CN114277446A - Method for synthesizing gene coding beta-lactam compound library by one-pot method - Google Patents

Abstract

The invention provides a method for constructing a gene coding beta-lactam compound library by oligomeric nucleic acid-terminal alkyne compound through a Kinugasa reaction. The method has good universality, simple operation and mild conditions, can efficiently realize the construction of the gene coding beta-lactam compound library, and provides a quick and practical effective way for enriching the diversity of the molecules of the gene coding beta-lactam compound library. The method is suitable for the synthesis of libraries of gene-encoded compounds in multiwell plates.

Description

Method for synthesizing gene coding beta-lactam compound library by one-pot method

Technical Field

The invention belongs to the technical field of gene coding compound libraries, and particularly relates to a method for applying a synthesis strategy of synthesizing oligonucleic acid-beta-lactam by a one-pot method to the construction of a gene coding compound library.

Background

Drug development relies on multiple biological screening methods to discover lead compounds^[1]. The origin analysis of 66 clinical candidate drugs by scientists of Aslicon between 2016 and 2017 finds that: 43% of clinical drug candidates were found based on known activity of compounds, endogenous ligands or previous projects of research basis. 29% were found by high throughput screening method^[2]. High throughput screening methods are important methods for the discovery of lead compounds, particularly for the discovery of unknown target protein ligands. The remaining screening methods include structure-based drug design^[3](Structure-based drug design, SBDD; 14%), targeting Screening (8%), lead compound discovery based on fragmented compounds (FBLG; 5%)^[4]And gene coding library screening (DELT; 1%). SBDD is an important way for finding a lead compound by guiding the structure design and screening of a small molecular compound according to the 3D structural characteristics of a target protein and relying on a computer algorithm^[5-6]. In the development of new drugs, scientists are constantly seeking more efficient screening methods to find superior active compounds among many compounds through differences in binding affinity to biological targets and/or pharmacological potency in a non-differential screening manner. The important role of highly automated and deeply optimized high throughput screening methods in the discovery of active compounds is undisputed. However, their high cost, low total number of included compounds, limited chemical space structure and limitations on biological screening methods are increasingly inadequate for the development of new drugs^[7]. In the practice of screening for many disease proteins, it is futile to use this traditional method. In order to break through the bottleneck of high-throughput screening method, the screened compound presents a geometric leap in quantity and chemical space structure, and simultaneously a plurality of biological screening modes and gene coding compound library technology (DELT) are used^[8]。

Brenner and Lerner proposed the DELT theory of originality in 1992 and envisioned that it would enable the synthesis and screening of large libraries of coding compounds in a much faster way than traditional chemistry^[9]. Gene-encoded compound libraries (DELs) greatly increase the number of compounds and the diversity of chemical spatial structures compared to traditional high-throughput screening^[10]. In reaction tubes of very small volume, e.g. tens of microliters, it is possible to synthesize tens of millions or even hundreds of millions of different compounds by a series of reactions^[11]. The principle of DELT is to label each small molecule compound in the reaction with gene fragments of different specific sequences. Large-scale synthesis of millions to billions of compound libraries labeled with specific gene sequences by using a split-and-pool (split-and-pool) method with limited cost and time using combinatorial chemistry strategies^[12]. The resulting mixture of compounds is then incubated with a protein target, and physical separation and finding of compounds with high affinity is achieved by washing away compounds that do not bind to the target protein^[13]. The library of gene-encoded compounds required to incubate the target protein requires only a very small dose (micrograms) and can be performed in a very short time (e.g., within 1 day). Can easily be used under different conditions^[14](e.g., pH of the solution, manner of mixing of sample proteins, different protein concentrations, presence or absence of competing compounds, presence of different buffers or cofactors, etc.) multiple bioscreening experiments are performed simultaneously. Because the gene sequences correspond to the structures of the compounds one by one, the chemical structural formula of the active compound can be obtained by decoding the gene sequences after Polymerase Chain Reaction (PCR) amplification and Next Generation Sequencing (NGS) reading. Further, the compounds with high affinity are individually synthesized and separated from the oligonucleotideDetermining the binding force of the compound without the oligonucleotide attached thereto to the target protein to confirm the biological activity thereof^[15]. This biological screening approach is fundamentally different from the traditional high-throughput biological activity screening in that target proteins are performed individually from a single compound.

The DEL screening method for live cells, which has emerged in this field in recent years, is an attractive achievement^[16-17]. It represents a compound that can be screened for cellular biological activity by the DELT platform. DEL live cell screens would eliminate the need for purified target proteins and for protein modifications. Therefore, the process of biological screening is simplified, and the original ecological structure of the protein is better maintained. Therefore, the pharmacologist can find better lead compound on the platform^[18]. Although the DELT platform has only a short history of development, it has gained wide acceptance and application, and is actively influencing the progress of new drug development.

The diversity in the chemical structure of construction is one of the important reasons DELT can successfully screen for biologically active compounds. The construction of coding libraries by synthesizing some parent compounds with novel structures through traditional organic chemistry and then linking the parent compounds to oligo-nucleic acids is a synthetic strategy for many DELs. Furthermore, it is also possible to directly find and develop new chemical reactions, particularly Multi-component reactions (DEL library syntheses), on oligo-nucleic acids^[19]. The gene coding library is synthesized by multi-component chemical reaction in one step, so that time and labor are saved, and irreversible damage of the chemical reaction to the oligomeric nucleic acid is reduced. In view of the importance of this work, the development of organic chemical reactions applied to DELT is an important part of our work.

Beta-lactams are an important class of compounds. Penicillin derivatives (penams), cephalosporins and cephamycins (cephems), monobactams, carbapenems and carbacephem compounds are typical representatives of beta-lactam compounds^[20]。

Beta. since the nineteen twentieth era in the alexander fleming was seeking an anti-staphylococcal phageThe emergence of lactam antibiotics has drastically changed the situation of human fight against bacterial infections. Beta-lactam antibiotics are one of three major classes of antibiotics. Beta-lactams are the most successful drug species for the past 60 years for treating bacterial infections caused by a variety of bacteria. Beta-lactam antibiotics are among the most commonly prescribed drugs. Statistically, the annual cost of these antibiotics is currently about $ 150 billion per year, 65% of the total antibiotic market^[21-22]。

From the structure-activity relationship of the drugs, the drugs have a common characteristic of having a chemically highly reactive 3-carbon 1-nitrogen ring (beta-lactam ring) chemical structure.

