CN115467029A - Chemical ring closure construction method of cyclic compound library and cyclic compound library - Google Patents

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

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CN115467029A
CN115467029A CN202110645996.1A CN202110645996A CN115467029A CN 115467029 A CN115467029 A CN 115467029A CN 202110645996 A CN202110645996 A CN 202110645996A CN 115467029 A CN115467029 A CN 115467029A
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ring
molecule
functional group
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closing
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魏巍
刘辰
白远超
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Yafei Shanghai Biolog Medicine Science & Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

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Abstract

The invention provides a chemical ring closure construction method of a cyclic compound library and the cyclic compound library, which are synthesized by the reaction of a solid phase carrier, a molecule containing a photocleavable group, a connecting molecule, a synthetic building block and a ring closure end molecule at one end. The method has mild ring closing conditions and stronger universality, and expands the chemical reaction types of the coding compound library and the diversity space of the compound library.

Description

Chemical ring closure construction method of cyclic compound library and cyclic compound library
Technical Field
The invention relates to the field of pharmaceutical chemistry, in particular to a chemical ring closure construction method of a cyclic compound library and the cyclic compound library.
Background
Advances in disease characterization and target identification continue to shift drug discovery to unknown areas, and from a pathological point of view, more and more emerging targets are attractive, but at the same time challenging to modulate with established low molecular weight compounds or biologics. One approach to solving this problem is to create a library of compounds, small molecule compounds derived from traditional libraries of compounds that in principle can be bioavailable, allow cellular penetration, and are suitable for incorporation into defined proteins (e.g., enzymes).
Compounds derived from DNA coding libraries (DEL) have the opportunity to screen as many as 10, similar to small molecules 9 One or more possible chemical entities. The opportunity to find new chemical starting points is greatly increased. However, small molecules often perform poorly in combating protein-protein interactions. Protein interactions tend to have large and extended interaction surfaces, on the other hand, biologies are more suitable for such applications and are often very selective and stronger binding.
Cyclic peptides have attracted considerable attention as a promising class of therapeutic candidates. They have a number of important advantages over linear congeneric products. Macrocyclization generally limits the conformational freedom of the cyclic peptide, allowing the cyclic molecule to bind more tightly and specifically to the target protein by minimizing the entropy of 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 may be suitable for effectively covering large and shallow interfaces involved in protein-protein interactions, whereas traditional drug-like small molecules are not easily targeted.
At present, reference 1 (Min Hyeon Shin, et al Bioconjugate Chemstry,2019,30,2931-2938) reports the establishment of a cyclic peptidomimetic library encoded by DNA and describes the establishment of a cyclic peptidomimetic library encoded by DNA, but the synthetic process of the method relies on chloroacetic acid to introduce synthetic building blocks at each step, and the synthetic building blocks are only a single active group-NH 2 The result of this is that the number and type of atoms between each synthetic building block remains constant and is a repeating-CO-CH 2 -N-building blocks, the diversity of the compound library is insufficient. A great number of previous drug development experiments prove that the length and the type of carbon atoms between drugs can cause great changes to the solubility and the permeability of the drugs; the cyclic peptide library is mainly used for screening the binding capacity of the PPI, the binding capacity of the PPI mainly depends on intramolecular or intermolecular hydrogen bonds formed by N atoms or oxygen atoms, and N atoms formed after the synthesis blocks in the molecules of the cyclic compound library of reference 1 are tertiary amines, which are not favorable for binding with protein or have insufficient binding force. Therefore, the compound library is limited in types and cannot meet the synthesis of a diverse cyclic compound library required by modern drug development. In addition, the difficulty in constructing cyclic compound libraries is that the long-chain structure is reacted at both ends head to tail to form a ring. Because the compound library is established, the premise of carrying DNA sequence is requiredThe ring closing is completed, so that the restriction on the chemical ring closing reaction is relatively high. Reference 1 uses metal ions in the ring-closing reaction, and the metal ions may chelate with DNA molecules, which adversely affects the stability of the DNA molecules and limits the stability and accuracy of the screening results.
Therefore, there is a need in the art to establish a ring closing method with milder and more universal ring closing reaction conditions and other cyclic molecular structures not limited to peptidomimetics, so as to obtain a richer cyclic compound library and increase the diversity of the compound library. In addition, there is a need in the art to establish compound library and target protein binding complex technology to directly screen targeted protein for research and increase the utility of compound library.
Disclosure of Invention
One of the technical problems to be solved by the present invention is to provide a method for constructing a chemical reaction closed loop of a cyclic compound library, which has the advantages of milder closed loop reaction conditions and strong universality, can be used for constructing monocyclic and bicyclic compound libraries, overcomes the limitation of the closed loop mode of the cyclic compound library in the prior art, and has few side reactions.
The second technical problem to be solved by the present invention is to provide a new cyclic compound library with a cyclic molecular structure to increase the diversity of the compound library and expand the application range of the compound library in new drug screening.
In order to solve the above technical problems, the present invention further provides a first technical solution:
a method for constructing a cyclic compound library, comprising the steps of:
1) Directly or indirectly connecting the solid-phase carrier G with a molecule M containing a photocleavable group to obtain G-M:
2) The method comprises the following steps:
a1. reacting and linking G-M with at least trifunctional linker molecule L1 to obtain G-M-L1;
b1. G-M-L1 reacts with an initial nucleotide molecule HP and an open primer OP in sequence to connect the initial nucleotide molecule HP with a solid phase carrier G to obtain OP-HP-G-M-L1;
c1. OP-HP-G-M-L1 and synthetic Block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 Connecting with OP to obtain tag 1 —OP—HP—G—M—L1—C 1 ；
d1. Defining a group of corresponding synthetic building blocks and DNA label connection reaction as an extension step, repeating the extension step to enable the synthetic building blocks to be sequentially spliced to form an extended chain, and enabling the DNA labels corresponding to the synthetic building blocks to be sequentially spliced to form an extended chain to obtain tag n —……—tag 1 —OP—HP—G—M—L1—C 1 ……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 2 and not more than 7, and n is an integer;
e1. reacting the product obtained in the last step with a blocking primer CP to enable the blocking primer CP to react with tag n In a linkage of which HP-OP-tag 1 —……—tag n Formation of the complete DNA coding sequence by-CP to obtain DNA-G-M-L1-C 1 ……—C n ；
f1. 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 DNA-G-M-L1 (-C) 1 ……—C n ) -a, i.e. library of compounds S1'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000031
3) Performing intramolecular cyclization reaction on the compound library S1' in HEPES buffer solution at the temperature of 20-40 ℃ to ensure that the final synthesized building block C n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure BDA0003109642370000032
Namely, cyclic compound library S1.
The invention providesIn one technical scheme, a ring-closing end molecule A, an extended chain formed by splicing synthetic building blocks and a solid phase carrier are respectively connected to the three ends of a connecting molecule L1, and the final synthetic building block C is utilized n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, a cyclic compound library is obtained, and DNA is coded on a solid phase carrier. All the synthesis steps in the scheme are completed on a solid phase carrier, and the quality of a compound library is further ensured. The compound library obtained by the scheme can be suitable for screening by a flow cytometry fluorescence sorting technology (FACS), the screened cyclic compound does not need high-throughput sequencing, the screened cyclic compound can be directly separated from a solid phase carrier by utilizing a decomposition reaction under illumination, the screening period is short, and the efficiency is high.
In order to solve the above technical problem, the present invention provides a second technical solution:
a method for constructing a cyclic compound library, comprising the steps of:
1) Directly or indirectly connecting the solid-phase carrier G with a molecule M containing a photocleavable group to obtain G-M:
2) The method comprises the following steps:
a2. reacting and linking G-M with a linking molecule L1 with at least four functional groups to obtain G-M-L1;
b2. G-M-L1, an initial nucleotide molecule HP and an open primer OP are sequentially reacted and connected, wherein the initial nucleotide molecule HP is connected with a connecting molecule L1, and G-M-L1-HP-OP is obtained;
c2. respectively mixing the product obtained in the last step with the synthesized building block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 To OP to obtain G-M-L1 (-HP-OP-tag) 1 )— C 1 ；
d2. Defining a group of corresponding synthetic building blocks and DNA labels to be connected and reacting as an extension step, repeating the extension step to enable the synthetic building blocks to be sequentially spliced to form an extended chain and enable the DNA labels corresponding to the synthetic building blocks to be sequentially spliced to form an extended chain to obtainG—M—L1(—HP—OP—tag 1 —……—tag n )—C 1 —……—C n (ii) a Wherein n is more than or equal to 2 and less than or equal to 7 and n is a positive integer;
e2. d, reacting the product obtained in the step d2 with a blocking primer CP to ensure that the blocking primer CP reacts with tag n In a linkage of which HP-OP-tag 1 —……—tag n The CP forming the complete DNA coding sequence, resulting in G-M-L1 (-DNA) -C 1 —……—C n ；
f2. Reacting the product obtained in the step e1 with a ring-closing end molecule A to connect the ring-closing end molecule A with L1 to obtain G-M-L1 (-DNA) (-C) 1 ……—C n ) -a, compound library S2'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000041
3) Performing intramolecular cyclization reaction on the compound library S2' in HEPES buffer solution at the temperature of 20-40 ℃ to ensure that the final synthesized building block C n Reacting with ring-closing terminal molecule A to form a ring
Figure BDA0003109642370000042
Namely, cyclic compound library S2.
In the second technical scheme provided by the invention, a ring-closing end molecule A, an extended chain formed by splicing synthetic building blocks and a solid-phase carrier are respectively connected to the three ends of a connecting molecule L1, and the final synthetic building block C is utilized n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and a cyclic compound library is obtained. The resulting cyclic compound can be detached from the solid support and the DNA encoded on the compound. The compound library obtained by the scheme can carry out secondary screening on library compounds by cracking the molecules M containing the photocleavable groups. The secondary screening comprises the following steps: the library of compounds obtained can be subjected to a second screening, after a first screening on the solid support and subsequent detachment of the compound from the solid support by cleavage of the photocleavable group-containing molecule M, at which time the DNA is encoded on the library compound. Secondary screening can be combinedDifferent screening technologies, such AS flow cytometry fluorescence sorting (FACS) screening, traditional target protein affinity screening, AS-MS screening, etc. are combined to improve the accuracy of the screening result. The library of compounds obtained by this protocol is suitable for the traditional DEL screening model, and the screened compounds require high-throughput sequencing.
The cyclic compound library S2 can be obtained by cracking a molecule M containing a photocleavable group through a light reaction
Figure BDA0003109642370000043
Namely, cyclic compound library S2'. This cyclic library S2' can be used for the second screening described above.
Further, in order to solve the above technical problems, the present invention also provides another method for constructing a compound library having a bicyclic structure, that is, a third technical solution:
a method for constructing a cyclic compound library, comprising the steps of:
1) Directly or indirectly connecting the solid-phase carrier G with a molecule M containing a photocleavable group to obtain G-M:
2) The method comprises the following steps:
a3. reacting and connecting G-M with a connecting molecule L1 with at least five functional groups to obtain G-M-L1;
G-M-L1, with an initiator nucleotide molecule HP and an open primer OP in sequence, to link the initiator nucleotide molecule HP with a solid support G to obtain OP-HP-G-M-L1;
c3. OP-HP-G-M-L1 and synthetic Block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 Is connected with OP to obtain tag 1 —OP—HP—G—M—L1—C 1 ；
d3. Defining a group of corresponding synthetic building blocks and DNA label connection reaction as an extension step, repeating the extension step to make the synthetic building blocks spliced in sequence to form an extended chain, and making the DNA labels corresponding to the synthetic building blocksThe tags are sequentially spliced to form an extended chain to obtain tag n —……—tag 1 —OP—HP—G—M—L1—C 1 ……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 0 and not more than 7, and n is an integer;
e3. the product obtained in the last step, a ring-closing end molecule A and a DNA label tag corresponding to the ring-closing end molecule A A Reacting to connect the ring-closing terminal molecule A with L1 and tag A And tag n Are connected to obtain tag A —tag n —……—tag 1 —OP—HP—G —M—L1(—C 1 ……—C n ) -A; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000051
f3. Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure BDA0003109642370000052
g3. The product obtained in the last step is subjected to the expansion step to synthesize a building block C n+1 ，……，C n+m And corresponding DNA label tag n+1 ，……，tag n+m Sequentially reacting to synthesize the building block C n+1 Linked to L1 and DNA tag n+1 And tag A Are connected to obtain
Figure BDA0003109642370000053
Wherein, the final composite building block C n+m Having a first ring-closing functional group: primary amino radical (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); m is more than or equal to 0 and less than or equal to 7, m is an integer, n + m is more than or equal to 2 and less than or equal to 7;
h3. the product obtained in the last step is reacted withThe blocking primer CP reacts to make the blocking primer CP react with tag n+m Are connected, wherein HP-OP-tag 1 —……—tag n+m -CP formation of the complete DNA coding sequence to obtain
Figure BDA0003109642370000061
i3. 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
Figure BDA0003109642370000062
Namely a compound library S3'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000063
3) Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n+m The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure BDA0003109642370000064
Namely, cyclic compound library S3 having a bicyclic structure.
