Bidirectional cyclic peptide DNA-encoded compound, and library construction method therefor and use thereof

Abstract

Provided are a bidirectional cyclic peptide DNA-encoded compound, and a library construction method therefor and a use thereof. Specifically, provided are a method for constructing a library of bidirectional cyclic peptide DNA-encoded compounds and a DNA-encoded cyclic peptide library constructed by using the method. The method is simple and convenient to operate, uses substrates that are widely applicable and easily available at low cost, has a wide application range, and is particularly suitable for searching for a cyclic peptide hit compound for a protein target.

Description

Bicyclic peptide type DNA encoded compound and its library construction method and application TECHNICAL FIELD

[0001] The present application relates to the fields of chemistry and biotechnology, in particular, the present application relates to a bicyclic peptide type DNA encoded compound and its library construction method and application. BACKGROUND

[0002] Cyclic peptides are a kind of "special" compound type, which is in the chemical space between small molecules and biological macromolecules, and has unique physicochemical properties and drug-making advantages. It has the advantages of traditional small molecule synthesis convenient operation, low cost, low toxicity, potential oral drug and transmembrane targeting intracellular target. It also has the advantages of high affinity and selectivity of biological macromolecules and target. Especially for protein-protein interaction target (PPI), cyclic peptide molecules show unique advantages. With the continuous development of chemical-biological technology, and the in-depth understanding of researchers on the physicochemical properties, metabolic stability and membrane permeability of cyclic peptides, cyclic peptides have ushered in a new wave of attention.

[0003] High-throughput screening based on entity compound library is one of the most important methods for discovering cyclic peptide lead compounds. DNA encoded compound library technology is a new generation of high-throughput screening technology platform, which uses combinatorial technology to quickly synthesize a large number of compounds, and combines with chemical biology technology to read out the structure of the compound with affinity from the mixed library molecule set. This technology platform has the advantages of low cost, short time and low labor cost, and has been adopted by pharmaceutical companies and universities to find lead compounds for target targets. Therefore, we design and develop cyclic peptide DNA encoded compound library with drug-like properties.

[0004] The traditional synthesis method of cyclic peptide type DNA encoded molecule library adopts a one-way synthesis method, which can only synthesize small size ring structures in a limited number of chemical synthesis steps. This will cause the overall diversity of the macrocycle to be insufficient, and the affinity to the target is not enough. Therefore, we design and develop a two-way synthesis method to construct a DNA encoded cyclic peptide molecule library. The advantage of this synthesis method is that it can synthesize large size ring structures using limited chemical steps. While improving the diversity of the macrocycle structure, it enhances the force of the target and increases the possibility of drug-making. SUMMARY

[0005] An object of the present application is to provide a novel bicyclic peptide type DNA encoded compound and a method for constructing a molecule library thereof.

[0006] In a first aspect of the present application, a preparation method of a bicyclic peptide type DNA encoded compound is provided, comprising the following steps:

[0007] (a) providing a compound shown as Formula M0, wherein the compound shown as Formula M0 contains Linker (L), A2 and A3, wherein the Linker has -A1-N fragment attached thereto;

[0008] wherein,

[0009] N (or ) is a single / double stranded deoxyribonucleotide sequence, a single / double stranded ribonucleotide sequence, or a combination thereof;

[0010] A1 is a chemical bond or a chemical structure connecting Linker and N;

[0011] Linker (or L) is a multifunctional linking backbone;

[0012] A2 and A3 are reactive groups;

[0013] (b) providing a synthetic building block shown as Formula M1;

[0014] wherein,

[0015] is a synthetic building block central structure;

[0016] G1, G2 are reactive groups;

[0017] (c) reacting the compound shown as Formula M0 with the synthetic building block shown as Formula M1 to form a compound shown as Formula Q containing a nucleic acid fragment;

[0018] wherein,

[0019] m, n are each independently an integer from 1 to 100;

[0020] each O1 and O2 is independently a chemical bond or a chemical structure;

[0021] N (or ), A1, Linker (or L), G2 is as defined above;

[0022] (d) ring-forming reaction of the compound shown as Formula Q with the synthetic building block shown as Formula M1 to form a compound shown as Formula R containing a nucleic acid fragment:

[0023] wherein,

[0024] P1 and P2 are chemical structures or chemical bonds formed by the reaction of two reactive groups G2 on the synthetic building block center structure of the compound of Formula Q and two reactive groups (G1 and G2) on the synthetic building block of Formula M1, respectively;

[0025] N (or ), A1, Linker (or L), m, n, O1, O2 are as defined above.

[0026] In another preferred embodiment, m and n are each independently an integer from 1 to 50, preferably, m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0027] In another preferred embodiment, m and n are the same.

[0028] In another preferred embodiment, m and n are not the same.