According to our knowledge, no report is made on the synthesis of beta-lactam rings on oligomeric nucleic acids. If the beta-lactam compound DEL can be synthesized, the research has scientific theoretical value and social and economic benefits.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for applying the synthesis strategy of the oligonucleic acid-beta-lactam to the construction of a gene coding compound library by a one-pot method. The method has good universality, simple operation and mild conditions, can efficiently realize the construction of the gene coding beta-lactam compound library, and provides a quick and practical effective way for enriching the diversity of the molecules of the gene coding beta-lactam compound library.

Among them, the strategy for synthesizing oligo-nucleic acid-beta-lactam is described in another patent application (named as: a method for synthesizing quaternary lactam on oligo-nucleic acid by one-pot method) filed on the same day as the present application, and it specifically describes a method for preparing oligo-nucleic acid-beta-lactam by using oligo-nucleic acid-terminal alkyne compound, and the reaction equation is as follows^[23]：

The invention applies the synthesis strategy of the oligomeric nucleic acid-beta-lactam to the construction of a gene coding compound library, which is specifically shown as follows:

the invention provides a method for constructing a gene coding beta-lactam compound library by oligomeric nucleic acid-terminal alkyne compound through a Kinugasa reaction^[23]Specifically, the oligonucleotide-terminal alkyne compound is used as a substrate, the oligonucleotide-terminal alkyne compound is converted into oligonucleotide-beta-lactam by a one-pot method in the presence of a nitrone reagent, and a gene coding beta-lactam compound library is constructed on the basis of the reaction.

In order to solve the technical problems, the invention provides the following technical scheme:

the application provides a construction method of a gene coding beta-lactam compound library, which comprises the steps of taking a mixture of X oligonucleotide-terminal alkyne marked by oligonucleotide chains (TagA) with a specific sequence as a substrate, reacting with Y nitrone reagents respectively under the catalysis of copper to convert into oligonucleotide-beta-lactam, then marking by the oligonucleotide chains (TagB) with the specific sequence to obtain a mixture of cis-isomers and trans-isomers of the oligonucleotide-beta-lactam marked by the (X Y) TagA-TagB, and realizing the construction of the gene coding beta-lactam cis-isomer and trans-isomer compound library with the size of X Y. The specific reaction equation is as follows:

wherein, the oligonucleotide in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers, and the length of the chain is not limited.

Wherein, the structural formula of the oligonucleotide-terminal alkyne compound marked by the oligonucleotide chain (TagA) with a specific sequence is as follows:

wherein, R in the structural formula¹Can be hydrogen, amino, nitro, cyano, hydroxyl, mercapto, aryl ketone, alkyl ketone, C₁-C₁₂Alkyl radical, C₂-C₆Alkylene radical, C₂-C₆Alkynyl, C₃-C₈Cycloalkyl radical, C₁-C₆Any one to more of alkyl oxygen, aryl, heterocyclic aryl, halogen (preferably, fluorine, chlorine, bromine, iodine), carboxyl, ester group, amide group or any combination thereof;

wherein, TagA in the structural formula is a single-stranded or double-stranded oligonucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers with known sequences, wherein the length of the chain is not limited;

wherein, the TagB is a single-stranded or double-stranded oligonucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers of known sequence, wherein the length of the chain is not limited;

wherein the nitrone structure is as follows:

wherein, Ar in the structural formula¹Is a five-membered, six-membered or larger aromatic ring or aromatic heterocycle;

wherein, Ar in the structural formula²Is a five-membered, six-membered or larger aromatic ring or aromatic heterocycle;

wherein, the nitrone can be a pure compound or a crude reaction product generated in situ;

wherein X is theoretically any positive integer;

wherein Y is theoretically any positive integer;

wherein the copper catalyst is copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, cuprous iodide, a copper-beta-cyclodextrin complex, copper bis (2, 4-pentanedionate), copper acetylacetonate, copper tetra (acetonitrile) tetrafluoroborate, copper dichloro (1, 10-phenanthroline), copper bis (8-hydroxyquinoline), copper trifluoromethanesulfonate, copper bis (trifluoro-2, 4-pentanedionate), copper perchlorate, copper tetra (acetonitrile) hexafluorophosphate, cuprous acetate, copper bromide, copper fluoride, cuprous bromide, cuprous chloride-bis (lithium chloride) complex, cuprous bromide dimethyl sulfide complex; preferably, the copper catalyst is cuprous iodide, copper tetrakis (acetonitrile) hexafluorophosphate, cuprous chloride-bis (lithium chloride) complex; more preferably, the copper catalyst is cuprous iodide.

Wherein the reaction is carried out in the presence of a base which is one or a mixture of more of potassium carbonate, sodium bicarbonate, potassium bicarbonate, lithium carbonate, lithium hydroxide, potassium hydroxide, sodium hydroxide, cesium hydroxide, sodium borate, potassium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, methylamine, ethylamine, propylamine, isopropylamine, N, N-diethylamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, N, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N' -tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine or dicyclohexylamine; preferably, the alkali is one or a mixture of tetramethyl guanidine, N-methyl dicyclohexyl amine or dicyclohexyl amine. More preferably, the base is N, N-dicyclohexylmethylamine.

Wherein the reaction is carried out in the presence of a solvent selected from the group consisting of water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, t-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2, 2-difluoroethanol, 2,2, 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, ethyl acetate, Cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer solution and organic alkali buffer solution; preferably, the solvent is a mixture of water and acetonitrile; in a more preferred embodiment, the solvent is a mixture of water and acetonitrile and the total acetonitrile content is not less than 50%.

In one embodiment, the molar concentration of the oligonucleotide-terminal alkyne mixture labeled with oligonucleotide strand of specific sequence (TagA) is 0.1-2 mmol/l; preferably, the molar concentration of the oligonucleotide-terminal alkyne mixture labeled by the oligonucleotide chain (TagA) with the specific sequence is 0.5-1.5 mmol/l; more preferably, the molar concentration of the oligonucleotide-terminal alkyne mixture labeled with oligonucleotide strand of specific sequence (TagA) is 1.0 mmol/l.

In one embodiment, 1 equivalent of oligonucleotide chain with a specific sequence (TagA) -labeled oligonucleotide-terminal alkyne is used as a reference, and the amount of the nitrone is 1 to 500 equivalents; in a preferred embodiment, the nitrone is used in an amount of 1 to 100 equivalents; in a more preferred embodiment, the nitrone is used in an amount of 50 equivalents.

