Chemical ring closure construction method of cyclic compound library and cyclic compound library

Abstract

The application provides a chemical ring-closing construction method of a cyclic compound library and the cyclic compound library, which is synthesized by using a solid carrier, a molecule containing a photo-cleavable group, a connecting molecule, a synthetic building block and a ring-closing end molecule at one end. The method of the application has mild ring-closing conditions and stronger universality, and expands the chemical reaction type of the coded compound library and the diversity space of the compound library.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicinal chemistry, in particular to a method for constructing a chemical ring-closing library of cyclic compounds and a library of cyclic compounds. BACKGROUND

[0002] Advances in disease characterization and target identification are continually moving drug discovery into uncharted territory, with an increasing number of emerging targets of interest from a pathological perspective, but with the challenge of modulating them with established low-molecular-weight compounds or biologies. One approach to address this is to build libraries of compounds, which can in principle be bioavailable, allow cell penetration, and be suitable for binding in defined proteins (e.g. enzymes) from traditional libraries of small molecules.

[0003] Compounds derived from DNA-encoded libraries (DEL) resemble small molecules, with the opportunity to screen up to 10 9 or more potential chemical entities. This greatly increases the chance of finding new chemical starting points. However, small molecules often perform poorly in countering protein-protein interactions. The interactions of proteins often have large and extended interaction surfaces, on the other hand, biologies are better suited for such applications and are often very selective and have stronger binding.

[0004] Cyclic peptides have attracted considerable attention as a promising class of therapeutic candidates. They have several important advantages over their linear counterparts. The conformational freedom of cyclic peptides is often restricted by macrocyclization, enabling the cyclic molecules to bind more tightly and specifically to target proteins by minimizing the entropy upon binding. In addition, cyclic peptides are more stable than linear molecules under conditions of hydrolysis by proteases. Furthermore, due to their relatively large size, cyclic peptides can be suitable for effectively covering large and shallow interfaces involved in protein-protein interactions, which are not easily targeted by traditional drug-like small molecules.

[0005] Currently, reference 1 (Min Hyeon Shin, et al. Bioconjugate Chemstry, 2019, 30, 2931-2938) reports the establishment of a DNA-encoded cyclic peptidomimetic library and describes the method for establishing a DNA-encoded cyclic peptidomimetic library, but the synthesis process needs to rely on chloroacetic acid to introduce synthetic building blocks at each step, and the synthetic building blocks are only primary amine small molecule compounds with a single active group -NH2. As a result, the number and type of atoms between each synthetic building block are fixed and repetitive -CO-CH2-N- structural units, and the diversity of the compound library is insufficient. A large number of previous drug research and development tests have shown that the length and type of carbon atoms between drugs can greatly change the solubility and permeability of drugs; and the cyclic peptide library is mainly used to screen the binding ability of PPI, and the binding ability of PPI mainly relies on intramolecular or intermolecular hydrogen bonds formed by N atoms or oxygen atoms. The N atom formed by the connection of the synthetic building blocks in the cyclic compound library of reference 1 is a tertiary amine, which is not conducive to protein binding or has insufficient binding force. Therefore, the compound library is very limited and cannot meet the synthesis of a diverse cyclic compound library required by modern drug research and development. In addition, the difficulty in constructing a cyclic compound library lies in the reaction of the long-chain structure at both ends to form a ring. Since the compound library is established under the premise of carrying a DNA sequence, the ring-closing reaction has high restrictions. Metal ions are used in the ring-closing reaction of reference 1, which may chelate with DNA molecules and adversely affect the stability of DNA molecules, limiting the stability and accuracy of the screening results.

[0006] Therefore, there is a need in the art to establish a ring-closing method with milder ring-closing reaction conditions and stronger universality, and other cyclic molecular structures other than peptidomimetics, to obtain a more diverse cyclic compound library and increase the diversity of the compound library. In addition, there is a need in the art to establish a compound library and target protein binding complex technology to directly screen targeted proteins for research and increase the use of the compound library. SUMMARY

[0007] One of the technical problems to be solved by the present application is to provide a chemical reaction ring-closing construction method for a cyclic compound library, which has the advantages of milder ring-closing reaction conditions and strong universality, can be used for the construction of single-ring and double-ring cyclic compound libraries, overcomes the limitations of the ring-closing method of the prior art cyclic compound library, and has fewer side reactions.

[0008] The second technical problem to be solved by the present application is to provide a new cyclic compound library with a cyclic molecular structure to increase the diversity of the compound library and expand its application range in new drug screening.

[0009] To solve the above technical problems, the present application further provides a first technical solution:

[0010] A method for constructing a library of cyclic compounds, comprising the following steps:

[0011] 1) Directly or indirectly connecting a solid support G with a molecule M containing a photocleavable group to obtain G-M:

[0012] 2) Proceeding as follows:

[0013] a1. Reacting G-M with a linking molecule L1 having at least three functional groups to obtain G-M-L1;

[0014] b1. Reacting G-M-L1 with a starting nucleotide molecule HP, an open primer OP in sequence to connect the starting nucleotide molecule HP with the solid support G to obtain OP-HP-G-M-L1;

[0015] c1. Reacting OP-HP-G-M-L1 with a synthetic building block C1, a DNA tag tag1 corresponding to the synthetic building block C1 to connect the synthetic building block C1 with L1 and the DNA tag tag1 with OP to obtain tag1-OP-HP-G-M-L1-C1;

[0016] d1. Defining a set of corresponding synthetic building blocks and DNA tag connection reactions as an extension step, repeating the extension step to make the synthetic building blocks sequentially spliced to form an extended chain and the DNA tags corresponding to the synthetic building blocks sequentially spliced to form an extended chain to obtain tag n ……—tag1-OP-HP-G-M-L1-C1……—C n ; wherein the last synthetic building block C n has a first ring-closing functional group: primary amino group (NH2-), secondary amino group (-NH-), hydroxyl group (OH-), or urea group (NH2CONH-); 2≤n≤7 and n is an integer;

[0017] e1. Reacting the product obtained in the previous step with a blocking primer CP to connect the blocking primer CP with tag n , wherein HP-OP —tag1—……—tag n -CP forms a complete DNA coding sequence to obtain DNA-G-M-L1-C1……—C n ;

[0018] f1. Reacting the product obtained in the previous step with a ring-closing end molecule A to connect the ring-closing end molecule A with L1 to obtain DNA-G-M-L1(-C1……—C n )-A, i.e. a compound library S1'; the ring-closing end molecule A has a second ring-closing functional group

[0019] 3) Compound library S1' was subjected to an intramolecular cyclization reaction in HEPES buffer solution at 20℃~40℃ to synthesize the final building block C. n The first ring-closed functional group reacts with the second ring-closed functional group of the ring-closed end molecule A to form a ring, yielding... That is, the cyclic compound library S1.

[0020] In the first technical solution provided by this invention, by connecting the three ends of the connecting molecule L1 to the ring-closed end molecule A, the extended chain formed by the splicing of the synthetic building blocks, and the solid support, the final synthetic building block C is utilized. n The first cyclic functional group reacts with the second cyclic functional group of the cyclic terminal molecule A to form a ring, obtaining a cyclic compound library, with DNA encoded on a solid-phase support. All synthetic steps in this scheme are performed on a solid-phase support, ensuring higher quality of the compound library. The compound library obtained by this scheme is suitable for screening using flow cytometry fluorescence sorting (FACS). The screened cyclic compounds do not require high-throughput sequencing; they can be directly detached from the solid-phase support using a photolysis reaction, resulting in a short screening cycle and high efficiency.

[0021] To solve the above-mentioned technical problems, the present invention provides a second technical solution:

[0022] A method for constructing a cyclic compound library includes the following steps:

[0023] 1) By directly or indirectly linking a solid support G to a molecule M containing a photolytically cleavable group, G-M is obtained:

[0024] 2) Follow these steps:

[0025] a2. G-M is reacted with a linker molecule L1 having at least four functional groups to obtain G-M-L1;

[0026] b2. G-M-L1 is sequentially reacted with the starting nucleotide molecule HP and the opening primer OP, wherein the starting nucleotide molecule HP is connected with the linker molecule L1 to obtain G-M-L1-HP-OP;

[0027] c2. React the product obtained in the previous step with synthetic building block C1 and the DNA tag tag1 corresponding to synthetic building block C1, respectively, so that synthetic building block C1 is linked to L1 and DNA tag1 is linked to OP, to obtain G—M—L1(—HP—OP—tag1)—C1.

[0028] d2. define a set of corresponding synthetic building blocks and DNA tags as an extension step, repeat the extension step to make the synthetic building blocks sequentially ligate to form an elongated chain and the DNA tags corresponding to the synthetic building blocks sequentially ligate to form an elongated chain, to obtain G-M-L1(—HP—OP—tag1—……—tag n )—C1—……—C n ; wherein 2≤n≤7 and n is a positive integer;

[0029] e2. react the product obtained in step d2 with a blocking primer CP to make the blocking primer CP ligate to tag n , wherein HP—OP —tag1—……—tag n —CP forms a complete DNA code sequence, to obtain G-M-L1(—DNA)—C1—……—C n ;

[0030] f2. react the product obtained in step e1 with a ring closure end molecule A to make the ring closure end molecule A ligate to L1, to obtain G-M-L1(—DNA)(—C1……—C n )—A, i.e. a compound library S2'; the ring closure end molecule A has a second ring closure functional group

[0031] 3) perform intramolecular ring closure reaction of the compound library S2' in HEPES buffer solution at 20-40°C to make the last synthetic building block C n react with the ring closure end molecule A to form a ring, to obtain i.e. a cyclic compound library S2.

[0032] In the second technical solution provided by the present application, the ring closure end molecule A, the elongated chain formed by the mutual ligation of the synthetic building blocks, and the solid carrier are connected at the three ends of the connecting molecule L1, respectively, the last synthetic building block C nThe first cyclic functional group reacts with the second cyclic functional group of the cyclic-terminal molecule A to form a ring, obtaining a cyclic compound library. The resulting cyclic compounds can be detached from the solid-phase support, and DNA is encoded in the compounds. The compound library obtained by this method can be further screened by cleaving molecules M containing photocleavable groups. The secondary screening involves: first, a first screening on the solid-phase support; then, cleaving molecules M containing photocleavable groups to detach the compounds from the solid-phase support, at which point DNA is encoded in the compound, and the resulting compound library can be screened a second time. Different screening techniques can be combined for the secondary screening, such as flow cytometry fluorescence sorting (FACS), traditional target protein affinity screening, AS-MS screening, or any combination of two, to improve the accuracy of the screening results. The compound library obtained by this method is suitable for the traditional DEL screening mode, and the screened compounds need to be sequenced using high-throughput sequencing.

[0033] The cyclic compound library S2 can be cleaved by photo-irradiation to obtain molecule M containing a photocleavable group, thus yielding...

[0034] This refers to the cyclic compound library S2'. This cyclic compound library S2' can be used for the aforementioned second screening.

[0035] Furthermore, to address the aforementioned technical problems, this invention also provides another method for constructing a compound library with a bicyclic structure, namely, the third technical solution:

[0036] A method for constructing a cyclic compound library includes the following steps:

[0037] 1) By directly or indirectly linking a solid support G to a molecule M containing a photolytically cleavable group, G-M is obtained:

[0038] 2) Follow these steps:

[0039] a3. G-M is reacted with a linker molecule L1 with at least five functional groups to obtain G-M-L1;

[0040] b3. G-M-L1 reacts sequentially with the starting nucleotide molecule HP and the opening primer OP, causing the starting nucleotide molecule HP to be linked to the solid support G, resulting in OP-HP-G-M-L1;

[0041] c3.OP—HP—G—M—L1 reacts with synthetic building block C1 and the DNA tag tag1 corresponding to synthetic building block C1, so that synthetic building block C1 is linked to L1 and DNA tag tag1 is linked to OP, resulting in tag1—OP—HP—G—M—L1—C1.

[0042] d3. Defining a set of corresponding ligation reactions of synthetic building blocks and DNA tags as an extension step, repeating the extension step to sequentially assemble the synthetic building blocks to form an elongated chain and to sequentially assemble the DNA tags corresponding to the synthetic building blocks to form an elongated chain, to obtain tag n —tag1—OP—HP—G—M—L1—C1……—C n ; wherein the last synthetic building block C n has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); 0≤n≤7 and n is an integer;

[0043] e3. Reacting the product obtained in the previous step with ring closure end molecule A, the DNA tag tag A corresponding to ring closure end molecule A, to link ring closure end molecule A to L1 and to link tag A to tag n , to obtain tag A —tag n —……—tag1—OP—HP—G —M—L1(—C1……—C n )—A; ring closure end molecule A has a second ring closure functional group

[0044] f3. Reacting the product obtained in the previous step in an intramolecular ring closure reaction at 20-40°C in a HEPES buffer solution to form a ring between the first ring closure functional group of the last synthetic building block C n and the second ring closure functional group of ring closure end molecule A, to obtain

[0045] g3. Reacting the product obtained in the previous step according to the extension step with synthetic building blocks C n+1 , …, C n+m and their corresponding DNA tags tag n+1 , …, tag n+m , to link synthetic building blocks C n+1 to L1 and to link DNA tags tag n+1 to tag A , to obtain wherein the last synthetic building block C n+m has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); 0≤m≤7 and m is an integer, 2≤n+m≤7;

[0046] h3. Reacting the product obtained in the previous step with blocking primer CP, to link blocking primer CP to tag n+mare connected, wherein HP— OP— tag1—……—tag n+m —CP forms a complete DNA coding sequence, obtaining

[0047]

[0048] i3. The product obtained in the previous step is reacted with a ring closure end molecule A, so that the ring closure end molecule A is connected to L1, obtaining

[0049] i.e. a compound library S3'; the ring closure end molecule A has a second ring closure functional group

[0050] 3) The product obtained in the previous step is subjected to an intramolecular ring closure reaction in a HEPES buffer solution at 20-40°C, so that the first ring closure functional group of the last synthetic building block C n+m reacts with the second ring closure functional group of the ring closure end molecule A to form a ring, obtaining i.e. a cyclic compound library S3 having a bicyclic structure.

[0051] In the construction method of the above-mentioned compound library having a bicyclic structure provided by the present application, the ring closure end molecule A and the extended chain formed by the mutual splicing of the synthetic building blocks from C1 to C n are connected to the two ends of the linking molecule L1 of at least five functional groups, respectively, they react to form a ring to constitute a first ring structure, and the ring closure end molecule A and the extended chain formed by the mutual splicing of the synthetic building blocks from C n+1 to C n+m are connected to the two ends of the linking molecule L1 of at least five functional groups, respectively, they react to form a ring to constitute a second ring structure, and one end is connected to a solid carrier and a DNA coding sequence, to obtain a cyclic compound library having a bicyclic structure. In this scheme, the DNA is coded on the solid carrier, and all the synthesis steps are completed on the solid carrier, so that the quality of the compound library is more guaranteed. The compound library obtained by this scheme can be applied to the flow cytometry fluorescence sorting technology (FACS) screening, and the screened cyclic compound does not need high-throughput sequencing, but can be directly separated from the solid carrier by a decomposition reaction under light, so that the screening cycle is short and the efficiency is high.

