Sample derivatization reagents and their application in detecting optical isomers

Abstract

The present invention provides a sample derivatization reagent and its application in detecting optical isomers. Specifically, the present invention provides a compound having a structure of formula I, and the compound of the present invention can enable the optical isomers of small molecule compounds such as amino acids to be separated and detected quickly, efficiently and accurately by ion mobility mass spectrometry or liquid chromatography mass spectrometry.

Description

Technical Field

[0001] The present invention relates to the field of instrumental analysis, and in particular to a sample derivatization reagent and its application in detecting optical isomers, and in particular to the application of a steroidal acid chloride sample derivatization reagent in separating and detecting optical isomers of compounds containing amino or hydroxyl groups. Background Art

[0002] α-Amino acid (α-AA) is an important small molecule chiral metabolite, and its content changes are closely related to the physiological activities and diseases of living organisms. Among α-AA, the physiological effects of D-AA and L-AA are completely different. Most D-AA must be converted into L-AA before it can be absorbed and utilized by the body. Excessive D-AA can cause adverse reactions such as diseases. Some studies in recent years have shown that the content of certain D-AA shows significant changes when living organisms are in a diseased state. Since the content of D-AA in the body is at trace levels and D-AA and L-AA have the same molecular weight, it is quite challenging to accurately qualitatively and quantitatively analyze D-AA in complex biological samples.

[0003] In addition, when preparing peptide drugs by solid phase synthesis, the racemization and optical isomerism of amino acids are important sources of impurities. At present, the control of racemic peptide impurities is usually to completely hydrolyze the synthetic peptide into free amino acids, and then indirectly determine them by performing enantiomeric separation on the mixture of chiral amino acids with different configurations and calculating the relative proportion of racemization. Therefore, the separation of chiral amino acids becomes a key step in the control of racemic peptide impurities.

[0004] The study of chiral amino acids is of great significance in the fields of life sciences, medicinal chemistry, and material chemistry, all of which involve the separation of chiral amino acids. The chiral amino acid analysis method based on liquid chromatography-mass spectrometry (LC-MS) technology has developed rapidly in recent years. It has not only become a basic analysis method for synthetic and natural peptides and proteins, but also suitable for disease diagnosis in clinical chemistry. At present, the pharmacopoeia has applied mass spectrometry technology to the analysis of allosteric chiral amino acids. This method is fast, accurate, and sensitive, and does not require racemic peptide reference substances. It can also monitor the possibility of the presence of other allosteric amino acids at the same time. However, it usually takes 5-100 minutes to complete the separation and analysis of a mixed amino acid sample using LC-MS technology, and there are still difficulties in achieving high-throughput analysis.

[0005] The emergence of ion mobility mass spectrometry (IM-MS) technology has made it possible to achieve high-throughput analysis of chiral amino acids. As a powerful separation and analysis tool, IM-MS can achieve separation and analysis of isomers at the millisecond level, and has shown unique advantages in the analysis of isomers of chiral drugs, peptides and complex organic compounds. However, most of the derivatization reagents reported for IM-MS have poor universality, and it is difficult to achieve chiral recognition of all 19 natural amino acids (including acidic, basic and neutral amino acids) by combining a derivatization reagent with IM-MS. In addition, some derivatization reagents have problems such as difficult preparation, severe reaction conditions, low reaction efficiency, many by-products, and high cost, which reveal the limitations of chiral derivatization combined with IM-MS in the analysis of biological samples.

[0006] Therefore, there is an urgent need in the art to provide a method for rapidly, efficiently and accurately separating and detecting optical isomers of compounds containing amino groups (such as amino acids) or hydroxy groups. Summary of the invention

[0007] The object of the present invention is to provide a sample derivatization compound for detecting optical isomers.

[0008] The object of the present invention is to provide a method for rapidly, efficiently and accurately separating and detecting optical isomers of compounds containing amino groups (such as amino acids) or hydroxy groups.

[0009] In a first aspect of the present invention, a compound for sample derivatization is provided, wherein the compound has a structure of Formula I:

[0010] AL 1 -BL 2 -M

[0011] I

[0012] Wherein, A is a reactive group, and A is selected from the following group: formyl chloride or sulfonyl chloride;

[0013] B is a steroid skeleton portion, and B is a divalent group including a substituted or unsubstituted structure:

[0014]

[0015] In B, ring W 1 having 0, 1, 2 or 3 double bonds;

[0016] Ring W 2 having 0, 1 or 2 double bonds;

[0017] Ring W 4 Has 0 or 1 double bond;

[0018] L 1 and L 2 is a linking group, and L 1 and L 2 Each is independently selected from the group consisting of a bond, -O-, -NH-, -NHCO-, -COO-, -C1-C4 alkylene-, -C1-C4 alkyleneoxy-;

[0019] In B, the term "substituted" refers to one or more hydrogen atoms (e.g., 2, 3, 4, etc.) on a group being independently replaced by a substituent selected from the group consisting of: -OH, oxo (=O), halogen, -CN, -NH 2 , di(C1-C6 alkyl)amino-, -COOH, -CONH 2 、-SO 2 NH 2 、-SO 2 C1-C6 alkyl, unsubstituted or halogenated C1-C12 alkyl, unsubstituted or halogenated C2-C12 alkenyl, unsubstituted or halogenated C2-C12 alkynyl, unsubstituted or halogenated C1-C12 alkoxy, unsubstituted or halogenated -COC1-C12 alkyl, unsubstituted or halogenated -OCOC1-C12 alkyl, unsubstituted or halogenated -NH-CO-C1-C8 alkyl, unsubstituted or substituted with one or more R a The substituent is selected from the group consisting of phenyl-C1-C6 alkyl-COO-, benzyl, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl having 1-3 heteroatoms selected from N, S and O, C6-C12 aryl, and 5-12 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, and the R a The substituents are independently selected from the group consisting of -OH, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy;

[0020] An unisotopically labeled or isotopically labeled tag portion M, wherein M is selected from the following group: H, unsubstituted or substituted C1-C12 alkyl, unsubstituted or substituted C2-C12 alkenyl, unsubstituted or substituted C2-C12 alkynyl, unsubstituted or substituted C1-C12 alkoxy, unsubstituted or substituted phenyl-C1-C6 alkyl-, unsubstituted or substituted C3-C8 cycloalkyl, unsubstituted or substituted 3-8 membered heterocycloalkyl having 1-3 heteroatoms selected from O, N, S, unsubstituted or substituted C6-C12 aryl, unsubstituted or substituted 5-12 membered heteroaryl having 1-3 heteroatoms selected from O, N, S;

[0021] In M, the term "substituted" means that one or more hydrogen atoms (e.g., 2, 3, 4, etc.) on the group are independently selected from the group R bSubstituents are substituted with: (deuterium) D, oxo (=O), halogen, unsubstituted or halogenated C1-C8 alkyl, unsubstituted or halogenated C2-C8 alkenyl, unsubstituted or halogenated C2-C8 alkynyl, unsubstituted or halogenated C1-C8 alkoxy, unsubstituted or halogenated -COC1-C8 alkyl, unsubstituted or halogenated -NH-C(O)-C1-C8 alkyl, unsubstituted or halogenated C3-C6 cycloalkyl, unsubstituted or halogenated 3-6 membered heterocycloalkyl having 1-3 heteroatoms selected from N, S and O, unsubstituted or halogenated C6-C12 aryl, unsubstituted or halogenated 5-6 membered heteroaryl having 1-3 heteroatoms selected from N, S and O; and

[0022] When M is isotopically labeled, C contains at least 2 (such as 3-12, preferably 4-8) atoms selected from the following group: 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl.

