CN117903044A - Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof - Google Patents

Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof Download PDF

Info

Publication number
CN117903044A
CN117903044A CN202311865218.9A CN202311865218A CN117903044A CN 117903044 A CN117903044 A CN 117903044A CN 202311865218 A CN202311865218 A CN 202311865218A CN 117903044 A CN117903044 A CN 117903044A
Authority
CN
China
Prior art keywords
compound
reaction
group
pyridin
dye
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311865218.9A
Other languages
Chinese (zh)
Inventor
刘春荣
陈林峰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Central China Normal University
Original Assignee
Central China Normal University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Central China Normal University filed Critical Central China Normal University
Priority to CN202311865218.9A priority Critical patent/CN117903044A/en
Publication of CN117903044A publication Critical patent/CN117903044A/en
Pending legal-status Critical Current

Links

Landscapes

  • Pyridine Compounds (AREA)

Abstract

The invention provides a pyridine-2-alkynyl carbonyl compound, a preparation method and application thereof, wherein the compound can realize amino acid, polypeptide and protein modification through efficient reaction with protein sulfhydryl. The reagent has important application value in the fields of antibody coupling drug development, covalent inhibitor development, functional proteomics analysis and the like by combining click reaction and other functional modification reactions.

Description

Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a pyridine-2-alkynyl carbonyl compound, and a preparation method and application thereof. More particularly, the invention relates to conjugates obtained by selective reaction of a boscalid or functional molecule-bearing alkynyl carbonyl compound with a protein sulfhydryl group for crosslinking, and a preparation method and application thereof.
Background
In life science research, protein modification has a very important meaning, so that the protein has more complex structure and more diversified functions. Protein modifications can be categorized into natural modifications and artificial chemical modifications. Recently, artificial chemical modification of proteins has been attracting attention of students, and the artificial modification can obtain a specific function of proteins, and has been widely used in research of active proteomics, targeted drugs, and the like.
At present, three methods exist for artificially modifying proteins, namely, a gene codon technology, which can accurately modify proteins, but requires complicated biological experiment operation, has technical barriers and has a narrow application range; 2. fusion protein technology, i.e., protein tagging, but polypeptide tags are generally at the end of a protein, with functions limited to improving solubility, subcellular localization and affinity adsorption, and may have an impact on protein structure and function; 3. the chemical modification method uses chemical reagent to selectively and directly react with N-segment, C-end and active functional group of amino acid residue side chain of protein, and is simple and convenient, and is the most widely used method at present. The ideal chemical modification method needs to meet the following requirements:
1. Is compatible with biological solvent conditions;
2. the reaction is tiny and efficient;
3. the product has certain stability;
4. The reaction is highly selective.
Cysteine is one of the common targets for chemical modification, but common sulfanyl alkylation reaction and Michael addition reaction modification have the problems of poor selectivity, unstable reaction products, low reaction rate, low reaction yield and the like.
Therefore, in both the fields of chemical and biological research and biological medicine, a cysteine labeling reagent with high speed, high efficiency, stable product and good selectivity is urgently needed, so that the defect of protein modification means is overcome.
Disclosure of Invention
The invention aims to provide a sulfhydryl labeling reagent with high reaction yield, high reaction rate, stable reaction product and good selectivity to sulfhydryl-containing species, a synthesis method thereof and application thereof in protein modification and coupling compounds.
In one aspect of the invention, a pyridine-2-alkynyl carbonyl compound is provided, and the structure is shown as a general formula I:
Wherein:
X is-CH 2 -, -O-, -NH-, phenyl or phenyl derivative;
L is- (CH 2)n -or- (CH 2CH2O)n) -linked to the X group, wherein n is selected from any integer from 0 to 20;
R is optionally selected from the group consisting of any one or more of the following: methyl, amino, carboxyl and reactive intermediates thereof, substituents for click reactions, fluorophores, subcellular localization groups, drugs with cytotoxicity, small molecule inhibitors, and the like.
As a preferred embodiment, the carboxyl group and its reactive intermediate include:
as a preferred embodiment, the substituents for click reaction include: the sequence of the sequences-N 3,
In a preferred embodiment, the fluorescent group is any one of rhodamine dye, fluorescein dye, coumarin dye, polycyclic aromatic hydrocarbon dye, NBD-amine dye, naphthalimide dye, BODIPY dye and cyanine dye fluorescent group.
