A method for the synthesis of DNA-encoded compounds oxysaccharides

Abstract

The present application relates to a kind of DNA encoding compound synthesis method of oxy-sugar compound, which uses On-DNA phenolic compound and 2-fluorosugar as raw material, obtains On-DNA oxy-sugar compound under the condition of calcium catalyst existence reaction.The synthesis method of DNA encoding compound oxy-sugar provided by the present application can be carried out in aqueous phase, simple post-processing, mild condition, can obtain high diversity DNA encoding compound library in a short time with high yield, and be suitable for the synthesis of DNA encoding compound in multi-well plate.

Description

Technical Field

[0001] This invention belongs to the field of encoded compound library technology, specifically relating to a method for constructing On-DNA oxy-glycoside compounds in a DNA-encoded compound library. 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] Developing methods for constructing on-DNA oxy-glycosides can enrich the application scenarios of on-DNA phenolic compounds and expand the application of sugar structures in DNA-encoded compound libraries, thereby further increasing the diversity of the compound libraries and improving the probability of screening effective compounds. However, no methods for constructing on-DNA oxy-glycosides have been reported to date. Therefore, it is hoped that a new method for synthesizing on-DNA oxy-glycosides suitable for high-volume multi-well plate operations can be developed to increase the diversity of DNA-encoded compound libraries and further enhance the application value of DNA-encoded compound library technology. Summary of the Invention

[0005] This invention develops 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 On-DNA phenolic compound libraries into On-DNA oxy-glycoside compound libraries in a single reaction.

[0006] This invention provides a method for synthesizing DNA-encoded oxy-glycosides, wherein the method uses On-DNA phenolic compounds and 2-fluoroglycosides as raw materials, and reacts them in the presence of a calcium catalyst to obtain On-DNA oxy-glycoside compounds.

[0007] The structural formula of the On-DNA phenolic compound is as follows: The structural formula of 2-fluorosaccharides is:

[0008]

[0009] In the structural formula, the DNA contains a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which is linked to R1 by one or more chemical bonds or groups.

[0010] The length of the DNA is 10 to 200.

[0011] In this structural formula, DNA and R1 are connected by one or more chemical bonds. A single chemical bond means that DNA and R1 are directly linked. Multiple chemical bonds mean that DNA and R1 are connected by several chemical bonds, such as a methylene group (-CH2-) linking the amino group of DNA to R1 (two chemical bonds); a carbonyl group (-CO-) linking the amino group of DNA to R1 (two chemical bonds); or a methylene carbonyl group (-CH2CO-) linking the amino group of DNA to R1 (three consecutive chemical bonds).

[0012] Preferably, the amino group of DNA is linked to R1 via a carbonyl group (-CO-).

[0013] Ar is selected from groups with a molecular weight of less than 1000 that are directly attached to R1 and a hydroxyl group;

[0014] R1 is selected from groups with a molecular weight of less than 1000 that are directly linked to DNA and Ar, or it does not exist.

[0015] Preferably, Ar is a 5-10 aryl or a 5-10 heteroaryl; the aryl or heteroaryl group can be independently converted by one or more hydrogens, halogens, or -C. 1～6 Alkyl, halogen-substituted C 1～6 Alkyl, nitro, cyano, NR a R b OR b -OCOR b -COOR b replace;

[0016] R1 is selected from key, -C 0～4 alkylene-, -C 0～4 Alkylene-C 5～10 Aryl-, -C 0～4 Alkylene-C 5～10 heteroaryl-, -C 0～4 Alkylene-C 3～10 cycloalkyl-, -C 0～4 Alkylene-C 3～10 Heterocyclic alkyl-, wherein one or more alkylene groups may be independently replaced by -O-, -NH-, -S-, or a carbonyl group; the alkylene group may be replaced by one or more independent hydrogen, halogen, -OH, or -C groups. 1～6 Alkyl, -NH(CO)R a OR b halogen-substituted C 1～6 Alkyl, nitro, cyano, NR a R b Substitution; the aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group may be replaced by one or more independent hydrogens, halogens, -OH, or -C. 1～6 Alkyl, halogen-substituted C 1～6 Alkyl, nitro, cyano, NR a R b replace;

[0017] R a R b For H, C 1～6 Alkyl, halogen-substituted C 1～6 alkyl.

