Oligonucleic acid-α-hydroxyphosphonic acid compound and synthesis method thereof

Abstract

The present invention provides an oligonucleic acid-α-hydroxyphosphonic acid compound shown in formula I and a synthesis method thereof. The synthesis method of the present invention uses an oligonucleic acid-aldehyde compound shown in formula III and a phosphite compound shown in formula II as raw materials, reacts in a solvent, and obtains an oligonucleic acid-α-hydroxyphosphonic acid compound shown in formula I. The method has little damage to DNA, good universality, simple operation, mild conditions, and can produce oligonucleic acid-α-hydroxyphosphonic acid compounds in high yield, providing an important basis for expanding and enriching the DELT compound library.

Description

Technical Field

[0001] The invention belongs to the field of gene encoding compound library construction, and specifically relates to an oligonucleic acid-alpha-hydroxyphosphonic acid compound and a synthesis method thereof. Background Art

[0002] At present, the exploration of lead compounds in medical research is constantly entering an unknown field. Therefore, it is increasingly difficult to find suitable chemicals to combine with existing compounds and screen them. Traditional high-throughput screening has the disadvantages of limited number of compound libraries, long time and high cost, which limits the timeliness of lead compound discovery (AA Shelat, RK Guy, Nat. Chem. Biol. 2007, 3, 442.). Brenner and Lerner proposed in 1992 a method for bioactivity screening using gene-encoded compound library technology (DELT). It combines molecular biology technology, adds a DNA tag to each compound at the molecular level, and can produce up to 100 million compound libraries in a relatively short period of time through combinatorial chemical synthesis methods. Directly screen the compound mixture labeled by this DNA one by one for biological activity (S. Brenner; RA Lener. Proc. Natl. Acad. Sci. USA, 1992, 89, 5381-5383.), use a very small amount of DNA-encoded compound library and target protein for biological screening, and then elute the adsorbed compound. Use PCR technology to replicate and amplify, sequence and decode the amplified DNA sequence, and find the corresponding specific compound. This technology not only breaks through the high cost of traditional high-throughput biological screening and the bottleneck of compound chemical space, and realizes a leap in the chemical structure space and quantity of the screened compounds, but also can simultaneously screen lead compounds concisely and efficiently under multiple biological screening conditions, becoming an increasingly popular method for discovering lead compounds.

[0003] In order to successfully screen small molecule compounds with affinity for biological target proteins, building diversity in chemical structure is an important guarantee for DELT to successfully screen. However, due to the particularity of DNA chemical structure, it can only be stable under certain conditions (temperature, pH, ion concentration, solvent, etc.). At the same time, the chemical reactions used in the construction of gene-encoded compound libraries also require a higher conversion rate, so the types of chemical reactions that can be used in gene-encoded compound libraries are very limited (RJ Fair, RT Walsh, CD Hupp. Bioorg. Med. Chem. Lett., 2021, 51, 128-339.). Therefore, the more types of chemical reactions that can be successfully applied to gene-encoded compound libraries, the richer the conditions, the more choices there are when designing and synthesizing gene-encoded compound libraries, and the richer the diversity of the resulting compound library. This technology has now become a new pillar of drug discovery. At the same time, there are more and more reports on new DNA synthesis reactions. Therefore, developing more synthesis methods that can be applied to gene-encoded compound libraries is also 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, BMPaegel. Chem. Rev. 2021, 121, 7155-7177; Y. Shi, YR Wu, WNZhang, et al. RSC Adv., 2021, 11, 2359-2376.).

[0004] Phosphonic acid is an analog of phosphoric acid and exists in large quantities in the human body. They are a class of endogenous substances that are harmless to the human body and are excellent small molecule carriers that can help drugs improve their water solubility and transport them into cells. The introduction of phosphonate groups on the basis of the original drug not only improves the selectivity of the drug and reduces its side effects, but also improves its solubility, provides a variety of administration routes, and greatly increases the bioavailability of the drug. The phosphonate prodrugs currently on the market mainly include antiviral drugs adefovir dipivoxil and tenofovir disoproxil fumarate.

