Quaternary ammonium salt compound and its use as mass spectrometry detection sensitizer, amino detection reagent

Abstract

The present disclosure provides a novel pyridinium salt type compound, which can be used as a sensitizing agent for MALDI mass spectrometry detection, and can be used for sensitizing amino group detection. The compound disclosed in the present disclosure can improve the detection ability of MALDI-MSI for amino group small molecule derivatives in biological samples.

Description

Technical Field

[0001] The present disclosure belongs to the field of mass spectrometry detection, and provides a pyridinium salt type compound and its use as a MALDI mass spectrometry detection sensitizer. The compound of the present disclosure can be used for sensitization in MALDI mass spectrometry detection of amino groups, and the present disclosure also provides an amino group detection reagent. Background Art

[0002] Amino compounds play an indispensable role in maintaining the structure, function and energy balance of organisms. Among them, amino acids (AAs), as the basic components of proteins, are the basic materials for building cells and repairing tissues. They are widely distributed in organisms and participate in many biological processes. They are essential for nutrition, immunity, health and disease. Therefore, based on the biological significance of amino acids, it is extremely necessary to obtain accurate and complete in situ distribution and abundance information of AAs in biological samples for studying the pathogenesis of amino acid-related diseases. The following are representative structures of amino metabolites in organisms.

[0003]

[0004] Mass spectrometry imaging (MSI) is a new in situ visualization technology based on MS technology. It can image thousands of molecules such as alkali metal ions, metabolites, lipids, peptides, proteins and glycans without labeling. It has the advantages of being compatible with both targeted and non-targeted research, high sensitivity, high resolution, and high salt tolerance. MALDI-MSI has been widely used in the in situ detection and imaging of various endogenous / exogenous biomolecules, greatly promoting the development of in situ multi-omics such as spatial metabolomics and lipidomics.

[0005] Although MALDI MSI technology has been developed to a very mature stage, the traditional MALDI matrix will produce a large number of fragments and cluster ions in the low molecular weight range (m / z <500) during use, resulting in background interference, which seriously affects the determination of small molecules. At the same time, due to the presence of other ionization / desorption inhibitors and lipids, this will greatly reduce the sensitivity of the method to compounds with low abundance and / or difficult to ionize. Therefore, the application of this technology in many important fields such as mass spectrometry imaging of small molecules, especially AAs, metabolomics analysis, etc. is limited.

[0006] Tissue-on-tissue chemical derivatization (OTCD) is an extremely important means to achieve in situ visualization research of the above-mentioned difficult-to-ionize compounds. This method changes the physical and chemical properties of the substrate by targeting the reactive groups of the derivatization reagent to react with specific functional groups of the molecules to be tested in the tissue, thereby improving the selectivity, sensitivity, and resolution of mass spectrometry analysis and eliminating the discrimination effect in multi-component analysis, thereby improving the application of small molecule mass spectrometry imaging. In recent years, the application of this technology in MSI research has received widespread attention. To date, many MALDI MS probes have been successfully developed and applied to the detection and imaging of amino compounds. FMP-10 has been designed to achieve full network coverage of the biosynthesis and degradation pathways of various neurotransmitters in brain tissue slices; oxonium and its derivative probes TMPy / DPP / TPP have been designed and applied to the detection of various neurotransmitters and glycine; CA probes have been designed and applied to endogenous amine metabolites; NBA and its derivative probes have been designed and applied to the detection sensitivity of various primary amine metabolites in cell culture medium and rat brain slices; TMPP probes optimized by 4-SPITC and 3-SBASE have been designed and applied to the analysis of tissue slices to obtain the location of various compounds including peptides and proteins. mTRAQ has been designed and applied to the study of protein and peptide analysis. TAHS probes have been designed and applied to the detection of various amino acids in human colon cancer xenografts and non-small cell lung cancer. The currently known commonly used probes and sensitization mechanisms are as follows:

[0007]

[0008] MALDI-MSI amino in situ derivatization probe

[0009]

[0010] Reaction Mechanism of MALDI-MSI In-situ Derivatization Probes and Amino Groups

