Synthesis method and application of DNA-ketoamide conjugate

Abstract

The invention relates to the technical field of DNA (deoxyribonucleic acid) coding molecular libraries, and particularly discloses a synthetic method and application of a DNA-ketoamide conjugate. Wherein an oxidizing agent is added into a solution containing the DNA-hydroxyamide conjugate as shown in the general formula (I) in the specification, reaction is performed for at least 1 hour under the condition of-5 DEG C to 5 DEG C, and the DNA-ketoamide conjugate as shown in the general formula (II) in the specification is obtained. The synthesis method has the characteristics of high selectivity, mild reaction conditions, biological system compatibility and the like, effectively ensures that other sensitive groups in DNA molecules are not influenced in the reaction process, and has a relatively high conversion rate.

Description

Technical Field

[0001] This application relates to the field of DNA-encoded molecular library technology, specifically to a method for synthesizing DNA-ketoamide conjugates and their applications. Background Technology

[0002] DNA-encoded molecular libraries are synthesized using combinatorial chemistry, assembling a large number of chemical building blocks into a giant library of compounds. Each compound molecule is attached with a unique DNA strand, serving as its "barcode." By amplifying and sequencing the DNA sequence of the bound compounds, the compounds can be effectively identified.

[0003] Constructing α-ketoamide structures on DNA molecules to form DNA-ketoamide conjugates offers unique geometric conformations that impart favorable pharmacokinetic properties and metabolic stability due to their combined nucleophilic and electrophilic reactivity. When the α-ketoamide structure acts as a non-electrophilic segment, it enhances target binding by strengthening hydrogen bonding; when acting as an electrophilic segment, its carbonyl group can form a covalent bond with cysteine ​​residues at the protease catalytic site, achieving highly efficient inhibition. This characteristic makes it widely used in antiviral drug design (such as coronavirus main protease inhibitors), antibacterial agents, and immunomodulators, with typical examples including the hepatitis C drug telaprevir and broad-spectrum antiviral compounds. The α-ketoamide structure, by regulating molecular rigidity and reaction specificity, provides a key chemical basis for developing highly active, low-toxicity targeted drugs. This offers more structural options for discovering novel drug molecules. However, the relatively mild and efficient reaction conditions and broad substrate applicability of DNA limit the expansion of the reaction toolbox, resulting in the lack of reported efficient synthetic methods for DNA-ketoamide conjugates.

[0004] Therefore, it is necessary to develop synthetic methods for DNA-ketoamide conjugates. Summary of the Invention

[0005] The purpose of this application is to provide a method for synthesizing DNA-ketoamide conjugates and their applications, in order to solve the problem that there is currently no effective method for synthesizing DNA-ketoamide conjugates.

[0006] To achieve the above-mentioned objectives, in a first aspect, this application provides a method for synthesizing a DNA-ketoamide conjugate.

[0007] The method for synthesizing the DNA-ketoamide conjugate of this application includes the following steps: An oxidant was added to a solution containing a DNA-hydroxyamide conjugate of general formula (I), and the mixture was reacted at -5 °C to 5 °C for at least 1 h to obtain a DNA-ketoamide conjugate of general formula (II). The structural formula of the DNA-hydroxyamide conjugate represented by general formula (I) is shown below: ; The structural formula of the DNA-ketoamide conjugate represented by general formula (II) is shown below: ; Wherein, R1 is selected from: alkyl primary amines with 3 to 6 carbon atoms, branched alkyl primary amines with 4 carbon atoms, six-membered cycloalkyl primary amines and amino acids, and substituted benzylamines, anilines, asymmetric secondary amines with 5 carbon atoms, and six-membered cycloalkyl secondary amines with mutually independent substituents selected from any one or more of alkyl, alkoxy, halogen, hydroxyl, trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl, with substituted groups ... trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl, with substituted groups selected from any one or more of alkyl, alkoxy, halogen, and six-membered cycloalkyl secondary amines; R2 is selected from: an aromatic benzene ring substituted with one or more substituents, wherein the substituents are independently selected from hydrogen, halogen, trifluoromethyl, carboxyl, amino, nitro, cyano, hydroxy, phenyl, ester, and amide; or isopropyl; or alkyl having 1 to 4 carbon atoms.

[0008] In some embodiments, the molar equivalent of the oxidant is 15 to 75.

[0009] In some embodiments, the oxidant is first dissolved in a 0.4-0.6 M alkaline solution and then added to the DNA-hydroxyamide conjugate solution.

