Stable isotope labeling reagent for amino / azide / carboxyl compound as well as preparation method and application of stable isotope labeling reagent

Abstract

The invention relates to the technical field of biochemical analysis, in particular to a stable isotope labeling reagent for amino / azide / carboxyl compounds as well as a preparation method and application of the stable isotope labeling reagent. The stable isotope labeling reagent is structurally characterized in that one end of the stable isotope labeling reagent is carboxyl, alkynyl and hydrazide groups and is connected with reactive groups of a target object, and the other end of the stable isotope labeling reagent is dimethyl-substituted tertiary amino, so that high-strength fragment ions are generated, and meanwhile, groups with different stable isotopes and poor quality are realized; therefore, the interference of matrix effect on quantification is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a stable isotope labeling reagent for amino / azide / carboxyl compounds, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is widely used in research fields such as life analysis, environmental testing, and food evaluation due to its high sensitivity and selectivity. The selected reaction monitoring (SRM) or multi-stage reaction ion monitoring (MRM) mode of HPLC-MS / MS can selectively monitor targeted and semi-targeted analytes in samples. However, the existence of matrix effects greatly reduces the analytical performance of HPLC-MS / MS.

[0004] In recent years, stable isotope coding technology has received great attention, especially stable isotope coding derivatization technology. With the help of light / heavy isotope coding derivatization reagents, isotope derivatives of a class of analytes with the same functional groups can be obtained. It usually uses heavy isotope derivatives as internal standards for HPLC-MS / MS quantitative analysis. However, most of the current labeling reagents are expensive and have a low degree of commercialization. In addition, there are low signal of labeled products, poor accuracy and sensitivity, etc., which limits their widespread use. Based on this, it is of great significance to develop a group of new stable isotope labeling reagents. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a stable isotope labeling reagent for amino / azide / carboxyl compounds and a preparation method and application thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a compound represented by formula (I), formula (II) and formula (III);

[0008]

[0009] In formula (Ⅰ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅱ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅲ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

[0010] The second aspect of the present invention provides a method for preparing the compound represented by formula (I), comprising the following steps:

[0011] (1) 8-amino-2-naphthol reacts with methyl bromoacetate in an alkaline environment to synthesize compound 1;

[0012] (2) Compound 1 is hydrolyzed under alkaline conditions to synthesize compound 2;

[0013] (3) Compound 2 is reacted with formaldehyde (CH2O) or isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain a compound represented by formula (I);

[0014] The structural formula of compound 1 is shown below:

[0015]

[0016] The structural formula of compound 2 is shown below:

[0017]

[0018] The third aspect of the present invention provides a method for preparing the compound represented by formula (II), comprising the following steps:

[0019] The compound represented by formula (I) undergoes a condensation reaction with propargylamine to obtain a compound represented by formula (II).

[0020] The fourth aspect of the present invention provides a method for preparing a compound represented by formula (III), comprising the following steps:

[0021] (1) Compound 1 reacts with formaldehyde (CH2O) or isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain compound 3;

[0022] (2) Compound 3 reacts with hydrazine hydrate to obtain a compound represented by formula (III);

[0023] The structural formula of compound 1 is shown below:

[0024]

[0025] The structural formula of compound 3 is shown below:

[0026]

[0027] In compound 3, the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

[0028] The fifth aspect of the present invention provides the use of the compound represented by formula (I) as a stable isotope labeling reagent for amino compounds.

[0029] When both R1 and R2 in formula (I) are -CH3, the compound represented by formula (I) is a stable isotope labeling reagent for light amino compounds.

[0030] When both R1 and R2 in formula (I) are -CHD2, the compound represented by formula (I) is a stable isotope labeling reagent for medium-sized amino compounds.

[0031] When both R1 and R2 in formula (I) are -CD3, the compound represented by formula (I) is a stable isotope labeling reagent for heavy amino compounds.

[0032] The sixth aspect of the present invention provides the use of the compound represented by formula (II) as a stable isotope labeling reagent for azide compounds.

[0033] When both R1 and R2 in formula (II) are -CH3, the compound represented by formula (II) is a stable isotope labeling reagent for light azide compounds.

