A method for the synthesis of DNA-encoded compounds furanose derivatives

Abstract

The present application relates to a kind of DNA encoding compound furanose derivative synthesis method, the reaction method is with On-DNA aryl ethylene compound and 2-carboxylic acid furanose compound as raw material, in alkaline environment by photocatalytic decarboxylation free radical addition reaction obtains On-DNA furanose derivative, the reaction can be carried out in the mixed aqueous phase of organic solvent / aqueous phase, simple operation, environment-friendly, suitable for the synthesis of DNA encoding compound library using multi-well plate.

Description

Technical Field

[0001] This invention belongs to the field of encoded compound library technology, specifically relating to a method for synthesizing On-DNA furanose derivatives in the construction of DNA encoded compound libraries. Background Technology

[0002] In drug development, especially in the development of new drugs, high-throughput screening targeting biological targets is one of the main methods for rapidly obtaining lead compounds. However, traditional high-throughput screening based on single molecules is time-consuming, requires huge equipment investment, and has a limited number of compounds (millions). Furthermore, the construction of compound libraries requires decades of accumulation, which limits the efficiency and possibility of lead compound discovery. In recent years, DNA-encoded compound library technology (WO2005058479, WO2018166532, CN103882532) has emerged. This technology combines combinatorial chemistry and molecular biology techniques, adding a DNA tag to each compound at the molecular level. It can synthesize compound libraries of up to hundreds of millions in a very short time, becoming the trend of next-generation compound library screening technology. It has begun to be widely used in the pharmaceutical industry, producing many positive effects (Accounts of Chemical Research, 2014, 47, 1247-1255).

[0003] DNA-encoded compound libraries enable the rapid generation of giant compound libraries through combinatorial chemistry, and allow for high-throughput screening of lead compounds, making lead compound screening faster and more efficient than ever before. One of the challenges in constructing DNA-encoded compound libraries is the need for high-yield synthesis of chemically diverse small molecules on DNA. Since DNA requires specific conditions (solvent, pH, temperature, ion concentration) to maintain stability, and the On-DNA reactions used in the construction of DNA-encoded compound libraries also need to achieve high yields, the types of reagents, reaction types, and reaction conditions of chemical reactions performed on DNA (referred to as On-DNA reactions) directly affect the richness and selectivity of DNA-encoded compound libraries. Therefore, developing DNA-compatible chemical reactions has become a long-term exploration and research direction for DNA-encoded compound library technology, directly impacting the application and commercial value of DNA-encoded compound libraries.

[0004] Furanose derivatives represent an important class of drug compound skeletal structures; however, no method has been reported for synthesizing On-DNA furanose derivatives from On-DNA alkenyl compounds. Therefore, this study aims to develop a novel synthetic method for On-DNA furanose derivatives suitable for high-volume multi-well plate operations. This method utilizes photocatalysis to introduce furanose derivative structures, thereby increasing the diversity of DNA-encoded compound libraries and further enhancing the application value of DNA-encoded compound library technology. Summary of the Invention

[0005] This invention provides a method for synthesizing DNA-encoded compound libraries that features stable raw material storage, mild reaction conditions, good substrate versatility, minimal DNA damage, and is suitable for batch processing using multi-well plates. It can rapidly convert DNA-encoded arylethylene compound libraries into On-DNA furanose derivative compound libraries in a single reaction.

[0006] This invention provides a method for synthesizing DNA-encoded furanose derivatives. The method uses On-DNA arylethylene compounds and 2-carboxylic acid furanose compounds as raw materials, and obtains On-DNA furanose derivatives through photocatalytic decarboxylation radical addition reaction under alkaline conditions.

[0007] Among them, the structure of the On-DNA arylethylene compound is as follows: The structural formula of 2-carboxylic acid furan ribose compounds is:

[0008] The DNA in the structural formula contains a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which is linked to R1 or alkenyl groups in the compound by one or more chemical bonds or groups.

