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Rotational relaxation dynamics of a β-carboline analogue in cyclodextrin nanocavity: How does the cavity size barricade the molecular rotation?
Journal of Molecular Structure 934 (2009) 91–95
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Rotational relaxation dynamics of a b-carboline analogue in cyclodextrin nanocavity: How does the cavity size barricade the molecular rotation? Arabinda Mallick Department of Chemistry, Kashipur Michael Madhusudan Mahavidyalaya, Purulia, West Bengal 723132, India
a r t i c l e
i n f o
Article history: Received 2 May 2009 Received in revised form 15 June 2009 Accepted 17 June 2009 Available online 23 June 2009 Keywords: Rotational dynamics Cyclodextrin Cavity size Anisotropy
a b s t r a c t In the present work the rotational dynamics of a polarity sensitive biologically active b-carboline derivative, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), have been discussed in three native cyclodextrin environments using ultrafast time-resolved fluorescence technique. AODIQ exhibited bi-exponential anisotropy decay in the cyclodextrin environments. The rotational motion of the probe was interpreted on the basis of two independent motion; local (internal motion of the guest within the aggregate) and global (overall rotational tumbling of the complexes). It is found that the cyclodextrin environment causes significant retardation of rotational motion of the probe. Experimental results reveal that relative host–guest size determines the character of the intermolecular host–guest dynamics. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Cyclodextrins (CDs) are attractive microvessels capable of embedding appropriately sized molecules and the resulting supramolecules can serve as an excellent miniature models for nano-bio conjugates [1–5]. These conjugates are drawing much attention of the chemists as well as biologists because of its widespread application in the pharmaceutical industry; especially due to their importance as micro vessel for the selected drug delivery. Spatially confined cyclodextrin nanocavity can modify the chemical reactivity as well as dynamics of the guest molecule significantly because of change in the micropolarity and steric rigidity inside the cavity compared to the situation in the bulk aqueous phase [1–10]. Molecular rotation, one of the most fundamental processes, too, is influenced by the prevalence of these interactions. Due to the presence of primary and secondary hydroxyl groups pointing outside the cavity, the outer surface is hydrophilic, whereas the inner surface, lined with inside pointing CAH groups and ether-like oxygens, is hydrophobic. Because of this particular property, CDs are able to form complex with various organic compounds in aqueous solution [1–10]. The resulting supramolecular structures have profound implications on the structural and dynamical aspects of many chemical and biological systems. Furthermore cyclodextrin complexation can give beneficial modification of guest molecules such as solubility enhancement, stabilization of labile guests, physical isolation of incompatible compounds and control of
E-mail address: [email protected] 0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.06.022
volatility and sublimation. The cyclodextrin molecules have internal cavity accessible to the guest molecules of proper dimension through an opening of 4.5–5.3, 6.0–7.0 and 7.5– 8.5 Å for a-CD, b-CD and c-CD, respectively [5,11]. The depths of all the CDs are more or less the same (7.9 Å). Thus depending on the cavity size, CDs are capable of encapsulating guest molecules of different dimensions. Ultrafast dynamics of a molecule confined in a nanocavity plays key role in many biological processes. The depolarization of fluorescence of probe molecules in cyclodextrins, micelles and lipids, is one of the most powerful methodology yielding structural and dynamical information concerning the tumbling or rotational motion of the probe molecule within the confined environments on the picosecond/nanosecond time scale [12–19]. The fluorophore, AODIQ, used in the present experiment has recently been shown to be an excellent fluorescent probe for biological systems [20–24], belong to the group of bioactive indole family. Molecules containing indole nucleus like b-carbolines, carbazoles, etc. are, by now, well established as bioactive molecules [25–28]. The single step synthesis of AODIQ from 1-methyl3,4-dihydro-b-carboline [25] also leads to the presumption that the molecule should have biological activity. In recent times, some progress towards understanding how the available space for the inclusion of the guest molecule effects the rotational dynamics of the solute molecule in confined systems have been addressed [12–19]. In spite of achieving a reasonable degree of success in that direction, considering the potential of the present molecule in biophysical applications, in the present work the rotational relaxation dynamics have been studied in three common cyclodextrins with a different cavity size.
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
2. Experimental 3-Acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ) (Scheme 1) was synthesized in the laboratory using the method mentioned elsewhere [29]. It was purified by column chromatography and the purity of the compound was checked by thin layer chromatography (TLC). The compound was further vacuum sublimed before use. a-, b- and c-cyclodextrins (Fluka) were used as received without further purification. Triply distilled water was used for making the experimental solutions. The steady state fluorescence anisotropy was performed with a Hitachi spectrofluorimeter F-4010 model. Excitation and emission bandwidths were 5 nm. Steady state anisotropy, r, was defined by:
r ¼ ðIVV G IVH Þ=ðIVV þ 2G IVH Þ
ðiÞ
where IVV and IVH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. The G factor was defined as:
G ¼ IHV =IHH
Steadystate fluorescence anisotropy
92
0.15 0.12 0.09 0.06 0.03 0.00
1.0 Water
1.5
α −2.0 CD
2.5
β −3.0 CD
3.5
γ -CD 4.0
Environment Fig. 1. Maximun change of steady state fluorescence anisotropy in different environments.
ðiiÞ
I terms refer to parameters similar to those mentioned above for the horizontal position of the excitation polarizer. For anisotropy decay, emissions at parallel III and perpendicular (I\) polarizations were collected by rotating the analyzer at regular intervals. The time-resolved anisotropy, r(t) was calculated using the following relation:
rðtÞ ¼ ½III ðtÞ GI? ðtÞ=½III ðtÞ þ 2GI? ðtÞ
ðiiiÞ
where G is the correction factor for the detector sensitivity to the polarization detection of the emission [16]. Apparent (average) rotational relaxation time (sr) for bi-exponential iterative fitting were calculated from the decay times and the pre-exponential factors using the following relation:
< sr >¼ a1r s1r þ a2r s2r :
ðivÞ
3. Results and discussion 3.1. Steady state absorption and emission The steady state absorption and fluorescence spectra of AODIQ (Scheme 1) in aqueous and different CD environments are already discussed in one of our previous report [24]. However, for the general readership and to make the base to understand the time-resolved data, here we highlight only the salient features of the steady state results. The absorption spectrum of an aqueous solution of AODIQ shows a broad and unstructured low energy band with a maximum at around 420 nm. Addition of CDs to the aqueous solution of AODIQ hardly changes the absorption spectra. Room temperature emission spectrum of AODIQ in aqueous medium is characterized by a broad unstructured band with a maximum around 520 nm ascribed to the intramolecular charge transfer (ICT) transition within the fluorophore [30]. Gradual addition of the CDs to the aqueous solution of the probe leads to a
N
N
O
H
Scheme 1. Structure of AODIQ.
3.2. Steady state and time-resolved fluorescence anisotropy The steady state anisotropy of AODIQ in water was found to be 0.04. While in the CD environments the steady state anisotropy increased with the CD concentration (Fig. 1). From the above figure it is evident that the in all the CD environments the steady state anisotropy value is increased compared to bulk aqueous phase. But from the quantitative point of view the c-CD is much different from that of the other two-CD (Table 1). Such high anisotropy value in c-CD environments indicate that rotational motion of the probe is much more restricted compared to the others. Similar high steady state anisotropy value in c-CD was reported for DPH and Coumarin 153 [31–33]. To get more information on the dynamic aspects of this biological molecule regarding the rotational motion time-resolved fluorescence anisotropy decay was studied in the CD environments. Time-resolved studies are much more sensitive than the steady state ones for exploring the local environment around a fluorophore [16]. The time-resolved decay of the fluorescence anisotropy [r(t)] of the organic dye molecule is directly related to the reorientation dynamics of excited molecules and hence best suited for the investigation of local molecular dynamics near the binding site. In order to see how the rotational relaxation dynamics of the probe is affected for changing the environments from bulk water to the cyclodextrin environments, time-resolved fluorescence anisotropy measurements have been performed. In pure aqueous medium the
Table 1 Some photophysical parameters of AODIQ in different cyclodextrin environments.
CH3 O
hypsochromic shift of the emission maximum, an enhancement in the fluorescence yield and a narrowing of the emission band. All these observations suggest that the polarity of the CD environment is less than the polarity of the bulk aqueous phase since similar effects have been observed in less polar solvents [21,22,24,30]. The increase in the fluorescence quantum yield in the CD environments is rationalized from the consideration of the relative stabilization of the ICT state of the probe [24].
Environments
Emission maxima
Quantum yield
Steady state anisotropy
Water a-CD (35 mM) b-CD (10 mM) c-CD (30 mM)
520 515 513 479
0.08 0.10 0.12 0.15
0.039 0.056 0.059 0.142
93
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
0.5
Water α-CD β-CD γ -CD
0.4
r(t)
0.3 0.2 0.1 0.0 -0.1
0
1000
2000
3000
4000
Time (ps) Fig. 2. Fluorescence anisotropy decay [r(t)] of AODIQ in aqueous CD environment.
anisotropy decay is single exponential. Addition of CDs changes the relaxation behavior of AODIQ significantly and it becomes completely bi-exponential in the cyclodextrin media. Fig. 2 illustrates the fluorescence anisotropy decays of AODIQ in aqueous and aqueous cyclodextrin environments. The functional form of the bi-exponential anisotropy decay r(t) in CDs can be represented as:
rðtÞ ¼ r0 ½a1 expðt=s1r Þ þ a2 expðt=s2r Þ
ðvÞ
where r0 is the limiting anisotropy, which describes the inherent depolarization of a given molecule [16]. s1r and s2r are the tworeorientation times of the probe in CDs, a1r and a2r are the pre-exponents that provide the fraction of the two-reorientation times. All the anisotropy decay parameters in aqueous as well as aqueous cyclodextrin environments are collected in Table 2. From the above Table it is clear that in all the CD cavity, the anisotropy decays are characterized by a fast component s1r and a slow component s2r; the faster component dominating over the slower one, (a1r > a2r) for a and b-CD while it is reversed in case of c-CD. The values of the anisotropy decays listed in Table 2 show that AODIQ molecule exhibits much slower fluorescence anisotropy decay in cyclodextrin environments as compared to that in pure water. In pure water the rotational relaxation of AODIQ probe occurs within 145–190 ps time scale. The shorter component is close to the reorientation time of the probe in aqueous medium, whereas the longer component is significantly higher (4–14 times depending on the CDs). This indicates that the AODIQ molecule faces motionally restricted environments in cyclodextrins. To compare the relative extent of restriction in CDs, average rotational relaxation time can roughly be used, because it is model independent [13]. The average rotational relaxation times () were calculated using Eq. (iv). The values of are also incorporated in Table 2. For AODIQ, reorientation seems to occur on similar time scales in a-CD and b-CD environments and the process is 2–3 times slower than the reorientation process in aqueous phase. Interestingly, in c-CD environments this
process is about eight times slower than that in water. This indicates that the probe is motionally restricted to a much greater extent in cCD environment compared to the other two-CD environment. Fig. 2 clearly reflects this trend. In cyclodextrin media, the observation of two distinctly different time constants in anisotropy decay implies the existence of two dynamical processes that occur on different time scales. Following the previous works by many workers [1,32,34–36] I attribute the anisotropy decay (in CD) is only due to the dynamics of the dye encapsulated in CD and not due to the free dye in solution. Balabai et al. explained this issue using chromophores having different charge (cationic, anionic, and neutral) and showed that the anisotropy decay in CD environments is only due to the bound dye only and the fast component of the decay cannot be attributed to the rotational relaxation of free guest [36]. Using resorufin (anion), oxazine-118 (cation) they found that the fast component reflects the internal motion of the guest inside the cavity of cyclodextrins and the slow component of the decay corresponds to the overall motion of the complexes. El-Kemary and Tormo et al explained the same issue in the light of same model using Milrinone drug and methyl 2-amino-4,5-dimethoxy bezoate as guest molecule [34,35]. Following the work of Balabai et al. [36] Roy et al. [32], Tormo et al. [35] and most recently El-Kemary et al. [34] attempts have been taken to explain the observed bi-exponential decay in terms of the probe experiencing different kinds of rotation in the CD environments. Such a behavior is often explained under the assumption that the long time components are due to the overall motion of the host–guest complex and the short time components are due to the independent internal angular motion of the probe molecules inside the cyclodextrin cavity. In their works Balabai and his co-workers have measured the rotational relaxation times of different probes in CD cavity and they showed that the rotational relaxation time for CD increases as a result of formation of inclusion complexes with the probe molecules [36]. The present experimental results follow similar trends; rotational relaxation time increases with the formation of inclusion complexes. As a matter of fact, confinement of a probe inside the CD cavity increases the hydrodynamic diameter of the system (the sum of the lengths of the host, the CD, and the guest) and this causes enhancement of the rotational relaxation time. Thus, the increase in rotational relaxation time of probe-CD inclusion complexes can be rationalized in terms of increase in the effective volume and dimension of the inclusion complexes as compared to the free probe or CD molecule (considering the measured very small difference in lifetime and assuming the macroscopic viscosity is same in all the solution containing CDs). The size of the probe is bigger than the CD cavity then it is obvious that some portion of the probe molecule remains exposed to the aqueous environments resulting in an increase in the effective volume of the inclusion complexes and this consideration will surely increases the effective volume. Consequently the latter will experience an increased friction during rotation in comparison to the free probe or CD. Considering the same average length of all the CDs and for the same probe the huge enhancement of the average rotational relaxation time in case of c-CD compared to the other CDs implies the larger volume of the inclusion complexes for the latter.
Table 2 Decay parameters of fluorescence anisotropy of AODIQ in different CD cavity. Environment
Conc. (mM)
r0
a1r
s1r (ns)
a2r
s2r (ns)
(ns)
v2
Water a-CD b-CD c-CD
– 30 10 30.40
0.40 0.40 0.39
1.0 0.787 0.746 0.396
0.145 0.186 0.170 0.141
0.212 0.253 0.603
1.289 0.848 1.858
0.145 0.419 0.341 1.139
1.02 1.03 1.37 1.27
94
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
N H
N O CH3 O
N H
N O CH3 O
N H
O N
N O CH3
H3C
N H
O
O
O N H3C O
N H
N H
N O CH3 O
Fig. 3. Schemetic representation (AODIQ-c-CD) aggregates.
From a quick look on the data presented in Tables 2 one can realize that both internal and overall motion of the host–guest complex is altered with the cavity diameter of the cyclodextrins. The time constants for the internal motion of the guest a-CD > bCD > c-CD while the slower component is in the order of cCD > a-CD > b-CD. The size of the CD cavity c > b > a. It is generally accepted that only one phenyl ring is able to penetrate the small cavity of a-CD; thus, rest of the molecule is reasonably outside the cavity. Since the cavity diameter of the b-CD and c-CD is higher than the a-CD so the probe molecule can enjoy more space available for its rotation and consequently experiences relatively free in motion that’s why the fast time constant increases when we move from a- to c-CD. To verify the dependence of the slower component in the different CD we have to look on the geometrical characteristics of the reactants. This dependence clearly indicates that the size of the inclusion complexes is also in the order of cCD > a-CD > b-CD. At this connection it is important to note down the interesting aspects of this present article. In pure water and for the other CDs the rotational relaxation of AODIQ probe occurs within 145– 190 ps time scale with no residual anisotropy decay. From the Fig. 2 it is evident that in the presence of 35 mM c-CD the fluorescence anisotropy decay of AODIQ exhibits a large residual decay which is totally absent in case of other CDs and also for the lower concentration of c-CD. This observation suggests that in high concentration (35 mM) of c-CD the hydrodynamic size of the host– guest complex is very large. Very recently the hydrodynamic size of this suprastructures measured by TEM and DLS studies have been reported; it is in the order of micrometer [37]. This size clarifies the dependence of slow component in different CDs. To extract and develop the high potential of the formed suprastructures, it is essential to demonstrate the possible conformation of the elemental molecules stranded on the molecular chain. In addition, since the conformation of the c-CD in a molecular necklace directly reflects the formation process, analysis of the c-CD conformation is also important from both fundamental and practical points of view. The probe forms the inclusion complexes with the c-CD molecule (at high concentrations) maintaining 1:2 stoichiometry [24]. So the initial building block is one probe molecule associated with two-CD molecules [24]. Regarding the orientation of the CD molecules, c-CDs are thought to be stranded one by one. Since c-CD has two different hydroxyl groups (primary and secondary) on the two rim-ends of its cavity, it can have three possible conformations, namely head to head (secondary to secondary), head to tail (secondary to primary) and tail to tail (primary to primary). From their nuclear magnetic resonance (NMR), Ultraviolet (UV) and X-ray diffraction studies on the inclusion complexes of polymeric probes Harada et al proposed 100% head to head (secondary to secondary) or tail to tail (primary to primary) orientation of the CD [38]. Unfortunately, these methods can only give the macroscopic and averaged information regarding the conformations of these supramolecules. Recently Miyake et al reframed the proposition by using scanning electron microscopy (SEM) and they concluded that about 20% head to tail (secondary to primary) conformation exist in such type of aggregates, the rest is head to head or tail to tail [39]. Considering coexistence of all orientational possibilities of the c-CD units following Li and McGown [40], Roy [32] and Pistolis
et al. [31] a possible model of the AODIQ-c-CD aggregates where AODIQ molecules are completely shielded by the CDs (Fig. 3) have been presented. Other possible orientations are also logically feasible. At this present moment I am unable to address one question properly that in the presence of the same molecule why a-CD and b-CD fail to form this type of aggregates? I believe that proper matching of the geometry of the probe and the cyclodextrins and also the geometry restricted condition is the key factor which provides energetic contribution as well as the order and directionality needed for the formation of very well defined nanostructures. Probably a- and b-CD cannot satisfy this condition as the c-CD so this CDs are not able to form such type of aggregates. 4. Conclusion The present study reports the rotational relaxation dynamics of a potential bioactive molecule, AODIQ, in three common CD environments differing in cavity size. The rotational relaxation dynamics of AODIQ are modified remarkably when it is encapsulated in the CD cavity. Significant increase in the average rotational correlation time in CDS as compared to that in water indicates that the rotational dynamics of AODIQ is substantially slowed down upon binding of the probe with the CDs. From the time dependent anisotropy decay it is inferred that the interaction of AODIQ with the wider cavity of the c-CD gives rise to a formation of nanotubular type linear aggregates of AODIQ: c-CD. The present report projects that time-resolved anisotropy may be a convenient tool (at primary level) for the detection of formation of nanoaggregates. Acknowledgements The author greatly indebted to Prof. N. Chattopadhyay for his encouragement, allround support providing laboratory facilities etc. and helpful discussion. The author greatly appreciate the cooperation received from Prof. N. Sarkar, Dr. A. Chakraborty, Dr. D. Seth of I.I.T. Kharagpur and Prof. S. Basak of SINP Kolkata, for their kind help in time-resolved and steady state fluorescence anisotropy measurements, respectively. The author is also thankful to his friend Dr. B. Haldar and all group members of Prof. Chattopadhyay. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
A. Douhal, Chem. Rev. 104 (2004) 1995. Y. He, P. Fu, X. Shen, H. Gao, Micron 39 (2008) 495. T. Shimizu, M. Masuda, H. Minamikawa, Chem. Rev. 105 (2005) 1401. B. Haldar, A. Mallick, N. Chattopadhyay, J. Chem. Edu. 89 (2008) 429. V.T.D. Souza, M.L. Bender, Acc. Chem. Res. 20 (1987) 146–152. A. Mallick, B. Haldar, N. Chattopadhyay, J. Photochem. Photobiol. B 78 (2005) 215. A. Munoz de la Pena, T. Ndou, J. Zung, I.M. Warner, J. Phys. Chem. 95 (1991) 3330–3334. S. Hamai, J. Phys. Chem. B 101 (1997) 1707. G.S. Cox, N.J. Turro, J. Am. Chem. Soc. 106 (1984) 422. D.W. Cho, Y.H. Kim, S.G. Kang, M. Yoon, J. Phys. Chem. 98 (1994) 558. S. Li, W.C. Purdy, Chem. Rev. 92 (1992) 1457. G.R. Fleming, Chemical Applications of Ultrafast Spectroscopy, Oxford University Press, New York, 1986. G.B. Dutt, J. Phys. Chem. B 108 (2004) 805. D. Chakrabarty, A. Chakrabarty, D. Seth, P. Hazra, N. Sarkar, J. Chem. Phys. 122 (2005) 184516.
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95 [15] P. Das, A. Mallick, A. Chakrabarty, B. Haldar, N. Chattopadhyay, J. Chem. Phys. 125 (2006) 044516. [16] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1999. [17] A. Chakraborty, D. Chakraborty, P. Hazra, D. Seth, N. Sarkar, Chem. Phys. Lett. 397 (2004) 216. [18] A. Chakraborty, D. Chakraborty, P. Hazra, D. Seth, N. Sarkar, Chem. Phys. Lett. 397 (2004) 469. [19] E.L. Quitevis, A.H. Marcus, F.D. Fayer, J. Phys. Chem. 97 (1993) 5762. [20] A. Mallick, B. Haldar, S. Maiti, S.C. Bera, N. Chattopadhyay, J. Phys. Chem. B 109 (2005) 14675. [21] A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem. B 108 (2005) 14683. [22] A. Mallick, B. Haldar, S. Maiti, N. Chattopadhyay, J. Colloid Interface Sci. 108 (2005) 14683. [23] (a) A. Mallick, M.C. Mandal, B. Haldar, A. Chakrabarty, P. Das, N. Chattopadhyay, J. Am. Chem. Soc. 128 (2006) 3126; (b) A. Mallick, M.C. Mandal, B. Haldar, A. Chakrabarty, P. Das, N. Chattopadhyay, J. Am. Chem. Soc. 128 (2006) 10629. [24] P. Das, A. Chakrabarty, B. Haldar, A. Mallick, N. Chattopadhyay, J. Phys. Chem. B 111 (2007) 7401. [25] P. Das, A. Mallick, D. Sarker, N. Chattopadhyay, J. Colloid Interface Sci. 320 (2008) 9. [26] A. Dias, A.P. Varela, M.G. Miguel, A.L. Maçanita, R.S. Becker, H.D. Burrows, J. Phys. Chem. 100 (1996) 17970.
95
[27] A. Chakrabarty, A. Mallick, B. Haldar, P. Das, N. Chattopadhyay, Biomacromolecules 8 (2007) 920. [28] A. Chakrabarty, A. Mallick, B. Haldar, P. Purkayastha, P. Das, N. Chattopadhyay, Langmuir 23 (2007) 4842. [29] V.S. Giri, B.C. Maity, S.C. Pakrashi, Heterocycles 22 (1984) 233. [30] A. Mallick, S. Maiti, B. Haldar, P. Purkayastha, N. Chattopadhyay, Chem. Phys. Lett. 371 (2003) 688. [31] G. Pistolis, I. Balomenou, J. Phys. Chem. B 100 (2006) 15562. [32] D. Roy, S.K. Mondal, K. Sahu, S. Ghosh, P. Sen, K. Bhattacharyya, J. Phys. Chem. A 109 (2005) 9716. [33] P. Sen, D. Roy, S.K. Mondal, K. Sahu, S. Ghosh, K. Bhattacharyya, J. Phys. Chem. A 109 (2005) 9716. [34] M. El-Kemary, J.A. Organero, L. Santos, A. Douhal, J. Phys. Chem. B 110 (2006) 14128. [35] L. Tormo, J.A. Organero, A. Douhal, J. Phys. Chem. B 109 (2005) 17848. [36] N. Balabai, B. Linton, A. Napper, S. Priyadarshi, A.P. Sukharevsky, D.H. Waldek, J. Phys. Chem. B 102 (1998) 9617. [37] P. Das, A. Mallick, D. Sarkar, N. Chattopadhyay, J. Phys. Chem. C 112 (2008) 9600. [38] A. Harada, J. LI, M. Kamachi, J. Am. Chem. Soc. 116 (1994) 3192. [39] K. Miyake, S. Yasuda, A. Harada, J. Sumaoka, M. Komiyama, H. Shigekawa, J. Am. Chem. Soc. 125 (2003) 5080. [40] G. Li, L.B. McGown, Science 264 (1994) 249.
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Rotational relaxation dynamics of a b-carboline analogue in cyclodextrin nanocavity: How does the cavity size barricade the molecular rotation? Arabinda Mallick Department of Chemistry, Kashipur Michael Madhusudan Mahavidyalaya, Purulia, West Bengal 723132, India
a r t i c l e
i n f o
Article history: Received 2 May 2009 Received in revised form 15 June 2009 Accepted 17 June 2009 Available online 23 June 2009 Keywords: Rotational dynamics Cyclodextrin Cavity size Anisotropy
a b s t r a c t In the present work the rotational dynamics of a polarity sensitive biologically active b-carboline derivative, 3-acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ), have been discussed in three native cyclodextrin environments using ultrafast time-resolved fluorescence technique. AODIQ exhibited bi-exponential anisotropy decay in the cyclodextrin environments. The rotational motion of the probe was interpreted on the basis of two independent motion; local (internal motion of the guest within the aggregate) and global (overall rotational tumbling of the complexes). It is found that the cyclodextrin environment causes significant retardation of rotational motion of the probe. Experimental results reveal that relative host–guest size determines the character of the intermolecular host–guest dynamics. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Cyclodextrins (CDs) are attractive microvessels capable of embedding appropriately sized molecules and the resulting supramolecules can serve as an excellent miniature models for nano-bio conjugates [1–5]. These conjugates are drawing much attention of the chemists as well as biologists because of its widespread application in the pharmaceutical industry; especially due to their importance as micro vessel for the selected drug delivery. Spatially confined cyclodextrin nanocavity can modify the chemical reactivity as well as dynamics of the guest molecule significantly because of change in the micropolarity and steric rigidity inside the cavity compared to the situation in the bulk aqueous phase [1–10]. Molecular rotation, one of the most fundamental processes, too, is influenced by the prevalence of these interactions. Due to the presence of primary and secondary hydroxyl groups pointing outside the cavity, the outer surface is hydrophilic, whereas the inner surface, lined with inside pointing CAH groups and ether-like oxygens, is hydrophobic. Because of this particular property, CDs are able to form complex with various organic compounds in aqueous solution [1–10]. The resulting supramolecular structures have profound implications on the structural and dynamical aspects of many chemical and biological systems. Furthermore cyclodextrin complexation can give beneficial modification of guest molecules such as solubility enhancement, stabilization of labile guests, physical isolation of incompatible compounds and control of
E-mail address: [email protected] 0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.06.022
volatility and sublimation. The cyclodextrin molecules have internal cavity accessible to the guest molecules of proper dimension through an opening of 4.5–5.3, 6.0–7.0 and 7.5– 8.5 Å for a-CD, b-CD and c-CD, respectively [5,11]. The depths of all the CDs are more or less the same (7.9 Å). Thus depending on the cavity size, CDs are capable of encapsulating guest molecules of different dimensions. Ultrafast dynamics of a molecule confined in a nanocavity plays key role in many biological processes. The depolarization of fluorescence of probe molecules in cyclodextrins, micelles and lipids, is one of the most powerful methodology yielding structural and dynamical information concerning the tumbling or rotational motion of the probe molecule within the confined environments on the picosecond/nanosecond time scale [12–19]. The fluorophore, AODIQ, used in the present experiment has recently been shown to be an excellent fluorescent probe for biological systems [20–24], belong to the group of bioactive indole family. Molecules containing indole nucleus like b-carbolines, carbazoles, etc. are, by now, well established as bioactive molecules [25–28]. The single step synthesis of AODIQ from 1-methyl3,4-dihydro-b-carboline [25] also leads to the presumption that the molecule should have biological activity. In recent times, some progress towards understanding how the available space for the inclusion of the guest molecule effects the rotational dynamics of the solute molecule in confined systems have been addressed [12–19]. In spite of achieving a reasonable degree of success in that direction, considering the potential of the present molecule in biophysical applications, in the present work the rotational relaxation dynamics have been studied in three common cyclodextrins with a different cavity size.
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
2. Experimental 3-Acetyl-4-oxo-6,7-dihydro-12H indolo-[2,3-a] quinolizine (AODIQ) (Scheme 1) was synthesized in the laboratory using the method mentioned elsewhere [29]. It was purified by column chromatography and the purity of the compound was checked by thin layer chromatography (TLC). The compound was further vacuum sublimed before use. a-, b- and c-cyclodextrins (Fluka) were used as received without further purification. Triply distilled water was used for making the experimental solutions. The steady state fluorescence anisotropy was performed with a Hitachi spectrofluorimeter F-4010 model. Excitation and emission bandwidths were 5 nm. Steady state anisotropy, r, was defined by:
r ¼ ðIVV G IVH Þ=ðIVV þ 2G IVH Þ
ðiÞ
where IVV and IVH are the intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. The G factor was defined as:
G ¼ IHV =IHH
Steadystate fluorescence anisotropy
92
0.15 0.12 0.09 0.06 0.03 0.00
1.0 Water
1.5
α −2.0 CD
2.5
β −3.0 CD
3.5
γ -CD 4.0
Environment Fig. 1. Maximun change of steady state fluorescence anisotropy in different environments.
ðiiÞ
I terms refer to parameters similar to those mentioned above for the horizontal position of the excitation polarizer. For anisotropy decay, emissions at parallel III and perpendicular (I\) polarizations were collected by rotating the analyzer at regular intervals. The time-resolved anisotropy, r(t) was calculated using the following relation:
rðtÞ ¼ ½III ðtÞ GI? ðtÞ=½III ðtÞ þ 2GI? ðtÞ
ðiiiÞ
where G is the correction factor for the detector sensitivity to the polarization detection of the emission [16]. Apparent (average) rotational relaxation time (sr) for bi-exponential iterative fitting were calculated from the decay times and the pre-exponential factors using the following relation:
< sr >¼ a1r s1r þ a2r s2r :
ðivÞ
3. Results and discussion 3.1. Steady state absorption and emission The steady state absorption and fluorescence spectra of AODIQ (Scheme 1) in aqueous and different CD environments are already discussed in one of our previous report [24]. However, for the general readership and to make the base to understand the time-resolved data, here we highlight only the salient features of the steady state results. The absorption spectrum of an aqueous solution of AODIQ shows a broad and unstructured low energy band with a maximum at around 420 nm. Addition of CDs to the aqueous solution of AODIQ hardly changes the absorption spectra. Room temperature emission spectrum of AODIQ in aqueous medium is characterized by a broad unstructured band with a maximum around 520 nm ascribed to the intramolecular charge transfer (ICT) transition within the fluorophore [30]. Gradual addition of the CDs to the aqueous solution of the probe leads to a
N
N
O
H
Scheme 1. Structure of AODIQ.
3.2. Steady state and time-resolved fluorescence anisotropy The steady state anisotropy of AODIQ in water was found to be 0.04. While in the CD environments the steady state anisotropy increased with the CD concentration (Fig. 1). From the above figure it is evident that the in all the CD environments the steady state anisotropy value is increased compared to bulk aqueous phase. But from the quantitative point of view the c-CD is much different from that of the other two-CD (Table 1). Such high anisotropy value in c-CD environments indicate that rotational motion of the probe is much more restricted compared to the others. Similar high steady state anisotropy value in c-CD was reported for DPH and Coumarin 153 [31–33]. To get more information on the dynamic aspects of this biological molecule regarding the rotational motion time-resolved fluorescence anisotropy decay was studied in the CD environments. Time-resolved studies are much more sensitive than the steady state ones for exploring the local environment around a fluorophore [16]. The time-resolved decay of the fluorescence anisotropy [r(t)] of the organic dye molecule is directly related to the reorientation dynamics of excited molecules and hence best suited for the investigation of local molecular dynamics near the binding site. In order to see how the rotational relaxation dynamics of the probe is affected for changing the environments from bulk water to the cyclodextrin environments, time-resolved fluorescence anisotropy measurements have been performed. In pure aqueous medium the
Table 1 Some photophysical parameters of AODIQ in different cyclodextrin environments.
CH3 O
hypsochromic shift of the emission maximum, an enhancement in the fluorescence yield and a narrowing of the emission band. All these observations suggest that the polarity of the CD environment is less than the polarity of the bulk aqueous phase since similar effects have been observed in less polar solvents [21,22,24,30]. The increase in the fluorescence quantum yield in the CD environments is rationalized from the consideration of the relative stabilization of the ICT state of the probe [24].
Environments
Emission maxima
Quantum yield
Steady state anisotropy
Water a-CD (35 mM) b-CD (10 mM) c-CD (30 mM)
520 515 513 479
0.08 0.10 0.12 0.15
0.039 0.056 0.059 0.142
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A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
0.5
Water α-CD β-CD γ -CD
0.4
r(t)
0.3 0.2 0.1 0.0 -0.1
0
1000
2000
3000
4000
Time (ps) Fig. 2. Fluorescence anisotropy decay [r(t)] of AODIQ in aqueous CD environment.
anisotropy decay is single exponential. Addition of CDs changes the relaxation behavior of AODIQ significantly and it becomes completely bi-exponential in the cyclodextrin media. Fig. 2 illustrates the fluorescence anisotropy decays of AODIQ in aqueous and aqueous cyclodextrin environments. The functional form of the bi-exponential anisotropy decay r(t) in CDs can be represented as:
rðtÞ ¼ r0 ½a1 expðt=s1r Þ þ a2 expðt=s2r Þ
ðvÞ
where r0 is the limiting anisotropy, which describes the inherent depolarization of a given molecule [16]. s1r and s2r are the tworeorientation times of the probe in CDs, a1r and a2r are the pre-exponents that provide the fraction of the two-reorientation times. All the anisotropy decay parameters in aqueous as well as aqueous cyclodextrin environments are collected in Table 2. From the above Table it is clear that in all the CD cavity, the anisotropy decays are characterized by a fast component s1r and a slow component s2r; the faster component dominating over the slower one, (a1r > a2r) for a and b-CD while it is reversed in case of c-CD. The values of the anisotropy decays listed in Table 2 show that AODIQ molecule exhibits much slower fluorescence anisotropy decay in cyclodextrin environments as compared to that in pure water. In pure water the rotational relaxation of AODIQ probe occurs within 145–190 ps time scale. The shorter component is close to the reorientation time of the probe in aqueous medium, whereas the longer component is significantly higher (4–14 times depending on the CDs). This indicates that the AODIQ molecule faces motionally restricted environments in cyclodextrins. To compare the relative extent of restriction in CDs, average rotational relaxation time can roughly be used, because it is model independent [13]. The average rotational relaxation times (
process is about eight times slower than that in water. This indicates that the probe is motionally restricted to a much greater extent in cCD environment compared to the other two-CD environment. Fig. 2 clearly reflects this trend. In cyclodextrin media, the observation of two distinctly different time constants in anisotropy decay implies the existence of two dynamical processes that occur on different time scales. Following the previous works by many workers [1,32,34–36] I attribute the anisotropy decay (in CD) is only due to the dynamics of the dye encapsulated in CD and not due to the free dye in solution. Balabai et al. explained this issue using chromophores having different charge (cationic, anionic, and neutral) and showed that the anisotropy decay in CD environments is only due to the bound dye only and the fast component of the decay cannot be attributed to the rotational relaxation of free guest [36]. Using resorufin (anion), oxazine-118 (cation) they found that the fast component reflects the internal motion of the guest inside the cavity of cyclodextrins and the slow component of the decay corresponds to the overall motion of the complexes. El-Kemary and Tormo et al explained the same issue in the light of same model using Milrinone drug and methyl 2-amino-4,5-dimethoxy bezoate as guest molecule [34,35]. Following the work of Balabai et al. [36] Roy et al. [32], Tormo et al. [35] and most recently El-Kemary et al. [34] attempts have been taken to explain the observed bi-exponential decay in terms of the probe experiencing different kinds of rotation in the CD environments. Such a behavior is often explained under the assumption that the long time components are due to the overall motion of the host–guest complex and the short time components are due to the independent internal angular motion of the probe molecules inside the cyclodextrin cavity. In their works Balabai and his co-workers have measured the rotational relaxation times of different probes in CD cavity and they showed that the rotational relaxation time for CD increases as a result of formation of inclusion complexes with the probe molecules [36]. The present experimental results follow similar trends; rotational relaxation time increases with the formation of inclusion complexes. As a matter of fact, confinement of a probe inside the CD cavity increases the hydrodynamic diameter of the system (the sum of the lengths of the host, the CD, and the guest) and this causes enhancement of the rotational relaxation time. Thus, the increase in rotational relaxation time of probe-CD inclusion complexes can be rationalized in terms of increase in the effective volume and dimension of the inclusion complexes as compared to the free probe or CD molecule (considering the measured very small difference in lifetime and assuming the macroscopic viscosity is same in all the solution containing CDs). The size of the probe is bigger than the CD cavity then it is obvious that some portion of the probe molecule remains exposed to the aqueous environments resulting in an increase in the effective volume of the inclusion complexes and this consideration will surely increases the effective volume. Consequently the latter will experience an increased friction during rotation in comparison to the free probe or CD. Considering the same average length of all the CDs and for the same probe the huge enhancement of the average rotational relaxation time in case of c-CD compared to the other CDs implies the larger volume of the inclusion complexes for the latter.
Table 2 Decay parameters of fluorescence anisotropy of AODIQ in different CD cavity. Environment
Conc. (mM)
r0
a1r
s1r (ns)
a2r
s2r (ns)
v2
Water a-CD b-CD c-CD
– 30 10 30.40
0.40 0.40 0.39
1.0 0.787 0.746 0.396
0.145 0.186 0.170 0.141
0.212 0.253 0.603
1.289 0.848 1.858
0.145 0.419 0.341 1.139
1.02 1.03 1.37 1.27
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A. Mallick / Journal of Molecular Structure 934 (2009) 91–95
N H
N O CH3 O
N H
N O CH3 O
N H
O N
N O CH3
H3C
N H
O
O
O N H3C O
N H
N H
N O CH3 O
Fig. 3. Schemetic representation (AODIQ-c-CD) aggregates.
From a quick look on the data presented in Tables 2 one can realize that both internal and overall motion of the host–guest complex is altered with the cavity diameter of the cyclodextrins. The time constants for the internal motion of the guest a-CD > bCD > c-CD while the slower component is in the order of cCD > a-CD > b-CD. The size of the CD cavity c > b > a. It is generally accepted that only one phenyl ring is able to penetrate the small cavity of a-CD; thus, rest of the molecule is reasonably outside the cavity. Since the cavity diameter of the b-CD and c-CD is higher than the a-CD so the probe molecule can enjoy more space available for its rotation and consequently experiences relatively free in motion that’s why the fast time constant increases when we move from a- to c-CD. To verify the dependence of the slower component in the different CD we have to look on the geometrical characteristics of the reactants. This dependence clearly indicates that the size of the inclusion complexes is also in the order of cCD > a-CD > b-CD. At this connection it is important to note down the interesting aspects of this present article. In pure water and for the other CDs the rotational relaxation of AODIQ probe occurs within 145– 190 ps time scale with no residual anisotropy decay. From the Fig. 2 it is evident that in the presence of 35 mM c-CD the fluorescence anisotropy decay of AODIQ exhibits a large residual decay which is totally absent in case of other CDs and also for the lower concentration of c-CD. This observation suggests that in high concentration (35 mM) of c-CD the hydrodynamic size of the host– guest complex is very large. Very recently the hydrodynamic size of this suprastructures measured by TEM and DLS studies have been reported; it is in the order of micrometer [37]. This size clarifies the dependence of slow component in different CDs. To extract and develop the high potential of the formed suprastructures, it is essential to demonstrate the possible conformation of the elemental molecules stranded on the molecular chain. In addition, since the conformation of the c-CD in a molecular necklace directly reflects the formation process, analysis of the c-CD conformation is also important from both fundamental and practical points of view. The probe forms the inclusion complexes with the c-CD molecule (at high concentrations) maintaining 1:2 stoichiometry [24]. So the initial building block is one probe molecule associated with two-CD molecules [24]. Regarding the orientation of the CD molecules, c-CDs are thought to be stranded one by one. Since c-CD has two different hydroxyl groups (primary and secondary) on the two rim-ends of its cavity, it can have three possible conformations, namely head to head (secondary to secondary), head to tail (secondary to primary) and tail to tail (primary to primary). From their nuclear magnetic resonance (NMR), Ultraviolet (UV) and X-ray diffraction studies on the inclusion complexes of polymeric probes Harada et al proposed 100% head to head (secondary to secondary) or tail to tail (primary to primary) orientation of the CD [38]. Unfortunately, these methods can only give the macroscopic and averaged information regarding the conformations of these supramolecules. Recently Miyake et al reframed the proposition by using scanning electron microscopy (SEM) and they concluded that about 20% head to tail (secondary to primary) conformation exist in such type of aggregates, the rest is head to head or tail to tail [39]. Considering coexistence of all orientational possibilities of the c-CD units following Li and McGown [40], Roy [32] and Pistolis
et al. [31] a possible model of the AODIQ-c-CD aggregates where AODIQ molecules are completely shielded by the CDs (Fig. 3) have been presented. Other possible orientations are also logically feasible. At this present moment I am unable to address one question properly that in the presence of the same molecule why a-CD and b-CD fail to form this type of aggregates? I believe that proper matching of the geometry of the probe and the cyclodextrins and also the geometry restricted condition is the key factor which provides energetic contribution as well as the order and directionality needed for the formation of very well defined nanostructures. Probably a- and b-CD cannot satisfy this condition as the c-CD so this CDs are not able to form such type of aggregates. 4. Conclusion The present study reports the rotational relaxation dynamics of a potential bioactive molecule, AODIQ, in three common CD environments differing in cavity size. The rotational relaxation dynamics of AODIQ are modified remarkably when it is encapsulated in the CD cavity. Significant increase in the average rotational correlation time in CDS as compared to that in water indicates that the rotational dynamics of AODIQ is substantially slowed down upon binding of the probe with the CDs. From the time dependent anisotropy decay it is inferred that the interaction of AODIQ with the wider cavity of the c-CD gives rise to a formation of nanotubular type linear aggregates of AODIQ: c-CD. The present report projects that time-resolved anisotropy may be a convenient tool (at primary level) for the detection of formation of nanoaggregates. Acknowledgements The author greatly indebted to Prof. N. Chattopadhyay for his encouragement, allround support providing laboratory facilities etc. and helpful discussion. The author greatly appreciate the cooperation received from Prof. N. Sarkar, Dr. A. Chakraborty, Dr. D. Seth of I.I.T. Kharagpur and Prof. S. Basak of SINP Kolkata, for their kind help in time-resolved and steady state fluorescence anisotropy measurements, respectively. The author is also thankful to his friend Dr. B. Haldar and all group members of Prof. Chattopadhyay. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
A. Douhal, Chem. Rev. 104 (2004) 1995. Y. He, P. Fu, X. Shen, H. Gao, Micron 39 (2008) 495. T. Shimizu, M. Masuda, H. Minamikawa, Chem. Rev. 105 (2005) 1401. B. Haldar, A. Mallick, N. Chattopadhyay, J. Chem. Edu. 89 (2008) 429. V.T.D. Souza, M.L. Bender, Acc. Chem. Res. 20 (1987) 146–152. A. Mallick, B. Haldar, N. Chattopadhyay, J. Photochem. Photobiol. B 78 (2005) 215. A. Munoz de la Pena, T. Ndou, J. Zung, I.M. Warner, J. Phys. Chem. 95 (1991) 3330–3334. S. Hamai, J. Phys. Chem. B 101 (1997) 1707. G.S. Cox, N.J. Turro, J. Am. Chem. Soc. 106 (1984) 422. D.W. Cho, Y.H. Kim, S.G. Kang, M. Yoon, J. Phys. Chem. 98 (1994) 558. S. Li, W.C. Purdy, Chem. Rev. 92 (1992) 1457. G.R. Fleming, Chemical Applications of Ultrafast Spectroscopy, Oxford University Press, New York, 1986. G.B. Dutt, J. Phys. Chem. B 108 (2004) 805. D. Chakrabarty, A. Chakrabarty, D. Seth, P. Hazra, N. Sarkar, J. Chem. Phys. 122 (2005) 184516.
A. Mallick / Journal of Molecular Structure 934 (2009) 91–95 [15] P. Das, A. Mallick, A. Chakrabarty, B. Haldar, N. Chattopadhyay, J. Chem. Phys. 125 (2006) 044516. [16] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum, New York, 1999. [17] A. Chakraborty, D. Chakraborty, P. Hazra, D. Seth, N. Sarkar, Chem. Phys. Lett. 397 (2004) 216. [18] A. Chakraborty, D. Chakraborty, P. Hazra, D. Seth, N. Sarkar, Chem. Phys. Lett. 397 (2004) 469. [19] E.L. Quitevis, A.H. Marcus, F.D. Fayer, J. Phys. Chem. 97 (1993) 5762. [20] A. Mallick, B. Haldar, S. Maiti, S.C. Bera, N. Chattopadhyay, J. Phys. Chem. B 109 (2005) 14675. [21] A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem. B 108 (2005) 14683. [22] A. Mallick, B. Haldar, S. Maiti, N. Chattopadhyay, J. Colloid Interface Sci. 108 (2005) 14683. [23] (a) A. Mallick, M.C. Mandal, B. Haldar, A. Chakrabarty, P. Das, N. Chattopadhyay, J. Am. Chem. Soc. 128 (2006) 3126; (b) A. Mallick, M.C. Mandal, B. Haldar, A. Chakrabarty, P. Das, N. Chattopadhyay, J. Am. Chem. Soc. 128 (2006) 10629. [24] P. Das, A. Chakrabarty, B. Haldar, A. Mallick, N. Chattopadhyay, J. Phys. Chem. B 111 (2007) 7401. [25] P. Das, A. Mallick, D. Sarker, N. Chattopadhyay, J. Colloid Interface Sci. 320 (2008) 9. [26] A. Dias, A.P. Varela, M.G. Miguel, A.L. Maçanita, R.S. Becker, H.D. Burrows, J. Phys. Chem. 100 (1996) 17970.
95
[27] A. Chakrabarty, A. Mallick, B. Haldar, P. Das, N. Chattopadhyay, Biomacromolecules 8 (2007) 920. [28] A. Chakrabarty, A. Mallick, B. Haldar, P. Purkayastha, P. Das, N. Chattopadhyay, Langmuir 23 (2007) 4842. [29] V.S. Giri, B.C. Maity, S.C. Pakrashi, Heterocycles 22 (1984) 233. [30] A. Mallick, S. Maiti, B. Haldar, P. Purkayastha, N. Chattopadhyay, Chem. Phys. Lett. 371 (2003) 688. [31] G. Pistolis, I. Balomenou, J. Phys. Chem. B 100 (2006) 15562. [32] D. Roy, S.K. Mondal, K. Sahu, S. Ghosh, P. Sen, K. Bhattacharyya, J. Phys. Chem. A 109 (2005) 9716. [33] P. Sen, D. Roy, S.K. Mondal, K. Sahu, S. Ghosh, K. Bhattacharyya, J. Phys. Chem. A 109 (2005) 9716. [34] M. El-Kemary, J.A. Organero, L. Santos, A. Douhal, J. Phys. Chem. B 110 (2006) 14128. [35] L. Tormo, J.A. Organero, A. Douhal, J. Phys. Chem. B 109 (2005) 17848. [36] N. Balabai, B. Linton, A. Napper, S. Priyadarshi, A.P. Sukharevsky, D.H. Waldek, J. Phys. Chem. B 102 (1998) 9617. [37] P. Das, A. Mallick, D. Sarkar, N. Chattopadhyay, J. Phys. Chem. C 112 (2008) 9600. [38] A. Harada, J. LI, M. Kamachi, J. Am. Chem. Soc. 116 (1994) 3192. [39] K. Miyake, S. Yasuda, A. Harada, J. Sumaoka, M. Komiyama, H. Shigekawa, J. Am. Chem. Soc. 125 (2003) 5080. [40] G. Li, L.B. McGown, Science 264 (1994) 249.