[0052] Further, to solve the above-mentioned technical problems, the present application further provides a construction method of a compound library having a bicyclic structure, i.e. a fourth technical solution:

[0053] A construction method of a cyclic compound library, comprising the following steps:

[0054] 1) A solid carrier G is directly or indirectly connected to a molecule M containing a light-cleavable group, obtaining G—M:

[0055] 2) The following steps are performed:

[0056] a4. G-M is reacted with a linker molecule L1 having at least six functional groups to obtain G-M-L1;

[0057] b4. G-M-L1 is sequentially reacted with the starting nucleotide molecule HP and the opening primer OP, wherein the starting nucleotide molecule HP is connected with the linker molecule L1 to obtain G-M-L1-HP-OP;

[0058] c4. React the product obtained in the previous step with synthetic building block C1 and the DNA tag tag1 corresponding to synthetic building block C1, respectively, so that synthetic building block C1 is linked to L1 and DNA tag1 is linked to OP, to obtain G—M—L1(—HP—OP—tag1)—C1.

[0059] d4. Define a set of corresponding synthetic building blocks and DNA tags as an extension step. Repeat the extension step to sequentially assemble the synthetic building blocks into an extended strand, and sequentially assemble the DNA tags corresponding to the synthetic building blocks into an extended strand, to obtain G—M—L1(—HP—OP—tag1—……—tag n )—C1—……—C n Among them, the final composite block C n It has a first cyclic functional group: primary amino (NH2—), secondary amino (—NH—), hydroxyl (OH—), or urea (NH2CONH—); 2≤n≤7 and n is an integer;

[0060] e4. The product obtained in step d4 is combined with the DNA tag corresponding to the closed-loop molecule A. A The reaction connects the ring-closed molecule A to L1, and also causes the tag to... A with tag n Connecting them, we get G—M—L1(—HP—OP—tag1—…… —tag n —tag A (—C1……—C) n —A; the ring-closed end molecule A has a second ring-closed functional group.

[0061] f4. The product obtained in the previous step is subjected to an intramolecular cyclization reaction in HEPES buffer solution at 20℃~40℃, so that the final building block C is synthesized. n The first ring-closed functional group reacts with the second ring-closed functional group of the ring-closed end molecule A to form a ring, yielding...

[0062] g4. Combine the product obtained in the previous step with the synthetic building block C according to the extended steps described above. n+1 , ..., Cn+m and its corresponding DNA tag tag n+1 ,..., tag n+m sequentially, to synthesize the building block C n+1 connected with L1 and DNA tag tag n+1 connected with tag A to obtain wherein the last building block C n+m has a first ring-closing functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); 0≤m≤7 and m is an integer, 2≤n+m≤7;

[0063] h4. reacting the product obtained in the last step with a blocking primer CP, to connect the blocking primer CP with tag n+m , wherein HP—OP—tag1—...—tag n+m —CP forms a complete DNA coding sequence, to obtain

[0064]

[0065] i4. reacting the product obtained in the last step with a ring-closing end molecule A, to connect the ring-closing end molecule A with L1, to obtain

[0066] i.e. a compound library S4'; the ring-closing end molecule A has a second ring-closing functional group

[0067] 3) performing intramolecular ring-closing reaction on the product obtained in the last step in HEPES buffer solution at 20℃-40℃, to react the first ring-closing functional group of the last building block C n+m with the second ring-closing functional group of the ring-closing end molecule A to form a ring, to obtain i.e. a cyclic compound library S4 having a double ring structure.

[0068] In the construction method of the above-mentioned compound library having a double ring structure provided by the present application, the ring-closing end molecule A and the extended chain formed by splicing the building blocks C1 to C n are connected at both ends of the connecting molecule L1 having at least six functional groups, respectively, and they react to form a first ring structure, and the ring-closing end molecule A and the extended chain formed by splicing the building blocks C n+1 to C n+mThe extended chains of the synthetic building blocks are spliced together to form an elongated chain, which reacts into a ring to form a second ring structure, and the two ends are connected to a solid support and a DNA coding sequence, respectively, to obtain a circular compound library with a double ring structure. The final obtained circular compound can be detached from the solid support, and the DNA code is on the compound. The compound library obtained by this scheme can be subjected to secondary screening by cleaving the molecule M containing the photolabile group. Secondary screening includes: first screening on a solid support, and then cleaving the molecule M containing the photolabile group to detach the compound from the solid support, at which time the DNA code is on the library compound, and the obtained compound library can be subjected to secondary screening. Secondary screening can be combined with different screening techniques, such as flow cytometry fluorescence sorting technology (FACS) screening, traditional target protein affinity screening, AS-MS screening, etc. Any two of them are combined to improve the accuracy of the screening results. The compound library obtained by this scheme is suitable for traditional DEL screening mode, and the screened compound needs to be subjected to high-throughput sequencing.

[0069] The circular compound library S4 can be subjected to a photoreaction to cleave the molecule M containing the photolabile group to obtain

[0070] That is, a circular compound library S4”. The circular compound library S4’ can be used for the aforementioned secondary screening.

[0071] In the four technical solutions provided in the present application, HEPES refers to 2-(4-(2-hydroxyethyl)piperazino)ethanesulfonic acid, CAS No. 7365-45-9.

[0072] In the four technical solutions provided in the present application, the solid support G is selected from any one or more of PEG resin, PEGA resin, TentaGel resin, and solid support CPG.

[0073] In the second and fourth technical solutions, the solid support G contains an active functional group R1. The solid support G can be represented by a general formula G0-R1, wherein R1 represents the active functional group of the solid support G; G0 represents the other solid support structure of the solid support G except the active functional group. The solid support G is connected to the molecule M containing the photolabile group directly or indirectly through the active functional group R1. R1 can be selected from: amino, carboxyl, or hydroxyl. In a preferred embodiment, the solid support G is selected from a solid support with an amino active functional group; that is, preferably, the active functional group R1 is selected from amino, such as primary amino, secondary amino. More preferably, the active functional group R1 is selected from primary amino. Preferably, the solid support is selected from PEGA.

[0074] In the first and third technical solutions, the solid support G contains two active functional groups R1 and R1'. The solid support G can be represented by the general formula R1'-G0-R1, wherein R1 represents an active functional group for directly or indirectly connecting the molecule M containing a photocleavable group, and R1' represents an active functional group for connecting the DNA coding sequence. In a preferred embodiment, R1 is an amino group, and R1' is a carboxyl group.

[0075] In the four technical solutions provided by the present application, the molecule M containing a photocleavable group contains at least two active functional groups, which are represented by R2 and R3, respectively. The molecule M containing a photocleavable group can be represented by the general formula R2-M0-R3, wherein R2 and R3 represent two active functional groups independent of each other. R2 is an active functional group responsible for connecting the solid support G, and R3 is an active functional group responsible for connecting the linker molecule L1. R2 and R3 exist in a form protected by a protecting group or a form not protected by a protecting group, and R2 and R3 do not interfere with each other's connection reaction. Among them, mainly R3 does not interfere with the reaction process of R2 connecting with the solid support G, or R3 is in a form protected by a protecting group when R2 connects with the solid support G.

[0076] In a preferred embodiment, R2 exists in a form not protected by a protecting group, and R3 exists in a form protected by a protecting group. After G-M is obtained by connecting R2 with the solid support G, the protecting group of R3 needs to be removed and then subsequent reactions are performed.

[0077] There are two ways for connecting the solid support G with the molecule M containing a photocleavable group. One way is that R2 and R1 directly complementarily pair to obtain G-M. Complementarily pairing refers to that two active functional groups react to connect the chemical structures in which the two active functional groups are located in the form of a covalent bond, such as connecting two molecules in the form of a covalent bond to form one molecule. Based on different reaction principles, R1 and R2 can be reacted to remove all or part of fragments, such as removing one molecule of water after R1 and R2 are connected; or R1 and R2 can be reacted without removing any fragments, such as addition reaction. When R1 and R2 directly complementarily pair, the following combinations can be selected: amino and carboxyl, amino and hydroxyl, amino and phosphoric acid group, amino and alkyl halide or aryl halide, etc.

[0078] In another way, R2is connected to R1via another bifunctional linker. For example, the bifunctional linker has two reactive groups; the first reactive group can react with the reactive group R1of the solid support G, and the second reactive group can react with the reactive group R2of the molecule M containing a photocleavable group. The reaction can be carried out in two ways: first, the first reactive group reacts with the reactive group R1of the solid support G, and then the second reactive group reacts with the reactive group R2of the molecule M containing a photocleavable group; second, the second reactive group reacts with the reactive group R2of the molecule M containing a photocleavable group, and then the first reactive group reacts with the reactive group R1of the solid support G, to obtain the indirect connection of G— M. In the indirect connection mode, it is no longer required that R2and R1have a complementary pairing relationship.

[0079] In the molecule M containing a photocleavable group, the photocleavable group is preferably:

[0080]

[0081] wherein R3is the C atom of the side chain directly connected to the benzene ring at the ortho position of the nitro group; R2is connected to the C atom of the benzene ring through one or more covalent bonds, or R2is connected to the C atom to which R3is connected through one or more covalent bonds; the benzene ring can contain zero, one or more side chains or substituents that do not interfere with the connection reaction of R2and R3. R2is connected to the C atom of the benzene ring through one or more covalent bonds, or R2is connected to the C atom to which R3is connected through one or more covalent bonds; the benzene ring can contain zero, one or more side chains or substituents that do not interfere with the connection reaction of R2and R3.

[0082] Under suitable light conditions, the chemical bond between R3and the C atom to which R3is connected in the molecule M containing a photocleavable group can be broken. The molecule M containing a photocleavable group can be cleaved at 365 nm.

[0083] In some preferred embodiments, the molecule M containing a photocleavable group can be selected from the following structures:

[0084]

[0085] In the above structures, R3may be selected from -OH, -NH2, -NHNH2, -N3, Cl, Br, etc.

[0086] In the above structures, R2is represented by a carboxyl group.

[0087] In some specific embodiments, the solid support G and the molecule M containing a photocleavable group can be indirectly connected via a small molecule compound having a bifunctional group.

[0088] In some embodiments, the solid support G has an active functional group of amino group, and the small molecule compound with dual functional groups is a small molecule compound with active functional groups of carboxyl group and amino group protected by a protecting group. The solid support G is connected with the small molecule compound with dual functional groups by reacting to form an amide bond; the reaction solvent is an organic solvent, such as dichloromethane, N,N-dimethylformamide; the reaction temperature is 15-30°C, preferably 20-25°C; and the reaction time is 1-12 hours, preferably 2-6 hours. Then, the amino group of the small molecule compound with dual functional groups is deprotected, and then connected with the molecule M containing a photocleavable group; the protecting group of the amino group is preferably a Fmoc protecting group; the deprotection reaction solvent is an organic solvent, such as dichloromethane, N,N-dimethylformamide, and piperidine is added to the reaction solvent; the reaction temperature is 15-30°C, preferably 20-25°C; and the reaction time is 1-12 hours, preferably 1-6 hours. Then, the deprotection is followed by connection with the molecule M containing a photocleavable group; the small molecule compound with dual functional groups after deprotection provides an active functional group of amino group, and the molecule M containing a photocleavable group provides an active functional group of carboxyl group, and the reaction is connected by forming an amide bond; the reaction solvent is an organic solvent, such as N,N-dimethylformamide; one or more of N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP) are added to the reaction solvent; the reaction temperature is 15-30°C, preferably 20-25°C; and the reaction time is 1-12 hours, preferably 1-6 hours.

[0089] In some embodiments, the solid support G can be directly connected with the molecule M containing a photocleavable group. The solid support G has an active functional group of amino group, and the molecule M containing a photocleavable group provides an active functional group of carboxyl group, and the reaction is connected by forming an amide bond; the reaction solvent is an organic solvent, such as dichloromethane, N,N-dimethylformamide; one or more of N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP) are added to the reaction solvent; the reaction temperature is 15-30°C, preferably 20-25°C; and the reaction time is 1-12 hours, preferably 1-6 hours.

[0090] In the four technical solutions provided by the present application, the connecting molecule L1 is a connecting molecule with multiple active functional groups. In the first technical solution, the connecting molecule L1 is a connecting molecule with at least three active functional groups, in the second technical solution, the connecting molecule L1 is a connecting molecule with at least four active functional groups, in the third technical solution, the connecting molecule L1 is a connecting molecule with at least five active functional groups, and in the fourth technical solution, the connecting molecule L1 is a connecting molecule with at least six active functional groups. Among them, the connecting molecule with four active functional groups can be spliced by two connecting molecules with three active functional groups; the connecting molecule with five active functional groups can be spliced by three connecting molecules with three active functional groups, or by one connecting molecule with three active functional groups and one connecting molecule with four active functional groups; the connecting molecule with six active functional groups can be spliced by four connecting molecules with three active functional groups, or by two connecting molecules with three active functional groups and one connecting molecule with four active functional groups, or by two connecting molecules with four active functional groups, or by one connecting molecule with three active functional groups and one connecting molecule with five active functional groups.

[0091] In the first technical solution, the connecting molecule L1 has at least three active functional groups R4, R5 and R6. The connecting molecule L1 can be represented by the following general formula: Wherein, R4, R5, R6 are three active functional groups. R4, R5 and R6 can exist in the form of protected by a protecting group or not protected by a protecting group respectively, and R4, R5 and R6 do not interfere with each other's connection reaction. In the first technical solution, R4 is an active functional group which is complementary paired with the active functional group R3 of the molecule M containing a photo-cleavable group, R5 is an active functional group which is reacted and spliced with the synthetic building block C1, and R6 is an active functional group which is spliced with the ring-closing end molecule A. The reaction sequence of the three active functional groups is: first, R4 is reacted and spliced with R3, so R5 and R6 are required not to interfere with the reaction process of R4 and R3; then, R5 is reacted and spliced with the synthetic building block C1, so R6 is required not to interfere with the reaction process of R5 and the synthetic building block C1; finally, after the completion of the sequential splicing of each synthetic building block and DNA tag, R6 is reacted and spliced with the ring-closing end molecule A. In a preferred embodiment, R4 exists in the form of not protected by a protecting group, and R5 and R6 exist in the form of protected by a protecting group which is different from the deprotection reaction mechanism. For example, R4 is a carboxyl group not protected by a protecting group, R5 is an amino group protected by a protecting group, and R6 is a carboxyl group protected by a protecting group; after R4 is reacted and connected with the active functional group R3 of the molecule M containing a photo-cleavable group to obtain G—M—L1, a deprotection reaction mechanism is first used to remove the protecting group of R5, R5 is reacted and spliced with the synthetic building block C1, and the sequential splicing of each DNA tag and each synthetic building block is simultaneously completed, then another deprotection reaction mechanism which is different from the first one is used to remove the protecting group of R6, R6 is reacted and spliced with the ring-closing end molecule A, and the subsequent reaction is carried out.