[0023] In another preferred embodiment, when M is isotopically labeled, C contains at least 2 (such as 3-12, preferably 4-8) atoms selected from the following group: 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl.

[0024] In another preferred embodiment, when M is isotopically labeled, C comprises at least 2 (such as 3-12, preferably 4-8) atoms selected from the following group: 2 H. 13 C and 15 N, preferably, 2 H.

[0025] In another preferred embodiment, when M is isotopically labeled, C is a group selected from the group consisting of: -CD 3 , CD 2 CD 3 、-C(CD 3 ) 3 , -d 5 -phenyl.

[0026] In another preferred embodiment, B is a substituted or unsubstituted divalent group selected from the structures B1-B9:

[0027] In each formula, the substitution is as defined above.

[0028] In another preferred embodiment, in any structure of Formula B or Formulas B1-B9,

[0029]

[0030] B and L 1 and L 2 The attachment sites of are independently selected from the group consisting of positions 1, 2, 3, 4, 6, 7, 11, 12, 15, 16 and 17.

[0031] In another preferred embodiment, any structure of Formula B or Formula B1-B9 and L 1 and L 2 The attachment sites of are independently selected from the group consisting of positions 2, 3, 4, 6, 11, and 17, preferably, positions 3 and 17.

[0032] In another preferred embodiment, in any of the structures of formula B or formula B1-B9, the H at position 17 is replaced by one or two R 3 Replace, and each R 3 independently selected from the group consisting of a bond, -OH, -C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 alkoxy, -COC1-C12 alkyl, OCOC1-C12 alkyl, -CN, -NH 2 , di(C1-C6 alkyl)amino-, -COOH, -CONH 2 、-SO 2 NH 2 、SO 2 C1-C6 alkyl, unsubstituted or substituted with one or more R a The substituent is selected from the group consisting of phenyl-C1-C6 alkyl-CO-, benzyl, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl having 1-3 heteroatoms selected from N, S and O, C6-C12 aryl, 5-12 membered heteroaryl having 1-3 heteroatoms selected from N, S and O; and said R a The substituents are independently selected from the group consisting of -OH, halogen, C1-C6 alkyl, C1-C6 alkoxy; or two R 3 Formation =O.

[0033] In another preferred embodiment, each R 3independently selected from the group consisting of H, -OH, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl; or one R 3 For the key, another R 3 Selected from the following group: H, -OH, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl.

[0034] In another preferred embodiment, the compound is selected from the following group:

[0035]

[0036] The compound is optionally non-isotopically labeled or isotopically labeled.

[0037] In a second aspect, the present invention provides a combination of mass spectrometry derivatization reagents, the combination comprising:

[0038] (a) a compound according to the first aspect of the present invention which is not isotopically labeled; and

[0039] (b) said compound which is isotopically labeled.

[0040] In a third aspect, the present invention provides a mass spectrometry derivatization kit, the kit comprising:

[0041] (a) a first container comprising the compound according to the first aspect of the present invention which is not isotopically labeled; and / or

[0042] (b) A second container comprising the isotope-labeled compound.

[0043] In another preferred embodiment, the kit further comprises instructions.

[0044] The fourth aspect of the present invention is the use of the compound as described in the first aspect of the present invention in the preparation of a sample derivatization reagent for qualitative and / or quantitative detection of optical isomers of a substance to be tested, wherein the substance to be tested is selected from the following group: amino acids, hydroxy acids, or a combination thereof.

[0045] In another preferred embodiment, in the test substance, the amino acid is an α-amino acid, such as a natural amino acid or a non-natural amino acid.

[0046] In another preferred embodiment, in the substance to be tested, the hydroxy acid is an α-hydroxy acid.

[0047] In another preferred embodiment, the sample is a biological sample, an environmental sample, a reaction solution, etc., typically, such as animal body fluids, secretions, tissues; plant tissues, plant extracts, etc.; polypeptide solid phase synthesis reaction solution, reaction products, protein hydrolysis samples, etc.

[0048] In another preferred embodiment, the sample is used for ion mobility mass spectrometry (such as electrospray ionization-U-type ion mobility analyzer-triple quadrupole mass spectrometry (ESI-UMA-QQQ MS)) detection and / or liquid chromatography mass spectrometry detection (such as high performance liquid chromatography-electrospray ionization-time of flight mass spectrometry (HPLC-ESI-Q-TOF MS), high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-QQQ), high performance liquid chromatography-ion hydrazine mass spectrometry, etc.).

[0049] In a fifth aspect, the present invention provides a method for detecting optical isomers of a substance to be tested in a sample, comprising the steps of:

[0050] (a) reacting a sample with the compound described in the first aspect of the present invention to obtain a derivatized sample; and

[0051] (b) separating and detecting the sample obtained in step (a) using ion mobility mass spectrometry or LC-MS; and

[0052] Wherein, in the sample, the substance to be tested is selected from the following group: amino acids, hydroxy acids, or a combination thereof.

[0053] In another preferred embodiment, the method is used for qualitative and / or quantitative detection of optical isomers of the substance to be tested.

[0054] In another preferred embodiment, in step (a), the reaction has one or more technical features selected from the following group:

[0055] (a) the reaction temperature is 25±15°C, preferably 25±10°C, more preferably 25±5°C;

[0056] (b) the reaction time is 1-30 min, preferably 3-15 min, more preferably 5-10 min;

[0057] (c) the pH of the reaction is 8-11, preferably, 9-10; and / or

[0058] (c) There is Na in the reaction system + , such as sodium carbonate, sodium bicarbonate, or a combination thereof.

[0059] In another preferred embodiment, the step (a) further comprises the step of: (aI) further pre-treating the derivatized sample to obtain a sample for detection.

[0060] In another preferred embodiment, the step (a) further includes the steps of: (a1) providing an isotope internal standard obtained by reacting a standard sample of the analyte with an isotope-labeled compound as described in the first aspect of the present invention (with the same structure as the compound in (a)); (a2) mixing the isotope internal standard at a certain concentration with the derivatized sample to obtain an isotope internal standard sample; (a3) ​​further pre-treating the isotope internal standard sample to obtain a sample for detection.

[0061] In another preferred embodiment, in the test substance, the amino acid is an α-amino acid, such as a natural amino acid or a non-natural amino acid.

[0062] In another preferred embodiment, in the substance to be tested, the hydroxy acid is an α-hydroxy acid.

[0063] In another preferred embodiment, the ion mobility mass spectrometer is electrospray ionization-U-type ion mobility analyzer-triple quadrupole mass spectrometer (ESI-UMA-QQQ MS).

[0064] In another preferred embodiment, the LC-MS is high performance liquid chromatography-electrospray ionization-time of flight mass spectrometry (HPLC-ESI-Q-TOF MS), high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-QQQ) or high performance liquid chromatography-ion hydrazine mass spectrometry.

[0065] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the ball-and-stick model diagram of the density functional theory calculation optimization of the labeling products of D-type and L-type serine with 17β-EBC.

[0067] Figure 2 It is the ion mobility spectra of the labeled products of each enantiomer of 19 natural amino acids and 4 unnatural amino acids with 17β-EBC under standard conditions (E, V / mm, represents the electric field).

[0068] Figure 3 The graphs are comparative ion mobility spectra of the labeled products of standard serine enantiomers with S-NAP and 17β-EBC under standard conditions.

