Dual Intramolecular Hydrogen Bond as a Switch for Inducing Ground

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Dual Intramolecular Hydrogen Bond as a Switch for Inducing Ground and Excited State Intramolecular Double Proton Transfer in Doxorubicin: An Excitation Wavelength Dependence Study Dipak Kumar Rana, Sayaree Dhar, Arindam Sarkar, and Subhash Chandra Bhattacharya* Department of Chemistry, Jadavpur University, Kolkata700032, India

bS Supporting Information ABSTRACT: This paper investigates how solution conditions, especially solvent polarity and hydrogen bonding, influence the fluorescence of an anticancer drug, doxorubicin hydrochloride (DOX). When excited at 480 nm, this molecule shows single fluorescence. However, when excited at 346 nm, it shows dual fluorescence. The ground and excited state intramolecular double proton transfer in DOX has been observed and investigated to shed light on their corresponding spectroscopy and dynamics in different protic and aprotic solvents. An increase in pH results in enhancement of emission from the ionic conformer with parallel dwindling of emission of the neutral species. Based on the experimental and theoretical studies on DOX, a ground and excited state intramolecular double proton transfer mechanism is proposed to explain the unusual excitationdependent dual fluorescence of DOX.

’ INTRODUCTION Fundamental investigation and application of organic molecules exhibiting ground and excited state intramolecular proton transfer (ESIPT) is of significant interest due to the ubiquity of this process in a large variety of biological and photochemical processes.16 The acidbase characteristics of many molecules are significantly modulated upon electronic excitation, and different pathways of excited state proton transfer reactions have been encountered in the literature.613 A few to mention are intramolecular proton transfer via H-bonded vicinal groups, distal proton transfer by proton relay involving solvent H-bonded bridges, concerted biprotonic transfer within a doubly H-bonded dimer, coupled proton and electron transfer, or intermolecular double proton transfer with solvent molecules.613 In recent years, ESIPT phenomenon has been the subject of very active research, especially due to its wide range of implications in energy/data storage devices and optical switching,1416 Raman filters and hard scintillation counters,17 polymer photostabilizers,18,19 and triplet quenchers.20 Other applications center around electroluminescent materials with photochemical stability, resistance to thermal degradation, and low self-absorption and light emitting diode materials.20 It has been suggested that ESIPTs have the potential for understanding the binding properties of protein,21 as well as optical probes for biomolecules.22,23 But excited state intramolecular double proton transfer (ESIDPT), which may be used to systematize and control the emissive properties of the drug, is rare in literature. DOX (Scheme 1), due to its symmetric two hydrogen bonding centers, has been observed to undergo the ESIDPT process. r 2011 American Chemical Society

Photophysical study of DOX in different solvents originates principally from two aspects. The first one arises from its novel biological applications in pharmaceuticals and the second one due to the presence of electron donors and acceptors in the moiety. Doxorubicin is a member of anthracycline group of antibiotics. It is known to show chemotherapeutic activity by intercalating between adjacent DNA base pairs and preventing replication by causing conformational changes in the DNA molecule.24 The drug is tetracyclic, containing three planar and aromatic hydroxy anthraquinonic rings that compose its chromophore and one nonplanar, nonaromatic ring attached to an aminoglycosidic side chain. Due to the presence of various functional groups, doxorubicin can assume various prototropic forms at different pH values.25 Recently, DOX and its derivatives have drawn renewed attention as new drug delivery technologies because of their capacity to improve in vitro the response of several resistant cell lines to the transported drug.26,27 The drug is widely used in chemotherapy, for example, for the treatment of Kaposi’s sarcoma,28 ovarian carcinoma,29 or breast cancer.30 Many anticancer drugs are intrinsically fluorescent, such as doxorubicin, which makes them convenient for probing and visualization with various microscopic imaging technologies.31 To characterize how the drug is transported to its target, it is useful to establish a relationship between the environment and the photophysical properties of DOX. This will enable one to Received: May 5, 2011 Revised: July 9, 2011 Published: July 18, 2011 9169

dx.doi.org/10.1021/jp204165j | J. Phys. Chem. A 2011, 115, 9169–9179

The Journal of Physical Chemistry A Scheme 1. Schematic Drawing of the DOX, Investigated in the Present Study