In one embodiment, the N, N-dicyclohexylmethylamine is used in an amount of 1 to 500 equivalents, based on 1 equivalent of oligonucleotide-terminal alkyne labeled with a specific sequence oligonucleotide chain (TagA); in a preferred embodiment, the N, N-dicyclohexylmethylamine is used in an amount of 10 to 200 equivalents; in a more preferred embodiment, the N, N-dicyclohexylmethylamine is used in an amount of 100 equivalents.

In one embodiment, the cuprous iodide is used in an amount of 0.1 to 100 equivalents based on 1 equivalent of oligonucleotide chain (TagA) -terminal alkyne labeled with oligonucleotide chain of specific sequence; in a preferred embodiment, the amount of the cuprous iodide is 0.1 to 50 equivalents; in a more preferred embodiment, the amount of cuprous iodide used is 10 equivalents.

In one embodiment, the reaction is carried out at a temperature of from 0 to 90 ℃; in a preferred embodiment, the temperature is 0 to 40 ℃; in a more preferred embodiment, the temperature is 25 ℃.

In one embodiment, the reaction is carried out for a time period ranging from 1 to 24 hours; in a preferred embodiment, the time is 5 to 24 hours; in a more preferred embodiment, the time is 12 hours.

In one embodiment, the reaction is carried out under an atmosphere which is one of air, nitrogen, argon, in a preferred embodiment, the atmosphere is one of nitrogen or argon, in a more preferred embodiment, the atmosphere is nitrogen.

In one embodiment, the amount of TagB is 1 to 10 equivalents based on 1 equivalent of oligonucleotide- β -lactam; in a preferred embodiment, the TagB is used in an amount of 1 to 2 equivalents; in a more preferred embodiment, the TagB is used in an amount of 1 equivalent.

The invention provides a method for constructing a gene coding beta-lactam compound library by taking oligomeric nucleic acid-terminal alkyne as a raw material. The method has good universality, simple operation and mild conditions, and is suitable for synthesizing the gene coding compound library by using a multi-hole plate.

Drawings

In FIGS. 1 to 28, "P" in all figures represents the reaction product.

FIG. 1a and FIG. 1b are schematic diagrams showing oligo-nucleic acid-NH according to the present invention₂Preparing the liquid chromatography mass spectrum detection result and extracting ion flow diagram of the oligomeric nucleic acid-terminal alkyne compound P1-1.

FIG. 2a and FIG. 2b are schematic diagrams showing oligo-nucleic acid-NH according to the present invention₂Preparing the liquid chromatography mass spectrum detection result and extracting ion flow diagram of the oligomeric nucleic acid-terminal alkyne compound P1-2.

FIG. 3a and FIG. 3b are schematic diagrams showing oligo-nucleic acid-NH according to the present invention₂Preparing the liquid chromatography mass spectrum detection result and extracting ion flow diagram of the oligomeric nucleic acid-terminal alkyne compound P1-3 for raw materials.

FIG. 4a and FIG. 4b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using oligo-nucleic acid-COOH as raw material to prepare oligo-nucleic acid-terminal alkyne compound P1-4, respectively.

FIG. 5a and FIG. 5b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the oligo-nucleic acid-terminal alkyne compound P1-5 prepared from oligo-nucleic acid-COOH as a raw material according to the present invention, respectively.

FIG. 6a and FIG. 6b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using oligo-nucleic acid-COOH as raw material to prepare oligo-nucleic acid-terminal alkyne compound P1-6, respectively.

FIG. 7a and FIG. 7b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using oligo-nucleic acid-COOH as raw material to prepare oligo-nucleic acid-terminal alkyne compound P1-7, respectively.

FIG. 8a and FIG. 8b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne compound P1-1 as the raw material to prepare the oligonucleotide-terminal alkyne compound P2-1 labeled with the oligonucleotide chain (TagA115) of a specific sequence.

FIG. 9a and FIG. 9b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne compound P1-2 as the raw material to prepare the oligonucleotide-terminal alkyne compound P2-2 labeled with the oligonucleotide chain (TagA116) of a specific sequence.

FIG. 10a and FIG. 10b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne compound P1-3 as the raw material to prepare the oligonucleotide-terminal alkyne compound P2-3 labeled with the oligonucleotide chain (TagA117) of a specific sequence.

FIG. 11a and FIG. 11b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne compound P1-4 as the raw material to prepare the oligonucleotide-terminal alkyne compound P2-4 labeled with the oligonucleotide chain (TagA118) of a specific sequence.

FIG. 12a and FIG. 12b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, for preparing an oligonucleotide-terminal alkyne compound P2-5 labeled with an oligonucleotide chain (TagA119) of a specific sequence, using the oligonucleotide-terminal alkyne compound P1-5 as a raw material.

FIGS. 13a and 13b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the oligonucleotide-terminal alkyne compound P2-6 labeled by the oligonucleotide chain (TagA120) with a specific sequence prepared from the oligonucleotide-terminal alkyne compound P1-6 of the present invention.

FIGS. 14a and 14b are a liquid chromatography mass spectrometry detection result and an extracted ion flow diagram of the oligonucleotide-terminal alkyne compound P2-7 labeled by the oligonucleotide chain (TagA121) with a specific sequence prepared from the oligonucleotide-terminal alkyne compound P1-7 according to the present invention.

FIGS. 15a and 15b are the liquid chromatography mass spectrometry detection results and the extracted ion flow diagram, respectively, of a mixture containing 7 oligonucleotide-terminal alkyne labeled with oligonucleotide chains (TagA) of specific sequence prepared according to the present invention.

FIGS. 16a and 16b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-1, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-1.

FIG. 17a and FIG. 17b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne mixture P3-1 as the raw material to prepare the oligonucleotide-terminal alkyne mixture P4-1 labeled with the oligonucleotide chain (TagB101) of a specific sequence.

FIGS. 18a and 18b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-2, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-2.

FIG. 19a and FIG. 19b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne mixture P3-2 as the raw material to prepare the oligonucleotide-terminal alkyne mixture P4-2 labeled with the oligonucleotide chain (TagB102) of a specific sequence.

FIGS. 20a and 20b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-3, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-3.

FIG. 21a and FIG. 21b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne mixture P3-3 as the raw material to prepare the oligonucleotide-terminal alkyne mixture P4-3 labeled with the oligonucleotide chain (TagB103) of a specific sequence.

FIGS. 22a and 22b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-4, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-4.

FIG. 23a and FIG. 23b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne mixture P3-4 as the raw material to prepare the oligonucleotide-terminal alkyne mixture P4-4 labeled with the oligonucleotide chain (TagB104) of a specific sequence.

