A naphto[2,1-b]furan as a new fluorescent label
Table 2. UV-visible absorption and fluorescence data for amino acids 2a-j
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- Bu səhifədəki naviqasiya:
- Spectrophotometric and spectrofluorimetric titrations with ions
- Figure 3.
- Table 3.
Table 2. UV-visible absorption and fluorescence data for amino acids 2a-j in ACN, ACN/H2O (9:1) and EtOH//H2O (9:1).
Spectrophotometric and spectrofluorimetric titrations with ions The new (bi)thienyl amino acids 2a-j were tested for their ability to act as fluorescent chemosensors in the recognition of biomedically relevant ions by performing spectrophotometric and spectrofluorimetric titrations in ACN and ACN/H2O (9:1), in the presence of relevant organic and inorganic anions (AcO-, F-, Cl-, Br-, I-, ClO4-, CN-, NO3-, BzO-, OH-, H2PO4- and HSO4-) and of alkaline, alkaline-earth and transition metal cations (Na+, K+, Cs+, Ag+, Cu+, Cu2+, Ca2+, Cd2+, Co2+, Pb2+, Pd2+, Ni2+, Hg2+, Zn2+, Fe2+, Fe3+ and Cr3+). As stated previously, the introduction of a UV-active and fluorescent heterocyclic unit at the side chain of the amino acid is expected to provide additional binding sites for a variety of ions. A preliminary evaluation of the chemosensing ability was performed by addition of 100 equiv of each cation/anion to solutions of amino acids 2a-j in acetonitrile and the changes in the intensity of the UV-vis absorption and fluorescence spectra were recorded. In the UV-vis absorption spectra of the various amino acids in the presence of each tested ion, no changes were seen in the bands corresponding to the maximum wavelength of absorption, except for methoxybithienyl amino acid 2i in the presence of Cu2+. It was found that this amino acid is a very sensitive and selective colorimetric chemosensor for Cu2+ as it displayed a marked colour change from pale yellow to pink. Among all the other cations tested, only Cu+ induced a minor pink coloration (Figure 3, top) that was negligible compared to that of Cu2+. The spectrophotometric titration with Cu2+ revealed that, upon addition of increasing amounts of the cation, the band at 327 nm decreased, accompanied by the appearance and increase of a new red-shifted band at 529 nm (Figure 3, bottom). The same preliminary test was carried out in order to assess the changes (band shift and/or intensity) in the fluorescence spectra of the various amino acids in the presence of each tested ion. The nitro derivative 2g was not tested since it was practically non-fluorescent. This test revealed the ability of compounds 2a-f,h-j to interact especially with the more basic anions F- and OH- and with Cu2+ and Fe3+, with different sensitivity (the amount of ion necessary to induce changes in the fluorescence spectra depending on the compound). The sensing ability for anions was lower (requiring more equivalents for a significant fluorescence quenching, ca. 80-90%) than for cations (which required less equivalents for a complete quenching). 
Figure 3. (top) Colour changes of bithienyl amino acid 2i in acetonitrile (1.0 × 10-4 mol dm-3) in the presence of 10 equiv of the various metal cations; (bottom) Spectrophotometric titration with Cu2+ (up to 5 equiv) in acetonitrile. In the case of methoxybithienyl amino acid 2i, chosen as representative example, in the spectrofluorimetric titrations with F- and OH-, upon addition of the anion it was visible the appearance and increase of a new band at 484 nm suggesting the formation of the deprotonated form of the amino acid due to the basicity of the anions. In the spectrofluorimetric titrations with Cu2+ and Fe3+, a considerable decrease of the fluorescence intensity was observed for (bi)thienyl amino acids 2a-f,h-j, with a small number of metal equivalents being necessary to completely quench fluorescence (Figure 4 for the titration of 2i with F-, OH-, Cu2+ and Fe3+). Also, for some amino acids the addition of much larger amounts (more than 100 equiv) of Hg2+ (2h) and Pd2+ (2b-f and 2h) induced considerable but incomplete quenching. 
Figure 4. Fluorimetric titrations of bithienyl amino acid 2i with F- (A), OH- (B), Cu2+ (C) and Fe3+ (D), in acetonitrile [λexc = 327 nm]. Inset: normalised emission at 402 nm and 484 nm, as a function of added ion equivalents. Association constants (Kass) between several amino acids and some selected ions were calculated from the spectrofluorimetric titration data with HypSpec program. The results suggested the formation of a ligand-metal(anion) complex with 2:1 stoichiometry (which was confirmed with Job’s plots) and it was found that the new amino acids bind preferentially to Fe3+ and Cu2+ (Table 3 for anions and table 4 for cations). Although it cannot be stated that the new amino acids are selective for any cation, they display higher sensitivity for iron and copper as seen by the larger association constants. Moreover, the Kass obtained for the bithienyl amino acids 2i and 2j are higher than the corresponding thienyl amino acids 2c (bearing a methoxy group) and 2f (bearing a cyano group), showing the effect of the additional sulphur donor atom on the coordination ability. Previous studies on other heterocyclic amino acids have shown that free carboxylic and amino terminals did not influence significantly the coordination process, which should preferably occur through the heteroatoms at the side chain of the amino acids (Esteves et al. 2010). Table 3. Logarithm of association constants (log Kass) for the interaction of (bi)thienyl amino acids 2c-f,h-j with several anions in acetonitrile (ligand:anion stoichiometry 2:1).
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