Screening Internal Donor-Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline-Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA.

Donor-acceptor biaryls serve as microenvironment fluorescent sensors with highly quenched intramolecular charge transfer (ICT) emission in polar protic solvents that turns on in aprotic media. In DNA, canonical donor-acceptor fluorescent base analogs can be prepared through on-strand Suzuki-Miyaura cross-coupling reactions involving 8-bromo-2'-deoxyguanosine (8-Br-dG) with an acceptor aryboronic acid. Herein, we demonstrate that replacement of 8-Br-dG with N-methyl-4-bromoaniline (4-Br-An) containing an acyclic N-glycol group can be employed in the on-strand Suzuki-Miyaura reaction to afford new donor-acceptor biaryl nucleobase surrogates with a 40-fold increase in emission intensity for fluorescent readout within single-strand oligonucleotides. Screening the best acceptor for turn-on fluorescence upon duplex formation afforded the carboxythiophene derivative [COOTh]An with a 7.4-fold emission intensity increase upon formation of a single-bulged duplex (-1) with the surrogate occupying a pyrimidine-flanked bulge. Insertion of the [COOTh]An surrogate into the lateral TT loops produced by the antiparallel G-quadruplex (GQ) of the thrombin binding aptamer (TBA) afforded a 4.1-fold increase in probe fluorescence that was accompanied by a 20 nm wavelength shift to the blue upon thrombin binding. The modified TBA afforded a limit of detection of 129 nM for thrombin and displayed virtually no emission response to off-target proteins. The fluorescence response of [COOTh]An to thrombin binding highlights the utility of the thienyl-aniline moiety for monitoring DNA-protein interactions.

Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines.

Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2'-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8-aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.