In the method for constructing the compound library with the bicyclic structure, ring-closing end molecules A and C are respectively connected to two ends of connecting molecules L1 with at least five functional groups 1 To C n The synthetic building blocks of (a) are spliced to form an extended chain, which reacts to form a ring to form a first ring structure, and two ends of the chain are respectively connected with a ring-closing end molecule A and a ring-closing end molecule C n+1 To C n+m The synthesized building blocks are spliced with each other to form an extended chain, the extended chain reacts to form a ring to form a second ring structure, and one end of the extended chain is connected with a solid phase carrier and a DNA coding sequence to obtain a cyclic compound library with a double-ring structure. In this scheme, the DNA is encoded on a solid support and all the synthetic steps are performed on the solid support, resulting in a library of compounds of greater qualityThe guarantee is ensured. The compound library obtained by the scheme can be suitable for screening by a flow cytometry fluorescence sorting technology (FACS), the screened cyclic compound does not need high-throughput sequencing, the screened cyclic compound can be directly separated from a solid phase carrier by utilizing a decomposition reaction under illumination, the screening period is short, and the efficiency is high.
Further, in order to solve the above technical problems, the present invention also provides a method for constructing a compound library having a bicyclic structure, that is, a fourth technical solution:
a method for constructing a cyclic compound library, comprising the steps of:
1) Directly or indirectly connecting the solid-phase carrier G with a molecule M containing a photocleavable group to obtain G-M:
2) The method comprises the following steps:
a4. reacting and linking G-M with a linker molecule L1 having at least six functional groups to obtain G-M-L1;
b4. G-M-L1, an initial nucleotide molecule HP and an open primer OP are sequentially reacted and connected, wherein the initial nucleotide molecule HP is connected with a connecting molecule L1, and G-M-L1-HP-OP is obtained;
c4. respectively mixing the product obtained in the last step with the synthesized building block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 To OP to obtain G-M-L1 (-HP-OP-tag) 1 )— C 1 ；
d4. Defining a group of corresponding synthetic building blocks and DNA labels to be connected and reacted as an extension step, repeating the extension step to make the synthetic building blocks spliced in sequence to form an extended chain and make the DNA labels corresponding to the synthetic building blocks spliced in sequence to form an extended chain to obtain G-M-L1 (-HP-OP-tag) 1 —……—tag n )—C 1 —……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 2 and not more than 7, and n is an integer;
e4. d, mixing the product obtained in the step d4 with DNA labels tag corresponding to the ring-closing end molecule A and the ring-closing end molecule A A Reacting to connect ring-closing terminal molecule A with L1 and tag A And tag n Are linked to obtain G-M-L1 (-HP-OP-tag) 1 —…… —tag n —tag A )(—C 1 ……—C n ) -A; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000071
f4. Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure BDA0003109642370000072
g4. The product obtained in the last step is subjected to the expansion step to synthesize a building block C n+1 ，……，C n+m And corresponding DNA label tag thereof n+1 ，……，tag n+m Sequentially reacting to synthesize the building block C n+1 Linked to L1 and DNA tag n+1 And tag A Are connected to obtain
Figure BDA0003109642370000081
Wherein the last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); m is more than or equal to 0 and less than or equal to 7, m is an integer, n + m is more than or equal to 2 and less than or equal to 7;
h4. reacting the product obtained in the last step with a blocking primer CP to enable the blocking primer CP to react with tag n+m Are connected, wherein HP-OP-tag 1 —……—tag n+m -CP formation of the complete DNA coding sequence, obtaining
Figure BDA0003109642370000082
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
Figure BDA0003109642370000083
Namely a compound library S4'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure BDA0003109642370000084
3) Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n+m The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure BDA0003109642370000085
Namely, cyclic compound library S4 having a bicyclic structure.
In the method for constructing the compound library with the bicyclic structure, the two ends of the connecting molecule L1 with at least six functional groups are respectively connected with the ring-closing end molecule A and the ring-closing end molecule C 1 To C n The synthetic building blocks are spliced to form an extended chain, the chain reacts to form a ring to form a first ring structure, and the two ends of the chain are respectively connected with a ring-closing end molecule A and a ring-closing end molecule C n+1 To C n+m The synthesized building blocks are spliced with each other to form an extended chain, the extended chain reacts to form a ring to form a second ring structure, and the two ends of the extended chain are respectively connected with a solid phase carrier and a DNA coding sequence to obtain the cyclic compound library with a double-ring structure. The cyclic compound ultimately obtained can be detached from the solid support and the DNA encoded on the compound. The compound library obtained by the scheme can carry out secondary screening on library compounds by cracking the molecules M containing the photocleavable groups. The secondary screening comprises the following steps: the library of compounds obtained may be subjected to a second screening step, in which the compounds are first screened on a solid support and then cleaved from the solid support by cleaving the photocleavable group-containing molecule M, in which case DNA is encoded on the library compounds. The secondary screening can be combined to use differentThe screening technology of (2), such AS flow cell fluorescence sorting (FACS) screening, traditional target protein affinity screening, AS-MS screening and the like, is combined to improve the accuracy of the screening result. The compound library obtained by the scheme is suitable for the traditional DEL screening mode, and the screened compounds need to be subjected to high-throughput sequencing.
The cyclic compound library S4 can be obtained by cracking a molecule M containing a photocleavable group through a light reaction
Figure BDA0003109642370000091
Namely the cyclic compound library S4". This library S4' of cyclic compounds can be used for the second screening described above.
In the four technical schemes provided by the invention, HEPES refers to 2- (4- (2-hydroxyethyl) piperazine) ethanesulfonic acid with CAS number 7365-45-9.
In the four technical schemes provided by the invention, the solid phase carrier G is selected from any one or more of PEG resin, PEGA resin, tentaGel resin and solid phase carrier CPG.
In the second and fourth embodiments, the solid support G contains a reactive functional group R1. The solid phase carrier G can be represented by the general formula G0-R1, wherein R1 represents an active functional group of the solid phase carrier G; g0 represents a solid-phase support structure other than the active functional group of the solid-phase support G. The solid support G is directly or indirectly connected with the molecule M containing the photocleavable group through the active energy-hanging group R1. R1 may be selected from: amino, carboxyl, or hydroxyl. In a preferred embodiment, the solid support G is selected from solid supports with amino-reactive functional groups; that is, preferably, the reactive functional group R1 is selected from amino groups, such as primary amino groups, secondary amino groups. More preferably, the reactive functional group R1 is selected from primary amino groups. It is preferred. The solid phase carrier is selected from PEGA.
In the first and third embodiments, the solid support G contains two reactive functional groups R1 and R1'. The solid support G can be represented by the general formula R1 '-G0-R1, wherein R1 represents a reactive functional group for direct or indirect attachment to the photocleavable group-containing molecule M and R1' represents a reactive functional group for attachment to a DNA coding sequence. In a preferred embodiment, R1 is amino and R1' is carboxy.
In the four technical schemes provided by the invention, the molecule M containing the photocleavable group contains at least two active functional groups, which are respectively represented by R2 and R3. The photocleavable group-containing molecule M may be represented by the general formula R2-M0-R3, wherein R2 and R3 represent two mutually independent reactive functional groups. R2 is the reactive functional group responsible for attachment to the solid support G, and R3 is the reactive functional group responsible for attachment to the linker molecule L1. R2 and R3 are independently present in a form protected by a protecting group or in a form not protected by a protecting group, and R2 and R3 do not interfere with a linking reaction of each other. Wherein, mainly R3 does not interfere the reaction process of the connection of R2 and the solid phase carrier G, or R3 is protected by a protecting group when the connection of R2 and the solid phase carrier G is carried out.
In a preferred embodiment, R2 is present in an unprotected form with a protecting group and R3 is present in a protected form with a protecting group. After R2 is connected with a solid phase carrier G to obtain G-M, R3 needs to remove a protecting group and then carries out subsequent reaction.
The solid-phase carrier G and the molecule M containing the photocleavable group are connected in two ways. In one embodiment, R2 and R1 are reacted in direct complementary pairing to give G-M. Complementary pairing means that two reactive functional groups react to covalently link the chemical structures of the two reactive functional groups, for example, two molecules are covalently linked to form a molecule. Based on different reaction principles, all or part of fragments can be removed when R1 and R2 react, for example, one molecule of water is removed after R1 is connected with R2; alternatively, R1 and R2 may react without removing any fragments, such as by addition. When R1 and R2 are directly complementary and paired, they can be selected from the following combinations: amino and carboxyl, amino and hydroxyl, amino and phosphate, amino and alkyl halide or aryl halide, etc.
Alternatively, R2 and R1 are indirectly linked via another bifunctional linker molecule to give G-M. For example, the bifunctional linker molecule has two reactive functional groups; wherein, the first active group can complementarily match and react with the active functional group R1 of the solid phase carrier G, and the second active group can complementarily match and react with the active functional group R2 of the molecule M containing the photocleavable group. The reaction process can have two forms: firstly, a first active group is firstly connected with an active functional group R1 of a solid-phase carrier G in a reaction way, and then a second active group is connected with an active functional group R2 of a molecule M containing a photocleavable group in a reaction way; and secondly, reacting and connecting a second active group with an active functional group R2 of the molecule M containing the photocleavable group, and reacting and connecting the first active group with an active functional group R1 of the solid-phase carrier G to obtain indirectly connected G-M. In the indirect connection mode, it is no longer required whether R2 and R1 have a complementary pairing relationship.
In the molecule M containing a photocleavable group, the photocleavable group is preferably:
Figure BDA0003109642370000101
wherein R3 is a C atom of a side chain positioned at the ortho-position of the nitro group and directly connected with a benzene ring; r2 is attached to
Figure BDA0003109642370000102
R2 is connected with the C atom on the benzene ring through one or more covalent bonds or R2 is connected with the C atom connected with R3 through one or more covalent bonds; the benzene ring may contain zero, one or more side chains or substituents which do not interfere with the reaction of R2 and R3.
Under suitable light conditions, in the photocleavable group-containing molecule M, the chemical bond between R3 and the C atom to which R3 is attached can be broken. The photocleavable group-containing molecule M can be cleaved at 365 nm.
In some preferred embodiments, the photocleavable group-containing molecule M may be selected from the following structures:
Figure BDA0003109642370000111
in the above structure, R3 can be selected from-OH and-NH 2 、-NHNH 2 、-N 3 Cl, br, etc.
In the above structures, R2 is represented by carboxyl.
In some embodiments, the solid support G and the photocleavable group-containing molecule M can be indirectly linked through a small molecule compound having bifunctional groups.
In some embodiments, the solid support G has a reactive functional group, the small molecule compound having a bifunctional group is a carboxyl group having a reactive functional group and an amino group protected by a protecting group. The solid phase carrier G reacts with the small molecular compound with the bifunctional group to generate an amido bond for connection; the reaction solvent is organic solvent such as dichloromethane and N, N-dimethylformamide; the reaction temperature is 15-30 ℃, preferably 20-25 ℃; the reaction time is from 1 to 12 hours, preferably from 2 to 6 hours. Then, deprotecting the amino group protected by the protecting group of the small molecular compound with bifunctional groups, and reacting and connecting the small molecular compound with a molecule M containing a photocleavable group; the protecting group for the amino group is preferably an Fmoc protecting group; the reaction solvent for removing the protecting group is an organic solvent such as dichloromethane and N, N-dimethylformamide, and piperidine is added into the reaction solvent; the reaction temperature is 15-30 ℃, preferably 20-25 ℃; the reaction time is 1 to 12 hours, preferably 1 to 6 hours. Then, carrying out deprotection and then carrying out reaction connection with a molecule M containing a photocleavable group; after deprotection, the small molecular compound with double functional groups provides active functional group amino, the molecule M containing the photocleavable group provides active functional group carboxyl, and amide bonds are generated through reaction for connection; the reaction solvent is an organic solvent such as N, N-dimethylformamide; adding one or more of N, N' -Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and 4-Dimethylaminopyridine (DMAP) into a reaction solvent; the reaction temperature is 15-30 ℃, preferably 20-25 ℃; the reaction time is 1 to 12 hours, preferably 1 to 6 hours.
In some embodiments, the solid support G and the photocleavable group-containing molecule M can be directly linked. The solid-phase carrier G has an active functional group amino, the molecule M containing the photocleavable group provides an active functional group carboxyl, and the active functional group carboxyl reacts to generate an amido bond for connection; the reaction solvent is an organic solvent such as dichloromethane and N, N-dimethylformamide; adding one or more of N, N' -diisopropyl carbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and 4-Dimethylaminopyridine (DMAP) into a reaction solvent; the reaction temperature is 15-30 ℃, preferably 20-25 ℃; the reaction time is 1 to 12 hours, preferably 1 to 6 hours.
In the four technical schemes provided by the invention, the connecting molecule L1 is a connecting molecule with multiple active functional groups. In a first technical scheme, the connecting molecule L1 is a connecting molecule with at least three active functional groups, in a second technical scheme, the connecting molecule L1 is a connecting molecule with at least four active functional groups, in a third technical scheme, the connecting molecule L1 is a connecting molecule with at least five active functional groups, and in a fourth technical scheme, the connecting molecule L1 is a connecting molecule with at least six active functional groups. Wherein, the connecting molecules of the four active functional groups can be spliced by the connecting molecules of two trifunctional groups; the connecting molecules with five active functional groups can be spliced by three trifunctional connecting molecules or spliced by one trifunctional connecting molecule and one tetrafunctional connecting molecule; the six active functional group connecting molecules can be obtained by splicing four trifunctional connecting molecules, or two trifunctional connecting molecules and a tetrafunctional connecting molecule, or two tetrafunctional connecting molecules, or a trifunctional connecting molecule and a pentafunctional connecting molecule.
