High-purity DNA-encoded library, preparation method therefor, and use thereof

Abstract

A method for constructing a DNA-encoded library, specifically, (s1) providing an HP module, the HP module comprising a nucleic acid; (s2) covalently attaching chemical structural units to the HP module to form chemical moieties attached to the HP module, while covalently attaching oligonucleotides that encode the chemical structural units to the HP module to form encoded nucleic acid moieties of the HP module, thereby forming a nucleic acid-compound conjugate; (s3) subjecting the nucleic acid-compound conjugate to a linking reaction with a compound shown in formula (II) to obtain a first mixture; S-R5 (II), wherein the S is a solid-phase carrier, and R5 is a second group participating in the linking reaction; (s4) subjecting the first mixture to filtration, washing, and purification, and then to cleavage to obtain a new compound; and (s5) repeating steps (s2)-(s4) x times, wherein x is an integer greater than 0, thereby forming a DNA-encoded library. The obtained DNA-encoded library has a larger load capacity, high purity, a larger peptide library, and wider applicable reactions.

Description

High purity DNA encoded library and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of compound purification, in particular to high purity DNA encoded library and preparation method and application thereof. BACKGROUND

[0002] DNA encoded library (DEL) is a widely used compound library construction and screening method. Since the first report of the synthesis method of DNA encoded compound, a plurality of screening methods based thereon have been reported, and a plurality of molecules entering clinical research have been found.

[0003] The construction of DEL is mostly based on headpiece (HP, a chemical reaction end with an amino group at one end and a coding end containing a DNA sequence at the other end), and a chemical structure module and a specific DNA sequence are connected correspondingly, thereby forming a one-to-one molecular library. The DNA coding end is generally connected to a specific sequence of DNA by a ligase. The reaction of the chemical end generally includes chemical coupling, impurity removal by precipitation, deprotection, etc. After multiple rounds of such repeated reactions, DEL with extremely high diversity can be synthesized. After affinity screening with target proteins fixed on a solid phase, DEL members with binding force are recovered, and the corresponding coding DNA is amplified, thereby discovering library members with binding force. This library construction and screening method has many advantages such as high library capacity, high screening throughput, rapidness, and low cost.

[0004] However, the existing DEL library has the problem of low purity. Low-purity DEL library can lead to a high false positive rate in DEL screening, thereby reducing the success rate of DEL screening. The general purification method of the existing DEL is to remove small molecular impurities by ethanol precipitation and ultrafiltration after the reaction of the chemical end. However, this method cannot remove large molecular raw materials that have not reacted, and large molecular impurities will continue to accumulate during the enzyme connection step.

[0005] Therefore, there is an urgent need in the art for a DEL preparation and purification method with high universality, high yield, good purification efficiency, and longer library construction rounds. SUMMARY

[0006] The purpose of the present application is to develop a method for preparing DEL with high purity, good universality, and high purification efficiency.

[0007] In a first aspect, the present application provides a method for preparing a nucleic acid encoded compound or a nucleic acid encoded library, the method comprising the steps of:

[0008] (s1) providing a HP module, the HP module comprising a chemical reaction end and a DNA coding end;

[0009] (s2) covalently linking a chemical moiety to the HP module to form a chemical moiety attached to the HP module; and covalently linking an oligonucleotide encoding the chemical moiety to the HP module to form an encoding nucleic acid moiety of the HP module, thereby forming a nucleic acid-compound conjugate (or a first intermediate);

[0010] (s3) subjecting the nucleic acid-compound conjugate to a ligation reaction with a solid support to obtain a mixture containing a reaction product;

[0011] (s4) isolating or purifying the reaction product from the mixture and subjecting the reaction product to cleavage, thereby cleaving the nascent compound from the solid support;

[0012] (s5) using the nascent compound as a HP module for a subsequent reaction, repeating steps (s2) - (s4) x times, x being an integer > 0, thereby obtaining a nucleic acid encoded compound or a nucleic acid encoded library.

[0013] In another preferred embodiment, the nucleic acid encoding is DNA encoding.

[0014] In another preferred embodiment, the nucleic acid encoded library is a DNA encoded library (DEL).

[0015] In another preferred embodiment, the method comprises the steps of:

[0016] (s1) providing a HP module, said HP module being provided with (a) a chemical reactive end (preferably an amino end) for ligation reaction with a chemical moiety, and a DNA encoding end for ligation with an encoding tag;

[0017] (s2) covalently linking a chemical moiety to the HP module to form a chemical moiety attached to the HP module; and covalently linking an oligonucleotide encoding the chemical moiety to the HP module to form an encoding nucleic acid moiety of the HP module, thereby forming a nucleic acid-compound conjugate;

[0018] (s3) subjecting the nucleic acid-compound conjugate to a ligation reaction with a reactant of formula (II) to obtain a mixture containing a reaction product; S-R 5b (II)

[0019] wherein S is a solid support;

[0020] R 5b is a second group participating in the ligation reaction;

[0021] (s4) isolating or purifying the reaction product from the mixture and subjecting the reaction product to cleavage, thereby cleaving the nascent compound from the solid support;

[0022] (s5) repeating steps (s2)-(s4) x times, x is an integer ≥ 0, using the new compound as a HP module for the subsequent reaction, thereby obtaining a nucleic acid encoded compound or a nucleic acid encoded library.

[0023] In another preferred embodiment, in step s3, the ligation reaction comprises a click chemistry reaction.

[0024] In another preferred embodiment, in step s3, the ligation reaction comprises amide bond formation, Suzuki coupling, Sonogashira coupling and copper catalyzed alkyne-azide cycloaddition.

[0025] In another preferred embodiment, in step s4, the mixture is filtered, washed, purified, thereby obtaining a compound.

[0026] In another preferred embodiment, x is an integer from 1 to 20, preferably from 2 to 10, more preferably from 3 to 8.

[0027] In another preferred embodiment, the solid support is selected from the group consisting of a particle, a microsphere, a microparticle, or a combination thereof.

[0028] In another preferred embodiment, the solid support is dispersed in a solvent or a solution.

[0029] In another preferred embodiment, the solid support has a particle size (or average particle size) from 1 nm to 5 mm, preferably from 10 nm to 100 um, more preferably from 1 um to 10 um.

[0030] In another preferred embodiment, the solid support comprises a magnetic bead.

[0031] In another preferred embodiment, the solid support is selected from the group consisting of a PS resin, a PEG resin, a silicate-based solid phase medium, graphene, agarose, mesoporous glass, or a combination thereof.

[0032] In another preferred embodiment, R 5b is a second group participating in a click chemistry reaction, preferably a reactive group comprising a C2-C 12 alkynyl group, BCN, BARAC, DIFC, or DIBO.

[0033] In another preferred embodiment, for each library member, the method comprises the following steps:

[0034] (s1) providing a HP module, said HP module being provided with (a) a chemical reactive end (preferably an amino end) for ligation reaction with a chemical structural unit, and a DNA coding end for ligation with a coding tag;

[0035] (s2) covalently linking a chemical moiety to the HP module to form a chemical moiety attached to the HP module, and covalently linking an oligonucleotide encoding the chemical moiety to the HP module to form an encoding nucleic acid moiety of the HP module, forming a nucleic acid-compound conjugate;

[0036] (s3) subjecting the nucleic acid-compound conjugate to a ligation reaction with a compound of formula (II) to form a mixture containing a reaction product; S-R 5b (II)

[0037] wherein S is a solid support;

[0038] R 5b is a second group involved in the ligation reaction;

[0039] (s4) isolating or purifying the reaction product from the mixture and subjecting the reaction product to cleavage to thereby cleave the nascent compound from the solid support and obtain the nascent compound;

[0040] (s5) using the nascent compound as a HP module for a subsequent reaction, repeating steps (s2)-(s4) x times, x being an integer > 0, to obtain the library member.

[0041] In another preferred embodiment, R 5b is a second group involved in the click chemistry reaction.

[0042] In another preferred embodiment, the nucleic acid-compound conjugate is attached to the solid support via a linker, and the method further comprises:

[0043] reacting one end of the linker with the chemical moiety and one end with a reactant comprising the solid support, thereby attaching the nucleic acid-compound conjugate to the solid support;

[0044] cleaving the linker, thereby releasing the library member or the nascent compound from the solid support.

[0045] In another preferred embodiment, the reaction of the reactant comprising the solid support comprises a click chemistry reaction.

[0046] In another preferred embodiment, the linker comprises a linking structure, a protecting group and a protected group, and is attached in that order.

[0047] In another preferred embodiment, the protected group is attached to the chemical moiety.

[0048] In another preferred embodiment, the linking structure is attached to the reactant comprising the solid support.

[0049] In another preferred embodiment, the chemical building blocks are sequentially added to the HP module to form the chemical moiety.

[0050] In another preferred embodiment, the cleavage is between a protecting group of a linker and a protected group.

[0051] In another preferred embodiment, the cleavage is performed using piperidine or piperazine / DBU.

[0052] In another preferred embodiment, the cleavage is performed by deprotection.

[0053] In another preferred embodiment, the nucleic acid-mixture conjugate comprises a deprotection structure, a linker, a chemical moiety, and a coding nucleic acid moiety, and are connected in order.

[0054] In another preferred embodiment, coding oligonucleotides encoding each chemical building block are sequentially added to the HP module to form the coding nucleic acid moiety, wherein the coding oligonucleotide encoding the chemical building block is added to the HP module before, after, or simultaneously with the addition of the chemical building block to the HP module.

[0055] In another preferred embodiment, the oligonucleotide encoding the chemical building block is linked to the HP module by a ligase.

[0056] In another preferred embodiment, the ligase comprises a nucleic acid ligase, such as T4 ligase.

[0057] In another preferred embodiment, a first coding oligonucleotide encoding a first chemical building block is covalently linked to the HP module.

[0058] In another preferred embodiment, the method comprises covalently linking a first coding oligonucleotide to the linking oligonucleotide to form a coding nucleic acid moiety linked to the HP module.

[0059] In another preferred embodiment, the method comprises covalently linking a second coding oligonucleotide encoding a second chemical building block to the coding nucleic acid moiety.

[0060] In another preferred embodiment, the method comprises linking one or more additional coding oligonucleotides encoding additional chemical building blocks to the coding nucleic acid moiety.

[0061] In another preferred embodiment, the method comprises cross-linking two or more chemical building blocks in the chemical moiety.

[0062] In another preferred embodiment, the method comprises the steps of:

[0063] (s1) providing a HP module, said HP module being provided with (a) a chemical reactive end (preferably an amino end) for a ligation reaction with a chemical structure unit, and a DNA coding end for ligation with a coding tag;

[0064] (s2) covalently linking a chemical structure unit Z to the chemical reactive end of said HP module, and covalently linking a coding tag of said chemical structure unit to the DNA coding end of said HP module, thereby forming a nucleic acid-compound conjugate of formula V1: 5a - Z1- HP - Z2 (V1)

[0065] wherein

[0066] HP is the reacted HP module;

[0067] Z1 is a chemical moiety corresponding to the chemical structure unit and linked to HP;

[0068] R 5a is a first group participating in click chemistry;

[0069] Z2 is a coding tag corresponding to Z1;

[0070] (s3) subjecting said nucleic acid-compound conjugate of the previous step to a click chemistry reaction with a reactant of formula (II): 5b (II)

[0071] wherein

[0072] S is a solid support;

[0073] R 5b is a second group participating in click chemistry;

[0074] thereby forming a reaction product of formula III: S - R5- Z1- HP - Z2 (III)

[0075] wherein

[0076] S, Z1, HP and Z2 are as defined above,

[0077] R5 is the first group R 5a and the second group R 5b forming a bivalent linker by the click chemistry reaction;

[0078] (s4) isolating the reaction product of the previous step and subjecting to a cleavage reaction, thereby forming a new chemical compound,

[0079] wherein the new chemical compound has a chemical reactive end (preferably an amino end) for further connection reaction with a chemical structure unit, and a DNA coding end for further connection reaction with a coding tag;

[0080] (s5) repeating the steps (s2) - (s4) x times, x is an integer ≥ 1, thereby forming a DEL library;

[0081] The difference is that:

[0082] In step (s2), the new chemical compound in the previous step (step (s4)) replaces the HP module in step (s2), thereby forming a nucleic acid-chemical compound conjugate of formula Vx

[0083] R 5a - (Z1) 1+x - HP - (Z2) 1+x (Vx)

[0084] wherein,

[0085] HP is the reacted HP module;

[0086] Z1 is each independently a chemical moiety corresponding to a chemical structure unit and connected to HP

[0087] R 5a is a first group participating in click chemistry;

[0088] Z2 is each independently a coding tag corresponding to Z1;

[0089] x is an integer ≥ 1;

[0090] and in step (s3), a reaction product of formula IIIx is formed; S - R5 - (Z1) 1+x - HP - (Z2) 1+x (IIIx)

[0091] wherein,

[0092] S, Z1, HP, Z2, R5 and x are as defined above.

