reduces the significant quantitative error caused by the purity of the chiral reagent, ensuring higher accuracy in chiral analysis results. Furthermore, when the chiral reagent possesses a large, rigid framework such as a naphthalene ring, fluorene group, or adamantane, it significantly optimizes the mobility separation of derivatized diastereomers.
[0021] More preferably, the chiral reagent is ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl4-azidobenzoate, with the following structural formula:
[0022]
[0023] More preferably, the chiral reagent and the labeled substrate undergo derivatization via a click chemistry reaction in the presence of a catalyst and a ligand; the catalyst is a monovalent copper catalyst.
[0024] Furthermore, the ratio of the catalyst to the ligand is 0.1 to 10:1 in molar ratio; the ratio of the catalyst to the chiral reagent is 0.01 to 10:1.
[0025] Furthermore, the monovalent copper catalyst is a cuprous halide, or a monovalent copper catalyst formed by the reaction of a divalent copper salt and a reducing agent.
[0026] Furthermore, the monovalent copper catalyst is at least one of cuprous fluoride, cuprous chloride, cuprous bromide, and cuprous iodide; or it is a monovalent copper catalyst formed by the reduction of copper sulfate and sodium ascorbate.
[0027] More preferably, the ligand comprises at least one of diisopropylethylamine, triethylamine, tri-n-butylamine, imidazole, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
[0028] Preferably, in step (3), the ratio of the chiral reagent to the labeled substrate is 0.1 to 10:1 in molar ratio.
[0029] Preferably, in step (3), the derivatization reaction is a click chemistry reaction, and the temperature of the click chemistry reaction is room temperature.
[0030] In this method, the chiral reagent and the labeled substrate correspond to alkynyl and azide groups, respectively, using a derivatization reaction (click chemistry) between them as the technical means. This reaction has extremely high selectivity and does not affect the chirality of the analyte. Furthermore, the reaction can be carried out rapidly at room temperature, completing within minutes, and the derivatization conditions are mild, ensuring that the target product is not racemized during the reaction. This diastereomeric strategy for chiral analysis is simple, rapid, and has good sensitivity and accuracy.
[0031] Preferably, the specific analysis process of step (4) is as follows: the derivatized reaction solution is analyzed by ion mobility mass spectrometry, the target product is qualitatively analyzed by the mass-to-charge ratio of diastereomers, the mass spectrometry yield is analyzed based on the signal intensity of the internal standard and the signal intensity of the diastereomers, the diastereomer ratio is calculated based on the mobility separation of the diastereomers and the ratio of the peak area of the corresponding extracted ion mobility map, and the corresponding enantiomer excess value is calculated.
[0032] Based on the above technical solutions, the principle and inventive concept of this invention are as follows:
[0033] Since it is currently impossible to directly analyze the enantiomeric excess (ee) value of asymmetric catalytic reaction products with a single chiral center using ion mobility mass spectrometry, the inventors have devised a solution by introducing a relatively inert labeling group into the reaction substrate, which can be subsequently modified and whose introduction has almost no impact on the stereoselectivity of the reaction. This allows the target product generated by the asymmetric reaction to undergo a derivatization process through a specific reaction, increasing the difference between the chiral target enantiomers. The diastereomerication strategy of this invention involves introducing a relatively inert alkynyl labeling group onto the asymmetric reaction substrate and conducting an asymmetric catalytic reaction. In a high-throughput reaction mixture, the labeled substrate undergoes a click chemistry reaction with a chiral reagent for efficient diastereomerication, transforming the resulting reaction product from an enantiomer into a diastereomer that can be distinguished by ion mobility mass spectrometry. The relative ratio of the diastereomers indirectly reflects the enantiomeric excess (ee) value of the target reaction product to be measured.
