Oligonucleotide-thiophosphonate compounds and methods for their synthesis

Abstract

The application provides an oligonucleotide-thiophosphonate compound shown in formula I and a synthesis method thereof, the synthesis method is as follows: taking an oligonucleotide-thiol compound shown in formula III and a phosphonate compound shown in formula II as raw materials, reacting in a solvent to obtain the oligonucleotide-thiophosphonate compound shown in formula I. The method has small damage to DNA, good universality, simple operation, mild conditions and can obtain the oligonucleotide-thiophosphonate compound in high yield, and provides a new method for expanding and enriching a DELT compound library.

Description

Technical Field

[0001] This invention belongs to the field of gene-encoded compound library construction, specifically relating to an oligonucleotide-thiophosphate compound and its synthesis method. Background Technology

[0002] Currently, the exploration of lead compounds in pharmaceutical research is increasingly venturing into uncharted territory. Therefore, finding suitable chemical substances to combine with existing compounds and screen them is becoming increasingly difficult. Traditional high-throughput screening suffers from drawbacks such as limited compound library size, long processing times, and high costs. This limits the timeliness of lead compound discovery (AA Shelat, RK Guy, Nat. Chem. Biol. 2007, 3, 442.). In 1992, Brenner and Lerner proposed a method for bioactivity screening using gene-encoded compound library technology (DELT). This method combines molecular biology techniques, adding a DNA tag to each compound at the molecular level. Through combinatorial chemistry synthesis, up to hundreds of millions of compound libraries can be generated in a short time, allowing direct bioactivity screening of this DNA-tagged mixture of compounds (S. Brenner; RALerner. Proc. Natl. Acad. Sci. USA, 1992, 89, 5381-5383.). This technique employs a very small library of DNA-encoded compounds and target proteins for biological screening, followed by PCR amplification and sequencing / decoding of the amplified DNA sequences to identify the corresponding specific compounds. This method not only overcomes the high cost and limitations of traditional high-throughput biological screening in terms of compound chemical spatiality, achieving a leap in the spatial range and quantity of screened compounds, but also allows for the simple and efficient screening of lead compounds under various biological screening conditions simultaneously, making it an increasingly popular method for lead compound discovery.

[0003] To successfully screen small molecule compounds with affinity for biological target proteins, the diversity of chemical structures is crucial for the success of DELT screening. However, due to the unique chemical structure of DNA, it is only stable under certain conditions (temperature, pH, ion concentration, solvent, etc.), and the chemical reactions used for constructing gene-encoded compound libraries require high yields. Therefore, the types of chemical reactions that can be used in gene-encoded compound libraries are quite limited (RJFair, RTWalsh, CDHupp. Bioorg. Med. Chem. Lett., 2021, 51, 128-339.). Only when the variety of chemical reactions that can be successfully applied to gene-encoded compound libraries is greater, and the conditions are more diverse, can there be more choices in the design and synthesis of gene-encoded compound libraries, resulting in a more diverse library. However, the number of organic chemical reactions that can be successfully applied to the synthesis of DELT remains very limited. Therefore, developing more synthetic methods applicable to gene-encoded compound libraries is one of the important tasks in the DELT field (RM Franzini, C. Randolph. J Med. Chem., 2016, 59, 6629-6644; P. Dickson, T. Kodadek. Org. Biomol. Chem., 2019, 17, 4676-4688; K. S. Chinese, A. Brunschweiger. Tetrahedron. Lett. 2020, 61, 151889; PRFitzgerald, BMP aegel. Chem. Rev. 2021, 121, 7155-7177; Y. Shi, YR Wu, WNZhang, et al. RSC Adv., 2021, 11, 2359-2376.).

[0004] Thiophosphates possess significant biological activity and are widely used in the skeletal structures of pharmaceuticals and pesticides. Therefore, research on the efficient synthesis of thiophosphates is of great scientific and practical value. In biomedicine, thiophosphates can be used to synthesize diethoxyphosphonothiocholine iodide (ACHE inhibitor), which can be used to treat chronic glaucoma; they can also be further used to synthesize amifostine, a cancer treatment that significantly reduces neurotoxicity in various parts of the body. Furthermore, they can be used to synthesize various types of hypertension drugs, such as the antihypertensive drug ivermectin hydrochloride, which was launched in Japan in 1994.

