2-Aminobenzene Derivatives as Unnatural Nucleobases and Their DNA Duplex Stabilities

Abstract

Scheme 1. Synthesis of aminobenzene derivatized nucleotidesThe thermal stability and faithful replication of a natural DNA duplex is based on the Watson-Crick hydrogen-bonding (H-bonding) patterns of dA:dT and dC:dG base pairs. Additional stable and selective unnatural base pairs would facilitate hybridization and encoding experiments in cases where natural sequences cross hybridize or where increased information storage is desirable.1 Recent studies have shown that H-bonds between unnatural nucleobases are not absolutely essential for selective base pairing.2 On the basis of this concept, a large number of nucleotides bearing large and predominantly hydrophobic nucleobases have been synthesized and characterized according to the isocarbostyril, napthyl, and indole scaffolds, whose pairing is mediated by hydrophobic interactions.2 From these studies, several self-pairs (formed by pairing of identical analogues) and heteropairs (formed by pairing of 2 different analogues) that rival natural base pairs in terms of thermal stability and selectivity against mispairing have been identified. A larger aromatic surface area increases the thermal stability and selectivity of unnatural base pairs, and a structural study suggested that analogues intercalate into the opposite strand between the pairing nucleobase and an adjacent nucleobase. 3 We therefore became interested in examining nucleobase analogues that lack a large aromatic surface area but are optimized to interact edgeon analogous to natural Watson-Crick base pairing.Recent works have focused on the systematic examination of the contribution to base pair stability of nucleobase shape, hydrophobicity, dipole moment, and polarizability within the benzene, pyridine, or pyridone scaffolds. 4 Our current focus is identifying combinations of methyl-group substitutions at the major groove position and heteroatom derivatizations at the ortho-position in the unnatural nucleobase scaffold responsible for the improved stability of these smaller unnatural nucleobases. Although 2-aminophenyl derivatives are good candidates as hydrophobic surrogates, relatively little work has been done on their deoxy-C-nucleosides. Some research groups have synthesized these derivatives by inducing a titanium aldol reaction/ O-H insertion reaction between diazoketone and aminobenzaldehyde 5 and C-C bond formation between aminophenyl derivative and 2'-deoxy-D-ribono-lactone. 6 However, these processes have the disadvantages of long steps and low stereoselectivity. Further, no previous study has examined the effect of 2-aminophenyl derivatives on the thermal stability of the DNA duplex. To further understand how 2-aminobenzene derivatives contribute to DNA stability, we synthesized and characterized the 2-aminobenzene nucleotide analogues AM and ADM (Figure 1, Scheme 1). We also synthesized the N-methylated aminobenzene derivatives MAN, MMAN, and DMMAN to evaluate the amino functional group at the 2-position for its role in optimizing DNA stability. A palladium-mediated Heck-type coupling reac...