FIGS. 24a and 24b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-5, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-5.

FIGS. 25a and 25b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the oligonucleotide-terminal alkyne mixture P4-5 labeled by the oligonucleotide chain (TagB105) with a specific sequence prepared from the oligonucleotide-terminal alkyne mixture P3-5 according to the present invention.

FIGS. 26a and 26b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention, respectively, using the oligonucleotide-terminal alkyne mixture containing 7 oligonucleotide chains (TagA) labeled with specific sequences as the substrate, and in the presence of copper catalyst and nitrone S2-6, the oligonucleotide-terminal alkyne is converted into the oligonucleotide-beta-lactam mixture P3-6.

FIG. 27a and FIG. 27b are the liquid chromatography mass spectrometry detection result and the extracted ion flow diagram of the present invention using the oligonucleotide-terminal alkyne mixture P3-6 as the raw material to prepare the oligonucleotide-terminal alkyne mixture P4-6 labeled with the oligonucleotide chain (TagB106) of a specific sequence.

FIGS. 28a and 28b are the liquid chromatography mass spectrometry detection result and the extracted ion flow graph of the oligonucleotide-terminal alkyne mixture containing 42 oligonucleotide chains (TagA-TagB) labeled with specific sequences prepared in the present invention, respectively.

FIG. 29 is a representative structural formula of a starting acid-terminal alkynyl compound and an amine-terminal alkynyl compound of the present invention.

FIG. 30 is a representative structural formula of a raw material nitrone of the present invention.

The oligonucleotide chains (TagA # ###, TagB # ####) with the specific sequences are specific oligonucleotide markers collected in the dragon-synthesizing chemical synthesis.

Detailed Description

The technical solution of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely a subset of the embodiments of the invention and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. All oligonucleotide starting materials in the embodiments of the present invention are double-stranded or single-stranded substrates for the oligonucleotides. The synthesis of the substrate for the oligo-nucleic acid-terminal alkyne compound is very similar and the general synthesis can be performed as described in example 1.

In the following examples, "/" in the sequences of the oligonucleotide strands referred to are intended to separate the two complementary strands of the oligonucleotide strands, with the sequence before the "/" referring to the sequence of one strand of the oligonucleotide strand and the sequence after the "/" referring to the sequence of the other strand of the oligonucleotide strand.

Example 1 Synthesis of oligonucleotide chain (TagA) -tagged oligonucleotide-terminal alkyne Compounds of specific sequences

1) Synthesis method 1

1. 10 nanomolar oligo-nucleic acid-NH₂Dissolved in deionized water to make a1 millimole/liter solution (10. mu.L, 10nmol, 1 eq). To this was added 80 equivalents of the acid-terminal alkynyl compound (200 mmol/l DMSO solution, 80 equivalents), 200 equivalents of N, N-diisopropylethylamine (200 mmol/l DMSO solution, 200 equivalents), 50 equivalents of 2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate (200 mmol/l DMSO solution, 50 equivalents). The mixture was mixed well with a vortex shaker. Adding the mixture into oligo-nucleic acid-NH₂After mixing uniformly, the mixture was reacted at room temperature for 1 hour. After the reaction is finished, adding a sodium chloride solution of 5 mol/L with the total volume of 10 percent into the reaction solution, then continuously adding absolute ethyl alcohol with the total volume of 3 times, after uniform oscillation, placing the reaction in a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water, and purifying by high performance liquid chromatography to obtain the oligonucleotide-terminal alkyne compound.

2. 5nmol of the oligo-nucleic acid-terminal alkyne compound was dissolved in deionized water to prepare a1 mmol/L solution (5. mu.L, 5nmol, 1 eq.) and 1 eq of TagA (0.76 mmol/L H) was added₂O solution, 1 eq), 3 microliters of T4DNA ligase buffer, 0.5 microliters of DNA ligase, and mixing the above solutions uniformly, reacting at room temperature for 1 hour to obtain a reaction solution containing the oligonucleotide-terminal alkyne compound labeled with the nucleotide chain of the specific sequence (TagA).

2) Synthesis method 2

1. 10nmol of oligo-nucleic acid-COOH was dissolved in deionized water to make a1 mmol/L solution (10. mu.L, 10nmol, 1 eq.). To this was added 50 equivalents of 2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate (200 mmol/l DMSO solution, 50 equivalents), 50 equivalents of N, N-diisopropylethylamine (200 mmol/l DMSO solution, 50 equivalents), 50 equivalents of the amine-terminal alkynyl compound (200 mmol/l DMSO solution, 50 equivalents). Then, the above solutions were mixed well and reacted at room temperature for 1 hour. After the reaction is finished, adding a sodium chloride solution of 5 mol/L with the total volume of 10 percent into the reaction solution, then continuously adding absolute ethyl alcohol with the volume of 3 times of the total volume, after uniform oscillation, placing the reaction in a refrigerator with the temperature of minus 80 ℃ for freezing for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. Dissolving the rest precipitate with deionized water, and purifying by high performance liquid chromatography to obtain the oligonucleotide-terminal alkyne compound.

2. 5nmol of the oligo-nucleic acid-terminal alkyne compound was dissolved in deionized water to prepare a1 mmol/L solution (5. mu.L, 5nmol, 1 eq), to which 1 eq of TagA (0.76 mmol/L H) was added₂O solution, 1 eq), 3 microliters of T4DNA ligase buffer, 0.5 microliters of DNA ligase, and the above solutions were mixed well. Reacting at room temperature for 1 hour to obtain a reaction solution containing the oligonucleotide-terminal alkyne compound labeled by the oligonucleotide chain (TagA) with a specific sequence.

Example 2 Synthesis of nitrones

0.08 mmol of an aromatic nitro compound was dissolved in 700. mu.L of acetonitrile, and 0.16 mmol of an aromatic aldehyde-based compound, 0.096 mmol of ammonium chloride, 0.08 mmol of zinc powder and 100. mu.L of water were added thereto. The reaction was stirred at 40 ℃ for 12 hours under nitrogen. After the reaction, the mixture was centrifuged at 4000rpm for 10 minutes, and the resulting supernatant containing nitrone was used directly for the cyclization reaction.

Example 3 Synthesis of oligonucleotide chain of specific sequence (TagA115, sequence GTCGTCATTCCACTGTT/AGTGGAATGA) labeled oligonucleotide-terminal alkyne Compound P2-1

The procedure was as in example 1.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-1 is 16316, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the yield is confirmed to be 70% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 1.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-1 labeled by the oligonucleotide chain with a specific sequence (TagA115 with the sequence of GTCGTCATTCCACTGTT/AGTGGAATGA) is 24683, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in figure 8.

