Using biomimetic chemical reduction or Clostridium perfringens cell extract containing azoreductase, the dimer-fluorescent probe 2,4-O-bisdansyl-6,7-diazabicyclooct-6-ene, which possesses a conformationally constrained cis-azo bridge, is reduced to the tetra-equatorial 2,4-O-bisdansyl-cyclohexyl-3,5-bisammonium salt which exhibits fluorescence indicative of a dansyl monomer.

The intramolecular heterodimer probe 4-O-dabsyl-2-O-dansyl-myo-inositol-1,3,5-orthoformate undergoes biomimetic reduction of the azo bond in the dabsyl group to give a fluorescence emission band at 560 nm. This is attributed to the loss of energy transfer between the fluorophore (dansyl) and quencher (dabsyl) groups as a result of the formation of 4-O-(4-aminobenzene sulfonyl)-2-O-dansyl-myo-inositol-1,3,5-orthoformate.

This research describes the effects of structural variation and medium effects for the novel split-oligonucleotide (tandem) probe systems for exciplex-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the exciplex partners and the consequent exciplex fluorescence emission on excitation. The model system consists of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide is equipped with functions able to form an exciplex on correct, contiguous hybridization. This study investigates the influence of a number of structural aspects (i.e. chemical structure and composition of exciplex partners, length and structure of linker groups, locations of exciplex partner attachment, as well as effects of media) on the performance of DNA-mounted exciplex systems. The extremely rigorous structural demands for exciplex formation and emission required careful structural design of linkers and partners for exciplex formation, which are here described. Certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplexes, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. The exciplexes formed emitted at ∼480 nm with large Stokes shifts (∼130–140 nm). Comparative studies with pyrene excimer systems were also carried out.

Organic intramolecular exciplexes, N-(4-dimethylaminobenzyl)-N-(1-pyrenemethyl)amine (1) and N′-4-dimethylaminonaphthyl-N-(1-pyrenemethyl)amine (2), were used as model systems to reveal major factors affecting their exciplex fluorescence, and thus lay the basis for developing emissive target-assembled exciplexes for DNA-mounted systems in solution. These models with an aromatic pyrenyl hydrocarbon moiety as an electron acceptor appropriately connected to an aromatic dimethylamino electron donor component (N,N-dimethylaminophenyl or N,N-dimethylaminonaphthyl) showed strong intramolecular exciplex emission in both non-polar and highly polar solvents. The effect of dielectric constant on the maximum wavelength for exciplex emission was studied, and emission was observed for 1 and 2 over the full range of solvent from non-polar hydrocarbons up to N-methylformamide with a dielectric constant of 182. Quantum yields were determined for these intramolecular exciplexes in a range of solvents relative to that for Hoechst 33258. Conformational analysis of 1 was performed both computationally and via qualitative 2D NMR using 1H-NOESY experiments. The results obtained indicated the contribution of pre-folded conformation(s) to the ground state of 1 conducive to exciplex emission. This research provides the initial background for design of self-assembled, DNA-mounted exciplexes and underpins further development of exciplex-based hybridisation bioassays.

Using biomimetic chemical reduction or Clostridium perfringens cell extract containing azoreductase, the dimer-fluorescent probe 2,4-O-bisdansyl-6,7-diazabicyclooct-6-ene, which possesses a conformationally constrained cis-azo bridge, is reduced to the tetra-equatorial 2,4-O-bisdansyl-cyclohexyl-3,5-bisammonium salt which exhibits fluorescence indicative of a dansyl monomer.

This research describes the effects of structural variation and medium effects for the novel split-oligonucleotide (tandem) probe systems for exciplex-based fluorescence detection of DNA. In this approach the detection system is split at a molecular level into signal-silent components, which must be assembled correctly into a specific 3-dimensional structure to ensure close proximity of the exciplex partners and the consequent exciplex fluorescence emission on excitation. The model system consists of two 8-mer oligonucleotides, complementary to adjacent sites of a 16-mer DNA target. Each probe oligonucleotide is equipped with functions able to form an exciplex on correct, contiguous hybridization. This study investigates the influence of a number of structural aspects (i.e. chemical structure and composition of exciplex partners, length and structure of linker groups, locations of exciplex partner attachment, as well as effects of media) on the performance of DNA-mounted exciplex systems. The extremely rigorous structural demands for exciplex formation and emission required careful structural design of linkers and partners for exciplex formation, which are here described. Certain organic solvents (especially trifluoroethanol) specifically favour emission of the DNA-mounted exciplexes, probably the net result of the particular duplex structure and specific solvation of the exciplex partners. The exciplexes formed emitted at ∼480 nm with large Stokes shifts (∼130–140 nm). Comparative studies with pyrene excimer systems were also carried out.