[0034] When both R1 and R2 in formula (II) are -CHD2, the compound represented by formula (II) is a stable isotope labeling reagent for medium-sized azide compounds.

[0035] When both R1 and R2 in formula (II) are -CD3, the compound represented by formula (II) is a stable isotope labeling reagent for heavy azide compounds.

[0036] The seventh aspect of the present invention provides the use of the compound represented by formula (III) as a stable isotope labeling reagent for carboxyl compounds.

[0037] When both R1 and R2 in formula (III) are -CH3, the compound represented by formula (III) is a stable isotope labeling reagent for light carboxyl compounds.

[0038] When both R1 and R2 in formula (III) are -CHD2, the compound represented by formula (III) is a stable isotope labeling reagent for medium-sized carboxyl compounds.

[0039] When both R1 and R2 in formula (III) are -CD3, the compound represented by formula (III) is a stable isotope labeling reagent for heavy carboxyl compounds.

[0040] The eighth aspect of the present invention provides a stable isotope labeling reagent, including a compound represented by formula (I) or a compound represented by formula (II) or a compound represented by formula (III).

[0041] The beneficial effects of the present invention are:

[0042] (1) The structural characteristics of the stable isotope labeling reagent provided by the present invention are that one end is a carboxyl group, an alkynyl group and a hydrazide group, which is connected to the reactive group of the target substance, and the other end is a dimethyl-substituted tertiary amine group to produce high-intensity fragment ions and simultaneously realize groups with different mass differences of stable isotopes, thereby reducing the interference of matrix effects on quantification.

[0043] (2) The stable isotope labeling reagent provided by the present invention has a moderate molecular weight, a relatively simple structure, relatively stable properties, and is easy to operate. The labeling reaction conditions are mild, and the sample does not need to be purified by chromatographic separation, thereby reducing operational errors.

[0044] (3) The stable isotope labeling reagent provided by the present invention can reduce noise to a certain extent, improve the signal-to-noise ratio, thereby improving sensitivity and ensuring the effectiveness of mass spectrometry signal acquisition. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] Figure 1 is the mass spectrum of compound 3-[d0];

[0047] Figure 2 The NMR of compound 3-[d0] 1 HNMR spectra;

[0048] Figure 3 is the mass spectrum of compound 3-[d6];

[0049] Figure 4 The NMR of compound 3-[d6] 1 HNMR spectra;

[0050] Figure 5 is the mass spectrum of compound 4-[d0];

[0051] Figure 6 The NMR of compound 4-[d0] 1 HNMR spectra;

[0052] Figure 7 is the mass spectrum of compound 4-[d6];

[0053] Figure 8 The NMR of compound 4-[d6] 1 HNMR spectra;

[0054] Fig. 9 is the mass spectrum of compound 6-[d0];

[0055] Fig.10 The NMR of compound 6-[d0] 1 HNMR spectra;

[0056] Fig.11 is the mass spectrum of compound 6-[d6];

[0057] Fig.12 The NMR of compound 6-[d6] 1 HNMR spectra;

[0058] Fig.13 The figure is a diagram showing the effect of using the compound represented by formula (II) as a stable isotope labeling reagent for azide compounds, wherein a is a mass spectrum of a blank control group, b, c, and d are respectively the mass spectra of a polypeptide modified with azidoacetic acid at the N-terminus after competition with DTT, and a polypeptide modified with azidoacetic acid at the N-terminus labeled with stable isotope chemically by compounds 4-[d0] and 4-[d6];

[0059] Fig.14 The figure shows the effect of using the compound represented by formula (III) as a stable isotope labeling reagent for carboxyl compounds, wherein a, b, c, and d are respectively the mass spectra of a polypeptide containing an exposed carboxyl group at the C-terminus, and the mass spectra of a polypeptide containing an exposed carboxyl group at the C-terminus labeled with stable isotope chemically of compounds 6-[d0], 6-[d4], and 6-[d6]. DETAILED DESCRIPTION

[0060] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0061] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0062] The first typical embodiment of the present invention provides a compound represented by formula (I), formula (II) and formula (III);

[0063]

[0064] In formula (Ⅰ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅱ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅲ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

[0065] A second typical embodiment of the present invention provides a method for preparing the compound represented by formula (I), comprising the following steps:

[0066] (1) 8-amino-2-naphthol reacts with methyl bromoacetate in an alkaline environment to synthesize compound 1;

[0067] (2) Compound 1 is hydrolyzed under alkaline conditions to synthesize compound 2;

[0068] (3) Compound 2 is reacted with formaldehyde (CH2O) or isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain a compound represented by formula (I);

[0069] The structural formula of compound 1 is shown below:

[0070]

[0071] The structural formula of compound 2 is shown below:

[0072]

[0073] In one or more embodiments, in step (1), the molar ratio of 8-amino-2-naphthol to methyl bromoacetate is 1:(1.1-1.35).

