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US-20260125413-A1 - UNIVERSAL SOLID SUPPORTS AND PHOSPHORAMIDITE BUILDING BLOCKS FOR OLIGONUCLEOTIDE SYNTHESIS

US20260125413A1US 20260125413 A1US20260125413 A1US 20260125413A1US-20260125413-A1

Abstract

Disclosed herein are chemical preparation of oligonucleotides, chemical entities useful in such preparation, processes for such preparation, and methods of use relating to the chemical preparation of oligonucleotides. Further disclosed herein are novel universal solid supports and phosphoramidite building blocks for oligonucleotide synthesis on solid phase that effect the removal of 3′-phosphate moieties during the course of oligonucleotide deprotection.

Inventors

  • Andrei Pavel GUZAEV
  • Khirud Gogoi
  • Suzie Sheng
  • Harri Oskari Loennberg
  • Mikhail A. Guzaev

Assignees

  • AM CHEMICALS LLC

Dates

Publication Date
20260507
Application Date
20230828

Claims (15)

  1. 1 . A compound of Formula I wherein: R 1 and R 2 either form an orthoester function —C(CH 3 )(OCH 3 )— or one of R 1 and R 2 is hydrogen, a trityl protecting group or a derivative thereof, or a xanthenyl protecting group or a derivative thereof, and the other of R 1 and R 2 is an acetyl, a propionyl, a n-butyryl, a benzoyl, or L 1 , wherein: L 1 is a linking moiety —C(═O)—Z—(C═O)-A 1 , wherein: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH 2 ) 2 —, —(CH 2 )—O—(CH 2 )—, and —(CH 2 )—O—C 6 H 4 —O—(CH 2 )—; A 1 is a hydroxy group, a salt of a hydroxy group and an inorganic cation or a tertiary amine, a covalent bond to SP 1 , or —NH(CH 2 ) n —OR 7 , wherein: SP 1 is an oxygen, amino, an aminoalkyl or a hydroxyalkyl covalently attached to a solid phase material comprising a controlled pore glass, a magnetic controlled pore glass, a silica-containing particle, a styrene-containing polymer or copolymer, a divinylbenzene-containing polymer or copolymer, a copolymer of styrene and divinylbenzene, a controlled pore glass grafted with a styrene-containing polymer, a controlled pore glass grafted with a copolymer of styrene and divinylbenzene, a copolymer of styrene and divinylbenzene grafted with polyethylene glycol, or a flat glass surface; n is an integer from 2 to 10; R 7 is hydrogen or PA, wherein: and wherein R 8 is methyl or 2-cyanoethyl group and R 14 is an alkyl, iso-alkyl, sec-alkyl, or tert-alkyl; R 3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone electron pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl, wherein when R 4 is other than a lone electron pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxylic functional group; R 5 and R 6 are independently hydrogen or methyl; Y is —(C═O)—, —CH(OR 9 )—, —CH(NR 10 R 11 )—, or —[C(OR 12 )(OR 13 )]—, wherein: R 9 is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, (t-butyl) diphenylsilyl, —(C═O)A 1 , L 1 , or PA wherein: m is an integer ranging from 2 to 10; R 10 is L 1 , —(C═O)-A 1 , or —(C═O)-W 1 -(CH 2 ) p —W 2 , wherein: W 1 is —(CH 2 )—, —(NH)—, or —(NH)(C═O)—; W 2 is a hydroxy, amino, —O—PA, (C═O)-A 1 , or —[NH(C═O)]-A 1 ; and p is an integer from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; and R 12 and R 13 together form ketal bridges —(CH 2 ) 2 — or —CH 2 [C(CH 3 ) 2 ]—CH 2 —; provided one and only one of R 1 , R 2 , and Y is or includes L 1 or PA.
  2. 2 . The compound of claim 1 wherein one of R 1 and R 2 is hydrogen, tris-(4-methoxyphenyl)methyl, bis-(4-methoxyphenyl)phenylmethyl, 9-phenylxanthen-9-yl, or 9-(4-methoxyphenyl)xanthen-9-yl, and the other of R 1 and R 2 is L 1 wherein: A 1 is a hydroxy group, a salt of a hydroxy group and an inorganic cation or a tertiary amine, or a covalent bond to SP 1 ; and (i) Y is —CH(OR 9 )—, wherein R 9 is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl; or (ii) Y is —[C(OR 12 )(OR 13 )]—.
