Quantitative method for chiral compound based on enantiomer internal standard

Abstract

The invention discloses a chiral compound quantification method based on an enantiomer internal standard, and belongs to the technical field of testing. According to the method, chiral distinguishing and enantiomer excess (ee) determination are carried out based on a diastereoisomerization strategy and ion mobility mass spectrometry, and absolute quantitative analysis of chiral compounds can be realized with extremely high flux by adding an enantiomer internal standard; the method can realize dual quantitative analysis of absolute concentration and stereocomposition of the chiral compound only by depending on mobility dimension, is successfully applied to high-throughput screening of yield and stereoselectivity of an enzyme catalytic reaction, can avoid interference of a matrix effect and an ion inhibition effect, and has the advantages of high analysis speed, high accuracy, simplicity, convenience and rapidness.

Description

Technical Field

[0001] The present invention relates to the field of testing technology, and in particular to a chiral compound quantification method based on an enantiomeric internal standard. Background Art

[0002] Chiral compounds are ubiquitous in organisms and in the structures of active pharmaceutical ingredients, holding significant research significance in fields such as synthetic chemistry, life sciences, and medicinal chemistry. High-throughput quantitative analysis of chiral compounds presents significant challenges. In addition to determining the isomeric composition of chiral compounds, it is also necessary to determine the absolute content of each isomer. Due to their structural similarities, the differentiation of chiral compounds often requires specialized analytical techniques such as chiral chromatography, nuclear magnetic resonance (NMR), circular dichroism (CD), and mass spectrometry. Chiral chromatography is currently the gold standard for chiral compound analysis, enabling accurate estimation of the enantiomeric excess (ee) of enantiomers. Chromatographic separations for chiral analysis of a single sample often require tens of minutes, severely limiting the efficiency of chiral compound analysis. Ion mobility mass spectrometry (IM-MS) is a novel mass spectrometry technique that enables isomer differentiation based on the collision cross section (CCS) of analyte gaseous ions under gas-phase conditions and on a millisecond timescale, effectively resolving isomers. However, since enantiomers in chiral isomers have the same CCS value, current IM-MS technology cannot achieve direct distinction of enantiomers.

[0003] The absolute quantitative analysis of compounds is of irreplaceable importance in research in fields such as biology, medicine, and environmental science, laying the foundation for revealing the essential laws of complex systems and promoting applied research. Stable isotope labeling quantification is the most accurate quantitative analysis method in mass spectrometry analysis. Mass spectrometry quantitative analysis is performed based on the difference in the mass-to-charge ratio between the target analyte and its isotope internal standard. Due to the low natural abundance of isotopes, they are very expensive and easily exchanged, which greatly limits the practicality of stable isotope labeling quantification methods. In addition, although absolute quantitative analysis of substances can be achieved based on optical detection methods, such as enzyme markers, it is unable to provide information on the isomeric composition of chiral compounds. Therefore, existing analytical technologies lack a new mass spectrometry method that can simultaneously and rapidly analyze the isomeric composition and absolute quantification of chiral compounds.

[0004] Chinese patent CN202311198476.6 discloses a high-throughput screening method for asymmetric catalytic reactions. By combining the pre-functionalization and diastereoisomerization strategies of asymmetric catalytic reaction substrates, high-throughput screening of chemical reactions for stereoselectivity can be achieved. However, this method requires functionalization of the reaction substrate (such as alkynyl), and the introduced modifying groups may affect the yield of the reaction. In addition, this method can only obtain the ee value of the chiral reaction product and cannot take into account the acquisition of the reaction yield. Although the use of a homologous internal standard can obtain relative mass spectrometry yields, the ion suppression effect and matrix effect present in mass spectrometry analysis will affect the accuracy of absolute quantification.

[0005] In 2024, Professor Christian Wolf's team reported an optical sensing strategy for chiral alcohols (Angew. Chem. Int. Ed. 2024, 63, e202409790), which enables rapid determination of chiral alcohol concentration and ee value. However, this optical analysis technique often requires the use of a standard sample to create a calibration curve for ee determination, and absolute quantitative analysis can be subject to interference from reducing substances in the test system, significantly limiting its potential for practical applications.

