StructureâBinding Effects: Comparative Binding of ... - ACS Publications

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Structure−Binding Effects: Comparative Binding of 2‑Anilino-6-naphthalenesulfonate by a Series of Alkyl- and Hydroxyalkyl-Substituted β‑Cyclodextrins Audrey Favrelle,†,∥ Géraldine Gouhier,*,† Frédéric Guillen,†,⊥ Claudette Martin,† Nadine Mofaddel,† Samuel Petit,‡ Kara M. Mundy,§ Spencer P. Pitre,§,# and Brian D. Wagner*,§ †

Normandie Université, COBRA, UMR 6014, FR 3038, INSA Rouen, CNRS, IRCOF, 1 rue Tesnière 76821 Mont-Saint-Aignan, France ‡ Normandie Université, Crystal Genesis Unit, SMS, EA 3233, Université de Rouen, 76821 Mont Saint-Aignan Cedex, France § Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince Edward Island CIA 4P3, Canada S Supporting Information *

ABSTRACT: Cyclodextrins (CDs) are the most widely used organic hosts for the inclusion of guest molecules. CDs can be readily modified through substitutions of the hydroxyl groups, and these modified CDs can have different host binding properties compared to those of parent CDs. However, only relatively few systematic studies of the effects of chemical substitution on CD binding ability have been reported thus far. In this paper, we report the study of the binding properties of five different analytically pure modified β-cyclodextrin (β-CD) hosts (substituted with alkyl and/or hydroxyalkyl groups) with 2-anilino-6-naphthalenesulfonate (2,6-ANS) as guest. Binding constants for the formation of the inclusion complex between 2,6-ANS and each CD were determined using both fluorescence spectroscopy and capillary electrophoresis. Addition of modified CDs to an aqueous solution of 2,6-ANS resulted in significant enhancement of the fluorescence intensity of 2,6-ANS, as well as a significant spectral blue shift, indicative of inclusion. Inclusion of 2,6-ANS within the CD cavity was confirmed by NMR spectroscopy. Substitution at position 3 decreased the magnitude of the binding constants, while alkyl or hydroxylalkyl substitution of the primary hydroxyl at position 6 increased the magnitude of the binding constant in all cases, in relation with increasing length of the alkyl chain linker. In addition, binding constants decreased with solvent polarity when increasing amounts of methanol were added. Structure−binding correlations for CDs based on these binding constant results are presented and discussed.

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INTRODUCTION Cyclodextrins (CDs) are natural cyclic oligomers of glucopyranose with a truncated cone shape in aqueous solution and a large and well-defined cavity.1−3 The presence of this cavity allows for CDs to act as host molecules for the formation of water-soluble supramolecular host−guest complexes.4,5 Their attractive properties including aqueous solubility, commercial availability, low cost, and excellent guest binding ability have made CDs by far the most popular molecular host. Furthermore, the presence of 3 hydroxyl groups per glucopyranose monomer in positions 2, 3, and 6 makes possible a wide range of chemical modifications,6 that modify CD properties.2,6,7 We are interested in the effects of chemical modifications on CD host properties. We have previously used a number of commercially available modified CDs, in particular 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD), and have shown in general that the binding constants of modified CDs compared to the parent CD are significantly larger.5,8−12 However, such commercial CDs are mixtures with different substitution numbers and patterns, making correlation between substitution and host © XXXX American Chemical Society

properties open to criticism. In this work we have synthesized pure known substituted CDs 2 and 4 based on literature methods13−19 and developed innovative methods to reach the new CD hosts 5 and 6 (Table 1) in order to determine accurate correlations between the binding constants and the modified CD host structures (CD 3 was commercially available). We report herein the results of the binding studies between parent native β-CD 1 and five different chemically modified β-CDs hosts 2−6 and 2-anilino-6-naphthalenesulfonate (2,6ANS) 7 as guest, in buffered aqueous solution as well as in mixed water−methanol solutions. The nature of the substituents and the substitution patterns for modified CDs, as well as their abbreviations used in this paper, are given in Table 1. Binding constants for the formation of the inclusion complex between 2,6-ANS 7 and each CD 1−6 were determined using both fluorescence spectroscopy and capillary electrophoresis. Received: July 23, 2015 Revised: September 11, 2015

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DOI: 10.1021/acs.jpcb.5b07157 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

a series of tethered modified β-CDs, including host molecules incorporating two or three β-CD moieties.30 Most relevant for this present work, Liu et al. also reported structure−binding relationships for per-methylated-β-CD with two azobenzene guests, and showed unique binding regioselectivity for this modified CD as compared to the parent β-CD.31 In addition to these experimental studies, a number of authors have reported computational studies of quantitative structure−binding relationships in CDs.32−36 Some binding studies of guests by CDs in nonaqueous or mixed organic−aqueous solvents have been reported.37−45 Organic solvent systems studied include DMSO,37,38 DMF,39,40 mixed alcohol−water,41−44 and various other organic solvents.45 In all these studies, a significant decrease of the binding constant for a specific guest was observed in organic solvents as compared to aqueous solvent, or upon increase in organic solvent proportion in organic−aqueous solvent mixtures. This paper reports the first study of both the effect of different substituents and substitution patterns on the host binding abilities of modified CDs with 2,6-ANS guest (structure−binding relationships as a function of host structure for a single guest), and the effect of solvent polarity on the host binding properties. It is complementary to the earlier work of Bright et al.,20−23 which reported structure−binding relationships as a function of guest structure for a single host, β-CD.

Table 1. Abbreviations and Substituents of the Modified β-CDs 1−6 Used in This Work, and Structure of 2,6-ANS 7

CD

no.