  3. 3 . The compound of claim 1 wherein one of R 1 and R 2 is hydrogen tris-(4-methoxyphenyl)methyl, or bis-(4-methoxyphenyl)phenylmethyl, and the other of R 1 and R 2 is acetyl, propionyl, n-butyryl, or benzoyl.
  4. 4 . The compound of claim 3 wherein (i) Y is —CH(OR 9 )—, wherein R 9 is L 1 , and wherein A 1 is a hydroxy group optionally forming a salt with an inorganic cation or a tertiary amine, or optionally forming a covalent bond to SP 1 ; or (ii) Y is —CH(NR 10 R 11 ), wherein R 11 is hydrogen, methyl, ethyl, or benzyl, and (a) R 10 is L 1 wherein A 1 is a hydroxy group, a salt of a hydroxy group and an inorganic cation or a tertiary amine, or a covalent bond to SP 1 ; or (b) R 10 is —(C═O)-W 1 -(CH 2 ) p —W 2 , wherein: W 2 is an amino, —(C═O)-A 1 , or —[NH(C═O)]-A 1 wherein A 1 is a hydroxy group, a salt of a hydroxy group and an inorganic cation or a tertiary amine, or a covalent bond to SP 1 ; and p is an integer from 3 to 10.
  5. 5 . The compound of claim 1 wherein one of R 1 and R 2 is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl and the other of R 1 and R 2 is L 1 wherein: (i) A 1 is-NH(CH 2 ) n —OR 7 ; and (ii) Y is —CH(OR 9 ), wherein R 9 is methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or phenyldimethylsilyl.
  6. 6 . The compound of claim 1 wherein one of R 1 and R 2 is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl and the other of R 1 and R 2 is acetyl, propionyl, n-butyryl, or benzoyl and Y is —CH(OR 9 )—, wherein: (i) R 9 is PA, or (ii) R 9 is —(CH 2 ) m —O-PA.
  7. 7 . The compound of claim 1 wherein one of R 1 and R 2 is tris-(4-methoxyphenyl)methyl or bis-(4-methoxyphenyl)phenylmethyl, the other of R 1 and R 2 is acetyl, propionyl, n-butyryl, or benzoyl, and Y is —CH(NR 10 R 11 ), wherein R 11 is hydrogen, methyl, ethyl, or benzyl, wherein: R 10 is —(C═O)-W 1 -(CH 2 ) p —W 2 , and wherein: W 1 is —(CH 2 )—, —(NH)—, or —(NH)(C═O)—, and W 2 is hydroxy or O-PA.
  8. 8 . The compound of claim 1 wherein R 1 and R 2 form an orthoester function —C(CH 3 )(OCH 3 )—, and wherein (i) Y is —CH(OR 9 )—; and (ii) R 9 is hydrogen, PA, or —(CH 2 ) m —O-PA.
  9. 9 . The compound of claim 1 , wherein the trityl protecting group or the derivative thereof or the xanthenyl protecting group or the derivative thereof is selected from the group consisting of 4-methoxytrityl, 4,4′-dimethoxytrityl, 4,4′,4″-trimethoxytrityl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.
  10. 10 . A method for functionalizing a solid phase material with a first monomeric subunit, comprising the steps of: (a) providing a solid phase material-bound compound of Formula I wherein: R 1 and R 2 either form an orthoester function —C(CH 3 )(OCH 3 )— or one of R 1 and R 2 is hydrogen, a trityl protecting group or a derivative thereof, or a xanthenyl protecting group or a derivative thereof and the other of R 1 and R 2 is an acetyl, a propionyl, a n-butyryl, a benzoyl, L 1 , wherein: L 1 is a linking moiety —C(═O)—Z—(C═O)-A 1 , wherein: Z is selected from the group consisting of a covalent bond, a methylene group, —(CH 2 ) 2 —, —(CH 2 )—O—(CH 2 )—, and —(CH 2 )—O—C 6 H 4 —O—(CH 2 )—; A 1 is a covalent bond to SP 1 or —NH(CH 2 ) n —OR 7 , wherein: SP 1 is an oxygen, amino, an aminoalkyl or a hydroxyalkyl covalently attached to a solid phase material comprising a controlled pore glass, a magnetic controlled pore glass, a silica-containing particle, a styrene-containing polymer or copolymer, a divinylbenzene-containing polymer or copolymer, a copolymer of styrene and divinylbenzene, a controlled pore glass grafted with a styrene-containing polymer, a controlled pore glass grafted with a copolymer of styrene and divinylbenzene, a copolymer of styrene and divinylbenzene grafted with polyethylene glycol, and a flat glass surface; n is an integer from 2 to 10; R 7 is PX, wherein: and wherein R 8 is methyl or 2-cyanoethyl group and X is oxygen or sulfur; R 3 