[0092] In the second technical solution, the connection molecule L1 has at least four active functional groups R4, R5, R6 and R7. The connection molecule L1 can be represented by the following general formula: R4, R5, R6, and R7 can exist independently in a form protected by a protecting group or a form not protected by a protecting group, and R4, R5, R6, and R7 do not interfere with each other's connection reaction. In the second technical solution, R4 is an active functional group that is complementarily paired with the active functional group R3 of the molecule M containing a photocleavable group, R5 is an active functional group that reacts and splices with the synthetic building block C1, R6 is an active functional group that splices with the ring-closing end molecule A, and R7 is an active functional group that reacts and splices with the starting nucleotide molecule HP. The reaction sequence of the four active functional groups is: first, R4 reacts and splices with R3 of the molecule M containing a photocleavable group, so R5, R6, and R7 are required not to interfere with the reaction process of R4 splicing with R3; then, R7 reacts and splices with the starting nucleotide molecule HP and the open primer OP (also called an open primer) in turn, and R5 reacts and splices with the synthetic building block C1, so R5 and R6 are required not to interfere with the splicing reaction process of R7; finally, after each synthetic building block and DNA tag is spliced in turn, R6 reacts and splices with the ring-closing end molecule A. In a preferred embodiment, R4 exists in a form not protected by a protecting group, and R5, R6, and R7 exist in a form protected by protecting groups that differ in deprotection reaction mechanism. The connection molecule of the four active functional groups can be obtained by splicing two trifunctional connection molecules.

[0093] In the third technical solution, the connection molecule L1 has at least five active functional groups R4, R5, R6, R8, and R9. The connection molecule L1 can be represented by the following general formula: R4, R5, R6, R8, and R9 can exist independently in a form protected by a protecting group or a form not protected by a protecting group, and R4, R5, R6, R8, and R9 do not interfere with each other's connection reaction. In the third technical solution, R4 is an active functional group that is complementarily paired with the active functional group R3 of the molecule M containing a photocleavable group, R5 is an active functional group that reacts and splices with the synthetic building block C1, R6 is an active functional group that splices with the ring-closing end molecule A, R8 is an active functional group that reacts and splices with the synthetic building block C1, and R9 is an active functional group that reacts and splices with the starting nucleotide molecule HP. The reaction sequence of the five active functional groups is: first, R4 reacts and splices with R3 of the molecule M containing a photocleavable group, so R5, R6, R8, and R9 are required not to interfere with the reaction process of R4 splicing with R3; then, R9 reacts and splices with the starting nucleotide molecule HP and the open primer OP (also called an open primer) in turn, and R5 reacts and splices with the synthetic building block C1, so R5, R6, and R8 are required not to interfere with the splicing reaction process of R9; finally, after each synthetic building block and DNA tag is spliced in turn, R6 reacts and splices with the ring-closing end molecule A, and R8 reacts and splices with the synthetic building block C1. In a preferred embodiment, R4 exists in a form not protected by a protecting group, and R5, R6, R8, and R9 exist in a form protected by protecting groups that differ in deprotection reaction mechanism. The connection molecule of the five active functional groups can be obtained by splicing three trifunctional connection molecules. n+1R9 is the active functional group for reaction splicing with the ring-closing end molecule A. The reaction sequence of the five active functional groups is: first, R4 reacts with R3 of the molecule M containing the photocleavable group to splice, thus requiring that R5, R6, R8, and R9 do not interfere with the reaction process of splicing R4 and R3; then R5 reacts with the synthetic building block C1 to splice, thus requiring that R6, R8, and R9 do not interfere with the reaction process of splicing R5; then R6 reacts with the ring-closing end molecule A to splice, thus requiring that R8 and R9 do not interfere with the reaction process of splicing R6; then, after the first ring structure is formed, R8 reacts with the synthetic building block C n+1 to splice, thus requiring that R9 does not interfere with the reaction process of splicing R8; finally, after the synthetic building blocks and the DNA tag are spliced in turn, R9 reacts with the ring-closing end molecule A to splice. In a preferred embodiment, R4 is in the form of being protected by a protecting group that is not protected, and R5, R6, R8, and R9 are in the form of being protected by protecting groups that have different deprotection reaction mechanisms. The five active functional groups of the linking molecule can be spliced from three trifunctional linking molecules, or from one trifunctional linking molecule and one four-functional linking molecule.

[0094] In the fourth technical solution, the linking molecule L1 has at least six active functional groups R4, R5, R6, R7, R8, and R9. The linking molecule L1 can be represented by the following general formula: wherein R4, R5, R6, R7, R8, and R9 are the six active functional groups. R4, R5, R6, R7, R8, and R9 can independently exist in the form of being protected by a protecting group or in the form of being protected by no protecting group, and R4, R5, R6, R7, R8, and R9 do not interfere with each other's connection reaction. In the fourth technical solution, R4 is an active functional group that is complementary to the active functional group R3 of the molecule M containing the photocleavable group, R5 is an active functional group that reacts with the synthetic building block C1 to splice, R6 is an active functional group that reacts with the ring-closing end molecule A to splice, R7 is an active functional group that reacts with the starting nucleotide molecule HP to splice, R8 is an active functional group that reacts with the synthetic building block C n+1 R9 is the active functional group for reaction splicing with the ring-closing end molecule A. The reaction sequence of the five active functional groups is: first, R4 reacts with R3 of the molecule M containing the photocleavable group to splice, thus requiring that R5, R6, R7, R8, and R9 do not interfere with the reaction process of splicing R4 and R3; then R7 reacts with the starting nucleotide molecule HP and the open primer OP in turn to splice, and R5 reacts with the synthetic building block C1 to splice, thus requiring that R5, R6, R8, and R9 do not interfere with the reaction process of splicing R7, and R6, R8, and R9 do not interfere with the reaction process of splicing R5; then, after the first ring structure is formed, R8 reacts with the synthetic building block C n+1The reaction is spliced, therefore R9 must not interfere with the splicing reaction of R8; finally, after all the synthetic building blocks and DNA tags are spliced ​​sequentially, R9 is reacted with the ring-closing molecule A for splicing. In a preferred embodiment, R4 exists in an unprotected form, while R5, R6, R7, R8, and R9 exist in a protected form with different deprotection reaction mechanisms.

[0095] In the second technical solution, a preferred embodiment is that the linker molecule L1 is composed of two trifunctional linker molecules L1' and L1” spliced ​​together, which can be represented by the following general formula: L1'—L1”; wherein, L1' has three active functional groups R4, R7 and R4', and L1” has three active functional groups R5, R6 and R5', and R4' and R5' are complementary pairing reaction linked together.

[0096] In the second technical solution, a preferred embodiment is that the connecting molecule L1 contains a decomposable functional group R. L R L During decomposition, the connecting molecule L1 splits into two molecular fragments: one containing R4 and R7, and the other containing R5 and R6.

[0097] In the preferred embodiment of the second technical solution provided by the present invention, the compound library obtained after secondary screening can be directly obtained by secondary segmentation cleavage of the library compounds through the decomposable functional group R in the linking molecule L1, achieving the same effect of short cycle and high efficiency. The cyclic compound library S2 is cleaved by photo-irradiation or acid degradation of the decomposable functional group R in the linking molecule L1. L The general structural formula of the obtained library compounds is:

[0098] That is, the cyclic compound library S2”'. Here, L1' is the linker molecule L1 cleaved from R. L And the remaining structural fragments after the R4 and R7 groups were removed.

[0099] In some specific embodiments, the linker molecule L1 may consist of two trifunctional linkers L1' and L1'', and a linker molecule L0 connecting L1' and L1'', with a decomposable functional group R. Lis a decomposable functional group located in the structure of the connecting molecule L0, or is a functional group connecting the connecting molecule L0 and the connecting molecule L1', or is a functional group connecting the connecting molecule L0 and the connecting molecule L1''. The connecting molecule L1 can be represented by the following general formula: L1'-L0-L1''. Wherein, L1' has three active functional groups R4, R7 and R4', L1'' has three active functional groups R5, R6 and R5', L0 has two active functional groups R4'' and R5''; R4' and R4'' are connected by complementary pairing reaction, R5' and R5'' are connected by complementary pairing reaction. That is, the decomposable functional group R L may be a functional group formed after the complementary pairing reaction of R4' and R4'', or may be a functional group formed after the complementary pairing reaction of R5' and R5'', or may be a decomposable functional group located in the structure of the connecting molecule L0.

[0100] In some specific embodiments, in the method, the decomposable functional group R L is an acid-cleavable group, or is a photo-cleavable group having a different cleavage wavelength from the molecule M containing the photo-cleavable group. The acid-cleavable group can be an ester bond, an amide bond, etc.

[0101] Specifically, the connecting molecule L0 is selected from the following structures:

[0102]

[0103] In the molecular structure of the decomposable connecting molecule L0 represented by formula 1 to formula 7, the hydroxyl group and the aldehyde group can be used to react and splice with the active functional groups of the connecting molecules L1' and L1'' to form the decomposable functional group R L . R L may be cleaved under acidic conditions or light. For example, the hydroxyl group can react with the carboxyl group to form an ester bond, which can be cleaved into two molecular fragments under acidic conditions; the aldehyde group can react with the amine group to form an amine bond; in the molecular structure of the decomposable connecting molecule L0 represented by formula 8-9, the hydroxyl group and the amine group respectively react with the carboxyl group of L1'' to form an ester bond and an amide bond, which can be cut by light, and the wavelength of the light is 290 nm.

[0104] Similarly, in the fourth technical solution, a preferred embodiment is that the connecting molecule L1 contains a decomposable functional group R L , R L decomposes to split the connecting molecule L1 into two molecular fragments, which are: a molecular fragment containing R4, R7 and a molecular fragment containing R5, R6, R8, R9.

[0105] In this way, in the above-mentioned preferred embodiments of the fourth technical solution, the compound library obtained after the secondary screening can be obtained by decomposing the decomposable functional group R L The secondary segmental cleavage of the library compound directly obtains the screened compound, and the same effect of short cycle and high efficiency is achieved. The cyclic compound library S4 is obtained by cleaving the decomposable functional group R L of the connecting molecule L1 through a light reaction or acid degradation, and the structure general formula of the obtained library compound is:

[0106] i.e. the cyclic compound library S4'". Wherein, L1' is the residual structure segment after the cleavage of the connecting molecule L1 L and the removal of the R4 and R7 groups.

[0107] In some preferred embodiments, the active functional group of the connecting molecule L1 and the active functional group complementary to the active functional group can be selected from the following combinations: amino and carboxyl, hydroxyl and carboxyl, phosphonic acid and hydroxyl, amino and alkyl halide or aryl halide, etc.

[0108] In the first technical solution, in some specific embodiments, the ring-closing end molecule A is directly connected with the connecting molecule L1. The active functional group R6 of the connecting molecule L1 is carboxyl, and the ring-closing end molecule A provides an active functional group amino, and the two are connected by reacting to generate an amide bond; the reaction solvent is an organic solvent, such as dichloromethane, N,N-dimethylformamide; one or more of N,N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP) are added to the reaction solvent; the reaction temperature is 15-35°C, preferably 20-30°C; the reaction time is 1-12 hours, preferably 1-6 hours.

[0109] In the above-mentioned four technical solutions, in some specific embodiments, the molecule M containing a photo-cleavable group is directly connected with the connecting molecule L1. The active functional group R4 of the connecting molecule L1 is carboxyl, and the molecule M containing a photo-cleavable group provides an active functional group hydroxyl, and the two are connected by reacting to generate an ester bond; the reaction solvent is an organic solvent, such as dichloromethane, N,N-dimethylformamide; one or more of N,N'-diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP) are added to the reaction solvent; the reaction temperature is 15-35°C, preferably 20-30°C; the reaction time is 1-12 hours, preferably 1-6 hours.

[0110] In the three technical solutions provided by the present application, the starting nucleotide molecule HP has an active functional group R10, which is complementary to the active functional group R7 of the linking molecule L1 or the active functional group R1' of the solid-phase carrier G.

[0111] In the first and third technical solutions, the solid-phase carrier G has two active functional groups R1 and R1', and the R1' of the solid-phase carrier G reacts with the active functional group R10 of the starting nucleotide molecule HP to splice, so that the starting nucleotide molecule HP is connected to the solid-phase carrier G. Further, the starting nucleotide molecule HP can be connected to the open primer OP under the action of DNA ligase, and further connected to more DNA tags tag under the action of DNA ligase to achieve the purpose of extending the DNA coding sequence. The starting nucleotide molecule HP refers to the starting connection end of the DNA chain in the DNA splicing technology. The open primer OP refers to the DNA chain that can be extended in the DNA splicing technology.

[0112] In the second and fourth technical solutions, the active functional group R7 of the linking molecule L1 reacts with R10 to splice, so that the starting nucleotide molecule HP is connected to the linking molecule L1. Further, the starting nucleotide molecule HP can be connected to the open primer OP under the action of DNA ligase, and further connected to more DNA tags tag under the action of DNA ligase to achieve the purpose of extending the DNA coding sequence.

[0113] In some specific embodiments, the active functional group R10 of the starting nucleotide molecule HP is selected from amino groups, and the active functional group R7 of the linking molecule L1 or the active functional group R1' of the solid-phase carrier G that is complementary to R10 is a carboxyl group. The carboxyl group reacts with R10 (amino group) to form an amide bond, so that the linking molecule L1 or the solid-phase carrier G is spliced with the starting nucleotide molecule HP. The carboxyl group can be a carboxyl group protected by a tert-butyl ester group; the deprotection reaction condition of the carboxyl tert-butyl ester is 95% trifluoroacetic acid deprotection. After the deprotection group is completed, the active functional group carboxyl reacts with the active functional group R10 amino group of the starting nucleotide molecule HP (H DNA) to form an amide bond for connection; first, react with N,N'-diisopropyl carbodiimide (DIC), N-hydroxysuccinimide (NHS) in an organic solvent such as dichloromethane, N,N-dimethylformamide for 1-12 hours, preferably 1-6 hours; then react with the starting nucleotide molecule HP (H DNA) in a HEPES buffer solution for 1-48 hours, preferably 12-24 hours, and the reaction temperature is 15-30°C, preferably 20-25°C.

[0114] In some specific embodiments, the starting nucleotide molecule HP is connected to the open primer OP under the action of T4 DNA ligase, so as to extend the DNA sequence and prepare for connecting the DNA tag tag.

