Inclusion complex of β-cyclodextrin with tetrabutylammonium bromide: Synthesis, characterization and interaction with calf thymus DNA

Journal Pre-proof Inclusion complex of β-cyclodextrin with tetrabutylammonium bromide: Synthesis, characterization and interaction with calf thymus DNA

Susama Chakraborty, Pranab Ghosh, Basudeb Basu, Amitava Mandal PII:

S0167-7322(19)31840-9

DOI:

https://doi.org/10.1016/j.molliq.2019.111525

Reference:

MOLLIQ 111525

To appear in:

Journal of Molecular Liquids

Received date:

30 March 2019

Revised date:

5 August 2019

Accepted date:

6 August 2019

Please cite this article as: S. Chakraborty, P. Ghosh, B. Basu, et al., Inclusion complex of β-cyclodextrin with tetrabutylammonium bromide: Synthesis, characterization and interaction with calf thymus DNA, Journal of Molecular Liquids(2019), https://doi.org/ 10.1016/j.molliq.2019.111525

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© 2019 Published by Elsevier.

Journal Pre-proof

Inclusion Complex of β-Cyclodextrin with Tetrabutylammonium bromide: Synthesis, Characterization and Interaction with Calf thymus DNA Susama Chakraborty1, Pranab Ghosh2, Basudeb Basu2,3, Amitava Mandal1* 1

Molecular Complexity Laboratory, Department of Chemistry, Raiganj University, Raiganj, Uttar

Dinajpur, West Bengal, India, Pin 733134. Department of Chemistry, University of North Bengal, Raja Rammohunpur, West Bengal, India,

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Department of Chemistry, Raiganj University, Raiganj, Uttar Dinajpur, West Bengal, India, Pin

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Pin 734013.

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733134. *

Abstract

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[email protected]

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Corresponding author Tel.: +91 03523-244039, Fax: +91 03523-242580, E-mail address:

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Surfactant molecules are now become indispensible in drug formulations to reduce drug-drug interactions and most often to enhance the bioavailability of poorly water soluble life saving drugs. Recently, tetrabutylammonium bromide (TBAB) is the cationic surfactant of choice. A further advancement could be achieved by using mixed surfactant-cyclodextrin (CD) systems in drug delivery. A study on the inclusion behaviour of TBAB with β-CD host would therefore, have high scientific value for the development of novel TBAB based pharmaceutical formulations. We report herein the synthesis and complete physicochemical and spectral (UV, IR, NMR, ROESY, XRD, TGA, SEM etc.) characterization of the inclusion complex (IC) between TBAB and β-CD. The stoichiometry of the formed IC was evaluated as 1:1 by Job’s plot. A comparison of 1H NMR spectra of TBAB, β-CD and IC and ROESY spectrum revealed that two out of four n-butyl chains were incorporated into CD cavity and the ammonium nitrogen atom stick out to the wider CD edge. The thermal stability of TBAB was found to increase after complexation. The experimental

Journal Pre-proof data were also correlated with the molecular docking study. In addition, the binding mode of the synthesized IC with Calf thymus DNA was also reported. Keywords: Inclusion complex, TBAB, β-CD, Job’s plot, ROESY, Calf thymus DNA 1. Introduction The spontaneous reorganisation or association of molecules by noncovalent interactions under equilibrium conditions into well-defined secure structures is termed as supramolecular selfassembly [1,2]. It provides an inexpensive and easier method for preparing ensembles of particles

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in a controllable manner to achieve any desired goal. Self-assembly always allows the creation of

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demanding molecular topologies [2,3]. Proper exploitation of their collective properties in making

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molecular functional devices is the key behind the present interest in supramolecular self-

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assembly [4,5]. It may be exploited to fabricate the mechanical properties of any composite

performed concurrently [6,7].

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material. In modern drug delivery supramolecular self-assembly allows multiple tasks to be

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One classic example of supramolecular self-assembly is the host-guest inclusion complexes (ICs)

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completed of cyclodextrins (CDs) and guest molecules. CDs are cyclic oligosaccharides of six to eight glucopyranose units interlinked via α-1,4-linkages forming truncated conical structures having the secondary hydroxyl groups at the wider side and the primary hydroxyl groups at the narrow side of the cone. This geometry offers a hydrophobic interior and hydrophilic peripheral surface. These are called α, β, and γ-CDs respectively. Such amazing quality of CDs has been utilized to form host-guest ICs with diverse organic guest molecules [8]. Due to this uniqueness, CDs have been used extensively in pharmaceutical formulations, foodstuffs, pesticides, toilet articles and in textile processing [9-12]. In addition, nowadays CDs have found applications in molecular recognition and self-assembly, molecular encapsulation, sensing and chemical stabilization [13-16]. In aqueous solutions, the hydrophobic internal cavity of CD is occupied by energetically unfavourable few water molecules that can be easily substituted by suitable non

