Analytical method based on the enantiomeric structure of amino acid derivatives of natamycin

Abstract

The present invention relates to an analysis method for the enantiomeric structure of amino acid derivatives based on natamycin, comprising the following steps: S1, mixing the standard of amino acid derivative enantiomers and natamycin or natamycin and p-toluenesulfonic acid, and then adding a solvent to prepare a binary or ternary mixture solution containing water; S2, using an ion source to generate corresponding non-covalent complex ions in the mixture solution; S3, using an ion mobility spectrometer to measure the ion mobility of the non-covalent complex ions, and according to the ion mobility spectrometer results, the structural information of the amino acid derivative enantiomer can be obtained. The analytical reagent and method provided by the present invention have the advantages of being simple, accurate, and having a fast analysis speed, and are simpler than the current analysis methods for enantiomers of benzyloxycarbonyl amino acids and amino acid benzyl esters.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and testing, and particularly relates to an analysis method based on the enantiomeric structure of an amino acid derivative of natamycin. Background Art

[0002] Chiral molecules refer to molecules with certain configurations or conformations that cannot be overlapped with their mirror images, just like our hands, the left hand cannot overlap with the mirror image of the right hand. The chirality of molecules is usually caused by asymmetric carbon. All chiral molecules are optically active. They have similar structures, but their biochemical activities have certain differences. Chirality is one of the basic properties of nature. Chirality is ubiquitous and has important connections with life phenomena, human health, and environmental safety. For chiral molecules, a pair of enantiomers have exactly the same physical and chemical properties in a non-chiral environment except for optical rotation, but they behave differently in a chiral environment. The living system is a typical chiral environment. There are many varieties of naturally occurring chiral compounds, and they usually contain only one of the enantiomers. Biomolecules such as amino acids, drug enzymes, proteins, receptors, etc. have asymmetric properties. Their two enantiomers usually show differences in biological activity, absorption, distribution, etc. in the living system.

[0003] Amino acids exist in all forms of life, and amino acids and their protective group derivatives have very wide applications. For example, amino acid protective groups, N-benzyloxycarbonyl (Cbz), benzyl (Bzl), tert-butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) can be used to synthesize various bioactive substances with other molecules. Benzyloxycarbonyl amino acids and amino acid benzyl esters are important bioactive substances for the synthesis of peptide sweeteners, nutritional supplements, cosmetic materials, surfactants and insulin precursors. Therefore, the chiral recognition of enantiomers of amino acid protective group derivatives has received great attention.

[0004] At present, the most commonly used chiral separation method is chromatography, which mainly includes gas chromatography (GC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), but chromatography requires complex sample pretreatment; HPLC requires expensive chiral columns, and the types of chiral columns are limited. In addition, ion mobility spectrometry (IMS) has been proven to be an effective method for rapid identification of isomeric ions. It is separated by neutral gas in an electric field based on the difference in ion mobility or ion collision cross section (CCS). However, for chiral molecules such as benzyloxycarbonyl amino acids and amino acid benzyl esters, due to the high structural similarity between the two enantiomers, it is impossible to separate them using the ion mobility spectrometry signal of IMS-MS. Therefore, it is very worthwhile to develop a simple and rapid method to separate benzyloxycarbonyl amino acids and amino acid benzyl esters, which is also one of the key issues in the current research on enantiomeric separation. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide an analytical method for the enantiomeric structure of amino acid derivatives based on natamycin, which has the advantages of simplicity, accuracy, fast analysis speed, etc. and can distinguish the enantiomers of chiral amino acid derivatives.

[0006] The technical solution to achieve the purpose of the present invention is: a method for analyzing the enantiomeric structure of an amino acid derivative of natamycin, comprising the following steps:

[0007] S1. mixing a standard of an amino acid derivative enantiomer and natamycin or natamycin and p-toluenesulfonic acid, and then adding a solvent to prepare a binary or ternary mixture solution containing water;

[0008] S2, using an ion source to generate corresponding non-covalent complex ions in the mixture solution;

[0009] S3. Use an ion mobility spectrometer to measure the ion mobility of the non-covalent complex ions. According to the ion mobility spectrometer results, the structural information of the amino acid derivative enantiomers can be obtained.

[0010] The amino acid derivative enantiomers in S1 of the above technical solution are benzyloxycarbonyl amino acid enantiomers and / or amino acid benzyl ester enantiomers.

[0011] The solvent in S1 described in the above technical solution is a mixture of water and alcohol or acetonitrile.

[0012] In the above technical solution, the content ratio of the standard substance of the amino acid derivative enantiomer to natamycin or the content ratio of the standard substance to natamycin and p-toluenesulfonic acid in S1 is greater than 0.

[0013] The ion source in S2 described in the above technical solution is an electrospray ionization ion source or a laser-assisted desorption ionization ion source or a desorption electrospray ionization ion source.

[0014] The ion mobility spectrometer in S3 of the above technical solution can measure the mobility or ion collision cross section of ions.

[0015] The ion mobility spectrometer device in S3 of the above technical solution is a combination of a mass spectrometer.