Example 4 Synthesis of an oligonucleotide chain of specific sequence (TagA116, sequence GTCGCTGTCAGTCTGTT/AGACTGACAG) labeled oligonucleotide-terminal alkyne Compound P2-2

The procedure was as in example 1.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-2 is 16329, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the yield is confirmed to be 70% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 2.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-2 labeled by the oligonucleotide chain (TagA116 with GTCGCTGTCAGTCTGTT/AGACTGACAG) with specific sequence is 24696, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 9.

Example 5 Synthesis of oligonucleotide chain of specific sequence (TagA117, sequence GTCGTTAGTGGCACGTT/GTGCCACTAA) labeled oligonucleotide-terminal alkyne Compound P2-3

The procedure was as in example 1.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-3 is 16283, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the yield is confirmed to be 70% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 3.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-3 labeled by the oligonucleotide chain with a specific sequence (TagA117 with the sequence of GTCGTTAGTGGCACGTT/GTGCCACTAA) is 24650, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 10.

Example 6 Synthesis of an oligonucleotide chain of specific sequence (TagA118, sequence GTCGTGGCATGCGAGTT/TCGCATGCCA) labeled oligonucleotide-terminal alkyne Compound P2-4

The procedure was as in example 1, synthesis 2.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-4 is 16283, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the yield is confirmed to be 65% by combining the detection result of the liquid chromatography mass spectrometry after the OD is determined by an ultramicro spectrophotometer, and the result is shown in figure 4.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-4 labeled by the oligonucleotide chain (TagA118 with the sequence of GTCGTGGCATGCGAGTT/TCGCATGCCA) with specific sequence is 24652, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 11.

Example 7 Synthesis of an oligonucleotide chain of specific sequence (TagA119, sequence GTCGTCGCCGGAGCGTT/GCTCCGGCGA) labeled oligonucleotide-terminated alkyne Compound P2-5

The procedure was as in example 1, synthesis 2.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-5 is 16345, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and after OD measurement is carried out by an ultramicro spectrophotometer, the yield is confirmed to be 80% by combining with the detection result of liquid chromatography mass spectrometry, and the result is shown in FIG. 5.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-5 labeled by the oligonucleotide chain with specific sequence (TagA119 with the sequence of GTCGTCGCCGGAGCGTT/GCTCCGGCGA) is 24712, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 12.

Example 8 Synthesis of an oligonucleotide chain of specific sequence (TagA120, sequence GTCGGAACTTGCCCGTT/GGGCAAGTTC) labeled oligonucleotide-terminal alkyne Compound P2-6

The procedure was as in example 1, synthesis 2.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-6 is 16331, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and after OD determination is carried out by an ultramicro spectrophotometer, the yield is confirmed to be 75% by combining with the detection result of liquid chromatography mass spectrometry, and the result is shown in FIG. 6.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-6 labeled by the oligonucleotide chain (TagA120 with a specific sequence of GTCGGAACTTGCCCGTT/GGGCAAGTTC) with a specific sequence is 24698, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 13.

Example 9 Synthesis of an oligonucleotide chain of specific sequence (TagA121, sequence GTCGCTCGGATCGAGTT/TCGATCCGAG) labeled oligonucleotide-terminal alkyne Compound P2-7

The procedure was as in example 1, synthesis 2.

Wherein, the molecular weight of the oligo-nucleic acid-terminal alkyne compound P1-7 is 16349, the molecular weight of the product is detected by liquid chromatography mass spectrometry, and after OD determination is carried out by an ultramicro spectrophotometer, the yield is confirmed to be 80% by combining with the detection result of liquid chromatography mass spectrometry, and the result is shown in FIG. 7.

Wherein, the molecular weight of the product of the oligonucleotide-terminal alkyne compound P2-7 labeled by the oligonucleotide chain (TagA121 with the sequence of GTCGCTCGGATCGAGTT/TCGATCCGAG) with specific sequence is 24716, and the molecular weight of the product is detected by liquid chromatography mass spectrometry, and the result is shown in FIG. 14.

Example 10 preparation of a mixture comprising 7 oligonucleotide-terminal alkynes labelled with oligonucleotide strands of a specific sequence (TagA)

The 7 reaction solutions of examples 3, 4, 5, 6, 7, 8, and 9 were combined, 5 mol/l sodium chloride solution of 10% of the total volume was added to the combined reaction solutions, and then anhydrous ethanol of 3 times the total volume was further added. After shaking uniformly, the reaction was frozen in a freezer at-80 ℃ for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. And dissolving the rest precipitate with deionized water to obtain a mixture containing 7 oligonucleotide chains (TagA) marked by specific sequences and terminal alkyne, wherein the molecular weight range of the product is 24647-24716. The liquid chromatography mass spectrometry detects the molecular weight range of the corresponding product, and the yield is confirmed to be 80% by combining the detection result of the liquid chromatography mass spectrometry after the OD is measured by an ultramicro spectrophotometer, and the result is shown in figure 15.

Example 11 Synthesis of oligonucleotide chain of specific sequence (TagB) -labeled oligonucleotide-lactam mixtures

1) Synthesis of oligo-nucleic acid-beta-lactam mixtures

1nM of the oligonucleotide-terminal alkyne mixture labeled with 7 specific sequences of oligonucleotide strands (TagA) from example 10 was dissolved in deionized water to prepare a1 mM solution (1. mu.L, 1nmol, 1 eq). To this were added 50 equivalents of the in situ generated nitrone in acetonitrile/water (7/1) (100 mmol/l, 50 equivalents), 100 equivalents of N, N-dicyclohexylmethylamine in acetonitrile (200 mmol/l, 100 equivalents) and 10 equivalents of cuprous iodide in acetonitrile (20 mmol/l, 10 equivalents). The solution is mixed evenly and reacted for 12 hours at 25 ℃ under the protection of nitrogen. After the reaction was completed, 100 equivalents of an aqueous solution of sodium diethyldithiocarbamate (1 mol/l, 100 equivalents) was added to the reaction solution to remove copper. After the copper removal, 5 mol/L sodium chloride solution with the total volume of 10 percent is added into the reaction solution, and then absolute ethyl alcohol with the volume of 3 times of the total volume is added. After shaking to homogeneity, the reaction was frozen in a freezer at-80 ℃ for 2 hours, then centrifuged at 4000rpm for half an hour and the supernatant decanted. Dissolving the rest precipitate with deionized water to obtain the oligo-nucleic acid-beta-lactam mixture.

