Method for producing oligonucleotide

Abstract

This method for producing an oligonucleotide comprises a step for reacting a phosphoramidite compound represented by formula (II), or an optical isomer thereof, and a compound represented by formula (III). (In formula (II), ring A, B, X, Y, Z, R1, R2, R5, R6, R7, R8, R9, R10, p, and m are as defined in the description. In formula (III), B, X, Y, Z, R9, R12, and q are as defined in the description.)

Description

Methods for producing oligonucleotides

[0001] The present disclosure relates to methods for making oligonucleotides.

[0002] DNA-encoded libraries (DELs) are chemical libraries in which artificially synthesized compounds are tagged with DNA barcodes. This technology, proposed in 1992 by S. Brenner and RA Lerner of the Scripps Research Institute, allows for the identification of structural information by linking artificial compounds to genetic information (Non-Patent Document 1). Their method combines split-and-pool techniques to construct libraries by alternating the synthesis of bioactive molecules with the chemical synthesis of DNA barcodes that record the synthesis information. KD Janda et al. and MA Gallop et al. independently reported the first examples of DELs in 1993 (Non-Patent Documents 2 and 3). Both of their results were achieved by alternately synthesizing peptides and DNA on a solid support. The first-generation DELs involved the chemical synthesis of protected DNA barcodes (hereinafter referred to as the "recording-by-synthesis" method), marking a monumental achievement toward the realization of DELs.

[0003] In the 2000s, the second generation of DEL, called "encoded ssDNA barcodes," emerged. In this method, unprotected ssDNA is prepared in advance, and libraries are constructed based on the ability of the library strand to catch and release its complementary strand through DNA hybridization.

[0004] In 2001, DNA-templated organic synthesis (DTS) was reported by Gartner and Liu et al. (Non-Patent Document 4). While DTS is extremely advantageous in that it can construct a diverse library of compounds with complex structures, it has several problems, including a very complicated process for constructing a DNA barcode library that takes into account sequence specificity in hybridization, the need to use reagents prepared for DTS reactions, which limits the number of applicable building blocks, and the limited synthetic yield at each step, resulting in a relatively small number of cycles and limited diversity in the libraries that can be constructed.

[0005] DNA routing, reported by P.B. Harbury et al. at Stanford University in 2004, involves capturing a library of encoded ssDNA barcodes with the anticodon of each building block, transferring them to a DEAE Sepharose resin, and then chemically synthesizing the corresponding building blocks on the resin (Non-Patent Documents 5-7). Unlike DTS, this method allows for the application of chemical reaction conditions in organic solvents using standard reagents. They actually constructed peptide or peptoid libraries using combinatorial chemistry with standard Fmoc amino acids or chloroacetic acid and amines. However, HPLC analysis of peptide-DNA conjugates synthesized in eight cycles revealed multiple by-products. Additionally, the presence of numerous hydroxyl groups and tertiary amines on DEAE Sepharose limits the applicable chemical reaction space.

[0006] In the second generation of DEL, many library construction methods using encoded ssDNA barcodes have been reported. However, each individual technique requires complex know-how and has problems such as the number of synthesis cycles, applicable chemical reaction conditions, yield and purity, structural complexity of the molecules that can be constructed, and library diversity.

[0007] In 2009, GlaxoSmithKline (GSK) reported a third-generation DEL construction method using enzymatic DNA recording (Non-Patent Document 8). This DNA recording method uses enzymatic ligation with T4 DNA ligase to extend the DNA barcode, and similar to the first-generation DEL recording-by-synthesis method, the library is constructed by alternating chemical synthesis of bioactive molecules and DNA barcode extension reactions. These methods do not require the preparation of an oligonucleotide library in advance, taking into account sequence specificity in hybridization, as is the case with the second-generation encoded ssDNA barcode strategy.

[0008] DEL construction using enzymatic DNA recording is currently the most widely used technique for DEL construction. DNA recording, which utilizes enzymatic ligation in a liquid phase, is widely used due to its simplicity. However, because each cycle requires ethanol precipitation and PAGE purification, it is difficult to maintain high library yields. In addition, the yield of the chemical reaction at each step is not quantitative.

[0009] Meanwhile, in 2015, BM Paegel et al. at UC Irvine applied enzymatic ligation-based DNA recording to construct DELs on solid supports (Non-Patent Document 9). This enabled purification at each step to be completed by washing procedures, similar to conventional solid-phase synthesis, making library construction easier. However, problems caused by the chemical fragility of DNA, such as DNA damage, persist, and library construction is typically limited to two or three cycles.

[0010] Brenner, S. et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383.Nielsen, J. et al, J. Am. Chem. Soc. 1993, 115, 9812-9813.Needels, MC et al, Proc. Natl. Acad. Sci. USA 1993, 90, 10700-10704.Gartner, ZJ et al., J. Am. Chem. Soc. 2001, 123, 6961-6963.Halpin, DR et al., PLoS Biol. 2004, 2, 1015-1021.Halpin, DR et al., PLoS Biol. 2004, 2, 1022-1030.Halpin, DR et al., PLoS Biol. 2004, 2, 1031-1038.Clark, MA et al., Nat. Chem. Biol. 2009, 5, 647-654.MacConnell, AB et al., ACS Comb. Sci. 2015, 17, 518-534.

[0011] As described above, the third generation of DELs has seen the emergence of DNA Recording by enzymatic ligation, which alternates between the extension of DNA barcodes by enzymatic ligation and the chemical synthesis of building blocks.

[0012] However, the biggest problem is the fragility of DNA. The current mainstream of DELs uses "unprotected" DNA, similar to that found in its "natural" state. Due to the physical and chemical fragility of DNA, DNA is damaged during the construction of DELs. As a result, restrictions are placed on the chemical reactions that can be applied to DELs, limiting chemical diversity. Furthermore, the results of drug screening using constructed DELs can be misleading.

[0013] For example, if we try to create a molecule (drug candidate) with structure ABC using DEL with ligation, as shown in Figure 1(A), we chemically link structure A to the end of the DNA via a linker, then enzymatically attach DNA A corresponding to structure A to the end of the DNA opposite to the end where structure A was attached, then chemically link structure B to structure A at the end of the DNA chain, then enzymatically attach DNA B corresponding to structure B to the end of DNA A opposite to the end where structure B was attached, then chemically link structure C to structure B at the end of the DNA chain, and then enzymatically attach DNA C corresponding to structure C to the end of DNA C opposite to the end where structure C was attached. Only when all three DNAs A, B, and C are in place can we identify the final molecule we want to create.

[0014] Thus, conventional DEL alternates between chemical and enzymatic reactions, but the DNA used is in its naturally occurring "unprotected" state. Unprotected DNA contains highly reactive functional groups, such as amino, hydroxyl, and phosphate groups, in its chemical structure, and the DNA itself can react chemically. This can lead to DNA damage during the DEL construction process, resulting in inaccurate DNA readings. For examples of actual damage that can occur, see Satz AL, et al., Nat. Rev. Methods Primers 2022, 2, 2-17. For an evaluation of the durability of unprotected DNA, see MacConnell, AB et al., ACS Comb. Sci. 2015, 17, 518-534.

[0015] This problem of DNA damage is the same whether in liquid phase or solid phase, and in DEL, which does not use ligation but uses unprotected DNA, the problem of DNA being broken by chemical reactions also occurs. Therefore, this problem applies to all DEL methods and is commonly recognized by those skilled in the art.

[0016] To avoid this DNA damage, the chemical reactions that can be applied to DEL are limited to conditions that minimize damage to unprotected DNA. The number of chemical reactions that can be performed to construct DEL is also limited (usually 2-4 synthesis cycles). As a result, the quality of the constructed library is reduced. The available chemical reactions are not necessarily those that can efficiently produce drug molecules. Furthermore, the components of the library tend to be small molecules containing fragments.

[0017] The purpose of the present disclosure is to provide a method for chemically synthesizing DNA barcodes (Recording-by-Synthesis method) that can solve one or more of the above-mentioned problems that current DELs have.

[0018] In order to achieve the above object, the present disclosure includes, for example, the following aspects.

[0019] Item 1. A method for producing an oligonucleotide, comprising a step of reacting a phosphoramidite compound represented by formula (II) or an optical isomer thereof with a compound represented by formula (III). (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11. (In formula (III), each B is independently any one of nucleobases, each X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and q is 0 or an integer from 1 to 50.

[0020] Item 2. The method according to Item 1, wherein ring A is a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle. Item 3. A method for deprotecting a phosphate group in an oligonucleotide obtained by the method according to Item 1, comprising heating the oligonucleotide obtained by the method according to Item 1 in an aqueous solution at a pH of 6.0 to 11.0 so as to deprotect the group represented by the following formula (XI) in the oligonucleotide: (In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

[0021] Item 4. A compound represented by the following formula (I): (In formula (I), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 12 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 13 are each independently —OH, —SH, or a protecting group for a phosphate group, and R 13 at least one of the groups is a group represented by the following formula (XI): (In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8is -H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; m is an integer from 0 to 11; r is an integer from 1 to 50;

[0022] Item 5. A method for producing a phosphoramidite compound represented by formula (II) or an optical isomer thereof, which comprises reacting a compound represented by formula (IV) with a compound represented by formula (X) in the presence of an activator. (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11. (In formula (IV), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and p is 0 or an integer from 1 to 50. (In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

[0023] Item 6. A phosphoramidite compound represented by the following formula (II) or an optical isomer thereof: (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10is -H, a hydroxyl protecting group, or a linker attached to a solid support; and m is an integer from 0 to 11. p is 0 or an integer from 1 to 50.

[0024] Item 7. An amidite-forming reagent containing a compound represented by formula (X). (In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

[0025] Item 8. A compound represented by the following (X-2) or an optical isomer thereof: Item 9. A method for producing a DNA-encoded library, comprising: (i) preparing a solid support or a soluble carrier having a first functional group reactive with a nucleoside and a second functional group reactive with a first compound; (ii) binding a first building block to the first functional group, wherein the first building block is a nucleoside monomer or an oligonucleoside; (iii) binding a first compound to the second functional group; (iv) binding a second building block to the first building block bound to the first functional group in step (ii), wherein the second building block is a nucleoside monomer or an oligonucleoside; and (v) binding a second compound to the first compound bound to the second functional group in step (iii), wherein the binding of the building block and the binding of the compound are alternately performed in steps (ii) to (v); The method, wherein the first building block bound to the first functional group is a compound represented by formula (III), and the second building block is a phosphoramidite compound represented by formula (II) or an optical isomer thereof. (In formula (III), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and q is 0 or an integer from 1 to 50. (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11.

[0026] Item 10. The method according to Item 9, wherein a set consisting of one step of coupling a nucleoside and one step of coupling a compound is considered to be one cycle, and the method is carried out in multiple cycles. Item 11. The method according to Item 9 or 10, wherein step (i) comprises a step of preparing a solid support having a first functional group reactive with a nucleoside and a second functional group reactive with a first compound, the solid support having an inner layer and an outer layer covering the inner layer, the DNA synthesis initiation site being located on the outer layer of the solid support, and linkers having an Fmoc group being attached to the inner and outer layers of the solid support.

[0027] (A) Schematic diagram of candidate molecules. (B) General schematic diagram of DEL using enzymatic ligation. Partially modified from Satz, AL et al., Nat. Rev. Methods Primers 2022, 2, 1-17. Chemical structure and deprotection mechanism of phosphate protecting groups. a) 2-cyanoethyl group, b) methyl group. Nuc.: Chemical structure of protected DNA with a general protecting group evaluated in the chemical resistance evaluation of nucleophile-protected DNA. Example of the chemical durability evaluation results of NAPM-type phosphate protecting groups and comparison with conventional protecting groups. Conditions: 20% piperidine / NMP, room temperature, 15 minutes. Absorbance at 265 nm was monitored. w / o dinucleotide was obtained from the reaction mixture without protected DNA. †: Peak of compounds 15, 16, or 21. ‡: Peak of degradation product. *: Peak of anthracene used as an internal standard. Temperature and time dependent changes in the deprotection of oligonucleotides synthesized using NAPM-type phosphoramidites with borate buffer. dT 10 C. Conditions: 250 mM borate buffer pH 8.5. Upper panel: UV chromatogram. Absorbance at 260 nm was monitored. The retention time of the target product was around 28.8 minutes. Lower panel: Results of mass spectrometry using MALDI-TOF MS. Temperature- and time-dependent changes during deprotection in aqueous solvent of oligonucleotides synthesized using NAPM-type phosphoramidites. dT 10C. Upper panel: UV chromatogram of the product treated with 100 mM bicine buffer, pH 8.5. Lower panel: UV chromatogram of the product treated with 100 mM potassium phosphate buffer, pH 7.4. Absorbance at 260 nm was monitored. The retention time of the target product was around 28.8 minutes. Deprotection of oligonucleotides synthesized using NAPM-type phosphoramidites in aqueous solvent. dT 10 C. Conditions: 90°C, 3 hours. Top: UV chromatogram. Bottom: Results of mass spectrometry using MALDI-TOF MS. Deprotection of oligonucleotides synthesized using NAPM-type phosphoramidites using water. Conditions: Sterile water, 90°C, 12 hours. Results of mass spectrometry using MALDI-TOF MS. Example of chemical resistance evaluation of protected DNA to expand the chemical reaction space. a) dC Bz a) Evaluation of the durability of dT against Mitsunobu alkylation reaction. b) Evaluation of the durability of dT against Mitsunobu alkylation reaction. c) Evaluation of the durability of the NAPM group against Mitsunobu alkylation reaction. Deprotection of benzyl protection in nucleic acid bases. Top: DNA-compatible heterogeneous catalytic reduction-like reaction. Bottom: Heat treatment with aqueous sodium borate solution. Results of Alternating Synthesis-1 based on Recording-by-Synthesis method: Analysis of N-methyl peptides and DNA barcodes after 10 cycles of alternate synthesis. a) Chemical structure and LC / MS chromatogram analysis of synthesized bioactive molecules. †: Peaks observed in off-ligand synthesis. ‡: Peak of target molecule. b) Schematic diagram of DNA barcode deprotection. c) Native PAGE of each step in the Recording-by-Synthesis method for DNA barcodes. R f7-10,Ac: Commercially available reference sample. d) Sequence and observed mass in MALDI-TOF MS analysis of each step in the Recording-by-Synthesis method. e) An example of a MALDI-TOF MS mass spectrum result. The spectrum is the result after DNA Recording-11 described in Scheme 7. Results after ssDNA amplification of DNA barcodes in Alternating Synthesis-1 based on the Recording-by-Synthesis method. Results of Alternating Synthesis-2 based on the Recording-by-Synthesis method: Quality check of the alternate synthesis of LUNA18 at DEL a) Chemical structure and LC / MS results of LUNA18 synthesized through alternate synthesis. b) Native PAGE and c) MALDI-TOF MS analysis of DNA barcodes.

[0028] As used herein, the singular forms "a," "an," and "the" are intended to include both the singular and the plural unless otherwise expressly stated herein or otherwise clearly contradicted by context.

[0029] In this specification, the terms "comprise" and "include" are concepts that encompass "consist essentially of" and "consist only of."

[0030] In the numerical ranges described in stages in this specification, the upper or lower limit of a certain numerical range can be arbitrarily combined with the upper or lower limit of another numerical range. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of the numerical range may be replaced with a value shown in an example or a value that can be unambiguously derived from an example. Furthermore, in this specification, a numerical value connected with "to" means a numerical range that includes the numbers before and after "to" as the upper and lower limits.

[0031] As used herein, Ca-Cb refers to a group having a carbon number ranging from a to b, including both a and b. For example, "C1-C6" refers to a group having a carbon number ranging from 1 to 6.

