Chapter 4. Fluorescence Studies of Hydroxy Indole Derivatives

Transcription

1 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives

2 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives Introduction Multiphoton excitation is particularly used to stimulate the same fluorescent species as obtained under single photon excitation but in the case of hydroxy indole derivatives the nature of luminescence from the two processes are entirely different. The single photon excitation of serotonin results in emission around 340 nm and its fluorescence mechanism is reasonably well understood. In contrast, the multiphoton excitation of serotonin and 5-hydroxytryptophan resulted in green luminescence around 500 nm. Shear et al. 132 first suggested that this multiphoton-induced hyperluminescence from excitation of serotonin might arise from the 5-indoloxyl radical absorbing at 420 nm consistent with the two photon excitation at 800 nm. This was further extended by Bisby and coworkers using both nanosecond ultraviolet laser and multiphoton excitation. In a two laser experiment 133 using a pump laser at 308 nm for photolysis of 5- hydroxytryptophan and a probe laser at 430 nm for further excitation, it has been shown by them an enhancement in the intensity of the green fluorescence with increasing pump-to-probe time delay. They have further investigated 135 the multiphoton excited fluorescence from serotonin complexed with β-cyclodextrin. The work of Bisby et al. 133 showed that the fluorescence in neutral solutions consists of a single component with a lifetime of 0.91 ns which is in good agreement with 0.8 ns reported by Gostkowski et al. 136 Identical emission spectra in several solvents were also reported 133 by them using multiphoton excitation at 740 nm and the fluorescence lifetime doubled in neutral buffered

3 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 82 D 2 O solutions and also increased in non-aqueous solvents. Contrary to the finding by Shear et al., 132 the Bisby group 133 reported that the 5-indoloxyl radical may not be the source of green fluorescence but suggested further detailed investigation of fluorescence lifetime measurements. In Chapter 3, results from the study of the free radical chemistry of hydroxy indole derivatives using pulse radiolysis and quantum chemical methods are discussed which provided an insight of its physicochemical properties such as kinetic parameters, characteristics of absorption spectra, relative stabilities of various intermediate products etc. In this Chapter, we report the work on multiphoton induced fluorescence of three hydroxy indoles: 5-hydroxyindole, 5-hydroxytryptamine and 5-hydroxytryptophan and Scheme 4.1 depicts their structures. 4 HO R 5 Hydroxyindole (HIn), R = H N 1 H 2 5 Hydroxytryptamine (HTpe), R = CH 2 CH 2 NH 2 5 Hydroxytryptophan (HTpn), R = CH 2 CH(COOH)NH 2 Scheme 4.1: Structures of 5-hydroxy indole derivatives used in this study. 4.2 Fluorescence from Multiphoton Excitation Excitation and Emission Spectra Emission spectra from multiphoton excitation (MPE) of three hydroxy indole derivatives by Ti-sapphire laser pulses were recorded in aqueous solutions and detected by CCD. The fluorescence emission at 500 nm was seen on multiphoton excitation at 750 nm in all three derivatives and Figure 4.1 depicts

4 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 83 their emission spectra on excitation of 5 x 10-3 mol dm -3 phosphate buffered solutions at ph 7. Also shown is the excitation spectrum with maximum at 750 nm in the case of HTpn. 1.0 b a Intensity (a.u.) 0.5 c d Excitation Wavelength/nm Figure 4.1: Emission spectra on multiphoton excitation of (a) HTpn, (b) HIn, (c) HTpe and (d) excitation spectrum of HTpn. As can be seen, the emission intensities are nearly identical in all three derivatives with maximum centred around nm, but the spectra in the case of HIn and HTpe have a shoulder at the lower and higher wavelengths suggesting that the emission may arise from different vibrational states. Our finding is in accord with the well known green fluorescence reported earlier on multiphoton excitation of these derivatives by Shear et al. 132 and Bisby et al Furthermore, the green emission was also recorded in both acidic and basic media. Figure 4.2 represents the spectra measured with HTpn at ph 1, 4, 7 and 9.

