CN115028552B - Azidation reagent and preparation method and application thereof - Google Patents

Azidation reagent and preparation method and application thereof Download PDF

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CN115028552B
CN115028552B CN202210525826.4A CN202210525826A CN115028552B CN 115028552 B CN115028552 B CN 115028552B CN 202210525826 A CN202210525826 A CN 202210525826A CN 115028552 B CN115028552 B CN 115028552B
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compound
azide
reagent
methyl
stirring
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CN115028552A (en
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王晓剑
李迅
王保川
王益万
徐乐
李月阳
曾平
姜绍丽
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Nanjing Tech University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C247/00—Compounds containing azido groups
    • C07C247/02—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C247/08—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being unsaturated
    • C07C247/10—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being unsaturated and containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/65—One oxygen atom attached in position 3 or 5
    • C07D213/66—One oxygen atom attached in position 3 or 5 having in position 3 an oxygen atom and in each of the positions 4 and 5 a carbon atom bound to an oxygen, sulphur, or nitrogen atom, e.g. pyridoxal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses an azide reagent and a preparation method and application thereof. The invention uses the characteristic structure R 1 According to the method described in literature, partial hydroxyl protected and subsequent easy-to-deprotect compound 2 is synthesized, the compound 2 reacts with azide compound 1 under alkaline conditions to obtain compound 3, the compound 3 can be rapidly deprotected to obtain compound 4 under dilute acid environment, the compound 4 reacts with bromoacetate esters to obtain compound 5 with better stability in short time in a competitive manner, and the post-hydroxymethyl is oxidized to obtain compound 6, and the ester group of compound 6 is easily and rapidly hydrolyzed under acid environment to obtain the target azide reagent. The preparation method is simpler, the material cost is low, the industrial production is easy, the azide reagent is used for the fixed-point azide of the protein, and the influence of other high-reactivity residues on the surface of the protein is effectively avoided.

Description

Azidation reagent and preparation method and application thereof
Technical Field
The invention belongs to the field of chemical biology, and particularly relates to an azide reagent, a preparation method and application thereof.
Background
Covalent modification of proteins is a powerful tool for protein research, and functional molecules can play important roles in recognition, labeling, modification, enrichment and the like of proteins after being covalently bound with side chain amino groups on the surfaces of the proteins, such as methylation, glycosylation, acetylation, phosphorylation, ubiquitination and the like, and are common covalent modification means. However, proteins often have a large number of highly reactive residues, and when such chemical modifications are made to a specific site residue, the residues tend to be disturbed by other residues such as epsilon amino groups of lysine or cysteine sulfhydryl groups. These side reactions often lead to undefined site binding, affect product homogeneity, reduce the selectivity of the conjugate, which is critical for some protein drug conjugates, and therefore, development of modification strategies for proteins resistant to interference by reactive residues, improving the specificity of binding of functional molecules to proteins, and protein azide is a good solution. The azido group is used as a commonly used functional handle for bioorthogonal modification, has small volume occupation, almost has no structural disturbance on biomacromolecules, can perform efficient [3+2] cycloaddition reaction with alkyne compounds, is not interfered by other groups, and is often the first choice functional group applied to in-vivo or in-vitro biomarkers. The chemical modification after the azido is introduced into biological macromolecules such as proteins and the like can effectively avoid the influence of other reactive residues, and has obvious specificity advantage in protein modification. However, the azido group cannot be spontaneously derived in an organism, and can only be introduced subsequently by other means, just like the modification of the protein, and the existing work generally obtains the effect of widely aziding the protein, so that the aim of site-specific azidation cannot be achieved.
Disclosure of Invention
The primary object of the present invention is to provide an azide reagent.
It is still another object of the present invention to provide a method for producing the above-mentioned azide reagent.
It is a further object of the present invention to provide the use of the above-described azide reagent for site-specific azide in a protein.
The invention is realized in such a way that the azide reagent comprises a reagent C1 and a reagent C2, and the chemical structural formula of the azide reagent is shown as the following formula (I):
among the reagents C1:
R 1 selected from any one of H group, F group, methyl, ethyl, nitro, trifluoromethyl, methoxy and ethoxy,
R 2 is characterized by the structure of-N 3 、-O-(CH 2 ) m -N 3 Or- (OCH) 2 CH 2 ) n -N 3 Wherein m=1 to 10, n=1 to 5,
R 3 is the hydroxyl radical of the formula-OH,
X 1 is a C atom or an N atom;
among the reagents C2:
R 1 selected from any one of H group, F group, methyl, ethyl, nitro, trifluoromethyl, methoxy and ethoxy,
R 2 is characterized by the structure of-OH, -O (CH) 2 ) a -CH 3 、-O(CH 2 ) a -OH、-O(CH 2 ) a -NH 2 、-(OCH 2 CH 2 ) b -OH or- (OCH) 2 CH 2 ) b -NH 2 Wherein a=1 to 10, b=1 to 5,
R 3 is characterized by the structure of-O (CH) 2 ) c -N 3 、-O(CH 2 CH 2 O) d -CH 2 CH 2 N 3 、-NH(CH 2 ) c -N 3 、-NH(CH 2 CH 2 O) d -CH 2 CH 2 N 3 Wherein c=1 to 10 and d=1 to 5.