[0032] As used herein, "nucleotide" refers to a compound comprising a nucleoside base, a sugar, and a phosphate bond. The nucleotide may be a naturally occurring nucleotide such as adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, cytidine triphosphate, or uridine triphosphate, or a modified nucleotide. The nucleoside base portion of the nucleotide may be a naturally occurring base such as adenine, guanine, cytosine, thymine, or uracil, or a modified, non-natural nucleoside base. The sugar portion of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D- or L-configuration. The phosphate portion may be, for example, phosphorothioate, phosphorodithioate, methylphosphonate, boranophosphate, or methylphosphate.

[0033] As used herein, "oligonucleotide" refers to 2 to 50 nucleotides.

[0034] As used herein, "nucleoside" refers to a compound comprising a nucleoside base and a sugar, which may be a naturally occurring nucleoside such as adenosine, thymidine, guanosine, cytidine, or uridine, or a modified nucleoside. The nucleoside base portion of the nucleotide may be a naturally occurring base such as adenine, guanine, cytosine, thymine, or uracil, or a modified, non-natural nucleoside base. The sugar portion of the nucleoside may be a naturally occurring deoxyribose or ribose, and may be in the D- or L-configuration.

[0035] As used herein, "oligonucleoside" refers to 2 to 50 nucleosides.

[0036] As used herein, the term "aromatic group" can be used interchangeably with "aryl group" and refers to a monovalent group formed by removing one hydrogen atom from an aromatic hydrocarbon nucleus. The aromatic hydrocarbon may be a monocyclic aromatic hydrocarbon or a polycyclic aromatic hydrocarbon, and the polycyclic aromatic hydrocarbon may be a fused polycyclic aromatic hydrocarbon. Examples of aromatic hydrocarbons include benzene, naphthalene, anthracene, and the like. In a preferred embodiment, the aromatic hydrocarbon is benzene. When the aromatic group is substituted, the substituent may be a C1-C6 alkyl, halogen, or the like.

[0037] As used herein, "halogen" or "halo" refers to fluorine, chlorine, bromine, or iodine.

[0038] As used herein, the term "solid support" refers to a resin, polymer, or inorganic oxide. Examples of inorganic oxides include those selected from the group consisting of silica, alumina, zeolite, and controlled pore glass. The solid support may take the form of a membrane, beads, gel, or the like.

[0039] As used herein, the term "soluble support" refers to a reaction field for liquid-phase synthesis of biologically relevant molecules, including peptides and oligonucleotides, as well as other physiologically active molecules. Soluble supports are also referred to as "liquid-phase supports," in contrast to "solid-phase supports." While not necessarily bound by the following, in a particularly preferred embodiment, the soluble support refers to a support that can reversibly alternate between a dissolved and an insoluble (solid) state, or that exhibits high solubility in a specific solvent, thereby facilitating the purification of synthesized molecules. As used herein, the term "hydrophobic tag" refers to a hydrophobic group derived from a soluble support, formed by binding a soluble support, which is a hydrophobic compound, to the hydroxyl group of a phosphoramidite, nucleotide, or nucleoside. The hydrophobic tag has the property of reversibly alternately alternating between a dissolved and an insoluble (solid) state, or that exhibits high solubility in a specific solvent, thereby facilitating the purification or identification of the molecule to which the hydrophobic tag is attached.

[0040] The present disclosure provides an amiditizing reagent containing a compound represented by formula (X). In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is —H, C1-C6 alkyl, or halogen; and m is an integer from 0 to 11.

[0041] The present inventors have found that the phosphorodiamidite compound represented by formula (X) has a 4- to 7-membered nitrogen-containing monocyclic heterocycle at a terminal different from the diamidite, and has a structure in which a carbonyl group is bonded to the 4- to 7-membered nitrogen-containing monocyclic heterocycle, making ring A less susceptible to decomposition during oligonucleotide synthesis than a linear structure, and is stable under the synthesis conditions for biologically active molecules such as peptides in DEL, and can form a protecting group for the phosphate of DNA that can be deprotected under mild conditions. While not wishing to be bound by theory, the present invention can be carried out by using R 6It is believed that the protecting group is removed via an intramolecular cyclization reaction caused by nucleophilic attack on an intramolecular carbon atom due to effective neighboring group participation by the -CO- group. Therefore, the protecting group generated by reacting a nucleoside or oligonucleoside with a compound represented by formula (X) is stable under the synthesis conditions of a physiologically active molecule, but can be removed under milder conditions by an intramolecular reaction.

[0042] In a preferred embodiment, ring A is a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle.

[0043] In a preferred embodiment, R 1 , R 2 , R 3 , and R 4 are the same or different C1-C6 alkyl.

[0044] In a preferred embodiment, ring A is a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 , R 2 , R 3 , and R 4 are the same or different C1-C6 alkyl, and m is 0.

[0045] More preferably, ring A contains one nitrogen and no other heteroatoms.

[0046] In some embodiments, the compound represented by formula (X) is a compound represented by formula (X-1): Ring A is a 4-7 membered nitrogen-containing monocyclic saturated heterocycle, R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

[0047] In a preferred embodiment, ring A is a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 ~R 5 are each independently C1-C6 alkyl, and R 6 is hydrogen or C1-C6 alkyl, and m is 0.

[0048] In a more preferred embodiment, the compound represented by formula (X) is a compound represented by the following (X-2) or an optical isomer thereof:

[0049] The present disclosure also provides a method for producing a phosphoramidite compound represented by formula (II) or an optical isomer thereof, which comprises reacting a compound represented by formula (IV) with a compound represented by formula (X) in the presence of an activator. In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle, each B is independently a nucleobase, each X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is -H, C1-C6 alkyl, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11. In formula (IV), each B is independently a nucleobase, X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; and p is 0 or an integer from 1 to 50. In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is —H, C1-C6 alkyl, or halogen; and m is an integer from 0 to 11.

[0050] The nucleoside phosphoramidite represented by formula (II) may be a monomer or an oligomer such as 2-mer to 50-mer, for example, a nucleoside phosphoramidite represented by formula (II) is a 2-mer to 25-mer.

[0051] In formula (II) and formula (IV), each B is independently a nucleobase. B may be a nucleobase protected with a protecting group or an unprotected nucleobase.

[0052] B can be, for example, a naturally occurring base such as adenine, guanine, cytosine, thymine, and uracil, or 7-deazaguanine, 7-deaza-8-azaguanine, 5-propynylcytosine, 4-N-methylcytosine, 5-methylcytosine, 5-propynyluracil, 7-deazaadenine, 7-deaza-8-azaadenine, 6-N-methyladenine, 7-deaza-6-oxopurine, 6-oxopurine, 3-deazaadenosine, Modified bases include, but are not limited to, 2-oxo-5-methylpyrimidine, 2-oxo-4-methylthio-5-methylpyrimidine, 2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine, 2-aminopurine, 5-fluorouracil, 2,6-diaminopurine, 8-aminopurine, 4-triazolo-5-methylthymine, and 4-triazolo-5-methyluracil.

[0053] When B is a nucleic acid base protected by a protecting group, the nucleic acid base is a nucleic acid base whose reactive functional group of the base is protected.Typically, nucleic acid base has an amine group protected by an amine protecting group, such as amide or carbamate.For example, the amine group of adenine and cytosine is typically protected by a benzoyl protecting group, and the amine group of guanine is typically protected by an acyl group such as isobutyryl group, acetyl group, or t-butylphenoxyacetyl group, or an ultramild protecting group.However, other protection schemes can also be used.

[0054] For example, nucleic acid bases may be protected with silyl-based or acetal-based protecting groups, and the protected sites are not limited to amines; carbonyl groups can also be protected. Silyl-based protecting groups protect amines and include, but are not limited to, trialkylsilyl groups such as 2-(trimethylsilyl)ethoxycarbonyl (Teoc) and 2'-O-triisopropylsilyl-oxy-methyl (TOM). Acetal-based protecting groups include, but are not limited to, substituted or unsubstituted tetrahydropyranyl (THP), substituted or unsubstituted tetrahydrofuranylethoxyethyl (EE), methoxymethyl (MOM), and benzyloxymethyl (BOM).

[0055] Preferred examples of the protecting group for the nucleic acid base of B include the following protecting groups (XIV-1) to (XIV-4).

[0056] In formula (XIV-1), R 1 each independently represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; m is an integer from 0 to 5; and * represents a bond to the nitrogen or oxygen of the nucleobase.

[0057] In formula (XIV-2), R 1 is an optionally substituted C1-C30 hydrocarbon group (e.g., a C5-30 linear or branched alkyl group or a C5-C30 linear or branched alkenyl group), -N(Ph)Ph (Ph is a phenyl group), or a tert-butoxy group, and * is a bond to the nitrogen or oxygen of the nucleobase.

[0058] The C1-C30 hydrocarbon group may be a straight-chain or branched-chain hydrocarbon group, for example, a C1-C20, preferably C1-C10, more preferably C1-C6 straight-chain or branched-chain alkyl group, or an aromatic ring (e.g., phenyl). 1 When is substituted, one or more of the carbon atoms may be substituted with halogen (eg, fluorine, chlorine, or bromine), hydroxy, or alkoxy.

[0059] In formula (XIV-3), R 1 is an optionally substituted C1-C30 hydrocarbon group (for example, a straight-chain or branched-chain alkyl group or a straight-chain or branched-chain alkenyl group), and * is a bond to the nitrogen or oxygen of the nucleobase.

[0060] The C1-C30 hydrocarbon group may be a linear or branched hydrocarbon group, for example, a C1-C30, preferably C1-C20, more preferably C1-C10 linear or branched alkyl group, or an aromatic alkyl group (e.g., benzyl). 1 When is substituted, one or more of the carbon atoms may be substituted with halogen (eg, fluorine, chlorine, or bromine), hydroxy, or alkoxy.

[0061] In formula (XIV-4), R 1 and R 2 each independently represents an optionally substituted linear or branched hydrocarbon group, such as C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or a halogen; R 1 and R 2 may be bonded to form a ring, R 1 and R 2 is substituted, one or more of the carbon atoms may be substituted with halogen (e.g., fluorine, chlorine, or bromine), hydroxy, amine, or alkoxy, and * is a bond to the nitrogen of a nucleobase.

[0062] In formulas (XIV-1) to (XIV-4), the aromatic group may be substituted or unsubstituted, and is preferably a phenyl group. When the aromatic group is substituted, the substituents include C1-C6 alkyl, alkoxy, halogen, etc.

[0063] Substitution with the above (XIV-1) to (XIV-4) makes deprotection less likely than in the unsubstituted case, allowing the stability of the oligonucleotide or oligonucleoside to be adjusted. Furthermore, multiple protecting groups may be used simultaneously as needed, and the conditions for removing the protecting groups depend on the protecting groups used.

[0064] In formula (II) and formula (IV), when Y is a protected hydroxyl group, each Y may independently be a hydroxyl group protected with an acyl-based protecting group, an ether-based protecting group, or a silyl-based protecting group. Examples of acyl-based protecting groups include acetyl, benzoyl, and pivaloyl. Examples of ether-based protecting groups include benzyl, p-methoxybenzyl (PMB), and allyl. Examples of silyl-based protecting groups include trialkylsilyl groups such as t-butyldimethylsilyl and triisopropylsilyl, and mono-, di-, or trialkylphenylsilyl groups such as t-butyldiphenylsilyl.

[0065] In a preferred embodiment, in formula (II) and formula (IV), R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support.

[0066] In a preferred embodiment, R 10 are hydroxyl-protecting groups, and preferably each independently is an acid-labile protecting group, an acyl-based protecting group, an ether-based protecting group, a silyl-based protecting group, or a hydrophobic group derived from a soluble carrier.

[0067] An acid-labile protecting group is a protecting group that can be removed by contacting the group with a protic acid or a Lewis acid. Acid-labile protecting groups are known to those skilled in the art. Examples of acid-labile protecting groups include a substituted or unsubstituted trityl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted tetrahydrofuranyl group, or a pixyl group. The trityl group is usually substituted with an electron-donating group such as an alkoxy group. In a more preferred embodiment, R 10 is substituted or unsubstituted trityl, 9-(phenyl)xanthenyl (hereinafter "pixyl"), or tetrahydropyranyl (hereinafter "THP"). In a more preferred embodiment, R 10 is unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, trialkoxytrityl, THR, or pixyl. 10 is 4,4'-dimethoxytrityl.

[0068] Examples of acyl protecting groups include acetyl, benzoyl, isobutyryl, pivaloyl, etc. Examples of ether protecting groups include benzyl, p-methoxybenzyl (PMB), allyl, etc. Examples of silyl protecting groups include trialkylsilyl groups such as t-butyldimethylsilyl or triisopropylsilyl, and mono-, di-, or trialkylphenylsilyl groups such as t-butyldiphenylsilyl.

[0069] R 10 For the hydrophobic group derived from the soluble carrier when R is a hydrophobic group derived from the soluble carrier, 12It is possible to use a hydrophobic group derived from a soluble carrier, which will be described in detail with respect to the above. Note that methods for synthesizing, isolating, and / or purifying oligonucleotides using phosphoramidite compounds starting from nucleosides having a soluble carrier bound to their hydroxyl groups are known (Volume 19, Issue 26, June 24, 2013, Pages 8615-8620, https: / / chemistry-europe.onlinelibrary.wiley.com / doi / full / 10.1002 / chem.201300655, and https: / / pubs.acs.org / doi / 10.1021 / acs.joc.1c01756), and those skilled in the art can produce, separate, and / or purify oligonucleotides within the scope of their ordinary skills.

[0070] In some embodiments, R 10 is a linker attached to the solid support. The linker can be any linker used in solid phase synthesis, such as a bifunctional linker, linear or branched, and can include: C1-10 alkyl, heteroalkyl of 1 to 10 atoms, C2-10 alkenyl, C2-10 alkynyl, C5-10 aryl, cyclic or polycyclic systems of 3 to 20 atoms, phosphodiester, peptide, oligosaccharide, oligonucleotide, oligomer, polymer, polyalkyl glycol (e.g., polyethylene glycol, -(CH2CHO) n CH2CH2-, where n is an integer from 1 to 50), or combinations thereof.

[0071] As the activator, any known activator that promotes the synthesis of oligonucleotides or oligonucleosides can be used, including tetrazole-type, imidazole-type, and imidazolium salt-type activators, and those skilled in the art can select an appropriate activator.

[0072] Examples of the activating agent include, but are not limited to, 1H-tetrazole, diisopropylammonium salt of 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole.

[0073] In some embodiments, the activator comprises an activator represented by formula (XIII): In the formula (XIII), R 1 is selected from the group consisting of H, -F, -Cl, -Br, -CN, methoxy, cyclobutadiene, adamantane, a linear or branched, saturated or unsaturated C1-C22 alkyl group, an optionally substituted aromatic ring; R 2 and R 3 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CN, methoxy, cyclobutadiene, adamantane, a straight or branched chain saturated or unsaturated C1-C22 alkyl group, and an optionally substituted aromatic ring, or R 2 and R 3 together form a phenyl ring with the two carbon atoms on the imidazole ring to which they are attached.

[0074] The present disclosure also provides a phosphoramidite compound represented by formula (II) or an optical isomer thereof. In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle, each B is independently a nucleobase, each X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is -H, C1-C6 alkyl, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11.

[0075] Ring A is preferably a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle.

[0076] Ring A is preferably a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle, and has no heteroatoms other than one nitrogen atom in the ring.

[0077] B, Y, and R in formula (II) 9 and R 10 The details are as explained above.

[0078] In some embodiments, in Formula (II), p=0 and R 10 is a substituted or unsubstituted trityl group.