[0010] In some embodiments, the reaction time is 1 h to 5 h.

[0011] In some embodiments, the reaction temperature is 0 ℃ ± 1 ℃.

[0012] In some embodiments, in the structural formula of the general formula (I) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-hydroxy acid by one or more chemical bonds or groups; And / or, In the structural formula of general formula (II) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-keto acid by one or more chemical bonds or groups.

[0013] In some embodiments, the nucleotide chain is selected from at least one of artificially modified nucleotide chains and unmodified nucleotide chains.

[0014] In some embodiments, the method also includes the step of detecting the product using ultra-high performance liquid chromatography-mass spectrometry.

[0015] A second aspect of this application provides a method for synthesizing DNA-ketoamide conjugates and the use of the synthesized DNA-ketoamide conjugates as components of antiviral drugs.

[0016] Beneficial technical effects: Compared to existing technologies, the synthetic method of this application can oxidize the α-hydroxyl group on the DNA molecule in a pure aqueous system, thereby enabling the oxidation of the products obtained by amidation of DNA-coupled primary and secondary amines with α-hydroxy acids of different structures as substrates. The reaction process of this application is characterized by high selectivity, specifically oxidizing only the hydroxyl group in the structure without affecting other sensitive groups in the DNA molecule; and the reaction conditions are mild and biocompatible, specifically, the entire reaction is carried out in a pure aqueous phase, the conditions are mild, compatible with biological systems, and effectively maintains the chemical integrity of DNA. Furthermore, it has broad substrate adaptability, specifically applicable to the coupling of diverse DNA-coupled primary and secondary amine compounds with α-hydroxy acids of different structures, including various natural and non-natural amino acids, as well as cyclic amines, secondary amines, and long-chain aliphatic amines, and most substrates exhibit excellent conversion efficiency. Attached Figure Description

[0017] To make the objectives, technical solutions, and advantages of this application clearer, a preferred description of this application will be provided below with reference to the accompanying drawings, wherein: Figure 1 This is a schematic diagram of the overall reaction equation involved in the synthesis method of this application embodiment; Figure 2 DNA conjugate generated in Example 1 L The spectrum of -phenylalanine a1, where A is the chromatogram and B is the mass spectrum; Figure 3 DNA conjugate generated in Example 1 L The spectrum of -phenylpropionic hydroxyamide ao1, where A is the chromatogram and B is the mass spectrum; Figure 4 DNA conjugate generated in Example 1 L The spectrum of phenylacetone amide AO1, where A is the chromatogram and B is the mass spectrum; Figure 5 This is a schematic diagram showing the oxidation reaction conversion rates of some of the α-hydroxy acid substrates o1~o20 involved in this application.

[0018] Figure 6 This is a schematic diagram showing the DNA-coupled amines involved in the reactions of Examples 1 to 27 of this application and the corresponding conversion rates. Detailed Implementation

[0019] To make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the following detailed description of this application is provided in conjunction with embodiments.

[0020] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0021] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0022] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0023] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0024] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0025] In the embodiments of this application, DMA represents N,N'-dimethylacetamide; HATU represents 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIPEA represents N,N-diisopropylethylamine; EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; S-NHS represents sodium N-hydroxysuccinimide sulfonate; HOAt represents N-hydroxy-7-azabenzotriazole; DMSO represents dimethyl sulfoxide; and K2RuO4 represents potassium ruthenium acid.

[0027] This application provides a method for synthesizing a DNA-ketoamide conjugate, comprising the following steps: An oxidant was added to a solution containing a DNA-hydroxyamide conjugate of general formula (I), and the mixture was reacted at -5 °C to 5 °C for at least 1 h to obtain a DNA-ketoamide conjugate of general formula (II). The structural formula of the DNA-hydroxyamide conjugate represented by general formula (I) is shown below: ; The structural formula of the DNA-ketoamide conjugate represented by general formula (II) is shown below: ; Wherein, R1 is selected from: alkyl primary amines with 3 to 6 carbon atoms, branched alkyl primary amines with 4 carbon atoms, six-membered alkyl primary amines and amino acids; substituted benzylamines, anilines, asymmetric secondary amines with 5 carbon atoms, and six-membered cycloamines with substituents independently selected from any one or more of alkyl, alkoxy, halogen, hydroxyl, trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl; R2 is selected from: aromatic benzene rings substituted with one or more substituents, wherein the substituents are independently selected from hydrogen, halogen, trifluoromethyl, carboxyl, amino, nitro, cyano, hydroxyl, phenyl, ester and amide; or isopropyl; or alkyl with 1 to 4 carbon atoms. The entire reaction equation is as follows. Figure 1 As shown.