[0115] In some specific embodiments of the four technical solutions above, the active functional group R5 of the connecting molecule L1 is an amino group protected by an Fmoc protecting group. The Fmoc protecting group can be removed under the condition of a piperidine-containing organic solvent, and then the connecting synthesis block is prepared.

[0116] In the four technical solutions above, the active functional groups R5 and R6 of the connecting molecule L1 have different protecting groups with different deprotection reaction mechanisms. In some specific embodiments, R6 is a carboxyl group protected by an allyl hydroxyl (AllO-) group, and R5 is an amino group protected by a fluorenylmethoxycarbonyl (Fmoc) group.

[0117] In the various technical solutions provided by the present application, the completion of the splicing of one synthesis block and its corresponding DNA tag is referred to as one extension step. By repeating the extension step, the sequential connection of the synthesis blocks and the sequential connection of the DNA tags are performed to expand the number of compounds in the compound library. Each synthesis block has a DNA tag corresponding thereto, the DNA tags corresponding to different synthesis blocks are different, and the synthesis block and the DNA tag corresponding thereto constitute a list. More synthesis blocks and DNA tags corresponding thereto are selected from the list, and the extension step is repeated to sequentially connect each synthesis block to the synthesis block of the previous extension step and to sequentially connect each DNA tag to the DNA tag of the previous extension step, that is, to respectively lengthen the chain of the synthesis block and the chain of the DNA tag.

[0118] In the first technical solution, by repeating the extension step, tag n ……—tag1—OP—HP—G—M—L1—C1……—C n The number n of the connected synthesis blocks and the DNA tags corresponding thereto is determined according to the size of the ring required. Generally, n is an integer and 2≤n≤7.

[0119] In the second technical solution, by repeating the extension step, G—M—L1(—HP—OP—tag1—……—tag n )—C1—……—C n ; wherein n is an integer and 2≤n≤7.

[0120] In the third technical solution, by repeating the extension step, G—M—L1(—HP—OP—tag1—……—tag

[0121] wherein 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7.

[0122] In a fourth technical solution, the extension step is repeated to obtain

[0123] wherein 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7.

[0124] In the present application, the synthetic building block is a small molecule compound having at least two active functional groups. The first active functional group and the second active functional group of the synthetic building block are in the form of an unprotected group, and the first active functional group and the second active functional group do not interfere with each other. The synthetic building block can further comprise a backbone structure (e.g., a backbone structure unit) which is connected in a ring or can be connected to a ring in the form of a side chain of the ring. The active functional groups of two adjacent synthetic building blocks are complementary, i.e., the two active functional groups are reacted together to form a covalent bond connection. The two active functional groups of the synthetic building block are independently selected from any one of an amino group, a carboxyl group, an aldehyde group, an alkenyl group, an alkyne group, a halogen, an azide, a hydroxyl group, and a thiol group.

[0125] In a preferred embodiment, the first active functional group is in the form of a protected group or an unprotected group, and the second active functional group is in the form of a protected group. When the first active functional group is in the form of a protected group, the deprotection mechanism of the first active functional group is different from the deprotection mechanism of the second active functional group.

[0126] In a preferred embodiment, the first active functional group is in the form of an unprotected group, and the second active functional group is in the form of a protected group. In the splicing reaction of the synthetic building block, the first active functional group in the form of an unprotected group is directly used for splicing reaction, and then the protecting agent of the second active functional group needs to be removed before the next splicing reaction of the synthetic building block.

[0127] When the extension step is repeated, the synthetic building blocks are spliced in sequence. In the first and second technical solutions, the synthetic building blocks are spliced in sequence, the first active functional group of the synthetic building block C1 is responsible for splicing with the linking molecule L1, and the first active functional groups of the subsequent synthetic building blocks are spliced with the second active functional groups of the previous synthetic building blocks in sequence, and the last synthetic building block C nThe second active functional group of the synthetic building block C is the first ring-closing functional group of the first ring structure, and the first ring-closing functional group is selected from the group consisting of: primary amino group (NH2-), secondary amino group (-NH-), hydroxyl group (OH-), and urea group (NH2CONH-), and the first ring-closing functional group is responsible for splicing with the ring-closing end molecule A. The first active functional group and the second active functional group of each synthetic building block do not exist in the form of being protected by a protecting group, and the first active functional group and the second active functional group do not interfere with each other. n The first active functional group of each synthetic building block between C n and C n is spliced with the second active functional group of the previous synthetic building block in sequence, and the second active functional group (i.e. the first ring-closing functional group of the first ring structure) of the synthetic building block C n+1 is responsible for splicing with the ring-closing end molecule A; and the first active functional group of the synthetic building block C n+1 is responsible for splicing with the other active functional group R8 of the linking molecule L1, and each synthetic building block between C n+m and C n+m is spliced with the second active functional group of the previous synthetic building block in sequence, and the first active functional group of the synthetic building block C n+m is responsible for splicing with the second ring-closing end molecule A; and the first active functional group and the second active functional group do not exist in the form of being protected by a protecting group, and the first active functional group and the second active functional group do not interfere with each other.

[0128] In a preferred embodiment, the adjacent synthetic building blocks are connected through an amide bond, an ester bond, an ether bond, an amine bond, or an imine bond

[0129] In a preferred embodiment, the first active functional group of each synthetic building block is the same active functional group, for example, all are carboxyl groups; the second active functional group of each synthetic building block is also the same active functional group, for example, all are amino groups; and the first active functional group and the second active functional group are different.

[0130] In a preferred embodiment, the double active functional groups of the synthetic building block are carboxyl and amino groups, respectively, and the carboxyl and amino groups exist independently in the form of being protected by a protecting group or in the form of not being protected by a protecting group.

[0131] In a preferred embodiment, the synthetic building blocks are bifunctional compounds having both amino and carboxyl groups. In some embodiments, the amino and carboxyl groups in the synthetic building blocks are attached to the same carbon atom, such as an alpha-amino acid. In other embodiments, the amino and carboxyl groups in the synthetic building blocks are attached to different atoms, such as a non-amino acid having both amino and carboxyl groups, or an N-substituted amino acid.

[0132] In other preferred embodiments, the synthetic building blocks are selected from the group consisting of substituted or unsubstituted dicarboxylic acids, substituted or unsubstituted diamines, substituted or unsubstituted diols, alpha, beta-unsaturated aldehydes, alpha, beta-unsaturated ketones, alpha, beta-unsaturated acids, alkenes or alkynes containing hydroxyl, amine, aldehyde, carboxyl, sulfonate, or halogen substituents, natural amino acids, or unnatural amino acids, N-substituted amino acids.

[0133] In a preferred embodiment, the first reactive functional group is a carboxyl group, which is present in an unprotected form.

[0134] In a preferred embodiment, the first reactive functional group is an amino group, which is present in a protected form. The protecting group for the amino group is a Fmoc protecting group.

[0135] In a preferred embodiment, the synthetic building blocks are Fmoc-amino acids.

[0136] In a preferred embodiment, among the synthetic building blocks, there are two synthetic building blocks having a third reactive functional group in addition to the first and second reactive functional groups. The third reactive functional groups of the two synthetic building blocks are capable of complementary pairing and reacting to form a cyclic structure. The two third reactive functional groups react to form a covalent bond. The two third reactive functional groups can undergo ring closure by any known ring closure reaction in the art, such as enzymatic ring closure or simple chemical ring closure. More preferably, the two synthetic building blocks are separated by at least one synthetic building block. In a preferred embodiment, the third reactive functional groups of the two synthetic building blocks are a carboxyl group and an amino group. The third reactive functional groups are present in a protected form, and the protecting groups are removed before the two third reactive functional groups undergo the ligation reaction.

[0137] As a preferred embodiment, in the first technical solution and / or the second technical solution, the synthetic building blocks C1 to C nAny two of the synthetic building blocks have a third reactive functional group, and the third reactive functional groups of the two synthetic building blocks can be complementary to pair and react to form a cyclic structure through the reaction of the respective third reactive functional groups. The two third reactive functional groups can be cyclized through any known cyclization reaction in the art, such as enzymatic catalysis or through a simple chemical reaction. In this way, a library of bicyclic structure compounds is obtained based on the first technical solution and / or the second technical solution. In the library of bicyclic structure compounds, the two cyclic structures share the two synthetic building blocks and the synthetic building blocks between the two synthetic building blocks, and the two cyclic structures are more compact in space, which is particularly significant for screening of emerging targets.

[0138] As a preferred embodiment, in the third technical solution and / or the fourth technical solution, the synthetic building block C1 to the synthetic building block C n or the synthetic building block C n+1 to the synthetic building block C n+m Any two of the synthetic building blocks have a third reactive functional group, and the third reactive functional groups of the two synthetic building blocks can be complementary to pair and react to form a cyclic structure through the reaction of the respective third reactive functional groups. The two third reactive functional groups can be cyclized through any known cyclization reaction in the art, such as enzymatic catalysis or through a simple chemical reaction. In this way, a library of bicyclic structure compounds is obtained based on the first technical solution and / or the second technical solution. In the library of bicyclic structure compounds, the two cyclic structures share the two synthetic building blocks and the synthetic building blocks between the two synthetic building blocks, and the two cyclic structures are more compact in space, which is particularly significant for screening of emerging targets. n or the synthetic building block C n+1 to the synthetic building block C n+m to the synthetic building block C

[0139] In a preferred embodiment, at least one of the synthetic building blocks contains a scaffold structure, and the scaffold structure has an E3 ligase substrate structure that can bind to an E3 ligase. The library of compounds with an E3 ligase substrate structure can be applied in PROTAC, and multiple screening of the library can be achieved.

[0140] In some specific embodiments, the scaffold structure is selected from:

[0141]

[0142] In a preferred embodiment, each of the synthetic building blocks in the synthetic building blocks contains at least one exocyclic side chain. The exocyclic side chain provides an extended function for further derivatization of the library compounds, so as to expand the diversity of the library of compounds.

[0143] In a preferred embodiment, each of the synthetic building blocks contains at least two exocyclic side chains, and the at least two exocyclic side chains are connected by chemical reaction to form a bicyclic structure. The cyclization reaction of the two exocyclic side chains can be performed before or after the enzymatic cyclization reaction. The method of forming a bicyclic structure from two exocyclic side chains can be performed in any of the three technical solutions provided in the present application.

[0144] The DNA tags are connected to each other in sequence by DNA ligase. In an extension step, a DNA tag and a synthetic building block corresponding to the DNA tag are subjected to ligation reaction to extend the chain of the synthetic building block and the chain of the DNA tag, respectively. In an extension step, the ligation reaction of the DNA tag can be performed first to extend the chain of the DNA tag, and then the ligation reaction of the synthetic building block corresponding to the DNA tag is performed to extend the chain of the synthetic building block; or the ligation reaction of the synthetic building block can be performed first to extend the chain of the synthetic building block, and then the ligation reaction of the DNA tag corresponding to the synthetic building block is performed to extend the chain of the DNA tag.

[0145] In the four technical solutions provided in the present application, after the DNA tags are ligated, a blocking primer CP (also referred to as a finishing primer) is connected by DNA ligase to form a complete DNA coding sequence. The blocking primer CP refers to the terminal chain of DNA that stops extension in the DNA ligation technology.

[0146] In the three technical solutions provided in the present application, the cyclization end molecule A is a compound having two active functional groups, one of which is i.e. the second cyclization functional group, which is responsible for cyclization reaction with the first cyclization functional group of the terminal synthetic building block to form a cyclic structure; and the other active functional group is R11, which is responsible for ligation reaction with the active functional groups R6 and R9 of the linking molecule L1. The cyclization end molecule A can be represented by the general formula R11-A0-SO2F. R11 is an active functional group for ligation reaction with the active functional groups of the linking molecule L1, and -SO2F (sulfonyl fluoride group) is an active functional group for cyclization reaction.

[0147] In the technical solutions provided in the present application, the second cyclization functional group of the cyclization end molecule A under mild conditions, so that the cyclization end molecule A and the terminal synthetic building block are connected to form a sulfonamide bond, a sulfonate ester bond or a sulfonyl urea bond in a ring. The cyclization reaction conditions are intramolecular cyclization reaction in a HEPES buffer solution at 20-40°C for 12-24 hours.

[0148] The application provides a construction method of a cyclic compound library, and intramolecular ring closure reactions of first ring closure functional groups and second ring closure functional groups are carried out, ring closure conditions are mild, universality is stronger, and the chemical reaction type of the coded compound library and the diversity space of the compound library are expanded.

[0149] The application provides a construction method of a cyclic compound library, and the number and types of atoms provided by each synthetic building block in the ring structure can be diversified and adjusted, splicing reactions of the synthetic building blocks are not limited, and various synthetic building blocks can be used for splicing, and universality is strong.

[0150] The application provides a method for cutting by illumination, reaction conditions are mild, and the influence of removal of a solid phase on the stability of a DNA coding sequence under other harsh conditions is effectively avoided.

[0151] The application provides a method for synthesizing a DNA coded compound by using a solid phase carrier, and post-treatment and purification operations of reactions are simple, and only a few simple filtration and washing operations are needed, so that post-treatment, separation and purification processes of each reaction are simplified, the synthesis period of the DNA coded compound library is significantly shortened, and costs are greatly saved.

[0152] In the compound library synthesis method, the linker connecting the DNA coding sequence and the compound library is first connected to the compound library, and then the DNA coding sequence is connected, the synthesis ratio of the starting nucleotide molecule HP and the docking compound is about 1:250, the DNA dosage ratio is greatly reduced, and costs are saved.

[0153] In the compound library synthesis method, 249 parts of DNA that are not connected are left after each DNA tag splicing, one of the double active functional groups of the synthetic building block has a protective group, and one step of deprotection of the synthetic building block is needed when the synthetic building block and the corresponding DNA tag are alternately connected, and the reaction point of the compound library of the unconnected DNA can be blocked when the deprotection group is removed, so that the excessive DNA is removed, interference is reduced, and the effect of purification is achieved, the efficiency and uniqueness of the DNA coding are improved, and the purity of the final product is improved.

[0154] The application further provides a cyclic compound library (i.e., the compound library S1) constructed by the foregoing method, and the structural general formula is as follows:

[0155]

[0156] 2≤n≤7 and n is an integer.

[0157] G represents a solid phase carrier, M represents a molecule containing a photo-cleavable group, L1 is at least a trifunctional linker molecule, and the DNA coding sequence is connected to the solid phase carrier G through an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence; A represents a ring-closing end molecule A, which has a second ring-closing functional group G and M, M and L1, and L1 and A are connected through covalent bonds; the last synthetic building block C n has a first ring-closing functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); A and C n are connected into a ring through a sulfonyl amide bond, a sulfonate ester bond, or a sulfonyl urea bond formed by the reaction of the first ring-closing functional group and the second ring-closing functional group.

