Exploring a novel class of fluorophores: the 2,5-dihydro-1,2,3-triazines
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The 1,2,3-triazines, also known as the v-triazines, are the least studied of the three classes of triazine. This is due to the ring’s lower stability and more limited synthetic routes. A new class of fluorophores, based on a 1,2,3-triazine core with substituted aryl groups, has been synthesized . Here we study the photophysical behaviour of one derivative, 5-methoxycarbonyl-5-(N-phenylformimidoyl)-2,4,6-triphenyl-2,5-dihydro-1,2,3-triazine (5MPFH TDT). In most solvents, 5MPFH TDT has a strong absorbance band at 310 nm (S2 excitation) with a weaker band at ~400 nm associated with excitation into S1. 5MPFH TDT fluorescence is complex with three distinct emission bands centred at 520 nm when excited at 405 nm. These three fluorescence emission bands are a consequence of three conformers (g0, g1, and g2) that are vibrationally accessible. Inter-conversion between these conformers occurs in the excited state. The shape and position of these three emission bands have been shown to be fairly constant and no strong solvatochromism is observed for a wide range of organic solvents . However, 5MPFH TDT is very hydrophobic and has a high tendency to aggregate in aqueous solutions, which is problematic as we are exploring the use of 5MPFH TDT as a protein label. To improve solubility in aqueous solutions we studied the use of a range of non-ionic, anionic, and cationic surfactants to solubilize 5MPFH TDT. 5MPFH TDT emission properties in the various micelles were studied in detail as a simple model for its behaviour in protein solutions. Its steady-state fluorescence emission was found to be very stable in all non-ionic and cationic surfactant solutions above the critical micelle concentration (CMC). Though the shape of each fluorescence emission band changed very little, the fluorescence intensity increased and the three fluorescence emission bands were slightly blue-shifted with higher surfactant concentration. The fluorescence anisotropy and fluorescence lifetime also increased significantly with increasing surfactant concentration indicating that the fluorophore had been incorporated within the micelle. Conversely, 5MPFH TDT fluorescence was found to be quenched in anionic surfactants. The hydrophobic effect seems to be the driving force behind the interaction between the triazine and the micelles and the strength of this interaction was found to be comparable with some much smaller, planar fluorophores. The strong interaction between 5MPFH TDT and the micelle aggregates meant it was a suitable fluorophore for the estimation of micelle size. 5MPFH TDT and SR101 were used as a FRET pair for the estimation of the size of CTAB micelles and the donor-acceptor distance calculated corresponded closely to the radius of a CTAB micelle. The quenching effect of SR101 on 5MPFH TDT fluorescence was modelled by a Perrin plot indicated that a quenching “sphere-of-action” was in operation. Since 5MPFH TDT interacted quite strongly with micelles it seemed likely that it would also interact well with BSA. A clear blue shift and high fluorescence anisotropy marked the interaction between fluorophore and protein. A BSA–5MPFH TDT binding stoichiometry of 2:1 was determined from a Job plot analysis, suggesting that the triazine was initiating the formation of BSA dimers by conferring a greater degree of hydrophobicity to the protein surface. However, the rotational correlation time of the triazine (15.51 (±7.07%) ns) was considerably less than the value reported in the literature for BSA  indicating that the mobility of the triazine, as part of the BSA–5MPFH-TDT complex, was not overly restricted. The location of 5MPFH TDT on the protein surface would explain the greater level of mobility indicated by the rotational correlation time calculated.
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