Journal Pre-proof polar organic guest molecules, which prefers to penetrate into the empty CD cavity, leading to the formation of an IC through noncovalent host-guest interactions. Surfactants are typical organic compounds having amphiphilic in nature, meaning they contain both polar hydrophilic head groups and hydrophobic hydrocarbon tails in their structure. Surfactants generally diffuse in water and adsorb at the interface between air and water or in cases where water is admixed with oil they adsorb at the interfaces between oil and water. Due to their exceptional functional properties, in modern drug formulations surfactants find a wide range of

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applications. Depending on the category of drug and its use, these include improving the aqueous

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solubility and/or stability of a drug in a liquid form, modifying and stabilizing the texture of a

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semisolid form, changing the flow properties of granulate, therefore improving the processing of

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an ultimate tablet dosage form. In addition to their blend as chemical excipients to aid both the physical and chemical properties of a drug formulation, surfactants may also be added to enhance

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the bio availability of a drug. The unique properties of a surfactant are such that it can alter the

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thermodynamic activity, diffusion or disintegration rate of dissolution of a drug. All these parameters affect the rate and extent of drug absorption. Besides, surfactants induce some direct

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effects on the surface of the biological cell membranes thus altering drug transport across cell membrane. The use of surfactants as solubilizers, emulsifiers, and dispersants for suspensions and as wetting agents in drug formulation can cause significant variation in the biological activity of drug in the formulation. The overall added effects, in general, of a surfactant in a drug formulation are complex and in most cases it offers the intended beneficial effects. Cationic surfactant like tetrabutylammonium bromide (TBAB) finds wide applications as phase transfer catalyst in the syntheses of diverse heterocyclic compounds [17,18] and to trap CO2 gas molecules by engaging in hydrate formation [19]. Pharmaceutically it is used in the cleaning of wounds [20]. It has also been successfully used in the pharmaceutical formulation of antifungal drug, such as echinocandin [21]. Accordingly, there remains ample scope of using TBAB as an ideal candidate for potential drug delivery systems or drug formulations. Previously, Mehendro et

Journal Pre-proof al. have reported [22] the interaction of tetrabutyl ammonium cation (TBA+) with - and -CD only on cellulose dissolution and interestingly, the computed apparent association constant of TBA+--CD system is very high (K = 1580 M−1). A detail study, therefore, specific on inclusion behaviour of TBAB with CD molecules might be helpful not only for developing TBAB based pharmaceutical formulations but also in understanding the mode of drug delivery actions. Such studies are hitherto unknown and the present study describes the synthesis, characterization and nature of possible binding mode of ICs of β-CD and TBAB with DNA (Figure 1). Our studies

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bind with the phosphate groups of Calf thymus DNA.

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clearly establish the formation of IC of β-CD and TBAB in 1:1 molar ratios and the resulting IC

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The IC of TBAB-CD was characterized by physicochemical studies such as conductance study;

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powder X-ray diffraction (XRD), TGA analyses, ultraviolet-visible (UV-VIS) spectrophotometry,

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Fourier transform-infrared (FT-IR) spectroscopy, 1H NMR, rotating frame nuclear Overhauser effect spectroscopy (ROESY), and SEM studies. Molecular docking was also carried out to

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identify the best orientation and the nature of interactions of included TBAB into β-CD cavity. In addition, interaction of IC with Calf Thymus DNA was also studied.

2.1 Materials

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2. Experimental Section

The studied compound, β-CD was procured from Spectrochem Pvt. Ltd., minimum purity 99 % (HPLC on dry basis), specific optical rotation []020 +159o to +165o and was used without further purification. The surfactant, TBAB was obtained from Merck, Minimum assay ≥ 99%. Water used for the conductance study was triply distilled and deionised. Calf thymus DNA (Sigma-Aldrich) was commercially purchased and used as received. DNA stock solution was prepared by dissolving 2 mg/mL accurately weighed solid calf thymus DNA in deionised triply distilled water with occasional shaking at room temperature and stored at 4 oC. Stock solution of IC was prepared by directly dissolving 10 mg/mL solid IC into deionised triply distilled water and stored at 4 oC. 2.2 Synthesis of Inclusion Complex