[0029] In another preferred embodiment, O1 is formed by a nucleic acid compatible chemical reaction of A2 in the compound of Formula M0 and G1 in the synthetic building block of Formula M1;

[0030] O2 is formed by a nucleic acid compatible chemical reaction of A3 in the compound of Formula M0 and another G2 in another synthetic building block of Formula M1.

[0031] In another preferred embodiment, when m and n are 1, O1 is a chemical bond formed by A2 in the compound of Formula M0 and G1 on the compound of Formula M1, and O2 is a chemical bond formed by A3 in the compound of Formula M0 and another G2 on the compound of Formula M1;

[0032] When m and n are both greater than 1, O1 is a chemical bond formed by G2 on the synthetic building block center structure of the compound of Formula Q and G1 on the compound of Formula M1, and O2 is a chemical bond formed by another G2 on the synthetic building block center structure of the compound of Formula Q and G1 on another compound of Formula M1.

[0033] In another preferred embodiment, P1 is formed by a nucleic acid compatible chemical reaction of G2 in the compound of Formula Q and G1 in the synthetic building block of Formula M1;

[0034] P2 is formed by a nucleic acid compatible chemical reaction of another G2 in the compound of Formula Q and G2 in the same synthetic building block of Formula M1.

[0035] In another preferred embodiment, in step (c), further comprising: reacting two G2 or one G2 in the compound of Formula Q obtained from the reaction with the synthetic building block of Formula M1, repeating multiple times, thereby obtaining a compound of Formula Q with multiple synthetic building blocks.

[0036] In another preferred embodiment, each of A1, O1, O2, P1, P2 is independently a chemical structure or a chemical bond selected from the group consisting of:

[0037] wherein, is an aromatic or heteroaromatic ring.

[0038] In another preferred embodiment, each of A2, A3, G1, and G2 is independently selected from the group consisting of H, -N3, aldehyde, hydroxyl, carboxyl, terminal alkene, terminal alkyne, chloro, bromo, iodo, substituted or unsubstituted thiol, substituted or unsubstituted -S-SH, substituted or unsubstituted phenyl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted amino, substituted or unsubstituted -NH-C(O)-OH, substituted or unsubstituted -NH-C(O)H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted C 3-7 cycloalkyl, 4-7 membered cycloalkenyl, substituted or unsubstituted C 5-9 cycloalkynyl, substituted or unsubstituted -C(O)O-C1-C6alkyl, substituted or unsubstituted sulfonamide;

[0039] said substitution means that one or more (1, 2, 3, 4, 5, 6) hydrogens on the group are replaced by a substituent selected from the group consisting of oxo (=O), cyano, halogen, -SO3Na, substituted or unsubstituted C 1-6 alkyl, C 1-4 alkyl, C 2-6 alkenyl, substituted or unsubstituted 4-7 membered heterocycloalkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted 4-7 membered heterocyclyl, substituted or unsubstituted 6-8 membered heterocycloalkynyl, substituted or unsubstituted 5-7 membered heteroaryl, halogenated C 1-4 alkyl, halogenated phenyl; wherein, said substitution means that one or more (1, 2, 3, 4, 5, 6) hydrogens on the group are replaced by a substituent selected from the group consisting of halogen, cyano, oxo (=O), -SO3Na, -SO2(C 1-6 alkyl), C 1-4 alkyl, C 1-4 alkyl, C 1-4 alkyl, C 1-6 amide.

[0040] In another preferred embodiment, each of A2, A3, G1, and G2 is independently selected from the group consisting of:

[0041] wherein X is F, Cl, Br or I;

[0042] is an aromatic ring or an aromatic heterocycle.

[0043] In another preferred embodiment, is a C6-20aromatic ring or a 5-20 membered aromatic heterocycle.

[0044] In another preferred embodiment, is a C6-10aromatic ring or a 5-9 membered aromatic heterocycle.

[0045] In another preferred embodiment, the substitution site of the substituent in is ortho, meta or para.

[0046] In another preferred embodiment, Linker (or L) is a trivalent group formed by a combination of chemical elements and / or chemical bonds selected from the group consisting of:

[0047] (1) chemical elements: C, H, O, N, P, S, Si; and

[0048] (2) chemical bonds: C-C, C=C, C-Y, C=Y, Y-Y, Y=Y; wherein each Y is independently selected from H, O, N, P, S, Si.

[0049] In another preferred embodiment, the Linker (or L) is selected from the group consisting of:

[0050] wherein q is 1, 2, 3, or 4.

[0051] In another preferred embodiment, the synthetic building block central structure is a structure formed by a combination of chemical elements and / or chemical bonds selected from the group consisting of:

[0052] (1) chemical elements: C, H, O, N, P, S, Si, halogen; and

[0053] (2) chemical bonds: C-C, C=C, C-Y, C=Y, Y-Y, Y=Y; wherein each Y is independently selected from H, O, N, P, S, Si.

[0054] In another preferred embodiment, the synthetic building block is a natural amino acid or an unnatural amino acid.

