Secondary Face-to-Face 2-2′ β-Cyclodextrin Dimers Linked with Fluorescent Rigid Spacer Arms: A Cyclodextrin-Based Ratiometric Sensor for Bile Salts

FULL PAPER

DOI: 10.1002/ejoc.201101598

Secondary Face-to-Face 2–2� β-Cyclodextrin Dimers Linked with FluorescentRigid Spacer Arms: A Cyclodextrin-Based Ratiometric Sensor for Bile Salts

Manuel C. Martos-Maldonado,[a] Indalecio Quesada-Soriano,[b] Juan M. Casas-Solvas,[a][‡]

Luis García-Fuentes,[b] and Antonio Vargas-Berenguel*[a]

Keywords: Cyclodextrins / Host-guest systems / Sensors / Fluorescent probes / Cross-coupling / Click chemistry

The synthesis of a series of β-cyclodextrin (CD) dimers inwhich the two macrocycle units are linked by rigid-rod phen-ylene-ethynylene tethers of different length and shapethrough the cyclodextrin secondary sides is reported. Thesynthesis involves Sonogashira coupling reactions for con-struction of the tethers and final grafting of the CD units byclick chemistry. The self-aggregation of the synthesized CDdimers in aqueous medium was investigated by fluorescencespectroscopy, isothermal titration calorimetry and dynamiclight scattering. Rigid-rod-tethered cyclodextrin dimers

Introduction

Cyclodextrins (CD) are a group of structurally relatedcyclic oligosaccharides that consist of d-glucopyranoseunits linked by α-(1�4) bonds. Natural α-, β- and γ-CDscontain six, seven, and eight units of such carbohydrateunits, respectively. They possess a relatively rigid torus-shaped structure that defines an inner hydrophobic cavityrimmed by two hydrophilic openings. These openings aredifferent in diameter, the narrower end, containing primaryhydroxy groups located on the C-6 of the glucopyranoseunits, and the wider end, containing secondary hydroxygroups on the C-2 and C-3 positions. CDs are well-knownto form inclusion complexes in aqueous solution with alarge variety of organic molecules of hydrophobic natureand suitable size and geometry.[1–3] Because of this abilityto behave as a host, CDs find a wide range of applicationsin areas such as artificial enzymes, analytical chemistry,food technology, drug delivery, and sensors.[4–12] In ad-dition, CDs are convenient building blocks for the prepara-tion of nanostructured functional materials.[13]

Bridged bis-β-CD compounds consisting of two CDunits tethered by linkers of different nature have received

[a] Area of Organic Chemistry, University of Almeria,Crta. de Sacramento s/n, 04120 Almería, SpainFax: +34-950-015481E-mail: [email protected]

[b] Area of Physical Chemistry, University of Almeria,Crta. de Sacramento s/n, 04120 Almería, Spain

[‡] Current Address: School of Chemistry, University of Bristol,Cantock’s Close, Bristol, BS8 1TS, United KingdomSupporting information for this article is available on theWWW under http://dx.doi.org/10.1002/ejoc.201101598.

© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2012, 2560–25712560

formed polydisperse nanoparticles of 240 to 480 nm in watersolution depending on the tether shape, and exhibited fluo-rescence. The binding and fluorescence sensing abilities ofthe CD dimers toward sodium cholate and deoxycholatewere studied. As a result, the newly synthesized rigid-rod-tethered cyclodextrin dimers proved to be very sensitive fluo-rescent chemosensors. A new cyclodextrin-based ratiometricfluorescence probe has thus been developed for the detec-tion of bile salts such as sodium cholate and deoxycholate.

much attention because of their enhanced binding abilitiesand molecular selectivity as compared with the nativeCDs.[14,15] In addition to the cooperative binding throughwhich the two adjacent CD cavities of these bridgedbis(CD)s can encapsulate guest molecules, the linker cancontribute to the stabilization of such complexes by provid-ing additional binding interactions or contributing to ex-tending the overall binding site. The face through whichthe CD moieties are bridged influences the supramolecularbehavior of the CD dimers towards ditopic guests. CD unitscan be linked through their primary faces, i.e., 6–6�bis(CD), through a head-to-tail fashion, i.e., 6–2� or 6–3�bis(CD), and through their secondary faces, i.e., 2–2� or 3–3� bis(CD). The supramolecular behavior of 6–6� bis(CD)has been extensively studied. Such studies claim that 6–6�bis(CD) form a sandwich-like supramolecular structure of1:1 stoichiometry with guest molecules or, alternatively, n:nsupramolecular assemblies involving intermolecular co-operative binding. The latter consists of a linear assemblyin which guest and host are interconnected by the simulta-neous penetration of the guest into the CD cavities of dif-ferent dimers through their secondary sides. Such supra-molecular behavior tends to occur when 6–6� bis(CD)s con-tain short-length linkers, whereas 6–6� bis(CD)s havinglong-length linkers prefer to form 1:1 complexes(Scheme 1).[14–17]

Interestingly, the least documented 2–2� bis(CD)s appearto have a different supramolecular behavior towards ditopicguests.[18–35] Secondary-face-linked β-CD dimers oftenshow higher complexing constants than those bridged byprimary faces,[18–21,35] probably due to the fact that guest

A Cyclodextrin-Based Ratiometric Sensor for Bile Salts

Scheme 1. Schematic representation of β-CD.

molecules preferentially penetrate the cavity through thewider secondary rim.[1–3] To the best of our knowledge, no2–2� CD dimers have shown a tendency to form supra-molecular polymers.

The linker that tethers the two CD units may help toprovide functional properties such as a sensing ability. In-deed, chemical sensors based on cyclodextrin derivativeshave received considerable attention.[10,13,15] We have re-cently reported some examples of electrochemical cyclodex-trin-based sensors,[34] but most of the reported chemicalsensors use CD derivatives with an appended photoactivemoiety, with fluorescent-based sensors occupying a promi-nent place among the optical sensors. In most cases, thesensing ability of CD-derived chemical sensors is based ona competition between self-inclusion of the active moietyand complexation of the guest. Because the sensing mecha-nism involves competition for occupation of the CD cavity,a decrease in the guest binding affinity of the CD deriva-tives compared with native β-CD normally occurs.[10] In ad-dition, displacement of the fluorophore from the cavity tothe bulk aqueous media causes a fluorescence intensity de-crease. Alternatively, so-called “turn-on” fluorescent chemi-cal sensors, having the fluorophore appended to CDthrough a rigid spacer, have been reported in which fluores-cence intensity increased upon host–guest complex forma-tion.[10]

Herein, we report the preparation of a series of CD di-mers in which the two CD units are linked by rigid-rodphenylene-ethynylene tethers of different length and shapethrough the CD secondary sides. Previously, we have inves-tigated the supramolecular behavior of 2–2� CD dimerslinked with short and rigid spacers towards multitopicguests such as bile salts and observed a distinctive behaviorwith respect to their 6–6� counterpart.[35] The rigid rod-likecharacter and conjugation of the oligo- and poly(phen-yleneethynylene) compounds are responsible of their inter-esting electronic and photophysical properties,[36–38] how-ever, the strong π–π interactions between the tether chainsalso results in a tendency to form aggregates.[39,40]