[0074] In one or more embodiments, in step (1), the reaction temperature is 57 to 65° C., preferably 60° C.; and the reaction time is 12 to 16 hours.

[0075] In one or more embodiments, in step (2), the reaction temperature is 65 to 80° C., preferably 70° C.; the reaction time is 2.5 to 4 hours, preferably 3 hours.

[0076] In one or more embodiments, in step (3), compound 2 reacts with formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN) to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CH3.

[0077] Preferably, the compound represented by formula (I) is as follows:

[0078]

[0079] In one or more embodiments, in step (3), compound 2 is reacted with isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CHD2.

[0080] Preferably, the compound represented by formula (I) is as follows:

[0081]

[0082] In one or more embodiments, in step (3), compound 2 reacts with isotope-substituted formaldehyde (CD2O) and sodium cyanoborodeuteride (NaBD3CN) to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CD3.

[0083] Preferably, the compound represented by formula (I) is as follows:

[0084]

[0085] In one or more embodiments, in step (3), the molar ratio of compound 2 to CH2O or CD2O, NaBH3CN or NaBD3CN is 1:(3-6):(1.2-1.6).

[0086] A third typical embodiment of the present invention provides a method for preparing a compound represented by formula (II), comprising the following steps:

[0087] The compound represented by formula (I) undergoes a condensation reaction with propargylamine to obtain a compound represented by formula (II).

[0088] In one or more embodiments, 1-hydroxybenzotriazole (HOBt) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (1-ethyl-(3-dimethylaminopropyl)carbodiimide) are used as condensation agents during the condensation reaction.

[0089] Preferably, the molar ratio of the compound represented by formula (I) to 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl)carbodiimide and propargylamine is 1:(1.1-1.35):(1.25-1.45).

[0090] A fourth typical embodiment of the present invention provides a method for preparing a compound represented by formula (III), comprising the following steps:

[0091] (1) Compound 1 reacts with formaldehyde (CH2O) or isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain compound 3;

[0092] (2) Compound 3 reacts with hydrazine hydrate to obtain a compound represented by formula (III);

[0093] The structural formula of compound 1 is shown below:

[0094]

[0095] The structural formula of compound 3 is shown below:

[0096]

[0097] In compound 3, the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

[0098] In one or more embodiments, in step (1), compound 1 reacts with formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN) to obtain compound 3, in which the substituents R1 and R2 have the same structure, both of which are -CH3.

[0099] Preferably, the compound represented by compound 3 is as follows:

[0100]

[0101] In one or more embodiments, in step (1), compound 1 is reacted with isotope-substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) to obtain compound 3, in which R1 and R2 substituents have the same structure, both of which are -CHD2.

[0102] Preferably, the compound represented by compound 3 is as follows:

[0103]

[0104] In one or more embodiments, in step (1), compound 1 reacts with isotope-substituted formaldehyde (CD2O) and sodium cyanoborodeuteride (NaBD3CN) to obtain compound 3, in which the substituents R1 and R2 have the same structure, both of which are -CD3.

[0105] Preferably, the compound represented by compound 3 is as follows:

[0106]

[0107] In one or more embodiments, in step (1), the molar ratio of compound 1 to CH2O or CD2O, NaBH3CN or NaBD3CN is 1:(3-6):(1.2-1.6).

[0108] In one or more embodiments, in step (2), the molar ratio of compound 3 to hydrazine hydrate is 1:23-25, preferably 1:24.

[0109] In one or more embodiments, in step (2), the temperature for the reaction of compound 3 with hydrazine hydrate is 65-75° C., preferably 70° C., and the reaction time is 2.5-4 h, preferably 3 h.

