Analysis and identification method of N-acylamino acid and application of analysis and identification method

Abstract

The invention discloses an analysis and identification method of N-acylamino acid and application of the analysis and identification method. According to the method, a one-pot method is adopted, amino acid and fatty acid are synthesized into a target object under the action of a coupling reagent, a derivatization reagent N, N-diisopropylethylenediamine (DIAAA) is directly added for in-situ chemical derivatization without separation, and then liquid chromatography-mass spectrometry (LC-MS) analysis is carried out. And carrying out structure identification according to the DIAAA characteristic fragment, the amino acid residue fragment and the acyl fragment. The method can be extensively used for systematic identification and quantitative analysis of complex biological samples, and is especially suitable for N-oleoyl amino acid. Experiments prove that N-oleoyl tyrosine, proline, leucine and tryptophan have remarkable inhibitory activity on hepatoma cells HepG2, which indicates that the N-oleoyl tyrosine, proline, leucine and tryptophan have application potential in preparation of anti-hepatoma drugs. According to the method, efficient and accurate analysis of the N-acylamino acid is realized.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and pharmaceutical technology, specifically relating to an analytical identification method for N-acyl amino acids and its application. Background Technology

[0002] Fermented soy products, such as soy sauce, miso, and fermented black beans, are an important part of Asian culinary traditions. In recent years, their potential health benefits have received increasing attention. Studies have shown that the fermentation process, through the action of microorganisms and enzymes, breaks down large molecules in soybeans, such as proteins and carbohydrates, into more easily absorbed peptides, amino acids, and sugars. This not only improves the flavor and texture of the products but may also generate new bioactive substances. Epidemiological surveys suggest an association between regular consumption of fermented soy products and a lower risk of certain cancers, making the exploration of their underlying functional components a research hotspot. Currently, the known bioactive components in fermented soy products mainly include soy isoflavones, bioactive peptides, and polyphenols. However, fermentation is a complex biochemical process that may produce a large number of secondary metabolites with unknown structures. A comprehensive understanding of these "dark matter" products remains a challenge and a frontier of current research.

[0003] N-Acylamino acids are a special class of metabolites formed by the linkage of the amino group of an amino acid to the carboxyl group of a fatty acid via an amide bond. These compounds exhibit diverse structures and can be classified into short-chain, medium-chain, and long-chain N-acylamino acids based on the length of their fatty acid chains. Recent studies have shown that endogenous N-acylamino acids play important roles in physiological and pathological processes such as energy metabolism, inflammatory responses, and nerve signal transduction. Some members have shown potential as biomarkers for metabolic diseases or lead compounds for novel drugs. For example, exercise-induced N-lactylamino acids have been confirmed as key signaling molecules regulating systemic energy balance. Furthermore, studies have identified some N-acylamino acids in soy sauce and miso, finding them to be novel flavor compounds contributing to umami and richness. This suggests that fermented foods may be an important source of dietary N-acylamino acids.

[0004] However, systematic research on the composition, content, and correlation with the fermentation process of N-acyl amino acids in fermented soybean products, especially the traditional Chinese fermented product "lightly fermented soybean paste," remains lacking. One fundamental technical bottleneck lies in the significant challenge of achieving efficient and accurate analysis of these compounds. Liquid chromatography-mass spectrometry (LC-MS) is the mainstream tool for metabolomics analysis, but its application in the comprehensive analysis of N-acyl amino acids faces the following prominent problems:

[0005] The N-acyl amino acid family is large, and combinations of different fatty acid chains (length, saturation, branching) with 20 protein-derived amino acids can produce hundreds of structures with significant differences in their physicochemical properties (especially polarity). Achieving simultaneous high-resolution separation under single chromatographic conditions is extremely challenging.

[0006] Many N-acyl amino acids are isomers of each other (e.g., isomeric fatty acid derivatives with the same molecular formula linked by different amino acids, or isomeric amino acid derivatives with the same molecular formula linked by different fatty acids). These isomers often have highly similar or even identical spectra in conventional tandem mass spectrometry databases, and accurate differentiation cannot be achieved by matching with public libraries alone; moreover, the biological activities of some isomers may differ significantly.

[0007] The lack of commercially available standards for the vast majority of N-acyl amino acids severely limits accurate quantitative analysis and absolute structural confirmation based on standard curves. Traditional chemical synthesis methods for preparing single standards are typically cumbersome, time-consuming, and have limited yields, making it difficult to meet the needs of high-throughput, systematic profiling analysis.

[0008] Therefore, developing a new method that can overcome the above limitations and achieve high coverage, high reliability, and accurate quantification of N-acyl amino acids in complex matrices (such as fermented soybean products) is of great scientific significance and application value for in-depth exploration of the functional components of traditional fermented foods such as light fermented soybeans, elucidating their health effects mechanisms, and promoting the development of functional foods. Summary of the Invention

[0009] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides an analytical identification method for N-acyl amino acids and its application.

[0010] According to a first aspect of the present invention, a method for identifying N-acyl amino acids is provided, comprising the following steps: S1. Reacting a test sample containing an amino acid with a fatty acid, a coupling reagent, and a first base in the same reaction vessel to synthesize an N-acyl amino acid; S2. Without separating the N-acyl amino acid, adding a derivatizing reagent N,N-diisopropylethylenediamine (DIAAA) and a second base to the same reaction vessel to perform in-situ chemical derivatization of the N-acyl amino acid synthesized in step S1 to obtain a derivatized product; S3. Analyzing the derivatized product by liquid chromatography-mass spectrometry (LC-MS) to obtain mass spectrometry data; S4. Identifying the structure of the N-acyl amino acid based on at least two of the characteristic fragment ions derived from DIAAA, characteristic fragment ions generated from the amino acid residues of the N-acyl amino acid, and characteristic fragment ions generated from the acyl group of the N-acyl amino acid in the mass spectrometry data.

[0011] Preferably, in step S1, the coupling reagent includes hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU) and 1-hydroxybenzotriazine (HOBt); and / or, the first base and the second base are both triethylamine (TEA).

[0012] Preferably, in step S4, the characteristic fragment ions derived from DIAAA include ions with m / z of 86.09 and 128.14, and / or include fragment ions formed by the loss of neutral fragments of the molecular ion of the derivatized product by 42.05 Da or 84.09 Da.

[0013] Preferably, in step S4, the characteristic fragment ions generated from the amino acid residues of the N-acyl amino acid are generated by breaking the bond between the carboxyl carbon and the α carbon of the amino acid moiety in the N-acyl amino acid molecule and / or breaking the amide bond between the amino acid moiety and the acyl moiety.

[0014] Preferably, in step S3, a reversed-phase chromatography column is used for liquid chromatography separation, the mobile phase includes an aqueous solution containing formic acid and an acetonitrile solution containing formic acid, and a gradient elution program is used; the mass spectrometry analysis is performed in electrospray ionization positive ion mode, and the mass number acquired by the mass spectrometer ranges from m / z 30 to 1700.

