Preparation, physicochemical analysis and molecular modeling investigation of 2,2′-Bipyridine: β-Cyclodextrin inclusion complex in solution and solid state

Accepted Manuscript Preparation, physicochemical analysis and molecular modeling investigation of 2,2'Bipyridine:β-Cyclodextrin inclusion complex in solution and solid state R. Periasamy, S. Kothainayaki, K. Sivakumar PII:

S0022-2860(15)30137-X

DOI:

10.1016/j.molstruc.2015.07.026

Reference:

MOLSTR 21681

To appear in:

Journal of Molecular Structure

Received Date: 10 April 2015 Revised Date:

26 June 2015

Accepted Date: 14 July 2015

Please cite this article as: R. Periasamy, S. Kothainayaki, K. Sivakumar, Preparation, physicochemical analysis and molecular modeling investigation of 2,2'-Bipyridine:β-Cyclodextrin inclusion complex in solution and solid state, Journal of Molecular Structure (2015), doi: 10.1016/j.molstruc.2015.07.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Preparation, physicochemical analysis and molecular modeling investigation of 2,2'-Bipyridine:β-Cyclodextrin inclusion complex in solution and solid state R. Periasamy1, S. Kothainayaki1* and K. Sivakumar2 2

Chemistry Section, FEAT, Annamalai University, Annamalainagar, Tamil Nadu, India – 608002 Department of Chemistry, Faculty of Science, SCSVMV University, Enathur, Kanchipuram 631 561,Tamilnadu

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Abstract

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Supramolecular interaction between 2,2'-Bipyridine (BPY) and β-Cyclodextrin (β-CD) has been investigated in solution and solid state. Non-covalent interaction between BPY and β-CD was studied in solution using absorption and fluorescence spectroscopy. Inclusion complex of BPY and β-CD was prepared in solid state by co-precipitation method and it was

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characterized using Fourier Transform Infra-red spectroscopy (FT-IR), Thermal analysis, Scanning Electron Microscopy (SEM), Powder X-ray diffractometry (XRD) and Atomic Force Microscopy (AFM). Binding constant values and 1:1 stoichiometry of the inclusion complex

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were calculated using Benesi-Hildebrand plots at 303 K. Using continuous variation method the 1:1 stoichiometry has been confirmed for BPY: β-CD complex. Thermodynamic parameter, ∆G

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of inclusion complex formation was determined and the negative value indicated that the inclusion process was an exergonic and spontaneous process. The most probable model of BPY: β-CD inclusion complex suggested by molecular docking studies was in good agreement with the results obtained by experimental methods.

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*Corresponding author Tel:- +91 9486456733 Email: [email protected] (S. Kothainayaki) 2,2'-Bipyridine, β-Cyclodextrin, Inclusion complex, Atomic Force Microscope,

UV - Fluorescence spectrum, Molecular Docking.

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Important abbreviations used in the manuscript

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Keyword:

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2,2'-Bipyridine

β-CD

-

β-Cyclodextrin

FT-IR

-

Fourier Transform Infra-red spectroscopy

DSC

-

Differential Scanning Calorimetry

SEM

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Scanning Electron Microscopy

XRD

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Powder X-ray diffractometry

AFM

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Atomic Force Microscopy

TCSPC

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Time-correlated single photon counting

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1.Introduction

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BPY

2,2'-Bipyridine (BPY) is found in natural products like collismycins or

caerulomycins [1-3]. Since from the discovery, BPY is extensively used in the complexation of metal ions. Ru and Os complexes of bipyridines are quite attractive because they are chemically, thermally and photo chemically stable and often show fluorescence. Two or more BPY units are used as ligands and they act as bridges to interconnect metal centers in a well-defined spatial

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arrangement. Generation of coordination polymers is the another interesting application of BPY containing macrocycles. In such a polymers, the specific sequence (orthogonal) of macrocycles would make their solubility higher than normal even without the introducing flexible chains

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which is the main method to increase the solubility of conformationally inflexible macromolecules. These coordination polymers are expected to have novel applications such as

complexes have also been [4] carried out and analyzed.

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magnetic and electronic materials. Synthesis of β-CD-functionalized BPY and their metal

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Host-guest chemistry is the most important subdivision of supramolecular chemistry. In host-guest chemistry a complex is formed when two or more molecules or ions are held together in a unique structural relationship through intermolecular forces like ion-pairing, hydrogen bonding, van der Waals forces and / or hydrophobic interactions rather than that of covalent bonds [5-7]. A “host-guest” complex is produced when a molecule (host) tend bind another

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molecule (guest). The interactions between them are found to be non-covalent [8]. The host normally is a large molecule or aggregate such as synthetic cyclic compound or an enzyme with a suitable size hole or cavity. The guest may be a neutral molecule or mono atomic species or

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simple inorganic ion. The host is defined as “the molecular entity possessing convergent binding

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site” and the guest is defined as “the molecular entity having divergent binding sites”. Host cavities include suitable guests and acts as the building blocks for large functional architectures [9]. The term “host-guest” chemistry designate the different type of processes occurring in various kinds of research fields, such as organometallic, biological, analytical and organic chemistry and involving ions and molecules with different properties dimensions and structures. Generally host-guest interactions are involved in the formation of multiple non-covalent bonds between a host (large and geometrically concave organic molecule) and a simple guest

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(inorganic/organic molecule/ion). Moreover the geometrical requirements are essential to fit the definition of host-guest chemistry [10]. Various types of macrocyclic ligands were synthesized which possess structures of enhancing complexity. They include cavitands, cryptands,

seems to be the most important one [11]. The

cyclodextrins

also

called

cycloamyloses,

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carcerands, calixarenes, cyclophanes etc., [10]. Among all potential hosts, the cyclodextrin (CD)

Schardinger

dextrins

or

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cycloglycopyranoses are cyclic oligosaccharides in which glucose units are linked by alpha 1-4

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glucoside bonds [12,13] The main interests of CDs are their capability to form inclusion complexes with number of compounds [14-18].

X-ray structural analysis reveals that in

cyclodextrins the primary hydroxyl groups (C6) are on the narrower edge and the secondary hydroxyl groups (C2 and C3) are on the wider edge of the ring . The ether-like oxygens and apolar C3, C5 hydrogens are present inside the torus-like molecules. Thus these molecules with

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a hydrophilic exterior can be dissolved in water and an apolar cavity can provide a hydrophobic matrix which is described as a micro heterogeneous environment [19]. As a result, cyclodextrins can able to form inclusion complexes with a wide variety of hydrophobic guest molecules.

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Guest molecules (one or two) can be encapsulated by one, two or three CDs. In this type of

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inclusion complexes, a guest molecule is held within the cavity of cyclodextrin host molecule. Formation of inclusion complex is a dimensional fit between guest and host cavity [20]. The hydrophobic cavity of cyclodextrin molecules provides a micro heterogeneous environment into which suitable sized non-polar guest molecules can enter to form inclusion complexes [21]. No covalent bonds are formed or broken during the formation of inclusion complex [22]. Driving force for these inclusion complex formations is the release of water molecules from the cyclodextrin cavity. Water molecules are replaced by hydrophobic guest molecules present in

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the solution and attain an apolar-apolar association, decreasing ring strain in CDs resulting in a more stable state with lower energy [23].

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The most suitable guest molecules for molecular encapsulation in cyclodextrins include branched or straight chain aliphatics, alcohols, ketones, aldehydes, fatty acids, organic acids, gases, aromatic and polar compounds such as amines, halogens and

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oxyacids [24].

From a microscopical point of view, the molecule is said to be micro-encapsulated when

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a guest molecule is individually surrounded by a cyclodextrin ( or its derivative). This may cause chancges in physical and chemical properties of the guest molecules. Therefore the ability of CDs to form host-guest complexes led to their use in a number of industries [25,26]. For example CDs have been used in the pharmaceutical industry as solubilizers, diluents and tablet ingredients which improve the stability, bio availability and

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pharmacokinetic properties of drugs [27-30]. In cosmetic field, the interaction of the guest with CDs produces a higher energy barrier to overcome to volatilize, thus producing long-lasting

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fragrance [31]. In food Industry β-CD is used to remove cholesterol from milk to produce dairy products low in cholesterol [26]. CDs can play a major role in environmental science in terms of

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solubilisation of organic contaminants, enrichment and removal of organic pollutants and heavy metals from soil, water and atmosphere [32]. CDs are also applied in water treatment to increase the stabilizing action, encapsulation and adsorption of contaminants [33]. Earlier the inclusion complexation of various aromatic and hetero cyclic molecules with

β-CD has been investigated [34-39]. Recently we have reported spectral investigation and structural characterization of inclusion complexes of β-CD with Dibenzalacetone [40], 4,4'-methylenebis(2-chloroaniline) [41] and 4,4'-methylenebis(N,N-dimethylaniline) [42]. In the

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present study we report the intermolecular complexation between 2,2'-Bipyridine (BPY) and β-Cyclodextrin in solution and solid state. We have utilized absorption and fluorescence spectral data to determine the stoichiometry and binding constant of BPY: β-CD complex.

Solid

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inclusion complex has been prepared and characterized by FT-IR, Thermal analysis, Scanning Electron Microscopy (SEM), Powder X-ray diffraction (XRD) and Atomic force Microscopy (AFM) methods. Molecular Docking study has also been applied to investigate the inclusion

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process of the guest within β-CD and most probable model of 1:1 inclusion complex has been

2. Materials and methods 2.1 Materials

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proposed.

β-cyclodextrin and 2,2'-Bipyridine were purchased from Sigma-Aldrich chemical company and used as such. All other chemicals and solvents used were of the highest grade

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(Spectral grade) commercially available. Triply distilled water was used for the preparation of aqueous solutions. Purity of the compound was checked by its melting point and also by obtaining identical fluorescence spectra at different excitation wavelength. The pH solution (6.8)

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was prepared by adding appropriate amount of NaOH and H3PO4. The experimental solutions

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were prepared just before taking each measurement.

2.2 Preparation of BPY: β-CD inclusion complex Solid BPY : β-CD inclusion complex was prepared using well established

co-precipitation method [43]. BPY and β-CD with 1:1 molar ratio were accurately weighed separately. Saturated β-CD solution was prepared in water. BPY solution in methanol was added to β-CD solution with continuous stirring for about 48 h till a white precipitate was formed. The precipitate was filtered, washed with triply distilled water and dried in an oven at

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50 °C for 12 h. The solid inclusion complex was obtained in the form of colorless powder and it was further analyzed using FT-IR, XRD, DSC-TG, SEM and AFM.

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2.3 Preparation of Physical Mixture A physical mixture of BPY and β-CD in 1:1 molar ratio was prepared by grinding the mixture in a mortar to obtain homogeneous blend.

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2.4. Instruments

The absorption spectral (UV-Visible spectrum) measurements were carried out on

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Hitachi model U-2001 spectrophotometer. Fluorescence measurements were made using a JASCO FP-750 spectrofluorimeter. pH values were measured on ELICO pH meter (model L1-10T). Fluorescence Lifetimes were recorded by using a time-resolved single photon counting picosecond spectrofluorimeter (Tsunami, Spectra physics).

FT-IR

between 4000 cm-1 and

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spectra of powder sample BPY, β-CD and the solid inclusion complex were measured 400 cm

-1

on Avtar-330 FT-IR spectroscopy using KBr pellet.

Powder X-ray diffraction patterns were obtained by Xpert PRO analytical diffractometer

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and the operating conditions at voltage - 40 kv, current- 30 mA, scanning- 5.08 Sec in continuous mode. Microscopic morphological structure measurements were performed

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with JEOL-JSM 5610LV scanning electron microscope. Thermal analysis was carried out in the temp range of 20-300 °C in steam of nitrogen atmosphere on DSC-50 thermal analyzer (shimadzu.Japan). Surface morphology was also recorded using AGILENT-N 9410 A SERIES 5500 AFM.

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2.5 Determination of stoichiometry of inclusion complex by Job’s method One of the first methods used for the determination of the stoichiometry of inclusion complex was Job’s method also known as the continuous variation method [44]. The experiment

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was carried out using stock solutions with equimolecular concentrations of β-CD and BPY. The samples were prepared by mixing different volume of these two solutions in such a way that the total concentration of [BPY] and [β-CD] remains constant and the molar fraction of the guest

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XBpy varies in the range of 0-1. The variation of the experimental measurement property, ∆OD in presence of the host with respect to the value of free guest is plotted against XBpy. Absorbance

2.6. Molecular docking study

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was recorded at different molar ratios by using UV spectrophotometer.

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The most probable structure of the BPY : β-CD inclusion complex was determined by molecular docking studies using the PatchDock server [45]. The 3D structural data of β-CD and BPY was obtained from crystallographic databases. The guest molecule (BPY) was docked in to

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the host molecule (β-CD) cavity using PatchDock server by submitting 3D coordinate data of BPY and β-CD molecules. Docking was performed with complex type configuration settings.

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PatchDock server follows a geometry-based molecular docking algorithm to find out docking transformations with good molecular shape complementarity score. PatchDock algorithm separates the Connolly dot surface representation [46, 47] of the molecules into concave, convex and flat patches. These divided complementary patches are matched in order to generate candidate transformations and evaluated by geometric fit and atomic desolvation energy scoring

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[48] function. RMSD (root mean square deviation) clustering is applied to the docked solutions to select the non-redundant results and to discard redundant docking structures.

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Semiempirical quantum mechanical calculations The ground state of BPY molecule was optimized using ArgusLab program by AM1 method. MolSoft MolBrowser tool was used to visualize the 3D structural data.

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3. Result and discussion

3.1.1 Absorption spectral analysis

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3.1 Interaction of BPY with β-Cyclodextrin in solution

The inclusion complex composed of BPY and β-CD in aqueous solution has been characterized by UV spectroscopy. In general the shape, position of peak maximum and molar

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absorption co-efficient of the guest molecule are strongly depend on the CD cavity environment. Absorption and fluorescence spectral data of BPY in different concentrations of β-CD are summarized in Table.1. Fig.1a. illustrates the absorption spectra of BPY in aqueous solution at

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pH.6.8 containing varying concentration of β-CD. The absorption spectrum of BPY exhibits two maxima at 231.8 and 285 nm. Upon increasing the concentration of β-CD from 0 M to 0.012 M,

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the absorption spectrum of BPY is slightly red shifted at longer wavelength ( 285 nm to 289 nm) with slight increase in its absorbance at both the maxima. These changes are attributed due to (i) nitrogen atom of pyridine ring interacts with secondary hydroxyl groups of

β-CD, because it is

well known that CDs are good hydrogen donors and (ii) decreasing polarity of the CD cavity environment experienced by the guest molecule during inclusion complex formation[50].

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The binding constant ‘K’ and stoichiometric ratio of the inclusion complex of BPY with β-CD can be determined from the changes observed in the absorbance of BPY with increasing concentration of β-CD using Benesi-Hildebrand equation [51]. Benesi-Hildebrand equations for

1/ (A-Ao) = 1/∆ε + 1/K[BPY]o ∆ε[β-CD]o

------------(1) ------------(2)

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1/ (A-Ao) = 1/∆ε + 1/K [BPY]o ∆ε[β-CD]o2

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1:1 and 1:2 complexes are shown by equation (1) and (2) respectively.

where (A-Ao) is the difference between the absorbance of BPY in the presence and absence of

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β-CD. ∆ε is the difference between the molar absorption co-efficient of BPY in the presence and absence of β-CD. [BPY]o and [β-CD]o are the initial concentrations of BPY and β-CD, respectively. ‘K’ is the Binding constant. A good linear correlation (R2 = 0.9948) is obtained when 1/(A-Ao) is plotted against 1/[β-CD] [Fig.1a inset] indicating that the stoichiometry of the

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inclusion complex is 1:1 [52]. Moreover, the plot of 1/(A-Ao) against 1/[β-CD]2 [Fig.1b] gives down ward curve. The non-linearity of the plot ruled out the possibility of 1:2 stoichiometry between BPY and β-CD [36]. Hence the stoichiometry of the inclusion complex was 1:1 as

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evidenced by fitting a double-reciprocal plot of 1/(A-Ao) Versus 1/[β-CD] . The binding constant value ‘K’ of the inclusion complex formation obtained from the slope of the Benesi-Hildebrand

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plot using eqn (3) and it is found to be 91 ±3 M-1 at 303 K. K = 1/ Slope [A-Ao]

----------- (3)

3.1.2. Fluorescence spectral analysis The effects caused by the addition of β-CD on the emission spectra of BPY in aqueous solution are more pronounced than the corresponding effects on absorption spectra. The emission spectra of BPY with increasing concentration of β-CD are shown in Fig.2a. Maximum

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absorption wavelength of the BPY has been chosen for excitation . When excited at 280 nm, BPY exhibits a broad structureless emission band in water at 326 nm. Upon the addition of β-CD, BPY exhibits a large bathochromic shift in its spectrum from 326 nm to 332 nm with a

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concomitant increase in the fluorescence intensity. When fluorescent molecules in aqueous solution are included in cyclodextrins, fluorescence spectra may be influenced which indicates the formation of inclusion complexes [53,54]. The above results suggest the formation of

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inclusion complex between BPY and β-CD [37]. The shifts observed in the spectral maximum and the changes obtained in the fluorescence intensity may be attributed due to formation of

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inclusion complex between BPY and β-CD. When the guest molecule was entrapped in the cavity of β-CD, the microenvironment around β-CD with lesser polarity and stronger rigidity would restrict the freedom of guest molecules inside the cavity and the steric hindrance of β-CD torus can enhance the fluorescence efficiencies of guest molecules [55].

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The β-CD dependence of BPY fluorescence can be analyzed using Benesi-Hildebrand equation [51] for 1:1 complex equation (4).

------------- (4)

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1/ I-Io =1/ I'-Io + 1/ K (I'-Io) [β-CD]

where “K” is the binding constant, Io is the intensity of fluorescence of BPY without β-CD.

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`I´ is the fluorescence intensity of BPY with a particular concentration of β-CD and I' is the fluorescence intensity of BPY with the highest concentration of β-CD. Fig. 2a (inset) shows the plot of

1/[I-Io] Vs 1/[β-CD]. The linearity of the plot with the

correlation co-efficient of R2 = 0. 9994 indicates the formation of 1:1 complex between BPY and β-CD. The binding constant “K” calculated from the slope of the Benesi-Hildebrand plot using eqn (5) and it is found to be 107 ± 2 M-1 at 303K.

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K

= 1/ Slope [I-Io]

------------- (5)

The B-H equation for 1:2 stoichiometry is given in equation (6) 1/ I-Io =1/ I'-Io+ 1/ K (I'-Io) [β-CD]2

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------------- (6)

where “K” represents the binding constant of 1:2 complex. The plot of 1/[I-Io] Vs 1/[β-CD]2 reveals a non-linear correlation as shown in Fig.2b (inset) indicating that BPY : β-CD complex

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does not possess 1:2 stoichiometry. 3.1.3. Job’s Plot

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The stoichiometry of the inclusion complex of BPY and β-CD is further confirmed by Job’s continuous variation method. Fig.2b shows the change in optical density ‘∆OD’ against mole fraction of BPY. In the case of 1:1 inclusion complex the maximum deviation will be observed for mole fraction 0.5 [56]. As shown in Job’s plot, the peak maximum is obtained at

stoichiometry.

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mole fraction 0.5 which indicates that the inclusion complex between BPY and β-CD has 1:1

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3.1.4. Analysis of fluorescence decay curves

The inclusion complexation between BPY and β-CD not only causes enhancement of

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fluorescence intensity and peak shifts [57], but also induces increased fluorescence lifetime in the hydrophobic environment [58,59]. Time-correlated single photon counting (TCSPC) [60-62] rules on the concept that the probability distribution for emission of a single photon after an excitation event yields the actual intensity versus time distribution of all the photons emitted as a result of the excitation. In the present study, picosecond time-resolved fluorescence experiments with BPY is performed in aqueous solution in the presence and absence of β-CD in order to assess the microenvironment polarity around the included guest molecule (Fig.3). In the absence

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of β-CD, the fluorescence decay curve observed for BPY in water is perfectly fitted to a single exponential function. In contrast the decay profile of BPY in the presence of β-CD could be analysed only by a linear combination of two exponential functions.

The short and long

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fluorescence lifetimes, relative amplitude and χ2 values for BPY and its inclusion complex are summarized in Table 2. The elongated lifetimes in the presence of β-CD clearly indicates that the environment around the β-CD molecule is more hydrophobic than the bulk water. The short

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(τs 1.83 ns - 1.46 ns) and long (τ1 is 8.08 ns-1.12 x10-8 s) life times observed in the presence of β-CD should originate from two different fluorescing species. A systematic change in the

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fluorescence lifetimes and relative amplitude values of the complex with an increase in the concentration of β-CD is an indication that a small fraction of the free BPY and a greater amount of BPY : β-CD inclusion complex are present in the solution. The marked decrease in the shorter lifetime is due to increased microviscosity caused by the added β-CD. In the excited state

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microviscosity, plays a predominant role compared to micro polarity [63]. The increase in the abundance of the species with a longer lifetime species in the presence of β-CD is due to the confinement effect offered to BPY within the β-CD cavity which confirms the existence of two

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emitting species in solution with different individual lifetimes.

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3.1.5 Spontaneity of inclusion process The determination of thermodynamic parameter, Gibbs free energy change for the

host - guest inclusion process can be calculated from the binding constant values, ‘K’ using the following equation (7)

∆G = - RT ln K

-------------- (7)

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∆G values for the binding of BPY with β-CD using absorption and fluorescence data are – 11.36 and -11.77 KJ mol-1 respectively.

303 K. 3.2. Characterization of inclusion complex in solid state

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3.2.1. FT-IR spectroscopy analysis

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Negative value of ∆G suggests the spontaneity of host-guest complexation reaction at

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The interaction between the guest and host in the inclusion complexation process has been proved by FT-IR spectral study [64]. When the guest molecule is encapsulated by means of inclusion complexation, the absorption bands resulted from the included part of guest molecule are generally shifted in their position or intensities may be altered. The FT-IR spectra of β-CD, BPY, physical mixture and solid complex are shown in Fig.4. The changes in the shape

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and position or intensities of absorption bands of β-CD, BPY, physical mixture and inclusion complex are observed. Since β-CD and BPY form a solid inclusion complex, the non-covalent interactions between β-CD and BPY such as hydrophobic interactions, van der Waals

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interactions and hydrogen bonding lower the energy of the included part of BPY, thus reducing

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the absorption intensities of the corresponding bands. The O-H stretching frequency appears at 3379 cm-1 in β-CD (Fig.4a). It also appears at 3379 cm-1 in physical mixture. It is shifted to 3383 cm-1 in the case of solid inclusion complex. Similarly ring C-H stretching vibration of BPY appeared at 3057 cm-1 is disappeared and merged with the broad band of β-CD in the solid inclusion complex. The C=C, C=N ring stretching frequencies appeared at 1566 and 1437 cm-1 in pure BPY (Fig.4b) is significantly shifted to 1591 and 1451 cm-1 in the solid complex (Fig.4d) with ∼75% reduced intensity. The out of plane ring stretching frequency is appeared at 750 cm-1

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in pure BPY and 759 cm-1 in the solid inclusion complex with significant reduction in its intensity, due to complexation. The stretching frequencies of BPY is well matched with values that are already reported [65]. Further the broader peak at 3383 cm-1 in solid complex is due to

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the presence of larger number of hydrogen bonded –OH groups introduced by β-CD during complex formation. The stretching frequency of C-O bonds of β-CD observed at 1028 cm-1 is shifted to 1036 cm-1 in the solid inclusion complex. These changes occur when the BPY is

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entrapped into the nano hydrophobic cavity of β-CD. On the other hand the FT-IR spectrum of physical mixture imitated the characteristics peaks of BPY and β-CD which can be regarded as a

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simple superimposition of host and guest molecules. During host-guest inclusion complexation, no new bonds are formed which strongly reveals the non-covalent interaction between host and guest [66]. This may be due to van der Waals interaction between BPY and β-CD. Thus the FT-IR spectral study singnificantly proves the strong interaction between BPY and β-CD during

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the formation of inclusion complex.

3.2.2. Scanning electron microscopy analysis

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Scanning electron microscopy is a qualitative method used to study the structural

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aspects of β-CD, guest and the solid product obtained using different methods of preparation [67,68]. The SEM photographs of BPY, β-CD, their physical mixture, and solid inclusion complex prepared by co-precipitation method are shown in Fig.5. The difference in crystalline state of the raw materials and the product seen under electron microscope indicates the formation of the inclusion complex [69,70]. β-CD (Fig.5a) is composed of parallelogram shaped, quite densed crystals which are well separated from each other whereas BPY (fig.5b) appeared as irregular shape crystals.