As a preferred embodiment, the subcellular localization group comprises: a subcellular localization peptide,
As a preferred embodiment, the cytotoxic drug is selected from the group consisting of: tubulin inhibitors, topoisomerase inhibitors, DNA binding agents.
As a preferred embodiment, the small molecule inhibitor comprises: enzyme inhibitors, ion channel blockers, receptor agonists, antagonists and inverse agonists.
As a preferred embodiment, the R group is a combination group of a substituent for click reaction and any one of a fluorescent group, a subcellular localization group, a drug with cytotoxicity or a small molecule inhibitor, i.e., a fluorescent group, a subcellular localization group, a drug with cytotoxicity or a small molecule inhibitor is coupled by click reaction.
The invention also provides a preparation method of the pyridine-2-alkynyl carbonyl compound, which uses 2-ethynyl pyridine andThe reaction is carried out in the presence of A catalyst and A solvent for the starting materials, wherein A is halogen, for example I, cl, F, br.
As a preferred embodiment, the catalyst is a lithium-based catalyst, such as n-butyllithium.
The invention also provides another preparation method of the pyridine-2-alkynyl carbonyl compound, which uses 2-halogenated pyridine andThe reaction is carried out in the presence of a catalyst and a solvent as starting materials.
As a preferred embodiment, the catalyst is a platinum catalyst and a copper catalyst, such as bis triphenylphosphine platinum dichloride, cuprous iodide.
As a preferred embodiment, the halogen element includes I, cl, F, br.
In a preferred embodiment, the solvent is selected from THF or DMF.
The invention also provides application of the pyridine-2-alkynyl carbonyl compound serving as a labeling reagent or a conjugate of the compound containing the reduced sulfhydryl group or serving as a labeling reagent or a conjugate for preparing the compound containing the reduced sulfhydryl group.
As a preferred embodiment, the reduced thiol-containing compound comprises a cysteine, polypeptide, protein, enzyme, antibody fragment, which is located either inside or outside the cell.
The reaction equation involved in the application is shown in a general formula II:
wherein the Subs are cysteines, polypeptides, proteins, enzymes, antibodies, antibody fragments, or other substrates containing reduced sulfhydryl groups.
As a preferred embodiment, the X group isThe L chain is- (CH 2)4 -.
As a preferred embodiment, the X group is-CH 2 -and the L chain is- (CH 2)3 -.
As a preferred embodiment, when the pyridine-2-alkynylcarbonyl compound is used as a labeling reagent, the R group is the above-mentioned fluorescent group or is further coupled to the fluorescent group via the above-mentioned substituent for click reaction.
In a preferred embodiment, when the pyridine-2-alkynyl carbonyl compound is used as a labeling reagent, the R group is a molecule further conjugated with an affinity tag or containing an affinity tag through the above substituent for click reaction, or is conjugated with the affinity tag or containing an affinity tag through other reaction, and the affinity tag includes biotin, desthiobiotin, his-tag, FLAG, strep-tag II, HA-tag.
As a preferred embodiment, the pyridine-2-alkynyl carbonyl compound is used for cell fluorescence imaging, and the related reaction is shown in a general formula II. Wherein the Subs are proteins having a reduced thiol group in the cell, and the R group is selected from the group consisting of: the sequence of the sequences-N 3,
And rhodamine dyes, fluorescein dyes, coumarin dyes, polycyclic aromatic hydrocarbon dyes, NBD-amine dyes, naphthalimide dyes, BODIPY dyes and cyanine dyes which are connected through click reaction or other reactions.
As a preferred embodiment, the X group is-CH 2 -, the L chain is- (CH 2)3 -, and the R group isAfter labeling the clickable tag to the cellular protein, a copper catalyzed click reaction is used to further attach a rhodamine dye molecule to the cellular protein, thereby fluorescently labeling the protein.
As a preferred embodiment, the X group is-CH 2 -, the L chain is- (CH 2)3 -, and the R group is attached by click reaction1, 8-Naphthalimide dye molecules, and directly completing fluorescent labeling of cellular proteins.
As a preferred embodiment, the pyridine-2-alkynyl carbonyl compound is used for protein subcellular localization, and the related reaction is shown in a general formula II. Wherein the Subs are proteins having reduced sulfhydryl groups; the R group is selected from: by click reaction or other reaction conjugation of subcellular localization peptides,
As a preferred embodiment, the X group is-CH 2 -, the L chain is- (CH 2)3 -, and the R group isTriphenylphosphine molecules are coupled to proteins via reaction formula II, and the proteins are localized in intracellular mitochondria due to the positive charge of triphenylphosphine in the physiological environment.
As a preferred embodiment, the pyridine-2-alkynyl carbonyl compound is used for cysteine protein enrichment and proteomics analysis, and the related reaction is shown as a general formula II. Wherein the Subs are total proteins of bacteria, fungi or cells; the R group is selected from: biotin, desthiobiotin, his-tag, FLAG, strep-tag II, HA-tag or other affinity tag attached by a click reaction or other reaction, and molecules comprising these affinity tags.
