Published: September 26, 2011

r 2011 American Chemical Society 20970 dx.doi.org/10.1021/jp206299y | J. Phys. Chem. C 2011, 115, 20970–20977

ARTICLE

pubs.acs.org/JPCC

Modulation of Small Molecule Induced Architecture of CyclodextrinAggregation by Guest Structure and Host SizePrasun Ghosh, Arnab Maity, Tarasankar Das, Jyotirmayee Dash,* and Pradipta Purkayastha*

Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, Mohanpur Campus, Mohanpur 741252,WB, India

bS Supporting Information

’ INTRODUCTION

Self-aggregation of cyclodextrins (CDs) in water is an aspectthat is still not completely understood. A number of reports havebeen published on this aspect using the concept of self-aggregationof CD with or without the aid of an externally addedcompound.1�5 In general, native CDs can form aggregates inwater with the size of about 200�300 nm, which considerablydepends on the type of CDs.6�8 According to Loftsson et al., theaggregates, having noninclusion complexation or micelle-likestructures, were able to solubilize lipophilic drugs.9 At low pHionization of the hydroxyl groups leads to dispersion of theseaggregates, leading in turn to increased solubility. It has also beennoticed that solubility of β-CD is increased in the presence ofstructure breaking solutes but with retention of aggregates.1 Thedriving force for the self-assembly of cyclodextrin molecules canthus be considerably ascribed to hydrogen bonding.7,10 The CDsprefer to line up in ideally parallel or staggered parallel arrange-ment with quadrupolar character.10

The six-, seven-, and eight-membered sugar residues consti-tuting α-, β-, and γ-CDs have been the focus of interest toresearchers in pure and applied fields.11,12 The hydrophobicinner cavities of the truncated conelike structures of the CDshave 4�8 Å diameters. Depending on the relative sizes ofthe CDs and the guest molecules, more than one guest canbe accommodated inside a single CD cavity.13 Formation ofsmall molecule induced CD nanotubes or nanorods has been thetopic of interest for many groups in applied fields of chemicalresearch.2�4,14,15 Our recent observations have proved that

formation of small molecule induced CD nanotubular supras-tructures depends significantly on the concentration of theguest.4,17,18 We have shown spectroscopically that the CDnanotubular aggregation takes place through primary and sec-ondary interactions via hydrogen bonding.18

The present report describes the importance of the structure ofguest molecule as well as the host on building up of the CDsuprastructures. The molecule that we have opted as the guestsystem is a bis-phenylethynyl amide meta-linked to 2,6-pyridine(I), as shown in Scheme 1, is a potentG-quadruplexDNAbindingsmall molecule.19,20 We have studied the impact of molecularstructure on CD aggregation motif. We observed that even thesmallest cavity sized α-CD can form well developed nanotubularsuprastructures depending on the concentration of the guestcompound through the basic 1:2 guest�host encapsulationmode.4,18 Because of restriction in the internal cavity size,α-CD leads to typically patterned guest binding. This is diffe-rent from the binding motif of β- and/or γ-CDs.4,17,21 From thestructural features of 6-bromo-2-phenylethylaminopyridine(BPEAP) (I) (Scheme 1) it is apparent that the hydrophobicamine portions can get encapsulated inside the hydrophobic CDcavities. The central pyridine ring also offers another possible sitefor encapsulation with the hydrophobic CD cavities. Thus, it canbe hypothesized that the initial encapsulation of the amine

Received: July 4, 2011Revised: August 24, 2011

ABSTRACT: A small molecule based on bisphenylethynyl amidemeta linked to 2,6-pyridine can induce cyclodextrin (CD) aggre-gation through a possible 1:3 guest�host motif. Most of the smallmolecules induce CDnanotubular bundles principally through 1:2guest�host unit capsules and aggregate through hydrogen bond-ing among the hydroxyl groups present on the rims of the CDmolecules. The N,N-dimethyl aminopropyl carboxamide sidechain and the central pyridine ring of 6-bromo-2-phenylethyla-minopyridine (BPEAP) induces nanotubular bundles with α-CDand laminar bundles with β- and γ-CDs, which, as a consequence,may result in formation of pores in the aggregates that can findwide applications in biological storage machinery. The stoichiom-etry of the ingredients of the unit capsules has been evidenced byJob’s plot. The size-dependent suprastructures induced by BPEAP have been studied by steady state and time-resolved fluorescencespectroscopy and atomic force microscopy.

