Preparation method of on-DNA cyclopeptide compound containing pyrrole structure

Abstract

The invention belongs to the technical field of DNA coding molecular libraries, and particularly relates to a preparation method of an on-DNA cyclopeptide compound containing a pyrrole structure. The preparation method of the on-DNA cyclopeptide compound comprises the following steps: dissolving a reaction substrate in a buffer solution, adding an organic solvent, the buffer solution, inorganic alkali, organic alkali, an oxidizing agent, furan, diamine and dithiol to carry out cyclization reaction, adding a separating agent into a final reaction solution, standing, centrifuging, removing supernatant, and freeze-drying to obtain a product. The reaction is not influenced by substituent groups on furan, thiol and an amine reagent, the substrate is wide in universality, low in price and easy to obtain, and development of cyclopeptide compounds is promoted.

Description

Technical Field

[0001] This invention belongs to the field of DNA-encoded molecular library technology, specifically relating to a method for preparing an on-DNA cyclic peptide compound containing a pyrrole structure. Background Technology

[0002] Cyclic peptide molecules have a cyclic backbone of at least 12 atoms and contain multiple amino acid residues. Their molecular weight ranges from 500 to several thousand, allowing them to occupy chemical spaces between small and large molecules. The advantage of cyclic peptides in drug development lies in their ability to combine the best properties at both ends: cyclic peptides can be administered orally like small molecules, act across cell membranes to target intracellular sites, and also possess the excellent specificity and binding affinity of large antibody molecules. Therefore, in addition to being used as therapeutic and diagnostic drugs, cyclic peptides can also serve as carriers to deliver payloads to specific locations. Consequently, academia and industry are increasingly investing in and developing cyclic peptides to obtain ligands for challenging targets, particularly protein-protein interactions. The conformation of peptides plays a crucial role in their physiological activity. Compared to flexible linear peptides, the more rigid conformation of cyclic peptides plays a vital role in enhancing metabolic stability and improving binding affinity. The initial batch of cyclic peptide drug molecules were mainly derived from natural products and their derivatives, such as polymyxin B, vasopressin, and cyclosporine. To construct diverse libraries of DNA-encoded cyclic peptides, researchers have developed various on-DNA cyclization methods, such as amide and disulfide bonds present in natural molecules, more stable thioether bonds, S-aromatic ether bonds mimicking the O-aromatic ether bonds in natural products, and CuAAC and Thiol-Ene reactions in click chemistry. Among these, utilizing nucleophilic groups in two natural amino acid residues, such as amino and thiol groups, allows for the efficient construction of on-DNA cyclic peptide molecules through multi-component cyclization reactions, such as the multi-component cyclization reaction involving o-phthalaldehyde (OPA).

[0003] Developing more cyclization reactions not only increases the diversity of chemical structures but also provides a wider range of cyclic peptide conformations, increasing the probability of discovering lead compounds. Therefore, there is an urgent need to provide more convenient, universally applicable, inexpensive, and readily available on-DNA cyclization methods to broaden the application scope of cyclic peptide DNA-encoded molecular libraries. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing on-DNA cyclic peptide compounds containing pyrrole structures, which can be used to construct DNA-encoded compound libraries and enrich the types of chemical reactions for constructing DEL libraries.

[0005] 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 cyclic peptide compound containing a pyrrole structure, wherein the reaction route of the preparation method is as follows: ; Where n represents the amount of pyrrole produced in the reaction; For DNA; Linker represents the peptide chain of a cyclic peptide compound.

[0006] Specifically, n is 1 or 2.

[0007] To be more specific, It can be a single-stranded deoxyribonucleotide sequence or a double-stranded deoxyribonucleotide sequence.

[0008] Specifically, the Linker is selected from linear framework structures composed of chemical elements or chemical bonds; the chemical elements include: C, H, O, N, P or S; the chemical bonds include: CC, C=C, CY, C=Y, YY or Y=Y; and Y is independently selected from H, O, N, P or S.

[0009] Specifically, in Formula 1, A1 / A2 is a combination of SH / SH, NH2 / NH2, SH / NH2, or NH2 / SH; where SH is a thiol functional group and NH2 is an amino functional group.

