Functionalized Tricyclic Cytosine Analogues Provide Nucleoside Fluorophores with Improved Photophysical Properties and a Range of Solvent Sensitivities

Abstract

Tricyclic cytosines (tC and tCO frameworks) have emerged as a unique class of fluorescent nucleobase analogues that minimally perturb the structure of B-form DNA and that are not quenched in duplex nucleic acids. Systematic derivatization of these frameworks is a likely approach to improve on and diversify photophysical properties, but has not so far been examined. Synthetic methods were refined to improve on tolerance for electron donating and electron withdrawing groups, resulting in a series of eight new, fluorescent cytidine analogues. Photophysical studies show that substitution of the framework results in a pattern of effects largely consistent across tC and tCO and provides nucleoside fluorophores that are brighter than either parent. Moreover, a range of solvent sensitivities is observed, offering promise that this family of probes can be extended to new applications that require reporting on the local environment.

Fluorescence Turn-On Sensing of DNA Duplex Formation by a Tricyclic Cytidine Analogue

“…This increase is larger than that of the parent tC for the same solvent change (4-fold) but smaller than what we have observed in a past study of the 8-methoxy-tC nucleoside (30-fold). 29 …”

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

“…[41,43,44] We developed syntheticm ethodst op reparet C (O) derivatives, some of which we have reported previously,b ut all are summarized here. [19,36] The amidites of 8-Cl-tC O ,a nd 8-MeO-tC are new compounds, as is the 8-CN-tC O nucleoside and itsp recursors. The targeted family of compounds spanned ar ange of electronic richness as indicated by Hammett s p from À0.83 to 0.66, comprising the -NEt 2 ,-OMe, -H, -Cl, and -CN groups (Figure 1).…”

“…[19,36] Thatw ork identified 8-Cl-tC O as the member of the tC family with the brightest fluorescence and 8-DEA-tC as the least bright nucleoside, and those results hinted at trends relatinga nalogues'e lectronics to photophysical properties such as Stokes shift. [36] Further studies in duplex DNA showedt hat 8-DEA-tC exhibits up to a2 0-foldi ncrease in fluorescencew hen it is base stacked and correctly base paired, making it the most powerful turn-onf luorescent nucleoside known. [19] Here, we report on the synthesis and properties of 8-CN-tC O ,anew,m ost electron-deficient tC O analogue, and we assess the fluorescencer esponses of the familyo ft C (O) analogues to base stacking and pairing in double-stranded DNA within the context of av aried set of nearest neighboring bases ( Figure 1; the abbreviation tC (O) is used to encompassb oth tC and tC O compounds).…”

“…[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. [32,34,35,37,38] But the application of computational work to rational design of novel fluorophores is mostly limited to substituente ffects.…”

Synthesis of Fluorescence Turn‐On DNA Hybridization Probe Using the DEAtC 2′‐Deoxycytidine Analog

“…The synthesis is related to methods for synthesizing other tC derivatives (Lin et al, 1995; Sandin et al, 2007; Rodgers et al, 2014). The protocol begins with the commercially available starting material 5-amino-2-methylbenzothiazole, which is first doubly ethylated at the amino group to provide the diethylamino-derivative.…”