An n-hydroxysuccinimide ester, its preparation method and use in the analysis of amino acids

Abstract

The application discloses a kind of N Hydroxysuccinimide ester and its preparation method and application in amino acid analysis, N Hydroxysuccinimide ester is DPMB or DPEB. The DPMB and DPEB provided by the application can be used for the analysis and detection of amino acids. Through the use of 2,5-dioxopyrrolidin-1-yl-4-(4-methylpiperazin-1-yl)benzoate (DPMB) and its structural analog 2,5-dioxopyrrolidin-1-yl-4-(4-ethylpiperazin-1-yl)benzoate (DPEB) alone or in combination as a pair of structural analog reagents, a substitution reaction occurs with amino acids, and the resulting derivatives can improve the hydrophobicity of amino acids and introduce easily ionizable groups, greatly improving the mass spectrometry response in ESI positive ion mode.

Description

Technical Field

[0001] This invention relates to the field of amino acid detection technology, specifically to a... N 2-hydroxysuccinimide ester, its preparation method, and its application in amino acid analysis. Background Technology

[0002] Amino acids are the basic building blocks of proteins and essential metabolites for life activities. They are not only precursors to protein synthesis but also participate in various physiological processes such as energy metabolism, signal transduction, and immune regulation. Therefore, accurately determining the amino acid composition and content in biological samples, food, pharmaceuticals, or the environment is of great significance for understanding life mechanisms, evaluating nutritional value, controlling product quality, and diagnosing diseases.

[0003] Chemical derivatization is an effective method; after reaction with derivatizing reagents, the hydrophobicity of amino acids increases, leading to improved chromatographic retention. Furthermore, derivatization can significantly improve the sensitivity of amino acids in mass spectrometry analysis, reduce background signals, and enable trace detection. However, existing derivatization reagents still have some drawbacks, such as matrix effects that can bias 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 amino acid derivatization reagents, combined with chromatographic and mass spectrometric techniques, have significant scientific and practical value for improving the accuracy and efficiency of amino acid analysis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an amino acid derivatization reagent.

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

[0007] A sort of N -Hydroxysuccinimide ester, with one of the following structural formulas: .

[0008] The present invention also proposes a method described above. N A method for preparing 4-hydroxysuccinimide ester includes the following steps: reacting 4-(4-methylpiperazine)benzoic acid or 4-(4-ethylpiperazine)benzoic acid... N -Hydroxysuccinimide, N,N' The product is obtained by reacting dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), and solvent. N -Hydroxysuccinimide ester.

[0009] Preferably, 4-(4-methylpiperazine)benzoic acid or 4-(4-ethylpiperazine)benzoic acid, N -Hydroxysuccinimide, N,N' - Dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were mixed, and then a solvent was added to react and obtain the desired product. N -Hydroxysuccinimide ester.

[0010] Preferably, the 4-(4-methylpiperazine)benzoic acid or 4-(4-ethylpiperazine)benzoic acid, N The mass ratio of -hydroxysuccinimide was 440:276; N-hydroxysuccinimide, N,N' The mass ratio of -dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 276:495:74.

[0011] Preferably, the solvent is dichloromethane.

[0012] Preferably, the reaction is carried out at room temperature for 10-15 hours.

[0013] The present invention also proposes a method described above. N Application of 1-hydroxysuccinimide ester in amino acid analysis and / or separation.

[0014] Preferably, the N -Hydroxysuccinimide ester as a derivatization reagent for amino acids.

[0015] The present invention also proposes a method using the aforementioned N A method for the analysis and / or separation of amino acids using hydroxysuccinimide esters includes the following steps: N 1-Hydroxysuccinimide ester was used as a labeling reagent to react with amino acids to obtain derivatives; then, the derivatives were detected by LC-MS.

[0016] Preferably, the amino acid comprises one or more of Arg, Trp, Phe, Leu, Met, Thr, Ile, Pro, Tyr, Ala, Nva, Val, Cit, Ser, Gln, Glu, Orn, and Asp.

[0017] Preferably, the amino acid solution is mixed with the triethylamine solution and N The hydroxysuccinimide ester mixture is reacted at 30-50℃ for 30-60 minutes to complete the labeling and obtain the derivative.

[0018] Preferably, the above-mentioned N Methods for the analysis and / or separation of amino acids using hydroxysuccinimide esters, N -Hydroxysuccinimide ester or The reagent is used as a labeling agent to react with amino acids to obtain derivatives; then the derivatives are detected using LC-MS; or... N -Hydroxysuccinimide ester As a labeling reagent, it reacts with amino acids to give derivative A. N -Hydroxysuccinimide ester As a labeling reagent, it reacts with 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.

