CN117758374A - On-DNA alpha-hydroxy olefin lead compound and synthesis method thereof - Google Patents

On-DNA alpha-hydroxy olefin lead compound and synthesis method thereof Download PDF

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CN117758374A
CN117758374A CN202211130945.6A CN202211130945A CN117758374A CN 117758374 A CN117758374 A CN 117758374A CN 202211130945 A CN202211130945 A CN 202211130945A CN 117758374 A CN117758374 A CN 117758374A
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dna
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薛丽俊
胡允金
刘伟杰
吴靖
李淑
杨珂新
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Kanglong Beijing New Drug Technology Ltd By Share Ltd
Kanglong Huacheng Ningbo Technology Development Co ltd
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Kanglong Beijing New Drug Technology Ltd By Share Ltd
Kanglong Huacheng Ningbo Technology Development Co ltd
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Abstract

The invention provides an On-DNA alpha-hydroxy olefin lead compound and a synthesis method thereof, belonging to the field of construction of gene coding compound libraries. The invention discloses a method for synthesizing alpha-hydroxy olefin lead compounds On DNA for the first time, which has the advantages of small damage to the DNA, good universality, simple operation and mild condition, and can prepare On-DNA alpha-hydroxy olefin compounds with high conversion rate. Under the reaction condition of the invention, the product On-DNA alpha-hydroxy olefin compound has high conversion rate and good DNA integrity in the product. The invention enriches the chemical reaction types of synthesizing the coding compound library on DNA, constructs a novel alpha-hydroxy olefin skeleton for the gene coding compound library, and has very good application prospect in the development of leading medicines.

Description

On-DNA alpha-hydroxy olefin lead compound and synthesis method thereof
Technical Field
The invention belongs to the field of construction of gene coding compound libraries, and particularly relates to an On-DNA alpha-hydroxy olefin lead compound and a synthesis method thereof.
Background
Through many years of application, development and perfection, high-throughput screening has established a perfect screening process, and is an important way for international mainstream drug research and development companies to obtain target protein lead compounds. However, the traditional high-throughput screening of single molecules has the defects of limited number of compound libraries, long time, high cost and the like, and cannot meet the requirements of developing new drugs.
Brenner and Lerner creatively proposed in 1992 methods that can use the gene-encoded compound library technique (DELT) for screening of bioactive compounds. The principle of DELT is to label each small molecule compound during the reaction with a different specific sequence of gene fragments, and mass synthesize millions to billions of libraries of compounds linked to a specific gene sequence by split and pool methods using combinatorial chemistry strategies, using limited costs and time. The resulting mixture of compounds is then incubated with the protein target, physical separation is achieved by washing away compounds that do not bind to the protein target and finding compounds with high affinity. The gene encoding the compound library required for incubation of the target protein requires only an extremely small dose (microgram scale) and can be performed in a short time (e.g., within 1 day). Meanwhile, the DELT technology can easily perform multiple biological screening experiments under different conditions. At present, screening of libraries of gene-encoded compounds (Dels) has become a popular method for discovering new protein ligands (Goodnow, R.A.; dumelin, C.E.; keefe, A.D. DNA-encoded chemistry: enabling the deeper sampling of chemical space. Nat. Drug discovery.2016; neri, D.; lerner, R.A. DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable information. Annu. Rev. Biochem.2018,87, 479-502). DELs have great advantages over traditional chemistry in terms of cost, yield, and construction of larger chemical space, greatly increasing the number and diversity of compounds through high throughput screening. In practical use, DEL bioscreen successfully found an affinity for many protein targets (Zimmermann, G.; neri, D.DNA-encoded chemical libraries: foundations and applications in lead discovery. Drug discovery. Today 2016,21,1828-1834), including small chemical molecules currently in clinical trials (Belyanskaya, S.L.; ding, Y.; calahan, J.F.; lazaar, A.L.; israel, D.I. ChemChem.2017, 18, 837-842).
One of the most important current efforts in gene-encoding compound library technology is the development of chemical reactions On DNA (called "On-DNA chemical reactions" for short), and some On-DNA chemical reactions have been reported. For example, the sea medicine Mingkang New drug development Co., ltd (patent application CN 201910609569.0) discloses a method for obtaining On-DNA aromatic compounds by Suzuki coupling reaction: the On-DNA aryl halide is used as a substrate and reacts with an organic potassium trifluoroborate reagent in the presence of Pd catalyst, ligand and alkali to prepare the On-DNA aromatic compound. The method increases the diversity of DNA coding compound libraries of On-DNA aryl halides, has high reaction yield, wide substrate universality, mild condition and convenient operation, and is suitable for the synthesis of DNA coding compound libraries by porous plates. The Chengdu lead medicine development Co., ltd (patent application CN 201910590679.7) discloses a method for synthesizing an On-DNA aryl benzyl substituted compound, which takes an On-DNA aldehyde compound as a raw material, reacts with indole under an alkaline condition to generate an On-DNA indolol compound, and then the On-DNA indolol compound is reduced into an indole alkylation compound by diethyl 1, 4-dihydro-2, 6-dimethyl-3, 5-pyridine dicarboxylic acid under an acidic condition. The more kinds of chemical reactions are carried out on the gene-encoded compound library, the more conditions are enriched, the more choices are made when designing and synthesizing the gene-encoded compound library, and the more diversity of the compound library is obtained. However, the types of On-DNA chemical reactions reported in the current publications are limited, and the wide demands for developing lead compound discovery cannot be met.