monitor the uptake of DOX by a given carrier and its release from the carrier to a target site by monitoring the change in absorption and emission spectra, as DOX migrates from the microenvironment of the carrier to that of the target site. Unfortunately, only a few scattered studies have reported the photophysical properties of DOX and its derivatives.32,33 Some studies have focused on the binding of DOX to DNA;3439 others have monitored the DOX fluorescence inside cultured cells.4045 These studies provided a limited understanding of the photophysical properties of DOX. The UV absorption and fluorescence emission of DOX were found to be highly dependent on solution pH. DOX exhibits a maximum of fluorescence at 593 nm in aqueous solution (excitation at 479 nm) but an emission maximum at 577 nm in ethanol (excitation at 480 nm). This dramatic spectral shift is attributed to the different structures adopted by DOX in different solvents.46 The observation of the neutral form of DOX in ethanol and its protonated form in a more polar solvent (water) suggests that one may use the solvatochromic effects exhibited by DOX to determine its local environment, a powerful investigation tool to describe the polarity of a medium in which the drug is located. Such a result would be expected to have immediate application to characterize the transfer mechanism of DOX in a given delivery system. In the present paper, we have investigated first time an interesting excitation-dependent dual fluorescence phenomenon of an anticancer drug Doxorubicin in different protic and aprotic solvents and buffer solution of different pH. This molecule shows single fluorescence (band A), dual fluorescence (bands A and B) as the excitation wavelength varies from 480 nm (around the first absorption band) to 346 nm (around the second absorption band). Both fluorescence bands show appreciable solvatochromic shifts with increasing solvent polarity. Finally, the mechanism of the unusual excitation-dependent dual fluorescence of DOX was discussed based on the experimental and theoretical investigations.

’ MATERIALS AND METHODS Experimental Details. The anticancer drug Doxorubicin was purchased from Fluka (Oakville, Canada) and used without any further purification. All the solvents dioxane (DX), tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile (ACN), ethanol (EtOH), methanol (MeOH), and ethylene glycol (EG) used

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were all from E. Merck (HPLC grade). The solvents were dried according to the method described elsewhere.47 The purified and dried solvents were transparent in the spectral region of interest. Buffer solutions were prepared by mixing appropriate volumes of aqueous solutions of Na2HPO4 and NaH2PO4 (0.1 M each) and different pH were maintained by adding appropriate amount of standard HCl or NaOH solution. The pH of the solutions was measured by pH meter (model ELICO LI614). Millipore water was used for the preparation of aqueous solutions. The stock solution of DOX (1.0 103 M) was prepared in water, and a fixed amount of this concentrated solution was added to each experimental solution. A Shimadzu (model UV-1700) UVvis spectrophotometer and a Spex fluorolog-2 (model FL3-11) spectrofluorimeter with an external slit width of 2.5 mm were used to collect absorption and fluorescence spectra, respectively. All measurements were done repeatedly and reproducible results were obtained. All fluorescence spectra were corrected for the instrumental response. Prior to the spectroscopic measurements solutions were deoxygenated by bubbling nitrogen through them. The fluorescence quantum yield (Φf) was measured relative to quinine sulfate (Φf = 0.54 in 0.1 M H2SO4).48 Fluorescence lifetimes were determined from time-resolved intensity decay by the method of time-correlated single-photon counting using a picosecond diode laser at 403 nm (IBH, picoLED-07) and at 375 nm (IBH, UK) as light source. The signal was detected at the magic angle (54.7) polarization using a Hamamatsu MCP PMT (3809U). The typical response time of laser system at 403 and 375 nm were 70 and 90 ps, respectively. The data stored in a multichannel analyzer was routinely transferred to IBH DAS-6 decay analysis software. For all the lifetime measurements the fluorescence decay curves were analyzed by biexponential iterative fitting program provided by IBH such as FðtÞ ¼

∑i Ri expð t=τi Þ

where Ri is a pre-exponential factor representing fractional contribution to the time-resolved decay of the component with a lifetime τi. Mean (average) lifetimes Æτæ for biexponential decays of fluorescence were calculated from the decay times and pre-exponential factors using the following equation hτ i ¼

R1 τ1 þ R2 τ2 R1 þ R2

Computational Details. The quantum chemical calculations were performed using the GAUSSIAN 03 package. The ground state geometries of two tautomers of DOX were optimized using Becke’s three-parameter hybrid exchange functional with the LeeYangParr correlation functional (B3LYP) method in conjunction with the 6-31+G** basis set. Computations of difference density plots, dipole moments, and optimization in ground and excited states were performed using DFT and time-dependent DFT, the B3LYP functional (TD-B3LYP), and the 6-31 +G** basis set. The HOMO and LUMO frontier orbital of the tautomers were calculated by TD-DFT methods at the B3LYP/ 6-31G** level.

’ RESULTS AND DISCUSSION Solvent-Assisted Absorption and Emission. The absorption and emission spectra of the investigated DOX were recorded 9170