[0055] In another preferred embodiment, in step (c) and step (d), the synthetic building blocks are connected via or with the connecting groups (i.e., A2and A3) in Formula M0 by a reaction selected from the group consisting of amidation, reductive amination, nucleophilic substitution, Suzuki coupling, Heck coupling, Sonogashira coupling, S- aromatization, CuAAC reaction, photoinduced reaction, preferably, by amidation.

[0056] In a second aspect of the present application, there is provided a bidirectional cyclic peptide type DNA encoded compound as shown in Formula R,

[0057] wherein,

[0058] N (or ) is a single / double stranded deoxyribonucleotide sequence, a single / double stranded ribonucleotide sequence, or a combination thereof;

[0059] A1is a chemical bond or a chemical structure connecting Linker and N;

[0060] Linker (or L) is a multifunctional connecting skeleton;

[0061] m, n are each independently an integer from 1 to 100;

[0062] each O1and O2is independently a chemical bond or a chemical structure;

[0063] is a synthetic building block central structure; and

[0064] P1and P2are each a chemical bond or a chemical structure.

[0065] In a third aspect of the present application, there is provided a method for constructing a library of bidirectional cyclic peptide type DNA encoded compounds,

[0066] The library of bidirectional cyclic peptide type DNA encoded compounds comprises t bidirectional cyclic peptide type DNA encoded compounds having a structure as shown in Formula R, t is a positive integer ≥ 1000;

[0067] wherein,

[0068] N (or ), A1, Linker (or L), m, n, O1, O2, P1, P2are as defined in the second aspect of the present application;

[0069] The method comprises:

[0070] (a) synthesizing the bidirectional cyclic peptide type DNA-encoded compounds of formula R using the method of claim 1; and

[0071] (b) combining t compounds of formula R to form the library of nucleic acid encoded compounds.

[0072] In another preferred embodiment, t is > 10,000, preferably > 100,000, more preferably > 1000,000, more preferably > 5000,000, most preferably > 10,000,000.

[0073] In a fourth aspect of the present application, there is provided a library of bidirectional cyclic peptide type DNA-encoded molecules comprising t bidirectional cyclic peptide type DNA-encoded compounds of formula R, wherein t is a positive integer > 1000;

[0074] wherein,

[0075] N (or ), A1, Linker (or L), m, n, O1, O2, P1, P2 are as defined in the second aspect of the present application.

[0076] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 shows the mass spectrum of the product in Example 1, step (1).

[0078] Figure 2 shows the mass spectrum of the product in Example 1, step (2).

[0079] Figure 3 shows the mass spectrum of the product in Example 1, step (3).

[0080] Figure 4 shows the mass spectrum of the product in Example 1, step (4).

[0081] Figure 5 shows the mass spectrum of the product in Example 1, step (5). DETAILED DESCRIPTION

[0082] The inventors have made extensive and in-depth research and for the first time developed a brand new bidirectional synthesis method, which can efficiently construct a large size cyclic peptide type DNA-encoded molecule library. The method provided by the present application is simple to operate, mild in conditions, high in substrate universality, and has a wide application field, and has a promoting effect on the development of the medical field. Based on this, the inventors have completed the present application.

[0083] Definitions

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0085] As used herein, the terms "comprising" or "including," or "having" can be open-ended, and allow for "consisting essentially of," or "consisting of." In other words, the terms "comprising" or "including," or "having" also include the terms "consisting essentially of," or "consisting of."

[0086] Group Definitions

[0087] Definitions of standard chemical terminology can be found in reference works, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of the art are employed in practicing the application, such as mass spectroscopy, NMR, IR and UV / VIS spectroscopy and pharmacological methods. Unless specific definitions are provided, the nomenclature utilized in connection with the description herein is consistent with that employed in the relevant art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical formulation, and delivery, and treatment of patients. For example, reactions and purification can be performed utilizing the instructions provided by manufacturers of reagents, or according to conventional methods well known in the art, or as described herein. The foregoing techniques and procedures can be readily implemented by the skilled artisan by following the teachings of the present specification and the examples which follow. In the present description, groups and substituents thereof can be selected by one skilled in the art to provide stable moieties and compounds.

[0088] Throughout this document, unless specifically stated otherwise, each chiral carbon atom in all compounds of the application can optionally be in the R or S configuration, or a mixture of the R and S configurations. Unless specifically stated otherwise, a structural formula depicted in the present application is intended to include all isomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomers: for example, the R, S configuration of asymmetric centers, the (Z), (E) isomers of double bonds, and the (Z), (E) conformational isomers. Accordingly, individual stereochemical isomers of the compounds of the present application, or mixtures of the enantiomeric, diastereomeric, or geometric (or conformational) isomers, are within the scope of the present application.