[0018] Furthermore, the Ar is selected from the following groups: The Ar can be independently substituted by one or more hydrogens, halogens, methyl groups, ethyl groups, or methoxy groups.

[0019] Furthermore, R1 is selected from the following groups:

[0020] More specifically: the On-DNA phenolic compounds mentioned are selected from, but not limited to:

[0021] As a preferred embodiment, the present invention provides a method for synthesizing a DNA-encoded compound, oxy-glycoside, comprising the following steps: adding 0.1 to 1000 molar equivalents of a 2-fluoroglycoside compound to an On-DNA phenolic compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5 mM, then adding 0.1 to 1000 molar equivalents of a calcium catalyst, and finally adding 0.1 to 10 times the solution volume of ethanol, and reacting at -50°C to 100°C for 0.1 to 24 hours until the reaction is complete.

[0022] Furthermore, the calcium catalyst is selected from, but is not limited to, calcium hydroxide, calcium chloride, and calcium carbonate.

[0023] Preferably, the calcium catalyst is Ca(OH)2.

[0024] Furthermore, the 2-fluorosaccharide compound is selected from, but is not limited to, α-D-2-fluoropyranose, 2-fluoromannose, and 2-fluoromaltose.

[0025] Preferably, the 2-fluoroglycoside is selected from α-D-2-fluoropyranose.

[0026] Further, 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, dimethylformamide, dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer. Preferably, the reaction solvent is a mixture of water and ethanol.

[0027] Further, the reaction temperature is -20℃, 0℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, or 100℃. Preferably, the reaction temperature is 20℃.

[0028] The reaction time is 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, or 24 hours. Preferably, the reaction time is 0.2 hours.

[0029] In the method, the molar equivalent of the On-DNA phenolic compound is 1, and the molar equivalent of the 2-fluorosaccharide compound is 0.1 equivalent, 1 equivalent, 5 equivalent, 10 equivalent, 50 equivalent, 100 equivalent, 200 equivalent, 300 equivalent, 400 equivalent, 500 equivalent, 600 equivalent, 800 equivalent, or 1000 equivalent, preferably the molar equivalent of the 2-fluorosaccharide is 1000 equivalent.

[0030] The molar equivalent of the calcium catalyst in the method is 0.1 equivalent, 1 equivalent, 5 equivalent, 10 equivalent, 50 equivalent, 100 equivalent, 200 equivalent, 300 equivalent, 400 equivalent, 500 equivalent, 700 equivalent, 800 equivalent, or 1000 equivalent. Preferably, the molar equivalent of the calcium catalyst is 200 equivalent.

[0031] In the method, the volume ratio of ethanol to solution is 0.1, 0.2, 0.5, 1, 2, or 3 times. Preferably, the volume ratio of ethanol to aqueous solution is 1.

[0032] Furthermore, the method is used for batch operations of perforated plates.

[0033] Furthermore, the method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

[0034] This invention provides a method for introducing On-DNA oxy-glycoside structures into DNA-encoded compound libraries by reacting On-DNA phenolic compounds with 2-fluoroglycosides in the presence of a calcium catalyst. This method has wide applicability to various On-DNA phenolic compounds. It offers high yields, produces a single product, 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.

[0035] 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.

[0036] The term "substitution" refers to the replacement of one or more hydrogen atoms in a molecule by other different atoms or molecules.

[0037] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix (Ca～b Alkyl groups indicate any alkyl group containing one to two carbon atoms ("a" to "b"). Therefore, for example, C 1～12 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 12 carbon atoms.

[0038] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon group consisting of a carbon atom, such as methyl-CH3, ethyl-CH2CH3, methylene-CH2-, or isopropyl; the alkyl group can also be part of other groups, such as C 1～6 Alkoxy, C 1～6 Alkylamino.