[0015] According to a second aspect of the present invention, a method for systematically identifying N-acyl amino acids from complex biological samples is provided, comprising the following steps: P1. After lyophilizing and grinding the biological sample, stepwise extraction is performed using at least two solvents of different polarities, the extracts are combined and centrifuged to obtain a test solution; P2. A portion of the test solution is taken, and the N-acyl amino acids contained therein are derivatized in situ using the method described in any one of claims 1 to 5 to obtain a derivatized product; P3. The derivatized product is analyzed by liquid chromatography-mass spectrometry, and the structure of the N-acyl amino acid is identified and confirmed based on the obtained mass spectrometry data and a standard with a mass error of less than 5 ppm.

[0016] Preferably, in step P1, the at least two solvents of different polarities are selected from water, methanol, and at least two of an aqueous methanol solution with a concentration of 10%-90% (v / v).

[0017] According to a third aspect of the present invention, a method for quantitative analysis of N-oleoyl amino acids is provided, comprising the following steps:

[0018] Q1. Prepare a series of standard solutions of at least one N-oleoyl amino acid standard. Add an internal standard to each standard solution and perform chemical derivatization. Then perform liquid chromatography-mass spectrometry analysis. Plot a standard curve with the ratio of analyte to internal standard peak area as the ordinate and the standard concentration as the abscissa.

[0019] Q2. Add the internal standard to the test sample solution containing N-oleoyl amino acids and perform the same chemical derivatization treatment as in step Q1;

[0020] Q3. Perform liquid chromatography-mass spectrometry analysis on the derivatized product obtained in step Q2 to obtain the ratio of the chromatographic peak area of ​​the analyte to the internal standard in the sample to be tested.

[0021] Q4. Based on the peak area ratio obtained in step Q3, use the standard curve established in step Q1 to calculate the content of N-oleoyl amino acids in the sample to be tested;

[0022] The chemical derivatization process involves using a derivatization reagent system containing HATU, HOBt, and DIAAA to derivatize the carboxyl group of N-oleoyl amino acids.

[0023] Preferably, in steps Q1 and Q2, the chemical derivatization treatment includes: (i) in a first stage, using HATU and HOBt as coupling agents to carry out a reaction under alkaline conditions; and (ii) in a second stage, adding DIAAA to carry out a derivatization reaction.

[0024] According to a fourth aspect of the present invention, there is provided the use of an N-oleoyl amino acid in the preparation of a medicament for the prevention and / or treatment of liver cancer, wherein the N-oleoyl amino acid is selected from at least one of N-oleoyltyrosine, N-oleoylproline, N-oleoylleucine and N-oleoyltryptophan.

[0025] This invention provides the above-mentioned method for the analysis of N-acyl amino acids based on one-pot synthesis and in-situ derivatization, and its application. Compared with the prior art, it has the following significant advantages:

[0026] The prior art heavily relies on a limited number of commercial standards, making it difficult to comprehensively analyze the structurally diverse N-acyl amino acid family. The present invention creatively combines "one-pot micro-synthesis" with "in-situ chemical derivatization". This method can, before analysis, in-situ synthesize target or potential N-acyl amino acids from the amino acids to be measured or known and fatty acids at a micro-scale, and immediately perform derivatization. Through the combined structural analysis of the highly regular characteristic mass spectrometry fragmentation fragments generated after derivatization (such as DIAAA tag ions, amino acid residue characteristic ions, acyl chain characteristic ions), it bypasses the absolute dependence on a large number of commercial standards, making it possible to identify a large number of unknown or known N-acyl amino acids in complex samples with high throughput and high confidence.

[0027] There are a large number of N-acyl amino acid isomers caused by different amino acid types or differences in fatty acid chain structures, which are difficult to distinguish by traditional mass spectrometry database matching. The derivatization strategy of the present invention induces a well-defined fragmentation path. Among them, the amino acid residue characteristic ions (resulting from the cleavage of Cα-COOH bonds and amide bonds) can directly indicate the amino acid type; the acyl chain characteristic ions can reflect the structural information of fatty acids. By correlating these two types of key information with DIAAA characteristic ions, the fragments can be clearly attributed to specific molecular skeletons, thus effectively distinguishing different fatty acid isomers connected to the same amino acid or different amino acid isomers connected to the same fatty acid, greatly improving the accuracy of structural identification.

[0028] N-acyl amino acids have a wide polarity range, and it is challenging to simultaneously analyze short-chain (hydrophilic) and long-chain (hydrophobic) members. The DIAAA derivatization reaction adopted in the present invention significantly enhances the hydrophobicity of low-polarity target compounds and the hydrophilicity of high-polarity target compounds by introducing diisopropylethylenediamine groups with a polarity reversal effect, improving their retention behavior on reversed-phase chromatography, enabling different chain-length N-acyl amino acids with large polarity differences to achieve good separation and simultaneous detection under an optimized gradient elution program. In addition, this derivatization reaction also significantly improves the mass spectrometry ionization efficiency of target compounds, thereby reducing the detection limit and enhancing the detection ability for low-abundance N-acyl amino acids.

[0029] The present invention first systematically applies the above high-efficiency analysis method to the research of traditional fermented food Douchi and its raw material black beans, successfully identifying up to 222 N-acyl amino acids and revealing significant differences before and after fermentation. This discovery greatly enriches the scientific understanding of the composition of bioactive substances in Douchi, establishing N-acyl amino acids as an important class of previously overlooked functional ingredient groups in this fermented product, providing a new material basis and research direction for in-depth interpretation of the health benefits of Douchi.

[0030] Based on the above findings, this invention further focuses on the abundant and significantly varied N-oleoyl amino acids in fermented soybeans, and through in vitro cell experiments, it has been clearly verified that N-oleoyltyrosine, N-oleoylproline, N-oleoylleucine, and N-oleoyltryptophan have significant dose-dependent inhibitory activity against the proliferation of HepG2 liver cancer cells (IC50 values ​​in the range of 15-19 μM). This result not only provides direct scientific evidence and specific active substances for the traditional or potential anti-tumor efficacy of fermented soybeans, but also provides clear lead compounds and experimental basis for the development of novel anti-liver cancer drugs, health products, or functional foods with these specific N-oleoyl amino acids as active ingredients, giving the technical achievements of this invention clear and important translational application value.

[0031] In summary, this invention not only provides a powerful N-acyl amino acid analysis tool that breaks through the bottlenecks of existing technologies, but also uses this tool to discover new functional component groups in important traditional foods, and further identifies specific compounds with clear pharmacological activities. It realizes a complete chain from analytical method innovation to bioactive substance discovery and functional verification, which has both important scientific significance and application potential. Attached Figure Description

[0032] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0033] Figure 1 A schematic diagram of a one-pot micro-synthesis-in-situ DIAAA derivatization strategy provided according to an embodiment of the present invention is shown.

[0034] Figure 2 The diagram shows a comparison of N-propionyl-histidine and N-oleoyl-tyrosine derivatization before and after the present invention.