[0069] Figure 4 The figure shows the extracted ion chromatograms of the labeled products of 18 natural amino acids enantiomers with 17β-EBC under standard conditions.

[0070] Figure 5It is the ion migration spectrum of the labeled products of 7 kinds of α-HAs enantiomers with 17β-EBC under standard conditions.

[0071] Figure 6 The graphs are comparative ion mobility spectra of the labeled products of standard lactic acid enantiomers with S-NAP and 17β-EBC under standard conditions.

[0072] Figure 7 The figure shows the extracted ion chromatograms of the labeled products of 7 enantiomers of α-HAs with 17β-EBC under standard conditions.

[0073] Figure 8 This is an analysis of the ion migration spectra and content differences of D-type and L-type serine in the brain of C57 mice of different ages.

[0074] Fig. 9 This is the growth curve and -galactosidase staining diagram of the oxidative stress-induced cell senescence model after D-serine treatment.

[0075] Fig.10 This is the ion mobility spectrum of amino acid component identification after the derivatization reaction of Deltorphin II acid hydrolysis solution with 17β-EBC under standard conditions.

[0076] Fig.11 It is the UMA ion migration spectrum of 17β-EBC-D-Ala detection limit under the condition of excess 17β-EBC-L-Ala.

[0077] Fig.12 It is the ion mobility spectra of the standard alanine enantiomers and the labeled products of other steroidal chiral selectors under standard conditions. DETAILED DESCRIPTION

[0078] After extensive and in-depth research, the inventors have provided a mass spectrometry derivatization reagent and its application in detecting optical isomers through a large number of screenings and tests. The present invention provides a sample derivatization reagent with a novel structure. Due to the steroid ring in the labeling reagent and the specific stereostructure formed together with the analytes of different configurations, the derivative products containing the analytes of different configurations can be separated by an ion mobility tube or a chromatographic column. Furthermore, when an isotope label is present, the substances passing through the ion mobility tube or the chromatographic column can be quantified using a mass spectrometry isotope internal standard method, thereby achieving separation and quantitative and qualitative detection of optical isomers at the same time. The present invention was completed on this basis.

[0079] the term

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0081] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0082] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0083] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0084] Where substituents are specified by their conventional chemical formula (written from left to right), they equally include chemically identical substituents resulting from the structure written from right to left, e.g., -CH 2 O-intended to include-OCH 2 -, -COO- are intended to include -OCO-.

[0085] As used herein, the term "alkyl" as a whole or as part of another group refers to a straight or branched chain alkyl group containing the specified number of carbon atoms, such as "C 1 -C 12 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 12 carbon atoms, including alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, preferably alkyl groups such as C 1 -C 2 , C 1 -C 3 , C 1 -C 4 , C 1 -C 5 , C 1 -C 6 , C 1 -C 7 , C 1 -C 8 , C 1 -C 9 , C 1 -C 10 , C 2 -C 3 , C 2 -C 4 , C 2 -C 5 , C 2 -C 6 , C 3 -C4 , C 3 -C 5 , C 3 -C 6 , C 3 -C 7 , C 3 -C 8 , C 4 -C 5 , C 4 -C 6 or C 5-6 Typical "alkyl" includes, but is not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, isopentyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, etc. In the present invention, alkyl also includes substituted alkyl. "Substituted alkyl" means that one or more positions in the alkyl are substituted, especially 1-4 substituents, which can be substituted at any position.

[0086] As used herein, the term “C 1 -C 12 "Alkoxy" refers to a straight or branched chain alkoxy group having 1 to 12 carbon atoms, having a C 1 -C 12 Alkyl-O-structure, C 1 -C 15 Alkoxy includes, but is not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, etc., preferably ethoxy. 1 -C 12 Alkoxy also includes substituted C 1 -C 15 Alkoxy.

[0087] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl may include any number of carbon atoms, wherein "C2-C12 alkenyl" refers to a straight or branched hydrocarbon having 2-12 carbon atoms and at least one double bond, such as C 2 , C 2- C 3 , C 2- C 4 , C 2- C 5 , C 2- C 6 , C 2- C 7 , C 2- C 8 , C 2- C 9 , C 2- C10 , C 3 , C 3- C 4 , C 3- C 5 , C 3- C 6 , C 4 , C 4- C 5 , C 4- C 6 , C 5 , C 5- C 6 and C 6 . The alkenyl group may have any suitable number of double bonds, including but not limited to 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include but are not limited to vinyl (vinyl group), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Like the above-mentioned alkyl groups, alkenyl groups may be substituted or unsubstituted.

[0088] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl groups may include any number of carbon atoms, and "C2-C12 alkynyl" refers to a straight or branched chain hydrocarbon having 2-12 carbon atoms and at least one triple bond, such as C 2 , C 2- C 3 , C 2- C 4 , C 2- C 5 , C 2- C 6 , C 2- C 7 , C 2- C 8 , C 2- C 9 , C 2- C 10 , C 3 , C 3- C 4 , C 3- C 5 , C 3- C 6 , C 4 , C 4- C 5 , C 4- C 6 , C5 , C 5- C 6 and C 6 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentenyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Like the above-mentioned alkyl groups, alkynyl groups may be substituted or unsubstituted.

[0089] The term "aryl" refers to an aromatic cyclic hydrocarbon compound group, wherein "C6-C12 aryl" refers to an aromatic cyclic hydrocarbon compound group containing 6, 7, 8, 9, 10, 11 or 12 ring carbon atoms, with 1-2 rings, especially monocyclic and bicyclic groups, such as phenyl, biphenyl or naphthyl. Where there are two or more aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group can be connected by a single bond (such as biphenyl) or fused (such as naphthalene, anthracene, etc.). "Substituted aryl" means that one or more positions in the aryl group are substituted, especially 1-3 substituents, which can be substituted at any position.

[0090] As used herein, the term "heteroaryl" refers to a heteroaromatic system containing 1-3 atoms selected from N, O, and S, wherein "5-12-membered heteroaromatic" refers to a 5-12-membered heteroaromatic system containing 1-3 atoms selected from N, O, and S. The heteroaryl is preferably a 5- to 10-membered ring, more preferably a 5- or 6-membered ring, and includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, and the like. "Heteroaryl" may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, deuterated alkyl, haloalkyl, alkoxy, haloalkoxy, alkenyl, alkynyl, alkylthio, alkylamino, halogen, amino, nitro, hydroxy, mercapto, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylthio, oxo, carboxyl and carboxylate.

[0091] As used herein, the term "cycloalkyl" refers to a fully saturated cyclic hydrocarbon group having a number of carbon atoms, wherein "C 3 -C 12 "Cycloalkyl" refers to a fully saturated or partially unsaturated cyclic hydrocarbon group having 3 to 8 carbon atoms, preferably C 3 -C 4 , C 3 -C 5 , C 3 -C6 , C 3 -C 7 , C 3 -C 8 , C 4 -C 6 , C 5 -C 6 . "Replace C 3 -C 8 "Cycloalkyl" means that one or more positions in the cycloalkyl are substituted, especially 1-4 substituents, which can be substituted at any position, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, etc. In the present invention, "cycloalkyl" is intended to include "substituted cycloalkyl".

