A stable isotope labeling reagent for amino / azido / carboxyl compounds, its preparation method and application

Abstract

The present application relates to the technical field of biochemical analysis, in particular to a stable isotope labeling reagent for amino / azido / carboxyl compounds and a preparation method and application thereof. The stable isotope labeling reagent provided by the present application has the structural characteristics that one end is a carboxyl group, an alkyne group and a hydrazide group, and is connected with a reactive group of a target object, and the other end is a dimethyl-substituted tertiary amine group, so as to generate high-intensity fragment ions, and at the same time, groups with different mass differences of stable isotopes are realized, so as to reduce the interference of matrix effect on quantification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical analysis, and particularly relates to a stable isotope labeling reagent for amino / azido / carboxyl compounds and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already known in this field.

[0003] High performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is widely used in the fields of life analysis, environmental detection, food evaluation, etc. due to its high sensitivity and high selectivity. The selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) mode of HPLC-MS / MS can selectively monitor target and semi-target analytes in samples. However, the presence of matrix effect greatly reduces the analysis performance of HPLC-MS / MS.

[0004] In recent years, stable isotope coding technology has attracted much attention, especially stable isotope coding derivatization technology. By means of light / heavy isotope coding derivatization reagents, isotope derivatives of a class of analytes with the same functional group can be obtained. The heavy isotope derivative is usually used as an internal standard for HPLC-MS / MS quantitative analysis. However, most of the current labeling reagents are expensive and have low commercialization degree. In addition, there are low signal of labeled products, poor accuracy and sensitivity, etc., so their widespread use is limited. Therefore, it is of great significance to develop a new type of stable isotope labeling reagent. SUMMARY

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

[0006] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0007] In a first aspect of the present application, a compound represented by formula (I), formula (II) and formula (III) is provided.

[0008]

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

[0010] In a second aspect of the present application, a preparation method of the compound shown in formula (I) is provided, comprising the following steps:

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

[0012] (2) Compound 1 is hydrolyzed in an alkaline environment to synthesize compound 2;

[0013] (3) Compound 2 is reacted with formaldehyde (CH2O) or isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain the compound shown in formula (I);

[0014] The structural formula of compound 1 is as follows:

[0015]

[0016] The structural formula of compound 2 is as follows:

[0017]

[0018] In a third aspect of the present application, a preparation method of the compound shown in formula (II) is provided, comprising the following steps:

[0019] The compound shown in formula (I) is subjected to condensation reaction with propargylamine to obtain the compound shown in formula (II).

[0020] In a fourth aspect of the present application, a preparation method of the compound shown in formula (III) is provided, comprising the following steps:

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

[0022] (2) Compound 3 is reacted with hydrazine hydrate to obtain the compound shown in formula (III);

[0023] The structural formula of compound 1 is as follows:

[0024]

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

[0026]

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

[0028] In a fifth aspect of the present application, the compound shown in formula (I) is provided as an application of a stable isotope labeled reagent of an amino compound.

[0029] When the two substituents R1 and R2 in formula (I) are both -CH3, the compound shown in formula (I) is a stable isotope labeled reagent of a light amino compound.

[0030] When the two substituents R1 and R2 in formula (I) are both -CHD2, the compound shown in formula (I) is a stable isotope labeled reagent of a medium amino compound.

[0031] When the two substituents R1 and R2 in formula (I) are both -CD3, the compound shown in formula (I) is a stable isotope labeled reagent of a heavy amino compound.

[0032] In a sixth aspect of the present application, the compound shown in formula (II) is provided as an application of a stable isotope labeled reagent of an azide compound.

[0033] When the two substituents R1 and R2 in formula (II) are both -CH3, the compound shown in formula (II) is a stable isotope labeled reagent of a light azide compound.

[0034] When the two substituents R1 and R2 in formula (II) are both -CHD2, the compound shown in formula (II) is a stable isotope labeled reagent of a medium azide compound.