Preferably, in the reagent C1:
X 1 is N-base, R 1 Is methyl, R 2 is-N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-OCH 2 CH 2 -N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 4 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-OCH 2 CH 2 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 4 -N 3 。
Preferably, in the reagent C2:
X 1 is N-base, R 1 Is methyl, R 2 is-OH, R 3 is-OCH 2 -N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-O (CH) 2 ) 2 OH，R 3 is-OCH 2 -N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-OCH 3 ，R 3 is-OCH 2 -N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 3 -CH 2 CH 2 OH，R 3 is-OCH 2 -N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-OH, R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-OCH 3 ，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 is-O (CH) 2 ) 2 OH，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is N-base, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 3 -CH 2 CH 2 OH，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-OH, R 3 is-OCH 2 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-O (CH) 2 ) 2 OH，R 3 is-OCH 2 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-OCH 3 ，R 3 is-OCH 2 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 3 -CH 2 CH 2 OH，R 3 is-OCH 2 -N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-OH, R 3 Is- (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-OCH 3 ，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 is-O (CH) 2 ) 2 OH，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 ；
Or X 1 Is C group, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 3 -CH 2 CH 2 OH，R 3 is-O (CH) 2 CH 2 O) 3 -CH 2 CH 2 N 3 。
The invention further discloses a preparation method of the reagent C1, which comprises the following steps:
(1) Dissolving the compound 2 in ultra-dry tetrahydrofuran THF, adding alkali under the nitrogen atmosphere and ice bath condition, stirring and reacting for 0.5-1.5 h, adding the azido compound 1, and stirring and reacting for 0.5-1.5 h; slowly heating to room temperature, stirring overnight, extracting, concentrating under reduced pressure to extract organic phase, and purifying to obtain compound 3; wherein,
the molar ratio of the compound 2 to the alkali 1 to the azido compound 1 is 1:1 to 1.5:1 to 1.5;
the compound 2 is a compound 1 according to the literature (Mao X, li W, zhu S, et al Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications [ J)]Chemical Communications,2020,56 (55): 7601-7604.) the compound 1 is a compound with the characteristic structure R 1 An aryl or a pyridylmethanol derivative selected from any one of commercially available benzyl alcohol and its derivatives, pyridoxine and its derivatives;
the base of step (1) is 1, 8-diazabicyclo [5.4.0] undec-7-ene or sodium hydride;
the azido compound 1 is selected from diphenyl azide phosphate and a containing characteristic structure- (CH) 2 ) m -N 3 Brominated azido alkanes of (C) and containing a characteristic structure- (CH) 2 CH 2 O) n -N 3 Wherein m=1 to 10 and n=1 to 5;
(2) Compound 3 was dissolved in a volume ratio of 1:1, heating and refluxing in a mixed THF and dilute acid solution with pH value of 4 for 4 hours, and concentrating under reduced pressure to obtain a compound 4;
(3) Dissolving the compound 4 in ultra-dry N, N-dimethylformamide, adding alkali, stirring at room temperature for reaction for 20-40 min, slowly adding bromoacetate compounds, stirring for 11-13 h, filtering after the reaction is finished, extracting the collected filtrate, concentrating under reduced pressure to extract an organic phase, and purifying to obtain a compound 5; wherein,
the molar ratio of the compound 4 to the alkali to the bromoacetate compound is 1:1 to 1.5:0.9 to 1;
the alkali in the step (3) is selected from any one of potassium carbonate, sodium carbonate and cesium carbonate;
the bromoacetate compound is ethyl bromoacetate or butyl bromoacetate;
(4) Dissolving the compound 5 in ultra-dry tetrahydrofuran, adding manganese dioxide, stirring and reacting for 17-19 h under the condition of nitrogen atmosphere and room temperature, centrifuging for 4-5 min at 8000r/min, collecting a supernatant organic phase, concentrating under reduced pressure, and purifying to obtain a compound 6; wherein,
the molar ratio of the compound 5 to the manganese dioxide is 1: 40-50 percent;
(5) Dissolving a compound 6 in a solvent, adding an ester-based hydrolysate, carrying out reflux reaction for 1.5-2.5 h, concentrating and purifying the reaction solution under reduced pressure to obtain an azide reagent C1;
the solvent in the step (5) is ultrapure water; the ester-based hydrolysate is trifluoroacetic acid; the molar ratio of compound 6 to trifluoroacetic acid (TFA) was 1:4 to 5.
Preferably, in step (1), the azide compound 1 is diphenyl azide phosphate, bromoazidoethane, bromoazidotrigine.
Preferably, in steps (1), (3), the extracted extractant is analytically pure Dichloromethane (DCM) or Ethyl Acetate (EA);
in the steps (1), (3), (4) and (5), the purification is separation and purification by silica gel column chromatography.
The invention further discloses a preparation method of the reagent C2, which comprises the following steps:
(1) Dissolving the compound 2 in ultra-dry tetrahydrofuran THF, adding alkali under the nitrogen atmosphere and ice bath condition, stirring and reacting for 0.5-1.5 h, adding brominated compound or di-tert-butyl dicarbonate, and stirring and reacting for 0.5-1.5 h; slowly heating to room temperature, stirring overnight, extracting, concentrating under reduced pressure to extract organic phase, and purifying to obtain compound 3; wherein,
the molar ratio of the compound 2, the base 1 and the bromo compound (or di-tert-butyl dicarbonate) is 1:1 to 1.5:1 to 1.5;
the compound 2 is a compound 1 according to the literature (Mao X, li W, zhu S, et al Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications [ J)]Chemical Communications,2020,56 (55): 7601-7604.) the compound 1 is a compound with the characteristic structure R 1 An aryl or a pyridylmethanol derivative selected from any one of commercially available benzyl alcohol and its derivatives, pyridoxine and its derivatives;
the base of step (1) is 1, 8-diazabicyclo [5.4.0] undec-7-ene or sodium hydride;
the bromo compound is selected from the group consisting of a feature-containing structure- (CH) 2 ) a -CH 3 Brominated alkanes of (C) and containing characteristic structures- (CH) 2 ) a Bromoalkyl tert-butyl carbonates of the formula-OCOOtBu, containing features- (CH) 2 ) a Bromoalkylamino tert-butyl carbonate of-NHCOOtBu and containing a characteristic structure- (CH) 2 CH 2 O) b Bromoethyleneglycol tert-butyl carbonate of the formula-OCOOtBu or of the formula- (CH) 2 CH 2 O) b -any one of the bromoethyleneglycolylamino-tert-butyl carbonates of NHCOOtBu, wherein a = 1 to 10 and b = 1 to 5;
(2) Compound 3 was dissolved in a volume ratio of 1:1, heating and refluxing in a mixed THF and dilute acid solution with pH value of 4 for 4 hours, and concentrating under reduced pressure to obtain a compound 4;
(3) Dissolving the compound 4 in ultra-dry N, N-dimethylformamide, adding alkali, stirring at room temperature for reaction for 20-40 min, slowly adding the azido compound 2, stirring for 11-13 h, filtering after the reaction is finished, extracting the collected filtrate, concentrating under reduced pressure to extract an organic phase, and purifying to obtain a compound 5; wherein,
the molar ratio of the compound 4 to the alkali to the azido compound 2 is 1:1 to 1.5:0.9 to 1;
the alkali in the step (3) is selected from any one of potassium carbonate, sodium carbonate and cesium carbonate;
the azido compound 2 is selected from BrCH containing characteristic structure 2 COO-(CH 2 ) c -N 3 、BrCH 2 CONH-(CH 2 ) c -N 3 、BrCH 2 COO-(CH 2 CH 2 O) d -CH 2 CH 2 N 3 、BrCH 2 CONH-(CH 2 CH 2 O) d -CH 2 CH 2 N 3 Wherein c=1 to 10 and d=1 to 5;
(4) Dissolving the compound 5 in ultra-dry tetrahydrofuran, adding manganese dioxide, stirring and reacting for 17-19 h under the condition of nitrogen atmosphere and room temperature, centrifuging for 4-5 min at 8000r/min, collecting a supernatant organic phase, concentrating under reduced pressure, and purifying to obtain a compound 6; wherein,
the molar ratio of the compound 5 to the manganese dioxide is 1: 40-50 percent;
(5) Dissolving the compound 6 in a solvent, adding an ester-based hydrolysate, reacting for 1.5-2.5 h, concentrating and purifying the reaction solution under reduced pressure to obtain an azide reagent C2
The solvent of the step (5) is ultra-dry Dichloromethane (DCM); the ester-based hydrolysate is trifluoroacetic acid; the volume ratio of trifluoroacetic acid (TFA) to solvent is 1:4, a step of;
wherein, feature structure R of azide reagent C2 2 The bromo-compound with the terminal hydroxyl or amino protected by tert-butyl ester is firstly reacted with the compound 2, and finally the tert-butyl ester is removed under the acidic condition for protection to obtain the product; feature R for azide reagent C2 2 is-CH 2 OH, the hydroxyl of the characteristic structure is protected in advance by di-tert-butyl dicarbonate, and finally the tert-butyl dicarbonate is removed under acidic condition for protection.
Preferably, in steps (1), (3), the extracted extractant is analytically pure Dichloromethane (DCM) or Ethyl Acetate (EA);
in the steps (1), (3), (4) and (5), the purification is separation and purification by silica gel column chromatography.
The invention further discloses application of the azide reagent in protein site-specific azide.
The invention overcomes the defects of the prior art and provides an azide reagent and a preparation method and application thereof. The azide reagent comprises a reagent C1 and a reagent C2 with the same chemical general formula, and the chemical structural formula of the azide reagent is shown as the following formula (I):
1. the synthetic route of the reagent C1 of the invention is as follows:
the preparation process comprises the following steps:
(1) With characteristic structure R obtained by commercially available or known synthetic methods 1 Firstly according to the literature (Mao X, li W, zhu S, et al Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications [ J)]Chemical Communications,2020,56 (55): 7601-7604.) the method of synthesis affords a partially hydroxy protected, subsequent readily deprotected compound 2, which compound 2 is a 3,4' -hydroxy protected aryl or pyridylmethanol derivative, comprising the following representative compound structural formula:
the compound 2 reacts with the azide compound 1 to obtain a compound 3 through a simple and easy-to-operate nucleophilic substitution reaction under alkaline conditions, wherein the compound 3 is a 3,4' -hydroxyl protected azide aromatic analogue, and the compound comprises the following representative compound structural formula:
(2) Compound 3 can be rapidly deprotected under dilute acid conditions to give compound 4, compound 4 being a deprotected azidoaromatic methanol analog, comprising the following structural formula:
(3) In weak base environment, compound 4 and bromoacetate ester compound undergo nucleophilic substitution reaction to obtain compound 5 with better stability in short time, wherein compound 5 is an ethylated azidoaromatic methanol analogue, and the compound comprises the following structural formula:
(4) The post-hydroxymethyl is oxidized to obtain a compound 6, wherein the compound 6 is an ethylated azidoaromatic aldehyde analogue, and comprises the following representative compound structural formula:
(5) In an acid environment, the ester group of the compound 6 is easy to hydrolyze rapidly to obtain a target azide reagent C1, wherein the azide reagent C1 is an azide aromatic aldehyde analogue, and the compound comprises the following structural formula:
2. the synthetic route of the reagent C2 of the invention is as follows:
route one:
route two:
the preparation process comprises the following steps:
(1) A commercially available aryl or pyridylmethanol derivative (Compound 1) was first synthesized according to the method described in the literature (Mao X, li W, zhu S, et al Bifunctical pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications [ J ]. Chemical Communications,2020,56 (55): 7601-7604.) to give a partially hydroxy-protected, subsequent readily deprotected compound 2, which compound 2 is a 3,4' -hydroxy-protected aryl or pyridylmethanol derivative, comprising the following chemical formula:
compound 2 and R containing characteristic structure under alkaline condition through nucleophilic substitution with simple and easy operation 3 To obtain a compound 3, wherein the compound 3 is a 3,4' -hydroxy protected aromatic analogue, and comprises the following structural formula:
(2) Compound 3 can be rapidly deprotected under dilute acid conditions to give compound 4, compound 4 being a deprotected aromatic methanol analog, comprising the following structural formula:
(3) In a weak base environment, the compound 4 and the azide compound 2 undergo nucleophilic substitution reaction to competitively obtain a compound 5 with better stability in a short time, wherein the compound 5 is an azide aromatic methanol analogue, and the compound comprises the following structural formula:
(4) And then carrying out hydroxymethyl oxidation reaction to obtain a compound 6, wherein the compound 6 is an azidated aromatic aldehyde analogue, and comprises the following structural formula:
(5) Under an acid environment, the protecting group of the compound 6 is easy to rapidly deprotect to obtain a target azide reagent C2, wherein the azide reagent C2 is an azide aromatic aldehyde analogue, and the structural formula is as follows:
the azide reagent can be used for protein fixed-point azide modification, specifically, the azide reagent uses aromatic aldehyde as a matrix, -CO-CH 2 O-is taken as an aldehyde group ortho substituent to assist the reagent of the invention to combine with protein at fixed points, so as to realize the introduction of azide in the protein.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects:
(1) The preparation of the azide reagent is simpler, the material cost is low, the equipment requirement is low, and the industrial production is easy;
(2) The azide reagent can be used for protein fixed-point azide, can effectively avoid the influence of other high-reactivity residues on the surface of the protein, and has no protein modification byproducts.
Drawings
FIG. 1 is a schematic illustration of an azide reagent C1-1 in an embodiment of the present invention 1 H NMR spectrum;
FIG. 2 is the presentThe inventive examples include the azide reagent C1-1 13 C NMR spectrum;
FIG. 3 is a reaction chromatogram of benzyl glycinate and azide reagent C1-1 in an example of the present invention;
FIG. 4 is a mass spectrum of benzyl glycinate azide isomer 1 in the example of the present invention;
FIG. 5 is a mass spectrum of benzyl glycinate azide isomer 2 in the example of the present invention;
FIG. 6 is a diagram of SDS-PAGE analysis and determination of insulin azide in the example of the present invention; wherein, lane 1: insulin-FITC-yne; lane 2: insulin; lane 3: insulin-C1-1-FITC-yne.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1 Synthesis of azidation reagent C1-1
1. The synthesis route of the azide reagent C1-1 is as follows:
2. the specific process of the synthesis comprises the following steps:
(1) Pyridoxine (compound 1) was prepared according to the literature [1] Compound 2 (1.881 g,9 mmol) obtained by synthesis was dissolved in ultra-dry tetrahydrofuran (THF, 30 mL), and 1, 8-diazabicyclo [5.4.0] was added under stirring under an ice-bath and nitrogen atmosphere]Undec-7-ene (DBU, 1.34mL,9 mmol) was stirred under nitrogen for 1h while maintaining an ice bath, and diphenyl azide phosphate (2.477 g,9 mmol) was added; after maintaining the ice bath and stirring under nitrogen atmosphere for 1 hour, the temperature was slowly raised to room temperature and stirred overnight, and the reaction was quenched by addition of ultrapure water (30 mL) under ice bath conditions.
The reaction solution was poured into a 250mL separating funnel, supplemented with ultrapure water (100 mL), extracted three times with dichloromethane (DCM, 30 mL), and the organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 3 in 90% yield.
(2) Compound 3 (500 mg,2.1 mmol) was dissolved in a mixed solution of THF and diluted hydrochloric acid (10 mL), the pH of the solution was 4, the temperature was raised to a reflux state, the reaction was carried out for 4 hours, and then compound 4 was obtained after concentration under reduced pressure.
(3) Compound 4 (320 mg,1.6 mmol) was added to N, N-dimethylformamide (DMF, 8 mL), followed by cesium carbonate (Cs) 2 CO 3 654.6mg,2 mmol), stirring at 25deg.C for 30min, slowly adding dropwise ethyl bromoacetate (Ethyl bromoacetate,267.2mg,1.6 mmol) solution, stirring at 25deg.C for 12 hr, filtering to remove cesium carbonate (Cs) 2 CO 3 )。
The resulting organic phase was poured into cold water (100 mL) and extracted three times with dichloromethane (DCM, 15 mL), the solvent N, N-Dimethylformamide (DMF) was mostly taken into the aqueous phase. The organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 5 in 40% yield.
(4) Compound 5 (388.2 mg,2 mmol) was dissolved in ultra-dry tetrahydrofuran (THF, 10 mL) and manganese dioxide (MnO) was added 2 6.95g,80 mmol), stirred for 18h at 25℃under nitrogen, centrifuged for 5min at 8000r/min, the supernatant organic phase collected, concentrated under reduced pressure to give the crude product, which was chromatographed on silica gel to give compound 6 in 60% yield.
(5) Compound 6 (333.8 mg,1.2 mmol) was poured into ultrapure water (6 mL), trifluoroacetic acid (TFA, 0.4mL,5.38 mmol) was added, stirring was performed under reflux for 4h, and the reaction solution was concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound C1-1 in 60% yield.
The characterization map of the compound C1-1 is shown in figure 1 and figure 2.
C1-1 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.59(s,1H),8.43(s,1H),4.74(s,2H),4.71(s,2H),2.55(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,170.22,154.99,153.94,145.98,132.28,127.25,70.55,48.53,19.65.HRMS m/z Found:251.0780,calculated:251.0780for C 10 H 11 N 4 O 4 [M+H] + .
EXAMPLE 2 Synthesis of azidation reagent C1-2
1. The synthesis route of the azide reagent C1-2 is as follows:
2. the specific process of the synthesis comprises the following steps:
(1) Pyridoxine (compound 1) was prepared according to the literature [1] Compound 2 (1.881 g,9 mmol) obtained by synthesis was dissolved in ultra-dry tetrahydrofuran (THF, 50 mL), sodium hydride (NaH, 0.216g,9 mmol) was added under stirring in an ice bath under nitrogen atmosphere, and after stirring in an ice bath under nitrogen atmosphere for 1 hour, bromoazide (1.34 g,9 mmol) was added; after maintaining the ice bath and stirring under nitrogen atmosphere for 1 hour, the temperature was slowly raised to room temperature and stirred overnight, and the reaction was quenched by addition of ultrapure water (50 mL) under ice bath conditions.
The reaction solution was poured into a 500mL separating funnel, supplemented with ultrapure water (100 mL), extracted three times with dichloromethane (DCM, 30 mL), and the organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 3 in 80% yield.
(2) Compound 3 (556 mg,2.0 mmol) was dissolved in a mixed solution of THF and diluted hydrochloric acid (20 mL) at pH 4, and the mixture was heated to reflux for 4 hours, and then concentrated under reduced pressure to give compound 4.
(3) Compound 4 (317 mg,1.5 mmol) was added to N, N-dimethylformamide (DMF, 10 mL), followed by cesium carbonate (Cs) 2 CO 3 654.6mg,2 mmol), stirring at 25deg.C for 30min, slowly dropwise adding ethyl bromoacetate (224 mg,1.35 mmol) solution, stirring at 25deg.C for 12 hr, and filtering to remove cesium carbonate (Cs) 2 CO 3 )。
The resulting organic phase was poured into cold water (100 mL) and extracted three times with dichloromethane (DCM, 15 mL), the solvent N, N-Dimethylformamide (DMF) was mostly taken into the aqueous phase. The organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 5 in 35% yield.
(4) Compound 5 (648 mg,2 mmol) was dissolved in ultra-dry tetrahydrofuran (THF, 10 mL) and manganese dioxide (MnO) was added 2 6.95g,80 mmol), stirred for 18h at 25℃under nitrogen, centrifuged for 5min at 8000r/min, the supernatant organic phase collected, concentrated under reduced pressure to give the crude product, which was chromatographed on silica gel to give compound 6 in 60% yield.
(5) Compound 6 (483 mg,1.5 mmol) was poured into ultrapure water (6 mL), trifluoroacetic acid (TFA, 0.459mL,6 mmol) was added, stirring was performed under reflux for 4h, and the reaction solution was concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound C1-2 in a yield of 70%.
C1-2 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.54(s,1H),8.53(s,1H),4.84(s,2H),4.78(s,2H),3.44(t,J＝7.9Hz,2H)2.55(s,3H),1.65(t,J＝7.2Hz,2H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,170.24,154.96,153.90,153.92,132.28,132.04,72.42,71.42,70.54,49.52,19.62.HRMS m/z Found:295.1042，calculated:295.1046for C 12 H 15 N 4 O 5 [M+H] + yield 60%
The synthesis of the azide reagents C1-3, C1-4, C1-5 and C1-6 is carried out by adopting the same steps of C1-2.
EXAMPLE 3 Synthesis of azidation reagent C1-3
The synthesis of the azide reagent C1-3 is substantially the same as the synthesis of C1-2, except that in step (1), the azide compound 1 used is bromoazide tetraethylene glycol.
The chemical structural formula of the azide reagent C1-3 is as follows:
c1-3 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.57(s,1H),8.43(s,1H),4.87(s,2H),4.72(s,2H),3.60(t,J＝4.3Hz,2H),3.57–3.51(m,8H),3.50–3.45(m,2H),3.43–3.37(m,4H),2.55(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)193.00,169.18,155.35,154.14,146.51,132.61,127.75,71.00,70.30,70.24,70.19,69.78,68.57,64.60,50.47,48.94,33.88,20.08.HRMS m/zFound:427.1830,calculated:427.1829for C 18 H 27 N 4 O 8 [M+H] + yield 58%
EXAMPLE 4 Synthesis of azidation reagent C1-4
The synthesis of azido reagent C1-4 is essentially the same as the synthesis of C1-2, except that in step (1), compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and azido compound 1 is diphenyl azide phosphate.
The chemical structural formula of the azide reagent C1-4 is as follows:
c1-4 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.40(s,1H),7.42(d,J＝7.6Hz,1H),7.02(d,J＝7.4Hz,1H),4.64(s,2H),2.78(s,2H),2.25(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,170.02,161.22,137.22,135.92,123.28,121.04,120.14,67.53,49.42,15.40.HRMS m/z Found:250.0816,calculated:250.8028for C 11 H 12 N 3 O 4 [M+H] + the yield is 65%.
EXAMPLE 5 Synthesis of azidation reagent C1-5
The synthesis of azido reagent C1-5 is essentially the same as the synthesis of C1-2, except that in step (1), compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and azido compound 1 is azidobromide.
The chemical structural formula of the azide reagent C1-5 is as follows:
c1-5 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.42(s,1H),7.41(d,J＝7.6Hz,1H),7.12(d,J＝7.4Hz,1H),4.84(s,2H),4.74(s,2H),3.24(t,J＝7.6Hz,2H),2.25(s,3H),1.68(t,J＝7.8Hz,2H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,169.88,161.02,135.68,132.96,125.52,118.54,118.38,72.24,69.70,67.86,48.88,19.26.HRMS m/z Found:294.1096,calculated:294.1090for C 13 H 16 N 3 O 5 [M+H] + the yield was 68%.
EXAMPLE 6 Synthesis of azidation reagent C1-6
The synthesis of the azidation reagent C1-6 is essentially the same as the synthesis of C1-2, except that in step (1), compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and azido compound 1 is bromoazido tetraethylene glycol.
The chemical structural formula of the azide reagent C1-6 is as follows:
c1-6 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.56(s,1H),7.46(d,J＝7.9Hz,1H),7.14(d,J＝7.6Hz,1H),4.86(s,2H),4.78(s,2H),3.66(t,J＝4.3Hz,2H),3.58–3.50(m,8H),3.49–3.43(m,2H),3.41–3.35(m,4H),2.25(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.69,169.98,161.12,135.58,132.86,125.82,118.64,118.58,72.86,70.37,70.32,70.28,70.21,69.79,67.82,60.72,50.48,44.30,19.98.HRMS m/z Found:426.1882,calculated:426.1876for C 19 H 28 N 3 O 8 [M+H] + the yield is 70%.