[0092] As used herein, the term "heterocycloalkyl" refers to a fully saturated or partially unsaturated cyclic group having a number (greater than or equal to 3) of ring atoms and 1-3 heteroatoms, wherein "3-8 membered heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having 3-8 ring atoms and 1-3 heteroatoms (including but not limited to 3-7 membered monocyclic, 7-8 membered bicyclic systems). Among them, the nitrogen atom or sulfur atom can be oxidized, and the nitrogen atom can also be quaternized. The heterocyclic group can be connected to the residue of any heteroatom or carbon atom of the ring or ring system molecule. Typical monocyclic heterocycles include, but are not limited to, azetidinyl, pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, hexahydroazepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxanyl and tetrahydro-1,1-dioxythiophene, and the like.

[0093] When the substituent is a non-terminal substituent or a related group removes a H atom, it is a subunit of the corresponding group, usually a divalent group, for example, an alkyl group removes a H atom to become an alkylene group (for example: methylene, ethylene, propylene, isopropylene (such as ), butylene (such as ), alkoxy corresponds to alkyleneoxy (-CH 2 O-、-CH 2 CH 2 -O-CH 2 -、-CH 2 OCH 2 CH 2 CH 2 -)wait.

[0094] As used herein, the term "plurality" refers to two or more, such as 2, 3, 4, 5 or 6.

[0095] As described herein, the compounds of the present invention can be replaced with any number of substituents or functional groups to expand their scope of inclusion. Generally, the term "substituted" appears before or after the term "optional", and the general formula of the substituent included in the present invention refers to the use of a specified structural substituent to replace the hydrogen free radical. When multiple positions in a specific structure are replaced by multiple specific substituents, each position of the substituent can be the same or different. The term "substituted" used herein includes all allowed organic compound substitutions. In a broad sense, allowed substituents include non-cyclic, cyclic, branched non-branched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, heteroatom nitrogen can have a hydrogen substituent or any allowed organic compound described above to supplement its valence state. In addition, the present invention is not intended to limit the allowed substitution of organic compounds in any way.

[0096] As used herein, the term "substituted" means that any one or more hydrogens on the designated atom are replaced with a substituent selected from the designated substituent, provided that the normal valency of the designated atom is not exceeded and that the compound produced by the substitution is stable, i.e., a compound that can be isolated, characterized, and tested for biological activity.

[0097] If not specifically stated, the substitution refers to that one or more H on each group is independently substituted by a substituted or unsubstituted group selected from the group A: -OH, oxo (=O), halogen, -CN, -NH 2 , di(C1-C6 alkyl)amino-, -COOH, -CONH 2 、-SO 2 NH 2 、-SO 2 C1-C6 alkyl, unsubstituted or halogenated C1-C12 alkyl, unsubstituted or halogenated C2-C12 alkenyl, unsubstituted or halogenated C2-C12 alkynyl, unsubstituted or halogenated C1-C12 alkoxy, unsubstituted or halogenated -COC1-C12 alkyl, unsubstituted or halogenated -OCOC1-C12 alkyl, unsubstituted or halogenated -NH-CO-C1-C8 alkyl, unsubstituted or substituted with one or more R a The substituent is selected from the group consisting of phenyl-C1-C6 alkyl-COO-, benzyl, C3-C8 cycloalkyl, 3-8 membered heterocycloalkyl having 1-3 heteroatoms selected from N, S and O, C6-C12 aryl, and 5-12 membered heteroaryl having 1-3 heteroatoms selected from N, S and O, and the R a The substituents are independently selected from the group consisting of -OH, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy.

[0098] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are intended to be encompassed within the scope of the present invention.

[0099] Compounds of formula I

[0100] The present invention provides a compound for sample derivatization, wherein the compound has a structure of Formula I:

[0101] AL 1 -BL 2 -M

[0102] I

[0103] Among them, A, L 1 , B, L 2 and M are as defined above.

[0104] Generally, in Formula I, A is a reactive group, preferably an acyl chloride or sulfonyl chloride group. It is the reactive site of the present invention, which can react quickly with the amino groups of amino acids and polypeptides under alkaline conditions. In addition, this group is also an important group for improving the separation degree of chiral amino acids.

[0105] B. The steroid skeleton part is selected from various substituted or unsubstituted steroid skeletons. For example (but not limited to) estradiol, cholesterol, and androsterone. It is the chiral selective part of the compound of the present invention. Introducing this single chiral structure into the molecule of the amino-containing chiral compound can introduce a new stereo (chiral) center, increase the difference in collision cross-sectional area of ​​a pair of isomers to be tested, and make it easier to distinguish in the process of chromatographic separation and ion mobility separation, thereby playing a role in chiral recognition.

[0106] In the compounds of the present invention, the connection sites of the steroid skeleton part B with A and C have little effect on sample detection, and any suitable position can be selected for connection. Unless otherwise specified, the steroid skeleton can be numbered as follows:

[0107] M is the label part. During the synthesis process, stable isotopes (such as deuterium) can be easily introduced for isotope comparison analysis based on mass spectrometry. The molecular weight difference between the light-labeled and heavy-labeled compounds is n Da, which is very helpful for distinguishing and identifying a group of peaks in the mass spectrum.

[0108] As used herein, the terms "non-isotopically labeled" or "lightly labeled" are used interchangeably to refer to each molecule atom having or substantially having the atomic mass or atomic composition under natural conditions.

[0109] As used in the present invention, the terms "isotopically labeled" or "heavy labeled" are used interchangeably and refer to a molecule (usually a tag portion M) containing at least 2 (such as 3-12, preferably 4-8) atoms selected from the following group: 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F and 36 Cl, preferably 2 H. 13 C or 15 N is more preferably 2 H (D, deuterium).

[0110] In the present invention, the atomic mass of the unlabeled compound and the isotope-labeled compound is preferably 1 , with the following relationship M 1 -M 0 ≥2Da, preferably, 12≥M 1 -M 0 ≥3Da, more preferably, preferably, 8Da≥M 1 -M 0 ≥4Da, such as M 1 -M 0 =5Da, 6Da or 7Da.

[0111] Method for detecting optical isomers

[0112] The compound of the present invention can be used as a sample derivatization reagent to detect (identify) the optical isomers (chirality) of small molecular compounds such as amino acids and hydroxy acids in the sample.

[0113] As used herein, the term "optical isomer" refers to stereoisomers formed due to different configurations of one or more chiral atoms contained in a molecule, including enantiomers and diastereomers. When a molecule contains n chiral carbon atoms, the number of optical isomers is 2. n .

[0114] Therefore, the present invention provides a method for detecting optical isomers of a substance to be tested in a sample, comprising the steps of:

[0115] (a) reacting the sample with the compound of formula I described above to obtain a derivatized sample; and

[0116] (b) separating and detecting the sample obtained in step (a) using ion mobility mass spectrometry or LC-MS.

[0117] Preferably, in the sample, the substance to be detected is selected from the following group: amino acids, hydroxy acids, or a combination thereof.

[0118] In the present invention, the derivatized sample is separated and detected by an ion migration tube or a chromatographic column before mass spectrometry detection, so that the substances to be tested of different configurations (especially small molecules that cannot be effectively separated by the ion migration tube or the chromatographic column) and the derivatized products of the compounds of the present invention are separated (such as in terms of time) before mass spectrometry detection, thereby solving the problem that mass spectrometry cannot detect optical isomers; in addition, mass spectrometry has the characteristics of high resolution and high sensitivity, and can detect complex samples with extremely low content of the substances to be tested.