R1

R2

R3

β-CD 2,3-DM-β-CD 2,6-DM-β-CD 2,3,6-TM-β-CD 6-HE-2,3-DM-β-CD 6-nHP-2,3-DM-β-CD

1 2 3 4 5 6

H CH3 CH3 CH3 CH3 CH3

H CH3 H CH3 CH3 CH3

H H CH3 CH3 CH2CH2OH CH2CH2CH2OH

The use of pure CD hosts with the same guest allows for the validation of our comparative study by correlating the measured binding constants to the substitution pattern of the host. The determination of host structure−binding constant relationships is required for the fundamental understanding of the host properties of CDs, and the CD inclusion process. This knowledge is essential for future rational design of modified CDs with specific target binding properties. There have been a number of previous studies, primarily fluorescence-based, about the inclusion complexation of ANS fluorescent probes in CD hosts. For example, Bright et al.20−23 have reported extensive studies of the inclusion of a series of anilinonaphthalenesulfonates in β-CD, including 2,6-ANS which is the guest of interest in this paper, and were able to draw a number of conclusions about the specific nature of the CD:1,8ANS inclusion complexes for the parent β-CD. For instance, they showed that the position of the sulfonate group relative to the aniline group was an essential factor in the magnitude of the resulting binding constant, with the strongest binding observed for the streamlined 2,6-ANS, and weaker binding observed when the two substituent groups were closer together on the naphthalene ring.20 They also showed that the CD binding site is heterogeneous, in that a range of guest penetration and geometry is possible.21,22 They also studied the effects of coincluded alcohols on the nature of the ANS-CD complexes, and showed that coinclusion of alcohol can stabilize the inclusion complexes.23 Schneider et al. studied ANS guests in α-, β-, and γ-CD, using both fluorescence and NMR, and reported that both the anilino and the naphthalene moieties show inclusion within the β-CD cavity.24 Recently, Sueishi et al. reported on the inclusion complexes of 1,8- and 2,6-ANS in parent (1) and modified (3,4) β-CDs as well as in cucurbit[7]uril, using both fluorescence and NMR.25 In addition to the above-described previous studies on ANS/CD inclusion, there have been a limited number of other experimental structure−binding studies of CDs reported previously; a few representative examples will be described hereafter.26−31 In many cases, the binding of differently shaped or substituted guests by different sized parent or modified CDs has been studied.26−29 For example, Xiang and Anderson26 measured the binding constants for the inclusion complexes between variously substituted purine guests and α-, β-, and HPβ-CD hosts. They correlated the magnitude of the binding constant and the geometry of the complex to the structural match between the guest and host. Other authors have further studied the effects of modification of the β-CD parent on its host properties. Liu et al. reported structure−binding relationships for

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EXPERIMENTAL SECTION Chemical Materials. Native β-cyclodextrin 1 was purchased from Wacker Chemicals (Germany), and CD 3 was obtained from Sigma-Aldrich. Other chemicals were purchased from SigmaAldrich, Acros (Belgium), and Fluka Analytical (Switzerland). All the solvents employed for the reactions were distilled once before use. Deuterated solvents were purchased from EurisoTop (France). 2-Anilino-6-naphthalenesulfonate (2,6-ANS) 7 was obtained from Molecular Probes, Inc., or Life Technologies (Invitrogen, Villebon sur Yvette, France). Potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), sodium hydroxide (+98% purity), and methanol (HPLC grade, >99%) were obtained from Sigma-Aldrich. Synthesis. Heptakis-(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose (2). Heptakis-(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose 2 was obtained in three steps from the native β-cyclodextrin 1 using the usual method as already described in the literature. Analytical data were identical to the literature.46 Heptakis-(2I−VII,3I−VII,6I−VII-tri-O-methyl)-cyclomaltoheptaose (4). Heptakis-(2I−VII,3I−VII,6I−VII-tri-O-methyl)-cyclomaltoheptaose 4 was synthesized and characterized as previously reported.47 Heptakis-(6-O-hydroxyethyl)-(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose (5, Scheme 1). The intermediate compound heptakis-(6-O-ethoxycarbonylmethyl)-(2I−VII,3I−VII-di-O-methyl)cyclomaltoheptaose 8 was obtained in one step from the heptakis(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose 2 using the usual method already described in the literature.13 Analytical data were identical to the literature. Heptakis-(6-O-hydroxyethyl)-(2I−VII,3I−VII-di-O-methyl)cyclomaltoheptaose 5 was synthesized as follows: lithium aluminum hydride (190.8 mg, 5.02 mmol, 35 equiv) and 8 (280 mg, 0.14 mmol, 1 equiv) were dissolved, respectively, in 20 and 55 mL of dry tetrahydrofuran under magnetic stirring and inert atmosphere. Lithium aluminum hydride solution was cooled to −30 °C, and the solution of 8 was added dropwise. The reaction was carried out for 17 h at room temperature under B


