CN-121991148-A - 5.́ Synthesis of terminal phosphorothioate nucleotide and application thereof in oligonucleotide

CN121991148ACN 121991148 ACN121991148 ACN 121991148ACN-121991148-A

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses synthesis of 5 ́ -terminal thiophosphonate nucleotide and application thereof in oligonucleotide, wherein a chemical modification strategy comprises preparation of a 5' -thiophosphonous acid modified nucleotide phosphoramidite monomer and a 5' -thiophosphonic acid modified nucleotide phosphoramidite monomer, and connection of the 5 ́ -terminal of a target oligonucleotide molecule prepared from the modified nucleotide phosphoramidite according to a carbon-thiophosphonate C-PSO 2 2‑ chemical bond, so that a non-natural 5' -terminal thiophosphonic acid structure is constructed, the modified oligonucleotide does not belong to a natural substrate of phosphatase, can resist nuclease degradation, can improve affinity with specific proteins and improve in vivo biological activity of siRNA.

Inventors

ZHANG YUFEN
SUN GANG
SUN HONGWEI
ZHU BIN
ZHENG ZHENSHENG
HUANG YUANSONG

Assignees

苏州盛诺维生物科技有限公司

Dates

Publication Date: 20260508
Application Date: 20251101

Claims (10)

1. The synthesis of 5 ́ -terminal thiophosphonate nucleotide and the application thereof in oligonucleotide are characterized in that the chemical modification strategy comprises that the 5' -terminal is connected with thiophosphonate PSO 2 2- through a carbon-thiophosphonate C-PSO 2 2- chemical bond, and the chemical modification strategy comprises a nucleotide structure shown in a formula 1 or a formula 2: Formula 1; Formula 2; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and a derivative thereof, Y is one or more of methylene CH 2 , alkyl, alkylene, alkylidene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl having a branched structure, R is an acyl group such as hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, thiocarbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, a silicon group such as thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, R ́, R ́ ́ is a silicon group such as hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl, seleno, alkylsulfamoyl or the like.
2. The synthesis of a 5 ́ -terminal phosphorothioate nucleotide and its use in oligonucleotides according to claim 1, wherein the chemical modification strategy further comprises preparing a modified nucleotide phosphoramidite monomer linked between the 5' -terminal and the phosphonite via a carbon-phosphonite chemical linkage, comprising a nucleotide structure of formula 3 or formula 4 linked to the 5 ́ -terminal of the nucleic acid sequence, and the vulcanization treatment yields a 5 ́ -terminal phosphorothioate modified oligonucleotide: Formula 3; Formula 4; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil containing a protecting group, and derivatives thereof; Y is one or more of methylene CH 2 , alkyl group with branched structure, alkylene group, alkylidene group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, alkoxyalkyl group, LG 1 、LG 2 、LG 3 is a silicon group such as cyanoethyl group, cyanopropyl group, cyanoisobutyl group, (CH 3 ) 2 CCH 2 CN, phenethyl group, allyl group, allyloxycarbonyl group, benzyl group, acyl group, trimethylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, LG 4 is a silicon group such as halogen, amino group, cyano group, azide group, or alkyloxy group, and R is an alkyl group, cycloalkyl group, alkoxyalkyl group, carbonylalkyl group, benzyl Bn, benzoyl Bz, acyl group such as acetyl group or propionyl group, thioacyl group, alkenyl group, cycloalkenyl group, alkynyl group, allyl group, allyloxycarbonyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, or tert-butyldiphenylsilyl group, R ́ is a group such as hydrogen, halogen, alkyl group, cycloalkyl group, alkoxy group, alkoxyalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, cyano group, azide group, amino group (amino group), mercapto group, alkylseleno group, etc.
3. The synthesis of a 5 ́ -terminal phosphorothioate nucleotide and its use in an oligonucleotide according to claim 2, said 5 ́ -terminal phosphonous modified nucleotide monomer comprising the structure of formula 5 or formula 6: Formula 5; Formula 6; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil containing a protecting group, and derivatives thereof; Y is one or more of methylene CH 2 , alkyl group with branched structure, alkylene group, alkylidene group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, alkoxyalkyl group, LG 1 、LG 2 is a silicon group such as cyanoethyl group, cyanopropyl group, cyanoisobutyl group, (CH 3 ) 2 CCH 2 CN, phenethyl group, allyl group, allyloxycarbonyl group, benzyl group, acyl group, trimethylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, etc., R is an acyl group such as alkyl group, cycloalkyl group, alkoxyalkyl group, carbonylalkyl group, benzyl Bn, benzoyl Bz, acetyl group or propionyl group, etc., a thioacyl group, alkenyl group, cycloalkenyl group, alkynyl group, allyl group, allyloxycarbonyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, etc., R ́, R ́ ́ is a hydrogen, halogen, alkyl group, cycloalkyl group, alkoxy group, alkoxyalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, cyano group, azide group, amino group (amino group), mercapto group, alkyl group, seleno group, alkylseleno group, etc., R 1 and R 2 are respectively alkyl group, cycloalkenyl group or aryl group.
