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Oct 30, 2014 - ABSTRACT: The photophysics and photochemistry of norfloxacin (NF) have been investigated in aqueous solutions of different pH using ...
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Femtosecond Transient Absorption Spectroscopy Study of the Early Events of Norfloxacin in Aqueous Solutions with Varying pH Values Tao Su,†,‡ Ming-De Li,*,† Jiani Ma,† Naikei Wong,† and David Lee Phillips*,† †

Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China Department of Molten Salt Chemistry and Engineering, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 2019 Jia Luo Road, Jiading District, Shanghai, P. R. China 201800

‡

S Supporting Information *

ABSTRACT: The photophysics and photochemistry of norfloxacin (NF) have been investigated in aqueous solutions of different pH using femtosecond transient absorption spectroscopy (fs-TA). Resonance Raman spectroscopic experiments on NF have also been conducted in aqueous solutions of different pH to characterize the vibrational and structural information on the initial forms of NF. The experimental results in combination with density functional theory calculations of the key intermediates help us to elucidate the early events for NF after photoexcitation in aqueous solutions with varying pH values. The fs-TA results indicate that NF mainly underwent photophysical processes on the early delay time scale (before 3 ns), and no photochemical reactions occurred on this time scale. Specifically, after the irradiation of NF, the molecule reaches a higher excited singlet Sn and then decays to the lowest-lying excited singlet state S1 followed by intersystem crossing to transform into the lowest-lying triplet state T1 with a high efficiency, with an exception that there is a lower efficiency observed in basic aqueous solution due to the generation of an intramolecular electron transfer as an additional pathway to waste energy.

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at the carbon-7 of quinolone,21 is a hydrophobic broadspectrum antibacterial drug with a half-life of 3.0−4.5 h.22 It shows higher antibacterial activity and a broader substrate scope due to the presence of the piperazinyl ring, compared with other members in the same drug family devoid of this ring.23 However, it has been reported that NF is able to induce phototoxicity in animals and humans,24 which was initially speculated to be mediated by photosensitization mediated by singlet oxygen but was recently revised to be the result of a defluorination reaction. Although the latter is an uncommon reaction for fluoroaromatics due to the fairly high dissociation energy of the carbon−fluorine bond (up to ∼120 kcal/ mol),25,26 it has been established that heterolytic C−F bond cleavage is more efficient for the tautomeric form than for the cationic or anionic form in some molecules.27 NF is regarded to be an amphoteric substrate with two proton binding sites, namely, the 4′-nitrogen of the piperazinyl ring and the quinolone carboxyl group, and hence, there are also two main protolytic equilibria, with pKa values of approximately 8.5 and 6.2, respectively.16,18,28 As such, the species present in solution can take four formsthe neutral form, NF(N); anionic form, NF(A); cationic form, NF(C); and tautomeric form, NF(T) (see Figure 1)depending on the pH of aqueous solution,

INTRODUCTION Fluoroquinolone compounds1,2 (FQs, 7-dialkylamino-6-fluoroquinol-4-one-3-carboxylic acids) are antibacterial agents that have been used widely in the treatment of respiratory and urinary tract infections. FQs are a family of molecules that were first introduced into clinical therapies in the 1980s. FQs are active against a wide range of aerobic Gram-positive and -negative organisms.1,2 The photophysical and photochemical behaviors of FQs have recently gained great interest in phototoxicity research because of emerging evidence for their photomutagenic and photocarcinogenic properties.3,4 In general, these drugs are responsible for the deleterious effects observed in sunlight-induced cutaneous reactions, and some of them have been demonstrated to operate as phototoxicity inducers.5,6 Although the photochemistry and photobiology of FQs has become a subject of intense scrutiny,7−19 some properties of FQs are not fully understood. Likewise, the mechanistic processes underlying FQs photoexcitation and photosensitization remain to be clarified. Decades of works in this area have established that photodegradation of FQs in aqueous solutions typically leads primarily to defluorination and cleavage of the cyclic amino substituent at C-77,9,20 and that the degradation reaction(s) occurs via a triplet excited state for monofluorine FQs and probably singlet/triplet excited state for difluorine FQs.11,14 Among FQs, norfloxacin [1-ethyl-6-fluoro1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid, NF], first synthesized in 1980 by introducing a piperazine ring © XXXX American Chemical Society

Received: July 5, 2014 Revised: October 3, 2014

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were used to characterize and determine the form(s) of NF that is/are photoexcited under different conditions. The highest photodegradation efficiency was found for NF in neutral water at pH 7.2 as determined by previous investigations. Therefore, more attention was focused on the characterization of NF in neutral water in this work. In order to elucidate the photophysical events, femtosecond transient absorption (fsTA) spectroscopy was first used to explore the very early stages of the photophysics of NF in aqueous solutions with varying pH values, which can supply important information for an improved understanding of the subsequent photochemical reactions.

which lead the photochemistry of NF also to present significant pH dependence.