[0093] In another preferred embodiment, in the DEL library, there are library members (i.e. compounds of the DEL library) of formula IV: (Z1) 1+x - HP - (Z2) 1+x (IV)

[0094] wherein,

[0095] Z1, HP, Z2, and x are as defined above.

[0096] In another preferred embodiment, the chemical reaction end of the new chemical compound is an amino end.

[0097] In another preferred embodiment, the chemical reaction end of the new chemical compound is an amino end.

[0098] In another preferred embodiment, the chemical reaction end of the new chemical compound is an amino end.

[0099] In another preferred embodiment, the chemical reaction end of the new chemical compound is an amino end.

[0100] In another preferred embodiment, the DEL library is a DNA-encoded polypeptide compound library.

[0101] In another preferred embodiment, the chemical structure unit Z is selected from the group consisting of amino acids, non-amino acid compounds.

[0102] In another preferred embodiment, the chemical structure unit Z comprises natural or unnatural amino acids, or amino acid analogs (such as dipeptides).

[0103] In another preferred embodiment, the chemical structure unit Z comprises D-type amino acids, L-type amino acids, or a combination thereof.

[0104] In another preferred embodiment, the R 5a is Y1-R4-R3-, wherein R4 and R3 are as defined above, and Y1 is a first functional group involved in the ligation reaction.

[0105] In another preferred embodiment, the R 5b is -L1-Y2, wherein L1 is a linker group, and Y2 is a second functional group involved in the ligation reaction.

[0106] In another preferred embodiment, the first and second functional groups are functional groups for click reaction, preferably, the first functional group is selected from the group consisting of -alkyne, BCN, BARAC, DIFC, or DIBO, and the second functional group is selected from the group consisting of -N3; or vice versa.

[0107] In another preferred embodiment, the R 5a is N3-R4-R3-, wherein R4 and R3 are as defined above.

[0108] In another preferred embodiment, the nucleic acid-compound conjugate has the structure shown in Formula (I):

[0109] wherein n is an integer > 0;

[0110] R1and R1' are each independently an amino acid or a chemical moiety capable of undergoing a coupling reaction with an amino group;

[0111] R2is the coding nucleic acid moiety;

[0112] R3is a protecting group (e.g., Fmoc);

[0113] R4is a linking structure.

[0114] In another preferred embodiment, R2has the structure -Ln-R 2a wherein R 2a is a coding nucleic acid sequence (i.e., polynucleotide), and Ln is a linking group (preferably a divalent linking group) between R2aand the nearest R1.

[0115] In another preferred embodiment, Ln is a divalent linking group.

[0116] In another preferred embodiment, the backbone of Ln has 3-20 (preferably 4-15) backbone chain atoms selected from the group consisting of C, O, N.

[0117] In another preferred embodiment, the backbone of Ln is composed of backbone chain elements selected from the group consisting of: -CH2-, -CO-, -O-, -NH-, or combinations thereof.

[0118] In another preferred embodiment, Ln has the structure -Ln1-Ln2-Ln3-,

[0119] wherein Ln1is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-;

[0120] Ln2is -(O-C2H4) w wherein w is a positive integer from 0 to 20 (preferably 1 to 10, more preferably 2 to 8);

[0121] Ln3is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-.

[0122] In another preferred embodiment, n is an integer from 1 to 20, preferably from 2 to 10, more preferably from 3 to 8.

[0123] In another preferred embodiment, the linking structure is selected from the group consisting of a polyethylene glycol chain, a fatty carbon chain, and a short polyamino acid.

[0124] In another preferred embodiment, the -(-N(R1')-R1) n - in the compound of formula (I) is the chemical moiety.

[0125] In another preferred embodiment, the protecting group is selected from the group consisting of Fmoc, Cbz, or a non-amino protecting group.

[0126] In another preferred embodiment, the reactant of formula II is S is as described above.

[0127] In another preferred embodiment, step (s4) further includes the step:

[0128] (s4a) After filtration, washing and purification of the first mixture, the compound shown in formula (IIIa) is obtained;

[0129] In the formula, n, R1, R1', R2, R3, R4 and S are as described above;

[0130] (s4b) The compound shown in formula (IIIa) is cleaved to obtain the compound shown in formula (IVa), thereby forming a DNA-encoded library;

[0131] In the formula, n, R1, R1' and R2 are as described above.

[0132] In another preferred embodiment, the purity of the DEL library prepared by the method is ≥60%, more preferably ≥70%, and even more preferably ≥80%.

[0133] In a second aspect, the present invention provides a DEL library, which is generated by the method described in the first aspect of the present invention.

[0134] In a third aspect, the present invention provides a compound of formula (Vx), R 5a -(Z1) 1+x -HP-(Z2) 1+x (Vx)

[0135] In the formula,

[0136] HP refers to the HP module after the reaction;

[0137] Z1 is an independent chemical moiety corresponding to a chemical structural unit and connected to HP;

[0138] R 5a The first functional group to participate in click chemistry;

[0139] Z2 is an independent encoded tag corresponding to Z1;

[0140] X is an integer ≥ 0.

[0141] In another preferred embodiment, the compound has the structure shown in formula (I):

[0142] wherein n is an integer greater than 0;

[0143] R1and R1' are each independently an amino acid or a chemical moiety capable of undergoing a coupling reaction with an amino group;

[0144] R2is the coding nucleic acid moiety;

[0145] R3is a protecting group (e.g., Fmoc);

[0146] R4is a linking structure.

[0147] In another preferred embodiment, R2has the structure -Ln-R 2a wherein R 2a is a coding nucleic acid (i.e., polynucleotide) and Ln is a linking group (preferably a divalent linking group) between R2aand the nearest R1.

[0148] In another preferred embodiment, Ln is a divalent linking group.

[0149] In another preferred embodiment, the backbone of Ln has 3-20 (preferably 4-15) backbone chain atoms selected from the group consisting of C, O, N.

[0150] In another preferred embodiment, the backbone of Ln is composed of backbone chain elements selected from the group consisting of -CH2-, -CO-, -O-, -NH-, or combinations thereof.

[0151] In another preferred embodiment, Ln has the structure -Ln1-Ln2-Ln3-,

[0152] wherein Ln1is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-;

[0153] Ln2is -(O-C2H4) w wherein w is a positive integer from 0 to 20 (preferably 1 to 10, more preferably 2 to 8);

[0154] Ln3is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-.

[0155] In another preferred embodiment, n is an integer from 1 to 20, preferably from 2 to 10, more preferably from 3 to 8.

[0156] In another preferred embodiment, the linking structure is selected from the group consisting of a polyethylene glycol chain, a fatty carbon chain, and a short polyamino acid.

[0157] In another preferred embodiment, the -(-N(R1')-R1) n - in the compound of formula (I) is the chemical moiety.

[0158] In another preferred embodiment, Z1 in the compound of formula (Vx) is -(-N(R1')-R1) in formula (I). n -.

[0159] In another preferred embodiment, R 5a is N3-R4-R3 in formula (I).

[0160] In another preferred embodiment, the compound is selected from the group consisting of:

[0161] wherein R 2a is an encoding nucleic acid.

[0162] In another preferred embodiment, the compound is prepared by the steps of:

[0163] (s1) providing a HP module, the HP module comprising a nucleic acid;

[0164] (s2) covalently linking a chemical structure unit to the HP module to form a chemical moiety linked to the HP module, and covalently linking an oligonucleotide encoding the chemical structure unit to the HP module to form an encoding nucleic acid moiety of the HP module, the compound of formula (I).

[0165] In a fourth aspect of the present application, a compound of formula (IIIx) is provided, S-R5-(Z1) 1+x -HP-(Z2) 1+x (IIIx)

[0166] wherein

[0167] HP is a reacted HP module;

[0168] Z1 is each independently a chemical moiety corresponding to a chemical structure unit and linked to the HP

[0169] R5 is a first group R 5a and a second group R 5b forming a bivalent linker by a click chemistry reaction;

[0170] Z2 is each independently an encoding tag corresponding to Z1;

[0171] X is an integer ≥ 0;

[0172] S, R 5a and R 5b as described in the first aspect of the present application.

[0173] In another preferred embodiment, the compound has the structure of Formula (III):

[0174] wherein,

[0175] n is an integer greater than 0;

[0176] R1and R1' are each independently an amino acid or a chemical group capable of undergoing a coupling reaction with an amino group;

[0177] R2is the coding nucleic acid moiety;

[0178] R3is a protecting group (e.g., Fmoc);

[0179] R4is a linking structure;

[0180] S is a solid support.

[0181] In another preferred embodiment, R2has the structure of -Ln-R 2a wherein R 2a is a coding nucleic acid (i.e., polynucleotide), and Ln is a linking group (preferably a divalent linking group) between R2aand the nearest R1.

[0182] In another preferred embodiment, Ln is a divalent linking group.

[0183] In another preferred embodiment, the backbone of Ln has 3-20 (preferably 4-15) backbone chain atoms selected from the group consisting of C, O, N.

[0184] In another preferred embodiment, the backbone of Ln is composed of backbone chain elements selected from the group consisting of -CH2-, -CO-, -O-, -NH-, or combinations thereof.

[0185] In another preferred embodiment, Ln has the structure of -Ln1-Ln2-Ln3-,

[0186] wherein Ln1is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-;

[0187] Ln2is -(O-C2H4) w wherein w is a positive integer from 0 to 20 (preferably 1 to 10, more preferably 2 to 8);

[0188] Ln3is a substituted or unsubstituted C1-C6alkylene (preferably C1-C4alkylene), or -CO-NH-.

[0189] In another preferred embodiment, the solid support comprises a particle, a microsphere.

[0190] In another preferred embodiment, the solid support comprises a magnetic bead.

[0191] In another preferred embodiment, the solid support is selected from the group consisting of a PS resin, a PEG resin, a silicate-based solid phase medium, graphene, agarose, mesoporous glass, or a combination thereof.

[0192] In another preferred embodiment, Z1 in the compound of formula (Vx) is -( -N(R1')-R1) in formula (I). n -.

[0193] In another preferred embodiment, R in the compound of formula (Vx) is N3-R4-R3 in formula (I). 5a

[0194] In another preferred embodiment, the compound is prepared by the following steps:

[0195] (s1) subjecting the compound of formula (I) to a click chemistry reaction with a compound of formula (II) to obtain a first mixture;

[0196] wherein m, n, R1, R1', R2 and R3 are as previously described; S-R5 (II)

[0197] wherein S, R5 are as previously described;

[0198] (s5) subjecting the first mixture to filtration, purification, and cleavage to obtain the compound of formula (III).

[0199] In another preferred embodiment, the compound is selected from the group consisting of:

[0200] wherein R2 is the coding nucleic acid moiety;

[0201] S is a solid support.

[0202] In a fifth aspect, the present application provides a compound of formula (VI), R6— R3— L1— R 5a (VII)

[0203] wherein,

[0204] R6 is an amino acid group;

[0205] R3 is a protecting group of the coupling group;

[0206] L1 is a linking structure;

[0207] R 5a is a group participating in a click chemistry reaction.

[0208] ​In another preferred embodiment, R6is a monovalent group derived from a compound selected from the group consisting of:

[0209] In another preferred embodiment, R3is an amino protecting group, preferably FMOC, Cbz, Boc, Pht, Tos, Tfa,, Trityl, oxycarbonyl-based (e.g., Nvoc), or sulfonamide-based (e.g., Ns) amino protecting group, more preferably R3is a divalent group.

[0210] In another preferred embodiment, L1is a divalent linking group having the structure of -(L3)x-, wherein x is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;

[0211] and each L3is independently selected from the group consisting of a bond, C, CH, CH2, C=0, O, NH, NHCO, O-C2H4.

[0212] In another preferred embodiment, one or more H in L1is optionally substituted with a substituent selected from the group consisting of deuterium, halogen, hydroxyl, amino.

[0213] In another preferred embodiment, one or more L3is a linking structure selected from the group consisting of a polyethylene glycol chain, an aliphatic carbon chain, and a short polyamino acid.

[0214] In another preferred embodiment, R 5a is selected from the group consisting of N3, BCN, BARAC, DIFC, DIBO, or an alkyne group.

[0215] In another preferred embodiment, the compound is selected from the group consisting of:

[0216] In another preferred embodiment, the compound is used for synthesizing a library as described in the second aspect of the present application.