dx.doi.org/10.1021/jp204165j |J. Phys. Chem. A 2011, 115, 9169–9179

The Journal of Physical Chemistry A

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in several solvents of different polarity and proton donating ability at room temperature. Figure 1 shows the absorption spectra in different nonpolar and polar solvents, while the corresponding spectral data in different solvents are summarized in Table 1. DOX exhibits two absorption bands, one strong band around 480 nm wavelength region, and other weak shoulder around 346 nm wavelength region. The λmaxabs value of strong absorption bands varies from 475 to 480 nm ongoing from ACN to EG. This absorption band may be due to charge transfer transition and the different photophysical parameters indicate that the absorption spectrum of DOX depends on the polarity of the medium. Therefore, it is reasonable to suppose that the two absorption bands (around 346 and 480 nm, respectively) may be due to the two tautomers of DOX. DOX exhibits interesting excitation-dependent dual fluorescence in different solutions. If the excitation varies from 420 to 550 nm, DOX will show single fluorescence (referred as “band A”) in all solvents (Figure 2a). The spectrum shows vibrational structure, suggesting partially local excitation character of the excited state. The fluorescence maximum is greatly affected by the solvent polarity where a much red-shifted fluorescence is observed on increasing the solvent polarity. As the most characteristic phenomenon of charge transfer (CT) excited state is the bathochromic shift with increasing solvent polarity, this result indicates the CT character of band A. However, if the excitation wavelength is shorter than 420 nm, DOX will exhibit dual fluorescence in all solvents (Figure 2b). In addition to band A, a new band (band B) emerges on the

short-wavelength side. This new band shows a slight blue shift with increasing solvent polarity but not as prominent as band A. Because the fluorescence spectra depend on the excitation wavelength, it is important to investigate the excitation spectra of both emission bands. Figure 3 shows the excitation spectra of DOX monitored at both fluorescence bands. As can be seen from Figure 3, the excitation spectra monitored at both emission bands are quite different from each other, indicating different species in ground state. The one monitored at band A is very similar to its absorption spectrum. However, the excitation spectrum monitored at band B is quite different from the absorption spectrum in the region above 346 nm. It is reported in literature49 that absorption and excitation spectra do not correspond to each other if there are more species in the ground state, or if the sole present species has different forms in the ground state (aggregates, complexes, tautomeric forms, etc.). However, in order to get the clear idea of band A, the solventinduced shift of the fluorescence maximum has been observed from the variation of the fluorescence energy E(F) with ET(30) and corresponding Stokes-shift (Δν̅ ) with ET(30) plot has been shown in Figure 4, where a double linear correlation is found. Polar protic solvents fall on a separate line (having a higher slope), indicating that the mode of solvation of the emitting state is different from that in the polar aprotic solvents. This is probably due to hydrogen bonding interactions. In fact, the correlations are much stronger and their slopes reflect the high sensitivity of E(F) to the

Figure 1. Absorption spectra of DOX in some solvents; [DOX] = 5.6 106 mol dm3.

Figure 2. Emission spectra of DOX in different solvents: (a) excited at 480 nm; (b) excited at 346 nm.

Table 1. Spectroscopic Parameters of DOX in Different Solvents fluorescencea

absorbance λabs max (nm)

εmax (dm3 mol1 cm1)

λAf (nm)

λBf (nm)

ET(30) (kcal mol1)

Δν̅ (cm1)

H2O

479

9001

594

421

63.1

4042

EG

481

9178

584

422

56.3

3667

MeOH

476

11119

578

422

55.5

3707

EtOH

480

11295

577

422

51.9

3502

ACN

475

10942

579

414

46.0

3781

DMF THF

480 481

10766 11472

582 578

426 431

43.9 37.4

3651 3489

DX

480

10413

580

422

36.3

3592

solvent

a

A represents cis-enol-A and B represents cis-enol-B. 9171



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solvent polarity. The enhanced solvent sensitivity of E(F) compared to absorption energy E(A) can be attributed to increased solutesolvent interactions in the excited state due to an increased dipole moment of the probe upon excitation. Ground State Equilibrium. In general, DOX may exist in three tautomeric forms in solution: keto and two cis-enol. Among these tautomers, cis-enol forms are considered to be the photochemical stable ones because they are stabilized by the intramolecular hydrogen bonding and the conjugated system. Theoretical studies have shown that the energies of keto form are much higher than their cis-enol isomers. Therefore, they can only be observed experimentally at very low temperatures.50 Because our experiments were all carried out at room temperature, it is reasonable to consider the existence of two cis-enol forms. There are actually two possible structures of cis-enol form of DOX, as shown in Scheme 2. In cis-enol-A (corresponding to emission band A), the intramolecular hydrogen bonding is

Figure 3. Excitation spectra of DOX in water monitored at different emission bands: [DOX] = 5.6 106 mol dm3.