[0011] However, full coverage detection of 20 common amino acids by OTCD-MSI on tissue samples is still unrealized, and the probe TAHS commonly used for amino acid visualization imaging has the risk of delocalization of metabolites in situ analysis due to long incubation time. Continuing to develop sensitizing reagents for substances containing amino groups in MALDI mass spectrometry imaging detection technology is undoubtedly of great significance for in-depth exploration of the mechanism of amino acids in complex biological processes. Summary of the invention

[0012] In the present disclosure, the inventors, after in-depth research, utilized the principle that pyrylium salts can easily and selectively react with primary amines to form pyridinium cations, designed a series of amino probes containing permanent pyridine nitrogen positive inner salts based on 2,4,6-TPP, and introduced a series of reactive warheads. DCT was obtained after basic modification of the specific strong reactive warhead and conjugated fluorophore. The probe has good reactivity to amino derivatization and MALDI MS positive ion detection signal. More importantly, we applied DCT to normal mouse tissues for the first time and achieved the detection of 20 common amino acids.

[0013] The inventors have conducted in-depth research to develop a highly reactive and highly sensitive sensitizing agent for substances containing amino groups in mass spectrometry imaging detection technology, and have successfully developed the technical solution of the present disclosure. Specifically, the present disclosure provides the use of a pyridinium salt compound represented by the following formula (1) as an amino sensitizing agent for mass spectrometry detection:

[0014]

[0015] G is a chloride ion or a tetrafluoroborate ion.

[0016] Compared with the prior art, the present invention can provide a method having the characteristics of high reactivity, high selectivity, and no generation of competitive ions, and can be used in MALDI-MSI to enhance the sensitivity of the detection of amino groups.

[0017] The present disclosure also provides a method for detecting amino acids by mass spectrometry based on the compound represented by the following formula (1), and a related kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a derivatization flow chart of the amino derivatization experiment;

[0019] Figure 2 is a diagram illustrating the structural formula of the pyridinium salt series probes disclosed herein;

[0020] Figure 3 is a diagram illustrating the reaction mechanism of the pyridinium salt series probes disclosed herein with amino groups;

[0021] Figure 4 It is the reaction group related rate curve and the MALDI MS detection signal bar graph after each probe reacts with the amino compound;

[0022] Figure 5 It is the application of probe DCT to amino acid imaging of mouse heart tissue;

[0023] Figure 6 It is the application of probe DCT to amino acid imaging of mouse liver tissue;

[0024] Figure 7It is the probe DCT applied to the amino acid imaging of mouse spleen tissue;

[0025] Figure 8 It is the application of probe DCT to amino acid imaging of mouse lung tissue;

[0026] Fig. 9 It is the probe DCT applied to the amino acid imaging of mouse kidney tissue;

[0027] Fig.10 It is a probe DCT applied to amino acid imaging of mouse brain tissue. DETAILED DESCRIPTION

[0028] According to the above contents of the present disclosure, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present disclosure, other various forms of modifications, replacements or changes may be made.

[0029] I. Definitions and Terminology

[0030] It should be noted that the terms "amino derivatization reagent", "sensitizer", "probe", etc. in the present disclosure refer to reagents that can react with the amino-containing compound to be detected and the derivatization product after the reaction can be more clearly observed in the mass spectrometry imaging detection technology. They are commonly present in the chemical reaction sites of the amino group and the chromophore part used for mass spectrometry imaging. In the present disclosure, "derivatization" sometimes specifically refers to the process of the reaction of the compound of the present disclosure with the amino group in the amino-containing compound. The term "MALDI" refers to matrix-assisted laser desorption ionization (Matrix-Assisted Laser Desorption Ionization). MALDI mass spectrometry is to disperse the analyte in the matrix molecules and form crystals. When the crystals are irradiated with laser, the energy absorbed by the matrix molecules by radiation leads to energy accumulation and rapid heat generation, thereby sublimating the matrix crystals, causing the matrix and analyte to expand and enter the gas phase. It is one of the core technologies of mass spectrometry imaging technology. "In situ" refers to a position corresponding to the actual position in the tissues and organs in the biological sample.