[0217] It should be understood that, within the scope of the present application, all combinations of the above-described technical features of the present application and the technical features specifically described hereinafter (e.g., in the examples) can be interchanged 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

[0218] Figure 1 shows the structure schematic of the compounds in the DEL library. Among them, the solid structure is the specific structure for the reaction to be coupled to the solid phase surface; the connecting structure is the chemical structure connecting the solid reaction group and the modified protecting group; the protecting group is the specific chemical structure of the protecting group required in the DEL synthesis process; the protected structure is the specific chemical structure required in the DEL synthesis process; the chemical moiety is the diverse chemical structure or combination synthesized by the DEL; the linker is the starting material for the synthesis of the DEL, one end is an amino group, and the other end contains a single-stranded or double-stranded DNA with a specific nucleotide sequence.

[0219] Figure 2 shows the chemical structure synthesis schematic in the DEL construction.

[0220] Figure 3 shows the schematic diagram of coupling DEL to the solid phase surface.

[0221] Figure 4 shows the schematic diagram of selectively eluting the target DEL structure from the washed solid phase surface.

[0222] Figure 5 shows the structure schematic of the DEL library with corresponding DNA tags through multiple rounds of chemical synthesis and coupling.

[0223] Figure 6 shows the DEL construction flowchart.

[0224] Figure 7 shows the coupling and purification method of the first round of polypeptide target product (Ile-AOP-HP).

[0225] Figure 8 shows the coupling and purification method of the second round of polypeptide target product (Lys(Boc)-Ile-AOP-HP).

[0226] Figure 9 shows the coupling and purification method of the third round of polypeptide target product (Ala-Lys(Boc)-Ile-AOP-HP).

[0227] Figure 10 shows the coupling and purification method of the fourth round of polypeptide target product (Val-Ala-Lys(Boc)-Ile-AOP-HP).

[0228] Figure 11 shows the coupling and purification method of the fifth round of polypeptide target product (Lue-Val-Ala-Lys(Boc)-Ile-AOP-HP).

[0229] Figure 12 shows the LCMS characterization of the crude product of the chemical coupling reaction in Figure 7, the supernatant obtained after fixing the reaction crude product on the solid phase carrier, and the first round of target product (Ile-AOP-HP) eluted from the solid phase with piperidine.

[0230] Figure 13 shows LCMS characterization of the crude chemical coupling reaction product of Figure 8, the supernatant obtained after immobilization of the crude reaction product on a solid support, and the second round target product (Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0231] Figure 14 shows LCMS characterization of the crude chemical coupling reaction product of Figure 9, the supernatant obtained after immobilization of the crude reaction product on a solid support, and the third round target product (Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0232] Figure 15 shows LCMS characterization of the crude chemical coupling reaction product of Figure 10, the supernatant obtained after immobilization of the crude reaction product on a solid support, and the fourth round target product (Val-Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0233] Figure 16 shows LCMS characterization of the crude chemical coupling reaction product of Figure 11, the supernatant obtained after immobilization of the crude reaction product on a solid support, and the fifth round target product (Leu-Val-Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0234] Figure 17 shows PAGE analysis of the compounds of Figure 7. From left to right: reaction starting material (AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, first round target product (Ile-AOP-HP) eluted from the solid phase with piperidine, DNA molecular weight standard.

[0235] Figure 18 shows PAGE analysis of the compounds of Figure 8. From left to right: DNA molecular weight standard, reaction starting material (Ile-AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, second round target product (Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0236] Figure 19 shows PAGE analysis of the compounds of Figure 9. From left to right: DNA molecular weight standard, reaction starting material (Lys(BOC)-Ile-AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, third round target product (Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0237] Figure 20 shows PAGE analysis of the compounds in Figure 10. From left to right: DNA molecular weight marker, reaction raw material (Ala-Lys(BOC)-Ile-AOP-HP), crude product of chemical coupling reaction, supernatant obtained after immobilization of the crude product of solid phase carrier, fourth round target product (Val-Ala-Lys(BOC)-Ile-AOP-HP) obtained after elution of the solid phase with piperidine.

[0238] Figure 21 shows a schematic diagram of the synthesis of the DEL5-pure library.

[0239] Figure 22 shows the application and characterization of mFmoc amino acids in the synthesis of the DEL5-pure library.

[0240] Figure 23 shows the distribution of amino acid DNA tags in the DEL5-native library and the DEL5-pure library.

[0241] Figure 24 shows SDS-PAGE analysis of purified TfRl and biotinylated TfRl. 1: purified TfRl protein; 2: biotinylated TfRl protein; 3: supernatant obtained after incubation of biotinylated TfRl protein with streptavidin magnetic beads; 4: wash buffer collected after incubation of biotinylated TfRl protein with streptavidin magnetic beads; 5: protein eluted from streptavidin magnetic beads after heating.

[0242] Figure 25 shows PAGE analysis of 12% of the NGS sequencing samples of DNA tags with Illumina sequencing adapters on both sides. From left to right: DEL5-native and DEL5-pure libraries without selection and the outputs of the first and second rounds of selection. The observed single bands represent their molecular weights and purities.

[0243] Figure 26 shows the characterization of TfRl-specific peptides. DETAILED DESCRIPTION

[0244] The inventors have made extensive and in-depth research and, through a large number of experiments and screening, first discovered a method for constructing a DNA-encoded library. The method of the present application is carried out in a liquid phase, and the newly synthesized compounds constructed using the method of the present application can be used as HP modules for the next round of reactions, thereby enabling the construction of the next round of newly synthesized compounds. The inventors optimized the conditions for raw material synthesis, amino acid coupling, and removal, thereby enabling the construction of DNA-encoded libraries with higher load, higher purity, longer polypeptide libraries, and wider application reactions. On this basis, the present application was completed.

[0245] TERMS

[0246] For the purposes of the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the present invention pertains. Before the present invention is described, it is to be understood that this invention is not limited to the particular methodologies and experimental conditions described, as such methodologies and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0247] As used herein, the terms "comprises", "comprising", "includes", "including" or "comprising" or "including" or "comprises" are to be construed as being inclusive (i.e. to one or more elements or components) and not exclusive (i.e. to one element or component).

[0248] The term "about" can refer to a value or a composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, the expression "about 100" includes within its intended scope all values between 99 and 101, and all values between 99.1, 99.2, 99.3, 99.4, and the like.

[0249] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is to be construed as including all values within the range and fraction of the value within the range (e.g. one tenth and one hundredth of an integer) where appropriate.

[0250] As used herein, the term "and / or" refers to any and all possible combinations of one or more of the associated listed items.

[0251] The term "C1-C 12 Alkynyl" refers to a straight-chain or branched-chain alkynyl group having from 1 to 12 carbon atoms. The term "C1-C 12 Alkyl preferably refers to C1-C8alkyl, preferably C1-C6alkyl, more preferably C1-C5alkyl, more preferably C1-C4alkyl. 10 Alkyl, preferably C1-C8alkyl, preferably C1-C6alkyl, more preferably C1-C5alkyl, more preferably C1-C4alkyl.

[0252] The terms "HP module", "HP primer" are used interchangeably and refer to a HP module provided with (a) a chemical reactive end (preferably an amino end) for performing a ligation reaction with a chemical structure unit, and a DNA coding end for ligation with a coding tag.

[0253] The term "chemical moiety" consists of one or more chemical structure units. The term "coding nucleic acid moiety" consists of one or more coding oligonucleotides.

[0254] The terms "coding oligonucleotide" and "Tag" are used interchangeably and both refer to the coding oligonucleotide encoding the chemical building block.

[0255] The terms "DEL5-pure", "the five-peptide library of the invention", "nucleic acid encoded chemical or nucleic acid encoded library" and "the DEL library of the invention" are used interchangeably.

[0256] A DNA encoded chemical library (DEL) is a collection of chemically distinct library members, each comprising (i) a chemical moiety formed of a set of chemically linked chemical building blocks and (ii) a nucleic acid encoding the chemical building blocks forming the chemical moiety. The number of distinct members in a library represents the complexity of the library and is defined by the number of building blocks forming each chemical moiety and the number of different variants of each building block.

[0257] A chemical moiety is a chemical entity or molecular structure displayed by a library member and comprises one, two or multiple chemical building blocks. A chemical moiety is covalently linked to a scaffold and is generated by the sequential covalent addition of one or more chemical building blocks to form a straight chain or backbone with a terminal end linked to the scaffold and a free terminal end. A cleavage group can be linked to a chemical moiety. Different chemical moieties displayed by members of a DEL library are formed by different combinations of chemical building blocks. The chemical moieties displayed by a DEL can be straight chain, macrocycle, bicyclic, polycyclic or branched compounds of different sizes (e.g. Lipinski-like small compounds and larger compounds). In some embodiments, a chemical moiety can be any small molecule (i.e. a molecule having a molecular weight of less than about 1,000 Daltons). In other embodiments, a chemical moiety can be any medium-sized molecule (i.e. a molecule having a molecular weight of less than about 5,000 Daltons). Small molecules can be organic or inorganic, isolated (e.g. from a library of compounds or a natural source) or obtained by derivatization of known compounds. A chemical moiety can be designed or constructed to have one or more desired properties, such as the ability to bind a biological target, solubility, availability of hydrogen bond donors and acceptors, rotational freedom of bonds, positive charge, negative charge, in vivo stability, cell permeability and / or oral availability.

[0258] A chemical moiety displayed in a nucleic acid encoded chemical library can be linked to a single strand of nucleic acid ("single pharmacophore library") or two different strands of nucleic acid that are hybridized together, one or more building blocks linked to each strand ("double pharmacophore library").

[0259] The coding nucleic acid moiety is a nucleic acid tag or a peptide nucleic acid tag that identifies a chemical building block in the chemical moiety. The coding nucleic acid moiety can be a linear nucleic acid molecule comprising a terminal end linked to the compound or member and a free end. Preferably, the coding nucleic acid moiety is linked to the scaffold. To record a previously or subsequently introduced chemical building block, the coding nucleic acid moiety can be elongated by covalently adding a coding oligonucleotide to the free end. The coding nucleic acid moiety can be used for identification of the purified compound after amplification and nucleic acid sequencing. In some embodiments, the cleavage group can be linked to the nucleic acid moiety, for example at the free end.

[0260] The coding nucleic acid moiety or fragment of the coding nucleic acid moiety can be linked to the HP module before, after or simultaneously with the linking of the scaffold to the solid support.

[0261] The chemical moiety or fragment of the chemical moiety, such as the chemical building block, can be linked to the HP module before, after or simultaneously with the linking of the scaffold to the solid support.

[0262] The solid support is an insoluble body that presents a surface on which a newly generated member or compound can be linked in the production process as described herein. Examples of suitable supports include resins, beads, nanoparticles and polymers such as polystyrene-polyethylene glycol (PEG) composites, PEG and poly-epsilon-lysine (ε-PL) (see, for example, Albericio F (2000). Solid-Phase Synthesis: A Practical Guide. Boca Raton: CRC Press). Conveniently, the support can be in the form of a particle, for example a bead. In some embodiments, the solid support can be a bead of a graft copolymer composed of a polystyrene matrix grafted with polyethylene glycol (PEG). The solid support can be produced using standard techniques or obtained from commercial suppliers (for example, Rapp Polymere GmbH, DE). Isolation of the compound on the solid support from solution can be achieved by any convenient method, such as filtration, by magnetic interaction (for magnetic beads), by centrifugation and the like. Rapp Polymere GmbH, DE). Isolation of the compound on the solid support from solution can be achieved by any convenient method, such as filtration, by magnetic interaction (for magnetic beads), by centrifugation and the like.

[0263] Other suitable solid supports can include polystyrene beads, cross-linked polystyrene beads, polymeric beads, glass beads, coated glass beads, controlled pore glass beads, beaded controlled pore glass beads, silica microparticles, coated silica microparticles, iron oxide particles, coated iron oxide particles, PEGA (polyethylene glycol-acrylamide) resins and other commercially available or custom made synthetic solid supports of varying sizes or combinations thereof. Suitable solid supports can be magnetic. Examples of magnetic solid supports include Magnefy™ and Microspheres (Bangs Laboratories, Inc.). Examples of solid supports can include copolymers such as acrylamide-PEG copolymers, polymeric particles additionally comprising paramagnetic or ferromagnetic materials, core-shell particles, porous particles, non-porous particles, or other combinations of organic chemical materials and ferromagnetic materials. Other suitable solid supports are known in the art (see, e.g., Pon, R.T. Curr. Protoc. Nucleic Acid Chem. (2000); Chaudhuri, R.G. and Paria, S., Chem. Rev. (2011); Wu, W., He, Q. and Jiang, C. Nanoscale Res. Lett. (2008); Hermanson, G.T., Bioconjugate Techniques: Third Edition (2013)).

[0264] DNA-encoded library

[0265] Compared with the traditional high-throughput screening of the million compound library, the DNA-encoded compound library connects the chemical structure with the DNA encoding sequence, and is established by using the mixed pool method, so that the DNA-encoded compound library can not only establish a compound library with various structures by using a large number of chemical reactions, but also can quickly identify the structure by using the characteristics of the DNA sequence easy to decode. The DNA-encoded compound library is interacted with the target protein through affinity screening, and a compound member with higher biological activity is enriched through a series of adsorption, washing, elution and other steps. Then, the corresponding DNA is amplified by PCR, the DNA sequence obtained by screening is found by using DNA sequencing, and decoding is performed to find the chemical structure of the binding compound and verification is performed, so that the purpose of discovering a lead compound by using the DNA-encoded compound library is achieved.