In addition to providing a rigid linker that prevents self-inclusion into the CD cavity, the phenylene-ethynylenetether provides a fluorescent moiety and therefore potentialsensing properties. We have investigated the sensing proper-ties of the newly synthesized fluorescent amphiphilic CD

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dimers towards multitopic guests such as bile salts sodiumcholate (NaC) and deoxycholate (NaDC). Bile salts are im-portant naturally occurring surfactant steroid compoundsthat play a significant role in the metabolism and excretionof cholesterol in mammals. Moreover, they are used asdrugs for nonsurgical treatment for gallstone diseases, andother pharmacological applications are under evaluation.[41]

Studies on the guest binding abilities of bile salts towards6–6� CD dimers have been widely undertaken,[42–51] al-though to the best of our knowledge only a few 2–2� CDdimers have been used as hosts for these molecules.[34,35,52]

The selected bile salts possess a similar framework contain-ing the four rings A–D and a side chain, and vary slightlyin the number of hydroxyl groups (Scheme 2).

Scheme 2. Bile salts used as guests.

Herein, we also describe a cyclodextrin-based ratiometricfluorescence sensor for the detection of NaC and NaDC.To the best of our knowledge, this is the first ratiometricfluorescence sensor developed for the detection of bile salts(Scheme 2).

Results and Discussion

We have reported the one-step synthesis of mono-2-O-propargyl-β-CD (13), which is a very convenient buildingblock for the construction of β-CD compounds function-alized on the secondary face. The terminal alkyne group of13 linked to the macrocycle at O-2 offers the possibility ofcoupling functional structures by using CuI-catalyzed alk-yne–azide cycloaddition (CuAAC)[34] or oxidative couplingreactions.[55] Thus, we performed the synthesis of bis-[(azidophenyl)ethynyl]benzene derivatives 9–12 containingsuitable azide groups for the coupling of CD derivative 13by CuAAC. Oxidative coupling of diethynylbenzenes 2 and3 with iodoanilines 1 and 4 in piperidine at room tempera-ture, using [Pd(PPh3)2Cl2] and CuI as catalysts under anatmosphere of hydrogen gas diluted with nitrogen, gavebis[(3�-aminophenyl)ethynyl]benzenes 5–8 in 85–93% yields.These reaction conditions led to an improved synthesis ofcompound 5 that had previously been synthesized in muchlower yield.[53,54] Diamine compounds 5–8 were then trans-formed through aromatic nucleophilic substitution of thecorresponding diazonium salts into diazide derivatives 9–12 in 79–93% yield. Finally, the coupling of CD 13 withbis[(azidophenyl)ethynyl]benzenes 9–12 was performed inN,N-dimethylformamide (DMF) at 105 °C with catalyticamounts of (EtO)3P·CuI, generating the phenylene-eth-ynylene-tethered bis(CD) compounds 14–17. Compounds

A. Vargas-Berenguel et al.FULL PAPER14–17 were very insoluble in solvents other than dimethylsulfoxide (DMSO) and DMF. The more water-soluble com-pounds 15 and 17 were purified by column chromatog-raphy, isolating the bis(CD) derivatives in 65 and 63% yield,respectively. Compounds 14 and 16 were purified by soxhletextraction with methanol, thus removing unreacted mono-propargyl 2I-O-β-cyclodextrin 13 and isolating the bis(CD)derivatives in 71 and 67% yield, respectively.

The molecular weights of compounds 14–17 were veri-fied by MALDI-TOF mass spectrometry and they werefully characterized by NMR spectroscopic techniques em-ploying 1H and 13C NMR, COSY and HSQC experiments.Inspection of 1H NMR spectra showed the absence of asignal at δ = 3.52 ppm, corresponding to alkyne proton of

Scheme 3. [a] Reagents and conditions: (a) [Pd(PPh3)2Cl2], CuI, piperidine, room temp., 1.5–6.5 h, 85–93%; (b) NaNO2, aq. HCl, NaN3,0 °C, 1.5 h, 79–93%. (c) CuI·EtO3P, DMF, 105 °C, 1.5–7.5 h, 63–71%.

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compound 13 and the appearance a singlet at δ = 8.75–8.93 ppm corresponding to the 1,2,3-triazole proton.Analysis of the HSQC spectra for compounds 14–17showed that the triazole methine carbon atoms resonate atδ = 122.4–123.6 ppm. Such signals as well as those at δ =145.0–145.6 ppm shown by the 13C NMR spectra and as-signed to the triazole C-4, confirm the presence of such aring in the molecule (Scheme 3).

Given the potential of the phenylene-ethynylene moietiesto aggregate due to π–π interactions, we investigated theself-aggregation properties by fluorescence spectroscopy.The fluorescence spectra of compounds 14–17 measured inwater solutions (see Figure S1 in the Supporting Infor-mation) show a band at 365 nm corresponding to emission


from the phenylene-ethynylene core. Compound 16 also hasan emission at 463 nm that may be assigned to the emissionof phenylene-ethynylene excimer due to π–π interactions be-tween adjacent phenylene-ethynylene groups as a result ofthe CD dimer aggregation. Careful observation of the emis-sion spectrum for 14 reveals a very slight shoulder ataround 460 nm that could be due to an excimer emission.Both compounds 14 and 16 share a linear phenylene-ethyn-ylene tether but the latter has the two CD units attached atthe meta position of the phenyl ring through a triazole moi-ety, instead of the para position as in compound 14. Suchemission at around 460 nm is not observed for 15 and 17,both of which share a nonlinear tether. Compounds 14 and16 showed low water solubility, which limited the type ofexperiments that could be performed (see below). The fluo-rescence spectra of compounds 15–17 measured in watersolutions at different concentrations (2 and 50 μm) showedboth a change of the emission intensity and a redshift withincreasing concentration.

As shown in Figure 1, from 1 to 100 μm, the intensity ofthe fluorescence emission for 17 increases with concentra-tion, and the emission band is bathochromically shifted.Such a bathochromic shift reflects changes in the water–solute interactions caused mainly by a reduced exposure ofthe solute to water. This behavior was also observed in thecase of compounds 15 and 16, and is indicative of the ten-dency of the CD dimers to self-aggregate in aqueous media.This is consistent with the behavior of other oligophenyl-ene-ethynylene-containing molecules. As mentioned above,most likely, such aggregation is driven by π–π interactionsbetween the aromatic systems.[36,39,40] The capacity of CDdimers 15–17 to undergo self-aggregation is consistent withthe reported tendency for self-aggregation of other amphi-philic cyclodextrins.[56–58]

Figure 1. Fluorescence spectra of compound 17 (λexc = 320 nm) inwater at concentrations from 1 to 100 μm. The intensity of thefluorescence emission (I) increases with concentration and theemission band is bathochromically shifted.