[0089] ​"Substituted amino" means an amino group substituted with one or two alkyl, alkylcarbonyl, arylalkyl, heteroarylalkyl groups as defined below, for example, mono-alkylamino, di-alkylamino, alkylamido, arylalkylamino, heteroarylalkylamino.

[0090] In the present application, the term "alkyl" as a group or part of a group (for example in the groups halo-substituted alkyl and the like) refers to a straight or branched chain hydrocarbon group, consisting only of carbon and hydrogen atoms, having, for example, 1 to 12 (preferably 1 to 8, more preferably 1 to 6) carbon atoms, and connected to the rest of the molecule by a single bond, for example including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, decyl and the like. For the purposes of the present application, the term "alkyl" preferably refers to alkyl groups having 1 to 6 carbon atoms.

[0091] In the present application, the term "alkenyl" as a group or part of a group means a straight or branched chain hydrocarbon group consisting only of carbon and hydrogen atoms, containing at least one double bond, having, for example, 2 to 14 (preferably 2 to 10, more preferably 2 to 6) carbon atoms and connected to the rest of the molecule by a single bond, for example, but not limited to, ethenyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-1,4-dienyl and the like.

[0092] The term "alkynyl" as a group or part of a group herein means a straight or branched chain hydrocarbon group consisting only of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, having, for example, 2 to 14 (preferably 2 to 10, more preferably 2 to 6) carbon atoms and connected to the rest of the molecule by a single bond, for example, but not limited to, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

[0093] In the present application, the term "carbocyclyl" as a group or part of another group means a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting solely of carbon and hydrogen atoms, which can include fused, bridged or spiro ring systems, having from 3 to 15 carbon atoms, preferably having from 3 to 10 carbon atoms, more preferably having from 3 to 8 carbon atoms, and which is saturated or unsaturated and which is attached to the rest of the molecule via a single bond through any available carbon atom. Preferably, the carbocyclyl group is a cycloalkyl group. The cycloalkyl group is preferably a C3-10cycloalkyl group. Unless specifically indicated otherwise in the specification, the carbon atoms in the carbocyclyl group can optionally be oxidized. Examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, indanyl, octahydro-4,7-methano-lH-indenyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, lH-indenyl, 8,9-dihydro-7H-benzo-cyclohepten-6-yl, 6,7,8,9-tetrahydro-5H-benzo-cycloheptenyl, 5,6,7,8,9,10-hexahydro-benzo-cyclooctenyl, fluorenyl, bicyclo[2.2.1]heptyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, bicyclo[2.2.2]octyl, bicyclo[3.1.1]heptyl, bicyclo[3.2.1]octyl, bicyclo[2.2.2]octenyl, bicyclo[3.2.1]octenyl, and octahydro-2,5-methano-indenyl, and the like.

[0094] In the present application, the term "heterocyclyl" as a group or as part of a group means a stable 3- to 20-membered non-aromatic ring radical consisting of two to fourteen carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, phosphorus, oxygen, and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or more cyclic ring system, which can include fused, bridged, or spiro ring systems; the nitrogen, carbon, or sulfur atoms in the heterocyclyl group can optionally be oxidized; the nitrogen atom can optionally be quaternized; and the heterocyclyl group can be partially or fully saturated. The heterocyclyl group can be attached to the remainder of the molecule via a carbon atom or a heteroatom and by a single bond. In a heterocyclyl group comprising fused rings, one or more rings can be an aromatic carbocyclyl or heteroaromatic carbocyclyl group as defined below, provided that the point of attachment to the remainder of the molecule is a non-aromatic ring atom. For the purposes of the present application, the heterocyclyl group is preferably a stable 4- to 11-membered non-aromatic monocyclic, bicyclic, bridged, or spiro radical comprising one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, more preferably a stable 4- to 8-membered non-aromatic monocyclic, bicyclic, bridged, or spiro radical comprising one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heterocyclyl groups include, but are not limited to, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2,7-diaza-spiro[3.5]nonan-7-yl, 2-oxa-6-aza-spiro[3.3]heptan-6-yl, 2,5-diaza-bicyclo[2.2.1]heptan-2-yl, azetidinyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolanyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinolizinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, indolinyl, octahydroindolyl, octahydroisoindolyl, pyrrolidinyl, pyrazolidinyl, phthalimido, and the like.

[0095] As used herein, the term "aromatic ring (group)" as a group or as part of a group means a conjugated hydrocarbon ring system radical having 6 to 18 carbon atoms, preferably having 6 to 10 carbon atoms. For the purposes of the present application, the aromatic ring (group) can be a monocyclic, bicyclic, tricyclic, or more cyclic ring system, which can also be fused with a carbocyclyl or heterocyclyl group as defined above, provided that the aromatic ring (group) is attached to the remainder of the molecule via an atom on the aromatic ring by a single bond. Examples of aromatic ring (group) include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, fluorenyl, 2,3-dihydro-1H-isoindolyl, 2-benzoxazolinonyl, 2H-1,4-benzoxazin-3(4H)-on-7-yl, and the like.