[0266] The method for constructing the DNA-encoded library of the application

[0267] The method for constructing the DNA-encoded library of the application is carried out in a liquid phase, so that the constructed DEL library has a larger load, a longer polypeptide library, higher diversity and higher purity. The method of the application can realize the construction of the DEL library for more than 5 rounds. The specific construction method is that the mutant Fmoc-protected amino acid is dehydrated and condensed with the HP module or the nucleic acid-compound conjugate to occur a dehydration condensation reaction, the azido group at the end of the mutant Fmoc is clicked with the solid support to occur a click chemistry reaction, the DEL is fixed on the solid surface, the mutant Fmoc protection group of the amino group in the DEL is removed by using piperidine, and thus the DEL library with high purity is obtained.

[0268] Referring to FIGS. 1-6, a method for preparing a nucleic acid-encoded compound or a nucleic acid-encoded library, the method comprising the steps of:

[0269] (s1) providing a HP module, said HP module comprising a chemical reactive end and a DNA encoding end;

[0270] (s2) covalently linking a chemical building block to said HP module to form a chemical moiety attached to said HP module, and covalently linking an oligonucleotide encoding said chemical building block to said HP module to form an encoding nucleic acid moiety of said HP module, thereby forming a nucleic acid-compound conjugate (or a first intermediate);

[0271] (s3) subjecting said nucleic acid-compound conjugate to a ligation reaction with a solid support, to obtain a mixture comprising a reaction product;

[0272] (s4) isolating or purifying said reaction product from said mixture, and subjecting the reaction product to a cleavage treatment, thereby cleaving the nascent chemical compound from the solid support;

[0273] (s5) using said nascent chemical compound as a HP module for a subsequent reaction, repeating steps (s2)-(s4) x times, x being an integer > 0, thereby obtaining a nucleic acid encoded chemical compound or a nucleic acid encoded library.

[0274] In a preferred embodiment, the method for constructing a DNA encoded library according to the present application comprises the steps of:

[0275] (s1) providing a HP module, said HP module comprising a chemical reactive end and a DNA encoding end;

[0276] (s2) covalently linking a chemical building block to said HP module to form a chemical moiety attached to said HP module, and covalently linking an oligonucleotide encoding said chemical building block to said HP module to form an encoding nucleic acid moiety of said HP module, thereby forming a nucleic acid-compound conjugate;

[0277] (s3) subjecting said nucleic acid-compound conjugate to a ligation reaction with a solid support, to obtain a mixture comprising a reaction product;

[0278] (s4) isolating or purifying said reaction product from said mixture, and subjecting the reaction product to a cleavage treatment, thereby cleaving the nascent chemical compound from the solid support;

[0279] (s5) repeating steps (s2)-(s4) x times, x being an integer > 0, thereby forming a DEL library.

[0280] In a preferred embodiment, the method according to the present application comprises the steps of:

[0281] (s1) providing a HP module, said HP module being provided with (a) a chemical reactive end (preferably an amino end) for a ligation reaction with a chemical structure unit, and a DNA coding end for ligation with a coding tag;

[0282] (s2) covalently linking a chemical structure unit Z to the chemical reactive end of said HP module, and covalently linking a coding tag of said chemical structure unit to the DNA coding end of said HP module, thereby forming a nucleic acid-compound conjugate of formula V1: 5a Z1-HP-Z2 (V1)

[0283] wherein

[0284] HP is the reacted HP module;

[0285] Z1 is a chemical moiety corresponding to the chemical structure unit and linked to HP

[0286] R 5a is a first group participating in click chemistry;

[0287] Z2 is a coding tag corresponding to Z1;

[0288] (s3) subjecting said nucleic acid-compound conjugate of the previous step to a click chemistry reaction with a reactant of formula (II): 5b (II)

[0289] wherein

[0290] S is a solid support;

[0291] R 5b is a second group participating in click chemistry;

[0292] thereby forming a reaction product of formula III: S-R5-Z1-HP-Z2 (III)

[0293] wherein

[0294] S, Z1, HP and Z2 are as defined above,

[0295] R5 is the first group R 5a and the second group R 5b forming a bivalent linker by the click chemistry reaction;

[0296] (s4) isolating the reaction product of the previous step and subjecting to a cleavage reaction, thereby forming a new chemical compound,

[0297] wherein the new chemical compound has a chemical reactive end (preferably an amino end) for further linkage reaction with a chemical structure unit, and a DNA coding end for further linkage reaction with a coding tag;

[0298] (s5) repeating the steps (s2) - (s4) x times, x being an integer > 1, thereby forming a DEL library;

[0299] The difference is that:

[0300] In step (s2), the new chemical compound in the previous step (step (s4)) replaces the HP module in step (s2), thereby forming a nucleic acid-chemical compound conjugate of formula Vx R 5a - (Z1) 1+x - HP - (Z2) 1+x (Vx)

[0301] wherein,

[0302] HP is the reacted HP module;

[0303] Z1 is each independently a chemical moiety corresponding to a chemical structure unit and linked to HP

[0304] R 5a is a first group participating in click chemistry;

[0305] Z2 is each independently a coding tag corresponding to Z1;

[0306] x is an integer > 1;

[0307] and in step (s3), a reaction product of formula IIIx is formed; S - R5 - (Z1) 1+x - HP - (Z2) 1+x (IIIx)

[0308] wherein,

[0309] S, Z1, HP, Z2, R5 and x are as defined above.

[0310] In a preferred embodiment, in the DEL library, there are library members of formula IV (i.e. compounds of the DEL library): (Z1) 1+x - HP - (Z2) 1+x (IV)

[0311] wherein,

[0312] Z1, HP, Z2, and x are as defined above.

[0313] In a preferred embodiment, the chemical building block Z is selected from the group consisting of Ala, Cys, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, Tyr.

[0314] In a preferred embodiment, the chemical building block Z is a D-amino acid.

[0315] In a preferred embodiment, the chemical building block Z comprises a non-natural amino acid selected from Table A.

[0316] Table A

[0317] Preferably, the chemical moiety is generated by sequentially adding chemical building blocks to a nascent member to generate a linear sequence (i.e. chain) of chemical building blocks attached proximal to the scaffold. The chemical building blocks in the chain can form the chemical moiety displayed by the member to which it is attached. For example, a first chemical building block can be covalently attached to a capture group of the scaffold. A second chemical building block can be attached to the first chemical building block to form a linear sequence or chain consisting of two chemical building blocks. The chain of chemical building blocks can have a proximal end attached to the scaffold and a distal end that is free. The first chemical building block can be in a proximal position and the second chemical building block can be in a distal position (i.e. the second chemical building block is an end or terminal chemical building block in the chain). The chain can be elongated by sequentially attaching further chemical building blocks to the distal end of the chain, for example by reacting with a chemical building block in a terminal position. After the chain of chemical building blocks is complete, a cleavage group can be attached to the distal end of the complete chain, for example by reacting with a chemical building block in the distal end (terminal chemical building block).

[0318] A chemical building block is a chemical group that forms a building block of a chemical moiety displayed by a library member. A chemical building block can be any chemical group comprising one, two or more binding groups. If a chemical building block is incorporated into a chain of chemical building blocks, such a chemical building block can be any chemical group comprising two or more binding groups.

[0319] Preferably, the chemical structure unit can comprise two or more binding groups that allow covalent attachment to a scaffold or other chemical structure unit. The two or more binding groups can exhibit different or orthogonal reactivity. For example, the chemical structure unit can comprise a proximal and distal binding group (e.g., a bifunctional structure unit). For example, the chemical structure unit can be covalently attached to the nascent member through the proximal binding group. The distal binding group of the chemical structure unit can be protected and / or used to attach additional chemical structure units to the nascent member. Each binding group can be a reactive functional group capable of reacting with a binding group from another chemical structure unit. The proximal and distal binding groups on two different structure units; or a binding group on a structure unit and a capture group of a scaffold should be complementary, i.e., capable of reacting together to form a covalent bond. Any reaction compatible with the coding system, solid support, and linker integrity can be employed. In some embodiments, any suitable DNA-compatible chemistry can be employed, such as amidation, Sonogashira coupling, Suzuki coupling, or copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or other click reactions. For example, one of the first and second binding groups can be a carboxyl group and the other can be an amine group.

[0320] The linking oligonucleotide is a nucleic acid to which the coding oligonucleotide encoding a chemical structure unit is attached to form the nucleic acid moiety. The linking oligonucleotide can have the same nucleotide sequence in different members of the library (i.e., a constant nucleotide sequence). The combination of coding oligonucleotides, and thus the sequence of the encoded nucleic acid moiety, can differ in different members of the library.

[0321] The linking oligonucleotide can have a terminal end that is attached to the nascent binding member and a free end to which the coding oligonucleotide is attached. The free end of the linking oligonucleotide can be compatible with the attachment of the coding oligonucleotide. For example, the free end can comprise a short 5' or 3' overhang ("sticky end") to facilitate attachment.

[0322] The linking oligonucleotide can be a natural nucleic acid, such as DNA or RNA, or it can be a nucleic acid analog, such as a peptide nucleic acid (PNA), phosphorodiamidite morpholino oligomer (PMO), phosphorothioate oligomer (PTO), locked nucleic acid (LNA), glycol nucleic acid (GNA), or threose nucleic acid (TNA).

[0323] The first coding oligonucleotide encoding the first chemical structure unit can be attached to the linking oligonucleotide. Suitable techniques for attachment of oligonucleotides are well established and include enzymatic ligation. Subsequent coding oligonucleotides encoding second and additional chemical structure units can be attached to the previous coding oligonucleotide to form an encoded nucleic acid comprising the coding oligonucleotides of the chemical structure units in the chain attached to the member.

[0324] In some embodiments, the ligation oligonucleotide can be double-stranded.

[0325] Double-stranded ligation oligonucleotides can be formed from intramolecular hybridization of a single nucleotide strand (i.e., a hairpin), or can be formed from intermolecular hybridization of two separate nucleotide strands. The double-stranded nucleotide sequence can be denatured to produce single-stranded nucleic acids prior to cleavage of the linker. Hybridization of the single-stranded nucleic acids of the first release member to the single-stranded nucleic acids of the second release member can be used, for example, to generate members of an ESAC library. Alternatively, double-stranded ligation oligonucleotides in which the two oligonucleotide strands are covalently linked can be employed.

[0326] Double-stranded coding oligonucleotides can be ligated to double-stranded ligation oligonucleotides by ligation using a ligase such as T4 DNA ligase according to standard techniques.

[0327] In other embodiments, the ligation oligonucleotide can be single-stranded. Single-stranded coding oligonucleotides can be ligated to ligation oligonucleotides using an adaptor oligonucleotide by splint ligation according to standard techniques.

[0328] A coding oligonucleotide is a nucleic acid molecule containing a nucleotide coding sequence that encodes a chemical structural unit and optionally a cleavage moiety, scaffold, and / or linker. The coding sequence (or coding region) can be any sequence of nucleic acid bases that is uniquely associated with a particular chemical structural unit. This allows the identity of the chemical moiety to be determined by sequencing or otherwise "reading" the coding sequence.

[0329] A coding sequence contains enough nucleotides to uniquely identify the chemical structural unit that it encodes. For example, if there are 20 variants of a chemical moiety, the coding sequence needs to contain at least 3 nucleotides (4 2 = 16, 4 3 = 64) to uniquely identify the chemical moiety. Coding sequences can be longer than necessary. The benefit of using longer coding sequences than necessary is that they provide the opportunity to distinguish the code by more than just a single nucleotide difference, which makes the decoding process more confident. For example, a first chemical structural unit from a population of 20 different chemical structural units (20 compounds) can be encoded by 6 nucleotides, and a second chemical structural unit from a population of 200 different moieties can be encoded by 8 nucleotides. Thus, the size of the coding sequence depends on the number of chemical structural units to be encoded (i.e., the number of different chemical structural units in the library). Nucleotide sequences and / or their complements can be used as coding sequences to encode chemical structural units. Suitable sequences for encoding chemical structural units in a library are well known in the art.

[0330] The coding sequence of a coding oligonucleotide can be flanked by constant regions. The constant regions can be long enough to allow efficient hybridization and ligation, for example 2-20 bases, preferably 9-15 bases.