The alpha-hydroxy olefin compound is a compound with an alpha-hydroxy olefin structure, has various biological activities, and can be found in various common drug molecules, such as Swertiamarin (Swertiamarin), cynaropicrin (Cnicin), cephalosporanic Acid (Cephalonic Acid), theophylline (Hygrophylline) and the like. Swertiamarin is an iridoid compound separated from swertia pseudochinensis of Gentianaceae, has inhibiting effect on autonomous rhythmic activities of isolated duodenum, uterus, gall bladder smooth muscle and bile duct sphincter of rat, and can resist excitation of acetylcholine and norepinephrine. Proved by clinic, the swertiamarin has obvious spasmolytic and analgesic effects on the smooth muscle spasmodic pains of the gastrointestinal tract and the biliary tract, has a certain sedative effect, and has no sensitization and irritation. The cynaropicrin, also called jeFall-ku-su, has antibacterial, antiinflammatory, skin whitening and antitumor effects, and has antibacterial effects on various bacillus such as Bacillus paradoxus.
However, since DNA must be stable under a certain water phase, pH, temperature, metal ion concentration and inorganic salt concentration, and a reaction for constructing a library of DNA-encoding compounds needs to have a high conversion rate, no report has been made on a method for constructing an alpha-hydroxyalkene gene-encoding compound on DNA. Therefore, the development of a method for synthesizing the On-DNA alpha-hydroxy olefin compounds has great significance for small DNA damage and high conversion rate.
Disclosure of Invention
The invention aims to provide an On-DNA alpha-hydroxy olefin lead compound and a synthesis method thereof, and application of the synthesis method in constructing a gene coding compound library.
The invention provides an On-DNA alpha-hydroxy olefin lead compound shown in a formula I:
wherein the DNA is a single-stranded or double-stranded nucleotide chain;
m is a connecting unit;
R 2 selected from nitro, cyano, and COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a ；
R 3 、R 4 Each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a Unsubstituted or substituted by R 3a The substituted following groups: c (C) 1 - 12 Alkyl, C 1-12 Alkoxy, C 2-8 Alkenyl, C 2-8 Alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-12 Alkyl, C 1-12 An alkoxy group; alternatively, R 3 、R 4 Connected into a ring;
R 2a selected from C 1-12 Alkyl, C 1-12 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-6 Alkylene group, R 2b Selected from C 1-12 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-12 Alkyl, C 1-12 An alkoxy group;
alternatively, R 4 Is hydrogen, R 2 、R 3 Connected into a ring.
Further, M is X 1 -Y 1 -P-Y 2 -X 2 -Q，X 1 、Y 1 、P、Y 2 、X 2 Q is each independently selected from the group consisting of unsubstituted, unsubstituted or substituted 1 SubstitutedThe following groups: NHCO, CONH, NH (V),5-6 membered aryl, 5-6 membered heteroaryl, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl, C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylene;
m is an integer of 1 to 10;
R 1 selected from hydrogen, halogen, nitro, cyano, mercapto, carboxyl, hydroxyl, halogenated or unhalogenated C 1-6 Alkyl, halogenated or non-halogenated C 1-6 An alkoxy group;
X 1 、Y 1 、P、Y 2 、X 2 q is not absent at the same time.
Further, the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula II-1:
wherein the DNA is a single-stranded or double-stranded oligonucleotide strand;
X 1 is NHCO or CONH, X 2 Is NHCO or CONH;
Y 1 is none or C 1-4 Alkylene group, Y 2 Is none or C 1-4 An alkylene group;
q is selected from unsubstituted or R 1 The substituted following groups: 5-6 membered aryl, 5-6 membered heteroaryl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, C 1-4 An alkylene group;
m is an integer of 1 to 8;
R 1 selected from hydrogen, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 An alkoxy group;
R 2 selected from nitro, cyano, and COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a ；
R 3 、R 4 Each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto、COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a Unsubstituted or substituted by R 3a The substituted following groups: c (C) 1 - 6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-6 Alkyl, C 1-6 An alkoxy group; alternatively, R 3 、R 4 Connected into a ring;
R 2a selected from C 1-6 Alkyl, C 1-6 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-4 Alkylene group, R 2b Selected from C 1-6 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-6 Alkyl, C 1-6 An alkoxy group;
alternatively, R 4 Is hydrogen, R 2 、R 3 Connected into a ring;
preferably, the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula II-2:
further, the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula III-a, a formula III-b, a formula III-c, a formula III-d, a formula III-e or a formula III-f:
wherein the DNA is a single-stranded or double-stranded oligonucleotide strand;
R 1 selected from hydrogen, halogen, C 1-3 Alkyl, C 1-3 An alkoxy group;
n is selected from 1,2 and 3;
R 2a selected from C 1-3 Alkyl, C 1-3 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-2 Alkylene group, R 2b Selected from C 1-3 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-3 Alkyl, C 1-3 An alkoxy group;
R 3 selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or substituted by R 3a Substituted phenyl, R 4 Selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or substituted by R 3a Substituted phenyl, or R 3 、R 4 Is linked to form unsubstituted or R 3b Substituted 4-membered saturated nitrogen heterocycles; r is R 3a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, R 3b Selected from Boc, C 1-3 Alkyl, C 1-3 An alkoxy group;
Y 3 is C 1-4 An alkylene group;
Y 4 is C 1-4 An alkylene group.