We also studied the self-aggregation of the bis(CD) byisothermal titration calorimetry (ITC). Thus, dilution ex-periments were carried out to measure the heat changewhen a concentrated solution of CD dimer was injectedinto the calorimeter cell initially containing phosphatebuffer. The heat arises from dimers or oligomers presentin the higher concentration solution that dissociate upon


dilution. ITC dilution experiments with 15 (450 μm) and 17(520 μm) gave rise to a series of endothermic heat pulsesthat decreased in a nonlinear fashion as the concentrationof such compounds increased in the calorimetric cell (Fig-ure 2). These results agree with the fluorescence experi-ments described above and suggest that both CD dimersare involved in a self-association equilibrium. The resultingthermal dilution profile is consistent with the simplestn2 � 2n dissociation model.[34] Therefore, analysis of theITC data by nonlinear regression procedures provided ag-gregation constant (Kagg) values of 1.9�104 and4.4 �103 m–1 for 15 and 17, respectively. However, whensimilar ITC experiments were performed for these com-pounds at lower concentration (20 μm), similar to that usedfor the titrations of the CD dimers with NaC and NaDC

Figure 2. Thermograms for dilution experiments conducted withcompounds 15 (a) and 17 (b). The studies involved injecting 25aliquots (10 μL each) of these compounds (stock concentration of0.45 mm for 15 and 0.52 mm for 17) into 10 mm phosphate buffer(pH 7.2) at 25 °C. The smooth solid line represents the best fit ofthe experimental data to a n2 �2n dissociation model.

A. Vargas-Berenguel et al.FULL PAPER(see below), no heat exchange was observed, indicating thatat such concentrations the size of the aggregate does notdepend on the concentration.

To confirm the self-aggregation of CD dimers 14–17,DLS measurements were performed in 10 mm phosphatebuffer at pH 7.2. It was found that these CD dimers forma polydisperse solution of aggregates with an average sizeof 218�42 nm for 14, 205� 41 nm for 15, 157 �9 nm for16, and 184 �14 nm for 17 (Table 1) at a concentration (2μm) in which the size would not depend on the concentra-tion as shown by the ITC dilution experiments (Figure 2).

Table 1. Average sizes expressed as hydrodynamic radius (Rh) ob-tained from DLS measurements of compounds 14–17 (2 μm) in theabsence and the presence of NaDC and NaC (20 μm) in 10 mmphosphate buffer (pH 7.2).

Bis(CD) Rh [nm]+ NaDC + NaC

14 218�42 196�35 163 �2815 205�41 181�26 150 �2216 157 �9 151�19 141�1517 184�14 142�21 216 �34

The sensing properties of CD dimers 14–17 towards bilesalts sodium cholate (NaC) and sodium deoxycholate(NaDC) were studied using fluorescence spectroscopy. Asillustrated in Figure 3 (see also Supporting Information),the fluorescence intensity of CD dimers 14–17 decreasedwith stepwise addition of NaC and NaDC. The decrease influorescence intensity as concentration of bile salt increased

Figure 3. Top: Fluorescence spectra of compound 15 (2 μm, λexc = 325 nm) in the presence of increasing amounts of NaC (a) and NaDC(b) from 0 to 20 μm. The fluorescence intensity decreases with the increase of bile salt concentration. Bottom: Variation of the fluorescenceintensities of compounds 14 (♦), 15 (�), 16 (�), and 17 (�) at 2 μm with the concentration of NaC (c) and NaDC (d). The smooth solidlines represent the trend of the variation.


(Figure 3, A and B) is indicative of an increase in the po-larity of the environment around the spacer moiety.

The presence of bile salt thus appears to induce a higherexposure of the spacer to the solvent. Given the rigidity ofthe spacers, which prevents self-inclusion of the fluorescentmoiety, these results suggest that bile salt binding interac-tion with CD dimers involves a rearrangement of the aggre-gates, thus reducing the shielding of the fluorophore fromthe surrounding water.[56] Dynamic light scattering (DLS)measurements performed with bis(CD) 14–17 in the pres-ence of bile salts NaC and NaDC showed that the presenceof bile salt did not cause any appreciable change in the sizedistribution of the aggregates (see Table 1).

We carried out ITC experiments in 10 mm phosphatebuffer (pH 7.2) to gain a deeper insight into the bindingabilities of the CD dimers. Stepwise addition of aliquots ofa solution of bile salt (NaC or NaDC) to an aqueous solu-tion containing CD dimers 14–17 led to a decrease in theextent of released heat (see Figure 4 and the SupportingInformation).

ITC data can be fitted using a nonlinear least square al-gorithm based on different models, the simplest of whichconsiders n equal and independent binding sites. We as-sumed that the distance between the CD units in CD dimers14–17 is long enough that guest binding to one of the CDunits does not affect the guest binding to the other CDmoiety. The experimental data provided a good fit to thismodel, affording the desired thermodynamic parameters (n,ΔH, and K) (Figure 5 and Table 2). ITC data for the bind-


Figure 4. Titration of compound 17 with NaDC in 10 mm phos-phate buffer (pH 7.2) at 25 °C. The smooth solid lines representthe best fit of the experimental data to a model of n equal andindependent binding sites.

ing of NaC to CD dimers 15–17 showed that it was exother-mic with a unfavorable entropic contribution and affordedstable complexes with apparent K values of 3.9–9.0� 104

m–1.The ITC experiments performed with bis(CD) 14 did not

provide reliable data, most likely due to its low water solu-bility. ITC data showed that the complexation of CD di-mers 14–17 with NaDC are enthalpy-driven, along with an

Figure 5. Free energy (–ΔG0, black), enthalpy (–ΔH, grey), and entropy changes (TΔS0, white) for the binding of conjugates 14–17 toNaC (a) and NaDC (b) in 10 mm phosphate buffer (pH 7.2) at 25 °C.

Table 2. Apparent thermodynamic parameters for the binding of bis(CD) 14–17 to bile salts NaC and NaDC obtained from ITC experi-ments in 10 mm phosphate buffer (pH 7.2) at 25 °C.

Bile salt Bis(CD) �10–4 [m–1] –ΔG0 [kJ/mol] –ΔH [kJ/mol] –TΔS0 [kJ/mol] n

NaC 14 – – – – –15 9.0�0.5 28.0 �0.4 30.6�4.6 2.1 �0.8 0.5� 0.116 4.9�0.5 26.8 �0.4 37.7�5.9 10.9 �5.9 0.5�0.117 3.9�0.4 25.9 �0.3 33.9�7.9 7.5 �7.9 0.5�0.1

NaDC 14 6.1�0.6 27.2 �0.4 84.1 �20.1 56.9 �18.1 1.1 �0.215 16.3�1.1 29.7 �0.3 39.7�1.3 10.0 � 2.3 0.8 �0.116 18.8�0.8 30.1 �0.4 54.8�1.3 24.7 �5.3 0.6 � 0.117 8.4�0.7 28.0 �0.4 43.1�2.1 15.1 �4.1 1.0�0.1


unfavorable entropic contribution. The apparent affinity (Kvalues) varies as 16 �15� 17�14. Such K values are forthe NaDC binding to CD dimers 15 and 16 one order ofmagnitude higher than those for the binding to CD dimers14 and 17. As shown in Table 2, n values were 0.5 for thethree bis(CD) compounds, which means that the NaC·CDdimer complexes with 15–17 are formed with a 1:2 stoichi-ometry. Furthermore, the complexation of NaDC with CDdimers 15 and 16 gave n values of 0.8 and 0.6, respectively,whereas those with 14 and 17 gave n values close to 1. Thissuggests that, in some cases, supramolecular structures forhosts 14–17 either contain unbound CD units or CD cavi-ties that are for some reason unavailable, perhaps due tosteric hindrance caused by aggregation. By comparing theK values for each CD dimer, those for NaC are lower thanthose for NaDC, which is consistent with the behavior ofother reported CD dimers.[34,52]

