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CN-122011070-A - 3.́ -Amino-5 ́ -nucleotide phosphoramide synthesis and application thereof in oligonucleotide

CN122011070ACN 122011070 ACN122011070 ACN 122011070ACN-122011070-A

Abstract

The invention discloses synthesis of 3' -amino-5 ́ -nucleotide phosphoramide and application thereof in oligonucleotide, wherein the phosphoramide modified oligonucleotide comprises at least one of nucleotide structural units shown in formula I, formula II, formula III or formula IV: ... Formula I, ... Formula II, ... Formula III, ... The invention is based on the specific definition of the structure of the nucleotide phosphoramide monomer, and the preparation of the oligonucleotide is coupled according to the direction of 5 ́ to 3 ́. Compared with the traditional oligonucleotide molecules, the oligonucleotide molecules provided by the invention contain the phosphoramide modification, have more excellent effects on pharmaceutical parameters such as enzyme degradation resistance, stability and the like, and in addition, the phosphoramide modification can improve the combination property between the modified oligonucleotide molecules and environmental ions, thereby providing a novel chemical modification strategy for research and development and application of oligonucleotide drugs.

Inventors

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

Assignees

  • 苏州盛诺维生物科技有限公司

Dates

Publication Date
20260512
Application Date
20251211

Claims (10)

  1. 1. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and the application thereof in oligonucleotide are characterized in that the phosphoramide modified oligonucleotide comprises at least one nucleotide structural unit shown in formula I, formula II, formula III or formula IV: ... ... In the formula I and the formula II: The Base is a nucleotide Base or a derivative thereof, and the nucleotide Base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methyl pseudouracil and other nucleotide bases and derivatives thereof; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 3 is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silicon-based oxymethylene, alkoxyalkyl, benzyl, or acyl; ... ... In formula III, formula IV: The Base is a nucleotide Base or a derivative thereof, and the nucleotide Base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methyl pseudouracil and other nucleotide bases and derivatives thereof; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 4 and R 5 are each independently hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, seleno, alkylseleno, or the like.
  2. 2. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotide synthesis according to claim 1, wherein the phosphoramide modified oligonucleotide synthesis comprises: ... Formula V; In formula V: 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; PG is trityl, 4-methoxytrityl, 4' -dimethoxytrityl, 4' ' -trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 3 is acyl such as alkyl, cycloalkyl, alkoxyalkyl, carbonylalkyl, benzyl Bn, benzoyl Bz, acetyl or propionyl, alkenyl, cycloalkenyl, alkynyl, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; ... In formula VI: 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; PG is trityl, 4-methoxytrityl, 4' -dimethoxytrityl, 4' ' -trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, trimethylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 4 and R 5 are each independently hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, seleno, alkylseleno, or the like.
  3. 3. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotides according to claim 2, wherein said nucleotide monomers comprise: 、 、 、 、 、 、 、 、 、 、 , Wherein: r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 3 is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silicon-based oxymethylene, alkoxyalkyl, benzyl, or acyl.
  4. 4. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotides according to claim 2, wherein said nucleotide monomers comprise: 、 、 、 、 、 、 、 、 、 、 , Wherein: r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; r 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl.
  5. 5. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotides according to claim 2, wherein said nucleotide monomers comprise: 、 、 、 、 、 、 、 、 、 、 , Wherein: r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; r 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl.
  6. 6. The phosphoramide-modified oligonucleotide of any of claims 3 to 5, wherein the nucleotide monomer is substituted with a trityl (Tr), 4-methoxytrityl (MMTr) or 4,4',4 "-trimethoxytrityl (TMTr) group.
  7. 7. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotide according to claim 2, wherein said nucleotide monomer is prepared by reacting a nucleotide molecule having a protecting group at N3' with phosphoramide dichloride.
  8. 8. The synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and its use in oligonucleotide according to claim 1, wherein the modified oligonucleotide comprises 3 ́ -amino and 5 ́ -phosphoramide modified nucleotide structures, which not only increases the biostability and the anti-enzymatic activity of the oligonucleotide, but also improves the binding properties between the modified oligonucleotide molecule and anions.
  9. 9. The process for synthesizing a phosphoramide-modified oligonucleotide according to any one of claims 1 to 8, wherein the coupling of nucleotide monomers in solid phase synthesis is in the direction of 5 ́ to 3 ́, the process comprising the steps of: (a) Loading the nucleotide monomer on a solid phase carrier, removing the protecting group of 3' on the nucleotide monomer, and washing; (b) Pumping nucleotide monomers into the washed system for coupling, and then removing 3 '-protecting groups on the nucleotide monomers through cap treatment and washing, wherein the nucleotide monomers are phosphoramide nucleotide monomers or phosphoramidite nucleotide monomers; if the nucleotide monomer is the nucleotide monomer of phosphoramidite, after the coupling reaction, the trivalent phosphite ester is converted into pentavalent phosphate ester through oxidation or sulfuration, and then capping is carried out, and the protecting group of 3' on the nucleotide monomer is removed and washed; (c) Repeating the operation of the step (b) to obtain the oligonucleotide with the phosphoramide modified nucleotide structure.
  10. 10. The oligonucleotide of claim 1, wherein the oligonucleotide is selected from any one or a combination of at least two of a small interfering nucleotide, an antisense oligonucleotide, a microrna, a small activating RNA, a small guide RNA, a transfer RNA, and an aptamer.