[0119] A possible mechanism of the present invention (but not intended to limit the present invention) is that α-AAs and α-HAs react with the acyl chloride groups of the compounds of the present invention in the presence of metal ions (such as Na + , K + ) forms a 7-membered ring in the presence of , and forms a derivatized product with a specific stereo configuration with the moderately rigid steroidal ring in the compound of the present invention. Compared with the chiral difference of the analyte, the compound of the present invention significantly increases the difference in the stereostructure between the two, so that the derivatized product can be effectively separated in the ion migration tube or chromatographic column separation.

[0120] For example, the derivatization product of the isotope-labeled compound of the present invention and the analyte standard can be used as an isotope internal standard, so that the isotope internal standard method can be used in the mass spectrometry to quantify the different isomers of the analyte. The mass spectrometry isotope internal standard method of treating the sample with a derivatization agent can be performed according to methods known in the art.

[0121] For example, the step (a) may include the following steps: (a1) providing an isotope internal standard obtained by reacting an isotope-labeled compound of formula I according to the present invention with a standard substance to be tested; (a2) mixing the isotope internal standard with the derivatized sample at a certain concentration (under non-reactive conditions) to obtain an isotope internal standard sample; (a3) ​​further pre-treating the isotope internal standard sample to obtain a sample for detection. Preferably, in step (a2), the mixing conditions are different from the reaction conditions of the standard substance to be tested and the compound of the present invention to avoid further reaction.

[0122] Ion Mobility Mass Spectrometry

[0123] Ion mobility mass spectrometry adds ion mobility as a new separation and measurement factor to the traditional organic mass spectrometer. It can distinguish isomers based on the differences in the spatial structure of the compounds through its unique ion mobility function, and can simultaneously obtain high-precision mass information and different ion mobility separation time information. It is very suitable for research on structural or compositional differences, such as the research of macromolecular compounds such as proteins and peptides in food, as well as small molecular compounds such as amino acids.

[0124] In the present invention, there is no special requirement for the type and model of the ion mobility mass spectrometer, and the ion mobility mass spectrometer commonly used in the art can be used.

[0125] Liquid chromatography-mass spectrometry (LC-MS)

[0126] Liquid chromatography-mass spectrometry analysis refers to the process in which the components in a sample are separated by liquid chromatography and then introduced into a mass spectrometer through a suitable interface. They are ionized by an ion source into fragment ions with a certain mass-to-charge ratio, which are then separated and detected by a mass analyzer.

[0127] It should be pointed out that, in general, conventional chromatographic columns (such as reverse phase chromatography and normal phase chromatography) cannot separate isomeric small molecules because the polarity and structure of the two isomeric molecules are basically the same. However, after the compounds of the present invention are derivatized, the derivatized products of different isomers can be separated by chromatographic columns, which makes it possible for liquid chromatography-mass spectrometry to be used for the detection of optical isomers of small molecules.

[0128] In the present invention, there is no particular requirement for the type and model of the liquid chromatography-mass spectrometer, and a liquid chromatography-mass spectrometer commonly used in the art can be used. For example, the liquid chromatography can be HPLC, UPLC, etc.; the ionization source of the mass spectrometer can be (but not limited to): API, ESI, APCI, etc., and the detector can be (but not limited to): QQQ, Q-TOF, ion trap, etc.

[0129] In liquid chromatography, the chromatographic column used for separation can be selected according to the sample, such as a reverse phase chromatographic column (C8 column, C18 column), a normal phase chromatographic column, etc. The compound of the present invention and the derivative products of amino acids and hydroxy acids are relatively easy to separate, which can be achieved by a C18 column, etc.

[0130] In addition, for the quantitative detection of samples, both the non-isotope-labeled and isotope-labeled compounds of the present invention can be used simultaneously, and the isotope quantification method and process using a derivatization reagent conventional in the art can be used.

[0131] Samples, substances to be tested

[0132] In the present invention, the compounds of the present invention can be applied to a variety of small molecule analytes, as long as the small molecule can react with the acyl chloride group of the compound. For example, the analyte is (but not limited to) an amino acid (such as α-amino acid, such as a natural amino acid, an unnatural amino acid), a hydroxy acid (such as α-hydroxy acid), or a combination thereof.

[0133] Preferably, the substance to be detected has one or more chiral carbon atoms, and more preferably, the carbon atom connected to the amino group or hydroxyl group of the amino acid or hydroxy acid is a chiral carbon atom.

[0134] Preferably, the amino acid is selected from the group consisting of (but not limited to) glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, lysine, arginine, histidine, glutamic acid, aspartic acid, glutamine, asparagine, cysteine, methionine, or a combination thereof. The amino acid may be D-type, L-type, or a mixture of D-type and L-type in any ratio.

[0135] Preferably, the hydroxy acid is selected from the group consisting of, but not limited to, glucuronic acid, tartaric acid, glycolic acid, malic acid, lactic acid, citric acid, 2-hydroxybutyric acid, mandelic acid, benzilic acid, hydroxyoctanoic acid, or a combination thereof.

[0136] In the present invention, there is no particular limitation on the sample, and the sample is applicable to any organic sample. The sample can be pre-treated by a commonly used method in the art to meet the sample loading requirements of ion mobility mass spectrometry or high performance liquid chromatography mass spectrometry.

[0137] Generally, the sample can be a biological sample, an environmental sample, a quality control sample, a reaction solution, etc., typically, such as animal body fluids (such as blood), tissues, secretions, excretions, etc.; plant extracts, secretions; polypeptide solid phase synthesis reaction solution or reaction products, protein or polypeptide hydrolysis samples; drugs, cosmetics, foods, etc. For sample processing, other pre-treatment steps except the derivatization reaction can be carried out according to methods commonly used in the art. For example, the sample can be processed by conventional methods before or after the reaction with the compound of the present invention, such as centrifugation, filtration, extraction, concentration, drying, etc.

[0138] Preferably, the test substance reacts with the compound of the present invention under the following reaction conditions: in a 0.02-0.1 M sodium carbonate-sodium bicarbonate buffer solution (pH 8-11, preferably 9.16-10.83), at room temperature for 5-30 minutes.

[0139] Preferably, after the reaction is completed, the sample can be blown dry and dissolved in a solvent (preferably methanol, ethanol, acetonitrile or a mixed solvent thereof with water (preferably, the water content ≤50% v / v, such as 30-10% water)) for further processing or injection.

[0140] Sample derivatization reagent kits

[0141] In the present invention, compounds of the same structure that are not isotopically labeled and those that are isotopically labeled can be used separately. For example, in ion mobility mass spectrometry, there is no need to introduce isotope labels. In addition, when using ordinary mass spectrometry (such as LC-MS / MS, etc.), isotope standards of the sample to be tested can also be used for isotope quantification.

[0142] Preferably, when using mass spectrometry other than ion mobility mass spectrometry, the un-isotope-labeled and isotope-labeled compounds of the present invention are used together to further achieve quantitative detection by mass spectrometry without the need to use isotope standards of the sample to be tested. This is very advantageous when simultaneously testing multiple amino acids or hydroxy acids because it is not necessary to use multiple corresponding isotope standards of the analytes.

[0143] Therefore, the present invention also provides a sample derivatization reagent combination, comprising:

[0144] (a) a compound of formula I according to the present invention which is not isotopically labeled; and

[0145] (b) isotope-labeled compounds (referring to compounds with the same structure as the compound in (a), the only difference being whether or not they are isotope-labeled).

[0146] In addition, a kit comprising the above reagent combination is also provided. In another preferred embodiment, the kit also includes instructions.

[0147] In the combination or kit, the compound not labeled with an isotope (a) and the compound labeled with an isotope (b) are packaged separately.