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EXPERIMENTAL AND COMPUTATIONAL METHODS Materials. NF was obtained from Aldrich (>98% purity) and was used as received. Sample solutions at different pHs of NF were adjusted by addition of sodium hydroxide and perchloric acid. The sample solutions used in the resonance Raman spectroscopy experiments were prepared at a concentration of 1.0 mM. Femtosecond Transient Absorption Spectroscopy (fsTA) Experiments. The fs-TA experiments were done by employing an experimental setup and methods detailed previously,29 and only a brief description is provided here. fsTA measurements were done using a femtosecond regenerative amplified Ti:sapphire laser system in which the amplifier was seeded with the 120 fs laser pulses from an oscillator laser system. The laser probe pulse was produced by utilizing ∼5% of the amplified 800 nm laser pulses to generate a white-light continuum (320−800 nm) in a CaF2 crystal, and then this probe beam was split into two parts before traversing the sample. One probe laser beam goes through the sample while the other probe laser beam goes to the reference spectrometer in order to monitor the fluctuations in the probe beam intensity. For the experiments discussed in this work, a 60 mL solution was flowed through a 2 mm path length cuvette. This flowing sample was then excited by a 267 nm pump laser beam. An absorbance of 1 at 267 nm was used for the sample solutions for the fs-TA experiments in order to maintain the same number of photons being absorbed for the same irradiating conditions for the samples. Resonance Raman Spectroscopy Experiments. Resonance Raman spectroscopy experiments were conducted with an established experimental apparatus and methods detailed in previous publications.30 Briefly, a 273.9 nm laser pulse generated from the second anti-Stokes hydrogen Raman shift laser line generated from the fourth harmonic of a Nd:YAG nanosecond pulsed laser was used for probe laser pulse. The energy for the laser pulse was typically in the 0.3−0.6 mJ range with a 10 Hz repetition rate. The laser beam was focused onto the flowing sample. By using reflective optics, the Raman scattered light was collected into a spectrometer whose grating dispersed the light onto a liquid nitrogen-cooled CCD detector. The Raman signal was accumulated for 30 s by the CCD before acquisition on an interfaced PC computer, and subsequently, 10−20 scans of the signal were added up to generate a resonance Raman spectrum. The Raman shifts of the spectra were calibrated by the known acetonitrile (MeCN) solvent Raman bands with an estimated accuracy of 5 cm−1. Density Functional Theory Computations. DFT calculations were performed by the Becke three-parameter hybrid method with the Lee−Yang−Parr correlation functional

Figure 1. Schematic diagrams of the structures of the four prototropic forms of NF present in aqueous solutions.

The complicated ground state forms and molecular structure of NF results in its exhibiting versatile photophysical and photochemical variety. Albini and co-workers10 found that NF could undergo different degradation routes in diverse conditions. NF will produce the corresponding 6-hydroxy derivatives by nucleophilic substitution of fluorine at position C-6 by a hydroxyl group (see Scheme 1)10 by irradiation in Scheme 1. Proposed Overall Photo-Defluorination Reaction of NF in Neutral Water

neutral water at pH 7.2 and with lower efficiency at pH 4.5 and 10. When the pH value is lower than 1, no defluorination occurs, but the piperazinyl side chain is degraded.27 It is thus essential to examine the early photophysical processes that can supply evidence for the direct determination of the precursors for the photochemical reactions under different pH conditions. In addition, through the use of absorption and emission experiments, Miranda and co-workers9,16 found that, other than the intersystem crossing (ISC) process for singlet excited-state, intramolecular electron transfer (IET) from the N(4′) atom of the piperazinyl ring to the FQ main system is also an efficient energy-wasting pathway that is dramatically enhanced in basic media and in nonprotic organic solvents such as acetonitrile.9,16 Bosca and co-workers reported the first clear example of excimer detection in aqueous media and studied electron transfer reactions between NF and naproxen, tryptophan, etc., and photophysical properties of NF.17 Although numerous studies have attempted to characterize the transient species and photophysical transformations of NF, there are several important issues that remain unaddressed, specifically, (1) there are few clear identifications of the form(s) of the NF molecule actually being excited in neutral aqueous solution and (2) femtosecond time-resolved spectroscopies have not yet been used to elucidate the initial photophysical events for NF in aqueous solutions with varying pH values after photoexcitation. In the present work, resonance Raman spectroscopy and density functional theory (DFT) calculations B