O5H6 3 3 3 O1, while in cis-enol-B (corresponding to emission band B), it is O1H6 3 3 3 O5. These two cis-enol form isomers are converted into each other through a transition state (TS). We have calculated the relative energies of the two tautomers and the transition state with B3LYP/6-31G** method. The results showed that cis-enol-B (relaxed form) lies only 3.0 kJ mol1 above cis-enol-A (relaxed form). This energy difference between the two tautomers is so small that they should be able to exist in solution simultaneously. Excited State Equilibrium. It is important to mention here that if the excited-state dipole moment vector is rotated within the molecular framework relative to the ground-state dipole moment vector, a large dipolar solvent relaxation could also be observable.51 This could occur for both the normal fluorescence and the proton-transfer fluorescence. The drug molecule, because of the delicate origin of its proton-transfer fluorescence, would offer the opportunity of separating the dipolar (protic) solvent cage relaxation from the protic-catalyzed proton transfer fluorescence. To confirm the proton transfer species in excited state, we observed the effect of dual fluorescence spectra of DOX in methanolwater mixture of different composition and the spectra are shown in Figure 5. As percentage of the water in the methanolwater mixture is increased continuously, the relative intensity of band B goes to maximum, whereas the intensity of band A decreases gradually with an isoemissive point at 512 nm. From this experimental result, one may suspect that the intramolecular H-bond of DOX in water is retarded by successive addition of methanol in the solution and ESIDPT process become embarrassed. As more water is added, ESIDPT process become facilitated and emits fluorescence in the 421 nm region, decreasing the intensity of band A. It is interesting that water

Figure 4. Variation of Stokes shift as a function of solvent polarity parameter.

Figure 5. Emission spectra of DOX in MeOH as a function of water concentration (v/v): (i) 0%, (ii) 10%, (iii) 20%, (iv) 40%, (v) 50%, and (vi) 60% water; inset: (vii) 60%, (viii) 80%, and (ix) 100% water. λexc = 346 nm.

Scheme 2. Equilibrium between the Two cis-enol Form of DOX

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Figure 6. Plot of emission intensity of DOX at 421 nm (cis-enol-B) against percentage of methanol.

addition (above 60%) to methanol solution of DOX shows decrease in intensity of the 421 nm energy band also. This dual behavior of the drug in watermethanol mixtures suggests that the drug exhibits two types of phenomena above and below of a certain percentage of water. One possible reason for decrement of band B could be the formation of intermolecular H-bond between water and the drug molecule with sufficient amount of water and it competes with intramolecular H-bonding and ESIDPT process become hindered. Zhao and Han beautifully demonstrated intermolecular hydrogen bond weakening and strengthening behavior in polar protic solvent in electronically excited state for carbonyl chromophores.52,53 It is commonly accepted that the intramolecular H-bond closing the six-membered ring cannot be readily disrupted by protic solvents, and therefore, a sufficient amount of water is needed for its competition. This implies that the ESIDPT process becomes favored only after attaining a certain polarity of the medium. For protonated aminopyrene, Pines and Fleming also reported similar observation where in a wateralcohol mixture the deprotonation rate actually increases with alcohol concentration up to about 6570% and at higher alcohol concentrations the deprotonation process is retarded.54 The variation of ESIDPT process with polarity of the medium has been depicted in Figure 6, where intensity of the cis-enol-B has been plotted against percentage of methanol. The break point in the plot is the threshold polarity value after which the ESIDPT process in DOX is retarded. In a basic solvent, like THF, the emission band B shows structures that are 10 times more intense than that formed in other solvents. The dramatic increase in fluorescence intensity observed in THF cannot be due to solvent polarity alone but due to specific interactions. In the excited state, polar aprotic solvents form solvation cage surrounding carbonyl group. As a consequence large stabilization of intramolecular H-bond that favors the proton transfer process. This is considerable as a consequence of larger stabilization of the cis-enol-B in excited state compared to the cis-enol-A. In DMF, DOX also experiences such stabilization but to a less extent due to the less basic nature of DMF than THF. Based on the Results and Discussions presented above, a possible mechanism (Scheme 3) of the excitation-dependent dual fluorescence of DOX has been illustrated. In ground state, the two tautomers are close in energy and can exist in solution simultaneously. The ground state intramolecular proton transfer