Further, the On-DNA alpha-hydroxy olefin lead compound is selected from one of the following compounds:
the invention also provides a method for synthesizing the On-DNA alpha-hydroxy olefin lead compound, which comprises the following steps: the method comprises the steps of reacting a compound shown in a formula I-a and a compound shown in a formula I-b in a solvent in the presence of alkali to obtain an On-DNA alpha-hydroxy olefin lead compound shown in the formula I:
。
further, the equivalent ratio of the compound shown in the formula I-a, the compound shown in the formula I-b and the base is 1: (500-2000): (500-2000);
the temperature of the reaction is 0-90 ℃ and the time is 1-24 hours;
the solvent is selected from water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, amyl alcohol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene;
the base is selected from one or more than two of triethylene diamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N' -tetramethyl ethylenediamine, 1, 3-tetramethyl guanidine, N, N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, carbonate, phosphate, borate, triethyl ammonium acetate and tris (hydroxymethyl) aminomethane.
Further, the equivalent ratio of the compound shown in the formula I-a, the compound shown in the formula I-b and the base is 1:1000:1000;
the temperature of the reaction is 25 ℃ and the time is 16 hours;
the solvent is selected from the group consisting of a mixture of water and acetonitrile, a mixture of water and N, N-dimethylformamide, and a mixture of water and ethanol;
the base is triethylenediamine.
Further, in the mixture of water and acetonitrile, the volume ratio of water to acetonitrile is (1-2): (1-2);
in the mixture of water and N, N-dimethylformamide, the volume ratio of the water to the N, N-dimethylformamide is (1-2): (1-2);
in the mixture of water and ethanol, the volume ratio of water to ethanol is (1-2): (1-2).
Further, the compound shown in the formula I-a isThe preparation method of the compound shown in the formula I-a comprises the following steps:
(1) Reacting the compound HP with a compound A1 to obtain a compound A2;
(2) Reacting the compound A2 with a deprotection agent to obtain a compound A3;
(3) The compound A3 reacts with the compound A4 to obtain the compound shown in the formula I-a.
Further, in step (1), the reaction is carried out in the presence of sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, the equivalent ratio of compound HP, compound A1, sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride being 1: (30-50): (200-300): (30-50), the solvent of the reaction is selected from one or more of water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, and the mixture of two or more thereof, and the temperature is 0-30 ℃, the reaction time is 0.5-4 hours; preferably, the equivalent ratio of the compound HP, the compound A1, the sodium tetraborate and the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride is 1:40:250:40, the solvent for the reaction is a mixed solution of water and dimethyl sulfoxide, the reaction temperature is 4 ℃, and the reaction time is 1 hour;
in the step (2), the deprotection agent is piperidine;
in step (3), the reaction is carried out in the presence of sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, the equivalent ratio of compound A3, compound A4, sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride being 1: (30-50): (200-300): (30-50), the solvent of the reaction is selected from one or more of water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, and the mixture of two or more thereof, and the temperature is 0-30 ℃, the reaction time is 0.5-4 hours; preferably, the equivalent ratio of the compound A3 to the compound A4 to the sodium tetraborate to the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride is 1:40:250:40, the solvent for the reaction is a mixed solution of water and dimethyl sulfoxide, the reaction temperature is normal temperature, and the reaction time is 2 hours.
The invention also provides application of the synthetic method in constructing a gene coding compound library.
Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.
The minimum and maximum values of the carbon atom content of the hydrocarbon groups are indicated by a prefix, e.g. prefix C a-b Alkyl means any alkyl group containing from "a" to "b" carbon atoms. For example, C 1-6 Alkyl refers to straight or branched chain alkyl groups containing 1 to 6 carbon atoms.
"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, such as phenyl and naphthyl. The aryl ring may be fused to other cyclic groups (including saturated and unsaturated rings) but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, while the point of attachment to the parent must be at a carbon atom on the ring with a conjugated pi-electron system. Aryl groups may be substituted or unsubstituted.
"heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. Heteroatoms as referred to herein include oxygen, sulfur and nitrogen. Such as furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring. Heteroaryl groups may be optionally substituted or unsubstituted.
"linking unit" includes chemical bonds, groups, which are capable of acting as links.
"halogen" includes fluorine, chlorine, bromine, iodine.
The invention discloses a method for synthesizing alpha-hydroxy olefin lead compounds On DNA for the first time, which has the advantages of small damage to the DNA, good universality, simple operation and mild condition, and can prepare On-DNA alpha-hydroxy olefin compounds with high conversion rate.
It is well known in the art that DNA must be stable under certain conditions and that reactions applied to the construction of libraries of DNA encoding compounds require higher conversion rates. Under the reaction condition of the invention, the product On-DNA alpha-hydroxy olefin compound has high conversion rate and good DNA integrity in the product. The integrity of the On-DNA alpha-hydroxy olefin compound obtained by the invention can be confirmed by liquid chromatography mass spectrometry, and can be verified by carrying out the next step of DNase catalytic coupling reaction. The invention enriches the chemical reaction types of synthesizing the coding compound library on DNA, constructs a novel alpha-hydroxy olefin skeleton for the gene coding compound library, and has very good application prospect in the development of leading medicines.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: structure of raw material HP.
Fig. 2: s is S 2 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 3: s is S 3 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 4: s is S 4-1 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 5: s is S 4-2 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 6: s is S 4-3 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 7: s is S 4-4 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 8: s is S 4-5 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 9:S 4-6 is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 10: s is S 4-7 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 11: s is S 4-8 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 12: s is S 4-9 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 13: s is S 4-10 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 14: s is S 4-11 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 15: s is S 4-12 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 16: p (P) 1 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 17: p (P) 2 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 18: p (P) 3 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 19: p (P) 4 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 20: p (P) 5 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 21: p (P) 6 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 22: p (P) 7 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 23: p (P) 8 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 24: p (P) 9 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 25: p (P) 10 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 26: p (P) 11 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 27: p (P) 12 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 28: p (P) 13 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 29: p (P) 14 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 30: p (P) 15 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 31: p (P) 16 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 32: p (P) 17 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 33: p (P) 18 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 34: p (P) 19 Is characterized by liquid chromatography mass spectrometry detection patterns.
Fig. 35: p (P) 1- P 19 Is a summary of structure and conversion.
Fig. 36: liquid chromatography mass spectrometry detection spectrum of Tag A-P7.
Fig. 37: real-time fluorescent quantitative polynucleotide chain reaction verification route patterns.
Fig. 38: qPCR product P-S 4-1 Is a target for the amplification curve of (a).
Fig. 39: qPCR product T-P 7 Is a target for the amplification curve of (a).
Fig. 40: qPCR product T-P 7 Is a standard curve of (2).
Fig. 41: qPCR product P-S 4-1 And T-P 7 Is a melting curve of (2).
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
The alpha-EWG-olefin reagent adopted in the embodiment of the invention is one of the following compounds, and is a commercial product:
the oligonucleotide-aldehyde compound adopted in the embodiment of the invention is S 4-1 -S 4-12 One of (S) 4-1 -S 4-12 The structure of (2) is as follows:
the following is S 4-1 -S 4-12 Is a synthetic method of (a).
(one) synthetic oligoPolynucleic acid-aldehyde compound S 4-1
Step 1, synthesis of DNA-NHFmoc (S 2 )
DNA-NHFmoc (S) was synthesized according to the following reaction scheme 2 )：
100 nanomoles of HP (HP, having the structure shown in FIG. 1, a commercially available product) was dissolved in deionized water to prepare a1 millimoles/liter solution (100 microliters, 1 equivalent). 40 equivalents of a DMSO solution (20. Mu.l, 200 mmol/l) of the starting headpiece compound (commercially available product), 250 equivalents of sodium tetraborate (Na) pH=9.5 2 B 4 O 7 ) Buffer (100. Mu.l, 250 mmol/l), 40 equivalents of aqueous 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) (20. Mu.l, 200 mmol/l) are mixed and the mixture is thoroughly mixed using a vortex shaker. The mixture was then added to the HP solution, and the mixture was mixed and reacted at 4℃for 1 hour. After the completion of the reaction, 5 mol/liter sodium chloride solution was added to the reaction solution in an amount of 10% by volume. Then, absolute ethanol with the total volume being 3 times is continuously added, after uniform oscillation, the reaction solution is placed in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After this, the supernatant was decanted off by centrifugation at 4000rpm for half an hour. Drying the rest precipitate to obtain oligonucleotide-NHFmoc, abbreviated as DNA-NHFmoc (S) 2 ). Detection of DNA-NHFmoc (S) using liquid chromatography Mass Spectrometry 2 ) As shown in fig. 2, the molecular weight was 5406.