In a first embodiment, the linker molecule L1 has at least three reactive functional groups R4, R5 and R6. Linker molecule L1 can be represented by the following general formula:
Figure BDA0003109642370000121
wherein R4, R5 and R6 are three active functional groups. R4, R5 and R6 may each independently be in the form protected with a protecting group or not protected with a protecting groupThe protected form of the group exists, and R4, R5 and R6 do not interfere with the connection reaction of each other. In a first technical scheme, R4 is a reactive functional group which is complementarily paired with a reactive functional group R3 of a molecule M containing a photocleavable group, R5 is a reactive functional group which is reacted and spliced with a synthetic block C1, and R6 is a reactive functional group which is spliced with a ring-closing end molecule A. The reaction sequence of the three reactive functional groups is: firstly, R4 and R3 are reacted and spliced, so that R5 and R6 are required not to interfere the reaction process of connecting R4 and R3; then R5 and the synthetic block C1 are spliced by reaction, so that the R6 is required not to interfere the process of splicing the R5 and the synthetic block C1 by reaction; and finally, after the synthetic building blocks and the DNA labels are spliced in sequence, reacting and splicing the R6 with the ring-closing end molecule A. In a preferred embodiment, R4 is in a form not protected by a protecting group, and R5 and R6 are in a form protected by protecting groups having different deprotection mechanisms. For example, R4 is an unprotected carboxyl group, R5 is an amino group protected with a protecting group, and R6 is a carboxyl group protected with a protecting group; after R4 reacts with an active functional group R3 of a molecule M containing a photocleavable group to obtain G-M-L1, a protective group of R5 is removed by utilizing a deprotection reaction mechanism, and R5 and a synthetic building block C are reacted 1 And (3) performing reaction splicing, continuously completing sequential splicing of each DNA label and each synthetic building block, removing a protecting group of R6 by using another different deprotection reaction mechanism, and performing reaction splicing of the R6 and the ring-closing end molecule A to perform subsequent reaction.
In a second embodiment, the linker molecule L1 has at least four reactive functional groups R4, R5, R6 and R7. The linker molecule L1 may be represented by the following general formula:
Figure BDA0003109642370000131
wherein R4, R5, R6 and R7 are four active functional groups. R4, R5, R6, and R7 may be independently present in a form protected with a protecting group or in a form unprotected with a protecting group, and R4, R5, R6, and R7 do not interfere with each other in a linking reaction with each other. In a second variant, R4 is a reactive function complementary to the reactive function R3 of the molecule M comprising a photocleavable group, and R5 is a reactive function complementary to the reactive function R3 of the molecule M comprising a photocleavable groupReactive functional group for reactive splicing with the synthetic block C1, R6 is reactive functional group for reactive splicing with the ring-closing end molecule A, and R7 is reactive functional group for reactive splicing with the starting nucleotide molecule HP. The reaction sequence of the four reactive functional groups is: firstly, R4 and R3 of a molecule M containing a photocleavable group are spliced in a reaction manner, so that R5, R6 and R7 are required not to interfere with the reaction process of splicing R4 and R3; then R7 is sequentially spliced with an initial nucleotide molecule HP and an open primer OP (also called an open primer) in a reaction way, and R5 and a synthetic building block C 1 Reaction splicing, so that R5 and R6 are required not to interfere with the splicing reaction process of R7; and finally, after the synthetic building blocks and the DNA labels are spliced in sequence, reacting and splicing the R6 with the ring-closing end molecule A. In a preferred embodiment, R4 is in a form without protection by a protecting group, and R5, R6 and R7 are in a form protected by a protecting group with a different deprotection reaction mechanism. The linking molecule with four active functional groups can be spliced by two trifunctional linking molecules.
In a third embodiment, the linker molecule L1 has at least five reactive functional groups R4, R5, R6, R8 and R9. Linker molecule L1 can be represented by the following general formula:
Figure BDA0003109642370000132
wherein R4, R5, R6, R8 and R9 are five reactive functional groups. R4, R5, R6, R8 and R9 may independently exist 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 a linking reaction of each other. In a third embodiment, R4 is a reactive function complementary to the reactive function R3 of the molecule M containing a photocleavable group, R5 is a reactive function complementary to the block C of synthesis 1 Reactive functional group for reactive splicing, R6 is reactive functional group for splicing with ring-closing terminal molecule A, and R8 is reactive functional group for splicing with synthetic block C n+1 Reactive functional group for reaction splicing, and R9 is a reactive functional group for splicing with the ring-closing terminal molecule A. The reaction sequence of the five reactive functional groups is: firstly, R4 reacts with R3 of a molecule M containing a photocleavable group for splicing, so that R5, R6, R8 and R9 are required not to interfere with the reaction process of splicing R4 and R3; then R5 and the synthesized building block C 1 Reaction splicing, so that R6, R8 and R9 are required not to interfere with the splicing reaction process of R5; then R6 reacts with ring-closing terminal molecule A for splicing, so that R8 and R9 are required not to interfere with the splicing reaction process of R6; then, after the first ring structure is formed, R8 is combined with the synthetic block C n+1 Reaction splicing, so that R9 is required not to interfere with the splicing reaction process of R8; and finally, after the synthesis building blocks and the DNA labels are sequentially spliced, reacting and splicing the R9 and the Guan Huanduan molecule A. In a preferred embodiment, R4 is in a form not protected by a protecting group, and R5, R6, R8 and R9 are in a form protected by a protecting group having a different deprotection reaction mechanism. The five reactive functional groups of linker molecules can be spliced from three trifunctional linker molecules or from one trifunctional linker molecule and one tetrafunctional linker molecule.
In a fourth embodiment, the linker molecule L1 has at least six reactive functional groups R4, R5, R6, R7, R8, R9. Linker molecule L1 can be represented by the general formula:
Figure BDA0003109642370000141
wherein R4, R5, R6, R7, R8 and R9 are six active functional groups. R4, R5, R6, R7, R8, R9 can be independently present in a form protected by a protecting group or in a form not protected by a protecting group, and R4, R5, R6, R7, R8 and R9 do not interfere with each other in a connection reaction with each other. In a fourth variant, R4 is a reactive function complementary to the reactive function R3 of the molecule M containing a photocleavable group, and R5 is a reactive function complementary to the block C of synthesis 1 Reactive functional group for reactive splicing, R6 is a reactive functional group for splicing with the ring-closing end molecule A, R7 is a reactive functional group for reactive splicing with the starting nucleotide molecule HP, R8 is a reactive functional group for reactive splicing with the synthetic block C n+1 Reactive functional group for reaction splicing, and R9 is a reactive functional group for splicing with the ring-closing terminal molecule A. The reaction sequence of the six reactive functional groups is: firstly, R4 reacts with R3 of a molecule M containing a photocleavable group for splicing, so that R5, R6, R7, R8 and R9 are required not to interfere with the reaction process of splicing R4 and R3; then R7 is sequentially opened with the initial nucleotide molecule HPPrimer OP reaction splicing, R5 and synthetic building block C 1 Reacting and splicing, so that R5, R6, R8 and R9 are required not to interfere with the splicing reaction process of R7, and R6, R8 and R9 are required not to interfere with the splicing reaction process of R5; then, after the first ring structure is formed, R8 is combined with the synthetic block C n+1 Reactive splicing, thus requiring that R9 does not interfere with the splicing reaction process of R8; and finally, after the synthetic building blocks and the DNA labels are spliced in sequence, reacting and splicing the R9 with the ring-closing end molecule A. In a preferred embodiment, R4 is in a form not protected by a protecting group, and R5, R6, R7, R8 and R9 are in a form protected by a protecting group with a different deprotection reaction mechanism.
In the second embodiment, the linker molecule L1 is preferably composed of two trifunctional linker molecules L1', L1", and can be represented by the following general formula: l1' -L1 "; wherein, L1 'has three reactive functional groups R4, R7 and R4', L1 'has three reactive functional groups R5, R6 and R5', and R4 'and R5' are connected by complementary pairing reaction.
In a second embodiment, the linker molecule L1 comprises a decomposable functional group R L ，R L During decomposition, the linker molecule L1 is split into two molecular fragments: a molecular fragment comprising R4, R7 and a molecular fragment comprising R5, R6.
In the above preferred embodiment of the second technical solution provided by the present invention, the compound library obtained after the secondary screening can be subjected to secondary segmental lysis on the library compound by linking the decomposable functional group R in the molecule L1, so as to directly obtain the screened compound, and the effects of short cycle and high efficiency are also achieved. The cyclic compound library S2 is cracked or acid-degraded by illumination reaction to connect the decomposable functional group R in the molecule L1 L The resulting library compound has the general structural formula:
Figure BDA0003109642370000142
i.e. cyclic compound library S2"'. Wherein L1' is the linker molecule L1 cleavage R L And the residual structural fragments after the R4 and R7 groups are removed.
In some embodiments, the linker molecule L1 can be composed of two trifunctional linker molecules L1', L1' and a linker molecule L0 linked between L1 'and L1', and having a decomposable functional group R L Is located within the structure of the linker molecule L0, or is a functional group linking the linker molecule L0 to the linker molecule L1'. Linker molecule L1 can be represented by the general formula: l1 '-L0-L1'. Wherein L1' has three reactive functional groups R4, R7 and R4', L1 "has three reactive functional groups R5, R6 and R5', L0 has two reactive functional groups R4" and R5"; r4 'is linked to R4' by complementary pairing reaction, and R5 'is linked to R5' by complementary pairing reaction. That is, the decomposable functional group R L The functional group may be a functional group formed by the complementary pairing reaction of R4 'and R4", a functional group formed by the complementary pairing reaction of R5' and R5", or an independently decomposable functional group located within the structure of the linker molecule L0.
In some embodiments, the method further comprises adding a decomposable functional group R to the reaction mixture L Is an acid cleavable group or a photocleavable group having a cleavage wavelength different from that of said photocleavable group-containing molecule M. The acid cleavable group can be an ester bond, an amide bond, and the like,
specifically, linker molecule L0 is selected from the following structures:
Figure BDA0003109642370000151
Figure BDA0003109642370000161
wherein, in the molecular structure of the decomposable connecting molecule L0 shown in the formulas 1 to 7, hydroxyl and aldehyde groups can be used for reacting and splicing with the active functional groups of the connecting molecules L1 'and L1' to form a decomposable functional group R L 。R L Cleavage can be performed under acidic conditions or under light. For example,hydroxyl can react with carboxyl to generate ester bond, and the ester bond can be cracked into two molecular fragments under acidic condition; the aldehyde group can react with the amine group to generate an amine bond; in the molecular structure of the decomposable linker molecule L0 shown in formulas 8-9, hydroxyl and amido react with carboxyl of L1' respectively to generate ester bond and amido bond which can be photo-cleaved, and the wavelength of light irradiation is 290nm during photo-cleavage.
Similarly, in a preferred embodiment of the fourth embodiment, the linker molecule L1 comprises a decomposable functional group R L ，R L During decomposition, the linker molecule L1 is split into two molecular fragments: molecular fragments comprising R4, R7 and molecular fragments comprising R5, R6, R8, R9.
In this way, in the above preferred embodiment of the fourth embodiment, the compound library obtained after the second screening can be linked to the decomposable functional group R in the molecule L1 through the linker molecule L The secondary staged cracking is carried out on the library compound to directly obtain the screened compound, and the effects of short period and high efficiency are also achieved. The cyclic compound library S4 is cracked or acid-degraded by illumination reaction to connect the decomposable functional group R in the molecule L1 L The resulting library compound has the general structural formula:
Figure BDA0003109642370000162
i.e. cyclic compound library S4"'. Wherein L1' is a linker molecule L1 cleavage R L And the residual structural fragments after the R4 and R7 groups are removed.
In some preferred embodiments, the reactive functional group that complementarily pairs any reactive functional group of linker molecule L1 with the reactive functional group may be selected from the following combinations: amino and carboxyl, hydroxyl and carboxyl, phosphate and hydroxyl, amino and alkyl halide or aryl halide, etc.
In a first embodiment, the ring-closing end molecule A is directly reacted with the linker molecule L1. The active functional group R6 of the connecting molecule L1 is carboxyl, the closed ring A end molecule A provides an active functional group amino, and the two react to generate an amido bond for connection; the reaction solvent is an organic solvent such as dichloromethane and N, N-dimethylformamide; adding one or more of N, N' -Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and 4-Dimethylaminopyridine (DMAP) into a reaction solvent; the reaction temperature is 15-35 ℃, preferably 20-30 ℃; the reaction time is 1 to 12 hours, preferably 1 to 6 hours.
In some embodiments of the four aforementioned embodiments, the photocleavable group-containing molecule M is directly reactive linked to the linker molecule L1. The active functional group R4 of the connecting molecule L1 is carboxyl, the molecule M containing the photocleavable group provides an active functional group hydroxyl, and the two react to generate an ester bond for connection; the reaction solvent is an organic solvent such as dichloromethane and N, N-dimethylformamide; adding one or more of N, N' -Diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and 4-Dimethylaminopyridine (DMAP) into a reaction solvent; the reaction temperature is 15-35 ℃, preferably 20-30 ℃; the reaction time is 1 to 12 hours, preferably 1 to 6 hours.
In the three technical schemes provided by the invention, the initial nucleotide molecule HP has an active functional group R10, and the R10 and the active functional group R7 of the connecting molecule L1 or the active functional group R1' of the solid phase carrier G are subjected to complementary pairing reaction.