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portions by two CD host molecules will control the approachof the third CD to encapsulate the pyridinyl moiety. Herein wehave investigated this hypothesis by employing fluorescencespectroscopy and atomic force microscopy (AFM) and proposeda structural schematic for the BPEAP-induced CD suprastructureformation.

’EXPERIMENTAL SECTION

Materials. BPEAP was prepared and purified by using literatureprocedure.19 Stock solution of the compound (1.001 � 10�3 M)was prepared in triple distilled water. The final concentration underdifferent conditions of BPEAP was 2 � 10�6 M. Cyclodextrinswere bought from Sigma-Aldrich, WI, USA and used as received.Methods. The absorption spectra were recorded using a

Varian Cary 300 Bio UV�vis spectrophotometer. Fluorescencemeasurements were performed using a PerkinElmer LS 55scanning spectrofluorimeter. The fluorescence lifetimes weremeasured by themethod of time-correlated single-photon count-ing using a picosecond spectrofluorimeter from Horiba JobinYvon IBH. The instrument was equipped with FluoroHub singlephoton counting controller, Fluoro3PS precision photomulti-plier power supply and FC-MCP-50SC MCP-PMT detectionunit. A laser head or a nano-LED pulsed diode powered by apulsed diode controller (IBH) was used as the excitation lightsource. The excitation wavelength was 281 nm. The typicalresponse time of this laser head was <1 ns. To calculate thelifetime, the fluorescence decay curves were analyzed by aniterative fitting program provided by IBH. The AFM studieswere made using an NT-MDT NTEGRA instrument procuredfrom NT-MDT, CA, USA. The steady state anisotropy, r, can berepresented as r = (IVV � GIVH)/(IVV + 2GIVH), where IVH andIVV are the intensities obtained from the excitation polarizer

oriented vertically and the emission polarizer oriented in hor-izontal and vertical positions, respectively. The factor G isdefined asG = (IHV)/(IHH). Semiempirical quantummechanicalAM1 calculations for geometry optimization were performedusing Gaussian 03 software.

’RESULTS AND DISCUSSION

CDs, with lipophilic inner cavities and hydrophilic outersurfaces, are capable of interacting with a large variety of guestmolecules to form noncovalent inclusion complexes. Chemicallythey are cyclic oligosaccharides containing at least six D-(+)glucopyranose units attached by α-(1, 4) glucosidic bonds.11,12

The three natural CDs, α-, β-, and γ-CDs (with 6, 7, or 8 glucoseunits, respectively), differ in their ring size and solubility.22

These compounds, until a few decades ago, were recognized toform 1:1 and/or 1:2 complexes with guest molecules dependingon the size of the latter. The formation of small moleculeimpregnated CD nanotubular suprastructures stemmed fromthe early studies of Li et al.15 Biologically important guests aregenerally used in such studies with the principal aim of applyingthe findings in drug delivery. In the present study we have used aDNA G-quadruplex binding molecule BPEAP that can controlthe structural motif of CD suprastructures.

We observe two distinct peaks at 260 and 350 nm in theabsorption spectrum of BPEAP as shown in Figure 1A. The bandat 260 nm is assigned to πfπ* transition while that at 350 nm isdue to nfπ* transition. The latter arises because of the existenceof the nonbonding electrons in the molecular system. Toascertain that the 350 nm band is from nfπ* transition, wetook the absorption spectra of BPEAP in dioxane�water mixedsolvents by varying their proportions. This changes the polarityof the medium through a vast range from very low to appreciably

Scheme 1. Equilibrium between the Neutral Form of BPEAP (I) and Its Ionic Counterpart (II)

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high as for water. A resulting bathochromic shift of the band at350 nm on increasing the concentration of dioxane, i.e., onlowering the polarity of the medium confirms the nfπ* band(see Supporting Information). Addition of CD to BPEAPsolution in steps of 1 mM does not show any remarkable changein the absorption spectrum. This may seem contradictory sinceCD also provides lower polarity to the fluorophore compared towater. This apparent contradiction may be averted by the factthat the polarity within the CDs is of the order of the lowermembers of alcohol series. Hence, the πfπ* and nfπ* bandsmay not show any appreciable shift in their positions.

We further applied Job’s method of continuous variation to theabsorbance spectra of BPEAP in CDs to determine the stoichio-metric ratios of the hosts to the guest.23 The correspondingabsorption spectra, which have been considered in the subsequentcalculations, are given as Supporting Information. The respectiveplots for BPEAP in α-, β-, and γ-CDs are shown in parts B�D ofFigure 1. The plots are bimodal in nature, and the breaks in thepattern denote the stoichiometries as indicated in the figures. Job’splot is generally used to determine the nature of interactionbetween two or more species during complexation.23 Sametype of interaction provides similar profiles in the plot. In thepresent case, the host�guest interaction does not change theabsorption spectral profiles for the samples on varying the CDs

(see Supporting Information). Thus, similar Job’s plots areexpected in all the cases. At a very low concentration of theCDs, where the proportion of the guest compound is higher thanthe host, we find 2:1 guest�host complexation, whereas, when theproportion reverses we observe a 1:3 guest�host interaction. Thismay be attributed to the insertion of the hydrophobic side-chainsof two BPEAP molecules inside the host cavity when there is alower concentration of hosts and higher concentration of guestmolecules in solution.24 The cavity size of all the three CDs is largeenough to accommodate the side-chains as confirmed by measur-ing the size of the moieties after optimizing the geometry ofBPEAP semiempirically. On the other hand, when the solution isenriched with host concentration, each carboxamide side-chainand central pyridene ring of BPEAP get encapsulated with hostmolecules and 1:3 guest�host complexation was observed. Onfurther enhancement of the host concentration, we observed asharp enhancement in the ΔA value in the Job’s plots suggestingthe development of nanotubular suprastructures. The correspond-ing binding constants (K) have been calculated from the respectiveJob’s plots and tabulated in Table 1 (also see Supporting In-formation). BPEAP shows high binding affinity for both 2:1 and1:3 guest�host complexation.

We used fluorescence spectroscopy to examine the guest�host complexation in the excited state. We considered the

Figure 1. (A) Absorption spectrum of BPEAP in water. The other plots are showing Job plots for complexation of BPEAP (guest) with (A) α-, (B) β-,and (C) γ-CDs determined by UV�vis spectrophotometry in water at ambient temperature. ΔA denotes the difference in the absorbance of eachsolution at 261 nm from that without any CD.

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fluorescence changes of BPEAP in presence of the three CDs.Parts A�C of Figure 2 show the changes in fluorescence ofBPEAP with sequential addition of aliquots of α-, β-, and γ-CDs.BPEAP shows two peaks at ∼380 nm and ∼490 nm. These canbe assigned to the neutral and the ionic species, respectively,according to Scheme 1. Because of higher solvation, the ionicspecies will fluoresce at higher wavelength compared to theneutral one. Increase in concentration of α-CD results in aremarkable hypsochromic shift of the 490 nm band by ∼50 nmaccompanied by its appreciable intensification. This indicates aprofuse change in the molecular environment due to lowering ofthe surrounding polarity. To obtain an idea about the extent ofpolarity change, we compared the data with those gathered from

a study on the behavior of BPEAP inwater�dioxanemixture (seeSupporting Information). The amount of spectral shift was dueto an imposed polarity comparable to 60�40% dioxane-watermixture.25

We compared the ratio of the change in intensity of the peaksat the two specified wavelengths for BPEAP (Figure 2D). Aconsiderable enhancement in the ratio was observed whenBPEAP was treated with α-CD. This indicates that the ionicspecies of BPEAP (II in Scheme 1) is affected considerably due tothe encapsulation over the neutral (I in Scheme 1); whereas uponaddition of β- and γ-CDs to BPEAP, there is significant decreasein change in intensity of the peaks that suggests differentialinteraction of BPEAP with CDs. On treatment with β- and γ-CD, we observe hypsochromic shifts of both the peaks by about10 nm. Decrease in the ratio of the peak intensities in these casesindicate that the fluorescence due to the neutral species increasesmore than that for the ionic ones, which is more pronounced inthe case of β-CD than that in γ-CD. We have performed steadystate anisotropy experiments and fluorescence quenching studiesto further understand the nature of interaction between BPEAPand the CDs (Figure 3). The anisotropy plot further suggests thedifference in interaction of BPEAP with α-CD compared to β-and γ-CDs. There is a remarkable enhancement in the anisot-ropy (r) of BPEAP when treated with the smaller cavities of α-CD. β- and γ-CDs behave somewhat similarly showing a small

Figure 2. Fluorescence spectra of BPEAP in (A) α- (0�25 MM), (B) β- (0�6 mM), and (C) γ-CD (0�10 mM). In all the cases the fluorescenceintensity increases with enhancement in CD concentration as indicated by the upward arrow. (D) shows the change in intensity ratio of 480 to 377 nmwith different concentrations of the CDs (data collected from parts A, B, and C).