Step 2, synthesizing DNA-NH 2 (S 3 )
DNA-NH was synthesized according to the following reaction scheme 2 (S 3 )：
100 nanomolar DNA-NHFmoc (S 2 ) 1 mmol/l (100. Mu.l, 1 eq.) was prepared by dissolving in deionized water, to which 10% was addedIs prepared by mixing uniformly piperidine (piperidine) aqueous solution (56 μl, 659 eq) and then reacting at room temperature for 1 hour. After the completion of the reaction, 5 mol/liter sodium chloride solution was added to the reaction solution in an amount of 10% by volume. Then, absolute ethanol with the total volume being 3 times is continuously added, after uniform oscillation, the reaction is placed in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After this, the supernatant was decanted off by centrifugation at 4000rpm for half an hour. Drying the rest precipitate to obtain oligonucleotide-NH 2 (S 3 ) Abbreviated as DNA-NH 2 . Detection of DNA-NH by liquid chromatography mass spectrometer 2 (S 3 ) As shown in fig. 3, the molecular weight was 5184.
Step 3, synthesizing the oligonucleotide-aldehyde compound S 4-1
Synthesis of oligonucleotide-aldehyde S according to the following reaction scheme 4-1 ：
10 nanomolar DNA-NH 2 (S 3 ) 1 mmol/l (10. Mu.l, 1 eq.) was prepared by dissolving in deionized water. 40 equivalents of 3-fluoro-5-formyl-benzoic acid (commercially available product) in DMSO (2. Mu.l, 200 mmol/l), 250 equivalents of sodium tetraborate (Na) at pH=9.5 2 B 4 O 7 ) Buffer (10. Mu.l, 250 mmol/l), 40 equivalents of aqueous 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMT-MM) (2. Mu.l, 200 mmol/l) are mixed and the mixture is thoroughly mixed using a vortex shaker. Adding the mixture to DNA-NH 2 Is mixed uniformly and then reacts for 2 hours at normal temperature. After the completion of the reaction, 5 mol/liter sodium chloride solution was added to the reaction solution in an amount of 10% by volume. Then, absolute ethanol with the total volume being 3 times is continuously added, after uniform oscillation, the reaction solution is placed in a refrigerator with the temperature of minus 80 ℃ for 2 hours. After this, the supernatant was decanted off by centrifugation at 4000rpm for half an hour. The rest precipitate is dried to obtain the oligonucleotide-aldehyde compound S 4-1 . Detection of S by liquid chromatography-mass spectrometer 4-1 Is shown in the figure, e.gAs shown in FIG. 4, the molecular weight was 5334 and the yield was 90%.
(II) Synthesis of oligonucleotide-aldehyde Compound S 4-2 -S 4-12
The same procedure as described above was followed, except that 3-fluoro-5-formyl-benzoic acid in step 3 was replaced with the corresponding starting material to synthesize the oligonucleotide-aldehyde compound S, respectively 4-2 -S 4-12 . Detection of S by liquid chromatography-mass spectrometer 4-2 -S 4-12 As shown in fig. 5-15.
Example 1: synthesis of oligonucleotide-alpha-hydroxy olefin Compound P 1
1.0 nanomolar oligonucleotide-aldehyde S 4-1 To an aqueous solution of (1.0 mmol/l, 1.0 eq, 1.0. Mu.l) was added 1000 eq DABCO (triethylenediamine, commercially available product, 1.0. Mu.l, 1.0 mol/l acetonitrile solution) and 1000 eq 2-cyclopentenone (commercially available product, 1.0. Mu.l, 1.0 mol/l acetonitrile solution), and the mixture was thoroughly mixed by vortexing and reacted at 25℃for 16 hours. After the reaction is finished, adding 5 mol/L sodium chloride solution with the total volume of 10 percent and absolute ethanol with the total volume of 3 times into the reaction solution, after shaking uniformly, putting the reaction solution into a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours, then carrying out high-speed freezing centrifugation (4 ℃ for 12000 revolutions per minute for 15 minutes), pouring out supernatant, and obtaining the product of the oligonucleotide-alpha-hydroxy olefin compound P after the rest precipitation and drying 1 . P detection by liquid chromatography-mass spectrometer 1 The results of the detection are shown in FIG. 16, the molecular weight is 5416, and the conversion rate is 78%. The invention can accurately detect the conversion rate of the target product by utilizing a liquid chromatography-mass spectrometer.
Example 2: synthesis of oligonucleotide-alpha-hydroxy olefin Compound P 2 -P 19
The synthesis method of reference example 1 is different from that of the oligonucleotide-aldehyde compound S 4-1 Correspondingly replace S 4-1 -S 4-12 In which 2-cyclopentenone is replaced by corresponding alpha-EWG-olefin reagent to synthesize oligonucleotide-alpha-hydroxy olefin compound P 2 -P 19 . P detection by liquid chromatography-mass spectrometer 2 -P 19 The detection results are shown in FIGS. 17-34.
