A chiral amine, its preparation method, and its application in the analysis and resolution of chiral amino acids.

Abstract

This invention discloses the application of chiral amines in the analysis and / or resolution of chiral amino acids, and a method for analyzing and / or resolving chiral amino acids. The method for analyzing and / or resolving chiral amino acids using chiral amines includes the following steps: using chiral amines... d One or two of 5-APPM and APPM are used as labeling reagents to react with chiral amino acids to obtain derivatives; the derivatives are then detected by LC-MS; wherein, the carbon atom on the pyrrole ring of the chiral amine has an R configuration. This invention relates to APPM and its deuterated stable isotope form. d 5-APPM can undergo a specific condensation reaction with chiral amino acids, and the resulting derivative contains easily ionized groups, thereby enhancing the mass spectrometry signal response and improving detection sensitivity in electrospray ionization (ESI) positive ion mode. This invention also achieves efficient separation of chiral amino acid enantiomers on conventional chromatographic columns.

Description

Technical Field

[0001] This invention relates to the field of chiral amino acid detection technology, specifically to a chiral amine, its preparation method, and its application in the analysis and resolution of chiral amino acids. Background Technology

[0002] Different enantiomers of chiral amino acids have significantly different physiological functions and metabolic pathways in organisms. For example, L-amino acids are the main building blocks of proteins in organisms, while D-amino acids also play important roles in certain biological processes, such as memory and learning. Therefore, separation techniques are particularly important when analyzing chiral amino acids in biological samples.

[0003] Chemical derivatization is an effective method for the efficient separation of chiral amino acids on chromatographic columns. By reacting with chiral derivatizing reagents, chiral amino acids can be converted into diastereomers, thus enabling separation on conventional columns. Furthermore, derivatization can significantly improve the sensitivity of chiral amino acids in mass spectrometry analysis, reduce background signals, and achieve trace detection. However, existing derivatization reagents still have some drawbacks, such as matrix effects that can lead to biased analytical results. While isotope internal standards can effectively correct mass spectrometry results, their availability is limited and their cost is high.

[0004] In conclusion, the development and application of novel chiral amino acid derivatization reagents, combined with chromatographic and mass spectrometric techniques, have significant scientific and applied value for improving the accuracy and efficiency of chiral amino acid analysis. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to optimize the detection sensitivity of chiral amino acids and improve their enantiomer separation efficiency.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] An application of a chiral amine in the analysis and / or resolution of chiral amino acids, wherein the chiral amine has one of the following structural formulas:

[0008] ;

[0009] In the structural formula, the carbon atom on the pyrrole ring connected to the amino group has an R configuration.

[0010] Preferably, the chiral amino acid is one or a mixture of alanine, valine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, and citrulline.

[0011] Preferably, the chiral amino acid includes D amino acids in configuration and L Amino acids with specific configurations.

[0012] Preferably, the chiral amino acid comprises one or more of D / L-Arg, D / L-Trp, D / L-Phe, D / L-His, D / L-Leu, D / L-Met, D / L-Thr, D / L-Ile, D / L-Pro, D / L-Tyr, D / L-Ala, D / L-Nva, D / L-Val, D / L-Cit, D / L-Ser, D / L-Gln, D / L-Asn, and D / L-Asp, or a mixture thereof.

[0013] Preferably, the chiral amino acid in D Configuration and L The configuration ratio is 1:1.

[0014] Preferably, the chiral amino acid is a mixture of 18 amino acids: alanine, valine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, and citrulline, with each amino acid comprising a 1:1 ratio. D Configuration and L Configuration.

[0015] Preferably, the method for preparing the chiral amine includes the following steps:

[0016] S1. Reaction of deuterated benzoic acid or benzoic acid and oxaloyl chloride in a solvent;

[0017] S2, mix the product obtained in S1 with ( R )-3-Boc-aminopyrrole reacts in the presence of a base and a solvent;

[0018] S3. The product obtained in S2 is reacted with trifluoroacetic acid in a solvent to obtain the chiral amine.