[0035] Figure 3 A schematic diagram illustrating the differentiation of N-acyl isoleucine / leucine isomers according to an embodiment of the present invention is shown.

[0036] Figure 4 The mass spectrometry fragmentation diagrams of N-propionyl-threonine and N-isobutyryl-serine provided according to embodiments of the present invention are shown.

[0037] Figure 5 The figure shows the results of HepG2 cells treated with a specified concentration of N-oleoyl amino acids for 24 hours according to an embodiment of the present invention. Detailed Implementation

[0038] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0039] Example 1: A one-pot micro-synthesis and in-situ chemical derivatization identification method for N-acyl amino acids

[0040] 1. Preparation of reagents and instruments

[0041] Amino acid standards: 19 natural amino acids other than cysteine.

[0042] Fatty acid standards: acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, oleic acid, linoleic acid, and linolenic acid.

[0043] Derivatization reagent: N,N-diisopropylethylenediamine (DIAAA, purity ≥98%).

[0044] Coupling reagents: O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea (HATU, purity ≥98%), 1-hydroxybenzotriazole (HOBt, purity ≥98%).

[0045] Alkaline reagent: Triethylamine (TEA, purity ≥99.5%).

[0046] Solvents: Acetonitrile (LC-MS grade), methanol (LC-MS grade), Milli-Q ultrapure water, concentrated hydrochloric acid.

[0047] Instruments: Agilent 1290 ultra-high performance liquid chromatograph (UHPLC), Agilent 6545 precision mass quadrupole time-of-flight mass spectrometer (Q-TOF) equipped with electrospray ionization (ESI) source; Waters ACQUITY UPLC® HSS T3 column (2.1 × 150 mm, 1.7 μm); micro vortex mixer; nitrogen blower.

[0048] 2. Preparation of the test solution

[0049] a) Weigh out 19 natural amino acid standards and dissolve them in 0.1 mol / L hydrochloric acid aqueous solution or 60% methanol aqueous solution (v / v) to prepare single amino acid stock solutions with a concentration of 1 mg / mL.

[0050] b) Mix the above 19 amino acid stock solutions and dilute with 60% methanol aqueous solution (v / v) to prepare an amino acid mixture stock solution with a total concentration of 200 μg / mL.

[0051] c) Weigh out the standards of acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, oleic acid, linoleic acid, and linolenic acid respectively, dissolve them in acetonitrile, and prepare a fatty acid standard solution with a concentration of 20 mg / mL.

[0052] d) All prepared solutions were aliquoted and stored in a -20°C refrigerator for later use.

[0053] 3. One-pot micro-synthesis—in-situ chemical derivatization of N-acyl amino acids

[0054] a) Take 10 μL of the amino acid mixture stock solution prepared in step 2 (containing a total of 2 μg of amino acids) into a 1.5 mL Eppendorf tube and blow the solvent dry with a gentle stream of nitrogen.

[0055] b) Add the following to the dried residue in sequence: 5 μL 20 mM HATU (dissolved in acetonitrile), 5 μL 20 mM HOBt (dissolved in acetonitrile), 5 μL 20 mg / mL oleic acid standard solution (dissolved in acetonitrile, i.e., oleic acid is selected as the example fatty acid), and 5 μL 20 mM TEA (dissolved in acetonitrile).

[0056] c) Tightly cap the Eppendorf tube, place it on a vortex mixer, and vortex at room temperature (approximately 25°C) for 20 minutes to synthesize N-acyl amino acids.

[0057] d) Without performing any separation or purification operations, add the following to the same reaction tube: 5 μL 20 mM HATU (dissolved in acetonitrile) and 5 μL 100 mM DIAAA (dissolved in acetonitrile solution containing 200 mM TEA).

[0058] e) Seal the reaction tube again and vortex the reaction at room temperature for 5 minutes to perform in-situ chemical derivatization of the N-oleoyl amino acid synthesized in step c).

[0059] f) After the reaction is complete, add 20 μL of acetonitrile to the reaction tube to make the total volume of the reaction system reach 50 μL, vortex to mix, and the derivatized product solution is obtained.

[0060] g) Take 1 μL of the derivatized product solution and use it directly for subsequent LC-MS analysis.

[0061] like Figure 1As shown, using a mixture of 19 natural amino acids and fatty acids as substrates, HATU / HOBt was used as a coupling agent under alkaline conditions to generate N-acylated amino acids by vortexing at room temperature. Subsequently, the original reaction mixture was directly added to the DIAAA derivatization reagent without separation, and finally, 19 in-situ DIAAA-derived N-acylated amino acids were synthesized in a one-pot micro-scale process within 25 min.

[0062] 4. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis

[0063] The following conditions were used to analyze the derivatized products:

[0064] Chromatographic conditions:

[0065] Column: Waters ACQUITY UPLC® HSS T3 (2.1 × 150 mm, 1.7 μm). Column temperature: 40 ℃. Flow rate: 0.3 mL / min. Injection volume: 1 μL. Mobile phase: A was Milli-Q ultrapure water containing 0.1% (v / v) formic acid, and B was acetonitrile containing 0.1% (v / v) formic acid. Gradient elution program: 0–0.5 min: 5% B; 0.5–2.5 min: 5%–10% B; 2.5–5 min: 10%–15% B; 5–11 min: 15%–25% B; 11–17 min: 25%–30% B; 17–19 min: 30%–35% B; 19–22 min: 35%–50% B; 22–22.5 min: 50%–70% B; 22.5–25 min: 70%–95% B; 25–27.9 min: 95% B; 28.0 min: 5% B (hold for 30 min to equilibrate the system).

[0066] Mass spectrometry conditions:

[0067] Ion source: Electrospray ionization (ESI), positive ion mode. Full scan range: m / z 30–1700. Drying gas temperature: 300 °C. Drying gas flow rate: 11 L / min. Sheath gas temperature: 325 °C. Sheath gas flow rate: 11 L / min. Nebulizer pressure: 35 psi. Capillary voltage: 3500 V. Nozzle voltage: 500 V.

[0068] 5. Mass Spectrometry Fragmentation Patterns and Structural Identification of N-Acyl Amino Acids

[0069] By analyzing mass spectrometry data obtained from LC-MS, especially the high-resolution mass spectrometry information of the parent ion and fragment ions of the derivatized products, the structure of N-acyl amino acids can be identified. This method mainly relies on the following three types of characteristic fragment ions:

[0070] ① Characteristic fragment ions introduced by DIAAA derivatization:

[0071] The characteristic ion peaks at m / z 86.0964 and 128.1434 (theoretical exact mass number) can be identified as N-isopropyl-N-vinylpropyl-2-ammonium ion and N-vinylpropyl-2-ammonium ion, respectively, which are the hallmark products of the DIAAA derivatization reaction.

[0072] The molecular ion [M+H] of the derivatization product was observed. + 42.0473 Da (propylene) was lost. ) or 84.0947 Da (two propylenes, 2× Characteristic fragment ions in neutral fragment formation and This further confirms the existence of the DIAAA structural unit.