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water. A comparison of this resonance Raman spectrum to the normal Raman spectra calculated for NF(N) and NF(T) is shown in Figure 1S of the Supporting Information. Our results reveal that the experimental vibrational frequency pattern is in relatively good agreement with the calculation results for NF(T) but not NF(N), except for the moderate discrepancies between the computational and experimental Raman spectral intensities, which can mainly be caused by the solvent effect on the experimental Raman spectra and by the experimental Raman spectrum being resonantly enhanced while the calculated one is a nonresonant Raman spectrum. Most of the Raman bands observed for NF(T) are due to vibrations associated with the ring CC, C−C, CO, and C−N stretching motions in the 800−1800 cm−1 region. For instance, the CC and C−C stretching modes contribute to the Raman bands of NF(T) at 1622 and 1328 cm−1. The bands related to the CC and C−C stretching modes in the calculated Raman spectra for NF(T) exhibit the same behavior as in the experimental Raman bands. Further details about the comparison between the experimental results and the calculated normal Raman spectrum for NF(T) are given in Table 1S (Supporting Information). The resonance Raman spectrum of NF obtained in pure MeCN is shown in Figure 2S (Supporting Information) along with the calculated normal Raman spectra of NF(N) and NF(T). More specific descriptions for comparison between the experimental results of Figure 2S and the calculated normal Raman spectrum of NF(N) are presented in Table 2S (Supporting Information). Inspection of Figures 1S and 2S (Supporting Information) suggests that the experimental resonance Raman spectra are significantly different from one another, with the spectrum obtained in neutral water (pH 7.2) being mainly contributed by the NF(T) form of the molecule, while the spectrum acquired in pure MeCN can be mostly accounted for by the NF(N) form of the molecule. These results strongly suggest that the hydrogen bonding and higher polarity of water substantially changes the relative population of the NF(N) and NF(T) forms of the molecules present in these solutions. Resonance Raman spectroscopy experiments were also conducted in acidic (pH 1) and basic (pH 13) aqueous solutions, and these spectra are shown in Figures 3S and 4S (Supporting Information). For the acidic and basic aqueous solutions, very different resonance Raman spectra were observed in each case, and these resonance Raman spectra also showed features distinct from those observed in the neutral aqueous (pH 7.2) and MeCN solutions. Analysis of the results of Figures 3S and 4S (Supporting Information) suggest that there is good agreement between the experimental results and the predicted NF(A) and NF(C) normal Raman spectra from DFT calculations. This is also consistent with our expectations based on the pKa values of the prototropic groups in NF, specifically, the NF(A) form is the predominant species in the basic (pH 13) aqueous solution and the NF(C) form is the predominant species in the acidic (pH 1) aqueous solution. In addition, the computational optimized structures of NF(N) and NF(T) shown in Figure 3 reveal that the tautomeric structure with an intriguing conformational change results in noticeable intramolecular hydrogen bonding at the position-6 F atom. This increased hydrogen bonding, in turn, substantially weakens the C−F bond at position-6. This could be a crucial factor for facilitating bond cleavage at C-6 when NF is photoexcited in neutral aqueous solutions.

approximation (U)B3LYP method with a 6-31G(d,p) basis set. Raman spectra were obtained by determination of the Raman intensities from the transition polarizabilities calculated by numerical differentiation and assuming a zero excitation frequency. A frequency scaling factor of 0.991 was used for comparison between the calculated results and the experimental spectra. No imaginary frequency modes were observed at the stationary states of the optimized structures. All of the calculations were done by employing the Gaussian 03 program31 installed in the High Performance Computing Cluster at the Computer Centre in The University of Hong Kong.

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RESULTS AND DISCUSSION UV/vis Absorption and Resonance Raman Spectroscopy Study for NF in Neutral Water. UV/vis experiments were performed in MeCN and neutral water whose pH was 7.2, as adjusted by sodium hydroxide, and the absorption spectra obtained are shown in Figure 2. The absorption spectrum of

Figure 2. UV−vis absorption spectra of NF in neutral water (pH 7.2) and pure MeCN.

