Synthesis and ion selectivity studies of potential fluorescent heavy metal ion indicators

Two new potential heavy metal ion indicators of the phenanthroline-based family, displaying distinct fluorescence profiles, were synthesized. Their ion selectivity is discussed in terms of their ionophore/fluorophore properties and the extent of conjugation in their framework. The 2-(2-hydroxyphenyl)benzazole containing fluorophores used in the construction of the probes, exhibit a unique fluorescence profile with a high Stokes shift that is attributed to an excited state intramolecular proton transfer (ESIPT).


Introduction
Fluorescent sensors are powerful tools, used in the in vitro and/or in vivo monitoring of biologically relevant species such as metal ions, because of the simplicity and high sensitivity of fluorescence spectroscopy. 1A typical fluorescent sensor contains a recognition site, in this case the ionophore, linked to a fluorophore which translates the recognition event into a fluorescence signal. 2][5][6][7] The selection of the fluorophore required more careful consideration: a large Stokes shift is a highly desired characteristic for a fluorescent probe since it permits an efficient separation of the light exciting the matrix and the light dispersed by the sample. 8Excited-state intramolecular proton transfer (ESIPT) is a reaction that can produce emissive species with sizeable, often by 100 nm or more, long wavelength shifts on fluorescence spectra.The ESIPT process generally involves a hydroxyl proton transfer to an acceptor in the excited state, resulting in tautomer emission with a large Stokes shift. 92-(2-Hydroxyphenyl)benzazoles are handy fluorescent molecules which show high Stokes shifts owing to this mechanism (Scheme 1).Scheme 1. Prototautomers involved in excited-state intramolecular proton transfer (ESIPT).

Results and Discussion
1][12] The amines were converted into the respective isothiocyanates 4a and 4b by treatment with excess thiophosgene in acetone at ambient temperature.Sensors 5 and 6 were prepared by mixing the isothiocyanates with 5-amino-1,10-phenanthroline in DMF in the presence of catalytic amounts of 4-diisopropylethylamine (Scheme 2).
Fluorescence studies on sensor 6 in the presence of increasing Pb 2+ concentrations revealed the profile of a photoinduced electron transfer (PET) indicator with a distinct "turn on" response in low micromolar Pb 2+ ion levels, with a large Stokes shift of 170 nm (Figure 1).Similar responses have been reported previously by our group 13 and others, 10 and are interpreted based on the analogous 2-(2-benzoxazolyl)-and 2-(2-benzothiazolyl)phenol systems that undergo an intramolecular proton transfer at the excited state (ESIPT).][16][17][18][19] This effect is responsible for the observed high Stokes shift.Upon excitation of the ion-free probe 6 an electron is transferred from the phenanthroline moiety to the fluorophore and fluorescence is quenched.At micromolar concentrations, the Pb 2+ ions coordinate with the nitrogen atoms of the phenanthroline system preventing the electron transfer, thus leading to a "turn on" response mode of the sensor 6 to the presence of Pb 2+ ions.][22]  M Pb +2 5x10 -5 M Pb +2 4x10 -5 M Pb +2 3x10 -5 M Pb +2 2x10 -5 M Pb +2 1x10 -5 M Pb +2 free Binding of sensor 6 to a host of metal ions including Cd 2+ , Cu 2+ , Fe 3+ , Hg 2+ , Mg 2+ and Mn 2+ was registered by strong fluorescence quenching.This "turn off" spectral response is known for phenanthroline-type probes such as Phen Green FL and Phen Green SK. 4 The only exception was that of Al 3+ interaction that resulted in a fluorescence decrease at 540 nm with a simultaneous increase at 450 nm and a clear isoemissive point at 480 nm.A possible explanation of this behavior is that the Al 3+ ions would coordinate with the phenolic oxygen influencing the chromophore and therefore cancelling the ESIPT mechanism.Owing to this effect, a blue shift in the emission spectrum was observed, the Stokes shift decreasing from 185 to 95 nm.This effect is shown on Figure 2 depicting a ratiometric-type fluorescence response of the sensor 6 at high micromolar Al 3+ ion concentrations.The fluorescence behavior of sensor 5 to a host of ions including Pb 2+ , Cd 2+ , Cu 2+ , Fe 3+ , Fe 2+ , Hg 2+ , Mg 2+ , Mn 2+ , Co 2+ and Al 3+ is presented on Table 1.The response of the sensor was in all cases a "turn off" one, with dissociation constants in the micromolar range.The ion-free probe exhibits a λ exc maximum of 360 nm with a respective λ em at 554 nm and a quantum yield of Φ = 0.025.Given the structural similarity of sensors 5 and 6, the large (194 nm) Stokes shift of the former sensor is, as expected, the result of the ESIPT mechanism.

Metal-ion response screening for ion probes 5 and 6
A graphical overview of the relative responses of the two sensors to 30 µM metal ion solutions is depicted in Figure 3.The results are plotted as fluorescence changes relative to those of the ionfree reference solutions of sensors 5 or 6, expressed as (F-Fo)/Fo, where F is the fluorescence intensity of ion-containing solutions and Fo is the fluorescence intensity of each reference solution.Blue bars indicate the response to 30 µM ion concentration.Worth noticing is the "turn-on" type of response of sensor 6 in the presence of Pb 2+ ions as opposed to the rest of the ions studied, a behavior that is not observed in the case of sensor 5.This difference in response should rest in the inherent properties of both sensors.Pb 2+ is a "soft" metal ion and would favor a stronger coordination with the "soft" sulfur atom in the benzothiazole moiety in sensor 5, whereas a weaker interaction of Pb 2+ with the "hard" oxygen in sensor 6 is expected.It is therefore, possible that the stronger interaction of Pb 2+ with the sulfur ion in sensor 5 would result in a decrease in binding with the phenanthroline nitrogens attenuating its interference with the electron transfer process.Moreover, the availability of either heteroatom is retained even upon excitation of the compounds as shown in Scheme 3.This selectivity towards the Pb 2+ ions in phenanthroline probes where O-donor atoms are replaced by other softer donor atoms such as S and N is well known. 23,24heme 3. Prototautomers involved in excited-state intramolecular proton transfer (ESIPT) of compounds 5 or 6.Structural changes that take place upon excitation do not alter the availability of heteroatom X for metal ion coordination.

Conclusions
Two new small-molecule sensors were prepared via short and efficient syntheses.The benzoxazole-phenanthroline type probe 6 exhibited a "turn-on" behavior selectively for Pb 2+ ions and a ratiometric profile response to Al 3+ ions; the latter response however, takes place at 45-fold higher concentrations.The response of probe 6 to the rest of metal ions studied is a "turn off" one with relatively smaller responses, thus rendering 6 a potential lead ion sensor in aqueous solutions.The benzothiazole congener 5 was a "turn off" probe responding to the presence of metal ions with up to 80% decrease in fluorescence intensity, with Cu 2+ , Fe 3+ , Fe 2+ , Hg 2+ , and Mg 2+ inducing the largest responses.All studies were performed in aqueous solutions, a fact implying that both sensors may be practical tools in analytical measurements and the detection of ions in biological studies.

Experimental Section
General.All reactions were carried out under anhydrous conditions in dry solvents, using argon or nitrogen in flame-dried glassware.Reactions were monitored by thin-layer chromatography (TLC) using silica gel plates from Merck (60F 254 ), which were visualized under a UV-vis Lamp (254 and 366 nm, respectively) or with a 7% ethanolic solution of phosphomolybdic acid.Flash column chromatography was performed in silica gel 60 from Merck (230-400 mesh).The Attenuated Total Reflection (ATR)-FTIR spectra were recorded on a Thermo-Electron Nicolet 6700 FTIR optical spectrometer with a DTGS KBr detector at a resolution of 4 cm -1 .NMR spectra were taken on an AMX500 Bruker FT-NMR or a MSL300 Bruker FT-NMR spectrometer; proton chemical shifts are reported in relative to tetramethylsilane.Fluorescence spectra were recorded on an Aminco Bowman spectrofluorimeter (Spectronics Co., USA).HRMS were taken at ProFI, Foundation for Research and Technology-Hellas (ITE), Heraklion, Greece.Ultra-pure water was collected from a PURELAB Ultra instrument by ELGA.

Preparation of indicator solutions containing adjusted ion concentrations
In 3 mL nanopure water (pH = 7) were added 15 µL aliquots of a 10 mM in DMSO (≥99.5%)dye solution to make a final indicator concentration of 5 µM.To this solution were added microliter aliquots of metal ion stock 10 -2 M solutions to yield a set of broad ion concentration range dye solutions.

Table 1 .
Spectral profile of sensors 5 and 6 in the presence of increasing ion concentrations a K d values were detected in cases where the decrease in emission intensity of sensors 5 or 6 in the presence of increasing ion concentrations was distinct.b nd = not detected.