[0005]

[0006] Several methods for the efficient synthesis of small thiophosphate esters have been reported in traditional chemistry. For example, in 1968, Murdock et al. reported the reaction of triethyl phosphite with thiophenol in bromochloroform to prepare aromatic-substituted thiophosphate esters. However, this method is not applicable to common thiols, and it requires anhydrous and oxygen-free conditions for small molecules, making it unsuitable for constructing thiophosphate esters from DNA. In 2013, Kaboudi et al. discovered that phosphite compounds and thiols can form thiophosphate esters through dehydrogenation under copper catalyst. However, this condition is only applicable to thiophenol compounds, exhibiting poor functional group compatibility. Additionally, Song et al. reported a method for obtaining thiophosphates from thiols and dialkyl phosphates under oxygen atmosphere and acetonitrile solvent conditions without transition metal catalysis. However, reactions on DNA are generally carried out in centrifuge tubes, making it difficult to ensure a sufficient oxygen atmosphere; therefore, this method is also unsuitable for constructing thiophosphate esters from DNA. (a)LLMurdock,TLHopkins.Synthesis of O,O-dialkyl S-aryl phosphorothiolates[J].Org.Chem.1968,33(2),907-908;b)Q.An,J.Shen.N.Butt.et al.ChemInform abstract:asymmetric domino double Michaeladdition of nitroolefins and aldehyde esters with trans-perthhydroindolic acidas an organocatalyst[J].Synthesis.2013,45(12),1612-1623; c)S.Song,YQZhang.etal.Cs2CO3-Catalyzed aerobic oxidative cross-dehydrogenative coupling of thiolswith phosphonates and arenas[J].Angew.Chem.Int.Ed.2017,56,2487-2491.)

[0007] Currently, there are no reported methods for constructing on-DNA phosphate thioester gene-encoded compounds on DNA. DNA chemistry differs from ordinary chemical reactions; many common small-molecule chemical reactions require harsh conditions that can easily cause DNA denaturation and damage its chemical structure, making them unsuitable for constructing gene-encoded compound libraries. Therefore, developing a method for synthesizing on-DNA phosphate thioester compounds with minimal DNA damage and high conversion efficiency is of great significance. Summary of the Invention

[0008] The purpose of this invention is to provide a method for synthesizing oligonucleotide-thiophosphate compounds.

[0009] This invention provides an oligonucleotide-thiophosphate compound having the structure shown in Formula I:

[0010]

[0011] Among them, L1 and L2 are connection units;

[0012] R1 and R2 are each independently selected from C 1～12 Alkyl, phenyl, or benzyl.

[0013] Furthermore, the above L1 is:

[0014] And / or, the L2 is substituted or unsubstituted: C 1～6 Alkylene or 5-6 aryl or heteroaryl;

[0015] The number of substituents in the substitution is 1 to 3, and each substituent is independently selected from halogens, C, etc. 1～5 Alkyl, C 1～5 Amide group, amino group, or protected amino group.

[0016] Furthermore, the L2 mentioned above is either substituted or unsubstituted: C 1～3 alkylene, phenyl, or pyridyl;

[0017] Wherein, the number of substituents is one, and the substituent is a fluorine, methyl, acetamino, or Fmoc-protected amino group.

[0018] Furthermore, R1 and R2 are each independently selected from C 1～5 Alkyl, phenyl, or benzyl, preferably, R1 and R2 are each independently selected from methyl, ethyl, phenyl, or benzyl.

[0019] Furthermore, the above-mentioned compound is one of the following compounds:

[0020]

[0021] This invention also provides a method for synthesizing oligonucleotide-thiophosphate compounds, which uses oligonucleotide-thiol compounds of formula III and phosphate compounds of formula II as raw materials, and reacts them in a solvent under the action of a catalyst to obtain oligonucleotide-thiophosphate compounds of formula I; the reaction formula is as follows:

[0022]

[0023] Among them, L1 and L2 are connection units;

[0024] R1 and R2 are each independently selected from C 1～12 Alkyl, phenyl, or benzyl.

[0025] Furthermore, the molar ratio of the oligonucleotide-thiol compound shown in Formula III, the phosphate ester compound shown in Formula II, and the catalyst is 1:(1-500):(1-1000), preferably 1:(50-250):(200-600);

[0026] And / or the temperature of the reaction is 0–90°C; preferably 4–50°C;

[0027] And / or the reaction time is 1 to 24 hours; preferably 2 to 20 hours;

[0028] And / or the catalyst is sodium tert-butoxide, potassium tert-butoxide, cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, lithium carbonate, lithium hydroxide, potassium hydroxide, sodium hydroxide, cesium hydroxide, sodium borate, potassium borate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, triethylenediamine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undecane The catalyst is one or more of the following: -7-ene, N,N,N',N'-tetramethylethylenediamine, 1,1,3,3-tetramethylguanidine, N,N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, inorganic salt buffer solution, and organic base buffer solution; in a preferred embodiment, the catalyst is one or more of the following: triethylenediamine, triethylamine, and N,N-diisopropylethylamine; in a more preferred embodiment, the catalyst is N,N-diisopropylethylamine.