[0158] The application also provides a second circular compound library (i.e., compound library S2) constructed by the foregoing method, which has a general structure as follows:

[0159]

[0160] wherein 2≤n≤7 and n is an integer;

[0161] G represents a solid phase carrier, M represents a molecule containing a photo-cleavable group, L1 is at least a trifunctional linker molecule, and the DNA coding sequence is connected to the solid phase carrier G through an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence; A represents a ring-closing end molecule A, which has a second ring-closing functional group G and M, M and L1, and L1 and A are connected through covalent bonds; the last synthetic building block C n has a first ring-closing functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); A and C n are connected into a ring through a sulfonyl amide bond, a sulfonate ester bond, or a sulfonyl urea bond formed by the reaction of the first ring-closing functional group and the second ring-closing functional group.

[0162] The application also provides a third circular compound library (i.e., compound library S3) constructed by the foregoing method, which has a general structure as follows:

[0163]

[0164] wherein 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7.

[0165] G represents a solid phase carrier, M represents a molecule containing a photocleavable group, L1 is at least a five functional group linker molecule, the DNA coding sequence is connected to the solid phase carrier G through an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence from head to tail, C n+1 are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group n+m are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group G and M, M and L1, L1 and A are connected through covalent bonds; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); two A are connected with C n , C n+m through the first ring closure functional group and the second ring closure functional group to form a sulfonyl amide bond, a sulfonic acid ester bond or a sulfonyl urea bond, and are connected into a ring to form a bicyclic structure.

[0166] The application also provides a fourth cyclic compound library (i.e. compound library S4) constructed by the above method, which has the following structure formula:

[0167]

[0168] Wherein, 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7;

[0169] G represents a solid phase carrier, M represents a molecule containing a photocleavable group, L1 is at least a six functional group linker molecule, the DNA coding sequence is connected to L1, and L1 and the DNA coding sequence are connected through an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence from head to tail, C n+1 are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group n+m are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group G and M, M and L1, L1 and A are connected through covalent bonds; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); two A are connected with C n , C n+m through the first ring closure functional group and the second ring closure functional group to form a sulfonyl amide bond, a sulfonic acid ester bond or a sulfonyl urea bond, and are connected into a ring to form a bicyclic structure.

[0170] The present application also provides a cyclic compound library (i.e. compound library S2") constructed by the aforementioned method, which has the following general structure:

[0171]

[0172] wherein 2≤n≤7 and n is an integer;

[0173] L1 is an at least trifunctional linker molecule, a DNA coding sequence is linked to L1, and L1 and the DNA coding sequence are connected by an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group L1 and A are connected by a covalent bond; the last synthetic building block C n has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); A and C n are connected into a ring by a sulfonyl amide bond, a sulfonyl ester bond, or a sulfonyl urea bond formed by the reaction of the first ring closure functional group and the second ring closure functional group.

[0174] The present application also provides a cyclic compound library (i.e. compound library S4") constructed by the aforementioned method, which has the following general structure:

[0175]

[0176] wherein 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7;

[0177] L1 is an at least pentafunctional linker molecule, a DNA coding sequence is linked to L1, and L1 and the DNA coding sequence are connected by an amide bond; C1 to C n are synthetic building blocks with double active functional groups connected in sequence from head to tail; C n+1 to C n+m are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring closure end molecule A, which has a second ring closure functional group L1 and A are connected by a covalent bond; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); two ring closure end molecules A are respectively connected to C n , C n+mThe sulfonamide bond, sulfonate bond or sulfonyl urea bond formed by the reaction between the first ring closure functional group and the second ring closure functional group is connected into a ring to form a bicyclic structure.

[0178] In addition, the application also provides a compound library S2 or a compound library S2" by degrading the degradable group R L A cyclic compound library is obtained, which has a general structure as follows:

[0179] That is, a cyclic compound library S2"'. Wherein, 2≤n≤7 and n is an integer;

[0180] L1' is a residual structural fragment after the cleavage of R L and the removal of R4 and R7 groups, L1 is a connecting molecule with at least four functional groups, the four functional groups are R4, R5, R6 and R7 respectively, and the connecting molecule L1 further comprises a decomposable functional group R L , R L decomposition, so that the connecting molecule L1 is split into two molecular fragments, the two molecular fragments are a molecular fragment comprising R4 and R7 and a molecular fragment comprising R5 and R6 respectively; C1 to C n are synthetic building blocks with double active functional groups connected in sequence; A represents a ring closure end molecule A, which has a second ring closure functional group L1' and A are connected by a covalent bond; the last synthetic building block C n has a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—) or urea group (NH2CONH—); A and C n The sulfonamide bond, sulfonate bond or sulfonyl urea bond formed by the reaction between the first ring closure functional group and the second ring closure functional group is connected into a ring.

[0181] In addition, the application also provides a compound library S4 or a compound library S4" by degrading the degradable group R L A cyclic compound library is obtained, which has a general structure as follows:

[0182] That is, a cyclic compound library S4"'. Wherein, 0≤n≤7, 0≤m≤7, n and m are integers, and 2≤n+m≤7;

[0183] L1' is a residual structural fragment after the cleavage of R L and the removal of R4 and R7 groups, the connecting molecule L1 comprises a decomposable functional group R L , R LThe connecting molecule L1 is split into two molecular fragments upon decomposition, the two molecular fragments are: a molecular fragment containing R4, R7 and a molecular fragment containing R5, R6, R8, R9; C1 to C n synthetic building blocks with dual reactive functional groups connected in sequence from head to tail, C n+1 to C n+m synthetic building blocks with dual reactive functional groups connected in sequence from head to tail; A represents a ring closure end molecule A with a second ring closure functional group L1' is connected to A by a covalent bond; the last synthetic building block C n , the last synthetic building block C n+m with a first ring closure functional group: primary amino group (NH2—), secondary amino group (—NH—), hydroxyl group (OH—), or urea group (NH2CONH—); the two ring closure end molecules A are connected to C n , C n+m by reaction of the first ring closure functional group and the second ring closure functional group to form a sulfonamide bond, a sulfonate ester bond or a sulfonyl urea bond, and are connected to form a ring to form a bicyclic structure.

[0184] Specifically, each of the synthetic building blocks (C1, …, C n , C n+1 , …, C n+m ) is independently selected from substituted or unsubstituted amino acids, substituted or unsubstituted dicarboxylic acids, substituted or unsubstituted diamines, substituted or unsubstituted diols, α,β-unsaturated aldehydes, α,β-unsaturated ketones, α,β-unsaturated acids, natural amino acids or unnatural amino acids.

[0185] Specifically, at least one of the synthetic building blocks contains a backbone structure, and the backbone structure has an E3 ligase substrate structure capable of binding to an E3 ligase. The backbone structure is connected to the ring or is connected to the ring in the form of a side chain of the ring.

[0186] Specifically, the backbone structure is selected from:

[0187]

[0188] Specifically, each of the synthetic building blocks contains at least one exocyclic side chain. The exocyclic side chain provides an extended function for further derivatization of the library compound, and at the same time can perform some functional modifications similar to lipidation on the side chain. The exocyclic side chain can further improve the membrane permeability and penetration properties of the molecule on the basis of the membrane permeability of the molecule itself, so as to expand the diversity of the compound library.

[0189] Specifically, each of the synthetic building blocks contains at least two exocyclic side chains, and the at least two exocyclic side chains are connected to form a bicyclic structure through chemical reaction. The bicyclic or polycyclic structure can stabilize the macrocyclic molecular conformation, improve the rigidity of the cyclic structure, improve the stability of the cyclic structure molecule, and prolong the half-life of the cyclic structure molecule drug.

[0190] The compound library of the present application provides a cyclic compound library, increases the diversity of the compound library, and expands the application range of the compound library in new drug screening.

[0191] In the compound library of the present application, the number and types of atoms in the ring structure can be diversified and adjusted by different synthetic building blocks, thereby expanding the diversity of the compound library.

[0192] In the compound library of the present application, the adjacent synthetic building blocks can be spliced by using an amide bond (—CO—NH—), and the ring structure is provided with more hydrogen bond combination potential, which is beneficial to improve the binding force of the ring structure and the protein.

[0193] In the cyclic compound library of the present application, the synthetic building blocks on the ring can contain a backbone structure, such as an E3 ligase substrate structure, thereby increasing the application of the compound library in PROTAC. DETAILED DESCRIPTION

[0194] The technical solutions of the present application will be described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0195] In the reaction route marks of each of the following embodiments, indicates a solid support (PEGA resin), and other indicates a synthetic building block. The English characters L, N, A, I, E, P, etc. in the formula (I) are one-letter abbreviations of amino acids, such as L for leucine (Leu), N for asparagine (Asn), A for alanine (Ala), I for isoleucine (Ile), E for glutamic acid (Glu), and P for proline (Pro). indicates that the synthetic building block is leucine, and other one-letter abbreviations are interpreted according to the usual one-letter abbreviations of amino acids in the art. The characters AA1, …, AA4 in the formula (I) all indicate that the synthetic building block is an amino acid, and are distinguished only by the serial numbers 1, …, 4, etc. The characters C1, …, C n , C n+1, …, C n+m , …, C

[0196] Synthesis of the library of monocyclic cyclic compounds

[0197]

[0198] Synthesis method:

[0199] 1. Take 100 mg of PEGA resin to get compounds 1-4 according to the synthesis method above.

[0200] 2. Linking molecule L1 and ring-closing end molecule A to splice

[0201] The resin, tetrakis triphenylphosphine palladium (2.89 mg, 0.2 eq) and phenylsilane (54.12 mg, 20 eq) were stirred in 5 mL of dichloromethane at room temperature for 1 hour under nitrogen protection. The resin was washed with DCM (3x3 mL), DMF (3x3 mL), and dried, and the resin was washed with DMF (3x3 mL), water (3x5 mL) and dried for standby, to get 65 mg of resin. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and 4-(2-aminoethyl) benzene sulfonyl fluoride (25 mg, 5 eq) were stirred in 5 mL of DMF for 2 min, then the resin was added, and the mixture was stirred at room temperature for 2 h, and the resin was washed with DMF (3x3 mL), water (3x5 mL) and dried for standby. 5 mL of 20% 2-methylpiperidine DMF solution was added to the resin, and stirred at room temperature for 1 h, and the resin was washed with DMF (3x5 mL), DCM (3x3 mL), DMF (3x5 mL) and dried for standby, to get 55 mg of resin.

[0202] 3. Ring-closing end molecule A and the last synthetic building block react to form a ring

[0203] The 20 mg of resin was dissolved in 1 mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 degrees overnight to get 18 mg of resin. The resin was washed with water (3x5 mL), DMF (3x3 mL), water (3x5 mL) and dried to get a DNA-encoded cyclic peptide library.

[0204] Verification of ring closure of the library of monocyclic cyclic compounds

[0205]

[0206] Synthesis method:

[0207] 1. Stir DIC (50 mg, 5 eq), HOBt (51 mg, 5 eq), Fmoc-4peg-COOH (190 mg, 5 eq) in 5 mL of DMF for 2 h. Wash the resin with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dry for use. Yield: 300 mg of resin.

[0208] 2. Synthesis of the last building block

[0209] Using 20% piperidine in DMF (5 mL), DIC (50 mg, 5 eq), HOBt (51 mg, 5 eq), N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (120 mg, 5 eq), follow the condensation procedure of Step 1 to get 280 mg of resin.

[0210] 3. Removal of the Alloc protecting group of the linker L1

[0211] Stir 280 mg of resin, tetrakis(triphenylphosphine)palladium (9 mg, 0.5 eq) and phenylsilane (165 mg, 20 eq) in 5 mL of dichloromethane at room temperature under nitrogen for 1 h. Wash the resin with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dry for use. Yield: 260 mg of resin.

[0212] 4. Reaction of the linker L1 with the cyclization end molecule A with a benzene ring structure

[0213] Free 4-(2-aminoethyl)benzenesulfonyl fluoride (110 mg) with a saturated solution of sodium carbonate, DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), 4-(2-aminoethyl)benzenesulfonyl fluoride (90 mg, 5 eq), follow the condensation procedure of Step 1 to get 250 mg of resin.

[0214] 5. Removal of the Fmoc protecting group of the amino group of the last building block

[0215] Stir 250 mg of resin in 20% 2-methylpiperidine in DMF (5 mL) for 3 h. Wash the resin with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dry for use. Yield: 230 mg of resin.

[0216] 6. Cyclization of the cyclization end molecule A with a benzene ring structure with the last building block

[0217] Dissolve 50 mg of resin in 1 mL of HEPES buffer (pH = 8.0) and incubate the mixture at 37 °C overnight. Yield: 40 mg of resin.

[0218] 7. Cleavage of the compound from the solid support under photolysis

[0219] The resin was filtered and the solvent was lyophilized to give 2 mg of compound 8.

[0220] LCMS: 631 (M+H)+.

[0221] Synthesis of the library of monocyclic cyclic compounds

[0222]

[0223] Synthetic method:

[0224] 1. Refer to the above synthetic method to obtain compounds 1-4 from 100 mg of PEGA resin.

[0225] 2. Ligation of the molecule L1 with the cyclization end molecule A with heterocyclic structure

[0226] The resin, tetrakis(triphenylphosphine)palladium (2.89 mg, 0.2 eq) and phenylsilane (54.12 mg, 20 eq) were stirred in 5 mL of dichloromethane at room temperature for 1 hour under nitrogen protection. The resin was washed with DCM (3 x 3 mL), DMF (3 x 3 mL), and dried by suction, and the resin was washed with DMF (3 x 3 mL), water (3 x 5 mL) and dried by suction for standby, to obtain 65 mg of resin. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and 2-(piperazin-1-yl)ethane-1-sulfonyl fluoride (25 mg, 5 eq) were stirred in 5 mL of DMF for 2 min, then the resin was added, and the mixture was stirred at room temperature for 2 h. The resin was washed with DMF (3 x 3 mL), water (3 x 5 mL) and dried by suction for standby. 5 mL of 20% 2-methylpiperidine in DMF was added to the resin, and the mixture was stirred at room temperature for 1 h. The resin was washed with DMF (3 x 5 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dried by suction for standby, to obtain 55 mg of resin.

[0227] 3. Cyclization of cyclization end molecule A with the last synthetic building block

[0228] The 50 mg of resin was dissolved in 5 mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 degrees overnight to obtain 40 mg of resin. The resin was washed with water (3 x 5 mL), DMF (3 x 3 mL), and water (3 x 5 mL) and dried by suction to obtain a DNA-encoded cyclic peptide library.

[0229] Verification of cyclization of the library of monocyclic cyclic compounds

[0230]

[0231] Synthesis method:

[0232] 1. Synthesis of the assembly of building blocks and linker molecule L1

[0233] DIC (50 mg, 5 eq), HOBt (51 mg, 5 eq), 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16- pentaoxa-4-azanonadecan-19-oic acid (190 mg, 5 eq) were stirred in 5 mL of DMF for 2 h. The resin was washed with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dried for use. The resin was obtained as 300 mg.