[0031] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0032] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure form or racemic form. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral raw materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of the present disclosure.

[0033] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring of said event or circumstance.

[0034] The term "substituted" or "substituted" means that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, as long as the valence state of the particular atom or group is normal and the substituted compound is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are replaced. Unless otherwise specified, the type and number of substituents can be any on the basis of chemically achievable.

[0035] When any variable (such as R n ) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with one to three R, the group may be optionally substituted with up to three R, and each occurrence of R is an independent choice. In addition, combinations of substituents and / or variants thereof are permitted only if such combinations result in stable compounds.

[0036] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, having the indicated number of carbon atoms. 1-6 "Alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. It may be divalent, such as methylene, ethylene.

[0037] The term "alkoxy" may be linear, branched or cyclic. The number of carbon atoms of the alkoxy group is not particularly limited, but is preferably 1 to 20. Specific examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, neopentoxy, isopentyloxy, n-hexyloxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, n-octyloxy, n-nonyloxy, n-decyloxy, etc., but are not limited thereto.

[0038] The term "carbonyl" or "carboxyl" includes structures in which a carbon of a compound or fragment is connected to an oxygen atom via a double bond. Examples of moieties containing a carbonyl group include aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, and the like.

[0039] The term "acyl" is a carbonyl structure in which the carbon atom of the carbonyl group is connected to hydrogen (i.e., formyl), an aliphatic group (C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, such as acetyl), a cycloalkyl group (C3-C8 cycloalkyl), a heterocyclic group (C3-C8 heterocycloalkyl and C5-C6 heteroaryl), an aryl group (C6 aryl, such as benzoyl). The acyl group may be unsubstituted or substituted acyl (e.g., salicyl).

[0040] In the present disclosure, examples of the halogen group may include fluorine, chlorine, bromine, or iodine.

[0041] In this disclosure, the term "cycloalkyl" refers to a monocyclic saturated hydrocarbon system without heteroatoms and double bonds. For example, the term "C 3-6 Examples of "cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0042] In the present disclosure, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system, which is obtained by removing a hydrogen atom from a single carbon atom of a parent aromatic ring system. It includes a saturated, partially unsaturated ring, or a bicyclic group of an aromatic ring fused with an aromatic carbocyclic ring. Specific examples thereof include phenyl or naphthyl, but are not limited thereto.

[0043] In the present disclosure, the term "heterocycloalkyl" refers to a 5-12 membered saturated non-aromatic system having ring carbon atoms and 1 to 2 ring heteroatoms. Specific examples of the heterocyclic group include piperidinyl or tetrahydropyrrolyl, but are not limited thereto.

[0044] In the present disclosure, the term "heteroaryl" refers to a monovalent aromatic group containing at least one heteroatom independently selected from nitrogen, oxygen and sulfur. The heteroaryl group may be a monocyclic ring or a polycyclic ring system, such as a bicyclic ring, wherein two or more rings exist in the form of a cyclic ring, a bridged ring or a spirocyclic ring, wherein at least one ring contains one or more heteroatoms. Specific examples of heteroaryl groups include pyridyl, thienyl, imidazolyl, pyrimidinyl, pyridyl, furanyl, pyrazinyl, thiazolyl, quinolyl, isoquinolyl, indolyl, benzimidazolyl, imidazopyridyl, benzofuranyl, pyridazinyl, isoindolyl, pyridonyl, but are not limited thereto.

[0045] The term "heterocycle" refers to a 5-12 membered saturated non-aromatic system having ring carbon atoms and 1 to 2 ring heteroatoms, wherein the heteroatoms are independently selected from nitrogen, sulfur or oxygen atoms. In heterocyclic groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as long as the valence permits. The heterocycle may be a monocyclic or polycyclic system, such as a bicyclic ring, wherein two or more rings exist in the form of a cyclic, bridged or spirocyclic ring, wherein at least one ring contains one or more heteroatoms. In a spirocyclic heterocyclic group, two different rings have one atom in common, and an example of a spirocyclic heterocyclic group is an azaspiropentyl group, but is not limited thereto.