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reaction controlled the equilibrium between the two tautomers and further modulated the charge separation direction during excitation. Because the excited state dipole moment of cis-enol-A is larger than that of cis-enol-B, the excited state energy of the former should be lower than the latter due to solvent stabilization. Thus, low-energy excitation can only generate cis-enol-A*. In this case, ESIDPT would not happen because extra energy is needed to overcome the energy barrier from cis-enol-A* to cisenol-B*. As a result, only single fluorescence corresponding to cisenol-A* is observed. If the excitation energy exceeds a certain threshold, both tautomers can be promoted to their corresponding excited states; moreover, there is enough extra energy for the two tautomers to transform into each other through ESIDPT. Dual fluorescence appears when both species relax to their corresponding ground states. During the period after excitation and before emission, ESIDPT actually controlled the charge recombination direction. As the solvent polarity increases, cisenol-A* will be stabilized by solvation more prominently than cis-enol-B* due to its larger dipole moment. As a result, the equilibrium will shift to the cis-enol-A* side and band A will become dominant. Fluorescence Quantum Yields and Time-Resolved Study. Fluorescence lifetime serves as a sensitive indicator of the local environment in which a given fluorophore is placed.55 Lifetimebased measurements are rich in information and provide unique insights into the systems under investigation.56 To investigate the effect of solvation on the dynamics of the excited state the radiative [kr = Φf/τf] and nonradiative [knr = (1 Φf)/τf] rate constant were calculated by using fluorescence quantum yields (Φf) and lifetimes (τf) of DOX in two different energy bands. The values of Φf, τf, kr and knr in different solvents are presented in Table 2. It appears that the radiative rate constants are practically insensitive to a change in solvation except THF which may be due to increased basicity. The nonradiative rate constant for DOX exhibits solvent sensitivity with polarity of the medium. An inspection of Table 2 shows that the fluorescence quantum yields and lifetimes show a relatively wide variation with solvent polarity. The fluorescence decay behavior of DOX has been studied in solvents of different polarities. Figure 7 shows some representative fluorescence decay curves of DOX in different solvents. The data in the table indicate that the lifetime of DOX falls in two categories. Most decay curves can be well fitted with a biexponential with a χ2 value near unity. The others are fitted with single exponentials. Without putting emphasis on the magnitude of individual decay constants in such biexponential decays, we desire to use the mean fluorescence lifetime (Table 2) as valuable parameter for exploring the nature of the interaction. The biexponential fitting does not necessarily indicate that the decay curve has only two discrete time constants; it may imply a distribution of time constants around two well separated values, one may speculate that there exist two different tautomeric forms of DOX, resulting from intramolecular hydrogen bonding. To further confirm whether the emission of DOX at the 421 and 593 nm peaks arise from two different species, a fluorescence decay experiment was carried out at 421 nm in addition to 593 nm. The decay time of DOX at 593 nm was found to be 1.11.6 ns and at 421 nm it was 1.540.085 ns in different solvents (Table 2). The lifetime of cis-enol-A is larger than that of the cis-enol-B, which is combined with the fact that cis-enol-A is more stable, and the stability comes only from the hydrogen bonding interaction. The fluorescence decay at 421 nm in DMF (1.54 ns) and THF 9173



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Scheme 3. Model of Ground and Excited-State Prototropic Transformations of DOX in Different Solvents

Table 2. Relative Quantum Yields, Lifetimes, and the Rate Constant of DOX in Different Solventsa

a

ΦfB

kr (ns1) knr (ns1) knr/kr

solvent τavA (ns) τavB (ns)

ΦfA

H2O

1.14

0.430

0.040 0.013

0.04

0.84

24.05

EG

1.53

0.790

0.076 0.073

0.05

0.60

12.09

MeOH EtOH

1.62 1.73

0.085 0.090

0.078 0.005 0.089 0.030

0.05 0.05

0.57 0.53

11.80 10.21

ACN

1.59

0.200

0.081 0.022

0.05

0.58

11.32

DMF

1.61

1.540

0.086 0.043

0.05

0.57

10.59

THF

1.63

1.180

0.161 0.274

0.10

0.51

5.21

DX

0.84

0.088

0.078 0.009

0.09

1.09

11.80

A represents cis-enol-A and B represents cis-enol-B.

(1.18 ns) solution is somewhat different from other solvent which is due to the cis-enol-B get stabilized in these two solvents. These results clearly show that the fluorescence emission at 421 and 593 nm comes from two different tautomeric form of DOX. On further investigation, the fluorescence decay experiments were also performed in different composition of watermethanol mixture in both the emission band and the data are presented in Table 3. With up to 60% water in the mixture, the decay time of cis-enol-A diminished and cis-enol-B enhanced, and above 60% water, a decrement of decay time in both tautomers was observed. Furthermore, the decay at 421 nm displayed a rise time, suggesting that the formation of the species and that emitted with a 1.6 ns decay time was delayed. These results clearly support that the 421 nm emission band arises at the expense of the 593 nm emission band. Above 60% water content in the mixture, interplay between intramolecular and intermolecular H-bonding has been demonstrated. The same result has also been obtained from

Figure 7. Typical fluorescence decay curves of DOX in some solvents: (a) instrument response function, (b) MeOH, (c) EG, (d) THF, and (e) DMF. Excitation wavelength is kept at 375 nm. Inset: Typical fluorescence decay curves of DOX associated with lamp profile in some solvents. Excitation wavelength is kept at 403 nm.

the steady state emission study. From the above piece of evidence we may conclude that the intramolecular double proton transfer process is further modulated in the excited state. KamletTaft Analysis. To obtain an insight into the various modes of solvation determining the absorption and fluorescence energies, the multiple linear regression analysis approach of Abraham et al.57 has been used. Correlation of E(A) and E(F) was found with Taft’s π* value, an index of the solvent dipolarity/ polarizability, and the R and β values58 representing the hydrogen bond donating and accepting ability of the solvent, respectively. The following regression equations were obtained 9174



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Table 3. Lifetime of DOX of Both cis-enol-A (A) and cisenol-B (B) in Different Compositions of WaterMethanol Mixture % of water in water χ2

τavA (ns)

methanol mixture

τavB (ns)

