Nucleotides: Part LXXVI : Synthesis and Properties of New Isoalloxazine (=Benzo[g]pteridine‐2,4(1H,3H)‐dione) Derivatives as Labels for Oligonucleotides

Abstract

A series of new fluorescing 8‐(6‐hydroxyhexyl)isoalloxazine (=8‐(6‐hydroxyhexyl)benzo[g]pteridine‐2,4(1H,3H)‐dione) derivatives 4–13 were synthesized from 6‐[(6‐hydroxyhexyl)amino]uracil (2) with 1‐chloro‐4‐nitrosobenzene via 8‐chloro‐10‐(6‐hydroxyhexyl)isoalloxazine (3) and subsequent substitution of the Cl‐atom of 3 by various amines (Scheme). Analogously, 8‐substituted 10‐{3‐[(2,2‐dimethyl‐1,3‐dioxolan‐4‐yl)methoxy]propyl}isoalloxazines 19, 20, and 23–25 were prepared which yielded on deprotection the corresponding 10‐[3‐(2,3‐dihydroxypropoxy)propyl]alloxazines 21, 22, and 26–28. Their conversion into the 3″‐O‐(4,4′‐dimethoxytrityl) derivatives 29–33 and subsequent transformation into the corresponding 2″‐(2‐cyanoethyl N,N‐diisopropylphosphoramidites) 34–38 led to new building blocks for oligonucleotide synthesis. A series of 21‐mer oligodeoxyribonucleotides carrying the fluorescing isoalloxazine 37 in various positions of the chain were assembled in a DNA synthesizer. Combination with the complementary sequence yielded the stable duplexes 40–54 showing by the melting temperatures Tm that the fluorophor (F) does not harm the stability of the unmodified duplex 39 (Table).