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3.70−3.40 (m, 21H, HI−VII3CD, HI−VII4CD, HI−VII5CD), 3.25−3.15 (dd, 7H, J = 3 and 9 Hz, HI−VII2CD). 13C NMR: (CDCl3, 75 MHz) δ (ppm) 134.9 (7C, Cb, CH), 116.7 (7C, Cc, CH2), 98.8 (7C, CI−VII1CD), 82.0 (7C, CI−VII2CD), 81.8 (7C, CI−VII3CD), 80.3 (7C, CI−VII4CD), 72.1 (7C, CH2CH, Ca), 70.9 (7C, CI−VII5CD), 69.0 (7C, CI−VII6CD), 61.5 (7C, O3CDCH3), 58.5 (7C, O2CDCH3). ESI-HRMS+: calcd for C77H126O35Na m/z 1633.7977, found 1633.7985. Analytical data were identical with that found in literature.13 Heptakis-(6-O-hydroxypropyl)-(2I−VII,3I−VII-di-O-methyl)cyclomaltoheptaose 6 was obtained as follows. Compound 9 (220 mg, 0.14 mmol, 1 equiv) was dissolved in 7 mL of dry tetrahydrofuran under magnetic stirring and inert atmosphere. Then borane−tetrahydrofuran complex (1 M) (2.3 mL, 2.3 mmol, 17 equiv) was added dropwise to the reaction medium at 0 °C. After 2 h of magnetic stirring at room temperature, sodium hydroxide (3 M, 7.2 mL, 21.5 mmol, 157 equiv) was added at 0 °C followed by the addition of hydrogen peroxide (7.2 mL, solution at 35%, 83.5 mmol, 610 equiv) and 3.3 mL of methanol. The reaction was carried out for 2 h at room temperature under magnetic stirring. In the end, the reaction mixture was poured into dichloromethane and washed with ammonium chloride (4 × 10 mL), and with brine (4 × 10 mL), and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated under vacuum and purified by column chromatography on a silica gel (CH2Cl2/MeOH 100:0 to 85:15). A 239 mg portion of compound 6 was isolated as a white powder (98% yield). C77H140O42 (MW = 1736 g mol−1); IR 3421 cm−1 (OH); Rf 0.50 (CH2Cl2/MeOH 9:1); mp 96 °C; [α]D +118° (20 °C, CHCl3, 0.312 g/dL). 1H NMR: (CDCl3, 300 MHz) δ (ppm) 5.08 (m, 7H, HI−VII1CD), 4.10−3.35 (m, 63H, Ha, Hc, HI−VII3CD, HI−VII4CD, HI−VII5CD, and HI−VII6CD), 3.63 (s, 21H, O3CDCH3), 3.51 (s, 21H, O2CDCH3), 3.20−3.10 (m, 7H, HI−VII2CD), 2.00− 1.70 (m, 14H, Hb). 13C NMR: (CDCl3, 75 MHz) δ (ppm) 99.8 (7C, CI−VII1CD), 82.1 (7C, CI−VII4CD), 81.4 (7C, CI−VII2CD), 81.0 (7C, CI−VII3CD), 71.1 (7C, CI−VII5CD), 69.6 (7C, CII−VII6CD), 68.5 (7C, Ca), 61.4 (7C, O3CDCH3), 59.6 (7C, Cc), 58.6 (7C, O2CDCH3), 32.0 (7C, Cb). ESI-HRMS+: calcd for C77H140O42Na m/z 1759.8717, found 1759.8744. Spectroscopic Analysis. The structure elucidation of the final products were confirmed by high-resolution mass spectrometry (HR-MS) using a lockspray electrospray (ESI) source performed in positive ion mode on a Synapt G2 HDMS (Waters, Manchester, U.K.). Products were dissolved in acetonitrile or methanol and infused into the electrospray ionization source. Accurate mass measurement was achieved using a protonated molecule of bombesine (m/z 1619.8229) as internal reference. The source and desolvation temperatures were kept at 90 and 250 °C, respectively. Nitrogen was used as the nebulizer and desolvation gas (flow rate of 500 L h−1, temperature of 250 °C). The capillary and cone voltages were 3 kV and 144 V, respectively. Scanning was performed in the range 50−2000 Da at a scan rate of 1 s/scan. Data acquisition and processing were performed with MassLynx 4.1 software. The purity of the final compounds were determined by LC/MS. A C18-bonded silica column (4.6 mm × 250 mm, 5 μm, Kromasil, Interchim) was used, and elution was performed at 1 mL/min using an isocratic eluent of acetonitrile/water 9:1. The effluent from the column was directed toward the ESI source of the Finnigan LCQ Advantage Max instrument. LC/ESI-MS data were recorded in the positive ion mode. The capillary temperature was kept at 220 °C, and nitrogen was used as a drying and nebulizing gas. The capillary and spray voltage were 41 V and 5.4 kV, respectively.

Scheme 1. Synthesis of Modified CDs 5 and 6

magnetic stirring and nitrogen atmosphere. The reaction mixture was poured into water (2 mL) at 0 °C to hydrolyze the excess of lithium aluminum hydride, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (CH2Cl2/MeOH 92:8 to 85:15). A 165 mg portion of compound 5 was isolated as a yellow oil (72% yield). C70H126O42 (MW = 1638 g mol−1); IR 3414 cm−1 (OH); Rf 0.41 (CH2Cl2/MeOH 9:1). 1H NMR: (CDCl3, 300 MHz) δ (ppm) 5.10 (m, 7H, HI−VII1CD), 3.61 (s, 21H, O3CDCH3), 3.48 (s, 21H, O2CDCH3), 3.95−3.35 (m, 63H, Ha, Hb, HI−VII3CD, HI−VII4CD, HI−VII5CD, and HI−VII6CD), 3.20−3.05 (m, 7H, HI−VII2CD). 13 C NMR: (CDCl3, 75 MHz) δ (ppm) 99.2 (7C, CI−VII1CD), 81.8 (7C, CI−VII4CD), 81.5 (7C, CI−VII2CD), 80.9 (7C, CI−VII3CD), 72.9 (7C, CI−VII5CD), 70.9 (7C, CII−VII6CD), 70.0 (7C, Ca), 61.4 (7C, O3CDCH3), 58.5 (7C, O2CDCH3), 53.4 (7C, Cb). ESI-HRMS+: calcd for C70H126O42Na m/z 1661.7621, found 1661.7651. Heptakis-(6-O-hydroxypropyl)-(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose (6, Scheme 1). The intermediate compound heptakis-(6-O-allyl)-(2I−VII,3I−VII-di-O-methyl)-cyclomaltoheptaose 9 was synthesized as follows: sodium hydride (2.59 g, 64.75 mmol, 21 equiv) dispersed in mineral oil (60%) was dissolved in 160 mL of anhydrous dimethylformamide under magnetic stirring and inert atmosphere. Then, 2 (4.19 g, 3.15 mmol, 1 equiv) was slowly added, and the mixture was stirred for 1 h at 0 °C and then for 3 h at room temperature. Allyl bromide (9.1 mL, 105 mmol, 34 equiv) was then added dropwise at 0 °C over 1 h, and the reaction was carried out for 72 h at room temperature under magnetic stirring and nitrogen atmosphere. At the end, methanol was added in order to hydrolyze the excess of sodium hydride, and the solvent was evaporated under reduced pressure. The residue was taken in ethyl acetate (150 mL), and the organic phase was washed with water (4 × 50 mL) and dried over magnesium sulfate. This intermediate 9 was obtained as a yellow powder (99% yield) and used for the next step without further purification. C77H126O35 (MW = 1610 g mol−1); IR 921 cm−1 (CHCH2); Rf 0.34 (CH2Cl2/MeOH 95:5). 1H NMR: (CDCl3, 300 MHz) δ (ppm) 5.96−5.83 (m, 7H, Hb), 5.30−5.05 (m, 21H, Hc and HI−VII1CD), 4.10−3.95 (m, 14H, Ha), 3.90−3.75 (m, 14H, HI−VII6CD), 3.64 (s, 21H, O3CDCH3), 3.50 (s, 21H, O2CDCH3), C