As a preferred embodiment, the X group is-CH 2 -, the L chain is- (CH 2)3 -, and the R group is(Biotin), labeling biotin molecules onto Hela cell total protein by reaction formula ii, enriching cysteine-containing proteins by streptavidin magnetic beads, and performing functional proteomic analysis using mass spectrometry.
As a preferred embodiment, the pyridine-2-alkynyl carbonyl compound is used for cysteine protein enrichment and proteomics analysis, and the related reaction is shown as a general formula II. Wherein the Subs are total proteins of bacteria, fungi or cells; the R group is selected from: tubulin inhibitors, topoisomerase inhibitors, DNA binding agents.
As a preferred embodiment, the X group is-CH 2 -, the L chain is- (CH 2)3 -, and the R group is
The MMAE molecules are coupled to antibodies that are reduced to break disulfide bonds via reaction formula ii, which can subsequently be used for antibody targeted therapy.
The labeling reagent of the invention can be divided into three types of pyridine-2-alkynyl ketone, pyridine-2-alkynyl formate and pyridine-2-alkynyl formate amide according to the difference of another substituent on carbonyl. All three types of marking reagents can realize amino acid, polypeptide and protein modification through efficient reaction with protein sulfhydryl. The reagent has important application value in the fields of antibody coupling drug development, covalent inhibitor development, functional proteomics analysis and the like by combining click reaction and other functional modification reactions.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the invention, the drawings needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a bar graph of conversion of PyYO-3 over time using HPLC for PyYO-3 to cysteine reactions;
FIG. 2 is a plot of the conversion of PyYO-3 over time for PyYO-3 reacted with cysteine as measured using HPLC and a fitted reaction profile;
FIG. 3 shows the conversion of PyYO-1 to different nucleophilic amino acids, pyYO-1;
FIG. 4 is a schematic illustration of the reaction of PyYO-2 with 14 peptide;
FIG. 5 is a graph of mass spectral results of PyYO-2 reacted with 14 peptide: DMSO control (left) and PyYO-2 modification (right);
FIG. 6 shows Western-Blot results of PyYO-Biotin on beta-lactoglobulin markers at different incubation times;
FIG. 7 shows Western-Blot results of β -lactoglobulin markers at various concentrations of PyYO-Biotin;
FIG. 8 is a graph of the mass spectrum of the reaction of PyYO-2 with a C2Am protein: DMSO control (left) and PyYO-2 modification (right);
FIG. 9 is a SDS-PAGE result of PyYO-2 binding to a click reaction for fluorescent labeling of proteins;
FIG. 10 is a photograph showing fluorescence of PyYO-2 binding click reactions for fluorescent labeling of intracellular proteins;
FIG. 11 is a SDS-PAGE result of PyYO-Fluor for fluorescence labeling of trastuzumab;
FIG. 12 is a fluorescence photograph of PyYO-Fluor coupled trastuzumab incubated Her2 positive/negative cells.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but is not to be construed as being limited to the present invention, and modifications or substitutions made in the method, steps or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
In the specification, pyYO is used for referring to the pyridin-2-ynylketone compound, and PyYO-1, pyYO-2, pyYO-3, pyYO-Biotin and pyYO-Fluor in the corresponding examples are all the names of a specific molecular structure of the pyridin-2-ynylketone compound.
PyYP denotes a pyridine-2-alkynoate compound, and PyYP-1 is a name belonging to a specific molecular structure of the pyridine-2-alkynoate compound.
PyYPA denotes a pyridin-2-ynyl amide compound, pyYPA-1 is a designation of a specific molecular structure belonging to the pyridin-2-ynyl amide compound.
Example 1 pyridin-2-ynylketones (PyYO) and Synthesis thereof
A pyridine-2-alkynyl ketone compound has a structure shown in a general formula I:
wherein X is-CH 2 -or phenyl and derivatives thereof;
L is- (CH 2)n -or- (CH 2CH2O)n) -linked to the X group, wherein n is selected from any integer from 0 to 20;
r is selected from the group consisting of: methyl, amino, carboxyl and reactive intermediates thereof, substituents for click reactions, fluorophores, subcellular localization groups or drugs with cytotoxicity, and the like.
(1) When X is-CH 2 -, R is methyl, the synthesis reaction is as follows:
taking n=0 in L as an example, the specific synthesis steps are as follows:
Under the protection of argon, 2-ethynyl pyridine (8 mmol,0.8 mL) and tetrahydrofuran (8 mL) which are strictly dehydrated are added into a 50mL three-port bottle, liquid nitrogen acetone is cooled to minus 78 ℃ in a bath, n-butyl lithium (8 mmol,3.3 mL) is slowly added dropwise for 30min, stirring is carried out for 1h at minus 78 ℃, the temperature of the system is slowly increased to minus 30 ℃, after reaction for 2h, tetrahydrofuran solution (5 mL) of ethyl propionate (4 mmol,0.43 mL) is added at minus 78 ℃ and reacted for 20min, BF 3·Et2 O (1 mL) is added at the same temperature, after reaction for 30min, the temperature is increased to 0 ℃ and stirring is continued for 2h. The reaction was quenched with saturated NH 4 Cl (5 mL), extracted with diethyl ether (10 mL. Times.3), washed with saturated NaCl solution (10 mL. Times.3), and dried over anhydrous NaSO 4. Concentrating under reduced pressure, and passing through chromatographic column to obtain brown solid PyYO-1.