[0005]

[0006] For example, estradiol mustard developed by Roche has poor selectivity for prostate cancer. After the introduction of phosphonate groups, estradiol mustard phosphate has significantly improved selectivity for prostate cancer and reduced toxic side effects. Clindamycin generally causes severe pain when injected, and tastes bitter when taken orally and is prone to gastrointestinal reactions. However, after clindamycin is made into clindamycin phosphate, its water solubility is improved, so it can be administered by intravenous injection. It is quickly hydrolyzed into clindamycin under the action of alkaline phosphatase in the blood, avoiding the taste discomfort and gastrointestinal reactions caused by oral administration. The introduction of phosphonate groups on the basis of phenytoin to obtain fosphenytoin sodium increases the water solubility of the original drug (140 mg·mL -1 ), and made it into 50 mg mL -1 The aqueous solution can be administered by intravenous injection or intramuscular injection, which greatly overcomes the adverse reactions caused by the clinical application of phenytoin and eliminates the drug-drug interaction of phenytoin.

[0007] The currently known method for forming phosphonic acid compounds on DNA is mainly developed by Dillon's research group using 4,4-di-tert-butyl bipyridine as a catalyst, and hypophosphite chloride is coupled with aryl iodide to obtain phosphonic acid lead compounds (Flood, Dillon T, et al. "RASS-Enabled S / PC and S-NBond Formation for DELSynthesis." Angewandte Chemie, 2020, 132, 19.). However, this synthesis method can only obtain phenylphosphonic acid compounds with a low conversion rate (<33%); and -α-hydroxyphosphonic acid compounds cannot be synthesized. Secondly, the reported reaction conditions must be controlled under strictly anhydrous conditions. Experiments show that even 1% water can easily cause reaction quenching and low reaction yield. In fact, it is precisely because of the difference between DNA chemistry and ordinary chemical reactions that many ordinary small molecule chemical reaction conditions are harsh, easily causing DNA denaturation and destroying DNA chemical structure, making them unable to be used in the construction of gene-encoded compound libraries.

[0008] Therefore, developing a method for synthesizing synthetic oligonucleic acid-α-hydroxyphosphonic acid compounds with little DNA damage and high conversion rate is of great significance for enriching DELT and further screening and developing active drugs with phosphonate groups. Summary of the invention

[0009] The purpose of the present invention is to provide a method for synthesizing oligonucleic acid-alpha-hydroxyphosphonic acid compounds.

[0010] The present invention provides an oligonucleic acid-α-hydroxyphosphonic acid compound having a structure shown in Formula I:

[0011]

[0012] Among them, L 1 , L 2 is a connection unit;

[0013] R 1 , R 2 Each independently selected from C 1～12 Alkyl, phenyl or benzyl.

[0014] Furthermore, the above L 1 for:

[0015] and / or, the L 2 Substituted or unsubstituted: C 1～6 Alkylene, 3-8 membered hydrocarbon ring, 5-6 membered aryl or heteroaryl, 5-6 membered and 5-6 membered fused aryl or fused heteroaryl, C 2～6 Alkenylene or C 2～6 Alkyne;

[0016] Wherein, the substituted substituent is halogen, hydroxyl, C 1～5 Alkyl or haloalkyl or C 1～5 Alkoxy.

[0017] Furthermore, L 2 Substituted or unsubstituted: C 1～2 Alkylene, 3-membered cyclic hydrocarbon, 5-6-membered aryl or heteroaryl, or 5-membered and 6-membered fused heteroaryl;

[0018] wherein the substituted substituent is fluorine, bromine, hydroxyl, methyl, trifluoromethyl or methoxy;

[0019] Preferably, L 2 is substituted or unsubstituted: ethylene, cyclopropane, phenyl, pyridyl, furanyl, pyrrolyl, thienyl, pyrazolyl, indolyl or indazolyl.

[0020] Furthermore, R 1 , R 2 Each independently selected from C 1～5 Alkyl or benzyl, preferably, R 1 , R 2 Each is independently selected from methyl, ethyl or benzyl.