Description

3.́ -Amino-5 ́ -nucleotide phosphoramide synthesis and application thereof in oligonucleotide Technical Field The invention relates to the technical field of oligonucleotides, in particular to synthesis of 3 ́ -amino-5 ́ -nucleotide phosphoramide and application thereof in oligonucleotide medicines. Background ‌ Oligonucleotide drugs bind specifically to the target gene of interest by base complementary pairing, expand the target to the upstream mRNA of the pathogenic protein, and affect the expression of the target gene from post-transcriptional levels. The oligonucleotide drug has the advantages of rich targets, high research and development success rate, short research and development period, lasting drug effect and the like, and provides a solution for treating a plurality of refractory diseases. Furthermore, oligonucleotides are widely used in ‌ molecular biology research, disease diagnosis and treatment ‌, for example as PCR primers, gene editing tools or targeting drugs. At present, the oligonucleotide sequences synthesized by traditional nucleotide monomers have the problem of poor stability and are easy to degrade in human serum and human liver microsomes. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a phosphoramide modified oligonucleotide and a synthesis process thereof, wherein the phosphoramide modified oligonucleotide has a specific phosphoramide modified nucleotide structure through the preparation of nucleotide phosphoramide, so that the stability of the synthesized oligonucleotide can be improved and the biological activity of the synthesized oligonucleotide can be improved. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: in a first aspect, the present invention provides a phosphoramide-modified oligonucleotide comprising at least one nucleotide building block of formula I, formula II, formula III or formula IV: ... ... In the formula I and the formula II: The Base is a nucleotide Base or a derivative thereof, and the nucleotide Base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methyl pseudouracil and other nucleotide bases and derivatives thereof; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 3 is hydrogen, alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); ... ... In formula III, formula IV: The Base is a nucleotide Base or a derivative thereof, and the nucleotide Base is adenine, guanine, cytosine, 5-methylcytosine, thymine, uracil, xanthine, hypoxanthine, pseudouracil or N1-methyl pseudouracil and other nucleotide bases and derivatives thereof; r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 4 and R 5 are each independently hydrogen, halogen, alkyl, cycloalkyl, alkoxyalkyl, alkenyl, cycloalkenyl, alkynyl, cyano, azide, amino (amino), mercapto, alkylmercapto, seleno, alkylseleno, or the like. The phosphoramide-modified oligonucleotide synthesis process adopts a nucleotide monomer shown as a formula V or a formula VI: ... Formula V; In formula V: 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; PG is trityl, 4-methoxytrityl, 4' -dimethoxytrityl, 4' ' -trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl or t-butyldiphenylsilyl); r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl, or aryl; R 1 and R 2 are each alkyl, alkenyl, alkynyl, cycloalkyl or aryl; R 3 is alkyl (including methyl, ethyl, long chain alkyl), cycloalkyl, alkenyl, cycloalkenyl, alkynyl, silyl (including trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl), silyloxymethylene, alkoxyalkyl, benzyl, or acyl (including acetyl, propionyl, butyryl, isobutyryl, benzoyl); ... In formula VI: 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; PG is trityl, 4-methoxytrityl, 4' -dimethoxytrityl, 4' ' -trimethoxytrityl, allyl, allyloxycarbonyl, benzyl, acyl, silyl (including trimethylsilyl, t-butyldimethylsilyl or t-butyldiphenylsilyl); r is hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkoxyalkyl