Reagents for universal site-specific labeling and modifications of nucleic acids

• US 9,856,285 B2
• Filed: 03/17/2015
• Issued: 01/02/2018
• Est. Priority Date: 03/17/2015
• Status: Active Grant

• Current Assignee: Kaohsiung Medical University
• Original Assignee: Kaohsiung Medical University
• Inventors: Wang, Tzu-Pin, Su, Yu-Chih
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US14/660,489
• Publication Number:
  US 20160272669A1
• Time in Patent Office: 1,022 Days
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present invention discloses a method of harnessing versatile phosphoramidation reactions to regioselectively link a group of alkynyl-/azido-containing compounds to post-synthetic nucleic acids primed with phosphate at the 5′ termini. The method includes steps of: (a) monophosphoramidating H₂N(CH₂)_nNH₂ to the 5′ end of the nucleic acid to form an intermediate 1 where n is an integer from 2 to 6, and the intermediate 1 has a formula of:

(b) amidating the free —NH₂ group of the intermediate 1 with

to form an intermediate 2; and (c) reacting the terminal —N₃ group of the intermediate 2 with 1-{3-{[4-(2-cyclooctyn-1-ylmethyl)benzoyl]amino}propyl}-4-{2-[4-(dimethylamino)phenyl]ethenyl}pyridinium hexafluorophosphate (Alkyne MegaStokes dye 608) by the SPAAC reaction to form modified nucleic acids. Without the presence of Cu(I), the modified nucleic acids were subjected to azide-alkyne cycloaddition in an aqueous solution to obtain various nucleic acid conjugates with a high yield.

Claims

1. A method for modifying a nucleic acid molecule, comprising:

(a) monophosphoramidating H₂N(CH₂)_nNH₂ to the 5′

end of the nucleic acid to form a conjugated product, wherein n is an integer from 2 to 6, and the conjugated product has a formula of;

2. The method of claim 1, wherein the cyclooctyne is 1-{3-{[4-(2-cyclooctyn-1-ylmethyl)benzoyl]amino}propyl}-4-{2-[4-(dimethylamino)phenyl]ethenyl}pyridinium hexafluorophosphate.

3. The method of claim 1, wherein the reacting step is a strain-promoted azide-alkyne cycloaddition reaction.