Amino chiral metabolite resolution reagent and application thereof

Abstract

The invention provides an amino chiral metabolite resolution reagent and application thereof, and belongs to the technical field of biochemistry. The compound TPP-R-BSA and TPP-S-BSA reagents provided by the invention contain triphenylphosphine groups with positive charges and a chiral center structure, and can be used for performing mass spectrum labeling on amino functional group chiral compounds. According to the present invention, the chiral resolution of 13 chiral amino compounds can be simultaneously performed under the neutral condition for the first time, and the mild degree of the derivatization is significantly higher than the mild degree of the existing chiral derivatization reagent; according to the reagent, an analysis method which is mild in amino functional group chiral compound resolution, high in sensitivity and high in selectivity can be established by utilizing a liquid chromatography-mass spectrometry detection technology. And an effective and reliable mass spectrum chiral derivatization reagent is provided for the resolution research of amino functional group chiral metabolites and the screening of chiral biomarkers.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and particularly to amino chiral metabolite resolution reagents and their applications. Background Art

[0002] Chirality is a special symmetry property of molecules in three-dimensional space and is a fundamental feature of biological systems, especially prominent in the field of pharmacy. In many cases, chiral molecules usually exist in the form of enantiomers (R- and S-configurations), and these two configurations may exhibit completely different pharmacological properties. In addition, chiral enantiomers may undergo different metabolic processes in vivo, which is an important feature of many endogenous metabolites. In order to accurately screen truly effective chiral biomarkers, it is of great significance to develop and establish highly sensitive chiral metabolite separation and analysis methods using liquid chromatography-mass spectrometry (LC-MS) technology for drug toxicity and mechanism research, disease diagnosis and gene function elucidation, safety evaluation of drug components, and in vivo chiral metabolomics research.

[0003] There are various low-molecular-weight metabolite optical isomers such as amino acids and lactic acid in living organisms. It has always been believed that only one of the optical isomers has physiological activity. However, with the progress and development of analysis techniques, D-amino acids existing in living organisms have been discovered. Multiple research reports have shown that D-amino acids are gradually recognized as endogenous metabolites and have attracted much attention as new physiological active substances and biomarkers for disease diagnosis in the fields of metabolomics and disease diagnosis. However, these known D-amino acids are limited to a few molecules. The screening of chiral metabolic biomarkers and the research on the correlation between optical isomers and diseases are still in their infancy.

[0004] Commonly used chiral separation and determination techniques include high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (HPCE), supercritical fluid chromatography (SFC), etc. Among them, HPLC is one of the most widely used and effective methods. The separation and analysis of enantiomers by HPLC can be divided into direct method and indirect method. The direct method has the advantages of simple operation, less racemization during the analysis process, a wide range of additive selection, but a longer system equilibrium time, a large consumption of additives, and poor versatility. The indirect method is that chiral enantiomers react with a highly optically pure derivatization reagent to form diastereomers before separation, and then chromatographic separation and determination are carried out. Although the indirect method involves derivatization reactions of samples, which prolongs the analysis time and has relatively high requirements for the derivatization reagent, such as rapid enantiomeric derivatization reactions and consistent reaction rates. However, non-chiral columns with low price and high column efficiency can be used during separation, and derivatization reagents can be selected during the reaction to improve detection sensitivity, so it is more popular in trace analysis of actual samples. However, so far, most chiral derivatization reagents need to react with the target amino functional group under alkaline conditions to form diastereomers. When screening biomarkers through non-targeted metabolomics, the alkaline conditions increase the probability of racemization of unknown chiral compounds during derivatization. In addition, it is difficult to confirm the adduct ion type of the derivatization reagent, which further increases the probability of false positives in the screening of derivatization products. Summary of the Invention

[0005] The object of the present invention is to provide an amino chiral metabolite resolution reagent and its application. The compounds used in the reagent all contain a positively charged triphenylphosphine group and a chiral center structure, which can label amino functional group chiral compounds, and use liquid chromatography-mass spectrometry (LC-MS) technology to establish a mild, highly sensitive, and highly selective analysis method for the resolution of amino functional group chiral compounds.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides a compound TPP-R-BSA having the structure of Formula I:

[0008]

[0009] The present invention also provides a compound TPP-S-BSA having the structure of Formula II:

[0010]

[0011] The present invention also provides a preparation method of the compound TPP-R-BSA, including the following steps:

[0012] Dissolve TPPP, mix it with H-PRO-OTBU and N-methylimidazole, then add N,N,N',N'-tetramethylchlorourea hexafluorophosphate for reaction. After removing the solvent, dissolve and wash the residue, retain the organic layer, dry, filter, and evaporate to dryness under reduced pressure to obtain Intermediate 1;

[0013] Dissolve the Intermediate 1, mix it with HCl for reaction, and dry to obtain Intermediate 2;

[0014] Dissolve the Intermediate 2, add MYTSA for reaction, and after removing the solvent and purification, obtain Compound TPP-R-BSA.

[0015] The present invention also provides a preparation method of Compound TPP-S-BSA, which is characterized by including the following steps:

[0016] Dissolve TPPP, mix it with H-D-PRO-OTBU and N-methylimidazole, then add N,N,N',N'-tetramethylchlorourea hexafluorophosphate for reaction. After removing the solvent, dissolve and wash the residue, retain the organic layer, dry, filter, and evaporate to dryness under reduced pressure to obtain Intermediate 1;

[0017] Dissolve the Intermediate 1, mix it with HCl for reaction, and dry to obtain Intermediate 2;

[0018] Dissolve the Intermediate 2, add MYTSA for reaction, and after removing the solvent and purification, obtain Compound TPP-S-BSA.

[0019] The present invention also provides the application of Compound TPP-R-BSA or Compound TPP-S-BSA in the resolution of chiral metabolites of amino functional groups.

[0020] The present invention also provides the application of Compound TPP-R-BSA or Compound TPP-S-BSA in the screening of disease biomarkers.

[0021] The present invention also provides a chiral derivatizing reagent composition, which is characterized by including Compound TPP-R-BSA and Compound TPP-S-BSA.

[0022] The present invention also provides a chiral detection kit, which is characterized by including the above-mentioned chiral derivatizing reagent composition.

[0023] The present invention also provides the application of the above-mentioned chiral derivatizing reagent composition or the above-mentioned chiral detection kit in drug metabolism research, toxicity evaluation or pharmacodynamic analysis.

[0024] The present invention also provides the application of the above-mentioned chiral derivatizing reagent composition or the above-mentioned chiral detection kit in the quality control of chiral components in drugs or biological samples.

[0025] Advantages of the present invention:

[0026] The compounds TPP-R-BSA and TPP-S-BSA reagents provided by the present invention contain a positively charged triphenylphosphine group and a chiral center structure, and can be used for mass spectrometry labeling of amino-functional group chiral compounds. Chiral resolution was simultaneously performed on 13 chiral amino acids, and the resolution (Rs) was 1.56 - 12.67. For the first time, chiral resolution of 13 chiral amino compounds was completed under neutral conditions. The mildness of derivatization is significantly higher than that of existing chiral derivatization reagents. This reagent can utilize liquid chromatography-mass spectrometry (LC-MS) technology to establish a mild, highly sensitive, and highly selective analytical method for the resolution of amino-functional group chiral compounds. It provides an effective and reliable mass spectrometry chiral derivatization reagent for the resolution research of amino-functional group chiral metabolites and the screening of chiral biomarkers. Description of the Drawings

[0027] Figure 1 It is the mass chromatogram of LC-ESI-MS of the TPP-R-BSA mass spectrometry derivatization reagent;

[0028] Figure 2 It is the mass spectrum (m / z = 669.25) of LC-ESI-MS of the TPP-R-BSA mass spectrometry derivatization reagent;

[0029] Figure 3 It is the mass chromatogram of LC-ESI-MS of the TPP-S-BSA mass spectrometry derivatization reagent;

[0030] Figure 4 It is the mass spectrum (m / z = 669.25) of LC-ESI-MS of the TPP-S-BSA mass spectrometry derivatization reagent;