χ2

100

1.14

1.22

0.43

1.21

80

1.15

1.26

0.49

1.46

60

1.16

1.24

0.79

1.39

50 40

1.18 1.24

1.19 1.12

0.75 0.67

1.25 1.46

30

1.27

1.00

0.62

1.27

20

1.29

1.02

0.58

1.13

0

1.62

1.40

0.09

1.03

for DOX EðFÞ ¼ 74:21 0:042R þ 0:167β 1:848π

Figure 8. Absorption spectra of DOX in buffer solution as a function of pH. Curves (ixii) correspond to pH 2.0, 5.0, 8.0, 9.0, 9.3, 9.6, 10.0, 10.25, 10.75, 11.0, 12.0, and 13.0, respectively. Inset shows titration curve.

EðAÞ ¼ 89:33 0:748R þ 0:196β 0:522π From the E(A) and E(F) values, it is observed that dipolar interactions (π*) predominate in the excited state properties. The intercept values indicate the E(F) and E(A) values of the drug in a purely nonpolar solvent like cyclohexane, where there is no specific interaction. The observed E(F) and E(A) values of the drug in cyclohexane are very close to the intercept values. The dipole moment of the drug was determined by the solvatochromic comparison method, using the LippertMataga equations:59 EðAÞ þ EðFÞ ¼

and

ðμ20 μ21 Þ½2ðε 1Þ þ 2ΔGðgasÞ þ ΔðspÞ a3 ð2ε þ 1Þ

"

ðμ1 μ0 Þ2 EðAÞ EðFÞ ¼ a3

#"

2ðε 1Þ 2ðn2 1Þ ð2ε þ 1Þ ð2n2 þ 1Þ

#

where ε and n are the dielectric constants and refractive indices of the solvents, respectively. ΔG(gas) is the value of ΔG in the gas phase and a is the Onsager cavity radius of the drug. These equations are valid for aprotic solvents where specific interactions (Δsp) are absent. [E(A) + E(F)] and [E(A) E(F)] were plotted against appropriate dielectric functions and the ratio of the dipole moment in the S1 state (μ1) to that in S0 state (μ0) was obtained from the ratio of the slopes from the two plots. From quantum chemical calculations by DFT methods at the B3LYP/ 6-31G** level involving complete geometry optimization of the ground state of DOX, the ground state dipole moment (μ0) obtained is 7.10 D. When the μ0 value from the DFT calculation was used, the (μ1) value obtained from the slope ratio is 10.78 D, which is very close to the value calculated by the TD-DFT method in excited state. Prototropic Equilibria and pH-Dependent Dual Absorption and Emission of DOX. The absorption spectra of DOX undergo drastic changes on increasing the pH. The basic absorption and emission features of DOX in buffer solutions having different pH values (from 2 to 13) are depicted in Figure 8. The spectral variations indicate the possibility of using of both phenolic OH for either absorption or fluorescence based pH sensing. As it is

Figure 9. Emission spectra of DOX in buffer solution as a function of pH; λexc = 346 nm.

observed from Figure 8, the intensity of both the long-wavelength absorption and emission bands are decreased with increasing pH. Increase of pH leads to disappearance of the absorption band at 480 nm along with the formation of a new band at 550 nm which can be attributed to the deprotonated form. The isosbestic points at 512 nm for DOX indicate a ground state acidbase equilibrium involving two species. It is pertinent to mention here that with increase of pH the peak corresponding to 346 nm band suffers blue shift with slightly increase in intensity. From the inflection point of the spectrophotometric titration curves (inset of Figure 8), the determined ground state pKa value is 9.88. The fluorometric titration curves yield very similar pKa values. The appearance of dual fluorescence of the acidic and basic species over a large pH range (9.510.5) as well as the similarity of the pKa values obtained from spectrophotometric and fluorometric titration curves point to a slow prototropic equilibrium in the excited singlet state. This means that the radiative deactivation competes successfully with the proton dissociation process. The room-temperature emission spectrum of DOX solution in buffer shows a single and unstructured band with peak at 593 nm ascribed to the neutral form of cis-enol-A. Gradual increase in pH of buffer solution changes the emission spectrum drastically. A new red-shifted emission band with a peak at 630 nm develops 9175