[0035] When the two substituents R1 and R2 in formula (II) are both -CD3, the compound shown in formula (II) is a stable isotope labeled reagent of a heavy azide compound.

[0036] In a seventh aspect of the present application, the compound shown in formula (III) is provided as an application of a stable isotope labeled reagent of a carboxyl compound.

[0037] When the two substituents R1 and R2 in formula (III) are both -CH3, the compound shown in formula (III) is a stable isotope labeled reagent of a light carboxyl compound.

[0038] When the two substituents R1 and R2 in formula (III) are both -CHD2, the compound shown in formula (III) is a stable isotope labeled reagent of a medium carboxyl compound.

[0039] When both R1 and R2 in the formula (III) are -CD3, the compound shown in the formula (III) is a stable isotope labeled reagent of heavy carboxyl compound.

[0040] In an eighth aspect, the present application provides a stable isotope labeled reagent, which comprises the compound shown in the formula (I), the compound shown in the formula (II) or the compound shown in the formula (III).

[0041] The present application has the following advantages:

[0042] (1) The stable isotope labeled reagent provided by the present application has the structural characteristics that one end is connected with the reactive group of the target object through carboxyl, alkyne and hydrazide groups, and the other end is dimethyl-substituted tertiary amine group, so as to generate high-intensity fragment ions and realize the groups with different mass differences of stable isotopes, thereby reducing the interference of matrix effect on quantification.

[0043] (2) The stable isotope labeled reagent provided by the present application has moderate molecular weight, relatively simple structure, stable property, simple operation, mild labeling reaction condition, and no need for sample purification through chromatographic separation, thereby reducing the operation error.

[0044] (3) The stable isotope labeled reagent provided by the present application can reduce noise and improve signal-to-noise ratio to some extent, thereby improving sensitivity and ensuring the effectiveness of mass spectrum signal acquisition. BRIEF DESCRIPTION OF DRAWINGS

[0045] The drawings constituting a part of the present application are used to provide further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

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

[0047] Figure 2 is a nuclear magnetic resonance of compound 3-[d0]; 1 HNMR chart;

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

[0049] Figure 4 is a nuclear magnetic resonance of compound 3-[d6]; 1 HNMR chart;

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

[0051] Figure 6 is a nuclear magnetic resonance of compound 4-[d0]; 1 HNMR chart;

[0052] Figure 7 Mass spectrum of compound 4-[d6];

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

[0054] Figure 9 Mass spectrum of compound 6-[d0];

[0055] Figure 10 NMR of compound 6-[d0] 1 HNMR chart;

[0056] Figure 11 Mass spectrum of compound 6-[d6];

[0057] Figure 12 NMR of compound 6-[d6] 1 HNMR chart;

[0058] Figure 13 Application effect chart of compound of formula (II) as stable isotope labeling reagent of azide compound, wherein a is mass spectrum chart of blank control group, b, c, d are mass spectrum charts of polypeptide with azide acetic acid modified at N terminal after DTT competition, compound 4-[d0] and 4-[d6] stable isotope chemical labeling polypeptide with azide acetic acid modified at N terminal, respectively;

[0059] Figure 14 Application effect chart of compound of formula (III) as stable isotope labeling reagent of carboxyl compound, wherein a, b, c, d are mass spectrum charts of polypeptide containing exposed carboxyl group at C terminal, compound 6-[d0], 6-[d4] and 6-[d6] stable isotope chemical labeling polypeptide containing exposed carboxyl group at C terminal, respectively. DETAILED DESCRIPTION

[0060] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, 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 application belongs.

[0061] It is also important to note that the terms "comprises", "comprising", "includes", "including", "contains", "containing" or variations thereof herein, do not specify an exhaustive or complete list of elements or steps as per the specific embodiment. Unless otherwise defined, 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 application belongs.

[0062] In a first exemplary embodiment of the present application, there is provided a compound of formula (I), formula (II) and formula (III):

[0063]

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

[0065] In a second exemplary embodiment of the present application, there is provided a method for preparing a compound of formula (I), comprising the following steps:

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

[0067] (2) Compound 1 is hydrolyzed in an alkaline environment to synthesize compound 2;

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

[0069] The structural formula of compound 1 is as follows:

[0070]

[0071] The structural formula of compound 2 is as follows:

[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 57-65°C, preferably 60°C; and the reaction time is 12-16h.