NF (dissociated form) in neutral water is characterized by an intense absorption band (around 272 nm maximum) and two weaker bands at 322 and 336 nm (with a long tail extending up to 400 nm). The solvent polarity affects the spectrum to a moderate extent, as shown by the major band at 272 nm becoming red-shifted to 284 nm in pure MeCN with decreasing solvent polarity. However, the absorption at λ > 300 nm did not experience an appreciable solvent shift. Compelling spectroscopic evidence has been reported elsewhere that indicates that polar solvents are able to stabilize (π, π*) states relative to (n, π*) states.32 Consistent with this view, the absorption band at 272 nm can be viewed to be mainly contributed by a (n, π*) transition. In the following resonance Raman spectroscopy experiments and fs-TA experiments, a 267 nm laser was used as the pump source to excite the sample. As reported in preliminary investigations, there are mainly four prototropic species of NF that can exist in aqueous solutions, and the relative populations in these forms may depend on the pH of the solution. The chemical structures of these four forms are shown in Figure 1.15 The efficiency of NF photodegradation was also found to be pH-dependent, as can be expected on the basis of the relative population changes among the different ground-state forms of the molecule.8,12,13,24 To clarify what form(s) of NF exist in neutral aqueous solutions, a resonance Raman spectrum was obtained in neutral C

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around 357 nm. Concomitantly, a negative band (around 426 nm) appeared while another broad absorption band from about 520−650 nm also began to increase in intensity. The contour of the fs-TA spectra of NF in neutral water clearly displays these spectral evolutions. Since the ground state of NF gave no absorption band in the 400−450 nm region (see Figure 2), the negative signal observed in such a region cannot be attributed to ground-state depletion. This demonstrates that a new species is generated with an emission band at around 426 nm, a strong absorption band at 357 nm, and a broad absorption band at 520−650 nm. Subsequently, both of the absorption bands begin to decrease in intensity gradually with a slight shift of the absorption band that is accompanied by another new absorption band emerging at around 600 nm. An isosbestic point can be observed at about 550 nm, and this indicates that the former species is the precursor to the latter one. The first change observed at very early delay times (before the 0.7 ps spectrum in Figure 4a) is assigned to the transition from the ground state (S0) to the higher excited state (Sn) as the energy of the 267 nm pump laser pulse is expected to be higher than that of the lowest transition from the HOMO− LUMO. The red shift of the Sn absorption peak is accompanied by a new broad band above 500 nm, and this variation in the spectra is mainly attributed to a rapid internal conversion (IC) from Sn to the lowest-lying excited singlet state (S1). The growth of the negative emission band also indicates the simultaneous generation of the emissive S1 state. These assignments are in good agreement with a time scale generally associated with a typical IC process.33 In terms of the general principles of photochemical and photophysical changes, the subsequent events involve either ISC to the lowest-lying excited triplet (T1) or a photochemical reaction (for example, photodefluorination). A strong reduction of the photodegradation quantum yield and sensitization by benzophenone have been reported as evidence for a long-lived excited state as the main precursor species.11 Nanosecond flash photolysis and picosecond spectroscopy of EX, reveal that ISC accounts for an

Figure 3. Optimized geometries of the ground states of the NF(N) (left) and NF(T) (right) forms of NF molecule are shown.

Study of the Photophysical Behaviors of NF in Neutral Water by fs-TA. A number of studies have addressed the characterization of transient species and photoproducts of NF, and they have indicated that NF has higher efficiency of photodefluorination in neutral water at 7.2. Therefore, the study of NF by fs-TA in neutral water is first discussed here and then followed by an elaboration of the experimental results in different pH aqueous solutions for comparison. Previously, picosecond transient absorption experiments have been conducted to investigate the photophysics and photochemistry of some FQs, including to lomefloxacin (LF) and enoxacin (EX), FQ compounds sharing some structural similarity with NF, but the analogous information on NF remains lacking.11 In this work, fs-TA, having a higher time resolution on the order of about 100 fs, enables us to observe and explore the ultrafast processes. Figure 4 shows fs-TA spectra of NF obtained in neutral water (pH 7.2). It has been determined from the resonance Raman results that the T form is the dominant species in neutral water, so the initial form observed in the fsTA experiments is the NF(T) form. To clearly describe the transformation of one species into another (which occurs on somewhat different time scales), the spectra obtained at early delay times (0−0.6 and 0.8−4 ps) and at later delay times (after 20 ps) are displayed separately. It can be seen that only one species was generated at very early delay times that has a band maximum at 354 nm growing in rapidly within 0.6 ps. Later, the maximum point of this band shifted to

Figure 4. Fs-TA spectra of NF obtained in neutral water (pH 7.2) after 267 excitation from 0.28 to 0.36 ps (a), 0.81 to 3.56 ps (b), and 21 to 2190 ps (c) are shown. (d) Shown is the contour of the fs-TA spectra of NF in neutral water. D

