CN-122011024-A - Phosphoramidite monomer containing bio-orthogonal groups and preparation method and application thereof

CN122011024ACN 122011024 ACN122011024 ACN 122011024ACN-122011024-A

Abstract

The invention discloses a phosphoramidite monomer containing bio-orthogonal group as shown in formula I, a preparation method and application thereof, belonging to the field of chemical modification of oligonucleotides and development of functional nucleic acid, the monomer can be used for preparing modified oligonucleotides through a DNA/RNA synthesizer, and the bioorthogonal groups are introduced into any positions of the oligonucleotides, so that the variety of phosphoramidite monomer modules containing the bioorthogonal groups is enriched, and a nucleic acid chemical modification raw material system is perfected. The obtained modified oligonucleotide can be suitable for biological orthogonal reaction, and is used for preparing nucleic acid medicaments and oligonucleotide probes, and the support function nucleic acid can be applied to the diversification of the fields of chemistry, biology and medicine.

Inventors

LIU MINGZHE
WANG ZIYIN
WANG HONGMEI
HE WENPENG

Assignees

沈阳药科大学

Dates

Publication Date: 20260512
Application Date: 20260209

Claims (7)

1. A phosphoramidite monomer containing bio-orthogonal groups, which is characterized in that the phosphoramidite monomer is a compound with a structure shown in a formula I: Where n=an integer between 1 and 8.
2. A method for preparing the bio-orthogonal group-containing phosphoramidite monomer according to claim 1, comprising the steps of: Wherein n and R 1 are as defined in claim 1; Step a, carrying out nucleophilic reaction on trans-4-hydroxymethyl piperidine-3-alcohol and N-benzyloxycarboryl amino bromoamine; B, the product obtained in the step a reacts with 4,4' -dimethoxy triphenylmethyl chloride under the protection of anhydrous and anaerobic inert gas and through catalysis of a catalyst; C, the product obtained in the step b reacts with palladium carbon and ammonium formate to remove the protecting group of the carbobenzoxy; Step d, the product obtained in the step c reacts with carboxylic acid containing bio-orthogonal groups under the protection of anhydrous oxygen-free inert gas and through catalysis of a catalyst; And e, under the protection of anhydrous and anaerobic inert gas, the product obtained in the step d is catalyzed by a catalyst, and 2-cyanoethyl N, N-diisopropyl chlorophosphamide is adopted to carry out phosphoramidite reaction, so that a phosphoramidite monomer containing biological orthogonal groups is obtained.
3. Use of a bio-orthogonal group containing phosphoramidite monomer according to claim 1 for the preparation of a bio-orthogonal group modified oligonucleotide, wherein the bio-orthogonal group is introduced into any position of the oligonucleotide by DNA/RNA solid phase synthesis techniques.
4. A bioorthogonal modified oligonucleotide, characterized in that the bioorthogonal group-containing phosphoramidite monomer according to claim 1 is introduced into any position of the oligonucleotide by means of DNA/RNA solid phase synthesis technology.
5. Use of the biorthogonal modified oligonucleotide of claim 4 in nucleic acid related research and detection.
6. Use of the biorthogonal modified oligonucleotide of claim 4 for the preparation of a biomimetic drug in medical treatment-related studies.
7. The use of the biorthogonal modified oligonucleotide of claim 4 for the preparation of a nanoprobe material.

Description

Phosphoramidite monomer containing bio-orthogonal groups and preparation method and application thereof Technical Field The invention belongs to the field of chemical modification of oligonucleotides and development of functional nucleic acid, relates to a phosphoramidite monomer containing bio-orthogonal groups, a preparation method and application thereof, and in particular relates to a phosphoramidite monomer containing bio-orthogonal groups such as norbornene, sydney ketone or cyclooctyne and the like by taking trans-4-hydroxymethyl piperidine-3-ol as a framework and application thereof in oligonucleotide chemical modification. Background Bioorthogonal reactions are chemical reactions that occur in cells or tissues within an organism that do not interfere with the biochemical reactions of the organism itself. This reaction allows for chemical labeling and imaging in living cells without interfering with the biological system. The [4+2] cycloaddition reaction between electron-deficient dienoic and electron-rich dienophiles is known as the "inverse electron-demand diels-alder reaction (IEDDA)". IEDDA has good biocompatibility and high reaction specificity, is the bio-orthogonal reaction with the fastest reaction rate, and is commonly used in the labeling coupling of nucleic acid. Norbornene (Norbornene) is a structural unit formed by the fusion of a cyclohexene ring and a methylene bridge, and the double bond in the ring of the norbornene gives higher reactivity and stability to the molecule. Can be rapidly bio-orthogonal reacted with tetrazine (Tz). With the development of nucleic acid chemical synthesis technology, nucleic acids and modified analogues thereof have been widely used in chemistry, biology and medicine. The comprehensive chemical modification or partial substitution of the oligonucleotide can be realized by a DNA/RNA solid phase synthesis method and a phosphoramidite method. The chemically modified oligonucleotide can be used as a bionic drug and can be widely used for preparing nano probe materials. Chemical modifications of oligonucleotides include modifications to sugar rings, base sites, modifications to phosphodiester backbones, substitution of nucleosides with unnatural monomers, and the like. In nucleic acid modification, the bio-orthogonal reaction can be used for introducing specific functional groups, so as to realize post-synthesis modification of DNA/RNA, and the method can be further used for labeling, imaging or drug delivery of nucleic acid. In recent years, chemists design and synthesize a series of bio-orthogonal group modified oligonucleotide probes for the fields of nucleic acid imaging, drug targeted delivery and the like. Most commonly, bioorthogonal groups are introduced as coupling handles onto natural nucleosides, but the synthetic procedure is cumbersome. The coupling handle modified nucleoside triphosphates can be introduced by polymerase method, but the objective of selective site-directed introduction in the oligonucleotide is not achieved. The strategy of introducing the coupling handle onto the natural nucleoside in the form of phosphoramidite monomer has few reports on the nucleoside types connected with the coupling handle, and the solid phase synthesis condition is strict and complex, and meanwhile, the introduction of the handle may influence the function of the natural nucleoside. Most of the reported bioorthogonal modified oligonucleotides are made from base site modified phosphoramidite monomers, and few have been reported to introduce bioorthogonal groups using nucleoside substitutes. Therefore, in order to enrich the variety of phosphoramidite monomer modules containing bio-orthogonal groups, it is necessary to design and synthesize a novel phosphoramidite monomer containing bio-orthogonal groups that is simple and practical. The invention takes trans-4-hydroxymethyl piperidine-3-alcohol as a framework to design and synthesize a phosphoramidite monomer containing a bioorthogonal group, prepares the oligonucleotide modified by the bioorthogonal group through a DNA/RNA solid phase synthesis technology, and provides a usable molecular tool for the development of subsequent functional nucleic acid. Disclosure of Invention The invention aims to provide a phosphoramidite monomer containing a bioorthogonal group and a bioorthogonal group modified oligonucleotide. The invention firstly discloses a phosphoramidite monomer containing bio-orthogonal groups, which is a compound with a structure shown in a formula I: Where n=an integer between 1 and 8. The invention provides a preparation method of phosphoramidite monomer containing bio-orthogonal group shown in formula I, comprising the following steps: (a) Nucleophilic reaction of trans-4-hydroxymethyl piperidin-3-ol with N-benzyloxycarboxamide bromide; (b) The product obtained in the step (a) reacts with 4,4' -dimethoxy triphenylmethyl chloride under the catalysis of 4-dimethylaminopyridine under the protection