5 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 84 For the entire ph range investigated, maximum spectral intensity was seen in neutral solutions, which was lowered in both acidic and basic media. For example, a red shift of 25 nm from 515 nm and a 25% lowering of intensity at ph 4 was observed. But the effects are more drastic at both ph 1 and 9. A reduction in intensity by 50% in basic solutions whereas at ph 1 virtually a featureless spectrum with marginal intensity (20%) was seen. 1.0 c Intensity (a.u.) 0.5 d b a Wavelength/nm Figure 4.2: Emission spectra on multiphoton excitation at 750 nm for HTpn in acidic, neutral and basic solutions. ph: (a) 1 (b) 4 (c) 7 and (d) Fluorescence Decay Kinetics The fluorescence decay kinetics in all three derivatives were measured and, as an example, the decay time profile at 500 nm obtained in neutral solutions of HTpn is given in Figure 4.3a. The analysis of fluorescence lifetime measurements has shown a triple exponential decay in all three derivatives. The

6 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 85 fastest component among them having a lifetime of 50 ps is not considered because similar feature was seen from the solvent only sample. Since this is multiphoton excitation experiment, such fast contributions may arise from some trace impurities. 1.0 Intensity (a.u.) 0.5 a b Wavelength/nm Figure 4.3: Fluorescence decay profiles measured at 500 nm in HTpn at ph 7 in the a) absence and b) presence of ABTS 2-. It may be pointed out that the fluorescence emission when analysed either for single exponential as was reported earlier 133 or biexponential decay gave χ 2 2 and the analysis has not been satisfactory. Therefore, only the two slower decaying components of triple exponential with χ were taken into account. The lifetimes and amplitudes of the two components measured in all derivatives at different ph and in the presence of the reductant ABTS 2- are presented in Table 4.1.

7 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 86 The fit of the data reveals that the emission at 500 nm is mostly dominated by a major component with a yield of about 95% and τ 1 ns and a minor slower decaying component having τ 2 ns. The τ values of the former component were found to be 460, 670 and 850 ps in neutral buffered solutions of HIn, HTpe and HTpn respectively. Our measured value in HTpn is in good agreement with τ = 910 ps reported earlier 133. Furthermore, the lifetime seems to increase with substitution on side chain with a lowering by nearly 50% on going from HTpn to unsubstituted HIn. Table 4.1: Fluorescence lifetimes and relative amplitudes in 5-hydroxy indole derivatives at different ph and in presence and absence of ABTS 2-. Compd ph λ em /nm [ABTS 2- ]/10-6 mol dm -3 a 1 τ 1 /ps a 2 τ 2 /ns χ HTpn HIn HTpe Fluorescence decay profiles were also measured in acidic (ph 4) and basic (ph 9) HTpe solutions but the yields and lifetimes of the major component remained more or less unaffected. In contrast, the major component decayed much faster with τ = 280 and 160 ps in highly acidic solutions (ph 1) of HTpn

8 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 87 and HTpe, respectively. This is also manifest in the weak fluorescence intensity spectrum measured at ph 1 in HTpn (Figure 4.2). A few experiments were carried out to study the effect of the reductant ABTS 2- on the intensity and lifetimes of green fluorescence in the case of HTpn. As can be seen from Table 4.1, τ 1 in the absence of ABTS 2- and τ 2 in its presence are similar. Thus, the addition of (3 5) x 10-4 mol dm -3 ABTS 2- seems to introduce a new fluorescence decay channel (20%) occurring much faster with rates of about (350 ps) -1 which is expected to be dependent on ABTS 2- concentration if the quenching is controlled by diffusion. Otherwise, most of the HTpn fluorescence (80%) still decays with the same time constant as in the absence of ABTS 2- which is evident from the decay profile shown in Figure 4.3b Time-Resolved Fluorescence Measurements Since the time-resolved fluorescence (TRF) spectra provide wealth of information on the spectral dynamics, TRF measurements were carried out in all three derivatives. Their reconstructed spectra from such profiles measured at different wavelengths and ph are shown in Figure 4.4. When the fast 50 ps component is excluded, TRF decays at different wavelengths are nearly the same indicating the absence of any spectral dynamics. In other words, the spectral changes observed at early times must be attributed to the initial fast component which is assigned to the solvent or other artefacts. The absence of spectral dynamics indicates that the green fluorescence following the multiphoton excitation arises from a single chemical species and that origin of the structures in the emission spectra should be vibrational.