[0021] Furthermore, the above compound is one of the following compounds:

[0022]

[0023] The present invention also provides a method for synthesizing an oligonucleic acid-α-hydroxyphosphonic acid compound, which uses an oligonucleic acid-aldehyde compound shown in formula III and a phosphite compound shown in formula II as raw materials, reacts in a solvent under the action of a base to obtain an oligonucleic acid-α-hydroxyphosphonic acid compound shown in formula I; the reaction formula is as follows:

[0024]

[0025] Among them, L 1 , L 2 is a connection unit;

[0026] R 1 , R 2 Each independently selected from C 1～12 Alkyl, phenyl or benzyl.

[0027] Furthermore, the molar ratio of the oligonucleic acid-aldehyde compound represented by the above formula III, the phosphite compound represented by the formula II and the base is: 1:(1-500):(1-500), preferably 1:(50-250):(100-300);

[0028] and / or the reaction temperature is 0 to 90°C; preferably 4 to 50°C;

[0029] And / or the reaction time is 1 to 24 hours; preferably 2 to 10 hours;

[0030] and / or the base is a mixture of one or more of 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, potassium dihydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, triethylamine, n-butylamine, isobutylamine, 4-dimethylaminopyridine, pyridine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, N,N,N',N'-tetramethylethylenediamine, 1,1,3,3-tetramethylguanidine, N,N-dicyclohexylmethylamine, dicyclohexylamine, tetrahydropyrrole, alkaline inorganic salt buffer solution, and organic base buffer solution; preferably, the base is a mixture of one or more of alkaline inorganic salt buffer solution, potassium carbonate, and N,N-diisopropylethylamine;

[0031] 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 , 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, or a mixture of any one or more thereof; preferably, the reaction solvent is a mixture of water and acetonitrile.

[0032] Furthermore, the molar ratio of the oligonucleic acid-aldehyde compound represented by the above formula III, the phosphite compound represented by the above formula II and the base is 1:200:250;

[0033] and / or the reaction temperature is 25°C;

[0034] And / or the reaction time is 4 hours;

[0035] and / or the base is a boric acid buffer solution with a pH of 9.5;

[0036] And / or the solvent is a mixture of water and acetonitrile and the content of acetonitrile is not higher than 50% of the total volume.

[0037] Furthermore, L 1 for:

[0038] and / or, the L 2 Substituted or unsubstituted: C 1～6 Alkylene, 3-8 membered hydrocarbon ring, 5-6 membered aryl or heteroaryl, 5-6 membered and 5-6 membered fused aryl or fused heteroaryl, C 2～6 Alkenylene or C 2～6 Alkyne;

[0039] Wherein, the substituted substituent is halogen, hydroxyl, C 1～5 Alkyl or haloalkyl or C 1～5 Alkoxy.

[0040] Furthermore, L 2 Substituted or unsubstituted: C 1～2 Alkylene, 3-membered cyclic hydrocarbon, 5-6-membered aryl or heteroaryl, or 5-membered and 6-membered fused heteroaryl;

[0041] wherein the substituted substituent is fluorine, bromine, hydroxyl, methyl, trifluoromethyl or methoxy;

[0042] Preferably, L 2 is substituted or unsubstituted: ethylene, cyclopropane, phenyl, pyridyl, furanyl, pyrrolyl, thienyl, pyrazolyl, indolyl or indazolyl.

[0043] Furthermore, R 1 , R 2 Each independently selected from C 1～5 Alkyl or benzyl, preferably, R 1 , R 2 Each is independently selected from methyl, ethyl or benzyl.

[0044] Furthermore, the compound of formula I is one of the following compounds:

[0045]

[0046] The present invention also provides the use of the above-mentioned synthesis method in constructing a gene-encoded compound library.

[0047] Beneficial effects of the invention: The invention provides a method for synthesizing oligonucleic acid-α-hydroxyphosphonic acid compounds on DNA, and successfully obtains oligonucleic acid-α-hydroxyphosphonic acid compounds under the action of a specific base, at a specific temperature and for a specific time. The method has little damage to DNA, good universality, simple operation, mild conditions, and can obtain oligonucleic acid-α-hydroxyphosphonic acid compounds in high yield, providing an important basis for expanding and enriching the DELT compound library.

[0048] The "DNA" in the structural formula of the present invention is a single-stranded or double-stranded oligonucleotide chain.