Journal Pre-proof Method of co-precipitation was applied to make the solid IC in 1:1 ratio between the host and guest molecules. A saturated solution of β-CD was prepared with deionised triply distilled water. Then a saturated solution of TBAB in methanol was added gradually at room temperature with constant stirring. Stirring was continued for another 48 hours. The obtained white mass was filtered through 0.45 µm filter, dried under vacuum, crushed to powder and was kept in sealed vial under vacuum. 2.3 Apparatus and Procedure

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Solubility of the β-CD in water (triply distilled, deionised and degassed water with a specific

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conductance of 1 x 10-6 S cm-1) and the title compound viz., TBAB in aqueous β-CD, were

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accurately checked prior to the set off the experimental work, and we observed that the particular

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surfactant, TBAB, was freely soluble in all proportions of aqueous CD. Stock solutions of TBAB were prepared by mass (weighed by Mettler Toledo AG-285 with uncertainty 0.0003 g), and the

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working solutions were obtained by mass dilution at 298.15 K. Conductance was taken in

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Systronic EU-TECH 304 CON 700 conductivity meter. NMR spectra were recorded in Brucker-Avance 400 MHz NMR spectrometer with 5 mm BBO

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probe using D2O as solvent. Powdered XRD was performed in Bruker D8 advance powder X-Ray diffractometer. TGA analyses were carried out in Perkin Elmer Diamond TG/DTA analyzer. UVVIS spectra were taken in Lasany LI-2700 spectrophotometer. IR spectra were recorded in Perkin Elemer FT-IR Spectrometer (RX-1) in KBr discs. SEM study was performed using a JSM-7500 F SEM (JEOL, Japan) at 5.0 k.v. 3. Results and Discussion 3.1 Conductance Study Conductivity measurement [23] is a frequently used method for studying inclusion phenomenon. It can also be used to elucidate the stoichiometry of the ICs formed [24]. If the freely water soluble TBAB forms an IC with β-CD, the conductivity of the solution would be markedly affected by the gradual addition of β-CD. The conductivity of various β-CD concentrations in

Journal Pre-proof aqueous TBAB was measured at 25

o

C, and the dependence of conductivity on β-CD

concentrations is shown in figure 2. When one or more hydrophobic tail of TBAB gets entered into the inner hydrophobic β-CD cavity and binds itself with the CD molecule through noncovalent interactions, the movement of the free ions gets arrested. As a result solution conductivity decreases since the number of free ions per unit volume has been decreased. Remarkable decrease of solution conductivity with gradual increase of β-CD concentration indicates the IC formation between TBAB and β-CD. At a definite β-CD concentration, there

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occurred a break point in the specific conductance curve of TBAB (Figure 2). After the break

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point with further addition of β-CD only a finite decrease in specific conductance values of TBAB

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was observed. Such a noticeable break point in the conductivity curve was observed at a

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concentration close to 5.0 mmol/L of β-CD. This suggested that the stoichiometry of the β-CDTBAB complex was equimolar [25]. Thus, a 1:1 IC has been formed between TBAB and β-CD

Inclusion complex

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β-CD + TBAB

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and a dynamic equilibrium exists between the host β-CD and guest TBAB molecules.

At the break point all guest molecules are encapsulated into hydrophobic β-CD cavity and after

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that the equlibrium shifts to the right hand side. The formation of IC can be accounted for the insertion of one or more hydrophobic group(s) of TBAB into the β-CD cavity. The formed IC may have different stoichiometric possibility such as 1:1 (linear), 2:1 (T-shaped or linear), 3:1 (pyramidal) and 4:1 (star like) ratios of β-CD and TBAB as depicted in scheme 1. 3.2 UV-VIS Studies In the study of host-guest supramolecular chemistry absorption spectra were regularly used to verify IC formation [26,27]. In the present study, the absorption spectra of isotonic solutions of βCD, TBAB and IC were taken into consideration. β-CD has almost no absorption (Figure 3) throughout the wavelength scanned, consequently its absorbance values can be neglected [28]. The absorption intensity of TBAB, which shows higher absorbance among the three (figure 3),

Journal Pre-proof showed a tiny blue shift accompanied with strong hypochromic effect after mixing with β-CD, thus suggesting the IC formation between TBAB and β-CD. A physical mixture of TBAB and CD prepared by grinding together in mortar pestle also showed similar UV spectral pattern, thus signifying the formation of IC (see supporting information). 3.3 FT-IR Analysis FT-IR spectroscopy is another primary analytical tool for confirming IC formation in solid state.

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In the present context, FT-IR spectra of TBAB and β-CD were compared to that of solid IC (Figure 4) to validate its formation. The FT-IR spectra of IC showed majority of the characteristic

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peaks for β-CD in almost similar wave numbers and in intensity to that of pure β-CD molecule;

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however, that for TBAB was found to absent or shift at different wave numbers [29,30], with

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profoundly lowered in intensity. Generally in solid IC, the non-covalent interactions such as van

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der waals interactions, hydrophobic interactions or hydrogen bonding between the host and guest molecules lead to lowering of energy of the included part of the guest molecule, thus reducing the

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peak intensities of the corresponding IR bands. The characteristic bands of pure TBAB showed CH asymmetric stretching vibration (νas CH3) at 2963 cm-1 and the symmetrical stretching vibration

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for methylene group (νs CH2) at 2849 cm-1. The symmetrical bending vibration for methyl group (δs CH3) appeared at 1379 cm-1 and the asymmetrical bending vibration (δas CH3) at 1456 cm-1. The scissoring band (δs CH2) for the hydrocarbon chain came into at 1465 cm-1, rocking vibration (ρCH2) at 718 cm-1 and methylene twisting and wagging vibrations are visible at 1378 cm-1 and 1160 cm-1 respectively. C-N stretching vibration is noticeable at 1027 cm-1. The characteristic bands of pure β-CD appeared at 3390 cm-1 (intermolecular H-bonded O-H stretching vibration); 2916 cm-1 for -CH or -CH2 stretching vibrations and 1017 cm-1 for primary alcohol C-O stretching vibrations. However, in IC the characteristic intermolecular H-bonded -OH stretching band of pure β-CD became very narrow and has been shifted to 24 cm-1 higher wave number (at 3414 cm1

). Narrowed down of hydroxyl peaks of pure β-CD is familiar [31-33], with the formation of

supramolecular host-guest complex. The C-H asymmetric stretching vibration for TBAB (νas CH3)

Journal Pre-proof has also been shifted to 2927 cm-1 after complexation with greatly reduced in intensity. Appearance of the C-H asymmetric stretching vibration band at lowered wave number with low intensity suggested that the vibration of the methyl groups is somehow restricted due to inclusion. The methylene scissoring vibration band (δs CH2) disappeared in IC and methylene twisting vibration band was shifted to 1358 cm-1. This is because of the trapping of TBAB hydrophobic tail into CD cavity. Interestingly, in IC the C-N stretching vibration appeared at 1026 cm-1 coinciding to that of the pure TBAB. This fact clearly signified that the positively charged nitrogenous head

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3.4 1H NMR Analysis

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group of TBAB just protrude outside the CD cavity.

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In modern day chemistry, 1H NMR is the most extensively used practice to study IC formation,

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deeper insight into the inclusion modes and geometries of ICs [34]. It is very helpful to provide direct evidence about the surfactant in host solution, since complexation affects the proton

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environments of the host as well as the guest. This effect will thus be reflected as variations in

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chemical shifts of protons from both species [35,36]. In the present case, a 1:1 IC has been formed between TBAB and β-CD. It is well known that β-CD molecule adopts the conformation of a

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torus where H-3 and H-5 protons are located inside the cavity, whereas H-2 and H-4 are outside the torus (Figure 5). Figure 6 shows the 1H NMR spectra of β-CD, TBAB, and the IC in D2O. It is clear from figure 6 that in IC some signals appeared as strong unresolved broad peaks. This observation is characteristic to that of IC formation between TBAB and β-CD [37]. Insertion of TBAB into the hydrophobic β-CD cavity would result in changes of chemical shifts of TBAB and β-CD in the 1H NMR spectra. Due to inclusion phenomena, in general, significant change in chemical shifts is observed for H-3 and H-5 protons, which are located at the inner CD cavity. The chemical shift change is normally represented as Δδ. This difference is used to monitor the interaction between the host and guest molecules. A positive sign signify a downfield shift and a negative sign means an up field shift. The change in chemical shift values for different protons of β-CD and TBAB after complxation has been tabulated in table 1 and 2 respectively. It is clear that

Journal Pre-proof presence of TBAB produce slightly up field chemical shift values for five CD protons except H-5. The negative values of chemical shift change (Δδ) have also been tabulated in table 1. This shift, therefore, provides the indication for the formation of an IC between β-CD and TBAB [38-42]. However, H-5 proton did not appear in the 1H NMR spectrum of IC. It is probably overlapped with the H-6 signal. This is happened because after complexation H-5 proton is too screened by the included TBAB to give a signal in NMR. The observed up field shift for H-3 proton was also appreciably larger in comparison to that of H-6 proton which is located at the primary face of β-

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CD. This larger up field shift of H-3 proton along with the absence of H-5 signal indicates that the

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TBAB molecule has been entered through the wider rim of β-CD (Scheme 2). Noticeably, in our

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case change in chemical shift values have also been found for the two exterior protons, H-2 and

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H-4 to lower  values. This observation suggested that all the four hydrophobic tails of TBAB

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might not been inserted into the hydrophobic CD cavity and the observed change in chemical shift values are due to some interaction between the two exterior CD protons and those of

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unincorporated hydrophobic TBAB tails. This fact is also supported by change in chemical shift values of TBAB protons (Table 2). The observed van der waal’s deshielding is supportive to the

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formation of IC between TBAB and β-CD. The area ratio of H-1 proton of β-CD to that of H-1/ proton of TBAB indicates the formation of a 1:1 complex between the host β-CD and the guest TBAB.

3.5 ROESY Experiment 1

H-1H ROESY is valuable for assigning correlations between non bonded protons that are close to

each other in space. A 2D ROESY spectrum always gives through-space correlations using spinspin relaxation. 1H-1H ROESY spectrum thus can further validate the configuration of formed IC between host and guest molecule. Figure 7 shows the 2D ROESY spectra of β-CD-TBAB complex. In the ROESY spectrum, the interactions between H-3, H-6, H-5 of β-CD and the -CH2 groups in the hydrocarbon chain of TBAB which resonate at a centre of 2.595 ppm and at 0.521 ppm, could be identified, suggesting that some hydrocarbon chain(s) of the guest molecule, TBAB

Journal Pre-proof was inside the CD cavity and the nitrogen group protrudes outside. Correlations were observed for the protons H-1, H-2, H-4 of β-CD to the H-4/ protons of TBAB. These correlations indicated the through space interactions of the exterior protons of β-CD to the unincorporated hydrophobic tail of TBAB. ROESY spectrum thus, confirmed the observations found from 1H NMR spectrum. An obvious correlation was also observed between the H-1 and H-2 protons of β-CD. This is an inevitable phenomenon ever since H-1 and H-2 protons are adjoining in the β-CD molecule. Similar correlations were also observed for the protons of H-2 and H-3, H-3 and H-4 as well as H-

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4 and H-5 of β-CD. Furthermore, the 2D ROESY spectrum also reveal that there are correlations

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among the -CH2 groups of TBAB resonating at 1.226 ppm and 0.934 ppm. In this case, we cannot

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exclude the possibility that in the binding condition the included long chains of TBAB have close

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contact. ROESY experiment along with 1H NMR spectrum indicated the spatial proximity among the -CH2 groups of TBAB hydrocarbon chain and the H-3 and H-5 β-CD protons located inside

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the cavity. Hence out of four, few n-butyl hydrocarbon chains of TBAB are inside the

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hydrophobic β-CD cavity and the rest are on the exterior side. If the four n-butyl chains adopt a zigzag conformation in the complex, that is, with the hydrocarbon chain completely extended, its

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calculated length on average is near about 3.82Å. The height of β-CD is, however, about 7.9 Å. So the guest molecule should be in permanent motion in the β-CD cavity and adopts a conformation which tends to occupy the whole space of the cavity [34]. But due to the conical/torus shape of βCD, insertion of four n-butyl chains together into the β-CD cavity is fully restricted and only two n-butyl chains from the guest molecule, TBAB can insert (occupying a space of 7.65 Å) at a time into β-CD cavity to form an IC with 1:1 inclusion ratio as depicted in Scheme 1. In contrast to our finding, Mehendro et al. have claimed, [22] that only one n-butyl chain of TBAB has been inserted into the β-CD cavity to form a 1:1 IC. 3.6 Stoichiometry for TBAB-β-CD Inclusion complex One of the best methods used in supramolecular host-guest chemistry to recognize the stoichiometry of the host-guest ICs is the continuous variation method, universally known as the