In the first and third embodiments, the solid support G comprises two reactive functional groups R1 and R1', and R1' of the solid support G is reacted with the reactive functional group R10 of the initiator nucleotide molecule HP to effect ligation, thereby linking the initiator nucleotide molecule HP to the solid support G. Furthermore, the initial nucleotide molecule HP can be connected with the open primer OP reaction under the action of DNA ligase, and further more DNA tags tag are connected in sequence under the action of the DNA ligase to achieve the purpose of extending the DNA coding sequence. The initial nucleotide molecule HP refers to the initial ligation end of a DNA strand in DNA splicing technology. The open primer OP refers to a DNA chain which can be extended in the DNA splicing technology.
In the second and fourth embodiments, the reactive functional group R7 of the linker molecule L1 is reacted with R10 to form a splice, such that the starting nucleotide molecule HP is linked to the linker molecule L1. Furthermore, the initial nucleotide molecule HP can be connected with an open primer OP reaction under the action of DNA ligase, and further more DNA label tags are connected in sequence under the action of the DNA ligase so as to achieve the purpose of prolonging the DNA coding sequence.
In some embodiments, the reactive functional group R10 of the starting nucleotide molecule HP is selected from amino, and the reactive functional group R7 of the linker molecule L1 or the reactive functional group R1' of the solid support G that is complementary to R10 is carboxyl. The carboxyl group reacts with R10 (amino group) to form amido bond, so that the linking molecule L1 or the solid phase carrier G is spliced with the initial nucleotide molecule HP. The carboxyl group may be a carboxyl group protected by forming a tert-butyl ester group; deprotection reaction conditions for carboxy t-butyl ester were 95% trifluoroacetic acid deprotection. The carboxyl group of the active functional group obtained after the deprotection is finished reacts with the amino group of the active functional group R10 of the initial nucleotide molecule HP (HDNA) to form an amide bond connection; firstly, reacting with N, N' -Diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (NHS) in an organic solvent such as dichloromethane and N, N-dimethylformamide for 1-12 h, preferably 1-6 h; and then reacting with a starting nucleotide molecule HP (HDNA) in a HEPES buffer solution for 1-48 h, preferably 12-24 h, wherein the reaction temperature is 15-30 ℃, preferably 20-25 ℃.
In some embodiments, the initiator nucleotide molecule HP is ligated to the open primer OP by T4 DNA ligase to extend the DNA sequence in preparation for ligation of the DNA tag.
In some embodiments of the four preceding embodiments, the reactive functional group R5 of linker molecule L1 is an amino group protected with an Fmoc protecting group. The Fmoc protecting group can be removed under piperidine-containing organic solvent conditions and then ready for ligation into building blocks.
In the four technical schemes, the reactive functional groups R5 and R6 of the linker molecule L1 have different protection groups for deprotection mechanisms. In some embodiments, R6 is carboxy protected with allylhydroxy (AllO-) and R5 is amino protected with fluorenylmethyloxycarbonyl (Fmoc).
In various technical schemes provided by the invention, the completion of the splicing of a synthetic building block and a corresponding DNA label is called as an expansion step. By repeating the extension step, sequential ligation of each synthetic building block and sequential ligation of DNA tags are performed to extend the number of compounds in the compound library. Each synthesis block has a unique corresponding DNA label, the DNA labels corresponding to different synthesis blocks are different, and the synthesis blocks and the corresponding DNA labels form a list. Optionally selecting more synthetic blocks and corresponding DNA tags from the list, repeating the expansion step, such that each synthetic block is sequentially linked to the synthetic block of the previous expansion step and each DNA tag is sequentially linked to the DNA tag of the previous expansion step, i.e. the strand of the synthetic block and the strand of the DNA tag are extended, respectively.
In a first technical solution, tag is obtained by repeating the extension step n —……—tag 1 —OP—HP—G— M—L1—C 1 ……—C n . The number n of connected synthetic building blocks and the corresponding DNA tags is determined according to the size of the required loop. Generally, n is an integer having a value of 2. Ltoreq. N.ltoreq.7.
In a second solution, G-M-L1 (-HP-OP-tag) is obtained by repeating the expansion step 1 —…… —tag n )—C 1 —……—C n (ii) a Wherein n is not less than 2 and not more than 7, and n is an integer.
In a third technical solution, the expansion step is repeated to obtain
Figure BDA0003109642370000181
Wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7, n and m are integers, and n + m is more than or equal to 2 and less than or equal to 7.
In a fourth technical solution, the expansion step is repeated to obtain
Figure BDA0003109642370000182
Wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7.
In the present application, the synthetic building blocks are small molecule compounds with at least a double active functional group. The first active functional group and the second active functional group of the synthetic building block do not exist in a form without protection of a protection group at the same time, and the first active functional group and the second active functional group do not interfere with each other. The synthetic segments may also include a skeletal structure (e.g., a skeletal structural unit) that is attached to the rings or may be attached to the rings in a side chain of the rings. The reactive functional groups of two adjacent synthetic building blocks are complementary, i.e., react together to form a covalent bond through two reactive functional groups that are complementary to each other. The double-active functional groups of the synthetic building block are respectively and independently selected from any one of amino, carboxyl, aldehyde group, alkenyl, alkynyl, halogen, azide, hydroxyl and sulfhydryl.
In a preferred embodiment, the first reactive functional group is present in protected or unprotected form with a protecting group and the second reactive functional group is present in protected form with a protecting group. When the first reactive functional group is present in a form protected by a protecting group, the deprotection mechanism of the first reactive functional group is different from that of the second reactive functional group.
In a preferred embodiment, the first reactive functional group is present in an unprotected form with a protecting group and the second reactive functional group is present in a protected form with a protecting group. In the splicing reaction of the synthesized building block, a first active functional group which is not protected by a protective group is directly utilized to carry out the splicing reaction, then a protective agent of a second active functional group needs to be removed, and then the splicing reaction of the synthesized building block in the next step is carried out.
And when the expanding step is repeated, all the synthetic building blocks are spliced in sequence. In the first and second technical schemes, the synthetic building blocks are spliced in sequence to form the building block C 1 The first active functional group is responsible for splicing with the connecting molecule L1, the first active functional group of each subsequent synthesized building block is sequentially spliced with the second active functional group of the previous synthesized building block, and the last synthesized building block C n The second active functional group of (a) is a first ring-closing functional group selected from: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy group (OH) -, or ureido (NH) 2 CONH) -) that is responsible for the splice-ring closure with the ring-closing terminal molecule A. The first active functional group and the second active functional group of each synthetic block do not exist in a form protected by a protective group at the same time, and the first active functional group and the second active functional group do not interfere with each other. In the third and fourth solutions, the building block C is synthesized 1 Is responsible for the splicing to one reactive functional group R5 of the linker molecule L1, C 1 To C n And synthetic blocks C n The first active functional group of the building block C is spliced with the second active functional group of the previous building block to synthesize the building block C n The second reactive functional group (i.e. the first ring-closing functional group of the first ring structure) is responsible for splicing with the ring-closing terminal molecule a; synthetic block C n+1 Is responsible for splicing with another reactive functional group R8 of the linker molecule L1, C n+1 To C n+m Each synthetic block and synthetic block C n+m The first active functional group is spliced with the second active functional group of the previous synthesized building block in sequence to synthesize the building block C n+m The second reactive functional group (i.e. the first ring-closing functional group of the second ring structure) is responsible for splicing with the second ring-closing terminal molecule a; the first active functional group and the second active functional group are not in the form of protection of a protection group at the same time, and the first active functional group and the second active functional group do not interfere with each other.
In a preferred embodiment, adjacent synthetic segments are connected by the following chemical bonds: amide, ester, ether, amine or imine bond
In a preferred embodiment, the first reactive functional group of each synthetic block is the same reactive functional group, for example both carboxyl groups; the second reactive functional group of each synthetic block is also the same reactive functional group, e.g., both are amino groups; and the first reactive functional group is different from the second reactive functional group.
In a preferred embodiment, the di-active functional groups of the synthetic building blocks are carboxyl and amino groups, respectively, and the carboxyl and amino groups are each independently present in protected form with or without a protecting group.
In a preferred embodiment, the synthetic building block is a dual active functional compound having both amino and carboxyl groups. In some embodiments, the amino and carboxyl groups in the synthetic building block are attached to the same carbon atom, e.g., an alpha-amino acid. In other embodiments, the amino and carboxyl groups in the synthetic building blocks are attached to different atoms, and may be non-amino acid compounds having both amino and carboxyl groups, or may be N-substituted amino acids.
In other preferred embodiments, 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 substitutions, natural or unnatural amino acids, N-substituted amino acids.
In a preferred embodiment, the first reactive functional group is a carboxyl group, which is present in a form unprotected by a protecting group.
In a preferred embodiment, the first reactive functional group is an amino group, the amino group is present in protected form with a protecting group, and the protecting group for the amino group is an Fmoc protecting group.
In a preferred embodiment, the synthetic building block is an Fmoc-amino acid.
In a preferred embodiment, in each synthetic block, there are two synthetic blocks having a third reactive functionality in addition to the first and second reactive functionalities, the third reactive functionalities of the two synthetic blocks being capable of complementary pairing reaction joining to form a cyclic structure by reaction splicing of the respective third reactive functionalities. The two third reactive functional groups react to form a covalent bond. The two third reactive functional groups may be ring-closed by any means known in the art for ring-closing reactions, such as by enzymatic reactions or by simple chemical reactions. More preferably, the two composite blocks are spaced apart by at least one composite block. In a preferred embodiment, the third reactive functional groups of the two synthetic building blocks are carboxyl groups and the other amino group. The third active functional groups exist in a form protected by the protecting groups, and the protecting groups are required to be respectively removed before the splicing reaction of the two third active functional groups.
As a preferred embodiment, in the first and/or second aspect, the block C is synthesized 1 To a synthetic block C n Any two of the two synthetic building blocks have a third active function, and the third active functional groups of the two synthetic building blocks can be connected in a complementary pairing reaction, and are spliced to form a ring structure through respective third active functional groups in a reaction manner. The two third reactive functional groups may be closed by any means known in the art for a ring closure reaction, such as by an enzymatic reaction or by a single pure chemical reaction. In this way, a library of bicyclic compounds is obtained based on the first and/or second embodiments. In the two ring structures of the double-ring structure compound library, the two synthesis building blocks and the synthesis building block between the two synthesis building blocks are shared to form a parallel-ring structure, and the space of the two ring structures is more compact, so that the double-ring structure compound library has a special significance for screening emerging targets.
As a preferred embodiment, in the third and/or fourth technical means, the synthetic block C 1 To a synthetic block C n Or synthetic block C n+1 To a synthetic block C n+m Any two of the two synthetic building blocks have a third active function, and the third active functional groups of the two synthetic building blocks can be connected by complementary pairing reaction, and are spliced to form a ring structure through respective third active functional group reactions. The two third reactive functional groups may be ring-closed by any means known in the art for ring-closing reactions, such as by enzymatic reactions or by simple chemical reactions. Thus, the building block C is synthesized 1 To a synthetic block C n Concrete or synthetic block C n+1 To a synthetic block C n+m The fused ring structure is obtained, and the method has special significance for screening emerging targets.
In a preferred embodiment, 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. The compound library with the E3 ligase substrate structure can be applied to PROTAC, and can realize multipurpose screening of the library.
In some embodiments, the skeletal structure is selected from:
Figure BDA0003109642370000211
in a preferred embodiment, the synthetic segments each comprise at least one exocyclic side chain in common. The exocyclic chain provides extended functionality for further derivatization of library compounds to expand compound library diversity.
In a preferred embodiment, each of the synthetic building blocks contains at least two exocyclic chains in total, and the at least two exocyclic chains are connected by a chemical reaction to form a bicyclic structure. The ring closure reaction of the two exocyclic chains may be performed before or after the enzymatic ring closure reaction. The method for forming the double-ring structure by two ring outside chains can be carried out in all three technical schemes provided by the invention.
The DNA tags (tag) are connected with each other by DNA ligase. In an extension step, a splicing reaction of a DNA tag and a synthetic block corresponding to the DNA tag is completed, so that the chain of the synthetic block and the chain of the DNA tag are respectively extended. In an extension step, the splicing reaction of the DNA label can be performed to extend the chain of the DNA label, and then the splicing reaction of the synthetic block corresponding to the DNA label is performed to extend the chain of the synthetic block; alternatively, the ligation reaction with the synthesis block is performed first to extend the strand of the synthesis block, and then the ligation reaction with the DNA tag corresponding to the synthesis block is performed to extend the strand of the DNA tag.
In the four technical schemes provided by the invention, after the DNA labels are spliced, the closed primers CP (also called end type primers) are connected through DNA ligase, so that a complete DNA coding sequence is formed. The blocking primer CP refers to the terminal strand of DNA that stops the extension in the DNA splicing technology.
In the three technical schemes provided by the invention, the ring-closing end molecule A is a compound with double active functional groups, wherein one active functional group is
Figure BDA0003109642370000221
Namely a second ring-closing functional group which is responsible for carrying out a cyclization reaction with the first ring-closing functional group of the terminal synthetic building block to form a ring-shaped structure; wherein the other active functional group is R11, and R11 is responsible for reacting and splicing with the active functional groups R6 and R9 of the connecting molecule L1. The ring-closing end molecule A can be represented by the general formula R11-A0-SO 2 And F represents. R11 is a reactive functional group, -SO for reactive splicing with a reactive functional group of the linker molecule L1 2 F (sulfonyl fluoride) is a reactive functional group for ring closure reactions.