The yield using the above synthesis was 42% and compound PyYO-1 was analyzed:
1-(pyridin-2-yl)pent-1-yn-3-one(PyYO-1)1H NMR(600MHz,Chloroform-d)δ8.59(d,J＝4.1Hz,1H),7.67(t,J＝7.3Hz,1H),7.52(d,J＝7.7Hz,1H),7.32-7.26(m,1H),2.67(q,J＝7.2Hz,2H),1.13(t,J＝7.3Hz,3H).13C NMR(150MHz,Chloroform-d)δ188.3,150.6,140.9,136.5,128.8,124.7,87.9,85.6,39.1,7.9.
(2) When X is-CH 2 -, R is ethynyl, the synthesis reaction is as follows:
taking L as- (CH 2)3 -as an example, the specific synthesis steps are as follows:
10mL of ultra-dry tetrahydrofuran and 0.3mL (0.3 mmol,1 e.q.) of 2-ethynylpyridine are added into a 50mL three-necked flask under the strict anhydrous and anaerobic conditions and nitrogen protection conditions, the reaction vessel is placed in a Dewar flask, cooled to-78 ℃ (liquid nitrogen-acetone), and 1.4mL of 2.4M n-butyllithium n-hexane solution is slowly dropped into the reaction system under magnetic stirring. After the addition of 5 minutes, the reaction was continued for 25 minutes, the temperature was increased to-45℃and the system changed from a yellow clear solution to a pale pink turbidity. Liquid nitrogen was again added to the dewar to reduce the temperature to-78 ℃, tetrahydrofuran solution (3 mL) of N, O-dimethylhept-6-ynylhydroxylamine (580 mg,0.3mmol,1 e.q.) was added dropwise to the reaction system, the reaction was continued for 45 minutes, the reaction temperature was increased to-10 ℃, TLC monitored that the starting material 2-ethynylpyridine was mostly reacted without change in concentration, and the reaction was quenched by addition of 10mL of saturated ammonium chloride solution. The reaction was transferred to a 100mL round bottom flask and the organic solvent was distilled off under reduced pressure, leaving a yellow oil insoluble in the aqueous layer. 20mL of diethyl ether was added, the organic phase was separated, the aqueous layer was extracted three times with 20mL of diethyl ether, and the organic phases were combined and distilled under reduced pressure. The solid residue was chromatographed on silica gel (ethyl acetate: petroleum ether=1:15) to give PyYO-2 as a yellow-black solid.
The yield using the above synthesis was 58% and compound PyYO-2 was analyzed:
1-(pyridin-2-yl)nona-1,8-diyn-3-one(PyYO-2)1H NMR(400MHz,Chloroform-d)δ8.71-8.63(m,1H),7.74(tt,J＝7.7,1.9Hz,1H),7.63-7.56(m,1H),7.39-7.33(m,1H),2.76(td,J＝7.3,1.9Hz,2H),2.26-2.21(m,2H),1.96(q,J＝2.6Hz,1H),1.90-1.83(m,2H),1.63-1.57(m,2H).13C NMR(150MHz,Chloroform-d)δ150.6,128.8,124.6,85.6,68.8,44.9,27.6,18.2.
(3) When X is phenylene and R is hydrogen, the synthesis reaction is as follows:
taking n=0 in L as an example, the specific synthesis steps are as follows:
Under the protection of argon, 2-ethynyl pyridine (5 mmol,0.5 mL) and tetrahydrofuran (6 mL) which are strictly dehydrated are added into a 50mL three-port bottle, liquid nitrogen acetone is cooled to minus 78 ℃ in a bath, n-butyllithium (7.5 mmol,3.2 mL) is slowly added dropwise, stirring is carried out for 1h at minus 78 ℃, the system is slowly heated up to minus 30 ℃ from minus 78 ℃, benzoyl chloride (7.5 mmol,0.85 mL) is added at minus 78 ℃ after reaction for 2h, reaction is carried out for 30min, and stirring is continued after heating to 0 ℃. The reaction was quenched with saturated NH 4 Cl (5 mL), extracted with diethyl ether (10 mL. Times.3), washed with saturated NaCl solution (10 mL. Times.3), and dried over anhydrous NaSO 4. Concentrating under reduced pressure, and passing through chromatographic column to obtain brown solid PyYO-3.
The yield using the above synthesis was 59% and compound PyYO-3 was analyzed:
1-phenyl-3-(pyridin-2-yl)prop-2-yn-1-one(PyYO-3)1H NMR(400MHz,Chloroform-d)δ8.72(d,J＝4.8Hz,1H),8.27(d,J＝7.8Hz,2H),7.78(tt,J＝7.7,1.9Hz,1H),7.71(d,J＝7.8Hz,1H),7.65(dd,J＝8.4,6.5Hz,1H),7.53(td,J＝7.9,1.9Hz,2H),7.40(t,J＝6.3Hz,1H).
example 2 pyridin-2-ynyl ester Compound (PyYP) and Synthesis thereof
A pyridine-2-alkynyl ester compound has a structure shown in a general formula I:
wherein X is-O-;
L is- (CH 2)n -or- (CH 2CH2O)n) -linked to the group X 2, wherein n is selected from any integer from 0 to 20;
(1) When R is methyl, the synthesis reaction is as follows:
Taking L as- (CH 2)2 -as an example, the specific synthesis steps are as follows:
Under the protection of argon, 2-ethynyl pyridine (5 mmol,0.5 mL) and tetrahydrofuran (15 mL) which are strictly dehydrated are added into a 50mL three-port bottle, liquid nitrogen acetone is cooled to minus 78 ℃ in a bath, n-butyllithium (7.5 mmol,3.2 mL) is slowly added dropwise, stirring is carried out for 1h at minus 78 ℃, the system is slowly heated up to minus 30 ℃ from minus 78 ℃, propyl chloroformate (7.5 mmol,0.85 mL) is added into the three-port bottle after reaction for 2h at minus 78 ℃, reaction is carried out for 30min, and stirring is continued after heating to 0 ℃ for 2h. The reaction was quenched with saturated NH 4 Cl (5 mL), extracted with diethyl ether (10 mLx 3), washed with saturated NaCl solution (10 mLx 3), and dried over anhydrous NaSO 4. Concentrating under reduced pressure, and passing through chromatographic column to obtain brown solid PyYP-1.
The yield using the above synthesis was 96% and compound PyYP-1 was analyzed:
ethyl 3-(pyridin-2-yl)propiolate(PyYP-1)1H NMR(600MHz,Chloroform-d)δ8.58(d,J＝4.6