[0073] ② Characteristic fragment ions of N-acyl amino acid residues:

[0074] The C-C bond between the carboxyl carbon (C-1) and the α carbon (C-α) of the amino acid moiety in an N-acyl amino acid molecule breaks, generating an N-acylimine ion. This ion can further undergo C-N bond cleavage to generate the characteristic amino acid imine ion ([ =CH-R] + Its precise mass number can be used to quickly identify the corresponding amino acid species (such as tyrosine, proline, leucine, tryptophan, etc.).

[0075] ③ Characteristic fragment ions of N-acyl amino acid acyl groups:

[0076] The cleavage of the amide bond between the acyl moiety and the amino acid, or the cleavage of the acyl moiety itself, can generate a characteristic N-acylacetylenium ion (m / z 43.0178) (e.g. (Series of ions). By analyzing the precise mass number of these ions, structural information such as the carbon chain length and degree of unsaturation of the acyl group can be determined, for example, to distinguish between oleoyl and linoleoyl groups.

[0077] 6. Appraisal Results

[0078] In this embodiment, oleic acid was reacted with a mixture of 19 amino acids. Through the aforementioned LC-MS analysis and fragment ion analysis, multiple N-oleoyl amino acids, including but not limited to N-oleoyltyrosine, N-oleoylproline, N-oleoylleucine, and N-oleoyltryptophan, were successfully identified from the derivatized product. The identification was based on the simultaneous detection of characteristic fragments from DIAAA (e.g., m / z 86.10, 128.14), characteristic imine ions from specific amino acid residues, and characteristic acyl ion fragments from the oleoyl group (C18:1). The quality error of all identification results was less than 5 ppm, confirming the high accuracy and reliability of this method.

[0079] like Figure 2 As shown, the in-situ DIAAA derivatization strategy can effectively regulate the retention behavior of short-chain and long-chain N-acylated amino acids in reversed-phase chromatography and improve the mass spectrometry response. After in-situ DIAAA derivatization, the retention time of hydrophilic N-propionyl-histidine increased from 1.7 min to 3.7 min, while the retention time of hydrophobic N-oleyl-tyrosine decreased from 26.9 min to 25.5 min, both accompanied by a significant improvement in mass spectrometry response.

[0080] like Figure 3 As shown, after in-situ DIAAA derivatization, all five pairs of N-acyl isoleucine / leucine isomers were baseline separated.

[0081] like Figure 4 As shown, N-propionylated-threonine and N-isobutylated-serine are isomers, differing only slightly in the N-acyl side chain and the amino acid backbone. The structures of threonine and serine can be confirmed by characteristic fragment ions at m / z 74.06 and 60.04, respectively, while fragment ions at m / z 57.03 and 71.04 indicate the structures of the N-propionylated and N-isobutylated groups, respectively.

[0082] Table 1. Characteristic fragment ions of N-acetylated amino acids

[0083]

[0084] (Continued from the table above)

[0085]

[0086] This embodiment demonstrates that the one-pot micro-synthesis and in-situ chemical derivatization combined with LC-MS analysis method provided by this invention can efficiently and sensitively identify N-acyl amino acids. This method avoids cumbersome intermediate product separation steps and achieves precise structural identification of the amino acid and acyl groups in N-acyl amino acids through characteristic fragment ions, laying a technical foundation for the systematic identification of N-acyl amino acids in complex samples.

[0087] Example 2: Systematic identification and differential analysis of N-acyl amino acid profiles in BS and FS samples

[0088] 1. Samples, reagents and instruments

[0089] Biological samples: BS samples (pre-fermentation samples) and FS samples (post-fermentation samples).

[0090] Internal standard: 4-chloro-DL-phenylalanine (4-Cl-Phe).

[0091] Derivatization reagent: N,N-diisopropylethylenediamine (DIAAA, purity ≥98%).

[0092] Coupling reagents: O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethylurea (HATU, purity ≥98%), 1-hydroxybenzotriazole (HOBt, purity ≥98%).

[0093] Alkaline reagent: Triethylamine (TEA, purity ≥99.5%).

[0094] Extraction solvents: methanol (LC-MS grade), Milli-Q ultrapure water.

[0095] Instruments: Vacuum freeze dryer, grinder, ultrasonic extractor, high-speed centrifuge, Agilent 1290 ultra-high performance liquid chromatograph (UHPLC) coupled with an Agilent 6545 precision mass quadrupole time-of-flight mass spectrometer (Q-TOF), nitrogen evaporator, vortex mixer. Data analysis software: Agilent Qualitative Analysis B.06.00, SIMCA-P (Umetrics).

[0096] 2. Preparation of the test solution

[0097] a) Sample pretreatment: The collected BS and FS samples were rapidly frozen in liquid nitrogen and then freeze-dried using a vacuum freeze dryer. The freeze-dried samples were then ground using a grinder and passed through a 50-mesh sieve to obtain a uniform dried powder.

[0098] b) Stepwise extraction: Accurately weigh approximately 150 mg of the dried powder and place it in a 5 mL centrifuge tube. Sequential extraction is performed using three solvents of different polarities:

[0099] Add 1.5 mL of methanol and extract by sonication at room temperature for 30 minutes.

[0100] Add 1.5 mL of 50% methanol aqueous solution (v / v) and sonicate for 30 minutes at room temperature.

[0101] Add 1.5 mL of Milli-Q ultrapure water and sonicate for 30 minutes at room temperature.

[0102] c) Combining of extracts: After each ultrasonic extraction, centrifuge the tubes at 13500 rpm for 10 minutes at 4°C and carefully aspirate the supernatant. Combine the supernatants obtained from the three extractions in the same container and mix well to obtain the total extract. This extract contains a broad spectrum of metabolites extracted with solvents of different polarities.

[0103] d) Aliquoting of the test solution: Aliquot the total extract and store it in a -80°C freezer for later use, avoiding repeated freeze-thaw cycles.

[0104] 3. In-situ chemical derivatization treatment

[0105] Take 50 μL of the total extract prepared in step 2 into a 1.5 mL Eppendorf tube, add 5 μL of 20 μg / mL 4-Cl-Phe internal standard solution (dissolved in methanol), mix well, and dry the solvent under a gentle nitrogen stream. In situ derivatization is then performed on the dried residue to label the N-acyl amino acids. The following reagents are added sequentially:

[0106] 5 μL 20 mM HOBt (dissolved in acetonitrile), 5 μL 20 mM HATU (dissolved in acetonitrile), 5 μL 100 mM DIAAA (dissolved in acetonitrile solution containing 200 mM TEA), 5 μL 20 mM HATU (dissolved in acetonitrile).

[0107] After the addition is complete, immediately cap the tube and vortex the reaction at room temperature (approximately 25°C) for 10 minutes. After the reaction is complete, add 30 μL of acetonitrile to bring the total volume to 50 μL, and vortex thoroughly to obtain the derivatized product solution to be tested. Take 1 μL of this solution directly for LC-MS analysis.