Table 1. Binding Constants of the Different Guest�HostComplexes as Calculated from the Job’s Plots

CD nature of guest�host complex binding constant

α 2:1 6.52 � 108M�1

1:3 4.94 � 1016M�3

β 2:1 9.53 � 108M�1

1:3 9.35 � 1016M�3

γ 2:1 23.71 � 108M�1

1:3 27.69 � 1016M�3

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enhancement in the parameter, β-CD being the better of thetwo. Such interactions generally enclose the guest within the

core, and thus external agents, such as a fluorescence quencher,cannot reach the captivated guest to quench its fluorescence.4

Figure 4. Fluorescence decay profiles of BPEAP in varying concentrations (A) α-, (B) β-, and (C) γ-CDs. The data correspond to those tabulated inTable 2. The samples were excited at 280 nm, and the emission monochromator was set to 490 nm.

Figure 3. (A) Change in the steady state anisotropy of BPEAP in different CDs in aqueous medium. The anisotropy has been calculated from theintensity at the higher wavelength emission in each case. (B) Quenching of BPEAP fluorescence by potassium iodide (KI) in aqueous medium: (black)without any CD, (red) in 10 mM α-CD, (green) in 6 mM β-CD, and (blue) in 6 mM γ-CD. I and I0 are the fluorescence intensities at a particularwavelength with and without KI, respectively.

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However, we have observed that iodide can easily reach thechromophore of BPEAP even after its interaction with the CDs.We have examined the change in fluorescence of free BPEAP andits bound states upon addition of KI (Figure 3B). This indicatesthat the guest�host interaction has created an “open type”superstructure that allows the iodide ions to percolate and persistfor collisional quenching.

Examination of the structure of BPEAP (I) (Scheme 1) showstwo immediately possible sites for CD encapsulation, i.e., thehydrophobic amine side chains. Besides these sites the centralpyridine ring also has probability to get encapsulated inside thehydrophobic CD core.26,27 Thus, there is always a possibility of

1:3 guest�host interaction. As a consequence, we may finddifferences in the spectral behavior of the guest compared to 1:2interaction.2�4,8,17,18 We are using CD molecules of threedifferent cavity sizes, which presumably may take a decisive rolein the final architecture of the guest�host aggregation. Toprovide further evidence toward the spectroscopic behavior ofBPEAP in CDs we collected time-resolved fluorescence decaydata of BPEAP in the three CD environments. The decay profilesare shown in Figure 4, and the calculated data are tabulated inTable 2.

As has been discussed above, it is now known that BPEAPexists in two forms: neutral and ionic. The ionic form is supposedto provide the faster decay component. Contribution of theneutral form of BPEAP is higher than its ionic counterpart inaqueous solution. In the α-CD environment, however, the ionicspecies contributes more. On addition of α-CD in the aqueoussolution of BPEAP we observe an enhancement in the excited-state lifetime of the ionic species by 334 ps, whereas there is asmall decrease in the parameter for the neutral form. The decayprofiles in Figure 5A also show that the faster decaying species isbecoming the major component with an increase in α-CDconcentration, and the decay time of the ionic species graduallyincreases on addition of more α-CD to the BPEAP solution. Thesteady state fluorescence spectral change under the same circum-stances showed a concomitant hypsochromic shift (Figure 3A).Thus, we can conclude that the ionic species is necessarilyinteracting with α-CD under reduced polarity. This may alterthe equilibrium shown in Scheme 1 and drive it toward the ionicform; hence we see a gradual decrease in the fluorescence lifetimeof the neutral species as also the contribution to the decayprofile. In β-CD, the decay times of both the species are gettingslightly enhanced with somewhat equal contributions. Presenceof γ-CD nearly replicates the observations of β-CD; only theenhancement in the decay time in this case is even smaller andcontribution of the neutral species is somewhat in the higher side.This reflects the notion that the bigger CDs allow some watermolecules inside its cavity.