[0031] Figure 5 It is the optimization of the derivatization conditions of the TPP-S-BSA reagent with amino acids;

[0032] Figure 6 It is the mass chromatogram of the TPP-S-BSA reagent for simultaneously resolving 13 amino acids;

[0033] Figure 7 It is the mass chromatogram of the TPP-S-BSA reagent for simultaneously resolving 13 amino acids. Detailed Embodiments

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] A mass spectrometry derivatization reagent labeled with an amino-functional chiral compound, 4-carboxybutyltriphenylphosphonium bromide (TPPP) and L-proline tert-butyl ester [H-PRO-OTBU], and N-ethynyl-N,4-dimethylbenzenesulfonamide (MYTSA) were used as starting materials, and (R)-(5-(2-(((1-((N,4-dimethylphenyl)sulfonamido)vinyl)oxy)carbonyl)pyrrolidin-1-yl)-5-oxopentyl)triphenylphosphonium (TPP-R-BSA) was synthesized by reaction. The specific structural formula is as follows:

[0036]

[0037] Chemical Formula:C 38 H 42 N2O5PS +

[0038] Exact Mass:669.25466

[0039] TPP-R-BSA

[0040] A mass spectrometry derivatization reagent labeled with an amino-functional chiral compound, 4-carboxybutyltriphenylphosphonium bromide (TPPP) and D-proline tert-butyl ester [H-D-PRO-OTBU], and N-ethynyl-N,4-dimethylbenzenesulfonamide (MYTSA) were used as starting materials, and (S)-(5-(2-(((1-((N,4-dimethylphenyl)sulfonamido)vinyl)oxy)carbonyl)pyrrolidin-1-yl)-5-oxopentyl)triphenylphosphonium (TPP-S-BSA) was synthesized by reaction. The specific structural formula is as follows:

[0041]

[0042] Chemical Formula:C38 H 42 N2O5PS +

[0043] Exact Mass:669.25466

[0044] TPP-R-BSA

[0045] Example

[0046] Synthesis of TPP-R-BSA:

[0047] 443.31 mg of TPPP was dissolved in 20 mL of acetonitrile. 175 μL of H-PRO-OTBU and 205 μL of N-methylimidazole were added and stirred at room temperature for 5 minutes. Then, 336.70 mg of N,N,N',N'-tetramethyluronium chloride hexafluorophosphate was added and the mixture was allowed to react overnight at room temperature on a thermostatic magnetic stirrer. The solvent was then evaporated under reduced pressure. The resulting residue was redissolved in 30 mL of dichloromethane and washed with an equal volume of water. After sufficient simmering, the aqueous layer was discarded, and the dichloromethane layer was washed three times with an equal volume of 0.25 M sodium carbonate solution. It was then washed twice with an equal volume of 1 M HCl. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure to yield a white solid. The resulting white solid was dissolved in 3 mL of acetonitrile, stirred overnight with 6 M HCl, filtered, and dried in a vacuum oven to yield 520.32 mg of a white powder. Dissolve 597 mg of the dried white powder in 5 mL of dichloromethane, add 210 mg of MYTSA, and allow to react overnight at room temperature on a thermostatic magnetic stirrer. The solvent is then evaporated under reduced pressure. Purification by preparative liquid chromatography yields 420.25 mg of a white solid. LC-ESI-MS spectroscopic data, (m / z): 669.25 [M] + ,t R =6.25min; 11H-NMR (300 MHz, CDCl3) δ 7.83–7.57 (m, 19H), 4.40–4.32 (m, 1H), 3.57 (s, 1H), 3.53 (d, J = 6.6 Hz, 1H), 3.22 (d, J = 13.3 Hz, 1H), 3.18–3.10 (m, 1H), 2.91 (s, 3H), 2.45 (d, J = 3.2 Hz, 2H), 2.43 (s, 3H), 2.39 (s, 1H), 2.00–1.83 (m, 5H), 1.64 (s, 2H), 1.47–1.18 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 171.47 (s), 169.90 (s), 146.46 (s), 144.44 (s), 135.35 (s), 135.31 (s), 133.61 (s), 133.48 (s), 130.80 (s), 130.63 (s), 129.72 (s), 128.15 (s), 118.65 (s), 117.51 (s), 101.64 (s), 58.87 (s), 47.16 (s), 36.80 (s), 32.78 (s), 28.89 (s), 25.16 (s), 24.95 (s), 22.55 (s), 21.79 (s), 21.70 (s). The yield was approximately 52.2%.