Hz,1H),7.66(d,J＝7.7Hz,1H),7.53(d,J＝7.8Hz,1H),7.32-7.27(m,1H),4.13(t,J＝6.7Hz,2H),1.67(d,J＝14.2Hz,2H),0.91(t,J＝7.4Hz,3H).13C NMR(150MHz,Chloroform-d)δ155.8,152.7,142.7,138.6,130.7,126.8,85.8,70.1,23.9,12.5.
example 3 pyridin-2-ynyl ester Compound (PyYPA) and Synthesis thereof
A pyridine-2-alkynyl ester compound has a structure shown in a general formula I:
Wherein X is imino;
L is- (CH 2)n -or- (CH 2CH2O)n) -linked to the group X 2, wherein n is selected from any integer from 0 to 20;
(1) When R is methyl, the synthesis reaction is as follows:
Taking L as- (CH 2)2 -as an example, the specific synthesis steps are as follows:
To a 50mL round bottom flask under argon was added, in order, propynylpropylamine (55.5 mg,0.5 mmol), 2-iodopyridine (10.3 mg,0.5 mmol), DMF (10 mL) with strict water removal, and 5%o mol of bis triphenylphosphine platinum dichloride and cuprous iodide. The reaction was carried out at room temperature for 8h, the progress of the reaction was checked by TLC, and after the complete consumption of the reactant, DMF was dried under reduced pressure and the product was a brown solid by column chromatography PyYPA-1.
The yield using the above synthesis was 72% and compound PyYPA-1 was analyzed:
N-propyl-3-(pyridin-2-yl)propiolamide(PyYPA-1)1H NMR(400MHz,Chloroform-d)δ8.67(d,J＝4.9Hz,1H),7.77(td,J＝7.7,1.6Hz,2H),7.63(d,J＝7.8Hz,1H),7.39(t,J＝6.4Hz,1H),6.18(s,1H),3.37(q,J＝6.8Hz,2H),1.63(h,J＝7.2Hz,2H),1.00(t,J＝7.4Hz,3H).
EXAMPLE 4 reaction Rate of pyridine alkynecarbonyl Compounds
The reaction is dynamically monitored by using high performance liquid chromatography, and the rate constant and the conversion rate of the reaction are obtained by fitting a reaction curve.
The specific experimental steps are as follows:
1) The products obtained by separation and purification through the organic synthesis means are respectively prepared into different concentrations (0.01 mM,0.05mM,0.10mM,0.20mM and 0.50 mM), and a standard curve of the concentration-absorption peak area of the products is drawn by using HPLC and a corresponding elution method;
2) Respectively preparing acetonitrile solution of a labeling reagent and 2 XPBS solution of acetylcysteine methyl ester, mixing and placing the mixture at 37 ℃ for reaction, taking out a certain amount of reaction system solution at intervals, and drawing a product conversion chart (figure 1) and a first-order kinetic chart (figure 2) of the reaction according to a formula of y=A-A x p (-k x) (y corresponds to time is the product peak area, x is the reaction time, A is the theoretical yield obtained by fitting, k is the first-order kinetic constant obtained by fitting) by using HPLC and a corresponding elution method.
The results are summarized in Table 1 below:
TABLE 1 rate constant of reaction and conversion results
EXAMPLE 5 reaction Selectivity of pyridine alkynecarbonyl compounds
Boc-tyrosine methyl ester, boc-phenylalanine methyl ester, boc-tryptophan methyl ester, boc-serine methyl ester, boc-cysteine methyl ester, boc-histidine methyl ester, 20 XPBS stock solution with Boc-lysine methyl ester concentration of 10mM, 10mM PyYP-1 acetonitrile solution and 50mM acetonitrile solution of p-methoxyacetophenone were respectively prepared. Respectively diluting an amino acid solution and PyYP-1 solution by 10 times, mixing and reacting at a ratio of 1:1, adding 1% of p-methoxy acetophenone solution as an internal reference, and starting timing. HPLC analysis was performed by feeding samples at 30min,150min,270min, respectively. In addition, the amino acid solution was replaced with 2×pbs as a control group. The results are shown in FIG. 3.
EXAMPLE 6 pyridine alkyne labeling polypeptide
A synthetic single letter sequence GTSWCYNQKRHDGP of 14 peptide was used as a labeled substrate, which sequence included various nucleophilic amino acids. The polypeptide powder was prepared as a 10. Mu.M PBS solution, while 5mM PyYO-2 in DMSO was prepared. To 100. Mu.L of the polypeptide solution, 1. Mu. LPyYO-2 was added and the mixture was allowed to react at 37℃for 30 minutes, as shown in FIG. 4, while a control group was set in which PyYO-2 was replaced with DMSO.
The reacted polypeptide solution was purified by solid phase extraction and concentrated by lyophilization. The lyophilized solid was redissolved in water and mixed with 20% sinapic acid 1:1 and mass spectrometry was performed using AB SCIEX TOF/TOF 5800, as shown in FIG. 5, with one and only one PyYO-2 tag on the polypeptide, indicating that the pyridinyne compound had good selectivity while completely labeling the polypeptide cysteine.
EXAMPLE 7 pyridine alkyne-based Compound marker protein
PyYO-2 was attached to azido Biotin by copper-catalyzed click reaction for protein labelling reactions, the reaction scheme is shown below, and PyYO-Biotin refers to the synthesized product.
Preparing a reduced beta-lactoglobulin solution: dissolving 10 mu M beta-lactoglobulin in PBS, adding TCEP with a final concentration of 30 mu M, reacting for 2 hours in a water bath at 37 ℃, and breaking disulfide bonds in the protein to fully expose sulfhydryl groups;
Preparing a C2Am protein solution with the concentration of 1mg/mL;
PyYO-Biotin solution with concentration of 50mM is prepared and dissolved in DMSO;
PyYO-2 solution was prepared at a concentration of 50mM and dissolved in DMSO.
(1) Influence of the incubation time of the pyridine alkyne compound on the protein labelling:
7 parts of 100. Mu.L of beta-lactoglobulin solution were placed in 7 EP tubes, respectively, a solution of 2. Mu. LPyYO-Biotin was added to one of the EP tubes, reacted in a water bath at 37℃and the same PyYO-Biotin solution was added after 60, 90, 110, 115, 117, 119 minutes, respectively. Then PyYO-Biotin were 100 equivalents each and the reaction times were 120, 60, 30, 10,5,3,1 minutes, respectively.
As a result of Western-Blot analysis using streptavidin-modified antibody, the WB band was not changed after 5 minutes of reaction time, which indicates that the reaction end point was reached after 5 minutes of labeling reaction of PyYO-Biotin under the reaction conditions as shown in FIG. 6.
(2) Influence of the incubation concentration of pyridine alkynes on protein labeling:
6 portions of 100. Mu.L of beta-lactoglobulin solution were placed in 6 EP tubes, respectively, and 4. Mu.L of PyYO-Biotin solution or diluent (less than 4. Mu.L of DMSO to 4. Mu.L) was added to make the concentration in each tube 100. Mu.M, 250. Mu.M, 500. Mu.M, 1mM,1.5mM and 2mM, respectively. The reaction was carried out for 1 hour in a 37℃water bath.
As a result of Western-Blot analysis using streptavidin-modified antibody, FIG. 7 shows that PyYO-Biotin at 500. Mu.M concentration has excellent labeling effect on beta-lactoglobulin under the reaction conditions.
(3) Marking efficiency of pyridine alkynes:
Two 1mL portions of the C2Am solution were placed in 2 EP tubes, respectively, and 2. Mu.L of PyYO-2 solution or 2. Mu.L of DMSO solution were added, respectively, and reacted in a 37℃water bath for 30 minutes. The reacted protein solution was precipitated using a methanol/chloroform method to obtain a clean protein precipitate. Mass spectrometry was performed using Thermo SCIENTIFIC Q Exactive after protein re-solubilization, resulting in deconvolution as shown in fig. 8, the mass of the tag group increased 211.23 compared to the DMSO group, conforming to the mass of PyYO-2, and the tag was almost complete.
(4) The pyridine alkyne compound combines with click reaction to carry out fluorescent marking on the protein:
6 parts of TCEP treated beta-lactoglobulin 100. Mu.L was placed in 6 EP tubes, respectively, to which PyYO-2 solutions of different concentrations or 1. Mu.L of DMSO were added, respectively, so that the PyYO-2 final equivalent ratios were 0, 25, 50, 100, 200 and 500, respectively. Copper sulphate, TBTA, azidonaphthoimide and sodium ascorbate were then added to each tube in this way and reacted for 2 hours at 37 ℃ in a water bath.
The SDS-PAGE analysis of the fluorescence intensity of the proteins showed that PyYO-2 can be used for fluorescence labelling of proteins, and the labelling reaction and the copper-catalyzed click reaction do not interfere with each other, and can be used for functional proteomic analysis as shown in FIG. 9.
EXAMPLE 8 application of pyridine alkynes in cell imaging
Cells were inoculated into glass-bottomed cell culture dishes and cultured for about 24 hours to allow cell attachment growth. Hydrogen peroxide at various concentrations or equal volumes of PBS were added to the medium. After 5 minutes, the medium was discarded and incubated with PBS containing PyYO-2 (50. Mu.M) for 5 minutes. The PBS solution containing PyYO-2 was discarded, washed three times with PBS, cells were fixed by adding 4% PFA fixative, and then the fixative was discarded. Finally, adding a catalyst for click reaction and naphthalimide-azide, and reacting for 2 hours at room temperature in a dark place. Nuclei were stained with DAPI prior to laser confocal.
The results of confocal laser excitation are shown in FIG. 10, which illustrates that pyridine alkynes can be used to label intracellular protein cysteines and can be used to attach functional molecules through subsequent click reactions.
Example 9 application of pyridine alkynes in antibody-coupled drugs
PyYO-2 was attached to azidofluorescein by copper-catalyzed click reaction for protein coupling reaction, pyYO-Fluor refers to the synthesized product.
The antibody was diluted to 1mg/mL (6.85 μm) with PBS containing 6.85mM dithiothreitol, ph=8.0 and incubated at 37 ℃ for 40 min. Dithiothreitol was removed by ultrafiltration and, after lyophilization, redissolved in 7.4 PBS containing 1mM EDTA. PyYO-Fluor or AF647 was added at a final concentration of 200. Mu.M and reacted for 2 hours. The antibodies were purified using Thermo Scientific Zeba columns and the SDS-PAGE analysis was shown in FIG. 11.
BT-474 cells and MCF-7 (negative control) after 4% PFA fixation were incubated with PyYO-Fluor or AF647 conjugated antibodies at 10. Mu.g/mL for 1.5 hours at 37℃protected from light, washed 3 times with PBST, and PBS one time. The tablets were then room temperature capped using DAPI containing cappers for 20min.
The laser confocal results are shown in fig. 12, which illustrates that the pyridine alkyne compound can be used for modifying antibody proteins, for example, fluorescein molecules in the experiment can be replaced by drug molecules, drugs can be stably modified on cysteine of antibodies, and synthesis of drugs coupled with the antibodies can be applied.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that numerous modifications and variations can be made without departing from the principles of the invention, and such modifications and variations are to be regarded as being within the scope of the invention.