[0079] In some embodiments, in Formula (II), p=0 and R 10 is 4,4'-dimethoxytrityl.

[0080] The present disclosure also provides a method for producing an oligonucleotide, comprising the step of reacting a compound that is a nucleoside phosphoramidite represented by formula (II) with a compound that is a nucleoside represented by formula (III). In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle, each B is independently a nucleobase, each X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is -H, C1-C6 alkyl, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11.

[0081] Ring A is preferably a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle.

[0082] Ring A is preferably a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle, and has no heteroatoms other than one nitrogen atom in the ring.

[0083] B, Y, and R in formula (II) 9 and R 10 The details are as explained above. In formula (III), each B is independently a nucleobase, X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a hydroxyl protecting group, or a linker attached to a solid support; and q is 0 or an integer from 1 to 50.

[0084] The method for producing the oligonucleotide may be carried out by liquid phase synthesis or solid phase synthesis.

[0085] In a preferred embodiment, in formula (III), R 12 is a protecting group for a hydroxyl group, preferably an acid-labile protecting group, or a trialkylsilyl group such as t-butyldimethylsilyl or triisopropylsilyl, or a t-butyldiphenylsilyl group. An acid-labile protecting group is a protecting group that can be removed by contacting the group with a protic acid or a Lewis acid. Acid-labile protecting groups are known to those skilled in the art. Examples of acid-labile protecting groups include a substituted or unsubstituted trityl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted tetrahydrofuranyl group, or a pixyl group. The trityl group is usually substituted with an electron-donating group such as an alkoxy group. In a more preferred embodiment, R 12 is substituted or unsubstituted trityl, 9-(phenyl)xanthenyl (hereinafter "pixyl"), or tetrahydropyranyl (hereinafter "THP"). In a more preferred embodiment, R 12 is unsubstituted trityl, monoalkoxytrityl, dialkoxytrityl, trialkoxytrityl, THP, or pixyl. 12 is 4,4'-dimethoxytrityl.

[0086] In some embodiments, R of formula (III)12 The hydroxyl-protecting group is a group containing polyethylene glycol (PEG) or methoxy PEG derived from a soluble carrier, a group containing an ionic liquid, or a hydrophobic group. Examples of hydrophobic groups derived from a soluble carrier include hydrophobic tags. The hydrophobic tag is a hydrophobic group used as an identification tag to select and / or purify the phosphoramidite compound represented by formula (II) or its optical isomer, or an oligonucleotide obtained by further reacting the phosphoramidite compound represented by formula (II) or its optical isomer with a nucleoside, from reaction residues during liquid-phase synthesis. Such groups are known.

[0087] The hydrophobic tag is a hydrophobic group used as an identification tag for selecting and / or purifying an oligonucleoside obtained by reacting a phosphoramidite compound represented by formula (II) or an optical isomer thereof, or a phosphoramidite compound represented by formula (IV) or an optical isomer thereof, with a nucleoside, from reaction residues in a liquid phase synthesis. Such groups are well known.

[0088] For example, R 12 is a group represented by the following formula (XII-1) or a group represented by the formula (XII-2) described in Japanese Patent No. 6770553. In formula (XII-1), each R independently represents a linear or branched alkyl group having 10 to 40 carbon atoms.

[0089] m represents an integer of 1 to 5. When m is 2 or more, the multiple ROs may be the same or different.

[0090] X is a direct bond, -(CH) n - (n is an integer from 1 to 5), -(CH) n It represents -NH- (n is an integer of 1 to 5), -NH-, or a hydrocarbon group (the total number of carbon atoms in the carbon chain is 1 to 45) which may contain an ester bond, an amide bond, an ether bond, or a carbonyl group in the carbon chain. In formula (XII-2), each R independently represents a linear or branched alkyl group having 10 to 40 carbon atoms.

[0091] m represents an integer of 1 to 5. When m is 2 or more, the multiple ROs may be the same or different.

[0092] X represents O, NH, or S.

[0093] n represents an integer of 1 to 4.

[0094] Formula (XII-1) may in particular be formula (XII-1a) below: In formula (XII-1a), R 1 ,R 2 is an alkyl chain, and X is a hydrocarbon group (the total number of carbon atoms in the carbon chain is 1 to 40) which may contain a direct bond, NH, an ester bond, an amide bond, an ether bond, or a carbonyl group in the carbon chain.

[0095] The formula (XII-2) may in particular be the following formula (XII-2a): In formula (XII-2a), R 1 , R 2 , and R 3 each independently represents a linear or branched alkyl group having 10 to 40 carbon atoms. X represents O or S. n represents an integer of 1 to 4. Alternatively, the hydrophobic tag may be a fluorine-containing hydrocarbon group, adamantyl methyl ester, or a long-chain alkyl chain group.

[0096] In some embodiments, R 12 is a linker attached to the solid support. 12 The linker attached to the solid support of 10 The linkers attached to the solid support described above can be used.

[0097] The present disclosure also provides a method for deprotecting a phosphate group in an oligonucleotide, the method comprising heating an oligonucleotide obtained by reacting a nucleoside phosphoramidite represented by formula (II) above with a nucleoside represented by formula (III) above under aqueous solution conditions at a neutral to weakly basic pH so as to deprotect a group represented by formula (XI) below in the oligonucleotide: In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is —H, C1-C6 alkyl, or halogen; and m is an integer from 0 to 11.

[0098] The neutral to weakly basic pH is preferably between pH 6.0 and 11.0, more preferably between pH 6.0 and 10.0. In certain preferred embodiments, the neutral to weakly basic pH is between pH 6.5 and 9.0.

[0099] To adjust the pH, a buffering agent used to prepare a buffer solution containing, for example, phosphoric acid, bicine, boric acid, or a salt thereof, or a salt thereof can be used. Water such as sterile water can also be used. By deprotecting the oligonucleotide under aqueous solution conditions at the above pH, the oligonucleotide can be deprotected while suppressing cleavage of the main chain.

[0100] In some embodiments, the group represented by formula (XI) is a group represented by formula (XI-1): In the formula, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

[0101] The present disclosure also provides a compound represented by formula (I): In formula (I), each B is independently a nucleobase, X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 12 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 13 are each independently —OH, —SH, or a protecting group for a phosphate group, and R 13 At least one of the above is a group represented by the following formula (XI), and r is an integer of 1 to 50. In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is —H, C1-C6 alkyl, or halogen; and m is an integer from 0 to 11.

[0102] In addition, R 10 and R 12 and when both are hydrophobic tags, R 10 and R 12 may be omitted if both are linkers attached to a solid support.

[0103] The oligonucleotide represented by formula (I), in which at least some of the phosphate groups have groups represented by formula (XI), is stable under the synthesis conditions for the physiologically active molecule in the production of DEL, and can be deprotected under mild conditions.

[0104] Furthermore, the oligonucleotides disclosed herein are chemically synthesized by extending DNA by one or several nucleosides at a time, synthesizing them in a single-stranded state while each functional group remains protected, and finally deprotecting them. The synthesis typically takes no more than 10 minutes per base, significantly shortening the time per cycle compared to ligation. Furthermore, this method makes it easier to adjust the length of the DNA barcode, thereby enabling the construction of DELs with short barcodes. Because fewer bases are required to read one sequence, misreading can be reduced.

[0105] The present disclosure also provides a method for producing a DNA-encoded library, comprising the steps of: (i) providing a solid support or a soluble carrier having a first functional group reactive with a nucleoside and a second functional group reactive with a first compound; (ii) binding a first building block to the first functional group, wherein the first building block is a nucleoside monomer or an oligonucleoside; (iii) binding a first compound to the second functional group; (iv) binding a second building block to the first building block bound to the first functional group in step (ii), wherein the second building block is a nucleoside monomer or an oligonucleoside; and (v) binding a second compound to the first compound bound to the second functional group in step (iii), wherein the steps (ii) to (v) of binding the building block and binding the compound are performed alternately; the first building block bound to the first functional group is a compound represented by formula (III), and the second building block is a phosphoramidite compound represented by formula (II) or an optical isomer thereof. (In formula (III), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and q is 0 or an integer from 1 to 50. (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is -H, C1-C6 alkyl, or halogen; R 8 is -H, C1-C6 alkyl, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11.

[0106] Conventional nucleic acid synthesis using a resin solid support is difficult to scale up due to the high cost of the resin and the need for relatively large amounts of expensive reagents. To overcome these limitations of solid supports, "liquid-phase synthesis" has been applied. However, when synthesizing long oligomers, the solubility of the solid support can hinder dissolution of the reaction mixture, and complicated purification procedures are required.

[0107] "Soluble-supported liquid-phase synthesis" combines the advantages of both liquid-phase and solid-phase synthesis, overcoming the aforementioned drawbacks. In this liquid-phase synthesis, the solid support is replaced with a soluble support, e.g., a soluble anchor molecule such as polyethylene glycol (PEG) chain or other organic tag, allowing homogeneous reactions using reaction reagents and facilitating separation of the synthesized nucleic acid from the reagents after the reaction. Such soluble supports are well known.

[0108] Examples of soluble supports include soluble supports based on PEG such as methoxy PEG (Tetrahedron Lett 32:3251-3254) (hereinafter, "based on" can be replaced with "produced using"); soluble supports based on ionic liquids such as imidazolium ions as identification tags (J Org Chem 71:7907-7910); fluorine supports based on fluorous tags using fluorine-containing hydrocarbon groups as identification tags (WO 2005 / 070859 A1); soluble supports based on anchor molecules such as adamantyl methyl ester, benzyl alcohol or diphenylmethyl alcohol (or amine) having a long-chain alkyl group (WO2009014176, WO 2009014177, WO 2010104169, WO 20100249374, WO 2011078295, Org Lett 14:4514-4517; Tetrahedron Lett 53:1936-1939; WO 201202979, WO 2012165545, WO 2012165546, WO 2013089241, specific examples include, but are not limited to, AJIPHASE (registered trademark), 4-branched PEG (J. Am. Chem. Soc. 2020, 142, 16610-16621), and tetrazine derived from pentaerythritol (Eur. J. Org. Chem. 2013, 6687-6693).

[0109] In some embodiments, the soluble carrier is, for example, a compound represented by any of (XII-3-1) to (XII-3-5) below. (wherein R represents a linear or branched alkyl group having 10 to 40 carbon atoms, and X is OH or NH2).

[0110] In some embodiments, the soluble carrier is a compound represented by the following formula (XII-4) or a compound represented by formula (I) described in Japanese Patent No. 6770553. In formula (XII-4), each R independently represents a linear or branched alkyl group having 10 to 40 carbon atoms, m represents an integer of 1 to 5, and when m is 2 or greater, multiple ROs may be the same or different, and X represents a direct bond, -(CH) n - (n is an integer from 1 to 5), -(CH) n represents -NH- (n is an integer of 1 to 5), -NH-, a hydrocarbon group (the total number of carbon atoms in the carbon chain is 1 to 45) which may contain an ester bond, an amide bond, an ether bond, or a carbonyl group in the carbon chain, and A is carboxy.

[0111] Compounds represented by formula (I) described in Japanese Patent No. 6770553 (In the formula, each R independently represents a linear or branched alkyl group having 10 to 40 carbon atoms; m represents an integer of 1 to 5; when m is 2 or greater, multiple R Os may be the same or different; X represents O, S, or an amide bond; and n represents an integer of 1 to 4.)

[0112] In some embodiments, the soluble carrier is a compound represented by formula (XII-5): wherein R is CH2N3. The first and second functional groups can be attached via a linker from a soluble support and used to prepare DNA-encoded libraries.

[0113] The first compound and the second compound may be, for example, an amino acid (e.g., α-, β-, γ-, δ-, and ε-amino acids, and derivatives of natural and unnatural amino acids), a compound having one or more reactive functional groups, or a compound having protected functional groups, such as cyanuric acid chloride, etc. The first compound and the second compound may be the same or different.

[0114] The method for producing the DNA-encoded library can be carried out in one or more cycles, with one cycle consisting of a set of one nucleoside binding step and one compound binding step.

[0115] The first building block, the second building block, ... the nth building block and the first compound, the second compound, ... the nth compound are alternately bonded to the first functional group and the second functional group, respectively.

[0116] In a preferred embodiment, the method for producing a DNA-encoded library is performed in multiple cycles, for example, 2, 3, 4, 5, or more cycles, where one cycle is a set consisting of one nucleoside binding step and one compound binding step.

[0117] In a preferred embodiment, the solid support comprises an inner layer and an outer layer covering the inner layer, the DNA synthesis initiation site is located on the outer layer of the solid support, and a linker having an Fmoc group is attached to the inner and outer layers of the solid support. However, the theory is not necessarily limited to this and may vary depending on the form of the solid support used.

[0118] This double-layer structure of the solid support allows efficient binding of both oligonucleosides and compounds on the same solid support.

[0119] The abbreviations used in this specification are as follows, unless there is an obvious contradiction in the context.

[0120] 1,2-DME: 1,2-Dimethoxyethane; Aib: Amino isobutyric; AMA: Ammonium Hydroxide / 40% aqueous MethylAmine 1:1 v / v; Abs.: Absorbance; Ac2O: Anhydrous acetic acid; BME: 2-mercaptoethanol; Bn: Benzyl; Boc: tert-butoxycarbonyl; Bz: BenzoylCE: 2-cyanoethyl; COMU: Ethyl 2-cyano-2-((dimethyliminio)(morpholino)methyloxyimino)acetate hexafluorophosphate; CPG: Controlled pore glass; CuAAc: Cu-Catalyzed Azide-Alkyne Cycloaddition; DABCO: 1,4-Diazabicyclo[2.2.2]octane; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DCI: 4,5-dicyanoimidazole; DEAE: Diethylaminoethyl; DIC: N,N'-Diisopropylcarbodiimide; DIAD: Diisopropyl Azodicarboxylate; DIEA: N,N-diisopropylethylamine; DMAP: 4-Dimethylaminopyridine; DMF: N,N-dimethylformamide; DMTr: 4,4´-Dimethoxytrityl; ESI: Electrospray ionization Fmoc: Fluorenylmethyloxycarbonyl; Fmoc-AEEA-OH: {2-[2-(FMOC-amino)ethoxy]ethoxy}acetic Acid; HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-Oxide Hexafluorophosphate;HPLC: High-performance liquid chromatography; HRMS: High Resolution Mass Spectrometer iBu: Isobutyryl; MALDI: Matrix-Assisted Laser Desorption / Ionization MS: Mass; NAPM: N-acetylpyrrolidin-2-ylmethyl; NGS: Next generation sequencer; NFW: Nuclease-free water; NMP: N-methylpyrrolidone; NMR: Nuclear magnetic resonance; Ns: 2-Nitrobenzenesulfonyl; Oxyma: Ethyl cyano(hydroxyimino)acetate; PAGE: Polyacrylamide gel electrophoresis; PBS: Phosphate-buffered saline; Phe: Phenyl; PPh3: Triphenylphosphine; PCR: Polymerase chain reaction; PyBOP: 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate; Pya: Methyl-pyrrolidine amidine; Sar: Sarcosine; SPOS: Solid-phase oligonucleotide synthesis; SPPS: Solid-phase peptide synthesis; Su: Succinimide; TBDPS: tert-Butyldiphenylchlorosilane; TCA: Trichloroacetic acid; TEAA: Triethyl ammonium acetate; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; TIPS: Triisopropylsilane; TMOF: Trimethyl Orthoformate; TOF: Time of flight;TPS: 2,4,6-Triisopropylbenzenesulfonyl;