[0046] The term "partially unsaturated bicyclic heterocycle" means a bicyclic group containing C atoms and at least one of heteroatoms such as N, O, S as ring members, the bicyclic group is partially unsaturated, and contains at least one C-C double bond. The largest unsaturated heterocycle contains the maximum number of C-C double bonds allowed by the ring size and double bonds between C atoms and heteroatoms. The partially unsaturated bicyclic heterocycle contains less double bonds than the number allowed by the ring size. Specific examples of partially unsaturated bicyclic heterocycles include, but are not limited to, benzofuranyl, benzothiophenyl, quinoxalinyl, quinazolinyl, isoindolinone, pteridinyl, and the like.

[0047] II. Examples of Synthesis Methods of Compounds of Formula (1)

[0048] The synthesis of the specific compound (MALDI derivatization reagent) used in the examples of the present disclosure was achieved by the following method. The structure of the synthesized derivatization reagent is 1 H and 13 The corresponding compounds are also marked in the following flow charts with abbreviated names, which are also used below. The instruments and reagents used can be found in the Examples section.

[0049]

[0050] Method 1: 2,4,6-triphenylpyrylium tetrafluoroborate (1188.6 mg, 3 mmol, 1 equivalent) was mixed with sodium pivalate (3 mmol, 1 equivalent), followed by addition of A: 5-amino-2-fluoropyridine (4.5 mmol, 1.5 equivalent) dissolved in 10 mL acetonitrile or B: p-aminobenzenesulfonic acid (4.5 mmol, 1.5 equivalent) or C: p-aminobenzoic acid (4.5 mmol, 1.5 equivalent) dissolved in 10 mL methanol and stirred at 85° C. in an oil bath for 12 h. The mixture was concentrated and eluted using a silica gel column for preliminary purification by different chromatographic elution systems. The product FTB, TSP or TCP was obtained after precipitation with ether.

[0051] Method 2: TSP (464.6 mg, 1 mmol, 1 equivalent) or TCP (428.5 mg, 1 mmol, 1 equivalent) was mixed with thionyl chloride (5 mmol, 5 equivalents) and dissolved in 1 mL of ultra-dry acetonitrile-ultra-dry N,N-dimethylformamide (1:1, v / v). The reaction was carried out at room temperature for 12 h, and the product CSP or CBP was obtained after rotary evaporation. Methyl esters were formed after dissolution in methanol and characterized by Q-TOF MS.

[0052] Method 3 (Synthesis of candidate derivatization reagents): CBP (447.0 mg, 1 mmol, 1 equivalent) and N-hydroxysuccinimide (1 mmol, 1 equivalent) were dissolved in 2 mL of ultra-dry acetonitrile-ultra-dry N,N-dimethylformamide (1:1, v / v), triethylamine (1 mmol, 1 equivalent) was added at room temperature for 12 h, and the product was concentrated and separated and purified by thin layer chromatography. The product DCT was obtained by precipitation with anhydrous diethyl ether.

[0053] Method1-Product:

[0054] Method2-Product:

[0055] Method3-Product:

[0056] Reagents FTB. 1 H NMR(600MHz, Acetonitrile-d3)δ8.46(s,2H),8.13(d,J=7.1Hz,2H),8.09(d,J=2.5Hz,1H),7.83-7.79(m, 1H),7.72(d,J=7.5Hz,1H),7.68(d,J=8.1Hz,2H),7.48-7.45(m,2H),7.45-7.40(m,8H),6.90-6.86(m,1H). 13C NMR (126MHz, Acetonitrile-d3) δ162.95,158.62,157.97,147.92,142.71,134.55,133.26,131.59,130.75,129.71,126.92,111.22,111.03.

[0057] ReagentsTSP. 1 H NMR (600MHz, Methanol-d4) δ8.50(s,2H),8.18-8.13(m,2H),7.71-7.64(m,3H),7.62-7.57(m,2H),7.46-7.32(m,12H). 13 C NMR (126MHz, Methanol-d4) δ158.75,158.75,158.32,148.33,148.33,141.51,135.28,134.27,133.77,131.60,130.92,130.01,127.67,126.95.