[0234] 2. Synthesis of the assembly of building blocks

[0235] DIC (50 mg, 5 eq), HOBt (51 mg, 5 eq), N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N- methylglycine (120 mg, 5 eq) were used in 5 mL of 20% piperidine in DMF following the condensation procedure of Step 1 to obtain the resin as 280 mg.

[0236] 3. Removal of the Alloc protecting group from linker molecule L1

[0237] The resin (280 mg), tetrakis(triphenylphosphine)palladium (9 mg, 0.5 eq) and phenylsilane (165 mg, 20 eq) were stirred in 5 mL of dichloromethane under nitrogen at room temperature for 1 h. The resin was washed with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dried for use. The resin was obtained as 260 mg.

[0238] 4. Reaction assembly of linker molecule L1 with the heterocyclic ring- closing end molecule A

[0239] DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), 2-(piperazin-1-yl)ethane-1-sulfonyl fluoride (90 mg, 5 eq) were used following the condensation procedure of Step 1 to obtain the resin as 250 mg.

[0240] 5. Removal of the Fmoc protecting group from the amino group of the final assembly of building blocks

[0241] The resin (250 mg) was stirred in 5 mL of 20% 2-methylpiperidine in DMF for 3 h. The resin was washed with DMF (3 x 3 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dried for use. The resin was obtained as 230 mg.

[0242] 6. The ring closure of the end molecule A with a heterocyclic structure is reacted with the last synthetic building block to form a ring

[0243] Dissolve 50 mg of resin in 1 mL of HEPES buffer (PH = 8.0), and mix the mixture at 37 degrees overnight to obtain 40 mg of resin.

[0244] 7. The compound is detached from the solid phase carrier under photolysis

[0245] Add 40 mg of resin to 1 mL of NMP, irradiate under 365 nm ultraviolet light for 1 h, filter the resin, and freeze dry the solvent to obtain 2 mg of compound 8.

[0246] LCMS: 624 (M+H)+.

[0247] Example 5 Synthesis of a library of cyclic compounds with side chains

[0248]

[0249] Step 1. Splicing of the synthetic building block and the linking molecule L1

[0250] Stir DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), Fmoc-Acp-OH (63 mg, 5 eq) in 3 mL of N,N-dimethylformamide for 10 min, then add 100 mg of swelled resin, stir the mixture at room temperature for 1 h, and wash the resin with N,N-dimethylformamide (3 x 3 mL), dichloromethane (3 x 3 mL), and N,N-dimethylformamide (3 x 5 mL) respectively, and dry for standby, to obtain 100 mg of resin.

[0251] Step 2. Splicing of the synthetic building block

[0252] Add 5 mL of 20% piperidine in N,N-dimethylformamide to the resin, stir at room temperature for 1 h, and wash the resin with N,N-dimethylformamide (3 x 5 mL), dichloromethane (3 x 3 mL), and N,N-dimethylformamide (3 x 5 mL) respectively, and dry for standby, to obtain 100 mg of resin. Refer to the condensation operation of Step 1 to obtain 95 mg of resin by splicing DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and Fmoc-Lys(Boc)-OH (59 mg, 5 eq).

[0253] Step 3. Introduce side chains in the manner of splicing of synthetic building blocks

[0254] To the resin was added 5 mL of 20% piperidine in N,N-dimethylformamide and stirred at room temperature for 1 h. The resin was washed with N,N-dimethylformamide (3 x 5 mL), dichloromethane (3 x 3 mL), and N,N-dimethylformamide (3 x 5 mL) and dried. The resin was 100 mg. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C1 (49 mg, 5 eq) were added to the resin and the condensation procedure of Step 1 was followed to give 95 mg of resin. The Fmoc deprotection and condensation procedure of Step 1 was followed using 5 mL of 20% piperidine in N,N-dimethylformamide, DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C2 (52 mg, 5 eq) to give 90 mg of resin.

[0255] Step 4. Ligation of molecule L1 to the cyclization end molecule A

[0256] To the resin was added 5 mL of 20% piperidine in N,N-dimethylformamide and stirred at room temperature for 1 h. The resin was washed with N,N-dimethylformamide (3 x 5 mL), dichloromethane (3 x 3 mL), and N,N-dimethylformamide (3 x 5 mL) and dried. The resin was 100 mg. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C1 (49 mg, 5 eq) were added to the resin and the condensation procedure of Step 1 was followed to give 95 mg of resin. The Fmoc deprotection and condensation procedure of Step 1 was followed using 5 mL of 20% piperidine in N,N-dimethylformamide, DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C2 (52 mg, 5 eq) to give 90 mg of resin.

[0257] Step 5. Removal of the Boc protecting group from the amino group of the last synthetic building block

[0258] To the resin was added 5 mL of 20% piperidine in N,N-dimethylformamide and stirred at room temperature for 1 h. The resin was washed with N,N-dimethylformamide (3 x 5 mL), dichloromethane (3 x 3 mL), and N,N-dimethylformamide (3 x 5 mL) and dried. The resin was 100 mg. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C1 (49 mg, 5 eq) were added to the resin and the condensation procedure of Step 1 was followed to give 95 mg of resin. The Fmoc deprotection and condensation procedure of Step 1 was followed using 5 mL of 20% piperidine in N,N-dimethylformamide, DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and synthetic building block C2 (52 mg, 5 eq) to give 90 mg of resin.

[0259] Step 6. Cyclization of the cyclization end molecule A to the last synthetic building block

[0260] Dissolve 70 mg of resin in 5 mL of HEPES buffer (PH=8.0), the mixture is reacted at 37 degrees overnight to get 50 mg of resin. The resin is washed with water (3x5 mL), DMF (3x3 mL), water (3x5 mL) and dried to get 65 mg of resin of cyclopeptide.

[0261] Step 7. Cleavage of the compound from the solid support under photolysis

[0262] Dissolve 65 mg of resin in 0.5 mL of acetonitrile and 0.5 mL of water, irradiate under 365 nm ultraviolet light for 5 h, filter the resin, dialyze the solvent and freeze-dry to get 5 mg of compound 8.

[0263] LCMS: 1205.8 (M+H)+.

[0264] Example 6. Ring closure verification of the library of bicyclic cyclic compounds

[0265]

[0266]

[0267] Step 1. Synthesis of the assembly of the building block and the linking molecule L1

[0268] Stir DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), Fmoc-Acp-OH (63 mg, 5 eq) in 3 mL of N,N-dimethylformamide for 10 min, then add 100 mg of swelled resin, stir the mixture at room temperature for 1 h, wash the resin with N,N-dimethylformamide (3x3 mL), dichloromethane (3x3 mL), N,N-dimethylformamide (3x5 mL) and dry to get 100 mg of resin for standby.

[0269] Step 2. Synthesis of the assembly of the building block

[0270] Add 5 mL of 20% piperidine in N,N-dimethylformamide to the resin, stir at room temperature for 1 h, wash the resin with N,N-dimethylformamide (3x5 mL), dichloromethane (3x3 mL), N,N-dimethylformamide (3x5 mL) and dry to get 100 mg of resin for standby. Refer to the condensation operation of step 1 to get 95 mg of resin of DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), Fmoc-Lys(Boc)-OH (59 mg, 5 eq).

[0271] Step 3. Introduction of the ring closure terminal molecule A

[0272] 95 mg of resin, tetrakis triphenylphosphine palladium (2.89 mg, 0.2 eq) and phenylsilane (54.12 mg, 20 eq) were stirred in 5 mL of dichloromethane at room temperature for 1 h under nitrogen. The resin was washed with N,N-dimethylformamide (3 x 3 mL), dichloromethane (3 x 3 mL), N,N-dimethylformamide (3 x 5 mL) and dried for use. This resulted in 85 mg of resin. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), and 2-(piperazin-l-yl)ethane-l-sulfonyl fluoride (25 mg, 5 eq) were stirred in 5 mL of DMF for 2 min, then the resin was added. The mixture was stirred at room temperature for 2 h. The resin was washed with DMF (3 x 3 mL), water (3 x 5 mL) and dried for use. This resulted in 75 mg of resin.

[0273] Step 4. Removal of Boc protecting group from the last synthetic building block

[0274] The 75 mg of resin was dissolved in 5 mL of (TFA / DCM = 1 :4) solution and the solution was stirred at room temperature for 30 min. The resin was washed with 10% DIPEA in N,N-dimethylformamide (3 x 10 mL) and dried for use. This resulted in 70 mg of resin.

[0275] Step 5. First ring closure

[0276] The 70 mg of resin was dissolved in 5 mL of HEPES buffer (pH = 8.0) and the mixture was reacted at 37 °C overnight. The resin was washed with water (3 x 5 mL), DMF (3 x 3 mL) and dried for use. This resulted in 65 mg of resin.

[0277] Step 6. Assembly of the synthetic building block and introduction of the second ring closure end molecule A

[0278] The resin 50 mg of compound 8 was obtained by following the standard solid phase synthesis procedure of step 2 and step 3.

[0279] Step 7. Second ring closure to form the bicyclic structure

[0280] The resin was dissolved in 5 mL of 20% 2-methylpiperidine in DMF and stirred at room temperature for 1 h. The resin was washed with DMF (3 x 5 mL), DCM (3 x 3 mL), DMF (3 x 5 mL) and dried for use. The resin was dissolved in 5 mL of HEPES buffer (pH = 8.0) and the mixture was reacted at 37 °C overnight. The resin was washed with water (5 x 5 mL) to obtain 45 mg of resin.

[0281] Step 8. Cleavage of the compound from the solid support by photolysis

[0282] The resin was washed with 0.5 mL of acetonitrile and 0.5 mL of water, irradiated under UV light at 365 nm for 5 h, filtered, and the solvent was dialyzed and lyophilized to give 3 mg of compound 10.

[0283] LCMS: 1277.9 (M+H)+.

[0284] Example 7: Splicing of the third reactive functional group from two synthetic building blocks to form a bicyclic structure

[0285]

[0286] Synthetic procedure:

[0287] Step 1. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), Fmoc-NH-PEG3CH2COOH (55 mg, 5 eq) were stirred in 3 mL of N,N-dimethylformamide for 10 min, then 100 mg of swollen resin was added, the mixture was stirred at room temperature for 1 h, the resin was washed with N,N-dimethylformamide (3 x 3 mL), dichloromethane (3 x 3 mL), N,N-dimethylformamide (3 x 5 mL), H2O (3 x 3 mL) and dried for use, to give 100 mg of resin.

[0288] Step 2. The resin was subjected to standard solid-phase peptide synthesis to give 90 mg of resin, in which DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq), Fmoc-protected amino acid (60 mg, 5 eq) were used.

[0289] Step 3. 5 mL of 50% trifluoroacetic acid in dichloromethane was added to the resin, which was stirred at room temperature for 1 h, the resin was washed with N,N-dimethylformamide (3 x 5 mL), dichloromethane (3 x 3 mL), N,N-dimethylformamide (3 x 5 mL) and dried for use, to give 87 mg of resin. PyBop (27 mg, 2 eq), DIPEA (17 mg, 5 eq) were stirred in 3 mL of N,N-dimethylformamide for 1 min, then added to the resin, the mixture was stirred at room temperature for 1 h, the resin was washed with N,N-dimethylformamide (3 x 3 mL), dichloromethane (3 x 3 mL), N,N-dimethylformamide (3 x 5 mL) and dried for use, to give 85 mg of resin. In this step, the side chain of the synthetic building block glutamic acid (E) reacted with the side chain of the synthetic building block lysine (K) to form a ring structure.

[0290] Step 4. 85 mg resin, tetrakis triphenylphosphine palladium (2.89 mg, 0.2 eq) and phenylsilane (54.12 mg, 20 eq) were stirred in 5 mL of dichloromethane at room temperature for 1 hour under nitrogen protection. The resin was washed with DCM (3 x 3 mL), DMF (3 x 3 mL), respectively, and then dried. DIC (16 mg, 5 eq), HOBt (17 mg, 5 eq) and 2-(piperazin-1-yl)ethane-1-sulfonyl fluoride (25 mg, 5 eq) were stirred in 5 mL of DMF for 2 min, and then the resin was added. The mixture was stirred at room temperature for 2 h. The resin was washed with DMF (3 x 3 mL), water (3 x 5 mL), respectively, and then dried to obtain 75 mg of resin.

[0291] Step 5. 20% 2-methylpiperidine in DMF solution 5 mL was added to 75 mg of resin, and the mixture was stirred at room temperature for 1 h. The resin was washed with DMF (3 x 5 mL), DCM (3 x 3 mL), DMF (3 x 5 mL), respectively, and then dried to obtain 70 mg of resin. The 70 mg of resin was dissolved in 5 mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 degrees overnight. The resin was washed with water (3 x 5 mL), DMF (3 x 3 mL), water (3 x 5 mL), respectively, and then dried to obtain 60 mg of resin of the cyclic peptide.

[0292] Step 6. 30 mg of resin was added to 1 mL of NMP, and the mixture was irradiated at 365 nm ultraviolet light for 5 h. The resin was filtered, dialyzed and lyophilized to obtain 5 mg of compound 8.

[0293] LCMS: 1271.2 (M+H) + .

[0294] From the above verification results, it can be seen that the construction method of the present application can construct a bicyclic compound library. Synthetic block E and synthetic block K each provide an exocyclic side chain, and the two exocyclic side chains are connected to form a ring structure through a chemical reaction. The bicyclic structure of the library compound obtained in this embodiment is a bridged ring, and the two rings share one or more synthetic blocks.