[0121] Test Example 1: Comprehensive Analysis of the Chemical Durability of Protected DNA and Search for Chemically Stable Protecting Groups for DEL Construction in Organic Solvents. 1. Materials and Methods. 1.1 General Information. Reagents were purchased from standard suppliers and used without further purification. Oligonucleotides were synthesized using synthetic phosphoramidites on an automated DNA synthesizer (M-2-MX, Nippon Techno Service Co., Ltd.). The synthesized oligonucleotides were analyzed by Prominence HPLC (Shimadzu Corporation) using a COSMOSIL 5C18-MS-II packed column (Nacalai Tesque, 4.6 x 150 mm, 5 μm). The oligonucleotide concentrations were determined by UV absorbance at 260 nm measured with a NanoDrop 1000 spectrophotometer (ThermoFisher Scientific). Preparative HPLC was performed on a Prominence HPLC system (Shimadzu Corporation) using a COSMOSIL 5C18-AR-II packed column (Nacalai Tesque, 20 x 250 mm, 5 μm). Mass spectrometry to evaluate chemical resistance was performed using MALDI-TOF-MS (Bruker Daltonics), ESI-MS using LC-MS with an ACQUITY UPLC H-Class / SQD2 (Waters) using an ACQUITY UPLC (registered trademark) BEH C18 1.7 mm Column (Waters, 2.1 ID x 50 mm), or ex-expression CMS (Advion Interchim Scientific). Nuclear magnetic resonance (NMR) spectra were measured using a JEOL ECS400 ( 1 H 400 MHz, 13 C 101 MHz, 31 P 160 MHz, 19 F 376 MHz) spectrometer. 1 H NMR chemical shifts are for CHCl in CDCl 3 (δ 7.26 ppm) or CHD2CN in CD3CN (δ 1.94 ppm). 13C NMR is CDC l3 CDCl in 3 (δ 77.16 ppm) or CHD2CN in CD3CN (δ 1.32 and 118.26 ppm). 31 P NMR was analyzed using H3PO4 as an external standard. Chemical shift, multiplicity (s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, sept. = septet, m = multiplet), coupling constant (Hz), and integration were recorded. HRMS data were obtained using a microTOF II (Bruker Daltonics) or ACQUITY TM The Premier system (Waters Corp., Milford, MA, USA) was used as a Waters Xevo TM An ACQUITY Premier Peptide BEH C18 column (130Å, 1.7 μm, 2.1 x 100 mm) was used coupled to a G3 QTof mass spectrometer and analyzed by MassLynx. TM Controlled by 4.2.

[0122] 1.2 Evaluation of Chemical Resistance of Protected DNA After filtering 10 μL of the sample, analysis was performed by analytical HPLC. Reference samples were prepared by diluting NMP stock solutions of various compounds with the solvent used in each evaluation. Ns-l-Ala-OBn and Hl-Ala-OBn were prepared as previously reported (Sci. Rep., 2020, 10, 1-15; Chem. - A Eur. J., 2021, 27, 5790-5795). Ns-N-Me-l-Ala-OBn was synthesized and used (see diagram below). These l-alanine derivatives were prepared as stock solutions in NMP.

[0123] dA Pya , dC Pya , and dG Bn PyaFor HPLC analysis, except for ( ), a binary solvent system consisting of water and acetonitrile as solvents A and B, respectively, was employed. The column was developed with the following gradient: 25% B for 5 min, a linear gradient from 25% B to 100% B for 30 min, 100% B for 5 min, a linear gradient from 100% B to 25% B for 1 min, and 25% B for 10 min. dA Pya , dC Pya , dG Bn Pya For the evaluation, a binary solvent system consisting of 100 mM TEAA pH 7.0 as solvent A and acetonitrile as solvent B was used. The column was developed with the following gradient: 25% B for 5 min, a linear gradient from 25% B to 100% B for 30 min, 100% B for 15 min, a linear gradient from 100% B to 25% B for 1 min, and 25% B for 10 min.

[0124] Anthracene was used as an internal standard, and the absorbance at 376 nm was monitored and evaluated under the following conditions: Fmoc deprotection, amide bond formation, reductive amination with NaBH4, Ns protection, and Ns deprotection.

[0125] To calculate the residual rate, the absorbance was monitored at the following wavelengths: DMTrO-dT-OTBDPS and protected dinucleotide: 265 nm; DMTrO-dT Bn -OTBDPS, DMTrO-dA Bz -OTBDPS, DMTrO-dA MeBz -OTBDPS: 280 nm; DMTrO-dA Pya -OTBDPS: 305 nm; DMTrO-dG iBu -OTBDPS: 254 nm; DMTrO-dG Bn iBu -OTBDPS: 270nm; DMTrO-dG Bn Pya -OTBDPS: 295 nm; DMTrO-dC Bz -OTBDPS, DMTrO-dC MeBz -OTfBDPS, DMTrO-dC Pya -OTBDPS: 310nm.

[0126] Evaluations of the following Fmoc deprotection steps 1 and 2, amide bond formation steps 1, 2, and 3, Ns protection, and Ns deprotection were carried out by mixing various reaction solutions in a 1:1 ratio with 4 mM anthracene in NMP, with or without 4 mM protected DNA. After incubation at the indicated temperature and time, the samples were diluted 10-fold with various solvents to prepare samples. When the peak of the residue in the reaction solution overlapped with the peak of the target compound, the area corresponding to the overlapping peak in the blank sample was subtracted to calculate the value.

[0127] Fmoc deprotection 1: Piperidine Reaction solution: 40% piperidine NMP solution 30μL (v / v), temperature: 25℃, time, 15 minutes, solvent: MeCN / 2 M TEAA (pH 5.5) (7:3 v / v) Fmoc deprotection 2: DBU reaction solution: 4% DBU NMP solution 30μL (v / v), temperature: 25 °C, time, 10 min, solvent: MeCN / 2 M TEAA pH 6.0 (7:3, v / v) amide bond formation 1: COMU Reaction solution: 0.4 M Fmoc-Ala-OH, 0.4 M COMU, and 0.8 M DIEA 30 μL of NMP solution, temperature: 25 °C, time: 1 h, solvent: MeCN / 100 mM TEAA pH 7.0 (7:3, v / v).

[0128] Amide bond formation 2: HATU reaction solution: 0.3 M benzoic acid, 0.3 M HATU, 0.6 M DIEA in NMP, temperature: 60°C, time: 3 hours, solvent: diluted with MeCN / 100 mM TEAA pH 7.0 (7 / 3, v / v).

[0129] Amide bond formation 3: DIC / Oxyma Reaction solution: 0.4 M Fmoc-l-Ala-OH, 0.8 M DIC, 0.4 M Oxyma, and 0.02 M DIEA in NMP, temperature: 60 °C, time: 1 hour, solvent: MeCN / 100 mM TEAA pH 7.0 (7:3, v / v).

[0130] Reductive amination 1: 10 μL of an 8 mM anthracene solution in MeCN containing 12% NaBH (v / v) (with or without 8 mM protected DNA) was mixed with 20 μL of a 45 mM benzaldehyde and 4.5 mM L-Ala-OBn solution in MeCN containing 0.45% NMP (v / v). After incubation at 25 °C for 1 h, 10 μL of a 200 mM NaBH solution in MeOH was added and further incubated at 25 °C for 0.5 h. The reaction mixture was then diluted with 360 μL of MeCN / 100 mM TEAA pH 7.0 (7:3, v / v).

[0131] Reductive amination 2: NaBH(OAc)3. 20 μL of TMOF containing 1% AcOH and 6% NMP (v / v) (with or without 6 mM protected DNA) was mixed with 10 μL of a 120 mM benzaldehyde and 12 mM L-Ala-OBn solution in TMOF containing 1% AcOH and 1.2% NMP (v / v). After 1 h of incubation at 25 °C, 30 μL of a 100 mM NaBH(OAc)3 suspension in TMOF containing 1% AcOH was added and further incubated for 0.5 h at 25 °C. The reaction mixture was then diluted with 480 μL of MeCN / 100 mM TEAA pH 7.0 (7:3, v / v) and 60 μL of NMP.

[0132] Ns protection: Reaction solution: 0.2 M NsCl, 0.5 M 2,4,6-collidine, 50 mM L-Ala-OBn in NMP. Temperature: 25°C, Time: 15 min, Solvent: MeCN / 100 mM TEAA pH 7.0 (7:3, v / v). Recovery was calculated with and without an internal standard, and the lower value was used.

[0133] Mitsunobu alkylation: 10 μL of a 1,4-dioxane solution containing 6% NMP (v / v) with or without 6 mM protected DNA was mixed with 10 μL of a 1,4-dioxane solution of 1.2 M MeOH, 0.6 M PPh3, 150 mM N-Ns-L-Ala-OBn, and 10 μL of a 1,4-dioxane solution of 1.2% NMP (v / v), 0.6 M DIAD. After incubation at 60 °C for 3 h, the reaction mixture was diluted with 270 μL of MeCN / 100 mM TEAA pH 7.0 (7:3, v / v).

[0134] Ns deprotection reaction solution: 0.1 M Ns-N-Me-l-Ala-OBn, 0.8 M BME, 0.4 mM DBU in NMP, temperature: 25°C, time: 15 min, solvent: MeCN / 100 mM TEAA pH 7.0 (7:3, v / v).

[0135] Deprotection of Bn groups 1: 2.5 μL of 100 mM Pd(OAc)2 / NaBH4 protected DNA, 1.2 μL of 1 M Pd(OAc)2 solution in DMA, 6 μL of 2 M NaBH4 solution in NMP, and 112.8 μL of D-PBS (1.47 mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4, 137 mM NaCl, pH 7.4) were combined and incubated at room temperature for 5 hours.

[0136] Deprotection of Bn Group 2: Borate Buffer A 2 mM solution of protected DNA in 250 mM borate buffer pH 8.5 containing 1% NMP (v / v) was incubated at 90° C. for 24 hours.

[0137] 1.3 Oligonucleotide Synthesis Using NAPM Phosphoramidites Oligonucleotide synthesis was performed on Ac-dC-CPG resin using an automated DNA synthesizer according to standard procedures. Each phosphoramidite was dissolved in anhydrous MeCN to prepare a 0.1 M solution. DCI was used as the activator and dissolved in anhydrous MeCN to prepare a 0.25 M solution. After all coupling steps were completed, the terminal DMTr protecting group was removed using standard procedures. The beads were then treated according to the deprotection procedure described below.

[0138] The NAPM-protected oligonucleotides were incubated in AMA at 65°C for 15 minutes to deprotect the acetyl group at cytosine and cleave them from the resin, after which the solution was evaporated under reduced pressure.

[0139] Subsequently, the residue was incubated in 250 mM borate buffer (pH 8.5), 100 mM bicine buffer, or 100 mM phosphate buffer at 25, 45, 60, or 90°C for 30, 90, or 180 minutes, respectively.

[0140] The products were analyzed by HPLC, and the absorbance at 260 nm was monitored. The HPLC gradient was set as follows: Solvent A was 100 mM TEAA (pH 7.0) and Solvent B was MeCN.

[0141] The run consisted of 1% B for 5 minutes, a linear gradient from 1% B to 30% B over 35 minutes, a linear gradient from 30% B to 99% B over 1 minute, a 5-minute hold at 99% B, a linear gradient from 99% B to 1% B over 1 minute, and a 3-minute hold at 1% B. For samples deprotected with aqueous solvents, water-substituted samples were used for MALDI-TOF MS analysis.

[0142] Similar experiments were also performed on double-layered beads 1, which are modified polystyrene-PEG resins intended for actual DEL construction. 11-mer oligothymidine was synthesized on double-layered beads 1 using an automated DNA synthesizer according to the method described in Table 1.

[0143] Synthesis of double-layered beads 1: TentaGel Microspheres (30 μm, 265 mg, 0.3 mmol / g) were swollen in water for 4 days. After removing the water, the beads were washed with CHCl / EtO (55 / 45). 3.9 mL of a solution of 0.2 mM Boc-Sar-OSu, 2 mM Fmoc-OSu, and 6.7 mM DIEA in CHCl / EtO (55 / 45) was added to the beads. After the reaction, the beads were washed with CHCl / EtO (1:1), CHCl, and DMF. 3.0 mL of a solution of 0.2 M Fmoc-OSu and 0.4 M DIEA in CHCl / EtO (55 / 45) was added. After 2 h, 3.5 mL of a solution of 0.2 M AcO and 0.4 M DIEA in DMF was added and the mixture was shaken for 12 h. After removing the Boc protecting group by acid treatment, the beads were added to a solution of 0.1 M DMTrO-dT-succinic acid triethylammonium salt, 0.1 M PyBOP, and 0.2 M DIEA in 1.65 mL of anhydrous DMF, shaken for 2 h, and washed with DMF and CHCl. ​​The beads were dried in vacuo and stored at -30°C. All operations were performed at room temperature.

[0144] After DNA synthesis, a small amount of resin was suspended in various aqueous solvents or NFW and incubated for 12 hours at 90°C. These samples were analyzed using MALDI-TOF MS.

[0145] 1.4 Chemical Synthesis General Procedure A for the Preparation of Alkyloxy N,N,N',N'-Tetraisopropylphosphorodiamidite Reagents A round-bottom flask containing a suspension of bis(diisopropylamino)chlorophosphine (1.0 eq) in anhydrous EtO (0.8 M) was purged with Ar and cooled to 0 °C. To the suspension was added dropwise a solution of alcohol (1.1 eq) and EtN (3.0 eq) in anhydrous EtO (1.6 M). The mixture was allowed to warm to room temperature and stirred for 5 h. After that, the mixture was filtered through Celite®, and the filtrate was evaporated under reduced pressure and dried in vacuo to give the desired product.

[0146] General Procedure B for Deoxynucleoside-Phosphoramidite Coupling: 1H-Tetrazolediisopropylamine salt was prepared as previously described ( Mol. Pharm., 2006, 3, 161-173 ). A round-bottom flask containing a suspension of N,N,N′,N′-tetraisopropylphosphorodiamidite reagent and 3A molecular sieves in anhydrous CHCl (0.2 M) was purged with Ar, and the DMTr-protected deoxynucleoside and 1H-tetrazolediisopropylammonium salt were added. After stirring at room temperature for the indicated time, the mixture was filtered through Celite, washed with EtN in CHCl and heptane, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography. Residual solvent was removed by azeotropy with benzene. The purified product was then lyophilized in benzene to give the desired crude product.

[0147] General Procedure C for the Preparation of Protected Dinucleotides: 3'-O-[(1,1-dimethylethyl)diphenylsilyl]thymidine was prepared as previously described (Biochemistry, 2004, 43, 6167-6181). A round-bottom flask containing a suspension of 3'-O-[(1,1-dimethylethyl)diphenylsilyl]thymidine and 3A molecular sieves in anhydrous MeCN (0.066 M) was stirred at room temperature for 1 h. After stirring, the DMTr-protected deoxynucleoside phosphoramidite and DCI were added to the mixture. After stirring at room temperature for 0.5 h, the mixture was cooled to 0 °C and a solution of iodine and pyridine in THF / water (5:1, v / v, iodine was 0.26 M) was slowly added. After stirring for an additional 0.5 h, the mixture was filtered, washed with a small amount of MeCN, cooled to 0 °C, and washed with a small amount of 6% NaSO. 3 aq. was added. After 0.5 h, EtOAc was added to the vigorously stirred mixture, and the mixture was stirred at 5 °C for 0.5 h. The resulting two layers were then separated, and the aqueous layer was extracted with EtOAc. The combined organic layer was washed six times with 5% NaHCO3 aq / DMF (9:1, v / v), washed with saturated brine, dried over anhydrous MgSO4, filtered, and the filtrate was evaporated under reduced pressure to give the crude product. The crude product was purified by preparative HPLC.