To analyze the sensing abilities of compounds 14–17 inwater, the (I – I0)/I0 value was used as a sensitivity param-eter (Ps), where I is the fluorescence intensity of the com-plex and I0 is that of the host in the absence of guest. Thisparameter would enable the variation of the fluorescenceintensity induced by the interaction of the bile salt with theCD dimers to be evaluated. Unlike the stability constantvalue, which indicates the stability of the complex, the Ps

value reflects the nature of the environment in which thefluorophore group remains upon interaction with the guest.Thus, if the complex is very stable but the polarity of theenvironment in which the phenylene-ethynylene group re-mains unchanged, then Ps will be very low. In contrast, the

A. Vargas-Berenguel et al.FULL PAPERhigher the exposure of the phenylene-ethynylene moiety towater upon bile salt binding interaction, the higher the Ps

value will be.The sensitivity parameters Ps of CD dimers 14–17 for

bile salts NaC and NaDC are illustrated in Figure 6. ThePs values are negative in all cases because the I0 values forthe CD dimers are higher than I values in the presence ofthe bile salts. This behavior is consistent with the abovementioned higher exposure of the fluorophore moiety tothe bulk water. As seen in Figure 6, Ps values for all CDdimers 14–17 increase with the addition of bile salts NaC(Figure 6, A) and NaDC (Figure 6, B). The sequence of thesensing factors of the four CD dimers 14–17 for guestNaDC is 16� 14�15 �17 over the range of concentrationsof bile salts used. The sensitivity of CD dimer 16 towardslow to moderate concentrations of NaDC was notable. Theorder of sensitivity of the CD dimers 14–17 for NaC was16� 17 � 14� 15. Again, CD dimer 16 was the most sensi-tive host, although the sensitivity differences were higher athigher concentrations of bile salt. These facts indicate thatthe geometry of the phenylene-ethynylene moiety, whichmost likely influence the mode of self-aggregation, and thegroup at C-7 of the steroidal framework, affect the sensitiv-ity parameters.

In all cases, the CD dimers allow detection of NaC andNaDC at [bile salt]/[host] ratios lower than 1. When com-paring the obtained values with those reported for otherfluorescent β-cyclodextrin-based sensors, the sensing fac-tors of CD dimers 14–17 for NaC and NaDC are higher,

Figure 6. Sensitivity parameters (Ps) of CD dimers 14–17 towards bile salts NaC and NaDC. Top: Variation of sensitivity parametervalues [Ps, (I – I0)/I0] of compounds 14 (♦), 15 (�), 16 (�), and 17 (�) at 2 μm with the addition of increasing amounts of NaC (A) andNaDC (B) in 10 mm phosphate buffer (pH 7.2). The smooth solid lines represent the trend of the variation. Bottom: Ps values ofcompounds 14–17 (2 μm) in the presence of different concentrations of NaC and NaDC [1 (black), 7 (grey) and 20 (white) μm].


particularly in the case of CD dimer 16.[52,60,61] Further-more, the required concentrations of the host for providingreliable sensing parameters are lower than in most reportedcases.[52,59–61]

When the obtained sensitivity parameter values forNaDC and NaC were compared, we observed that whereasCD dimer 17 showed a similar sensitivity towards the twobile salts, CD dimers 14–16 were more selective towardsNaDC (Figure 6). Such selectivity increases with the con-centration of bile salts, which indicates that NaDC inducesa larger change in the fluorophore environment, and mostlikely a larger structural change in the mode of aggregationof compounds 14–16. These observations are consistentwith results obtained from ITC measurements discussedabove.

In addition to the high sensitivity of 16 towards guestsNaC and NaDC, as Figure 7 illustrates, upon gradual ad-dition of NaC and NaDC to CD dimer 16, the intensity ofthe emission bands at 348 and 364 nm significantly de-creased but that of the emission band at 463 nm slightlyincreased. The ratio of the emission intensities (I463/I348) in-creased as the concentration of NaC and NaDC was in-creased, rendering the supramolecular complex a sensitiveratiometric fluorescent sensor for both bile salts. Most fluo-rescent chemosensors are based on fluorescence measure-ments at a single wavelength, which may be influenced byvariations of the sample environment. However, wave-length-ratiometric probes are desirable because the ratiosare independent of the probe concentration. Thus, ratiomet-


Figure 7. Top: Fluorescence spectra of compound 16 (2 μm, λexc = 305 nm) in the presence of increasing amounts of NaC (A) and NaDC(B) ranging from 0 to 20 μm. The fluorescent intensities of the peaks at 348 and 364 nm decreased but the fluorescent intensity of thepeak at 463 nm increased as the concentration of bile salt was increased. Bottom: Variation of the ratio of the emission intensities (I463/I348) with the addition of increasing amounts of NaC (�) and NaDC (�) in 10 mm phosphate buffer (pH 7.2). The smooth solid linesrepresent the trend of the variation and do not correspond to a fit to a model.

ric fluorescent probes allow the measurement of emissionintensities at two wavelengths, which should provide a built-in correction for environmental effects. Therefore, the de-sign of ratiometric fluorescent sensors is of great currentinterest.[62] There are very few examples of cyclodextrin-based ratiometric fluorescence chemosensors, mostly for thedetection of ions,[63–66] and, to the best of our knowledge,CD dimer 16 is the first ratiometric fluorescent chemo-sensor for the detection of bile salts.

Conclusions

We have reported the synthesis of a series of CD dimersin which the two CD units are linked by rigid-rod phenyl-ene-ethynylene tethers of different length and shape throughthe CD secondary sides. The synthesis involves oxidativeSonogashira coupling reactions for the construction of thetethers and final grafting of the β-cyclodextrin units byclick chemistry. The resulting CD dimers self-aggregated inwater, forming polydisperse nanoparticles. Such CD di-meric nanoparticles formed stable complexes with sodiumcholate and deoxycholate, with the latter bile salt affordingstronger complexes. In addition, we demonstrated the highfluorescence sensing abilities of the synthesized CD dimerstoward those bile salts through the use of sensitivity param-eters. Finally, we have demonstrated that CD dimer 16 can


be used as a cyclodextrin-based ratiometric fluorescencesensor for the detection of sodium cholate and deoxychol-ate.