[0108] 4. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis

[0109] The derivatized test solution was analyzed using the same chromatographic and mass spectrometric conditions as in Example 1.

[0110] Chromatographic conditions: Waters ACQUITY UPLC® HSS T3 column (2.1 × 150 mm, 1.7 μm); mobile phase: water (A) containing 0.1% formic acid and acetonitrile (B) containing 0.1% formic acid; gradient elution program as in Example 1. Mass spectrometry conditions: electrospray ionization (ESI) positive ion mode; full scan range m / z 30–1700; ion source parameters as in Example 1.

[0111] 5. Data Analysis and Systematic Identification of N-Acyl Amino Acids

[0112] a) Data preprocessing: Import the collected raw LC-MS data into Agilent Qualitative Analysis B.06.00 software.

[0113] b) Characteristic fragment ion screening: First, use software to extract all chromatographic peaks in the chromatogram that produce the following characteristic fragment ions: (i) m / z 86.10 and / or 128.14 (DIAAA characteristic ions); (ii) those meeting the criteria... and The pattern of ions (DIAAA neutral loss characteristic). This step can quickly screen out candidate peaks that may be DIAAA-derived N-acyl amino acids.

[0114] c) High-precision identification: The candidate peaks selected in the previous step are subjected to precise molecular weight identification and secondary mass spectrometry (MS / MS) analysis. The specific procedure is as follows:

[0115] 1. By the molecular ion peak ([M+H]) + Find the exact mass number of and calculate its possible elemental composition.

[0116] 2. Compare the calculated precise molecular weight with the self-built "N-acyl amino acid-DIAAA derivative database". This database contains theoretical molecular formulas, precise molecular weights, corresponding N-acyl amino acid names, and predicted or known characteristic fragment ion information (such as amino acid residue imine ions, acylacetylenium ions, etc.).

[0117] 3. Examine the MS / MS spectra of the candidate peaks to confirm whether characteristic fragments from DIAAA and characteristic fragments from amino acid residues (such as […]) are present simultaneously. =CH-R] + ) and characteristic fragments from the acyl chain.

[0118] d) Identification and confirmation criteria: For a compound to be definitively identified as a specific N-acyl amino acid, it must meet all of the following conditions:

[0119] 1. Its [M+H] + The measured accurate mass number of ions has a mass error of less than 5 ppm compared with the theoretical value in the database.

[0120] 2. Its retention time is reasonable and the chromatographic peak shape is symmetrical.

[0121] 3. At least two types of characteristic fragment ions (such as DIAAA characteristic fragments + amino acid characteristic fragments, or DIAAA characteristic fragments + acyl characteristic fragments) can be detected in its MS / MS spectrum, and the measured mass number of these fragment ions matches the theoretical value well, indicating a reasonable fragmentation pathway.

[0122] Through the above systematic identification process, a total of 222 different N-acyl amino acids were successfully identified in BS and FS samples.

[0123] 6. Differential analysis of N-acyl amino acids

[0124] To investigate the effect of fermentation on the N-acyl amino acid composition of the samples, statistical difference analysis was performed on the BS and FS groups.

[0125] a) The names of the 222 identified N-acyl amino acids and their LC-MS peak areas in each sample group (normalized by internal standard 4-Cl-Phe) were compiled into a data matrix.

[0126] b) Import the data matrix into SIMCA-P software and perform principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to observe the overall metabolic profile differences between the BS group and the FS group, and screen variables that contribute significantly to the distinction between the groups (i.e., N-acyl amino acids).

[0127] c) For each identified N-acyl amino acid, perform Student's t-test between the BS and FS groups. Calculate the fold change (FC) of its content.

[0128] d) N-acyl amino acids that simultaneously meet the following two conditions are considered to have significant differences before and after fermentation: a p-value of less than 0.05 in the t-test, indicating that the difference is statistically significant; or a fold change in content. A value greater than 1.5 indicates that the magnitude of the change is biologically significant.

[0129] This analysis successfully screened out N-acyl amino acids whose content changed significantly between BS and FS samples. These compounds may be closely related to the biochemical transformation during fermentation.

[0130] This embodiment demonstrates the entire process of applying the in-situ derivatization combined with LC-MS method provided by this invention to the systematic identification and differential analysis of N-acyl amino acids in complex real biological samples (BS and FS). This method ensures broad coverage of metabolites through multi-solvent extraction, guarantees the reliability of results through data screening based on characteristic fragment ions and high-precision identification criteria, and effectively uncovers differential compounds among different samples through a combination of multivariate and univariate statistical methods. The results show that this method can efficiently and systematically identify a large number of N-acyl amino acids from complex matrices and reveal their dynamic changes in biological processes, demonstrating good practicality and potential for wider application.

[0131] Example 3: Quantitative analysis method of N-oleoyl amino acids in BS and FS samples

[0132] 1. Reagents, standards and instruments

[0133] Amino acids: L-alanine, L-phenylalanine, L-tryptophan, L-leucine, L-proline, L-tyrosine.

[0134] Acylation reagent: oleoyl chloride.

[0135] Quantitative analysis targets (standards): N-oleoyl-L-alanine (N-oleoyl-Ala), N-oleoyl-L-phenylalanine (N-oleoyl-Phe), N-oleoyl-L-tryptophan (N-oleoyl-Trp), N-oleoyl-L-leucine (N-oleoyl-Leu), N-oleoyl-L-proline (N-oleoyl-Pro), and N-oleoyl-L-tyrosine (N-oleoyl-Tyr). These standards must be obtained through synthesis and purification, or purchased from certified commercially available standards.

[0136] Internal standard (IS): 4-chloro-DL-phenylalanine (4-Cl-Phe).

[0137] Derivatizing reagents: N,N-diisopropylethylenediamine (DIAAA), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU), 1-hydroxybenzotriazole (HOBt).

[0138] Alkaline reagents: triethylamine (TEA), sodium hydroxide.

[0139] Solvents: Tetrahydrofuran (THF, anhydrous), ethyl acetate, acetonitrile (LC-MS grade), methanol (LC-MS grade), Milli-Q ultrapure water, hydrochloric acid.

[0140] Instruments: analytical balance, magnetic stirrer, rotary evaporator, silica gel column chromatography system, nuclear magnetic resonance spectrometer (NMR), Agilent 1290 ultra-high performance liquid chromatograph (UHPLC) coupled with Agilent 6545 Q-TOF mass spectrometer, nitrogen evaporator, vortex mixer.

[0141] 2. Synthesis and Characterization of N-Oleoyl Amino Acid Standards

[0142] To ensure the accuracy of quantitative analysis, a high-purity standard of the target N-oleoyl amino acid was first synthesized. Taking the synthesis of N-oleoyltyrosine (N-oleoyl-Tyr) as an example, the steps are as follows:

[0143] a) Weigh L-tyrosine (1.0 equivalent) into a round-bottom flask, add an appropriate amount of 2 mol / L sodium hydroxide (NaOH) aqueous solution, and stir to dissolve for 5 minutes in an ice-water bath (0 °C).