[0331] The encoding oligonucleotides are added to the members or compounds in a sequential manner, while incorporating the building blocks, to produce a nucleic acid molecule (i.e. nucleic acid moiety) containing a linear series of encoding oligonucleotides that encode the combination of chemical building blocks present in the members or compounds. A first encoding oligonucleotide encoding a first chemical building block can be linked to a linking oligonucleotide, and additional encoding oligonucleotides can each be linked to a preceding encoding oligonucleotide in the series to form the nucleic acid molecule (i.e. nucleic acid moiety). The sequence of the nucleic acid moiety of a library member encodes the chemical building blocks of the library member. Thus, sequencing the encoding nucleic acid moiety allows the chemical building blocks of the member to be identified.

[0332] Preferably, if desired, the collection of solid support particles can be readily suspended in solution to allow separation and pooling. In some embodiments, small solid support particle sizes can be preferred for ease of synthesis of libraries having a large number of different members. For example, microparticles or nanoparticles can be used.

[0333] New chemical compounds of the present application

[0334] A new chemical compound of the present application refers to a member of a DEL library prepared by the method of the first aspect of the present application.

[0335] In one preferred example, the compounds of the present application used for DEL library construction during a five-round construction process have the following structure: amino acid moiety - overhanging nucleotide moiety - Tag moiety of each round;

[0336] wherein for a round, the amino acid moiety is different (e.g. 5a to 5v); the overhanging nucleotide moiety is the same (e.g. ag for the first round); and the Tag moiety is a different 9-length DNA strand for each round.

[0337] The DNA Tag sequences and the corresponding amino acids for DEL library construction in the present application are shown in Table 4.

[0338] Table 4

[0339] The main advantages of the present application include:

[0340] 1. The DEL library constructed using the method of the present application has high purity, with purity > 90%.

[0341] 2. The method of the present application for constructing a DEL library breaks through the limitation of 4 rounds, and can achieve the construction of 5 rounds or more of DEL.

[0342] 3. The DEL library constructed using the method of the present application has higher diversity, longer polypeptide library, larger load, and wider application reactions.

[0343] 4. The method of the present application selectively removes unreacted starting materials after the chemical coupling, removes by-products generated in the chemical coupling, and results in a single target product.

[0344] 5. The new chemical compounds in the DEL prepared by the method of the present application can be used as HP modules for a new round of reactions.

[0345] The present application is further described in connection with the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the application. Unless otherwise indicated, the experimental procedures in the following examples were carried out under conventional conditions, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise specified.

[0346] General Methods

[0347] Amino-modified double-stranded nucleotide headpiece (HP): 5’- / 5Phos / GAGTCA / iSp9 / iUniAmM / iSp9 / TGACTCCC-3’, internal spacer 9 modification, iUniAmM: internal Uni-Link TM Amino Modifier internal Uni-Link TM Amino modification.

[0348] LC-MS analysis: The samples were separated by an ACQUITY Arc liquid chromatography equipped with an XBridge Pep B EHCl 8 30 μ 2.5 ft m 2.1 x 50 mm column, mobile phase A (water, with 0.05% ammonia and 0.01% formic acid) and B (acetonitrile, with 0.05% ammonia, 0.01% formic acid and 10% water), elution gradient of B from 3% to 95% in 8 minutes, and mass spectrometry detection by an ESIS Q Detector 2 system (Waters).

[0349] Nucleic acid ethanol precipitation: 2 μΐ of 5 molar sodium chloride solution (volume / volume = 10%) and 55 μΐ of -80 °C frozen ethanol (volume / volume = 250%) were added to 20 μΐ of the system, mixed and shaken uniformly, and then frozen at -80 °C for 30 minutes. The frozen sample was centrifuged at 4 °C and 10,000 rpm for 30 minutes, and the supernatant was removed after centrifugation. The remaining liquid of the sample was dried in a -80 °C freeze dryer.

[0350] Nucleic acid purification method (centrifugation method): The nucleic acid was dissolved in pure water, and a filter membrane with a molecular weight of 3000 or 10000 was used to centrifuge the ultrafiltration tube at 10000 rpm for 30 minutes. The liquid in the ultrafiltration tube was removed, and the same condition was used to centrifuge twice. After centrifugation, the supernatant was collected.

[0351] Nucleic acid quantification method (optical density method): 1 microliter of centrifuged supernatant was diluted with 99 microliters of pure water, and the nucleic acid concentration was measured using a microspectrophotometer. The concentration was calculated according to the following formula. The amount of substance n = 3 times the average value of the nucleic acid concentration times the dilution factor / molar mass.

[0352] Polyacrylamide gel electrophoresis analysis method: A 12% polyacrylamide gel was prepared, and the reactants and raw materials were diluted with pure water to a concentration of 100 nanograms / microliter aqueous solution. After mixing 6 microliters of water, 2 microliters of loading buffer and 2 microliters of diluent, 8.5 microliters of the mixture was taken, and the reaction yield was estimated according to the depth and total number of bands of the target band and the reactant band.

[0353] When the amount of nucleic acid primer is 3.3 microliters, the coupling product of 1-amino-3,6,9,12-tetraoxypentadecanoic acid and nucleic acid linker reacts with nucleic acid primer essentially completely. At this time, the nucleic acid primer is 1.3 times the amount of the coupling product of 1-amino-3,6,9,12-tetraoxypentadecanoic acid and nucleic acid linker. Ice bath to 1000 microliters of 2 millimolar concentration of 1-amino-3,6,9,12-tetraoxypentadecanoic acid and nucleic acid linker coupling product, add 1300 microliters of 2 millimolar concentration of nucleic acid primer, 800 microliters of 10 times T4 buffer, 48000 units of 240 microliters of T4 ligase and 4660 microliters of water. After shaking and mixing evenly, the reaction was carried out at 16 degrees Celsius on a shaker for 16 hours. The reaction result was analyzed by mass spectrometry or polyacrylamide gel electrophoresis. After the reaction, 800 microliters of 5 molar concentration of sodium chloride and 22 milliliters of negative 80 degrees Celsius frozen ethanol were added to the system, mixed and shaken evenly, and then frozen at negative 80 degrees Celsius for 30 minutes. The frozen sample was centrifuged at 4 degrees Celsius and 10000 rpm for 30 minutes, and the supernatant was removed after centrifugation. The remaining liquid in the 96-well plate or centrifuge tube was dried in a freeze-drying machine at negative 80 degrees Celsius. After freeze-drying, the nucleic acid was dissolved in water and the concentration was determined using a microspectrophotometer.

[0354] Example 1

[0355] Amino acid coupling in the synthesis of nucleic acid coding library: The solution of nucleic acid coding library containing N-terminal amino group (400 μL, 200 nmoles) was dissolved in sodium borate buffer (500 mM sodium borate buffer, pH 9.1). The protected amino acid (100 μL, 200 nmoles, 100 eq, dissolved in N,N-dimethylacetamide), 1-[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (100 μL, 200 nmoles, 100 eq, dissolved in N,N-dimethylacetamide) and N,N-diisopropylethylamine (100 μL, 200 nmoles, 100 eq, dissolved in N,N-dimethylacetamide) were mixed to generate the activated protected amino acid and transferred to the test tube containing the conjugate of 1-amino-3,6,9,12-tetraoxopentadecanoic acid and nucleic acid linker. After shaking at room temperature for 2 hours, the coupling reaction was monitored by liquid chromatography-mass spectrometry. After the liquid chromatography-mass spectrometry result showed that the conversion of the coupling reaction was over 90%, sodium chloride (5 molar, 1 / 10 volume) and cooled ethanol (stored at -80 °C, 3 times volume) were added to the test tube. Then the test tube was frozen at -80 °C for more than one hour and centrifuged at 10000 rpm for 30 minutes at 4 °C to obtain the nucleic acid precipitate. The supernatant was carefully discarded and the remaining precipitate was freeze-dried to obtain the crude product and analyzed by gel and liquid chromatography-mass spectrometry.

[0356] Solid phase attachment in nucleic acid encoding library synthesis: The crude product from the one step chemical coupling in the previous step was dissolved in water (1500 μl), tetrahydrofuran (400 μl) and 2 molar triethylammonium acetate (100 μl, pH 7) were added and the solution was preheated in a 30°C water bath for 5 minutes. 100 mg of controlled pore glass-bicyclo[6.1.0]nonyne beads were added and the solution was shaken at room temperature for 10 minutes. The reaction supernatant was separated from the beads by centrifugation and the beads were washed 2-3 times with 400 μl of wash buffer (20% tetrahydrofuran, 0.1 molar triethylammonium acetate, pH 7). The wash supernatant was combined with the reaction supernatant and transferred to a new test tube containing 100 mg of controlled pore glass-bicyclo[6.1.0]nonyne beads and shaken at room temperature for 10 minutes. The supernatant was then separated from the beads by centrifugation and the beads were washed 2-3 times with 400 μl of wash buffer. The wash supernatant and reaction supernatant were transferred to another new test tube containing 100 mg of controlled pore glass-bicyclo[6.1.0]nonyne beads. After shaking at room temperature for 10 minutes, the reaction supernatant was separated from the beads by centrifugation and transferred to a 15 ml centrifuge tube. All test tubes containing controlled pore glass-bicyclo[6.1.0]nonyne beads were washed once with 0.2 molar acetic acid-sodium acetate buffer, pH 5.46, once with acetonitrile and once with water. The wash supernatant was combined with the reaction supernatant and lyophilized. The sample was analyzed by gel and liquid chromatography-mass spectrometry.

[0357] Solid phase elution in the synthesis of nucleic acid encoded library: The entire controlled pore glass beads (300 mg total) from the previous step were collected into a test tube and dissolved in water (1.9 mL) with the addition of 100 μL of piperidine (5% v / v). After shaking at room temperature for 30 minutes, the supernatant was separated from the beads and transferred to a 15 mL centrifuge tube. Then, 1 mL of 5% aqueous piperidine was added to the beads and shaken at room temperature for an additional 15 minutes. The supernatant was separated from the beads by centrifugation and collected in a 15 mL centrifuge tube. The beads were then resuspended in 1 mL of 5% aqueous piperidine. After shaking at room temperature for 15 minutes, the beads were separated from the reaction supernatant by centrifugation and washed with water 3-5 times. The washes were combined with the reaction supernatant and transferred to a 15 mL centrifuge tube and lyophilized. The solid was dissolved in water (400 μL) and passed through an Amicon ultrafiltration device (400 μL, 3000 molecular size) at 10,000 rpm for 30 minutes to reduce the volume to less than 40 μL, then water was added to the Amicon ultrafiltration device to 400 μL. This was repeated three times to remove impurities and the remaining solution was collected and quantified using a UV spectrophotometer based on the absorbance of nucleic acids. The purified chemically extended nucleic acid encoded library was then analyzed by gel and liquid chromatography-mass spectrometry.

[0358] Example 2

[0359] Preparation of (9H-fluoren-9-yl)acetate: (9H-fluoren-9-yl)methanol (1.96 g, 10 mmol) was dissolved in dichloromethane (20 mL) and pyridine (1.05 mL, 13 mmol) and acetic anhydride (1.23 mL, 13 mmol) were slowly added to the solution at ice bath conditions. After the addition was complete, the resulting solution was allowed to warm to room temperature and stirring was continued for 16 hours. After the reaction was complete as determined by thin layer chromatography, the reaction was quenched with the addition of 1 M aqueous hydrochloric acid (20 mL) and extracted with ethyl acetate (20 mL, 3 times). The organic layer was dried over anhydrous sodium sulfate and concentrated to yield the product as a white solid (2.2 g, 92.6%).

[0360] 1 H NMR (500 MHz, CDC13) δ 7.78 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 4.38 (d, J = 7.3 Hz, 2H), 4.22 (t, J = 7.3 Hz, 1H), 2.15 (s, 3H).

[0361] LC / MS (ESI) m / z: 261.34 (M + Na) +

[0362] Preparation of 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)-4-oxobutanoic acid: Succinic anhydride (1.01 g, 10.1 mmol) and aluminum trichloride (3.6 g, 30.3 mmol) were dissolved in anhydrous dichloromethane (35.6 mL) and operated at 0 °C. In a 500 mL round bottom flask, (9H-fluoren-9-yl) acetate (2.2 g, 10.1 mmol) was dissolved in dichloromethane (6.25 mL) and added dropwise to the reaction solution, dropwise process lasted 20 minutes. The reaction mixture was then stirred at room temperature for 24 hours. After confirming the completion of the reaction by thin layer chromatography (TLC), the reaction was quenched to pH 1 with 1 molar aqueous hydrochloric acid solution at 0 °C. The reaction mixture was extracted with ethyl acetate (20 mL, 3 times), and the organic layer was washed with 1 molar aqueous hydrochloric acid solution (20 mL, 3 times) and saturated brine (20 mL, 3 times). The organic phase was dried with anhydrous sodium sulfate and recrystallized with petroleum ether to obtain a gray-white solid product (1.9 g, 61.1%).