Experimental SectionGeneral Methods: TLC analysis was performed on Merck SilicaGel 60 F254 aluminium sheets and visualized by UV light, and bydeveloping with either ethanolic sulfuric acid (5% v/v) or ethanolicphosphomolybdic acid (7% w/v). Flash column chromatographywas performed on Merck Silica Gel (230–400 mesh, ASTM). Melt-ing points were measured with a Büchi B-450 melting point appara-tus and are uncorrected. Optical rotations were recorded with aJasco P-1030 polarimeter at room temperature. [α]D values aregiven in 10–1 deg cm2 g–1. IR spectra were recorded with a MattsonGenesis II FTIR spectrometer. 1H, 13C and 2D NMR spectra(gCOSY and gHSQC) were recorded with a Bruker AvanceDPX300 spectrometer equipped with a QNP 1H/13C/19F/31P probe.Standard Bruker software was used for acquisition and processingroutines. Chemical shifts are given in ppm and referenced to in-ternal TMS (δH and δC = 0.00 ppm); J values are given in Hz.HRMS (FAB) were recorded with a Micromass Autospec spec-trometer using glycerol as matrix. HRMS (ESI) were acquired withan Agilent Technologies LC/MSD-TOF spectrometer using purineas internal reference. MALDI-TOF mass spectra were recordedwith a Perspective Biosystems Voyager DR-RP spectrometer using2,5-dihydroxybenzoic acid as matrix. Pure water (MilliQ, 18.2MΩcm) was obtained from a Millipore MilliQ Plus system. β-

A. Vargas-Berenguel et al.FULL PAPERCyclodextrin was purchased from Fluka and dried at 50 °C invacuo in the presence of P2O5 until constant weight. Bile salts so-dium cholate (NaC) and sodium deoxycholate (NaDC) were pur-chased from Sigma and Fluka. These compounds were dried atroom temperature in vacuo in the presence of P2O5 for 24 h. Otherreagents were purchased from Aldrich and used as received. 2I-O-Propargyl-β-cyclodextrin (13) was prepared as reported.[34] Sol-vents were dried according to literature procedures.[67]

Fluorescence Measurements: UV absorption and fluorescence emis-sion were measured with a Perkin–Elmer LS-50B spectrophotome-ter at room temperature using a 1 cm conventional quartz cell. Ex-citation slits were 3 nm for all conjugates, and emission slits were3 nm for conjugates 14–16 and 9 nm in the case of 17. Excitationand emission wavelengths used were 300 and 363.1 nm for 14,325 and 368.1 nm for 15, 305 and 348.3 nm for 16, and 320 and363.3 nm for 17, respectively. The emission of conjugates 15–17were studied at concentrations varying from 2 to 50–100 μm inMilliQ water. A concentration of 2 μm was used for 14. These ex-periments were repeated for all conjugates (2 μm) in the presenceof increasing amounts of bile salts NaC and NaDC ranging from0 to 20 μm in 2 mm phosphate buffer (pH 7.2) by mixing stocksolutions of these compounds (4 μm for conjugates, 1 mm for bilesalts).

ITC Experiments: Isothermal titration calorimetry (ITC) experi-ments were performed with a MCS isothermal titration calorimeterfrom Microcal, Inc. (Northampton, MA).[68] Volumes of samplecell and injection syringe were 1.38 mL and 250 μL, respectively.The reference cell was filled with MilliQ water. All solutions wereprepared in 10 mm phosphate buffer (pH 7.2) and degassed for10 min under vacuum prior to the titration experiments. Duringthe titrations, the reaction mixture was continuously stirred at400 rpm. The raw experimental data are presented as the amountof heat produced per second following each injection (correctedfor the bile salts heats of dilution in the case of the interactionexperiments) as a function of time. The amount of heat producedper injection was calculated by integration of the area under indi-vidual peaks by using the Origin software. The errors are providedfrom the best fit of the experimental data to the most suitablemodel in each case, and corresponds to the standard deviation inthe fitting of the curves.

For the dilution experiments, solutions of conjugates 15 (450 μm)and 17 (520 μm) were injected into buffer solution in 10 μL por-tions. Experimental data were fitted to a simple dissociation model.

For the binding experiments, solutions of bile salts NaC and NaDC(1–2 mm) were injected into solutions of conjugates 15–17 (25 μm)in 5 μL portions. A 7 μm solution was used in the case of 14 dueto its low water solubility. The dilution profiles, under identicalexperimental conditions, were obtained by injecting each bile saltinto buffer solution. The dilution heats were similar to the heatsignals detected after the saturation was reached and were sub-tracted from heats detected during the binding experiment. Amodel of n equal and independent sites was adequate to fit theexperimental data. From measurements of K, the free energy ofbinding, ΔG°, could be calculated and hence the entropy of bind-ing, ΔS°, determined from ΔG° = ΔH – TΔS° = –RT ln K. Thecalculation of thermodynamic functions implies the usual approxi-mation of setting standard enthalpies equal to the observed values.

DLS Measurements: Dynamic light scattering (DLS) measurementswere conducted with a Zetasizer nanoseries Nano-ZS-ZEN3600 in-strument equipped with a laser of 633 nm using a ZEN-112 cuvetteat 15 °C. For each measurement the number of scans was 25, therun duration was 35 s, the equilibration time was 140 s, and the


time delay was 4 s. Before measurements, the samples were incu-bated for 2 h, then filtered through a 0.45 μm nylon filter, centrifu-gated (15000 rpm, 45 min), and finally sonicated for 1 min.

Synthesis of Bis[(aminophenyl)ethynyl]benzenes 5–8: Exhaustive de-gassing of reaction mixtures were performed three times by apply-ing high vacuum for 5 min after freezing with liquid N2.

1,4-Bis[(4�-aminophenyl)ethynyl]benzene (5): A solution of p-iodoaniline (170 mg, 0.761 mmol), [Pd(PPh3)2Cl2] (6 mg,0.008 mmol), and CuI (6 mg, 0.031 mmol) in anhydrous piperidine(3 mL) was exhaustively degassed. 1,4-Diethynylbenzene (50 mg,0.380 mmol) was then added under a H2/N2 (1:1) atmosphere andthe mixture was stirred for 1.5 h. The solvent was removed by evap-oration under vacuum and the crude material was purified by col-umn chromatography (CH2Cl2/hexane, 5:1) to yield 5 (100 mg,85%) as a pale-yellow solid. NMR spectroscopic data for this com-pound were consistent with those reported in the literature.[53,54]

1,3-Bis[(4�-aminophenyl)ethynyl]benzene (6): A solution of p-iodoaniline (170 mg, 0.761 mmol), [Pd(PPh3)2Cl2] (6 mg,0.008 mmol), and CuI (6 mg, 0.031 mmol) in anhydrous piperidine(3 mL) was exhaustively degassed. 1,3-Diethynylbenzene (50 μL,0.365 mmol) was then added under a H2/N2 (1:1) atmosphere andthe mixture was stirred at room temp. for 3.5 h. The solvent wasremoved by evaporation under vacuum and the crude material waspurified by column chromatography (CH2Cl2/hexane, 5:1) to yield6 (105 mg, 93%) as a pale-yellow solid; m.p. 183 °C (dec.). IR(KBr): νmax = 3444, 3340, 1604, 1582, 813 and 692 cm–1. 1H NMR(300 MHz, CDCl3): δ = 7.64 (t, 4J2,4 = 1.8 Hz, 1 H, 2-H), 7.42 (dd,3J4,5 = 7.7, 4J2,4 = 1.8 Hz, 2 H, 4-H), 7.35 (d, 3J2�,3� = 8.8, 4 H, 2�-H), 7.29 (t, 3J4,5 = 7.7 Hz, 1 H, 5-H), 6.65 (d, 3J2�,3� = 8.8 Hz, 4 H,3�-H), 3.85 (br. s, 4 H, NH2) ppm. 13C NMR (75 MHz, CDCl3): δ= 146.9 (C-4), 134.2 (C-2), 133.2 (C-2�), 130.6 (C-5), 128.4 (C-1),124.3 (C-2), 114.9 (C-3�), 112.6 (C-1�), 90.7, 86.9 (C�C) ppm.HRMS (FAB): calcd. for [C22H16N2 + H]+ 309.1392; found309.1391.