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The Journal of Physical Chemistry B Scanning was performed in the range 300−2000 Da. 1H NMR and 13C NMR spectra were recorded with a Bruker AVANCE DPX300 spectrometer at 300.13 and 75.47 MHz, respectively, in deuterated chloroform (CDCl3) or deuterated benzene (C6D6) at 20 °C. All compounds were characterized by 1H and 13 C spectroscopy as well as by 1H−1H (COSY) and 1H−13C (HMBC) correlation experiments. Chemical shifts are given in δ-units measured downfield from Me4Si at 0 ppm using the residual solvent signal as a secondary reference. Optical rotations ([α]D) were measured with a PerkinElmer 341 digital micropolarimeter, using a sodium lamp (λ = 589 nm) at 20 °C. Solutions were prepared by dissolution of 2−10 mg of product in 2 mL of solvent (HPLC quality) and placed in a vessel of 1 dm length. The concentrations were expressed in grams per 100 mL of solvent. Infrared spectra were recorded using a PerkinElmer Spectrum 100 FITR spectrometer. Melting points (mp) were determined on a Kofler type WME system (Heizbank-Wagner & Munz). Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 plates (Merck, Germany), and all compounds were visualized by UVabsorption (254 nm) and charring with ethanol−H2SO4 solution followed by heating. Solutions for fluorescence-based binding studies were prepared in K2HPO4−H2KPO4 aqueous buffer. Absorption spectra of 2,6-ANS and CD solutions were measured on a Varian Cary Bio-5 UV−vis absorption spectrophotometer. Fluorescence spectra were measured on a Photon Technologies International PTI RF-M2004 spectrometer using 325 nm excitation with monochromator band passes of 1 nm. Fluorescence titrations were performed by measuring and integrating the fluorescence spectrum in the absence (Fo) and presence (F) of various concentrations of CD; F/Fo was calculated as the fluorescence enhancement as a result of a specific concentration of added CD.5 Values of the binding constant K were extracted from the fluorescence titration plots of F/Fo versus [CD] using nonlinear leastsquares fitting to the following eq 1:5,48 F K[CD] = 1 + (Fmax /Fo − 1) Fo 1 + K[CD]

The capillary was conditioned for 10 min with the electrolyte before running and for 2 min between each run. The pH of the running solutions was measured before each experiment by using a Model IQ240 pH meter (IQ Scientific Instruments Inc., San Diego, CA). A Branson 2510 sonication apparatus (Branson, Danbury) was used for degassing all solutions. In CE mode, the sample contains a fixed amount of 2,6-ANS, and the running buffer contains various amounts of β-CD. The electrophoretic mobility of the injected analyte is dependent on the β-CD concentration. The binding constant could be estimated using several linear least-squares plotting methods (x-reciprocal, y-reciprocal, or double-reciprocal plot). The double-reciprocal plot is known as the Benesi−Hildebrand plot in spectrophotometry. For 1:1 association complex, the change in solute mobility with changing ligand concentration is related to the eq 2:49 1 1 1 = + μi − μf (μc − μf )K[CD] μc − μf

(2)

Here μi is the experimentally measured electrophoretic mobility of the solute, μf is the mobility of the free (uncomplexed) solute, μc is the electrophoretic mobility of the solute−ligand complex, K is the binding constant, and [CD] is the equilibrium ligand concentration. Molecular Modeling. Molecular modeling calculations on the predicted cyclodextrin-2,6-ANS inclusion complexes were performed using Sybyl-X software, Version 1.3, 2011 (Tripos, L.P.). The molecular structure of 2,6-ANS was derived from the crystal structure of the potassium salt of the para-methyl 2,6-ANS derivative (ref code KTNOSA10 in the CSD, Cambridge Structural Database). Structures of native and permethylated β-CDs were extracted from crystallographic data also available in the CSD or from our own X-ray structural investigations. The other modified β-CDs were built by suitable substitutions from these experimental structures. Atomic coordinates were smoothly optimized by using molecular modeling tools available in the Sybyl environment. The standard Tripos force field was selected with partial atomic charges calculated using the Gasteiger−Marsili algorithm. After geometry optimization of single components, hypothetical inclusion complexes were obtained by manual insertion of the guest inside the cavity according to the best host−guest relative orientation. Energy minimization of the resulting complex was then performed in two steps: first the components were treated as rigid bodies so as to improve the global inclusion geometry, and then the whole molecular geometry was optimized to confirm the reliability of the produced complex.

(1)

Capillary electrophoretic (CE) experiments were performed using a P/ACE 2100 capillary electrophoresis system (Beckman Coulter, Fullerton, CA). An uncoated fused silica capillary (Thermo Electron SA, Courtaboeuf, France), 27.5 cm long (20 cm to the detector), 50 μm i.d., was used. The capillaries were thermostated at 21.0 ± 0.1 °C. The samples were pressureinjected by 20 mbar for 6 s at the inlet side of the capillary (anode). Electrophoretic runs were performed with a 10 or 20 kV potential. The UV detection was cathodic (λ = 214 nm). The P/ACE System 2000 software (Version 2.64, Beckman Coulter) piloted the electrophoretic system and was used for controlling the data. All running electrolytes were prepared, without any prerequisite purification, using ultrapure water produced by means of a Milli-Q-System water purification apparatus (Millipore France, Montigny-le-Brotonneux, France). The solutions were sonicated just before use for 10 min. The running electrolytes contained phosphate salts (K2HPO4−H2KPO4 according to the target pH value 6.80) with an ionic strength equal to 30 mM, and CD concentrations ranging from 0 to 5 mM were used. Samples were prepared in phosphate solution at a concentration equal to 10−3 M. New uncoated capillaries were activated by performing the following washing process: water for 2 min, sodium hydroxide (1 M) for 30 min, and water for 10 min.