[0363] 1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 8.07 (dd, J = 8.0, 1.6 Hz, 1H), 7.83 (d, J = 7.8 Hz, 2H), 7.63 (dd, J = 7.5, 1.1 Hz, 1H), 7.45 (td, J = 7.4, 1.2 Hz, 1H), 7.40 (td, J = 7.4, 1.2 Hz, 1H), 4.44 (dd, J = 10.8, 7.1 Hz, 1H), 4.37 (dd, J = 10.8, 7.3 Hz, 1H), 4.27 (t, J = 7.2 Hz, 1H), 3.38 (t, J = 6.5 Hz, 2H), 2.86 (t, J = 6.5 Hz, 2H), 2.15 (s, 3H).

[0364] 13C NMR (126 MHz, CDCl3) δ 197.53, 178.08, 171.13, 146.39, 145.10, 144.33, 140.17, 135.27, 128.64, 128.61, 128.27, 125.35, 124.87, 121.24, 120.10, 66.05, 46.88, 33.44, 28.19, 21.13.

[0365] LC / MS (ESI) m / z: 339.42 (M + H)+

[0366] 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)butanoic acid: To a solution of 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)-4-oxobutanoic acid in trifluoroacetic acid (1 mL) was added triethylsilane (796.44 μL, 2.5 mmol) dropwise. After stirring at room temperature for 12 h, the reaction mixture was poured into ice water and adjusted to pH 7 with saturated Na2C03solution at 0 °C. The reaction mixture was extracted with ethyl acetate (20 mL, 3 times), and the organic layer was washed with 1 M aqueous HC1 (20 mL, 3 times) and saturated brine (20 mL, 3 times). The organic phase was dried over Na2S04and recrystallized to give the product (432.6 mg) as a white solid.

[0367] 1 H NMR (600 MHz, CDC13) δ 7.73 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 7.4 Hz, 1H), 7.41 (s, 1H), 7.39 (t, J = 7.6 Hz, 2H), 7.29 (td, J = 7.4, 1.1 Hz, 1H), 7.23 (dd, J = 7.8, 1.6 Hz, 1H), 4.41 (dd, J = 10.8, 7.1 Hz, 1H), 4.32 (dd, J = 10.8, 7.5 Hz, 1H), 4.18 (t, J = 7.3 Hz, 1H), 2.75 (t, J = 7.6 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.15 (s, 3H), 2.06 - 1.96 (m, 2H).

[0368] 13 C NMR (126 MHz, CDC13) δ 178.31, 171.23, 144.22, 144.02, 141.38, 140.55, 139.53, 128.30, 127.89, 126.90, 125.30, 125.17, 120.08, 119.95, 66.63, 46.80, 35.35, 33.22, 26.58, 21.15.

[0369] LC / MS (ESI) m / z: 325.39 (M+H) +

[0370] 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)butanoic acid: To a solution of 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)-4-oxobutanoic acid in trifluoroacetic acid (1 mL) was added triethylsilane (796.44 μL, 2.5 mmol) dropwise. After stirring at room temperature for 12 h, the reaction mixture was poured into ice water and adjusted to pH 7 with saturated Na2C03solution at 0 °C. The reaction mixture was extracted with ethyl acetate (20 mL, 3 times), and the organic layer was washed with 1 M aqueous HC1 (20 mL, 3 times) and saturated brine (20 mL, 3 times). The organic phase was dried over Na2S04and recrystallized to give the product (432.6 mg) as a white solid.

[0371] Preparation of N-(14-azido-3,6,9,12-tetraoxatetradecyl)-4-(9-(hydroxymethyl)- 9H-fluoren-2-yl)butanamide: 4-(9-(hydroxymethyl)-9H-fluoren-2-yl)butanoic acid (200 mg, 0.617 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate (469 mg, 1.23 mmol) and N,N- diisopropylethylamine (430 μΐ, 2.46 mmol) were dissolved in dry dichloromethane and after pre-activation at 0 °C added to a solution of 2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethanamine (243 mg, 0.93 mmol) in dichloromethane. After stirring at room temperature for 12 h the reaction mixture was quenched with 1 molar aqueous hydrochloric acid solution. The reaction mixture was extracted with ethyl acetate (20 ml, 3 times), the organic layer was washed with saturated brine (20 ml, 3 times), dried over sodium sulfate and concentrated and purified by column chromatography (dichloromethane:methanol, 12:1 v / v) to give the product (206 mg, 63.5%) as a white solid.

[0372] 1 H NMR (600 MHz, CDC13) δ 7.72 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.50 (s, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.29 (td, J = 7.4, 1.1 Hz, 1H), 7.20 (dd, J = 7.8, 1.6 Hz, 1H), 6.46 (t, J = 5.6 Hz, 1H), 4.16 (dd, J = 10.6, 5.8 Hz, 1H), 4.08 (t, J = 6.4 Hz, 1H), 3.85 (dd, J = 10.6, 7.0 Hz, 1H), 3.65 - 3.58 (m, 14H), 3.52 (q, J = 4.9 Hz, 2H), 3.42 (td, J = 7.8, 6.8, 3.4 Hz, 2H), 3.34 (t, J = 5.1 Hz, 2H), 2.80 - 2.66 (m, 2H), 2.25 - 2.13 (m, 2H), 2.13 - 2.06 (m, 1H), 1.98 - 1.88 (m, 1H).

[0373] 13C NMR (151 MHz, CDC13) δ 173.41, 145.28, 144.34, 141.68, 140.58, 139.45, 127.98, 127.64, 126.80, 125.75, 124.73, 120.00, 119.90, 70.74, 70.66, 70.61, 70.57, 70.56, 70.22, 70.17, 70.11, 65.32, 50.75, 50.35, 39.35, 35.51, 35.23, 27.26. LC / MS (ESI) m / z: 527.41 (M+H) +

[0374] Preparation of (2-(l-azido-16-oxo-3,6,9,12-tetraoxo-15-aza-nonadecyl)-9H-fluoren-9- yl)methyl 4-nitrophenyl carbonate: N-(14-azido-3,6,9,12-tetraoxatetradecyl)-4-(9-(hydroxymethyl)- 9H-fluoren-2-yl)butanamide (662.6 mg, 1.26 mmol), piperidine (76 μL, 1.51 mmol) and 4- nitrophenyl chloroformate (760 mg, 3.8 mmol) were dissolved in dry dichloromethane under argon protection and ice bath. Stirring at room temperature for 12 hours, the reaction was quenched by adding 1 M hydrochloric acid, the reaction mixture was extracted with dichloromethane (20 mL, 3 times), the organic layer was washed with saturated brine (20 mL, 3 times), dried over sodium sulfate and concentrated, purified by column chromatography (dichloromethane:methanol, 15:1 v / v) to give the product (526 mg, 60%) as a white solid.

[0375] 1H NMR (600 MHz, CDC13) δ 8.27 (d, J = 9.1 Hz, 2H), 7.75 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.44 (s, 1H), 7.42 (t, J = 7.4 Hz, 1H), 7.35 (d, J = 9.1 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.25 (d, J = 4.0 Hz, 1H), 6.19 (m, 1H), 4.63 (dd, J = 10.5, 6.8 Hz, 1H), 4.57 (dd, J = 10.6, 7.1 Hz, 1H), 4.29 (t, J = 6.9 Hz, 1H), 3.66 - 3.60 (m, 14H), 3.56 (t, J = 5.0 Hz, 2H), 3.46 (q, J = 5.2 Hz, 2H), 3.37 (t, J = 5.1 Hz, 2H), 2.74 (t, J = 7.6 Hz, 2H), 2.24 (t, J = 7.5 Hz, 2H), 2.06 - 1.98 (m, 2H). 13 C NMR (151 MHz, CDC13) δ 173.09, 155.64, 152.57, 145.58, 143.24, 143.03, 141.51, 141.27, 139.50, 128.62, 128.23, 127.06, 125.46, 125.25, 125.15, 122.01, 120.21, 120.11, 71.07, 70.79, 70.73, 70.66, 70.60, 70.60, 70.31, 70.30, 70.14, 70.07, 53.57, 50.78, 46.72, 39.35, 35.96, 35.59, 27.55. LC / MS (ESI) m / z: 692.58 (M+H) +

[0376] Preparation of ((2-(l-azido-16-oxo-3,6,9,12-tetraoxo-15-azanonadecyl)-9H-fluoren-9-yl)methoxy)carbonyl)-D-alanine: (2-(l-azido-16-oxo-3,6,9,12-tetraoxo-15-azononanedioyl)-9H-fluoren-9-yl)methyl (4-nitrophenyl) carbonate (200 mg, 0.289 mmol) was dissolved in 10 mL of acetonitrile, followed by the addition of 3 equivalents of D-alanine (77 mg, 0.867 mmol) and 10 mL of an aqueous sodium bicarbonate solution (146 mg, 1.737 mmol). After stirring at room temperature for 48 to 72 hours, the reaction was quenched with 1 M aqueous hydrochloric acid. The mixture was extracted with ethyl acetate three times, and the combined ethyl acetate layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography. The target product (85 mg, 46% yield) was obtained as a light yellow oil.

[0377] The above procedure was repeated using different amino acids to obtain the corresponding products, as shown in Table 1. The starting material used was (2-(l-azido-16-oxo-3,6,9,12-tetraoxo-15-azononanedioyl)-9H-fluoren-9-yl)methyl (4-nitrophenyl) carbonate, which has the structure

[0378] Table 1

[0379] Preparation of (2-(4-oxo-4-((2-(2-(prop-2-yn-l-yloxy)ethoxy)ethyl)amino)butyl)-9H-fluoren-9-yl) acetate: 4-(9-(acetyloxymethyl)-9H-fluoren-2-yl)butanoic acid (861.3 mg, 2.65 mmol), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (764 mg, 3.98 mmol), and N,N-diisopropylethylamine (925.7 μL, 5.3 mmol) were dissolved in dry dichloromethane under ice bath cooling. After activation, amino-dipolyethylene glycol-azide (494.6 mg, 3.45 mmol) in dichloromethane was transferred. After stirring at room temperature for 12 hours, the reaction was quenched by the addition of 1 M aqueous hydrochloric acid. The reaction mixture was extracted with ethyl acetate (20 mL, 3 times), and the organic layer was washed with saturated brine (20 mL, 3 times). The organic phase was dried over sodium sulfate and concentrated. The product (851.2 mg, 71.35%) was obtained as an oily solid after purification by column chromatography (dichloromethane:methanol, 12:1 v / v).

[0380] 1 H NMR (500 MHz, CDC13) δ 7.72 (dt, J = 7.6, 0.9 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.58 (dd, J = 7.5, 1.0 Hz, 1H), 7.42 - 7.35 (m, 2H), 7.29 (td, J = 7.5, 1.1 Hz, 1H), 7.22 (dd, J = 7.8, 1.5 Hz, 1H), 6.07 (s, 1H), 4.42 (dd, J = 10.8, 6.9 Hz, 1H), 4.34 (dd, J = 10.8, 7.4 Hz, 1H), 4.18 (d, J = 7.2 Hz, 1H), 4.16 (d, J = 2.4 Hz, 2H), 3.70 - 3.61 (m, 4H), 3.56 (dd, J = 5.6, 4.6 Hz, 2H), 3.47 (q, J = 5.2 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 2.4 Hz, 1H), 2.22 (t, J = 7.5 Hz, 2H), 2.13 (s, 3H), 2.08 - 1.96 (m, 2H).

[0381] 13 C NMR (151 MHz, CDC13) δ 172.83, 171.10, 144.01, 143.90, 141.29, 140.82, 139.31, 128.20, 127.76, 126.75, 125.17, 125.02, 119.91, 119.80, 74.77, 70.03, 69.92, 69.04, 66.39, 58.41, 53.44, 46.71, 39.14, 35.82, 35.42, 27.36, 21.05.

[0382] LC / MS (ESI) m / z: 450 (M+H) +

[0383] Synthesis of 4-(9-(hydroxymethyl)-9H-fluoren-2-yl)-N-(2-(2-(prop-2-yn-1- yloxy)ethoxy)ethyl)butanamide: (2-(4-oxo-4-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)butyl)-9H-fluoren-9-yl) acetate (752.4 mg), methanol (16.75 mL), triethylamine (3.35 mL) was dissolved in water (3.35 mL). Refluxed for 3 hours, neutralized with 1 molar hydrochloric acid, extracted with ethyl acetate, neutralized with sodium bicarbonate, dried with sodium sulfate and concentrated, purified by column chromatography (dichloromethane:methanol, 99:1 volume / volume) to give the product (210.7 mg) as an oily solid.