[0006] Therefore, it is extremely challenging and of great research significance to develop a new method based on enantiomeric internal standards that can simultaneously achieve relative and absolute quantitative analysis of chiral compounds. Summary of the Invention

[0007] In view of the limitations of the above-mentioned prior art, the present invention provides a new method for quantifying chiral compounds based on an enantiomeric internal standard, comprising the following steps:

[0008] (1) Diastereoisomerization of chiral analytes:

[0009] The target chiral analytes are chemically derivatized using chiral derivatization reagents to convert them into diastereomers that can be distinguished by ion mobility mass spectrometry;

[0010] (2) Rapid chiral analysis by ion mobility mass spectrometry:

[0011] performing ion mobility mass spectrometry analysis on the diastereomers generated after the derivatization to obtain isomer composition information (enantiomeric excess (ee) or enantiomeric ratio (er)) of the chiral compound;

[0012] (3) Addition of enantiomeric internal standard:

[0013] An enantiomeric internal standard of known concentration and isomeric composition is added to the chiral analyte, the analyte containing the internal standard is diastereomerized, and analyzed by ion mobility mass spectrometry;

[0014] (4) Isomer composition and absolute quantitative analysis of chiral compounds:

[0015] The isomer composition and absolute quantitative analysis of the chiral compound are calculated based on the results of the two ion mobility mass spectrometry analyses performed with or without the addition of an enantiomeric internal standard.

[0016] Preferably, in step (1), a chiral derivatization reagent reacts with a chiral analyte, and the reaction system selected depends on the reactivity of the analyte. The corresponding derivatization reaction and chiral derivatization reagent are selected based on the inherent chemical functional groups in the analyte, and the chemical functional groups may be carboxyl, amino, hydroxyl, sulfhydryl, aldehyde, ester, carbon-carbon double bond, alkynyl, azide, etc.

[0017] Preferably, in step (1), the chiral reagent is used to convert the enantiomer analyte into a diastereomer that can be distinguished by IM-MS; the chiral derivatization reagent should have any one of a rigid skeleton such as fluorenyl, adamantane, etc.

[0018] According to a specific embodiment of the present invention, the above method is divided into two aspects for explanation. The first aspect is the rapid determination of the ee value of a chiral compound, and the second aspect is the absolute quantitative analysis using an enantiomeric internal standard. The analyte needs to be divided into two parts, one for chiral analysis and the other for absolute quantitative analysis.

[0019] In a first aspect of the present invention, a rapid analysis method for the ee value of a chiral compound is provided, comprising the following steps:

[0020] (1) Diastereoisomerization of chiral analytes:

[0021] Using a chiral derivatization reagent to chemically derivatize a chiral carboxylic acid analyte, the chiral carboxylic acid analyte is converted into diastereomers that can be distinguished by IM-MS, and a derivatization reaction solution is obtained;

[0022] (2) Rapid chiral analysis by ion mobility mass spectrometry:

[0023] The derivatization reaction solution is analyzed by ion mobility mass spectrometry to achieve rapid chiral differentiation;

[0024] (3) Calculation of ee value of chiral carboxylic acid:

[0025] The ion mobility mass spectrometry analysis results are processed, and the chiral carboxylic acid is qualitatively analyzed based on the accurate molecular weight. The corresponding extracted mobility map (EIM) is extracted based on the characteristic ions of the diastereomers, and the ee value of the chiral carboxylic acid is calculated based on the two mobility peak areas in the EIM results.

[0026] Preferably, the molar ratio of the chiral derivatization reagent to the target chiral analyte is 0.1 to 10:1, more preferably 1:1.

[0027] Further preferably, in step (1), the optical purity of the chiral derivatization reagent (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid should be ≥98%. The higher the optical purity, the more accurate the quantitative analysis of the ee value of the chiral carboxylic acid. In addition, the R-configured enantiomers are also used as chiral derivatization reagents.