[0096] As used herein, the term "aromatic heterocyclic ring (group) or heteroaryl group" as a group or part of another group means a 5- to 16-membered conjugated ring system having 1 to 15 carbon atoms (preferably having 1 to 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless specifically indicated otherwise in the specification, the aromatic heterocyclic ring (group) can be a monocyclic, bicyclic, tricyclic or more ring ring system, and can also be fused with a carbocyclic ring or heterocyclic ring as defined above, provided that the aromatic heterocyclic ring (group) is connected to the rest of the molecule via an atom on the heteroaromatic ring by a single bond. The nitrogen, carbon or sulfur atom in the aromatic heterocyclic ring (group) can be optionally oxidized; the nitrogen atom can be optionally quaternized. For the purposes of the present application, the heteroaromatic ring (group) is preferably a stable 5- to 12-membered aromatic group comprising 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably a stable 5- to 10-membered aromatic group comprising 1 to 4 heteroatoms selected from nitrogen, oxygen and sulfur or a 5- to 6-membered aromatic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur. Examples of aromatic heterocyclic ring (group) include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolyl, isoquinolyl, naphthylidinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothienyl, oxatriazolyl, cinnolinyl, quinazolinyl, thiophenyl, indolizinyl, phenanthrolinyl, isoxazolyl, phenoxazinyl, phenothiazinyl, 4,5,6,7-tetrahydrobenzo[b]thienyl, naphthopyridinyl, [1,2,4]triazolo[4,3-b]pyridazine, [1,2,4]triazolo[4,3-a]pyrazine, [1,2,4]triazolo[4,3-c]pyrimidine, [1,2,4]triazolo[4,3-a]pyridine, imidazo[1,2-a]pyridine, imidazo[1,2-b]pyridazine, imidazo[1,2-a]pyrazine, and the like.

[0097] Bipartite cyclic peptide type DNA encoded compounds and methods of making the same

[0098] The present invention provides bipartite cyclic peptide type DNA encoded compounds of the structure shown in Formula R and methods of making the same. Typically, the methods of the present invention comprise the steps of: reacting two reactive groups A2 and A3 in a compound of Formula M0 with a synthetic building block of Formula M1 bearing reactive groups (G1 and G2) in a nucleic acid compatible reaction environment to obtain a compound of Formula Q; reacting two reactive groups in a synthetic building block of Formula M1 with one reactive group present on a different synthetic building block in a compound of Formula Q to obtain a bipartite cyclic peptide type DNA encoded compound of Formula R.

[0099] In particular, a representative construction method comprises the following steps:

[0100] (a) providing a compound of formula M0, wherein the compound of formula M0 contains a Linker (L) having -A1-N attached thereto, A2 and A3;

[0101] (b) providing a synthetic building block of formula M1;

[0102] (c) reacting the compound of formula M0 with the synthetic building block of formula M1 to form a compound of formula Q1 having a nucleic acid fragment;

[0103] (d) reacting the compound of formula Q1 with the synthetic building block of formula M1 to form a compound of formula Q2 having a nucleic acid fragment:

[0104] (e) repeating the reaction of (d) 1 to r times to form a nucleic acid-linked compound of formula Q3, Q4, Q5:

[0105] wherein O1, O2, O3, O4, O5, O6, O7, O8, O9 and O 10 each independently is the same or different chemical structure (there are at least two attachment sites);

[0106] r is an integer from 2 to 100; preferably an integer from 3 to 50; more preferably an integer from 3 to 5.

[0107] (f) the compound of formula Q1, Q2, Q3, Q4, Q5 can be reacted with the synthetic building block of formula M1, respectively, to form a bidirectional cyclic peptide type DNA-encoded compound of formula R1, R2, R3, R4, R5, respectively:

[0108] wherein N (or ), A1, A2, A3, Linker (or L), m, n, O1, O2 are as defined above; O3, O4, O5, O6, O7, O8, O9, O 10 11 12 are defined similarly to O1, O2;

[0109] For example, O 11 ​​G2 and G1 through a nucleic acid compatible chemical reaction; O 12 G2 and G2 through a nucleic acid compatible chemical reaction.

[0110] An exemplary reaction flow chart of the present application is shown below:

[0111] wherein R1 and R2 are not particularly limited, determined by the specific selected compound, and are common groups or combinations thereof in the art, such as alkyl, alkenyl, alkynyl, amino, cyano, thio, mercapto, hydroxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, etc.

[0112] Applications

[0113] The bidirectional cyclic peptide type DNA encoded compound of formula R of the present application can also be used to prepare a bidirectional cyclic peptide type DNA encoded molecular library for large-scale and high-throughput screening work.

[0114] Typically, the high-throughput screening flow of the bidirectional cyclic peptide type DNA encoded molecular library of the present application includes:

[0115] (S1) A method for constructing a nucleic acid compatible bidirectional cyclic peptide type DNA encoded compound molecular library is developed.