[0049] The minimum and maximum values ​​of the carbon atom content in the hydrocarbon groups of the present invention are indicated by prefixes, for example, the prefix C a～b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. 1～6 The alkyl group refers to a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms.

[0050] The term "cycloalkyl" in the present invention refers to a saturated or unsaturated pure carbon ring (the atoms constituting the ring skeleton are all C) or a heterocycle (a heterocycle is a pure carbon ring in which some carbon atoms are replaced by heteroatoms such as O, N, S to form a stable ring structure (for example, a methylene group -CH 2 - is replaced by O to form a stable -O- structure, or replaced by N to form a stable -NH- structure; or replaced by S to form a stable -SO 2- etc.)) loses 1 or 2 hydrogen atoms to form a group.

[0051] The "condensed ring aromatic group" of the present invention refers to a group formed by a polycyclic aromatic compound losing 1 or 2 hydrogen atoms, wherein two rings in the polycyclic aromatic compound share two adjacent carbon atoms, such as naphthyl (i.e., a 6-membered aromatic ring and a 6-membered aromatic ring, or a benzene ring and a benzene ring), anthracenyl, and phenanthryl.

[0052] The "fused heteroaryl" of the present invention refers to a group formed by a polycyclic aromatic ring compound containing at least one heteroatom (O, N or S) losing 1 or 2 hydrogen atoms, wherein two rings in the aromatic ring compound share two adjacent carbon atoms or heteroatoms. For example, a 5-membered aromatic ring or heteroaromatic ring (e.g., furan, thiophene, pyrrole, pyridine ring) and a 6-membered aromatic ring or heteroaromatic ring (e.g., benzene, pyridine, pyridazine, pyrimidine, pyrazine ring), such as indole, or a 6-membered aromatic ring or heteroaromatic ring and a 6-membered aromatic ring or heteroaromatic ring, such as quinoline; or more than two rings, such as acridine.

[0053] The "organic solvent" of the present invention includes, but is 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, 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, 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.

[0054] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0055] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 For oligonucleotide-NHFmoc raw material (S 2 ) of the liquid chromatography-mass spectrum.

[0057] Figure 2 Oligonucleotide-NH 2 Raw materials (S 3 ) of the liquid chromatography-mass spectrum.

[0058] Figure 3 Oligonucleotide-NH 2 Raw materials (S 4 ) of the liquid chromatography-mass spectrum.

[0059] Figure 4 For oligonucleic acid-aldehyde compound raw material S 5-1 Liquid chromatography mass spectrum.

[0060] Figure 5 For each oligonucleic acid-aldehyde compound (S 5-1 ~S 5-21 ) structure diagram.

[0061] Figure 6 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-2 Liquid chromatography mass spectrum.

[0062] Figure 7 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-3 Liquid chromatography mass spectrum.

[0063] Figure 8 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-4 Liquid chromatography mass spectrum.

[0064] Fig. 9 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-5 Liquid chromatography mass spectrum.

[0065] Fig.10 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-6 Liquid chromatography mass spectrum.

[0066] Fig.11 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-7 Liquid chromatography mass spectrum.

[0067] Fig.12 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-8 Liquid chromatography mass spectrum.

[0068] Fig.13 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-9 Liquid chromatography mass spectrum.

[0069] Fig.14 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-10 Liquid chromatography mass spectrum.

[0070] Fig.15 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-11 Liquid chromatography mass spectrum.

[0071] Fig.16 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-12 Liquid chromatography mass spectrum.

[0072] Fig.17 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-13 Liquid chromatography mass spectrum.

[0073] Fig.18 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-14 Liquid chromatography mass spectrum.

[0074] Fig.19 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-15 Liquid chromatography mass spectrum.

[0075] Fig. 20 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-16 Liquid chromatography mass spectrum.

[0076] Fig.21 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-17 Liquid chromatography mass spectrum.

[0077] Fig. 22 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-18 Liquid chromatography mass spectrum.

[0078] Fig.23 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-19 Liquid chromatography mass spectrum.

[0079] Fig.24 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-20 Liquid chromatography mass spectrum.