[0039] The term "alkylene" refers to a saturated, straight-chain or branched, non-bridged divalent alkyl group containing multiple carbon atoms.

[0040] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0041] The term "alkoxy" refers to an alkyl group that is attached to an oxygen atom to form a substituent, such as methoxy, which is -OCH3.

[0042] The term "cycloalkyl" refers to a saturated or partially saturated cyclic group having a number of carbon atoms and no heterocyclic atoms, and having a single ring or multiple rings (including fused, bridged and spirocyclic systems).

[0043] The term "heterocyclic alkyl" refers to a non-aromatic cyclic group consisting of a carbon atom and a heteroatom selected from nitrogen, oxygen, sulfur, boron, silicon, etc., that is saturated or partially unsaturated (containing one or two double bonds). This cyclic group can be monocyclic or polycyclic.

[0044] The term "aryl" refers to an aromatic monocyclic or polycyclic group consisting of carbon atoms and lacking heteroatoms.

[0045] The term "heteroaryl" refers to a single or multiple cyclic aromatic group formed by replacing a carbon atom on at least one ring with a heteroatom selected from nitrogen, oxygen or sulfur, including monocyclic heterocyclic alkyl rings fused to phenyl or heteroaryl.

[0046] 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.

[0047] 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

[0048] Figure 1 The conversion rate distribution of the 18 On-DNA oxy-glycoside compounds obtained in Example 1 of this invention is shown in the figure. Detailed Implementation Plan

[0049] 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.

[0050] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0051] 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:

[0052]

[0053] Where A is adenine, T is thymine, C is cytosine, and G is guanine.

[0054] Example 1: Method for synthesizing On-DNA oxy-glycoside compounds

[0055] Step 1: Synthesis of On-DNA Phenolic Compounds

[0056]

[0057] HP(DNA-NH2) was dissolved in 250 mM, pH 9.4 borate buffer to prepare a 1 mM DNA solution (1 equivalent). Eighteen different H2NC(O)R1ArOH (50 equivalents, 0.2 M dissolved in dimethylacetamide), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (50 equivalents, 0.4 M dissolved in dimethylacetamide), and N,N-diisopropylamine (50 equivalents, 0.4 M dissolved in dimethylacetamide) were mixed thoroughly and then added to the DNA solution. The mixture was stirred thoroughly and reacted at 25°C for 1 hour.

[0058] After the reaction, 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 at low temperature (4℃) for half an hour. The supernatant was discarded, and the residual precipitate was freeze-dried and then dissolved in deionized water to obtain solutions of 18 different compounds 1. After quantification by OD, the conversion rate of compound 1 was confirmed by LC-MS to be 80-95%.

[0059] Step 2: Synthesis of On-DNA O-glycosides

[0060]

[0061] Compound 1 was prepared into a 1 mM DNA dd-H2O solution (1 equivalent). Fluorinated sugar (1000 equivalent, 1 M dissolved in dd-H2O), Ca(OH)2 catalyst (200 equivalent, 0.2 M dissolved in dd-H2O), and EtOH (1 volume) were added to the solution in sequence. The mixture was thoroughly mixed and reacted at 20 °C for 0.2 hours.

[0062] After the reaction was complete, ethylenediaminetetraacetic acid (200 equivalents, 0.2M dissolved in dd-H2O) was added to the reaction system, mixed thoroughly, and incubated at 20°C with shaking for 2 minutes. After the reaction was complete, 10% of the total volume of 5M sodium chloride solution was added to the resulting solution, followed by 3 times the total volume of anhydrous ethanol. After shaking thoroughly, the reaction mixture was placed on dry ice for 2 minutes, then centrifuged at 12000 rpm at low temperature (4°C) for 30 minutes. The supernatant was discarded, and the remaining precipitate was freeze-dried and then dissolved in deionized water to obtain the On-DNA product solution. After quantification by OD using a microplate reader, the conversion rate was confirmed by LCMS.