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Scheme 4. Model of Prototropic Equilibria in Ground and Excited States of DOX

due to the emission corresponding to the anionic species excited at 550 nm. The fluorescence spectra of the drug monitoring at 346 nm exhibit similar pattern as obtained in solvatometric titration curves. With increase in pH from 2 to 13, the peak with the maximum at 593 nm (band A) disappeared and a new peak with a maximum at 421 nm (band B) arose (Figure 9). Our results can most plausibly be explained by postulating the existence of two distinct excited state species (Scheme 4). The first of these is cis-enol-A* of DOX, which is responsible for the emission band at 593 nm, and cis-enol-B*, resulting from intramolecular double proton transfer and a virtual equilibrium that exists between them. In the ground state there is very little existence of the cis-enolB, which becomes favored in the excited state because of the large variation of dipole moment. Due to less aromatic character of the cis-enol-B*, it is energetically less favorable and remains in lower wavelength region. On the other hand, absorption spectra in different prototropic equilibrium exhibit three distinct species in ground state. Panoply of evidence favors the assignments that the 480 peak is responsible for cis-enol-A, while the large Stokes-shifted peak at 550 nm is for anionic species (cis-enol-A). The phenolate anion is capable of considerable resonance stabilization by delocalization of their formal charges and forming a new resonating species, which is also obtained in the excited state by intramolecular proton transfer process. The cis-enol-A, on the other hand, undergoes emission at 593 nm only upon excitation at 480 nm. By adding base, the intermolecular hydrogen bond is ruptured due to the creation of an anion, and this decreases the intensity of cis-enol-A fluorescence. But with an increase in pH as the formation of anion becomes facilitated, so by resonance stabilization, the more tautomer species it forms in the ground state and after excitation, the emission intensity of cis-enol-B also increases. Quantum Chemical Calculation. To have a better understanding of different excited state properties and to predict the

nature and mechanism of proton transfer, the energy of electronic transitions, heat of formation, and dipole moments were calculated using DFT methods at the B3LYP/6-31G** level for both the normal and the tautomer form in the ground and excited states. In the ground state, the OH bond length is 0.96320 Å, but in the optimized excited state, the OH bond length becomes 0.96584 Å and the COH angle increases from 108.9 to 109.7. This structural change of DOX in its excited state indicates the possibility of proton transfer in its excited state. The increase of the dipole moment (7.10 to 10.78 D) from ground state to excited state indicates possible redistribution of charge of DOX in its excited state, and this is only possible by intramolecular charge transfer from the acid moiety to the basic moiety of DOX.60,61 The variation of dipole moment as well as the heat of formation with OH bond length, where the other parameters remain unaltered, has been determined. The increase of the heat of formation with the OH bond length indicates that some external energy is required for the ESIDPT from the OH group of the DOX, which is only possible by exciting the molecule. After examination of the computed charge distribution using the Mulliken scheme, it is evinced that there is an increase of charge distribution on the oxygen atom of the CdO group (basic moiety) and a simultaneous decrease in charge on the oxygen atom of the OH group (acid moiety) on going from the ground to the excited state, which indicates a possible proton translocation in the excited state. The origin of the geometric difference introduced by excitation can be explained, at least in qualitative terms, by analyzing the change in the bonding character of the orbital involved in the electronic transition for each pair of bonded atoms. An electronic excitation results in some electron density redistribution that affects the molecular geometry. When the HOMO f LUMO 9176



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Scheme 5. Optimized Structure and Frontier Orbitals of cis-enol-A (top) and That of cis-enol-B (bottom) of DOXa

a

The bond lengths are in angstroms (Å). Energy of the frontier HOMO and LUMO orbital of the tautomers are given below.

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The Journal of Physical Chemistry A transition involves the loss of the bonding character of a bond, the bond concerned is lengthened and vice versa. The isodensity surface plots of HOMO and LUMO of both cis-enol-A and cisenol-B are shown in Scheme 5. The electronic distribution in HOMO in ground state of cis-enol-A shows that more electron cloud is projected toward the second and third benzene moiety of the parent planar anthracycline skeleton than that of first benzene ring. LUMO electron density of cis-enol-A shows the electronic distribution over whole anthracycline ring. When compared with the excited state profile of HOMO and LUMO of cis-enol-A, a subtle change in the density distribution is noticed. Electron distribution in HOMO of cis-enol-B in ground state shows the major contribution on hydroxyl group (acid moiety) of anthracycline skeleton of DOX, whereas in LUMO the electron cloud shifts more toward the keto group (basic moiety) which facilitates proton transfer from acid moiety to the basic moiety. The HOMO and LUMO energies of the corresponding cisenol-A and cis-enol-B are given in Scheme 5. A close inspection on this moiety also conveys that this affects the overall electronic arrangement to some extent. Changing electronic density at that region may also alter the bond length and dihedral angle. We have presented here the calculated bond distance and they are presented in Scheme 5. It is pertinent to mention here that the relative placing/position of hydroxyl group in the anthracycline skeleton of DOX substantially induces to bring the change of the overall electronic charge distribution of different forms which further modulates to their energies and relative populations. From the theoretical viewpoint and the calculations mentioned above it can be concluded that excitation of DOX will achieve a delocalized excited state and then relaxes to the proton transfer configuration by transferring the proton from the acid moiety to the basic moiety. With this piece of information, we get the mechanistic details of proton transfer in ground and excited state as obtained from the steady state absorption and emission and also from time-resolved study.