Oligonucleotide-alpha-hydroxy olefin compound P 1 -P 19 The structure and conversion of (c) are summarized in FIG. 35.
The following experiments prove the beneficial effects of the invention.
Experimental example 1: conditional screening experiments for the Synthesis of oligonucleotide-alpha-hydroxy olefin compounds
Synthesis of oligonucleotide-alpha-hydroxyalkene Compound P by reference example 2 19 The only difference is that the following parameters are controlled according to table 1: the type of base, the equivalent of the alpha-EWG-olefin reagent (i.e., acrylonitrile), the reaction solvent and the proportion. Calculating P under different parameters 19 Is a product conversion of (2). The results are shown in Table 1.
TABLE 1 Synthesis of oligonucleotide-alpha-hydroxy olefin compounds P under different conditions 19 Product conversion of (2)
It can be seen that the product conversion of the resulting oligonucleotide- α -hydroxyalkene compounds is 0 under the reaction conditions of numbers 1, 3-5; under the reaction conditions of No. 9-10 and No. 12-15, the product conversion rate of the obtained oligonucleotide-alpha-hydroxy olefin compound is up to more than 49 percent; under the reaction conditions of number 10, the product conversion of the obtained oligonucleotide- α -hydroxyalkene compound is highest. It is demonstrated that the product conversion of the oligonucleotide- α -hydroxyalkene compounds obtained using the methods of the present invention under the specific parameters of the examples is highest.
Experimental example 2: integrity verification of oligonucleotides in the oligonucleotide-alpha-hydroxy olefin compounds of the invention
1) Enzyme-linked reaction validation
The following uses an oligonucleotide-alpha-hydroxyalkene compound P 7 Linking experiments with Tag a (short-chain oligonucleotide, molecular weights of both strands 4064, 5884, respectively) to verify the integrity of the oligonucleotides:
the steps are as follows: 1.0 nanomole of P 7 1.0 mmol/l solution (1.0. Mu.l, 1.0 eq.) was prepared in deionized water, to which 1.2 eq. Tag A (1.0 mmol/l aqueous solution, 1.2. Mu.l), 1.0. Mu.l 10 XT 4 DNA ligation buffer solution and 0.5. Mu.l T4 DNA ligase were added. Then the solution is mixed uniformly and reacted for 1 hour at room temperature. After the reaction was completed, 5.0 mol/liter sodium chloride solution was added to the reaction solution in an amount of 10% by volume, then, absolute ethanol was continuously added in an amount of 3.0 times by volume, and after shaking uniformly, the reaction was frozen in a refrigerator at-80℃for 2.0 hours. After which the supernatant was decanted off by centrifugation at 4000rpm for half an hour. The remaining precipitate was collected and designated Tag A-P7. Tag A-P7 was dissolved in deionized water and then detected by liquid chromatography mass spectrometry to confirm the molecular weight of the product, the molecular weight was 15346, and the mass spectrometry detection result is shown in FIG. 36.
LCMS analysis showed that the oligonucleotide-alpha-hydroxyalkene compound P 7 Can be successfully coupled with Tag A. This demonstrates that the DNA strand integrity of the oligonucleotide-alpha-hydroxyalkene compounds obtained according to the synthesis method of the invention is good. The molecular weight can be accurately shown by LCMS mass spectrum, and the nucleotide is further proved to be not damaged. Thus, the reaction method of the present invention does not cause damage to the basic structure and activity of DNA.
2) Real-time fluorescent quantitative polynucleotide chain reaction (qPCR) validation
According to the route shown in FIG. 37, the method of the invention is used for synthesizing On-DNA alpha-hydroxy olefin lead compound P-P 7 The full length is obtained by enzyme linking reaction with Tag E and reverse primerDNA target fragment T-P 7 。T-P-P 7 The molecular weight can be accurately shown by LCMS mass spectrometry. Then for T-P 7 qPCR experiments were performed to verify the integrity of the DNA.
T-P 7 Gradually diluting for 10 times, diluting for 8 times, and obtaining a standard curve through qPCR experiments. The first 7 dilutions were used as templates for qPCR, and fluorescent dye kits (SYBR Green Master Mix kit, zemoeid) were added to the reaction solutions, respectively, and the final volume was controlled to 20 μl, and amplification was performed using a real-time fluorescence quantitative instrument (Quant studio 3). All samples were repeated for 3 replicates and the qPCR cycling program was set to: heating at 50 ℃ for 2 minutes, and thermally activating at 95 ℃ for 10 minutes; then, 40 cycles of denaturation at 95℃for 15 seconds, annealing at 60℃and extension for 1 minute were performed. Melting curve stage: 95℃for 15 seconds, 60℃for 1min,95℃for 15 seconds.
qPCR results showed that P-S 4-1 And T-P 7 No significant CT shift was seen in serial dilutions of (a) (fig. 38, 39), indicating that the synthetic method of the invention did not cause DNA damage. No multiple peaks were observed in the melting curve of qPCR (FIG. 41), indicating no significant change in DNA species after the reaction.
