Bioorthogonal Functionalization in the Minor Groove of DNA: Inverse-Electron Demand Diels Alder Reactions with Cyclopropenyl and Heptenoyl Protected 2-Amino-2'-deoxyadenosine as Target Site.

Abstract

Bioorthogonal functionalization in the minor groove of DNA with protected 2-amino-2'-deoxyadenosine (2-amino-dA, 1) as target site is described. Cyclopropene and heptene as part of 2-aminoacyl protecting groups served as dienophiles in inverse-electron demand Diels-Alder (iEDDA) reactions with 3,6-dipyridyl-1,2,4,5-tetrazine as diene. For the purpose, bis-protection of 1 with cyclopropenyl and heptenoyl groups was conducted. Selective removal of the 6-amino protecting groups gave mono-protected 1. 2-Amino-dA pyridazine conjugates were accessed by iEDDA cycloaddition with tetrazine. Second order rate constants disclosed faster reaction kinetics for cyclopropenyl than for heptenoyl 2-amino-dA. To access protected oligonucleotides, phosphoramidites prepared from bis-protected 2-amino-dA were used in standard solid-phase synthesis. 2-Amino protecting groups were retained using mild deprotection conditions. Hybridization experiments disclosed increased duplex stability when protected 2-amino-dA-dT pairs replaced 1-dT pairs. Mismatch discrimination of cyclopropenyl and heptenoyl 2-amino-dA was superior to 2-amino-dA. iEDDA reactions on protected oligonucleotides furnished oligonucleotide pyridazine conjugates. In base pairs with dT, 2-amino-dA pyridazine conjugates connected by a short rigid cyclopropenyl ring led to reduced thermal stability, whereas pyridazine conjugates attached by a long flexible linker retained duplex stability. The findings of this investigation pave the way for functionalization and labelling in the minor groove of DNA using copper free iEDDA cycloaddition.