[0029] And / or the solvent 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, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, glycerol, ethyl 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. The reaction is carried out by any one or a mixture of several of the following: diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, and xylene; preferably, the solvent for the reaction is a mixture of water and dimethyl sulfoxide.

[0030] Furthermore, the molar ratio of the oligonucleotide-thiol compound shown in Formula III, the phosphate ester compound shown in Formula II, and the base is 1:200:400;

[0031] And / or the temperature of the reaction is 25°C;

[0032] And / or the reaction time is 12 hours;

[0033] And / or the catalyst is N,N-diisopropylethylamine.

[0034] Furthermore, the above L1 is:

[0035] And / or, the L2 is substituted or unsubstituted: C 1～6 Alkylene or 5-6 aryl or heteroaryl;

[0036] The number of substituents in the substitution is 1 to 3, and each substituent is independently selected from halogens, C, etc. 1～5 Alkyl, C 1～5 Amide group, amino group, or protected amino group.

[0037] Furthermore, the L2 mentioned above is either substituted or unsubstituted: C 1～3 alkylene, phenyl, or pyridyl;

[0038] Wherein, the number of substituents is one, and the substituent is a fluorine, methyl, acetamino, or Fmoc-protected amino group.

[0039] Furthermore, R1 and R2 are each independently selected from C 1～5Alkyl, phenyl, or benzyl, preferably, R1 and R2 are each independently selected from methyl, ethyl, phenyl, or benzyl.

[0040] Furthermore, the compound of formula I is one of the following compounds:

[0041]

[0042] The present invention also provides the use of the above-described synthesis method in constructing gene-encoded compound libraries.

[0043] The beneficial effects of this invention are as follows: This invention provides a method for synthesizing oligonucleotide-thiophosphate compounds on DNA. Under the action of a specific catalyst, in a specific solvent system, at a specific temperature, and for a specific time, oligonucleotide-thiophosphate compounds are successfully prepared. This method causes minimal damage to DNA, has good universality, is simple to operate, operates under mild conditions, and can produce oligonucleotide-thiophosphate compounds in high yield, providing an important foundation for expanding and enriching the DELT compound library.

[0044] In the structural formula of this invention, "DNA" refers to a single-stranded or double-stranded oligonucleotide chain.

[0045] In this invention, the minimum and maximum carbon atom content in the hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～b Alkyl groups refer to any alkyl group containing one to two carbon atoms ("a" to "b"). For example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0046] In this invention, "cyclic hydrocarbon group" refers to a group formed by losing one or two hydrogen atoms from a saturated or unsaturated pure carbon ring (where all atoms constituting the ring skeleton are C) or a heterocycle (a heterocycle is a stable ring structure formed by replacing some carbon atoms in a pure carbon ring with heteroatoms such as O, N, or S; for example, a methylene group on a ring, -CH2-, is replaced by an O atom to form a stable O- structure, or replaced by an N atom to form a stable NH- structure; or replaced by an S atom to form a stable SO2- structure, etc.)).

[0047] The "organic solvents" of this invention include, but are not limited to, 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, 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, and diethyl ether. Thioethers, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, N-methylpyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), acetonitrile, acetone, cyclohexanone, dichloromethane, chloroform, chlorobenzene, 1,2-dichloroethane, ethyl acetate, n-hexane, cyclohexane, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 4-methoxypyridine, toluene, xylene.

[0048] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0049] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0050] Figure 1 This is a liquid chromatography-mass spectrum of oligonucleotide-NHFmoc raw material (S2).

[0051] Figure 2 This is a liquid chromatography-mass spectrum of oligonucleotide-NH2 raw material (S3).

[0052] Figure 3 This is a liquid chromatography-mass spectrum of oligonucleotide-COOH raw material (S4).

[0053] Figure 4 For oligonucleotide-thiol compound raw material S 5-1 The liquid chromatography-mass spectrum.

[0054] Figure 5 The present invention relates to oligonucleotide-thiophosphate compound S. 5-2 The liquid chromatography-mass spectrum.

