Preparation method of on-DNA polypeptide compound containing acryloyl structure and product
By generating on-DNA polypeptide compounds containing acryloyl groups under nucleic acid-compatible conditions, the problem of unstable covalent structures in DNA-encoded molecular libraries has been solved, enabling the efficient application of covalent inhibitor discovery.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2026-03-10
AI Technical Summary
The highly reactive covalent structures in existing DNA-encoded molecular libraries are unstable and have short shelf lives, making them difficult to apply to the discovery of covalent inhibitors.
Under nucleic acid-compatible conditions, an on-DNA polypeptide compound containing an acrylyl group is generated through an elimination reaction. This compound then acts as a covalent linker and undergoes an addition reaction with nucleophilic substituents in the target protein to form covalent bonds.
This provides a simple, universally applicable, and inexpensive preparation method that expands the application field of DNA-encoded molecular libraries and improves the efficiency of covalent inhibitor discovery.
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Abstract
Description
[0001] The present application claims priority to the Chinese patent application with the application number 2024112633210 and the title “Preparation method and product of on-DNA polypeptide compound containing acryloyl structure” filed on September 10, 2024. The entire content of the application is incorporated by reference. TECHNICAL FIELD
[0002] The present application belongs to the technical field of DNA-encoded molecule library, and specifically relates to a preparation method and product of on-DNA polypeptide compound containing acryloyl structure which can be used in DNA-encoded molecule library. BACKGROUND
[0003] In recent years, covalent inhibitors have received renewed interest as a drug discovery strategy. The approval of KRAS G12C drug Sotorasib marks a major advance in targeting “undruggable” protein targets through covalent binding. The discovery and development of covalent drugs is a rapidly expanding field, with new covalent binding modes and screening technologies emerging.
[0004] DNA-encoded molecule library technology (DELT) is a powerful tool that can identify compounds with affinity to target structures in a fast and economical way. DEL technology uses the split-and-pool synthesis strategy in combinatorial chemistry, i.e. m+n+i chemical reactions produce MxNxI compounds, which can quickly synthesize hundreds of millions of compounds for affinity screening in a short period of time. In addition, through the development of various nucleic acid-compatible chemical tools, the chemical structures in the DNA-encoded molecule library can cover a more diverse chemical space, improving the success rate of screening. DEL technology, as a mature method, combined with the corresponding covalent screening system, has been applied to the discovery of reversible and irreversible covalent inhibitors. Currently, the most commonly used method for synthesizing covalent DNA-encoded molecule libraries is to connect covalent linkers such as acrylamide, chloroacetyl chloride, propylene oxide, sulfonyl fluoride, etc. to on-DNA compounds in the last step of chemical synthesis.
[0005] However, these covalent structures with high reactivity are unstable and have a short shelf life during application. Therefore, it is possible to consider introducing covalent precursors to generate electrophilic end-olefin structures in situ by deprotection before screening. This chemical structure can act as a covalent linker to undergo addition reaction with nucleophilic substituents in the target protein to form a covalent bond, constructing an on-DNA covalent compound library for subsequent covalent screening with target.
[0006] Therefore, there is an urgent need to provide an on-DNA polypeptide compound with acryloyl structure and a preparation method thereof, which is easy to operate, has strong substrate universality and is cheap and easy to obtain, and applies DNA coded molecular library technology to new fields such as covalent inhibitor discovery. SUMMARY
[0007] In view of the above problems, the present application aims to provide a preparation method and product of an on-DNA polypeptide compound with acryloyl structure. The preparation method of the on-DNA polypeptide compound with acryloyl structure provided by the present application comprises the following steps: dissolving an on-DNA compound in a buffer to obtain an on-DNA compound solution, carrying out an elimination reaction, adding a separation agent, standing, centrifuging, discarding the supernatant, and freeze-drying to obtain a product. The preparation method of the present application causes a specific structure in the on-DNA polypeptide substrate to undergo an elimination reaction under nucleic acid compatible conditions to generate an end olefin structure. This chemical structure can form a covalent bond with a nucleophilic substitution group in a target protein through an addition reaction. The construction method of the present application is easy to operate, has strong substrate universality and is cheap and easy to obtain, and applies DNA coded molecular library technology to new fields such as covalent inhibitor discovery.
[0008] Terminology: In the present application, the term "Pd(PPh3)4" is also referred to as tetra(triphenylphosphine)palladium.
[0009] In the present application, the term "Pd(OAc)2" is also referred to as palladium(II) acetate.
[0010] In the present application, the term "DMA" is also referred to as dimethylacetamide.
[0011] In the present application, the term "MeCN" is also referred to as acetonitrile.
[0012] In the present application, the term "TBPH" is also referred to as tert-butyl hydroperoxide.
[0013] In the present application, the term "scavenger" is also referred to as sodium diethyldithiocarbamate, which has the following structure:
[0014] Scavenger structure.
[0015] In the present application, the term "Mukaiyama's reagent" is also referred to as 2-chloro-1-methylpyridinium iodide, which has the following structure:
[0016] Mukaiyama's reagent structure.
[0017] Group definitions: Definitions of standard chemical terms can be found in the references (including Carey and Sundberg, "Advanced Organic Chemistry 4th Edition." Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, IR, and UV / VIS spectroscopy and pharmacological methods, are used. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. For example, reactions and purifications may be carried out using the manufacturer's instructions for use of kits, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally carried out according to conventional methods well known in the art, based on the descriptions in the various summary and more specific references cited and discussed in this specification. In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds.
[0018] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides a method for preparing an on-DNA polypeptide compound containing an acryloyl group, characterized in that the preparation method includes the following steps: S1. Dissolve the on-DNA compound in a buffer solution to obtain an on-DNA compound solution; S2 and on-DNA compound solution were subjected to elimination reaction to obtain final reaction solution; S3. After the final reaction solution is cooled, a separating agent is added, the mixture is allowed to stand, centrifuged, the supernatant is discarded, and the product is obtained by freeze-drying. The on-DNA compound described in step S1 has the structure shown in Formula I:
[0019] Formula I.
[0020] Specifically, the aforementioned It is one or more of the following: single-stranded deoxyribonucleic acid sequence, double-stranded deoxyribonucleic acid sequence, single-stranded ribonucleic acid sequence, and double-stranded ribonucleic acid sequence; Preferably, the A single-stranded or double-stranded nucleotide chain obtained by polymerizing nucleotide monomers, whether artificially modified or unmodified.
[0021] Specifically, the aforementioned It has a linear or ring-shaped skeleton structure; More specifically, the aforementioned Selected from linear or cyclic framework structures composed of chemical elements or chemical bonds: The chemical elements mentioned include: C, H, O, N, P, or S; The chemical bonds include: C=C, C=C, CY, C=Y, YY, or Y=Y; wherein Y is independently selected from H, O, N, P, or S.
[0022] Preferably, the It has a linear peptide or cyclic peptide structure.
[0023] Specifically, R is a hydrogen atom, an aliphatic chemical structure, or an aromatic chemical structure.
[0024] More specifically, R is a hydrogen atom, benzyl, ethylamino, phenyl, , , , or .
[0025] Preferably, R is a hydrogen atom, a benzyl group, or a phenyl group.
[0026] Preferably, X is an oxygen atom, a sulfur atom, or a selenium atom.
[0027] Specifically, the buffer solution mentioned in step S1 includes a buffer solution with a pH value of 7.0-10.0.
[0028] Preferably, the buffer solution described in step S1 includes, but is not limited to, one or more of the following: HEPES buffer, MOPS buffer, Tris-HCl buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, sodium dihydrogen phosphate-sodium hydroxide buffer, and guanidine hydrochloride buffer.
[0029] More preferably, the buffer solution in step S1 is: disodium hydrogen phosphate-potassium dihydrogen phosphate buffer and / or Tris-HCl buffer.
[0030] Specifically, the elimination reaction described in step S2 includes: adding one or more of component 1, component 2, and component 3 to the on-DNA compound solution to obtain the final reaction; More specifically, component 1 is a solvent containing a catalyst; component 2 is a solvent containing an additive; and component 3 is a solvent containing a reagent.
[0031] Preferably, the catalyst is Pd(PPh3)4 and / or Pd(OAc)2.
[0032] Preferably, the additive is one or more of NaBH4, K2CO3, Et3N, NaOH, H2O2, and TBPH.
[0033] Preferably, the reagent is one or more of Mukaiyama's reagent and dihalogen alkylating reagent.
[0034] Preferably, the solvent in component 1, component 2 or component 3 described in step S2 is independently selected from: organic solvents and / or water.
[0035] More preferably, the organic solvent includes one or more of the following: acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, and 1,4-dioxane.
[0036] More preferably, the organic solvent is acetonitrile or dimethylacetamide.
[0037] Specifically, in step S2, component 1 contains 5-20 mM catalyst; component 2 contains 5-200 mM additive; and component 3 contains 5-200 mM reagent.
[0038] Preferably, in step S2, component 1 contains a 10 mM catalyst; component 2 contains a 10-100 mM additive; and component 3 contains a 10-100 mM reagent.
[0039] Specifically, the reaction conditions for the elimination reaction described in step S2 include: reaction at 0-70℃ for 10-180 minutes.
[0040] Specifically, the reaction formula for the preparation method is as follows: ; in, This indicates the electrophilic structure of the acryloyl group formed after the reaction.
[0041] Secondly, the present invention provides an on-DNA polypeptide compound containing an acryloyl group prepared by the above preparation method.
[0042] The beneficial effects of this invention are as follows: This invention is the first to develop methods for preparing various on-DNA polypeptide compounds containing acryloyl groups. Under nucleic acid-compatible conditions, a specific structure in the on-DNA polypeptide substrate undergoes an elimination reaction to generate a terminal olefin structure. This chemical structure acts as a covalent linker, capable of undergoing an addition reaction with nucleophilic substituents in the target protein to form a covalent bond. The construction method of this invention is simple to operate, has broad substrate applicability, and is inexpensive and readily available, applying DNA-encoded molecular library technology to new fields such as the discovery of covalent inhibitors. Detailed Implementation
[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.
[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0045] Example 1: Preparation of on-DNA polypeptide compounds containing acryloyl groups
[0046] On-DNA compound 1 was dissolved in 100 mM, pH 7.5 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM solution. Pd(PPh3)4 (10 mM / DMA) and NaBH4 (50 mM / MeCN) were added, and the reaction was carried out at 20°C for 1 hour. After the reaction was complete, 100 mM Scavenger solution was added to the reaction solution, and the mixture was heated at 70°C for 20 minutes. After cooling, 5 M sodium chloride solution and cold ethanol were added to the reaction solution, and the mixture was incubated at -78°C for 0.5 hours. Then, the mixture was centrifuged at 4°C to remove the supernatant, and lyophilized at room temperature for 60 minutes to remove residual solvent, yielding the product.
[0047] The Pd(PPh3)4 (10 mM / DMA) mentioned above means: Pd(PPh3)4 is dissolved in DMA solution to make the concentration of Pd(PPh3)4 10 mM.
[0048] The NaBH4 (50mM / MeCN) mentioned means that NaBH4 is dissolved in MeCN solution to make the NaBH4 concentration 50mM.
[0049] In the On-DNA compound 1 The structure is shown in Formula II. The structure and reaction conversion rate of the representative compound before and after the reaction in Example 1 are shown in Table 1.
[0050]
[0051] Formula II.
[0052] Table 1. Structure and conversion rate of representative compounds in Example 1 before and after the reaction.
[0053] Example 2: Preparation of on-DNA polypeptide compounds containing acryloyl groups
[0054] On-DNA compound 2 was dissolved in 100 mM, pH 7.5 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM solution. Mukaiyama's reagent (10 mM / H2O) and NaOH (100 mM / H2O) were added, and the reaction was carried out at 4°C for 1 hour. After the reaction was complete, 5 M sodium chloride solution and cold ethanol were added to the reaction solution, and the mixture was incubated at -78°C for 0.5 hours, then centrifuged at 4°C to remove the supernatant. The product was then lyophilized at room temperature for 60 minutes to remove residual solvent.
[0055] The Mukaiyama's reagent (10 mM / H2O) means that Mukaiyama's reagent is dissolved in H2O solution to make the concentration of Mukaiyama's reagent 10 mM.
[0056] The NaOH (100 mM / H2O) mentioned means: dissolving NaOH in H2O solution to make the NaOH concentration 50 mM.
[0057] In the On-DNA compound 2 The structure is shown in Formula II. The structures and reaction conversion rates of the representative compounds in Example 2 before and after the reaction are shown in Table 2.
[0058] Table 2. Structure and conversion rate of representative compounds in Example 2 before and after the reaction.
[0059] Example 3 Preparation of on-DNA polypeptide compounds containing acryloyl groups
[0060] On-DNA compound 2 was dissolved in 100 mM, pH 7.5 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM On-DNA compound 2 solution. A dihalogen alkylating agent (10 mM / DMA) and K₂CO₃ (100 mM / H₂O) were added, and the reaction was carried out at 20°C for 1 hour. After the reaction was complete, 5 M sodium chloride solution and cold ethanol were added to the reaction solution, and the mixture was incubated at -78°C for 0.5 hours, followed by centrifugation at 4°C to remove the supernatant. The product was then lyophilized at room temperature for 60 min to remove residual solvent.
[0061] The dihalogen alkylating agent (10 mM / DMA) refers to the following: the dihalogen alkylating agent is dissolved in a DMA solution to achieve a concentration of 10 mM. The structure of the dihalogen alkylating agent is shown below:
[0062] The K2CO3 (100 mM / H2O) mentioned means that K2CO3 is dissolved in H2O solution to make the K2CO3 concentration 100 mM.
[0063] In the On-DNA compound 2 The structure is shown in Formula II. Table 3 shows the structure and conversion rate of representative compounds before and after the reaction in Example 3.