[0016] After adopting the above technical solution, the present invention has the following positive effects:

[0017] The method for analyzing the target enantiomer by using the mobility-based mass spectrometer is simple and does not require pre-separation or pre-treatment of the target object. The chemical samples used are easy to obtain, inexpensive, non-toxic and harmless. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments and in conjunction with the accompanying drawings, wherein

[0019] Figure 1 Schematic diagram of the chemical structures of 17 groups of benzyloxycarbonyl amino acids and amino acid benzyl esters;

[0020] Figure 2 Schematic diagram of the chemical structure of natamycin and p-toluenesulfonic acid;

[0021] Figure 3 Figure a is a mass spectrometry diagram of a mixture of benzyloxycarbonyl amino acid and natamycin; Figure b is a mass spectrometry diagram of a mixture of amino acid benzyl ester, natamycin and p-toluenesulfonic acid;

[0022] Figure 4 TIMS separation diagrams of the mixture of 6 groups of benzyloxycarbonyl amino acids and natamycin according to the present invention; TIMS separation diagrams of the mixture of 6 groups of amino acid benzyl esters and natamycin; TIMS separation diagrams of the mixture of 5 groups of amino acid benzyl esters, natamycin and p-toluenesulfonic acid. DETAILED DESCRIPTION

[0023] (Example 1)

[0024] The present invention is based on 17 groups of benzyloxycarbonyl amino acids and amino acid benzyl ester enantiomers, such as Figure 1 As shown, the differences in chemical properties and effects of Cbz-His, Cbz-Lys, Cbz-Arg, Cbz-Gln, Cbz-Met, Cbz-Asn, Bzl-Pro, Bzl-Trp, Bzl-Thr, Bzl-Tyr, Bzl-Val, Bzl-Ser, Bzl-Glu, Bzl-Leu, Bzl-Phe, Bzl-Asp, and Bzl-Ala, as well as the defects and shortcomings of the existing methods for analyzing these 17 groups of amino acid derivative samples, are given, and a new chiral analysis method for benzyloxycarbonyl amino acids and amino acid benzyl ester enantiomers is given.

[0025] The method comprises the following steps: adding solvent to 6 groups of different enantiomer standards of benzyloxycarbonyl amino acids (Cbz-His, Cbz-Lys, Cbz-Arg, Cbz-Gln, Cbz-Met, Cbz-Asn) and natamycin (Nat) to prepare a binary mixture solution of "natamycin-benzyloxycarbonyl amino acids" containing water; adding 6 groups of different enantiomer standards of amino acids (Bzl-Pro, Bzl-Trp, Bzl-Thr, Bzl-Tyr, Bzl-V A binary mixture solution containing water of "natamycin-amino acid benzyl ester" is prepared by adding solvent to 5 groups of amino acid benzyl ester enantiomer standard substances (Bzl-Glu, Bzl-Leu, Bzl-Phe, Bzl-Asp, Bzl-Ala), natamycin (Nat) and p-toluenesulfonic acid (PTS) and adding solvent to prepare a ternary mixture solution containing water of "natamycin-amino acid benzyl ester-p-toluenesulfonic acid". The solvent used can be a mixture of water and other liquids, such as methanol, ethanol, acetonitrile and the like, and the preparation method is not limited.

[0026] The chiral selector and ligand used are natamycin (Nat) and p-toluenesulfonic acid (PTS), respectively. The molecular formulas of natamycin and p-toluenesulfonic acid are C 33 H 47 NO 13 and C7H8O3S, whose structural formula is shown in Figure 2 middle.

[0027] Then, an electrospray ionization source was used to generate monovalent, divalent, and ternary non-covalent complex ions of "natamycin-benzyloxycarbonyl amino acid", "natamycin-amino acid benzyl ester", and "natamycin-amino acid benzyl ester-p-toluenesulfonic acid", namely [Nat+(Cbz-AA)+H] + , [Nat+(Cbz-AA)2+H] + , [Nat+(Cbz-AA)3+H] + , [Nat2+(Cbz-AA)+H] + , [Nat+(Bzl-AA)+H] + and [Nat+(Bzl-AA)2+PTS2+H] + .

[0028] Finally, the experimental setup including ion mobility spectrometry was used to measure the monovalent binary and ternary complex ions [Nat+(Cbz-AA)+H] + , [Nat+(Cbz-AA)2+H] + , [Nat+(Cbz-AA)3+H] + , [Nat2+(Cbz-AA)+H]+ , [Nat+(Bzl-AA)+H] + and [Nat+(Bzl-AA)2+PTS2+H] + The ion mobility of the amino acid can be measured by ion mobility spectra to obtain the structural information of benzyloxycarbonyl amino acid and amino acid benzyl ester molecules.

