Dynamic Control of the Self-Assembling Properties of Cyclodextrins by the Interplay of Aromatic and Host-Guest Interactions

ORIGINAL RESEARCH published: 25 February 2019 doi: 10.3389/fchem.2019.00072 Frontiers in Chemistry | www.frontiersin.org 1February 2019 | Volume 7 | Article 72 Edited by: Leyong Wang, Nanjing University, China Reviewed by: Cheng Yang, Sichuan University, China Lihua Yuan, Sichuan University, China Dong-Sheng Guo, Nankai University, China *Correspondence: Carmen Ortiz Mellet [email protected] Francisco Mendicuti [email protected] José M. García Fernández [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Supramolecular Chemistry, a section of the journal Frontiers in Chemistry Received: 03 November 2018 Accepted: 28 January 2019 Published: 25 February 2019 Citation: Neva T, Carmona T, Benito JM, Przybylski C, Ortiz Mellet C, Mendicuti F and García Fernández JM (2019) Dynamic Control of the Self-Assembling Properties of Cyclodextrins by the Interplay of Aromatic and Host-Guest Interactions. Front. Chem. 7:72. doi: 10.3389/fchem.2019.00072 Dynamic Control of the Self-Assembling Properties of Cyclodextrins by the Interplay of Aromatic and Host-Guest Interactions Tania Neva1†, Thais Carmona2†, Juan M. Benito1, Cédric Przybylski 3, Carmen Ortiz Mellet4*, Francisco Mendicuti2*and José M. García Fernández1* 1Instituto de Investigaciones Químicas (IIQ), CSIC - University of Sevilla, Sevilla, Spain, 2Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Chemistry, University of Alcalá, Alcalá de Henares, Madrid, Spain, 3Sorbonne Université, CNRS, Institut Parisien de Chimie Moléculaire, IPCM, Paris, France, 4Department of Organic Chemistry, Faculty of Chemistry, University of Seville, Seville, Spain The presence of a doubly-linked naphthylene clip at the O-2Iand O-3II positions in the secondary ring of β-cyclodextrin (βCD) derivatives promoted their self-assembly into head-to-head supramolecular dimers in which the aromatic modules act either as cavity extension walls (if the naphthalene moiety is 1,8-disubstituted) or as folding screens that separate the individual βCD units (if 2,3-disubstituted). Dimer architecture is governed by the conformational properties of the monomer constituents, as determined by NMR, fluorescence, circular dichroism, and computational techniques. In a second supramolecular organization level, the topology of the assembly directs host-guest interactions and, reciprocally, guest inclusion impacts the stability of the supramolecular edifice. Thus, inclusion of adamantane carboxylate, a well-known βCD cavity-fitting guest, was found to either preserve the dimeric arrangement, leading to multicomponent species, or elicit dimer disruption. The ensemble of results highlights the potential of the approach to program self-organization and external stimuli responsiveness of CD devices in a controlled manner while keeping full diastereomeric purity. Keywords: cyclodextrins, self-assembly, supramolecular chemistry, naphthalene, host-guest chemistry, fluorescence, circular dichroism INTRODUCTION The incorporation of functional moieties into pre-existing cage molecules and macrocycles represents a powerful strategy to tailor their architectural, inclusion, self-assembling, stimulus responsiveness and biorecognition properties (Sansone and Casnati, 2013; Shetty et al., 2015; Gropp et al., 2017; Liu et al., 2017; Matsuoka and Nabeshima, 2018). A range of sophisticated molecular machines with programmed supramolecular behaviors have been designed by implementing this strategy, with applications in the fields of sensors, electronic or optical devices, natural ligand mimics, artificial catalysts or (bio) molecular carriers, among others (Jiménez Blanco et al., 2017; Webber and Langer, 2017; Yin et al., 2017; Guo et al., 2018; van Dijk et al., 2018; Yu et al., 2018; Zeng et al., 2018). The natural cyclooligosaccharides of the cyclodextrin family are paradigmatic Neva et al. Self-Assembling Cyclodextrins examples in this context (Crini, 2014). The circular arrangement of α-(1→4)-linked D-glucopyranosyl units in the commercially available hexa-, hepta-, and octameric representatives (α,β, and γCD, respectively) draws a hollow truncated-cone frame with a hydrophobic interior that can host complementary guests in a size-conditional basis (Simões et al., 2015; Ryzhakov et al., 2016; Álvarez-Lorenzo et al., 2017; García-Moreno et al., 2017; Carreño et al., 2018). The volume constraint can be overcome by the incorporation of shaping and recognition modules leading to cavity extension, cooperativity effects or self-assembly, allowing several levels of organization to be implemented. The design of non-viral gene delivery systems (Ortiz Mellet et al., 2011; Gallego-Yerga et al., 2015; Evenou et al., 2018; Hong et al., 2018), antitoxins (Díaz-Moscoso et al., 2011; Joshi et al., 2011), nanocontainers (Gallego-Yerga et al., 2014b, 2017; Varan et al., 2017; Engel et al., 2018) and actuators (Smiljanic et al., 2006; Tan et al., 2014; Yang et al., 2014) highlights the potential of this tactic. Appending aromatic walls at the CD rims has proven particularly beneficial to promote self-association and reinforce interactions with third species in a predictable manner (Benkovics et al., 2017; Zhu et al., 2017; Zhang et al., 2018), eventually enabling additional non-inclusion or interfacial complexing modes (McNally et al., 2009; de Jesus et al., 2012). Most reported examples of CD-aromatic conjugates focus on single position-linked derivatives that keep considerable mobility, which limits accurate three-dimensional definition, however (Gamieldien et al., 2010). Covalently bridging CD building blocks with aromatic tethers has been advantageously exploited for preorganization purposes in some cases (Carmona et al., 2015; Sun et al., 2015; Gallego-Yerga et al., 2018). Alternatively, prototypes featuring a two-point connected aromatic component at either the primary (Yang et al., 2008; Le Gac et al., 2016;Ménand et al., 2016, 2018; Yan et al., 2017) or the secondary rim (Balbuena et al., 2007, 2013) in a monomeric CD derivative have been reported, allowing unprecedented control over the topological and supramolecular attributes. The later architecture is particularly appealing: an aromatic component attached to the wider entrance of the inner cavity can act as gatekeeper, guest selector and/or aggregation promoter, the impact in the recognition abilities depending on the conformational bias (González-Álvarez et al., 2011, 2013). Thus, “hinge-type” o-xylylene segments connecting the vicinal secondary O-2 and O-3 positions in the same glucopyranosyl unit (e.g., 1) adopt a cap-like orientation that hinders inclusion and elicits instead the formation of supramolecular head-to-head (HH) dimers in which the individual constituents are closely packed (González-Álvarez et al., 2008; Mayordomo et al., 2013; Gallego-Yerga et al., 2014a). Differently, “clip-type” positional isomers in which the o-xylylene group intertwines the O-2Iand O-3II positions in consecutive monosaccharide units (e.g., 2) preferentially take semi-open arrangements due to the reduced flexibility at the cyclic benzylic area. As a result, dimeric species with higher interfacial distances that readily dissociate in the presence of a suitable CD cavity-fitting guest are formed, which can be exploited for the spatiotemporal control of the recognition properties (Neva et al., 2018). Interestingly, the supramolecular abilities were drastically influenced by the type of joint between the aromatic and the cyclooligosaccaride parts: replacing the oxylylene into a m-xylylene moiety in the clip-type geometry (e.g., 3) led to a fully open topology that abolished self-recognition and favored instead non-inclusion complexation of planar guests (Figure 1). It was anticipated that non-covalent interactions in this family of CD conjugates could be further tuned by modifying the nature of the aromatic clip, providing a flexible scheme to dynamically mold different self-assembling and host-guest fitting modes. The incorporation of naphthalene modules is particularly appealing at this respect: several relative orientations between the cyclooligosaccharide macroring and the aromatic surface can be preselected as a function of the naphthalene substitution pattern without altering the structure and inclusion capabilities of the CD cavity. We conceived that this concept could be implemented in the design of monomeric prototypes with the ability to form supramolecular dimers in which the CD cavities can be either interconnected, forming a single compartment, or separated by the aromatic walls. To probe this hypothesis, the new 1,8-disubstituted or 2,3-disubstituted naphthylene-equipped βCD isomers 4and 5(Figure 2) have been synthesized and their conformational, self-assembling and inclusion properties have been monitored by fluorescence spectroscopy, circular dichroism, NMR, MS, and computational techniques. The results underline the potential of strategies based on precise synthesis to drive aggregation of macromolecular entities and program their responsiveness to molecular recognition events, a critical aspect in the construction of functional devices. EXPERIMENTAL AND COMPUTATIONAL METHODS Synthesis and Compound Characterization Reagents and solvents were purchased from commercial sources and used without further purification. 1H (13C) NMR spectra were recorded at 600, 500 and 400 (150.8, 125.7, and 100.6) MHz. 2D COSY, 1D and 2D TOCSY, NOESY, ROESY, HSQC, and HMBC experiments were used to assist assignments. Thinlayer chromatography (TLC) was carried out on aluminum sheets coated with Kieselgel 60 F245 (E. Merck), with visualization by UV light and by charring with 10% H2SO4. Flash column chromatography was carried out on silica gel (230–400 mesh). Elemental analyses were performed at the Institute for Chemical Research (Sevilla, Spain). The target cyclic ether conjugates 4 and 5were readily obtained by alkylation of the diol precursor 7, obtained from 6by regioselective bis-demethylation with diisobutylaluminiun hydride (DIBAL·H) (Roizel et al., 2002; Xiao et al., 2010), with ether 1,8- or 2,3-bis(bromomethyl)naphthalene (Kemp et al., 1980) (Figure 3; see the Supplementary Data and Supplementary Figures S1–S16 for experimental details and full characterization data). Mass Spectrometry The electrospray ionization-high resolution mass spectrometry (ESIHRMS) experiments were performed using a LTQ-Orbitrap XL ETD instrument from Thermo Scientific operated in positive ionization mode with a spray voltage at +3.6 kV. Samples were prepared at 20 µg·mL−1in methanol/water (20:80 v/v). Once Frontiers in Chemistry | www.frontiersin.org 2February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins FIGURE 1 | Structures of the doubly-linked isomeric βCD-xylylene conjugates 1–3 (Left) and 3D molecular models of the corresponding minima binding energy dimer (for 1and 2) or monomer species (for 3) present in water solution (Right). Note that the aromatic ring (in green) orientation, relative to the CD cavity, shifts from cap-like to semi-open to fully open on moving from 1to 3(González-Álvarez et al., 2013; Neva et al., 2018). prepared, the fresh solutions were continuously infused at 3 µL·min−1using a 250-µL syringe. The applied voltages were 45 and 120 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 275◦C. The resolution level was set to 30,000 (m/z=400) for all studies, while the m/zrange was set to m/z200–2,000 in the profile mode and in the normal mass ranges. The spectra were analyzed using XCalibur 2.0.7 acquisition software (Thermo Scientific) without smoothing and background subtraction. The automatic gain control (AGC) allowed accumulation up to 2 ×105ions. The maximum injection time was set to 500 ms using 1 µ scan count. Fluorescence Spectroscopy Steady-state fluorescence measurements were performed by using a high sensitivity spectrofluorimeter, the SLM 8100C Aminco, equipped with a cooled photomultiplier and a double (single) monochromator in the excitation (emission) path. Excitation and emission slit widths were selected at 8-nm for both channels. Polarizers were set at the magic angle conditions. Frontiers in Chemistry | www.frontiersin.org 3February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins FIGURE 2 | Structures of the new βCD conjugates equipped with naphthalene clips prepared in this work. The fluorescence decay measurements were achieved on a Time Correlated Single Photon Counting (TCSPC) FL900 Edinburgh Instruments Spectrometer. A NanoLed (Horiba), which emits short repetitive optical pulses at 296nm, was used as a light source to excite the naphthyl groups. The system was equipped with two concave grating monochromators at both the excitation and emission paths and a red sensitive photomultiplier also immersed in a Peltier cooled housing. No polarizers were used. Data acquisition was carried out by using a multichannel time detector and a time window width of 200 ns with a total of 10,000 counts at the intensity maximum. The instrumental response function was regularly obtained by measuring the scattering of a Ludox solution. The cells housing temperature for both instruments were thermostatically controlled by using a Pt-100 probe with digital temperature processors. Right angle geometry and cylindrical quartz 2-mm inner path cells of 120 µL capacity were used for most of the experiments. Fluorescence intensities due to the inner effect were corrected (Icorr) according to Icorr =Iobs 10Aex+Aem 2(1) Where Aex and Aem are the absorption at the wavelength of excitation and emission, respectively. However, for some of the checking typical rectangular (square cross-section) quartz 1-cm path cells were used. Decay intensity profiles were fitted to a sum of exponential decay functions as I(t) = n X i=1 Aie−t/τi(2) by the iterative deconvolution method (O’Connor et al., 1979). The average lifetime of a multiple-exponential decay function was then defined as, hτi= n P i=1 Aiτ2 i n P i=1 Aiτi (3) where Aiis the pre-exponential factor of the component with a lifetime τiof the multi-exponential function intensity decay. The fractional contribution fiof each decay time to the steadystate intensity, which represents the fraction of total fluorescence intensity Iof the i-component at the wavelengths of observation, is given by fi=Aiτi n P i=1 Aiτi =Ii n P i=1 Ii (4) and the intensity weighted average lifetime <τ> from a dilute solution of a pair of emitting species, 1 and 2, that do not interact during the excited state lifetime can be obtained as, <τ >=f1τ1+f2τ2(5) From the fluorescence depolarization measurements, the anisotropy ris typically defined as: r=(IVV −GIVH)/(IVV +2GIVH) (6) where Ixy is the intensity of the emission that is measured when the excitation polarizer is in position x (V for vertical, H for horizontal), the emission polarizer is in position y, and the G factor (=IHV/IHH) corrects for any depolarization produced by the optical system (Lakowicz, 2008). For a single isolated excited chromophore which is dynamically quenched by a quencher Q, the τ/τ0ratio (with/without Q) is related with [Q] by the linear Stern-Volmer equation. For more complicated systems, the Stern-Volmer representations of <τ>/<τ0>are linear at the lowest [Q] region. Frontiers in Chemistry | www.frontiersin.org 4February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins Circular Dichroism Circular dichroism spectra were obtained by using a JASCO-715 spectropolarimeter. Recorded spectra were the average of three scans taken at a speed of 50 nm min−1with a time response of 0.125s. The sensitivity and resolution were fixed at 20 mdeg and 0.5 nm, respectively. Measurements were performed in 1 cm or 0.1 cm path quartz cells at 25◦C. Molecular Mechanics (MM) and Molecular Dynamics (MD) Simulations All calculations were performed with Sybyl X2.0 (SYBYL- X 2.0; Tripos Associates; ed. St. Louis, Missouri, USA, 2012) and the Tripos force field (Clark et al., 1989). A relative permittivity ε=3.5 (ε=1) was used in the vacuum (in the presence of water). Charges for βCD derivatives were obtained by MOPAC (Frisch et al., 2004). The starting modified βCD derivatives were built with the macroring in the non-distorted form (φ=0◦, ψ= −3◦,τ=121.7◦and side chain χangles in the trans conformation) (Pozuelo et al., 1996) and the naphthyl substituent for 4and 5in the most probable conformations for the chain that links the naphthalene moiety to a glucopyranose unit of the βCD macroring. The selected conformations were those of the minima potential energies obtained by placement of the four torsional angles that describe the rotation around C(3)-O-CH2- Car(1) and C(2)-O-CH2-Car(8) ether bonds according to the procedures described elsewhere (González-Álvarez et al., 2011). Optimizations were carried out by the simplex algorithm, and the conjugate gradient was used as a termination method with gradients of 0.2 (0.5) kcal·mol−1Å−1for the calculations carried out in vacuum (water) (Brunel et al., 1975; Press et al., 1988). Non-bonded cut-off distances were set at 12 Å. The Molecular Silverware algorithm (MS) and periodic boundary conditions (PBC) were used for system solvation when water was used as a solvent (Blanco, 1991). The methods used for the conformational study of the monomeric 4and 5and for their dimer formation were similar to those described previously for 1(González-Álvarez et al., 2008) and 2or 3(Neva et al., 2018). 2-ns MD simulations in the vacuum were performed at several temperatures ranging from 250 to 600 K on initial 4and 5conformations. Emulation of dimerization processes were performed starting from the optimized conformations of 4and 5. A single head- to-head orientation (HH) for the CD-to-CD approaching, in agreement with experimental findings, was considered (see Supplementary Figure S30 for the coordinate systems describing dimerization). Critical analysis of the structures generated by scanning the θ[O(4)-o-o’-O(4′)] dihedral angle in the −180 to +180orange (initially at 30◦intervals and 10olater) and the ycoordinate (oo’ distance) from 2 to 0.7 nm (0.05nm intervals) in the presence of water (fixed the ε[o-o’-O(4′)] angle at 90o) followed by optimization (gradient 1.5 kcal·mol−1Å−1) provided the most favorable θangle for approaching. Supplementary Figure S29 depicts an example for dimerization of 5. Once θwas fixed, the dimerization was emulated by approaching along the ycoordinate, in 0.05nm steps, from 2 to 0.7 nm (Supplementary Figure S30). This was also done in the presence of water (MS and PBC). Every structure was optimized (1.5 kcal ·mol−1Å−1) and saved for further analysis. Minima binding energy (MBE) structures for dimers were optimized once again (gradient 0.5 kcal ·mol−1Å−1) and used as the starting conformations for the 1.0 ns MD simulations following the same strategy described earlier (González-Álvarez et al., 2008; Gonzalez-Álvarez et al., 2009b; González-Álvarez et al., 2011; Neva et al., 2018). For computing complexation of 4-dimer with sodium adamantane-1-carboxylate (AC), we started from the most stable HH MBE 4-dimer structure. The anionic AC guest with the most favorable orientation (Supplementary Figure S41) was approached along the y coordinate from 2 to −2 nm (0.05 nm intervals) in the presence of water, in a way that is similar to the dimerization processes. Each structure generated and optimized in the presence of water was analyzed. In a similar manner, anionic AC complexation with 5monomer (MBE structure) was computed. NMR Titration Experiments Association constants (Ka) for the inclusion complexes were determined in phosphate-buffered D2O (pD 7.4, 0.1 M) at 298K by measuring the chemical shift variations in the 1H NMR spectra (500 MHz) of a solution of the CD derivatives 4or 5in the absence and in the presence of increasing amounts of AC. Typically, a stock solution of CD (ca. 0.5–1mM) was prepared. A 500 µL aliquot of this solution was transferred to a NMR tube and the initial NMR spectrum was recorded. Then, a solution of AC (15–20mM) was prepared in the stock CD solution (it is critically important to maintain a constant host concentration, pD and ionic strength throughout the titration experiment). Aliquots of the guest solution were sequentially added to the NMR tube and the corresponding spectrum was recorded after