Design, synthesis and fluorescent characterisation of 2,5-dihydro-1,2,3-triazines

The extrinsic labelling of biomolecules for identification and/or quantification is a common methodology used in the lifesciences. This labelling, for example, can be carried out with (i) radioactive markers or (ii) compounds with absorption and/or fluorescence properties. With the advent of confocal microscopy and live cell imaging, fluorescence offers the tools to visualise and quantify the chemical and biochemical processes in living cells. The subject of this thesis concerns the synthesis and investigation of the fluorescence of a new class of organic fluorophores; the 2,5-dihydro-1,2,3-triazines. A series of fluorescent 2,5-dihydro-1,2,3-triazines were synthesized via an interesting Huisgen 1,3-dipolar cycloaddition between 2,4,5-triphenyl-1,2,3-triazolium-1-arylaminide 1,3-dipoles and terminal alkyne propiolates. The initial cycloadduct undergoes a sigmatropic rearrangement followed by a ring expansion to generate the 2,5-dihydro-1,2,3-triazine fluorophore. The synthesis of 2,5-dihydro-1,2,3-triazine fluorophores with different structural changes, and the affect these changes had on the photophysics of the fluorophores was investigated. This was undertaken with a view to better understanding the fluorescence of this new class of fluorophore and to developing a fluorophore capable of conjugating to biomolecules. Several modifications were explored including (i) substituting the imine phenyl and N2 phenyl rings with heavy atoms, (ii) changing the methyl ester to a methyl ketone or 4-substituted phenyl ester, (iii) varying the substituent on the imine carbon and (iv) removing the phenylimine group. In collaboration with Dr. Alan G. Ryder at NUI Galway and Prof. Luke A. Burke at Rutgers University, New Jersey; a full fluorescence charaterisation was performed building on preliminary photophysical studies. Steady state ultraviolet absorption, fluorescence spectroscopy and time domain lifetime measurements were conducted in order to elucidated a model of fluorescence for the 2,5-dihydro-1,2,3-triazines. These measurements in combination with TD-DFT calculations (relative energies, dipole moments, oscillator strength and calculated transition wavelength), courtesy of Prof. Luke A. Burke, allowed us to generate a photophysical model that explains the fluorescence of the 2,5-dihydro-1,2,3-triazines. It was found that three structures may exist for the 2,5-dihydro-1,2,3-triazines: g0 (optimized for S0), g1 (optimized for S1) and, g2 (optimized for S2) and that the positioning of the imine and carbonyl ester on the triazine is very important in dictating the fluorescent and electronic properties. It was found that the phenylimine group is essential for the long lived fluorescence of the 2,5-dihydro-1,2,3-triazines. Furthermore, we propose that in order to maintain fluorescence the phenylimine group must have substituents small enough to allow the imine group to remain in plane with the carbonyl group. The energies of the excited states appeared to be affected by the interaction of the carbonyl with the imine hydrogen and by changes in electron density at the carbonyl group. A significant red shift was observed in the fluorescence emission in both cases where the methoxy group on the ester carbonyl was replaced with a less potent electron donating group or an electron withdrawing group e.g. methyl or benzotriazole group. An extensive solvent fluorescent study was performed; ultraviolet absorption, fluorescence spectroscopy and time domain lifetime measurements. It was found that the 2,5-dihydro-1,2,3-triazine fluorophores were not affected by the solvent or microenvironment as no solvatochromism was observed in both the ground and excited state. The photostability of triazine 3a in toluene was proven to be exceptional when placed under constant irradiation at 310 nm excitation in a spectrometer. When 3a in toluene was exposed to intense UV illumination in the dark over a 6 hour period, the photostability was slightly greater than when compared to a standard, quinine sulphate in 0.1M sulphuric acid. The fluorophores are also not susceptible to collisional quenching by molecular oxygen. Degassing of a solution of triazine 3a in toluene and measurement of its time correlated photon counting lifetime over a 35 minute only resulted in a 3 % increase in the intensity weighted average lifetime, as was also the case for triazine 10a. Substituting the 4-position of the imine phenyl and N2 phenyl rings with heavy atoms had very little effect on the molecule’s energy levels, and the heavy atom effect was observed to be small in these cases. Changing from a methyl ester to a family of 4-substituted phenyl esters appeared to have only minor influence on the photophysics, supported by computational calculations by Dr. Elisa Fadda (NUI Maynooth). Although, the 4-NO2 phenyl ester did display some solvent sensitivity, we believe that this is due to the NO2 group itself interacting with the solvent system as opposed to its affect on the ester group. The synthesis of propiolates containing stable activating groups (for coupling reactions) was performed. Their subsequent use in the 1,3-dipolar cycloaddition provided access to 2,5-dihydro-1,2,3-triazines containing the activating groups. These activated triazines are capable of conjugation to biomolecules. This approach generated an acylbenzotriazole and an isothiocyanate activated 2,5-dihydro-1,2,3-triazine. The acylbenzotriazole 2,5-dihydro-1,2,3-triazine was successfully coupled with L-alanine