[0029] In order to verify the feasibility and advancement of the method provided by the present invention, a commercial ion mobility spectrometry-mass spectrometry instrument, TIMS-TOFMS instrument produced by Bruke, was used to analyze 17 groups of benzyloxycarbonyl amino acids and amino acid benzyl ester enantiomers. The primary mass spectrum results are shown in FIG. Figure 3 As shown, it can be seen that the binary and ternary complex ions [Nat+(Cbz-D- / L-AA)+H] formed by benzyloxycarbonyl amino acid and natamycin, amino acid benzyl ester and natamycin, amino acid benzyl ester and natamycin and p-toluenesulfonic acid + 、[Nat+(Cbz-D- / L-AA)2+H] + 、[Nat+(Cbz-D- / L-AA)3+H] + 、[Nat2+(Cbz-D- / L-AA)+H] + 、[Nat+(Bzl-D- / L-AA)+H] + ,[Nat+(Bzl-D- / L-AA)2+PTS2+H] + No separation was obtained.

[0030] The content ratios of the 6 groups of benzyloxycarbonyl amino acid standards and natamycin are all any values ​​greater than 0; the content ratios of the 6 groups of amino acid benzyl ester standards and natamycin are all any values ​​greater than 0; the content ratios of the 5 groups of amino acid benzyl ester standards, natamycin and p-toluenesulfonic acid are all any values ​​greater than 0.

[0031] (Example 2)

[0032] This embodiment mainly includes the following two steps:

[0033] 1. Preparation of complex standard samples

[0034] Take 6 groups of benzyloxycarbonyl amino acid standards (Cbz-His, Cbz-Lys, Cbz-Arg, Cbz-Gln, Cbz-Met, Cbz-Asn) and natamycin in a ratio of 1:1 to a concentration of 10 -5 mol L -1The mixed solution is 50% methanol and 50% formic acid water, and the formic acid aqueous solution is 0.1% formic acid aqueous solution; 6 groups of amino acid benzyl ester standards (Bzl-Pro, Bzl-Trp, Bzl-Thr, Bzl-Tyr, Bzl-Val, Bzl-Ser) and natamycin are mixed in a ratio of 1:1 to a concentration of 10 - 5 mol L -1 A mixed solution was prepared, the solvent was 50% methanol and 50% formic acid water, and the formic acid aqueous solution was 0.1% formic acid aqueous solution; 5 groups of amino acid benzyl ester standards (Bzl-Glu, Bzl-Leu, Bzl-Phe, Bzl-Asp, Bzl-Ala), natamycin and p-toluenesulfonic acid were mixed in a ratio of 1:1:1 to a concentration of 10 -5 molL -1 The mixed solution comprises 50% methanol and 50% formic acid water as the solvent, and the formic acid aqueous solution is 0.1% formic acid aqueous solution.

[0035] 2. Detection and analysis of complex samples using TIMS-TOF

[0036] The prepared solution was directly detected and analyzed using TIMS-TOF, where the TIMS-TOF ion source voltage was 2.5-4.5 kV and the injection flow rate was 0-5 μL min -1 ; Atomizer pressure 0.3 bar; Dry gas 3 L min at 200 °C -1 , and then analyze the test results.

[0037] After TIMS, 17 groups of enantiomers were well separated. Figure 4 .

[0038] It can be clearly seen from the experimental results that 17 groups of chiral enantiomers of benzyloxycarbonyl amino acids and amino acid benzyl esters can be easily identified and distinguished, thereby obtaining the structural characteristics of the benzyloxycarbonyl amino acids and amino acid benzyl esters contained in the samples.

[0039] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing the enantiomeric structure of an amino acid derivative of natamycin, characterized in that: The following steps are involved: S1. Mixing a standard of amino acid derivative enantiomers and natamycin or natamycin and p-toluenesulfonic acid, and then adding a solvent to prepare a binary or ternary mixture solution containing water; the amino acid derivative enantiomers are benzyloxycarbonyl amino acid enantiomers and / or amino acid benzyl ester enantiomers; S2, using an ion source to generate corresponding non-covalent complex ions in the mixture solution; S3. Use an ion mobility spectrometer to measure the ion mobility of the non-covalent complex ions. According to the ion mobility spectrometer results, the structural information of the amino acid derivative enantiomers can be obtained.

2. The method for analyzing the enantiomeric structure of the amino acid derivative of natamycin according to claim 1, characterized in that: The solvent in S1 is a mixture of water and alcohol or acetonitrile.

3. The method for analyzing the enantiomeric structure of the amino acid derivative of natamycin according to claim 1, characterized in that: The content ratio of the standard substance of the amino acid derivative enantiomer in S1 to natamycin or the content ratio of the standard substance of the amino acid derivative enantiomer to natamycin and p-toluenesulfonic acid is greater than 0.

4. The method for analyzing the enantiomeric structure of the amino acid derivative of natamycin according to claim 1, characterized in that: The ion source in S2 is an electrospray ionization ion source, a laser-assisted desorption ionization ion source, or a desorption electrospray ionization ion source.

5. The method for analyzing the enantiomeric structure of the amino acid derivative of natamycin according to claim 1, characterized in that: The ion mobility spectrometer in S3 can measure the mobility or ion collision cross section of ions.

6. The method for analyzing the enantiomeric structure of the amino acid derivative of natamycin according to claim 1, characterized in that: The ion mobility spectrometer in S3 is a combination of a mass spectrometer.

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