On-DNA macrocyclic compound and preparation method thereof

Abstract

The invention provides an on-DNA macrocyclic compound and a preparation method thereof, and belongs to the technical field of DNA coding molecular libraries. The preparation method of the on-DNA macrocyclic compound provided by the invention comprises the following steps: dissolving an on-DNA compound in a buffer solution to obtain an on-DNA compound solution, and carrying out intramolecular deprotection-nucleophilic substitution / addition reaction under illumination of 300-400 nm to generate the on-DNA macrocyclic compound containing thioether bonds. Compared with a linear precursor of the macrocyclic molecule, the macrocyclic molecule can provide more rigid conformation and higher affinity with a protein target. The preparation method disclosed by the invention is compatible with nucleic acid, does not need to add extra reagents, also avoids a tedious protection-deprotection process, is simple and convenient to operate, uses cheap and easily available substrates, has strong universality, and can be applied to discovery of macrocyclic seedling compounds.

Description

Technical Field

[0001] This invention pertains to macrocyclic compound production technology, specifically DNA-encoded molecular library technology, and relates to an on-DNA macrocyclic compound and its preparation method. Background Technology

[0002] Cyclic peptides occupy an important position among drug molecule types due to their unique structure and biological activity. Furthermore, the intrinsic conformational constraints of cyclic peptides have attracted attention in supramolecular chemistry and molecular recognition. Due to their cyclic structure, cyclic peptides possess a certain degree of conformational constraint, exhibiting relative stability compared to linear peptides and stronger resistance to enzymatic degradation. This characteristic endows cyclic peptides with many important properties. Pharmacists have discovered that certain physiologically active linear peptides or their analogues, after head-tail or side-chain cyclization, yield cyclic peptide analogues that restrict and fix the molecular conformation, thereby enhancing their activity. The elucidation of the structures and successful synthesis of physiologically active cyclic peptides such as insulin, oxytocin, antibodies, and fungal toxins have led to rapid development in cyclic peptide medicinal chemistry. Some physiologically active cyclic peptides, such as insulin and cyclosporine A, have already been successfully applied clinically. With further in-depth research into the structure and properties of cyclic peptides, they will find even wider applications in supramolecular chemistry, bioorganic chemistry, and medicinal chemistry.

[0003] Cyclic peptides, through their diverse substituents and restricted conformations, can target deep pockets and superficial surface regions of target proteins with higher specificity and affinity, showing great potential, especially in regulating protein-protein interaction targets that are difficult to drug. With continuous advancements in synthetic techniques, various non-natural amino acids can be introduced into the cyclic peptide backbone, thereby enhancing its stability, membrane permeability, specificity, and affinity. Using traditional compound libraries for high-throughput screening to obtain lead compounds requires storing and processing thousands of compounds, and identifying molecules that specifically bind to target proteins is typically extremely labor-intensive. In contrast, the chemical structures of library molecules in DNA-encoded chemical libraries (DELs) are small organic molecules covalently linked to unique DNA tags. This technology can construct and screen unprecedentedly large-scale combinatorial compound libraries, enabling the efficient and low-cost discovery of many different affinity ligand molecules through affinity screening of protein targets and high-throughput sequencing decoding.

[0004] In the research of cyclic peptide drugs, DNA-encoded library technology (DELT) is an important tool. DELT uses DNA "tags" to label each small molecule to be screened, acting as "barcodes" to record the compound's structural information. This allows researchers to efficiently and accurately screen numerous candidate molecules, quickly identify lead compounds, and thus accelerate the drug discovery process. This technology can screen large libraries of small molecules in an efficient and cost-effective manner and can identify corresponding ligands for targets that are difficult to study using traditional methods.

[0005] The principle of DEL screening technology is to use a specific DNA sequence to label each small molecule compound in the reaction process, and to use combinatorial chemistry strategies to synthesize libraries of millions to tens of billions of compounds linked with the DNA sequence. DEL is then used for affinity screening with the target protein, eluting and removing compounds that do not bind to the target protein, thus obtaining a set of compounds that can bind to the protein. The DNA-labeled sequences of these small molecule compounds are amplified by PCR and then detected by gene sequencing. Because there is a one-to-one correspondence between DNA sequences and small molecule compounds, researchers only need to read the DNA sequence on the molecule that binds to the target protein to decode the corresponding molecular structure. This technology can quickly and efficiently complete the construction and screening of molecular libraries of hundreds of millions of molecules, demonstrating significant time and cost advantages in new drug discovery, and has attracted widespread interest and attention from academia and the pharmaceutical industry.