[0295] In conclusion, the above embodiments are only preferred embodiments of the present application and do not limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the construction of a library of cyclic compounds by chemical reaction- induced ring closure, characterized in that, Comprising the following steps: 1) connecting the solid support G directly or indirectly with the molecule M containing a photocleavable group to obtain G—M; 2) performing any one of Method 1, Method 2, Method 3 or Method 4; Method 1: performing steps a1~f1; a1. connecting G—M with a linking molecule L1 having at least three functional groups to obtain G—M—L1; b1. sequentially reacting G—M—L1 with a starting nucleotide molecule HP, an open primer OP to connect the starting nucleotide molecule HP with the solid support G to obtain OP—HP—G—M—L1; c1. reacting OP—HP—G—M—L1 with a synthesis block C1, a DNA tag tag1 corresponding to the synthesis block C1 to connect the synthesis block C1 with L1 and the DNA tag tag1 with OP to obtain tag1—OP—HP—G—M—L1—C1; d1. defining a set of corresponding synthetic building blocks and DNA tags as one extension step, repeating said extension step to sequentially assemble the synthetic building blocks to form elongated chains and to sequentially assemble the DNA tags corresponding to the synthetic building blocks to form elongated chains, obtaining tags n — tag1— OP— HP— G— M— L1— C1...— C n ; wherein the last synthetic building block C n has a first ring-closing functional group: a primary amino group (NH2—), a secondary amino group (— NH—), a hydroxyl group (OH—), or a urea group (NH2CONH—); 2≤ n≤ 7 and n is an integer; e1. The product from the previous step is reacted with blocking primer CP, which hybridizes to tag n , wherein HP—OP—tag1—……—tag n —CP forms a complete DNA coding sequence, resulting in DNA—G—M—L1—C1……—C n ; f1. reacting the product from the previous step with a ring closure end molecule A to attach ring closure end molecule A to L1, to give DNA—G—M—L1(—C1...—C n )—A, i.e. the compound library S1'; said ring closure end molecule A having a second ring closure function ; Method 2: performing steps a2~f2; a2. connecting G—M with a linking molecule L1 having at least four functional groups to obtain G—M—L1; b2. sequentially reacting G—M—L1 with a starting nucleotide molecule HP, an open primer OP to connect the starting nucleotide molecule HP with the linking molecule L1 to obtain G—M—L1—HP—OP; c2. reacting the product obtained in the previous step with a synthesis block C1, a DNA tag tag1 corresponding to the synthesis block C1 to connect the synthesis block C1 with L1 and the DNA tag tag1 with OP to obtain G—M—L1(—HP—OP—tag1)—C1; d2. Defining a set of corresponding synthetic building blocks and DNA tag ligation reactions as an extension step, repeating the extension step to sequentially assemble the synthetic building blocks to form elongated chains and the DNA tags corresponding to the synthetic building blocks to form elongated chains, resulting in G—M—L1(—HP—OP—tag1—……—tag n )—C1—……—C n ; wherein the last synthetic building block C n has a first ring-closing functional group: primary amino (NH2—), secondary amino (—NH—), hydroxyl (OH—), or urea (NH2CONH—); 2≤n≤7 and n is a positive integer; e2. The product from step d2 is reacted with blocking primer CP to allow the blocking primer CP to hybridize to tag n wherein HP—OP—tag1—...—tag n —CP forms a complete DNA coding sequence to give G—M—L1(—DNA)—C1—...—C n ; f2. reacting the product from step e2 with a ring closure end molecule A to attach ring closure end molecule A to L1 to give G—M—L1(—DNA)(—C1...—C n )—A, i.e. the compound library S2'; said ring closure end molecule A having a second ring closure function ; Method 3: performing steps a3~i3; a3. connecting G—M with a linking molecule L1 having at least five functional groups to obtain G—M—L1; b3. sequentially reacting G—M—L1 with a starting nucleotide molecule HP, an open primer OP to connect the starting nucleotide molecule HP with the solid support G to obtain OP—HP—G—M—L1; c3. reacting OP—HP—G—M—L1 with a synthesis block C1, a DNA tag tag1 corresponding to the synthesis block C1 to connect the synthesis block C1 with L1 and the DNA tag tag1 with OP to obtain tag1—OP—HP—G—M—L1—C1; d3. Defining a set of corresponding synthetic building blocks and DNA tag ligation reactions as an extension step, repeating said extension step to sequentially assemble the synthetic building blocks into elongated chains and the DNA tags corresponding to the synthetic building blocks into elongated chains, to obtain tag n — tag1— OP— HP— G— M— L1— C1...— C n ; wherein the last synthetic building block C n has a first ring-closing functional group: a primary amino group (NH2—), a secondary amino group (— NH—), a hydroxyl group (OH—), or a urea group (NH2CONH—); 2≤ n≤ 7 and n is an integer; e3. The product from the previous step is reacted with a cyclization end molecule A, a DNA tag tag corresponding to the cyclization end molecule A A to connect cyclization end molecule A to L1 and to connect tag A to tag n to obtain tag A —tag n —……—tag1—OP—HP—G—M—L1(—C1……—C n )—A; the cyclization end molecule A has a second cyclization functional group ; f3. The product from the previous step is subjected to intramolecular cyclization reaction at 20 °C to 40 °C in HEPES buffer solution to allow the first cyclization functional group of the last synthetic building block C n to react with the second cyclization functional group of cyclization end molecule A to form a ring to give ; g3. The product obtained in the previous step is reacted according to the extension step with the synthesis block C n+1 ,..., C n+m and the corresponding DNA tag tag n+1 ,..., tag n+m in sequence, so that the synthesis block C n+1 is linked to L1 and the DNA tag tag n+1 is linked to tag A , obtaining ; wherein the last synthesis block C n+m has a first ring-closing functional group: a primary amino group (NH2—), a secondary amino group (—NH—), a hydroxyl group (OH—) or a urea group (NH2CONH—); 1≤m≤7 and m is an integer, 3≤n+m≤7. h3. The product from the previous step is reacted with blocking primer CP, which forms a complete DNA coding sequence with HP—OP—tag1—...—tag n+m linked, wherein HP—OP—tag1—...—tag n+m —CP to give ； i3. reacting the product obtained in the previous step with a ring closure end molecule A to connect the ring closure end molecule A with L1 to obtain i.e. the compound library S3'; the ring closure end molecule A has a second ring closure function ; Method 4: performing steps a4~i4; a4. connecting G—M with a linking molecule L1 having at least six functional groups to obtain G—M—L1; b4. sequentially reacting G—M—L1 with a starting nucleotide molecule HP, an open primer OP to connect the starting nucleotide molecule HP with the linking molecule L1 to obtain G—M—L1—HP—OP; c4. reacting the product obtained in the previous step with a synthesis block C1, a DNA tag tag1 corresponding to the synthesis block C1 to connect the synthesis block C1 with L1 and the DNA tag tag1 with OP to obtain G—M—L1(—HP—OP—tag1)—C1; d4. Defining a set of corresponding synthetic building blocks and DNA tag ligation reactions as an extension step, repeating said extension step to sequentially assemble the synthetic building blocks to form elongated chains and the DNA tags corresponding to the synthetic building blocks to form elongated chains, resulting in G—M—L1(—HP—OP—tag1—……—tag n )—C1—……—C n ; wherein the last synthetic building block C n has a first ring-closing functional group: primary amino (NH2—), secondary amino (—NH—), hydroxyl (OH—), or urea (NH2CONH—); 2≤n≤7 and n is an integer; e4. The product from step d4 is reacted with a cyclization end molecule A, a DNA tag tag A corresponding to cyclization end molecule A, to attach cyclization end molecule A to L1 and tag A to tag n to obtain G—M—L1(—HP—OP—tag1—……—tag n —tag A )(—C1……—C n )—A; said cyclization end molecule A having a second cyclization functional group ; f4. The product from the previous step is subjected to intramolecular cyclization reaction at 20 °C to 40 °C in HEPES buffer solution to allow the first cyclization functionality of the last synthetic building block C n to react with the second cyclization functionality of the cyclization end molecule A to form a ring to give ; g4. The product obtained in the previous step is reacted according to the extension step with the synthesis block C n+1 ,..., C n+m and the corresponding DNA tag tag n+1 ,..., tag n+m in sequence, so that the synthesis block C n+1 is linked to L1 and the DNA tag tag n+1 is linked to tag A , obtaining ; wherein the last synthesis block C n+m has a first ring-closing functional group: a primary amino group (NH2—), a secondary amino group (—NH—), a hydroxyl group (OH—) or a urea group (NH2CONH—); 1≤m≤7 and m is an integer, 3≤n+m≤7. h4. The product from the previous step is reacted with blocking primer CP, which forms a complete DNA coding sequence with HP—OP—tag1—...—tag n+m —CP, resulting in n+m ​ ； i4. The product obtained in the previous step is reacted with the cyclization end molecule A, so that the cyclization end molecule A is connected to L1, to obtain i.e. the compound library S4'; the ring closure end molecule A has a second ring closure function ; 3) intramolecular cyclization reaction of the compound library S1', S2', S3' or S4' in HEPES buffer solution at 20-40 °C, so that the first cyclization functional group of the last synthetic building block reacts with the second cyclization functional group of the cyclization terminal molecule A to form a ring, to obtain i.e. the cyclic compound library S1; or obtained i.e. the library of cyclic compounds S2; or obtained i.e. a library of cyclic compounds S3 having a bicyclic structure; or obtained i.e. a library of cyclic compounds S4 having a bicyclic structure.

2. The method of claim 1, wherein, The solid phase carrier G is selected from any one or more of PEG resin, PEGA resin, TentaGel resin, and solid phase carrier CPG.

3. The method of claim 2, wherein, The solid phase carrier G has one active functional group R1, and R1 is an amino group; or the solid phase carrier G has two active functional groups R1 and R1', R1 is an amino group, and R1' is a carboxyl group.

4. The method of claim 1, wherein, The molecule M containing a photocleavable group contains at least two active functional groups, which are represented by R2 and R3, respectively; R2 is an active functional group responsible for connecting the solid phase carrier G, and R3 is an active functional group responsible for connecting the connecting molecule L1.

5. The method of claim 4, wherein, R2 exists in the form of an unprotected group, and R3 exists in the form of a protected group.

6. The method of claim 4, wherein, In the molecule M containing a photocleavable group, the photocleavable group is: ， wherein R3is the C atom of the side chain directly attached to the phenyl ring at the ortho position to the nitro group; R2is attached to the phenyl ring at a C atom spaced from R3by one or more covalent bonds or R2is attached to the C atom to which R3is attached by one or more covalent bonds; and the phenyl ring can contain 0, 1 or more side chains or substituents which do not interfere with the attachment of R2, R3. wherein R3is the C atom of the side chain directly attached to the phenyl ring at the ortho position to the nitro group; R2is attached to the phenyl ring at a C atom spaced from R3by one or more covalent bonds or R2is attached to the C atom to which R3is attached by one or more covalent bonds; and the phenyl ring can contain 0, 1 or more side chains or substituents which do not interfere with the attachment of R2, R3.

7. The method of claim 6, wherein, The molecule M containing a photocleavable group can be selected from the following structures: ， In the molecule M containing a photocleavable group, R3 can be selected from -OH, -NH2, -NHNH2, -N3, Cl, and Br; and R2 is represented by a carboxyl group.

8. The method of claim 1, wherein, In the method 1, the connecting molecule L1 has at least three active functional groups R4, R5, and R6; R4, R5, and R6 exist independently in the form of a protected group or the form of an unprotected group, and R4, R5, and R6 do not interfere with each other's connection reaction; R4 is an active functional group for reacting and splicing with the molecule M containing a photocleavable group, R5 is an active functional group for reacting and splicing with the synthetic building block C1, and R6 is an active functional group for reacting and splicing with the cyclization end molecule A.

9. The method of claim 1, wherein, In the method 2, the connecting molecule L1 has at least four active functional groups R4, R5, R6, and R7; R4, R5, R6, and R7 exist independently in the form of a protected group or the form of an unprotected group, and R4, R5, R6, and R7 do not interfere with each other's connection reaction; R4 is an active functional group for reacting and splicing with the molecule M containing a photocleavable group, R5 is an active functional group for reacting and splicing with the synthetic building block C1, R6 is an active functional group for reacting and splicing with the cyclization end molecule A, and R7 is an active functional group for reacting and splicing with the starting nucleotide molecule HP.

10. The method of claim 1, wherein, In the method 3, the connecting molecule L1 has at least five active functional groups R4, R5, R6, R8 and R9; R4, R5, R6, R8 and R9 are respectively in a form protected by a protecting group or a form not protected by a protecting group, and R4, R5, R6, R8 and R9 do not interfere with each other's connection reaction; R4 is an active functional group for splicing with the molecule M containing a photocleavable group, R5 is an active functional group for splicing with the synthetic building block C1, R6 is an active functional group for splicing with the ring-closing end molecule A, R8 is an active functional group for splicing with the synthetic building block C n+1 R9 is an active functional group for splicing with another ring-closing end molecule A.

11. The method of claim 10, wherein, In the method 4, the connecting molecule L1 has at least six active functional groups R4, R5, R6, R7, R8, R9; R4, R5, R6, R7, R8 and R9 are respectively in the form of a protected group or in the form of an unprotected group, and R4, R5, R6, R7, R8 and R9 do not interfere with each other's connection reaction; R4 is an active functional group for splicing with the molecule M containing a photocleavable group, R5 is an active functional group for splicing with the synthetic building block C1, R6 is an active functional group for splicing with the ring-closing end molecule A, R7 is an active functional group for splicing with the starting nucleotide molecule HP, R8 is an active functional group for splicing with the synthetic building block C n+1 R9 is an active functional group for splicing with another ring-closing end molecule A. In the method 4, the connecting molecule L1 has at least six active functional groups R4, R5, R6, R7, R8, R9; R4, R5, R6, R7, R8 and R9 are respectively in the form of a protected group or in the form of an unprotected group, and R4, R5, R6, R7, R8 and R9 do not interfere with each other's connection reaction; R4 is an active functional group for splicing with the molecule M containing a photocleavable group, R5 is an active functional group for splicing with the synthetic building block C1, R6 is an active functional group for splicing with the ring-closing end molecule A, R7 is an active functional group for splicing with the starting nucleotide molecule HP, R8 is an active functional group for splicing with the synthetic building block C n+1 R9 is an active functional group for splicing with another ring-closing end molecule A.

12. The method of claim 9, wherein, In the method 2, the linking molecule L1 comprises a decomposable functional group R L , R L decomposes to split the linking molecule L1 into two molecular fragments, the two molecular fragments being: a molecular fragment comprising R4, R7 and a molecular fragment comprising R5, R6, respectively.

13. The method of claim 11, wherein, In the method 4, the linking molecule L1 comprises a decomposable functional group R L , R L decomposes to split the linking molecule L1 into two molecular fragments, the two molecular fragments being: a molecular fragment comprising R4, R7 and a molecular fragment comprising R5, R6, R8, R9, respectively.

14. The method of claim 12 or 13, wherein, said decomposable functional group R L is an acid cleavable group or a photocleavable group having a different cleavage wavelength than the molecule M comprising the photocleavable group.

15. The method of claim 9, wherein, In the method 2, the linking molecule L1 can be composed of two trifunctional linking molecules L1', L1'', and one linking molecule L0 linking between L1' and L1'', and the decomposable functional group R L is located within the structure of the linking molecule L0, or is the functional group connecting the linking molecule L0 and the linking molecule L1', or is the functional group connecting the linking molecule L0 and the linking molecule L1''. L1' has three active functional groups R4, R7 and R4', L1'' has three active functional groups R5, R6 and R5', and L0 has two active functional groups R4'' and R5''. R4' and R4'' are complementary to each other and react to form a pair, and R5' and R5'' are complementary to each other and react to form a pair.

16. The method of claim 15, wherein, The connecting molecule L0 is selected from the following structures: ， ， ， ， ， ， ， ， 。 17. The method of claim 1, wherein, The starting nucleotide molecule HP has an active functional group R10, and R10 can complementarily pair react with the active functional group of the connecting molecule L1 or the active functional group of the solid phase carrier G.