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

[0076] In one or more embodiments, in step (3), compound 2 is reacted with formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN) to obtain a compound represented by formula (I), wherein R1 and R2 are the same and 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 isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) to obtain a compound represented by formula (I), wherein R1 and R2 are the same and 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 is reacted with isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) to obtain a compound represented by formula (I), wherein R1 and R2 are the same and are -CHD2.

[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, CH2O or CD2O, NaBH3CN or NaBD3CN is 1: (3-6): (1.2-1.6).

[0086] In a third typical embodiment of the present application, a method for preparing a compound represented by formula (II) is provided, comprising the following steps:

[0087] The compound represented by formula (I) is subjected to 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 (EDC) are used as condensing agents in the condensation reaction.

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

[0090] In a fourth exemplary embodiment of the present application, a method for preparing a compound of formula (III) is provided, comprising the steps of:

[0091] (1) reacting compound 1 with formaldehyde (CH2O) or isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) or sodium cyanoborodeuteride (NaBD3CN) to obtain compound 3;

[0092] (2) reacting compound 3 with hydrazine hydrate to obtain a compound of formula (III);

[0093] The structure of compound 1 is shown below:

[0094]

[0095] The structure of compound 3 is shown below:

[0096]

[0097] In one or more embodiments, in step (1), compound 1 is reacted with formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN) to obtain compound 3, and the two substituents R1and R2in compound 3 are the same and are -CH3.

[0098] In one or more embodiments, in step (1), compound 1 is reacted with formaldehyde (CH2O) and sodium cyanoborohydride (NaBH3CN) to obtain compound 3, and the two substituents R1and R2in compound 3 are the same and are -CH3.

[0099] Preferably, the compound of formula (III) is shown below:

[0100]

[0101] In one or more embodiments, in step (1), compound 1 is reacted with isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBH3CN) to obtain compound 3, and the two substituents R1and R2in compound 3 are the same and are -CHD2.

[0102] Preferably, the compound of formula (III) is shown below:

[0103]

[0104] In one or more embodiments, in step (1), compound 1 is reacted with isotopically substituted formaldehyde (CD2O) and sodium cyanoborohydride (NaBD3CN) to obtain compound 3, wherein R1 and R2 are both -CD3.

[0105] Preferably, compound 3 is as shown below:

[0106]

[0107] In one or more embodiments, in step (1), the molar ratio of compound 1, CH2O or CD2O, and 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 and hydrazine hydrate is 1:23-25, preferably 1:24.

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

[0110] In a fifth typical embodiment of the present application, there is provided a use of a compound represented by formula (I) as a stable isotope-labeled reagent for an amino compound.

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

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

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

[0114] In a sixth typical embodiment of the present application, there is provided a use of a compound represented by formula (II) as a stable isotope-labeled reagent for an azido compound.

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

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

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

[0118] In one or more embodiments, the application comprises the application in the labeling of polypeptides modified at the N-terminus with azidoacetic acid groups.

[0119] Preferably, the application comprises:

[0120] (1) adding the peptide chain modified at the N-terminus with azidoacetic acid to a gold nanoparticle (AuNPs) solution, after the reaction, centrifuging to remove the unbound polypeptides;

[0121] (2) adding the compound represented by formula (II) to perform stable isotope labeling;

[0122] (3) adding dithiothreitol (DTT) to the solution after centrifugation, after the reaction, centrifuging to collect the supernatant, and then diluting 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 at a final concentration of 0.5-1.5% (mass fraction), and preferably the surfactant is sodium dodecyl sulfate (SDS). The addition of surfactant can prevent the aggregation of gold nanoparticles, thereby improving the stability of gold nanoparticles.

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

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

[0127] The seventh typical embodiment of the present application provides the application of the compound represented by formula (III) as a stable isotope labeling reagent of carboxyl compound.

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

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

[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 the use of a tag in which the C-terminus of the polypeptide contains an exposed carboxyl group.

[0132] Preferably, the application includes:

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

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

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

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

[0137] The eighth typical embodiment of the present application provides a stable isotope labeling reagent, which includes a compound represented by formula (I), 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 solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[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 uniformly, it was stirred at 60°C for 12 h under reflux. The solution was cooled to room temperature, filtered, and concentrated under reduced pressure. After being extracted with ethyl acetate, washed with water and saturated sodium chloride solution, and dried over anhydrous Na2SO4, it was rotary evaporated. The crude product was separated by column chromatography to obtain compound 1.

[0144] Compound 1 (0.30 mmol, 69.37 mg) was taken in a 25 mL round-bottom flask, and methanol (6 mL) was added to dissolve it completely. Then, NaOH solution (2 mL, 2M) 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. After being 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 (0.36 mmol, 80.00 mg) was taken in a 25 mL round-bottom flask, and anhydrous methanol was added to dissolve it completely. Then, 800 μL of 7.4% CH2O and 1200 μL of NaBH3CN (0.6 mol / L) were added, and formic acid (FA) was used to adjust the pH to 5-6. The reaction was stirred for 1 h. After column chromatography, compound 3-[d0] (i.e., R1 and R2 in formula (I) are both -CH3) was obtained, which is a stable isotope-labeled reagent for light amino compounds. Figure 1 The mass spectrum of compound 3-[d0] is shown in 1 The HNMR spectrum of compound 3-[d0] is shown in Figure 2

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

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

[0149] ​(4) R1and R2are both -CD3:

[0150] Compound 2 (0.36 mmol, 80.00 mg) was added to a 25 mL round bottom flask, and 8 mL of methanol was added to fully dissolve it. Then 210 μL of 20% CD20 and 980 μL of NaBD3CN (0.6 mol / L) were added, and the pH was adjusted to 5-6 using FA. The reaction was stirred on a magnetic stirrer for 2 h. After column chromatography, compound 3-[d6] (i.e., R1and R2are both -CD3in formula (I)) was obtained. Compound 3-[d6] is a stable isotope-labeled reagent for heavy amino compounds. The mass spectrum of compound 3-[d6] is shown in Figure 3 1 The HNMR chart of compound 3-[d6] is shown in Figure 4

[0151] Example 2

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

[0153] The specific synthesis route is as follows:

[0154]

[0155] (1) R1and R2are both -CH3:

[0156] To a solution of HOBt (48.00 mg, 0.35 mmol) and EDC (68.40 mg, 0.35 mmol) in anhydrous DMF, compound 3-[d0] (78.75 mg, 0.32 mmol) was added, propargylamine (25 μL, 0.40 mmol) was added, and the reaction 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 carried out at ambient temperature for 13 h. After the reaction was completed, ethyl acetate was extracted, washed with water and brine, the organic layers were combined, dried, and rotary evaporated. The crude product was separated by column chromatography to obtain compound 4-[d0] (i.e., R1and R2are both -CH3in formula (II)). Compound 4-[d0] is a stable isotope-labeled reagent for light azido compounds. The mass spectrum of compound 4-[d0] is shown in Figure 5 1 The HNMR chart of compound 4-[d0] is shown in Figure 6

[0157] (2) R1and R2are both -CHD2:

[0158] ​​​​Using the same preparation method as in step (1), compound 3-[d0] is replaced by compound 3-[d4] to obtain compound 4-[d4] (i.e. both R1and R2substituents in formula (II) are -CHD2). Compound 4-[d4] is a stable isotope-labeled reagent for medium azido compounds.