The SEM image of their physical mixture (Fig.5c) exhibited the

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characteristic BPY crystals, mixed with the cyclodextrin particles or adhered to their surface, thus confirming the presence of crystalline BPY. In contrast, a change in the morphology and shape of the particles is observed in the BPY/β-CD solid inclusion complex (Fig.5d) prepared by

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co-precipitation method. The original morphology of the raw materials has been disappeared and it is no longer possible to differentiate BPY and β-CD, revealing an apparent interaction between

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BPY and β-CD [71].

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3.2.3. Powder XRD analysis

Evidence for host-guest inclusion complexation of BPY with β-CD is also obtained from Powder X-ray diffractometry. XRD is a useful method for the detection of β-CD encapsulation and it has been used to assess the degree of crystallinity of the given sample.

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Generally the crystalline nature of the guest molecule is reduced and more number of amorphous structures are increased in the solid inclusion complex [72,73]. The powder XRD patterns of β-CD, BPY, physical mixture and solid inclusion complex

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are displayed in Fig.6. The XRD Pattern of BPY shows a high degree of crystallinity and

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exhibits characteristic peaks at 2θ values of 15.63˚, 16.93˚, 17.02˚, 17.26˚, 34.36˚, 34.63˚, 34.74˚ and 72.89˚ [Fig.6b]. The diffraction pattern of β-CD exhibited important peaks at 2θ values of 8.95°, 10.56°, 12.45°, 18.60°, 22.94°, 31.8°, and 35.01° [Fig.6a]. Most of the characteristic peaks of BPY and β-CD are present in the diffraction pattern of physical mixture [Fig.6c] (eg. peaks at 2θ values 10.55˚, 12.46˚, 15.60˚, 17.02˚, 17.28˚, 18.92˚, 22.92˚, 35.02˚ and 72.80˚). The X-ray diffraction pattern of the solid inclusion complex [Fig.6d] is evidently different from that of pure BPY, and β-CD. XRD pattern of BPY and β-CD displayed crystalline pattern whereas in the

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complex the diffraction peaks are larger in number but it is not possible to distinguish the characteristic peaks, especially 2θ values of peaks after 25˚. Diffractogram of BPY: β-CD exhibits amorphous pattern lacking crystallinity. The peak intensities are reduced significantly in

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the diffraction pattern of the complex which also suggested the reduction in crystallinity. For example the more intense peak in XRD pattern of BPY with almost 70150 counts [2θ value 17.26˚] is reduced and it appears only with 295 counts in the solid complex(counts not shown).

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The substantial decrease in the crystallinity of solid complex in comparison with β-CD and BPY, and the amorphous character exhibited by the inclusion complex proves the

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nano hydrophobic interaction between β-CD and BPY [74,75]. 3.2.4. Thermal analysis

It is a very useful tool to investigate thermal properties of cyclodextrin complexes [76]. It can provide both qualitative and quantitative information about the physicochemical state of

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the guest inside the CD cavity. Inclusion of the guest in to CD cavity may cause disappearance of endothermic peak, appearance of new peak, broadening or shifting to different temperature

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indicating a change in the crystal lattice, melting, boiling or sublimation points. Thermograms of β-CD, BPY and BPY : β-CD inclusion complex are illustrated in the

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Fig.7. The thermogram of β-CD (Fig. 7a) shows a characteristic endothermic peak at 118 oC associated to crystal water losses (15%) from β-CD cavity. Dehydration of β-CD occurs through a temperature range from 75-125 oC with the maximum at 118 oC. Endothermic effect nearing 300oC is related to thermal destruction of β-CD. The thermogram of BPY (fig.7b) exhibiting pronounced endothermic effect of melting process without loss of mass is recorded. The experimental melting point of BPY at 76 oC is in good agreement with the literature value (~ 78 oC) [77]. Endothermic effect at 295 oC corresponds to thermal decomposition of BPY. In

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the physical mixture (Fig7c), absence of notable differences in the thermal degradation properties of β-CD confirms that there is no interaction between BPY and β-CD. In solid inclusion complex (Fig. 7d), the endothermic effect related to release of water molecules from

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the inner cavity of β-CD is shifted to 132 oC. There is no endothermic effect related to melting of BPY observed in the thermogram of inclusion complex. However the characteristic thermal degradation properties of β-CD are registered. The loss of mass observed from 50-300 oC is

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equal to 30 % whereas that of similar one in β-CD is only 15 %. So the thermal decomposition of BPY would have taken place in this temperature range (50-300

o

C). The thermal

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decomposition of encapsulated BPY proceeds at high temperature than uncomplexed BPY. Hence, thermal stability of BPY increases when it is encapsulated by β-CD. But the thermal stability of β-CD is decreased since it precedes its decomposition at 248 oC which is shown by

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exothermic peak.

3.2.5. Atomic force microscopy analysis

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The atomic force microscope (AFM) is one kind of Scanning probe microscopes (SPM). It is a high resolution microscopy technique which produces precise topographic images of a

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sample by scanning the surface with a nano meter scale probe (lateral resolution ~1 nm, vertical ~0.1nm). AFM uses a mechanical probe to magnify surface features up to 100,000,000 times and it produces 3-D images of the sample.

The knowledge of the surface topography at

nanometric resolution has made possible to probe dynamic biological [78], mechanical, manufacturing porous [79] and mainly thin film [80,81]. Using adequate software it is possible to evaluate characteristics such as roughness, porosity, average size and particle size distribution, which influence directly the optical, mechanical, surface, magnetic and electrical properties of

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thin films.

Fig.8 shows the 3D AFM images of BPY and solid inclusion complex of

BPY and β-CD. The nodules are seen as bright high peaks where as the pores are seen as dark depressions. Visualization of surface topography of BPY (fig.8a) and BPY : β-CD solid

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inclusion complex (Fig.8b) using AFM analysis has revealed a significant change in the morphology of the surface when BPY molecules formed inclusion complex with β-CD (Fig.8b). It is now known that when the surface consists of deep depressions that characterize pores and

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high peaks that correspond to nodules, high roughness parameters results [82]. As observed by Idris et al., [82] it can be noted that membranes with high surface roughness indicates high flux

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and a compound with smooth surface exhibits low flux. Further this change in roughness parameters is proportional to the change in the pore size of a compound. The surface texture of BPY and the solid inclusion complex was measured with horizontal length scale of 4µm and a vertical scale of 203 nm and 104 nm respectively. Hence it reveals that BPY has a rough surface

cavity.

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and the roughness of BPY is changed into a smooth surface when it is encapsulated into β-CD

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3.3. Molecular docking study of inclusion process

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The 3D structure of β-CD and BPY obtained from crystallographic databases are shown in Figs. 9a and 9b. The guest molecule, BPY was docked into the cavity of β-CD using PatchDock server. The PatchDock server program gave several possible docked models for the most probable structure based on the energetic parameters; geometric shape complementarity score [83], approximate interface area size and atomic contact energy [84] of the BPY:β-CD inclusion complex (Table 3). The docked BPY:β-CD 1:1 model(Fig.9c)with the highest geometric shape complementarity score 2700, approximate interface area size of the complex

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324.06 Å2 and atomic contact energy -210.01 kcal/mol was the highly probable and energetically favourable model and it is in good agreement with results obtained through experimental

3.4. Semiempirical quantum mechanical calculations

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methods.

The internal diameter of the β-CD is approximately 6.5 Å and its height is 7.8 Å

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(Scheme 1). The overall height of BPY is 9.1 Å (i.e., the vertical distance between H14 – H19), Considering the shape and dimensions of β-CD, it is clear that the BPY molecule cannot be

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completely accommodated inside β-CD. Hence there is possibility for only partial inclusion of BPY molecule into β-CD cavity as interpreted using experimental data (low binding constant

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value) and it is clearly shown in scheme.1.

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Scheme 1

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BPY:β-CD (1:1) host - guest mechanism

4. Conclusion :

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A survey of the literature related to usage of cyclodextrins for obtaining inclusion complexes with various fluorophores was carried out. Supramolecular interaction of BPY with

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β-CD has been investigated in solution and solid state. Taking into account the results obtained during these investigations, it was observed that the interaction between BPY and β-CD through the formation of inclusion complex leads to important modifications in the physicochemical

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properties of the guest molecule. The observed fluorescence intensity of BPY was enhanced largely when it was encapsulated in to β-CD cavity. The Benesi−Hildebrand and Job’s plots

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illustrated the formation of a 1:1 host-guest complex. In XRD analysis, reduction in crystallinity was observed due to inclusion complex formation. Morphological study using SEM and AFM confirmed that particles of the inclusion complex prepared by co-precipitation method showed a distinct morphology from the ones observed in the raw materials. Molecular docking study

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Figure captions:

1. Fig.1a. Absorption spectra of BPY with increasing concentration of β-CD

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(1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β-CD) ( Inset Benesi-Hildebrand absorption plot of BPY with β-CD (λabs = 232 nm) 2. Fig.1b. Benesi-Hildebrand absorption plot of 1/(A-Ao) against 1/[β-CD]2 .for BPY

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with β-CD (λabs = 232 nm)

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3. Fig.2a Fluorescence spectra of BPY with increasing concentration of β-CD (1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β-CD), ( Inset Benesi-Hildebrand fluorescence plot of BPY with β-CD)

4. Fig. 2b. Job’s plot of BPY with β-CD (inset. Benensi-Hildebrand fluorescence plot of 1/[I-Io] against 1/[β-CD]2 .for BPY) 5. Fig.3. Fluorescence decay curves of BPY with increasing concentration of β-CD

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6. Fig. 4. FT-IR spectra of a) β-CD, b) BPY c) physical mixture of BPY with β-CD and d) solid complex of BPY with β-CD. 7. Fig. 5. SEM images of a) β-CD, b) BPY (× 500), c) physical mixture of BPY with

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β-CD (× 500) and d) solid complex of BPY with β-CD (× 500).

8. Fig. 6. XRD pattern of a) β-CD, b) BPY c) physical mixture of BPY with β-CD and d) solid complex of BPY with β-CD.

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9. Fig. 7. Thermograms of a) β-CD, b) BPY c) physical mixture of BPY with β-CD and d) solid complex of BPY with β-CD.

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10. Fig. 8. Atomic force microscope photographs of (a) BPY and (b) solid complex of BPY

with β-CD.

11. Fig. 9. Ball and stick representation of (a) β-CD (b) BPY (c) 1:1 inclusion complex; the oxygen atoms are shown as red, nitrogen as blue, chlorine as green balls, carbon atoms

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are shown as golden balls and sticks, and hydrogen atoms are not shown.

Table.1 The absorption and fluorescence spectral maxima of BPY in different concentrations of β-CD at pH 6.8

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Concentration of β-CD [M]

λmax

abs

(nm)

λmax

abs

(nm)

λflu (nm)

231.8

0.267

285.0

0.002

232.0

0.324

285.0

0.004

232.0

0.342

285.4

0.006

232.0

0.372

286.2

0.308

328

0.008

232.0

0.413

287.0

0.324

328

0.010

232.0

0.444

288.4

0.346

330

0.012

232.0

0.522

289.0

0.380

332

326

0.270

327

0.288

328

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0.247

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Table 2. Fluorescence lifetime and amplitude of BPY with increasing concentration of β-CD (Excitation wavelength 280 nm, detection wavelength 326nm) Lifetime

Relative amplitude

χ2

(sec)

Standard deviation

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Concentration of β-CD (M)

1.89×10-9

100

1.11

1.15×10-11

0.002

1.83×10-9

87.22

1.10

8.91×10-12

8.08×10-9

12.78

1.62×10-9

83.67

9.01×10-9

16.33

0.006

1.54×10-9 1.03×10-8

0.008

1.50×10-9

0.012

75.14

65.86

1.08×10-8

34.14

1.46×10-9

52.42 47.58

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1.20

24.86

1.48×10-9

1.12×10-8

1.19

23.59

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0.010

76.41

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1.06×10-8

1.15

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0.004

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0

1.23

1.49×10-10

9.47×10-12 1.62×10-10 1.06×10-11 1.68×10-10 1.12×10-11 1.65×10-10 1.52×10-11 1.41×10-10

1.06

1.82×10-11 1.75×10-10

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Table 3: Scores of the top 10 docked models of BPY:β-CD inclusion complex computed using

Model

Geometric shape complementarity score

Approximate interface area size of the complex Å2

1

2700

324.06

2

2688

328.10

3

2658

323.40

4

2590

5

2550

6

2502

7

2486

8

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PatchDock server.

Atomic contact energy kcal/mol

-203.53 -210.30 -214.81

263.93

-197.46

308.00

-216.54

320.70

-215.38

2460

311.21

-211.06

9

2460

301.15

-197.03

10

275.90

-195.20

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- 210.01

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2176

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Fig.1a. Absorption spectra of BPY with increasing concentration of β-CD (1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β-CD) ( Inset: Benesi-Hildebrand absorption plot of BPY with β-CD

(λabs = 232 nm)

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Fig.1b. Benesi-Hildebrand absorption plot of 1/(A-Ao) against 1/[β-CD]2 .for BPY with β-CD (λabs = 232 nm)

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Fig.2a. Fluorescence spectra of BPY with increasing concentration of β-CD

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(1. Without, 2. 0.002, 3. 0.004, 4. 0.006, 5. 0.008, 6. 0.010 and 7. 0.012 M β-CD)( Inset Benesi-Hildebrand fluorescence plot of BPY with β-CD)

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Fig. 2b. Job’s plot of BPY with β-CD (inset. Benensi-Hildebrand fluorescence plot of

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1/[I-Io] against 1/[β-CD]2 .for BPY)

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Fig.3. Fluorescence decay curves of BPY with increasing concentration of β-CD

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Fig. 4. FT-IR spectra of a) β-CD, b) BPY c) physical mixture of BPY and β-CD and d) solid complex of BPY and β-CD.

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Fig. 5. SEM images of a) β-CD, b) BPY, c) physical mixture of BPY and β-CD and d) solid complex of BPY and β-CD.

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Fig. 6. XRD pattern of a) β-CD, b) BPY c) physical mixture of BPY and β-CD and d) solid complex of BPY and β-CD.

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Fig. 7. Thermo grams [TG-DSC] of a) β-CD, b) BPY c) physical mixture of BPY and β-CD and d) solid complex of BPY and β-CD.

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Fig. 8. Atomic force microscope photographs of (a) BPY and (b) solid complex of BPY and β-CD

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Fig. 9. Ball and stick representation of (a) β-CD (b) BPY (c) 1:1 inclusion complex; the

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oxygen atoms are shown as red, nitrogen as blue, carbon atoms are shown as golden

balls and sticks, and hydrogen atoms are not shown.

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Highlights: 1. The 1:1 stoichiometry was confirmed by Benesi-Hideband double reciprocal plots. 2.

FT-IR, Molecular docking studies strongly confirmed the formation of inclusion

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complex.

3. In XRD analysis, reduction of crystallinity in inclusion complex was observed.

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4. Life time studies shows that BPY has single and bi-exponential in water and β-CD.