EXAMPLE 7 Synthesis of azidation reagent C2-1
1. The synthesis route of the azide reagent C2-1 is as follows:
2. the specific process of the synthesis comprises the following steps:
(1) Pyridoxine (compound 1) was prepared according to the literature [1] Compound 2 (1.672 g,8 mmol) was dissolved in ultra-dry dichloromethane (DCM, 30 mL), and triethylamine (Et) was added under stirring in an ice-bath under nitrogen 3 N,0.91g,9 mmol), 4-dimethylaminopyridine (DMAP, 48.8mg,0.4 mmol) was kept in an ice bath and stirred under nitrogen for 15min, and then di-tert-butyl dicarbonate (1.963 g,9 mmol) was added; after maintaining the ice bath and stirring under nitrogen atmosphere for 15min, slowly heating to room temperature and stirring overnight, the reaction was quenched by addition of ultrapure water (30 mL) under ice bath conditions.
The reaction solution was poured into a 250mL separating funnel, supplemented with ultrapure water (100 mL), extracted three times with dichloromethane (DCM, 30 mL), and the organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 3 in 98% yield.
(2) Compound 3 (618 mg,2.0 mmol) was dissolved in a mixed solution of THF and diluted hydrochloric acid (10 mL), the pH of the solution was 4, the temperature was raised to a reflux state, the reaction was performed for 4 hours, and then the solution was concentrated under reduced pressure to obtain compound 4.
(3) Compound 4 (404 mg,1.5 mmol) was added to N, N-dimethylformamide (DMF, 10 mL), followed by cesium carbonate (Cs) 2 CO 3 654.6mg,2 mmol), stirring at 25deg.C for 30min, slowly dropwise adding a solution of azidomethyl 2-bromoacetate (270 mg,1.4 mmol), stirring at 25deg.C for 12 hr, and filtering to remove cesium carbonate (Cs) 2 CO 3 )。
The resulting organic phase was poured into cold water (150 mL) and extracted three times with dichloromethane (DCM, 15 mL), the solvent N, N-Dimethylformamide (DMF) was mostly taken into the aqueous phase. The organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 5 in 30% yield.
(4) Compound 5 (774 mg,2 mmol) was dissolved in ultra-dry tetrahydrofuran (THF, 20 mL) and manganese dioxide (MnO) was added 2 6.95g,80 mmol), stirred for 18h at 25℃under nitrogen, centrifuged5 minutes at a rotational speed of 8000r/min, collecting an organic phase of a supernatant, concentrating under reduced pressure to obtain a crude product, and separating by silica gel column chromatography to obtain the compound 6 with a yield of 62%.
(5) Compound 6 (458 mg,1.2 mmol) was poured into ultra-dry dichloromethane (DCM, 10 mL), trifluoroacetic acid (TFA, 2 mL) was added, and the reaction solution was concentrated under reduced pressure to give the crude product, which was separated by silica gel column chromatography to give compound C2-1 in 95% yield.
C2-1 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.14(s,1H),8.83(s,1H),4.94(s,2H),4.62(s,2H),3.99(s,2H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,169.24,154.00,150.90,143.02,128.28,126.04,75.42,65.34,48.52,19.69.HRMS m/z Found:281.0889,calculated:281.0886for C 11 H 13 N 4 O 5 [M+H] + .
EXAMPLE 8 Synthesis of azidation reagent C2-5
The synthesis of the azide reagent C2-5 is substantially the same as the synthesis of C2-1, except that in step (3), the azide compound 2 is 2-bromoacetic acid azidotrigidide.
The chemical structural formula of the azide reagent C2-5 is as follows:
c2-5 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.24(s,1H),8.53(s,1H),4.96(s,2H),4.60(s,2H),3.62(t,J＝7.3Hz,2H),3.54–3.50(m,8H),3.52–3.44(m,2H),3.40–3.34(m,4H),2.55(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,169.45,153.27,147.71,142.95,126.65,125.05,74.87,72.47,72.23,70.41,70.25,69.87,65.34,65.31,58.53,40.05,20.04.HRMS m/zFound:427.1831,calculated:427.1829for C 18 H 27 N 4 O 8 [M+H] + the yield is 90%.
EXAMPLE 9 Synthesis of azidation reagent C2-9
The synthesis of azido reagent C2-9 is essentially the same as the synthesis of C2-1, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and in step (3) azide compound 2 is 2-bromoacetic acid azidomethyl ester.
The chemical structural formula of the azide reagent C2-9 is as follows:
c2-9 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.30(s,1H),7.40(d,J＝7.2Hz,1H),6.88(d,J＝7.3Hz,1H),4.74(s,2H),4.44(s,2H),3.78(s,2H),2.25(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.74,170.00,161.32,136.22,135.82,125.62,120.28,120.04,76.96,65.53,48.42,15.62.HRMS m/zFound:280.0937,calculated:280.0933for C 12 H 14 N 3 O 5 [M+H] + the yield is 90%.
EXAMPLE 10 Synthesis of azidation reagent C2-13
The synthesis of azido reagent C2-13 is essentially the same as the synthesis of C2-1, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and in step (3) azide compound 2 is 2-bromoacetic acid azidotriginetetraglycol ester.
The chemical structural formula of the azide reagent C2-13 is as follows:
c2-13 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.54(s,1H),7.48(d,J＝7.3Hz,1H),7.24(d,J＝7.3Hz,1H),4.96(s,2H),4.88(s,2H),3.64(t,J＝4.3Hz,2H),3.56–3.52(m,8H),3.48–3.42(m,2H),3.40–3.32(m,4H),2.25(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.68,170.02,160.12,136.08,135.86,125.25,118.44,118.28,72.04,70.47,70.35,70.25,70.21,69.89,67.86,60.52,50.98,44.50,20.04.HRMS m/z Found:426.1880,calculated:426.1876for C 19 H 28 N 3 O 8 [M+H] + the yield is 95%.
EXAMPLE 11 Synthesis of azidation reagent C2-2
1. The synthesis route of the azide reagent C2-2 is as follows:
2. the specific process of the synthesis comprises the following steps:
(1) Pyridoxine (compound 1) was prepared according to the literature [1] Compound 2 (1.463 g,7 mmol) obtained by synthesis was dissolved in ultra-dry tetrahydrofuran (THF, 25 mL), sodium hydride (NaH, 0.216g,9 mmol) was added under stirring in an ice bath under nitrogen atmosphere, and after stirring in an ice bath under nitrogen atmosphere for 1h, 2-bromoethyl tert-butyl carbonate (2.016 g,9 mmol) was added; after maintaining the ice bath and stirring under nitrogen atmosphere for 1 hour, the temperature was slowly raised to room temperature and stirred overnight, and the reaction was quenched by addition of ultrapure water (30 mL) under ice bath conditions.
The reaction solution was poured into a 500mL separating funnel, supplemented with ultrapure water (100 mL), extracted three times with dichloromethane (DCM, 30 mL), and the organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 3 in 92% yield.
(2) Compound 3 (706 mg,2.0 mmol) was dissolved in a mixed solution of THF and diluted hydrochloric acid (10 mL), the temperature was raised to reflux, the pH of the solution was 4, the reaction was continued for 4 hours, and then the solution was concentrated under reduced pressure to give Compound 4.