In the technical scheme provided by the invention, the second ring-closing functional group of the ring-closing terminal molecule A
Figure BDA0003109642370000222
Primary amino (NH) of first ring-closing functional group for synthesizing building block with terminal 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH) under mild conditions, the ring-closing end molecule A and the terminal synthesis building block react to form sulfonamide bond, sulfonate bond or sulfonylurea bond, and then are connected to form a ring. The ring closing reaction is carried out in HEPES buffer solution at 20-40 ℃ for 12-24 h.
According to the construction method of the cyclic compound library, the ring closing reaction in molecules is generated through the first ring closing functional group and the second ring closing functional group, the ring closing condition is mild, the universality is stronger, and the chemical reaction type of the coded compound library and the diversity space of the compound library are expanded.
The invention provides a method for constructing a cyclic compound library, wherein the number and the types of atoms provided by each synthetic block in a ring structure can be adjusted in a diversified manner, the splicing reaction of each synthetic block is not limited, various synthetic blocks can be used for splicing, and the universality is strong. For example, the synthetic building blocks can be spliced by adopting amido bonds (-CO-NH-), so that more potential of hydrogen bond combination can be provided for the ring structure, and the bonding force between the ring structure and the protein can be improved.
The invention provides a method for cutting by utilizing illumination, the reaction condition is mild, and the adverse effect of removing solid phase synthesis under other severe conditions on the stability of a DNA coding sequence is effectively avoided.
The invention provides a method for synthesizing a DNA coding compound loaded by a solid phase carrier, the post-treatment and purification operation of the reaction is simple, and usually only a few times of simple filtration and washing operations are needed, so that the post-treatment and separation and purification processes of each step of reaction are simplified, the synthesis period of a DNA coding compound library is obviously shortened, and the cost is greatly saved.
According to the method for synthesizing the compound library, a connecting molecule (linker) for connecting a DNA coding sequence and the compound library is connected to the compound library, and then the connecting molecule is connected to the DNA coding sequence, wherein the synthesis ratio of the initial nucleotide molecule HP to the docking compound is about 1 to 250, so that the DNA dosage ratio is greatly reduced, and the cost is saved.
In the compound library synthesis method, 249 parts of DNA which is not connected is remained after DNA labels are spliced in each step and needs to be removed, one of double-active functional groups of a synthesis building block is provided with a protecting group, the protecting group removal of the synthesis building block in one step needs to be carried out when the synthesis building block and the corresponding DNA labels are connected in one-time alternate reaction, the reaction points of a compound library of missed DNA can be sealed while the protecting group removal is carried out, the removal of excessive DNA is facilitated, the interference is reduced, the purification effect is achieved, and the efficiency and uniqueness of DNA coding and the purity of final products are improved.
The invention also provides a cyclic compound library (namely a compound library S1) constructed by the method, and the structural general formula of the cyclic compound library is as follows:
Figure BDA0003109642370000231
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
g represents a solid-phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least trifunctional connecting molecule, and a DNA coding sequence is connected to the solid-phase carrier G through an amido bond; c 1 To C n The building blocks are sequentially connected end to end and have double active functional groups; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000232
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); a and C n The first ring-closing functional group and the second ring-closing functional group react to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring.
The invention also provides a second cyclic compound library (i.e. compound library S2) constructed by the method, which has the structural general formula:
Figure BDA0003109642370000233
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
g represents a solid phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least four-functional group connecting molecule, a DNA coding sequence is connected to L1, and L1 is connected with the DNA coding sequence through an amido bond; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000241
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (O)H) -, or ureido (NH) 2 CONH-); a and C n The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring.
The present invention also provides a third cyclic compound library (i.e., compound library S3) constructed by the aforementioned method, which has the general structural formula:
Figure BDA0003109642370000242
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
g represents a solid-phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least five-functional group connecting molecule, and a DNA coding sequence is connected to the solid-phase carrier G through an amido bond; c 1 To C n Is a synthetic building block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A having a second ring-closing functional group
Figure BDA0003109642370000243
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n The last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); two A are respectively connected with C n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonic ester bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonic ester bond or the sulfonylurea bond are connected to form a ring, so that a bicyclic structure is formed.
The present invention also provides a fourth cyclic compound library (i.e., compound library S4) constructed by the aforementioned method, having the general structural formula:
Figure BDA0003109642370000251
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
g represents a solid phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least six-functional-group connecting molecule, a DNA coding sequence is connected to L1, and L1 is connected with the DNA coding sequence through an amido bond; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000252
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic Block C n The last synthetic block C n+m Having a first ring-closing functional group: primary amino (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); two A are respectively connected with C n 、C n+m And a sulfonamide bond, a sulfonate bond or a sulfonylurea bond formed by the reaction of the first ring-closing functional group and the second ring-closing functional group are connected to form a ring, so that a bicyclic structure is formed.
The invention also provides a cyclic compound library (namely a compound library S2) constructed by the method, and the structure formula of the cyclic compound library is as follows:
Figure BDA0003109642370000253
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
l1 is an at least trifunctional connecting molecule, a DNA coding sequence is connected with L1, and L1 is connected with the DNA coding sequence through an amide bond; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A having a second ring-closing functional group
Figure BDA0003109642370000254
L1 is connected with A through a covalent bond; last synthetic block C n Has a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); a and C n The first ring-closing functional group and the second ring-closing functional group react to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring.
The invention also provides a cyclic compound library (namely a compound library S4) constructed by the method, and the structure formula of the cyclic compound library is as follows:
Figure BDA0003109642370000261
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
l1 is at least five functional groups connecting molecule, DNA coding sequence is connected with L1, L1 is connected with DNA coding sequence through amide bond; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000262
L1 is connected with A through a covalent bond; last synthetic Block C n The last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); two ring-closing terminal molecules A and C n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonic ester bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonic ester bond or the sulfonylurea bond are connected to form a ring, so that a bicyclic structure is formed.
In addition, the invention also provides a compound library S2 or a compound library S2' which is connected with the degradable group R in the molecule L1 through degradation L A ring obtainedThe structural general formula of the compound library is as follows:
Figure BDA0003109642370000263
i.e. cyclic compound library S2"'. Wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
l1' is the linker molecule L1 cleavage R L And removing residual structural fragments after removing R4 and R7 groups, wherein L1 is at least four-functional group connecting molecule, the four functional groups are respectively R4, R5, R6 and R7, and the connecting molecule L1 also comprises a decomposable functional group R L ， R L During decomposition, the linker molecule L1 is split into two molecular fragments: molecular fragments comprising R4, R7 and molecular fragments comprising R5, R6; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000264
L1' is connected with A through a covalent bond; last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 -, -di-amino (-NH-) hydroxy (OH) -, or ureido (NH) 2 CONH-); a and C n Through sulfonamide bond, sulfonic ester bond or sulfonylurea bond formed by the reaction of the first ring-closing functional group and the second ring-closing functional group.
In addition, the invention also provides that the compound library S4 or the compound library S4' is connected with the degradable group R in the molecule L1 through degradation L The obtained cyclic compound library has a structural general formula as follows:
Figure BDA0003109642370000265
i.e. cyclic compound library S4"'. Wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
l1' is the linker molecule L1 cleavage R L And the residual structural fragments after the removal of the R4 and R7 groups, the linker molecule L1 comprises a decomposable functional groupGroup R L ，R L During decomposition, the linker molecule L1 is split into two molecular fragments: molecular fragments comprising R4, R7 and molecular fragments comprising R5, R6, R8, R9; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure BDA0003109642370000271
L1' is connected with A through a covalent bond; last composite block C n The last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); two ring-closing terminal molecules A and C respectively n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring, so that a double-ring structure is formed.
Specifically, each of the synthetic blocks (C) 1 ，……，C n ，C n+1 ，……，C n+m ) Each 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.
Specifically, at least one of the synthetic building blocks contains a framework structure, and the framework structure has an E3 ligase substrate structure and can be combined with E3 ligase. The backbone structure is attached to the ring or to the ring in the form of a side chain to the ring.
Specifically, the skeletal structure is selected from:
Figure BDA0003109642370000272
specifically, in each of the synthetic blocks, each synthetic block contains at least one exocyclic side chain. The outer side chain of the ring provides an extended function of further derivatization of the library compound, and meanwhile, functional modification similar to lipidation can be carried out on the branched chain, so that the membrane permeability and permeability of the molecule can be further improved on the basis of the membrane permeability of the molecule, and the diversity of the compound library can be expanded.
Specifically, in each of the synthetic building blocks, each synthetic building block contains at least two cyclic outer chains, and the at least two cyclic outer chains are connected through a chemical reaction to form a double-ring structure. The double-ring or multi-ring structure can stabilize the conformation of the large ring molecule, improve the rigidity of the ring structure, improve the stability of the ring structure molecule and prolong the half-life period of the ring structure molecule drug.
The compound library provided by the invention provides a cyclic compound library, so that the diversity of the compound library is increased, and the application range of the compound library in new drug screening is expanded.
In the compound library, the number and the types of atoms in a ring structure can be adjusted in a diversified manner by different synthetic building blocks, so that the diversity of the compound library is expanded.
In the compound library, the splicing of adjacent synthetic building blocks can be carried out by utilizing amido bonds (-CO-NH) -, more hydrogen bond binding potentials are provided for a ring structure, and the improvement of the binding force of the ring structure and protein is facilitated.
In the cyclic compound library, the synthesis building blocks on the ring can contain a framework structure, such as an E3 ligase substrate structure, so that the application of the compound library in PROTAC is increased.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
In the reaction scheme designations for the following examples,
Figure RE-GDA0003212705090000281
represents a solid phase carrier (PEGA resin), other than the above
Figure RE-GDA0003212705090000282
All represent composite blocks.
Figure RE-GDA0003212705090000283
The English letters L, N, A, I, E, P and the like in the interior are one-letter symbols for amino acids, such as L for leucine (Leu), N for aspartic acid (Asn), A for alanine (Ala), I for isoleucine (Ile), E for glutamic acid (Glu), and P for proline (Pro).
Figure RE-GDA0003212705090000284
The synthetic block is denoted leucine and other one-letter symbol abbreviations are also explained by the one-letter symbol abbreviations for amino acids common in the art.
Figure RE-GDA0003212705090000285
With character AA therein 1 ，……，AA 4 All indicate that the block is an amino acid, and only the numbers 1 … …, etc. are used for distinction.
Figure RE-GDA0003212705090000286
Having a character C therein 1 ，……，C n ， C n+1 ，……，C n+m The composite blocks are all of any type of composite blocks referred to in this application and are identified only by the numbers 1 … … n, n +1 … … n + m. Photo linker denotes a photocleavable group. Ahx represents 6-aminocaproic acid.
EXAMPLE 1 Synthesis of monocyclic Cyclic Compound library
Figure BDA0003109642370000291
The synthesis method comprises the following steps:
1. compounds 1-4 were obtained by taking 100mg of PEGA resin according to the above synthesis method.
2. Reaction splicing of linker molecule L1 and ring-closing end molecule A
The resin, palladium tetratriphenylphosphine (2.89mg, 0.2eq) and phenylsilane (54.12mg, 20eq) were stirred under nitrogen atmosphere in 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with DCM (3X 3 mL), DMF (3X 3 mL) and dried, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried to obtain 65mg of resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), and 4- (2-aminoethyl) benzenesulfonyl fluoride (25mg, 5eq) were stirred in 5mL of DMF for 2min, then the resin was added, and the mixture was stirred at room temperature for 2h, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried. To the resin was added 5mL of 20% 2-methylpiperidine in DMF, and the mixture was stirred at room temperature for 1h, and the resin was washed with DMF (3X 5 mL), DCM (3X 3 mL), DMF (3X 5 mL) and suction dried to give 55mg of resin.
3. The ring-closing end molecule A reacts with the last synthesized building block to form a ring
20mg of the resin was dissolved in 1mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 18mg of the resin. The resin was washed with water (3X 5 mL), DMF (3X 3 mL), and water (3X 5 mL) and then dried by suction to give a library of DNA-encoded cyclic peptides.
Example 2 close-loop validation of monocyclic Cyclic Compound libraries
Figure BDA0003109642370000301
The synthesis method comprises the following steps:
1. DIC (50mg, 5eq), HOBt (51mg, 5eq), fmoc-4peg-COOH (190mg, 5eq) were stirred in 5mL of DMF for 2h, and the resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and then drained to dryness to give 300mg of resin.
2. Splicing of synthetic blocks
The condensation procedure of step 1 was followed using 20% piperidine in DMF 5mL, DIC (50mg, 5eq), HOBt (51mg, 5eq), N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine (120mg, 5eq) to give 280mg of resin.
3. Removal of the Alloc protecting group from linker molecule L1
280mg of resin, palladium tetratriphenylphosphine (9mg, 0.5eq) and phenylsilane (165mg, 20eq) were stirred under nitrogen at 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and suction dried to give 260mg of resin.
4. The connection molecule L1 reacts and splices with a ring-closing end molecule A with a benzene ring structure
The resin 250mg was obtained by liberating 4- (2-aminoethyl) benzenesulfonyl fluoride (110 mg) as DIC (1695g, 5eq), HOBt (17mg, 5eq), 4- (2-aminoethyl) benzenesulfonyl fluoride (90mg, 5eq) with a saturated solution of sodium carbonate according to the condensation procedure of step 1.