[0028] Further preferably, a chiral carboxylic acid compound is used as the target chiral analyte, whose active reactive group is a carboxyl group, and the chiral carboxylic acid is diastereomerized by selecting a specific condensation reaction; (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid, whose rigid skeleton is a fluorenyl group, is selected as the chiral derivatization reagent, and its structural formula is as follows:

[0029] .

[0030] More preferably, the chiral carboxylic acid is derivatized by an amide condensation reaction with a derivatizing agent in the presence of a condensing agent and an organic base.

[0031] The condensing agent is selected from at least one of carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), 2-chloro-1-methylpyridinium iodide (CMPI), 2-(7-azobenzotriazole)-N,N,N',N'tetramethyluronium hexafluorophosphate (HATU), tetramethylchloroformamidine hexafluorophosphate (TCFH), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), 5-nitro-4,6-dithiocyanatopyrimidine (NDTP), N-methylethynyl-p-toluenesulfonamide (MYTsA), and carbonyldiimidazole (CDI).

[0032] The concentration of the condensing agent solution is 10-50 mM, preferably 20 mM.

[0033] The organic base is selected from at least one of diisopropylethylamine, triethylamine, tri-n-butylamine, N-methylimidazole, pyridine, 1-hydroxybenzotriazole, and 2,6-lutidine.

[0034] The concentration of the organic base solution is 10-50 mM, preferably 20 mM.

[0035] Furthermore, in the step (1), the molar ratio of the condensing agent to the organic base is 0.1 to 10:1, preferably 1:1.

[0036] In terms of molar ratio, the ratio of the condensing agent to the chiral derivatizing agent is 0.1 to 10:1, preferably 1:1.

[0037] Furthermore, in step (1), a combination of CMPI and DIPEA is selected for chemical derivatization, and the derivatization reaction temperature is 0-60°C, preferably 40°C.

[0038] The derivatization reaction system is at least one of acetonitrile, methanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, chloroform, tetrahydrofuran, and toluene, preferably acetonitrile.

[0039] Preferably, in step (2), the derivatization reaction solution is subjected to mass spectrometry analysis using ion mobility mass spectrometry. The ion mobility mass spectrometry model can be any one of drift time ion mobility spectrometry (DTIMS), traveling wave ion mobility mass spectrometry (TWIMS), field asymmetric waveform ion mobility mass spectrometry (FAIMS), trapped ion mobility mass spectrometry (TIMS), and differential ion mobility mass spectrometry (DIMS).

[0040] Further preferably, the ion mobility mass spectrometer uses an electrospray-trap ion mobility-time-of-flight mass spectrometer Pro (Bruker, Germany) to perform ion mobility mass spectrometry analysis.

[0041] Further preferably, the ion mobility mass spectrometry analysis only needs to optimize the mobility range (mobility range: 0.55~1.90 V·s / cm 2 ), drift time (ramp time: 0~959 ms) and adduct ion form (H + 、Na + , K + 、Cu 2 + ……).

[0042] Preferably, in step (2), the derivatization reaction solution is injected and analyzed via an automatic injection pump or ultra-high performance liquid chromatography (UPLC).

[0043] Preferably, in step (3), the data processing software used is Compass Data Analysis 6.2 (Bruker, Germany), and the data processing of mobility peaks with poor separation can be performed with the help of peak fitting software.