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fit the kinetics at 363 nm, and two time constants, τ1 = 2.7 ps and τ2 = 1563 ps, were obtained. The shorter rise time constant (2.7 ps) should be attributed to a fast IC from Sn to S1 based on a reasonable time scale for the photophysical variation,33 while the longer decay time constant (1563 ps) is assigned to ISC from S1 to T1 accordingly. Furthermore, the fitting for the kinetics of the absorption changes at 620 nm by a biexponential function also gave two similar ascending time constants of τ4 = 3.6 ps and τ5 = 1671 ps, which are almost equal to those acquired by fitting the absorption changes at 363 nm. These time constants derived from the temporal dependence at 363 and 620 nm reflect the distinct growth of S1 and T1. In addtion, the time constant τ3 = 1486 ps from fitting result of the absorption peak at 426 nm also is close to τ2 and τ5. Albini and co-workers determined NF’s fluorescence lifetime to be 1.5 ns,11 which is similar to the lifetime of S1 found from our fs-TA experimental data (e.g., the time constant of 1563 ps), and this provides further support to the S1 assignment observed in the fs-TA spectra. Investigation of the Early Photophysical Events of NF in Different pH Aqueous Solutions by fs-TA. To further understand the photodegradation behavior, the fs-TA experiments also were conducted in different pH aqueous solutions for comparison. It has been reported that 6-hydroxy derivatives will be generated by nucleophilic substitution of fluorine at position C-6 by irradiation in neutral water with pH 7.2; however, the efficiency of this defluorination reaction will decrease in acidic and basic conditions, such as at pH 4.5 and 10.8,18 Furthermore, when the pH value is lower than 1, no defluorination occurs. The profile of the early-stage photophysical changes of NF in neutral water has been illustrated, which can give a clear clue for the subsequent photochemical reactions. However, the detailed information toward the earlyphase variations of NF in acidic and basic aqueous conditions has received sparse attention. In the following section, the fsTA experimental results in the aqueous solution at pH 1, 4.5, and 13 will be presented, and the corresponding photophyscial or photochemical variations will be discussed. Figure 6 shows the fs-TA results in aqueous solution at pH 1. It can be seen that there are three transformation periods, from 0 to 1.5 ps, 1.5 to 3.0 ps, and 10 ps to 2.5 ns, respectively. The contour of fs-TA of NF in pH 1 solution displays the profile changes from 0 to 3.0 ns (see Figure 6d). Obviously, the locations of absorption bands are different from those obtained in neutral water, which may be attributed to the different initial forms of the molecule being mainly present under the two diffuerent pH conditions. A strong absorption band at about 363 nm was observed in Figure 6a at very early delay times, which can be assigned to the generation of the higher excited state Sn based on the similar profile and position of that obtained in neutral water. Sn is a higher energy state that will relax to the lower excited state S1 via internal conversion. A negative feature assigned to the fluorescence emission was produced at about 445 nm, which verifies the assignment of an S1 species being present when this feature appears. These two changes are almost the same as those observed in neutral water. However, the subsequent absorption spectrum presents a completely different profile, where no new absorption band was generated with the depletion of the positive band at 363 nm and the negative feature at 445 nm. There are three possible reasons to account for this change in the photophysics taking place in the pH 1 solution. First, it may due to the high fluorescence emission efficiency for NF in the aqueous solution

efficient deactivation process from the excited singlet state. Effective oxygen quenching [rate constants in the range of (2 ± 3) × 109 M−1 s−1] is consistent with the effect of air equilibration on the photodegradation quantum yields. Therefore, it is reasonable to conclude that the new absorption band at around 600 nm can probably be attributed to the formation of T1 through ISC from S1. The above observations can be generalized into a scheme for describing the photophysical conversion occurring in the early time-delay fs-TA spectra (see Scheme 2); a higher-lying excited Scheme 2. Schematic Diagram for the Overall Photophysical Conversions Taking Place at Early Time Delaysa

a

The absorption band maxima in wavelengths observed in the fs-TA spectra for the Sn, S1, and T1 species are indicated in the diagram.

singlet Sn state was populated by the excitation of the 267 nm pump laser, followed by rapid IC to the lowest-lying excited singlet S1, and then ISC to the lowest-lying excited triplet T1 took place. To gain more mechanistic insights into the dynamical changes of the photophysical processes involved, the temporal dependence of the transient absorption intensity for NF at 363, 426, and 620 nm was monitored in neutral aqueous solution. The kinetics results and fitting plots are shown in Figure 5. A biexponential function was employed to