5. Fmoc protecting group for removing amino group from final synthesis block
Add 250mg of resin to a 20-cent solution of 2-methylpiperidine in DMF (5 mL) and stir for 3h. The resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and dried by suction to give 230mg of resin.
6. The ring-closing end molecule A with a benzene ring structure reacts with the last synthesized building block to form a ring
50mg of the resin was dissolved in 1mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 40mg of the resin.
7. Removing compound from solid phase carrier under photo-cleavage
40mg of the resin was added to 1ml of NMP, irradiated for 1h under 365nm UV light, the resin was filtered and the solvent was lyophilized to give 2mg of Compound 8.
LCMS:631(M+H)+。
Example 3 Synthesis of monocyclic Cyclic Compound library
Figure BDA0003109642370000321
The synthesis method comprises the following steps:
1. compounds 1-4 were obtained by taking 100mg of PEGA resin according to the above synthesis method.
2. The connection molecule L1 reacts with the ring-closing end molecule A with a heterocyclic structure for splicing
The resin, palladium tetratriphenylphosphine (2.89mg, 0.2eq) and phenylsilane (54.12mg, 20eq) were stirred under nitrogen atmosphere in 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with DCM (3X 3 mL), DMF (3X 3 mL) and dried, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried to obtain 65mg of resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), and 2- (piperazin-1-yl) ethane-1-sulfonyl fluoride (25mg, 5eq) were stirred in 5mL of DMF for 2min, then the resin was added, the mixture was stirred at room temperature for 2h, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried. 5mL of a 20-percent DMF solution of 2-methylpiperidine was added to the resin, and the mixture was stirred at room temperature for 1h, and the resin was washed with DMF (3X 5 mL), DCM (3X 3 mL), and DMF (3X 5 mL) and then dried by suction to obtain 55mg of the resin.
3. The ring-closing end molecule A reacts with the last synthesized building block to form a ring
50mg of resin was dissolved in 5mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 40mg of resin. The resin was washed with water (3X 5 mL), DMF (3X 3 mL), and water (3X 5 mL) and then dried by suction to obtain a library of DNA-encoding cyclic peptides.
Example 4 close-loop validation of monocyclic Cyclic Compound library
Figure BDA0003109642370000331
The synthesis method comprises the following steps:
1. splicing of synthetic building blocks with linker molecules L1
DIC (50mg, 5eq), HOBt (51mg, 5eq), 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16-pentaoxa-4-azanonacan-19-oic acid (190mg, 5eq) were stirred in 5mL of DMF for 2H and the resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and dried to afford 300mg of resin.
2. Splicing of synthetic blocks
The condensation procedure of step 1 was followed using 20% piperidine in DMF 5mL, DIC (50mg, 5eq), HOBt (51mg, 5eq), N- (((9H-fluoren-9-yl) methoxy) carbonyl) -N-methylglycine (120mg, 5eq) to give 280mg of resin.
3. Removal of the Alloc protecting group from linker molecule L1
280mg of resin, palladium tetratriphenylphosphine (9mg, 0.5eq) and phenylsilane (165mg, 20eq) were stirred under nitrogen protection at 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and dried by suction to give 260mg of resin.
4. The connection molecule L1 reacts with the ring-closing end molecule A with a heterocyclic structure for splicing
DIC (1695g, 5eq), HOBt (17mg, 5eq), 2- (piperazin-1-yl) ethane-1-sulfonyl fluoride (90mg, 5eq) were subjected to the condensation procedure in reference to step 1 to obtain 250mg of a resin.
5. Fmoc protecting group for removing amino group from final synthesis block
Add 250mg of resin to a 20-cent solution of 2-methylpiperidine in DMF (5 mL) and stir for 3h. The resin was washed with DMF (3X 3 mL), DCM (3X 3 mL), DMF (3X 5 mL) and dried by suction to give 230mg of resin.
6. The ring-closing end molecule A with heterocyclic structure reacts with the last synthesized building block to form a ring
50mg of the resin was dissolved in 1mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 40mg of the resin.
7. Removing compound from solid phase carrier under photo-cleavage
40mg of resin was added to 1ml of NMP, irradiated under 365nm UV for 1h, filtered and the solvent lyophilized to give 2mg of Compound 8.
LCMS:624(M+H)+。
EXAMPLE 5 Synthesis of side chain-bearing Cyclic Compound library
Figure BDA0003109642370000351
Step 1, splicing the synthetic building block with a connecting molecule L1
DIC (1695g, 5eq), HOBt (17mg, 5eq), fmoc-Acp-OH (63mg, 5eq) were stirred in 3mL of N, N-dimethylformamide for 10min, then 100mg of the swollen resin was added, the mixture was stirred at room temperature for 1h, and the resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), and N, N-dimethylformamide (3X 5 mL) and then dried by suction to obtain 100mg of the resin.
Step 2. Splicing of the synthetic blocks
To the resin was added 5mL of a 20% piperidine solution in N, N-dimethylformamide, and the mixture was stirred at room temperature for 1h, and the resin was washed with N, N-dimethylformamide (3X 5 mL), dichloromethane (3X 3 mL), and N, N-dimethylformamide (3X 5 mL), respectively, and then dried by suction to obtain 100mg of the resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), fmoc-Lys (Boc) -OH (59mg, 5eq) were subjected to the condensation procedure of step 1 to give 95mg of a resin.
Step 3, introducing side chains according to the splicing mode of the synthetic building blocks
To the resin was added 5mL of a 20% piperidine solution in N, N-dimethylformamide, and the mixture was stirred at room temperature for 1h, and the resin was washed with N, N-dimethylformamide (3X 5 mL), dichloromethane (3X 3 mL), and N, N-dimethylformamide (3X 5 mL), respectively, and then dried by suction to give 100mg of the resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), and Synthesis of Block C1 (49mg, 5eq) resin 95mg was obtained by the condensation procedure of step 1. Using a 20% piperidine in N, N-dimethylformamide solution 5ml, dic (1695g, 5eq), HOBt (17mg, 5eq), (synthesis block C2) (52mg, 5eq) resin 90mg was obtained by reference to the Fmoc removal and condensation procedure of step 1.
Step 4, the connection molecule L1 reacts with the ring-closing end molecule A for splicing
90mg of resin, palladium tetratriphenylphosphine (2.89mg, 0.2eq) and phenylsilane (54.12mg, 20eq) were stirred under nitrogen at 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL) and then dried by suction to obtain 85mg of resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), and 2- (piperazin-1-yl) ethane-1-sulfonyl fluoride (25mg, 5eq) were stirred in 5mL of DMF for 2min, then the resin was added, the mixture was stirred at room temperature for 2h, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried. 5mL of a 20-percent DMF solution of 2-methylpiperidine was added to the resin, and the mixture was stirred at room temperature for 1h, and the resin was washed with DMF (3X 5 mL), DCM (3X 3 mL), and DMF (3X 5 mL) and then dried by suction to obtain 75mg of the resin.
Step 5, removing Boc protecting group of amino of the final synthesized building block
Resin 75mg was added to a 5mL (TFA/DCM = 1:4) solution, which was stirred at room temperature for 1h. To the resin was added a 10% solution of DIPEA in N, N-dimethylformamide (3 mL) and the mixture was stirred at room temperature for 2 hours. The resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL) and then dried by suction to give 70mg of the resin.
Step 6, the ring closing end molecule A reacts with the last synthesized building block to form a ring
70mg of the resin was dissolved in 5mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 50mg of the resin. The resin was washed with water (3X 5 mL), DMF (3X 3 mL), and water (3X 5 mL) and dried by suction to give a cyclic peptide resin 65mg.
Step 7. Removing the compound from the solid support under photocleavage
65mg of the resin was added to a mixed solution of 0.5mL acetonitrile and 0.5mL water, irradiated under 365nm UV light for 5h, the resin was filtered, and the solvent was dialyzed and lyophilized to give 5mg of Compound 8.
LCMS:1205.8(M+H)+。
Example 6 close-loop validation of bicyclic Cyclic Compound library
Figure BDA0003109642370000371
Figure BDA0003109642370000381
Step 1, splicing the synthetic building block with a connecting molecule L1
DIC (1695g, 5eq), HOBt (17mg, 5eq), fmoc-Acp-OH (63mg, 5eq) were stirred in 3mL of N, N-dimethylformamide for 10 minutes, then 100mg of the swollen resin was added, the mixture was stirred at room temperature for 1 hour, and the resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), and N, N-dimethylformamide (3X 5 mL) and then dried by suction to obtain 100mg of the resin.
Step 2. Splicing of the synthetic blocks
To the resin was added 5mL of a 20% piperidine solution in N, N-dimethylformamide, and the mixture was stirred at room temperature for 1h, and the resin was washed with N, N-dimethylformamide (3X 5 mL), dichloromethane (3X 3 mL), and N, N-dimethylformamide (3X 5 mL), respectively, and then dried by suction to give 100mg of the resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), fmoc-Lys (Boc) -OH (59mg, 5eq) were subjected to the condensation procedure of step 1 to give 95mg of a resin.
Step 3, introducing a ring-closing end molecule A
95mg of the resin, palladium tetratriphenylphosphine (2.89mg, 0.2eq) and phenylsilane (54.12mg, 20eq) were stirred under nitrogen at 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL) and dried to give 85mg of resin. DIC (1695g, 5eq), HOBt (17mg, 5eq), and 2- (piperazin-1-yl) ethane-1-sulfonyl fluoride (25mg, 5eq) were stirred in 5mL of DMF for 2min, then the resin was added, the mixture was stirred at room temperature for 2h, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL) and dried to obtain 75mg of resin.
Step 4, removing Boc protecting group of amino of the final synthesized building block
Resin 75mg was added to a 5mL (TFA/DCM = 1:4) solution, and the solution was stirred at rt for 30min. The resin was washed with 10% DIPEA in N, N-dimethylformamide (3X 10 mL) and dried to give 70mg of resin.
Step 5, closing the ring for the first time
70mg of resin was dissolved in 5mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight to give 65mg of resin. The resin was washed with water (3X 5 mL), DMF (3X 3 mL) and suction dried to give 65mg of resin.
Step 6, splicing of the synthesized building block and introduction of a second closed-loop terminal molecule A
Standard solid phase synthesis procedures described with reference to step 2 and step 3 gave 50mg of compound 8 as resin.
Step 7, forming a double-ring structure by the second closing ring
To the resin was added 5mL of a DMF solution of 20% 2-methylpiperidine, stirred at room temperature for 1h, the resin was washed with DMF (3X 5 mL), DCM (3X 3 mL), DMF (3X 5 mL) and dried before use, the resin was dissolved in 5mL of HEPES buffer (pH = 8.0), the mixture was reacted overnight at 37 degrees, and the resin was washed with water (5X 5 mL) to give 45mg of resin.
Step 8. Removing the compound from the solid support by photocleavage
45mg of the resin was added to a mixed solution of 0.5mL acetonitrile and 0.5mL water, irradiated under 365nm UV light for 5h, the resin was filtered, and the solvent was dialyzed and lyophilized to give 3mg of Compound 10.
LCMS:1277.9(M+H)+。
Example 7 construction of a double Ring Structure from the third reactive functional group splice of two synthetic building blocks
Figure BDA0003109642370000401
The synthesis method comprises the following steps:
step 1. Mixing DIC (1695g, 5eq), HOBt (17mg, 5eq), fmoc-NH-PEG 3 CH 2 COOH (55mg, 5eq) was stirred in 3mL of N, N-dimethylformamide for 10min, then 100mg of the swollen resin was added, and the mixture was stirred at room temperature for 1H, and the resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL), and H2O (3X 3 mL), respectively, and then dried by suction to obtain 100mg of the resin.
And 2, obtaining 90mg of resin by the resin according to standard solid-phase polypeptide synthesis operation, wherein DIC (1695g, 5eq), HOBt (17mg, 5eq) and Fmoc protected amino acid (60mg, 5eq).
Step 3. Add 5mL of 50% trifluoroacetic acid in dichloromethane to the resin, stir at room temperature for 1h, wash the resin with N, N-dimethylformamide (3X 5 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL) and pump dry to get 87mg of resin. PyBop (27mg, 2eq), DIPEA (17mg, 5eq) in 3mL of N, N-dimethylformamide was stirred for 1 min, then added to the resin, the mixture was stirred at room temperature for 1h, and the resin was washed with N, N-dimethylformamide (3X 3 mL), dichloromethane (3X 3 mL), N, N-dimethylformamide (3X 5 mL) and dried to obtain 85mg of resin. In this step, the side chain of the synthetic block glutamic acid (E) reacts with the side chain of the synthetic block lysine (K) to form a ring structure.
Step 4, 85mg of the resin, palladium tetratriphenylphosphine (2.89mg, 0.2eq) and phenylsilane (54.12mg, 20eq) were stirred under nitrogen at 5ml of dichloromethane at room temperature for 1 hour. The resin was washed with DCM (3X 3 mL), DMF (3X 3 mL), and dried, DIC (1695g, 5eq), HOBt (17mg, 5eq), and 2- (piperazin-1-yl) ethane-1-sulfonyl fluoride (25 mg, 5eq) were stirred in 5mL of DMF for 2min, then the resin was added and the mixture stirred at room temperature for 2h, and the resin was washed with DMF (3X 3 mL), water (3X 5 mL), and dried to give 75mg of resin.