2) Synthesis of oligonucleotide-beta-lactam mixtures labelled with oligonucleotide chains of specific sequence (TagB)

1 nanomole of oligo-nucleic acid-beta-lactam mixture was dissolved in deionized water to prepare a1 mmol/L solution (1. mu.L, 1nmol, 1 eq.) and 1 eq of TagB (0.76 mmol/L H) was added₂O solution, 1 equivalent), 1 microliter of T4DNA ligase buffer solution and 0.2 microliter of DNA ligase, and the solution is uniformly mixed and reacted for 1 hour at room temperature to obtain solution of the oligonucleotide-beta-lactam mixture marked by the oligonucleotide chain (TagB) with a specific sequence.

Example 12 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB101, sequence TAACAGCCTC/GAGGAGGCTGTTAAAC)

1) Synthesis of nitrone S2-1

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-1 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-1 product is 24888-24957. The liquid chromatography mass spectrometry detects the corresponding molecular weight range of the product, and after the OD is measured by an ultramicro spectrophotometer, the reaction yield is confirmed to be 58% by combining the detection result of the liquid chromatography mass spectrometry, and the result is shown in figure 16.

3) Synthesis of oligonucleotide chain (TagB101, sequence TAACAGCCTC/GAGGAGGCTGTTAAAC) labeled oligonucleotide-beta-lactam mixture P4-1 of specific sequence

The procedure was as in step 2 of example 11. Wherein the molecular weight range of the P4-1 product is 32953-33022. The liquid chromatography mass spectrometry detects the molecular weight of the product, and the result is shown in FIG. 17. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 13 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB102, sequence GCTTGGGTTC/GAGGAACCCAAGCAAC)

1) Synthesis of nitrone S2-2

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-2 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-2 product is 24862-24931. The liquid chromatography mass spectrometry detects the corresponding product molecular weight range, and the reaction yield is confirmed to be 47% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 18.

3) Synthesis of oligonucleotide chain with specific sequence (TagB102, its sequence is GCTTGGGTTC/GAGGAACCCAAGCAAC) labeled oligonucleotide-beta-lactam mixture P4-2

The procedure was as in step 2 of example 11. Wherein the molecular weight range of the P4-2 product is 32927-32996, and the result is shown in FIG. 19 when the molecular weight of the product is detected by liquid chromatography mass spectrometry. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 14 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB103, sequence GGCTTTATTG/GAGCTTTAAAGCCAAC)

1) Synthesis of nitrone S2-3

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-3 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-3 product is 24852-24921. The liquid chromatography mass spectrometry detects the corresponding product molecular weight range, and the reaction yield is confirmed to be 52% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 20.

3) Synthesis of oligonucleotide chain with specific sequence (TagB103, its sequence is GGCTTTATTG/GAGCTTTAAAGCCAAC) labeled oligonucleotide-beta-lactam mixture P4-3

The procedure was as in step 2 of example 11. Wherein the molecular weight range of the P4-3 product is 32917-32986. The liquid chromatography mass spectrometry detects the molecular weight of the product, and the result is shown in figure 21. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 15 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB104, sequence TAATAGCCGG/GAGCCGGCTATTAAAC)

1) Synthesis of nitrone S2-4

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-4 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-4 product is 24930-24999. The liquid chromatography mass spectrometry detects the corresponding molecular weight range of the product, and the result is shown in figure 22, after the OD determination is carried out by an ultramicro spectrophotometer, the reaction yield is confirmed to be 48% by combining the detection result of the liquid chromatography mass spectrometry.

3) Synthesis of oligonucleotide chain with specific sequence (TagB104, its sequence is TAATAGCCGG/GAGCCGGCTATTAAAC) labeled oligonucleotide-beta-lactam mixture P4-4

The procedure was as in step 2 of example 11. Wherein, the molecular weight range of the P4-4 product is 32995-33064. The liquid chromatography mass spectrometry can detect the molecular weight of the product, and the result is shown in FIG. 23. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 16 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB105, sequence CCACCGGCAA/GAGTTGCCGGTGGAAC)

1) Synthesis of nitrone S2-5

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-5 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-5 product is 24936-25005. The liquid chromatography mass spectrometry detects the corresponding molecular weight range of the product, and the reaction yield is confirmed to be 43% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer, and the result is shown in figure 24.

3) Synthesis of oligonucleotide chain with specific sequence (TagB105, its sequence is CCACCGGCAA/GAGTTGCCGGTGGAAC) labeled oligonucleotide-beta-lactam mixture P4-5

The procedure was as in step 2 of example 11. Wherein the molecular weight range of the P4-5 product is 33001-33070. The liquid chromatography mass spectrometry detects the molecular weight of the product, and the result is shown in FIG. 25. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 17 Synthesis of oligonucleotide-beta-lactam mixture labeled with an oligonucleotide chain of specific sequence (TagB106, sequence GCCTCACATA/GAGTATGTGAGGCAAC)

1) Synthesis of nitrone S2-6

The procedure is as in example 2.

2) Synthesis of oligo-nucleic acid-beta-lactam P3-6 mixture

The procedure was as in step 1 of example 11. Wherein the molecular weight range of the P3-6 product is 24927-24996. The liquid chromatography mass spectrometry detects the corresponding molecular weight range of the product, and the result is shown in figure 26, wherein the reaction yield is 46% by combining the detection result of the liquid chromatography mass spectrometry after the OD determination is carried out by an ultramicro spectrophotometer.

3) Synthesis of oligonucleotide chain with specific sequence (TagB106, its sequence is GCCTCACATA/GAGTATGTGAGGCAAC) labeled oligonucleotide-beta-lactam mixture P4-6

The procedure was as in step 2 of example 11. Wherein the molecular weight range of the P4-6 product is 32992-33061. The liquid chromatography mass spectrometry can detect the molecular weight of the product, and the result is shown in FIG. 27. The reaction solution obtained in the step is not separately subjected to post-treatment, and is combined with other similar products and then precipitated together.