[0048] Synthesis of TPP-S-BSA:

[0049] 443.31 mg of TPPP was dissolved in 20 mL of acetonitrile solution. 175 μL of H-D-PRO-OTBU and 205 μL of N-methylimidazole were added, and the mixture was stirred at room temperature for 5 min. Then, 336.70 mg of N,N,N',N'-tetramethylchlorourea hexafluorophosphate was added, and the reaction was carried out overnight at room temperature on a thermostatic magnetic stirrer. The solvent was removed under reduced pressure. The resulting residue was redissolved in 30 mL of dichloromethane, washed with an equal volume of water, allowed to stand fully, and the aqueous layer was discarded. The dichloromethane layer was washed three times with an equal volume of 0.25 M sodium carbonate solution. Then, it was washed twice with an equal amount of 1 M HCl. The organic layer was taken, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure to obtain a white solid. The obtained white solid was dissolved in 3 mL of acetonitrile solution, 6 M HCl was added, and the mixture was stirred overnight and then filtered by suction, and dried in a vacuum drying oven to obtain 532.32 mg of a white powder. 597 mg of the dried white powder was dissolved in 5 mL of dichloromethane, 210 mg of MYTSA was added, and the reaction was carried out overnight at room temperature on a thermostatic magnetic stirrer. The solvent was removed under reduced pressure. After purification by preparative liquid chromatography, 445.25 mg of a white solid was obtained. LC-ESI-MS spectral data, (m / z): 669.25 [M]+, tR = 6.24 min; 1H-NMR (300 MHz, CDCl3) δ 7.89 (t, J = 14.2 Hz, 1H), 6.00 (d, J = 8.2 Hz, 1H), 4.14 (t, J = 6.0 Hz, 1H), 4.04 (s, 1H), 2.90 (d, J = 7.6 Hz, 6H), 2.60–2.47 (m, 1H), 2.35–2.16 (m, 3H), 1.76 (s, 3H); 13C-NMR (75 MHz, CDCl3) δ 178.54 (s), 138.69 (s), 103.39 (s), 77.58 (s), 76.74 (s), 68.60 (s), 53.83 (s), 42.11 (s), 37.99 (s), 23.16 (s), 21.63 (s). The yield was approximately 55.3%.

[0050] From Figure 1 , and Figure 2 From the mass chromatogram and mass spectrum of LC-ESI-MS, it can be seen that the molecular weight of the synthesized TPP-R-BSA reagent is consistent with the detected mass-to-charge ratio m / z = 669.25. It can be judged that the synthesized compound is indeed the TPP-R-BSA reagent. Specifically 1 The 1H-NMR data can be referred to the synthesis part of TPP-R-BSA.

[0051] From Figure 3 , Figure 4It can be seen from the mass chromatogram and mass spectrum of LC-ESI-MS that the molecular weight of the synthesized TPP-S-BSA reagent is consistent with the detected mass-to-charge ratio of 669.25. It can be judged that the synthesized compound is indeed the TPP-S-BSA reagent. Specifically 1 The 1H-NMR data can be referred to the TPP-S-BSA synthesis section.

[0052] UPLC-MS analysis conditions: Chromatographic column: ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm, Waters, USA); Chromatographic conditions: Mobile phase A: 0.1% formic acid aqueous solution, Mobile phase B: 0.1% formic acid methanol solution; Gradient: B% = 25 - 28 - 40 - 40% (0 - 15 - 20 - 30 min); Flow rate: 0.30 mL·min -1 ; Column temperature: 40°C; Injection volume: 1.0 μL; Ionization mode: ESI + .