4. The synthesis of a 5 ́ -terminal phosphorothioate nucleotide and its use in oligonucleotides according to claim 1, wherein the chemical modification strategy further comprises preparing a modified nucleotide phosphoramidite monomer linked between the 5' -terminal and the phosphorothioate via a carbon-phosphorothioate chemical linkage comprising a nucleotide structure of formula 7 or formula 8: Formula 7; Formula 8; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil containing a protecting group, and derivatives thereof; Y is one or more of methylene CH 2 , alkyl group with branched structure, alkylene group, alkylidene group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, alkoxyalkyl group, LG 1 、LG 2 、LG 3 is a silicon group such as cyanoethyl group, cyanopropyl group, cyanoisobutyl group, (CH 3 ) 2 CCH 2 CN, phenethyl group, allyl group, allyloxycarbonyl group, benzyl group, acyl group, trimethylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, LG 4 is a silicon group such as halogen, amino group, cyano group, azide group, or alkyloxy group, and R is an alkyl group, cycloalkyl group, alkoxyalkyl group, carbonylalkyl group, benzyl Bn, benzoyl Bz, acyl group such as acetyl group or propionyl group, thioacyl group, alkenyl group, cycloalkenyl group, alkynyl group, allyl group, allyloxycarbonyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, or tert-butyldiphenylsilyl group, R ́ is a group such as hydrogen, halogen, alkyl group, cycloalkyl group, alkoxy group, alkoxyalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, cyano group, azide group, amino group (amino group), mercapto group, alkylseleno group, etc.
5. The synthesis of a 5 ́ -terminal phosphorothioate nucleotide and its use in an oligonucleotide according to claim 4, said 5 ́ -terminal phosphorothioate modified nucleotide monomer comprising one of the structures of formula 9 or formula 10: Formula 9; Formula 10; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil containing a protecting group, and derivatives thereof; Y is one or more of methylene CH 2 , alkyl group with branched structure, alkylene group, alkylidene group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, alkoxyalkyl group, LG 1 、LG 2 is a silicon group such as cyanoethyl group, cyanopropyl group, cyanoisobutyl group, (CH 3 ) 2 CCH 2 CN, phenethyl group, allyl group, allyloxycarbonyl group, benzyl group, acyl group, trimethylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, etc., R is an acyl group such as alkyl group, cycloalkyl group, alkoxyalkyl group, carbonylalkyl group, benzyl Bn, benzoyl Bz, acetyl group or propionyl group, etc., a thioacyl group, alkenyl group, cycloalkenyl group, alkynyl group, allyl group, allyloxycarbonyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, etc., R ́, R ́ ́ is a hydrogen, halogen, alkyl group, cycloalkyl group, alkoxy group, alkoxyalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, cyano group, azide group, amino group (amino group), mercapto group, alkyl group, seleno group, alkylseleno group, etc., R 1 and R 2 are respectively alkyl group, cycloalkenyl group or aryl group.
6. The synthesis of a 5 ́ -terminal thiophosphonate nucleotide and its application in oligonucleotide, characterized in that the synthesis of the 5' -phosphonite modified nucleotide phosphoramidite monomer is characterized in that hypophosphorous acid replaces the leaving group of the 5' -end, after phosphonite at the position is esterified, O3 ́ -hydroxy is released to combine with phosphoramidite to obtain the 5' -phosphonite modified nucleotide phosphoramidite monomer.
7. The synthesis of 5 ́ -terminal thiophosphonate nucleotide and its application in oligonucleotide, characterized in that the synthesis of 5' -thiophosphonate modified nucleotide phosphoramidite monomer, the substitution of phosphinic acid for leaving group at 5' -end, the esterification of phosphinic acid at the position, the conversion of phosphite ester into thiophosphonate ester by sulfuration treatment, releasing O3 ́ -hydroxy and phosphoramidite combination to obtain 5' -thiophosphonate modified nucleotide phosphoramidite monomer.
8. The synthesis of a 5 ́ -terminal phosphorothioate nucleotide and its use in oligonucleotides according to claim 1, wherein the chemical modification strategy comprises synthesis of a 5 ́ -terminal carbon-phosphinic acid or carbon-phosphorothioate chemical bond linked nucleotide phosphoramidite monomer to produce a 5 ́ -terminal phosphorothioate modified oligonucleotide of the same sequence as INCLISIRAN. Compared with INCLISIRAN, the dissociation constant of the Ago2 protein is combined, and the terminal thiophosphonate modification provided by the invention improves the affinity between modified siRNA and Ago2 whether C5 ́ is directly connected with thiophosphonic acid or C5 ́ is connected with thiophosphonic acid spacer methylene.
9. The synthesis of a5 ́ -terminal phosphorothioate nucleotide and its use in oligonucleotide according to claim 1, wherein the 5 ́ -terminal phosphorothioate modified oligonucleotide is prepared in sequence with INCLISIRAN, and wherein the 5 ́ -terminal phosphorothioate modified siRNA is capable of significantly reducing PCSK9 mRNA levels and LDL-C levels in model animals compared to INCLISIRAN, whether the C5 ́ is directly linked to phosphorothioate or the C5 ́ is linked to phosphorothioate spacer methylene.
10. A 5 ́ -terminal phosphorothioate modified oligonucleotide comprising in its structural units a nucleotide comprising the modified structure of any one of claims 1 to 7, said oligonucleotide being selected from the group consisting of a small interfering nucleotide, an antisense oligonucleotide, a microrna, a small activating RNA, a guide RNA, a transfer RNA and an aptamer, or a combination of at least two.