1,4-Bis[(3�-aminophenyl)ethynyl]benzene (7): A solution of[Pd(PPh3)2Cl2] (9 mg, 0.013 mmol) and CuI (9 mg, 0.046 mmol) inanhydrous piperidine (4 mL) was exhaustively degassed. m-Iodoan-iline (145 μL, 1.195 mmol) and 1,4-diethynylbenzene (75 mg,0.571 mmol) were then added under a H2/N2 (1:1) atmosphere andthe mixture was stirred at room temp. for 3.5 h. The solvent wasremoved by evaporation under vacuum and the crude material waspurified by column chromatography (CH2Cl2/hexane, 5:1 � 10:1)to yield 7 (164 mg, 89 %) as a pale-yellow solid; m.p. 182 °C (dec.).IR (KBr): νmax = 3434, 3349, 1614, 1599, 1578, 836, 786 and685 cm–1. 1H NMR (300 MHz, CDCl3): δ = 7.48 (s, 4 H, 2-H), 7.14(t, 3J = 7.8 Hz, 2 H, 5�-H), 6.95 (d, 3J5�,6� = 7.7 Hz, 2 H, 6�-H),6.86 (s, 2 H, 2�-H), 6.67 (d, 3J4�,5� = 7.8 Hz, 2 H, 4�-H), 3.70 (br. s,4 H, NH2) ppm. 13C NMR (75 MHz, CDCl3): δ = 146.5 (C-3�),131.7 (C-2), 129.5 (C-5�), 123.9, 123.3 (C-1,1�), 122.3 (C-6�), 117.9(C-2�), 115.7 (C-4�), 91.6, 88.7 (C�C) ppm. HRMS (FAB): calcd.for [C22H16N2 + H]+ 309.1392; found 309.1391.

1,3-Bis[(3�-aminophenyl)ethynyl]benzene (8): A solution of[Pd(PPh3)2Cl2] (6 mg, 0.008 mmol) and CuI (6 mg, 0.032 mmol) inanhydrous piperidine (3 mL) was exhaustively degassed. m-Iodoan-iline (100 mL, 0.814 mmol) and 1,3-diethynylbenzene (50 μL,0.365 mmol) were then added under a H2/N2 (1:1) atmosphere andthe mixture was stirred at room temp. for 6.5 h. The solvent wasremoved by evaporation under vacuum and the crude material waspurified by column chromatography (CH2Cl2/hexane, 5:1) to yield8 (105 mg, 90%) as a pale-yellow solid; m.p. 105 °C (dec.). IR(KBr): νmax = 3421, 3347, 1617, 1596, 1576, 870, 790 and 684 cm–1.1H NMR (300 MHz, CDCl3): δ = 7.73 (s, 1 H, 2-H), 7.50 (d, 3J4,5


= 7.8 Hz, 2 H, 3�-H), 7.33 (t, 3J4,5 = 7.8 Hz, 1 H, 5-H), 7.17 (t, 3J= 7.7 Hz, 2 H, 5�-H), 6.99 (d, 3J5�,6� = 7.5 Hz, 2 H, 6�-H), 6.87 (s,2 H, 2�-H), 6.68 (d, 3J4�,5� = 8.0 Hz, 2 H, 4�-H), 3.64 (br. s, 4 H,NH2) ppm. 13C NMR (75 MHz, CDCl3): δ = 146.4 (C-3�), 134.6(C-2), 131.2 (C-4), 129.4 (C-5�), 128.5 (C-5), 123.7, 123.7 (C-1,1�),120.1 (C-6�), 117.8 (C-2�), 115.6 (C-4�), 90.4, 88.1 (C�C) ppm.HRMS (FAB): calcd. for [C22H16N2 + H]+ 309.1392; found309.1391.

General Procedure for the Synthesis of Bis[(azidophenyl)ethynyl]-benzenes 9–12: Bis[(aminophenyl)ethynyl]benzenes 5–8 (50 mg,0.162 mmol) were suspended in a 15% (w/w) HCl solution (3.3 mL)at 0 °C. A solution of NaNO2 (31 mg, 0.435 mmol) in water(2.5 mL) was added dropwise and the mixture was stirred at thesame temperature for 50 min. A solution of NaN3 (27 mg,0.415 mmol) in water (2.5 mL) was then added dropwise and themixture was stirred at 0 °C for 45 min in the dark. The resultingprecipitate was filtered off and washed with water to yield the cor-responding bis[(azidophenyl)ethynyl]benzene as a solid. Com-pounds 9–12 are photosensitive.

1,4-Bis[(4�-azidophenyl)ethynyl]benzene (9): Starting from 5, use ofthe general procedure yielded 9 (48 mg, 82%) as a pale-orange so-lid; m.p. 151 °C. IR (KBr): νmax = 2123, 1592, 1510, 1297, 1261,1100, 1064, 833, 799, 525 cm–1. 1H NMR (300 MHz, [D8]THF): δ= 7.57 (d, 3J2�,3� = 8.7 Hz, 4 H, 2�-H), 7.54 (s, 4 H, 2-H), 7.14 (d,3J2�,3� = 8.7 Hz, 4 H, 3�-H) ppm. 13C NMR (300 MHz, [D8]THF):δ = 138.5 (C-4�), 131.0, 129.4 (C-2,2�), 121.2, 117.6 (C-1,1�), 117.2(C-3�), 88.6, 87.0 (C�C) ppm. HRMS (ESI): calcd. for[C22H12N6 – N2 + 3 H]+ 335.1297; found 335.1292.

1,3-Bis[(4�-azidophenyl)ethynyl]benzene (10): Starting from 6, use ofthe general procedure yielded 10 (46 mg, 79%) as a pale-orangesolid; m.p. 165 °C. IR (KBr): νmax = 2131, 1586, 1503, 1309, 1284,832, 796, 685, 532 cm–1. 1H NMR (300 MHz, CDCl3): δ = 7.69 (s,1 H, 2-H), 7.52 (d, 3J2�,3� = 8.5 Hz, 4 H, 2�-H), 7.48 (d, 3J4,5 =7.8 Hz, 2 H, 4-H), 7.34 (t, 3J4,5 = 7.8 Hz, 1 H, 5-H), 7.02 (d, 3J2�,3�

= 8.5 Hz, 4 H, 3�-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 140.3(C-4�), 134.6 (C-2), 133.3 (C-2�), 131.4 (C-4), 128.7 (C-5), 123.6 (C-1), 119.7 (C-1�), 119.3 (C-3�), 89.5, 89.0 (C�C) ppm. HRMS (ESI):calcd. for [C22H12N6 – N2 + 3 H]+ 335.1297; found 335.1292.

1,4-Bis[(3�-azidophenyl)ethynyl]benzene (11): Starting from 7, use ofthe general procedure yielded 11 (54 mg, 93%) as a pale-orangesolid; m.p. 168 °C. IR (KBr): νmax = 2128, 1593, 1566, 1470, 1425,1319, 1295, 889, 839, 793 cm–1. 1H NMR (300 MHz, CDCl3): δ =7.52 (s, 4 H, 2-H), 7.38–7.28 (m, 4 H, 5�,6�-H), 7.21–7.18 (m, 2 H,2�-H), 7.01 (dt, 3J4�,5� = 7.2, 4J = 1.9 Hz, 2 H, 4�-H) ppm. 13C NMR(75 MHz, CDCl3): δ = 140.5 (C-3�), 131.8 (C-2), 129.9, 128.3 (C-5�,6�), 124.8, 123.1 (C-1�,1), 122.0 (C-2�), 119.4 (C-4�), 90.4, 90.1(C�C) ppm. HRMS (ESI): calcd. for [C22H12N6 – N2 + 3 H]+

335.1297; found 335.1292.

1,3-Bis[(3�-azidophenyl)ethynyl]benzene (12): Starting from 8, use ofthe general procedure yielded 12 (48 mg, 82%) as a pale-orangesolid; m.p. 139 °C. IR (KBr): νmax = 2127, 1598, 1565, 1480, 1424,1315, 1295, 1248, 896, 785, 681 cm–1. 1H NMR (300 MHz, CDCl3):δ = 7.74 (s, 1 H, 2-H), 7.52 (d, 3J4,5 = 7.9 Hz, 2 H, 4-H), 7.40–7.29(m, 5 H, 5,5�,6�-H), 7.22–7.19 (m, 2 H, 2�-H), 7.02 (dt, 2 H, 3J4�,5�

= 7.3, 4J = 2.0 Hz, 4�-H) ppm. 13C NMR (75 MHz, CDCl3): δ =140.5 (C-3�), 134.9 (C-2), 131.8 (C-4), 129.9, 128.7, 128.3 (C-5,5�,6�), 124.8, 123.4 (C-1,1�), 122.0 (C-2�), 119.4 (C-4�), 89.4, 89.1(C�C) ppm. HRMS (ESI): calcd. for [C22H12N6 – N2 + 3 H]+

335.1297; found 335.1292.

Synthesis of Bis[(4�-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-1-yl}phenyl)ethynyl]benzenes 14–17


1,4-Bis[(4�-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-1-yl}phenyl)ethynyl]benzene (14): CuI·(EtO)3P (10 mg, 0.028 mmol)was added to a solution of 9 (25 mg, 0.069 mmol) and 13 (171 mg,0.146 mmol) in anhydrous DMF (6 mL) under N2 atmosphere andthe mixture was stirred at 105 °C for 1.5 h. The solution waspoured into acetone (100 mL) and the resulting precipitate was fil-tered off, dissolved in water, and freeze dried. The solid was washedwith methanol using a Soxhlet extraction apparatus (overnight) toyield 14 (132 mg, 71%) as a pale-yellow solid; m.p. 263 °C (dec.).[α]D = +90 (c = 0.25 in DMF). IR (KBr): νmax = 3396, 2924, 1644,1522, 1156, 1080, 1028 cm–1. 1H NMR (300 MHz, [D6]DMSO): δ= 8.92 (s, 2 H, 5-H-C2HN3), 8.02 (d, 3J2�,3� = 8.3 Hz, 4 H, 3�-H),7.83 (d, 3J2�,3� = 8.3 Hz, 4 H, 2�-H), 7.68 (s, 4 H, 2-H), 6.00–5.90(m, 4 H, OH), 5.85–5.60 (m, 20 H, OH), 5.10–4.75 (m, 18 H, 1I–VII-H, CH2O), 4.60–4.40 (m, 16 H, OH), 3.88 (t, 3J = 8.8 Hz, 2 H, 3I-H), 3.70–3.49 (m, 59 H, 2I,3II–VII,4I,5I–VII,6I–VII,6�I–VII-H), 3.48–3.20 (m, 2II–VII,4II–VII-H, overlapped with HDO) ppm. 13C NMR(75 MHz, [D6]DMSO): δ = 145.2 (C-4-C2HN3), 136.4 (C-4�), 133.1(C-3�), 131.9 (C-2), 122.4 (C-5-C2HN3), 122.4, 122.1 (C-1,1�), 120.2(C-2�), 102.0–101.7 (C-1II–VII), 100.1 (C-1I), 90.5, 90.3 (C�C),82.0–81.4 (C-4I–VII), 79.7 (C-2I), 73.2–71.7 (C-2II–VII,3I–VII,5I–VII),64.3 (CH2O), 60.1–59.8 (C-6I–VII) ppm. MALDI-TOF-MS: calcd.for [C112H156N6O70 + Na + 2 H]+ 2729.9 and [C112H156N6O70 – N4

+ Na + 4 H]+ 2675.9; found 2729.5 and 2675.6.

1,3-Bis[(4�-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-1-yl}phenyl)ethynyl]benzene (15): CuI·(EtO)3P (10 mg, 0.028 mmol)was added to a solution of 10 (25 mg, 0.069 mmol) and 13 (171 mg,0.146 mmol) in anhydrous DMF (6 mL) under N2 atmosphere andthe mixture was stirred at 105 °C for 4.5 h. The solution waspoured into acetone (100 mL) and the resulting precipitate was fil-tered off, dissolved in water, and freeze dried. The crude materialwas purified by column chromatography (MeCN/water, 2:1) toyield 15 (121 mg, 65%) as a pale-yellow solid; m.p. 283 °C (dec.).[α]D = +102 (c = 0.25 in DMF). IR (KBr): νmax = 3383, 2926, 1638,1595, 1155, 1040, 1027 cm–1. 1H NMR (300 MHz, [D6]DMSO): δ= 8.92 (s, 2 H, 5-H-C2HN3), 8.03 (d, 3J2�,3� = 8.5 Hz, 4 H, 3�-H),7.83 (d, 3J2�,3� = 8.5 Hz, 4 H, 2�-H), 7.84 (s, 1 H, 2-H), 7.68 (d, 3J4,5

= 7.3 Hz, 2 H, 4-H), 7.56 (t, 3J4,5 = 7.3 Hz, 1 H, 5-H), 6.00–5.90(m, 4 H, OH), 5.85–5.60 (m, 20 H, OH), 5.05–4.70 (m, 18 H, 1I–VII-H, CH2O), 4.60–4.40 (m, 16 H, OH), 3.88 (t, 3J = 8.8 Hz, 2 H, 3I-H), 3.70–3.49 (m, 59 H, 2I,3II–VII,4I,5I–VII,6I–VII,6�I–VII-H), 3.45–3.20 (m, 2II–VII,4II–VII-H, overlapped with HDO) ppm. 13C NMR(75 MHz, [D6]DMSO): δ = 145.2 (C-4-C2HN3), 136.4 (C-4�), 134.1(C-2), 133.1 (C-2�), 132.6 (C-1), 131.9 (C-4), 129.6 (C-5), 122.4 (C-5-C2HN3), 122.1 (C-1�), 120.2 (C-3�), 102.0–101.6 (C-1II–VII), 100.1(C-1I), 89.7, 89.2 (C�C), 82.0–81.3 (C-4I–VII), 79.6 (C-2I), 73.1–71.7 (C-2II–VII,3I–VII,5I–VII), 64.3 (CH2O), 60.1–59.8 (C-6I–VII) ppm.MALDI-TOF-MS: calcd. for [C112H156N6O70 + Na + 2 H]+ 2729.9and [C112H156N6O70 – N4 + Na + 4 H]+ 2675.9; found 2729.5 and2675.6.

1,4-Bis[(3�-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-1-yl}phenyl)ethynyl]benzene (16): CuI·(EtO)3P (10 mg, 0.028 mmol)was added to a solution of 11 (25 mg, 0.069 mmol) and 13 (171 mg,0.146 mmol) in anhydrous DMF (6 mL) under N2 atmosphere andthe mixture was stirred at 105 °C for 3.5 h. The solution waspoured into acetone (100 mL) and the resulting precipitate was fil-tered off, dissolved in water, and freeze dried. The solid was washedwith methanol using a Soxhlet extraction apparatus (overnight) toyield 16 (125 mg, 67%) as a pale-yellow solid; m.p. 251 °C (dec.).[α]D = +121 (c = 0.25 in DMF). IR (KBr): νmax = 3388, 2925, 1642,1606, 1157, 1080, 1027 cm–1. 1H NMR (300 MHz, [D6]DMSO): δ= 8.75 (s, 2 H, 5-H-C2HN3), 8.09 (s, 2 H, 2�-H), 8.00–7.90 (m, 2H, 4�-H), 7.80–7.60 (m, 8 H, 2,5�,6�-H), 6.00–5.90 (m, 4 H, OH),

A. Vargas-Berenguel et al.FULL PAPER5.80–5.60 (m, 20 H, OH), 5.10–4.80 (m, 18 H, 1I–VII-H, CH2O),4.60–4.40 (m, 16 H, OH), 3.88 (t, 3J = 8.8 Hz, 2 H, 3I-H), 3.75–3.45 (m, 59 H, 2I,3II–VII,4I,5I–VII,6I–VII,6�I–VII-H), 3.45–3.20 (m,2II–VII,4II–VII-H, overlapped with HDO) ppm. 13C NMR (75 MHz,[D6]DMSO): δ = 145.1 (C-4-C2HN3), 136.9 (C-3�), 132.0, 131.9,131.5 (C-2,5�,6�), 130.6 (C-1�), 123.6 (C-1), 122.5, 122.4 (C-4�, C5-C2HN3), 120.6 (C-2�), 102.0–101.6 (C-1II–VII), 100.1 (C-1I), 90.3,90.1 (C�C), 82.0–81.3 (C-4I –V II ), 79.6 (C-2I) , 73.2–71.7(C-2II–VII,3I–VII,5I–VII), 64.3 (CH2O), 60.0–59.8 (C-6I–VII) ppm.MALDI-TOF-MS: calcd. for [C112H156N6O70 + Na + 2 H]+ 2729.9and [C112H156N6O70 – N4 + Na + 4 H]+ 2675.9; found 2729.5 and2675.6.

1,3-Bis[(3�-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-1-yl}phenyl)ethynyl]benzene (17): CuI·(EtO)3P (10 mg, 0.028 mmol)was added to a solution of 12 (25 mg, 0.069 mmol) and 13 (171 mg,0.146 mmol) in anhydrous DMF (6 mL) under N2 atmosphere andthe mixture was stirred at 105 °C for 7.5 h. The solution waspoured into acetone (100 mL) and the resulting precipitate was fil-tered off, dissolved in water, and freeze dried. The crude materialwas purified by column chromatography (MeCN/water, 5:2) toyield 17 (120 mg, 63%) as a pale-yellow solid; m.p. 273 °C (dec.).[α]D = +98 (c = 0.25 in DMF). IR (KBr): νmax = 3379, 2928, 1639,1608, 1175, 1006, 1029 cm–1. 1H NMR (300 MHz, [D6]DMSO): δ= 8.93 (s, 2 H, 5-H-C2HN3), 8.16 (s, 2 H, 2�-H), 8.04–7.97 (m, 2H, 4�-H), 7.87 (s, 1 H, 2-H), 7.74–7.65 (m, 6 H, 4,5�,6�-H), 7.56 (t,3J4,5 = 7.7 Hz, 1 H, 5-H), 6.05–5.90 (m, 4 H, OH), 5.85–5.60 (m,20 H, OH), 5.10–4.75 (m, 18 H, 1I–VII-H, CH2O), 4.51–4.49 (m,16 H, OH), 3.87 (t, 3J = 8.8 Hz, 2 H, 3I-H), 3.70–3.45 (m, 59 H,2I,3II–VII,4I,5I–VII,6I–VII,6�I–VII-H), 3.44–3.20 (m, 2II–VII,4II–VII-H,overlapped with HDO) ppm. 13C NMR (75 MHz, [D6]DMSO): δ= 145.0 (C-4-C2HN3), 136.8 (C-2), 134.2 (C-3�), 132.1, 131.5, 130.6,129.6, 129.6 (C-1,4,5,5�,6�), 123.6 (C-5-C2HN3), 122.6, 122.5 (C-1�,2�), 120.6 (C-4�), 101.9–101.7 (C-1II–VII), 100.1 (C-1I), 89.5, 89.0(C�C), 82.0–81.3 (C-4I–VII), 79.9 (C-2I), 73.2–71.7 (C-2II–VII,3I–-

VII,5I–VII), 64.3 (CH2O), 60.1–59.8 (C-6I–VII) ppm. MALDI-TOF-MS: calcd. for [C112H156N6O70 + Na]+ 2727.9 and[C112H156N6O70 – N4 + Na + 2 H]+ 2673.9; found 2727.2 and2673.7.

Supporting Information (see footnote on the first page of this arti-cle): Fluorescence titrations, ITC titrations, 13C NMR spectra ofcompounds 6–8, 10–12, and 14–17, and DLS measurements.

Acknowledgments

We acknowledge the Andalusian Government (Consejería de Econ-omía, Innovaciónn y Ciencia, Junta de Andalucía) and the EUEuropean Regional Development Fund (FQM3141 and in partFQM06903). Financial support by the EU through a Marie CurieITN program (CYCLON 237962) and the Spanish Ministry ofEducation (PhD fellowship to M. C. M.-M.) is also acknowledged.

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Published Online: March 21, 2012

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