Step 5. Add 5mL of a 20% solution of 2-methylpiperidine in DMF to 75mg of resin, stir at room temperature for 1h, wash the resin with DMF (3X 5 mL), DCM (3X 3 mL), DMF (3X 5 mL) and drain to dryness to give 70mg of resin. 70mg of resin was dissolved in 5mL of HEPES buffer (pH = 8.0), and the mixture was reacted at 37 ℃ overnight. The resin was washed with water (3X 5 mL), DMF (3X 3 mL), and water (3X 5 mL) and dried by suction to give 60mg of the cyclic peptide resin.
And step 6, adding 1ml of NMP into 30mg of resin, irradiating for 5 hours under 365nm ultraviolet light, filtering the resin, dialyzing the solvent, and freeze-drying to obtain 5mg of compound 8.
LCMS:1271.2(M+H) + 。
From the above verification results, the construction method of the present application can construct a bicyclic compound library. The synthetic building block E and the synthetic building block K respectively provide an outer side chain, and the two outer side chains are connected through chemical reaction to form a ring structure. The library compound obtained in this example had a bridge ring as its double ring structure, and one or more synthetic building blocks were shared between the two rings.
In summary, the above embodiments are only preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (46)

1. A chemical reaction ring closure construction method of a cyclic compound library is characterized by comprising the following steps:
1) Directly or indirectly connecting a solid-phase carrier G with a molecule M containing a photocleavable group to obtain G-M;
2) Performing any one of method 1, method 2, method 3, or method 4;
the method comprises the following steps: according to the steps a 1-f 1;
a1. reacting and linking G-M with at least trifunctional linker molecule L1 to obtain G-M-L1;
b1. G-M-L1 reacts with an initial nucleotide molecule HP and an open primer OP in sequence to connect the initial nucleotide molecule HP with a solid phase carrier G to obtain OP-HP-G-M-L1;
c1. OP-HP-G-M-L1 and synthetic Block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 Connecting with OP to obtain tag 1 —OP—HP—G—M—L1—C 1 ；
d1. Defining a group of corresponding synthetic building blocks and DNA label connection reaction as an extension step, repeating the extension step to enable the synthetic building blocks to be sequentially spliced to form an extended chain and enable the DNA labels corresponding to the synthetic building blocks to be sequentially spliced to form the extended chain to obtain tag n —……—tag 1 —OP—HP—G—M—L1—C 1 ……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 2 and not more than 7, and n is an integer;
e1. reacting the product obtained in the last step with a blocking primer CP to enable the blocking primer CP to react with tag n Are connected, wherein HP-OP-tag 1 —……—tag n Formation of the complete DNA coding sequence by-CP to obtain DNA-G-M-L1-C 1 ……—C n ；
f1. 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 DNA-G-M-L1 (-C) 1 ……—C n ) -a, compound library S1'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000011
The method 2 comprises the following steps: according to the steps a 2-f 2;
a2. reacting and linking G-M with a linking molecule L1 with at least four functional groups to obtain G-M-L1;
b2. G-M-L1, an initial nucleotide molecule HP and an open primer OP are sequentially reacted and connected, wherein the initial nucleotide molecule HP is connected with a connecting molecule L1, and G-M-L1-HP-OP is obtained;
c2. respectively mixing the product obtained in the last step with the synthesized building block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Linked to L1, DNA tag 1 To OP to obtain G-M-L1 (-HP-OP-tag) 1 )—C 1 ；
d2. Defining a group of corresponding synthetic building blocks and DNA labels as an extension step, repeating the extension step, so that the synthetic building blocks are sequentially spliced to form an extended chain, and the DNA labels corresponding to the synthetic building blocks are sequentially spliced to form an extended chain, thereby obtaining G-M-L1 (-HP-OP-tag) 1 —……—tag n )—C 1 —……—C n (ii) a Wherein n is more than or equal to 2 and less than or equal to 7 and n is a positive integer;
e2. d, reacting the product obtained in the step d2 with a blocking primer CP to ensure that the blocking primer CP reacts with tag n Are connected, wherein HP-OP-tag 1 —……—tag n The CP forming the complete DNA coding sequence, resulting in G-M-L1 (-DNA) -C 1 —……—C n ；
f2. Reacting the product obtained in the step e1 with a ring-closing end molecule A to link the ring-closing end molecule A with L1 to obtain G-M-L1 (-DNA) (-C) 1 ……—C n ) -a, compound library S2'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000021
The method 3 comprises the following steps: according to the steps a 3-i 3;
a3. reacting and connecting G-M with a connecting molecule L1 with at least five functional groups to obtain G-M-L1;
G-M-L1, with an initiator nucleotide molecule HP and an open primer OP in sequence, to link the initiator nucleotide molecule HP with a solid support G to obtain OP-HP-G-M-L1;
c3. OP-HP-G-M-L1 and synthetic Block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 Connecting with OP to obtain tag 1 —OP—HP—G—M—L1—C 1 ；
d3. Defining a group of corresponding synthetic building blocks and DNA label connection reaction as an extension step, repeating the extension step to enable the synthetic building blocks to be sequentially spliced to form an extended chain and enable the DNA labels corresponding to the synthetic building blocks to be sequentially spliced to form the extended chain to obtain tag n —……—tag 1 —OP—HP—G—M—L1—C 1 ……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 0 and not more than 7, and n is an integer;
e3. the product obtained in the last step, a ring-closing end molecule A and a DNA label tag corresponding to the ring-closing end molecule A A Reacting to connect ring-closing terminal molecule A with L1 and tag A And tag n Are connected to obtain tag A —tag n —……—tag 1 —OP—HP—G—M—L1(—C 1 ……—C n ) -A; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000022
f3. Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the product is obtained
Figure FDA0003109642360000023
g3. The product obtained in the last step is subjected to the expansion step to synthesize the building block C n+1 ，……，C n+m And corresponding DNA label tag thereof n+1 ，……，tag n+m Sequentially reacting to synthesize the building block C n+1 Linked to L1 and DNA tag n+1 And tag A Are connected to obtain
Figure FDA0003109642360000031
Wherein the last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); m is more than or equal to 0 and less than or equal to 7, m is an integer, n + m is more than or equal to 2 and less than or equal to 7;
h3. the product obtained in the last step reacts with the blocking primer CP to ensure that the blocking primer CP reacts with tag n+m Are connected, wherein HP-OP-tag 1 —……—tag n+m -CP formation of the complete DNA coding sequence to obtain
Figure FDA0003109642360000032
i3. 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
Figure FDA0003109642360000033
Namely a compound library S3'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000034
The method 4 comprises the following steps: according to the steps a 4-i 4;
a4. reacting and linking G-M with a linker molecule L1 having at least six functional groups to obtain G-M-L1;
b4. G-M-L1, an initial nucleotide molecule HP and an open primer OP are sequentially reacted and connected, wherein the initial nucleotide molecule HP is connected with a connecting molecule L1, and the G-M-L1-HP-OP is obtained;
c4. respectively mixing the product obtained in the last step with the synthesized building block C 1 Synthetic block C 1 Corresponding DNA tag 1 Reacting to synthesize the building block C 1 Ligation to L1, DNA tag 1 To OP to obtain G-M-L1 (-HP-OP-tag) 1 )—C 1 ；
d4. Defining a group of corresponding synthetic building blocks and DNA labels as an extension step, repeating the extension step, so that the synthetic building blocks are sequentially spliced to form an extended chain, and the DNA labels corresponding to the synthetic building blocks are sequentially spliced to form an extended chain, thereby obtaining G-M-L1 (-HP-OP-tag) 1 —……—tag n )—C 1 —……—C n (ii) a Wherein the last synthetic block C n Having a first ring-closing functional group: primary amino group (NH) 2 (-) amino), a secondary amino group (-NH) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); n is not less than 2 and not more than 7, and n is an integer;
e4. d4, mixing the product obtained in the step d4 with the closed-loop terminal molecule A and the DNA label tag corresponding to the closed-loop terminal molecule A A Reacting to connect ring-closing terminal molecule A with L1 and tag A And tag n Are linked to obtain G-M-L1 (-HP-OP-tag) 1 —……—tag n —tag A )(—C 1 ……—C n ) -A; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000041
f4. Performing intramolecular cyclization reaction on the product obtained in the last step in HEPES buffer solution at the temperature of 20-40 ℃ to finally synthesize the building block C n The first ring-closing functional group reacts with the second ring-closing functional group of the ring-closing terminal molecule A to form a ring, and the ring is obtained
Figure FDA0003109642360000042
g4. The product obtained in the last step is subjected to the expansion step to synthesize the building block C n+1 ，……，C n+m And corresponding DNA label tag thereof n+1 ，……，tag n+m Sequentially reacting to synthesize the building block C n+1 Linked to L1 and DNA tag n+1 And tag A Are connected to obtainTo
Figure FDA0003109642360000043
Wherein the last synthetic block C n+m Having a first ring-closing functional group: primary amino group (NH) 2 - (O)' secondary amino (-NH-) -) hydroxy (OH) -, or ureido (NH) 2 CONH-); m is more than or equal to 0 and less than or equal to 7, m is an integer, n + m is more than or equal to 2 and less than or equal to 7;
h4. the product obtained in the last step reacts with the blocking primer CP to ensure that the blocking primer CP reacts with tag n+m In a linkage of which HP-OP-tag 1 —……—tag n+m -CP formation of the complete DNA coding sequence, obtaining
Figure FDA0003109642360000044
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
Figure FDA0003109642360000051
Namely a compound library S4'; the ring-closing terminal molecule A has a second ring-closing functional group
Figure FDA0003109642360000052
3) Performing intramolecular ring closure reaction on a compound library S1', S2', S3 'or S4' in HEPES buffer solution at the temperature of 20-40 ℃, so that a first ring closure functional group of the finally synthesized building block reacts with a second ring closure functional group of a ring closure end molecule A to form a ring, and respectively obtaining
Figure FDA0003109642360000053
Namely cyclic compound library S1;
or to obtain
Figure FDA0003109642360000054
Namely cyclic compound library S2;
orTo obtain
Figure FDA0003109642360000055
Namely cyclic compound library S3 having a bicyclic structure;
or to obtain
Figure FDA0003109642360000056
Namely, cyclic compound library S4 having a bicyclic structure.
2. The method of claim 1, wherein the solid phase support G is selected from one or more of PEG resin, PEGA resin, tentaGel resin and solid phase support CPG.
3. The method of claim 2, wherein the solid support G has one reactive functional group R1, R1 being an amino group; or the solid phase carrier G has two active functional groups R1 and R1', R1 is amino, and R1' is carboxyl.
4. The method of claim 1, wherein said photocleavable group-containing molecule M contains at least two reactive functional groups, 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 ring A terminal molecule A or the connecting molecule L1.
5. The method of claim 4, wherein R2 is present in an unprotected form with a protecting group and R3 is present in a protected form with a protecting group.
6. The method of claim 4, wherein in the photocleavable group-containing molecule M, the photocleavable group is:
Figure FDA0003109642360000061
wherein R3 isThe side chain positioned at the ortho-position of the nitro is positioned on the C atom directly connected with the benzene ring; r2 is attached to
Figure FDA0003109642360000062
R2 is connected with the C atom on the benzene ring by one or more covalent bonds or R2 is connected with the C atom connected with R3 by one or more covalent bonds; the benzene ring may contain 0,1 or more side chains or substituents which do not interfere with the reaction of linking R2 and R3.
7. The method of claim 6, wherein the photocleavable group-containing molecule M may be selected from the following structures:
Figure FDA0003109642360000063
wherein R3 can be selected from-OH and-NH 2 、-NHNH 2 、-N 3 Cl, br; r2 are all represented by carboxyl.
8. The method of claim 1, wherein in method 1, linker molecule L1 has at least three reactive functional groups R4, R5, and R6; r4, R5 and R6 are respectively and independently in a form protected by a protecting group or a form not protected by the protecting group, and the R4, R5 and R6 do not interfere with the connection reaction of each other; r4 is an active functional group for reaction and splicing with a molecule M containing a photocleavable group, R5 is an active functional group for reaction and splicing with a synthetic block C1, and R6 is an active functional group for reaction and splicing with a ring-closing end molecule A.
9. The method of claim 1, wherein in method 2, linker molecule L1 has at least four reactive functional groups R4, R5, R6 and R7; r4, R5, R6 and R7 can be respectively and independently in a form protected by a protecting group or a form not protected by the protecting group, and the R4, R5, R6 and R7 do not interfere with the connection reaction of each other; r4 is an active functional group for reactive splicing with a molecule M containing a photocleavable group, R5 is an active functional group for reactive splicing with a synthetic block C1, R6 is an active functional group for reactive splicing with a ring-closed terminal molecule A, and R7 is an active functional group for reactive splicing with a starting nucleotide molecule HP.
10. The method of claim 1, wherein in method 3, linker molecule L1 has at least five reactive functional groups R4, R5, R6, R8 and R9; r4, R5, R6, R8 and R9 can independently exist in a form protected by a protecting group or a form not protected by the protecting group, and the R4, R5, R6, R8 and R9 do not interfere with the connection reaction of each other; r4 is an active functional group which is reacted and spliced with a molecule M containing a photocleavable group, and R5 is a synthetic block C 1 Reactive functional group for reactive splicing, R6 is reactive functional group for splicing with ring-closing terminal molecule A, and R8 is reactive functional group for splicing with synthetic block C n+1 Reactive functional group for reaction splicing, and R9 is a reactive functional group for splicing with the ring-closing terminal molecule A.