[0148] Compound Preparation Method

[0149] In the present invention, there is no special requirement for the preparation method of the compound, and the compound can be prepared by organic synthesis method using commercially available raw materials in the art.

[0150] Specifically, a typical synthesis method includes the following steps:

[0151] (a) reacting the compound HO-B-OH with M-CO-Cl to obtain the compound HO-B-OCO-M; and

[0152] (b) The compound HO-B-OCO-M reacts with triphosgene to form Cl-CO-B-OCO-M, wherein B and M are as defined above.

[0153] More specifically, an exemplary method for synthesizing a compound comprises the following steps:

[0154] a) Dissolve estradiol, tetrabutylammonium hydrogen sulfate and potassium carbonate in dichloromethane / water (2:1-3:1), slowly add benzoyl chloride / deuterated benzoyl chloride dropwise, and react at room temperature for 24-48 hours;

[0155] b) Extract with dichloromethane and spin dry, and recrystallize with methanol to obtain [d 0 ]-or [d 5 ]-Crude estradiol benzoate;

[0156] c) Under argon protection,0 ]-or [d 5 ]-Estradiol benzoate was dissolved in dry deoxygenated dichloromethane, and an appropriate amount of pyridine was added at zero degrees, followed by triphosgene, and the mixture was returned to room temperature and stirred at room temperature for 24 to 48 hours to obtain [d 0 ]-or [d 5 ]-estradiol benzoate-17-carbonyl chloride is the reagent.

[0157] Preferably, the concentration of estradiol is recommended to be 0.1 to 1 M. The molar ratio of potassium carbonate to estradiol is recommended to be 2:1. The amount of tetrabutylammonium hydrogen sulfate is recommended to be 2%. The molar ratio of benzoyl chloride / deuterated benzoyl chloride to estradiol is recommended to be 1.1:1. 0 ]-or [d 5 The recommended molar ratio of ]-estradiol benzoate is 0.333:1. The recommended amount of pyridine is 20-50 μL. The recommended extraction solvent is dichloromethane. The recommended solvent for recrystallization is methanol.

[0158] The main advantages of the present invention include:

[0159] The present invention provides a derivatization reagent for chiral analysis of α-AAs and α-HAs, which can react rapidly with the amino group of amino acids and the hydroxyl group of hydroxy acids at room temperature, and the operation is safe, simple and rapid. The medium-rigid steroid group in the reagent molecule can increase the separation and resolution of labeled α-AAs and α-HAs, especially the method combining liquid chromatography mass spectrometry and ion mobility mass spectrometry to achieve rapid, efficient and accurate mass spectrometry analysis. The derivatized test substance is more targeted in the mass spectrometry analysis process, and the complexity of mass spectrometry analysis is also reduced to a certain extent.

[0160] The reagent and method of the present invention provide a good means for high-throughput research of endogenous small molecule metabolites, and provide an effective strategy for mass spectrometry analysis of biomarkers in diagnostic medicine, disease diagnosis involving chiral small molecules, and quality control of polypeptide drugs. For example, in the quality control of polypeptide drugs, each hydrolyzed amino acid can react with light and heavy labeling reagents with high efficiency, and generate a single or a pair of signal peaks in the extracted ion chromatograms or ion migration spectra corresponding to the accurate mass numbers of the light and heavy labeled amino acids, respectively, and their relative intensities reflect the degree of racemization of each amino acid in the actual polypeptide drug.

[0161] Secondly, compared with the reported chiral acyl chloride derivatization reagents (FLEC-Cl, S-NAP, S-CAM and APOC, etc.), the raw materials of the present invention are cheap and easy to obtain, and the synthesis steps are simple; compared with the fluorenyl group in FLEC-Cl, the moderately rigid steroid group in the SC molecule can increase the separation and resolution of labeled α-AAs and α-HAs, which is more conducive to enhancing the sensitivity and separation of chiral recognition of α-AAs and α-HAs.

[0162] Finally, the present invention can adopt two analytical methods: (1) direct ESI-UMA-QQQ MS analysis method, which is easy to operate and does not require the sample to undergo chromatographic separation and purification steps, greatly improving the analysis efficiency and enabling high-throughput analysis; (2) HPLC-ESI-Q-TOF MS analysis method, which can achieve accurate qualitative and quantitative analysis of allosteric chiral α-AAs and α-HAs.

[0163] The present invention will be further described below in conjunction with specific implementation. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0164] Example 1: Preparation of [d0]- / [d5]-17β-EBC

[0165] Dissolve 1 eq estradiol (0.1 M), 2% tetrabutylammonium hydrogen sulfate, and 2 eq potassium carbonate in DCM / H2O (2:1), slowly add 1.1 eq benzoyl chloride / deuterated benzoyl chloride dropwise, and react at room temperature for 24 h. Extract with DCM and spin dry, then recrystallize in methanol to obtain crude [d0]- or [d5]-estradiol benzoate;

[0166] Under argon protection, [d0]- or [d5]-estradiol benzoate (0.333M) was dissolved in dry deoxygenated DCM, 20 μL of pyridine was added at 0°C, and then 0.333 eq of triphosgene was added, and the mixture was returned to room temperature. The mixture was stirred at room temperature for 24 hours to obtain a colorless liquid [d0]- or [d5]-estradiol benzoate-17β-carbonyl chloride, which is the isotope labeling reagent of the present invention.

[0167] The reaction formula is as follows:

[0168]

[0169] Example 2: Density functional theory (DFT) verification.

[0170] The DFT analysis results are as follows:

[0171] The results of 17β-EBC labeling D-type and L-type serine are as follows Figure 1 As shown in the figure, it can be seen that the structures of 17β-EBC-D-Ser and 17β-EBC-L-Ser are significantly different. The carbonyl oxygen atom of the acyl chloride part and the carboxyl carbonyl oxygen atom of the serine part are coordinated with the same sodium ion to form a relatively stable seven-membered ring structure, which increases the effect of the chiral selector and is beneficial to the separation of 17β-EBC-D-AA and 17β-EBC-L-AA; and forms a relatively rigid structure, which improves the resolution.

[0172] Example 3: Application of the isotope-labeled reagent ([d0]- / [d5]-17β-EBC) of the present invention

[0173] The application principle is as follows:

[0174]

[0175]

[0176] 1. Chiral recognition of α-AAs

[0177] 1. Weigh the amino acid standard and dissolve it in 0.1M sodium carbonate-sodium bicarbonate buffer solution (pH 9.16-10.83) to prepare a 50mM amino acid mother solution for subsequent derivatization reactions;

[0178] 2. Take 1 mL of the above amino acid mother solution in two groups, add 500 μL of light labeling reagent 17β-EBC (333 nmol / μL) to one group and 500 μL of S-NAP (333 nmol / μL) to the other group under nitrogen protection, and stir at room temperature for 5 min;

[0179] 3. After the reaction solution was dried with nitrogen, an acetonitrile-water solution with a volume ratio of 1:1 was added, and the solution was filtered through a 0.22 μm PTFE filter membrane to remove impurities;

[0180] 4. ESI-UMA-QQQ MS and HPLC-ESI-Q-TOF MS analysis were performed. The ESI-UMA-QQQ MS analysis conditions were as follows: data acquisition was performed in SIM mode, all UMA spectra were acquired in filter scan mode, drift gas: nitrogen; drift gas flow rate: 1 L / min; scan time: 2000 ms; detector: U-shaped mobility analyzer (UMA). The HPLC-ESI-Q-TOF MS analysis conditions were as follows: data acquisition was performed in positive ion mode, a 100 mm*2.1 mm*1.7 um C8 column was used, 2 μL of the reaction solution was taken, and the mobile phase was acetonitrile-water (containing 0.1% formic acid) with a volume ratio of (15:85) to (80:20).