[0159] (3) both R1and R2substituents are -CD3:

[0160] Using the same preparation method as in step (1), compound 3-[d0] is replaced by compound 3-[d6] to obtain compound 4-[d6] (i.e. both R1and R2substituents in formula (II) are -CD3). Compound 4-[d6] is a stable isotope-labeled reagent for heavy azido compounds. The mass spectrum of compound 4-[d6] is shown in Figure Figure 7 1 HNMR spectrum of compound 4-[d6] is shown in Figure Figure 8

[0161] Example 3

[0162] Preparation of compound shown in formula (III):

[0163] The specific synthesis route is shown as follows:

[0164]

[0165] (1) Using the same method as in steps (2), (3) and (4) of Example 1, compound 5-[d0], compound 5-[d4] and compound 5-[d6] are synthesized by replacing raw material compound 2 with compound 1.

[0166] (2) Compound 5-[d0] is dissolved in methanol, and hydrazine hydrate (500 μL, 7.5 mmol) is added and mixed uniformly. After reflux stirring at 70 °C for 3 h, the solution is cooled to room temperature and concentrated under vacuum. Dichloromethane is extracted, washed with deionized water, dried over anhydrous Na2SO4, and the crude product is purified by silica gel chromatography to obtain compound 6-[d0] (i.e. both R1and R2substituents in formula (III) are -CH3). Compound 6-[d0] is a stable isotope-labeled reagent for light carboxyl compounds. The mass spectrum of compound 6-[d0] is shown in Figure Figure 9 1 HNMR spectrum of compound 6-[d0] is shown in Figure Figure 10

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

[0168] ​​​​(4) The same preparation method as step (2) in this example was used, and compound 5-[d0] was replaced by compound 5-[d6] to synthesize compound 6-[d6], which is a stable isotope-labeled reagent of heavy carboxyl compound. The mass spectrum of compound 6-[d6] is shown in Figure 11 1 The HNMR spectrum is shown in Figure 12

[0169] Experimental Example 1

[0170] Application of the compound represented by formula (I) as a stable isotope-labeled reagent of an amino compound:

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

[0172] (1) In the above-mentioned Example 2, the carboxyl group in formula (I) was subjected to condensation reaction with the amino group in propargylamine to prepare formula (II), and the synthesis method is not described here.

[0173] (2) The results are shown in Figure 5 and Figure 7 The compound 3-[d0] and compound 3-[d6] were reacted with propargylamine to remove one molecule of H2O, indicating that the condensation was successfully carried out. Further compared with Figure 1 and Figure 3 The spectrum shown in Figure 5 and Figure 7 has fewer impurity peaks and lower mass spectral signals of impurity peaks.

[0174] Experimental Example 2

[0175] Application of the compound represented by formula (II) as a stable isotope-labeled reagent of an azide compound:

[0176] (1) 1 mL of AuNPs (13±2 nm) solution containing 0.1% SDS was added to the sample bottle, stirred at room temperature for 0.5 h, and then 100 μL of 1 mM N-terminal modified azidoacetic acid polypeptide solution was added. After 36 h of reaction, light and heavy stable isotope-labeled reagents (final concentration of 200 μM), i.e. 4-[d0] and 4-[d6], were added after centrifugation. The stable isotope labeling process was carried out by Click reaction, and an excess of dithiothreitol (DTT) was added after centrifugation to compete with the labeled polypeptide, and stirred overnight at room temperature. Finally, the supernatant was centrifuged and diluted for mass spectrometry detection. The amino acid sequence of the N-terminal modified azidoacetic acid polypeptide is azidoacetic acid-Lys-Glu-Ala-Met-Ile-Glu-Glu-Pro-Ala-mercaptoethylamine.

[0177] ​​(2) In the process of mass spectrometry detection, Thermo Scientific Q-Exactive Orbitrap mass spectrometer was used, data acquisition was carried out in positive ion mode, and the mobile phase was methanol-water solution (containing 0.1% formic acid) with a volume ratio of 1:1. The results are shown in Figure 13 Figure 2, when the polypeptide modified with azidoacetic acid at the N-terminus was detected alone, there were many surrounding impurity peaks, and after stable isotope labeling, the detected spectrum showed a relatively clean mass spectrum, and had good stable isotope characteristics.