[0055] Figure 6 The present invention relates to oligonucleotide-thiophosphate compound S. 5-3 The liquid chromatography-mass spectrum.

[0056] Figure 7 The present invention relates to oligonucleotide-thiophosphate compound S. 5-4 The liquid chromatography-mass spectrum.

[0057] Figure 8 The present invention relates to oligonucleotide-thiophosphate compound S. 5-5 The liquid chromatography-mass spectrum.

[0058] Figure 9 The present invention relates to oligonucleotide-thiophosphate compound S. 5-6 The liquid chromatography-mass spectrum.

[0059] Figure 10 The present invention relates to oligonucleotide-thiophosphate compound S. 5-7 The liquid chromatography-mass spectrum.

[0060] Figure 11 The present invention relates to oligonucleotide-thiophosphate compound S. 5-8 The liquid chromatography-mass spectrum.

[0061] Figure 12 The present invention relates to oligonucleotide-thiophosphate compound S. 5-9 The liquid chromatography-mass spectrum.

[0062] Figure 13 The present invention relates to oligonucleotide-thiophosphate compound S. 5-10 The liquid chromatography-mass spectrum.

[0063] Figure 14 The present invention relates to oligonucleotide-thiophosphate compound S. 5-11 The liquid chromatography-mass spectrum.

[0064] Figure 15 The present invention relates to oligonucleotide-thiophosphate compound S. 5-12 The liquid chromatography-mass spectrum.

[0065] Figure 16 The present invention relates to oligonucleotide-thiophosphate compound S. 5-13 The liquid chromatography-mass spectrum.

[0066] Figure 17 The present invention relates to oligonucleotide-thiophosphate compound S. 5-14 The liquid chromatography-mass spectrum.

[0067] Figure 18 The present invention relates to oligonucleotide-thiophosphate compound S. 5-15 The liquid chromatography-mass spectrum.

[0068] Figure 19 This is the liquid chromatography-mass spectrum of the oligonucleotide-thiophosphate compound P1 of the present invention.

[0069] Figure 20 This is the liquid chromatography-mass spectrum of the oligonucleotide-thiophosphate compound P2 of the present invention.

[0070] Figure 21 This is the liquid chromatography-mass spectrum of the oligonucleotide-thiophosphate compound P3 of the present invention.

[0071] Figure 22 This is the liquid chromatography-mass spectrum of P4, an oligonucleotide-thiophosphate compound of the present invention.

[0072] Figure 23 This is the liquid chromatography-mass spectrum of P5, an oligonucleotide-thiophosphate compound of the present invention.

[0073] Figure 24 This is the liquid chromatography-mass spectrum of P6, an oligonucleotide-thiophosphate compound of the present invention.

[0074] Figure 25 This is the liquid chromatography-mass spectrum of P7, an oligonucleotide-thiophosphate compound of the present invention.

[0075] Figure 26 This is the liquid chromatography-mass spectrum of P8, an oligonucleotide-thiophosphate compound of the present invention.

[0076] Figure 27 This is the liquid chromatography-mass spectrum of P9, an oligonucleotide-thiophosphate compound of the present invention.

[0077] Figure 28 The present invention relates to oligonucleotide-thiophosphate compound P. 10 The liquid chromatography-mass spectrum.

[0078] Figure 29 The present invention relates to oligonucleotide-thiophosphate compound P. 11 The liquid chromatography-mass spectrum.

[0079] Figure 30 The present invention relates to oligonucleotide-thiophosphate compound P. 12 The liquid chromatography-mass spectrum.

[0080] Figure 31 The present invention relates to oligonucleotide-thiophosphate compound P. 13 The liquid chromatography-mass spectrum.

[0081] Figure 32 The present invention relates to oligonucleotide-thiophosphate compound P. 14 The liquid chromatography-mass spectrum.

[0082] Figure 33 The present invention relates to oligonucleotide-thiophosphate compound P.15 The liquid chromatography-mass spectrum.

[0083] Figure 34 The present invention relates to oligonucleotide-thiophosphate compound P. 16 The liquid chromatography-mass spectrum.

[0084] Figure 35 The present invention relates to oligonucleotide-thiophosphate compound P. 17 The liquid chromatography-mass spectrum.

[0085] Figure 36 The present invention relates to oligonucleotide-thiophosphate compound P. 18 The liquid chromatography-mass spectrum.