[0028] In some embodiments, the method further includes detecting the product using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to confirm the formation of carbonyl groups. Specifically, after the reaction is complete, primary water, 3 M to 6 M sodium chloride aqueous solution, and wastewater ethanol at -20 °C are added sequentially, mixed well, and then frozen at -80 °C for 2 to 4 hours. A first centrifugation is then performed at 4 °C. After centrifugation, the supernatant is removed, and a -20 °C ethanol aqueous solution is added for a second centrifugation, after which the supernatant is removed. The precipitate is then freeze-dried in a vacuum environment at -80 °C. Finally, the freeze-dried material is dissolved in primary water and analyzed by UHPLC-MS. In some embodiments, the first centrifugation is performed at 13,000 rpm to 14,000 rpm for 20 to 60 minutes. The second centrifugation is performed at 13,000 rpm to 14,000 rpm for 10 to 30 minutes.

[0029] In some embodiments, in the structural formula of the general formula (I) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-hydroxy acid by one or more chemical bonds or groups; and the nucleotide chain is selected from at least one of artificially modified nucleotide chains and unmodified nucleotide chains.

[0030] In some embodiments, in the structural formula of the general formula (II) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-keto acid by one or more chemical bonds or groups, and the nucleotide chain being selected from at least one of artificially modified nucleotide chains and unmodified nucleotide chains.

[0031] In some embodiments, the ratio of the DNA-hydroxyamide conjugate represented by general formula (I) to the oxidant, calculated in molar equivalents, is 1:15 to 75. A feed ratio within this range promotes a complete reaction. Typical but non-limiting molar equivalent ratios include 1:15, 1:16, 1:18, 1:20, 1:22, 1:25, 1:28, 1:30, 1:31, 1:33, 1:35, 1:40, 1:45, 1:50, 1:52, 1:55, 1:60, 1:65, 1:70, 1:72, and 1:75, or any range between two molar equivalent ratios. Within these ranges, a chemical reaction can occur effectively.

[0032] In some embodiments, the oxidant is first dissolved in a 0.4-0.6 M alkaline solution, mixed thoroughly to obtain an oxidant solution, and then the oxidant solution is added to the DNA-hydroxyamide conjugate solution. This helps to improve the uniformity of the reaction and promotes mild reaction conditions. For example, the oxidant can be dissolved in a 0.4 M sodium hydroxide (NaOH) solution, or a 0.5 M NaOH solution, or a 0.6 M NaOH solution. Potassium hydroxide solution or other alkaline solutions can also be used.

[0033] In some embodiments, the oxidant is selected from at least one of potassium ruthenate, dimethyl phthalate, oxamine salts, 2,2,6,6-tetramethylpiperidine oxide, tetrapropylammonium perruthenate, sodium periodate, iodobenzene acetate, 2-iodobenzoic acid, ruthenium trichloride, and ferric hydroxide. Potassium ruthenate is preferred because it has higher oxidation efficiency than other oxidants, a broader substrate range, mild reaction conditions, and does not damage DNA.

[0034] In some implementations, the reaction time is 1 h to 5 h. For example, it can be a typical but non-limiting reaction time such as 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, and 5 h, or any range between two reaction times, with the reaction stopping when it is complete.

[0035] In some embodiments, the reaction temperature is a typical but non-limiting reaction temperature such as -5 °C, or -4 °C, or -3 °C, or -2 °C, or -1 °C, or 0 °C, or 1 °C, or 1 °C, or 3 °C, or 4 °C, or 5 °C, or a range between any two reaction temperatures, as long as it is conducive to the occurrence and continuation of the reaction.

[0036] In some embodiments, the DNA of the DNA conjugate is a single-stranded or double-stranded nucleotide chain, which is obtained by sequentially coupling Fmoc-Cys(StBu)-OH with one or more chemical bonds or groups, followed by deaminoacetylation of Fmoc and cysteine. In some embodiments, a commercially available headpiece with a molecular weight of 4937 and the following structural formula can be used: In some embodiments, the DNA-hydroxyamide conjugate solution represented by general formula (I) is an aqueous solution.