[0009] The length of the DNA is 10–200;

[0010] In this structural formula, the DNA and R1 or alkenyl groups are connected by one or more chemical bonds. A single chemical bond means the DNA and R1 or alkenyl groups are directly connected; multiple chemical bonds mean the DNA and R1 or alkenyl groups are connected by several chemical bonds. For example, the DNA and R1 or alkenyl groups are connected by a methylene group (-CH2-) to the amino group of the DNA, i.e., connected by two chemical bonds; or the DNA and R1 or alkenyl groups are connected by a carbonyl group (-CO-) to the amino group of the DNA, also connected by two chemical bonds; or the DNA and R1 or alkenyl groups are connected by a methylene carbonyl group (-CH2CO-) to the amino group of the DNA, also connected by three consecutive chemical bonds.

[0011] Preferably, the amino group of DNA is linked to R1 or alkenyl group via a carbonyl group (-CO-).

[0012] R1 is selected from groups with a molecular weight of less than 1000 that are directly attached to DNA and alkenyl carbon atoms;

[0013] R2 is selected from groups with a molecular weight of less than 1000 that are directly attached to an alkenyl carbon atom;

[0014] R3 is selected from groups with a molecular weight of less than 1000 that are directly bonded to an oxygen atom.

[0015] Preferably, R1 and R2 are each selected from 5- to 10-membered aryl or heteroaryl groups, and the number of substituents in R1 and R2 is one or more; the substituents in R1 and R2 are independently selected from one or more of hydrogen, halogen, nitro, cyano, alkyl, alkoxy, and halogen alkyl.

[0016] R3 is selected from benzoyl, benzyl, acetyl, and alkyl groups; wherein the alkyl group is C1 to C2. 10 alkyl.

[0017] Further: R1 and R2 are selected from the following groups:

[0018] X is any one of O, S, and NH, and the substituents of R1 and R2 are independently selected from one or more of hydrogen, trifluoromethyl, methyl, ethyl, and methoxy.

[0019] More specifically: the On-DNA arylethylene compounds are selected from, but not limited to:

[0020] As a preferred option, the 2-carboxylic acid furanose compounds are selected from:

[0021] A method for synthesizing a DNA-encoded compound, a furanose derivative, includes the following steps: adding 10 to 1000 molar equivalents of a 2-carboxylic acid furanose compound and 10 to 1000 molar equivalents of a base to a solution of an On-DNA arylethylene compound with a molar equivalent of 1 and a molar concentration of 0.1-5 mM, and finally adding 1 to 10 molar equivalents of a catalyst, and reacting under light at 10°C to 100°C for 0.5 to 16 hours.

[0022] Further, the base is selected from one or more of sodium borate, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium phosphate, potassium phosphate, sodium hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate, N-methylmorpholine, triethylamine, diisopropylethylamine, DBU (1,8-diazabicycloundec-7-ene), 4-dimethylaminopyridine, 2,6-dimethylpyridine, or N-methylimidazole. Preferably, the base is dipotassium hydrogen phosphate.

[0023] Preferably, the reaction is carried out in a solvent, which is any one or a mixture of several aqueous solvents selected from water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer. Preferably, the reaction solvent contains water and dimethyl sulfoxide.

[0024] Furthermore, the pH of the reaction solvent is 5-11; preferably, the pH is 9.

[0025] Further, the catalyst for the reaction is selected from Ir[dF(CF3)ppy]2(dtbbpy)PF6, ([Ir(dtbbpy)(ppy)2][PF6]), Ir[pF(Me)ppy]2(dtbbpy)PF6, Ir(ppy)2(bpy)PF6 or 4-CzIPN; preferably, the catalyst is Ir[dF(CF3)ppy]2(dtbbpy)PF6.

[0026] Furthermore, the reaction needs to be carried out under a nitrogen atmosphere.

[0027] Further, the light output voltage of the reaction is 0 volts, 7.5 volts, or 13.8 volts; preferably, the light output voltage is 13.8 volts. Preferably, the wavelength of the light is 470 nm; preferably, the distance between the light source and the reactor is 0.5 cm.

[0028] Furthermore, the reaction time is 0.5 to 16 hours; preferably, the reaction time is 2 hours.