[0086] Figure 37 This is a liquid chromatography-mass spectrum of TagA-P9, the product of the oligonucleotide-α-hydroxyphosphonic acid compound P9 linked with TagA according to the present invention.

[0087] Figure 38 For various oligonucleotide-thiol compounds (S 5-1 ~S 5-15 (Structure diagram of )

[0088] Figure 39 The oligonucleotide-thiophosphate compounds (P1~P) of this invention 18 (Structure diagram of ) Detailed Implementation

[0089] All raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products. In this invention specification, "equivalent" refers to molar equivalent.

[0090] 1. Synthesis of oligonucleotide-NH2 precursor (S3)

[0091] 1.1 Synthesize oligonucleotide-NHFmoc raw material (S2) according to the following reaction formula:

[0092]

[0093] The structure of 100 nanomolar HP(S1,HP) is as follows: Figure 1As shown, a commercially available product was dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). 40 equivalents of a DMSO solution (20 μL, 200 mmol / L) of the starting head fragment compound (commercially available product), 250 equivalents of a sodium tetraborate (Na₂B₄O₇) buffer solution (pH 9.5, 100 μL, 250 mmol / L), and 40 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (20 μL, 200 mmol / L) were mixed and thoroughly vortexed. This mixture was then added to the HP solution, mixed thoroughly, and reacted at 4°C for 1 hour. After the reaction was complete, 10% (by total volume) of a 5 mol / L sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, shaken thoroughly, and the reaction solution was frozen at -80°C for 2 hours. Next, centrifuge at 4000 rpm for half an hour, and discard the supernatant. Dissolve the remaining precipitate in deionized water to obtain a DNA-NHFmoc solution. The chromatogram of DNA-NHFmoc (S2) was detected using liquid chromatography-mass spectrometry (LC-MS / MS) as shown below. Figure 1 As shown, its molecular weight is 5406.

[0094] 1.2 Synthesize oligonucleotide-NH2 precursor (S3) according to the following reaction formula:

[0095]

[0096] 100 nanomolars of DNA-NHFmoc(S2) were dissolved in deionized water to prepare a 1 mmol / L (100 μL, 1 equivalent) solution. 56 μL of a 10% piperidine aqueous solution was added, and the mixture was thoroughly mixed and reacted at room temperature for 1 hour. After the reaction was complete, 10% (by total volume) of a 5 mol / L sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, and the mixture was shaken thoroughly. The reaction solution was then frozen at -80°C for 2 hours. Afterward, the solution was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water to obtain a solution of oligonucleotide-NH2(S3, abbreviated as DNA-NH2). The chromatogram of DNA-NH2(S3) was detected using liquid chromatography-mass spectrometry (LC-MS / MS) as shown below. Figure 2 As shown, its molecular weight is 5184.

[0097] 2. Synthesis of oligonucleotide-COOH raw material (S4)

[0098] 2.1 Synthesize oligonucleotide-COOH raw material (S4) according to the following reaction formula:

[0099]

[0100] The structure of 100 nanomolar HP(S1,HP) is as follows: Figure 1 As shown, a commercially available product was dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 100 nanomoles, 1 equivalent). 40 equivalents of a DMSO solution (concentration: 200 mmol / L) of the starting head fragment compound (commercially available product), 250 equivalents of a sodium tetraborate (Na₂B₄O₇) buffer solution (concentration: 250 mmol / L) at pH 9.5, and 40 equivalents of an aqueous solution of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) (concentration: 200 mmol / L) were mixed and thoroughly vortexed. This mixture was then added to the HP solution, mixed thoroughly, and reacted at 4°C for 1 hour. After the reaction was complete, 10% by volume of a 5 mol / L sodium chloride solution was added to the reaction solution. Then, three times the total volume of anhydrous ethanol was added, the mixture was shaken thoroughly, and the reaction solution was frozen at -80°C for 2 hours. Next, centrifuge at 4000 rpm for half an hour, and discard the supernatant. Dissolve the remaining precipitate in deionized water to obtain a DNA-COOH solution. The chromatogram of DNA-COOH (S4) was detected using liquid chromatography-mass spectrometry (LC-MS / MS) as shown below. Figure 3 As shown, its molecular weight is 5229.