Claims (10)

1. The structure of the pyridine-2-alkynyl carbonyl compound is shown as a general formula I:
Wherein:
X is-CH 2 -, -O-, -NH-, phenyl or phenyl derivative;
L is- (CH 2)n -or- (CH 2CH2O)n) -linked to the X group, wherein n is selected from any integer from 0 to 20;
r is optionally selected from the group consisting of any one or more of the following: methyl, amino, carboxyl and reactive intermediates thereof, substituents for click reactions, fluorophores, subcellular localization groups, drugs with cytotoxicity, small molecule inhibitors.
2. The pyridin-2-ynylcarbonyl-like compound according to claim 1, wherein the carboxy group and its reactive intermediate comprises:
3. The pyridin-2-ynylcarbonyl-like compound according to claim 1, wherein the substituents for the click reaction comprise: the sequence of the sequences-N 3,
4. The pyridin-2-ynylcarbonyl compound according to claim 1, wherein the fluorescent group is selected from any one of rhodamine dye, fluorescein dye, coumarin dye, polycyclic aromatic hydrocarbon dye, NBD-amine dye, naphthalimide dye, BODIPY dye or cyanine dye fluorescent group.
5. The pyridin-2-ynylcarbonyl-like compound of claim 1, wherein the subcellular localization group comprises: a subcellular localization peptide,
6. The pyridin-2-ynylcarbonyl-like compound according to claim 1, wherein the cytotoxic drug comprises: tubulin inhibitors, topoisomerase inhibitors, and DNA binding agents.
7. The pyridin-2-ynylcarbonyl-like compound of claim 1, wherein the small molecule inhibitor comprises: enzyme inhibitors, ion channel blockers, receptor agonists, antagonists and inverse agonists.
8. A process for the preparation of a compound as claimed in any one of claims 1 to 7, characterized in that 2-ethynylpyridine andOr 2-halopyridine and/>The reaction is carried out as starting material in the presence of a catalyst, wherein A is halogen.
9. Use of a pyridin-2-ynylcarbonyl-like compound according to any one of claims 1 to 7 as a labelling agent or conjugate for a compound comprising a reduced thiol group or for the preparation of a labelling agent or conjugate for a compound comprising a reduced thiol group.
10. The use according to claim 9, wherein the reduced thiol-containing compound comprises cysteine, a polypeptide, a protein, an enzyme, an antibody fragment.
CN202311865218.9A 2023-12-28 2023-12-28 Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof Pending CN117903044A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311865218.9A CN117903044A (en) 2023-12-28 2023-12-28 Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311865218.9A CN117903044A (en) 2023-12-28 2023-12-28 Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN117903044A true CN117903044A (en) 2024-04-19