[0116] (S2) The method developed in (S1) is used to prepare a bidirectional cyclic peptide type DNA encoded molecular library.

[0117] (S3) The molecular library prepared in (S2) is incubated with a target protein, and bidirectional cyclic peptide type DNA encoded compounds with certain affinity to the target are collected, the nucleotide sequences carried by the compounds are amplified by PCR, high-throughput sequencing is performed, and the specific structure of the collected compounds is determined.

[0118] (S4) The encoded compounds collected in (S3) are chemically synthesized, and the activity of the compounds is verified by pharmacological experiments.

[0119] Compared with the prior art, the main advantages of the present application include:

[0120] (1) A method for constructing a bidirectional cyclic peptide type DNA encoded molecular library in a nucleic acid compatible environment is developed for the first time, the synthesis method has mild conditions, simple operation, strong substrate universality, and is cheap and easy to obtain, and has a wide application range.

[0121] (2) The method is applied to construct a DNA encoded molecular library for the first time, the size of the macrocycle is expanded, the diversity of the macrocycle structure is increased, the range of the covered chemical space is expanded, and the probability of discovering a lead compound is improved.

[0122] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturers. Unless otherwise specified, percentages and parts are by weight.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are merely illustrative.

[0124] Example 1: Preparation of a DNA-encoded compound library of 96 x 96 library

[0125] In this exemplary DNA-encoded compound library, a fixed encoded nucleic acid sequence (referred to as the starting primer, custom-made by Suzhou Jw Biotech Co., Ltd., HPLC purified) is connected to the starting fragment HP of the compound library.

[0126] HP is simplified as:

[0127] Top strand of the starting primer: 5'-PO4 2- -AAATCGATGTG-3' (SEQ ID NO: 01)

[0128] Bottom strand of the starting primer: 5'-PO4 2- -CATCGATTTGG-3' (SEQ ID NO: 02)

[0129] Step (1): Linking HP and Linker under nucleic acid compatible conditions

[0130] The HP was dissolved in 8 μL of sodium borate buffer solution (pH = 9.4, 250 mM), 3 μL of 200 mM N-Fmoc-N'-Boc-L-2,3-diaminopropionic acid (dissolved in DMA, prepared fresh) was added, 3 μL of 200 mM 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviated as HATU) (dissolved in DMA, prepared fresh) was added, 3 μL of 200 mM N,N-diisopropylethylamine (abbreviated as DIPEA) (dissolved in DMA, prepared fresh) was added, and the reaction was carried out at room temperature. After the reaction, 5 M aqueous sodium chloride solution and chilled ethanol were added, and the mixture was placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant was removed. The obtained DNA precipitate was dissolved in 8 μL of phosphate buffer solution (pH = 5.5, 250 mM), and heated at 60°C for 12 hours. After the reaction, 5 M aqueous sodium chloride solution and chilled ethanol were added, and the mixture was placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant was removed. The obtained DNA ligation product was dissolved in distilled water. The identification results are shown in FIG. 1.

[0131] Step (2): Ligation of the product of step (1) with a starting primer by enzymatic reaction

[0132] A 600 μL aqueous solution of 1 mM of the product of step (1) was mixed uniformly with 368 μL of an aqueous solution of 660 nmol of a starting primer that had been annealed, and T4 buffer solution, T4 ligase were added at 0°C, and the reaction was carried out at 16°C for 16 hours. After the reaction was complete, 300 μL of 5 M aqueous sodium chloride solution and 8500 μL of chilled ethanol were added, and the mixture was placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant was removed. The obtained DNA precipitate was dissolved in 585 μL of distilled water. The identification results are shown in FIG. 2.

[0133] Step (3): Synthesis of the first cycle of the DNA encoded compound library

[0134] Ninety-six ligation reactions were set up, and a solution of the product of step (2) at a concentration of 1 mM was dispensed into 96 consecutive wells of a 96-well plate, 5.5 μL per well, and 3.1 μL each of the upper and lower strands of the first cycle of labeled nucleotide double strands shown in Table A (abbreviated as first cycle labeled nucleotide double strands, custom-made by Suzhou Genewiz Biotech Co., Ltd., HPLC purified) at a concentration of 1.8 mM were added to each well, respectively.

[0135] X is one of the four deoxyribonucleotides A, T, C, and G.

[0136] According to step (2), T4 buffer solution, T4 ligase are added, after the reaction is completed, ethanol precipitation is carried out as described above, and the DNA precipitate is dissolved in 8 μL of sodium borate buffer solution (pH = 9.4, 250 mM), 3 μL of 200 mM Fmoc-amino acid (dissolved in DMA, prepared on demand), 3 μL of 200 mM 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviated as HATU) (dissolved in DMA, prepared on demand), 3 μL of 200 mM N,N-diisopropylethylamine (abbreviated as DIPEA) (dissolved in DMA, prepared on demand) are added, and the reaction is carried out at room temperature to connect the corresponding small molecule compound in the first cycle.