[0101] 3. Oligonucleotide-thiol compound raw material (S) 5-1 Synthesis of

[0102]

[0103] 100 nanomolars of DNA-NH₂ were dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). Then, 200 equivalents of dithiol dihydroxyacetic acid (commercially available, 100 μL, 200 mmol / L dimethyl sulfoxide solution), 1200 equivalents of N,N-diisopropylethylamine (120 μL, 1 mol / L acetonitrile solution), 1200 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 200 μL, 600 mmol / L dimethyl sulfoxide solution), and 200 equivalents of 1-hydroxy-7-azobenzotriazole (HOAt, 100 μL, 200 mmol / L dimethyl sulfoxide solution) were added. After thorough mixing, the mixture was reacted at room temperature for 2 hours. (This condensation reaction is referenced from: Li Y.; Gabriele E.; Samain F.; Favalli N.; Sladojevich) F.; Scheuermann.; Optimized Reaction Conditions for Amide BondFormation in DNA Encoded Combinatorial Libraries, Neri D. ACS Comb. Sci. 2016, 18, 8, 438–443.). After the reaction was complete, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution, followed by 3 times the total volume of anhydrous ethanol. After shaking well, the reaction mixture was placed in a freezer at -80°C for 2 hours. Then, the mixture was centrifuged at 4000 rpm at 4°C for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain an aqueous solution of oligonucleotide-disulfide. 100 nanomolar oligonucleotide-disulfide was dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). Then, 100 μL of 3-cyclohexylaminopropanesulfonic acid (CAPS) buffer (pH 8.0, 100 mmol / L) and 100 equivalents of DL-1,4-dithiothreitol (DTT, 50 μL, 200 mmol / L acetonitrile solution) were added. After thorough mixing, the mixture was reacted at 25°C for 0.5 hours (Reference: Kaori Sakurai; Thomas M. Snyder; and David R. Liu*; DNA-Templated Functional Group Transformations Enable Sequence-Programmed Synthesis Using Small-Molecule Reagents, J. Am. Chem. Soc. 2005, 127, 1660-1661).After the reaction was complete, 10% of the total volume of 5 mol / L sodium chloride solution and 3 times the total volume of anhydrous ethanol were added to the reaction solution. After shaking well, the reaction mixture was placed in a freezer at -80°C for 2 hours. Then, the mixture was centrifuged at 4000 rpm at 4°C for half an hour, the supernatant was discarded, and the precipitate was the oligonucleotide-thiol compound (S). 5-1 The remaining precipitate was dissolved in deionized water to obtain an oligonucleotide-thiol compound (S). 5-1 An aqueous solution was used directly in the next reaction. The oligonucleotide-thiol compound S was detected using liquid chromatography-mass spectrometry. 5-1 The spectrum is as follows Figure 4 As shown, its molecular weight is 5259 and the conversion rate is 83%.

[0104] Example 2 Oligonucleotide-Thiol Compound Raw Material (S) 5-6 Synthesis of

[0105]

[0106] Dissolve 100 nanomolars of DNA-COOH in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). Add 200 equivalents of cystamine dihydrochloride (commercially available, 100 μL, 200 mmol / L dimethyl sulfoxide solution), 1200 equivalents of N,N-diisopropylethylamine (120 μL, 1 mol / L acetonitrile solution), 1200 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 200 μL, 600 mmol / L dimethyl sulfoxide solution), and 200 equivalents of 1-hydroxy-7-azobenzotriazole (HOAt, 100 μL, 200 mmol / L dimethyl sulfoxide solution). Mix thoroughly and react at room temperature for 2 hours. (This condensation reaction is referenced from: Li Y.; Gabriele E.; Samain F.; Favalli.) N.; Sladojevich F.; Scheuermann.; Optimized Reaction Conditions for Amide Bond Formation in DNAEncoded Combinatorial Libraries, Neri D. ACS Comb. Sci. 2016, 18, 8, 438–443.). After the reaction was complete, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution, followed by 3 times the total volume of anhydrous ethanol. After shaking well, the reaction mixture was placed in a freezer at -80°C for 2 hours. Then, the mixture was centrifuged at 4000 rpm at 4°C for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain an aqueous solution of oligonucleotide-disulfide. 100 nanomolar oligonucleotide-disulfide was dissolved in deionized water to prepare a 1 mmol / L solution (100 μL, 1 equivalent). Then, 100 μL of 3-cyclohexylaminopropanesulfonic acid (CAPS) buffer (pH 8.0, 100 mmol / L) and 100 equivalents of DL-1,4-dithiothreitol (DTT, 50 μL, 200 mmol / L acetonitrile solution) were added. After thorough mixing, the mixture was reacted at 25°C for 0.5 hours (Reference: Kaori Sakurai; Thomas M. Snyder; and David R. Liu*; DNA-Templated Functional Group Transformations Enable Sequence-Programmed Synthesis Using Small-Molecule Reagents, J. Am. Chem. Soc. 2005, 127, 1660-1661). After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution and 3 times the total volume of anhydrous ethanol were added to the reaction solution. After shaking evenly, the reaction solution was placed in a freezer at -80°C for 2 hours.Then, centrifuge at 4000 rpm and 4°C for half an hour, discard the supernatant, and the precipitate is the oligonucleotide-thiol compound (S). 5-6 The remaining precipitate was dissolved in deionized water to obtain an oligonucleotide-thiol compound (S). 5-6 An aqueous solution was used directly in the next reaction. The oligonucleotide-thiol compound S was detected using liquid chromatography-mass spectrometry. 5-6 The spectrum is as follows Figure 10 As shown, its molecular weight is 5286, and the conversion rate is 86%. Other oligonucleotide-thiol compound raw materials (S) synthesized using the methods of Examples 1 and 2... 5-2 -S5-15, ), the test results are shown in Figures 5-18 S 5-1 The structural diagram of -S5-15 is shown below. Figure 38 .