[0006] The importance of thioether bonds in cyclic peptides cannot be ignored. As a substitute for disulfide bonds, thioether bonds can improve the stability and bioactivity of cyclic peptides, and extend their half-life in vivo. The introduction of thioether bonds can be achieved through chemical synthesis or enzymatic reactions, providing more options for the structural optimization of cyclic peptides. However, the introduction and stability of thioether bonds remain a challenge in DNA-encoded compound library technology. Significant differences exist in the reaction conditions required between organic synthesis and on-DNA synthesis, which limits the application and development of thioether cyclic peptides in DELT. The former typically uses organic solvents with relatively high peptide concentrations (mM levels), while the latter must be carried out at lower peptide concentrations (μM levels), orthogonal protecting group systems, additives (reducing agents, etc., deprotecting agents), and aqueous buffers.

[0007] Existing technology CN114276411B discloses a solid-phase synthesis method for cyclic peptide compounds containing trisulfide bonds. This method, based on solid-phase synthesis, first couples a resin to the carboxyl group of an N-terminal amino acid protected by Fmoc. Then, following the polypeptide sequence, the amino acids are sequentially coupled via amide bonds. Subsequently, the thiol protecting group of cysteine ​​is removed, and the mixture is solid-phase cyclized with a sulfurizing reagent to form the target product. This method can obtain high-purity cyclic peptide compounds containing trisulfide bonds in high yield. Furthermore, the method is simple to operate, has a short synthesis cycle, and is low in cost, making it suitable for large-scale production of cyclic peptides containing trisulfide-bridged rings or polypeptide derivatives with trisulfide bonds as the key backbone in their core structure. The disadvantages of this method are that it is a solid-phase synthesis method, the synthesis process is cumbersome, and it is not easily compatible with nucleic acids.

[0008] Prior art CN104144695A provides novel peptidomyomorph macrocyclic compounds that can be used in competitive binding assays to identify substances that can bind to natural ligands of proteins or peptides mimicked by the peptidomyomorph macrocyclic compounds; it also provides the generation of antibodies against the peptidomyomorph macrocyclic compounds, which specifically bind to the peptidomyomorph macrocyclic compounds and their associated precursor peptides, such as p53; furthermore, this technology provides preventive and therapeutic methods for treating subjects at risk of or suffering from diseases related to abnormal (e.g., insufficient or excessive) expression or activity of molecules (including p53, MDM2, or MDMX). The disadvantages of this method are the complexity of the reaction substrate, the cumbersome reaction process, and the need to add additional reagents. Summary of the Invention

[0009] This invention addresses the shortcomings of existing technologies, such as the incompatibility of reaction systems with nucleic acids, the use of cumbersome protecting group strategies, and low reaction yields with many impurities. Through a new synthetic strategy and reaction conditions, utilizing liquid-phase reaction and photo-cleavable protecting groups, thioether bonds are stably and efficiently introduced into cyclic peptides, preparing on-DNA thioether cyclic peptide molecules with high chemoselectivity and reactivity, thereby promoting the development of cyclic peptide drugs.

[0010] This invention aims to provide a method for preparing on-DNA macrocyclic compounds and a product thereof. The method for preparing on-DNA macrocyclic compounds provided by this invention includes the following steps: dissolving the on-DNA compound in a buffer solution to obtain an on-DNA compound solution; performing a deprotection-nucleophilic substitution / conjugated addition tandem reaction under light of a certain wavelength; adding a separating agent; allowing to stand; centrifuging; discarding the supernatant; and lyophilizing to obtain the product. The preparation method of this invention, under nucleic acid-compatible conditions, allows a specific structure in the on-DNA polypeptide substrate to undergo a deprotection-nucleophilic substitution / conjugated addition tandem reaction, generating an intramolecular thioether bond to obtain the target on-DNA cyclic peptide compound. The preparation method of this invention is simple to operate, uses inexpensive and readily available substrates, and has strong universality. It can apply macrocyclic DNA-encoded molecular libraries to various targets, including screening for protein-protein interaction targets that are difficult to drug, to find cyclic peptide lead compounds with high affinity, high specificity, and high drugability.

[0011] Group definition: See reference (Carey, Francis A. Advanced organic chemistry / -5) th Definitions of standard chemical terms can be found in [ed[M].]. 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 can generally be carried out according to conventional methods well known in the art, based on the descriptions in the various summary and more specific literatures 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.

[0012] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0013] On one hand, the present invention provides a method for preparing an on-DNA macrocyclic compound containing a thioether structure, characterized in that the preparation method includes the following steps:

[0014] S1. Dissolve the on-DNA compound in a buffer solution to obtain an on-DNA compound solution;

[0015] S2. Under light irradiation, the on-DNA compound undergoes a deprotection-nucleophilic substitution / conjugated addition tandem reaction to obtain the final reaction solution;

[0016] S3. Add separating agent to the final reaction solution, let stand, centrifuge, discard the supernatant, freeze dry to obtain the product;

[0017] The on-DNA compound described in step S1 has the structure shown in Formula I:

[0018]

[0019] Preferably, the A single-stranded or double-stranded nucleotide chain obtained by polymerizing nucleotide monomers, whether artificially modified or unmodified.

[0020] Preferably, the 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.

[0021] Preferably, the It is a linear structure.

[0022] Preferably, the A linear framework structure composed of chemical elements or chemical bonds:

[0023] 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 includes any one of H, O, N, P or S.

[0024] Preferably, the It has a linear peptide structure.

[0025] Specifically, the S mentioned is a sulfur atom.

[0026] Preferably, R is a photocutable protective substrate.

[0027] Photosensitive protecting groups, also known as photoinstantaneous protecting groups (PPGs), are protecting groups that can be cleaved under light. Deprotection of photosensitive protecting groups does not require the addition of chemical reagents, and the process is usually very fast and clean, making it particularly suitable for biochemical and microbiological fields. There are various types of photoinstantaneous protecting groups, the most common being nitrobenzyl derivatives, quinolinyl PPGs, and anthraquinone photosensitive protecting groups. In addition, PPGs include other types such as acyl groups, ethers, acetals, and silyl ethers. These types are mainly used to protect functional groups such as hydroxyl and carboxyl groups, and have wide applications in organic synthesis, biomedicine, and other fields.

[0028] Preferably, R includes

[0029]

[0030] Preferably, R is

[0031]

[0032] Preferably, X includes

[0033]

[0034] Preferably, X is

[0035]

[0036] Preferably, the buffer solution in step S1 includes 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, or guanidine hydrochloride buffer, and the pH range of the buffer solution is 7.0-10.0.

[0037] Specifically, the buffer solution in step S1 is a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0038] Preferably, the wavelength range of the light in step S2 is 300nm-400nm.

[0039] Preferably, the reaction conditions for the deprotection-nucleophilic substitution / conjugation tandem reaction in step S2 include a reaction temperature of 10-30°C for 10-180 min.

[0040] Preferably, the reaction formula of the preparation method is as follows:

[0041]

[0042] in, This indicates that a cyclic structure of thioether is formed after the reaction.

[0043] On the other hand, the present invention provides an intramolecular thioether cyclic on-DNA cyclic peptide compound prepared by the above preparation method.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] This invention provides an on-DNA macrocyclic compound and its preparation method. The method involves a light-mediated deprotection-nucleophilic substitution / nucleophilic addition tandem reaction. The reaction conditions are mild, compatible with nucleic acids, require no additional reagents, and avoid the cumbersome protection-deprotection process. The operation is simple, and the substrates used are inexpensive, readily available, and widely applicable. The DNA-encoded cyclic peptide library constructed using this method can be used in fields such as the discovery of high-affinity lead compounds targeting difficult-to-drug targets, such as protein-protein interactions. Attached Figure Description

[0046] Figure 1 Schematic diagram of the reaction process.

[0047] Figure 2 In the embodiments A structural image.

[0048] Figure 3 Schematic diagram of the reaction process. Detailed Implementation

[0049] 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.