Journal Pre-proof Job’s method [43]. By using UV-visible spectroscopy this continuous variation method was applied in the present study to determine the stoichiometry of the prepared IC. Different set of solutions for each TBAB and β-CD was prepared by varying the mole fraction of the guest in the range 0-1. Job’s plot was generated by plotting Δ A × R against R, where Δ A is the difference in absorbance of TBAB without and with β-CD and R = [TBAB]/([TBAB] + [β -CD]) [44,45]. Absorbance values were precisely measured at respective λmax for every solution at 298.15 K. The stoichiometry of IC can be found out from the value of R at the highest deviation, i.e., ratio of

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guest and host is 1:2 if R = 0.33; 1:1 if R = 0.5; 2:1 if R = 0.66 etc. In the present work, maxima

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for the Job’s plot was found at R = 0.5, which evidently imply 1:1 stoichiometry of the formed

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host-guest IC between TBAB and β-CD (Figure 8). Efforts were then made to find out the

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equilibrium constant for the inclusion of TBAB with -CD with the help of Bensi-Hildebrand

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equation. Unfortunately, Bensi-Hildebrand plots were random, scatter and data are not reproducible (see supporting information). Since TBAB is a highly substituted quaternary amine,

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all the attempts to detect the equilibrium constant for the present system with the help of Bensi-

3.7 SEM study

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Hildebrand equation were failed [46].

SEM analysis is regularly used to obtain accurate information about the surface topography and composition of molecules. Here through SEM study we compared the surface morphological structure of β-CD and solid IC, since modification of crystal morphology can be unambiguously taken as an evidence for the formation of a solid IC. Selected microphotographs of β-CD and IC are illustrated in figure 9. SEM photographs clearly elucidated the morphological difference between pure β-CD and IC. These photographs undoubtedly revealed that pure β-CD exhibits crystalline parallelogram structure [47]. On the other hand, the original parallelogram morphology of β-CD was completely disappeared in the solid IC, where only amorphous aggregates with irregular shapes were observed and also it was no longer feasible to distinguish the initial

Journal Pre-proof components. This change in crystalinity and morphology is obvious about the formation of IC between TBAB and β-CD. 3.8 X-ray diffractometry Study X-ray diffractometry (XRD) is a widely used practice in the study of ICs for assessing the structure and to ensure the formation of a new solid compound from the parent molecules [48]. According to Harada et al. the crystal structures of CD complexes can be classified chiefly into three categories: channel-type, cage-type, and layer-type [49]. The powder XRD pattern of

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TBAB, β-CD and IC are presented in figure 10. The results suggested that the obtained IC has

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fine crystalline powdery structure. In Figure 10a, major sharp and intense peaks are observed at

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diffraction angles (2θ) of 9.09°, 10.67°, 12.65°, 14.75°, 18.83°, and 20.92°, indicating that β-CD

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represents a typical cage structure [50,51]. Figure 10a, b and c showed that, the synthesized IC have generated different diffraction patterns relative to their parents, with key diffraction signals

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at 9.5o, 23.13°, 27.32o, and 28.31o indicating a head-to-head channel-type structure [51], which is

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different from that of β-CD and TBAB . The XRD results revealed that the solid IC is isomorphous with channel-type structure rather than the so-called “cage” type structure.

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Many investigations were reported in literature to elucidate the mechanism of formation of such a channel-type structure [50-52]. For guest molecules with long chain arm, the ICs adopt a head-tohead channel-type arrangement in which β-CD molecules self assembled through H-bonds between hydroxyl groups of neighbouring CD molecules along an axis to form a cylindrical structure [52]. These intermolecular hydrogen bonds between adjacent CDs play the vital role in stabilizing the complexes. Furthermore, in addition to the geometric compatibility, van der waals interactions and release of cyclodextrin strain energy upon complexation are considered to contribute to the formation of IC [53]. 3.9 TGA Study The thermal stability of IC was evaluated by using TGA analyses and was compared with that of the pure β-CD and TBAB. Figure 11 shows the weight loss curves for IC, β-CD and TBAB and

Journal Pre-proof figure 12 shows the DTA curve. It is clear from the figures that the synthesized IC has higher decomposition temperature than TBAB itself, suggesting that inclusion has increased the thermal stability of TBAB. The gain of extra thermal stability of TBAB after complexation with β-CD increases its potential to be used in drug formulations [54]. 3.10 Docking study In order to rationalize the conformation of TBAB-β-CD IC, 3D molecular docking was performed [55]. The guest molecule, TBAB was docked into the β-CD cavity using Autodock 4. Detail

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docked views of IC is presented in figure 13. Autodock 4 server program produced several

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probable docked models of the IC for the most probable structure based on the energetic

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parameters, geometric shape complementarity score, approximate interface area size and atomic

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contact energy [56,57]. The binding energies of the ten docked conformations were tabulated in table 3. From the docking study it is clear that two hydrophobic arms of TBAB were inserted into

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the β-CD cavity and the rest two are just outside of the wider edge. Thus it is obvious that these

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two unincorporated TBAB arms are in contact with the exterior protons (H-2 and H-4) of β-CD, thereby supporting our finding from the NMR studies that as against one, [22] two n-butyl chains

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of TBAB have been inserted into the hydrophobic CD cavity. 3.11 Interaction with DNA

The interaction of drug molecules with DNA has been of prime focus in the design of new efficient drug targeted to DNA. If TBAB-β-CD inclusion complex finds its application in a DNA targeted drug formulation, it would be of prime importance to know whether the prepared IC binds with DNA or not and if it binds with DNA, what would be its mode of binding which would accelerate the use of TBAB-β-CD IC in different drug formulations. This study may also serve as a model for interaction of present IC to proteins and enzymes, because small molecules often interact similarly with DNA. In general, small molecules interact with DNA through three noncovalent modes: intercalation, groove binding and external binding.

Also if the guest

molecule interacts with DNA, the presence of CD would necessarily affect the interaction, which

Journal Pre-proof should be embodied in the change of the spectroscopic properties [58]. Different techniques are reported in literature to investigate the nature of interaction of complex molecules with DNA. This includes UV-VIS spectrophotometry [9], fluorescence [60], cyclic voltammetry [61] and circular dichroism spectropolarimetry [62]. Figure 14 represents the UV absorption spectra of Calf thymus DNA with varying concentration of IC. Hyperchromism has been observed for the interaction of IC with DNA. This hyperchromic effect might be attributed to external contact which is purely electrostatic in nature [63,64], originating from non-specific, outside edge stacking interactions

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with the phosphate backbone of DNA [65].

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4. Conclusion

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The inclusion behaviours of β-CD and TBAB have been effectively studied by physicochemical

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and spectral (UV, IR, NMR, ROESY, XRD, TGA, SEM, etc.) techniques. The combined results of physicochemical and spectral studies indicated that β-CD and TBAB can form an IC with a

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molecular ratio of 1:1. The 1:1 (TBAB:β-CD) stoichiometry of the synthesized IC was confirmed

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by Job’s plot. Based on 1H NMR and 2D ROESY spectra, possible inclusion structures were speculated. It is clearly indicated from NMR (1H and ROESY) and docking studies that only two

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hydrophobic arms of TBAB (as against to one as claimed by Mehendro et al.) were inserted into the hydrophobic CD cavity and the nitrogenous head group was just outside of the wider rim. XRD data showed that the synthesized IC is isomorphous with head-to-head channel type structure, which is different from its parents. From TGA we found that inclusion has increased the thermal stability of TBAB. This would certainly increase its potential to be used in future drug formulations. Docking studies also support the findings of NMR studies that the two hydrophobic arms of TBAB were inserted into the β-CD cavity at a time and the rest two are just outside of the wider edge. The UV absorption spectra of Calf thymus DNA with changing concentration of IC showed hyperchromism for the interaction of IC with DNA. This hyperchromic effect is due to electrostatic external contact with the phosphate backbone of DNA. Thus, the present work

Journal Pre-proof successfully communicates both qualitative and quantitative idea about the formation of IC of βCD with TBAB, suggesting its potential as an adjuvant in pharmaceutical formulations. Acknowledgement The authors are grateful to the Department of Chemistry, Raiganj University for infrastructural facilities. SC is thankful to Government of West Bengal for awarding Swami Vivekananda Merit cum Means fellowship and is thankful to Miss. Koyeli Das (Dutta), post doc fellow, Department of Chemistry, IIT Madras for her support.

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[1] H. Jiao, S.H. Goh, S. Valiyaveettil, Inclusion complexes of poly (4-vinylpyridine)-

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Journal Pre-proof

Figure 1. The molecular structure of the studied (1) β-CD and (2) TBAB Figure 2. Plot of variation of conductance of TBAB corresponding to the added concentration of aqueous β-CD. Figure 3. Absorption spectra of (a) β-CD; (b) TBAB and (c) Inclusion complex Figure 4. FTIR- Spectra of (a) β-CD; (b) TBAB (c) Inclusion Complex Figure 5. (a) Truncated structure of free β-CD (b) Structure of free β-CD

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Figure 6. 1H NMR spectra of TBAB, β-CD and Inclusion Complex in D2O.

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Figure 7. (a) ROESY spectrum of IC in D2O, red circles are showing the correlations between βCD and TBAB; (b) pdb image of TBAB showing the calculated distance of n-butyl chains in

-p

zigzag conformation.

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Figure 8. Job’s plot of TBAB-β-CD system at 298.15 K. λ max = 215 nm and R =

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[TBAB]/([TBAB] + [β-CD]), Δ A = absorbance difference of the TBAB without and with β-CD.

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Figure 9. SEM images of different views of β-Cd in (a) 200 µm , (c) 2 µm and IC at (b) 200 µm , (d) 2 µm. Figure 10. X-ray powder diffraction pattern of (a) β-CD, (b) TBAB and (c) Solid IC of TBAB:β-

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CD

Figure 11. TGA diagram for (a) β-CD, (b) IC and (c) TBAB Figure 12. DTA diagram for (a) β-CD, (b) Inclusion Complex and (c) TBAB Figure 13. Docked view of inclusion complex (a) hydrophobic interaction view of docked complex of run 1 and (b) of run 9.

Figure 14: The absorption spectra of DNA with varying concentrations of IC at pH = 7.0. The concentrations of IC in (a) 10 µL; (b) 20 µL; (c) 30 µL; (d) 40 µL; (e) 50 µL and (f) 0 µL. Scheme 1: Possible stoichiometries of ICs with predicted shape Scheme 2: Favourable and unfavourable mode of insertion of TBAB into the β-CD cavity.

Journal Pre-proof Tables Table 1 1

H NMR chemical shifts of β-CD in free and complexed state determined in D2O at 300K β-CD ( in ppm)

H1 H2 H3 H4 H5 H6

4.875 3.453 3.775 3.391 3.365 3.686

Inclusion (in ppm) 4.646 3.225 3.514 3.163 Absent 3.454

in



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Table 2

H NMR chemical shifts of TBAB in free and complexed state determined in D2O at 300K TBAB ( in ppm)

H1 H2 H3 H4

2.762 1.210 0.926 0.506

in



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Table 3

Inclusion Complex (in Difference ppm) value 2.776 +0.014 1.226 +0.016 0.934 +0.008 0.521 +0.015

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Proton

-p

1

Complex Difference value -0.229 -0.227 -0.261 -0.228 --------0.232

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Proton

Run Estimated Inhibition Constant Ki mM

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Calculated binding energies of different docked conformations of IC Estimated Final VDW+HElectrostatic Torsional Free Intermolecular bond+DS Energy Free Energy of Energy Energy Kcal/mol Energy Binding Kcal/mol Kcal/mol Kcal/mol Kcal/mol 1 7.54 -2.90 -6.48 -6.58 +0.11 +3.58 2 13.88 -2.53 -6.11 -6.15 +0.04 +3.58 3 10.35 -2.71 -6.29 -6.36 +0.08 +3.58 4 11.49 -2.65 -6.23 -6.31 +0.08 +3.58 5 11.29 -2.66 -6.24 -6.32 +0.08 +3.58 6 10.35 -2.71 -6.29 -6.38 +0.09 +3.58 7 11.10 -2.67 -6.25 -6.34 +0.10 +3.58 8 10.59 -2.69 -6.27 -6.37 +0.10 +3.58 9 8.52 -2.82 -6.40 -6.47 +0.07 +3.58 10 11.68 -2.64 -6.22 -6.27 +0.06 +3.58 VDW, Vander waal energy; H-bond, hydrogen bond energy; DS, desolve energy.

Final Total Internal Energy Kcal/mol -1.52 -1.58 -1.42 -1.63 -1.41 -1.56 -1.48 -1.57 -1.41 -1.38

Journal Pre-proof Highlights  First report on the inclusion behaviour of TBAB with β-CD host.  Formation of inclusion complex (IC) was confirmed by UV, IR, NMR, ROESY, XRD, TGA etc. studies.  Stoichiometry of the synthesized IC was evaluated as 1:1 by Job’s plot and docking studies.  The IC binds with the phosphate backbone of Calf thymus DNA.

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 Keeps a high scientific value for the development of novel TBAB based pharmaceutical

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na

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-p

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formulations.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14