[0137] X is one of A, T, C, G, four deoxyribonucleotides.

[0138] After the reaction is completed, 5M sodium chloride solution and cold ethanol are added to the reaction solution, and the mixture is placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant is removed to obtain the DNA precipitate which is dissolved in 5% piperidine aqueous solution. After the reaction is completed, ethanol precipitation is carried out as described above to obtain the precipitate. After the reaction is completed, all the reaction solutions are mixed together, and the product is desalted and purified by using a 500 μL size 10K ultrafiltration tube (Amicon Ultra Centrifugal), and the identification result is shown in FIG. 3.

[0139] X is one of A, T, C, G, four deoxyribonucleotides.

[0140] The small molecule in the first cycle and the corresponding nucleotide double-stranded information are shown in Table A as follows:

[0141] Table A

[0142] Step (4): Synthesis of the second cycle of the DNA coded compound library

[0143] The connection of the nucleic acid sequence in the second cycle and the connection reaction step of the amino acid monomer are similar to step (3). After the reaction is completed, all the reaction solutions are mixed together, and ethanol precipitation is carried out as described above to obtain the DNA precipitate which is dissolved in 200 μL of distilled water. The product is desalted and purified by using a 500 μL size 10K ultrafiltration tube (Amicon Ultra Centrifugal), and the identification result is shown in FIG. 4.

[0144] X is one of the four deoxyribonucleotides A, T, C, G; wherein R1 and R2 are not particularly limited to the groups commonly used in the art, and are determined by the specific compound being linked.

[0145] The second cycle of small molecules and corresponding nucleotide double strand information is shown in Table B below:

[0146] Table B

[0147] Step (5): Nucleic acid compatible cyclization reaction

[0148] A solution of the product of step (4) at a concentration of 1 mM was dissolved in 8 μL of a sodium borate buffer solution (pH = 9.4, 250 mM), 3 μL of 200 mM N-acetyl-S-(tert-butylthio)cysteine (dissolved in DMA, prepared fresh) was added, 3 μL of 200 mM 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviated as HATU) (dissolved in DMA, prepared fresh) was added, 3 μL of 200 mM N,N-diisopropylethylamine (abbreviated as DIPEA) (dissolved in DMA, prepared fresh) was added, and the reaction was carried out at room temperature.

[0149] X is one of the four deoxyribonucleotides A, T, C, G.

[0150] After the reaction was completed, 5 M aqueous sodium chloride solution and chilled ethanol were added, and the mixture was placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant was removed. The obtained DNA precipitate was dissolved in a sodium borate buffer solution (pH = 9.4, 250 mM), 100 mM tris(2-carboxyethyl)phosphine hydrochloride (dissolved in distilled water, prepared fresh) and 100 mM 1,4-bis(bromomethyl)benzene (dissolved in DMA, prepared fresh) were added, and the reaction was carried out at room temperature. After the reaction was completed, 5 M aqueous sodium chloride solution and chilled ethanol were added, and the mixture was placed at -78°C for 0.5 hours, then centrifuged at 4°C, and the supernatant was removed. The obtained DNA precipitate was dissolved in distilled water, and the product was desalted and purified using an ultrafiltration tube (Amicon Ultra Centrifugal) with a size of 500 μL and a size specification of 10K, to obtain a DNA encoded compound library (the identification results are shown in FIG. 5) for subsequent protein affinity screening.

[0151] X is one of the four deoxyribonucleotides A, T, C, G.

[0152] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to readers of the disclosure and the appended claims.

Claims

1. A method for preparing a bi-directional cyclic peptide-type DNA encoded compound, characterized in that, comprises the following steps: (a) providing a compound of Formula M0, wherein the compound of Formula M0 comprises Linker (L), A2, and A3, wherein the Linker comprises an -A1-N moiety attached thereto; wherein, N (or ) is a single / double stranded deoxyribonucleotide sequence, a single / double stranded ribonucleotide sequence, or a combination thereof; A1is a chemical bond or a chemical structure connecting Linker and N; Linker (or L) is a multi-functional linker; A2and A3are reactive groups; (b) providing a synthetic building block of formula M1; in which, is a synthetic building block central structure; G1, G2are reactive groups; (c) reacting the compound of Formula MO with the synthetic building block of Formula Ml to form a compound of Formula Q comprising a nucleic acid fragment; in which, m, n are each independently an integer from 1 to 100; each O1and O2is independently a chemical bond or a chemical structure; N (or ), A1, Linker (or L), G2is as defined above; (d) ring closure of a compound of Formula Q with a synthetic building block of Formula Ml to form a compound of Formula R containing a nucleic acid fragment: in which, P1and P2are respectively a chemical structure or a chemical bond formed by the reaction of two reactive groups G2on the synthetic building block central structure in the compound of formula Q with two reactive groups (G1and G2) on the synthetic building block of formula M1; N (or ), A1, Linker (or L), m, n, O1, O2are as defined above.