[0110] A fifth typical embodiment of the present invention provides the use of the compound represented by formula (I) as a stable isotope labeling reagent for amino compounds.

[0111] When both R1 and R2 in formula (I) are -CH3, the compound represented by formula (I) is a stable isotope labeling reagent for light amino compounds.

[0112] When both R1 and R2 in formula (I) are -CHD2, the compound represented by formula (I) is a stable isotope labeling reagent for medium-sized amino compounds.

[0113] When both R1 and R2 in formula (I) are -CD3, the compound represented by formula (I) is a stable isotope labeling reagent for heavy amino compounds.

[0114] A sixth typical embodiment of the present invention provides the use of the compound represented by formula (II) as a stable isotope labeling reagent for azide compounds.

[0115] When both R1 and R2 in formula (II) are -CH3, the compound represented by formula (II) is a stable isotope labeling reagent for light azide compounds.

[0116] When both R1 and R2 in formula (II) are -CHD2, the compound represented by formula (II) is a stable isotope labeling reagent for medium-sized azide compounds.

[0117] When both R1 and R2 in formula (II) are -CD3, the compound represented by formula (II) is a stable isotope labeling reagent for heavy azide compounds.

[0118] In one or more embodiments, the application includes: application in labeling a polypeptide in which the N-terminus is modified with an azidoacetic acid group.

[0119] Preferably, the application includes:

[0120] (1) A peptide chain modified with azidoacetic acid at the N-terminus is added to a gold nanoparticle (AuNPs) solution. After the reaction, the unbound peptide is removed by centrifugation;

[0121] (2) adding a compound represented by formula (II) for stable isotope labeling;

[0122] (3) After centrifugation, dithiothreitol (DTT) is added thereto. After the reaction, the supernatant is collected by centrifugation and then diluted for mass spectrometry detection.

[0123] Further preferably, in step (1), the size of the gold nanoparticles is 13±2 nm.

[0124] Further preferably, in step (1), the gold nanoparticle solution contains a surfactant with a final concentration of 0.5-1.5% (mass fraction), preferably, the surfactant is sodium dodecyl sulfate (SDS). Adding a surfactant can prevent aggregation between gold nanoparticles, thereby improving the stability of gold nanoparticles.

[0125] Further preferably, in step (2), a stable isotope labeling reagent for a light azide compound and a stable isotope labeling reagent for a heavy azide compound are added.

[0126] Further preferably, in step (3), during the mass spectrometry detection, a Thermo Scientific Q-Exactive Orbitrap mass spectrometer is used, data is collected in positive ion mode, and the mobile phase is a methanol-water solution (containing 0.1% formic acid) in a volume ratio of 1:1.

[0127] A seventh typical embodiment of the present invention provides the use of the compound represented by formula (III) as a stable isotope labeling reagent for carboxyl compounds.

[0128] When both R1 and R2 in formula (III) are -CH3, the compound represented by formula (III) is a stable isotope labeling reagent for light carboxyl compounds.

[0129] When both R1 and R2 in formula (III) are -CHD2, the compound represented by formula (III) is a stable isotope labeling reagent for medium-sized carboxyl compounds.

[0130] When both R1 and R2 in formula (III) are -CD3, the compound represented by formula (III) is a stable isotope labeling reagent for heavy carboxyl compounds.

[0131] In one or more embodiments, the application includes: application in labeling a polypeptide having an exposed carboxyl group at the C-terminus.

[0132] Preferably, the application includes:

[0133] A mixed solvent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / (1-hydroxy-7-azabenzotriazole) (HOAT) / 2-(N-morpholine)ethanesulfonic acid monohydrate (MES) was added to the polypeptide salt sample, and then the compound represented by formula (III) was added. After the reaction, mass spectrometry analysis was performed.

[0134] Further preferably, the volume ratio of EDC to HOAT and MES in a mixed solvent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) / (1-hydroxy-7-azabenzotriazole) (HOAT) / 2-(N-morpholine)ethanesulfonic acid monohydrate (MES) is (0.8-1.2):(0.8-1.2):(6.5-8), preferably 1:1:7.