[0144] b) Dissolve oleoyl chloride (1.05 equivalents) in anhydrous tetrahydrofuran (THF), and slowly add the solution dropwise to the above-mentioned amino acid alkaline solution using a constant pressure dropping funnel while in an ice bath with continuous stirring.

[0145] c) After the addition is complete, remove the ice bath and allow the reaction mixture to react overnight (approximately 12-16 hours) at room temperature with stirring. The reaction progress is monitored by thin-layer chromatography (TLC).

[0146] d) After the reaction is complete, cool the reaction mixture in an ice-water bath and carefully acidify it with 5 mol / L hydrochloric acid (HCl) to a pH of approximately 2-3.

[0147] e) Extract the reaction mixture with ethyl acetate (10 mL each time, for a total of 3 times), and combine all organic phases.

[0148] f) The combined organic phases were dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure using a rotary evaporator to obtain the crude product.

[0149] g) The crude product is purified by silica gel column chromatography (the eluent is usually a petroleum ether / ethyl acetate or a dichloromethane / methanol gradient system).

[0150] h) The purified product was analyzed by proton NMR spectroscopy. and carbon spectrum Structural verification was conducted.

[0151] Following similar steps, N-oleoyl-L-alanine (N-oleoyl-Ala), N-oleoyl-L-phenylalanine (N-oleoyl-Phe), N-oleoyl-L-tryptophan (N-oleoyl-Trp), N-oleoyl-L-leucine (N-oleoyl-Leu), and N-oleoyl-L-proline (N-oleoyl-Pro) were synthesized, and their structures were confirmed by NMR characterization.

[0152] 3. Establishment and validation of quantitative analysis methods

[0153] 3.1 Preparation of Standard Solutions and Internal Standard Solutions

[0154] a) Standard stock solution (1 mg / mL): Accurately weigh approximately 1.0 mg of each of the six purified and identified N-oleoyl amino acid standards, place them in a 1 mL Eppendorf tube, and dissolve and mix with 80% acetonitrile aqueous solution (v / v) to obtain a single standard stock solution with a concentration of 1 mg / mL. Store at -20°C protected from light.

[0155] b) Mixed standard intermediate solution: As needed, take an appropriate amount of each single standard stock solution and dilute it with 80% acetonitrile to prepare a series of mixed standard intermediate solutions of different concentrations.

[0156] c) Internal standard (IS) stock solution (1 mg / mL): Accurately weigh approximately 1.0 mg of 4-Cl-Phe and place it in a 1 mL Eppendorf tube. Dissolve and dilute to volume with methanol. Store at -20°C.

[0157] d) Internal standard working solution (20 μg / mL): Accurately measure an appropriate amount of internal standard stock solution, dilute with acetonitrile, and prepare an internal standard working solution with a concentration of 20 μg / mL.

[0158] 3.2 Chemical derivatization treatment (key step)

[0159] This quantitative method employs a derivatization strategy consistent with that of Examples 1 and 2 to enhance detection sensitivity and specificity. For both the standard curve sample and the actual sample, derivatization is performed in the following two stages:

[0160] (i) First stage (activation and coupling): Take an appropriate amount of standard working solution or test solution (containing the target N-oleoyl amino acid) into an EP tube and dry it under nitrogen. Add 5 μL of 20 mM HATU, 5 μL of 20 mM HOBt, and 5 μL of 20 mM TEA (all dissolved in acetonitrile) sequentially to the residue, vortex to mix, and react at room temperature for 10 minutes. This step aims to "activate" any possible free carboxyl groups (for the standard, it is already an amide; this step mainly targets the sample matrix) and ensure the efficiency of subsequent derivatization.

[0161] (ii) Second stage (DIAAA derivatization): To the reaction system from the previous step, add 5 μL of 20 mM HATU and 5 μL of 100 mM DIAAA (dissolved in acetonitrile solution containing 200 mM TEA), vortex to mix, and react at room temperature for 20 minutes. This step covalently links DIAAA to the carboxyl group of the target molecule, forming a derivative that is easily ionized and produces characteristic fragments.

[0162] (iii) Volume adjustment: After the reaction is complete, add an appropriate amount of acetonitrile to make the final volume 50 μL, vortex to mix, and take 1 μL for LC-MS analysis. All standard curve points and samples should be added with an equal volume (e.g., 5 μL) of internal standard working solution (20 μg / mL 4-Cl-Phe) before analysis, and dried under nitrogen before derivatization to correct for deviations in pretreatment and instrument response.

[0163] 3.3 Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis Conditions

[0164] The chromatographic and mass spectrometric conditions were exactly the same as in Example 1. Chromatographic conditions: Waters ACQUITY UPLC® HSS T3 column; mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid; gradient elution program was the same as in Example 1. Mass spectrometric conditions: ESI positive ion mode, full scan range m / z 30–1700. To improve quantitative reliability, extractable ion chromatogram (EIC) or selected reaction monitoring (SRM) mode can be used to monitor specific molecular ions and characteristic fragment ions (e.g., m / z 86.10) of each target N-oleoyl amino acid-DIAAA derivative.

[0165] 3.4 Standard Curve Plotting and Analysis Methodology Validation

[0166] a) Standard Curve: The mixed standard intermediate solution was serially diluted with 80% acetonitrile to prepare a series of standard working solutions with at least 6 different concentration points (the concentration range covering the expected content in the analyte sample, e.g., 0.1 ng / mL to 1000 ng / mL). For each concentration point, the internal standard was added and derivatized according to the procedure in “3.2 Chemical Derivatization”, followed by LC-MS analysis. The standard curve was plotted using a weighted least squares linear regression with the peak area ratio of the analyte N-oleoyl amino acid derivative to the internal standard derivative as the ordinate (y) and the analyte concentration as the abscissa (x).

[0167] b) Linear range, limit of detection, and limit of quantitation: Record the linear range, regression equation, and correlation coefficient (R²) of the standard curve. Estimate the limit of detection (LOD) and limit of quantitation (LOQ) of the method using concentrations corresponding to signal-to-noise ratios (S / N) of approximately 3 and 10, respectively.

[0168] c) Precision and Accuracy: Prepare quality control (QC) samples at three concentration levels: low, medium, and high, with at least five samples per concentration level. Perform derivatization and analysis on the same day, and calculate intra-day precision (expressed as relative standard deviation (RSD%)) and accuracy (expressed as the ratio of measured concentration to theoretical concentration %). Repeat this process on different days (at least three days) and calculate inter-day precision and accuracy.

[0169] 4. Quantitative analysis of N-oleoyl amino acids in BS and FS samples

[0170] a) Preparation of test solution: The collection, freeze-drying, grinding and stepwise extraction of BS and FS samples were carried out in strict accordance with the method described in "2. Preparation of test solution" in Example 2 to obtain total extract.