It is further demonstrated by qPCR that the reaction method of the present invention does not cause damage to the basic structure and activity of DNA.
In summary, the invention discloses a method for synthesizing alpha-hydroxy olefin lead compounds On DNA for the first time, which has the advantages of small damage to DNA, good universality, simple operation, mild condition and capability of preparing On-DNA alpha-hydroxy olefin compounds with high conversion rate. Under the reaction condition of the invention, the product On-DNA alpha-hydroxy olefin compound has high conversion rate and good DNA integrity in the product. The invention enriches the chemical reaction types of synthesizing the coding compound library on DNA, constructs a novel alpha-hydroxy olefin skeleton for the gene coding compound library, and has very good application prospect in the development of leading medicines.

Claims (11)

1. An On-DNA alpha-hydroxy olefin lead compound shown in the formula I:
wherein the DNA is a single-stranded or double-stranded nucleotide chain;
m is a connecting unit;
R 2 selected from nitro, cyano, and COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a ；
R 3 、R 4 Each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a Unsubstituted or substituted by R 3a The substituted following groups: c (C) 1 - 12 Alkyl, C 1-12 Alkoxy, C 2-8 Alkenyl, C 2-8 Alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-12 Alkyl, C 1-12 An alkoxy group; alternatively, R 3 、R 4 Connected into a ring;
R 2a selected from C 1-12 Alkyl, C 1-12 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-6 Alkylene group, R 2b Selected from C 1-12 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-12 Alkyl, C 1-12 An alkoxy group;
alternatively, R 4 Is hydrogen, R 2 、R 3 Connected into a ring.
2. The On-DNA α -hydroxyalkene lead compound according to claim 1, characterized in that: the M is X 1 -Y 1 -P-Y 2 -X 2 -Q，X 1 、Y 1 、P、Y 2 、X 2 Q is each independently selected from the group consisting of unsubstituted, unsubstituted or substituted 1 The substituted following groups: NHCO, CONH, NH (V),5-6 membered aryl, 5-6 membered heteroarylAryl, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl, C 1-6 Alkylene, C 2-6 Alkenylene, C 2-6 Alkynylene;
m is an integer of 1 to 10;
R 1 selected from hydrogen, halogen, nitro, cyano, mercapto, carboxyl, hydroxyl, halogenated or unhalogenated C 1-6 Alkyl, halogenated or non-halogenated C 1-6 An alkoxy group;
X 1 、Y 1 、P、Y 2 、X 2 q is not absent at the same time.
3. The On-DNA α -hydroxyalkene lead compound according to claim 2, characterized in that: the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula II-1:
wherein the DNA is a single-stranded or double-stranded oligonucleotide strand;
X 1 is NHCO or CONH, X 2 Is NHCO or CONH;
Y 1 is none or C 1-4 Alkylene group, Y 2 Is none or C 1-4 An alkylene group;
q is selected from unsubstituted or R 1 The substituted following groups: 5-6 membered aryl, 5-6 membered heteroaryl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, C 1-4 An alkylene group;
m is an integer of 1 to 8;
R 1 selected from hydrogen, halogen, nitro, cyano, C 1-6 Alkyl, C 1-6 An alkoxy group;
R 2 selected from nitro, cyano, and COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a ；
R 3 、R 4 Each independently selected from hydrogen, halogen, carboxyl, nitro, cyano, hydroxyl, mercapto, COR 2a 、NHR 2a 、CONHR 2a 、NHCOR 2a Unsubstituted or substituted by R 3a The substituted following groups: c (C) 1 - 6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, 3-8 membered cycloalkyl, 5-6 membered aryl, 5-6 membered heteroaryl, R 3a Selected from C 1-6 Alkyl, C 1-6 An alkoxy group; alternatively, R 3 、R 4 Connected into a ring;
R 2a selected from C 1-6 Alkyl, C 1-6 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-4 Alkylene group, R 2b Selected from C 1-6 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-6 Alkyl, C 1-6 An alkoxy group;
alternatively, R 4 Is hydrogen, R 2 、R 3 Connected into a ring;
preferably, the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula II-2:
4. the On-DNA α -hydroxyalkene lead compound according to claim 3, wherein: the structure of the On-DNA alpha-hydroxy olefin lead compound is shown as a formula III-a, a formula III-b, a formula III-c, a formula III-d, a formula III-e or a formula III-f:
wherein the DNA is a single-stranded or double-stranded oligonucleotide strand;
R 1 selected from hydrogen, halogen, C 1-3 Alkyl, C 1-3 An alkoxy group;
n is selected from 1,2 and 3;
R 2a selected from C 1-3 Alkyl, C 1-3 Alkoxy, LR 2b 、OLR 2b The method comprises the steps of carrying out a first treatment on the surface of the L is C 1-2 Alkylene group, R 2b Selected from C 1-3 Alkyl, unsubstituted or substituted by R 2c Substituted phenyl, R 2c Selected from C 1-3 Alkyl, C 1-3 An alkoxy group;
R 3 selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or substituted by R 3a Substituted phenyl, R 4 Selected from hydrogen, C 1-3 Alkyl, C 1-3 Alkoxy, unsubstituted or substituted by R 3a Substituted phenyl, or R 3 、R 4 Is linked to form unsubstituted or R 3b Substituted 4-membered saturated nitrogen heterocycles; r is R 3a Selected from C 1-3 Alkyl, C 1-3 Alkoxy, R 3b Selected from Boc, C 1-3 Alkyl, C 1-3 An alkoxy group;
Y 3 is C 1-4 An alkylene group;
Y 4 is C 1-4 An alkylene group.