11. The method of claim 10, wherein in method 4, linker molecule L1 has at least six reactive functional groups R4, R5, R6, R7, R8, R9; r4, R5, R6, R7, R8 and R9 can independently exist in a form protected by a protecting group or a form not protected by the protecting group, and the R4, R5, R6, R7, R8 and R9 do not interfere with the connection reaction of each other; r4 is an active functional group which is spliced by reaction with a molecule M containing a photocleavable group, and R5 is a reactive functional group which is spliced by reaction with a synthetic block C 1 Reactive functional group for reactive splicing, R6 is a reactive functional group for splicing with the ring-closing end molecule A, R7 is a reactive functional group for reactive splicing with the starting nucleotide molecule HP, R8 is a reactive functional group for reactive splicing with the synthetic block C n+1 Reactive functional group for reaction splicing, and R9 is a reactive functional group for splicing with the ring-closing terminal molecule A.
12. The method of claim 9, wherein in method 2, linker molecule L1 comprises a decomposable functional group R L ，R L Splitting the linker molecule L1 into two molecular fragments upon decomposition, the two molecular fragments being: bag (CN)Molecular fragments containing R4 and R7 and molecular fragments containing R5 and R6.
13. The method of claim 11, wherein in method 4, linker molecule L1 comprises a decomposable functional group R L ，R L Upon decomposition, the linker molecule L1 is split into two molecular fragments, which are: molecular fragments comprising R4, R7 and molecular fragments comprising R5, R6, R8, R9.
14. The method of claim 12 or 13, wherein the decomposable functional group R is L Is an acid-cleavable group or a photocleavable group having a different cleavage wavelength than said photocleavable group-containing molecule M.
15. The method of claim 9, wherein in the method 2, the linker L1 is composed of two trifunctional linker molecules L1', L1", and a linker molecule L0 linked between L1' and L1", and the decomposable functional group R L Is located in the structure of the linker molecule L0, or is a functional group linking the linker molecule L0 with the linker molecule L1'; wherein L1' has three reactive functional groups R4, R7 and R4', L1 "has three reactive functional groups R5, R6 and R5', L0 has two reactive functional groups R4" and R5"; r4 'is linked to R4' by complementary pairing reaction, and R5 'is linked to R5' by complementary pairing reaction.
16. The method of claim 15, wherein the linker molecule L0 is selected from the following structures:
Figure FDA0003109642360000081
17. the method as claimed in claim 1, wherein the starting nucleotide molecule HP has a reactive function R10, R10 being capable of complementary pairing reaction with the reactive function of the linker molecule L1 or with the reactive function of the solid support G.
18. The method according to claim 17, wherein the reactive functional group R10 of the starting nucleotide molecule HP is an amino group and the reactive functional group of the linker molecule L1 or of the solid support G that reacts complementarily to 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 reactive functional groups.
20. The method of claim 19, wherein in method 1 and/or method 2, the composite blocks are spliced in sequence to form composite block C 1 The first active functional group of the building block C is responsible for splicing with the connecting molecule L1, 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 last synthetic building block C n The second active functional group is a first ring-closing functional group which is responsible for splicing and ring-closing end molecule A; the first active functional group and the second active functional group are not in the form of protection of the protection group at the same time, 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 block C is synthesized 1 Is responsible for the splicing to one reactive functional group of the linker molecule L1, C 1 To C n Each synthetic block and synthetic block C n The first active functional group of the building block C is spliced with the second active functional group of the previous building block to synthesize the building block C n The second active functional group is a first ring-closing functional group and is responsible for splicing with a ring-closing terminal molecule A to form a first ring structure; synthetic block C n+1 The first reactive functional group of (a) is responsible for the attachment of the linker moietySplicing of the other reactive function of the seed L1, C n+1 To C n+m Each synthetic block and synthetic block C n+m The first active functional group of the building block C is spliced with the second active functional group of the previous building block to synthesize the building block C n+m The second active functional group is another first ring-closing functional group and is responsible for splicing with another ring-closing terminal molecule A to form a second ring structure; the first active functional group and the second active functional group are not in the form of protection of the protection group 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 adjacent composite blocks are connected by the following chemical bonds: 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 amino and carboxyl groups of a double reactive functional group.
24. The method of claim 19, wherein the synthetic building blocks are selected from the group consisting of 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 substitutions, natural or unnatural amino acids, N-substituted amino acids.
25. The method of claim 19, wherein two of the synthetic blocks present in each synthetic block have a third reactive functionality in addition to the first reactive functionality and the second reactive functionality, the third reactive functionalities of the two synthetic blocks capable of complementary mating reactive joining, the third reactive functionalities of the two synthetic blocks reactive joining to form a cyclic structure.
26. The method of claim 25, wherein the two composite blocks are separated by at least one composite block.
27. The method of claim 25, wherein the third reactive functional groups of the two synthetic building blocks are carboxyl groups and the other is amino groups.
28. The method of claim 19, wherein the composite block further comprises a backbone structure attached to the ring or attached to the ring in 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 having an E3 ligase substrate structure capable of binding to an E3 ligase.
30. The method of claim 25, wherein the skeletal structure is selected from the group consisting of:
Figure FDA0003109642360000101
31. the method of claim 28, wherein each of said synthetic segments comprises a total of at least one exocyclic side chain.
32. The method of claim 28, wherein each of the synthetic blocks comprises a total of at least two exocyclic chains, and wherein the at least two exocyclic chains are joined by a chemical reaction to form a bicyclic structure.
33. The method of claim 1, wherein the DNA tags are sequentially linked to each other by a DNA ligase; in an extension step, a splicing reaction of a DNA tag and a synthetic block corresponding to the DNA tag is completed, so that the chain of the synthetic block and the chain of the DNA tag are respectively extended.
34. The method of claim 1, wherein in step 3), the second ring-closing functional group of ring-closing terminal molecule a
Figure FDA0003109642360000102
Performing a ring closing reaction with a primary amino group, a secondary amino group, a hydroxyl group or a carbamido group of a first ring closing functional group of the terminal synthetic block under mild conditions, so that a ring closing terminal molecule A reacts with the terminal synthetic block to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring; the condition of the ring closure reaction is that the intramolecular ring closure reaction is carried out in HEPES buffer solution at the temperature of 20-40 ℃ for 12-24 h.
35. The method of claim 1, wherein in method 2, the photocleavable group-containing molecule M is cleaved from the library S2 by a photoreaction to yield the photocleavable group-containing molecule M
Figure FDA0003109642360000103
Namely, cyclic compound library S2'.
36. The method of claim 1, wherein in method 4, the photocleavable group-containing molecule M is cleaved from the library S4 by a photoreaction to yield a photocleavable group-containing molecule M
Figure FDA0003109642360000111
I.e. cyclic compound library S4".
37. A cyclic compound library, which is characterized in that the structural general formula is one of the following six structural general formulas:
(1) The general formula of the structure is
Figure FDA0003109642360000112
Wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
g represents a solid-phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least trifunctional group connecting molecule, and a DNA coding sequence is connected to the solid-phase carrier G through an amido bond; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000113
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; a and C n The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonic ester bond or a sulfonylurea bond which are connected to form a ring;
or (2) the general structural formula is as follows:
Figure FDA0003109642360000114
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
g represents a solid phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least four-functional group connecting molecule, a DNA coding sequence is connected to L1, and L1 is connected with the DNA coding sequence through an amido bond; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000115
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; a and C n Through the first ring-closing functional group and the second ring-closing functional groupThe sulfonamide bond, the sulfonate bond or the sulfonylurea bond formed by the reaction of the two ring-closing functional groups are connected to form a ring;
or (3) the general structural formula is as follows:
Figure FDA0003109642360000121
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
g represents a solid-phase carrier, M represents a molecule containing a photocleavable group, L1 is an at least five-functional-group connecting molecule, and a DNA coding sequence is connected to the solid-phase carrier G through an amido bond; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000122
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n The last synthetic block C n+m Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; two A are respectively connected with C n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring, so that a double-ring structure is formed;
or (4) the general structural formula is as follows:
Figure FDA0003109642360000123
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
g represents a solid support, M represents a molecule containing a photocleavable group, L1 is an at least hexafunctional linker molecule, a DNA coding sequence is linked to L1, and a DNA coding sequence is linked between L1 and the DNA coding sequence throughAmide bond linkage; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000124
G and M, M and L1, and L1 and A are connected through covalent bonds; last synthetic block C n The last synthetic block C n+m Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; two A are respectively connected with C n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring, so that a double-ring structure is formed;
or (5) the general structural formula is:
Figure FDA0003109642360000131
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
l1 is an at least trifunctional connecting molecule, a DNA coding sequence is connected with L1, and L1 is connected with the DNA coding sequence through amido bond; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000132
L1 is connected with A through a covalent bond; last synthetic Block C n Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; a and C n The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonic ester bond or a sulfonylurea bond which are connected to form a ring;
or (6) the general structural formula is:
Figure FDA0003109642360000133
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
l1 is a connecting molecule with at least five functional groups, a DNA coding sequence is connected with L1, and L1 is connected with the DNA coding sequence through amido bond; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000134
L1 is connected with A through a covalent bond; last synthetic block C n Finally, the synthetic block C n+m Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; two ring-closing terminal molecules A and C respectively n 、C n+m The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonate bond or a sulfonylurea bond, and the sulfonamide bond, the sulfonate bond or the sulfonylurea bond are connected to form a ring, so that a double-ring structure is formed.
38. A cyclic compound library, characterized in that the general structural formula is one of the following two general structural formulas:
(1) The general structural formula is as follows:
Figure FDA0003109642360000135
wherein n is more than or equal to 2 and less than or equal to 7, and n is an integer;
l1' is the linker molecule L1 cleavage R L And removing residual structural fragments after removing R4 and R7 groups, wherein L1 is at least four-functional group connecting molecule, the four functional groups are respectively R4, R5, R6 and R7, and the connecting molecule L1 also comprises a decomposable functional group R L ，R L The decomposition causes the linker molecule L1 to split into two molecular fragments: a molecular fragment comprising R4, R7 and a molecular fragment comprising R5, R6; c 1 To C n Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000141
L1' is connected with A through a covalent bond; last synthetic block C n Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; a and C n The first ring-closing functional group and the second ring-closing functional group are reacted to form a sulfonamide bond, a sulfonic ester bond or a sulfonylurea bond which are connected to form a ring;
or (2) the general structural formula is:
Figure FDA0003109642360000142
wherein n is more than or equal to 0 and less than or equal to 7,0 and less than or equal to 7,n, m is an integer, and n + m is more than or equal to 2 and less than or equal to 7;
l1' is the linker molecule L1 cleavage R L And the residual structural fragments after the removal of the R4 and R7 groups, the linker molecule L1 comprises a decomposable functional group R L ，R L During decomposition, the linker molecule L1 is split into two molecular fragments: molecular fragments comprising R4, R7 and molecular fragments comprising R5, R6, R8, R9; c 1 To C n A synthetic block with double active functional groups connected end to end in sequence, C n+1 To C n+m Is a synthetic building block with double active functional groups which are connected end to end in sequence; a represents a ring-closing terminal molecule A which has a second ring-closing functional group
Figure FDA0003109642360000143
L1' is connected with A through a covalent bond; last synthetic block C n The last synthetic block C n+m Having a first ring-closing functional group: a primary amino group, a secondary amino group, a hydroxyl group, or a ureido group; two ring-closing terminal molecules A and C respectively n 、C n+m Through said first ring-closing functional group andand a sulfonamide bond, a sulfonate bond or a sulfonylurea bond formed by the reaction of the second ring-closing functional group is connected to form a ring, so that a bicyclic structure is formed.
39. The cyclic compound library of claim 37 or 38, wherein in each synthetic block, there are two synthetic blocks having a third reactive function in addition to the first reactive function and the second reactive function, and wherein the two third reactive functions can be cyclized by any means known in the art, such as by enzymatic reaction or by simple chemical reaction, and the two synthetic blocks are reacted and joined to form a covalent bond via the respective third reactive functions to obtain a cyclic structure.
40. The cyclic compound library of claim 39, wherein C 1 To C n Any two of the two synthetic building blocks have a third active function, the third active functional groups of the two synthetic building blocks can be connected in a complementary pairing reaction, and the third active functional groups of the two synthetic building blocks react and splice to form a covalent bond, so that a ring structure is obtained.
41. The library of cyclic compounds of claim 39, wherein C is n+1 To C n+m Any two of the two synthetic building blocks have a third active function, and the third active functional groups of the two synthetic building blocks can be connected in a complementary pairing reaction manner, and the third active functional groups react to splice covalent bonds to obtain a ring structure.
42. The cyclic compound library of claim 37 or 38, wherein each of the synthetic building blocks is independently selected from 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 a non-natural amino acid, an N-substituted amino acid.
43. The cyclic compound library of claim 42, wherein at least one of the synthetic building blocks comprises a backbone structure having an E3 ligase substrate structure capable of binding an E3 ligase.
44. The library of cyclic compounds of claim 42, wherein the backbone structure is selected from the group consisting of:
Figure FDA0003109642360000151
45. the cyclic compound library of claim 42, wherein each of the synthetic blocks comprises at least one exocyclic side chain in common.
46. The cyclic compound library of claim 42, wherein each of the synthetic building blocks contains a total of at least two exocyclic chains, and wherein the at least two exocyclic chains are joined by chemical reaction to form a bicyclic structure.
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