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.

Bioorthogonal functionalization in the minor groove of DNA with protected 2‐amino‐2′‐deoxyadenosine (2‐amino‐dA, 1) as a 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. [ABSTRACT FROM AUTHOR]