[0010] In some embodiments, the preparation method of the on-DNA cyclic peptide compound containing a pyrrole structure includes the following steps: (1) Dissolve the on-DNA compound in a buffer solution to obtain an on-DNA compound solution; (2) Dissolve the reagent in a solvent to prepare a reagent solution, and then add it to the on-DNA compound solution to carry out the cyclization reaction to obtain the final reaction solution; (3) Add a separating agent to the final reaction solution, let it stand, centrifuge, discard the supernatant, freeze dry, and obtain the product.

[0011] Specifically, the preparation method also includes organic solvents, buffer solutions, inorganic bases, organic bases, oxidants, furans, diamines, and dithiols.

[0012] More specifically, the organic solvent includes one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, and 1,4-dioxane.

[0013] Preferably, the organic solvent is acetonitrile, and the volume ratio of acetonitrile to water is 5:1.

[0014] More specifically, the buffer solution includes 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.

[0015] Preferably, the buffer solution is a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0016] More specifically, the inorganic base includes one or more of NaOH, Na2CO3, NaHCO3, LiOH, KOH, K2CO3, and KHCO3.

[0017] Preferably, the inorganic base is NaHCO3.

[0018] More specifically, the organic base includes one or more of TEA, DIPEA, DBU, piperidine, pyridine, DABCO, morpholine, and N-Me morpholine.

[0019] Preferably, the organic base is pyridine.

[0020] More specifically, the oxidant includes one or more of O2, NBS, DMDO, cytochrome P450 enzyme, Mn(OAc)3, CoCl2, TPP and RB.

[0021] Preferably, the oxidant is NBS.

[0022] Specifically, the furan reagent is .

[0023] Specifically, the diamine reagent is one or more of the following chemical reagents containing two alkyl primary amine functional groups: , , , , , , , , , , , , , , , , , .

[0024] Specifically, the dithiol reagent is one or more of the following chemical reagents containing two alkylthiol functional groups: , , , , , .

[0025] Specifically, the preparation method contains 5-200mM furan, 5-200mM NaHCO3 solution, 5-200mM NBS, 5-200mM pyridine, 5-200mM diamine reagent and 5-200mM dithiol reagent.

[0026] Preferably, the preparation method contains 100-200mM furan, 100-200mM NaHCO3 solution, 100-200mM NBS, 100-200mM pyridine, 100-200mM diamine reagent and 100-200mM dithiol reagent.

[0027] Specifically, the reaction conditions for the preparation method are 0-30℃ for 10-180 minutes.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to develop an intramolecular multi-component cyclization reaction to generate on-DNA macrocyclic compounds containing pyrrole structures and its preparation method. Under nucleic acid-compatible conditions, an intramolecular multi-component cyclization reaction is carried out based on the nucleophilicity of amino and thiol groups and the electrophilicity of furan, generating stable pyrrole structures while simultaneously constructing cyclic peptide backbones. This reaction is unaffected by substituents on furan, thiol, and amine reagents, has broad substrate universality, and is inexpensive and readily available, making it suitable for constructing DNA-encoded cyclic peptide libraries and for the discovery of cyclic peptide lead compounds. Attached Figure Description

[0029] Figure 1 In the embodiments A structural image.

[0030] Figure 2 A schematic diagram of the reaction process in Example 1.

[0031] Figure 3 A schematic diagram of the reaction process in Example 2.

[0032] Figure 4 A schematic diagram of the reaction process in Example 3.

[0033] Figure 5 A schematic diagram of the reaction process in Example 4. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] Experimental reagents: On-DNA compounds 1-4 (synthesized in the laboratory, see Table 1-4 for specific structures).

[0037] Example 1: Preparation of On-DNA macrocyclic compound 1 containing pyrrole structure Equal amounts of furan and NaHCO3 were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. The above solution was then subjected to a 0... o The reaction was carried out at C for 15 min. Equal amounts of NBS were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. o Under C conditions, NBS solution was slowly added to furan / NaHCO3 solution and maintained at 0°C. o The reaction was continued at C for 10 min. Subsequently, twice the amount of pyridine was added to the above solution, and the reaction was carried out at room temperature for 4 hours.

[0038] 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 On-DNA compound 1 solution. This solution was then added to the above mixture, and the reaction was carried out at room temperature for 3 hours. After the reaction was complete, 5 M sodium chloride solution and cold ethanol were added to the reaction mixture. 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 minutes to remove residual solvent, yielding On-DNA product 1.

