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Chemical Physics Letters 474 (2009) 88–92

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Excited state proton transfer promoted fluorescence resonance energy transfer: Modulation within cyclodextrin nanocavity Deboleena Sarkar, Atanu Mahata, Paramita Das, Agnishwar Girigoswami, Nitin Chattopadhyay * Department of Chemistry, Jadavpur University, Kolkata 700 032, India

a r t i c l e

i n f o

Article history: Received 10 February 2009 In final form 12 April 2009 Available online 18 April 2009

a b s t r a c t Excited state proton transfer (ESPT) and fluorescence resonance energy transfer (FRET) have been linearly coupled, leading to an efficient pH-sensitive energy transfer from carbazole to a potentially bioactive ketocyanine dye, 2-[3-(N-methyl-N-phenylamino)-2-propenylidene]indanone (MPAPI). The prototropic product produced exclusively from the photoexcited carbazole in the presence of alkali serves as the energy donor. The efficiency of the energy transfer process has been enhanced through the introduction of cyclodextrins of suitable dimensions. The imperative feature of the present model system involving the linear coupling of ESPT and FRET processes lies in its simplicity for designing pH sensitive molecular switches. Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction Photoexcitation may initiate intramolecular as well as intermolecular transformations in a molecule. Among such excited state photoprocesses the prominent and important ones are electron transfer [1,2], proton transfer [3,4] and conformational transformations like twisting, isomerization, etc. [5,6]. Excited state photoprocesses may sometimes be coupled to one another. When such a coupling is done within a molecular system, the excited state events may compete with each other dynamically. Therefore proper pairing of excited state processes can provide a score of structural/dynamical information about the participating molecules as well as the medium in which these coupling transformations take place [7,8]. In the present Letter, excited state (intermolecular) proton transfer (ESPT) and fluorescence resonance energy transfer (FRET) have been linearly coupled, leading to an efficient pH-sensitive energy transfer from carbazole to a potentially bioactive ketocyanine dye, 2-[3-(N-methyl-N-phenylamino)-2-propenylidene]indanone (MPAPI). To the best of our knowledge, this is the first endeavor of this sort. The prototropic product produced exclusively from the photoexcited carbazole in the presence of alkali serves as the energy donor. The efficiency of the energy transfer process has been controlled through the introduction of cyclodextrins of varying dimensions. The imperative feature of the present model system involving the coupled processes lies in its simplicity for designing pH sensitive molecular switches [9]. Fluorescence resonance energy transfer (FRET) is a powerful technique, and because of its explicit distance dependence [10], * Corresponding author. Fax: +91 33 2414 6266. E-mail address: [email protected] (N. Chattopadhyay). 0009-2614/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2009.04.032

it is used to characterize a wide variety of macromolecular assemblies such as biological membranes [11], proteins [12], and also bichromophoric molecular systems [13]. FRET has also been used to elucidate the structure of DNA [14]. The efficiency of FRET depends on the inverse sixth power of the distance of separation between the donor and acceptor molecules [9]. Because of such unique properties of FRET and also the high sensitivity to proximities in the nanometer range both in vitro and in vivo it is coined as ‘spectroscopic ruler’ [10]. Small amount of material, even a single cell that contains only picomoles of fluorescent material, may be examined. Another important photoprocess relevant to the biosystems is the state proton transfer (ESPT). This elementary reaction plays a mammoth role in several vital chemical, physical and biological processes including acid–base neutralization [15,16], electrophilic addition and a score of enzymatic reactions [17,18]. Many molecular machines, reported so far, are powered by proton transfer processes [19]. Molecular electronic/photonic devices refer to systems in which spatially directed electron, proton or energy-transfer processes take place in the photoexcited state [20]. As miniaturization of technology continues to progress, the long-standing fundamental problem of identifying and understanding the smallest physical systems that are capable of switching attracts growing interest. Molecular switching is also important in biology since many biological functions like allosteric regulation, vision, etc. are based on it [21]. A modular construction of light driven molecular switches is quite difficult to carry out. It would therefore be useful to exploit viable strategies for using light to operate ‘stand alone’ photochemically driven molecular switches [20]. Proton transfer is thus an interesting alternative in this aspect. A fluorescence induced molecular switch can be realized based on coupling of photoinduced proton transfer and energy transfer