Example 18 preparation of a mixture comprising 42 oligo-nucleic acids-lactams

The 6 reaction solutions of examples 12, 13, 14, 15, 16 and 17 were combined, and a 5 mol/l sodium chloride solution having a total volume of 10% was added to the combined reaction solutions. Then, anhydrous ethanol was further added in an amount of 3 times the total volume. After shaking uniformly, the reaction was frozen in a freezer at-80 ℃ for 2 hours. After that, the mixture was centrifuged at 4000rpm for half an hour, and the supernatant was decanted. The remaining precipitate was dissolved in deionized water to obtain a mixture containing 42 oligonucleotide strands (TagA-TagB) labeled with specific sequences. The molecular weight range of the product is 32992-33061. The molecular weight range of the corresponding product is detected by liquid chromatography mass spectrometry, and after the OD is measured by an ultramicro spectrophotometer, the reaction yield of the step is confirmed to be 46 percent by combining the detection result of the liquid chromatography mass spectrometry, and the result is shown in figure 28.

We have verified the universality and versatility of the conversion of different oligo-nucleic acid-terminal alkynes into oligo-nucleic acid-beta-lactam compounds, which is described in another patent application (title: a method for one-pot synthesis of beta-lactam on oligo-nucleic acid) filed on the same day as this application, and constructed a novel library of gene-encoded beta-lactam compounds with this strategy. Through a liquid chromatography mass spectrum, the method has the advantages of simple operation, good universality, mild conditions and high efficiency.

In summary, the above embodiments and drawings are only illustrative of the broad scope of the present invention, and should not be construed as limiting the scope of the present invention, and in theory the size of the library constructed in this way may be unlimited, and therefore any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.

Reference to the literature

[1]Holenz J.；Stoy P.Advances in lead generation[J].Bioorg.Med.Chem.Lett.2019,29,517-524.

[2]Brown D.G.；Jonas B.Where do recent small molecule clinical development candidates come from？[J].J.Med.Chem.,2018,61(21),9442-9468.

[3]Moitessier N.；Pottel J.；Therrien E.；et al.Medicinal chemistry projects requiring imaginative structure-based drug design methods[J].Acc.Chem.Res.,2016,49,1646-1657.

[4]Erlanson D.A.；Fesik S.W.；Hubbard R.E.；et al.Twenty years on:the impact of fragments on drug discovery[J].Nat.Rev.Drug Discov.,2016,15,605-619.

[5]Lavecchia A.；Giovanni C.Virtual screening strategies in drug discovery:a critical review[J].Curr.Med.Chem.,2013,20,2839-2860.

[6]Fuller N.；Spadola L.；Cowen S.；et al.An improved model for fragment-based lead generation at AstraZeneca[J].Drug Discov.Today,2016,21,1272-1283.

[7]Goodnow R.A.Jr.；Dumelin C.E.；Keefe A.D.DNA-encoded chemistry:enabling the deeper sampling of chemical space[J].Nat.Rev.Drug Discov.,2017,16,131–147.

[8]Neri D.；Lerner R.A.DNA-encoded chemical libraries:aselection system based on endowing organic compounds with amplifiable information[J].Annu.Rev.Biochem.,2018,87,479–502.

[9]Brenner S.；Lerner R.A.Encoded combinatorial chemistry[J].Proc.Natl.Acad.Sci.U.S.A.,1992,89,5381-5383.

[10] Leading in section rain water and success, innovation leading compound technology is 'speed up' for new drug research and development [ J ]. China scientific and technological industry, 2014,3,70-71.

[11]Clark M.A.；Acharya R.A.；Arico-Muendel C.C.；et al.Erratum:Design,synthesis and selection of DNA-encoded small-molecule libraries[J].Nat.Chem.Biol.,2009,5(10),772-772.

[12] Soaking in slow force; zdonna; try of sinus quality, et al study and application of library of gene-encoded compounds in drug screening and discovery [ J ]. J. International pharmaceutical research, 2018,45(10), 736-.

[13] Liu Kai; zhang Peng, Yao Ming kang De, New drug development and enabling platform [ J ]. Enterprise management, 2020(2).

[14]Machutta C.A.；Kollmann C.S.；Lind K.E.；et al.Prioritizing multiple therapeutic targets in parallel using automated DNA-encoded library screening[J].Nat.Commun.,2017,8,16081.

[15]Clark M.A.；Acharya R.A.；Arico-Muendel C.C.Design and synthesis and selection of DNA-encoded small-molecule libraries[J].Nat.Chem.Biol.,2009,5(9),647-654.

[16]Huang Y.；Meng L.；Nie Q.；et al.Selection of DNA-encoded chemical libraries against endogenous membrane proteins on live cells[J].Nat.Chem.,2021,13(1),77-88.

[17]Bo C.；Dongwook K.；Saeed A.；et al.Selection of DNA-Encodedencoded Libraries libraries to protein targets within and on living cells[J].J.Am.Chem.Soc.,2019,141(43),17057-17061.

[18]Zining W.；Todd L.G.；Xin Z.；et al.Cell-based selectionexpands the utility of DNA-encoded small-molecule library technology tocell surface drug targets:identification of novel antagonists of the NK3Tachykinin receptor[J].ACS Comb.Sci.,2015,17(12),722-731.

[19]Shi Y.；Wu Y.-r.；Zhang W.-n.et al.DNA-encoded libraries(DELs):a review of on-DNA chemistries and their output,RSC Adv.,2021,11,2359-2376

[20]Leone S.；Cascella M.；Pezone I.；et al.New antibiotics for thetreatment of serious infections in intensive care unit patients[J].Curr.Med.Res.Opin.2019,35,1331-1334.

[21]Thakuria B.；Lahon K.The beta lactam antibiotics as anempirical therapy in a developing country:an update on their currentstatus and recommendations to counter the resistance against them[J].J.Clin.Diagn.Res.2013,7,1207-1214.

[22]Gaynes R.The discovery of penicillin—new insights after morethan 75 years of clinical use[J].Emerging Infectious Diseases.2017,23,849-853.

[23]Malig T.C.；Yu D.；Hein J.E.A revised mechanism for theKinugasa reaction[J].J.Am.Chem.Soc.2018,140,9167-9173.