’ CONCLUSIONS Undoubtedly, the significant essence of this work is the fact that in different solvents, the unusual excitation-dependent dual fluorescence originates from the intramolecular double proton transfer in both ground and excited states. The diketone moiety of DOX induces intramolecular ketoenol tautomerism, which is responsible for highly solvent sensitive fluorescence properties. The foregoing results evince a possible existence of the ground state species: one due to cis-enol-A (480 nm), the second due to the formation of phenolate anion (open conformer 550 nm), and the third due to cis-enol-B (346 nm). The emission spectrum of DOX in THF is different from that in other solvents. This is due to the basic nature of the solvent, resulting larger stabilization of the intramolecular H-bond. The interplay between the intramolecular H-bond induced ESIDPT and intermolecular H-bond formation in water has been demonstrated as percentage of water increases in methanolwater mixture. In basic medium, the higher energy emission band gets pronounced with simultaneous disappearance of the lower energy band. The theoretical results based on density functional theory are in good agreement with experimental data. Thus, systematic investigation of the dependence of photophysics of anticancer drug, DOX on microenvironment as well as in different pH, may be crucial to gain

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fundamental insights into research fields of current interest regarding transport mechanism inside cultured cells.

’ ASSOCIATED CONTENT

bS

Supporting Information. Time-resolved emission spectra (TRES) of DOX in EG and THF are given. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 91 (033) 24146223. Fax: 91 (033) 24146584. E-mail: [email protected]; [email protected].

’ ACKNOWLEDGMENT Authors D.K.R and A.S acknowledges CSIR and S.D acknowledges UGC for providing fellowship. The authors are thankful to Prof. S. Bhattacharya, Burdwan University and Mr. D. Mandal, IACS, Kolkata, for theoretical calculation. The authors are also thankful to Prof. N. Sarkar and Ms. S. Sarkar, IIT Kharagpur, for their kind help in picosecond time-resolved fluorescence measurements and Prof. T. Ganguly and Mr. S. Das, IACS, Kolkata, for time-resolved emission spectra (TRES) measurement, respectively. The authors are also thankful to Prof. S. Bhar, Jadavpur University, for his valuable discussion. We sincerely thank the reviewers for their meticulous inspection of our manuscript and constructive suggestions. ’ REFERENCES (1) Mohammed, O. F.; Pines, D.; Dryer, J.; Pines, E.; Nibbering, E. T. J. Science 2005, 310, 83. (2) Liang, F.; Wang, L.; Ma, D.; Jing, X.; Wang, F. Appl. Phys. Lett. 2002, 81, 4. (3) Hillebrand, S.; Segala, M.; Buckup, T.; Correia, R. R. B.; Horowitz, F.; Stefani, V. Chem. Phys. 2001, 273, 1. (4) Kim, S.; Park, S. Y. Adv. Mater. 2003, 15, 1341. (5) Tong, H.; Zhou, G.; Wang, L.; Jing, X.; Wang, F.; Zhang, J. Tetrahedron Lett. 2003, 44, 131. (6) Agmon, N. J. Phys. Chem. A 2005, 109, 13. (7) Nie, D.; Bian, Z.; Yu, A.; Chen, Z.; Liu, Z.; Huang, C. Chem. Phys. 2008, 348, 181. (8) Balamurali, M. M.; Dogra, S. K. Chem. Phys. 2004, 305, 95. (9) Tanner, C.; Manca, C.; Leutwyler, S. Science 2003, 302, 1736. (10) Bardez, E.; Chatelain, A.; Larrey, B.; Valeur, B. J. Phys. Chem. 1994, 98, 237. (11) Kim, T. G.; Lee, S. I.; Jang, D.; Kim, Y. J. Phys. Chem. 1995, 99, 12698. (12) Chou, P.-T.; Martinez, M. L.; Clements, J. H. J. Phys. Chem. 1993, 97, 2618. (13) Nakagawa, T.; Kohtani, S.; Itoh, M. J. Am. Chem. Soc. 1995, 117, 7952. (14) Kuldova, K.; Corval, A.; Trommsdorff, H. P.; Lehn, J. M. J. Phys. Chem. A 1997, 101, 6850. (15) Nishiya, T.; Yamauchi, S.; Hirota, N.; Baba, M.; Hanazaki, I. J. Phys. Chem. 1986, 90, 5730. (16) Chou, P. T.; McMorrow, D.; Aartsma, T. J.; Kasha, M. J. Phys. Chem. 1984, 88, 4596. (17) Martinez, M. L.; Cooper, W. C.; Chou, P. T. Chem. Phys. Lett. 1992, 193, 151. (18) Heller, H. J.; Blattmann, H. R. Pure Appl. Chem. 1973, 36, 141. (19) Werner, T.; Woessner, G.; Kramer, H. E. A. In Photodegradation and Photostabolization of Coating; Pappas, S. P., Winslow, F. H., Eds.; American Chemical Society: Washington, DC, 1981; Vol. 151, p 1. 9178


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Dual Intramolecular Hydrogen Bond as a Switch for Inducing Ground

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