[0019] Preferably, in S1, the ratio of deuterated benzoic acid or benzoic acid to oxaloyl chloride is 500 mg: 0.42 mL; in S2, the amount of ( R The molar ratio of 3-Boc-aminopyrrole to deuterated benzoic acid or benzoic acid used in S1 is 1:1; in S3, the molar ratio of the product obtained in S2 to trifluoroacetic acid is 1:20.

[0020] Preferably, in S1, the reaction process includes reacting under ice bath conditions for half an hour, then reacting at room temperature for 4 hours; in S2, the reaction includes reacting under ice bath conditions for 15 minutes, then reacting at room temperature for 1 hour, followed by quenching with water; in S3, the reaction includes reacting at room temperature for 2 hours; in S1, the solvent is dichloromethane; in S2, the solvent is dichloromethane, and the base is triethylamine; in S3, the solvent is dichloromethane.

[0021] Preferably, the chiral amine is used as a labeling reagent for chiral amino acids.

[0022] Preferably, the ratio of chiral amino acid to chiral amine is 36ug:0.04mmol.

[0023] This invention also proposes a method for analyzing and / or resolving chiral amino acids using chiral amines, comprising the following steps: using chiral amines... , One or two of the following are used as labeling reagents to react with chiral amino acids to obtain derivatives; then the derivatives are detected by LC-MS; wherein, the carbon atom on the pyrrole ring in the chiral amine structure is in the R configuration.

[0024] Preferably, chiral amines or As a labeling reagent, it reacts with chiral amino acids to obtain derivatives; then the derivatives are detected by LC-MS; or, using chiral amines... As a labeling reagent, it reacts with chiral amino acids to give derivative A, which is then labeled with a chiral amine. As a labeling reagent, it reacts with chiral amino acids to obtain derivative B, which is used as an internal standard; derivative A and derivative B are mixed and then detected by LC-MS.

[0025] Preferably, the reaction process further includes the addition of a catalyst; the catalyst is a mixture of 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBT), or a mixture of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT), or a mixture of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-7-azobenzotriazole (HOAT).

[0026] Preferably, a mixture of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-7-azobenzotriazole is used as a catalyst during the reaction.

[0027] Preferably, the reaction temperature is 25-45°C and the reaction time is 30-60 minutes.

[0028] Preferably, during LC-MS detection, the chromatographic column is a SHIMADZU shim pack GIST C. 18 The chromatographic column was 2.1 × 100 mm, 2.0 μm in diameter, and the column temperature was 40℃. Mobile phases A and B were 0.1% formic acid aqueous solution and acetonitrile, respectively. The chromatographic gradient was as follows: 0-5 min, 2-7% mobile phase B; 5-9 min, 7-12% mobile phase B; 9-13 min, 12-15% mobile phase B; 13-17 min, 15-20% mobile phase B; 17-20 min, 20-30% mobile phase B; 20-22 min, 30-90% mobile phase B; 22-25 min, 90% mobile phase B; 25-27 min, 90-2% mobile phase B; 27-30 min, 2% mobile phase B. The flow rate was 0.3 mL / min.

[0029] Preferably, the chiral amino acid solution is mixed with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, N-hydroxy-7-azobenzotriazole and a chiral amine, and the mixture is reacted at 25-45°C for 30-60 minutes to complete the labeling and obtain the derivative.

[0030] The advantages of this invention are:

[0031] 1. This invention introduces APPM and its deuterated stable isotope forms. d 5-APPM can be used for the analysis and detection of chiral amino acids, through ( R 3-Aminopyrrolidone-1-ylbenzophenone (APPM) and its stable isotopic forms d 5-APPM, used alone or in combination as a pair of isotope labeling reagents, can undergo specific condensation reactions with chiral amino acids, generating derivatives containing easily ionized groups, thereby enhancing the mass spectrometry signal response and improving detection sensitivity in electrospray ionization (ESI) positive ion mode.

[0032] 2. In addition to enhancing detection sensitivity, this invention also achieves efficient separation of chiral amino acid enantiomers on conventional chromatographic columns. This achievement is of great significance for the enantiomer purity analysis and quality control of chiral amino acids, and helps to improve the accuracy of quantitative analysis.