[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0035] This invention provides a high-throughput screening method for asymmetric catalytic reactions. This method is based on a diastereomeric strategy of click chemistry between labeled substrates and chiral reagents, enabling rapid chiral analysis of a variety of chiral compounds. This invention is the first to apply a diastereomeric strategy to the rapid chiral analysis of asymmetric catalytic reactions, avoiding the cumbersome and time-consuming process of chiral chromatographic separation and analysis. It achieves high-throughput screening of asymmetric catalytic reactions and depicts the complex chemical reaction space information of asymmetric catalytic reactions, with the advantages of high accuracy and simplicity. Attached Figure Description
[0036] Figure 1 The ion mobility diagrams for compounds with different ee values in Example 1 are as follows: 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)pentan-4-yneic acid.
[0037] Figure 2 The substrate for high-throughput screening of the asymmetric α-alkylation reaction of photocatalysts in Example 2 is a combination of photocatalyst and organic catalyst;
[0038] Figure 3 This is a stereoselectivity heatmap corresponding to the high-throughput screening experiment of asymmetric α-alkylation reactions of 1430 photocatalytic aldehydes in Example 2.
[0039] Figure 4 This is an ion mobility diagram corresponding to the reactions with different enantiomeric excess (ee) values in the high-throughput experiment of Example 2. Detailed Implementation
[0040] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.
[0041] Model of ion mobility mass spectrometer: Electrospray-trapping ion mobility-time-of-flight mass spectrometer Pro (Bruker Daltonics, Germany) with a captive spray ion source.
[0042] Example 1
[0043] To verify the reliability of the chiral analysis and quantitative capabilities of the strategy of this invention, this embodiment uses alkyne standard compounds with known configurations for derivatization and performs rapid chiral analysis by ion mobility mass spectrometry:
[0044] (1) Prefunctionalization of the substrate:
[0045] Two alkynyl standard compounds, R-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentan-4-alkynyl acid and S-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentan-4-alkynyl acid, were selected as the alkynyl standard compounds to be analyzed. They were used as labeled substrates and prepared with hexanilide to form standard solutions of R-configuration labeled substrate and S-configuration labeled substrate, respectively, with a concentration of 10 mM.
[0046] (2) Diastereoisomerization of the product:
[0047] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl4-azidobenzoate was prepared into a standard solution of chiral reagent with a concentration of 10 mM using dichloromethane; cuprous iodide and diisopropylethylamine were prepared into standard solutions of catalyst and ligand with a concentration of 10 mM each using acetonitrile;
[0048] As shown in Table 1, according to different volume ratios (standard solution of R-configuration labeled substrate: standard solution of S-configuration labeled substrate), a total volume of 100 μL of the labeled substrate standard solution was transferred into a 1.5 mL centrifuge tube using a pipette. Then, 100 μL of chiral reagent standard solution and 100 μL of catalyst and ligand standard solution were added to each group, respectively. The reaction was carried out by sonication at room temperature for 10 min. Derivatization was completed by click chemistry to convert it into diastereomer. After the reaction, the copper catalyst was precipitated by centrifugation or static precipitation, and the supernatant was taken to obtain the derivatization reaction solution.
[0049] (3) Rapid chiral analysis by ion mobility mass spectrometry:
[0050] The derivatized reaction solution was diluted with chromatographic grade acetonitrile and injected for ion mobility mass spectrometry analysis. To reduce residual contamination in the mass spectrometer while ensuring good mass spectrometry response, the injection concentration in this embodiment was 100 μM. By setting and optimizing the ion mobility mass spectrometry parameters, the mass-to-charge ratio (m / z) of the target diastereomer was determined, and the diastereomer ratio was determined based on the corresponding extracted ion mobility diagram to calculate the enantiomeric excess (ee) value of the analyte. The mass spectrometry analysis time for each sample was 30 s.