[0044] The second aspect of the present invention provides an enantiomeric internal standard quantitative strategy for absolute quantitative analysis of chiral compounds, comprising the following steps:

[0045] (1) Distribution of chiral analyte samples:

[0046] The analyte solution was divided into two parts, one part was used for ee value determination, and the other part was used for absolute quantitative analysis;

[0047] (2) Determination of ee value of chiral analytes:

[0048] According to the first aspect of the present invention, the ee value of the chiral compound is quickly analyzed and the ee value is calculated. The calculation formula is as follows:

[0049] (1)

[0050] (2)

[0051] Among them, A S and A R represent the mobility peak areas corresponding to the S and R isomers, respectively, and m is the mobility peak area ratio;

[0052] (3) Addition of enantiomeric internal standard:

[0053] For absolute quantitative analysis of chiral analytes, refer to the ee value determination results, select the appropriate enantiomeric internal standard concentration and isomer composition, mix the internal standard with the analyte solution, perform chemical derivatization and second ion mobility mass spectrometry analysis. R Taking the R-configuration enantiomer internal standard as an example, the area ratio n of the two mobility peaks is calculated. The calculation formula 2 is as follows:

[0054] (3)

[0055] Among them, A S and A R Represent the mobility peak areas corresponding to S and R isomers, respectively. R' represents the mobility peak area of the enantiomeric internal standard, and n is the ratio of the mobility peak areas measured after adding the enantiomeric internal standard;

[0056] (4) Absolute quantitative analysis of chiral analytes:

[0057] Combining the two ion mobility analysis results, the absolute content of the chiral analyte is calculated, and the calculation formula 3 is as follows:

[0058] (4)

[0059] Among them, C S and C R Represent the absolute contents of S and R isomers, respectively. R' represents the concentration of the enantiomeric internal standard.

[0060] Preferably, in step (1), the chiral analysis sample is prepared into a certain molar concentration and then distributed according to a volume ratio. There is no requirement for the sample volume ratio for ee value and absolute quantitative analysis, and equal volume distribution is optimal.

[0061] Preferably, in step (2) and step (3), the reaction conditions of the chemical derivatization are controlled to be consistent.

[0062] In step (3), in order to ensure the accuracy of quantification, the ratio of the concentration of the internal standard of the enantiomer to the concentration of the target analyte to be measured is in the range of 0.01 to 100:1, preferably 1:1.

[0063] In the step (3), the isomer composition of the enantiomeric internal standard is −100% ee to 100% ee, but the isomer composition of the enantiomeric internal standard cannot be the same as the isomer composition of the chiral analyte.

[0064] Furthermore, in step (3), different configurations of enantiomer internal standards are used, and the above equation needs to be adjusted accordingly. If the concentration C S' If the internal standard is an S configuration, then Formula 2 should be adjusted to Formula 4:

[0065] (5)

[0066] Among them, A S and A R Represent the mobility peak areas corresponding to S and R isomers, respectively. S' Represents the mobility peak area of the enantiomeric standard.

[0067] Preferably, in step (4), the equation is based on the concentration C R' The formula for the derivation of the R-configuration internal standard is: if the concentration C S' If the internal standard is an S configuration, the absolute quantitative formula should be adjusted to:

[0068] (6)

[0069] Among them, C S and C R Represent the absolute contents of S and R isomers, respectively. R' represents the concentration of the enantiomeric internal standard.

[0070] Based on the above technical solutions, the principles and inventive concepts of the present invention are as follows:

[0071] Since ion mobility mass spectrometry currently cannot directly analyze the ee values of enantiomers with a single chiral center, the inventors designed a corresponding derivatization reaction system and chiral derivatization reagent based on the reactivity of the functional groups inherent in the chiral analyte structure. This converts the enantiomer analyte into diastereomers distinguishable by ion mobility mass spectrometry, thereby obtaining the ee value of the chiral analyte, thereby achieving relative quantitative analysis of the enantiomers. Building on diastereoisomerization and ion mobility mass spectrometry separation, the present invention proposes for the first time a strategy for quantifying chiral compounds based on an enantiomeric internal standard. By adding an enantiomeric internal standard, followed by further chemical derivatization and ion mobility mass spectrometry analysis, absolute quantitative analysis of the enantiomers can be achieved, including determination of the absolute content of the two configurations.