Sequence listing

<110> Kanglong chemical (Ningbo) science and technology development Co., Ltd; kanglong chemical (Beijing) New drug technology corporation

<120> a method for synthesizing gene coding beta-lactam compound library by one-pot method

<130> CP1210944/CB

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<170> PatentIn version 3.5

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Claims (12)

1. A method for synthesizing a gene coding beta-lactam compound library by a one-pot method comprises the steps of taking a mixture of X kinds of oligonucleotide-terminal alkyne marked by oligonucleotide chains (TagA) with specific sequences as a substrate, reacting with Y kinds of nitrone reagents under the catalysis of copper to convert the mixture into oligonucleotide-beta-lactam, and then marking by the oligonucleotide chains (TagB) with specific sequences to obtain a mixture of (X kinds of X Y kinds of) TagA-TagB marked oligonucleotide-beta-lactam; the reaction equation is as follows:

wherein, the oligonucleotide in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers, wherein the length of the chain is not limited;

wherein, the structural formula of the oligonucleotide-terminal alkyne compound marked by the oligonucleotide chain (TagA) with a specific sequence is as follows:

wherein, R in the structural formula¹Is hydrogen, amino, nitro, cyano, hydroxyl, mercapto, aryl ketone, alkyl ketone, C₁-C₁₂Alkyl radical, C₁-C₆Alkylene radical, C₃-C₈Cycloalkyl radical, C₁-C₆Any one to more of alkyl oxygen, aryl, heterocyclic aryl, halogen, carboxyl, ester group or any combination thereof; preferably, the halogen is selected from one of fluorine, chlorine, bromine and iodine;

wherein, TagA in the structural formula is a single-stranded or double-stranded oligonucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers with known sequences, wherein the length of the chain is not limited;

wherein, the TagB is a single-stranded or double-stranded oligonucleotide chain obtained by polymerizing artificially modified or unmodified nucleotide monomers of known sequence, wherein the length of the chain is not limited;

wherein the nitrone structure is as follows:

wherein, Ar in the structural formula¹Is a five-membered, six-membered or larger aromatic ring or aromatic heterocycle;

wherein, Ar in the structural formula²Is a five-membered, six-membered or larger aromatic ring or aromatic heterocycle;

wherein, the nitrone can be a pure compound or a crude reaction product generated in situ;

wherein X is theoretically any positive integer;

wherein Y is theoretically any positive integer.

2. The method according to claim 1, wherein the molar concentration of the oligonucleotide-terminal alkyne mixture labeled with oligonucleotide strand of specific sequence (TagA) is 0.1-2 mmol/l; preferably, the molar concentration of the oligonucleotide-terminal alkyne mixture after labeling with the oligonucleotide chain (TagA) of a specific sequence is 0.5-1.5 mmol/l; more preferably, the molar concentration of the oligonucleotide-terminal alkyne mixture labeled with the oligonucleotide strand of specific sequence (TagA) is 1.0 mmol/l.

3. The method according to claim 1, wherein the amount of said nitrone is 1-500 equivalents based on 1 equivalent of oligonucleotide-terminal alkyne labeled with oligonucleotide chain (TagA) of specific sequence; preferably, the dosage of the nitrone is 1-100 equivalent; more preferably, the nitrone is used in an amount of 50 equivalents.

4. The process according to claim 1, wherein the reaction is carried out in the presence of a base which is one or a mixture of more of potassium carbonate, sodium bicarbonate, potassium bicarbonate, lithium carbonate, lithium hydroxide, potassium hydroxide, sodium hydroxide, cesium hydroxide, sodium borate, potassium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, methylamine, ethylamine, propylamine, isopropylamine, N-diethylamine, triethylamine, N-butylamine, isobutylamine, 4-dimethylaminopyridine, N-diisopropylethylamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N' -tetramethylethylenediamine, tetramethylguanidine, pyridine, N-methyldicyclohexylamine or dicyclohexylamine; preferably, the alkali is one or a mixture of tetramethyl guanidine, N-methyl dicyclohexyl amine or dicyclohexyl amine; more preferably, the base is N, N-dicyclohexylmethylamine.

5. The method of claim 4, wherein the N, N-dicyclohexylmethylamine is used in an amount of 1 to 500 equivalents, based on 1 equivalent of oligonucleotide-terminal alkyne labeled with a specific sequence of oligonucleotide strands (TagA); preferably, the dosage of the N, N-dicyclohexylmethylamine is 10-200 equivalent; more preferably, the N, N-dicyclohexylmethylamine is used in an amount of 100 equivalents.

6. The process according to claim 1, characterized in that the copper catalyst is copper acetate, copper sulfate, copper chloride, copper nitrate, copper carbonate, cuprous iodide, copper- β -cyclodextrin complex, copper bis (2, 4-pentanedionate), copper acetylacetonate, tetrakis (acetonitrile) copper tetrafluoroborate, copper dichloro (1, 10-phenanthroline), copper bis (8-hydroxyquinoline), copper trifluoromethanesulfonate, copper bis (trifluoro-2, 4-pentanedionate), copper perchlorate, tetrakis (acetonitrile) copper hexafluorophosphate, cuprous acetate, copper bromide, copper fluoride, cuprous bromide, cuprous chloride-bis (lithium chloride) complex, cuprous sulfide bromide complex; preferably, the copper catalyst is cuprous iodide, copper tetrakis (acetonitrile) hexafluorophosphate, cuprous chloride-bis (lithium chloride) complex; more preferably, the copper catalyst is cuprous iodide.

7. The method according to claim 6, wherein the amount of cuprous iodide is 0.1 to 100 equivalents based on 1 equivalent of oligonucleotide-terminal alkyne labeled with oligonucleotide chain (tagA) of specific sequence; preferably, the dosage of the cuprous iodide is 0.1-50 equivalent; more preferably, the amount of cuprous iodide used is 10 equivalents.

8. The process according to claim 1, characterized in that the reaction is carried out in the presence of a solvent which is water, methanol, ethanol, propanol, isopropanol, N-butanol, isobutanol, tert-butanol, pentanol, cyclohexanol, 2-fluoroethanol, 2, 2-difluoroethanol, 2,2, 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, acetic acid chloride, acetic, One or more of dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1, 2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methoxypyridine, toluene, xylene, inorganic salt buffer solution and organic base buffer solution; preferably, the solvent is a mixture of water and acetonitrile; more preferably, the solvent is a mixture of water and acetonitrile and the total acetonitrile content is not less than 50%.

9. The method according to claim 1, wherein the reaction is carried out at a temperature of 0 to 90 ℃; preferably, the temperature is 0-40 ℃; more preferably, the temperature is 25 ℃.

10. The process according to claim 1, characterized in that the reaction is carried out for a time of 1 to 24 hours; preferably, the time is 5-24 hours; more preferably, the time is 12 hours.

11. Method according to claim 1, characterized in that the reaction is carried out under an atmosphere which is one of air, nitrogen or argon, preferably the atmosphere is one of nitrogen or argon, more preferably the atmosphere is nitrogen.

12. The method of claim 1, wherein the TagB is used in an amount of 1 to 10 equivalents based on 1 equivalent of the oligonucleic acid- β -lactam; preferably, the equivalent weight of the TagB is 1-2 equivalent weight; more preferably, the equivalent weight of TagB is 1 equivalent.

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