[0148] General Procedure D for Silylation: TBDPSCl was added to a DMTr-protected deoxynucleotide and imidazole in anhydrous DMF. After stirring the mixture at room temperature for at least 2 hours, the mixture was diluted with EtOAc and water. The two layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was evaporated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography.

[0149] General Procedure E for Methylation of DNA Bases: A round-bottom flask containing a solution of DMTr-protected deoxynucleotide and DBU in anhydrous MeCN (0.15 M) was cooled to 0 °C. The mixture was treated dropwise with MeI, and the atmosphere was purged with nitrogen. The mixture was allowed to warm to room temperature and stirred for 22.5 h. The mixture was then concentrated. The residue was purified by silica gel column chromatography to give the desired product.

[0150] General Procedure F for TPS Protection: A round-bottom flask containing a solution of the protected nucleoside in anhydrous CHCl (0.3 M) was cooled to 0 °C, and DMAP, EtN, and TPSCl were added. The atmosphere was then purged with nitrogen. The mixture was allowed to warm to room temperature, and after the indicated time, the mixture was diluted with CHCl, washed with saturated brine, dried over NaSO, filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the product.

[0151] General Procedure G for Bn Protection of DNA Bases: DMAP, DABCO, and BnOH were added to a round-bottom flask containing a suspension of TPS-protected nucleoside and 3A molecular sieves in 1,2-DME (0.07 M). After stirring at room temperature for 0.5 h, DBU was added to the mixture. After the indicated time, the mixture was diluted with CHCl3 and water, extracted with CHCl3, washed with brine, dried over Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the desired product. DMTrO-dA Bz -OTBDPS (1), DMTrO-dC Bz -OTBDPS (2), DMTrO-dG iBu-OTBDPS (3) and DMTrO-dT-OTBDPS (4) were prepared as previously reported (Biochemistry, 2004, 43, 6167-6181.).

[0152] Synthesis of Compounds 5-9 Compounds 5-9 in Table A were prepared according to General Procedure A above.

[0153] Synthesis of Compounds 10-14 Compounds 10-14 in Table B (DMTrO-dT-Et phosphoramidite (10), DMTrO-dT-Pr phosphoramidite (11), DMTrO-dT-iButyl phosphoramidite (12), DMTrO-dT-CyP phosphoramidite (13), DMTrO-dT-NAPM phosphoramidite (14)) were prepared according to General Procedure B above.

[0154]

[0155] Analytical data for DMTrO-dT-iButyl phosphoramidite (12) 1 H NMR (400 MHz, CDCl3): δ0.82 (dd, J = 6.8, 1.2 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H), 1.01-1.23 (m, 12H), 1.40 (dd, J = 5.2, 0.8 Hz, 3H), 1.64-1.91 (m, 1H), 2.24-2.35 (m, 1H), 2.43-2.58 (m, 1H), 3.13-3.68 (m, 6H), 3.75-3.82 (s, 6H), 4.18 (dd, J = 20.4, 2 Hz, 1H), 4.60-4.70 (m,1H), 6.38-6.45 (m, 1H), 6.78-6.86 (m, 4H), 7.20-7.33 (m, 7H), 7.37-7.43 (m, 2H), 7.63 (dd, J = 16.4, 1.2 Hz, 3H), 8.05-8.15 (brs, 1H). 13C NMR (101 MHz, CDCl3) δ163.76, 158.84, 150.32, 144.44, 135.90, 135.56, 135.45, 130.28, 128.35, 128.08, 127.25, 113.36, 111.26, 87.01, 84.95, 84.87, 77.48, 77.16, 76.84, 70.27, 63.52, 63.40, 55.36, 43.22, 43.15, 43.10, 43.03, 29.84, 24.66, 19.34, 19.30, 11.81. 31 P NMR (160 MHz, CDCl3): δ147.0, 147.3.HRMS (ESI) m / z calcd for C 41 H 54 N3NaO8P [M+Na] + : 770.3541, found 770.3537.

[0156] Analysis of DMTrO-dT-CyPホスホロアミダイト(13) 1 H NMR (400 MHz, CDCl3): δ1.05 (d, J = 6.8 Hz, 3H), 1.11-1.76 (m, 20H), 1.95-2.23 (m, 1H), 2.24-2.35 (m, 1H), 2.42-2.59 (m, 1H), 3.25-3.64 (m, 6H), 3.79 (s, 6H), 4.17 (dd, J = 23.2, 2 Hz, 1H), 4.59-4.70 (m, 1H), 6.37-6.45 (m, 1H), 6.78-6.87 (m, 4H), 7.20-7.33 (m, 7H), 7.37-7.43 (m, 2H), 7.63 (dd, J = 15.2, 1.2 Hz, 1H), 7.94 (brs, 1H). 13C NMR (101 MHz, CDCl3) δ163.84, 158.82, 150.37, 144.47, 144.44, 135.95, 135.86, 135.60, 135.56, 135.54, 135.45, 130.38, 130.30, 130.14, 128.19, 128.03, 127.94, 127.32, 127.11, 113.39, 113.31, 111.22, 86.99, 86.08, 85.60, 84.98, 84.90, 84.83, 73.55, 73.38, 73.33, 67.97, 67.82, 67.67, 63.41, 55.35, 43.18, 43.12, 43.06, 40.85, 40.48, 40.38, 29.46, 25.62, 25.55, 25.15, 24.67, 11.87, 11.75. 31 P NMR (160 MHz, CDCl3): δ 146.8, 147.1. HRMS (ESI) m / zcalcd for C 43 H 56 N3NaO8P [M+Na] + : 796.3697, found 796.3693.DMTrO-dT-NAPMホスホロアミダイト(14)のANALYSISデータ 1 H NMR (400 MHz, CDCl3): δ 0.99-1.07 (m, 4H), 1.09-1.21 (m, 8H), 1.36-1.44 (m, 3H), 1.74-2.16 (m, 7H), 2.24-2.38 (m, 1H), 2.40-2.61 (m, 1H), 3.26-3.82 (m, 14H), 3.91-4.27 (m, 2H), 4.57-4.70 (m, 1H), 6.36-6.46 (m, 1H), 6.76-6.87 (m, 4H), 7.20-7.32 (m, 7H), 7.35-7.42 (m, 2H), 7.58-7.66 (m, 1H), 8.04 (brs, 1H). 13C NMR (101 MHz, CDCl3): δ 169.61, 169.53, 163.93, 158.84, 158.80, 150.46, 144.41, 144.37, 144.31, 135.86, 135.65, 135.50, 135.41, 135.32, 130.21, 128.28, 128.06, 127.26, 127.20, 113.33, 111.40, 111.35, 111.28, 111.23, 87.04, 86.97, 86.02, 85.63, 85.57, 84.93, 84.80, 74.02, 73.82, 73.64, 73.47, 64.18, 64.04, 63.46, 63.22, 63.08, 62.87, 62.73, 58.74, 57.31, 57.23, 55.34, 48.41, 48.21, 46.00, 45.81, 43.30, 43.18, 43.06, 40.23, 28.68, 27.58, 27.46, 24.62, 24.55, 24.28, 24.10, 23.06, 22.99, 22.43, 22.32, 22.05, 21.97, 11.79. 31 P NMR (160 MHz, CDCl3): δ147.5, 147.7, 148.2, 148.4. HRMS (ESI) m / z calcd for C 44 H 57 N4NaO9P [M+Na] + : 839.3755, found 839.3739. Synthesis of Compounds 15-21. Following General Procedure C above, the protected dinucleotides 15-21 in Table C (CE-protected dinucleotide (15), Me-protected dinucleotide (16), Et-protected dinucleotide (17), Pr-protected dinucleotide (18), iButyl-protected dinucleotide (19), CyP-protected dinucleotide (20), and NAPM-protected dinucleotide (21) were prepared.

[0157] Synthesis of compounds 22-24 DMTrO-dA Pya -OH(22), DMTrO-dC Pya -OH(23), DMTrO-dG Pya-OH(24) was prepared as previously reported (J. Am. Chem. Soc., 1986, 108, 2040-2048).

[0158] Synthesis of Compounds 25-35: Protected nucleoside compounds 25-35 (DMTrO-dA) of Table D can be prepared according to any of the general procedures D-G above. Pya -OTBDPS(25), DMTrO-dA MeBz -OTBDPS(26), DMTrO-dC Pya -OTBDPS(27), DMTrO-dC MeBz -OTBDPS(28), DMTrO-dG TPS iBu -OTBDPS(29), DMTrO-dG Bn iBu -OTBDPS(30), DMTrO-dG Pya -OTBDPS(31), DMTrO-dG TPS Pya -OTBDPS(32), DMTrO-dG Bn Pya -OTBDPS(33), DMTrO-dT TPS -OTBDPS(34), DMTrO-dT Bn -OTBDPS(35)) was manufactured.

[0159] DMTrO-dA MeBz -OTBDPS(26) identification data 1 H NMR (400 MHz, CD3CN): δ 1.03 (s, 9H), 2.38-2.49 (m, 1H), 2.38-2.49 (m, 1H), 2.60-2.71 (m, 1H), 2.93-3.01 (m, 1H), 3.07-3.14 (m ,1H), 3.64 (s, 3H), 3.72 (s, 6H), 4.15-4.26 (m, 1H), 4.65-4.75 (m, 1H), 6.39 (t, J = 6.4 Hz, 1H), 6.66-6.73 (m, 4H), 6.98-7.49 (m, 20H), 7.56-7.67 (m, 4H), 7.96 (s, 1H), 8.29 (s, 1H). Synthesis of N-Me-N-Ns-l-Ala-OBn (36) The hydroxychloride salt of N-Me-l-Ala-OBn was prepared as previously reported (Bull. Chem. Soc. Jpn., 2022, 95, 621-627). A round-bottom flask containing a solution of the hydroxychloride salt of N-Me-l-Ala-OBn (273 mg, 1.19 mmol, 1.0 eq) in anhydrous THF (2.4 mL) was cooled to 0 °C. The solution was then treated dropwise with EtN (500 μL, 3.59 mmol, 3.0 eq). After stirring for 15 min, a solution of NsCl (387 mg, 1.75 mmol, 1.5 eq) in anhydrous THF (1.5 mL) was slowly added at 0 °C and allowed to warm to room temperature. After 25 h, the mixture was diluted with EtOAc and saturated aqueous NH4Cl. The two layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated brine, dried over Na2SO4, filtered, and evaporated. The residue was purified by silica gel column chromatography to give the desired product (349 mg, 78%) as a colorless sticky oil. 1 H NMR, 13 Identification was achieved by C NMR and HRMS.

[0160] HRMS (ESI) m / z calculation for C 54 H 56 N2O7Si [M+H] + : 379.0963, found 379.0953.TBSO-dT Bn Synthesis of -OTBS (37) 3´,5´-bis-O-TBS-thymidine was prepared as previously reported (Nucleic Acids Res., 2013, 41, 6774-6781). Following the general procedure G, 3´,5´-bis-O-TBS-thymidine (204 mg, 0.276 μmol, 1.0 eq), DABCO (64.4 mg, 574 μmol, 2.0 eq), BnOH (129 μL, 1.24 mmol, 4.5 eq), and DBU (62 μmol, 415 μmol, 1.5 eq) were used to obtain the desired compound (127 mg, 53%) as a white solid. The desired product was 1 Identification was achieved by 1 H NMR.

[0161] dT Bn Synthesis of (38) TBSO-dT Bn A round-bottom flask containing a solution of -OTBS (75.4 mg, 135 μmol, 1.0 eq) in anhydrous THF (0.7 mL) was cooled to 0 °C, and AcOH (23 μL, 40.2 μmol, 3.0 eq) and 1 M TBAF in THF (40.2 μL, 40.2 μmol, 3.0 eq) were added. The mixture was allowed to warm to room temperature and stirred for 3 days. The mixture was then diluted with EtOAc and water. The two layers were separated, and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered, and evaporated. The residue was purified by silica gel column chromatography to give dT Bn (29.1 mg, 44%) was obtained as a colorless sticky oil. 1 Identification was achieved by 1 H NMR.

[0162] Synthesis of compound 39 　(S)-2,2,2-Trifluoro-1-(2-(hydroxymethyl)pyrrolidin-1-yl)ethan-1-one was prepared as previously described (WO2013 / 077446). A round-bottom flask containing (S)-2,2,2-trifluoro-1-(2-(hydroxymethyl)pyrrolidin-1-yl)ethan-1-one (354 mg, 1.80 mmol, 1.0 eq) and a solution of EtN (501 μL, 3.59 mmol, 2.0 eq) in EtO (4.5 mL) was cooled to 0 °C and purged with argon. Bis(diisopropylamino)chlorophosphine (726 mg, 2.72 mmol, 1.5 eq) was then added, the mixture was purged with argon again, and stirred for 2.5 h. The mixture was filtered and washed with a mixture of EtO and EtN and heptane. The solvent was reduced by evaporation under reduced pressure so as not to completely evaporate the heptane in the filtrate, and the resulting residue was purified by silica gel column chromatography to give compound 39 (447 mg, 50%) as a colorless oil.

[0163] 1 H NMR (400 MHz, CDCl3): δ 1.10-1.24 (m, 24H), 1.81-2.19 (m, 4H),3.34-3.79 (m, 8H),2.88 (brs, 1H), 4.25-4.35 (m, 1H). 19 F NMR (376 MHz, CDCl): δ -70.4, -72.3. Synthesis of DMTrO-dT-NAPMCF phosphoramidite (40) According to general procedure B, compound 39 (447 mg, 1.05 mmol, 1.4 eq), DMTrO-dT-OH (411 mg, 0.755 mmol, 1.0 eq), and 1H-tetrazolediisopropylammonium salt (462.4 mg, 2.70 mmol, 3.6 eq) were mixed and stirred at room temperature for 6 hours. The reaction solution was then diluted with ethyl acetate and washed with saturated brine. The aqueous layer was further extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography to give compound 40 (621.3 mg) as a white solid.

[0164] 19 F NMR (376 MHz, CDCl): δ -70.3, -70.4, -72.4, -72.5. Synthesis of NAPMCF3-protected dinucleotide (41) Following general procedure C, 3'-O-[(1,1-dimethylethyl)diphenylsilyl]thymidine (103.5 mg, 0.22 mmol, 1.0 eq), compound 40 (242 mg, 0.28 mmol, 1.3 eq), and DCI (70.8 mg, 0.60 mmol, 2.7 eq) gave compound 41 (119 mg, 44%) as a white solid. 1 H NMR, 13 C NMR, 31 P NMR, 19 Identification was carried out by F NMR and HRMS.

[0165] HRMS (ESI) m / z calculation for C 64 H 71 F3N5O 15 PSi [M+Na] + : 1288.4298, found 1288.4333.DMTrO-dT Bn Synthesis of -NAPM phosphoramidite (42) DMTrO-dT BnThe -OH was prepared according to the method described (Bioorganic & Medicinal Chemistry 1995, 3, 101-8.). Compound 9 (2.29 g, 6.13 mmol, 1.3 eq) was prepared according to general procedure B using DMTrO-dT. Bn Using —OH (2.99 g, 4.71 mmol, 1.0 eq), and 1H-tetrazolediisopropylammonium salt (2.85 g, 16.6 mmol, 3.5 eq), compound 42 (3.77 g) was obtained as a white solid.