[0181] The analysis results of ESI-UMA-QQQ MS are as follows:

[0182] 1. 17β-EBC labeling results are as follows Figure 2 As shown in the fourth group of ion migration spectra, it can be seen from this figure that the derivatization products of D-serine and L-serine are well separated in UMA and can achieve baseline separation;

[0183] The results of S-NAP labeling are as follows Figure 3 As shown in the figure, it can be seen that the S-NAP labeling efficiency is low, the concentration of the derivatized products of D-serine and L-serine is small, and only a half-peak separation effect can be achieved in UMA.

[0184] In addition, if Figure 2 As shown in the figure, under standard reaction conditions, 19 natural amino acids and 4 unnatural amino acids labeled with 17β-EBC can be quickly separated by UMA, and most of them achieve baseline separation effect, which illustrates the significant chiral recognition ability of the labeling reagent, which is very beneficial for high-throughput chiral analysis of α-AAs in biological samples.

[0185] The analysis results of HPLC-ESI-Q-TOF MS are as follows:

[0186] 1. As shown in Table 1, the high-resolution mass spectrometry information of [d0]- / [d5]-17β-EBC-AAs:

[0187] Table 1

[0188]

[0189]

[0190] like Figure 4As shown, at least 18 natural amino acids labeled with 17β-EBC can be separated in the chromatographic column, which illustrates the significant chiral recognition ability of the labeling reagent, which is very beneficial for the accurate quantitative analysis of allosteric chiral α-AAs in biological samples.

[0191] 2. α-HAs chiral recognition

[0192] 1. Weigh the α-HAs standard and dissolve it in 0.1M sodium carbonate-sodium bicarbonate buffer solution (pH 9.16-10.83) to prepare a 50mM α-HA mother solution for subsequent derivatization reactions;

[0193] 2. Take 1 mL of the above α-HA mother solution, mix with 500 μL of light labeling reagent 17β-EBC and S-NAP (both 333 nmol / μL), and stir at room temperature for 5 min;

[0194] 3. After the reaction solution was dried with nitrogen, an acetonitrile-water solution with a volume ratio of 1:1 was added, and the solution was filtered through a 0.22 μm PTFE filter membrane to remove impurities;

[0195] 4. Perform ESI-UMA-QQQ MS and HPLC-ESI-Q-TOF MS analysis. ESI-UMA-QQQ MS analysis conditions are: data acquisition in SIM mode, all UMA spectra are acquired in filter scan mode, drift gas: nitrogen; drift gas flow rate: 1L / min; scan time: 2000ms; detector: UMA. HPLC-ESI-Q-TOF MS analysis conditions are: data acquisition in positive ion mode, using a 150mm*2.1mm*3.5um C18 column, taking 2μL of the reaction solution, and the mobile phase is acetonitrile-water (containing 0.1% formic acid) with a volume ratio of (15:85) to (80:20).

[0196] The analysis results of ESI-UMA-QQQ MS are as follows:

[0197] 1. 17β-EBC labeling results are as follows Figure 5 As shown in the sixth group of ion migration spectra, it can be seen from this figure that the derivatized products of D-lactic acid and L-lactic acid are well separated in UMA and can achieve baseline separation;

[0198] The results of S-NAP labeling are as follows Figure 6 As shown in the figure, it can be seen that the S-NAP labeling efficiency is low, the concentrations of the derivatized products of D-lactic acid and L-lactic acid are small, and they are almost impossible to separate in UMA or can only achieve a half-peak separation effect.

[0199] In addition, if Figure 5As shown in the figure, under standard reaction conditions, the seven α-hydroxy acids labeled with 17β-EBC can be quickly separated by UMA, and most of them achieve baseline separation effect, which illustrates the significant chiral recognition ability of the labeling reagent, which is very beneficial for high-throughput chiral analysis of α-hydroxy acid compounds in biological samples and cosmetics.

[0200] The analysis results of HPLC-ESI-Q-TOF MS are as follows:

[0201] 1. As shown in Table 2, the high-resolution mass spectrometry information of [d0]-17β-EBC-HAs:

[0202] Table 2

[0203]

[0204] 2. If Figure 7 As shown in the figure, the seven α-hydroxy acids labeled with 17β-EBC can be separated in the chromatographic column, and most of them can achieve baseline separation, which illustrates the significant chiral recognition ability of the labeling reagent. The baseline separation effect of each enantiomer α-hydroxy acid is very beneficial to the accurate quantitative analysis of allosteric chiral α-hydroxy acids in biological samples and cosmetics.

[0205] 3. Disease diagnosis based on chiral small molecules

[0206] 1. Freeze-dry the mouse brain tissue free amino acid extract in vacuum, weigh an appropriate amount of freeze-dried powder, and dissolve it in 500 μL 0.1 M sodium carbonate-sodium bicarbonate buffer solution (pH 9.16-10.83);

[0207] 2. Add 500 μL of 17β-EBC labeling reagent to the above solution and perform the same post-processing steps as in Example 3;

[0208] 3. Perform ESI-UMA-QQQ MS analysis. The ESI-UMA-QQQ MS analysis conditions are: data acquisition in SIM mode, all UMA spectra are acquired in filter scan mode, drift gas: nitrogen; drift gas flow rate: 1 L / min; scan time: 2000 ms; detector: UMA.

[0209] The analysis results of ESI-UMA-QQQ MS are as follows:

[0210] 1. 17β-EBC labeling results are as follows Figure 8 As shown by Figure 8 It can be seen that there are significant differences in the free D-serine in the brains of mice of different ages, and the concentration of D-serine in the brain tissue of old mice is extremely low; there is no significant difference in the concentration of L-serine in the brain tissue of young and old mice.

[0211] 2. If Fig. 9 As shown in the results, in the oxidative stress-induced cell senescence model, the degree of cell senescence was reduced after treatment with D-serine, and the growth ability was between that of normal group cells and oxidative stress-induced senescent cells, indicating that D-serine is closely related to aging. This illustrates the potential application of 17β-EBC chiral derivatization combined with ion migration method in disease diagnosis based on chiral small molecules.

[0212] 4. Quality Control of Peptide Drugs

[0213] 1. Weigh 5 mg of Deltorphin II standard and place it in a vacuum hydrolysis tube. Pass nitrogen for 15 minutes and add 1 mL of 6MDCl / D 2 O, repeated freezing and thawing 3 times, heated at 110 °C for 24 h under nitrogen protection;

[0214] 2. After the hydrolysis reaction is completed, evaporate the solvent in a 70-80℃ water bath, then add 1mL D2O, repeat 3 times to remove excess hydrochloric acid;

[0215] 3. Add 500 μL of 0.1 M sodium carbonate-sodium bicarbonate buffer solution (pH 9.16-10.83) to the residue after evaporation, then add 500 μL of 17β-EBC labeling reagent, and perform the same post-treatment steps as in Example 3;

[0216] 4. Perform ESI-UMA-QQQ MS analysis. The ESI-UMA-QQQ MS analysis conditions are: data acquisition in SIM mode, all UMA spectra are acquired in filter scan mode, drift gas: nitrogen; drift gas flow rate: 1 L / min; scan time: 2000 ms; detector: UMA.