9 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 88 Intensity A 10 ps B 50 ps C 1 ps Wavelength/nm Figure 4.4: Time-resolved fluorescence spectra measured on multiphoton excitation of HTpn at (A) ph 4 (B) ph 7 and (C) ph Emission Mechanism Scheme 4.2 briefly depicts the general multiphoton emission mechanism in 5-hydroxy indole derivatives. Since our TRF data indicates the involvement of a single species, we propose that the origin of green fluorescence is due to the indoloxyl radical formed from the radical cation on multiphoton excitation (reactions 1 and 2). This is also in accord with the suggestion of Shear et al. 132 Our earlier work on radiation chemical oxidation of serotonin by one electron oxidant, Br 2 radical has also shown the direct formation of indoloxyl radical absorbing at 420 nm. The measured lifetimes of green fluorescence at 500 nm (reaction 3) were found to be in the range ns for the three derivatives in neutral solutions indicating their dependence on the substituent. In highly acidic solutions at ph 1, considerable quenching of the fluorescence was seen. The similar behaviour observed by Bisby et al. 133 was

10 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 89 attributed to the dynamic quenching process by proton. At ph 1, the indoloxyl radical (pk a ~ 3) is in the protonated form due to the stabilization of the radical cation and the quenching due to this alternative process (reaction 4) is a distinct possibility. This also supports the assignment of the green fluorescence to the indoloxyl radical. As pointed out before, the addition of ABTS 2- does not quench the green fluorescence significantly, because the indoloxyl radical remains largely unaffected (reaction 5). This is not surprising because reduction potentials of ABTS 2 (0.63 V) 122 and the indoloxyl radical (0.64) 72 are nearly the same. Accordingly, our earlier radiation chemical work has shown only marginal scavenging of indoloxyl radical by ABTS 2-. Green Fluorescence τ ~ 0.35 ns τ ~ 1 ns ~20% Encounter complex ~80% 5 +ABTS 2- HO N R HO R MPE/-e - + -H + 1 N 2 O N R H 4 H 3 H Quenching of Emission Green Fluorescence τ ~ ns Scheme 4.2: Fluorescence mechanism in hydroxy indole derivatives following multiphoton excitation

11 Chapter 4 Fluorescence Studies of Hydroxy Indole Derivatives 90 The results on the TRF measurement have shown that the fluorescence decay at all wavelengths is nearly identical suggesting the involvement of a single species. Since the dimer radical formation from the reaction of indoloxyl radical with the substrate molecule occurs at least on a few tens of microsecond timescale, it is reasonable to assume that the green fluorescence originates from the monomeric indoloxyl radical. 4.4 Conclusions Ultrafast laser spectroscopy studies were conducted to study the features of the green fluorescence at 500 nm on multiphoton excitation of 5-hydroxy indole derivatives. The emission in neutral solutions is characterized by a major component (95%) with a time constant of 460 ps in unsubstituted indole which increased to 850 ps in substituted HTpn. Significant fluorescence quenching was seen on going from neutral to highly acidic solutions (ph 1). Neither fluorescence intensities nor lifetimes were affected on addition of reductant ABTS 2-. The fluorescence seems to originate from the indoloxyl radical formed on multiphoton excitation.