[0166] 1 H NMR (400 MHz, CDCl3): δ 0.98-1.19 (m, 12H), 1.46-1.54 (m, 3H), 1.68-2.14 (m, 7H), 2.19-2.33 (m, 1H), 2.61-2.75 (m, 1H), 3.27-3.79 (m, 14H), 3.91-4.27 (m, 2H), 4.53-4.67 (m, 1H), 5.39-5.48 (m, 2H), 6.37-6.46 (m, 1H), 6.80-6.85 (m, 4H), 7.18-7.44 (m, 14H) 7.86-7.95 (m, 1H).2. Results and discussion 2.1 Comprehensive analysis of the chemical durability of protected DNA under conditions for bioactive ligand synthesis in organic solvents. In the phosphoramidite method, DNA is extended one base at a time while protecting various highly reactive functional groups within the DNA itself. Therefore, DNA barcodes constructed by the recording-by-synthesis method exhibit higher chemical stability than DNA barcodes constructed from unprotected DNA, suggesting that general conditions for the synthesis of bioactive molecules using organic solvents can be applied. In this study, we focused on the Fmoc method, the most general method for solid-phase combinatorial chemistry, as the synthesis conditions for bioactive molecules to construct a compound library.

[0167] The Fmoc method uses strong organic bases such as piperidine and DBU for the deprotection of the key Fmoc group. However, DNA protecting groups are generally deprotected under such basic conditions or in the presence of nucleophiles. In particular, the 2-cyanoethyl group, a phosphate protecting group commonly used in the phosphoramidite method, is unstable under such basic conditions and rapidly deprotects (Figure 2(a)). Therefore, in the early recording-by-synthesis method developed by Janda et al. and Gallop et al. (J. Am. Chem. Soc., 1993, 115, 9812-9813; Proc. Natl. Acad. Sci. USA, 1993, 90, 10700-10704), a methyl group, known to be more stable under basic conditions, was used as the phosphate protecting group (Figure 2(b)). However, according to previous reports (Nucleic Acids Res., 1985, 13, 573-584, Tetrahedron Lett., 1980, 21, 861-864), the methyl group is also not completely stable under the conditions for synthesizing biologically active molecules using the general Fmoc method. Therefore, we first attempted to closely examine the chemical durability of protected DNA under the conditions for synthesizing biologically active molecules using the general Fmoc method.

[0168] Using protected nucleosides or dinucleotides, we evaluated the chemical durability of each cycle of DEL construction under the synthesis conditions of biologically active molecules in a solution-phase system (Figure 3). Common acyl protecting groups, such as the Bz group and the isobutyryl iBu group, were employed. In addition, the most commonly used 2-cyanoethyl (CE) group was selected as the phosphate protecting group and evaluated using dinucleotides. Furthermore, for all DNA protectors, the hydroxyl group at position 3 was protected with a tert-butyldiphenylsilyl (TBDPS) group, and the hydroxyl group at position 5 was protected with a dimethoxytrityl (DMTr) group. Therefore, the durability of the DMTr group was evaluated based on the results of the T protector.

[0169] The evaluation results are shown in Table 2, which lists the survival rates of various protected DNAs. Under the experimental conditions of this study, it was revealed that DMTr-protected hydroxyl groups, acyl-protected DNA bases, and unprotected thymine bases were stable under amide-forming conditions, including the general Fmoc deprotection conditions used in the Fmoc method, and heating conditions using condensing agents such as COMU, HATU, and DIC / Oxyma.

[0170] On the other hand, as expected, it was experimentally demonstrated that the 2-cyanoethyl group, which is the protecting group for phosphate, is rapidly deprotected under various basic conditions or in the presence of nucleophiles.

[0171]

[0172] 2.2. Search for a chemically stable and mild phosphate protecting group Based on the experimental results in 2.1, we attempted to search for a protecting group for DNA phosphate that is stable under the synthesis conditions of biologically active molecules and can be deprotected under mild conditions. The methyl group, the phosphate protecting group used in the early Recording-by-Synthesis method, is thought to be easily deprotected because its small structure makes it easy for nucleophiles to access the protecting group. Therefore, we attempted to improve the stability of the protecting group under the synthesis conditions of biologically active molecules by expanding the chemical structure starting from this smallest unit, the methyl group.

[0173] Prior to the actual evaluation, various dinucleotides with protected phosphate groups were synthesized in a liquid phase system (Scheme 1).

[0174]

[0175] Next, using the synthesized dinucleotides 16-20, we evaluated the chemical durability of the methyl (Me), ethyl (Et), propyl (Pr), isobutyl (iButyl), and cyclopentyl (CyP) groups used as protecting groups for the phosphate group under Fmoc deprotection conditions (Table 3). As a result, it was experimentally demonstrated that the alkyl groups, which are the next largest after the methyl group, and larger than the ethyl group, are sufficiently stable under typical Fmoc deprotection conditions.

[0176] On the other hand, when we attempted deprotection by heating in aqueous ammonia using a 25-mer oligothymidine synthesized on a CPG resin using compound 10, multiple peaks were observed on the HPLC chromatogram (not shown). This suggests that alkyl groups with carbon numbers greater than or equal to ethyl cannot be deprotected under typical conditions using aqueous ammonia. This result is thought to be due to the slow progress of the deprotection reaction of phosphate triesters with alkyl groups greater than or equal to ethyl because they are too chemically stable, and at the same time, competition exists between cleavage of the main chain triester by nucleophiles and hydrolysis.

[0177] Therefore, we focused on the NAPM group, whose alkyl group skeleton is derived from N-acetylprolinol, similar to that of the CyP group. Because the NAPM group has a similar structure to the CyP protecting group, it is expected to be bulky and have a more rigid structure than linear groups. Furthermore, it is thought that deprotection occurs via an intramolecular cyclization reaction caused by nucleophilic attack from the electron on the oxygen atom of the N-acetyl group on an intramolecular carbon (J. Am. Chem. Soc., 2002, 124, 4962-4963) (see reaction below). This suggests that deprotection can be performed under milder conditions. First, we attempted to examine the stability of the NAPM group under physiologically active conditions. dT amidite compound 14, which has a NAPM group as a phosphorus protecting group, and dinucleotide compound 21 were synthesized using the same synthetic method as in Scheme 1, according to the synthetic scheme shown in Scheme 2. 1 H, 13 C, and 31 Identification was performed by P NMR.

[0178] Next, we evaluated the chemical durability of compound 21 under the conditions for synthesizing biologically active molecules using the Fmoc method (Table 4, Figure 4). The results in Table 4 show that the NAPM group, like other alkyl protecting groups, is stable under the conditions for synthesizing biologically active molecules using the general Fmoc method.

[0179]

[0180] Next, we investigated the conditions for deprotecting the NAPM group. Under harsh conditions, such as prolonged heating at high pH in an ammonia solution, hydroxide ions act as nucleophiles, causing competition between deprotection and main chain cleavage. Therefore, we conducted heat treatment under milder conditions, such as neutral to weakly basic conditions.

[0181] First, compound 14 was coupled to Ac-dC-CPG resin using an automated DNA synthesizer to generate the 11-mer oligonucleotide d(T 10 C) was synthesized.

[0182] The synthesized protected oligonucleotides were then subjected to heat treatment in 0.1 M phosphate buffer (pH 7.4), 0.1 M bicine buffer (pH 8.5), and 0.25 M sodium biborate solution (pH 8.5) (Figures 5-7). The deprotection reaction proceeded in a temperature- and time-dependent manner in various buffers (Figures 5 and 6). As expected, it was demonstrated that oligonucleotides protected with NAPM groups at the phosphate group could be deprotected without backbone cleavage (Figure 7). Furthermore, a similar deprotection treatment was performed on an 11-mer oligothymidine synthesized on polystyrene-PEG resin. It was demonstrated that the NAPM group could be deprotected without backbone cleavage, including by heat treatment in sterile water, suggesting that deprotection is possible in water (Figure 8).

[0183] The NAPM group was explored as a protecting group for phosphate groups that can be deprotected under relatively mild conditions, and it was suggested that by chemically protecting the DNA barcode, the synthesis of biologically active molecules using the Fmoc method with common organic solvents could be applied to DEL.

[0184] 2.3. Expanding the Chemical Space of DNA-Encoded Libraries Based on Chemically Stable Phosphate Protecting Groups 2.3.1 Fukuyama-Mitsunobu Amine Synthesis To further expand the chemical space of DEL using the NAPM-based Recording-by-Synthesis method, we evaluated the chemical durability of various protected DNAs under the Fukuyama-Mitsunobu Amine synthesis, an alternative chemical reaction method to the Fmoc method, which is difficult to apply in conventional aqueous solvents or in the presence of unprotected DNA (Table 5). In addition to the compounds listed in Figure 3, compound 21, which contains an NAPM group, was also evaluated. As a result, various protected DNA bases generated by-products under Mitsunobu alkylation conditions, and MS analysis revealed that the DNA bases were alkylated (Figure 9(a) and Figure 9(b)).

[0185] When a dinucleotide in which the phosphate group was protected with an NAPM group was used, a by-product peak was observed on the HPLC chromatogram only under the Mitsunobu alkylation conditions (Figure 9(c)). Analysis determined that this was due to methylation of the thymine base moiety in compound 21. These results suggest that although the DNA base moiety can be alkylated by the Mitsunobu alkylation reaction, the NAPM group is sufficiently stable under the chemical reaction conditions shown in Table 5.

[0186] 2.3.2 Improving chemical durability by selecting protecting groups for DNA bases As shown in the results in Table 5 above, it is not possible to completely suppress side reactions of DNA bases under various chemical reaction conditions. Therefore, we attempted to obtain resistance to various reaction conditions by optimizing the protecting groups for DNA bases.

[0187] The evaluation results are shown in Table 6. The alkylation of the oxygen atoms in the carbonyl group at the 4-position of T and the 6-position of G was resolved by protecting these oxygen atoms with Bn. In addition, experiments using nucleosides suggested that this Bn protecting group, like the NAPM protecting group, can be removed by heat treatment in aqueous sodium borate or a DNA-compatible heterogeneous catalytic hydrogenation-like reaction while minimizing DNA damage (Figure 10). On the other hand, protection of the amino groups at the 4-position of C, the 6-position of A, and the 2-position of G with diamidine was not possible. Pya or C Pya As can be seen from the results (Table 6, entries 1 and 2), there is a problem of reduced resistance to reductive amination. On the other hand, both 4-N-Me-C and 6-N-Me-A are recognized by DNA polymerase in the same way as natural A and C, and 4-N-Me-C in particular is known to be more resistant to deamination than natural C. Based on the above, we have developed 4-N-Me-N-Bz-A (A) in which these bases are Bz-protected and the amino groups are completely masked. MeBz ) and 6-N-Me-N-Bz-C (C MeBz The chemical durability of the methylated A and C protected bases was also evaluated. The results suggested that these protected A and C bases were stable under the conditions of reductive amination and the Fukuyama-Mitsunobu amine synthesis. Furthermore, it was shown that these various protected DNA bases were relatively stable under the conditions for the synthesis of biologically active molecules using the Fmoc method.

[0188] 2.3.3 Chemical Durability of NAPM Group Derivatives Next, the durability of NAPMCF3, a derivative of the NAPM group, was evaluated using compound 41 (Table 7). The results showed a trend similar to that of compound 21. Reductive amination using NaBH4 suggested a decrease in durability. This is thought to be due to the substitution of the acetyl group with a trifluoroacetyl group, resulting in a decrease in durability to basicity. Furthermore, in Mitsunobu alkylation, a change similar to that observed in compound 21 was observed on the chromatogram, suggesting that a methylated form of T was observed, and this was not due to decomposition of the NAPMCF3 group.

[0189]

[0190] The recovery rate (residual rate) of the protected form of nucleic acid with each protecting group is shown. The conditions were the same as those in Tables 2 and 5. a. A suspension was used. b. No by-product peaks were observed on the chromatogram. c. A phenomenon similar to that of compound 21 was observed on the chromatogram, so it was determined that a methylated form of T was detected.

[0191] 3. Conclusions In this study, we focused on the NAPM group as a chemically stable protecting group for phosphate. We experimentally demonstrated that this protecting group is stable under the synthesis conditions of various biologically active molecules and can be deprotected under mild conditions. This will enable the construction of stable oligonucleotides that maintain their chemical structure during the synthesis of biologically active molecules, and is expected to enable an increase in the number of synthesis cycles in DEL and the application of synthetic conditions for biologically active molecules in common organic solvents. Furthermore, we demonstrated that the chemical reaction space in DEL can be expanded by using the NAPM group as a basis to tailor-make protecting groups for DNA bases.

[0192] Example 2: Alternating Synthesis Based on Recording-by-Synthesis Method 1. Materials and Methods 1.1 General Information Preparation of various reagents, synthesis of oligonucleotides, measurement of oligonucleotide concentrations, measurement of nuclear magnetic resonance (NMR) spectra, and measurement of HRMS were performed as described in Example 1. Oligonucleotides for single-stranded DNA amplification were purchased from standard suppliers. Analysis of oligonucleotides was performed using MALDI-TOF MS (Bruker Daltonics) with 3-hydroxypicolinic acid and triammonium citrate as the matrix. qPCR was performed using an Applied Biosystems StepOne Real Time PCR System (ThermoFisher Scientific). PCR amplicons were analyzed by polyacrylamide gel electrophoresis.

[0193] 1.2 Chemical Synthesis of NAPM-phosphoramidite The NAPM-amidite reagent (9) was prepared according to Test Example 1. 1H-Tetrazolediisopropylammonium salt was prepared according to a previous report (Mol. Pharm., 2006, 3, 161-173). DMTrO-dA Bz -NAPM-phosphoramidite (43), DMTrO-dC Bz -NAPM-phosphoramidite (44), DMTrO-dG iBu DMTrO-dT-NAPM-phosphoramidite (45) and DMTrO-dI-NAPM-phosphoramidite (46) were prepared according to the synthesis procedure for DMTrO-dT-NAPM-phosphoramidite (14) described in Test Example 1.

[0194] DMTrO-dA Bz -NAPM-phosphoramidite (43) Compound 9 (3.02 g), 1H-tetrazolediisopropylammonium salt (3.27 g) and DMTrO-dA Bz —OH (3.59 g) to give the desired compound as a white solid (4.9 g, 96%).

[0195] 1 H NMR (400 MHz, CDCl3): δ 1.06-1.29 (m, 12H), 1.77-2.21 (m, 7H), 2.60-2.77 (m, 1H), 2.88-3.03 (m, 1H), 3.29-3.67 (m, 7H), 3.68-3.86 (m, 7H), 4.13-4.42 (m, 2H), 4.69-4.82 (m, 1H), 6.47-6.61 (m, 1H), 6.73-6.85 (m, 4H), 7.15-7.34 (m, 7H), 7.34-7.45 (m, 2H), 7.48-7.65 (m, 3H), 8.02 (d, J = 7.2 Hz, 2H), 8.16-8.27 (m, 1H), 8.72 (d, J = 8.8 Hz, 1H), 8.94 (brs, 1H). 13C NMR (101 MHz, CDCl3): δ 169.57, 164.63, 158.64, 158.60, 152.66, 152.58, 151.61, 149.49, 144.63, 144.56, 142.20, 141.80, 141.75, 135.82, 135.69, 133.91, 132.80, 130.13, 128.95, 128.27, 127.92, 127.02, 126.96, 123.63, 113.21, 86.55, 86.45, 86.14, 86.08, 85.16, 84.97, 74.06, 73.87, 73.58, 73.40, 63.70, 63.57, 63.16, 63.03, 62.93, 58.69, 57.39, 57.32, 55.32, 48.39, 48.25, 45.78, 43.26, 43.13, 43.06, 39.70, 39.29, 28.66, 27.54, 24.73, 24.70, 24.66, 24.62, 24.21, 24.16, 23.10, 23.04, 22.48, 22.04. 31 P NMR (160 MHz, CDCl3): δ 147.2, 147.9, 148.1, 148.2. HRMS (ESI) m / z calcd for C51H60N7NaO8P [M+Na]+: 952.4133, found 952.4129.