(3) Compound 4 (284 mg,1.7 mmol) was added to N, N-dimethylformamide (DMF, 10 mL), followed by cesium carbonate (Cs) 2 CO 3 654.6mg,2 mmol), stirring at 25deg.C for 30min, slowly dropwise adding a solution of azidomethyl 2-bromoacetate (308 mg,1.6 mmol), stirring at 25deg.C for 12 hr, and filtering to remove cesium carbonate (Cs) 2 CO 3 )。
The resulting organic phase was poured into cold water (100 mL) and extracted three times with dichloromethane (DCM, 15 mL), the solvent N, N-Dimethylformamide (DMF) was mostly taken into the aqueous phase. The organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 5 in 30% yield.
(4) Compound 5 (853 mg,2 mmol) was dissolved in ultra-dry tetrahydrofuran (THF, 15 mL) and manganese dioxide (MnO) was added 2 6.95g,80 mmol), stirred for 18h at 25℃under nitrogen, centrifuged for 5min at 8000r/min, the supernatant organic phase collected, concentrated under reduced pressure to give the crude product, which was chromatographed on silica gel to give compound 6 in 60% yield.
(5) Compound 6 (424 mg,1 mmol) was poured into ultra-dry dichloromethane (DCM, 10 mL), trifluoroacetic acid (TFA, 2 mL) was added, and the reaction solution was concentrated under reduced pressure to give the crude product, which was separated by silica gel column chromatography to give compound C2-2 in 90% yield.
C2-2 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.54(s,1H),8.53(s,1H),4.84(s,2H),4.78(s,2H),3.44(t,J＝7.9Hz,2H)2.55(s,3H),1.65(t,J＝7.2Hz,2H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,170.24,154.96,153.90,153.92,132.28,132.04,72.42,71.42,70.54,49.52,19.62.HRMS m/z Found:294.0962,calculated:294.0964for C 12 H 15 N 4 O 5 [M+H] + .
EXAMPLE 12 Synthesis of azidation reagent C2-4
The synthesis of the azidation reagent C2-4 is substantially the same as the synthesis of C2-2, except that in step (1), the bromo compound is 2-bromotetraethyleneglycol-t-butyl carbonate.
The chemical structural formula of the azide reagent C2-4 is as follows:
c2-4 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.24(s,1H),8.73(s,1H),4.89(s,2H),4.88(s,2H),4.28(s,2H),3.72(m,2H),3.52(m,14H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,169.24,154.06,148.90,143.92,130.28,126.04,76.42,72.66,71.42,71.40,71.22,71.18,71.16,71.14,70.98,66.43,59.98,19.62.HRMS m/z Found:457.1940,calculated:457.1935for C 19 H 29 N 4 O 9 [M+H] + the yield is 95%.
EXAMPLE 13 Synthesis of azidation reagent C2-7
The synthesis of the azide reagent C2-7 is substantially the same as the synthesis of C2-2, except that in step (3), the azide compound 2 is 2-bromoacetic acid azidotrigidide.
The chemical structural formula of the azide reagent C2-7 is as follows:
c2-7 characterization data 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.55(s,1H),8.33(s,1H),4.97(s,2H),4.62(s,2H),3.80(t,J＝7.3Hz,2H),3.60(m,4H),3.52(m,14H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.80,169.38,155.27,150.14,144.51,130.61,126.75,72.60,72.48,70.42,70.40,70.24,70.22,70.20,69.76,68.88,65.72,62,30,49.94,20.04.HRMS m/z Found:471.2091,calculated:471.2099for C 20 H 31 N 4 O 9 [M+H] + The yield is 90%.
EXAMPLE 14 Synthesis of azidation reagent C2-8
The synthesis of the azidation reagent C2-8 is substantially the same as the synthesis of C2-2, except that in step (1), the bromo-compound is 2-bromotetraethyleneglycol-t-butyl carbonate, and in step (3), the azide compound 2 is 2-bromoacetic acid azidotriglycol-ester.
The chemical structural formula of the azide reagent C2-8 is as follows:
c2-8 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.50(s,1H),8.43(s,1H),4.87(s,2H),4.32(s,2H),4.01(t,J＝7.3Hz,2H),3.70(m,6H),3.52(m,24H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.82,169.18,153.27,151.14,147.51,132.61,125.75,72.80,72.46,72.44,70.42,70.40,70.38,70.36,70.34,70.24,70.22,70.21,70.20,70.19,69.56,68.88,64.56,61.78,44.04,19.88.HRMS m/z Found:603.2885,calculated:603.2877for C 26 H 43 N 4 O 12 [M+H] + the yield is 95%.
EXAMPLE 15 Synthesis of azidation reagent C2-10
The synthesis of the azide reagent C2-10 is substantially the same as the synthesis of C2-2, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol.
The chemical structural formula of the azide reagent C2-10 is as follows:
c2-10 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.54(s,1H),7.58(d,J＝7.2Hz,1H),7.20(d,J＝7.2Hz,1H),4.68(s,2H),4.58(s,2H),4.23(s,2H),3.22(t,J＝7.8Hz,2H)2.52(s,3H),1.68(t,J＝7.2Hz,2H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,170.24,159.96,144.90,141.92,122.38,116.04,115.96,74.96,72.44,71.42,69.54,48.52,19.82.HRMS m/z Found:324.1201,calculated:324.1196for C 14 H 18 N 3 O 6 [M+H] + the yield is 90%.
EXAMPLE 16 Synthesis of azidation reagent C2-12
The synthesis of the azidation reagent C2-12 is essentially the same as the synthesis of C2-2, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and the bromo compound is 2-bromotetraethyleneglycol t-butyl carbonate.
The chemical structural formula of the azide reagent C2-12 is as follows:
c2-12 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.44(s,1H),7.48(d,J＝7.2Hz,1H),7.28(d,J＝7.2Hz,1H),4.88(s,2H),4.84(s,2H),4.32(s,2H),3.72(m,2H),3.52(m,14H),2.54(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.70,170.24,159.96,144.90,141.92,122.38,116.04,115.96,76.42,72.66,71.42,71.40,71.22,71.18,71.16,71.14,70.98,66.43,50.98,19.62.HRMS m/z Found:456.1988,calculated:456.1982for C 20 H 30 N 3 O 9 [M+H] + the yield was 92%.
EXAMPLE 17 Synthesis of azidation reagent C2-15
The synthesis of the azidation reagent C2-15 is essentially the same as the synthesis of C2-2, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and in step (3) the azido compound is 2-bromoacetic acid azido tetraglycol ester.
The chemical structural formula of the azide reagent C2-15 is as follows:
c2-15 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.56(s,1H),7.28(d,J＝7.2Hz,1H),7.12(d,J＝7.2Hz,1H),4.67(s,2H),4.61(s,2H),3.84(t,J＝7.3Hz,2H),3.62(m,4H),3.51(m,14H),2.51(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.71,170.26,159.86,146.90,143.92,121.38,117.04,116.96,72.62,72.58,70.46,70.41,70.34,70.22,70.20,69.66,67.88,65.52,62,50,40.94,20.54.HRMS m/z Found:470.2145,calculated:471.2139for C 21 H 32 N 3 O 9 [M+H] + the yield was 92%.
EXAMPLE 18 Synthesis of azidation reagent C2-16
The synthesis of the azidation reagent C2-16 is substantially the same as the synthesis of C2-2, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol, the bromo compound is 2-bromotetraethyleneglycol t-butyl carbonate, and in step (3) the azide compound is 2-bromoacetic acid azidotrigglycol.