[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 directly react with the carboxylic acid, the carboxylic acid is activated with EDC and HOAT to form a stable labeled product. For labeling of polypeptides containing exposed carboxyl groups at the C-terminus, the polypeptide solution (50 μL, 5 mM) is placed in a sample bottle, then a mixed solvent of EDC / HOAT / MES with a volume ratio of 1:1:7 is added, and the concentrations of EDC and HOAT are both 50 mM, then light, medium and heavy stable isotope labeling reagents (100 μL, 10 mM) are added, i.e. 6-[d0], 6-[d4] and 6-[d6]. After mixing evenly, the solution is shaken at 20°C for 3h. Finally, after concentration and reconstitution, it is stored at 4°C. The amino acid sequence of the polypeptide containing exposed carboxyl groups at the C-terminus is NH2-Gly-Phe-Lys-Gly-COOH.

[0181] (2) The mass spectrometry conditions are the same as described in Experimental Example 2. The results are shown in Figure 14 Figure 3, the exposed carboxyl groups at the C-terminus of the polypeptide reacted with light, medium and heavy stable isotope labeling reagents, and mass spectrum peaks with mass increases of 241 Da, 245 Da and 247 Da were produced. Through comparative analysis, it was found that the signal intensity of the labeled peptide segment was significantly enhanced, the noise was reduced, and the signal-to-noise ratio was improved, thus embodying the excellent performance of the stable isotope labeling reagent designed in the present application in detecting polypeptides.

Claims

1. A compound represented by formula (I), formula (II) and formula (III). Formula (I); Formula (II); Formula (III); In formula (I), R1 and R2 are the same, and both are -CD3; in formula (II), R1 and R2 are the same, and both are CD3; in formula (III), R1 and R2 are the same, and both are -CD3.

2. The method for preparing the compound of formula (I) according to claim 1, characterized in that, The method comprises the following steps: (1) 8-amino-2-naphthol reacts with methyl bromoacetate in an alkaline environment to synthesize compound 1; (2) Compound 1 is hydrolyzed in an alkaline environment to synthesize compound 2; (3) Compound 2 reacts with isotopically substituted formaldehyde and sodium cyanoborohydride to obtain a compound represented by formula (I); The structural formula of compound 1 is as follows: ； The structural formula of compound 2 is as follows: 。 3. The method for preparing the compound of formula (II) according to claim 1, characterized in that, The method comprises the following steps: The compound represented by formula (I) is condensed with propargylamine to obtain a compound represented by formula (II).

4. The method for preparing the compound of formula (III) according to claim 1, characterized in that, The method comprises the following steps: (1) Compound 1 reacts with isotopically substituted formaldehyde and sodium cyanoborohydride 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 as follows: ； The structural formula of compound 3 is as follows: ； In compound 3, R1 and R2 are the same, and both are -CD3.

5. The compound represented by formula (I) in claim 1 is used as a stable isotope-labeled reagent of an amino compound. When R1 and R2 in formula (I) are both -CD3, the compound represented by formula (I) is a stable isotope-labeled reagent of a heavy amino compound.

6. The compound represented by formula (II) in claim 1 is used as a stable isotope-labeled reagent of an azide compound; when R1 and R2 in formula (II) are both -CD3, the compound represented by formula (II) is a stable isotope-labeled reagent of a heavy azide compound.

7. Use according to claim 6, wherein The application includes the use in labeling of a polypeptide N-terminally modified with an azide acetic acid group.

8. The compound represented by formula (III) in claim 1 is used as a stable isotope-labeled reagent of a carboxyl compound. When R1 and R2 in formula (III) are both -CD3, the compound represented by formula (III) is a stable isotope-labeled reagent of a heavy carboxyl compound.

9. Use according to claim 8, wherein the compound is ###0002### The application includes the use in labeling of a polypeptide C-terminally containing an exposed carboxyl group.

10. A stable isotope labeled reagent, characterized in that, The method comprises the compound represented by formula (I), formula (II) or formula (III) in claim 1.

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