[0196] DMTrO-dC Bz -OPro-phosphoramidite (44) Compound 9 (1.6 g), 1H-tetrazolediisopropylammonium salt (1.7 g) and DMTrO-dC Bz —OH (1.8 g) to give the desired compound as a white solid (2.4 g, 92%).

[0197] 1H NMR (400 MHz, CDCl3): δ 0.96-1.37 (m, 12H), 1.75-2.15 (m, 7H), 2.22-2.37 (m, 1H), 2.68-2.88 (m, 1H), 3.28-3.84 (m, 14H), 3.92-4.33 (m, 2H), 4.51-4.72 (m, 1H), 6.23-6.36 (m, 1H), 6.79-6.91 (m, 4H), 7.12-7.44 (m, 10H), 7.47-7.55 (m, 2H), 7.56-7.66 (m, 1H), 7.88 (d, J= 7.6 Hz, 2H), 8.21-8.39 (m, 1H), 8.57 (brs, 1H). 13 C NMR (101 MHz, CDCl3): δ169.50, 162.06, 158.77, 144.84, 144.66, 144.26, 144.20, 135.57, 135.53, 135.45, 135.34, 135.25, 133.21, 133.15, 130.24, 130.15, 129.09, 128.30, 128.08, 127.60, 127.26, 127.20, 113.37, 96.45, 87.31, 87.22, 87.01, 86.15, 85.88, 85.83, 72.77, 72.53, 72.34, 71.70, 71.53, 64.05, 63.91, 63.00, 62.93, 62.86, 62.79, 62.68, 62.51, 62.35, 58.73, 58.65, 57.25, 57.17, 55.31, 48.41, 48.21, 45.85, 45.76, 43.22, 43.15, 43.09, 43.04, 41.46, 41.17, 28.71, 27.56, 27.49, 24.70, 24.62, 24.59, 24.51, 24.30, 24.12, 23.07, 23.00, 22.40, 22.32, 22.03, 21.97. 31P NMR (160 MHz, CDCl3): δ 147.6, 147.9, 148.3, 148.4. HRMS (ESI) m / z calcd for C50H60N5NaO9P [M+Na]+: 928.4021, found 928.4002.

[0198] DMTrO-dG iBu -NAPM-phosphoramidite (45) Compound 9 (5.4 g), 1H-tetrazole diisopropylammonium salt (5.9 g) and DMTrO-dG Bz Using —OH (6.2 g), the desired compound was obtained as a white solid (7.5 g, 84%).

[0199] 1 H NMR (400 MHz, CDCl3): δ 0.99-1.39 (m, 18H), 1.72-2.29 (m, 7H), 2.34-2.55 (m, 2H), 2.64-2.98 (m, 1H), 3.13-4.41 (m, 16H), 4.51-4.69 (m, 1H), 6.11-6.31 (m, 1H), 6.69-6.85 (m, 4H), 7.10-7.50 (m, 9H), 7.68-7.83 (m, 1H), 11.70(brs, 1H). 13C NMR (101 MHz, CDCl3): δ 180.34, 179.70, 179.50, 179.14, 171.57, 170.23, 169.68, 169.63, 158.67, 155.91,155.82, 155.76, 148.67, 148.37, 148.32, 148.04, 147.90, 147.71, 144.81, 144.67, 144.62, 144.46, 137.50, 136.79, 136.61,135.94, 135.79, 135.74, 135.57, 135.52, 130.11, 130.04, 128.17, 128.12, 128.06, 127.99, 127.04, 122.00, 121.85, 121.38, 113.30, 113.26, 86.67, 86.55, 86.41, 86.19, 86.12, 85.94, 85.89, 85.74, 85.64, 85.30, 85.19, 84.51, 83.98, 75.03, 74.81, 74.07, 73.89, 73.62, 65.04, 64.91, 63.98, 63.87, 63.66, 63.45, 63.31, 59.07, 59.00, 58.75, 58.67, 57.56, 57.49, 57.31, 57.23,55.32, 48.48, 48.40, 46.18, 46.07, 45.19, 43.32, 43.25, 43.20, 43.12, 43.01, 42.88, 41.35, 40.31, 39.40, 36.00, 35.94, 35.83, 35.69, 28.76, 28.55, 27.61, 27.38, 24.88, 24.80, 24.76, 24.68, 24.62, 24.31, 24.23, 23.00, 22.94, 22.80, 22.67, 22.52, 22.36, 22.24, 19.21, 19.13, 19.06, 18.93. 31P NMR (160 MHz, CDCl3): δ 142.8, 144.9, 146.5, 147.0. HRMS (ESI) m / z calcd for C48H62N7NaO9P [M+Na]+: 934.4239, found 934.4212.

[0200] DMTrO-dI-NAPM-phosphoramidite (46) Using compound 9 (1.1 g), 1H-tetrazolediisopropylammonium salt (1.8 g) and DMTrO-dI-OH (1.7 g), the target compound was obtained as a white solid (1.6 g, 67%).

[0201] 1 H NMR (400 MHz, CDCl3): δ1.05-1.39 (m, 12H), 1.78-2.18 (m, 7H), 2.53-2.85 (m, 2H), 2.64-2.98 (m, 2H), 3.30-3.84 (m, 14H), 3.86-4.39 (m, 2H), 4.63-4.76 (m, 1H), 6.36-6.48 (m, 1H), 6.74-6.84 (m, 4H), 7.14-7.46 (m, 9H), 7.94-8.07 (m, 2H). 13C NMR (101 MHz, CDCl3): δ169.64, 169.57, 159.22, 159.14, 158.61, 158.57, 148.78, 145.17, 145.07, 144.93, 144.58, 144.52, 138.86, 138.60, 138.50, 135.77, 135.71, 135.61, 130.12, 128.22, 127.89, 126.93, 125.30, 125.24, 113.19, 86.51, 86.33, 85.96, 84.84, 84.67, 74.03, 73.83, 73.59, 64.07, 63.75, 63.66, 63.17, 63.03, 62.87, 62.73, 58.74, 57.26, 55.28, 48.36, 48.22, 46.04, 45.79, 43.21, 43.09, 39.93, 39.73, 28.61, 27.51, 24.64, 24.58, 24.21, 24.11, 23.04, 22.99, 22.86, 22.41, 22.34, 21.99. 31 P NMR (160 MHz, CDCl3): δ 148.1, 147.8, 147.3. HRMS (ESI) m / z calcd for C 44 H 55 N6O8P [M+Na]+: 849.3711 , found 849.3713.

[0202] Preparation of DMTrO-dT-succinic acid triethylammonium salt DMTrO-dT-succinic acid triethylammonium salt was prepared as described in WO2013 / 122236A1.

[0203] 1.3 Alternating Synthesis 1.3.1 General Procedure for Alternating Synthesis Based on Recording-by-Synthesis Method Fmoc-ANP-OH was prepared as previously reported (J. Am. Chem. Soc., 2008, 130, 10474-10475). Double-layer beads 1 were swollen in DMF for 30 minutes. After Fmoc deprotection, a solution of 0.2 M Fmoc-ANP-OH, 0.2 M COMU, and 0.4 M DIEA in DMF or NMP was added to the beads. After shaking for 1 hour, the beads were washed three times with DMF. Similarly, Fmoc deprotection and condensation of Fmoc-AEEA-OH (0.2 M Fmoc-AEEA-OH, 0.2 M COMU, 0.4 M DIEA, NMP) were repeated twice, followed by alternating synthesis. Each cycle included peptide synthesis and DNA recording. For peptide synthesis, each amino acid was coupled after Fmoc deprotection. The beads were then washed three times with DMF, anhydrous CHCl, and anhydrous MeCN. After the washing procedure, DNA recording was performed on an automated DNA synthesizer without cleaving the terminal DMTr group, unless otherwise noted. The beads were then washed with DMF before the next cycle. All peptide syntheses were performed in 150-300 μL reaction volumes.

[0204] Alternating synthesis based on the Recording-by-Synthesis method - 1 In this experiment, double-layered beads 1 (9.0 mg) were used. Fmoc deprotection was performed as follows.

[0205] Fmoc deprotection-1 method (20% piperidine in DNF, 2 and 8 min) was used in cycles 1, 2, 4, 6 and 8.

[0206] Fmoc deprotection-2 method (2% DBU in DMF, 10 min) was used in cycles 3, 5, 7, 9, and 10.

[0207] Unless otherwise specified, peptide synthesis was carried out at room temperature for 1 hour for cycles 1 to 6 and at 60°C for 1 hour for cycles 7 to 10.

[0208] Cycle 1: Peptide synthesis was performed in a solution of 0.2 M Fmoc-N-Me-L-Ala-OH, 0.2 M COMU, and 0.4 M DIEA in anhydrous NMP. Next, 5´-GT-3´ was synthesized as DNA recording-1.

[0209] Cycle 2: Peptide synthesis was performed using a solution of 0.2 M Fmoc-L-Phe(4-Cl)-OH, 0.2 M COMU, and 0.4 M DIEA in anhydrous NMP. The coupling reaction was carried out for 1.5 hours, yielding DNA recording-2 (5´-CA-3´).

[0210] Cycle 3: Peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-N-Me-D-Ala-OH, 0.2 M HATU, and 0.4 M DIEA. Next, 5´-GT-3´ was synthesized as DNA recording-3.

[0211] Cycle 4: Peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-D-Phe-OH, 0.2 M HATU, and 0.4 M DIEA. Next, 5´-CA-3´ was synthesized as DNA recording-4.

[0212] Cycle 5: Peptide synthesis was performed in anhydrous NMP solution containing 0.2 M Fmoc-N-Me-Leu-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA. Next, 5´-GT-3´ was synthesized as DNA recording-5.

[0213] Cycle 6 peptide synthesis was performed using a solution of 0.2 M Fmoc-L-Phe(4-OMe)-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA in anhydrous NMP. Next, 5´-CA-3´ was synthesized as DNA recording-6.

[0214] Cycle 7 peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-N-Me-D-Phe-OH, 0.2 M HATU, and 0.4 M DIEA. The condensation reaction was carried out for 80 minutes. Next, 5´-GT-3´ was synthesized as DNA recording-7.

[0215] Cycle 8 peptide synthesis was performed using an anhydrous NMP solution of 0.2 M Fmoc-L-Nva-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA. Next, 5´-CA-3´ was synthesized as DNA recording-8.

[0216] Cycle 9 peptide synthesis was performed using an anhydrous NMP solution of 0.2 M Fmoc-N-Me-L-Ala-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA. Next, 5´-GT-3´ was synthesized as DNA recording-9.

[0217] Cycle 10: Peptide synthesis was performed in anhydrous NMP containing 0.2 M Fmoc-N-Me-D-Ala-OH, 0.2 M COMU, and 0.4 M DIEA. The 5´-CA-3´ fragment was then recorded as DNA recording-10.

[0218] After cycle 10, the Fmoc group was deprotected with 2% DBU in DMF (10 min). Next, a solution of 0.2 M AcO and 0.4 M DIEA in anhydrous NMP was added. After 0.5 h, the beads were washed three times with DMF, anhydrous CHCl, and anhydrous MeCN. Subsequently, 5´-GATCT-3´ was synthesized as DNA recording-11. After DNA recording, the terminal DMTr group was cleaved using the deblocking procedure listed in Table 1 on an automated DNA synthesizer. The beads were then washed with DMF and CHCl and dried in vacuo. The beads were stored at -30 °C.

[0219] 1.3.3 Alternating synthesis based on the Recording by Synthesis method - 2 Fmoc-N-Me-Asp(OAll)-OH, Fmoc-Hph(3,5-F2,4-CF3)-OH, and Fmoc-N-Et-Phe(4-Me)-OH were prepared as previously reported (J. Am. Chem. Soc., 2023, 145, 24035-24051, WO2023 / 190283 A1).

[0220] Double-layered beads 1 (8.6 mg) were used in this experiment. Fmoc deprotection was performed as follows.

[0221] Fmoc deprotection-1 method (20% piperidine in DNF, 2 and 8 minutes) was used in cycles 1, 2, 4, 6, 8 and 10.

[0222] Fmoc deprotection-2 method (2% DBU in DMF, 10 min) was used in cycles 3, 5, 7, 9, and 11.

[0223] Peptide synthesis was carried out at room temperature for 1 hour unless otherwise noted.

[0224] Cycle 1: Peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-Sar-OH, 0.2 M COMU, and 0.4 M DIEA. Next, 5´-CA-3´ was synthesized as DNA recording-1.

[0225] Cycle 2: Peptide synthesis was performed in anhydrous NMP solution containing 0.2 M Fmoc-N-Me-Asp(OAll)-OH, 0.2 M COMU, and 0.4 M DIEA. Next, 5´-GT-3´ was synthesized as DNA recording-2.

[0226] Cycle 3: Peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-Gly(cPent)-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA at 60 °C for 1 h. Next, 5´-CA-3´ was synthesized as DNA recording-3.

[0227] Cycle 4 peptide synthesis was performed using anhydrous NMP solution of 0.2 M Fmoc-cycloleucine-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA at 60 °C for 3 h. This coupling reaction was repeated once. Subsequently, 5´-GT-3´ was synthesized as DNA recording-4.

[0228] Cycle 5: The peptide was synthesized in anhydrous NMP solution containing 0.2 M Fmoc-L-Pro-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA at 60 °C for 2 h. Next, 5´-CA-3´ was synthesized as DNA recording-5.

[0229] Cycle 6: Peptide synthesis was performed in anhydrous NMP containing 0.2 M Fmoc-Hph(3,5-F2,4-CF3)-OH and 0.35 M DIC. Next, 5´-GT-3´ was synthesized as DNA recording-6.

[0230] Cycle 7 peptide synthesis was carried out in anhydrous NMP solution containing 0.2 M Fmoc-Sar-OH, 0.2 M HATU, and 0.4 M DIEA. Next, 5´-CA-3´ was synthesized as DNA recording-7.

[0231] Cycle 8 peptide synthesis was performed using a solution of 0.2 M Fmoc-N-Et-L-Phe(4-Me)-OH, 0.2 M HATU, and 0.4 M DIEA in anhydrous NMP. Next, 5´-GT-3´ was synthesized as DNA recording-8.

[0232] Cycle 9: The peptide was synthesized in anhydrous NMP solution containing 0.2 M Fmoc-L-Aze(2)-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA at 60 °C for 3 h. Next, 5´-CA-3´ was synthesized as DNA recording-9.

[0233] Cycle 10 peptide synthesis was carried out using a solution of 0.2 M Fmoc-N-Me-L-Ala-OH, 0.4 M DIC, 0.2 M Oxyma, and 0.02 M DIEA in anhydrous NMP at 60 °C for 1 hour. Next, 5´-GT-3´ was synthesized as DNA recording-10.

[0234] Cycle 11: Peptide synthesis was performed in anhydrous NMP solution containing 0.2 M Fmoc-Ile-D-Ala-OH, 0.2 M COMU, and 0.4 M DIEA. Next, 5´-CA-3´ was synthesized as DNA recording-11.

[0235] Cycle 12: Peptide synthesis was performed in anhydrous NMP containing 0.2 M Fmoc-N-Me-Leu-OH, 0.2 M COMU, and 0.4 M DIEA. Next, 5´-GATCT-3´ was synthesized as DNA recording-12.

[0236] After 12 cycles, the allyl protecting group was deprotected as previously reported (ACS Comb. Sci., 2020, 22, 649-655.).

[0237] After deprotection of the allyl group, the Fmoc group was deprotected with 2% DBU in DMF (10 min). The beads were then washed with DMF, anhydrous CHCl, and anhydrous MeCN. The linear peptide on the beads was then cyclized by adding 300 μL of 0.20 M HATU, 0.021 M HOAt, and 0.033 M DIEA in NMP and reacting at room temperature for 4 hours. The beads were then washed with DNF. The DMTr protecting group was then removed using the automated DNA synthesizer using the deblocking procedure described in Table 1. The beads were then washed with DMF and CHCl and dried in vacuo. The beads were stored at -30°C.

[0238] 1.4 Cleavage and deprotection of DNA barcodes DNA barcodes were cleaved from the beads, deprotected, and analyzed as described below.

[0239] For DNA barcodes derived from the recording-by-synthesis method (alternate synthesis-1), a small number of beads were incubated in AMA for 15 min at 65°C in cycles 7–10. The final product after DNA recording-11 was incubated in AMA for 30 min at room temperature, followed by an additional 15 min at 65°C. The solution was then evaporated under reduced pressure, and the residue was incubated in 250 mM borate buffer, pH 8.5, at 90°C for 12 h. The solution was then directly analyzed by native PAGE (20%, 200V, 40 min). The buffer was exchanged with NFW for desalting before MALDI-TOF MS analysis. The product after DNA recording-11 was directly used for ssDNA amplification without any special purification procedures. The resulting PCR amplicons were analyzed by native PAGE.

[0240] For DNA barcodes derived from alternate synthesis based on Recording-by-Synthesis-4, a small number of beads were incubated in AMA at 65°C for 15 min. After concentration, the residue was incubated in 250 mM borate buffer, pH 8.5, at 90°C for 12 h, and the solution was directly analyzed by native PAGE (20%, 200V, 40 min). The buffer was exchanged into NFW for desalting before mass spectrometry analysis by MALDI-TOF MS.

[0241] 1.5 Peptide Analysis The peptides synthesized by the alternate synthesis were cleaved by UV irradiation (365 nm) in CH2Cl2 at room temperature for 0.5 h, and after concentration, the residue was redissolved in MeCN and analyzed by LC / MS.

[0242] 1.6 Single-stranded DNA amplification and NGS analysis This experiment was performed based on a previous report ( Nat. Protoc. 2020, 15, 2279-2300 ) with the following modifications.

[0243] The following sequence was used for the qPCR standard: 5′- ACACTCTTTCCTACGACGCTTCCGATCTN 20AGATCGGAAGCACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 1). N at positions 29 to 48 randomly contains any of A, C, G, and T.

[0244] In this experiment, we used DNA barcodes derived from Alternating Synthesis-1 based on the Recording-by-Synthesis method. PCR primers for index PCR were performed using the following sequences as Fw and Fv primers. PCR amplicons were analyzed using native PAGE.

[0245] Fw: 5'-AATGATACGGCGACCACCGAGATCTACACCCTGCGAACACTCTTTCCCTACACGACGCTCTT-3' (SEQ ID NO: 2) Rv: 5'-CAAGCAGAAGACGGCATACGAGATCCTGCGAGTGACTGGAGTTCAGACGTGT-3' (SEQ ID NO: 3)

[0246] 2. Results and Discussion 2.1 Recording-by-Synthesis-Based Alternating Synthesis - 1: 10-Cycle Synthesis of DNA-Encoded N-Methyl-Peptides Including Heating Conditions The Fmoc method is a chemical reaction method widely used in DEL, both in solid and solution phases. The first DEL experiments by Janda et al. and Gallop et al. also employed the Recording-by-Synthesis method (ibid.). In particular, Gallop et al. synthesized a 7-residue linear peptide and a corresponding long DNA barcode (>60 nt) based on a seven-cycle Recording-by-Synthesis method using an amidite with a methyl group as the phosphorus protecting group. Based on these results, we investigated the usefulness of Recording-by-Synthesis-based DEL construction, which uses an amidite with a NAPM group as the phosphorus protecting group to construct a short DNA barcode.

[0247] First, the phosphoramidites used in DNA synthesis required for alternate synthesis were synthesized (Scheme 3). dT, dA, dC, and dG amidites, each having an NAPM group as a phosphorus protecting group, were prepared according to the procedures described in Test Example 1.5 Chemical Synthesis and Test Example 2, 1.2 Chemical Synthesis.

[0248] Next, we performed alternating synthesis using these bilayer beads and amidites bearing NAPM groups as phosphorus protecting groups. Scheme 7(a) shows the alternating synthesis scheme for linear peptides containing functional groups such as azide and alkynes and DNA barcodes based on the recording-by-synthesis method of the present invention (Recording-by-Synthesis-1). Linear peptides were synthesized at room temperature using the Fmoc method commonly used in solid-phase peptide synthesis (SPPS). Biologically active molecules were supported on a solid support via a photocleavable ANP linker, allowing for confirmation of synthetic purity. DNA barcodes were synthesized using an automated DNA synthesizer using amidites bearing NAPM groups as protecting groups, according to the method described in Table 1.

[0249] The DNA recording method using the Recording-by-Synthesis method allows the DNA barcode to be extended one base at a time by chemical synthesis. The codon corresponding to each building block is two bases long. In addition, a GATCT NGS linker was added to the 5' end of the DNA barcode for NGS analysis.

[0250] The synthesized molecules consisted of alternating amino acids, including unnatural amino acids, and N-methyl amino acids, with the N-terminus capped with acetic anhydride. The first six cycles of peptide synthesis were performed at room temperature, while the last four cycles were performed at 60°C. For Fmoc deprotection, we used not only 20% piperidine / NMP solution but also 2% DBU / NMP solution (Scheme 7(b)).

[0251] The results for peptides synthesized by alternate synthesis are shown in Figure 11(a)-(e). When the products excised by 365 nm UV irradiation were analyzed using LC / MS, the peak of the desired product was confirmed as the main peak on the UV chromatogram (Figure 11(a)). This demonstrated that bulky, highly N-substituted linear peptides can be synthesized with very high purity using DEL. Furthermore, no degradation was observed at the N-terminus, where N-methylalanine residues are consecutive, suggesting that N-substituted peptides are resistant to the acidic conditions used for deprotection of DMTr groups within the timescale of DNA synthesis.

[0252] Next, the DNA barcodes from each step of the alternating synthesis were heated in AMA at 65 °C for 15 min (hereafter referred to as AMA treatment). The solution was then evaporated under reduced pressure, treated with borate buffer, and the purity was confirmed using polyacrylamide gel electrophoresis without further purification (Figure 11(c)). As a result, the DNA barcodes at each step were confirmed to be of the expected length. Furthermore, when these DNA barcodes were analyzed using MALDI-TOF MS, the error range was generally less than 0.1%, confirming that they were indeed barcodes of the desired length (Figure 11(d)). Since no masses other than the desired length were detected in the mass spectrometry results, we concluded that the DNA barcodes constructed through the Recording-by-Synthesis method during the alternating synthesis maintained a very high purity (Figure 11(e)). Furthermore, it was revealed that PCR amplicons could be successfully obtained from the DNA barcodes using ssDNA amplification technology (Figure 12).

[0253]

[0254] 2.5 Alternating Synthesis Based on Recording-by-Synthesis Method - 2: Synthesis of LUNA18 in DEL Macrocyclic peptides exhibit high binding specificity for biologically relevant targets due to their structural complexity. In particular, highly N-substituted macrocyclic peptides, such as cyclosporine A, have recently been recognized as promising bioactive molecules for medium-sized drug discovery due to their excellent pharmacokinetics, including membrane permeability and metabolic stability. However, rational design of macrocyclic peptides targeting specific targets is challenging, and in many cases, large-scale compound library searches, such as those using phage display and mRNA display, are employed. However, conventional library construction methods relying on in vitro or in vivo translation systems are often limited to natural building blocks, making it difficult to construct libraries of highly N-substituted macrocyclic peptides. Furthermore, the structural complexity of macrocyclic peptide hit compounds obtained through conventional selection systems poses a significant challenge, requiring extensive effort for structure-activity relationship studies.

[0255] On the other hand, unlike the library construction that relies on translation systems, DEL chemically synthesizes biologically active molecules, allowing for the construction of highly purified compound libraries incorporating a wide variety of building blocks. However, conventional methods limit the number of cycles in library construction to 2-4 cycles, and the applicable chemical reaction conditions and reagents are also limited, making it impossible to synthesize the highly N-substituted, bulky macrocyclic peptide libraries mentioned above.

[0256] Based on the above, at the end of this study, we attempted to carry out an alternating synthesis modeled on the macrocyclic peptide LUNA18, a KRAS inhibitor optimized for clinical application (Scheme 8).

[0257] In the previously reported synthesis of LUNA18, the main chain carboxyl group of N-methylaspartic acid was N-dimethylated, the side chain carboxylic acid was supported on chlorotrityl resin, and then a linear peptide was synthesized. Finally, the linear peptide was excised and cyclized in a solution-phase system to obtain the desired compound. In contrast, the alternating synthesis in this study requires that all steps, including cyclization, be carried out on DNA. Therefore, we adopted a strategy of condensing sarcosine to the end of a PEG spacer followed by N-methylaspartic acid, mimicking the C-terminal N-dimethyl of LUNA18, and cyclizing the side chain carboxylic acid and the N-terminal N-methylleucine. The LUNA synthesis process involves multiple condensation steps involving bulky N-methylamino acids, α,α-disubstituted amino acids, and bulky amino acids with a β-branched side chain on the α-carbon. Therefore, the synthesis process involved multiple long heating conditions and double coupling.

[0258] The results of the alternating synthesis are shown in Figure 13(a)-(c). After 365 nm UV irradiation, the product was analyzed using LC / MS. The dehydrogenated and chlorinated products were confirmed by ESI-MS (Figure 13(a)). Furthermore, the dimer formation observed on the solid phase during cyclization was not detected, nor was the mass of the unreacted linear peptide. These results suggest that the macrocyclic peptide LUNA18 can be synthesized. The DNA barcode was then deprotected using AMA and borate buffer treatment, and its mass was confirmed by native PAGE and MALDI-TOF MS (Figure 13(b) and (c)). Native PAGE confirmed a band believed to be the target length, and MALDI-TOF MS detected a mass close to that of the target DNA barcode (with an error of less than 0.1%). Furthermore, the absence of any incomplete-length masses suggested that the DNA barcode was synthesized with high purity. These results suggest that the DNA barcodes produced by the Recording-by-Synthesis method are sufficiently stable even under extreme chemical reaction conditions, and that the purity of the library depends on the efficiency of off-DNA ligand synthesis while minimizing the risk of DNA damage.

[0259] 3. Conclusions In this study, we performed an alternating synthesis of DNA barcodes based on the recording-by-synthesis method using amidite reagents bearing NAPM groups as phosphate protecting groups. Experimental results demonstrated that this method can synthesize DNA barcodes with high purity, even over 10 cycles. This result suggests that constructing protected short DNA barcodes physically and chemically reduces the chance of DNA damage, enabling the application of synthetic conditions for bioactive molecules in conventional organic solvents. The method developed in this study enables the construction of DELs with high purity, including complex compounds of not only small molecules but also medium and larger sizes, suggesting the potential for expanding the chemical space, structural diversity, and quality of DELs.

Claims

1. A method for producing an oligonucleotide, comprising the step of reacting a phosphoramidite compound represented by formula (II) or an optical isomer thereof with a compound represented by formula (III). (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11. (In formula (III), each B is independently any one of nucleobases, each X is an unshared electron pair, H, O, S, or BH3, each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy, each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y, and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and q is 0 or an integer from 1 to 50.

2. The method according to claim 1, wherein ring A is a 5- or 6-membered nitrogen-containing monocyclic saturated heterocycle.

3. A method for deprotecting a phosphate group in an oligonucleotide obtained by the method of claim 1, comprising heating the oligonucleotide obtained by the method of claim 1 in an aqueous solution at a pH of 6.0 to 11.0 so as to deprotect the group represented by the following formula (XI) in the oligonucleotide: (In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

4. A compound represented by the following formula (I): (In formula (I), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 12 is -H, a hydroxyl protecting group, or a linker attached to a solid support; R 13 are each independently —OH, —SH, or a protecting group for a phosphate group, and R 13 at least one of the groups is a group represented by the following formula (XI): (In formula (XI), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is -H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; m is an integer from 0 to 11; r is an integer from 1 to 50; 5. A method for producing a phosphoramidite compound represented by formula (II) or an optical isomer thereof, which comprises reacting a compound represented by formula (IV) with a compound represented by formula (X) in the presence of an activator. (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11. (In formula (IV), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and p is 0 or an integer from 1 to 50. (In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic saturated heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

6. A phosphoramidite compound represented by the following formula (II) or an optical isomer thereof: (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a hydroxyl protecting group, or a linker attached to a solid support; and m is an integer from 0 to 11. p is 0 or an integer from 1 to 50.

7. An amiditizing reagent containing a compound represented by formula (X). (In formula (X), ring A is a 4- to 7-membered nitrogen-containing monocyclic heterocycle, and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 3 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 4 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 3 and R 4 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; and m is an integer from 0 to 11.

8. A compound represented by the following (X-2) or an optical isomer thereof:

9. A method for producing a DNA-encoded library, comprising: (i) preparing a solid support or a soluble carrier having a first functional group reactive with a nucleoside and a second functional group reactive with a first compound; (ii) binding a first building block to the first functional group, wherein the first building block is a nucleoside monomer or an oligonucleoside; (iii) binding a first compound to the second functional group; (iv) binding a second building block to the first building block bound to the first functional group in step (ii), wherein the second building block is a nucleoside monomer or an oligonucleoside; and (v) binding a second compound to the first compound bound to the second functional group in step (iii), wherein the binding of building blocks and the binding of compounds are alternately performed in steps (ii) to (v); The method, wherein the first building block bound to the first functional group is a compound represented by formula (III), and the second building block is a phosphoramidite compound represented by formula (II) or an optical isomer thereof. (In formula (III), each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 12 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support, and q is 0 or an integer from 1 to 50. (In formula (II), ring A is a 4-7 membered nitrogen-containing monocyclic heterocycle; each B is independently a nucleobase; each X is an unshared electron pair, H, O, S, or BH3; each Y is independently selected from the group consisting of -H, halogen, a protected hydroxyl group, -CN, -CF3, and -C1-C6 alkoxy; each Z is independently -H, C1-C6 alkyl, or halogen, or forms a Z-Y bond with said Y; and R 1 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 2 is a C1-C6 alkyl, a C3-C7 cycloalkyl, or a substituted or unsubstituted aromatic group; R 1 and R 2 may be bonded to form a ring, R 5 represents C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, a substituted or unsubstituted aromatic group, or halogen; R 6 is hydrogen, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 7 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 8 is —H, C1-C6 alkyl, C1-C6 haloalkyl, a substituted or unsubstituted aromatic group, or halogen; R 9 are each independently —OH, —SH, or a protecting group for a phosphate group; R 10 is -H, a protecting group for a hydroxyl group, or a linker attached to a solid support; p is 0 or an integer from 1 to 50; and m is an integer from 0 to 11.

10. The method according to claim 9, wherein a set consisting of one step of binding a nucleoside and one step of binding a compound is performed in multiple cycles.

11. The method of claim 9 or 10, wherein step (i) includes a step of preparing a solid support having a first functional group reactive with a nucleoside and a second functional group reactive with a first compound, the solid support having an inner layer and an outer layer covering the inner layer, the DNA synthesis initiation site being located on the outer layer of the solid support, and linkers having an Fmoc group being attached to the inner and outer layers of the solid support.

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