5. The On-DNA α -hydroxyalkene lead compound according to any one of claims 1 to 4, characterized in that: the On-DNA alpha-hydroxy olefin lead compound is selected from one of the following compounds:
6. a method for synthesizing the On-DNA alpha-hydroxyalkene lead compound according to any one of claims 1 to 5, characterized in that: the method comprises the following steps: the method comprises the steps of reacting a compound shown in a formula I-a and a compound shown in a formula I-b in a solvent in the presence of alkali to obtain an On-DNA alpha-hydroxy olefin lead compound shown in the formula I:
7. the method according to claim 6, wherein: the equivalent ratio of the compound shown in the formula I-a to the compound shown in the formula I-b to the base is 1: (500-2000): (500-2000);
the temperature of the reaction is 0-90 ℃ and the time is 1-24 hours;
the solvent is selected from water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, amyl alcohol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene;
the base is selected from one or more than two of triethylene diamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N' -tetramethyl ethylenediamine, 1, 3-tetramethyl guanidine, N, N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, carbonate, phosphate, borate, triethyl ammonium acetate and tris (hydroxymethyl) aminomethane.
8. The method according to claim 7, wherein: the equivalent ratio of the compound shown in the formula I-a to the compound shown in the formula I-b to the base is 1:1000:1000;
the temperature of the reaction is 25 ℃ and the time is 16 hours;
the solvent is selected from the group consisting of a mixture of water and acetonitrile, a mixture of water and N, N-dimethylformamide, and a mixture of water and ethanol;
the base is triethylenediamine.
9. The method according to any one of claims 6-8, wherein: the compound shown in the formula I-a isThe preparation method of the compound shown in the formula I-a comprises the following steps:
(1) Reacting the compound HP with a compound A1 to obtain a compound A2;
(2) Reacting the compound A2 with a deprotection agent to obtain a compound A3;
(3) The compound A3 reacts with the compound A4 to obtain the compound shown in the formula I-a.
10. The method according to claim 9, wherein: in step (1), the reaction is carried out in the presence of sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, the equivalent ratio of compound HP, compound A1, sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride being 1: (30-50): (200-300): (30-50), the solvent of the reaction is selected from one or more of water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, and the mixture of two or more thereof, and the temperature is 0-30 ℃, the reaction time is 0.5-4 hours; preferably, the equivalent ratio of the compound HP, the compound A1, the sodium tetraborate and the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride is 1:40:250:40, the solvent for the reaction is a mixed solution of water and dimethyl sulfoxide, the reaction temperature is 4 ℃, and the reaction time is 1 hour;
in the step (2), the deprotection agent is piperidine;
in step (3), the reaction is carried out in the presence of sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, the equivalent ratio of compound A3, compound A4, sodium tetraborate and 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride being 1: (30-50): (200-300): (30-50), the solvent of the reaction is selected from one or more of water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2-difluoroethanol, 2-trifluoroethanol, hexafluoroisopropanol, benzyl alcohol, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, diethyl ether, propylene oxide, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, tetrahydropyran, 1, 4-dioxane, anisole, dimethyl sulfide, diethyl sulfide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, methylene chloride, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, N-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, and the mixture of two or more thereof, and the temperature is 0-30 ℃, the reaction time is 0.5-4 hours; preferably, the equivalent ratio of the compound A3 to the compound A4 to the sodium tetraborate to the 4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride is 1:40:250:40, the solvent for the reaction is a mixed solution of water and dimethyl sulfoxide, the reaction temperature is normal temperature, and the reaction time is 2 hours.
11. Use of the synthetic method of any one of claims 6-10 for constructing a library of genetically encoded compounds.
CN202211130945.6A 2022-09-16 2022-09-16 On-DNA alpha-hydroxy olefin lead compound and synthesis method thereof Pending CN117758374A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114920791A (en) * 2022-04-28 2022-08-19 康龙化成(宁波)科技发展有限公司 Synthesis method of oligo-nucleic acid-succinimide compound

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