[0135] Further preferably, a stable isotope labeling reagent for a light carboxyl compound, a stable isotope labeling reagent for a medium carboxyl compound, and a stable isotope labeling reagent for a heavy carboxyl compound are added.

[0136] Further preferably, during the mass spectrometry detection, a Thermo Scientific Q-Exactive Orbitrap mass spectrometer is used to collect data in the positive ion mode, and the mobile phase is a methanol-water solution (containing 0.1% formic acid) with a volume ratio of 1:1.

[0137] An eighth typical embodiment of the present invention provides a stable isotope labeling reagent, including a compound represented by formula (I) or a compound represented by formula (II) or a compound represented by formula (III).

[0138] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0139] Example 1

[0140] Preparation of the compound represented by formula (I):

[0141] The specific synthesis route is as follows:

[0142]

[0143] (1) In a 100 mL round-bottom flask, 8-amino-2-naphthol (0.84 mmol, 133.73 mg) and K2CO3 (1.74 mmol, 240.00 mg) were dissolved in 30 mL of acetone, and methyl bromoacetate (1.18 mmol, 112 μL) was added to the mixed solution. After stirring evenly, reflux and stir at 60°C for 12 h. The solution was cooled to room temperature, filtered, and concentrated under reduced pressure. Extracted with ethyl acetate, washed with water and saturated sodium chloride solution, dried over anhydrous Na2SO4, and then spin-dried. The crude product was separated by a chromatographic column to obtain compound 1.

[0144] Compound 1 (0.30 mmol, 69.37 mg) was placed in a 25 mL round-bottom flask, methanol (6 mL) was added to fully dissolve it, and then NaOH solution (2 mL, 2 M) was added. The reaction solution was stirred at 70 ° C for 3 h. The solution was concentrated under reduced pressure, and the residue was dissolved in H2O and acidified with hydrochloric acid. The sample was centrifuged to collect the lower precipitate to obtain compound 2.

[0145] (2) When both R1 and R2 are -CH3:

[0146] Compound 2 (0.36mmol, 80.00mg) was placed in a 25mL round-bottom flask, anhydrous methanol was added to fully dissolve it, and then 800μL 7.4% CH2O and 1200μL NaBH3CN (0.6mol / L) were added, and the pH was adjusted to 5-6 with formic acid (FA), and the reaction was stirred for 1h. After column chromatography, compound 3-[d0] (i.e., both R1 and R2 in formula (Ⅰ) are -CH3) was obtained. Compound 3-[d0] is a stable isotope labeling reagent for light amino compounds. The mass spectrum of compound 3-[d0] is shown in Figure 1 As shown, NMR 1 HNMR images Figure 2 shown.

[0147] (3) Both R1 and R2 are -CHD2:

[0148] Compound 2 (0.32 mmol, 80.00 mg) was placed in a 25 mL round-bottom flask, anhydrous methanol was added to dissolve it, and then 250 μL 20% CD2O and 1000 μL NaBH3CN (0.6 mol / L) were added, pH was adjusted to 5-6 with FA, and the mixture was reacted on a magnetic stirrer for 1.5 h. After column chromatography, compound 3-[d4] (i.e., both R1 and R2 in formula (I) are -CHD2) was obtained. Compound 3-[d4] is a stable isotope labeling reagent for medium-sized amino compounds.

[0149] (4) Both R1 and R2 substituents are -CD3:

[0150] Take compound 2 (0.36mmol, 80.00mg) in a 25mL round-bottom flask, add 8mL methanol to fully dissolve it, then add 210μL 20% CD2O and 980μL NaBD3CN (0.6mol / L), adjust pH to 5-6 with FA, and react on a magnetic stirrer for 2h. After column chromatography, compound 3-[d6] (i.e., both R1 and R2 substituents in formula (Ⅰ) are -CD3) is obtained. Compound 3-[d6] is a stable isotope labeling reagent for heavy amino compounds. The mass spectrum of compound 3-[d6] is shown in Figure 3 As shown, NMR 1 HNMR images Figure 4 shown.

[0151] Example 2

[0152] Preparation of the compound represented by formula (II):

[0153] The specific synthetic route is as follows:

[0154]

[0155] (1) Both R1 and R2 are -CH3:

[0156] Compound 3-[d0] (78.75 mg, 0.32 mmol) was added to a solution of HOBt (48.00 mg, 0.35 mmol) and EDC (68.40 mg, 0.35 mmol) in anhydrous DMF, propargylamine (25 μL, 0.40 mmol) was added, and the reaction solution was stirred at room temperature for 0.5 h. N,N-diisopropylethylamine (DIEA) (160 μL, 0.97 mmol) was added at 0°C and stirring was continued for 0.5 h. Finally, stirring was continued at ambient temperature for 13 h. After the reaction was completed, extraction with ethyl acetate was performed, the mixture was washed with water and brine, the organic layers were combined, dried and then spin-dried. The crude product was separated by a chromatographic column to obtain compound 4-[d0] (i.e., both R1 and R2 substituents in formula (II) are -CH3). Compound 4-[d0] is a stable isotope labeling reagent for light azide compounds. The mass spectrum of compound 4-[d0] is shown in FIG. Figure 5 As shown, NMR 1 HNMR images Figure 6 shown.

[0157] (2) Both R1 and R2 are -CHD2:

[0158] The same preparation method as in step (1) is used to replace compound 3-[d0] with compound 3-[d4] to obtain compound 4-[d4] (i.e., both R1 and R2 substituents in formula (II) are -CHD2). Compound 4-[d4] is a stable isotope labeling reagent for medium-sized azide compounds.

[0159] (3) Both R1 and R2 are -CD3:

[0160] The same preparation method as in step (1) is used to replace compound 3-[d0] with compound 3-[d6] to obtain compound 4-[d6] (i.e., both R1 and R2 in formula (II) are -CD3). Compound 4-[d6] is a stable isotope labeling reagent for heavy azide compounds. The mass spectrum of compound 4-[d6] is shown in FIG. Figure 7 As shown, NMR 1 HNMR images Figure 8 shown.

[0161] Example 3

[0162] Preparation of the compound represented by formula (III):

[0163] The specific synthesis route is as follows:

[0164]

[0165] (1) Compound 5-[d0], Compound 5-[d4], and Compound 5-[d6] were synthesized by the same method as in steps (2), (3), and (4) of Example 1, except that the raw material Compound 2 was replaced by Compound 1.

[0166] (2) Compound 5-[d0] was dissolved in methanol, hydrazine hydrate (500 μL, 7.5 mmol) was added and mixed evenly, and then refluxed and stirred at 70°C for 3 h. The solution was cooled to room temperature and concentrated under vacuum. The mixture was extracted with dichloromethane, washed with deionized water, dried over anhydrous Na2SO4, and the crude product was purified by silica gel chromatography to obtain compound 6-[d0] (i.e., both R1 and R2 in formula (III) are -CH3). Compound 6-[d0] is a stable isotope labeling reagent for light carboxyl compounds. The mass spectrum of compound 6-[d0] is shown in FIG. Fig. 9 As shown, NMR 1 HNMR images Fig.10 shown.

[0167] (3) Using the same preparation method as step (2) in this example, compound 5-[d0] is replaced by compound 5-[d4] to synthesize compound 6-[d4]. Compound 6-[d4] is a stable isotope labeling reagent for a medium-sized carboxyl compound.

[0168] (4) Using the same preparation method as step (2) in this example, compound 5-[d0] was replaced by compound 5-[d6] to synthesize compound 6-[d6], where compound 6-[d6] is a stable isotope labeling reagent for heavy carboxyl compounds. The mass spectrum of compound 6-[d6] is shown in Fig.11 As shown, NMR 1 HNMR images Fig.12 shown.

[0169] Experimental Example 1

[0170] Application of the compound represented by formula (I) as a stable isotope labeling reagent for amino compounds:

[0171] The stable isotope labeling reagent described in the first aspect of the present invention has successfully labeled the amino group in propargylamine.

[0172] (1) In the above-mentioned Example 2, the carboxyl group in formula (I) undergoes a condensation reaction with the amino group in propargylamine to obtain formula (II). The synthesis method is not described in detail here.

[0173] (2) The results are as follows Figure 5 and Figure 7 As shown, compound 3-[d0] and compound 3-[d6] reacted with propargylamine to remove one molecule of H2O, indicating that the condensation was successful. Figure 1 and Figure 3 Compared with the shown Figure 5 and Figure 7 The spectrum shown has fewer impurity peaks and lower mass spectrometry signals.

[0174] Experimental Example 2

[0175] Application of the compound represented by formula (II) as a stable isotope labeling reagent for azide compounds:

[0176] (1) Take 1mL of AuNPs (13±2nm) (containing 0.1% SDS) solution and add it to the sample bottle. Stir at room temperature for 0.5h, then add 100μL of 1mM N-terminally modified peptide solution with azidoacetic acid. After 36h of reaction, centrifuge and add light and heavy stable isotope labeling reagents (final concentration is 200μM), namely 4-[d0] and 4-[d6]. The stable isotope labeling process is carried out by Click reaction. After centrifugation, an excess of dithiothreitol (DTT) is added to it to compete with the labeled peptide, and stir at room temperature overnight. Finally, centrifuge and take the supernatant, dilute and enter mass spectrometry detection. The amino acid sequence of the N-terminally modified peptide with azidoacetic acid is azidoacetic acid-Lys-Glu-Ala-Met-Ile-Glu-Glu-Pro-Ala-mercaptoethylamine.

[0177] (2) During mass spectrometry, a Thermo Scientific Q-Exactive Orbitrap mass spectrometer was used to collect data in the positive ion mode, and the mobile phase was a 1:1 volume ratio of methanol-water (containing 0.1% formic acid). Fig.13 As shown, when the peptide modified with azidoacetic acid at the N-terminus is detected alone, there are many miscellaneous peaks around it. However, after stable isotope labeling, the detected spectrum presents a relatively clean mass spectrum with good stable isotope characteristic peaks.

[0178] Experimental Example 3

[0179] Application of the compound represented by formula (III) as a stable isotope labeling reagent for carboxyl compounds:

[0180] (1) Since the hydrazide group does not react directly with carboxylic acid, EDC and HOAT are used to activate the carboxylic acid to form a stable labeled product. For the labeling of the peptide with an exposed carboxyl group at the C-terminus, the peptide solution (50 μL, 5 mM) was placed in a sample bottle, and then a mixed solvent of EDC / HOAT / MES with a volume ratio of 1:1:7 was added, in which the concentrations of EDC and HOAT were both 50 mM, and then light, medium, and heavy stable isotope labeling reagents (100 μL, 10 mM), namely 6-[d0], 6-[d4], and 6-[d6], were added. After mixing evenly, the solution was shaken at 20°C for 3 h. Finally, it was concentrated and re-dissolved and stored at 4°C. The amino acid sequence of the peptide with an exposed carboxyl group at the C-terminus is NH2-Gly-Phe-Lys-Gly-COOH.

[0181] (2) Mass spectrometry analysis conditions were the same as those described in Experimental Example 2. Fig.14 As shown, the exposed carboxyl group at the C-terminus of the polypeptide reacted with the light, medium and heavy stable isotope labeling reagents, and produced mass spectrometry peaks with increased masses of 241Da, 245Da and 247Da. After comparative analysis, it was found that the mass spectrometry signal intensity of the labeled peptide segment was significantly enhanced, while the noise was reduced and the signal-to-noise ratio was improved, which reflects the excellent performance of the stable isotope labeling reagent designed by the present invention in detecting polypeptides.

Claims

1. A compound represented by formula (I), formula (II) and formula (III); In formula (Ⅰ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅱ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3; in formula (Ⅲ), the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

2. The method for preparing the compound represented by formula (I) according to claim 1, characterized in that: The steps include: (1) 8-amino-2-naphthol reacts with methyl bromoacetate in an alkaline environment to synthesize compound 1; (2) Compound 1 is hydrolyzed under alkaline conditions to synthesize compound 2; (3) Compound 2 is reacted with formaldehyde or isotope-substituted formaldehyde and sodium cyanoborohydride or sodium cyanoborodeuteride to obtain a compound represented by formula (I); The structural formula of compound 1 is shown below: The structural formula of compound 2 is shown below:

3. The preparation method according to claim 2, characterized in that: In step (3), compound 2 reacts with formaldehyde and sodium cyanoborohydride to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CH3; preferably, the compound represented by formula (I) is as follows: Alternatively, in step (3), compound 2 is reacted with isotope-substituted formaldehyde and sodium cyanoborohydride to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CHD2; preferably, the compound represented by formula (I) is as follows: Alternatively, in step (3), compound 2 reacts with isotope-substituted formaldehyde and sodium cyanoborodeuteride to obtain a compound represented by formula (I), wherein the substituents R1 and R2 in formula (I) have the same structure, both of which are -CD3; preferably, the compound represented by formula (I) is as follows:

4. The method for preparing the compound represented by formula (II) according to claim 1, characterized in that: The steps include: The compound represented by formula (I) undergoes a condensation reaction with propargylamine to obtain a compound represented by formula (II).

5. The method for preparing the compound represented by formula (III) according to claim 1, characterized in that: The steps include: (1) Compound 1 reacts with formaldehyde or isotope-substituted formaldehyde and sodium cyanoborohydride or sodium cyanoborodeuteride to obtain compound 3; (2) Compound 3 reacts with hydrazine hydrate to obtain a compound represented by formula (III); The structural formula of compound 1 is shown below: The structural formula of compound 3 is shown below: In compound 3, the two substituents R1 and R2 have the same structure, and R1 and R2 are selected from one of -CH3, -CHD2 or -CD3.

6. The preparation method according to claim 5, characterized in that: In step (1), compound 1 reacts with formaldehyde and sodium cyanoborohydride to obtain compound 3, wherein the substituents R1 and R2 in compound 3 have the same structure, both of which are -CH3; preferably, the compound represented by compound 3 is as follows: Alternatively, in step (1), compound 1 is reacted with isotope-substituted formaldehyde and sodium cyanoborohydride to obtain compound 3, wherein the substituents R1 and R2 in compound 3 have the same structure, both being -CHD2; preferably, the compound represented by compound 3 is as follows: Alternatively, in step (1), compound 1 reacts with isotope-substituted formaldehyde and sodium cyanoborodeuteride to obtain compound 3, wherein the substituents R1 and R2 in compound 3 have the same structure, both of which are -CD3; preferably, the compound represented by compound 3 is as follows:

7. Use of the compound represented by formula (I) as claimed in claim 1 as a stable isotope labeling reagent for an amino compound; When both R1 and R2 in formula (Ⅰ) are -CH3, the compound represented by formula (Ⅰ) is a stable isotope labeling reagent for light amino compounds; When both R1 and R2 in formula (Ⅰ) are -CHD2, the compound represented by formula (Ⅰ) is a stable isotope labeling reagent for medium-sized amino compounds; When both R1 and R2 in formula (I) are -CD3, the compound represented by formula (I) is a stable isotope labeling reagent for heavy amino compounds.

8. Use of the compound represented by formula (II) according to claim 1 as a stable isotope labeling reagent for azide compounds; When both R1 and R2 in formula (II) are -CH3, the compound represented by formula (II) is a stable isotope labeling reagent for light azide compounds; When both R1 and R2 in formula (II) are -CHD2, the compound represented by formula (II) is a stable isotope labeling reagent for medium-sized azide compounds; When both R1 and R2 in formula (II) are -CD3, the compound represented by formula (II) is a stable isotope labeling reagent for heavy azide compounds; Preferably, the application includes: Application in labeling of polypeptides with N-terminal modified with azidoacetic acid group.

9. Use of the compound represented by formula (III) according to claim 1 as a stable isotope labeling reagent for carboxyl compounds; When both R1 and R2 in formula (III) are -CH3, the compound represented by formula (III) is a stable isotope labeling reagent for light carboxyl compounds; When both R1 and R2 in formula (III) are -CHD2, the compound represented by formula (III) is a stable isotope labeling reagent for medium-sized carboxyl compounds; When both R1 and R2 in formula (III) are -CD3, the compound represented by formula (III) is a stable isotope labeling reagent for heavy carboxyl compounds; Preferably, the application includes: Application of the labeling to peptides containing an exposed carboxyl group at the C-terminus.

10. A stable isotope labeling reagent, characterized in that: It includes a compound represented by formula (I), a compound represented by formula (II), or a compound represented by formula (III).

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