Fluorescent Biaryl Uracils with C5-Dihydro- and Quinazolinone Heterocyclic Appendages in PNA

Abstract

There has been much effort to exploit fluorescence techniques in the detection of nucleic acids. Canonical nucleic acids are essentially nonfluorescent; however, the modification of the nucleobase has proved to be a fruitful way to engender fluorescence. Much of the chemistry used to prepare modified nucleobases relies on expensive transition metal catalysts. In this work, we describe the synthesis of biaryl quinazolinone-uracil nucleobase analogs prepared by the condensation of anthranilamide derivatives and 5-formyluracil using inexpensive copper salts. A selection of modified nucleobases were prepared, and the effect of methoxy- or nitro- group substitution on the photophysical properties was examined. Both the dihydroquinazolinone and quinazolinone modified uracils have much larger molar absorptivity (~4–8×) than natural uracil and produce modest blue fluorescence. The quinazolinone-modified uracils display higher quantum yields than the corresponding dihydroquinazolinones and also show temperature and viscosity dependent emission consistent with molecular rotor behavior. Peptide nucleic acid (PNA) monomers possessing quinazolinone modified uracils were prepared and incorporated into oligomers. In the sequence context examined, the nitro-substituted, methoxy-substituted and unmodified quinazolinone inserts resulted in a stabilization (∆Tm = +4.0/insert; +2.0/insert; +1.0/insert, respectively) relative to control PNA sequence upon hybridization to complementary DNA. All three derivatives responded to hybridization by the “turn-on” of fluorescence intensity by ca. 3-to-4 fold and may find use as probes for complementary DNA sequences.

Chimeric GFP–uracil based molecular rotor fluorophores

“…We have previously used 5-formyluracil, Scheme 2, readily prepared from uracil, in the synthesis of uracil–quinazolinone biaryl structures. 28 In the test of this approach, N 1 -unsubstituted 5-formyluracil (Scheme 2, compound 4 ) was condensed with a selection of anilines by refluxing in EtOH with catalytic tolunenesulfonic acid (pTSA) to produce the Schiff bases 5 and 6 shown in Scheme 2. The Schiff bases 5 and 6 were reacted with MEEA and compound 2 under literature conditions 27 used for similar condensations, but to no avail.…”

Isomorphic Fluorescent Nucleosides

“…Intriguingly, the 5-amino derivative ( 7 ) is a FRET acceptor for Trp’s indole (Figure c). This facilitates the monitoring of RNA–peptide/protein interactions by relying on native, intrinsically fluorescent Trp residues, which are disproportionally abundant within RNA binding domains. , The potential of this FRET pair has been demonstrated by studying the association of the HIV-1 Rev peptide with the Rev response element (RRE), its endogenous RNA target …”

Exploring Nucleobase Modifications in Oligonucleotide Analogues for Use as Environmentally Responsive Fluorophores and Beyond

“…Substituted quinazoline C5‐derivatives of uracil [77] exhibited molecular rotor properties ascribed to the biaryl bond. The fluorescence quantum yield increased in the more viscous solvent, glycerol, as compared to the lower viscosity solvents EtOH and THF.…”