[0080] Fig.25 The oligonucleic acid-α-hydroxyphosphonic acid compound S of the present invention 5-21 Liquid chromatography mass spectrum.

[0081] Fig.26The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 1 Liquid chromatography mass spectrum.

[0082] Fig. 27 The oligonucleic acid-α-hydroxyphosphonic acid compound (P 1 ~P 24 ) structure diagram.

[0083] Fig.28 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 2 Liquid chromatography mass spectrum.

[0084] Fig.29 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 3 Liquid chromatography mass spectrum.

[0085] Fig.30 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 4 Liquid chromatography mass spectrum.

[0086] Fig.31 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 5 Liquid chromatography mass spectrum.

[0087] Fig.32 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 6 Liquid chromatography mass spectrum.

[0088] Fig.33 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 7 Liquid chromatography mass spectrum.

[0089] Fig.34 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 8 Liquid chromatography mass spectrum.

[0090] Fig.35 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 9 Liquid chromatography mass spectrum.

[0091] Fig.36 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 10 Liquid chromatography mass spectrum.

[0092] Fig.37 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 11 Liquid chromatography mass spectrum.

[0093] Fig.38The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 12 Liquid chromatography mass spectrum.

[0094] Fig.39 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 13 Liquid chromatography mass spectrum.

[0095] Fig.40 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 14 Liquid chromatography mass spectrum.

[0096] Fig.41 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 15 Liquid chromatography mass spectrum.

[0097] Fig.42 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 16 Liquid chromatography mass spectrum.

[0098] Fig.43 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 17 Liquid chromatography mass spectrum.

[0099] Fig.44 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 18 Liquid chromatography mass spectrum.

[0100] Fig.45 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 19 Liquid chromatography mass spectrum.

[0101] Fig.46 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 20 Liquid chromatography mass spectrum.

[0102] Fig.47 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 21 Liquid chromatography mass spectrum.

[0103] Fig.48 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 22 Liquid chromatography mass spectrum.

[0104] Fig.49 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention 23 Liquid chromatography mass spectrum.

[0105] Fig.50 The oligonucleic acid-α-hydroxyphosphonic acid compound P of the present invention6 The product after connecting with TagA is TagA-P 6 Liquid chromatography mass spectrum. DETAILED DESCRIPTION

[0106] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products. All "equivalents" in the present specification refer to molar equivalents.

[0107] 1. Oligonucleotide-NH 2 Raw materials (S 3 )

[0108] 1.1 According to the following reaction formula, the oligonucleotide-NHFmoc raw material (S 2 ):

[0109]

[0110] 100 nanomoles HP(S 1 , the structure of HP is as follows Figure 1 40 equivalents of the starting head fragment compound (commercial product) in DMSO (20 μl, 200 mmol / l), 250 equivalents of sodium tetraborate (Na4PO4) at pH = 9.5 were added. 2 B 4 O 7 ) buffer (100 μL, 250 mmol / L), 40 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (20 μL, 200 mmol / L) were mixed, and the mixture was thoroughly mixed with a vortex oscillator. Then the above mixture was added to the HP solution, mixed evenly and reacted at 4°C for 1 hour. After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction solution was placed in a -80°C refrigerator and frozen for 2 hours. After that, centrifuged at 4000 rpm for half an hour, and the supernatant was poured off. The remaining precipitate was dissolved in deionized water to obtain a solution of DNA-NHFmoc. DNA-NHFmoc (S 2 ) is shown in the spectrum Figure 1 As shown, its molecular weight is 5406.

[0111] 1.2 Synthesize oligonucleotide-NH according to the following reaction formula 2 Raw materials (S 3 ):

[0112]

[0113] 100 nanomoles of DNA-NHFmoc(S 2 ) was dissolved in deionized water to prepare 1 mmol / L (100 μL, 1 equivalent), 56 μL of 10% piperidine aqueous solution was added thereto, the two were mixed evenly and reacted at room temperature for 1 hour. After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction was placed in a refrigerator at -80°C for 2 hours. After that, centrifuged at 4000 rpm for half an hour, and the supernatant was poured off. The remaining precipitate was dissolved in deionized water to obtain oligonucleic acid-NH 2 (S 3 , abbreviated as DNA-NH 2 ) solution. Detection of DNA-NH by liquid chromatography-mass spectrometry 2 (S 3 ) is shown in the spectrum Figure 2 As shown, its molecular weight is 5184.