Family

ID=90683065

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311865218.9A Pending CN117903044A (en) 2023-12-28 2023-12-28 Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN117903044A (en)

Similar Documents

Publication Publication Date Title
Shiu et al. Electron‐deficient alkynes as cleavable reagents for the modification of cysteine‐containing peptides in aqueous medium
Wang et al. Water-soluble allyl sulfones for dual site-specific labelling of proteins and cyclic peptides
US5686261A (en) Xanthylium dyes that are well retained in mitochondria
US7595198B2 (en) Photocleavable isotope-coded affinity tags
US20080287710A1 (en) Methods for the preparation of chemically misaminoacylated tRNA via protective groups
Durek et al. Synthesis of fluorescently labeled mono-and diprenylated Rab7 GTPase
Piloto et al. A naphtho [2, 1-b] furan as a new fluorescent label: synthesis and spectral characterisation
JP2002536428A (en) Improved compounds for protein binding
CN104877982A (en) Method and reagent for preparing peptide/protein conjugate
CN114685463A (en) Isorhamnetin photoaffinity probe and synthetic method and application thereof
Fedorowicz et al. Synthesis and evaluation of dihydro-[1, 2, 4] triazolo [4, 3-a] pyridin-2-ium carboxylates as fixed charge fluorescent derivatization reagents for MEKC and MS proteomic analyses
CN113549125A (en) Method for chemical modification of photo-catalytic biomacromolecule
CN115594649B (en) Cysteine residue specific chemical probe and preparation method and application thereof
CN117903044A (en) Pyridine-2-alkynyl carbonyl compound and preparation method and application thereof
Sok et al. Design of new sensitive α, β-unsaturated carbonyl 1, 8-naphtalimide fluorescent probes for thiol bioimaging
JP3425623B2 (en) DNA fluorescently labeled probe, fluorescently labeled plasmid
CN114262356B (en) Method for modifying tryptophan residues of protein
US8053194B2 (en) Labeling substance and chimeric substance and method for preparing the same and method for capturing, structurally analyzing and/or identifying biological substance by means of the labeling substance
Temming et al. Protein enrichment by capture–release based on strain-promoted cycloaddition of azide with bicyclononyne (BCN)
Johannes et al. SNAP-tagging the retrograde route
CN113683658A (en) Method for modifying histidine residues of protein
US20020147354A1 (en) Fluorescent dyes for the labeling of biological substrates
CN111808006A (en) Nitrogen heterocyclic ring allyl compound and preparation method and application thereof
US20060051811A1 (en) Site-specific labelling of proteins using acridone and quinacridone lifetime dyes
Narita et al. Clickable gold nanoparticles for streamlining capture, enrichment and release of alkyne-labelled proteins

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination