Encapsulation of rhodamine-6G within p-sulfonatocalix[n]arenes: NMR, photophysical behaviour and biological activities

Abstract

The inclusion complexes of rhodamine-6G (Rh6G) dye within p-sulfonatocalix[4]arene (SCX4), p-sulfonatothiacalix[4]arene (TSCX4), p-sulfonatocalix[6]arene (SCX6) hosts has been characterized using the ¹H NMR, 2D NMR NOESY, infrared, fluorescence experiments and scanning electron microscopy analysis. The inclusion complexes show encapsulation of the phenyl ring of the Rh6G guest within the SCXn (n = 4 and 6) or TSCX4, with its ester functionality protruding outside the cavity along the direction bisecting two adjacent sulfonato groups of the macrocyclic host; the inference that has been corroborated through the density functional theory. The aromatic protons of Rh6G confined within the host cavity of the complex reveal shielded signals in the measured NMR spectra. The formation of inclusion complexes has been confirmed through 2D NMR NOESY experiments. Fluorescence experiments demonstrated that the quenching constant of the SCX6 complex was ∼10⁴ times larger than those for the SCX4 and TSCX4 complexes. Biological activity measurements further revealed that the Rh6G⊂SCX4 complex possesses remarkable antiproliferative and antimicrobial activities.

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Introduction

Calixarenes represent a fascinating class of macrocyclic hosts, which have attracted growing attention in the recent literature owing to their ubiquitous applications in a variety of areas such as catalysis,¹ enzyme mimetic,² sensors,^3,4 chromatography.⁵ The vase shaped calixarenes in particular, those comprising four, six or eight phenol rings are synthesized using the base catalyzed condensation of substituted phenols with formaldehyde.⁶ The novel calix[n]arenes possess a hydrophobic interior (cavity) with hydrophilic portals; the solubilities of which can be enhanced significantly by modifying hydroxyl groups at the upper rim⁷ of the host. Accordingly the modified sulfonato substituted calix[4]arene (SCX4) or calix[6]arene (SCX6) hosts and sulfonatothiacalix[4]arene (TSCX4),⁷ showed increased water solubilities. The host–guest interactions in supramolecular complexes are known to be governed by dimensions of the host cavity and the charge distributions therein. The sulfonated calixarenes with varying cavity size are commercially available.⁸ Galindo-Murillo et al.⁹ studied binding of substituted CXn (n = 4–8) with guest molecules comprising pyrazole moieties. These authors concluded that stronger cation binding of sulfonato derived hosts over the ethoxy substituted cavitands which was facilitated through a large negative charge density at the upper rim of CXn. Binding of fluorescent dye to water soluble calixarene has thus emerged as an active area of research in supramolecular chemistry.¹⁰ Besides these investigations the p-sulfonatocalixarene macrocycles have shown a remarkable and selective binding ability toward biologically significant amino acids, nucleotide bases, transition metal complexes¹¹ and small organic molecules/ions.¹²

Interestingly the interactions of fluorescent dye with electron-rich hydroxy or alkoxy substituted aryl rings of SCXn or TSCX4 modified hosts have demonstrated significant fluorescence quenching. Pursuance to this fluorescence measurements on the complexes of acridine red, neutral red and rhodamine B guests with a variety of supramolecular hosts have been carried out by Liu et al.¹³ These authors concluded that the formation of inclusion complexes with β-cyclodextrin, cucurbit[7]uril or calix[4]arene sulfonate hosts exhibit 1 : 1 stoichiometric proportions. Nau and co-workers¹⁴ further reported that addition of cucurbituril to rhodamine B conduces the stable inclusion complex which finds potential applications as dye laser and bimolecular labeling. The reports on encapsulation of different dye molecules encapsulated macrocycles such as calixarene or modified calixarenes have appeared in the recent literature.¹⁵ Rajagopal and coworkers¹⁶ have shown that encapsulation of curcumin within SCX4 increases its bioavailability and further leading to enhanced water solubility. The host–guest binding of acridine in neutral and cationic forms with SCX6 was analyzed by Mohanty and others.¹⁷ From absorption spectroscopy and steady state fluorescence experiments these authors concluded that the cationic form binds strongly to the macrocycle via noncovalent interactions. The observed shifts in pK_a values further suggested that SCX6 can be explored in acid catalyzed enzymatic reactions. It has also been realized that conducing noncovalent complexes of Rh6G leads to self-quenching of the dye. In this regard photostability and photophysical properties of rhodamine dyes are explored in a variety of applications as laser dyes, molecular switches¹⁸ and chemosensors for transition metal ions^19–21 and hence, stimulated growing interest in the recent years. The 1 : 1 inclusion complex of Rh6G with β-cyclodextrin showed a remarkable increase in its fluorescence. Subsequent investigations on complexation of fluorescent dyes viz., Rh6G, acridine, fluorescein, with calixarenes or derivatives thereof, have become the focus of attention in the recent years.²² With this view, the present endeavour focuses on understanding the complexation behaviour of the Rh6G with SCX4, TSCX4 and SCX6 macrocycles. The molecular recognition behaviour of these macrocycles demonstrate varying binding strengths and the fluorescence behaviour of the encapsulated dye significantly.²³ The calixarene hosts endowed with distinct structural features and anticancer activities of their complexes have been explored. Interestingly the antibacterial, antifungal, antiviral²⁴ data on the aminocalix[n]arenes²⁵ are now available. Despite of extensive experimental investigations outlined above understanding complexation of dye molecule with modified calixarene derivatives are rather limited. To this direction the molecular insights derived from the density functional theory, accompanying complexation of the calixarene derivatives and anticancer drug imatinib guided the controlled delivery of drug molecule into tumor cells.

The present work therefore focuses on understanding complexation of Rh6G with SCX4, TSCX4 and SCX6 synthetic macrocycles, possessing similar void space, displayed schematically in Scheme 1 along with the isolated Rh6G.

Scheme 1 (a) SCX4, (b) TSCX4, (c) SCX6, hosts and (d) Rh6G.

The fluorescence, ¹H NMR, 2D COSY and NOESY, FTIR experiments are carried out to characterize the inclusion complexes of SCX4, TSCX4 and SCX6. The photophysical properties, morphological behaviour and antimicrobial and the antiproliferative activities of these complexes have also been investigated. The structures of SCX4 and TSCX4 complexes are corroborated through the use of density functional theory.

Results and discussion

¹H NMR experiments

¹H NMR spectra of Rh6G⊂SCX4, Rh6G⊂TSCX4 and Rh6G⊂SCX6 complexes along with those of individual host and Rh6G were acquired using the DMSO-d₆ as solvent. NMR spectrum of Rh6G in DMSO-d₆ along with the labeling of protons shown in Fig. 1S† further elucidated the fine structure along with chemical shifts and the spin–spin coupling constants. The triplet signal in spectrum belongs to –CH₃(a)(d) (δ = 1.26 ppm), the singlet due to –CH₃(c) (δ = 2.09) ppm, quintet (δ = 3.48 ppm) and singlet (δ = 3.57 ppm) assigned to –CH₂(b) and –CH₃(e) was also observed. The peaks at δ = 6.78, 6.94 ppm were assigned to A and A′ protons. Peak due to –NH and aromatic region appear at 7.33 and H_f, H_g, H_h and H_j led to doublet of doublet δ = 8.35, 7.62, doublet of triplet 7.47 and triplet around 6.72, respectively. The NMR spectrum of neat SCX4 exhibit two signals at δ = 3.96 ppm (Ar-CH₂: methylene hydrogen) and δ = 7.39 ppm (Ar-H: aromatic hydrogen) (Fig. 2S†). The complexation with Rh6G influences the nature and position of NMR peaks of SCX4. As shown in Fig. 1, the doublet of H_j (7.47 ppm) merged into the aromatic SCX4 proton signal (7.37 ppm) for the Rh6G⊂SCX4 complex. The spin–spin splitting due to H_f displays a doublet of doublet whereas the H_g, H_h protons engender triplet of doublet in ¹H NMR spectra. The –CH₃(a,d) appears as a triplet at the δ = 1.29 ppm.

Fig. 1 ¹H NMR spectrum of 1 : 1 mole ratio of Rh6G⊂SCX4 in DMSO-d₆ (*solvent peak).

TSCX4 has more electron density than SCX4 and SCX6 with vacant 3d orbitals of the sulfide linkages that renders a resonance-like interaction with the π-orbital of adjacent aromatic rings which results in delocalized negative charge as displayed in Fig. 2.²⁶ Thus, substitution of bridging –CH₂ functionalities by the sulfide group brings about expansion of cavity as evidenced from separation for the phenyl group and bringing –S of the TSCX4 macrocycle.⁸ The measured ¹H NMR spectrum of TSCX4 macrocycle has been given in Fig. 3S of the ESI.† The average distance between the phenyl and –CH₂ turns out to be 1.53 Å in SCX4 compared to that of 1.78 Å in TSCX4 (Table 1S of the ESI†) the structural variations herein reflect the Δδ of 0.66 ppm corresponding to Ar-OH.²⁷ It is evident from Fig. 4S of the ESI† that ¹H NMR spectra of SCX6 led to the peak at δ = 4.58 ppm which was assigned to Ar-OH. The complexation with Rh6G (Fig. 3) engenders a broadened singlet for Ar-CH₂ consequent to mobile conformations (1,2,3-alternate and double cone etc.).²⁸ As summarized in Table 2S† the change in chemical shifts (Δδ) accompanying the formation of inclusion complex display the signals arising from the H_j (−0.31) protons in Rh6G and Ar-OH (0.63) of SCX6 are largely sensitive to encapsulation. The influence of SCX6 on the aromatic proton of Rh6G is further evident.

Fig. 2 ¹H NMR spectrum of 1 : 1 mole ratio of Rh6G⊂TSCX4 in DMSO-d₆ (*solvent peak).

Fig. 3 ¹H NMR spectrum of 1 : 1 equivalent of Rh6G⊂SCX6 in DMSO-d₆ at room temperature (*solvent peak).

As pointed out earlier, the cavity-size of sulfonated calixarene host is one of the key factors governing the stability of the inclusion complex.²⁹ The Δδ parameters of the TSCX4 imply shielding owing to the presence of sulfide group with diminutive electrostatic repulsions.

Association constants and stoichiometries of complexes

Association constants of Rh6G guest with the SCX4 and TSCX4 macrocycles were measured from the ¹H NMR titration experiments using the DMSO-d₆ as solvent. As shown in Fig. 2S and 3S,† the –OH protons of SCXn show up near 5.38 ppm and 3.38 ppm, respectively. The hydroxy protons led to upfield signals with addition of Rh6G up to 30 mM concentration. The association constants of the Rh6G⊂SCX4 and Rh6G⊂TSCX4 complexes were calculated by the linear fitting of Δδ_H, which refers to change in chemical shift accompanying the complexation, as a function of guest to host mole ratio as shown in Fig. 5S and 6S,† respectively. The association constants of the SCX4 and TSCX4 complexes turn out to be 2.5 and 1.32 M⁻¹, respectively.

A method of Job's continuous variations was applied³⁰ to determine the binding stoichiometry of Rh6G and SCXn in DMSO-d_6. An equimolar (0.01 mM) solutions of Rh6G and SCXn were mixed to a fixed volume by varying the molar ratio (Rh6G : SCXn = 1 : 5, 1 : 3, 1 : 2, 1 : 1, 2 : 1, 3 : 1 and 4 : 1) keeping the total solute concentration as constant. To elucidate stoichiometry of the complexes NMR titration experiments were carried out. Thus stoichiometric ratios (0.3, 0.4, 0.6, and 0.7) of Rh6G (0.01 mM) and SCX4 (0.01 mM) were confirmed to be in proportions of 1 : 1 (Fig. 4 and 7S†). A merging of –CH₂ and –OH observed (Table 1) was attributed to hydrogen bonding between bridging –CH₂ and –OH of SCX4 on incorporation of Rh6G guest. It may, therefore be inferred that such shielding is more prominent for the 1 : 1 complexes.³⁰ A disaggregation of the guest and formation of 1 : 1 complex (Fig. 5) is transparent.³¹ These inferences are in consonance with conclusions drawn³² for the SCX4–crystal violet complexes. The effect of an increase in the concentration of Rh6G on SCX6 (Fig. 8S†) was monitored through the ¹H NMR experiments. Noteworthy enough, a peak assigned to Ar-OH shifts in shielded region consequent to increased cavity size of the SCX6 and the lower rim hydroxyl group thus is affected largely on complexation with the Rh6G guest.

Fig. 4 ¹H NMR spectra (500 MHz, 298 K) recorded in DMSO-d6 for (a) SCX4, (b) 1 : 5 Rh6G⊂SCX4, (c) 1 : 3 Rh6G⊂SCX4, (d) 1 : 2 Rh6G-SCX4, (e) 1 : 1 Rh6G⊂SCX4, (f) 2 : 1 Rh6G⊂SCX4, (g) 3 : 1 Rh6G⊂SCX4 and (h) 4 : 1 Rh6G⊂SCX4.

Table 1 Chemical shifts along with Δδ for SCX4 (0.01 mM), Rh6G (0.01 mM) and Rh6G⊂SCX4in DMSO-d6 (negative values indicate upfield shift)

Proton	δ0	Δ					(Δδ = δ − δ0) (ppm)
		0.3	0.4	0.5	0.6	0.7	0.3	0.4	0.5	0.6	0.7
Hf	8.27	8.27	8.27	8.27	8.27	8.27	0	0	0	0	0
Hg	7.91	7.91	7.91	7.91	7.91	7.91	0	0	0	0	0
Hh	7.84	7.84	7.85	7.84	7.84	7.84	0	0.01	0	0	0
Hj	7.72	7.73	7.72	7.73	7.73	7.73	–0.03	–0.04	–0.03	–0.03	–0.03
NH	7.46	7.46	7.45	7.46	7.46	7.46	0	—	0	0	0
A	6.94	6.93	6.93	6.94	6.94	6.94	–0.01	–0.01	0	0	0
A′	6.78	6.78	6.78	6.78	6.78	6.78	0	0	0	0	0
CH2(e)	3.57	3.57	3.57	3.57	3.57	3.57	0	0	0	0	0
CH2(b)	3.48	3.49	3.49	3.48	3.49	3.50	0.01	0.01	0	−0.01	0.02
CH3(c)	2.08	2.09	2.09	2.09	2.09	2.09	–0.01	–0.01	–0.01	–0.01	–0.01
CH3(a)(d)	1.27	1.26	1.24	1.26	1.26	1.25	–0.01	−0.03	0.06	–0.01	–0.02
Ar-H	7.25	7.38	7.38	7.38	7.38	7.37	0.01	0.01	0.01	0.01	0
Ar-OH	5.38	4.42	4.26	4.09	4.08	—	–1.13	–1.08	–1.29	−1.3	Peak merged
Ar-CH2-Ar	3.94	3.94	3.94	3.94	3.94	—	0	0	0	0

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Fig. 5 Job's plot for the (Rh6G⊂SCX4) complex of 0.1 mM where Δδ = change in chemical shifts in ppm, χ = mole ratio of host and guest.

2D NOESY

A nuclear overhauser effect (NOESY) cross-peaks are indicative of specific proximity relationships between host and guest protons which usually extend up to 4 Å. The space intermolecular interactions between calixarene inclusion complexes³³ are evident. For small molecules the 2D NOESY give rise to positive cross peaks whereas the diagonal signals are phase negative. As shown in Fig. 6 the NOE cross peaks of Rh6G⊂SCX4 reveal that the guest binding is facilitated through interactions from H_f, H_g, H_h and H_j protons with Ar-H of SCX4, which are portrayed in Fig. 9S.† The methylene protons are equivalent and exhibit no geminal coupling. The electron withdrawing (–CO₂CH₂CH₃) ability of the Rh6G, engenders π–π interactions between guest and the SCX4 macrocycle.³⁴ 2D NOESY relationships of protons in SCX4, TSCX4 and SCX6 are shown shematically in Scheme 2. From ¹H and 2D NMR correlations it may as well be deciphered that the relatively large size of SCX6 cavity engender weak interactions with the guest compared to those of SCX4 or TSCX4. The electron donating effect of thiol group affects the intensity of –CH₃(d) of ester group on the Rh6G concomitantly. The ester group protruding outside the host cavity facilitates hydrogen bonding interactions which further affect the intensity of –CH₃(d) significantly. It may as well be inferred that the aromatic protons showed strong NOESY enhancements than those bound to the xanthene core of Rh6G. Likewise the coupling was also observed for the Rh6G⊂SCX4 complex. Lastly the Rh6G⊂TSCX4 and Rh6G⊂SCX6 complexes led to similar inferences.

Scheme 2 2D NOESY relationships of protons in SCX4, TSCX4 and SCX6 are shown schematically.

Fig. 6 Selected region of the 2D NOESY NMR spectra of (a) Rh6G⊂SCX4 (b) Rh6G⊂TSCX4 and (c) Rh6G⊂SCX6 in DMSO-d₆ and schematic representation of complex.

DFT investigations

To delve further into structure of Rh6G complexes with SCX4 and TSCX4 macrocycles the DFT calculations employing ωB97x/6-31G(d,p) level of theory were carried out. Atomic labelling scheme for the Rh6G⊂SCX4 is given in Fig. 7. Conformers exhibiting distinct host–guest binding patterns were optimized at this level of theory. Theoretical calculations predicted that encapsulation of xanthene core within the SCX4 or TSCX4 cavities led to stationary point structures, shown in Fig. 10S of the ESI.† These conformers are destabilized up to ∼20 kJ mol⁻¹ and ∼33 kJ mol⁻¹ over their lowest energy structures (Fig. 8), those possess the pendent phenyl ring encapsulated within the host cavity and the xanthene core protruding along the direction midway between adjacent –SO₃H groups on the host rendering the O–H⋯O hydrogen bonding interactions with the ester substituent. The hydrogen bonding interactions from methylene as well as methyl group of the ester have also been inferred (Fig. 9).

Fig. 7 (Rh6G⊂SCX4) complex structure with atom labelling scheme.

Fig. 8 ωB97x/6-31G(d,p) optimized structures of stable conformers of individual guest, hosts and their complexes.

Fig. 9 Hydrogen bonding distances in (a) Rh6G⊂SCX4 and (b) Rh6G⊂TSCX4.

The host–guest binding patterns in the Rh6G⊂SCX4 and Rh6G⊂TSCX4 complexes predicted from the present theory are consistent with the 2D NMR experiments. Selected geometrical parameters in the SCX4, TSCX4, Rh6G, and their lowest energy inclusion complexes are summarized in Table 2. It may be conjectured that complexation of Rh6G with SCX4 and TSCX4 macrocycles is governed by O5–H⋯O_b hydrogen bonding and other noncovalent interactions. Selected hydrogen bonding distance parameters are given along with. Calculated binding energies of the Rh6G to SCX4 as well as to the TSCX4 macrocycle are marginally different, which turn out to be ∼108 kJ mol⁻¹. Net atomic charges derived from the Hirshfeld population analysis in different complexes are compared with the free SCX4, TSCX4 in Table 3. The hydrogen bonding interactions from two sulfonato groups are evident from the electron deficient O_b and O_c centres. The net Hirshfeld charges on non-bonded O_a atoms are nearly unchanged on complexation. To characterize the charge distribution within these complexes we portray the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbitals (LUMO) (isosurfaces of +0.02 au) in Fig. 10. The electron-rich regions in HOMO of the isolated Rh6G and in SCX4 or TSCX4 complexes are strikingly similar. It has further been transparent that both SCX4 and TSCX4 complexes display electron-rich regions in the HOMO localized near xanthene core of Rh6G. Besides the HOMO and LUMO gap of the isolated Rh6G guest was predicted to be 6.79 eV which increases to 9.88 eV on partial encapsulation of Rh6G within SCX4 and to 9.79 eV in TSCX4 cavitand. Calculated ¹H NMR chemical shifts revealed that A and A′ protons in Rh6G excluding the host cavity are nearly insensitive to encapsulation of the guest within SCX4 or TSCX4 as also observed in the measured ¹H NMR spectra. On the other hand, H_f and H_g guest protons on encapsulation within the SCX4 cavity revealed shielded signals with the deeply penetrated H_g exhibiting the largest shielding of 4.4 ppm relative to the isolated Rh6G in the calculated spectra. The δ_H parameters in the isolated host, guest and complex from theory are compared with experiment in Table 3S of the ESI.† The δ_H signals from the experiment, except for the H_g proton are nearly insensitive to the host guest binding. Similar inferences are drawn for the TSCX4 complex, which exhibit a shielding of 3.9 ppm for the H_g protons in the calculated spectra.

Table 2 Comparison of optimized structural parameters (bond distances in Å and angle) in SCX4, TSCX4 hosts, Rh6G guest and their inclusion complex

	SCX4	Rh6G⊂SCX4	TSCX4	Rh6G⊂TSCX4
a H-Bonded.
S1–O5	1.622	1.611	1.622	1.609
S1=O6	1.451	1.452	1.450	1.451
S2=O10	1.443	1.445	1.443	1.444
A(S1–O5–H)	107.08	109.65	106.98	110.04
A(H–O1–C)	111.18	110.67	110.83	109.38
O–H⋯O–H (lower rim)	1.718	1.705	1.896	2.014

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	Rh6G	Rh6G⊂SCX4	Rh6G⊂TSCX4
Ob–C	1.213	1.225	1.225
Oc–COb	1.331	1.319	1.318
CH2(e)^a	1.094	1.091	1.092
CH3(d)^a	1.093	1.093	1.093

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Table 3 Hirshfeld charges in isolated SCX4, TSCX4, Rh6G and their complexes

Atom	SCX4	Rh6G⊂SCX4	TSCX4	Rh6G⊂TSCX4
S1	0.504	0.494	1.203	0.497
S2	0.504	0.497	1.203	0.497
S3	0.504	0.509	1.203	0.514
S4	0.504	0.503	1.203	0.505
O1	−0.032	−0.020	−0.571	−0.003
O2	−0.032	−0.027	−0.571	−0.015
O3	−0.032	−0.043	−0.571	0.030
O4	−0.032	−0.011	−0.571	0.004
O5	−0.005	−0.069	−0.552	−0.066
O6	−0.321	−0.303	−0.522	−0.300
O7	−0.307	−0.308	−0.481	−0.304
O8	−0.005	−0.030	−0.553	−0.038
O10	−0.307	−0.299	−0.481	−0.296

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Atom	Rh6G	Rh6G⊂SCX4	Rh6G⊂TSCX4
Oa	−0.064	−0.066	−0.065
Ob	−0.272	−0.252	−0.250
Oc	−0.118	−0.093	−0.093

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Fig. 10 Frontier orbitals (isosurfaces of + 0.02 au) in HOMO and LUMO of (a) SCX4, Rh6G, Rh6G⊂SCX4, (b) TSCX4, Rh6G and Rh6G⊂TSCX4.

FT-IR analysis

FTIR spectra of Rh6G, SCX4 and Rh6G⊂SCX4 complexes are depicted in Fig. 11S.† It is evident that the complexation is accompanied by a shift of –NH stretching from 3099 cm⁻¹ in the isolated Rh6G guest to 2979 cm⁻¹ with a concomitant decrease in its intensity, which can be attributed to NH⋯O hydrogen bonding interactions from –NH group of the Rh6G. On the contrary, the –CN stretching vibration on complexation exhibits a shift in the opposite direction from 1600 cm⁻¹ to 1649 cm⁻¹. The direction of frequency shifts here serves as a signature of the formation of Rh6G⊂SCX4 complex. The 1700–1100 cm⁻¹ region further emerges with an intense band at the 1187 cm⁻¹ assigned to carbonyl stretching of the isolated Rh6G that corresponds to the 1149 cm⁻¹ vibration in the complex. The measured infrared spectra revealed the C=O stretching frequency shift in the Rh6G⊂SCX4 complex to be larger (∼7 cm⁻¹) than that observed for the TSCX4 complex. It may therefore, be conjectured that host–guest binding in 1 : 1 inclusion complexes is governed by the C=O⋯H hydrogen binding interactions in addition to those from the sulfonato and methylene group protruding outside the host cavity. The change Δν = ν_host/guest − ν_complex were calculated. A largest red shift of –NH stretching on complexation of Rh6G with the TSCX4 implies deeper penetration of the guest within the TSCX4 cavity of the complex. The –CN stretching has nearly been unchanged. As may readily be noticed, the frequency downshift of –NH stretching in Rh6G⊂SCX4 and Rh6G⊂SCX6 complexes was nearly twice as large as the Rh6G⊂TSCX4 complex. A large cavity of the SCX6 leads to the 3337 cm⁻¹ band assigned to the Ar-OH vibration. The direction of frequency shifts of the –NH and –CO stretching accompanying the complexation of Rh6G with SCX6 led to similar inferences. Selected vibrations of the individual hosts, isolated Rh6G, and the complexes are compared in Table 4. The numbers in parentheses refer to the frequency shift accompanying partial encapsulation of the guest; the positive and negative values refer to the ‘red’ and ‘blue’ shift, respectively. Furthermore the frequency downshift of 28 cm⁻¹ for carbonyl stretching in the SCX4 complex compared to that of 12 cm⁻¹ for the SCX6 complex is evident. As far as the host vibrations are concerned the complexation of Rh6G is accompanied by a frequency upshift of –SO₃H vibrations. A largest ‘blue shift’ of 89 cm⁻¹ was noticed for the Rh6G⊂TSX4 complex. As opposed to this, the Ar-OH vibrations revealed frequency ‘blue shift’ for the SCX4 and SCX6 complexes. The TSCX4 complex displays a shift in the opposite direction which amounts to ∼5 cm⁻¹.

Table 4 Selective vibrational frequencies of Rh6G and its complexes

	Rh6G	SCX4	TSCX4	SCX6	Rh6G⊂SCX4	Rh6G⊂TSCX4	Rh6G⊂SCX6
ν(–NH)	3145	—	—	—	2979(166)	3064(81)	2980(165)
ν(–CN)	1648	—	—	—	1649(−1)	1647(1)	1646(2)
ν(–CO)	1728	—	—	—	1700(28)	1707(21)	1716(12)
ν(Ar-OH)	—	3153	3352	3268	3179(−26)	3347(5)	3337(−69)
ν(–SO3H)	—	1118	1209	1267	1147(−29)	1298(−89)	1272(−5)

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Steady-state fluorescence studies

The steady-state fluorescence spectra of Rh6G and its SCX4 complex reveal the fluorescence quenching in the complexes. As shown in Fig. 12S,† a shift in fluorescence is less pronounced and results in Stoke shift which suggests the relaxation of the dye through geometrical and solvent effect is smaller as expected for inclusion in a more confined environment. The fluorescence spectra further revealed a strong and sharp emission peak at λ_em = 563 nm. A steady state fluorescence characteristic of Rh6G has been accompanied by significant changes upon addition of SCX4, TSCX4 and SCX6 indicating strong interactions between Rh6G and the macrocycle (Fig. 11).

Fig. 11 (a) Fluorescence spectra and (b) Stern–Volmer plot of Rh6G (0.001 M) in DMSO containing SCXn (4.6 × 10⁻³M) (i) Rh6G⊂SCX4, (ii) Rh6G⊂TSCX4 and (iii) Rh6G⊂SCX6 λ_ex = 535 nm, λ_em = 563 nm.

With an increase of concentration of SCXn added to Rh6G results in fluorescence quenching, the electron-rich sulfonato rings here prone to act as electron donors.³⁵ Quantum yield was calculated from eqn (1)

here m (RhB = 39 999.41, Rh6G⊂SCX4 = 20 830.53, Rh6G⊂TSCX4 = 29 021.40 and Rh6G⊂SCX6 = 25 204.71) was determined from the slope of a plot of integrated fluorescence intensity vs. absorbance, n being the refractive index of solvent taken to be unity and subscript R as the reference fluorophore with its quantum yield (Q = 0.5) being known.³⁶

The estimates for fluorescence quantum yield for the isolated dye as guest and the complex are given in Table 5. The observed decrease in quantum yield corroborates the inference on electron transfer accompanying the fluorescence that engenders the hole within the LUMO facilitating subsequent non-radiative de-excitation of the excited dye.³⁷ The change in fluorescence intensity has been portrayed as I₀/I vs. concentration of calixarene which yield a straight line. The Stern–Volmer constants (K_SV) were estimated from the slope using eqn (2)

I₀/I = 1 + K_SV[Q] (2)

with I and I₀ being the fluorescence intensities for the Rh6G in the presence and absence of SCX4, TCX4 and SCX6. From Fig. 13S,† the life time of dye (Rh6G) was determined (τ₀ = 3.73 ns), which agrees well with the reported value.³⁸ Bimolecular quenching constant (k_q) was obtained subsequently using the following equation.

K_SV = k_qτ₀ (3)

Table 5 Stern–Volmer quenching constant (K_SV) (in M⁻¹), bimolecular quenching constant (k_q) (in M⁻¹ s⁻¹) and quantum yield (Φ) for Rh6G and complexes in DMSO

	KSV	kq	Φ	R^2
Rh6G	—	—	0.95	—
Rh6G⊂SCX4	9.67 × 10^−5	2.43 × 10^8	0.260	0.99
Rh6G⊂TSCX4	7.32 × 10^−5	1.9 × 10^8	0.362	0.98
Rh6G⊂SCX6	9.82 × 10^−5	3.11 × 10^12	0.315	0.99

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Table 5 summarizes quenching constants (k_q) of Rh6G inclusion complexes of SCX4, TSCX4 and SCX6. The value k_q is small for Rh6G⊂SCX4 and Rh6G⊂TSCX4 may be due to steric shielding or low quenching efficiency whereas for Rh6G⊂SCX6 larger k_q was noticed.^39,40

Scanning electron microscopy

SEM analysis has emerged as a quantitative technique for measuring surface morphology. The structural difference of parent calixarenes and complexes can readily be inferred from Fig. 12. The physicochemical interactions within the complex are evident. The uniformity of amorphous solid at the microscopic level, all host, guest and complexes were studied upto 2–5 μm width. Rh6G shows an irregular shape whereas SCX4 and SCX6 show rectangular one, SCX6 is akin to SCX4 whereas TSCX4 appear as a network of cubical rods. The morphology of complexes Rh6G⊂SCX4 changed to stacked layers. On the other, hand TSCX4 shows 2D rectangular stacked structure.⁴¹

Fig. 12 SEM micrographs of (a) pure Rh6G, (b) SCX4, (c) SCX6, (d) TSCX4, (e) Rh6G⊂SCX4 and (f) Rh6G⊂TSCX4 in 1 : 1 mole ratio in acetonitrile.

Antimicrobial and antiproliferative activities

Three different sulfonated calixarenes with varying cavity size were tested for their antibacterial effect against B. subtilis ATCC 6633 and E. coli ATCC 8739 at different concentrations (Fig. 14S†). The present investigations showed that all three derivatives of calixarenes Rh6G⊂SCX4, Rh6G⊂TSCX4 and Rh6G⊂SCX6 at 1 : 2 ratio possess antibacterial activity against Gram positive bacteria and not against Gram negative bacteria E. coli ATCC 8739. The investigations further suggest the antibacterial activity of complexes follows the order: Rh6G⊂SCX6 > Rh6G⊂SCX4 > Rh6G⊂TSCX4 (Table 6). The antibacterial activities of Rh6G⊂SCX6 complex at specified concentrations are higher than other complexes which can possibly be attributed to its bulky structure and more –SO₃H groups. Perret and Coleman⁴² reported earlier the important role of –SO₃H group in SCX4 in determining the antibacterial activities. The increase in association constants of the complexes (Rh6G⊂SCX4 > Rh6G⊂TSCX4) increases their antibacterial activities.⁴³

Table 6 Antimicrobial activity (zone of inhibition in cm) of sulfonated calixarenes derivatives at different concentrations against B. subtilis ATCC 6633 and E. coli ATCC 8739^a

Complex	Zone of inhibition
	B. subtilis			E. coli
Concentration mg L^−1	25	50	100	25	50	100
a — No zone of inhibition.
Rh6G⊂SCX4	1.4 ± 0.1	1.7 ± 0.1	1.8 ± 0.1	—	—	—
Rh6G⊂TSCX4	1.1 ± 0.1	1.3 ± 0.3	1.5 ± 0.1	—	—	—
Rh6G⊂SCX6	1.6 ± 0.2	1.8 ± 0.1	2.0 ± 0.1	—	—	—
Ampicillin (+ve control)		2.3 ± 1.0			1.4 ± 0.1

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The antiproliferative activity of complexes, Rh6G⊂SCX4, Rh6G⊂TSCX4 and Rh6G⊂SCX6 in 1 : 2 proportions assayed on HeLa (cervical carcinoma) cell line. The observed IC₅₀ values were 4.9 ± 0.39, 7.53 ± 0.7 and 6.63 ± 0.54 μg mL⁻¹ respectively (Fig. 13), significantly lower than positive control methotrexate (8.25 ± 0.36). The calixarenes derivatives with Rh6G showed efficient antiproliferative activity. Grare et al.⁴⁴ reported the absence of cytotoxicity of p-guanidinoethylcalix[4]arene against HaCaT (human keratinocytes) and MRC-5 (human pulmonary embryonic fibroblasts) cell line. Besides Paclet et al.⁴⁵ studied the cytotoxicity of p-sulfonatocalix[n]arenes towards human neutrophils cells and reaffirmed the SCXn (with n = 4, 6 or 8) not to be cytotoxic. It may as well be conjectured that higher association constant of Rh6G⊂SCX4 than Rh6G⊂TSCX4 reveals large antiproliferative activity which reflects in its lower IC₅₀ value. The reciprocal relation of association constant to IC₅₀ values have earlier been established in the literature.⁴⁶ To the best of our knowledge, use of the p-sulfonatocalix[n]arene complexes with rhodamine-6G as antiproliferative and antimicrobial agents has been reported.

Fig. 13 IC₅₀ values of (Rh6G⊂SCX4), (Rh6G⊂TSCX4) and (Rh6G⊂SCX6) on HeLa (cervical carcinoma) cell line.

Experimental

Material and methods

Rhodamine 6G (Rh6G), p-sulfonatocalix[4]arene (SCX4), p-sulfonatothiacalix[4]arene (TSCX4) and p-sulfonatocalix[6]arene (SCX6) (Scheme 1) were obtained commercially and used without further purification. All solutions were prepared in DMSO (Sigma Aldrich) AR grade.

¹H and 2D NMR were obtained on the 500 MHz Bruker spectrophotometer using DMSO-d₆. ¹H NMR titrations of (Rh6G⊂SCX4), (Rh6G⊂SCX6) and (Rh6G⊂TSCX4) were carried out by keeping the concentration of host and guest being 0.1 mM and added in equivalents viz. 1 : 1, 1 : 2, etc. For determination of stoichiometry, solutions were taken with the varying host to guest mole ratios keeping sum of their concentrations to be constant and the final volume being 400 μL. The 2D NMR spectra were measured for 1 : 1 mole proportions of the (Rh6G⊂SCX4), (Rh6G⊂TSCX4) and (Rh6G⊂SCX6). To determine the association constants ¹H NMR titrations were performed with solutions of constant concentration of host (SCX4 and TSCX4) with the concentration of Rh6G being varied from 0–30 mM. The association constant were obtained from global fitting method.⁴⁷

FT-IR spectra were measured on the TENSOR-37 equipped with ATR. The complexes were prepared with 1 : 1 mole ratio of (Rh6G⊂SCX4), (Rh6G⊂TSCX4) and (Rh6G⊂SCX6) diluted in methanol, stirred well and evaporated subsequently. Powder was collected mixed with KBr pellets and the FT-IR analysis was carried out in the 5000–500 cm⁻¹ region. Finally the powdered complexes were sonicated in acetonitrile for the FESEM analysis and analyzed on (FEI, NOVANANOSEM, 450).

Fluorescence spectral measurements were performed for Rh6G, SCX4, TSCX4, and SCX6 by JASCO spectrofluorometer (FP-8300) using 1 cm × 1 cm quartz cell at 25 °C temperature. The concentration of guest Rh6G is kept constant at 0.001 M and the concentration of the host is varied for SCX4 (0.0046 M to 0.092 M), SCX6 (0.0046 M to 0.092 M), and TSCX4 (0.0046 M to 0.099 M). The emission spectra of Rh6G were recorded in the absence and presence of SCX4, TSCX4, and SCX6. The measurements of quenching constants were derived from knowing the change in fluorescence intensity as a function of the host concentrations. Further fluorescence quantum yields accompanying the (Rh6G⊂SCX4), (Rh6G⊂TSCX4) and (Rh6G⊂SCX6) complexes are compared with the rhodamine B (reference) which showed fluorescence quantum yield of 0.5.³⁶ Fluorescence lifetime decays were collected by a time-correlated single photon counting (TCSPC) setup from IBH Horiba Jobin Yvon (U.S.) using a 530 nm diode laser (IBH, U.K., NanoLED-375 L, with a λ_max = 530 nm) with a FWHM of 89 ps as a sample excitation source.

Computational details

The individual SCX4 and TSCX4 hosts, rhodamine-6G and their complexes were optimized within the framework of density functional theory (DFT) based on the ωB97x functional^48,49 theory employing the GAUSSIAN-09 program.⁵⁰ The internally stored 6-31G(d,p) basis set⁵¹ was used. This level of theory is known to simulate adequately the effects of long range dispersion corrections empirically. Different conformers of the host guest complexes with (i) substituted phenyl ring encapsulated within SCX4 cavity and ester substituent protruding outside directing along the midway between two sulfonate functionalities (conformer A) and N-alkyl chain substituent on xanthene moiety penetrating inside the SCX4 cavity with the phenyl ring of the guest being excluded (conformer B), were considered. Binding energies were obtained by subtracting the sum of the individual host and guest SCF energies from that of the complex. To characterize charge distribution within the individual host, guest and their inclusion complexes net atomic charges were derived using the population analysis based on the Hirshfeld partitioned scheme. ¹H NMR chemical shifts (δ_H) were obtained by subtracting the nuclear magnetic shielding tensors of protons in hosts, Rh6G and their complexes from those of the tetramethylsilane (reference) using the gauge-independent atomic orbital (GIAO)⁵² method. The effect of solvent (DMSO) on the ¹H NMR chemical shifts was modeled through the self-consistent reaction field (SCRF)⁵³ calculations by incorporating the polarizable continuum model (PCM).⁵⁴

Antimicrobial and antiproliferative activities

The antibacterial activity of calixarenes complexes, (Rh6G⊂SCX4), (Rh6G⊂TSCX4) and (Rh6G⊂SCX6) at 1 : 1 ratio was assessed against Gram positive (Bacillus subtilis ATCC 6633) and Gram negative (Escherichia coli ATCC 8739) bacterial species. To obtain data on antibacterial activities the complexes (Rh6G⊂SCX4), (Rh6G⊂SCX6) and (Rh6G⊂TSCX4) in 1 : 1 mole ratio were considered. Since the Rh6G revealed prominent effect over SCXn 1 : 2 mole ratio was chosen for the measurements of antibacterial studies.⁵⁵ The activity was measured with the nutrient agar plate containing (g L⁻¹) peptone, 5; NaCl, 5; beef extract, 1.5; yeast extract, 1.5 and agar 25. The 24 h grown bacterial suspension (70 μL) was spread uniformly on nutrient agar plate. Different concentrations of Rh6G, (Rh6G⊂SCX4), (Rh6G⊂SCX6) and (Rh6G⊂TSCX4) were used for antibacterial activity against Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739, ampicillin (50 ppm) as positive control. Plates were kept at 10 °C for 30 min for uniform diffusion of compounds in agar; which were incubated subsequently at 37 °C for 24 h followed by measurements of zone of inhibition of calixarene complexes with respect to positive control, ampicillin.

To obtain data on antiproliferative activity of calixarenes complexes (Rh6G⊂SCX4), (Rh6G⊂SCX6) and (Rh6G⊂TSCX4) in 1 : 1 proportions were studied using the MTT assay on HeLa (human cervical carcinoma) cell lines. The MTT assay in vitro cell proliferation assay has widely been used for assessing proliferation activity of both synthetic derivatives and natural products. For this study, cells were maintained in minimum eagle medium (1×) containing 2 mM L-glutamine; 10 mM sodium pyruvate; non-essential amino acids (NEAA) and 1.5 g L⁻¹ sodium bicarbonate at 37 °C. The varying concentrations of calixarenes complexes with Rh6G (25, 50 and 100 ppm) were incubated with HeLa cell line (50 000 cells per well) for 24 h at 37 °C with 5% CO₂ in 96 well micro titer plates. After 24 h incubation, culture media was removed from the 96 well culture plates then 40 μg mL⁻¹ MTT solution in phosphate buffer saline was added per well and incubated for 4 h in CO₂ incubator. After incubation, the formazan crystals formed in wells which were solubilized in 100 μL of DMSO and kept for 5–10 min for complete dissolution of formazan. The absorbance measurements were carried out at 570 nm on multimode plate reader (MultiskanEnspire, Perkin Elmer).

Conclusions

A systematic analyse of binding of SCX4, TSCX4, SCX6 with Rh6G fluorescent dye have been carried out combining NMR, FTIR, SEM with density functional theory. The inclusion complex formed revealed 1 : 1 complexations with phenyl ring of Rh6G encapsulating within the host cavity while the ester substituent protruding outside the host cavity. It has been shown complexation is conduced through interactions between Ar-OH of SCXn and aromatic protons in addition to hydrogen bonding interaction of methylene of ester substituent with the –SO₃H functionalities of the host. Computer modelling studies predicted that the conformer with the side chain of Rh6G protruding outside is favoured for Rh6G⊂SCX4 and Rh6G⊂TSCX4 complexes which concur with the inference drawn from the NMR experiments. Fluorescence measurements further showed that the Rh6G⊂SCX6 complex exhibit larger quenching compared to the remaining complexes. ¹H NMR along with COSY and NOESY experiments predicted that host–guest binding is brought about via noncovalent interactions between Ar-OH of SCXn and aromatic protons of Rh6G. The NMR titration studies further confirmed 1 : 1 stoichiometry of the complexes. Remarkably, enough antibacterial activity against Gram positive bacteria suggested that these complexes should prove useful for the design of new drug molecules for specific bacterial infections. The association constants of the Rh6G⊂SCX4 > Rh6G⊂TSCX4 complexes correlate qualitatively with their antibacterial and antiproliferative activities. The present investigations should pave a way to design biocompatible complexes with effective antiproliferative and antimicrobial agents at the low concentrations.

Acknowledgements

SVP and DDM are grateful to University Grants Commission, New Delhi for the support in the form of a research project (F. No. 42-289/2013). SVA acknowledges the research fellowship from the Savitribai Phule Pune University disbursed through the university potential excellence scheme. SPG and SVA thank the National Param Supercomputing Facility at the Centre for Development of Advanced Computing (CDAC), Pune, India where the molecular modelling studies were carried out.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23614f

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