[0072] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0073] The present invention provides a method for quantifying chiral compounds based on enantiomeric internal standards. This method uses ion mobility mass spectrometry and a quantitative strategy based on diastereoisomerization and enantiomeric internal standard addition to enable rapid chiral analysis of the ee value and absolute content of chiral compounds. This method not only avoids the tedious and time-consuming process of chiral chromatographic separation and analysis in chiral analysis, but also allows for highly accurate, simple, and rapid determination of the absolute content of chiral isomers. This method is unaffected by matrix effects and ion suppression effects in complex systems, providing a novel approach for the quantitative analysis of chiral compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 IM-MS analysis results of chiral carboxylic acid C2 with different ee values in Example 1;

[0075] Figure 2 To achieve absolute quantitative analysis of different concentrations of S-C2 using 5 mM R-C2 as the enantiomeric internal standard in Example 1;

[0076] Figure 3 To achieve absolute quantitative analysis of different concentrations of S-C2 using 5 mM racemized internal standard C2 as the enantiomeric internal standard in Example 1;

[0077] Figure 4 The reaction equation for the enzymatic hydrolysis of ester compounds and the screening components in Example 2;

[0078] Figure 5 The heat map results of the enzyme-catalyzed reaction yield and ee value in Example 2;

[0079] Figure 6 The figure shows the principle of using different types of enantiomeric internal standards for ee value and absolute quantitative analysis;

[0080] Figure 7 This is the general process for simultaneous analysis of ee value and concentration of chiral compounds. DETAILED DESCRIPTION

[0081] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0082] Ion mobility mass spectrometer model: timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) with a CaptiveSpray ion source.

[0083] Example 1

[0084] To verify the reliability of the chiral analysis and quantitative capabilities of the strategy of the present invention, this example used chiral carboxylic acid standards for derivatization and rapid chiral analysis by ion mobility mass spectrometry. Ion mobility mass spectrometry and an enantiomeric internal standardization strategy enabled dual quantitative analysis of the concentration and isomer composition of the chiral carboxylic acid:

[0085] (1) Preparation of chiral carboxylic acid sample solution:

[0086] N-Boc-phenylalanine with R and S configurations was selected as the chiral carboxylic acid standard compound to be analyzed. When performing enantiomer ee value analysis, acetonitrile was used as the solvent to prepare a 20 mM concentration of R-configuration carboxylic acid standard solution and an S-configuration carboxylic acid standard solution. According to the sample composition ratio in Table 1, 10 sets of standard solutions with different ee values and concentrations were prepared with a total volume of 100 μL. The solutions were then divided into two equal volumes, one for ee value determination and the other for absolute quantitative analysis.

[0087] (2) Diastereoisomerization of chiral carboxylic acids and determination of ee values:

[0088] Prepare a 20 mM chiral reagent standard solution of (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid in acetonitrile (partially dissolved); prepare a 20 mM condensing agent and organic base standard solution of CMPI and DIPEA in acetonitrile;

[0089] Use a pipette to transfer a total volume of 50 μL of the 10 groups of sample solutions in step (1) above into 10 0.6 mL centrifuge tubes. Then, add 50 μL of the chiral derivatization reagent standard solution, 50 μL of the condensation agent standard solution, and 50 μL of the organic base standard solution to each centrifuge tube in sequence, and ultrasonically react at 40°C for 10 min. Derivatization is completed by amide condensation to convert the target analyte into a diastereoisomer. The derivatized reaction solution is diluted 100 times with methanol and injected by needle pump or liquid phase autosampler for ion mobility mass spectrometry analysis. The ramp time is set to 600 ms and the mobility range is set to 1.20-1.30 V·s / cm 2 The mass spectrometry analysis time for each sample was 30 s. + (m / z: 660.2656) The corresponding EIM results were used to determine the diastereoisomer ratio, and the ratio m was calculated. The ee value of each group of samples was calculated according to Formula 2;

[0090] (3) Addition of enantiomeric internal standard:

[0091] Add 50 μL of 5 mM R-configuration N-Boc-phenylalanine internal standard solution to the remaining 10 test sample solutions in step (1) above, perform chemical derivatization and ion mobility mass spectrometry analysis according to step (2) above, and calculate the corresponding ratio n in equation 3;

[0092] (4) Absolute quantitative analysis of chiral analytes:

[0093] Substitute the ratios m and n obtained from the two ion mobility analysis results into Equation 4 for calculation, where C R' = 5 mM, the concentration of each group of samples was calculated, and the analysis results are shown in Table 1. Figures 1-3 The results of ee value and absolute quantitative analysis using N-Boc-phenylalanine as a model analyte are presented, indicating that the quantitative method of the enantiomeric internal standard of this chiral compound has the advantages of simple and rapid operation and accurate quantitative analysis.

[0094] Table 1: Volume ratio of standard solution of labeled substrate

[0095]

[0096] The analytical results for each sample group in this Example are shown in Table 1. This dual quantitative strategy enables the quantitative analysis of chiral analytes with varying concentrations and isomeric compositions. The mean absolute errors of concentration and ee values measured using the enantiomeric internal standard method based on IM-MS were 0.8 mM and 3.0%, respectively. For high-concentration samples, for example, an N-Boc-phenylalanine sample with a concentration of 20 mM and an ee of -43% was measured by IM-MS to have a concentration of 17.7 mM and an ee of -43% (No. 4). For samples with high ee values, an N-Boc-phenylalanine sample with a concentration of 7 mM and an ee of 80% was measured by IM-MS to have a concentration of 7.6 mM and an ee of 80% (No. 9). Furthermore, by adding an internal standard of known concentration and enantiomeric composition, this Example not only determines the concentration and enantiomeric composition of the target analyte, but also allows the absolute configuration of the target analyte to be determined based on the mobility behavior of the internal standard. In summary, the analysis results in Table 1 fully demonstrate the advantages of the enantiomeric internal standard strategy proposed in this study in the dual analysis of the isomer composition and absolute content of chiral carboxylic acids.

[0097] Example 2

[0098] Simultaneous quantitative analysis of yield and ee value of enzyme-catalyzed reactions:

[0099] (1) High-throughput experiments of enzyme-catalyzed reactions:

[0100] In this example, the enzyme-catalyzed ester hydrolysis reaction system was used as the research object. Nine representative ester substrates and 12 commercial industrial hydrolases were selected for orthogonal reaction screening, totaling 108 enzyme-catalyzed reactions ( Figure 4 The ester reaction substrate was prepared using a DMSO / 1×PBS (1:9, v / v) solution, and the enzyme catalyst was prepared using a 1×PBS solution. 50 μL of each standard substrate solution and enzyme solution were pipetted into a 96-well plate and shaken at 37°C and 800 rpm for 16 h. 20 μL of each of the reaction solutions was pipetted into two new 96-well plates (plate A and plate B). Plate A was used to determine the ee value of the enzyme-catalyzed reaction, while plate B was used to determine the yield of the enzyme-catalyzed reaction.

[0101] (2) Diastereoisomerization of chiral carboxylic acids and determination of ee values:

[0102] Prepare a 20 mM chiral reagent standard solution of (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid in acetonitrile (partially dissolved); prepare a 20 mM condensing agent and organic base standard solution of CMPI and DIPEA in acetonitrile;

[0103] To 96-well plate A, 20 μL of chiral derivatization reagent standard solution, 20 μL of condensing agent standard solution, and 20 μL of organic base standard solution were added sequentially. The reaction was sonicated in a water bath at 40°C for 10 minutes. Derivatization of the target analyte into diastereomers was completed by amide condensation. The derivatized reaction solution was diluted 20-fold with methanol and injected via a liquid phase autosampler and analyzed by ion mobility mass spectrometry. Parameters such as ramp time and mobility range were adjusted based on the different substrates. The mass spectrometry analysis time for each sample was 30 seconds. Based on the EIM results corresponding to each reaction, the ratio m and the ee value of each reaction were calculated.