The chemical structural formula of the azide reagent C2-16 is as follows:
c2-16 characterization data; 1 H NMR(400MHz,DMSO-d 6 ):10.56(s,1H),7.48(d,J＝7.2Hz,1H),7.22(d,J＝7.2Hz,1H),4.57(s,2H),4.22(s,2H),3.98(t,J＝7.3Hz,2H),3.72(m,6H),3.42(m,24H),2.55(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.73,169.26,158.86,145.90,144.92,120.38,117.04,116.98,72.60,72.56,72.46,70.42,70.41,70.40,70.38,70.36,70.34,70.32,70.22,70.21,70.19,69.46,68.38,64.96,61.28,40.04,19.98.HRMS m/z Found:602.2930,calculated:602.2925for C 27 H 44 N 3 O 12 [M+H] + the yield was 96%.
EXAMPLE 19 Synthesis of azidation reagent C2-3
1. The synthesis route of the azide reagent C2-3 is as follows:
2. the specific process of the synthesis comprises the following steps:
(1) Pyridoxine (compound 1) was prepared according to the literature [1] Compound 2 (1.463 g,7 mmol) obtained by synthesis was dissolved in ultra-dry tetrahydrofuran (THF, 25 mL), sodium hydride (NaH, 0.216g,9 mmol) was added under stirring in an ice bath and nitrogen atmosphere, and after stirring in an ice bath and nitrogen atmosphere for 1h, methyl bromide (254 mg,9 mmol) was added; maintaining ice bathAfter stirring under nitrogen for 1 hour, the mixture was slowly warmed to room temperature and stirred overnight, and then ultrapure water (30 mL) was added under ice bath conditions to quench the reaction.
The reaction solution was poured into a 500mL separating funnel, supplemented with ultrapure water (100 mL), extracted three times with dichloromethane (DCM, 30 mL), and the organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 3 in 90% yield.
(2) Compound 3 (4476 mg,2.0 mmol) was dissolved in a mixture of THF and dilute hydrochloric acid (10 mL), the temperature was raised to reflux and the pH of the solution was 4, and after reaction for 4h, compound 4 was obtained after concentration under reduced pressure.
(3) Compound 4 (329.76 mg,1.8 mmol) was added to N, N-dimethylformamide (DMF, 10 mL), followed by cesium carbonate (Cs) 2 CO 3 654.6mg,2 mmol), stirring at 25deg.C for 30min, slowly dropwise adding a solution of azidomethyl 2-bromoacetate (308 mg,1.6 mmol), stirring at 25deg.C for 12 hr, and filtering to remove cesium carbonate (Cs) 2 CO 3 )。
The resulting organic phase was poured into cold water (100 mL) and extracted three times with dichloromethane (DCM, 15 mL), the solvent N, N-Dimethylformamide (DMF) was mostly taken into the aqueous phase. The organic phase was collected, concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography to give compound 5 in 35% yield.
(4) Compound 5 (592 mg,2 mmol) was dissolved in ultra-dry tetrahydrofuran (THF, 15 mL) and manganese dioxide (MnO) was added 2 6.95g,80 mmol), stirred for 18h at 25℃under nitrogen, centrifuged for 5min at 8000r/min, the supernatant organic phase collected, concentrated under reduced pressure to give a crude product, which was chromatographed on silica gel to give compound C2-3 in 60% yield.
C2-3 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.44(s,1H),8.63(s,1H),4.82(s,2H),4.72(s,2H),3.99(s,2H),3.25(s,3H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,169.26,154.20,150.98,143.52,128.58,125.04,76.42,74.52,65.34,45.52,20.18.HRMS m/z Found:295.1048calculated:295.1042for C 12 H 15 N 4 O 5 [M+H] + .
EXAMPLE 20 Synthesis of azidation reagent C2-6
The synthesis of the azide reagent C2-6 is substantially the same as the synthesis of C2-2, except that in step (3), the azide compound 2 is 2-bromoacetic acid azidotrigidide.
The chemical structural formula of the azide reagent C2-6 is as follows:
c2-6 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.34(s,1H),8.63(s,1H),4.86(s,2H),4.58(s,2H),3.61(t,J＝7.3Hz,2H),3.55–3.52(m,8H),3.46–3.42(m,2H),3.40–3.34(m,4H),3.26(s,3H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.71,169.46,153.17,147.61,142.85,126.55,125.85,74.57,72.42,72.21,70.43,70.21,69.89,65.37,65.32,65.21,58.53,40.05,20.04.HRMS m/z Found:441.1992,calculated:441.1985for C 19 H 29 N 4 O 8 [M+H] + the yield is 60%.
EXAMPLE 21 Synthesis of azidation reagent C2-11
The synthesis of azidation reagent C2-11 is essentially the same as the synthesis of C2-2, except that in step (1), compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol.
The chemical structural formula of the azide reagent C2-11 is as follows:
c2-11 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.44(s,1H),7.44(d,J＝7.2Hz,1H),7.20(d,J＝7.2Hz,1H),4.78(s,2H),4.76(s,2H),3.89(s,2H),3.22(s,3H),2.52(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.72,170.02,161.42,136.52,135.92,125.72,120.38,120.24,76.86,65.63,65.33,40.62,16.62.HRMS m/z Found:294.1098calculated:294.1090for C 13 H 16 N 4 O 5 [M+H] + the yield was 58%.
EXAMPLE 22 Synthesis of azidation reagent C2-14
The synthesis of azido reagent C2-14 is essentially the same as the synthesis of C2-2, except that in step (1) compound 1 is (3-hydroxy-4-methyl-1, 2-phenylene) dimethanol and in step (3) azido compound 2 is 2-bromoacetic acid azido tetraglycol ester.
The chemical structural formula of the azide reagent C2-14 is as follows:
c2-14 characterization data: 1 H NMR(400MHz,DMSO-d 6 ):δ(ppm)10.34(s,1H),7.24(d,J＝7.2Hz,1H),7.18(d,J＝7.2Hz,1H),4.84(s,2H),4.56(s,2H),3.60(t,J＝7.3Hz,2H),3.55–3.50(m,8H),3.47–3.42(m,2H),3.39–3.34(m,4H),3.28(s,3H),2.51(s,3H). 13 C NMR(100MHz,DMSO-d 6 ):δ(ppm)192.69,170.00,160.32,136.58,135.96,125.35,118.24,118.20,74.51,72.40,72.23,70.44,70.25,69.99,65.39,65.34,65.22,56.53,41.05,19.98.HRMS m/z Found:44.2035,calculated:440.2033for C 20 H 30 N 3 O 8 [M+H] + the yield was 62%.