[0114] 2. Oligonucleic acid-COOH raw material (S 4 )

[0115] 2.1 According to the following reaction formula, the oligonucleotide-COOH raw material (S 4 ):

[0116]

[0117] 100 nanomoles of HP (S1, HP has the structure Figure 1 40 equivalents of the starting head fragment compound (commercial product) in DMSO solution (concentration: 200 mmol / L), 250 equivalents of sodium tetraborate (Na4PO4) with a pH of 9.5 were added. 2 B 4 O 7) buffer (concentration: 250 mmol / L), 40 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200 mmol / L) are mixed, and the mixture is thoroughly mixed with a vortex oscillator. Then the above mixture is added to the HP solution, mixed evenly and reacted at 4°C for 1 hour. After the reaction is completed, 10% of the total volume of 5 mol / L sodium chloride solution is added to the reaction solution. Then, 3 times the total volume of anhydrous ethanol is added, and after oscillation, the reaction solution is placed in a -80°C refrigerator and frozen for 2 hours. After that, centrifuge at 4000rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain a solution of DNA-COOH. DNA-COOH (S 4 ) is shown in the spectrum Figure 3 As shown, its molecular weight is 5241.

[0118] 3. Oligonucleic acid-aldehyde compound raw material (S 5-1 )

[0119]

[0120] 10 nanomoles of DNA-COOH(S 4 ) was dissolved in deionized water to prepare 1 mmol / L (10 μL, 1 equivalent), and 160 μL of DMSO, 100 equivalents of aminoacetaldehyde in DMSO (concentration: 200 mmol / L, 10 μL), 80 equivalents of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, concentration: 250 mmol / L, 10 μL) and 200 equivalents of N,N-diisopropylethylamine (DIEA, concentration: 250 mmol / L, 10 μL) were added, and the mixture was fully mixed with a vortex oscillator and reacted at 25°C for 2 hours. After the reaction was completed, 10% of the total volume of 5 mol / L sodium chloride solution was added to the reaction solution. Then, 3 times the total volume of anhydrous ethanol was added, and after oscillation, the reaction solution was placed in a refrigerator at -80°C for 2 hours. Afterwards, the mixture was centrifuged at 4000 rpm for half an hour, and the supernatant was discarded. The remaining precipitate was dissolved in deionized water to obtain the oligonucleic acid-aldehyde compound raw material (S 5-1 ) solution. The oligonucleic acid-aldehyde compound raw material (S 5-1 ) is shown in the spectrum Figure 4 As shown, its molecular weight is 5282 and the yield is 86%.

[0121] 4. Oligonucleic acid-aldehyde compound raw material (S 5-2 )

[0122]

[0123] 10 nmol DNA-NH 2 (S 3 ) was dissolved in deionized water to prepare 1 mmol / L (10 μL, 1 equivalent), and 200 equivalents of 2-formyl-1-cyclopropanecarboxylic acid ethyl ester (commercial product) in DMSO (concentration: 200 mmol / L), 250 equivalents of sodium tetraborate (Na 2 B 4 O 7 ) buffer (concentration: 250 mmol / L), 150 equivalents of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) aqueous solution (concentration: 200 mmol / L), mix well and react at 25°C for 12 hours. After the reaction is completed, add 10% of the total volume of 5 mol / L sodium chloride solution to the reaction solution. Then, continue to add 3 times the total volume of anhydrous ethanol, shake evenly, and freeze the reaction solution in a -80°C refrigerator for 2 hours. After that, centrifuge at 4000rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water to obtain the oligonucleotide-aldehyde compound raw material (S 5-2 ) solution. The oligonucleic acid-aldehyde compound raw material (S 5-2 ) is shown in the spectrum Figure 4 As shown, its molecular weight is 5279 and the yield is 86%. 5-1 -S 5-21 , the structure is as follows Figure 5 As shown in the liquid chromatography mass spectrum Figure 6-Figure 25 shown).