18. The method of claim 17, wherein, The active functional group R10 of the starting nucleotide molecule HP is an amino group, and the active functional group of the connecting molecule L1 or the active functional group of the solid phase carrier G for complementarily pairing reaction with R10 is a carboxyl group.

19. The method of claim 1, wherein, The synthetic building block is a small molecule compound having at least two active functional groups.

20. The method of claim 19, wherein, In the method 1 and / or method 2, each synthetic building block is sequentially spliced, the first active functional group of the synthetic building block C1 is responsible for splicing with the connecting molecule L1, and the first active functional group of each subsequent synthetic building block is sequentially spliced with the second active functional group of the previous synthetic building block, and the second active functional group of the last synthetic building block C n is the first ring closure functional group, which is responsible for splicing and ring closure with the ring closure terminal molecule A; the first active functional group and the second active functional group do not exist in the form of an unprotected group, and the first active functional group and the second active functional group do not interfere with each other.

21. The method of claim 19, wherein, In method 3 and / or method 4, the first active functional group of the synthetic building block C1 is responsible for splicing with an active functional group of the connecting molecule L1, C1 to C n The various composite blocks and composite block C between them n The first active functional group is sequentially joined to the second active functional group of the previous synthetic building block, and synthetic building block C n The second active functional group is the first ring-closing functional group, responsible for splicing with the ring-closing end molecule A to form the first ring structure; synthetic building block C n+1 The first active functional group is responsible for splicing with another active functional group of the linker molecule L1, C n+1 To C n+m The various composite blocks and composite block C between them n+m The first active functional group is sequentially joined to the second active functional group of the previous synthetic building block, and synthetic building block C n+m The second active functional group is another first ring-closing functional group, which is responsible for splicing with another ring-closing end molecule A to form a second ring structure; the first active functional group and the second active functional group do not exist in the form of unprotected groups at the same time, and the first active functional group and the second active functional group do not interfere with each other.

22. The method of claim 20 or 21, wherein, The adjacent synthetic building blocks are connected by an amide bond, an ester bond, an ether bond, an amine bond, or an imine bond.

23. The method of claim 19, wherein, The synthetic building block is a compound having both an amino group and a carboxyl group as double active functional groups.

24. The method of claim 19, wherein, The synthetic building blocks are selected from substituted or unsubstituted dicarboxylic acids, substituted or unsubstituted diamines, substituted or unsubstituted diols, α, β-unsaturated aldehydes, α, β-unsaturated ketones, α, β-unsaturated acids, alkenes or alkynes containing hydroxyl, amine, aldehyde, carboxyl, sulfonate or halogen substituents, natural amino acids or unnatural amino acids, and N-substituted amino acids.

25. The method of claim 19, wherein, In each of the synthetic building blocks, two of the synthetic building blocks have a third active functional group in addition to the first active functional group and the second active functional group, the third active functional groups of the two synthetic building blocks can be complementarily paired to react and join to form a cyclic structure.

26. The method of claim 25, wherein, The two synthetic building blocks are separated by at least one synthetic building block.

27. The method of claim 25, wherein, The third active functional groups of the two synthetic building blocks, one of which is a carboxyl group and the other of which is an amino group.

28. The method of claim 19, wherein, The synthetic building blocks further comprise a backbone structure, which is connected to the ring or is connected to the ring in the form of a side chain of the ring.

29. The method of claim 25, wherein, At least one of the synthetic building blocks comprises a backbone structure, and the backbone structure has an E3 ligase substrate structure capable of binding to an E3 ligase.

30. The method of claim 28, wherein, The backbone structure is selected from the following: ， ， ， 。 31. The method of claim 28, wherein, In the synthetic building blocks, each of the synthetic building blocks comprises at least one side chain outside the ring.

32. The method of claim 28, wherein, In the synthetic building blocks, each of the synthetic building blocks comprises at least two side chains outside the ring, and at least two of the side chains outside the ring are connected by a chemical reaction to form a bicyclic structure.

33. The method of claim 1, wherein, The DNA tags are sequentially connected to each other by a DNA ligase; in an extension step, a DNA tag and a synthetic building block corresponding to the DNA tag are joined to extend the chain of the synthetic building block and the chain of the DNA tag, respectively.

34. The method of claim 1, wherein, In step 3), the second ring-closing functional group of the ring-closing end molecule A The first cyclic functional group of the final synthetic building block, either primary amino, secondary amino, hydroxyl, or urea, undergoes a cyclic closure reaction under mild conditions, causing the cyclic-closed molecule A to react with the final synthetic building block to form a sulfonamide bond, sulfonate bond, or sulfonylurea bond to form a ring. The cyclic closure reaction is carried out intramolecularly in HEPES buffer solution at 20℃~40℃ for 12~24h.

35. The method of claim 1, wherein, In the method 2, the cyclic compound library S2 is subjected to a photolysis reaction to cleave molecules M containing a photolysis cleavable group, to obtain i.e. the library of cyclic compounds S2".

36. The method of claim 1, wherein, In the method 4, the cyclic compound library S4 is subjected to a photolysis reaction to cleave molecules M containing a photolysis cleavable group, to obtain i.e. the library of cyclic compounds S4".

37. A library of cyclic compounds, characterized in that, The structural general formula is one of the following six structural general formulae: (1) the structural general formula of which is , wherein 2≤n≤7 and n is an integer; G represents a solid support, M represents a molecule containing a photocleavable group, L1 is at least a trifunctional linker molecule, the DNA coding sequence is linked to the solid support G by an amide bond; C1 to C n are synthetic building blocks with dual reactive functionalities linked in a head-to-tail fashion; A represents a cyclized end molecule A having a second cyclization functionality ; the connections between G and M, M and L1, L1 and A are by covalent bonds; the last synthetic building block C n has a first cyclization functionality: a primary amino group, a secondary amino group, a hydroxyl group, or a urea group; A and C n are connected in a ring by a sulfonyl amide bond, a sulfonyl ester bond, or a sulfonyl urea bond formed by the reaction of the first cyclization functionality and the second cyclization functionality. or, (2) the structural general formula is: ， wherein 2≤n≤7 and n is an integer; G represents a solid support, M represents a molecule containing a photocleavable group, L1 is an at least tetrafunctional linker molecule, a DNA coding sequence is attached to L1, the attachment between L1 and the DNA coding sequence is via an amide bond; C1 to C n are synthetic building blocks with dual reactive functionalities connected end to end; A represents a ring closure end molecule A having a second ring closure functionality ; the attachment between G and M, M and L1, L1 and A is via a covalent bond; the last synthetic building block C n has a first ring closure functionality: a primary amino group, a secondary amino group, a hydroxyl group or a urea group; A and C n are connected into a ring via a sulphonamide bond, a sulphonate bond or a sulphonyl urea bond formed by reaction of the first ring closure functionality and the second ring closure functionality; or, (3) the structural general formula is: ， wherein 2≤n≤7, 1≤m≤7, n and m are integers, and 3≤n+m≤7; G represents a solid support, M represents a molecule containing a photo-cleavable group, L1 is at least a five functional group linker molecule, the DNA coding sequence is linked to the solid support G through an amide bond; C1 to C n are synthetic building blocks with dual reactive functional groups connected end to end, C n+1 to C n+m are synthetic building blocks with dual reactive functional groups connected end to end; A represents a ring closure end molecule A, which has a second ring closure functional group ; G and M, M and L1, L1 and A are connected through covalent bonds; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: primary amino group, secondary amino group, hydroxyl group or urea group; two A are connected through a sulfonyl amide bond, a sulfonyl ester bond or a sulfonyl urea bond formed by the reaction of the first ring closure functional group and the second ring closure functional group between C n , C n+m to form a ring, constituting a bicyclic structure; or, (4) the structural general formula is: ， wherein 2≤n≤7, 1≤m≤7, n and m are integers, and 3≤n+m≤7; G represents a solid support, M represents a molecule containing a photolytically cleavable group, L1 is a linker molecule with at least six functional groups, the DNA coding sequence is linked to L1, and L1 and the DNA coding sequence are linked by an amide bond; C1 to C n C is a synthetic building block with two active functional groups connected end to end. n+1 To C n+m It is a synthetic building block with two active functional groups connected end to end; A represents the ring-closed end molecule A, which has a second ring-closed functional group. G and M, M and L1, and L1 and A are connected by covalent bonds; the final composite building block C n The final composite block C n+m It has a first cyclic functional group: primary amino, secondary amino, hydroxyl, or urea group; the two A groups are respectively connected to C. n C n+m The rings are connected by sulfonamide bonds, sulfonate bonds or sulfonylurea bonds formed by the reaction of the first ring-closed functional group and the second ring-closed functional group to form a bicyclic structure; or, (5) the structural general formula is: ， wherein 2≤n≤7 and n is an integer; L1 is an at least trifunctional linker molecule, to which a DNA coding sequence is attached, the attachment between L1 and the DNA coding sequence being by an amide bond; C1 to C n are synthetic building blocks with dual reactive functionalities connected end to end; A represents a ring-closing end molecule A, which has a second ring-closing functionality ; the attachment between L1 and A is by a covalent bond; the last synthetic building block C n has a first ring-closing functionality: a primary amino group, a secondary amino group, a hydroxyl group, or a urea group; A and C n are connected into a ring by a sulphonamide bond, a sulphonate ester bond, or a sulphonyl urea bond formed by reaction of the first ring-closing functionality and the second ring-closing functionality. or, (6) the structural general formula is: ， wherein 2≤n≤7, 1≤m≤7, n and m are integers, and 3≤n+m≤7; L1 is at least a five functional group linker, a DNA coding sequence is linked to L1, and L1 is linked to the DNA coding sequence by an amide bond; C1 to C n are synthetic building blocks with dual reactive functional groups connected end to end, C n+1 to C n+m are synthetic building blocks with dual reactive functional groups connected end to end; A represents a ring closure end molecule A, which has a second ring closure functional group ; L1 is linked to A by a covalent bond; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a urea group; two ring closure end molecules A are connected to C n , C n+m by a sulfonamide bond, a sulfonate bond, or a sulfonyl urea bond formed by the reaction of the first ring closure functional group and the second ring closure functional group, and form a ring structure, which constitutes a bicyclic structure.

38. A library of cyclic compounds, characterized in that, The structural general formula is one of the following two structural general formulae: (1) the structural general formula is: wherein 2≤n≤7 and n is an integer; L1' is a connecting molecule L1 cleaved R L and the residual structure fragment after R4, R7 groups are removed, L1 is at least a four functional group connecting molecule, the four functional groups are R4, R5, R6, R7 respectively, and the connecting molecule L1 further comprises a decomposable functional group R L , R L decomposition causes the connecting molecule L1 to split into two molecular fragments, the two molecular fragments are: a molecular fragment comprising R4, R7 and a molecular fragment comprising R5, R6 respectively; C1 to C n are synthetic building blocks with double active functional groups connected in sequence from head to tail; A represents a ring-closing end molecule A, which has a second ring-closing functional group ; L1' is connected to A by a covalent bond; the last synthetic building block C n has a first ring-closing functional group: primary amino group, secondary amino group, hydroxyl group or urea group; A is connected to C n by a sulfonyl amide bond, a sulfonyl ester bond or a sulfonyl urea bond formed by the reaction of the first ring-closing functional group and the second ring-closing functional group. or, (2) the structural general formula is: wherein 2≤n≤7, 1≤m≤7, n and m are integers, and 3≤n+m≤7; L1' is a linking molecule L1 cleaved R L and the residual structure fragment after R4, R7 groups are removed, L1 is at least six functional groups linking molecule, six functional groups are R4, R5, R6, R7, R8 and R9 respectively, linking molecule L1 contains a decomposable functional group R L , R L Decomposition makes linking molecule L1 split into two molecular fragments, two molecular fragments are R4, R7 containing molecular fragment and R5, R6, R8, R9 containing molecular fragment respectively; C1 to C n is a synthetic building block with double active functional groups connected in sequence from head to tail, C n+1 to C n+m is a synthetic building block with double active functional groups connected in sequence from head to tail; A represents a ring closure terminal molecule A, which has a second ring closure functional group ; L1' is connected with A through covalent bond; the last synthetic building block C n , the last synthetic building block C n+m has a first ring closure functional group: primary amino group, secondary amino group, hydroxyl group or urea group; two ring closure terminal molecules A are connected into a ring through sulfonyl amide bond, sulfonyl ester bond or sulfonyl urea bond formed by reaction of the first ring closure functional group and the second ring closure functional group between C n , C n+m , constituting a bicyclic structure.

39. The library of cyclic compounds of claim 37 or 38, wherein, In each of the synthetic building blocks, two of the synthetic building blocks have a third active functional group in addition to the first active functional group and the second active functional group, and the two third active functional groups can be ring-closed by enzyme-catalyzed reaction or by simple chemical reaction, and the two synthetic building blocks are joined by the respective third active functional groups to form a covalent bond to obtain a cyclic structure.

40. The library of cyclic compounds of claim 37 or 38, wherein, C1 to C n Any two of the synthetic building blocks have a third reactive function, and the third reactive functions of the two synthetic building blocks can complementarily pair to react to form a covalent bond by reaction of the third reactive functions of the two synthetic building blocks to form a ring structure.

41. The library of cyclic compounds of claim 37 or 38, wherein C n+1 to C n+m Any two of the synthetic building blocks have a third reactive function, and the third reactive functions of the two synthetic building blocks can complementarily pair to react to join a covalent bond by reaction of the respective third reactive functions to splice a covalent bond to yield a cyclic structure.

42. The library of cyclic compounds of claim 37 or 38, wherein, Each of the synthetic building blocks is independently selected from the group consisting of a substituted or unsubstituted amino acid, a substituted or unsubstituted dicarboxylic acid, a substituted or unsubstituted diamine, a substituted or unsubstituted diol, an α,β-unsaturated aldehyde, an α,β-unsaturated ketone, an α,β-unsaturated acid, a natural amino acid, or an unnatural amino acid, an N-substituted amino acid.

43. The library of cyclic compounds of claim 42, wherein In each of the synthetic building blocks, at least one of the synthetic building blocks contains a backbone structure, and the backbone structure has an E3 ligase substrate structure that is capable of binding to an E3 ligase.

44. The library of cyclic compounds of claim 43, wherein The backbone structure is selected from the group consisting of: ， ， ， 。 45. The library of cyclic compounds of claim 42, wherein In each of the synthetic building blocks, each of the synthetic building blocks contains at least one exocyclic side chain.

46. The library of cyclic compounds of claim 42, wherein In each of the synthetic building blocks, each of the synthetic building blocks contains at least two exocyclic side chains, and the at least two exocyclic side chains are linked by a chemical reaction to form a bicyclic structure.

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