2. The method of claim 1, wherein, A1, O1, O2, P1, P2 are each independently a chemical structure selected from the group consisting of: In the formulae, is an aromatic ring or an aromatic heterocycle.

3. The method of claim 1, wherein, A2, A3, G1, and G2 are each independently selected from the following groups: H, -N3, aldehyde, hydroxyl, carboxyl, terminal alkene, terminal alkyne, chlorine, bromine, iodine, substituted or unsubstituted mercapto, substituted or unsubstituted -S-SH, substituted or unsubstituted phenyl, substituted or unsubstituted 5-7 heteroaryl, substituted or unsubstituted amino, substituted or unsubstituted -NH-C(O)-OH, substituted or unsubstituted -NH-C(O)H, substituted or unsubstituted C 1-4 Alkyl, substituted or unsubstituted C 2-6 alkenyl, substituted or unsubstituted C 2-6 Alkyne, substituted or unsubstituted C 3-7 Cycloalkyl, 4-7 membered cycloalkenyl, substituted or unsubstituted C 5-9 Cycloalkynyl, substituted or unsubstituted -C(O)O-C1-C6 alkyl, substituted or unsubstituted sulfonamide; The substituent is one or more (1, 2, 3, 4, 5, 6) hydrogens on the group replaced by a substituent selected from the group consisting of oxo (=0), cyano, halogen, -SO3Na, substituted or unsubstituted C 1-6 alkyl, C 1-4 alkoxy, C 2-6 alkenyl, substituted or unsubstituted 4-7 membered heterocycloalkenyl, substituted or unsubstituted C 2-6 alkynyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted 4-7 membered heterocyclyl, substituted or unsubstituted 6-8 membered heterocycloalkynyl, substituted or unsubstituted 5-7 membered heteroaryl, halogenated C 1-4 alkyl, halogenated phenyl; wherein the substituent is one or more (1, 2, 3, 4, 5, 6) hydrogens replaced by a substituent selected from the group consisting of halogen, cyano, oxo (=0), -SO3Na, -SO2(C 1-6 alkyl), C 1-4 alkoxy, C 1-4 alkoxy, nitro, phenyl, C 1-4 alkoxy-substituted phenyl, C 1-6 amido.

4. The method of claim 1, wherein, A2, A3, G1, and G2 are each independently selected from the group consisting of: wherein, X is F, Cl, Br or I; is an aromatic ring or an aromatic heterocycle.

5. The method of claim 1, wherein, The Linker (or L) is selected from the group consisting of: wherein, q is 1, 2, 3, or 4.

6. The method of claim 1, wherein, In step (c) and step (d), the synthetic building blocks or the synthetic building blocks and the linking groups (i.e., A2and A3) in formula M0are connected by a reaction selected from amidation, reductive amination, nucleophilic substitution, Suzuki coupling, Heck coupling, Sonogashira coupling, S- aromatization, CuAAC reaction, photoinduced reaction, preferably, by an amidation reaction.

7. A bi-directional cyclic peptide-type DNA encoded compound of the formula R in which, N (or ) is a single / double stranded deoxyribonucleotide sequence, a single / double stranded ribonucleotide sequence, or a combination thereof; A1is a chemical bond or a chemical structure connecting Linker and N; Linker (or L) is a multi-functional linker; m, n are each independently an integer from 1 to 100; each O1and O2is independently a chemical bond or a chemical structure; is a synthetic building block central structure; and P1and P2are respectively a chemical bond or a chemical structure.

8. A method for constructing a library of bi-directional cyclic peptide type DNA encoded compounds, characterized in that, The bidirectional cyclic peptide type DNA coded compound library comprises t bidirectional cyclic peptide type DNA coded compounds with the following formula R, t is a positive integer ≥1000. wherein, N (or ), A1, Linker (or L), m, n, O1, O2, P1, P2, G2are as defined in claim 7; the method comprises: (a) synthesizing a bi-directional cyclic peptide type DNA encoded compound of formula R using the method of claim 1; and (b) combining t compounds of formula R to thereby construct the library of nucleic acid encoded compounds.

9. The method of claim 8, wherein, t is > 10,000, preferably > 100,000, more preferably > 1000,000, more preferably > 5000,000, most preferably > 10,000,000.

10. A library of bi-directional cyclic peptide-like DNA encoded molecules, characterized in that, The bidirectional cyclic peptide type DNA coded molecular library comprises t bidirectional cyclic peptide type DNA coded compounds having the structure shown in the following formula R, t is a positive integer ≥ 1000; wherein, N (or ), A1, Linker (or L), m, n, O1, O2, P1, P2are as defined in claim 7.

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