[0064] Table 3. Structure and conversion rate of representative compounds in Example 3 before and after the reaction.
[0065] Example 4: Preparation of on-DNA polypeptide compounds containing acryloyl groups
[0066] On-DNA compound 3 was dissolved in 100 mM, pH 7.5 Tris-HCl buffer to prepare a 0.5 mM On-DNA compound 3 solution. H2O2 (10 mM / H2O) was added, and the reaction was carried out at 0°C for 0.5 hours. After the reaction was complete, 5 M sodium chloride solution and cold ethanol were added to the reaction solution, and the mixture was incubated at -78°C for 0.5 hours, then centrifuged at 4°C to remove the supernatant. The solution was then lyophilized at room temperature for 60 min to remove residual solvent, yielding the product.
[0067] The H2O2 (10 mM / H2O) mentioned means: H2O2 is dissolved in H2O solution so that the H2O2 concentration is 10 mM.
[0068] In the On-DNA compound 3 The structure is shown in Formula II. Table 4 shows the structure and conversion rate of representative compounds before and after the reaction in Example 4.
[0069] Table 4. Structure and conversion rate of representative compounds in Example 4 before and after the reaction.
[0070] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.
Claims
1. A method for preparing an on-DNA polypeptide compound containing an acryloyl structure, characterized by, The preparation method comprises the following steps: S1, dissolving the on-DNA compound in a buffer solution to obtain an on-DNA compound solution; S2, performing an elimination reaction on the on-DNA compound solution to obtain a final reaction solution; S3, after cooling the final reaction solution, adding a separation agent, standing, centrifuging, discarding the supernatant, and freeze-drying to obtain a product; The on-DNA compound in step S1 has a structure shown in formula I: Formula I; wherein, is one or more of a single-stranded deoxyribonucleotide sequence, a double-stranded deoxyribonucleotide sequence, a single-stranded ribonucleotide sequence, a double-stranded ribonucleotide sequence; The linear or cyclic backbone structure consisting of chemical elements selected from the group consisting of: C, H, O, N, P, or S; and chemical bonds selected from the group consisting of: C-C, C=C, C-Y, C=Y, Y-Y, or Y=Y; wherein Y is independently selected from the group consisting of: H, O, N, P, or S; X is an oxygen atom, a sulfur atom or a selenium atom; R is a hydrogen atom, an aliphatic chemical structure or an aromatic chemical structure; The elimination reaction in step S2 comprises: adding one or more of component 1, component 2 and component 3 to the on-DNA compound solution, and reacting to obtain a final reaction solution; The component 1 is a solvent containing a catalyst; the component 2 is a solvent containing an additive; and the component 3 is a solvent containing a reagent; The catalyst is Pd(PPh3)4 and / or Pd(OAc)2; The additive is one or more of NaBH4, K2CO3, Et3N, NaOH, H2O2 and TBPH; The reagent is one or more of Mukaiyama's reagent, a dihalogen alkylating agent and a scavenger.
2. The production method according to claim 1, characterized by, The solvent in the component 1, the component 2 or the component 3 in step S2 is independently selected from an organic solvent and / or water; The organic solvent comprises one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran and 1,4-dioxane.
3. The preparation method according to claim 2, characterized in that, The organic solvent is acetonitrile or dimethylacetamide.
4. The preparation method according to claim 1, characterized in that, The component 1 in step S2 contains 5-20 mM of the catalyst; the component 2 contains 5-200 mM of the additive; and the component 3 contains 5-200 mM of the reagent.
5. The method of claim 1, wherein, The component 1 in step S2 contains 10 mM of the catalyst; the component 2 contains 10-100 mM of the additive; and the component 3 contains 10-100 mM of the reagent.
6. The method of claim 1, wherein, The reaction condition of the elimination reaction in step S2 comprises: 0-70℃ for 10 min-180 min.
7. The preparation method according to claim 1, characterized in that, The buffer solution in step S1 comprises one or more of HEPES buffer solution, MOPS buffer solution, Tris-HCl buffer solution, sodium phosphate dibasic-sodium phosphate monobasic buffer solution, sodium phosphate dibasic-potassium phosphate monobasic buffer solution, sodium phosphate monobasic-sodium hydroxide buffer solution and guanidine hydrochloride buffer solution; and the pH value of the buffer solution is 7.0-10.
0.
8. The preparation method according to claim 5, characterized in that, The buffer solution in step S1 is sodium phosphate dibasic-potassium phosphate monobasic buffer solution and / or Tris-HCl buffer solution.
9. The method of claim 1, wherein, R in step S1 is a hydrogen atom, a benzyl group, an ethylamine group, a phenyl group, , , , or .
10. An on-DNA polypeptide compound containing an acryloyl structure prepared by the preparation method in any one of claims 1-9.