Figure 5. Shown are the normalized kinetics and their fitting plots for the NF in neutral water (pH 7.2) at 363, 426, and 620 nm. The time constants derived from the fittings are also displayed in each panel. See the text for more details. E

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Figure 6. fs-TA spectra of NF obtained in pH 1 acidic solution after 267 excitation from 1.0 to 1.5 ps (a), 1.8 to 3.0 ps (b), and 12 to 2391 ps (c) are shown. (d) Shown is the contour of the fs-TA spectra of NF in pH 1.0 acidic solution. The asterisks mark regions affected by the laser with 400 nm wavelength.

at pH 1. The S1 may primarily decay this way so that no new transient species was produced. Second, the ISC efficiency may become too low under the pH 1 condition to be observed. Third, a photophysical and/or photochemical reaction different from defluorination takes place to give rise to new intermediates whose absorption bands are not located in the experimental observational range, so they cannot be detected. This last case is consistent with the one reported by Albini and co-workers, in which no defluorination reaction took place in very acidic solution like the pH 1 solution examined here. A laser flash photolysis study on EX also found that the photodegradation quantum yield also depends on pH, being at a maximum in neutral aqueous conditions. Nanosecond flash photolysis experiments confirmed that the yield of absorbing transients is at a maximum at neutral pH while it decreases to zero at strong acid and alkaline pH.8 Here, the fs-TA results for NF in a pH 1 solution also indicate that the absorbing transients rapidly decrease to zero within about 2 ns, while there are still strong transient absorptions of NF in the neutral aqueous solution. A resonance Raman study on the initial substrate of NF indicated that the NF(C) form is the predominant species present in the strongly acidic aqueous solution, and the significantly different fs-TA results obtained in the acidic and neutral aqueous solutions indicate that both the dissociation of the carboxylic group and the protonation of the piperazinyl residues are key steps for the formation of the photochemically active form of NF. In the pH 1 solution, the protonation of the piperazinyl residues can account for the fast decay of the transient absorption to the zero within 2 ns. The influence of the dissociation of the carboxylic group will be discussed in the following section describing the results obtained in a strong basic solution. To gain a more clear understanding of the early stages of the photophysics and photochemistry, the dynamics of NF in acidic condition was obtained and this is displayed in Figure 7 with three representative wavelengths used for the kinetics fittings. A triexponential function was employed to fit the kinetics at 363 nm, and this resulted in three time constants, τ1 = 3.7 ps, τ2 =

Figure 7. Shown are the normalized kinetics and their fitting plots at 363, 446, and 620 nm for the NF in aqueous solution at pH 1. The time constants derived from the fittings are also displayed in each panel. See the text for more details.

22 ps, and τ3 = 545 ps, being determined. The shortest time constant (3.7 ps) should be assigned to generation of S1 from Sn based on the fs-TA results being similar to those obtained in a neutral water solution. The 22 ps time constant is likely associated with the intramolecular electron transfer (IET) process from the N(4′) atom of the piperazinyl ring to the fluoroquinolone (FQ) main system,18 while the 545 ps process is responsible for the fluorescence radiation from S1 to the ground state. Therefore, in the strongly acidic solution (pH 1), the S1 relaxes its energy by the intramolecular charge transfer (22 ps) and fluorescence radiation rather than by an ISC process; this is dramatically different from the higher efficiency of ISC for NF in a neutral aqueous solution. The results of the fs-TA in a pH 1 solution are consistent with the observation that NF cannot undergo photodefluorination in the aqueous soluion at pH 1. From the spectra (see Figure 6c), it can be seen that it indeed decayed to zero within 2 ns. Furthermore, the fitting for the kinetics of the absorption changes at 446 nm F

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Figure 8. fs-TA spectra of NF obtained in aqueous solution at pH 4.5 after 267 excitation from 1.0 to 1.4 ps (a), 1.5 to 2.1 ps (b), and 3 to 2791 ps (c) are shown. (d) Shown is the contour of fs-TA of NF in pH 4.5 acidic solution.

The kinetics results and fitting plots using three different wavelengths of NF in an aqueous solution at pH 4.5 are shown in Figure 9. It can be seen in Figure 9 that the spectra did not

by a biexponential function also produced one rise time constant of τ4 = 3.7 ps and a decy time constant of τ5 = 347 ps. It has been learned that this negative band was induced by the fluorescence emission, so the decay time constant τ5 close to τ3 was the decay time of fluorescence that corresponded to the decay of S1. Futhermore, the kinetics of the absorption band at 620 nm was also studied. Two similar time constants of τ6 = 25.6 ps and τ7 = 436 ps, which are almost equal to those acquired by fitting the absorption changes at 363 nm, were determined. Subsequently, the fs-TA spectra, shown in Figure 8, obtained in aqueous solution at pH 4.5 will be presented to illustrate the transformations during the early time delays. Figure 8d also shows the contour of the fs-TA spectra of NF in a pH 4.5 acidic solution. The first absorption band at about 360 nm (see Figure 8a) has a slight shift in wavelength compared with that seen in aqueous solutions at pH 7.2 and 1, which were mainly induced by the different ground-state forms present at pH 7.2 and 1 respectively. Figure 8b displayed the spectra between delay times of 1.5 and 2.1 ps, and this has an almost identical profile with those obtained in an aqueous solution at pH 7.2, so we can probably make a similar assignment of an S1 generation process. As expected, the spectral bands and their kinetics appear different from those observed at pH 1 (see Figure 8c) in the later delay time spectra for pH 4.5 aqueous solution. During the 3 ns window of observation, the absorption band did not fully decay to zero. Examination of Figure 8c shows that there should be a new absorption generated at between 500 and 600 nm, because the broad absorption band between 500 and 600 nm did not completely decrease with the depletion of the negative feature due to S1 emission. This can be interpreted as being due to the overlap of decay of old precursor and the generation of a new intermediate, which makes the absorption not change very much in intensity. The new species formed can be assigned to a T1 species with absorption bands at about 359 nm and between 500 and 600 nm on the basis of the intermediate having almost same profile of the spectra that were obtained at pH 7.2.

Figure 9. Shown are the normalized kinetics and their fitting plots at 363, 448, and 620 nm for the NF in aqueous solution at pH 4.5. The time constants derived from the fittings are also displayed in each panel. See the text for more details.

decay to zero at the late delay times, especially for the band between 500 and 600 nm, which appears to almost not change further, indicating that T1 did not decay completely within the 3 ns observation window for the fs-TA experiments. A biexponential function was used to fit the kinetics at 363 nm, and two time constants, τ1 = 2.6 ps and τ2 = 2315 ps, were found. The shorter one (2.6 ps) can be assigned to the transformation from Sn to S1 via IC, and the longer one (2315 ps) may be atrributed to S1 undergoing an ISC process to produce T1. The fitting for the kinetics of the absorption changes at 448 nm by a monoexponential function also produced one rise time constant of τ4 = 2383 ps, which is close to that of τ2, which was also assigned to the decay of S1. Similarly, the kinetics of the absorption band at 620 nm was also fitted by a biexponential function, and two time constants G

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Figure 10. fs-TA spectra of NF obtained in aqueous solution at pH 13 after 267 excitation from 1.0 to 1.4 ps (a), 1.5 to 2.5 ps (b), and 5 to 2700 ps (c) are shown. (d) Shown is the contour of the fs-TA spectra of NF in pH 13 basic solution.

of τ5 = 6.0 ps and τ6 = 1311 ps were determined, which are attributed to the growth of S1 and T1, respectively, on the basis of the preceding spectra analysis. The relatively large differences of τ4 and τ5 from τ1 and τ2 may be induced by the effect of water. These results are most similar to those obtained in neutral water with only the time constant values being a little different. This can be explained by the diverse initial forms of NF that may be photoexcited in the pH 4.5 aqueous solution (namely, the cationic and tautomeric forms of NF, NF(C) and NF(T), respectively). Miranda, Bosca, and co-workers carried out steady-state and time-resolved fluorescence measurements at different pH values to determine that the IET from the N(4′) atom of the piperazinyl ring to the FQ main system becomes dramatically enhanced for S1 to waste energy in basic media.16,18 Acetylation at N(4′) (as in N-acetylnorfloxacin) decreases the availability of the lone pair, making observable its fluorescence and the transient absorption spectrum of its triplet excited state even at high pH.16,18 But the direct ultrafast experimental evidence was missing. So we have also performed the fs-TA experiment in an aqueous solution at pH 13, and the spectra and the contour of the fs-TA spectra are shown in Figure 10. Inspection of Figure 10 shows that the spectra exhibit similar variations with those acquired in acidic solutions, but there are two differences for them. One is that the spectra did not decay to zero within 3 ns, and the other difference is that almost no negative feature was produced on the time scale of the fs-TA experiments. This is consistent with the results from a previous time-resolved spectroscopy study on enoxacin that the found that the absorption and emission properties of enoxacin are strongly affected by pH of the aqueous solution. The fluorescence quantum yield at pH 3.5 increases by a factor of 2 upon going to a neutral pH while a strong reduction was observed for alkaline pH.8 According to the results of previous investigations, the photodefluorination reaction can take place in basic solution with a low efficiency, and T1 as the precursor will decay to give another new transient species; however, no new absorption band was generated within 3 ns, indicating that T1 did not