[0029] Further, in the method, the molar equivalent of the On-DNA arylethylene compound is 1, the molar equivalent of the 2-carboxylic acid furan ribose compound is 10-1000, the molar equivalent of the base is 10-1000, and the molar equivalent of the catalyst is 1-10; preferably, the molar equivalent of the 2-carboxylic acid furan ribose compound is 50, 100, 200, 300, 400, 500, 600, 800, or 1. The molar equivalents of the base are 50, 100, 120, 200, 300, 400, 500, 600, 800, and 1000 equivalents, and the molar equivalents of the catalyst are 1, 4, 5, and 10 equivalents; most preferably, the molar equivalent of the 2-carboxylic acid furan ribose compound is 400 equivalents, the molar equivalent of the base is 120 equivalents, and the molar equivalent of the catalyst is 4 equivalents.

[0030] Furthermore, the above method is used for batch operations of multi-hole plates.

[0031] Furthermore, the above method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

[0032] This invention provides a method for obtaining On-DNA furanose derivatives from On-DNA alkenyl compounds within a DNA-encoded compound library. This method has broad applicability to various On-DNA alkenyl compounds and allows for the large-scale introduction of 2-carboxylic acid furanose compounds as synthetic modules. The method offers high yields, produces single products, can be carried out in a mixed aqueous phase using organic solvents and water, is simple to operate, environmentally friendly, and suitable for the synthesis of DNA-encoded compound libraries using multi-well plates.

[0033] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0034] "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0035] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes, for example, the prefix Ca~ b Alkyl groups indicate any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, C 1～12 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 12 carbon atoms.

[0036] Alkyl refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-. The alkyl group can also be part of other groups, such as C1-C6 alkoxy groups or C1-C6 alkylamino groups.

[0037] The halogen is fluorine, chlorine, bromine or iodine.

[0038] Alkoxy refers to an alkyl group that is attached to an oxygen atom to form a substituent, such as methoxy, which is -OCH3.

[0039] Aryl / aromatic ring refers to an aromatic single or multiple cyclic group composed of carbon atoms and without heteroatoms.

[0040] Aromatic heterocyclic groups refer to single or multiple cyclic groups composed of multiple C, O, S, N atoms that have aromatic properties.

[0041] 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.

[0042] 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

[0043] Figure 1 The conversion rate distribution of 15 On-DNA furanose derivatives obtained in Example 1 of this invention is shown in the figure. Detailed Implementation

[0044] The following detailed embodiments further illustrate the above-described content of the present invention, but should not be construed as limiting the scope of the subject matter 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.

[0045] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0046] In this invention, DNA-NH2 refers to a DNA structure with a -NH2 linker formed by single-stranded or double-stranded DNA and a linker group, such as the DNA-NH2 structure of "compound 1" in WO2005058479. Other examples include the following DNA structures:

[0047]

[0048] Where A is adenine, T is thymine, C is cytosine, and G is guanine.

[0049] DIPEA: N,N-diisopropylethylamine; DIC: N,N'-diisopropylcarbodiimide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DMA: dimethylacetamide; DMSO: dimethyl sulfoxide; DDTC: sodium diethyldithiocarbamate.

[0050] Example 1: Method for synthesizing on-DNA furanose ribose

[0051] Step 1: Synthesis of On-DNA alkenyl compounds

[0052]

[0053]

[0054] (1) Dissolve solution 1 in 250 mM borate buffer (pH 9.4) to prepare a 1 mM solution. Then, sequentially mix 50 equivalents of HATU (0.4 M, dissolved in DMA), 50 equivalents of aromatic carboxylic acid reagent (0.4 M, dissolved in DMA), and 50 equivalents of DIPEA (0.4 M, dissolved in DMA) at 0°C. Vortex the mixture thoroughly and then store it at 0°C for 5 minutes. Add the mixture to solution 1, mix well, and react at room temperature for 0.5–1 hour. Ar represents different aromatic rings.

[0055] After the reaction was complete, ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride solution was added to the solution after the reaction, followed by 3 times the total volume of anhydrous ethanol. After shaking and mixing, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at 12,000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain the solution of compound 2. After quantification by OD using an ELISA reader, the reaction conversion rate was confirmed to be 60%–90% by LCMS.