[0019] Preferably, during LC-MS detection, the chromatographic column type is ACQUITY UPLC BEH C. 18 The chromatographic column has dimensions of 2.1 × 100 mm and a diameter of 1.7 μm. The column temperature is 40℃. The mobile phase consists of mobile phase A and mobile phase B. 0.05% formic acid-2 mM ammonium bicarbonate aqueous solution and acetonitrile are used as mobile phase A and mobile phase B, respectively, for chromatographic detection. The chromatographic gradient is as follows: 0–5 min, 3–10% mobile phase B; 5–10 min, 10–15% mobile phase B; 10–15 min, 15% mobile phase B; 15–20 min, 15–30% mobile phase B; 20–23 min, 30–80% mobile phase B; 23–25 min, 80% mobile phase B; 25–27 min, 80–3% mobile phase B; 27–30 min, 3% mobile phase B; and the flow rate is 0.3 mL / min.

[0020] The advantages of this invention are: 1. The 2,5-dioxopyrrolidone-1-yl-4-(4-methylpiperazin-1-yl)benzoate DPMB and its structural analogue 2,5-dioxopyrrolidone-1-yl-4-(4-ethylpiperazin-1-yl)benzoate DPEB introduced in this invention can undergo specific substitution reactions with amino acids, and the resulting derivatives contain easily ionized groups, thereby enhancing the mass spectrometry signal response and improving the detection sensitivity in electrospray ionization (ESI) positive ion mode.

[0021] 2. This invention further provides a method for preparing DPMB and DPEB, which has simple synthesis steps and low cost, and can reduce the detection cost of amino acids to a certain extent, providing a practical and economical solution for the analysis and detection of amino acids. Attached Figure Description

[0022] Figure 1 The NMR spectrum of the DPMB prepared in Example 1 of this invention; Figure 2 The NMR spectrum of the DPEB prepared in Example 1 of this invention; Figure 3 The images show the secondary mass spectra of the labeled products of different amino acids (arginine, tryptophan, and phenylalanine) in Example 2 of this invention after being labeled with DPMB and DPEB; where a and d represent the results of arginine, b and e represent the results of tryptophan, and c and f represent the results of phenylalanine. Figure 4 The images show the secondary mass spectra of the labeled products of different amino acids (leucine, isoleucine, and methionine) after being labeled with DPMB and DPEB in Example 2 of this invention; where a and d represent the results of leucine; b and e represent the results of isoleucine; and c and f represent the results of methionine. Figure 5 The images show the secondary mass spectra of the labeled products of different amino acids (threonine, proline, and tyrosine) in Example 2 of this invention after being labeled with DPMB and DPEB; where a and d represent the results of threonine, b and e represent the results of proline, and c and f represent the results of tyrosine. Figure 6 The images show the secondary mass spectra of the labeled products of different amino acids (alanine, valine, and valine) after being labeled with DPMB and DPEB in Example 2 of this invention; where a and d represent the results of alanine, b and e represent the results of valine, and c and f represent the results of valine. Figure 7 The images show the secondary mass spectra of the labeled products of different amino acids (citrulline, ornithine, and serine) in Example 2 of this invention after being labeled with DPMB and DPEB; where a and d represent the results of citrulline, b and e represent the results of ornithine, and c and f represent the results of serine. Figure 8 The images show the secondary mass spectra of the labeled products of different amino acids (aspartic acid, glutamine, and glutamic acid) after being labeled with DPMB and DPEB in Example 2 of the present invention; where a and d represent the results of aspartic acid, b and e represent the results of glutamine, and c and f represent the results of glutamic acid. Figure 9 The images show the chromatograms of products of different amino acids labeled with DPMB in Example 3 of the present invention; where ar represents the chromatograms of Arg, Trp, Phe, Leu, Met, Thr, Pro, Tyr, Ala, Val, Cit, Orn, Ser, Gln, Asp, Glu, Ile, and Nva products in sequence. Figure 10The images show the chromatograms of products containing different amino acids labeled with DPEB in Example 3 of this invention; where ar represents the chromatograms of Arg, Trp, Phe, Leu, Met, Thr, Pro, Tyr, Ala, Val, Cit, Orn, Ser, Gln, Asp, Glu, Ile, and Nva products in sequence. Figure 11 The chromatograms are shown in Example 3 of the present invention before different amino acid labels are applied; where ar represents the results of Arg, Trp, Phe, Leu, Met, Thr, Pro, Tyr, Ala, Val, Cit, Orn, Ser, Gln, Asp, Glu, Ile, and Nva in sequence. Figure 12 The image shows the chromatograms of the amino acid isomers after being labeled with DPMB and DPEB in Example 3 of the present invention; where a and b are DPMB-labeled Ile / Leu and Val / Nva, respectively; and c and d are DPEB-labeled Ile / Leu and Val / Nva, respectively. Figure 13 The images show chromatograms of the products separated by DPMB labeling of amino acid isomers under modified chromatographic conditions in Example 3 of this invention; where a is the chromatogram of Ile / Leu and b is the chromatogram of Val / Nva. Figure 14 This is a chromatogram of the labeled products after DPMB (red) and DPEB (blue) labeling in Example 3 of the present invention; wherein, ac represents Pro, Asp, and Orn labeled by DPMB in sequence; and df represents Pro, Asp, and Orn labeled by DPEB in sequence. Detailed Implementation