[0124] Raw material HP(S 1 ) is represented by:

[0125]

[0126] However, it should be noted that the method of the present invention is not limited to the above-mentioned specific S 1 The structural DNA chain can be a single-stranded or double-stranded deoxyribonucleotide chain obtained by polymerization of other artificially modified or unmodified deoxyribonucleotide monomers, and the length of the chain is not limited.

[0127] Example 1: Using oligonucleic acid-aldehyde compound raw material (S 5-1 )Synthetic oligonucleic acid-α-hydroxyphosphonic acid compounds (P 1 )

[0128]

[0129] In 1 nanomole oligonucleic acid-aldehyde compound (S 5-1 , 1 mmol / L, 1 equivalent, 1 μl) was added to an aqueous solution of 250 equivalents of boric acid (Na 2 B 4 O 7 ) buffer (1 μl, 250 mmol / l) and diethyl phosphite (commercially available, 200 mmol / l acetonitrile solution, 200 equivalents, 1 μl). The mixture was thoroughly mixed by vortexing and reacted at 25°C for 4 hours. After the reaction was completed, 10% of the total volume of 5 mol / l sodium chloride solution and 3 times of anhydrous ethanol were added to the reaction solution. After oscillation, it was placed in a -80°C refrigerator for 2 hours, and then high-speed refrigerated centrifugation (4°C, 4000 rpm, 15 minutes), the supernatant was poured off, and the remaining precipitate was the product oligonucleic acid-α-hydroxyphosphonic acid compound (P 1 ), detected by liquid chromatography-mass spectrometry, the test results are shown in Fig.26 , molecular weight is 5421, and conversion rate is 73%.

[0130] Referring to the above method, other representative oligonucleic acid-α-hydroxyphosphonic acid compounds (P 2 -P 23 ), structure see Fig. 27 The corresponding LCMS test results are shown in Figure 28-Figure 49 .

[0131] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0132] Experimental Example 1: Screening of Synthesis Conditions of Oligonucleic Acid-α-Hydroxyphosphonic Acid Compounds of the Present Invention

[0133] Referring to the synthetic oligonucleic acid-α-hydroxyphosphonic acid compound P of Example 1 12 The method is different only in that the following parameters are controlled according to Table 1: base, diethyl phosphite equivalent, co-solvent with water, and reaction time.

[0134]

[0135] Calculation of oligonucleic acid-α-hydroxyphosphonic acid compounds P obtained under different parameters 12 The results are shown in Table 1. According to the experimental results, under the reaction conditions of No. 5, the obtained product P 12 The yield is the highest.

[0136] Table 1 Synthesis of oligonucleic acid-α-hydroxyphosphonic acid compounds P under different conditions 12 Conversion rate

[0137]

[0138] Experimental Example 2: Integrity verification experiment of oligonucleotides in oligonucleotide-α-hydroxyphosphonic acid compounds of the present invention

[0139]

[0140] Oligonucleic acid-α-hydroxyphosphonic acid compounds P 6 Linked with Oligo 1 (short-chain oligonucleotide, the molecular weights of the two chains are 4064 and 5884 respectively): 1 nanomole P 6 Dissolve in deionized water to prepare a 1 mmol / L solution (1 μl, 1 equivalent), add 1.2 equivalents of Tag A (1 mmol / L aqueous solution, 1.2 μl), 1 μl of 10×T4 DNA ligation buffer solution and 0.5 μl of T4 DNA ligase. Then mix the above solutions evenly and react at room temperature for 1 hour. After the reaction is completed, add 10% of the total volume of 5 mol / L sodium chloride solution to the reaction solution, and then continue to add 3 times the total volume of anhydrous ethanol. After shaking evenly, place the reaction in a -80°C refrigerator and freeze for 2 hours. Then centrifuge at 4000 rpm for half an hour and pour off the supernatant. The remaining precipitate is dissolved in deionized water and confirmed by liquid chromatography-mass spectrometry to obtain the product Tag AP 6 The molecular weight is 15384, and the mass spectrometry results are shown in Fig.50 .