decay during this delay time, so no photochemical reaction occurred within 3 ns. Due to the IET wasting most of the energy of S1, there was almost no population left to generate the fluorescence that induced the negative feature in the fs-TA spectra. This scenario is in agreement with the present fs-TA results. The kinetics and fitting plots using three different wavelengths of NF in an aqueous solution at pH 13 were performed to give a deeper understanding for the photophysical variations, and these results are shown in Figure 11. Similarly, seven time

Figure 11. Shown are the normalized kinetics and their fitting plots at 363, 442, and 620 nm for the NF in aqueous solution at pH 13.

constants, τ1 = 1.0 ps, τ2 = 13.8 ps, and τ3 = 352 ps (from the absorption band at 363 nm), τ4 = 1.1 ps and τ5 = 18 ps (from the absorption band at 442 nm), and τ6 = 11.9 ps and τ7 = 405 ps (from the absorption band at 620 nm), were obtained from these fitting results. τ1 = 1.0 ps and τ4 = 1.1 ps were assigned to the IC process from Sn to S1; τ2 = 13.8 ps, τ5 = 18 ps, and τ6 = 11.9 ps were attributed to the intramolecular charge transfer from the N(4′) atom of the piperazinyl ring to the FQ main system, which is consistent with the conclusion that the IET H

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has a higher efficiency in basic aqueous solutions.16,18 The τ3 = 352 and τ7 = 405 ps are attributed to the ISC process from S1 to T1. Therefore, the dissociation of the carboxylic group causes the NF to be populated as the anion form, which may account for the high IET process of NF in basic aqueous solutions. In summary, the spectra of fs-TA obtained in different pH conditions were significantly different from each other. These differences mainly arose from the deprotonation and protonation of the initial forms of NF in the various pH aqueous solutions.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Research Grants Council of Hong Kong (HKU 7048/11P) and the University Grants Committee Special Equipment Grant (SEG-HKU-07) to D.L.P. Support from the University Grants Committee Areas of Excellence Scheme (AoE/P-03/08) is also gratefully acknowledged.

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CONCLUSION The present study mainly investigated the early time scale photophysical behaviors of NF in aqueous solutions of different pH by employing femtosecond transient absorption spectroscopy. The diverse ground-state forms of NF were confirmed and characterized by resonance Raman spectra and results from DFT calculations. The NF(T) form, which was the initial substrate form after excitation by 267 nm for the subsequent photophysical reactions, was determined to be the predominant species observed in neutral water. On the ultrafast time scale, a strong transient absorption at 354 nm involving NF(T) S0 → Sn excitation was observed that then experienced a swift internal conversion to the S1 species that had a very strong transient absorption band at 357 nm and a broad absorption band from about 520 to 650 nm. Efficient ISC caused S1 to transform into T1, whose characteristic bands were mainly located at around 355 and 600 nm. Collectively, our study here provides an investigation and characterization of the excited states and intermediates from initial excitation on the femtosecond time scale and clear evidence for establishing the nature of the precursors for the subsequent NF photodefluorination mechanism and other reactions. In the other different pH aqueous solutions, the spectra and corresponding kinetics were also obtained to better understand the photophysical variations as a function of the pH of the aqueous solutions. These results are in good agreement with results found in the literature. The new information and insight into the photophysical behavior for NF molecular forms photoexcited in different pH aqueous solutions may be helpful for future investigations of the subsequent photochemical reactions of NF and related FQ compounds.

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ASSOCIATED CONTENT

S Supporting Information *

The experimental Raman spectra obtained in neutral aqueous solutions at pH 1, 7.2, and 13 and in MeCN and the calculated predicted Raman spectra of NF(C), NF(T), NF(A), and NF(N); comparisons of the experimental resonance Raman spectra vibrational frequencies with the DFT-calculated vibrational frequencies for the NF(T) and NF(N) species with preliminary vibrational assignments and qualitative descriptions of the vibrational modes in the 800−1800 cm−1 region; and Cartesian coordinates used in the calculations of all the intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*M.-D.L.: fax, (+852) 2597-1586; tel, (+852) 2859 2160; email: [email protected]. *D.L.P.: fax: (+852) 2597-1586. Tel: (+852) 2859 2160; email: [email protected]. I

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