[0050] It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited. 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 to or equivalent to those described herein can 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.

[0051] Experimental reagents: On-DNA compounds 1 and 2 (synthesized in the laboratory; see Tables 1 and 2 for their specific structures).

[0052] Example 1: Preparation of on-DNA cyclic peptide molecules by deprotection-nucleophilic substitution tandem reaction under light irradiation

[0053] On-DNA compound 1 was dissolved in 100 mM, pH 10.0 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM On-DNA compound 1 solution. The solution was reacted at 25°C for 60 min under 365 nm light. After the reaction, 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 On-DNA product 1. The structural formulas of the compounds before and after the reaction are shown in Table 1, and the schematic diagram of the reaction process is shown in [Figure 1]. Figure 1 In On-DNA compound 1, Structure such as Figure 2 As shown.

[0054] Example 2: Preparation of on-DNA cyclic peptide molecules by deprotection-conjugated addition tandem reaction under light conditions

[0055] On-DNA compound 2 was dissolved in 100 mM, pH 10.0 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer to prepare a 0.5 mM On-DNA compound 2 solution. The solution was reacted at 25°C for 60 min under 365 nm light. After the reaction, 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 On-DNA product 2. The structural formulas of the compounds before and after the reaction are shown in Table 2, and the schematic diagram of the reaction process is shown in [Figure number missing]. Figure 3 In On-DNA compound 2, Structure such as Figure 2 As shown.

[0056] Example 1: Determination of On-DNA product conversion rate

[0057] The reaction conversion rates of the On-DNA products in Examples 1 and 2 were determined using the following methods.

[0058] 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 (…). 1.7 μm (1 mm x 100 mm), monitored 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.

[0059] Data visualization and integration were performed using Mass Lynx V4.1 software, followed by reaction 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.

[0060] Following the above method, the reaction conversion rate of Example 1 was calculated, and the results are shown in Table 1. It can be seen that the reaction conversion rates of different precursors are different, but all can achieve a high conversion rate.

[0061] Table 1. Structure and conversion rate of compounds before and after reaction.

[0062]

[0063]

[0064] Following the above method, the reaction conversion rate of Example 2 was calculated, and the results are shown in Table 2. It can be seen that the reaction conversion rates of different reaction precursors are different, but they can all achieve a high conversion rate.

[0065] Table 2. Structure and conversion rate of compounds before and after reaction.

[0066]

[0067]

[0068] 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 on-DNA macrocyclic compounds containing sulfide structures, 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. Under light irradiation, the on-DNA compound undergoes a deprotection-nucleophilic substitution / addition reaction to obtain the final reaction solution; S3. Add separating agent to the final reaction solution, let stand, centrifuge, discard the supernatant, freeze dry to obtain the product; The on-DNA compound described in step S1 has the structure shown in Formula I: Among them, the Including single-stranded deoxyribonucleic acid sequences, double-stranded deoxyribonucleic acid sequences, single-stranded ribonucleic acid sequences, or double-stranded ribonucleic acid sequences; The aforementioned It is a linear structure 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; the Y includes any one of H, O, N, P or S; S stands for sulfur atom; R is a photocutable protective group; X is the reaction site.

2. The preparation method according to claim 1, characterized in that, R in step S1 includes 3. The preparation method according to any one of claims 1 or 2, characterized in that, The R mentioned is 4. The preparation method according to claim 1, characterized in that, The X includes 5. The preparation method according to any one of claims 1 or 4, characterized in that, The X mentioned is 6. The preparation method according to claim 1, characterized in that, The wavelength range of the illumination in step S2 is 300nm-400nm.

7. The preparation method according to claim 1, characterized in that, The buffer solution mentioned in step S1 includes 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, or guanidine hydrochloride buffer, and the pH range of the buffer solution is 7.0-10.

0.

8. The preparation method according to claim 7, characterized in that, The buffer solution mentioned in step S1 is a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer.

9. The preparation method according to claim 1, characterized in that, The reaction conditions for the deprotection-nucleophilic substitution / addition reaction described in step S2 include a reaction temperature of 10-30℃ for 10-180 min.

10. The on-DNA macrocyclic compound prepared by the preparation method according to any one of claims 1-9.

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