[0033] 3. This invention further provides a method for preparing APPM and dThe 5-APPM method has simple synthesis steps and low cost, which can reduce the detection cost of chiral amino acids to a certain extent, providing a practical and economical solution for the analysis and detection of chiral amino acids. Attached Figure Description

[0034] Figure 1 The NMR spectrum of the APPM prepared in Example 1 of this invention;

[0035] Figure 2 The sample prepared in Example 1 of this invention d 5-APPM NMR;

[0036] Figure 3 Different chiral amino acids in Example 2 of this invention were processed with APPM and d Secondary mass spectrum of the 5-APPM labeled product;

[0037] Figure 4 The chromatograms are of the products separated by APPM labeling of different chiral amino acids in Example 3 of the present invention;

[0038] Figure 5 These are chromatograms of different chiral amino acids before labeling in Example 3 of the present invention;

[0039] Figure 6 This is a chromatogram of the product separation of chiral amino acids labeled with APPM under different chromatographic conditions in Example 3 of the present invention;

[0040] Figure 7 In Embodiment 3 of the present invention, APPM and d Chromatogram of the labeled product after simultaneous labeling with 5-APPM;

[0041] Figure 8 Comparison of mass spectrometry responses of labeled products (Arg, Trp, His, Cit, Gln and Asp) using different catalysts in the APPM-labeled chiral amino acid reaction of Example 4 of this invention;

[0042] Figure 9 Comparison of mass spectrometry responses of labeled products (Phe, Met, Thr, Tyr, Ser, and Asn) using different catalysts in the APPM-labeled chiral amino acid reaction of Example 4 of this invention;

[0043] Figure 10 This is a comparison of the mass spectrometry responses of the labeled products (Leu, Ile, Pro, Ala, Nva, and Val) of different catalysts used in the APPM-labeled chiral amino acid reaction in Example 4 of this invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0046] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0047] It should be noted that the chemical reagents used in this invention are commercially available reagents. The LC-MS analysis of this invention was performed on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ionization source (ESI) (Turbo Ion Spray) and a Shimadzu LC-30AD UPLC system. The chromatographic column model was SHIMADZU shim pack GIST C. 18 (2.1 × 100 mm, 2.0 μm).

[0048] The chiral amino acids in the embodiments of the present invention include equal masses of alanine (Ala), valine (Nva), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), arginine (Arg), histidine (His), and citrulline (Cit).

[0049] Example 1

[0050] The reagent APPM and the present invention d Synthesis of 5-APPM

[0051] Benzoic acid (500 mg) was dissolved in dichloromethane (5 mL), and oxaloyl chloride (0.42 mL) was slowly added dropwise. After reacting in an ice bath for half an hour, the mixture was transferred to room temperature and reacted for 4 hours. After the reaction was complete, the solution was evaporated to dryness, dissolved in dichloromethane (5 mL), and then added (…). R760 mg of 3-Boc-aminopyrrole was added, followed by slow dropwise addition of 0.7 mL of triethylamine. After the addition was complete, the reaction was carried out in an ice bath for 15 min, then at room temperature for 1 h. After the reaction was completed, 1 mL of water was added to quench the reaction. Ethyl acetate and saturated brine (volume ratio 1:2) were added for extraction, and the organic layer was dried with anhydrous sodium sulfate. Purification by column chromatography (petroleum ether / ethyl acetate, volume ratio 1:1) yielded a white intermediate.

[0052] The obtained intermediate (500 mg) was dissolved in 10 mL of dichloromethane, and then trifluoroacetic acid (2.6 mL) was slowly added dropwise. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the solution was evaporated to dryness and extracted with dichloromethane. The product APPM was obtained by purification using a chromatography column (dichloromethane / methanol, v / v 15:1). In the structural formula, the carbon atom on the pyrrole ring bonded to the amino group has an R configuration, and its NMR spectrum is shown below. Figure 1 As shown;

[0053] By replacing the initial substrate in the above steps with deuterated benzoic acid, the following preparation was obtained. d 5-APPM In the structural formula, the carbon atom on the pyrrole ring bonded to the amino group has an R configuration, and its NMR spectrum is shown below. Figure 2 As shown.