[0023] 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.

[0024] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0025] 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.

[0026] It should be noted that the chemical reagents used in this invention are commercially available reagents. The LC-MS analysis in 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 ACQUITY UPLC BEH C. 18 (2.1 × 100 mm, 1.7 μm).

[0027] The amino acids in the embodiments of this invention include a mixture of the following by mass: alanine (Ala), valine (Nva), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), glutamine (Gln), threonine (Thr), aspartic acid (Asp), arginine (Arg), citrulline (Cit), ornithine (Orn), and glutamic acid (Glu).

[0028] Example 1 Synthesis of the reagents DPMB and DPEB described in this invention 4-(4-methylpiperazine)benzoic acid (440 mg), N 276 mg hydroxysuccinimide N,N' Dicyclohexylcarbodiimide (495 mg) and 4-dimethylaminopyridine (74 mg) were dissolved in dichloromethane (10 mL) and reacted at room temperature for 12 hours. After the reaction was complete, the mixture was extracted with dichloromethane, and the organic layer was dried with anhydrous sodium sulfate. The product DPMB was obtained by column chromatography (dichloromethane / methanol, v / v 30:1). Its NMR spectrum is as follows Figure 1 As shown; By replacing the initial substrate in the above steps with 4-(4-methylpiperazine)benzoic acid, DPEB was prepared. Its NMR spectrum is as follows Figure 2 As shown.

[0029] Example 2 Chemical labeling of DPMB / DPEB Take 5 μL of the above amino acid acetonitrile solution (the concentration of each amino acid in the solution is 4 μg / mL), and add triethylamine TEA (10 mmol / L, 186 μL), DPMB or DPEB (100 mmol / L, 9 μL) dissolved in acetonitrile in sequence, with a total volume of 200 μL. React at 35℃ for 30 minutes to obtain amino acid derivatives, in which the amino acids include alanine (Ala), valine (Nva), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), phenylalanine (Phe), glutamine (Gln), threonine (Thr), aspartic acid (Asp), arginine (Arg), citrulline (Cit), ornithine (Orn), and glutamic acid (Glu) in equal mass mixtures.

[0030] The labeling reaction formula is shown below:

[0031] Figures 3-8 The images show the secondary mass spectra of the labeled products after amino acids were effectively labeled by DPMB and DPEB, respectively. As can be seen from the images, different labeled products can produce characteristic secondary fragments, which can help with qualitative analysis.

[0032] Example 3 Splitting isomers In Example 2 N LC-ESI-MS analysis of DPMB and DPEB-labeled amino acid derivatives and underrivatized 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 amino acid derivatives and underrivatized amino acids were optimized in positive ion mode to achieve the best analytical performance. The analysis mode was MRM mode. MRM parameters were optimized using direct injection to obtain the best analytical performance. The column model was ACQUITY UPLC BEH C. 18(2.1 × 100 mm, 1.7 μm), column temperature 40℃. A mobile phase of 0.05% formic acid-2 mM ammonium bicarbonate aqueous solution (phase A) and acetonitrile (phase B) was used for chromatographic analysis. The optimized chromatographic gradient was: 0–5 min, 3–10% (v / v) phase B; 5–10 min, 10–15% (v / v) phase B; 10–15 min, 15% (v / v) phase B; 15–20 min, 15–30% (v / v) phase B; 20–23 min, 30–80% (v / v) phase B; 23–25 min, 80% (v / v) phase B; 25–27 min, 80% (v / v) phase B; 27–30 min, 3% (v / v) phase B; flow rate 0.3 mL / min. Figure 9 and 12 The ab diagram in the figure is a chromatogram of amino acid separation by DPMB. As can be seen from the figure, the isomers of the two pairs of amino acids are effectively separated, with good separation. Figure 10 and 12 The chromatogram in the figure shows the product chromatograms of different amino acids labeled with DPEB under the same chromatographic conditions. As can be seen from the figure, DPEB can effectively label different amino acids. Figure 11 The figures show chromatograms of different amino acids before labeling. As can be seen, unlabeled amino acids have earlier retention times and weaker retention on the reversed-phase column. Changing the chromatographic conditions does not yield ideal separation results. Figure 13 As shown, the chromatographic conditions were: column type SHIMADZU shim pack GIST C 18 (2.1 × 100 mm, 2.0 μm), column temperature 40℃, chromatographic gradient: 0-5 min, 3%-10% B phase, 5-10 min, 10%-30% B phase, 10-15 min, 30%-50% B phase, 15-20 min, 50%-80% B phase, 20-23 min, 80% B phase, 23-25 ​​min, 80%-3% B phase, 25-28 min, 3% B phase; flow rate 0.3 mL / min. The sample from Example 2... N The amino acid derivatives labeled with DPMB and DPEB were mixed in a 1:1 molar ratio and detected under the chromatographic conditions described above that yielded good resolution. Figure 14 The chromatograms show the separation of the labeled products after DPMB (shown in red) and DPEB (shown in blue). As can be seen from the figure, the amino acids labeled with DPMB and DPEB have similar retention times and consistent peak shapes.