[0141] LCMS analysis results showed that oligonucleic acid-α-hydroxyphosphonic acid compounds P 6 It can be successfully coupled with Tag A. This shows that the DNA chain integrity of the oligonucleic acid-α-hydroxyphosphonic acid compound obtained according to the synthesis method described in this application is good. The molecular weight can be accurately displayed by LCMS mass spectrometry, further confirming that the reaction method of the present invention does not cause substantial damage to the basic structure and activity of DNA.

[0142] In summary, the present invention provides a method for synthesizing oligonucleic acid-α-hydroxyphosphonic acid compounds on DNA, and the oligonucleic acid-α-hydroxyphosphonic acid compounds are successfully prepared by reacting in a system of specific base, raw material equivalent and specific solvent for a specific time. The method has little damage to DNA, good universality, simple operation, mild conditions, and can prepare oligonucleic acid-α-hydroxyphosphonic acid compounds with high conversion rate, providing an important basis for expanding and enriching the DELT compound library.

Claims

1. A method for synthesizing an oligonucleic acid-α-hydroxyphosphonic acid compound, characterized in that: It uses the oligonucleic acid-aldehyde compound shown in formula III and the phosphorous acid compound shown in formula II as raw materials, reacts in a solvent under the action of a base to obtain the oligonucleic acid-α-hydroxyphosphonic acid compound shown in formula I; the reaction formula is as follows: Among them, L1 and L2 are connection units; R1, R2 are independently selected from C 1～12 Alkyl, phenyl or benzyl; The molar ratio of the oligonucleic acid-aldehyde compound represented by formula III, the phosphite compound represented by formula II and the base is: 1:(1-500):(1-500); The reaction temperature is 0 to 90°C; The reaction time is 1 to 24 hours; The alkali is an alkaline inorganic salt buffer solution.

2. The synthesis method according to claim 1, characterized in that The solvent is water, methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, amyl alcohol, 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 di Methyl ether, ethylene glycol diethyl ether, 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, xylene, any one or a mixture of several thereof.

3. The synthesis method according to claim 2, characterized in that The molar ratio of the oligonucleic acid-aldehyde compound represented by formula III, the phosphite compound represented by formula II and the base is 1:(50-250):(100-300); and / or the reaction temperature is 4 to 50°C; And / or the reaction time is 2 to 10 hours; And / or the solvent is a mixture of water and acetonitrile.

4. The synthesis method according to claim 3, characterized in that The molar ratio of the oligonucleic acid-aldehyde compound represented by formula III, the phosphite compound represented by formula II and the base is 1:200:250; and / or the reaction temperature is 25°C; And / or the reaction time is 4 hours; and / or the base is a boric acid buffer solution with a pH of 9.5; And / or the solvent is a mixture of water and acetonitrile and the content of acetonitrile is not higher than 50% of the total volume.

5. The synthesis method according to claim 1, characterized in that L1 is: And / or, said L2 is substituted or unsubstituted: C 1～6 Alkylene, 3-8 membered hydrocarbon ring, 5-6 membered aryl or heteroaryl, 5-6 membered and 5-6 membered fused aryl or fused heteroaryl, C 2～6 Alkenylene or C 2～6 Alkynylidene; Wherein, the substituted substituent is halogen, hydroxyl, C 1～5 Alkyl or haloalkyl or C 1～5 Alkoxy.

6. The synthesis method according to claim 5, characterized in that L2 is substituted or unsubstituted: C 1～2 Alkylene, 3-membered cyclic hydrocarbon, 5-6-membered aryl or heteroaryl, or 5-membered and 6-membered fused heteroaryl; Wherein, the substituted substituent is fluorine, bromine, hydroxyl, methyl, trifluoromethyl or methoxy.

7. The synthesis method according to claim 6, characterized in that L2 is substituted or unsubstituted: ethylene, cyclopropane, phenyl, pyridyl, furanyl, pyrrolyl, thienyl, pyrazolyl, indolyl or indazolyl.

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

9. The synthesis method according to claim 8, characterized in that R1 and R2 are independently selected from methyl, ethyl or benzyl.

10. The synthesis method according to any one of claims 1 to 9, characterized in that: The compound of formula I is one of the following compounds:

11. Use of the synthesis method according to any one of claims 1 to 10 in constructing a library of gene-encoded compounds.

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