Phenyl-imidazolo-cytidine Analogues: Structure–Photophysical Activity Relationship and Ability To Detect Single DNA Mismatch

Abstract

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized para-substituted phenyl-imidazolo-cytidine ((Ph)ImC) analogues 5a-g and established a relationship between their structure and fluorescence properties. These analogues were more emissive than cytidine (λem 398-420 nm, Φ 0.009-0.687), and excellent correlation was found between Φ of 5a-g and σp(-) of the substituent on the phenyl-imidazolo moiety (R(2) = 0.94). Calculations suggested that the dominant tautomer of (Ph)ImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected (Ph)ImC analogues suggested a relationship between the HOMO-LUMO gap and Φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF3-(Ph)ImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A 17-fold reduction of fluorescence was observed for the G-matched duplex vs ODN(CF3-(Ph)ImdC), while for A-mismatched duplex, only a 2-fold decrease was observed. Furthermore, since the quantum yield of ODN(CF3-(Ph)ImdC):ODN(G) was reduced 17-fold vs that of a single strand, whereas that of ODN(CF3-(Ph)ImdC):ORN(G) was reduced only 3.8-fold, ODN(CF3-(Ph)ImdC) appears to be a DNA-selective probe. We conclude that the ODN(CF3-(Ph)ImdC) probe, exhibiting emission sensitivity upon single nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing.

Visibly Emissive and Responsive Extended 6-Aza-Uridines

“…This provides a useful design tool enabling the use of established linear free energy parameters to confidentially anticipate select photophysical properties. 29 , 30 …”

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing

“…These studies have generally used substituent effects to tune the photophysical properties of modified nucleobases and characterized their fluorescencea sf ree nucleosides, sometimes including Stern-Volmer quenching measurements with natural nucleosides. [12,13,[32][33][34][35] Our group has focused on elaborating the tricyclic cytidine scaffold by the introductiono fe lectron-donating groups( EDGs) and electron-withdrawing groups (EWGs), extending the conjugation, and studying the effects of substituent placement. [36] Recent works by Wilhelmssona nd others have applied time-dependent density functional theory (TDDFT) calculations to the prediction of absorption and emission spectra of nucleobase analogues with somes uccess.…”

“…[36] Recent works by Wilhelmssona nd others have applied time-dependent density functional theory (TDDFT) calculations to the prediction of absorption and emission spectra of nucleobase analogues with somes uccess. [32,34,35,37,38] But the application of computational work to rational design of novel fluorophores is mostly limited to substituente ffects. [37] While there are examples of series of structurally related fluorescent nucleosidest hat have been studied for their photophysics as free molecules, [32][33][34] systematic studies of nucleobase substituent effects on fluorescencei nt he base stack have been rarely performed.…”

“…[32,34,35,37,38] But the application of computational work to rational design of novel fluorophores is mostly limited to substituente ffects. [37] While there are examples of series of structurally related fluorescent nucleosidest hat have been studied for their photophysics as free molecules, [32][33][34] systematic studies of nucleobase substituent effects on fluorescencei nt he base stack have been rarely performed. Wilhelmssonh as reported that parent tricyclic cytidines have fluorescencei nsensitive to neighboring bases, and neighboringb ase effects have been studied for 2-aminopurine quenching in duplex DNA.…”

“…A number of research groups have studied the relationships between structure and fluorescence of nucleoside analogues, using a combination of empirical observations and computation. These studies have generally used substituent effects to tune the photophysical properties of modified nucleobases and characterized their fluorescence as free nucleosides, sometimes including Stern–Volmer quenching measurements with natural nucleosides . Our group has focused on elaborating the tricyclic cytidine scaffold by the introduction of electron‐donating groups (EDGs) and electron‐withdrawing groups (EWGs), extending the conjugation, and studying the effects of substituent placement .…”