[0104] (3) Addition of enantiomeric internal standard:

[0105] On the basis of step (2), the corresponding enantiomeric internal standard was selected according to the analysis results of the ee values of different reaction products. 20 μL of 5 mM enantiomeric internal standard, 20 μL of chiral derivatization reagent standard solution, 20 μL of condensation agent standard solution, and 20 μL of organic base standard solution were added to 96-well plate B in sequence, and the reaction was ultrasonicated in a water bath at 40°C for 10 min. Among them, based on the analysis results of the ee value of substrate S1, 5 mM of R-configuration product was used as internal standard for E4, and 5 mM of S-configuration carboxylic acid product was used as enantiomeric internal standard for absolute quantitative analysis of the remaining 11 enzyme-catalyzed reaction solutions. For the hydrolysis reactions involving substrates S2 and S8, 5 mM of S-configuration carboxylic acid product was used as enantiomeric internal standard. For the hydrolysis reactions involving substrates S3, S4, S6, and S7, 5 mM of R-configuration carboxylic acid product was used as enantiomeric internal standard. For the hydrolysis reaction involving substrate S5, 5 mM of the S-configuration carboxylic acid product was used as the internal standard for E2, and 5 mM of the R-configuration carboxylic acid product was used as the enantiomeric internal standard for the absolute quantitative analysis of the remaining 11 enzyme-catalyzed reaction solutions. For the hydrolysis reaction involving substrate S9, 5 mM of the S-configuration carboxylic acid product was used as the internal standard for E5 and E12, and 5 mM of the R-configuration carboxylic acid product was used as the enantiomeric internal standard for the absolute quantitative analysis of the remaining 10 enzyme-catalyzed reaction solutions. The enzyme-catalyzed reaction mixture after the addition of the enantiomeric internal standard was subjected to chemical derivatization and ion mobility mass spectrometry analysis according to the above step (2), and the corresponding ratio n in Equation 3 was calculated for the reaction in each well plate;

[0106] (4) Absolute quantitative analysis of enzyme-catalyzed reaction yield:

[0107] Substitute the ratios m and n obtained from the two ion mobility analysis results in steps (2) and (3) above into equation 4 or equation 5 to calculate the concentration of each reaction.

[0108] The high-throughput analysis results of this example are as follows Figure 5 As shown. By plotting the heat map of the yield and ee value of the enzyme-catalyzed ester hydrolysis reaction, the catalytic performance of the enzyme can be quickly and accurately revealed, and the structure-activity relationship between the enzyme catalyst and different substrates can be deeply revealed. Figure 5 Screening results indicate that most hydrolases exhibit high stereoselectivity when catalyzing the hydrolysis of ester substrates. Stereoselectivities vary significantly between different substrate and hydrolase combinations, sometimes exhibiting diametrically opposed stereo preferences. For substrate S6, the E3 hydrolase appears to be a promising biocatalyst for selective hydrolysis (28.9% yield, 97% ee). Furthermore, the E4 hydrolase selectively hydrolyzes racemized ibuprofen methyl ester to efficiently produce S-ibuprofen (26.2% yield, 88% ee), promising applications in the kinetic resolution and asymmetric biosynthesis of drugs such as ibuprofen. Most importantly, screening only 12 commercially available hydrolase tools resulted in the successful identification of three potential complementary enzyme pairs. The chiral compound quantification method developed in this study enables accurate and rapid simultaneous analysis of yield and stereoselectivity in high-throughput reactions. This technology is expected to be further applied in synthetic biology and fulfills the demand for high-throughput chiral screening techniques for directed enzyme evolution, demonstrating its promising application prospects.

[0109] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for quantifying chiral compounds based on an enantiomeric internal standard, characterized in that: The steps include: (1) Diastereoisomerization of chiral analytes: The target chiral analytes are chemically derivatized using chiral derivatization reagents to convert them into diastereomers that can be distinguished by ion mobility mass spectrometry; (2) Rapid chiral analysis by ion mobility mass spectrometry: performing ion mobility mass spectrometry analysis on the diastereomers generated after derivatization to obtain isomer composition information of the chiral analyte after chemical derivatization; (3) Addition of enantiomeric internal standard: An enantiomeric internal standard of known concentration and isomer composition is added to a target chiral analyte to obtain an analyte containing the internal standard, the analyte containing the internal standard is diastereomerized to obtain a derivatized reaction solution, and ion mobility mass spectrometry analysis is performed; (4) Isomer composition and absolute quantitative analysis of chiral compounds: The isomer composition and absolute quantitative analysis of the chiral compound are calculated based on the results of the two ion mobility mass spectrometry analyses performed with or without the addition of an enantiomeric internal standard.

2. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: In step (1), a specific chiral derivatization reaction and chiral derivatization reagent are selected based on the inherent functional groups in the structure of the target chiral analyte to perform diastereoisomerization on the target chiral analyte.

3. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: The molecular structure of the chiral derivatizing reagent contains a rigid skeleton including any one of a naphthalene ring, a fluorenyl group, and an adamantane ring, and comprises a reactive group corresponding to an inherent group in the target chiral analyte.

4. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: In the step (3), the isomer composition of the enantiomeric internal standard is −100% ee to 100% ee, and the ratio of the concentration of the enantiomeric internal standard to the concentration of the analyte is 0.01 to 100:

1.

5. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: In the step (1), the ratio of the chiral derivatization reagent to the target chiral analyte is 0.01-100:

1.

6. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: The specific analysis process of step (4) is as follows: for chiral analysis, the reaction solution after derivatization is subjected to ion mobility mass spectrometry analysis, and the target product is qualitatively analyzed by the precise molecular weight of the diastereoisomers. The diastereoisomer ratio is calculated based on the mobility separation of the diastereoisomers and the ratio of the peak areas of the corresponding extracted ion mobility graph, thereby indirectly obtaining the ee value of the analyte; for absolute quantitative analysis, an enantiomeric internal standard of known concentration and isomer composition is added to the analysis system, and then diastereoisomerization and ion mobility mass spectrometry analysis are performed. The absolute content of the analyte can be calculated based on the analysis results of the above two measurements.

7. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 1, characterized in that: In step (1), the target chiral analyte is a chiral carboxylic acid compound, and the chiral carboxylic acid compound and a chiral derivatizing reagent are subjected to an amide condensation reaction in the presence of a condensing agent and an organic base to complete diastereoisomerization.

8. The method for internal standardization and quantitative determination of the enantiomers of chiral compounds according to claim 7, wherein: In terms of molar ratio, the ratio of the condensing agent to the organic base is 0.1-10:1; and the ratio of the condensing agent to the chiral derivatization reagent is 0.01-10:

1.

9. The method for quantifying chiral compounds based on enantiomeric internal standard according to claim 7, characterized in that: The chiral derivatizing agent can be (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-aminohexanoic acid, the rigid skeleton of which is a fluorenyl group, and its structural formula is as follows: ； The condensing agent includes at least one of carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), 2-chloro-1-methylpyridinium iodide (CMPI), 2-(7-azobenzotriazole)-N,N,N',N'tetramethyluronium hexafluorophosphate (HATU), tetramethylchloroformamidine hexafluorophosphate (TCFH), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), 5-nitro-4,6-dithiocyanatopyrimidine (NDTP), N-methylethynyl-p-toluenesulfonamide (MYTsA), and carbonyldiimidazole (CDI); The organic base includes at least one of diisopropylethylamine, triethylamine, tri-n-butylamine, N-methylimidazole, pyridine, 1-hydroxybenzotriazole, and 2,6-lutidine.

10. The use of the method for quantifying chiral compounds based on enantiomeric internal standards according to claims 1-6, characterized in that: The enantiomeric internal standard quantitative method for chiral compounds is applicable to chiral compounds that can be distinguished in mobility by chemical derivatization strategies.

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