[0063] In summary, this invention, by controlling the solvent, temperature, pH, and other conditions during the reaction, achieves the introduction of On-DNA oxy-glycoside structures into a DNA-encoded compound library through the reaction of On-DNA phenolic compounds and 2-fluoroglycosides in the presence of a calcium catalyst. 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, oxy-glycoside, characterized in that: This method uses On-DNA phenolic compounds and 2-fluoroglycosides as raw materials, reacting them in the presence of a calcium catalyst to obtain On-DNA oxy-glycoside compounds; the structural formula of the On-DNA phenolic compounds is as follows: ; The structural formula of 2-fluorosaccharides is ; In the structural formula, the DNA contains a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and / or unmodified nucleotide monomers, which is linked to R1 by one or more chemical bonds or groups. Ar is selected from 5-10 aryl and 5-10 heteroaryl groups; the aryl and heteroaryl groups can be independently converted by one or more hydrogens, halogens, or -C. 1~6 Alkyl, halogen-substituted C 1~6 Alkyl, nitro, cyano, NR a R b OR b -OCOR b -COOR b replace; R1 is selected from key, -C 0~4 alkylene-, -C 0~4 Alkylene-C 5~10 Aryl-, -C 0~4 Alkylene-C 5~10 heteroaryl-, -C 0~4 Alkylene-C 3~10 cycloalkyl-, -C 0~4 Alkylene-C 3~10 Heterocyclic alkyl-, wherein one or more alkylene groups may be independently replaced by -O-, -NH-, -S-, or a carbonyl group; the alkylene group may be replaced by one or more independent hydrogen, halogen, -OH, or -C groups. 1~6 Alkyl, -NH(CO)R a OR b halogen-substituted C 1~6 Alkyl, nitro, cyano, NR a R b Substitution; the aryl, heteroaryl, cycloalkyl, or heterocycloalkyl group may be replaced by one or more independent hydrogens, halogens, -OH, or -C. 1~6 Alkyl, halogen-substituted C 1~6 Alkyl, nitro, cyano, NR a R b replace; R a R b H, -C 1~6 Alkyl, halogen-substituted C 1~6 alkyl; The method includes the following steps: adding 0.1 to 1000 molar equivalents of a 2-fluorosaccharide compound to an On-DNA phenolic compound solution with a molar equivalent of 1 and a molar concentration of 0.5-5 mM, then adding 0.1 to 1000 molar equivalents of a calcium catalyst, and finally adding 0.1 to 10 times the solution volume of ethanol, and reacting at -50℃ to 100℃ for 0.1 to 24 hours; The calcium catalyst mentioned therein is selected from calcium hydroxide.

2. The method according to claim 1, characterized in that: The Ar is selected from , , , , The Ar can be independently substituted by one or more hydrogen, halogen, methyl, ethyl, or methoxy groups. R1 is selected from: , , , , , , .

3. The method according to claim 1, characterized in that: The 2-fluorosaccharide compounds are selected from α-D-2-fluoropyranose, 2-fluoromannose, and 2-fluoromaltose.

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, dimethylformamide, dimethylacetamide, inorganic salt buffer, organic acid buffer, and organic base buffer.

5. The method according to claim 1, characterized in that: The reaction temperatures are -20℃, 0℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃.

6. The method according to claim 1, characterized in that: The reaction times are 0.1 hours, 0.2 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 16 hours, 18 hours, and 24 hours.

7. The method according to claim 1, characterized in that: The molar equivalents of the On-DNA phenolic compounds in the reaction are 0.1, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 800, and 1000 equivalents of the 1,2-fluorosaccharide compounds; the molar equivalents of the calcium catalyst are 0.1, 1, 5, 10, 50, 100, 200, 300, 400, 500, 700, 800, and 1000 equivalents.

8. The method according to claim 1, characterized in that: The volume ratios of ethanol to solution are 0.1, 0.2, 0.5, 1, 2, and 3.

9. The method according to any one of claims 1-8, characterized in that, The method is used for batch operations of multi-hole plates.

10. The method according to any one of claims 1-8, characterized in that, The method is used for the synthesis of DNA-encoded compound libraries for multi-well plates.

Images