Correlating with Figure 2D, it appears that in β- and γ-CDsthe equilibrium between the neutral and the ionic form present inwater is experiencing rather small perturbation. Thus, we statethat neither of the species reacts directly with these CDs besidestheir existence in somewhat more hydrophobic region thanwater. This effect is even lesser in case of γ-CD. We supposethat this is due to the larger size of these two CDs compared to

Table 2. Decay Parameters of BPEAP in α-, β-, and γ-CDsMonitored at 490 nm Exciting the Sample at 280 nma

[α-CD] (mM) τ1 (ps) τ2 (ns) χ2

0 555 (39.3) 4.1 (60.7) 1.19

2 779 (64.0) 4.1 (36.0) 1.21

4 796 (73.4) 4.1 (26.6) 1.07

6 821 (77.6) 4.0 (22.4) 1.16

8 836 (79.9) 3.9 (20.1) 1.21

10 837 (80.3) 3.7 (19.7) 1.19

12 840 (79.9) 3.4 (20.1) 1.17

15 889 (79.2) 3.4 (20.2) 1.15

[β-CD] (mM) τ1 (ps) τ2 (ns) χ 2

0 555 (39.3) 4.1 (60.7) 1.19

1 563 (44.2) 4.4 (55.8) 1.18

2 580 (47.0) 4.7 (52.0) 1.17

3 596 (49.3) 5.2 (50.7) 1.21

5 620 (51.1) 5.8 (48.9) 1.21

6 612 (51.2) 5.9 (48.8) 1.22

[γ-CD] (mM) τ1 (ps) τ2 (ns) χ 2

0 555 (39.3) 4.1 (60.7) 1.19

2 578 (39.0) 4.5 (61.0) 1.19

4 604 (41.2) 4.6 (58.9) 1.20

6 608 (42.7) 4.7 (57.3) 1.14aThe χ2 values are provided to reciprocate the goodness of the fits. Thenumbers in parentheses indicate the percentage contribution of eachcomponent.

Figure 5. Atomic force micrographs of BPEAP induced (A) α-, (B) β-, and (C) γ-CD aggregates.

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α-CD. Most probably, the responsible component in the overallphenomenon is the CD molecule that approaches the centralpyridine ring of BPEAP. The proposed model is represented inScheme 2. The scheme indicates more compactness in BPEAPinduced CD aggregation in case of α-CD. The bigger size of β-and γ-CDs affects the compactness of the aggregation and thusalso the progression in the aggregate formation.

We anticipate a rather laminar distribution in case of β- and γ-CDs, whereas the BPEAP-induced α-CD may form tubularsuprastructures.4 We used AFM to reinforce the effect of themolecular structure and the size of the CD hosts on BPEAPinduced CD aggregate formation (Figure 5). Scheme 2 and AFMimages suggest the existence of voids of varying sizes throughoutthe distribution of the CDs anchored by BPEAP molecules.

Scheme 2. Different Proposed Motifs of Aggregation of the Three Different CDs with BPEAP based on the Spectral Evidence

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’CONCLUSION

In summary, we have observed that the structure of the guestand size of the host (CD) determine the motif of guest-inducedhost aggregation. The host size is responsible for the nature of theaggregation. Particular aggregation behavior can be monitoredquite explicitly using fluorescence spectroscopy if the guestmolecule fluoresces. In the present article, we have chosen abiologically potent guest that can bind to the G-quadruplex DNAand contains three potential sites for host encapsulation. Theguest compound used here is extremely sensitive to polarity ofthe environment and thus becomes an extraordinary probe tomonitor the whole process. We have proposed a schematic forhost�guest binding based on the spectral evidence. We also haveproposed that the CD aggregates may possess voids throughoutthe distribution, which could be used as molecular sieves andmayfind immense biological applications such as storage. Guest�host chemistry is extremely important in drug delivery. In thisaspect we have been able to formulate a size dependent trendtoward guest�host binding.

’ASSOCIATED CONTENT

bS Supporting Information. Normalized absorption spectraof the band due to nfπ* transition in dioxane�water mixtures;fluorescence spectra of BPEAP in dioxane�water mixtures;transmission electron microscopy image of BPEAP-induced α-CD nanotubular aggregates; three-dimensional representation ofatomic force electron micrograph of BPEAP-induced α-CD(upper panel) and β-CD (lower panel) nanoaggregates; absorp-tion spectra of BPEAP in presence of the different CDs; method ofcalculation of the binding constants from Job’s plots. This materialis available free of charge via the Internet at http://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*E-mail: pradiptp@gmail.com (P.P.), jyotidash@iiserkol.ac.in(J.D.). Phone: +91-33-64513294. Fax: +91-33-25873020.

’ACKNOWLEDGMENT

The work is supported by Council of Science and IndustrialResearch, New Delhi, through Grant No. 01(2261)/08/EMR-II.J.D. thanks Department of Science and Technology, Govern-ment of India, for funding. P.G. and T.D. thank CSIR, and A.M.thanks UGC for their fellowships.

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