[0039] On-DNA compound 1 and On-DNA product 1 Structure such as Figure 1As shown in the diagram, the reaction is illustrated below. Figure 2 As shown, the structural formulas of the compounds before and after different reactions are shown in Table 1.

[0040] Example 2: Preparation of On-DNA macrocyclic compound 2 containing pyrrole structure Equal amounts of furan and NaHCO3 were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. The above solution was then subjected to a 0... o The reaction was carried out at C for 15 min. Equal amounts of NBS were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. o Under C conditions, NBS solution was slowly added to furan / NaHCO3 solution and maintained at 0°C. o The reaction was continued at C for 10 min. Subsequently, twice the amount of pyridine was added to the above solution, and the reaction was carried out at room temperature for 4 hours.

[0041] 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. This solution was then added to the above mixture, and the reaction was carried out at room temperature for 3 hours. After the reaction was complete, 5 M sodium chloride solution and cold ethanol were added to the reaction solution. 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 minutes to remove residual solvent, yielding On-DNA product 2.

[0042] On-DNA compound 2 and On-DNA product 2 Structure such as Figure 1 As shown in the diagram, the reaction is illustrated below. Figure 3 As shown, the structural formulas of the compounds before and after different reactions are shown in Table 2.

[0043] Example 3: Preparation of On-DNA macrocyclic compound 3 containing pyrrole structure Equal amounts of furan and NaHCO3 were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. The above solution was then subjected to a 0... o The reaction was carried out at C for 15 min. Equal amounts of NBS were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. o Under C conditions, NBS solution was slowly added to furan / NaHCO3 solution and maintained at 0°C. o The reaction was continued at C for 10 min. Subsequently, twice the amount of pyridine was added to the above solution, and the reaction was carried out at room temperature for 4 hours.

[0044] On-DNA compound 3 was dissolved in 100 mM, pH 7.5 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM solution. After adding the above mixture, the reaction was carried out at room temperature for 30 min. 1,3-Phenyldimethylamine was dissolved in an acetonitrile / water (5 / 1) mixture to prepare a 200 mM solution, which was added to the above reaction solution, and the reaction was continued at room temperature for 3 hours. After the reaction was completed, 5 M sodium chloride solution and cold ethanol were added to the reaction solution, and the mixture was placed 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 On-DNA product 3.

[0045] On-DNA compound 3 and On-DNA product 3 Structure such as Figure 1 As shown in the diagram, the reaction is illustrated below. Figure 4 As shown, the structural formulas of the compounds before and after different reactions are shown in Table 3.

[0046] Example 4: Preparation of On-DNA macrocyclic compound 4 containing pyrrole structure Equal amounts of furan and NaHCO3 were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. The above solution was then subjected to a 0... o The reaction was carried out at C for 15 min. Equal amounts of NBS were dissolved in a 5 / 1 mixture of acetonitrile and water to prepare a 100 mM solution. o Under C conditions, NBS solution was slowly added to furan / NaHCO3 solution and maintained at 0°C. o The reaction was continued at C for 10 min. Subsequently, twice the amount of pyridine was added to the above solution, and the reaction was continued at room temperature for 4 hours. 1,3-propanedithiolamine was dissolved in a 200 mM solution in a 5 / 1 mixture of acetonitrile and water, and added to the above reaction solution. The reaction was continued at room temperature for 30 min.

[0047] On-DNA compound 4 was dissolved in 100 mM, pH 7.5 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM solution of On-DNA compound 3. After adding the above mixture, the reaction was carried out at room temperature for 3 hours. After the reaction was completed, 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 minutes to remove residual solvent, yielding On-DNA product 4.

[0048] On-DNA compound 4 and On-DNA product 4 Structure such as Figure 1 As shown in the diagram, the reaction is illustrated below. Figure 5As shown, the structural formulas of the compounds before and after different reactions are shown in Table 4.

[0049] Example 1: Determination of On-DNA product conversion rate The reaction conversion rate of the On-DNA product was determined using the following method: On-DNA product sample preparation: The on-DNA product was dissolved in 15 μL of H2O to prepare a 0.03 mM aqueous solution. The on-DNA product sample was detected using a time-of-flight LC-MS / MS system (Agilent 6230B). 8 μL of the 0.03 mM solution was injected into an ACQUITY UPLCBEH C18 column (130 Å, 1.7 μm, 1 mm x 100 mm), and monitoring was performed at 260 nm. Solvent A: Deionized water containing 2.25% hexafluoroisopropanol (v / v) and 0.114% triethylamine (v / v). Solvent B: 90% methanol aqueous solution containing 2.25% hexafluoroisopropanol (v / v) and 0.114% triethylamine (v / v). Flow rate: 0.45 mL / min; Time: 6.00 min.

[0050] Data visualization and integration were performed using Mass Lynx V4.1 software, followed by conversion calculation via deconvolution. Specifically, the DNA product conversion rate was determined by integrating the UV absorbance (260 nm) peak area using the formula "Conversion Rate % = Target Product UV Peak Area / Total Recovered DNA UV Peak Area", ignoring differences in UV extinction coefficients among DNA species and assuming a 100% DNA recovery rate. Any non-oligonucleotide substances with UV absorbance (260 nm) were subtracted from the conversion rate calculation.

[0051] Following the above method, the reaction conversion rate of Example 1 was calculated, and the results are shown in Table 1. The reaction conversion rates of different precursors were different, but all of them could achieve a high conversion rate.

[0052] Table 1. Structures and reaction conversion rates of compounds before and after different reactions in Example 1.

[0053] Following the above method, the reaction conversion rate of Example 2 was calculated, and the results are shown in Table 2. The reaction conversion rates of different reaction precursors are different, but all can achieve a high conversion rate.

[0054] Table 2. Structures and reaction conversion rates of compounds before and after different reactions in Example 2.

[0055] Following the above method, the reaction conversion rate of Example 3 was calculated, and the results are shown in Table 3. The reaction conversion rates of different precursors were different, but all of them could achieve a high conversion rate.

[0056] Table 3. Structures and reaction conversion rates of compounds before and after different reactions in Example 3.

[0057] Following the above method, the reaction conversion rate of Example 4 was calculated, and the results are shown in Table 4. The reaction conversion rates of different reaction precursors are different, but all can achieve a high conversion rate.

[0058] Table 4. Structures and reaction conversion rates of compounds before and after different reactions in Example 4.

[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an on-DNA cyclic peptide compound containing a pyrrole structure, characterized in that, The reaction route of the preparation method is as follows: ； Where n represents the amount of pyrrole produced in the reaction, and n is 1 or 2; For DNA; Linker represents the peptide chain of a cyclic peptide compound.

2. The preparation method according to claim 1, characterized in that, The Linker is selected from linear framework structures composed of chemical elements or chemical bonds; The chemical elements include: C, H, O, N, P or S; the chemical bonds include: C=C, C=C, CY, C=Y, YY or Y=Y; and Y is independently selected from H, O, N, P or S.

3. The preparation method according to claim 1, characterized in that, In Formula 1, A1 / A2 is a combination of SH / SH, NH2 / NH2, SH / NH2, or NH2 / SH; where SH is a thiol functional group and NH2 is an amino functional group.

4. The preparation method according to claim 1, characterized in that, The preparation method further includes organic solvents, buffer solutions, inorganic bases, organic bases, oxidants, furans, diamines, and dithiols.

5. The preparation method according to claim 4, characterized in that, The organic solvents include one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butanol, isopropanol, tetrahydrofuran, and 1,4-dioxane.

6. The preparation method according to claim 4, characterized in that, The buffer solution includes 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.

7. The preparation method according to claim 4, characterized in that, The inorganic base includes one or more of NaOH, Na2CO3, NaHCO3, LiOH, KOH, K2CO3, and KHCO3.

8. The preparation method according to claim 4, characterized in that, The organic bases mentioned include one or more of TEA, DIPEA, DBU, piperidine, pyridine, DABCO, morpholine, and N-Me morpholine.

9. The preparation method according to claim 4, characterized in that, The oxidizing agents include one or more of O2, NBS, DMDO, cytochrome P450 enzyme, Mn(OAc)3, CoCl2, TPP and RB.

10. The preparation method according to claim 1, characterized in that, The reaction conditions for the preparation method include: reaction at 0-30℃ for 10-180 min.

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