[0054] Example 2

[0055] APPM / d Chemical labeling of 5-APPM

[0056] Take the above-mentioned chiral amino acid ACN solution (each amino acid concentration is 4 ug / mL, 5 μL), and sequentially add 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC 20 mmol / L, 4 μL) and N-hydroxy-7-azobenzotriazole (HOAT 100 mmol / L, 8 μL) dissolved in acetonitrile, APPM or d 5-APPM (100 mmol / L, 4 μL) was added, followed by the addition of acetonitrile to bring the total volume of the labeling reaction to 200 μL. The reaction was carried out at 30 °C for 30 minutes to obtain chiral amino acid derivatives. For each chiral amino acid, 5-APPM (100 mmol / L, 4 μL) was added simultaneously. D and LThe configuration, in a 1:1 ratio, comprises a mixture of chiral amino acids including alanine (Ala), valine (Nva), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), arginine (Arg), histidine (His), and citrulline (Cit).

[0057] The labeling reaction formula is shown below:

[0058]

[0059] in, The markings indicate chirality.

[0060] Figure 3 For Phe, Met and Cit D / L The mixture of configurations was respectively subjected to APPM and d The secondary mass spectra of the corresponding labeled products after effective labeling with 5-APPM show that different labeled products can produce characteristic secondary fragments, which can help with qualitative analysis.

[0061] Example 3

[0062] Split D / L configuration

[0063] In Example 2, the chiral amine APPM and d LC-ESI-MS analysis of 5-APPM-labeled chiral amino acid derivatives and underrivatized chiral amino acids was performed on a Shimadzu MS-8050 mass spectrometer (Shimadzu, Japan), equipped with an electrospray ionization source and a Shimadzu LC-30AD UPLC system. Mass spectrometry parameters for both chiral amino acid derivatives and underrivatized chiral amino acids were optimized in positive ion mode to achieve the best analytical performance. The analysis mode was MRM mode. Direct injection was used to optimize the MRM parameters for optimal analytical performance. The column model was SHIMADZU shim pack GIST C. 18(2.1 × 100 mm, 2.0 μm), column temperature 40℃. A 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B) were used as the mobile phases for chromatographic analysis. The optimized chromatographic gradient was: 0–5 min, 2–7% B; 5–9 min, 7–12% B; 9–13 min, 12–15% B; 13–17 min, 15–20% B; 17–20 min, 20–30% B; 20–22 min, 30–90% B; 22–25 min, 90% B; 25–27 min, 90–2% B; 27–30 min, 2% B; flow rate 0.3 mL / min. Figure 4 and Figure 5 The chromatograms of chiral amino acids show the following: after labeling, the chromatograms of the 18 chiral amino acids are... D / L The configurations are all effectively separated, exhibiting good decoupling. d 5-APPM and APPM have the same physicochemical properties, and their separation spectra are completely consistent. However, if the chromatographic conditions are changed, the separation effect is not ideal, and the 18 chiral amino acids cannot be separated. D / L Effectively decompose the configuration, such as Figure 6 As shown, the chromatographic conditions were: column type SHIMADZU shim pack GIST C 18 (2.1 × 100 mm, 2.0 μm), column temperature 30 ℃, chromatographic analysis was performed using 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B) as mobile phases. The chromatographic gradient was: 0-5 min, 3%-15% B, 5-10 min, 15%-30% B, 10-15 min, 30%-40% B, 15-17 min, 40%-50% B, 17-20 min, 50%-90% B, 20-25 min, 90% B, 25-27 min, 90%-3% B, 27-30 min, 3% B, where the fractions are volume fractions; the flow rate was 0.3 mL / min. The chiral amine APPM from Example 2 and... d 5-APPM-labeled chiral amino acid derivatives were mixed in a 1:1 molar ratio and detected under the chromatographic conditions described above that yielded good resolution (i.e., Figure 4 and Figure 5 (corresponding detection conditions) Figure 7 For APPM (shown in red) and dThe partial chromatograms of the separated labeled products after 5-APPM labeling (shown in blue) show that the chromatographic separation and retention behavior of the lightly and heavily labeled products are basically consistent.

[0064] Example 4

[0065] The only difference from Example 2 is that 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBT) are used, or 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) are used instead of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-7-azobenzotriazole (HOAT) as catalysts.

[0066] Figure 8-10 To compare the mass spectrometric responses of labeled products from different catalysts used in the APPM labeling of chiral amino acids, three different catalyst systems were compared: 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) and 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT), and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-7-azobenzotriazole (HOAT). The figure shows that the labeled product responses obtained from the TBTU / HOBT and EDC / HOBT catalytic systems in Example 4 were lower than those obtained from the EDC / HOAT group in Example 2.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a chiral amine in the analysis and / or resolution of chiral amino acids, wherein the chiral amine has one of the following structural formulas: ； in, In the structural formula, the carbon atom on the pyrrole ring connected to the amino group has an R configuration.

2. The application of the chiral amine according to claim 1 in the analysis and / or resolution of chiral amino acids, characterized in that: The chiral amino acid is one or a mixture of alanine, valine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, and citrulline.

3. The application of the chiral amine according to claim 1 in the analysis and / or resolution of chiral amino acids, characterized in that: The chiral amino acids include D amino acids in configuration and L Amino acids with specific configurations.

4. The application of the chiral amine according to claim 1 in the analysis and / or resolution of chiral amino acids, characterized in that: The method for preparing the chiral amine includes the following steps: S1. Reaction of deuterated benzoic acid or benzoic acid and oxaloyl chloride in a solvent; S2, mix the product obtained in S1 with ( R )-3-Boc-aminopyrrole reacts in the presence of a base and a solvent; S3. The product obtained in S2 is reacted with trifluoroacetic acid in a solvent to obtain the chiral amine.

5. The application of the chiral amine according to claim 1 in the analysis and / or resolution of chiral amino acids, characterized in that: The chiral amine is used as a labeling reagent for chiral amino acids.

6. A method for analyzing and / or resolving chiral amino acids using chiral amines, characterized in that: Includes the following steps: With chiral amines , One or two of the following are used as labeling reagents to react with chiral amino acids to obtain derivatives; then the derivatives are detected by LC-MS; wherein, the carbon atom on the pyrrole ring in the chiral amine structure is in the R configuration.

7. The method for analyzing and / or resolving chiral amino acids using chiral amines according to claim 6, characterized in that: With chiral amines or As a labeling reagent, it reacts with chiral amino acids to obtain derivatives; then the derivatives are detected by LC-MS; or, using chiral amines... As a labeling reagent, it reacts with chiral amino acids to give derivative A, which is then labeled with a chiral amine. As a labeling reagent, it reacts with chiral amino acids to obtain derivative B, which is used as an internal standard; derivative A and derivative B are mixed and then detected by LC-MS.

8. The method for analyzing and / or resolving chiral amino acids using chiral amines according to claim 6, characterized in that: During the reaction, a mixture of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-7-azobenzotriazole was used as a catalyst.

9. The method for analyzing and / or resolving chiral amino acids using chiral amines according to claim 6, characterized in that: The reaction is carried out at a temperature of 25-45°C for 30-60 minutes.

10. The method for analyzing and / or resolving chiral amino acids using chiral amines according to any one of claims 6-9, characterized in that: During LC-MS detection, the column type was SHIMADZU shim pack GIST C. 18 The chromatographic column was 2.1 × 100 mm, 2.0 μm in diameter, and the column temperature was 40℃. Mobile phases A and B were 0.1% formic acid aqueous solution and acetonitrile, respectively. The chromatographic gradient was as follows: 0-5 min, 2-7% mobile phase B; 5-9 min, 7-12% mobile phase B; 9-13 min, 12-15% mobile phase B; 13-17 min, 15-20% mobile phase B; 17-20 min, 20-30% mobile phase B; 20-22 min, 30-90% mobile phase B; 22-25 min, 90% mobile phase B; 25-27 min, 90-2% mobile phase B; 27-30 min, 2% mobile phase B. The flow rate was 0.3 mL / min.

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