[0217] The analysis results of ESI-UMA-QQQ MS are as follows:

[0218] 1. 17β-EBC labeling results are as follows Fig.10 As shown in the figure, it can be seen that the amino acid composition and configuration in the polypeptide can be accurately identified.

[0219] 2. If Fig.11 As shown, 17β-EBC labeled enantiomeric amino acids combined with UMA analysis method can detect at least 0.5% of another configuration amino acid in a large amount of one configuration amino acid, such as at least 0.5% D-Ala can be detected in the figure. This shows the application value of 17β-EBC chiral derivatization combined with ion migration method in the quality control of peptide drugs, which is conducive to the discovery of racemic peptide impurities.

[0220] Example 4: Application of other steroidal acyl chloride labeling reagents.

[0221] The application principle is as follows (such as cholesteryl chloroformate, CC):

[0222]

[0223] 1. Chiral recognition of α-AAs

[0224] 1. Weigh the amino acid standard and dissolve it in 0.1M sodium carbonate-sodium bicarbonate buffer solution (pH 9.16-10.83) to prepare a 50mM amino acid mother solution for subsequent derivatization reactions;

[0225] 2. Take 1 mL of the above D-, L-alanine mother solution respectively, mix with 500 μL of labeling reagent CC or AC, 17α-EBC, 16α-PC, TC (333 nmol / μL), and stir at room temperature for 5 min;

[0226] 3. After the reaction solution was dried with nitrogen, an acetonitrile-water solution with a volume ratio of 1:1 was added, and the solution was filtered through a 0.22 μm PTFE filter membrane to remove impurities;

[0227] 4. Perform ESI-UMA-QQQ MS analysis. The ESI-UMA-QQQ MS analysis conditions are: data acquisition in SIM mode, all UMA spectra are acquired in filter scan mode, drift gas: nitrogen; drift gas flow rate: 1 L / min; scan time: 2000 ms; detector: UMA.

[0228] The analysis results of ESI-UMA-QQQ MS are as follows:

[0229] The labeling results of CC or AC, 17α-EBC, 16α-PC, and TC are as follows Fig.12 As shown in the ion migration spectrum, it can be seen from the figure that the derivatization products of CC (or TC, 17α-EBC, 16α-PC)-D-Ala and CC (or TC, 17α-EBC, 16α-PC)-L-Ala can be separated in UMA, which illustrates the significant chiral recognition ability of the labeling reagent, which is very beneficial for the accurate qualitative and quantitative analysis of allosteric chiral α-AAs in biological samples.

[0230] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a compound of formula I in the preparation of a sample derivatization reagent for qualitative and / or quantitative detection of optical isomers of a substance to be tested, in, The substance to be tested is selected from the following group: amino acids, hydroxy acids, or a combination thereof: A-L 1 -B-L 2 -M I Wherein, A is a reactive group, and A is selected from the following group: formyl chloride or sulfonyl chloride; B is a steroid skeleton portion, and B is a divalent group of the following structure: And the divalent group has the following structure: L 1 and L 2 is a linking group, and L 1 is the key; Among them, B and L 1 The attachment site of is 17; B and L 2 The attachment site is selected from the group consisting of: 2, 3, and 4; L 2 are each independently selected from the group consisting of a bond, -O-, -NHCO-, -COO-; and an unlabeled or isotopically labeled tag moiety M, wherein M is selected from the group consisting of H, C1-C12 alkyl, phenyl-C1-C6 alkyl-, C3-C8 cycloalkyl, C6-C12 aryl; and When M is isotopically labeled, it contains at least 2 atoms selected from the group consisting of: 2 H. 3 H. 13 C and 14 C; Or the compound of formula I is:

2. The use according to claim 1, It is characterized in that When M is isotopically labeled, it contains at least 2 atoms selected from the group consisting of: 2 H and 13 C.

3. The use according to claim 1, It is characterized in that The compound of formula I is selected from the following group: The compound is optionally non-isotopically labeled or isotopically labeled.

4. The use according to claim 1, It is characterized in that The sample is a biological sample, an environmental sample, or a reaction solution.

5. The use according to claim 4, It is characterized in that The samples are animal body fluids, secretions, tissues; plant tissues, plant extracts; peptide solid phase synthesis reaction solutions, reaction products or protein hydrolyzate samples.

6. The use according to claim 1, It is characterized in that The amino acid is an α-amino acid, and the hydroxy acid is an α-hydroxy acid.

7. A mass spectrometry derivatization kit for qualitative and / or quantitative detection of optical isomers of a substance to be tested, It is characterized in that The substance to be tested is selected from the following group: amino acids, hydroxy acids, or a combination thereof: And the kit comprises: (a) a first container comprising a compound of formula I which is not isotopically labeled; and / or (b) a second container comprising the isotope-labeled compound; A-L 1 -B-L 2 -M I Wherein, A is a reactive group, and A is selected from the following group: formyl chloride or sulfonyl chloride; B is a steroid skeleton portion, and B is a divalent group of the following structure: And the divalent group has the following structure: L 1 and L 2 is a linking group, and L 1 is the key; Among them, B and L 1 The attachment site of is 17; B and L 2 The attachment site is selected from the group consisting of: 2, 3, and 4; L 2 are each independently selected from the group consisting of a bond, -O-, -NHCO-, -COO-; and The tag part M is not isotopically labeled, and M is selected from the following group: H, C1-C12 alkyl, phenyl-C1-C6 alkyl-, C3-C8 cycloalkyl, C6-C12 aryl; Or the compound of formula I is: Wherein having isotope labeling means that M is isotope labeled, wherein at least 2 atoms are selected from the following group: 2 H. 3 H. 13 C and 14 C.

8. The kit according to claim 7, It is characterized in that The compound of formula I is selected from the following group:

9. A method for detecting optical isomers of a substance to be tested in a sample, It is characterized in that Includes steps: (a) reacting a sample with a compound of formula I to obtain a derivatized sample; and (b) separating and detecting the sample obtained in step (a) using ion mobility mass spectrometry or LC-MS; and Wherein, in the sample, the substance to be tested is selected from the following group: amino acids, hydroxy acids, or a combination thereof; A-L 1 -B-L 2 -M I Wherein, A is a reactive group, and A is selected from the following group: formyl chloride or sulfonyl chloride; B is a steroid skeleton portion, and B is a divalent group of the following structure: And the divalent group has the following structure: L 1 and L 2 is a linking group, and L 1 is the key; Among them, B and L 1 The attachment site of is 17; B and L 2 The attachment site is selected from the group consisting of: 2, 3, and 4; L 2 are each independently selected from the group consisting of a bond, -O-, -NHCO-, -COO-; and a tag part M which is not isotopically labeled, and M is selected from the group consisting of H, C1-C12 alkyl, phenyl-C1-C6 alkyl-, C3-C8 cycloalkyl, C6-C12 aryl; and Or the compound of formula I is:

10. The method according to claim 9, It is characterized in that The step (a) further comprises the steps of: (a1) providing an isotope internal standard obtained by reacting a standard substance to be tested with the isotope-labeled compound of formula I; (a2) mixing a certain concentration of the isotope internal standard with the derivatized sample to obtain an isotope internal standard sample; (a3) ​​further pre-treating the isotope internal standard sample to obtain a sample for detection; Wherein having isotope labeling means that M in the compound of formula I is isotope labeled, wherein at least 2 atoms are selected from the following group: 2 H. 3 H. 13 C and 14 C.

Images