equilibration (Supplementary Figures S37, S38). Additions and measurements were continued until 90–100% complexation had been achieved. The chemical shifts of selected host resonances obtained at approximately ca. 15 different host-guest concentration ratios were plotted against the concentration of CD and used in an iterative leastsquares fitting procedure using a 1:1 stoichiometry binding model (Bisson et al., 1998, 2000). Errors are estimated in ±15%. RESULTS AND DISCUSSION Synthesis and Characterizations Imposing distance restrictions in CDs through covalent bonds can lead to significant alterations in their primary structure, usually by provoking conformational changes in one or several of the monosaccharide building blocks, which may substantially perturb the inclusion capabilities (Fujita et al., 1988; Fukudome et al., 2007b; Álvarez-Dorta et al., 2015, 2016;Immel et al., 2001; Fukudome et al., 2007; León et al., 2018). Compounds 4and 5were purposely conceived to avoid this and keep the toroidal βCD cavity characteristic of βCD undistorted, so that any effect in their supramolecular properties can be ascribed to the presence of the naphthalene component, devoid from Frontiers in Chemistry | www.frontiersin.org 5February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins FIGURE 3 | Synthesis of compounds 4and 5. additional effects on the βCD macroring topology. According to the available X-ray data for permethylated βCD 6(Caira et al., 2004), the through-space distance between the O-2I and O-3II positions in the cyclooligosaccharide backbone is 3.4 Å, which can be accommodated by benzylic positions in either 1,8- (for 4) or 2,3-disubstituted (for 5) naphthylene clips (separation distance <4.1 Å) without provoking any significant steric strain. Indeed, alkylation of the βCD diol precursor 6with 1,8- or 2,3-bis(bromomethyl)naphthalene, conducted in dry N,N-dimethylformamide (DMF) in the presence of sodium hydride (NaH), afforded the target cyclic naphthylene ethers 4and 5, respectively, as single products, as seen from thin layer chromatography (TLC) and mass spectrometry (MS) monitoring of the crude reaction mixtures (Figure 3). No side-products resulting from cross-linking or single-point etherification reactions were detected, pointing to a fast ringclosure once the bis(bromomethyl)naphthalene tether first reacts at either O-2Ior O-3II. Analytically pure samples of 4and 5, were isolated in 56 and 48%, respectively, after column chromatography on silica gel using 100:1→20:1 EtOAc/EtOH as eluent. The 1H and 13C NMR data for 4and 5confirmed the presence of the dual-bridged joint involving consecutive glucopyranosyl subunits, with all the pyranoid rings in the ground state 4C1 chair conformation, in agreement with full preservation of the basket-shaped architecture. Selective irradiation experiments (1D TOCSY, Supplementary Figures S7, S15 for 4and 5in the Supplementary Materials, respectively) featured 3JH,H vicinal coupling constants in the 9–10 Hz range for the non-anomeric sugar protons (4.1–3.1 ppm), indicative of all-axial arrangements for the H-2, H-3, H-4, and H-5 protons about the six-membered pyranoid rings. The anomeric H-1 protons (5.32–5.14 ppm) exhibited coupling constant values in the 3.8–4.1 Hz, in agreement with the αconfiguration. ROESY spectra further evidenced the expected intra-monosaccharide H-3—H-5 and inter-monosaccharide H-1—H-4 spatial correlations characteristic of undistorted cyclodextrin derivatives (Supplementary Figures S3, S10 for 4 and 5, respectively, in the Supplementary Materials). In the case of 4, the 1,8-dimethylnaphthylene clip (1H NMR resonances in the 8.0–7.5 ppm region) draws a 12-membered macrocycle with a five-carbon segment flattened region that imposes considerable rigidity, which is reflected in a broad dispersion of the benzylic proton resonances. The 2,3-dimethylnaphthylene clip in 5(1H NMR resonances in the 7.9–7.5 ppm region), in which the flattened segment in the bridging area is reduced to four-carbon centers, is expected to keep substantially higher flexibility. Indeed, the corresponding proton NMR spectrum closely resembled the spectrum of the o-xylylenated βCD derivative 2 (Supplementary Figure S16). Self-Assembling The 2,3-naphthylene-equipped βCD derivative 5, but not the 1,8-naphthylene positional isomer 4, exhibited a very apparent negative solubility coefficient in water: when heated, clear (25◦C) solutions became immediately turbid, returning to the limpid original state upon cooling. A negative water solubility coefficient is a characteristic feature of methylated cyclodextrin derivatives bearing aromatic appendages and can be ascribed to reversible dimerization processes (Filippone et al., 2002): in the dimer state, the hydrophobic aromatic moiety is shielded from the bulk, facilitating solvation and preventing unspecific clusterization at low temperatures, whereas heating results in dimer disruption, uncovering of the aromatic ring and precipitation. The dissimilar behavior of 4and 5was, however, intriguing. In principle, it could be interpreted in terms of differences in the propensity of 4and Frontiers in Chemistry | www.frontiersin.org 6February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins 5to dimerize. Notwithstanding, the high resolution electrospray ionization (HRESI)-mass spectra of 4and 5, recorded from aqueous solutions, both exhibited prominent pseudomolecular peaks for the dimer species (Supplementary Figure S17). The data can be concealed assuming that the aromatic surface in the more rigid derivative 4is similarly exposed to the solvent in the monomer and dimer entities, possibly acting as cavity extension module, whereas in 5-dimer the naphthalene walls are probably sandwiched between the two βCD constituents, in agreement with our starting hypothesis. The possibility to drive the architecture of the CD dimer species by changing the type of joint to the naphthylene clip was further substantiated by fluorescence spectroscopy. Figure 4 shows the emission spectra for water solutions of 4and 5at increasing concentrations (from 0 to ∼0.2 mM) at 25◦C upon excitation of the naphthalene chromophore (295 nm). The spectrum for 4exhibits a single band located at ∼340 nm and a shoulder at ∼380nm, whereas the spectrum for 5shows a double band with two maxima placed at ∼330 and ∼340 nm and a shoulder at ∼350nm. The low intensity shoulders located at longer wavelengths suggest the presence of intermolecular aromatic excimers, that is, the formation of shortlived dimeric complexes in an electronically excited state (Saigusa and Lim, 1996; Shirai et al., 2016). Conclusive confirmation of dimer formation was obtained from the analysis of the corresponding fluorescence decay profiles obtained from timeresolved fluorescence measurements, selecting the maximum of emission (340nm in both βCD derivatives 4and 5). The data were fitted to three-exponential decay functions at any concentration and temperature: a short-lived component (<0.5 ns) ascribed to the inherent scattering of the cylindrical cuvettes (not taken into account in the analysis), a second intermediate component in the 2–9 ns (for 4) or 3–12 ns (for 5) range that was attributed to the free dissociated monomer and a third slowest component in the 10–15 ns (for 4) or 20–60 ns (for 5), whose contribution increased with concentration and decreased with temperature, which was attributed to the dimeric entity (4-dimer or 5-dimer). In full agreement, the lifetime averages (<τ>) of the excited electronic states (obtained from Equation S3) increased with the concentration of 4or 5(Figure 5), a consequence of the increase of the fraction of the dimer species in the corresponding monomer/dimer equilibria. Plots of the corrected fluorescence intensity (Icorr, Equation 1 in the Experimental section), measured as the area under the emission spectra, against the concentration of 4and 5 in water at 25◦C (Figure 4 inserts) were linear in the whole range of concentrations used, meaning that the fluorescence quantum yield φ, i.e., the ratio of the number of photons emitted to the number of photons absorbed, does not change upon dimerization (φdimer ∼ =φmonomer;see Equation 8 in the Experimental section). In other words, the fluorescence intensity is not sensitive to the association process of 4or 5. Under this assumption, the dimerization isotherms fit the experimental data to Equation (9) (Experimental section). From them, the respective dimerization constants (KD), as well as the lifetimes for the monomeric (τ0=τmonomer) and dimeric (τ∞=τdimer) forms of both dimethylnaphthylene-clipped βCD derivatives 4 FIGURE 4 | Emission spectra for 4 (Left) and 5 (Right) in water in the 0 to ∼0.2mM range of concentrations (yellow to blue solid lines) at 25◦C. Variation of the corrected fluorescence intensity (Equation S1) with compound concentration measured at the intensity maxima are superimposed (dashed lines). and 5, were derived. Table 1 collects the ensemble of data for the whole range of temperatures. Remarkably, the propensity to form dimers is about 2-to-3 orders of magnitude higher as compared with the previously reported o-xylylene clipped βCD derivative 2(Neva et al., 2018). As expected from the shape of the isotherm plots in Figure 5, where a plateau is reached at much lower concentrations for 4than for 5, the KDvalues for 4-dimer formation (4.1–8.0 ·105M−1) were found to be above one order of magnitude higher as compared with the corresponding values for 5-dimer formation (1.4–2.2 ·104M−1). On the other hand, the significantly larger average lifetimes at infinite concentration (ascribed to the dimer form) for 5(67.5–79.8 ns) than for 4 (11.0–11.9 ns) point to considerable differences in the structure of the corresponding dimers, reinforcing the notion that the naphthylene moiety is probably better shielded from the solvent in 5-dimer than in 4-dimer. From the van’t Hoff linear representations shown in Figure 6, the 1Hand 1Sthermodynamic parameters associated to the dimerization process were found to be 1H= −2.5 ±5.1 kJ·mol−1,1S= +102.7 ±17.3 J·K−1mol−1for 4and 1H = +4.5 ±3.6 kJ·mol−1,1S= +97.3 ±12.2 J·K−1mol−1 for 5. Van der Waals or electrostatics favorable intermolecular interactions are usually characterized by relatively large 1H< 0 values, whereas hydrophobic forces, which tend to reduce the hydrophobic surface of non-polar moieties, are instead accompanied by near zero or slightly positive 1Hvalues (Southall et al., 2002; Meyer et al., 2006; Kronberg, 2016). The experimental 1Hdata are therefore consistent with a major role of the dimethylnaphthylene appended group in the dimerization process. The rather large and favorable 1Svalues accompanying monomer-to-dimer transformation for 4or 5 are also consistent with hydrophobic interactions as the driving force. They can be ascribed to a substantial loss of solvating shells during dimerization due to the large size of the naphthylene moiety, which overcomes the intrinsic entropy decrease due to the loss of degrees of freedom accompanying the association Frontiers in Chemistry | www.frontiersin.org 7February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins FIGURE 5 | Variation of the weighted average lifetime <τ> with the concentration of 4 (Left) or 5 (Right) in water in the 5–45◦C temperature range. TABLE 1 | Dimerization constant (KD) values and lifetime of the electronic excited state for the monomer (τmonomer) and dimer (τdimer) species obtained from analysis of decay profiles at different concentrations and temperatures for CDs 4 an 5. Compound Temperature (◦C) 10−5KD(M−1)τmonomer (ns) τdimer (ns) 4 5 8.0 ±1.5 2.7 ±0.4 11.9 ±0.2 4 15 5.6 ±0.9 2.8 ±0.3 11.9 ±0.2 4 25 4.1 ±0.6 2.3 ±0.3 11.6 ±0.2 4 35 5.6 ±1.3 2.0 ±0.4 11.4 ±0.2 4 45 6.8 ±1.2 2.4 ±0.3 11.0 ±0.2 5 5 0.18 ±0.02 3.2 ±0.8 77.9 ±3.1 5 15 0.14 ±0.02 2.6 ±0.8 79.8 ±3.8 5 25 0.21 ±0.05 2.4 ±1.3 74.4 ±4.6 5 35 0.22 ±0.03 2.3 ±0.8 69.7 ±2.6 5 45 0.19 ±0.03 2.6 ±0.8 67.5 ±3.0 of monomeric entities. The preponderant role of hydrophobic forces was further confirmed by conducting fluorescent decay measurements for solutions of 4and 5in solvents of decreasing dielectric constant (water, water-methanol mixtures, methanol and ethanol; Supplementary Figures S18, S19): a steep decrease in <τ> values was observed, which correlates with an increase in the proportion of the shorter lifetime monomer component at the expenses of the dimer. For instance, whereas a 1.5 ·10−4M solution of 4in water at 25◦C contains 90% of 4-dimer, in ethanol the dimer proportion drops to 40%. Structure and Conformation In order to obtain information on the topology of the 1,8- and 2,3-dimethylnaphthylene βCD conjugates 4and 5and on the changes that may occur in the location of the naphthyl chromophore upon dimer formation, fluorescence quenching experiments using diacetyl as the quencher (q) were next conducted. Measurements were carried out at 25◦C in water for two different concentrations of 4(0.02 and 0.2 mM, meaning 4- dimer molar fractions of 0.69 and 0.88, respectively, for KD= 4.1·105M−1;Table 1) or 5(0.03 and 0.2mM, meaning 5-dimer FIGURE 6 | Van’t Hoff linear representations for the dimerization processes of 4and 5obtained from the association constants collected in Table 1. molar fractions of 0.25 and 0.53, respectively, for KD=0.21·105 M−1;Table 1). From the Stern–Volmer (< τ >q=0/< τ > vs. [q]) plots (Lakowicz, 2008) (Supplementary Figure S24), which were linear in the range of quencher concentrations used (0 to 8 mM), the Stern-Volmer constants (Ksv) and bimolecular quenching constants (kq) were obtained (Table 2). For species bearing the same chromophore unit, kqvalues provides a qualitative assessment of the differences in the accessibility to the quencher: the lower the kqvalue, the less accessible is the chromophore. Changes in the concentration of 4did not affect kq, suggesting that the 1,8-naphthylene group was similarly exposed in the monomer and in the dimer (kq=1.4 ·109M−1s−1). Differently, the kqvalue for water solutions of 5decrease by about 15% on going from the lower (kq=0.6 ·109M−1s−1) to the higher concentration (kq=0.5 ·109M−1s−1), indicating that the 2,3-naphthylene moiety is less accessible to the quencher in 5-dimer as compared to the monomer form. The data could be interpreted as the shielding of the aromatic clip between the macrorings of two βCD units upon dimerization in the case of 5 Frontiers in Chemistry | www.frontiersin.org 8February 2019 | Volume 7 | Article 72 Neva et al. Self-Assembling Cyclodextrins TABLE 2 | Stern-Volmer plot parameters for the quenching 4and 5by diacetyl of at different concentrations at 25◦C. Compound Concentration (mM) Xdimer <τ >q=0 (ns) KSV (M−1)kq×10−9(M−1s−1) 4 0.02 0.69 10.5 ±0.1 15.0 ±0.2 1.4 ±0.1 4 0.2 0.88 10.3 ±0.1 14.3 ±0.6 1.4 ±0.1 5 0.03 0.25 45.6 ±0.1 29.7 ±0.7 0.6 ±0.3 5 0.2 0.53 49.5 ±0.1 24.7 ±0.5 0.5 ±0.2 but not in the case of 4, which points out to substantial differences in the architecture of 4-dimer and 5-dimer. Additionally, the lower kqvalues for 5than for 4whatever the concentration indicates that the 2,3-naphthylene moiety in 5is less exposed to the quencher than the 1,8-naphthylene module in 4, both in monomer and dimer form, which further advocates for strong differences in the orientation of the aromatic appendage relative to the cyclooligosaccharide scaffold. The presence of the naphthylene chromophore in 4and 5enables circular dichroism as a useful technique for conformational analyses: βCD is a chiral molecule; although it does not absorb above 200nm, a chromophore appended to βCD (or included in the cavity) exhibits induced circular dichroism (ICD) (Superchio et al., 2004; Berova et al., 2007). Most interestingly, circular dichroism also provides precious information on intermolecular interactions and the structure of supramolecular assemblies: when two (or more) chromophores are positioned close in space and adopt a proper (chiral) mutual orientation, the interactions between their electronic transition dipoles result in large rotational strengths, often surpassing those connected with the perturbations on each chromophore exerted by the chiral non-chromophoric frame. The most significant case arises when chromophores with strong electric-dipole allowed transitions couple to each other (exciton coupling; EC) (Pescitelli et al., 2011). It has been established that the sign of ICD for a chromophore inside the βCD cavity is positive when the electron transition dipole moment is parallel to the axis of CD while the one perpendicular to the axis is negative (Harata and Hisashi, 1975; Shimizu et al., 1979, 1981, 1982). This rule has been extended to derive a general law that the sign of ICD is reversed when the guest moves from inside of the cavity to outside and the direction of the transition is fixed, and it changes at the angle between the transition moment and the CD axis 54.7◦(Kodaka and Fukaya, 1989; Kodaka, 1991, 1993). The major UV transitions of the naphthyl group are 1Bb(∼230 nm), 1La(∼275 nm), and 1Lb(∼320nm), and they do not change substantially with substitution (Kodaka, 1998). In full agreement, the ordinary absorption spectra for 4or 5in water exhibit two intense main bands whose maxima are placed at ∼220 or ∼227 nm and ∼285 or ∼287nm and a rather weak shoulder located at ∼315 or ∼339 nm, which were ascribed to the 1Bb,1La, and 1Lbelectronic transitions, respectively (Figure 7, top panels). Here we follow the broadly accepted Platt’s notation, where the electronic state is designated by A, B, C, ...or K, L, M, ... in accordance with its total momentum number Q(or total ring quantum number) and the superscript “1” indicates a singlet state, whereas the subscripts “a” and “b” denote the location of the nodes of the wave function: with a and b, the nodes are indicated to be present on C–C bonds and on carbon atoms, respectively (Platt, 1949; Michl, 1978). The electron transition dipole moment of the short- and the high-wavelengths 1Bband 1Lbtransitions are nearly parallel and perpendicular, respectively, to the long axis of naphthalene, while that of the 1Latransition is tilted about 10– 40◦from the short axis (Kodaka, 1998; González-Álvarez et al., 2009a) (Figure 7, bottom panels). In the next paragraphs, the assignment of the ICD bands for compounds 4and 5conforms to these literature reports. The circular dichroism spectrum (expressed as molar ellipticity; [θ]) of a 0.11 mM solution of 4in water (4-dimer molar fraction 0.84; Figure 8, left panel) exhibits EC in the electronic 1Bbtransition band, which represents a strong evidence for the presence of aggregates facing the naphthylene-bearing βCD secondary rims (head-to-head; HH dimers), likely stabilized through the mutual interaction between the aromatic groups. In addition, the 1Laand 1Lbtransitions provide negative and slightly positive bands, respectively. The intensities of all the three bands (expressed as ellipticity; θ, mdeg) in the ICD spectrum increased with the concentration of 4(Figure 8, middle and right panels). At the lower concentrations used (0.67 ·10−4and 0.022 ·10−4 M for the 0.1- and 1 cm path cell, respectively) the 4-dimer molar fractions were ∼0.81 and 0.37, respectively, which means that the ICD signals at the highest concentration were mostly due to the dimer species. Negative and slightly positive 1Laand 1Lbbands then agree with the respective transition moments adopting a nearly parallel (for 1La) and forming an angle slightly larger than 54.7◦(for 1Lb) with respect to the 7-fold macroring axis. These geometrical requirements could be fulfilled for a fully open conformation of the 1,8-dimethylnaphthylene-βCD conjugate where the naphthylene group, slightly rotated about his short axis, essentially prolongs the βCD walls. The ICD spectra of a 0.3 mM solution of 5in water (5-dimer molar fraction ∼0.59; Figure 9, left panel) likewise exhibited EC in the 1Bbband, with peaks of similar intensity but opposite sign as compared with 4, consistent with an HH dimer. This EC was still visible at lower concentrations down to 0.17 mM (5-dimer molar fraction ∼0.51; Figure 10, right panel). The 1La and 1Lbtransition bands were both positive and rather weak, especially the last one that was hardly visible. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Chemistry | www.frontiersin.org 18 February 2019 | Volume 7 | Article 72