Squaramide‐Based 5’‐Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A

Abstract

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N ‐oxyamide and squaramide modifications at the 5’‐end of oligonucleotides as phosphate replacements and their biological evaluation using the 5’‐exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5’‐phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid‐phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5’‐phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5’‐terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

A hydroxamic-acid-containing nucleoside inhibits DNA repair nuclease SNM1A

“…This material was coupled with acetic acid and formic acid in 71% and 29% yield respectively using EDCI/HOAt-mediated coupling. 15 Deprotection of the silyl ethers 3 and 4 using TBAF gave the hydroxy compounds 5 and 6 in excellent yields (92% and 93% respectively).…”

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

“…[13] Diamides (squaramides) have been used as ap hosphate surrogate in nucleotide [14] or oligonucleotide (ON) analogues. [15] Ac hemically synthesized 2'-sugar-linked squaramate-RNAc onjugate,p repared through reaction of 2'amino-modified RNAwith diethyl squarate,w as reported to cross-link to aminoacyl-transferase FemX Wv , [16] as the only example of its use in nucleic-acid conjugation. Within the framework of our program aimed at base-functionalized nucleic acids for applications in chemical biology, [17] we designed novel squaramate-linked cytosine 2'-deoxyribonucleoside triphosphate (dNTP) for the enzymatic synthesis of modified DNAa nd cross-linking with proteins.…”

Probing the Binding Requirements of Modified Nucleosides with the DNA Nuclease SNM1A

“…SNM1A (698-1040) was stored as a 1.0 µM solution in reaction buffer (20 mM HEPES-KOH, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.05% Triton-X, 0.1 mg/mL BSA, 5% glycerol, 0.5 mM DTT). An oligonucleotide of the sequence 5′-TTA GCA GTC AGT CAG TCA TCG-Cy3-3′ was phosphorylated using T4 PNK (New England Biolabs) [ 44 ] and used as the substrate. Modified nucleosides were added to the assay in 40% DMSO solution (10 mM or as specified).…”