[0051] Table 1: Volume ratio of standard solutions for labeled substrates
[0052] Grouping Standard solutions of R-configuration labeled substrates: Standard solutions of S-configuration labeled substrates (v / v) 1 2：8 2 3：7 3 4：6 4 5：5 5 6：4 6 7：3 7 8：2 8 9：1
[0053] The analysis results of each group of samples in this embodiment are as follows: Figure 1 As shown, Figure 1The analytical results of analytes with different enantiomeric excess (ee) values are presented. The ion mobility mass spectrometry analysis results of (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentan-4-acetylic acid and (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentan-4-acetylic acid in different ratios after diastereomeric isomerization are also presented. 4-Alkyno acids can be rapidly and efficiently separated chirally by ion mobility mass spectrometry. Furthermore, the ratio of the mobility peak areas of the diastereomers generated by diastereomeric separation corresponds one-to-one with the R and S configurations of 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)pentane-4-alkynyl acids. This indicates that we can achieve chiral analysis of analytes with different enantiomeric excess values through a diastereomeric strategy, reflecting the high efficiency, rapid chiral analysis capability, and precise quantitative ability of this diastereomeric strategy. This demonstrates that this method possesses both high efficiency and rapid chiral analysis capabilities and precise quantitative ability.
[0054] Example 2
[0055] High-throughput screening methods for asymmetric catalytic reactions:
[0056] (1) Prefunctionalization of the substrate:
[0057] This example selects the visible light-guided organocatalytic asymmetric α-alkylation reaction of aldehydes published by MacMillan (Nicewicz, DA & MacMillan, DWCMerging photoredox catalysis with organocatalysis: The direct asymmetric alkylation of aldehydes. Science 322, 77-80 (2008)).
[0058] (2) High-throughput experiments on asymmetric reactions:
[0059] like Figure 3As shown, high-throughput experiments were conducted to screen a total of 1430 reactions involving 10 substrates (S1-S10), 13 photocatalysts (P1-P13), and 11 chiral organic catalysts (L1-L11). Reaction solutions for each component were prepared using DMF according to the feed ratios described in the literature. 25 μL of each of the four components was added to a 450 μL polypropylene 96-well plate, with a reactant concentration of 0.5 M. The reaction solution was then irradiated under white light for 8 hours. 4 μL of the resulting mixture was transferred to another 96-well plate, diluted with 36 μL of acetonitrile, and the corresponding labeled substrate standard solutions were obtained, which constituted the high-throughput reaction mixture.
[0060] (3) Diastereoisomerization of the product:
[0061] (S)-2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl4-azidobenzoate was prepared into a standard solution of chiral reagent with a concentration of 150 mM using dichloromethane; cuprous iodide and diisopropylethylamine were prepared into standard solutions of catalyst and ligand with a concentration of 400 mM each using acetonitrile; 1-phenylpropyl-2-ynyl-1-ol was used as an internal standard (IS) and prepared into a standard solution of internal standard with a concentration of 20 mM using acetonitrile;
[0062] Add 40 μL of standard solutions of chiral reagents, catalysts and ligands, and internal standards to the standard solutions of each labeled substrate. Then, seal the 96-well plate with a sealing film and sonicate it at room temperature for 10 min. Derivatization was completed by click chemistry to convert it into diastereomers. After the reaction, the copper catalyst was separated to obtain the derivatization reaction solution.
[0063] (4) Rapid chiral analysis by ion mobility mass spectrometry:
[0064] The derivatized reaction solution was diluted with chromatographic grade acetonitrile and injected for analysis. In this example, the injection concentration was 100 μM. The corresponding ion mobility mass spectrometry parameters were optimized according to different structures. The automatic sampler of liquid chromatography was used for injection analysis. The injection time (including needle washing) for each sample was 50 s, and the analysis time was 30 s. Therefore, the chiral analysis time for a single reaction was about 1.5 min.
[0065] The analysis results were processed using Compass Data Analysis software to calculate the diastereomer ratio (er) after derivatization and the corresponding enantiomeric excess (ee) value for the asymmetric catalytic reaction.
[0066] The high-throughput analysis results of this embodiment are as follows: Figure 3 , 4 As shown. Figure 3A heatmap of high-throughput, rapid chiral ee values for 1430 asymmetric reactions performed in Example 2 is presented, along with specific ee values for some of the heatmaps. The stereoselectivity of the selected aldehyde's asymmetric alkylation reaction primarily depends on the structure of the organocatalyst. L3, L4, and L10 exhibited high selectivity in different combinations of photocatalysts and substrates. This pre-functionalization of the substrate followed by diastereomericization of the product enables multi-parameter investigation of asymmetric catalytic reactions, broadening the reaction horizons for synthetic chemists. Figure 4 The analysis results of some reactions are presented, along with the stereoselectivity (ee) values and corresponding ion mobility mass spectrometry results. Compared to traditional chiral chromatography, this method enables in-situ direct analysis of complex reaction mixtures on a timescale of seconds or even less, demonstrating its unique advantage in high-throughput screening of asymmetric reactions. Combining the workflow and analysis time of this invention, this method avoids the cumbersome and time-consuming process of chiral chromatographic separation and analysis, achieving high-throughput screening of asymmetric catalytic reactions and depicting the complex chemical reaction spatial information of asymmetric catalytic reactions, while also being highly accurate, simple, and rapid.
[0067] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.
Claims
1. A high-throughput screening method for asymmetric catalytic reactions, characterized in that, Includes the following steps: (1) Prefunctionalization of the substrate: By selecting an asymmetric catalytic reaction system and introducing labeling groups into the substrate molecule structure, labeled substrates can be obtained. (2) High-throughput experiments on asymmetric reactions: The labeled substrate was subjected to high-throughput screening of asymmetric reactions to obtain a high-throughput reaction mixture; (3) Diastereoisomerization of the product: A chiral reagent is added to the high-throughput reaction mixture. The chiral reagent and the labeled substrate in the high-throughput reaction mixture undergo a derivatization reaction to convert the substrate into a diastereomer, thus obtaining a derivatization reaction solution. (4) Rapid chiral analysis by ion mobility mass spectrometry: The derived reaction solution was analyzed by ion mobility mass spectrometry to complete rapid chirality analysis.
2. The method according to claim 1, characterized in that: In step (1), the labeling group is a group that can be subsequently modified for derivatization, and the introduction of the labeling group will not have a substantial impact on the stereoselectivity of the selected reaction.
3. The method according to claim 1, characterized in that: In step (3), an internal standard is also added to the high-throughput reaction mixture.
4. The method according to claim 3, characterized in that: The internal standard contains the same terminal labeling group as the labeled substrate; the molar ratio of the labeled substrate to the internal standard is 0.1 to 10:
1.
5. The method according to claim 2, characterized in that: In step (3), the chiral reagent has an optical purity of ≥98%, and its molecular structure contains a rigid skeleton including any one of naphthalene ring, fluorenyl, and adamantane, as well as a reactive group corresponding to the labeling group in the labeling substrate.
6. The method according to claim 5, characterized in that, The labeling group in the labeled substrate is an alkynyl group, and the chiral reagent is ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl4-azidobenzoate, with the following structural formula:
7. The method according to claim 6, characterized in that: The chiral reagent and the labeled substrate undergo derivatization via a click chemistry reaction in the presence of a catalyst and a ligand; the catalyst is a monovalent copper catalyst; the ligand includes at least one selected from diisopropylethylamine, triethylamine, tri-n-butylamine, imidazole, and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; the molar ratio of the catalyst to the ligand is 0.1 to 10:1; the molar ratio of the catalyst to the chiral reagent is 0.01 to 10:
1.
8. The method according to claim 7, characterized in that: The monovalent copper catalyst is a cuprous halide, or a monovalent copper catalyst formed by the reaction of a divalent copper salt and a reducing agent.
9. The method according to claim 1, characterized in that: In step (3), the ratio of the chiral reagent to the labeled substrate is 0.1 to 10:1; the derivatization reaction is a click chemistry reaction, and the temperature of the click chemistry reaction is room temperature.
10. The method according to claim 1, characterized in that, The specific analysis process of step (4) is as follows: the derivatized reaction solution is analyzed by ion mobility mass spectrometry, and the target product is qualitatively analyzed by the mass-to-charge ratio of the diastereomers. The mass spectrometry yield is analyzed based on the signal intensity of the internal standard and the signal intensity of the diastereomers. The diastereomer ratio is calculated based on the mobility separation of the diastereomers and the ratio of the peak area of the corresponding extracted ion mobility map, and the corresponding enantiomer excess value is calculated.