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processes. Since FRET intensity is proportional to the inverse sixth power of the donor–acceptor distance [9], a change in their spatial relation could be an added route to modulate molecular switches. Not only that, one can also demonstrate the utility of such a study in finding suitable luminescent solar collector (LSC) materials [22]. Coupling of the two important photoprocesses, namely, ESPT and FRET, projects a viable expansion of the field. Because of the inherent character of ESPT, pH of the medium regulates the functioning of the process. Therefore controlling the pH of the medium can regulate transfer of energy. Further, ESPT promoted FRET has the potential to overrule a restriction on the direct energy transfer between the two interacting partners. In the present study the two dyes considered are carbazole and 2-[3-(N-methyl-N-phenylamino)-2-propenylidene]indanone (MPAPI); the latter one being a potentially bioactive ketocyanine dye. Carbazole, upon photo-excitation at 295 nm (where MPAPI has insignificant absorption) in aqueous alkaline solution undergoes an excited state proton transfer (ESPT) reaction [23,24]. Carbazole anion, produced exclusively in the photoexcited state, emits at 420 nm (Scheme 1). MPAPI is a ketocyanine dye (Scheme 2) used as indicator solute for studying solvation interaction in homogeneous and heterogeneous media [25,26]. In aqueous medium it yields a broad absorption peak at 460 nm and a broad charge transfer emission band peaking at 525 nm that remains, to a large extent, invariant to the pH of the medium. Considering the fact that the dye absorbs mostly at a region where the ESPT induced carbazole anion emits, the fluorophores are judged as a promising pair for an ESPT promoted FRET phenomenon (Fig. 1). The present study is unique and important in the sense that the donor species, carbazole anion, is generated exclusively in the photoexcited state from its neutral counter part through ESPT in alkaline pH only. The anion is nonexistent in the dark. Produced in the photoexcited state, it transfers its energy to the acceptor (MPAPI) in the ground state through a long-range dipole interaction. Thus, ESPT promoted FRET is a promising and convenient bypass route in circumstances where direct energy transfer between the partners is restricted. With the present system, an acidic pH will turn off the energy transfer completely while the process will be turned on in alkaline pH. It is strange that ESPT promoted FRET, as studied and reported in this Letter, in spite of its huge potential utility, has not been

*

* + OH-

-

N H

Abs = 295 nm

+ H2O

N

Emission = 420 nm

Emission = 360 nm

+OH -

+H2O

N H

Fluorescence Intensity (A.U.)

D. Sarkar et al. / Chemical Physics Letters 474 (2009) 88–92

(i)

(ix) (ix) (i)

350

400

450 500 550 Wavelength (nm)

600

Fig. 1. Fluorescence spectra of carbazole in the presence of 0.01 M NaOH as a function of MPAPI concentration (kexc = 295 nm). For curves (i)-(ix), MPAPI concentrations are 0, 1.1, 3.3, 5.4, 9.6, 15.0, 21.0 31.0 and 40.0 lM respectively. The black line indicates the emission spectrum of carbazole in absence of NaOH. Concentration of carbazole is 1  105 M.

explored. However, a recent work of Misra and Mishra has documented ESIPT promoted FRET [22]. The reverse process, i.e., FRET promoted ESPT was reported long ago [27], although little pursuance of the study is noted thereafter. This study therefore presents a new strategy that can be unequivocally applied to many suitable partners in sync. Notwithstanding the above significance we have also endeavored to enhance the FRET efficiency by modulating the environment. This has been achieved via trapping of carbazole in the rim region of cyclodextrins (CDs) basically to enhance the ESPT process. CDs are interesting microvessels capable of embedding appropriately sized molecules and the resulting supramolecules can serve as excellent miniature models for enzyme-substrate complexes. The cyclodextrin molecules have internal cavity accessible to the guest molecules of proper dimension through an opening of 4.5–5.3 Å, 6.0–7.0 Å and 7.5–8.5 Å for a-CD, b-CD and c-CD respectively [24,25]. The depths of all the CDs are more or less the same (7.9 Å). It is known that cyclodextrin complexation can give beneficial modification of the guest molecules, such as solubility enhancement, physical isolation of incompatible compounds, control of volatility and sublimation, stabilization of labile guests in terms of long-term protection of color, odor, flavor, etc. [28]. Furthermore, the chemical reactivity of the guest molecule can be modified remarkably through its incorporation into a CD cavity [29]. These unique properties of cyclodextrins have been exploited to improve the FRET efficiency in the present case over that in aqueous medium. 2. Experimental

N

-

Scheme 1. Proton transfer of carbazole in ground and excited states.

O N

CH3

Scheme 2. Structure of MPAPI.

Carbazole (Aldrich) was purified by repeated crystallization from 75% alcohol. The recrystallised sample was vacuum sublimed; column chromatographed using a neutral alumina column and 20% benzene–petroleum ether as eluent, then recrystallised once more. Its purity was checked by melting point determination and spectroscopic measurements. Sodium hydroxide (E. Merck, A.R.) was used without further purification. MPAPI was a kind gift from Prof. S. Bagchi of IISER, Kolkata [25]. a-CD, b-CD and c-CD were purchased from Fluka (USA). Triply distilled water was used to make the experimental solutions. The concentration of carbazole was 1  105 mol dm3 throughout the experiment.

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Absorption and steady-state fluorescence measurements were carried out using a Shimadzu MPS 2000 spectrophotometer and a Spex fluorolog-2 spectrofluorimeter equipped with DM3000F software respectively. All the experiments were performed at ambient temperature (27 °C) with air-equilibrated solutions. 3. Results and discussion The fluorescence spectrum of aqueous solution of carbazole at pH 7 shows two closely spaced peaks with a maximum at around 360 nm. As the pH of the solution is gradually increased, there occurs an excited state proton transfer resulting in a decrease in the intensity of the 360 nm emission band with a concomitant development of a new broad band at 420 nm. In this pH range the absorption as well as the excitation spectra remains unaltered indicating that the proton transfer in carbazole is exclusively an excited state phenomenon [23,24]. This new band at 420 nm is described to be due to emission from the carbazole anion produced in the excited state [23]. The absorption spectrum of MPAPI in aqueous medium shows broad unstructured band with maximum at around 460 nm. Effect of solvent on the fluorometric behavior of MPAPI has been studied in homogeneous and heterogeneous media [25]. It has been found that the longest wavelength absorption and emission bands of MPAPI arise due to the S0 M S1 transitions. The transitions originate from the intermolecular charge transfer from the amine nitrogen to the carbonyl group. In aqueous medium gradual addition of MPAPI to carbazole solution at pH 12.0 depicted a gradual depletion of the emission intensity of the carbazole anion band at 420 nm with a concomitant generation of a new band at 525 nm corresponding to MPAPI through an isoemissive point at 495 nm (Fig. 1). In the absence of the added base, excitation of the system at the same wavelength (295 nm) does not yield the 420 nm emission thereby barring the energy transfer process. The role of pH of the solution in the conjugated process is thus established. In the presence of MPAPI the energy of the photo-produced carbazole anion is transferred to the added acceptor leading to a diminution of the anionic fluorescence at 420 nm (Scheme 3). Blank experiments conducted with MPAPI in the absence of carbazole gave negative results and confirmed the occurrence of the FRET process between the chosen pair. Absence of any new absorption or emission band other than those of carbazole and MPAPI in the respective spectra negates the formation of any ground state or excited state complex between the interacting partners [30,31]. While addition of a-CD results in a lowering in the energy transfer efficiency, relative to that in aqueous medium, addition of b- and c-CD visibly produces a marked enhancement in the efficiency as evident from the degree of quenching in the 420 nm

(i)

(ix)

Fluorescence Intensity (A.U.)

D. Sarkar et al. / Chemical Physics Letters 474 (2009) 88–92

Fluorescence Intensity (A.U.)

90

(i)

(ix)

(ix) (i)

350

400 450 500 550 Wavelength (nm)

600

(ix) (i) 350

400

450 500 550 Wavelength (nm)

600

Fig. 2. Fluorescence spectra of carbazole in the presence of 6 mM b-CD as a function of concentration of MPAPI (kexc = 295 nm, pH 12). For curves (i)-(ix) concentrations of MPAPI are 0, 1.1, 3.3, 5.4, 9.6, 15.0, 21.0, 31.0, and 40.0 lM respectively. Inset shows the corresponding curves in the presence of 6 mM a-CD solution at pH 12.

emission with the addition of MPAPI as well as the yield of the 525 nm emission band (Fig. 2). Quenching of the steady-state fluorescence of the donor (carbazole anion) in the absence and in the presence of different cyclodextrins with the addition of the acceptor (MPAPI) was followed by Stern–Volmer equation, where the terms have their usual meanings [9]

F 0 =F ¼ 1 þ K SV ½Q 

ð1Þ

Slopes of the plots give KSV values in the respective media. The values of KSV are presented in Table 1. Linearity of the plots indicates one type of quenching (Fig. 3) [9]. Insignificant change in the emission band of the neutral form of carbazole (at 360 nm) upon addition of MPAPI confirms that it is not the active partner in the energy transfer and reinforces the impact of ESPT on the FRET process.

Table 1 Some parameters for the energy transfer process in various environments (see text). Environment

KSV  104 (M1)

E

R0 (Å)

r (Å)

Water (pH 12.0) 6 mM a-CD (pH 12.0) 6 mM b-CD (pH 12.0) 6 mM c-CD (pH 12.0)

3.1 ± 0.1 2.9 ± 0.1 4.5 ± 0.1 4.5 ± 0.1

0.37 ± 0.05 0.35 ± 0.05 0.49 ± 0.05 0.49 ± 0.05

29.6 30.0 27.2 26.5

32.2 33.3 27.3 26.7

ESPT of Carbazole Neutral

S1

MPAPI

Anion

FRET

(Donor) S1’ hν ν

295 nm

420 nm S0’

(Acceptor)

S1’’ 525 nm S0’’

S0 Scheme 3. Coupling of ESPT with FRET. Neutral carbazole undergoes ESPT to form anion in the excited state, which then transfers energy to the acceptor MPAPI.

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(F0 / F - 1)

6mM βCD 6mM γCD

0.5

0.0 0

5

10 15 [MPAPI] (μM)

20

25

Fig. 3. Stern–Volmer plots for the quenching of fluorescence of carbazole anion by MPAPI. Inset gives the respective environments at pH 12.

The determined KSV values fall in the normal range reported earlier for the FRET process and are order of magnitude higher than that observed for a normal diffusion controlled quenching process [9,30,31]. This observation suggests that the dominant mechanism of the fluorescence quenching is the resonance energy transfer through long-range dipole–dipole interaction rather than the simple diffusion limited process between the excited donor and the ground state acceptor molecule. 4. Energy transfer efficiency The efficiencies (E) of energy transfer in aqueous and aqueous cyclodextrin media have been determined by Eq. (2) [32–34]

E ¼ 1  F DA =F D

ð2Þ

where FDA and FD are the fluorescence intensities of the donor in the presence and in the absence of the acceptor. The values at 1:1 concentration of the donor–acceptor pair in different environments are provided in Table 1. It is evident from Table 1 that the energy transfer process is remarkably more efficient in b- and c-CD environments while it is retarded marginally in the presence of a-CD. For a FRET process it is important to read the process through a measure of the energy transfer efficiency (E). It is pertinent to mention here that even in presence of the cyclodextrins the system does not deviate appreciably from the classical homogeneous energy transfer model. CD concentrations being order of magnitude higher than that of carbazole in water almost all the probes are encapsulated within CDs [35]. The location of attachment of the probe is the rim region of b- and c-CDs (in a-CD there is hardly any inclusion effect). The interaction with the CDs basically leads to a promotion of the ESPT process [35]. According to Förster’s non-radiative energy transfer theory [32], the energy transfer efficiency E depends not only on the distance (r) between the donor and the acceptor, but also on the Förster distance (R0) (at which the efficiency of energy transfer is 50%). They are related by the equation:

E ¼ ½1 þ ðr=R0 Þ6 1

ð3Þ

The value of R0 depends on the spectral parameters related to both the donor and the acceptor molecular systems. R0 is expressed as follows:

R60 ¼ 8:8  1023 ½j2 n4 /D JðkÞ in Å

6

ð4Þ

where j2 is the factor expressing the relative orientation of the donor to the acceptor molecule, n is the refractive index of the

JðkÞ ¼

Z

1

F D ðkÞeA ðkÞk4 dk

ð5Þ

0

where FD(k) is the corrected fluorescence intensity of the donor at wavelengths k to (k + Dk), with the total intensity normalized to unity and eA(k) is the molar extinction coefficient of the acceptor at wavelength k. The overlap spectrum is shown in Fig. 4. We have determined both Förster distance (R0) and the distance between the donor and acceptor (r) acknowledging that there is an uncertainty in the distance measurement arising principally due to the orientation factor, which is an accepted problem in exploiting this technique. Förster distance has been calculated using the relation 4; the values are tabulated in Table 1. Knowing the values of E and R0, and using Eq. (3) the distance between the donor and the acceptor are estimated (Table 1). The higher values of r compared to R0 is reflected in the values of E, being lower than 0.5. A computed estimate of r can be had from the dimensions and concentrations of the acceptor molecules using nearest neighbor distribution [35]. A glance at Table 1 indicates that the values of R0 and r are minimum for encapsulation in b- and c-CD. The role of cyclodextrins on the efficacy of the ESPT coupled FRET process can be justified from the consideration of the relative dimensions of the CD cavities. Carbazole is encapsulated in b- and c-CD due to their appropriate cavity dimensions with binding constants of 63.3 and 1.14  102 M1 for b- and c-CD respectively. a-CD fails to

Absorbance of MPAPI

6mM αCD

1.0

medium, uD is the quantum yield of the donor in the absence of the acceptor. Generally, j2 is assumed to be 2/3, which is the value for donors and acceptors that randomize by rotational diffusion prior to energy transfer [9]. The quantum yield of carbazole in its neutral form in aqueous solution was determined relative to the quantum yield of the fluorophore in ethanolic medium (u = 0.349) [36]. The quantum yield of the anion of carbazole in aqueous alkaline medium (which is the actual donor in the FRET process here) was determined relative to the quantum yield of the neutral carbazole in water (uWater = 0.346). While doing this the refractive indices of water and aqueous NaOH solution (sufficiently concentrated) were taken as 1.3325 and 1.3628 respectively [37]. J(k) is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor in units of M1 cm3. The overlap integral expresses the degree of spectral overlap between the emission spectrum of the donor with the absorption spectrum of the acceptor. The greater the overlap, higher is the value of R0 and hence higher the energy transfer efficiency [9]. The spectral overlap integral J(k) is calculated by numerical integration method. The overlap integral J(k) for a donor–acceptor pair is defined as [9,30,31]

Fl. of carbazole

Water

350

400

450 500 Wavelength (nm)

550

Fig. 4. Normalized absorption spectrum of MPAPI (dashed line) and fluorescence spectrum of carbazole (solid line) in aqueous solution at pH 12.

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encapsulate carbazole due to its smaller size [35]. The efficiency of the ESPT process is enhanced upon inclusion of carbazole into these two cyclodextrins [35]. The enhanced fluorescence yield of the donor species in the presence of b- and c-CD therefore favors the coupled process. MPAPI, being a long molecule hardly has any binding effect with the cyclodextrins. Thus an enhancement in the FRET efficiency can be achieved by simply introducing cyclodextrins of appropriate dimensions depending on the geometry and dimensions of the interacting dyes or drugs. 5. Conclusion In conclusion, we have successfully coupled excited state proton transfer to fluorescence resonance energy transfer. Thus an efficient energy transfer has been accomplished that has been promoted exclusively through ESPT. The efficacy of the energy transfer process has further been enhanced by the use of cyclodextrins of appropriate cavity dimensions. With a proper choice of the two partners, the coupled reaction (ESPT promoted FRET) has the potential to be utilized conveniently in pH sensitive molecular switching. Extension of this strategy to couple excited state intramolecular proton transfer (ESIPT) with FRET has the potential to be exploited in exploring the protic nature of the medium explicitly. Acknowledgements The authors sincerely thank Prof. S. Bagchi of IISER, Kolkata, for his kind gift of the dye, MPAPI. Financial support from CSIR and DBT, Government of India, is gratefully acknowledged. D.S., A.M. and A.G. thank CSIR for their research fellowships/associateship. References [1] R. Marcus, N. Sutin, Biochem. Biophys. Acta. 811 (1985) 265. [2] J.D. Simon, Acc. Chem. Res. 21 (1988) 128.

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