[0033] 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. A kind N -Hydroxysuccinimide ester, characterized in that: Its structural formula is one of the following: 。 2. A device as described in claim 1 N The method for preparing 1-hydroxysuccinimide ester is characterized by: Includes the following steps: 4-(4-methylpiperazine)benzoic acid or 4-(4-ethylpiperazine)benzoic acid, N -Hydroxysuccinimide, N,N' The reaction of dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and solvent yields the aforementioned product. N -Hydroxysuccinimide ester.

3. The method according to claim 2 N The method for preparing 1-hydroxysuccinimide ester is characterized by: The 4-(4-methylpiperazine)benzoic acid or 4-(4-ethylpiperazine)benzoic acid, N The mass ratio of -hydroxysuccinimide is 440:276; N -Hydroxysuccinimide, N,N' - The mass ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 276:495:

74.

4. The method according to claim 2 N The method for preparing 1-hydroxysuccinimide ester is characterized by: The solvent is dichloromethane.

5. The claim 2-4 N The method for preparing 1-hydroxysuccinimide ester is characterized by: The reaction was carried out at room temperature for 10-15 hours.

6. A device as described in claim 1 N Application of 1-hydroxysuccinimide ester in amino acid analysis and / or separation.

7. A method employing the method described in claim 1 N A method for the analysis and / or separation of amino acids from 1-hydroxysuccinimide esters, characterized in that: Includes the following steps: by N 1-Hydroxysuccinimide ester was used as a labeling reagent to react with amino acids to obtain derivatives; then, the derivatives were detected by LC-MS.

8. The method of use according to claim 7 N A method for the analysis and / or separation of amino acids from 1-hydroxysuccinimide esters, characterized in that: The amino acid solution was mixed with the triethylamine solution and N The hydroxysuccinimide ester mixture is reacted at 30-50℃ for 30-60 minutes to complete the labeling and obtain the derivative.

9. The method of use according to claim 7 N A method for the analysis and / or separation of amino acids from 1-hydroxysuccinimide esters, characterized in that: by N -Hydroxysuccinimide ester or The reagent is used as a labeling agent to react with amino acids to obtain derivatives; then the derivatives are detected using LC-MS; or... N -Hydroxysuccinimide ester As a labeling reagent, it reacts with amino acids to give derivative A. N -Hydroxysuccinimide ester As a labeling reagent, it reacts with 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.

10. The use of the method described in any one of claims 7-9 N A method for the analysis and / or separation of amino acids from 1-hydroxysuccinimide esters, characterized in that: During LC-MS analysis, the column type was ACQUITY UPLC BEH C. 18 The chromatographic column has dimensions of 2.1 × 100 mm and a diameter of 1.7 μm. The column temperature is 40℃. The mobile phase consists of mobile phase A and mobile phase B. A 0.05% (v / v) formic acid-2 mM ammonium bicarbonate aqueous solution and acetonitrile are used as mobile phase A and mobile phase B, respectively, for chromatographic detection. The chromatographic gradient is as follows: 0–5 min, 3–10% (v / v) mobile phase B; 5–10 min, 10–15% (v / v) mobile phase B; 10–15 min, 15% (v / v) mobile phase B; 15–20 min, 15–30% (v / v) mobile phase B; 20–23 min, 30–80% (v / v) mobile phase B; 23–25 min, 80% (v / v) mobile phase B; 25–27 min, 80% (v / v) mobile phase B; 27–30 min, 3% (v / v) mobile phase B; the flow rate is 0.3 mL / min.

Images