[0171] b) Sample pretreatment and derivatization: Accurately measure 50 μL of total extract into an EP tube, add 5 μL of internal standard working solution (20 μg / mL 4-Cl-Phe), mix well, and dry under nitrogen. Subsequently, strictly follow the steps in “3.2 Chemical Derivatization Treatment” to perform a two-stage derivatization reaction on the dried residue, and bring the volume to 50 μL.

[0172] c) LC-MS Analysis and Content Calculation: 1 μL of the derivatized sample solution was analyzed by LC-MS. By comparing the peak area ratio of the target N-oleoyl amino acid-DIAAA derivative and the internal standard derivative in the sample, and substituting this ratio into the standard curve equation established in step “3.4”, the concentration of the N-oleoyl amino acid in the original extract was calculated. Combining the sample amount, extraction volume, and sample weight, the content (e.g., ng / g or μg / g) in the original BS or FS sample was finally calculated.

[0173] d) Data analysis: Statistical description (mean, standard deviation) of the N-oleoyl amino acid content of each quantitatively determined group in the BS group and FS group, and appropriate statistical tests (such as t test) can be used to compare the significance of the difference in content between the two groups.

[0174] This embodiment establishes an accurate quantitative analytical method for multiple N-oleoyl amino acids based on in-situ chemical derivatization combined with LC-MS. The method ensures the accuracy of the analytical target through standard synthesis and characterization, and ensures the reliability of the quantitative results through systematic methodological validation (linearity, sensitivity, precision, and accuracy). It has been successfully applied to the determination of target analytes in real complex biological samples (BS and FS). This method exhibits high specificity and sensitivity, providing a powerful analytical tool for in-depth research on the distribution, changes, and correlations of N-oleoyl amino acids in biological samples with physiological and pathological processes.

[0175] Example 4: Evaluation of the inhibitory activity of N-oleoyl amino acids identified in FS against the proliferation of hepatocellular carcinoma cells.

[0176] 1. Experimental Materials

[0177] Cell line: Human hepatoblastoma cell line (HepG2), purchased from the American Type Culture Collection (ATCC).

[0178] Test compounds (N-oleoyl amino acids): Four N-oleoyl amino acid standards identified in post-fermentation samples (FS) by the methods described in Examples 1 and 2, and synthesized and purified by the method in Example 3, were selected for activity testing. These are:

[0179] N-Oleoyl-L-Tyrosine

[0180] N-Oleoyl-L-Proline

[0181] N-Oleoyl-L-Leucine

[0182] N-Oleoyl-L-Tryptophan

[0183] Main reagents:

[0184] Dimethyl sulfoxide (DMSO, cell culture grade)

[0185] High-glucose DMEM medium

[0186] Fetal bovine serum (FBS)

[0187] Penicillin-streptomycin dual antibiotic solution (100X)

[0188] 0.25% trypsin-EDTA digestion solution

[0189] Cell Counting Kit-8 (CCK-8) Cell Proliferation and Toxicity Assay Kit

[0190] Main instruments and equipment: Cell culture incubator, biosafety cabinet, inverted microscope, low-speed centrifuge, ELISA reader, 96-well cell culture plate, pipette, etc.

[0191] 2. Preparation of the test solution

[0192] a) Preparation of stock solutions: Accurately weigh approximately 2.0 mg of each of the four N-oleoyl amino acid standards and place them in a 1.5 mL sterile EP tube. Add an appropriate amount of DMSO to dissolve and dilute to volume to prepare a 100 mM single-compound stock solution. After filtering the stock solution through a 0.22 μm sterile filter membrane, dispense it into individual containers and store at -20°C protected from light.

[0193] b) Preparation of working solutions: On the day of the experiment, the stock solutions of each compound were serially diluted in DMEM complete medium (containing 10% FBS) containing 1% penicillin-streptomycin (v / v). First, the 100 mM stock solution was diluted to an initial concentration of 100 μM, and then serially diluted in two-fold increments to obtain a series of working solutions of various concentrations (e.g., 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM). Throughout the dilution process, the final volumetric concentration of DMSO in all working solutions was ensured to be below 0.1% (v / v) to rule out any potential toxic effects of DMSO on cells.

[0194] 3. Drug activity assay (CCK-8 assay)

[0195] a) Cell seeding: HepG2 cells in logarithmic growth phase were digested with 0.25% trypsin and centrifuged (300 × g, 5 min) to collect the cell pellet. The cells were resuspended in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin, thoroughly mixed, and then counted. The cell suspension concentration was adjusted to... Cells / mL were seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well (i.e., per well). (cells). The culture plate was placed at 37°C, 5%... The cells were cultured in a saturated humidity incubator for 24 hours to allow them to adhere fully to the incubator wall.

[0196] b) Experimental grouping and drug administration: 24 hours later, carefully discard the old culture medium in each well and proceed with the following grouping treatment:

[0197] Blank group: only 100 μL of cell-free complete culture medium was added (for background subtraction).

[0198] Control group: 100 μL of complete culture medium containing the same amount of DMSO (<0.1%) as the highest concentration drug group was added.

[0199] Treatment Groups: 100 μL of working solution containing different concentrations (e.g., 100 μM, 50 μM, ..., 3.125 μM) of N-oleoyl amino acids was added.

[0200] Each concentration was tested in triplicate, and the entire experiment was repeated three times independently.

[0201] c) Cell incubation and detection: After drug treatment, the 96-well plate was returned to the incubator and incubated at 37°C and 5%... Incubate for 24 hours under the specified conditions. After incubation, carefully add 10 μL of CCK-8 solution to each well (except for the blank wells). Gently shake the culture plate to mix, then return it to the incubator and continue incubation for 1 hour.

[0202] d) Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0203] 4. Data Processing and Statistical Analysis

[0204] a) Cell viability calculation: Cell viability is expressed as a percentage, with the cell viability of the control group (untreated) set at 100%. The relative cell viability of each drug-treated group was calculated using the following formula: Cell viability (%) = [(OD_drug-treated group - OD_blank group) / (OD_control group - OD_blank group)] × 100%

[0205] OD_drug group, OD_control group, and OD_blank group represent the average OD values ​​of the drug group, control group, and blank group, respectively.

[0206] b) Value Calculation and Statistical Analysis: Data processing and analysis were performed using GraphPad Prism software (version 8.0 or later). The drug concentration (logarithm) was used as the x-axis (X), and the corresponding cell viability percentage as the y-axis (Y). A nonlinear regression model was used to fit the data, calculating the concentration required for each N-oleoyl amino acid to inhibit HepG2 cell proliferation by 50%, i.e., the half-maximal inhibitory concentration (MCC). Experimental results are expressed as mean ± standard error (Mean ± SEM) of three independent experiments.

[0207] 5. Experimental Results

[0208] CCK-8 assay and data analysis showed that all four tested N-oleoyl amino acids dose-dependently inhibited the proliferative activity of HepG2 liver cancer cells. Compared with the solvent control group, the viability of HepG2 cells decreased significantly with increasing drug concentration.