[0107] The representative structure of raw material HP(S1) is as follows:

[0108]

[0109] However, it should be noted that the method of the present invention is not limited to the DNA strand with the specific S1 structure described above. It can be a single-stranded or double-stranded deoxyribonucleotide chain obtained by polymerizing other artificially modified or unmodified deoxyribonucleotide monomers, and there is no limit to the length of the chain.

[0110] Example 1: Using oligonucleotide-thiol compound raw material (S) 5-1 Synthesizing oligonucleotide-thiophosphate compounds (P1)

[0111]

[0112] In 10 nanomolar oligonucleotide-thiol compounds (S 5-1 400 equivalents of N,N-diisopropylethylamine (20 μL, 200 mmol / L dimethyl sulfoxide solution) and 200 equivalents of diethyl phosphite (commercially available product, 200 mmol / L acetonitrile solution, 10 μL) were added to an aqueous solution of 1 mmol / L (10 μL). The mixture was thoroughly mixed by vortexing and reacted at 25°C for 12 hours. After the reaction was complete, 10% of the total volume of 5 mol / L sodium chloride solution and 3 times the volume of anhydrous ethanol were added to the reaction solution. After thorough mixing, the solution was placed in a -80°C freezer for 2 hours, followed by high-speed refrigerated centrifugation (4°C, 12000 rpm, 15 minutes). The supernatant was discarded, and the remaining precipitate was the product, an On-DNA phosphate thioester compound (P1), which was detected by liquid chromatography-mass spectrometry. The results are shown in the figure. Figure 19The molecular weight was 5396, and the conversion rate was 69%. Other representative on-DNA phosphate thioester compounds (P2-P) synthesized using the method described in Example 3... 18 The test results are shown below. Figures 20-36 Compound P1-P 18 See the structural diagram Figure 39 .

[0113] The following experimental examples demonstrate the beneficial effects of the present invention.

[0114] Experimental Example 1: Screening of Synthetic Conditions for Oligonucleotide-Thiophosphate Compounds of the Present Invention

[0115] Referring to Example 1, the synthesized oligonucleotide-thiophosphate compound P 10 The method differs only in that the following parameters are controlled according to Table 1: catalyst, diethyl phosphite equivalent, co-solvent with water, and reaction time.

[0116]

[0117] Calculate the oligonucleotide-thiophosphate compound P obtained under different parameters 10 The conversion rate was calculated. The results are shown in Table 1. Based on the experimental results, it can be seen that under reaction conditions #9, the product P obtained... 10 The yield is the highest.

[0118] Table 1. Synthesis of oligonucleotide-thiophosphate compounds P under different conditions 10 conversion rate

[0119]

[0120]

[0121] Experimental Example 2: Verification Experiment of the Integrity of Oligonucleotides in the Oligonucleotide-Thiophosphate Compounds of the Present Invention

[0122]

[0123] Linking on-DNA phosphate thioester compound P9 with oligo A (a short-chain oligonucleotide with molecular weights of 4064 and 5884 for the two chains): Dissolve 1 nanomolar of P9 in deionized water to prepare a 1 mmol / L solution (1 μL, 1 equivalent). Add 1.2 equivalents of oligo A (1 mmol / L aqueous solution, 1.2 μL), 1 μL of 10×T4 DNA ligation buffer, and 0.5 μL of T4 DNA ligase. Mix the solutions thoroughly and react at room temperature for 1 hour. After the reaction, add 10% (total volume) of 5 mol / L sodium chloride solution to the reaction mixture, followed by 3 times the total volume of anhydrous ethanol. After shaking thoroughly, freeze the mixture at -80°C for 2 hours. Then centrifuge at 4000 rpm for half an hour and discard the supernatant. The remaining precipitate was dissolved in deionized water, and the molecular weight of the product oligoA-P9 was confirmed by liquid chromatography-mass spectrometry (LC-MS / MS) to be 15367. The mass spectrometry results are shown below. Figure 37 .