[0058] Reagent TCP. 1 H NMR (600MHz, Methanol-d4) δ8.51 (s, 2H), 8.17 (d, J = 6.4Hz, 2H), 7.72 (d, J = 8.5Hz, 2H), 7.68~7.66 (m, 2H), 7.43 (d, J = 7.1Hz, 4H), 7.41~7.24 (m, 9H). 13 CNMR(126MHz,Methanol-d4)δ172.60,158.56,158.36,141.55,141.00,135.30,134.40,133.74,131.51,130.91,129.33,126.84.

[0059] Reagent CSP. High-resolution mass spectrometry identification of methyl ester compounds formed by CSP: C 30 H 24 NO3S + (Da,calc. / found)478.1471 / 478.1486,(error)3.1ppm.

[0060] Reagent CBP. High-resolution mass spectrometry identification of methyl ester compounds formed by CBP: C 31 H 24 NO2 + (Da,calc. / found)442.1802 / 442.1802,(error)0ppm.

[0061] Reagent DCT: 1 H NMR (600MHz, Acetonitrile-d3) δ8.44(s,2H),8.12(d,J=7.3Hz,2H),7.90(d,J=8.6Hz,2 H), 7.68 (d, J=7.9Hz, 2H), 7.47 (dd, J=28.1, 8.7Hz, 4H), 7.40~7.37 (m, 9H), 2.81 (s, 4H). 13 C NMR (126MHz, Acetonitrile-d3) δ170.82,161.53,158.31,157.37,145.08,134.59,133.81,133.36,131.71,131.55,130.70,129.53,126.92,26.37.

[0062] Example

[0063] The present disclosure is further explained below with reference to examples. The description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made according to the teachings of the present disclosure specification. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0065] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0066] Reagents and instruments

[0067] Reagents: 2,4,6-triphenylpyranium tetrafluoroborate was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., 5-amino-2-fluoropyridine was purchased from Shanghai Titan Technology Co., Ltd., p-aminobenzenesulfonic acid and thionyl chloride were purchased from Shanghai MacLean Biochemical Technology Co., Ltd., p-aminobenzoic acid was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., sodium pivalate and N-hydroxysuccinimide were purchased from Jiangsu Aikang Biopharmaceutical Research and Development Co., Ltd. The solvents used in analytical-grade reactions were purchased from Sinopharm Chemical Reagent Co., Ltd. The ultra-dry solvents used in anhydrous reactions were purchased from Beijing Bailingwei Technology Co., Ltd. Amino acid standards, phenylalanine (Phe), glycine (Gly), methionine (Met), threonine (Thr), glutamic acid (Glu), alanine (Ala), valine (Val), aspartic acid (Asp), leucine (Leu), isoleucine (IIe), asparagine (Asn), proline (Pro), glutamine (Gln), tyrosine (Tyr), serine (Ser), arginine (Arg), histidine (His), lysine (Lys) were purchased from Sigma-Aldrich, USA, tryptophan (Trp) was purchased from Beijing Bailingwei Technology Co., Ltd., and cysteine ​​(Cys) was purchased from Shanghai Yien Chemical Technology Co., Ltd. Dopamine (DA), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich, USA. HPLC grade methanol (MeOH) and acetonitrile (ACN) were purchased from Merck, USA. Deionized ultrapure water (<18.2 MΩ·cm resistivity) was obtained using a Millipore Milli-QAdvantage A10 purification system. Indium tin oxide (ITO) coated conductive slides (75 mm*25 mm, surface resistivity 8-12 Ω / m 2 ) were purchased from Bruker, Germany. Individual stock solutions of amino acid standards were prepared in 200 μM (methanol: water = 1: 1 v / v) and stored at -20°C.

[0068] instrument:

[0069] Heidolph or IKA magnetic stirrer;

[0070] BUCHI rotary evaporator;

[0071] UPLC-Q / TOF MS mass spectrometry (Synapt G2-Si Q / TOF MS Waters, USA);

[0072] Leica CM1950 cryostat;

[0073] HTX-TM nebulizer (HTX technologies, USA);

[0074] Equipped with MALDI 10kHz SmartBeam TM timsTOF flex mass spectrometer with 3D UV laser ion source;

[0075] timsControl (version 4.0.4, Bruker Daltonics) data acquisition software;

[0076] Data Analysis (version 5.3, Bruker Daltonics) data processing software;

[0077] flexImaging (version 7.1, Bruker Daltonics) imaging analysis software;

[0078] SCiLS Lab software (2024a Pro, Bruker Daltonics) imaging analysis software.

[0079] Animal tissue sections

[0080] Mouse tissues were obtained from 6-week-old male C57 BL / 6 mice, and heart, liver, spleen, lung, kidney, and brain tissues were collected. After resection, the desired tissue area was selected, and the tissue was quickly embedded in 2% CMC-Na solution and slowly frozen in -80°C isopentane (about 3-5 minutes). The samples were stored at -80°C and cut into 15μm thick slices before detection; the tissue slices were thawed and mounted on microscope slides and stored in a sealed container at -80°C. Frozen mouse tissues were cut into 15μm thickness using a cryostat and attached to a conductive indium tin oxide (ITO) slide. Used for derivatization analysis. Before analysis, the slices were dried in a vacuum for 10 minutes.

[0081] Mass spectrometry imaging sample preparation and data acquisition

[0082] Before amino derivatization, the tissue sections were dried in vacuum for 10 min. DCT probe 15 mM (8.4 mg) was dissolved in 1 mL 70% ACN containing 1% 0.1 M NaHCO3 and the reagent was sprayed on the dried tissue sections using HTX TM-Sprayer. The spray temperature was set to 55 °C, the flow rate was 80 μL / min, the linear nozzle speed was 120 cm / min, the track spacing was 3 mm, the nozzle height was 40 mm, the nitrogen pressure was 10 psi and the flow rate was 10 psi. The spray was applied 3 times, the drying time was 10 s, and the density was about 0.6 μg / mm2. After spraying, the sections were incubated in a steam chamber at 60 °C in MeOH:H2O (1:1, v / v) solution for 30 min (drying for 2 min every 10 min to avoid delocalization). Subsequently, 10 mg of the matrix CHCA was dissolved in 1 mL of 70% ACN containing 0.1% TFA. The spraying temperature was set to 75 °C and the flow rate was 120 μL / min. Other parameters were consistent with the DCT probe. The spraying was performed 4 times with a drying time of 0 s. The density was about 1.3 μg / mm 2 After the above treatment, the tissue sections were ready for MALDI MS data acquisition and analysis.

[0083] All MALDI MS experiments were performed on a timsTOF flex instrument equipped with a 10 kHz SmartBeam TM The 3D UV laser source performed MALDI ionization in positive mode. The MALDI laser was set to a frequency of 10,000 Hz, 50% laser power, 400 cumulative laser shots, a spatial resolution of 50 μm for data acquisition, and acquisition in the mass range of m / z 50-1200 Da. Agilent tuning mixtures were used to calibrate the instrument. MALDI-Q-TOF MS and MS / MS data were analyzed using DataAnalysis (version 5.3, Bruker Daltonics, Germany). These parameters of the ion source and mass spectrometer were set using timsControl (version 4.0.4, Bruker Daltonics, Germany), and optical images of tissue sections were uploaded to flexImaging (version 7.1, Bruker Daltonics, Germany) to divide the scan range. MALDI-MSI raw data were analyzed by SCiLS Lab (2024a Pro, Bruker Daltonics, Germany). The root mean square was used as the normalization method.

[0084] Experimental Example 1 Screening of Reactive Groups and Enhanced Detection Signal of Conjugated Fluorophores

[0085] In order to achieve targeted analysis of amino metabolites in mass spectrometry imaging experiments, the MALDI derivatization reagents used in tissue samples must have sufficient reactivity and good selectivity. The inventors used the chemical principle that pyrylium salts easily and selectively react with primary amines to form pyridinium cations, and designed a series of probes containing permanent pyridine nitrogen positive salts based on 2,4,6-TPP, introducing a series of reactive groups. The reactive groups known to react with amino groups were selected, and the specific experiment was as follows (experimental process as shown in the figure). Figure 1 ): The inventors intend to select reactive groups reported in the design of classical covalent inhibitors including N-hydroxysuccinimide lactone, fluoropyridine, sulfonyl chloride and acyl chloride (structural formula such as Figure 2 ) was used as a fragment of MALDI derivatization reagent for detecting amino metabolites. Five derivatization reagents were designed and synthesized (reaction mechanism as shown in Figure 3 ), the synthesis process is as described above (structure and spectral data as above).

[0086] The inventors first evaluated the reaction efficiency of the warhead with the representative substrate glycine ( Figure 4 A). As a result, among all the probes, DCT has the fastest and most thorough reaction rate, and the yield is close to 100% at 5 minutes. For other probes, CSP reaches a maximum yield of 96% at 2 hours; TPP reaches a maximum yield of 82% at 2 hours; and FTB reaches a maximum yield of 46% at 2 hours. However, we also found that the probe CBP with acyl chloride as the reactive warhead is not suitable for MALDI MS analysis because it is insoluble in non-protonic solvents and reacts with the solvent in protic solvents. After the above evaluation, the probe DCT with a high-efficiency reactive warhead performed the best.

[0087] From the above, we know that the N-hydroxysuccinimide ester DCT probe with a highly efficient reactive group has very high reliability and selectivity in the design of derivatization reagents for amino metabolites, and therefore it has been identified as the reactive group probe for the next step of development.

[0088] The inventors further compared the sensitivity of each probe to DA of aromatic amino groups or Gly-derivatized products of non-aromatic amino groups. The results showed that the response of DCT to the derivatized products of DA and Gly was the highest ( Figure 4 B).

[0089] In summary, the DCT probe has a very high sensitivity in the design of derivatization reagents for amino metabolites and was therefore determined as the probe to be used in the final reaction.

[0090] Experimental Example 2DCT achieves full coverage detection of common amino acids

[0091] After determining DCT as the optimal probe, the inventors explored whether DCT could achieve full coverage of derivatization detection of 20 common amino acids in biological samples and compared it with the original probe TPP. The inventors sprayed the probes DCT and TPP on mouse tissues including heart, liver, spleen, lung, kidney, and brain, and then sprayed the auxiliary universal matrix CHCA to observe and compare the differences in the types of amino acids ( Figures 5 to 10 ).

[0092] The results showed that, since DCT has good reactivity to both primary and secondary amines, the inventors achieved in-situ detection of all essential amino acids on tissue samples for the first time, and the actual measured m / z values ​​of all amino acid derivatization products were less than 10 ppm, and the secondary mass spectrometry fragments were consistent with the standard (see Table 1 below). DCT obtained better positive ion detection signals for amino acid derivatization products than TPP.

[0093] Table 1 Measured amino acid values ​​and secondary fragment information of liver tissue and standard products

[0094]

[0095] Based on the above analysis strategy, it is shown that the compounds disclosed in the present invention have a good sensitization effect on amino group detection and are good amino acid derivatization reagents.

[0096] However, the present disclosure is not limited to the above embodiments, which does not mean that the present disclosure must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement to the present disclosure, equivalent replacement of raw materials of the present product, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present disclosure.

[0097] The preferred embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0098] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

Claims

1. A compound represented by formula (I), G is a chloride ion or a tetrafluoroborate ion.

2. An amino sensitizing reagent for mass spectrometry detection, comprising a compound represented by formula (I), G is a chloride ion or a tetrafluoroborate ion.

3. The sensitizing reagent according to claim 2, which is a sensitizing reagent for sensitizing the detection of 20 common amino acids in matrix-assisted laser desorption ionization mass spectrometry (MALDI).

4. A mass spectrometry detection sensitivity enhancement kit, comprising a compound represented by formula (I), G is a chloride ion or a tetrafluoroborate ion.

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