[0056] (2) Dissolve solution 1 in 250 mM borate buffer (pH 9.4) to prepare a 1 mM solution. Then, sequentially mix 50 equivalents of N-hydroxysuccinimide (NHS) (0.4 M, dissolved in DMA), 40 equivalents of 2-bromoacrylic acid (0.2 M, dissolved in DMA), and 20 equivalents of N,N'-diisopropylcarbodiimide (DIC) (0.4 M, dissolved in DMA) at 0°C. Vortex the mixture thoroughly and then store it at 0°C for 5 minutes. Divide the mixture into two portions. Add one portion to solution 1, mix thoroughly, and react at 0°C for 5 minutes. Then add the remaining mixture to the solution, mix thoroughly, and react at 0°C for 5 minutes. Finally, allow the reaction mixture to react at room temperature for 5 minutes.

[0057] After the reaction was complete, ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride solution was added to the solution after the reaction, followed by 3 times the total volume of anhydrous ethanol. After shaking and mixing, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at 12000 rpm for half an hour, the supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain the solution of compound 4. After quantification by OD of the enzyme-linked immunosorbent assay (ELISA) reader, the reaction conversion rate was confirmed to be 56% by LCMS.

[0058] (3) Compound 2 was dissolved in water to prepare a 1 mM solution. Then, 10 equivalents of vinylboronic acid pinacol ester (0.2 M, dissolved in DMSO), 100 equivalents of cesium hydroxide (0.5 M, dissolved in water), and 2.5 equivalents of chlorine (sodium-2-dicyclohexylphosphine-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (sSPhos Pd G2) (0.01 M, dissolved in DMSO) were added sequentially. The mixture was thoroughly mixed and reacted at 100°C for 30 minutes. After the reaction was complete, 100 equivalents of sodium diethyldithiocarbamate (DDTC) (0.4 M, dissolved in water) were added to the reaction system, and the mixture was thoroughly mixed and reacted at 100°C for 10 minutes.

[0059] After the reaction was complete, the supernatant was collected by centrifugation and then ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride solution was added to the solution after the reaction, followed by 3 times the total volume of anhydrous ethanol. After shaking and mixing, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at 12,000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a solution of On-DNA arylethylene compound 3. After quantification by OD using an ELISA reader, the reaction conversion rate was confirmed to be 70%–90% by LCMS.

[0060] (4) Compound 4 was dissolved in water to prepare a 1 mM solution. Then, 100 equivalents of arylboronic acid compound (0.2 M, dissolved in DMSO), 100 equivalents of cesium hydroxide (0.5 M, dissolved in water), and 2.5 equivalents of chlorine (sodium-2-dicyclohexylphosphine-2',6'-dimethoxy-1,1'-biphenyl-3'-sulfonate)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (sSPhos Pd G2) (0.01 M, dissolved in DMSO) were added sequentially. The mixture was thoroughly mixed and reacted at 90°C for 2 hours. After the reaction was complete, 100 equivalents of DDTC (0.4 M, dissolved in water) were added to the reaction system, mixed thoroughly, and reacted at 90°C for 10 minutes. R 1 They are different aromatic rings.

[0061] After the reaction was complete, the supernatant was collected by centrifugation and then ethanol precipitation was performed: 10% of the total volume of 5M sodium chloride solution was added to the solution after the reaction, followed by 3 times the total volume of anhydrous ethanol. After shaking and mixing, the reaction was placed in dry ice and frozen for 0.5 hours. Then, it was centrifuged at 12,000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a solution of On-DNA arylethylene compound 5. After quantification by OD using an ELISA reader, the reaction conversion rate was confirmed to be 70%–90% by LCMS.

[0062] Step 2: Visible light-catalyzed reaction of 2-carboxylic acid ribofuranosyl furanose with On-DNA olefins

[0063] Dissolve On-DNA arylethylene compound 3 or 5 in water to prepare a 1 mM solution. Then, add 400 equivalents of 2-carboxylic acid furanose compound (0.5 M, dissolved in DMSO), 120 equivalents of dipotassium hydrogen phosphate (0.3 M, dissolved in water), and 4 equivalents of catalyst Ir[dF(CF3)ppy]2(dtbbpy)PF6 (0.01 M, dissolved in DMSO). Add DMSO to the reaction solution to make the organic phase:water phase volume ratio of the reaction system 3:2. Mix thoroughly, remove air for 2 hours, and replace with nitrogen for 2 hours. Set the light output voltage of the light irradiation device to 13.8 volts, wavelength to 470 nm, and distance between the light source and the reactor to 0.5 cm. Irradiate for 2 hours.