[0124] LCMS analysis showed that the on-DNA phosphate thioester compound P9 could be successfully coupled with oligo A. This indicates that the on-DNA phosphate thioester compound obtained according to the synthesis method described in this application has good DNA chain integrity. Furthermore, LCMS mass spectrometry accurately revealed its molecular weight, further confirming that the reaction method in this invention causes minimal damage to the basic structure and activity of DNA.

[0125] In summary, this invention provides a method for synthesizing oligonucleotide-thiophosphate compounds on DNA. By reacting these compounds in a specific system with specific base, reactant equivalences, and solvent for a specific time, the method successfully yields oligonucleotide-thiophosphate compounds. This method causes minimal DNA damage, has good versatility, is simple to operate, operates under mild conditions, and can produce oligonucleotide-thiophosphate compounds with high conversion rates, providing a novel approach for expanding and enriching the DELT compound library.

Claims

1. A method for synthesizing oligonucleotide-thiophosphate compounds, characterized in that, It uses oligonucleotide-thiol compounds represented by Formula III and phosphate ester compounds represented by Formula II as raw materials, reacting them in a solvent under the action of a catalyst to obtain oligonucleotide-thiophosphate ester compounds represented by Formula I; the reaction formula is as follows: Wherein, L1 and L2 are connection units; L1 is: or The L2 is either substituted or unsubstituted: C 1 ~ 6 Alkylene or 5-6 aryl or heteroaryl; wherein the number of substituents is 1-3, and each substituent is independently selected from halogens, C 1 ~ 5 Alkyl, C 1 ~ 5 Amide group, amino group, or protected amino group; R1 and R2 are each independently selected from C 1 ~ 12 Alkyl, phenyl, or benzyl; The molar ratio of the oligonucleotide-thiol compound shown in Formula III, the phosphate ester compound shown in Formula II, and the catalyst is 1:(1 ~ 500):(1 ~ 1000); The reaction temperature is 0 ~ 90℃; the reaction time is 1 ~ 24 hours; The catalyst is cesium carbonate, potassium carbonate, sodium carbonate, triethylenediamine, N,N-diisopropylethylamine, or a borate buffer solution with a pH of 9.5; The solvent is a mixture of water, dimethyl sulfoxide, and acetonitrile.

2. The synthesis method according to claim 1, characterized in that, The molar ratio of the oligonucleotide-thiol compound shown in Formula III, the phosphate ester compound shown in Formula II, and the catalyst is 1:(50 ~ 250):(200 ~ 600); And / or the temperature of the reaction is 4 ~ 50°C; And / or the reaction time is 2 to 20 hours; And / or the catalyst is triethylenediamine or N,N-diisopropylethylamine.

3. The synthesis method as described in claim 2, characterized in that, The molar ratio of the oligonucleotide-thiol compound shown in Formula III, the phosphate ester compound shown in Formula II, and the base is 1:200:400; And / or the temperature of the reaction is 25°C; And / or the reaction time is 12 hours; And / or the catalyst is N,N-diisopropylethylamine.

4. The synthesis method according to claim 1, characterized in that, L2 is substituted or unsubstituted: C 1 ~ 3 alkylene, phenyl, or pyridyl; Wherein, the number of substituents is one, and the substituent is a fluorine, methyl, acetamino, or Fmoc-protected amino group.

5. The synthesis method according to claim 1, characterized in that, R1 and R2 are each independently selected from C 1 ~ 5 Alkyl, phenyl, or benzyl.

6. The synthesis method as described in claim 5, characterized in that, R1 and R2 are each independently selected from methyl, ethyl, phenyl, or benzyl.

7. The synthesis method according to any one of claims 1 to 6, characterized in that, The compound of formula I is one of the following compounds: 。 8. Use of the synthetic method according to any one of claims 1 to 7 in constructing a gene-encoded compound library.

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