[0037] In the above reaction process, the α-hydroxyl group on the DNA molecule can be oxidized in a pure aqueous system. This allows for the oxidation of the products obtained by amidation of DNA-coupled primary and secondary amines with α-hydroxy acids of different structures as substrates. The reaction process of this application is characterized by high selectivity, specifically oxidizing only the hydroxyl group in the structure without affecting other sensitive groups in the DNA molecule; and the reaction conditions are mild and biocompatible, specifically, the entire reaction is carried out in a pure aqueous phase, the conditions are mild, compatible with biological systems, and effectively maintains the chemical integrity of the DNA. Furthermore, it has broad substrate adaptability, specifically applicable to the coupling of diverse DNA-coupled primary and secondary amine compounds with α-hydroxy acids of different structures, including various natural and non-natural amino acids, as well as cyclic amines, secondary amines, and long-chain aliphatic amines, and most substrates exhibit excellent conversion efficiency.

[0038] The following examples illustrate the synthesis method of the DNA-ketoamide conjugate in this application.

[0039] Example 1 The synthesis method of DNA-ketoamide conjugate (AO1) is as follows: Step S11: Add Fmoc-L-phenylalanine (25 μL, 200 mM dissolved in DMA), HATU (12.5 μL, 400 mM dissolved in DMA), and DIPEA (12.5 μL, 400 mM dissolved in DMA) sequentially to the EP tube, mix thoroughly, and activate for 10 min.

[0040] Step S12: Dissolve DNA-NH2 in sodium borate buffer solution with a concentration of 250 mM and pH 9.4 to prepare a 1 mM solution for later use.

[0041] Step S13: Take another EP tube, take 50 μL of DNA-NH2 sodium borate buffer from step S12, add the activation solution from step (1), mix well, and react at room temperature for 2 h.

[0042] Step S14: After the reaction is complete, ethanol precipitation is performed, and the recovery rate is confirmed by microplate reader. UHPLC-MS is used to confirm the correctness and conversion rate of the condensation product.

[0043] Step S15: The condensation product was redissolved in 100 μL of primary water and 10 μL of piperidine was added. The mixture was reacted at 25 °C for 30 min. After the reaction, ethanol was used for precipitation. The recovery rate was confirmed by microplate reader. UHPLC-MS was used to confirm the correctness of the DNA conjugate a1 structure and the conversion rate. The conversion rate was 95% and the recovery rate was 85%.

[0044] The specific synthesis path of a1 is shown below: .

[0045] The test results for a1 are as follows: Figure 2 As shown, A is a chromatogram and B is a mass spectrum. From Figure 2 As can be seen, the raw materials have been completely converted into the predicted product a1.

[0046] Step S16: Dissolve 1 nmol of a1 in 10 μL of 500 mM, pH 8.5 3-morpholinopropanesulfonic acid buffer. Add 4 μL of HOAt (100 mM dissolved in DMSO), 4 μL of DIPEA (400 mM dissolved in DMSO), 8 μL of S-mandelic acid (200 mM dissolved in DMSO), and 4 μL of EDCI (400 mM dissolved in DMSO), for a total reaction volume of 30 μL. Mix thoroughly and react at 25°C for 8 h. After the reaction is complete, ethanol precipitation is performed, and the recovery rate is confirmed by microplate reader. DNA coupling is confirmed by UHPLC-MS. L The correctness and conversion rate of the phenylpropionic hydroxyamide ao1 structure were verified, with a conversion rate of 95%.

[0047] The specific synthesis path of ao1 is shown below: .

[0048] The detection results of ao1 are as follows Figure 3 As shown, A is a chromatogram and B is a mass spectrum. From Figure 3 As can be seen from the data, the raw material a1 has been completely converted into the predicted product ao1.

[0049] Step S17: Add 19 μL of primary water to 400 pmol of ao1, then add 1 μL of K2RuO4 (15 mM dissolved in 0.5 M NaOH) and react at 0 °C for 1 h. After the reaction is complete, add 30 equivalents of copper-zinc chelating metal, let stand for 30 min, then centrifuge at 13,500 rpm for 30 min. Collect the supernatant for ethanol precipitation, confirm the recovery rate using an ELISA reader, and confirm DNA coupling using UHPLC-MS. L The correctness and conversion rate of the α-phenylacetone amide AO1 structure were verified, with a conversion rate of 95%.

[0050] The specific synthesis path of AO1 is shown below: .

[0051] The detection results of AO1 are as follows Figure 4 As shown, A is a chromatogram and B is a mass spectrum. From Figure 4As can be seen, the raw material ao1 is efficiently converted into the predicted product AO1.