[0064] After the reaction was complete, ethanol precipitation was performed: 10% (total volume) of 5M sodium chloride solution was added to the reaction solution, followed by 3 times the total volume of anhydrous ethanol. After shaking and mixing, the reaction mixture was placed in dry ice for 0.5 hours, then centrifuged at 12000 rpm for half an hour. The supernatant was discarded, and the remaining precipitate was dissolved in deionized water to obtain a solution of 15 On-DNA products. The conversion rate was quantified by OD using a microplate reader and then sent to LCMS to confirm the conversion efficiency. The conversion rates are shown in the attached figure. Figure 1 As shown.

[0065] In summary, this invention, by controlling the solvent, temperature, pH, and other conditions during the reaction, and in the presence of a base, utilizes visible light catalysis to yield on-DNA furanose derivatives from on-DNA alkenyl compounds and 2-carboxylic acid furanose compounds. This method has a wide substrate applicability, can be carried out in a mixed aqueous phase of organic solvent / aqueous phase, is simple to operate, environmentally friendly, and suitable for the synthesis of DNA-encoded compound libraries using multi-well plates.

Claims

1. A method for synthesizing a DNA-encoded compound, a furanose ribose derivative, characterized in that: This method uses On-DNA arylethylene compounds and 2-carboxylic acid furanose compounds as raw materials to obtain On-DNA furanose derivatives through photocatalytic decarboxylation radical addition reaction under alkaline conditions. The On-DNA arylethylene compound has the following structure: or The structural formula of 2-carboxylic acid furan ribose compounds is: ; The DNA in the structural formula contains a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which is linked to R1 or alkenyl groups in the compound by one or more chemical bonds or groups. R1 is selected from 5- to 10-membered aryl or heteroaryl groups; R2 is selected from 5- to 10-membered aryl or heteroaryl groups; R3 is selected from benzyl; The number of substituents in R1 and R2 is one or more; the substituents in R1 and R2 are independently selected from one or more of hydrogen, halogen, nitro, cyano, alkyl, alkoxy, and halogen alkyl. The base is selected from dipotassium hydrogen phosphate; The catalyst was selected from Ir[dF(CF3)ppy]2(dtbbpy)PF6; The output voltage of the reaction under light is 13.8 volts.

2. The method according to claim 1, characterized in that: R1 and R2 are respectively selected from , , , , , , , , or X is any one of O, S, and NH, and the substituents of R1 and R2 are independently selected from one or more of hydrogen, trifluoromethyl, methyl, ethyl, and methoxy.

3. The method according to claim 1, characterized in that: The method includes the following steps: adding 10 to 1000 molar equivalents of a 2-carboxylic acid furan ribose compound and 10 to 1000 molar equivalents of a base to an On-DNA arylethylene compound solution with a molar equivalent of 1 and a molar concentration of 0.1-5 mM, and finally adding 1 to 10 molar equivalents of a catalyst, and reacting under light at 10℃ to 100℃ for 0.5 to 16 hours.

4. The method according to claim 1, characterized in that: The reaction is carried out in a solvent, which is any one or a mixture of several of the following aqueous solvents: water, methanol, ethanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer.

5. The method according to claim 1, characterized in that: In the method, the molar equivalents of the On-DNA arylethylene compound are 50, 100, 200, 300, 400, 500, 600, 800, and 1000 equivalents for the 1,2-carboxylic acid furan ribose compound, the molar equivalents of the base are 50, 100, 120, 200, 300, 400, 500, 600, 800, and 1000 equivalents, and the molar equivalents of the catalyst are 1, 4, 5, and 10 equivalents.

6. The method according to any one of claims 1-5, characterized in that, The method is used for batch operations of multi-hole plates.

7. The method according to any one of claims 1-5, characterized in that, The method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

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