[0053]

[0054] Reaction structural formula of TPP-R-BSA reagent with amino acid

[0055]

[0056] Reaction structural formula of TPP-S-BSA reagent with amino acid

[0057] The above represents the reaction structural formulas of TPP-R-BSA and TPP-S-BSA mass spectrometry chiral derivatization reagents with chiral amino acids. Both mass spectrometry derivatization reagents can carry out derivatization reactions with amino groups under physiological conditions to generate diastereomers with two chiral centers of the same structure, which can be chiral separated in reverse-phase chromatography with the same separation effect.

[0058] Figure 5 Specifically, it represents the optimization of the derivatization conditions of the TPP-S-BSA mass spectrometry chiral derivatization reagent with chiral amino acids. The results show that the optimal conditions for the derivatization reaction are 70°C and 1 h.

[0059] From Figure 6 and Figure 7 it can be seen that good chiral separation is obtained after derivatization of 13 chiral amino acids on UHPLC-MS.

[0060] Table 1 Separation efficiency of 13 amino acids

[0061]

[0062]

[0063] Mobile phase I: (A) 0.1% FA in H2O, (B) 0.1% FA in MeOH, B Conc 25 - 28 - 40 - 40 - 90% (0 - 15 - 20 - 35 - 40 min);

[0064] k' = (t R - t0) / t0; α = (t R2 - t0) / (t R1 - t0); Rs = 2(t R2 - t R1 ) / (W2 + W1).

[0065] As can be seen from Table 1, the resolution of 13 chiral amino acids is 1.22 - 14.50, showing good chiral resolution performance.

[0066] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Compound TPP-R-BSA with an inclusion type I structure:

2. Compound TPP-S-BSA with an inclusion type II structure:

3. The preparation method of the compound TPP-R-BSA according to claim 1, characterized in that, It includes the following steps: Dissolve TPPP, mix it with H-PRO-OTBU and N-methylimidazole, then add N,N,N',N'-tetramethylchlorourea hexafluorophosphate for reaction. After removing the solvent, dissolve the residue, wash it, retain the organic layer, dry, filter, and evaporate to dryness under reduced pressure to obtain Intermediate 1; Dissolve the Intermediate 1, mix it with HCl for reaction, and dry to obtain Intermediate 2; Dissolve the Intermediate 2, add MYTSA for reaction, and after removing the solvent and purification, obtain Compound TPP-R-BSA.

4. The preparation method of the compound TPP-S-BSA according to claim 2, characterized in that, It includes the following steps: Dissolve TPPP, mix it with H-D-PRO-OTBU and N-methylimidazole, then add N,N,N',N'-tetramethylchlorourea hexafluorophosphate for reaction. After removing the solvent, dissolve the residue, wash it, retain the organic layer, dry, filter, and evaporate to dryness under reduced pressure to obtain Intermediate 1; Dissolve the Intermediate 1, mix it with HCl for reaction, and dry to obtain Intermediate 2; Dissolve the Intermediate 2, add MYTSA for reaction, and after removing the solvent and purification, obtain Compound TPP-S-BSA.

5. Use of the Compound TPP-R-BSA according to Claim 1 or the Compound TPP-S-BSA according to Claim 2 in the resolution of chiral metabolites of amino functional groups.

6. Use of the Compound TPP-R-BSA according to Claim 1 or the Compound TPP-S-BSA according to Claim 2 in the screening of disease biomarkers.

7. A chiral derivatizing reagent composition, characterized in that, It includes the Compound TPP-R-BSA according to Claim 1 and the Compound TPP-S-BSA according to Claim 2.

8. A chiral detection kit, characterized in that, It includes the chiral derivatization reagent composition according to Claim 7.

9. Use of the chiral derivatization reagent composition according to Claim 7 or the chiral detection kit according to Claim 8 in drug metabolism research, toxicity evaluation, or pharmacodynamic analysis. 1O. Use of the chiral derivatization reagent composition according to Claim 7 or the chiral detection kit according to Claim 8 in the quality control of chiral components in drugs or biological samples.

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