[0052] Examples 2-27 The reaction processes of Examples 2 to 27 are the same as those in Example 1. To save space, they will not be described in detail here. The specific reaction products and conversion rates are as follows: Figure 5 and Figure 6 As shown.

[0053] Notice, Figure 5 In this context, the conversion rate of the starting materials coupled with HP (commercially available amine-modified DNA double strands), i.e., O1~O20 oxidized to uppercase O1~O20, is used to demonstrate the substrate universality of the reaction described in this application in different DNA-coupled hydroxyamide substrates. Figure 6 This describes the substrate generality of different DNA amine conjugates, hence the labeling and... Figure 5 To distinguish them, the letter 'a' was added.

[0054] Depend on Figure 5 and Figure 6 It can be seen that the oxidation method proposed in this application has excellent substrate universality.

[0055] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for synthesizing a DNA-ketoamide conjugate, characterized in that, Includes the following steps: An oxidant was added to a solution containing a DNA-hydroxyamide conjugate of general formula (I), and the mixture was reacted at -5 °C to 5 °C for at least 1 h to obtain a DNA-ketoamide conjugate of general formula (II). The structural formula of the DNA-hydroxyamide conjugate represented by general formula (I) is shown below: ； The structural formula of the DNA-ketoamide conjugate represented by general formula (II) is shown below: ； Wherein, R1 is selected from: alkyl primary amines with 3 to 6 carbon atoms, branched alkyl primary amines with 4 carbon atoms, six-membered cycloalkyl primary amines and amino acids, and substituted benzylamines, anilines, asymmetric secondary amines with 5 carbon atoms, and six-membered cycloalkyl secondary amines with mutually independent substituents selected from any one or more of alkyl, alkoxy, halogen, hydroxyl, trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl, with substituted groups ... trifluoromethyl, amino, ester, amide, nitro, cyano or phenyl, with substituted groups selected from any one or more of alkyl, alkoxy, halogen, and six-membered cycloalkyl secondary amines; R2 is selected from: an aromatic benzene ring substituted with one or more substituents, wherein the substituents are independently selected from hydrogen, halogen, trifluoromethyl, carboxyl, amino, nitro, cyano, hydroxy, phenyl, ester, and amide; or isopropyl; or alkyl having 1 to 4 carbon atoms.

2. The method for synthesizing the DNA-ketoamide conjugate as described in claim 1, characterized in that, The oxidant is selected from at least one of potassium ruthenate, dimethyl phthalate, oxamine salt, 2,2,6,6-tetramethylpiperidine oxide, tetrapropylammonium perruthenate, sodium periodate, iodobenzene acetate, 2-iodobenzoic acid, ruthenium trichloride, and iron hydroxyoxide.

3. The method for synthesizing the DNA-ketoamide conjugate as described in claim 1, characterized in that, The ratio of the DNA-hydroxyamide conjugate represented by the general formula (I) to the oxidant is 1:15~75, based on the molar equivalent ratio.

4. The method for synthesizing the DNA-ketoamide conjugate as described in claim 1, characterized in that, The oxidant is first dissolved in a 0.4-0.6 M alkaline solution and then added to the DNA-hydroxyamide conjugate solution.

5. The method for synthesizing the DNA-ketoamide conjugate as described in claim 1, characterized in that, The reaction time is 1 h to 5 h.

6. The method for synthesizing the DNA-ketoamide conjugate as described in claim 1, characterized in that, The reaction temperature is 0℃±1℃.

7. The method for synthesizing the DNA-ketoamide conjugate according to any one of claims 1 to 6, characterized in that, In the structural formula of the general formula (I) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-hydroxy acid by one or more chemical bonds or groups; And / or, In the structural formula of general formula (II) shown, the DNA comprises a single-stranded or double-stranded nucleotide chain obtained by polymerization of nucleotide monomers, the nucleotide chain being linked to an α-keto acid by one or more chemical bonds or groups.

8. The method for synthesizing the DNA-ketoamide conjugate as described in claim 7, characterized in that, The nucleotide chain is selected from at least one of artificially modified nucleotide chains and unmodified nucleotide chains.

9. The method for synthesizing the DNA-ketoamide conjugate according to any one of claims 1 to 6, characterized in that, It also includes the step of detecting the product using ultra-high performance liquid chromatography-mass spectrometry.

10. The use of the DNA-ketoamide conjugate synthesized by the method of any one of claims 1 to 9 as an antiviral drug component.

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