[0384] 1 H NMR (600 MHz, CDC13 ) δ 7.73 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.56 (d, J = 7.4 Hz, 1H), 7.50 (s, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 6.25 (s, 1H), 4.19 - 4.16 (m, 3H), 4.09 (t, J = 6.4 Hz, 1H), 3.84 (dd, J = 10.7, 7.1 Hz, 1H), 3.74 - 3.59 (m, 4H), 3.59 - 3.50 (m, 2H), 3.44 (d, J = 5.1 Hz, 2H), 2.75 (dp, J = 27.3, 6.9 Hz, 2H), 2.42 (t, J = 2.4 Hz, 1H), 2.26 - 2.18 (m, 1H), 2.14 (dp, J = 21.8, 7.2 Hz, 2H), 2.00 - 1.90 (m, 1H).

[0385] 13 C NMR (126 MHz, CDC13 ) δ 173.33, 145.25, 144.22, 141.69, 140.48, 139.48, 128.01, 127.68, 126.83, 125.80, 124.70, 120.06, 119.95, 75.08, 70.20, 70.05, 69.20, 65.41, 58.59, 50.34, 39.36, 35.44, 35.20, 27.26.

[0386] LC / MS (ESI) m / z: 408 (M+H) +

[0387] Preparation of 2,5-dioxopyrrolidin-l-yl ((2-(4-oxo-4-((2-(2-(prop-2-yn-l- yloxy)ethoxy)ethyl)amino)butyl)-9H-fluoren-9-yl)methyl) carbonate: Argon atmosphere and ice bath cooling, 4-(9-(hydroxymethyl)-9H-fluoren-2-yl)-N-(2-(2-(prop-2-yn-l- yloxy)ethoxy)ethyl)butanamide (843.6 mg, 2.07 mmol), pyridine (183.3 μΐ, 2.28 mmol) and bis(2,5-dioxopyrrolidin-l-yl) carbonate (1.07 g, 4.14 mmol) were dissolved in dry acetonitrile, stirred at room temperature for 12 h, the reaction mixture was extracted with dichloromethane (20 mL, 3 times), washed with saturated brine (20 mL, 3 times) successively, dried over sodium sulfate and concentrated, purified by column chromatography (dichloromethane:methanol, 15:1 v / v) to give the product (763.4 mg, 67%) as a white solid.

[0388] 1 H NMR (500 MHz, CDC13) δ 7.73 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.45 - 7.38 (m, 2H), 7.32 (td, J = 7.5, 1.1 Hz, 1H), 7.25 (d, J = 6.2 Hz, 1H), 6.16 (t, J = 5.5 Hz, 1H), 4.62 (dd, J = 10.4, 7.1 Hz, 1H), 4.56 (dd, J = 10.4, 7.4 Hz, 1H), 4.31 (t, J = 7.2 Hz, 1H), 4.15 (d, J = 2.3 Hz, 2H), 3.69 - 3.60 (m, 4H), 3.55 (t, J = 5.1 Hz, 2H), 3.45 (q, J = 5.1 Hz, 2H), 2.81 (s, 4H), 2.78 - 2.70 (m, 2H), 2.40 (t, J = 2.4 Hz, 1H), 2.23 (t, J = 7.6 Hz, 2H), 2.10 - 1.98 (m, 2H).

[0389] 13 C NMR (126 MHz, CDC13) δ 173.33, 145.25, 144.22, 141.69, 140.48, 139.48, 128.01, 127.68, 126.83, 125.80, 124.70, 120.06, 119.95, 75.08, 70.20, 70.05, 69.20, 65.41, 58.59, 50.34, 39.36, 35.44, 35.20, 27.26.

[0390] LC / MS (ESI) m / z: 549 (M+H) +

[0391] Preparation of 2,5-dioxopyrrolidin-1-yl ((2-(4-oxo-4-((2-(2-(prop-2-yn-1- yloxy)ethoxy)ethyl)amino)butyl)-9H-fluoren-9-yl)methyl)carbonyl)-D-alanine: 2,5- dioxopyrrolidin-1-yl ((2-(4-oxo-4-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)butyl)- 9H-fluoren-9-yl)methyl) carbonate (200 mg, 0.342 mmol) was dissolved in 18 mL of acetonitrile, followed by the addition of 1.5 equivalents of D-alanine (43 mg, 0.514 mmol) and 2 mL of 10% aqueous sodium carbonate solution. After stirring at room temperature for 3 hours, the reaction was quenched with 1 M aqueous hydrochloric acid. The mixture was extracted with ethyl acetate three times, and the combined ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography. The target product (140 mg, 60% yield) was obtained as a light yellow oil.

[0392] The above same method can be used to obtain the corresponding product using different amino acids.

[0393] 1-Amino-3,6,9,12-tetraoxopentadecanoic acid was linked to a nucleic acid linker: 1000 microliters of a 2 millimolar aqueous nucleic acid linker solution was added with an equal volume of 500 millimolar borate buffer at pH 9.1. Equal volumes of 120 microliters each of 200 millimolar N,N-dimethylacetamide solutions of 1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16-pentaoxo-4- azanonadecanoic acid, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and N,N-diisopropylethylamine were mixed. After mixing, 300 microliters of the mixture was added to the nucleic acid linker solution and shaken well, and the reaction was allowed to proceed at room temperature for 1 hour. After the reaction was completed, the product was precipitated using the ethanol precipitation method and lyophilized. The lyophilized sample was dissolved in 4 milliliters of water by shaking, and after the precipitate was completely dissolved, 200 microliters of piperidine was added to the system, which was mixed well and shaken at room temperature for 1 hour. After the reaction was completed, 420 microliters of a 5 molar sodium chloride solution was added to the system, which was mixed well with 12 milliliters of 80 degrees Celsius cold ethanol and shaken at -80 degrees Celsius for 30 minutes. The frozen sample was centrifuged at 4 degrees Celsius and 10,000 rpm for 30 minutes, and after the supernatant was removed, the sample was lyophilized at -80 degrees Celsius. After redissolving in water, the product was purified by ultrafiltration centrifugation, and the concentration was quantified by ultraviolet absorbance to obtain 1000 microliters of the product at a concentration of 1.8 millimolar.

[0394] mFomc amino acids are obtained for the construction of DNA encoded compound or nucleic acid encoded libraries.

[0395] Example 3

[0396] The DEL library construction is shown in Figs. 7-11. The specific construction method is shown in Examples 1 and 2.

[0397] As shown in Fig. 7, the first round of DEL library construction. Mutated Fmoc protected isoleucine dehydrates and condenses with HP connected with AOP (AOP-HP), and the first chemical module insertion occurs; through the azido group at the end of the mutated Fmoc, click chemistry reaction with BCN on the surface of the solid phase carrier occurs, and the target DEL is fixed on the solid phase surface, so as to wash away the impurities; the piperidine is used to remove the mutated Fmoc protecting group of the amino group in the DEL, and the first round of DEL library (Ile-AOP-HP) is obtained after purification, and the chemical part is extended by one step.

[0398] As shown in Fig. 8, the second round of DEL library construction. Mutated Fmoc protected lysine dehydrates and condenses with the product of Fig. 7 (Ile-AOP-HP), and the second chemical module insertion occurs; through the azido group at the end of the mutated Fmoc, click chemistry reaction with BCN on the surface of the solid phase carrier occurs, and the target DEL is fixed on the solid phase surface, so as to wash away the impurities; the piperidine is used to remove the mutated Fmoc protecting group of the amino group in the DEL, and the second round of DEL library (Lys(BOC)-Ile-AOP-HP) is obtained after purification, and the chemical part is extended by one step.

[0399] As shown in Fig. 9, the third round of DEL library construction. Mutated Fmoc protected alanine dehydrates and condenses with the product of Fig. 8 (Lys(BOC)-Ile-AOP-HP), and the third chemical module insertion occurs; through the azido group at the end of the mutated Fmoc, click chemistry reaction with BCN on the surface of the solid phase carrier occurs, and the target DEL is fixed on the solid phase surface, so as to wash away the impurities; the piperidine is used to remove the mutated Fmoc protecting group of the amino group in the DEL, and the third round of DEL library (Ala-Lys(BOC)-Ile-AOP-HP) is obtained after purification, and the chemical part is extended by one step.

[0400] As shown in Fig. 10, the fourth round of DEL library is constructed. Mutated Fmoc-protected valine is subjected to dehydrative condensation reaction with the product of Fig. 9 (Ala-Lys(BOC)-Ile-AOP-HP), and the fourth round of chemical module insertion is performed; the azido group at the end of the mutated Fmoc is subjected to click chemistry reaction with BCN on the surface of the solid phase carrier, and the target DEL is fixed on the surface of the solid phase, so as to remove impurities by washing; the mutated Fmoc protecting group of the amino group in the DEL is removed by using piperidine, and the fourth round of DEL library (Val-Ala-Lys(BOC)-Ile-AOP-HP) after purification is obtained, and the chemical moiety is extended by one step.

[0401] As shown in Fig. 11, the fifth round of DEL library is constructed. Mutated Fmoc-protected leucine is subjected to dehydrative condensation reaction with the product of Fig. 10 (Val-Ala-Lys(BOC)-Ile-AOP-HP), and the fifth round of chemical module insertion is performed; the azido group at the end of the mutated Fmoc is subjected to click chemistry reaction with BCN on the surface of the solid phase carrier, and the target DEL is fixed on the surface of the solid phase, so as to remove impurities by washing; the mutated Fmoc protecting group of the amino group in the DEL is removed by using piperidine, and the fifth round of DEL library (Leu-Val-Ala-Lys(BOC)-Ile-AOP-HP) after purification is obtained, and the chemical moiety is extended by one step.

[0402] Figs. 12-20 are LCMS characterization and PAGE analysis diagrams of the target product.

[0403] Fig. 12 shows the LCMS characterization of the crude product of the chemical coupling reaction in Fig. 7, the supernatant obtained after fixing the crude product of the solid phase carrier reaction, and the first round of target product (Ile-AOP-HP) eluted from the solid phase by piperidine.

[0404] Fig. 13 shows the LCMS characterization of the crude product of the chemical coupling reaction in Fig. 8, the supernatant obtained after fixing the crude product of the solid phase carrier reaction, and the second round of target product (Lys(BOC)-Ile-AOP-HP) eluted from the solid phase by piperidine.

[0405] Fig. 14 shows the LCMS characterization of the crude product of the chemical coupling reaction in Fig. 9, the supernatant obtained after fixing the crude product of the solid phase carrier reaction, and the third round of target product (Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase by piperidine.

[0406] Fig. 15 shows the LCMS characterization of the crude product of the chemical coupling reaction in Fig. 10, the supernatant obtained after fixing the crude product of the solid phase carrier reaction, and the fourth round of target product (Val-Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase by piperidine.

[0407] Figure 16 shows LCMS characterization of the crude chemical coupling reaction product in Figure 11, the supernatant obtained after immobilization of the crude reaction product on a solid support, and the fifth round of target product (Leu-Val-Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0408] Figure 17 shows PAGE analysis of the compound in Figure 7. From left to right: reaction starting material (AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, and the first round of target product (Ile-AOP-HP) eluted from the solid phase with piperidine.

[0409] Figure 18 shows PAGE analysis of the compound in Figure 8. From left to right: reaction starting material (Ile-AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, and the second round of target product (Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0410] Figure 19 shows PAGE analysis of the compound in Figure 9. From left to right: reaction starting material (Lys(BOC)-Ile-AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, and the third round of target product (Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0411] Figure 20 shows PAGE analysis of the compound in Figure 10. From left to right: reaction starting material (Ala-Lys(BOC)-Ile-AOP-HP), crude chemical coupling reaction product, supernatant obtained after immobilization of the crude reaction product on a solid support, and the fourth round of target product (Val-Ala-Lys(BOC)-Ile-AOP-HP) eluted from the solid phase with piperidine.

[0412] Figure 21 shows a schematic of the flow for constructing the DEL5-pure library.

[0413] Figure 22 shows the use and characterization of mFmoc amino acids in the synthesis of the DEL5-pure library.

[0414] Example 4

[0415] The magnetic bead type and its amount, reaction buffer, reaction condition and catalyst in the construction of DEL library were optimized, and the results are shown in Table 2. The amino derivative resin was coupled with azidoacetic acid or BCN according to the supplier's instructions, 4.5 micromoles of resin (300 times the amount) was weighed, and 15 nanomoles of BCN or azido mutated Fmoc-lysine-HP coupling product was incubated in the corresponding buffer. The solid phase was separated, washed and eluted with piperidine solution under the corresponding conditions. After drying the eluent, the size, purity and yield of the obtained product were analyzed by gel. The experimental results showed that the solid phase carrier derivatized BCN was beneficial to the improvement of yield, the mixture solution of organic solvent and water was beneficial to the improvement of yield, and the solution containing tetrahydrofuran and triethylamine acetate was beneficial to the improvement of yield. The yield could reach 79% under the reaction at room temperature for 12 hours.