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, and benzyl; R 4 is selected from the group consisting of a lone electron pair, hydrogen, methyl, ethyl, propyl, isopropyl, and benzyl, wherein when R 4 is other than a lone electron pair, N has a positive charge and forms a salt with a halide anion or with an intramolecular carboxylic functional group; R 5 and R 6 are independently hydrogen or methyl; Y is —(C═O)—, —CH(OR 9 )—, —CH(NR 10 R 11 ), or —[C(OR 12 )(OR 13 )]—, wherein: R 9 is hydrogen, methyl, ethyl, benzyl, acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, t-butyldimethylsilyl, triisopropylsilyl, or (t-butyl) diphenylsilyl group, —(C═O)A 1 L 1 , or PX, wherein: m is an integer ranging from 2 to 10; R 10 is L 1 , —(C═O)-A 1 , or —(C═O)-W 1 (CH 2 ) p —W 2 , wherein: W 1 is —(CH 2 )—, —(NH)—, or —(NH)—(C═O)—; W 2 is —O—PX, or —[NH(C═O)]-A 1 ; and p is an integer ranging from 2 to 10; R 11 is hydrogen, methyl, ethyl, or benzyl; and R 12 and R 13 together form ketal bridges —(CH 2 ) 2 — or —CH 2 —[C(CH 3 ) 2 ]—CH 2 —; provided one and only one of R 1 , R 2 , and Y is or includes L 1 or PX; (b) selectively removing one of the protecting groups of Formula I to form a reactive hydroxyl group; (c) providing a first monomeric subunit comprising an activated phosphorus group and a protected hydroxy group, and reacting the activated phosphorus group of the first monomeric subunit with the reactive hydroxyl group of the compound of Formula I to form a monomer-functionalized solid support comprising a phosphite group; (d) treating said monomer-functionalized solid support with a capping agent and/or treating said monomer-functionalized solid support with an oxidizing solution or a sulfurizing agent to convert the phosphite triester group to a phosphotriester or phosphothioate triester, thereby forming an oxidized or sulfurized functionalized solid support; (e) optionally repeating steps (b), (c), and (d) one or more times for the oxidized or sulfurized functionalized solid support to form an oligomeric-functionalized solid support, wherein the monomeric subunit is the same or different each time steps (b), (c), and (d) are repeated.
  11. 11 . The method of claim 10 , further comprising deprotecting the oligomeric-functionalized solid support of step (e) and cleaving the oligomeric-functionalized solid support to form an oligomeric compound separate from the solid phase material, wherein the cleaving forms a terminal hydroxy group on the oligomeric compound at the site of cleavage.
  12. 12 . The method of claim 10 , wherein one of R 1 or R 2 is L 1 .
  13. 13 . The method of claim 10 , wherein R 7 is PX.
  14. 14 . The method of claim 10 , wherein the activated phosphorus group comprises a phosphoramidite, an H-phosphonate, or a phosphate triester.
  15. 15 . The method of claim 11 , wherein the oligomeric compound is an oligonucleotide, optionally comprising unnatural sugar-modified nucleotide residues, unnatural base-modified nucleotide residues, or non-nucleotide monomeric units.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a national stage application, filed under 35 U.S.C. § 371, of International Patent Application No. PCT/US2023/073030, filed on Aug. 28, 2023, which claims the benefit of U.S. Provisional Patent Application No. 63/373,619, filed on Aug. 26, 2022, each of which is incorporated by reference herein in its entirety. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Feb. 26, 2025, is named AM Chemicals-PCT-US.xml and is 8,192 bytes in size. FIELD The disclosure herein provides teaching of compounds, compositions and methods of use relating to synthesis of oligonucleotides. For example, the disclosure provides universal solid supports and phosphoramidite building blocks based on non-nucleosidic linkers for synthesis of standard and modified oligonucleotides, compositions comprising such non-nucleosidic solid supports, phosphoramidite building blocks, and methods of using such supports and building blocks in the synthesis of modified oligonucleotides. BACKGROUND A number of innovations have been introduced to the art of oligonucleotide synthesis. Amongst these innovations have been the development of excellent orthogonal protecting groups, activators, reagents, and synthetic conditions. The oligonucleotides themselves have been subject to a variety of modifications and improvements. Amongst these are chemistries that deliver the properties that are not present in naturally occurring oligonucleotides i.e. reduced negative charge, hydrophobicity, ability to emit fluorescence, protein and receptor binding properties, etc. These novel chemistries generally involve modification of building blocks of non-nucleosidic nature that become the constituent parts of the oligonucleotide. Oligonucleotides with a free 3′-hydroxy group essential for the enzymatic extension are the ones used most frequently in life sciences. Until late 1990's, the routine synthesis of these oligonucleotides has almost exclusively been carried out on nucleosidic solid supports containing 3′-terminal nucleosides attached via a readily cleavable ester linkage. An alternative approach uses universal solid supports. A universal solid support is one in which the 3′-terminal nucleoside residue is coupled to the support as a phosphoramidite building block in the first cycle of oligonucleotide synthesis. The oligonucleotide chain assembly then continues until the completion, and the support bound material is deprotected. It is important that, over the course of the final deprotection, the phosphate bridge formed between a universal linker and the 3′-terminal nucleoside can be cleaved in such a manner that the phosphate bridge remains with the universal linker. The net result of the deprotection is the oligonucleotide with the free, unprotected 3′-terminal hydroxy group identical to that prepared on nucleosidic solid supports is formed. The process of deprotection of synthetic oligonucleotides assembled on universal solid supports involves several relatively independent reactions, including: 1) removal of 2-cyanoethyl protecting group from internucleosidic phosphate or phosphorothioate residues; 2) deprotection of amino groups in nucleic bases that, in the standard and the most robust protecting scheme are protected with acetyl or benzoyl groups for cytidine (Cac and Cbz, respectively), benzoyl group for adenosine (Abz), isobutyryl group for guanosine (Gib) and for their 2′-deoxy, 2′-O-alky, and other analogs used in the art; 3) release of solid support-bound oligonucleotide in solution, and 4) 3′-dephosphorylation of the 3′-hydroxy group attached, via a phosphate or a phosphorothioate linkage to the universal linker. Of these processes, the removal of the isobutyryl group from Gib residues and 3′-dephosphorylation are the most time consuming and hence controls the length of the deprotection time, and has been reported as the rate limiting step (Schwartz, M. E., Breaker, R. R., Asteriadis, G. T., and Gough, G. R. 1995) Analysis of the prior art reveals that all universal solid supports disclosed to date share one common disadvantage in that the 3′-dephosphorylation of oligonucleotides synthesized on said solid supports remains the rate-limiting step in the course of deprotection of oligonucleotides. SUMMARY OF THE INVENTION One object of this disclosure is to provide novel compounds which may serve as solid supports and phosphoramidite building blocks for preparation of oligomeric compounds, analogs of natural and chemically modified oligonucleotides, wherein a non-nucleosidic moiety together with the phosphate moiety it is attached to is cleaved from the target oligonucleotides during the course of the final deprotection thereby releasing the free 3′-hydroxy group in sai