Application examples
(1) Benzyl glycinate hydrochloride (Benzyl glycinate hydrochloride,1.5mg, 9. Mu. Mol) was dissolved in sodium bicarbonate solution (NaHCO) 3 buffer,0.1M,pH 8.4,0.667mL) C1-1 (18 mg, 72. Mu. Mol) was added and stirred at 25℃for 24h, the reaction proceeds as follows:
diluting the reaction solution, and subjecting to high performance liquid chromatography mass spectrometryThe reaction results were determined by a combined instrument (LCMS). Benzyl glycinate was used to test the ability of an azide reagent to achieve the azide of a particular amino acid residue. As shown in FIG. 3, after the excessive azide reagent C1-1 and benzyl glycinate are reacted, two isomer products can be obtained, the peak time of the isomer product 1 is 21.40min, and the peak time of the isomer product 1 is 23.19min. Fig. 4 and 5 show mass spectrum data of two isomer products, and specific mass spectrum data of isomer product 1 is ESI-MS (positive): 416.20 (observed, [ M+H ]] + ) The calculated molecular mass of isomer product 1 was 415.15; specific mass spectrometry data for isomer product 2 was ESI-MS (positive): 416.21 (observed, [ M+H ]] + ) The calculated molecular mass of isomer product 2 was 415.15;
(2) Insulin (insulin, 3mg, 0.52. Mu. Mol) was dissolved in sodium bicarbonate solution (NaHCO 3 To buffer,0.1M, pH 8.4,3 mL) was added C1-1 (1.3 mg, 5.2. Mu. Mol), and after stirring at 25℃for 24 hours, a fluorescent alkyne compound (FITC-yne, 0.035. Mu. Mol) was added to the reaction mixture (10. Mu.L) and the mixture was stirred according to the literature (Yang M, jalloh A S, wei W, et al biocompatible click chemistry enabled compartment-specific pH measurement inside E.coli [ J ]]Nature Communications,2014,5 (1): 4981.) copper sulphate-sodium ascorbate-ligand solution (3.91 mM, 1. Mu.L) was added, stirred for 1h at 25℃and 10% (v/v) trifluoroacetic acid (TFA, 1. Mu.L) was added and stirred for 1h, after which the analysis by gel electrophoresis (SDS-PAGE) was carried out, see FIG. 6.
Insulin was used as a protein azide model, as shown in fig. 6, under ultraviolet irradiation, the fluorescence-labeled PLAA site-specific modified insulin showed clear bright spots (lane 3), and no fluorescence labeling could be achieved without the PLAA site-specific modified insulin (lane 1) and without the insulin itself (lane 2).
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (6)

1. An azide reagent, characterized in that the azide reagent comprises a reagent C1, and the chemical structural formula of the azide reagent is shown as the following formula (I):
（Ⅰ）
among the reagents C1:
R 1 selected from any one of H group, F group, methyl, ethyl, nitro, trifluoromethyl, methoxy and ethoxy,
R 2 is characterized by the structure of-N 3 、-O-（CH 2 ） m -N 3 Or- (OCH) 2 CH 2 ） n -N 3 Wherein m=1 to 10, n=1 to 5,
R 3 is the hydroxyl radical of the formula-OH,
X 1 is a C atom or an N atom.
2. The azide reagent as claimed in claim 1, wherein, in the reagent C1:
X 1 is N atom, R 1 Is methyl, R 2 is-N 3 ；
Or X 1 Is N atom, R 1 Is methyl, R 2 is-OCH 2 CH 2 -N 3 ；
Or X 1 Is N atom, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 4 -N 3 ；
Or X 1 Is C atom, R 1 Is methyl, R 2 is-N 3 ；
Or X 1 Is C atom, R 1 Is methyl, R 2 is-OCH 2 CH 2 -N 3 ；
Or X 1 Is C atom, R 1 Is methyl, R 2 Is- (OCH) 2 CH 2 ) 4 -N 3 。
3. A process for the preparation of reagent C1 according to claim 1, characterized in that it comprises the following steps:
(1) Dissolving the compound 2 in ultra-dry tetrahydrofuran THF, adding alkali under the nitrogen atmosphere and ice bath condition, stirring and reacting for 0.5-1.5 h, adding the azido compound 1, and stirring and reacting for 0.5-1.5 h; slowly heating to room temperature, stirring overnight, extracting, concentrating under reduced pressure to extract organic phase, and purifying to obtain compound 3; wherein,
the molar ratio of the compound 2 to the alkali 1 to the azido compound 1 is 1: 1-1.5: 1-1.5;
the chemical structural formula of the compound 2 is as follows:
；
the structural formula of the compound 3 is as follows:
；
base 1 of step (1) is 1, 8-diazabicyclo [5.4.0] undec-7-ene or sodium hydride;
the azido compound 1 is selected from diphenyl azide phosphate and a containing characteristic structure- (CH) 2 ） m -N 3 Brominated azido alkanes of (C) and containing a characteristic structure- (CH) 2 CH 2 O） n -N 3 Any one of bromoazide ethylene glycol, wherein m=1 to 10, n=1 to 5;
(2) Compound 3 was dissolved in a volume ratio of 1:1 and dilute acid solution with pH value of 4, heating and refluxing 4h, and concentrating under reduced pressure to obtain a compound 4;
the structural formula of the compound 4 is as follows:
；
(3) Dissolving a compound 4 in ultra-dry N, N-dimethylformamide, adding alkali 2, stirring at room temperature for reaction for 20-40 min, slowly adding bromoacetate compounds, stirring for 11-13 h, filtering after the reaction is finished, extracting the collected filtrate, concentrating under reduced pressure to extract an organic phase, and purifying to obtain a compound 5;
the structural formula of the compound 5 is as follows:
；
wherein,
the molar ratio of the compound 4 to the alkali 2 to the bromoacetate compound is 1: 1-1.5: 0.9-1;
the alkali 2 in the step (3) is selected from any one of potassium carbonate, sodium carbonate and cesium carbonate;
the bromoacetate compound is ethyl bromoacetate or butyl bromoacetate;
(4) Dissolving the compound 5 in ultra-dry tetrahydrofuran, adding manganese dioxide, stirring and reacting for 17-19 h under the condition of nitrogen atmosphere and room temperature, centrifuging for 4-5 min at 8000r/min, collecting a supernatant organic phase, concentrating under reduced pressure, and purifying to obtain a compound 6;
the structural formula of the compound 6 is as follows:
；
wherein,
the molar ratio of the compound 5 to the manganese dioxide is 1: 40-50 parts;
(5) Dissolving a compound 6 in a solvent, adding an ester-based hydrolysate, carrying out reflux reaction for 1.5-2.5 h, concentrating and purifying the reaction solution under reduced pressure to obtain an azide reagent C1;
the solvent in the step (5) is ultrapure water; the ester-based hydrolysate is trifluoroacetic acid; the molar ratio of compound 6 to trifluoroacetic acid (TFA) was 1: 4-5.
4. A method of preparation according to claim 3 wherein in step (1) the azide compound 1 is diphenyl azide phosphate, bromoazidoethane, bromoazidotrigine.
5. A process according to claim 3, wherein in steps (1), (3) the extracted extractant is analytically pure Dichloromethane (DCM) or Ethyl Acetate (EA);
in the steps (1), (3), (4) and (5), the purification is separation and purification by silica gel column chromatography.
6. Use of an azide reagent according to claim 1 or 2 in protein site-specific azide.
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Bifunctional pyridoxal derivatives as efficient bioorthogonal reagents for biomacromolecule modifications;Xianxian Mao et al.;《Chem. Commun.》;20200602;第56卷;第7601-7604页 *
Post-translational site-specific protein azidolation with an azido pyridoxal derivative;Wang, Baochuan et al.;《Chemical Communications (Cambridge, United Kingdom)》;20220606;第58卷(第53期);第7408-7411页 *
Rapid Catalyst-Free Hydrazone Ligation: Protein-Pyridoxal Phosphoramides;Xiaojian Wang et al.;《Bioconjugate Chem.》;20121127;第23卷(第12期);第2329-2334页 *
Single-site glycine-specific labeling of proteins;Landa Purushottam et al.;《Nat. Commun.》;20190610;第10卷;第2539页 *

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