[0416] Table 2

[0417] Example 5

[0418] The high-purity pentapeptide library (DEL5-pure) synthesized using the mFomc amino acid in Example 1 was compared with the pentapeptide DEL library (DEL-native) synthesized using the same D-type amino acid with natural Fmoc protecting group.

[0419] 5.1

[0420] In order to evaluate the nucleotide composition of the library, second-generation sequencing analysis was performed. The results are shown in Figure 23. The DEL5-native library has a relatively uniform distribution of DNA tags during T4 ligation and ethanol precipitation, resulting in a uniform sequence distribution. In contrast, sequencing of the DEL5-pure library shows uneven distribution of amino acids, with a significant lack of serine and threonine. This indicates that the mFmoc technology can effectively construct high-purity DNA-encoded peptide libraries and provides a more reliable method for monitoring the coupling efficiency of building blocks during the synthesis of DNA-encoded peptide libraries.

[0421] 5.2

[0422] In order to evaluate the effectiveness of the DNA-encoded peptide library of the present application in peptide ligand discovery, high-throughput screening was performed using transferrin receptor protein 1 (TfR1). High-throughput screening was performed using DEL5-native and DEL5-pure libraries on biotinylated TfR1 extracellular domain immobilized on magnetic beads. As shown in Figure 24, the pentapeptide library of the present application can specifically bind to the peptide ligand of TfR1, providing a valuable strategy for targeted delivery.

[0423] The polypeptides obtained by screening DEL5-native library (a) and DEL5-pure library (b) against TfR1 are shown in Table 3.

[0424] Table 3

[0425] Highly enriched peptide sequences identified in the DEL5-pure library screen showed strong consistency, while sequences identified in the DEL5-native library screen were relatively random.

[0426] 5.3

[0427] Following the screening of 5.2, DNA was recovered from both libraries and the DNA tags of the recovered peptides were PCR amplified, Illumina sequencing adapters were ligated, NGS sequencing and bioinformatics analysis were performed, and the results are shown in Figure 25. 97% and 96% of the sequences had the expected length of 57 base pairs, which corresponded to the five sticky-end bases of the Tag. The results indicated high efficiency of Tag ligation during library construction.

[0428] 5.4

[0429] The top-ranking peptides identified in the second round of screening from the DEL5-pure library were synthesized on a peptide synthesizer using Rink amide MHBC PS resin. Due to the hydrophobicity of the selected peptide sequences, amino-4,7,10,13-tetraoxypentadecanoic acid (AOP) and lysine (biotin) were attached to the C-terminus to enhance water solubility and introduce a binding tag for affinity assays. Enzyme-linked immunosorbent assay (ELISA) was first used to detect the binding of the peptides to the transferrin receptor 1 (TfR1) extracellular domain. TfR1 was immobilized on an ELISA plate, and biotinylated peptides were added to assess binding. After multiple washes, streptavidin-peroxidase was used to detect the bound peptides.

[0430] Although not all polypeptides were soluble at a 5 mM concentration, a significant portion of the peptides (11 out of 20) exhibited strong binding to TfR1, which was confirmed by chromogenic substrate detection (Figure 26a). In contrast, the control peptide (GGGGG-AOP-Lys(biotin)) did not show binding to TfR1, confirming the specificity of the interaction between the selected peptides and TfR1.

[0431] To further verify the binding affinity, the best peptide binders with sufficient water solubility were subjected to a biolayer interferometry (BLI) binding test. Peptides TR06, TR13, and TR17, which exhibited strong responses in the ELISA assay, were further quantified for their binding to the TfR1 protein using a BLI assay (Figure 26b). Since the biotin-labeled peptides had low solubility at micromolar concentrations, they were first immobilized on a streptavidin sensor surface. Streptavidin (SA) biosensor and the binding of different concentrations of transferrin receptor 1 (TfR1) protein was detected. These peptides showed strong binding to TfR1 in the low nanomolar range.

[0432] Since TfR1 is involved in the internalization of transferrin-related ligands, it is hypothesized that these peptides can facilitate cellular uptake. To verify this, fluorescein was attached to the peptide TR17, which has the best water solubility among the tested peptides, and its ability to penetrate stable cell lines and primary cell lines expressing TfR1 was evaluated (c in Fig. 26). Confocal microscopy imaging showed that the attached peptide significantly enhanced the internalization of fluorescein, confirming its ability to promote peptide-based drug delivery and cell penetration by binding to TfR1 (c in Fig. 26).

[0433] Discussion

[0434] In the prior art, to improve the purity of DEL, Liu D. team coupled a biotin molecule at the end of each reaction module, adsorbed all molecules connected with biotin through streptavidin, purified the product coupled with the raw material, and then broke the connection between biotin and the desired product under alkaline conditions to achieve the purpose of purification. Dario N. team purified the coupling product of each chemical reaction separately after each module of chemical reaction, thereby achieving the purpose of purification. Recently, Dario N. team reported a method for improving the purity of DEL by applying double-connection solid-phase synthesis.

[0435] These reported methods, although to some extent can improve the purity of the library, but there are low recovery rate, small scope of application, cumbersome process, and limited rounds of library construction, etc.

[0436] The applicant developed a strategy of using modified protecting groups, synthesized new protection forms of chemical reaction modules, and used them for chemical coupling in DEL library construction, thereby selectively purifying library members that successfully coupled the chemical modules. Taking Fmoc as an example, a long-chain polyethylene glycol linker was introduced on its benzene ring, and a click chemistry reaction group was inserted at the other end, and in the example, the azide and BCN groups were introduced, and the mutant Fmoc connected with azide was used to protect the amino acid, which was used to synthesize 5 peptides coupled with nucleotides, and the final product with a purity of 96% was obtained.

[0437] SEQUENCE LISTING Note: Pal is phenylalanine ammonia lyase; Pip is phthalimidooxy proline ester; Chg is cyclohexyl glycine; Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Nle is norleucine; Orn is ornithine.

[0438] 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 those skilled in the art upon consideration of this disclosure or can be learned from practice of the application. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.

Claims

A method of preparing a nucleic acid encoded compound or a nucleic acid encoded library, characterized in that, The method comprises the steps of: (s1) providing a HP module, said HP module comprising a chemical reactive end and a DNA encoding end; (s2) covalently linking a chemical building block to said HP module to form a chemical moiety attached to said HP module, and covalently linking an oligonucleotide encoding said chemical building block to said HP module to form an encoding nucleic acid moiety of said HP module, thereby forming a nucleic acid-compound conjugate (or first intermediate); (s3) subjecting said nucleic acid-compound conjugate to a ligation reaction with a solid support, thereby obtaining a mixture comprising reaction products; (s4) isolating or purifying said reaction products from said mixture and subjecting the reaction products to cleavage, thereby cleaving the nascent chemical compound from the solid support; (s5) using said nascent chemical compound as a HP module for a subsequent reaction, repeating steps (s2) to (s4) x times, x being an integer > 0, thereby obtaining a nucleic acid encoded chemical compound or a nucleic acid encoded library. The method of claim 1, wherein The method comprises the steps of: (s1) providing a HP module, said HP module comprising a chemical reactive end and a DNA encoding end; (s2) covalently linking a chemical building block to said HP module to form a chemical moiety attached to said HP module, and covalently linking an oligonucleotide encoding said chemical building block to said HP module to form an encoding nucleic acid moiety of said HP module, thereby forming a nucleic acid-compound conjugate; (s3) subjecting said nucleic acid-compound conjugate to a ligation reaction with a compound of formula (II), thereby obtaining a mixture comprising reaction products; S - R 5b (II) wherein S is a solid support; R 5b a second group for participating in a ligation reaction; (s4) isolating or purifying said reaction products from said mixture and subjecting the reaction products to cleavage, thereby cleaving the nascent chemical compound from the solid support; (s5) using said nascent chemical compound as a HP module for a subsequent reaction, repeating steps (s2) to (s4) x times, x being an integer > 0, thereby obtaining a nucleic acid encoded chemical compound or a nucleic acid encoded library. The method of claim 1, wherein For each library member, the method comprises the steps of: (s1) providing a HP module, said HP module comprising a chemical reactive end and a DNA encoding end; (s2) covalently linking a chemical building block to said HP module to form a chemical moiety attached to said HP module, and covalently linking an oligonucleotide encoding said chemical building block to said HP module to form an encoding nucleic acid moiety of said HP module, thereby forming a nucleic acid-compound conjugate; (s3) subjecting said nucleic acid-compound conjugate to a ligation reaction with a compound of formula (II), thereby obtaining a mixture comprising reaction products; S - R 5b (II) wherein S is a solid support; R 5b a second group for participating in a ligation reaction; (s4) isolating or purifying said reaction products from said mixture and subjecting the reaction products to cleavage, thereby cleaving the nascent chemical compound from the solid support; (s5) repeating steps (s2) - (s4) x times, x being an integer > 0, with the new chemical compound as HP module for the subsequent reaction, to obtain the library member. The method of claim 1, wherein The nucleic acid-compound conjugate is linked to the solid support via a linker, and the method further comprises: one end of the linker is linked to a chemical moiety and the other end is reacted with a reactant comprising a solid support, thereby linking the nucleic acid-compound conjugate to the solid support; cleaving the linker, thereby releasing the library member or the new chemical compound from the solid support. The method of claim 4, wherein The linker comprises a linking structure, a protecting group and a protected group, and is linked in this order. The method of claim 1, wherein The nucleic acid-mixture conjugate comprises a deprotection structure, a linker, a chemical moiety and a coding nucleic acid moiety, and is linked in this order. The method of claim 1, wherein The coding oligonucleotide encoding each chemical building block is added sequentially to the HP module to form the coding nucleic acid moiety, wherein the coding oligonucleotide encoding the chemical building block is added to the HP module before, after or simultaneously with the addition of the chemical building block to the HP module. The method of claim 1, wherein comprises the steps of: (s1) providing a HP module, said HP module being provided with (a) a chemical reactive end (preferably an amino end) for a linking reaction with a chemical building block, and a DNA coding end for a linking reaction with a coding tag; (s2) covalently linking a chemical building block Z to the chemical reactive end of the HP module, and covalently linking a coding tag encoding the chemical building block to the DNA coding end of the HP module, thereby forming a nucleic acid-compound conjugate according to formula VI: R 5a -Z1-HP-Z2 (V1) wherein HP is the reacted HP module; Z1 is a chemical moiety corresponding to a chemical building block and linked to HP R 5a a first group for participating in click chemistry; Z2 is a coding tag corresponding to Z1; (s3) subjecting the nucleic acid-compound conjugate of the previous step to a click chemistry reaction with a reactant according to formula (II), S - R 5b (II) wherein S is a solid support; R 5b a second group for participating in click chemistry; thereby forming a reaction product according to formula III; S-R5-Z1-HP-Z2 (III) wherein S, Z1, HP and Z2 are as defined above, (s4) isolating the reaction product of the previous step and subjecting to a cleavage reaction, thereby forming a new chemical compound, R5is a first group R 5a and a second group R 5b forming a divalent linking group by a click chemistry reaction; wherein the new chemical compound is provided with a chemical reactive end (preferably an amino end) for a linking reaction with a chemical building block, and a DNA coding end for a linking reaction with a coding tag; (s5) repeating steps (s2) - (s4) x times, x being an integer > 1, thereby forming a DEL library; the difference being that: in step (s2) the new chemical compound of the previous step (step (s4)) is used to replace the HP module in step (s2), thereby forming a nucleic acid-compound conjugate according to formula Vx wherein R 5a - (Z1) 1+x - HP - (Z2) 1+x (Vx) HP is the reacted HP module; Z1 is a chemical moiety corresponding to a chemical building block and linked to HP Z2 is a coding tag corresponding to Z1; R 5a a first group for participating in click chemistry; X is an integer > 1; and in step (s3) a reaction product according to formula IIIx is formed; wherein S - R5- (Z1) 1+x - HP - (Z2) 1+x (IIIx) S, Z1, HP and Z2 are as defined above, S, Z1, HP, Z2, R5, and x are as defined above. The method of claim 1, wherein The nucleic acid-compound conjugate has a structure shown in Formula (I): wherein n is an integer > 0; R1and R1' are each independently an amino acid or a chemical group capable of undergoing a coupling reaction with an amino group; R2is the coding nucleic acid moiety; R3is a protecting group (e.g., Fmoc); R4is a linking structure. A DEL library, characterized in that The DEL library is generated by the method of claim 1. A compound of formula (VII) characterized in that, R6— R3— L1— R 5a (VII) wherein R6is an amino acid group; R3is an amino protecting group; L1is a linking structure; R 5a a group that participates in a click chemistry reaction. The compound of claim 11, wherein R6is a monovalent radical derived from a compound selected from the group consisting of: The compound of claim 11, wherein L1is a divalent linking group having the structure -(L3)x- wherein x is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each L3is independently selected from the group consisting of a chemical bond, C, CH, CH2, C=0, O, NH, NHCO, O-C2H4.

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