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RESULTS AND DISCUSSION Synthesis. In addition to the known methylated CDs 2−4, two new compounds 5 and 6 with, respectively, a hydroxylated chain of two and three carbon atoms linked to the seven primary alcohols have been synthesized (Scheme 1). Such functionalizations have been selected to increase the cavity size of the host while keeping the hydrophilic properties of the CD. CD 5 was obtained in 72% yield by reduction with lithium aluminum hydride of the known14 6-ethoxycarbonylmethyl-2,3dimethyl-CD 8. CD 6 has been synthesized from the known 6-allyl-2,3dimethyl-CD 9.50 Reduction of allylic functions has been only published on 2,3-per-allylated-CD derivatives by direct action of 9-BBN51 or in three steps by formations of diol followed by oxidative cleavage and reduction of the resulting aldehyde.52 D


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The Journal of Physical Chemistry B Direct hydroboration reaction under Ortega-Caballero’s conditions did not lead to the desired compound 6. After optimization, the use of simple borane tetrahydrofuran complex followed by oxidation with hydrogen peroxide in basic medium led to the host 6 in 98% yield after column chromatography. Effects of Modified β-CDs 2−6 on 2,6-ANS. Fluorescence and Extraction of Binding Constants. In all cases, addition of modified β-CDs 2−6 to an aqueous solution of 2,6-ANS resulted in significant enhancement of the measured fluorescence intensity. For example, Figure 1 shows the

Figure 2. Fluorescence titration of 2,6-ANS 7 with CD 6 in aqueous buffer. The curve shows the fit to eq 1 with K = 1570 M−1.

titration data for 7 upon addition of CD 6, and the fit of this data to eq 1. For this trial, this fit yielded a binding constant K of 1570 M−1 and a maximum enhancement Fmax/Fo (i.e., extrapolated to complete inclusion of all guests) of 55. The average values over the three trials are given in Table 2. Table 2. Maximum Fluorescence Enhancement and Binding Constants for 2,6-ANS 7 Inclusion into Various Modified β-CDs 1−6 in Aqueous Buffer CD β-CD 1

Figure 1. Fluorescence spectrum of 2,6-ANS 7 in the presence of various concentrations of CD 6 in aqueous buffer, ranging from 0 (bottom spectrum) to 2 mM (top spectrum).

2,3-DM-β-CD 2

fluorescence spectrum of 2,6-ANS upon addition of various concentrations of 6, clearly illustrating the very large increase in fluorescence that results upon addition of this CD. Upon addition of 2 mM of 6, the total fluorescence intensity (F, the integrated area of the fluorescence spectrum) increased by a factor of 42 as compared to the total fluorescence intensity in the absence of this CD (Fo); i.e., the measured fluorescence enhancement F/Fo is 42 in the presence of 2 mM 6. This extremely large enhancement is indicative of the formation of a host−guest inclusion complex, and is a result of the lower polarity environment experienced inside the CD cavity as compared to that of the aqueous solution.5 There is a significant spectral blue shift accompanying this enhancement: λF,max = 464 nm in the absence of CD and 422 nm in the presence of 6 at a concentration of 2 mM, a blue shift of 42 nm. Even in the case of the parent β-CD 1, a blue shift of 21 nm was observed. This observation is consistent with our previous studies of ANS probes,8,9 as well as those of Bright et al.,20−23 and provides evidence for the formation of an inclusion complex, in which the 2,6-ANS guest experiences a significantly less polar environment. By comparison, in the case of the inclusion of 2,6-ANS within the cavity of the host molecule cucurbit[7]uril previously reported by our group, the 1H NMR studies showed that the complexation involved only inclusion of the phenyl group, and smaller spectral shifts of less than 10 nm were observed.53 The large shifts on the order of 20−40 nm in the current studies suggest some degree of participation of the naphthyl ring in the inclusion process. The fluorescence titration results of F/Fo versus [CD] can be used to extract the binding constant K as well as the maximum fluorescence enhancement, Fmax/Fo (the fluorescence enhancement if all 2,6-ANS guests are included within the CD host, which will be higher than the F/Fo value discussed above for 2 mM CD), using the fit to eq 1. Figure 2 shows the fluorescence

2,6-DM-β-CD 3

2,3,6-TM-β-CD 4

6-HE-2,3-DMβ-CD 5

6-nHP-2,3-DMβ-CD 6

solvent

Fmax/Fob

K/M‑1b

K/M‑1c

Wa 25% M/W 35% M/W W 25% M/W 35% M/W W 25% M/W 35% M/W W 25% M/W 30% M/W 35% M/W W

20 ± 2 11.2 ± 0.3 7.1 ± 0.5 39 ± 2 9.6 ± 1.2 6.0 ± 1.7 32 ± 1 14.0 ± 0.3 8.3 ± 0.6 50 ± 5 12.0 ± 1.2 9.7 ± 0.7 6.2 ± 1.2 39 ± 2

2220 ± 430 530 ± 50 220 ± 15 430 ± 20 230 ± 40 140 ± 50 8550 ± 360 2410 ± 210 1020 ± 80 940 ± 80 480 ± 60 320 ± 40 230 ± 50 930 ± 60

1820 ± 70 440 ± 20 69 ± 10 120 ± 20 140 ± 70 110 ± 50 7300 ± 230 1560 ± 160 480 ± 20 900 ± 7 480 ± 40 N/A 69 ± 3 915 ± 25

25% M/W 35% M/W W

15 ± 2 N/A 53 ± 2

370 ± 50 N/A 1690 ± 110

140 ± 30 96 ± 21 1130 ± 110

25% M/W 35% M/W 45% M/W

17 ± 3 9.4 ± 1.9 N/A

710 ± 150 400 ± 100 N/A

460 ± 150 210 ± 20 65 ± 4

a W = water, M = methanol. bDetermined using fluorescence spectroscopy. The data reported are an average and standard deviation of four trial runs. cDetermined using capillary electrophoresis.

The 1:1 stoichiometry of the complexes assumed with the use of eq 1 can be verified by plotting the double reciprocal plot of 1/(F/Fo − 1) versus 1/[CD]; this plot will be linear in the case of 1:1 complexation, but will be curved if higher order complexes occur. Figure 3 shows the double reciprocal plot in the case of 6, demonstrating the highly linear plot, with R2 = 0.9992, confirming 1:1 complexation for this CD; similar plots and linearity were obtained for all six CDs 1−6 used. The fluorescence titration results for each of the five modified β-CDs 2−6 as well as β-CD 1 itself are tabulated in Table 2, as averages of four independent trials for each CD for the binding constant K as well as the maximum enhancement Fmax/Fo. E


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Capillary Electrophoresis Studies of the Inclusion Complexation and Extraction of Binding Constants. The binding constants for the inclusion complexation of 2,6-ANS by β-CDs 1−6 were also determined using capillary electrophoresis, through the fit of the data to eq 2, and are also listed in Table 2. The same overall trends in the binding constant values observed from the fluorescence titration results are also observed in these capillary electrophoresis-based results: substitution at both the 2 and 3 positions reduces the binding constant (even with substitution at the 6 position), but substitution at just the 2 and 6 positions increases the binding constant. Again, 3 is the only modified CD that shows a binding constant larger than that of the parent β-CD: K = 7300 M−1 for 3 compared to 1820 M−1 for 1. Examination of the binding constants K in Table 2 shows a systematic difference between the values obtained by the two experimental techniques: the fluorescence-based measurements are consistently larger than those obtained using capillary electrophoresis, with an increase ranging from just 4% in the case of 4 (K values are actually the same within experimental error) to a difference of 72% in the case of 2. In addition, the difference between the K values for the two methods is generally more pronounced in the mixed aqueous−methanol solvents. One reason for this difference is that the fluorescence experiments directly probe the S1 excited state of the guest (the fluorescence occurs from this excited state). If the excited state lifetime is long relative to the exit and entrance rate of the guest into and out of the cavity, the fluorescence results will yield the binding constant of the guest in its excited state, rather than that in its ground state.54 By contrast, the electrophoresis results are definitely determining the binding of the ground state of 2,6-ANS. We are not able to measure the dynamics of the inclusion of 2,6-ANS into these modified β-CDs 2−6 in order to determine these entrance and exit rates to compare to the fluorescence lifetime. However, the larger value of K obtained from the fluorescence experiments in the case of most of the CDs suggests that there is indeed some involvement of the excited state in the binding that is being measured.54 We postulate that the observed increase in binding constant for 3 using fluorescence as compared to CE is a result of stronger hydrogen bonding between the more polar excited fluorescent state and a secondary hydroxyl group of the CD (see below) as compared to that in the case of the ground state studied in CE. It should be noted that this is the first report which provides a systematic comparison of the determination of binding constants using fluorescence versus capillary electrophoresis techniques. There was a previous report on the utility of these two techniques (plus UV absorption and liquid chromatography) for the determination of cyclodextrin binding constants,55 but it did not report binding constants measured under the same experimental conditions for comparison. NMR Studies of the Inclusion of 2,6-ANS 7 into the Modified β-CD Cavity. The dipolar interactions of 2,6-ANS with the modified β-CD were characterized by 1D 1H NMR. In fact, NMR is the experimental technique that provides the most direct evidence for the inclusion of a guest into the hydrophobic β-CD cavity in solution.56 Inclusion of 2,6-ANS into the modified β-CD cavity is evidenced by the change in chemical shifts of some of the guest and host protons, in comparison with the chemical shifts of the same protons in the free components. These changes in chemical shifts upon inclusion are shown in Figure 4 as stacked spectra for each of the modified CDs studies, and the positions of the peaks for 2,6-ANS in the absence and presence of the 6 CDs are listed in Table 3. It is clear from Figure 4 that the observed chemical shift variations upon mixing of the host and guest are

Figure 3. Double reciprocal plot for the fluorescence titration of 2,6-ANS 7 with CD 6 shown in Figure 2; the solid line is the linear fit (R2 = 0.9992).

As can be seen, substitution of β-CD by alkyl or hydroxyalkyl groups has a significant impact on the binding constant for inclusion of 2,6-ANS. Somewhat surprisingly, the binding constant decreased for all of the substituted β-CDs with the exception of 3, which is the only modified CD to remain unsubstituted at the secondary hydroxyl position 3. The specific effects of substitution on the magnitude of the binding constant will be discussed below. The magnitude of the maximum fluorescence enhancement listed in Table 2 is indicative of the relative polarity of the cavity of each of these modified CDs. Unlike the case of the magnitude of the binding constant K, the maximum fluorescence enhancement was found to increase for all the modified β-CDs 2−6 as compared to β-CD itself. In general, the hosts which are modified at all three positions 2, 3, and 6 (CDs 4−6) showed the largest enhancements, indicating the lowest polarity cavities, with CDs 4 and 6 giving maximum enhancements of the 2,6-ANS fluorescence on the order of 50-fold. The modified β-CD which showed the lowest enhancement of 2,6-ANS fluorescence was CD 3 (Fmax/Fo = 32), which indicates that its cavity is not as low in polarity as that of the other CDs, although it still showed an enhancement significantly higher than with the parent β-CD (Fmax/Fo = 20). From these results, it is clear that full alkyl or hydroxyalkyl substitution yields the lowest polarity CD cavities, and that substitution at the secondary 2 and 3 positions (upper CD cavity rim) leads to a greater decrease in cavity polarity than does substitution at the primary position 6 (2 has a significantly lower polarity than does 3, and CDs 2 and 5 show similar enhancement). In addition, longer substituents at the 6 position lead to lower CD cavity polarity (HP- vs HE-substitution). It is not clear however why CD 5 showed a lower enhancement of 2,6-ANS fluorescence than did CD 4, in spite of having a longer substituent at the 6 position; this may be due to the nature of the hydroxyl groups on this substituent as compared to the methyl substituents in CD 4. This effect of alkyl and hydroxylalkyl substitution on the effective cavity polarity is postulated to be a result of both the physical extension of the cavity by these groups, and the increased nonpolar nature of the cavity by the complete removal of the polar hydroxyl groups (in the case of alkyl substitution) or the separation of these hydroxyl groups away from the cavity rims (in the case of hydroxylalkyl substitution). It is interesting to note, however, that the modified CD with the highest enhancement (lowest polarity cavity), 6, did not give the strongest binding constant. This indicates that the hydrophobic effect is not the only driving force for inclusion in this host−guest inclusion system; this will be further discussed below. F


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but with a minor degree of inclusion of the naphthyl group, consistent with the fluorescence results. The magnitude of the binding constant, and the presence of at least some degree of naphthyl binding, is related to the cavity volume of the individual CDs, and to the ability of the various CDs to form hydrogen bonds. Indeed binding constant values K observed are consistently lower for 3-methylated CDs than for 3-unsubstituted CDs (e.g., 4 vs 3), a trend that is consistent with a different binding mode for the 3-OMe and 3-OH cyclodextrins. Modeling results confirmed the deeper inclusion of 2,6-ANS inside CD 3 due to its larger cavity compared to those of CD 2 and 4. The change in CD cavity size and shape with differing chemical modification is shown in Figure 5. Clearly CD 3 has the largest available cavity volume.

Figure 4. Stacked 1H NMR spectra of the guest 2,6-ANS in the absence and presence of the 6 CD hosts of interest.

significant relative to the bandwidths of the peaks and the uncertainty of the measurements, and that inversion in the order of the signals occurs. The absence of new peaks that can be assigned to the complex suggests that complexation is a dynamic process, with the included 2,6-ANS being in a fast exchange between the free and bound states. This result provides support for the involvement of the excited state in the binding process proposed above. Several trends in the modification of the 1H chemical shifts of 2,6-ANS upon complexation with the different CDs can be observed (Table 3). First, the chemical shifts of the phenyl ring protons are shifted consistently farther downfield upon complexation with 3-methylated CDs 2 and 4−6 (entries 3 and 5−7) than upon complexation with 3-OH CDs 1 and 3 (entries 2 and 4). Moreover, the maximum downfield shift in each complex is observed for the ortho proton with 3-OH CDs, and for the meta protons with 3-methylated CD. This indicates that the phenyl ring is probably included in the 3-methylated CDs’ cavities, whereas it may be less included in 3-unsubstituted CDs’ cavities. Weaker shift variations of the naphthyl protons of 2,6-ANS were observed (Table 3), providing evidence of some degree of inclusion of the naphthyl moiety of the guest into the CD cavity, at least in the case of CDs 2 and 3. Thus, the 1H NMR spectral modification provides direct evidence of the presence of an inclusion complex and therefore inclusion of 2,6-ANS into the CD cavity; this occurs mainly via the phenyl group of the guest (with the exception of CD 3), with only minor involvement of the naphthyl group. Therefore, the significant fluorescence spectral blue shifts indicated some involvement of the naphthyl ring, while the NMR results show that this inclusion process primarily involves the insertion of the phenyl group within the CD cavity,

Figure 5. Calculated structure of the cavity size and shape for CDs 2 (left), 3 (center), and 4 (right).

In addition, the formation of a weak hydrogen bond with the SO3− group of 2,6-ANS with a free hydroxyl group at the C3 position is also predicted by our calculations, as shown in Figure 6 for inclusion into CD 3, with a hydrogen bond distance

Figure 6. Hypothetical structure of the inclusion complex formed between 2,6-ANS 7 and CD 3. The predicted hydrogen bond is indicated by a dashed green line.

of 2.2 Å (H···N) or 2.5 Å (H···O). This figure shows the predicted inclusion of the naphthalene moiety within the CD cavity, in agreement with the NMR results described above. The calculations show a likely geometry of the CD 3:2,6-ANS, with the naphthalene included and the aniline group near the cavity opening, but still interacting with the CD. It is useful to compare the NMR results obtained in our work with those reported by Sueishi et al.25 for the inclusion of

Table 3. 2,6-ANS 7 (See Table 1 for H Labeling) 1H Chemical Shift Variation in the Presence of CD 1−6 entry 1 2 3 4 5 6 7

CD

3

4

5

7

8

o

m

p

1 2 3 4 5 6

7.23 0.09 −0.12 0.00 0.01 −0.06 −0.02

7.80 −0.02 0.15 −0.02 0.04 0.00 0.09

8.17 −0.05 −0.08 −0.13 −0.07 −0.09 −0.10

7.69 0.07 −0.11 0.00 0.02 −0.04 −0.02

7.64 −0.02 −0.02 −0.11 0.01 −0.07 0.00

7.23 −0.21 −0.29 −0.14 −0.16 −0.18 −0.21

7.37 −0.02 −0.55 −0.08 −0.27 −0.27 −0.35

7.07 0.06 −0.41 −0.08 −0.25 −0.24 −0.25

G


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Figure 7. Calculated structure of CDs 4 (left), 5 (center), and 6 (right).

2,6-ANS into CDs 3 and 4. Using fluorescence, they reported K = 8150 M−1 (3) and 1080 M−1 (4), which compare extremely well to our values of K = 8550 M−1 (3) and 940 M−1 (4) obtained via fluorescence. In terms of NMR results, they conclude that the anilino group is included within the CD cavity, whereas our work shows binding of the naphthalene group in the case of 3. We postulate that this is a result of the interaction between the sulfonate anion and one of the unsubstituted hydroxyl on the cavity rim for 3. Structure−Binding Relationships for the Inclusion Complexes of 2,6-ANS in Modified CDs. A number of interesting trends can be identified by comparing the binding constants for β-CDs 1−6 in aqueous buffer solutions (see Table 2). For convenience, the binding constants obtained from the fluorescence measurements will be used in the following comparisons, but the trends described are the same in the set of electrophoresis-derived binding constants. Alkyl substitution at both secondary hydroxyl positions 2 and 3 results in a decrease in the binding constant: 430 M−1 for 2 compared to 2220 M−1 for unsubstituted β-CD 1, and also 940 M−1 for 4 (per-Me) compared to 8550 M−1 for 3 (2,6-Me). Substitution at just one of these positions, namely, position 2 in the case of 3, leads to significantly increased binding. In fact, CD 3 is the only one of the five substituted β-CDs that shows a higher bonding to 2,6-ANS than does β-CD itself, and is the only one which has an unsubstituted secondary hydroxyl group (position C3). We propose that there is significant hydrogen bonding occurring between the 2,6-ANS guest and one secondary OH group on the β-CD, which is prevented by methylation of both the 2 and 3 positions. 1H NMR results indicate that the nonmethylated 3 position (3) is the only case in which strong phenyl binding is not indicated (insignificant chemical shifting of the phenyl protons), proving that inclusion occurs preferentially via the naphthyl, rather than the phenyl ring, consistent with hydrogen bonding occurring with the sulfonate group of 2,6-ANS in this case only. The modeling studies show the steric influence of the presence of the carbon chains grafted on the secondary face of the cyclodextrin; this is illustrated in Figure 7. The cavity size increases from CD 4 to 6, respectively, modifying the shape and the polarity of the host cavity. Substitution of the primary hydroxyl at position 6 resulted in an increased binding constant: 940 M−1 for 4 compared to 430 M−1 for 2. Furthermore, substitution at position 6 by a hydroxypropyl group resulted in a further increase in binding constant: 1690 M−1 for 6 compared to 940 M−1 for 4, while the binding constant for 5 is similar to that for 4 (hydroxyethyl vs methyl). Thus, a longer hydroxyalkyl group at the 6 position results in stronger binding due to the increased size of the CD cavity and to an overall decrease in cavity polarity by removal (in the case of alkyl substitution) or increased distance to the cavity (in the case of hydroxyalkyl substitution) of hydroxyl groups on the cavity rims.

As was discussed above, the cavity polarity (as indicated by the maximum fluorescence enhancement) was found to decrease with all substitutions. Thus, if the binding of 2,6-ANS was purely a result of the hydrophobic effect based on the difference in polarity of the cavity and the bulk solvent, then the binding would follow the same trend, and would be larger than the parent β-CD for all the modified CDs. The fact that this was not the case indicates that the cavity polarity is indeed not the only driving force for inclusion, and supports our proposal that hydrogen bonding between the 2,6-ANS and the secondary 3 position hydroxyl group is important in this specific inclusion process. Effect of Solvent Polarity on the Binding of 2,6-ANS 7 to Modified CDs. The results in Table 2 also clearly show the effect of solvent polarity on 2,6-ANS binding, since the binding constant decreases with decreasing polarity of the solvent mixture, using either technique. For example, in the case of CD 3 (which showed by far the strongest binding of 2,6-ANS), the binding constant decreased from 8550 M−1 in aqueous buffer to 1020 M−1 in 35% methanol/water. This trend was consistent for all six CDs studied. This decrease in binding as the solvent polarity decreases is attributed to a lower hydrophobic driving force, due to the increased similarity between the polarity inside the cavity and that of the bulk solvent. Linear regression analysis was performed on the data of K versus % methanol for each CD, and excellent linearity was obtained. For example, for CD 3, correlation coefficients R2 of 0.9993 (fluorescence data) and 0.9998 (CE data) were obtained. Similar results have been reported previously for other guests in CDs. For example, Gasull et al. determined the binding constant for the drugs methyl and ethyl salicylate in β-CD in mixed water−methanol solvents.44 They determined a low value of K for methyl salicylate in 55% MeOH:H2O of 16 M−1, but this value dropped to 7 M−1 in 70% MeOH:H2O. They reported a linear relationship between the binding constant and the dielectric constant of the solvent, and attributed this to the prevalence of the hydrophobic effect as a driving force for inclusion for this host− guest pair.

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CONCLUSION In this work, the binding constants for the inclusion of 2,6-ANS in β-CD and five alkyl- or hydroxyalkyl-substituted β-CDs were determined using both fluorescence spectroscopy and capillary electrophoresis. Good general agreement was obtained between the two methods, with the same trends observed upon substitution. The binding constants from the fluorescence studies were larger than those from capillary electrophoresis; this was attributed to the fluorescence studies probing the binding of the excited state of 2,6-ANS while capillary electrophoresis probes the binding of the ground state. A number of important structure−binding relationships were determined for these modified CDs. First, alkyl substitution at both secondary positions 2 and 3 was found to result in a decrease of the binding constant, whereas substitution at position 2 only resulted in an increase H



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in the binding constant. This was proposed to be the result of hydrogen bonding between the 2,6-ANS and the secondary hydroxyl of the CD host. Substitution of the primary hydroxyl at position 6 resulted in an increased binding constant in all cases, with increased binding obtained with increasing length of the substituent. This is suggested to be a result of the decreased polarity of the modified CD cavity upon alkyl and hydroxyalkyl substitution at this primary position. The fluorescence studies showed that higher substitution led to an increase of fluorescence enhancement, and thus lower cavity polarity; this is true for substitution at all positions. However, the magnitude of the binding constant did not always increase with increasing substitution, if both secondary positions 2 and 3 were substituted, again indicating the important role of hydrogen bonding in addition to hydrophobic effects in the stabilization of these complexes. Moreover, binding constants were lower when solvent polarity decreased by the addition of methanol. These structure−binding relationships were determined in the rational design of modified cyclodextrins for specific applications, and should allow the tuning of the binding constant of a specific guest with a modified β-CD. This will be potentially useful in a variety of CD inclusion applications, such as in supramolecular chromatography,57 fluorescence-based trace analysis,58 and drug delivery.59

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b07157. More detailed structural analysis and electrophoresis details (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Fax: 33-235522959. Phone: 33-235522909. E-mail: geraldine. [email protected]. *Fax: 1 902 566 0632. Phone: 1 902 628 4351. E-mail: [email protected]. Present Addresses ∥

Unity of Catalysis and Solid State Chemistry, UMR CNRS 8181, University of Lille 1 Science and Technology, Bat C7, Cité Scientifique, 59652 Villeneuve d’Ascq Cedex, France. ⊥ CNRS-UMR 5068, SPCMIB, University Paul Sabatier, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France. # Centre for Catalysis Research and Innovation and Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the University of Prince Edward Island and the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support, and the Region Haute-Normandie for financial support of the postdoctoral position for AF. I


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