[0209] Nonlinear fitting calculations were used to determine the effects of four N-oleoyl amino acids on HepG2 cells. The values ​​are as follows:

[0210] N-Oleoyl-L-Tyrosine: = 18.76 μM

[0211] N-Oleoyl-L-Proline: = 15.39 μM

[0212] N-Oleoyl-L-Leucine: = 16.42 μM

[0213] N-Oleoyl-L-Tryptophan: = 16.18 μM

[0214] This embodiment confirms through in vitro cell experiments that the four specific N-oleoyl amino acids identified from the fermentation sample—N-oleoyltyrosine, N-oleoylproline, N-oleoylleucine, and N-oleoyltryptophan—are present at micromolar concentrations (μmol level). Values ​​ranging from 15.39 μM to 18.76 μM effectively inhibited the proliferation of human hepatocellular carcinoma HepG2 cells, and the inhibitory effect showed a significant dose-dependent relationship. Results are as follows... Figure 5 The study showed that N-oleoyl amino acids reduced HepG2 cell viability in a dose-dependent manner, revealing potential anti-hepatocellular carcinoma activity.

[0215] Comparative example: Explanation of the effects of cysteine

[0216] To more clearly illustrate the scope of application and technical boundaries of the "one-pot micro-synthesis and in-situ chemical derivatization identification method" described in this invention, the following comparative cases of poor experimental results are added to demonstrate that the selection of this method scheme cannot be obtained by those skilled in the art through conventional reasoning, and that there are specific synergistic effects between each step and reagent.

[0217] The same reagents, instruments, and procedures as in Example 1 were used. In step S1, an amino acid mixture containing cysteine ​​(the other amino acids were the same) was reacted with oleic acid, followed by the same in-situ DIAAA derivatization and LC-MS analysis.

[0218] In the final LC-MS analysis, the characteristic chromatographic peak and mass spectrometric signal corresponding to the expected N-oleoylcysteine ​​DIAAA derivative were not detected. The inventors speculate that this may be because the thiol group (-SH) of the cysteine ​​side chain has high reactivity in the reaction system, readily undergoing oxidation, disulfide bond formation, or side reactions with other reagents, thereby competitively inhibiting or interfering with the acylation reaction of the target amino group, or leading to instability of the generated N-acylcysteine. This underscores the high efficiency and specificity of the method described in this invention for the vast majority of other amino acids, and is not a simple reaction stacking process.

[0219] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for identifying N-acyl amino acids, characterized in that, Includes the following steps: S1. The test sample containing amino acids is placed in the same reaction vessel with fatty acids, coupling reagents and a first base to synthesize N-acyl amino acids; S2. Without separating the N-acyl amino acid, add the derivatizing reagent N,N-diisopropylethylenediamine (DIAAA) and a second base to the same reaction vessel to perform in-situ chemical derivatization of the N-acyl amino acid synthesized in step S1 to obtain the derivatized product; S3. Perform liquid chromatography-mass spectrometry (LC-MS) analysis on the derivatized product to obtain mass spectrometry data; S4. The structure of the N-acyl amino acid is identified based on at least two of the following: characteristic fragment ions derived from DIAAA, characteristic fragment ions generated from the amino acid residues of the N-acyl amino acid, and characteristic fragment ions generated from the acyl group of the N-acyl amino acid.

2. The identification method according to claim 1, characterized in that, In step S1, the coupling reagent includes hexafluorophosphate O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU) and 1-hydroxybenzotriazole (HOBt); and / or, the first base and the second base are both triethylamine (TEA).

3. The identification method according to claim 1, characterized in that, In step S4, the characteristic fragment ions derived from DIAAA include ions with m / z of 86.09 and 128.14, and / or include fragment ions formed by the loss of neutral fragments of the molecular ion of the derivatized product by 42.05 Da or 84.09 Da.

4. The identification method according to claim 1, characterized in that, In step S4, the characteristic fragment ions generated from the amino acid residues of the N-acyl amino acid are produced by breaking the bond between the carboxyl carbon and the α carbon of the amino acid moiety in the N-acyl amino acid molecule and / or breaking the amide bond between the amino acid moiety and the acyl moiety.

5. The identification method according to claim 1, characterized in that, In step S3, liquid chromatography separation is performed using a reversed-phase column. The mobile phase includes an aqueous solution containing formic acid and an acetonitrile solution containing formic acid, and a gradient elution program is used. The mass spectrometry analysis was performed in electrospray ionization positive ion mode, and the mass number acquired by the mass spectrometer ranged from m / z 30 to 1700.

6. A method for systematically identifying N-acyl amino acids from complex biological samples, characterized in that, Includes the following steps: P1. After lyophilizing and grinding the biological sample, extract it stepwise using at least two solvents of different polarities, combine the extracts and centrifuge to obtain the test solution; P2. Take a portion of the test solution and, using the method described in any one of claims 1 to 5, perform in-situ derivatization of the N-acyl amino acid contained therein to obtain the derivatized product; P3. The derivatized product was analyzed by liquid chromatography-mass spectrometry, and the structure of the N-acyl amino acid was identified and confirmed based on the obtained mass spectrometry data and the standard with a mass error of less than 5 ppm.

7. The method according to claim 6, characterized in that, In step P1, the at least two solvents of different polarities are selected from water, methanol, and at least two of an aqueous methanol solution with a concentration of 10%-90% (v / v).

8. A method for the quantitative analysis of N-oleoyl amino acids, characterized in that, Includes the following steps: Q1. Prepare a series of standard solutions of at least one N-oleoyl amino acid standard. Add an internal standard to each standard solution and perform chemical derivatization. Then perform liquid chromatography-mass spectrometry analysis. Plot a standard curve with the ratio of analyte to internal standard peak area as the ordinate and the standard concentration as the abscissa. Q2. Add the internal standard to the test sample solution containing N-oleoyl amino acids and perform the same chemical derivatization treatment as in step Q1; Q3. Perform liquid chromatography-mass spectrometry analysis on the derivatized product obtained in step Q2 to obtain the ratio of the chromatographic peak area of ​​the analyte to the internal standard in the sample to be tested. Q4. Based on the peak area ratio obtained in step Q3, use the standard curve established in step Q1 to calculate the content of N-oleoyl amino acids in the sample to be tested; The chemical derivatization process involves using a derivatization reagent system containing HATU, HOBt, and DIAAA to derivatize the carboxyl group of N-oleoyl amino acids.

9. The quantitative analysis method according to claim 8, characterized in that, In steps Q1 and Q2, the chemical derivatization process includes: (i) In the first stage, HATU and HOBt are used as coupling agents and the reaction is carried out under alkaline conditions; (ii) In the second stage, DIAAA is added to carry out a derivatization reaction.

10. The use of an N-oleoyl amino acid in the preparation of a medicament for the prevention and / or treatment of liver cancer, characterized in that, The N-oleoyl amino acid is selected from at least one of N-oleoyltyrosine, N-oleoylproline, N-oleoylleucine, and N-oleoyltryptophan.

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