Description

5.́ Synthesis of terminal phosphorothioate nucleotide and application thereof in oligonucleotide Technical Field The invention relates to the technical field of biological medicine, in particular to a 5 ́ -terminal thiophosphorylation modified nucleotide monomer and application thereof in oligonucleotide. Background The oligonucleotide drug expands the drug target to the upstream mRNA of the pathogenic protein through base complementation pairing, specifically binds with the target gene of interest, regulates or knocks down the target gene, and affects the expression of the target gene from the post-transcriptional level. The oligonucleotide drug has the advantages of rich targets, lasting drug effect, short research and development period, high research and development success rate and the like, and provides a solution for the treatment of a plurality of refractory diseases. Chemical modification is one of the effective strategies for enhancing the stability and delivery of oligonucleotide drugs, and is widely used to improve the biological activity of drugs. Exonuclease acts on the phosphate group at the end of a nucleic acid chain, so that the small nucleic acid drug is degraded, and therefore, the 5 ́ -end modification of the nucleic acid sequence can improve the degradation resistance of the drug to the nuclease. In siRNA duplex, the 5' -end of antisense strand must carry a phosphate to specifically bind to side chain residues of MID domain in the RNA-induced silencing complex Argonaute2 (Ago 2) effector protein, which is critical for RNAi activity. Currently, 5 ́ -phosphorylation strategies, previously synthesized by chemical synthesis, are available as metabolically stable phosphate analogues. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide synthesis of 5 ́ -terminal thiophosphonate nucleotide and application thereof in oligonucleotide, in particular to preparation of nucleic acid medicaments by performing thiophosphonate modification on the 5' -terminal of the nucleotide through a novel modification strategy, wherein the modified phosphoramidite monomer is applied to nucleic acid medicaments. In order to solve the problems in the prior art, the invention is realized by the following technical scheme: The synthesis of 5 ́ -terminal phosphorothioate modified nucleotides and their use in oligonucleotides, the applied chemical modification strategy comprises: Preparing a nucleotide phosphoramidite monomer with C4' connected with phosphonite through a spacer group Y; Preparing a nucleotide phosphoramidite monomer with C4' connected with thiophosphonic acid through a spacer group Y; The two modified nucleotide phosphoramidite monomers are applied to the preparation of oligonucleotide to construct a5 ́ -terminal thiophosphonate modified oligonucleotide molecule; The C4 'at the 5 ́ -terminal end of the phosphate backbone of the oligonucleotide molecule is linked to a phosphorothioate through a spacer group Y to form a C4' -Y-PSO 22- fragment as shown in formula 1 or formula 2: Formula 1; Formula 2; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil, and a derivative thereof, Y is one or more of methylene CH 2, alkyl, alkylene, alkylidene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl having a branched structure, R is an acyl group such as hydrogen, alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, thiocarbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, a silicon group such as thioacyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl, R ́, R ́ ́ is a silicon group such as hydrogen, halogen, alkyl, cycloalkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkyl, seleno, alkylsulfamoyl or the like. Preparing a nucleotide monomer in which C4' is linked to a phosphonite P (III) through a spacer group represented by Y, comprising a structure represented by formula 3 or formula 4: Formula 3; Formula 4; Wherein: Base is a nucleotide Base such as adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methylpseudouracil containing a protecting group, and derivatives thereof; Y is one or more of methylene CH 2, alkyl group with branched structure, alkylene group, alkylidene group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, alkoxyalkyl group, LG 1、LG2、LG3 is a silicon group such as cyanoethyl group, cyanopropyl group, cyanoisobutyl group, (CH 3)2CCH2 CN, phenethyl group, allyl group, allyloxycarbonyl group, benzyl group, acyl group, trimethylsilyl group, tert-butyldimethylsilyl group or tert-butyldiphenylsilyl group, LG 4 is a silicon group such as halogen, amino group, cyano group, azide group, or alkyl