Secondary face-to-face 2-2' β-cyclodextrin dimers linked with fluorescent rigid spacer arms: A cyclodextrin-based ratiometric sensor for bile salts

Article abstract of DOI:10.1002/ejoc.201101598

The synthesis of a series of β-cyclodextrin (CD) dimers in which the two macrocycle units are linked by rigid-rod phenylene-ethynylene tethers of different length and shape through the cyclodextrin secondary sides is reported. The synthesis involves Sonog

Full text of DOI:10.1002/ejoc.201101598

FULL PAPER
DOI: 10.1002/ejoc.201101598
Secondary Face-to-Face 2–2Ј β-Cyclodextrin Dimers Linked with Fluorescent
Rigid 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 in
which the two macrocycle units are linked by rigid-rod phen-
ylene-ethynylene tethers of different length and shape
through the cyclodextrin secondary sides is reported. The
formed polydisperse nanoparticles of 240 to 480 nm in water
solution depending on the tether shape, and exhibited fluo-
rescence. The binding and fluorescence sensing abilities of
the CD dimers toward sodium cholate and deoxycholate
synthesis involves Sonogashira coupling reactions for con- were studied. As a result, the newly synthesized rigid-rod-
struction of the tethers and final grafting of the CD units by
click chemistry. The self-aggregation of the synthesized CD
dimers in aqueous medium was investigated by fluorescence
spectroscopy, isothermal titration calorimetry and dynamic
light scattering. Rigid-rod-tethered cyclodextrin dimers
tethered cyclodextrin dimers proved to be very sensitive fluo-
rescent chemosensors. A new cyclodextrin-based ratiometric
fluorescence 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 abilities
and molecular selectivity as compared with the native
CDs.[14,15] In addition to the cooperative binding through
which the two adjacent CD cavities of these bridged
bis(CD)s can encapsulate guest molecules, the linker can
contribute to the stabilization of such complexes by provid-
ing additional binding interactions or contributing to ex-
tending the overall binding site. The face through which
the CD moieties are bridged influences the supramolecular
behavior of the CD dimers towards ditopic guests. CD units
can be linked through their primary faces, i.e., 6–6Ј
bis(CD), through a head-to-tail fashion, i.e., 6–2Ј or 6–3Ј
bis(CD), and through their secondary faces, i.e., 2–2Ј or 3–
3Ј bis(CD). The supramolecular behavior of 6–6Ј bis(CD)
has been extensively studied. Such studies claim that 6–6Ј
bis(CD) form a sandwich-like supramolecular structure of
1:1 stoichiometry with guest molecules or, alternatively, n:n
supramolecular assemblies involving intermolecular co-
operative binding. The latter consists of a linear assembly
in 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 6–6Ј bis(CD)s con-
tain short-length linkers, whereas 6–6Ј bis(CD)s having
long-length linkers prefer to form 1:1 complexes
(Scheme 1).[14–17]
Introduction
Cyclodextrins (CD) are a group of structurally related
cyclic oligosaccharides that consist of d-glucopyranose
units linked by α-(1Ǟ4) bonds. Natural α-, β- and γ-CDs
contain six, seven, and eight units of such carbohydrate
units, respectively. They possess a relatively rigid torus-
shaped structure that defines an inner hydrophobic cavity
rimmed by two hydrophilic openings. These openings are
different in diameter, the narrower end, containing primary
hydroxy groups located on the C-6 of the glucopyranose
units, and the wider end, containing secondary hydroxy
groups on the C-2 and C-3 positions. CDs are well-known
to form inclusion complexes in aqueous solution with a
large variety of organic molecules of hydrophobic nature
and suitable size and geometry.[1–3] Because of this ability
to behave as a host, CDs find a wide range of applications
in 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 CD
units tethered by linkers of different nature have received
[a] Area of Organic Chemistry, University of Almeria,
Crta. de Sacramento s/n, 04120 Almería, Spain
Fax: +34-950-015481
E-mail: avargas@ual.es
Interestingly, the least documented 2–2Ј bis(CD)s appear
to have a different supramolecular behavior towards ditopic
guests.[18–35] Secondary-face-linked β-CD dimers often
show higher complexing constants than those bridged by
primary faces,[18–21,35] probably due to the fact that guest
[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 Kingdom
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101598.
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A Cyclodextrin-Based Ratiometric Sensor for Bile Salts
dimers towards multitopic guests such as bile salts sodium
cholate (NaC) and deoxycholate (NaDC). Bile salts are im-
portant naturally occurring surfactant steroid compounds
that play a significant role in the metabolism and excretion
of cholesterol in mammals. Moreover, they are used as
drugs for nonsurgical treatment for gallstone diseases, and
other pharmacological applications are under evaluation.[41]
Studies on the guest binding abilities of bile salts towards
6–6Ј CD dimers have been widely undertaken,[42–51] al-
though to the best of our knowledge only a few 2–2Ј CD
dimers 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 slightly
in the number of hydroxyl groups (Scheme 2).
Scheme 1. Schematic representation of β-CD.
molecules preferentially penetrate the cavity through the
wider secondary rim.[1–3] To the best of our knowledge, no
2–2Ј CD dimers have shown a tendency to form supra-
molecular polymers.
The linker that tethers the two CD units may help to
provide functional properties such as a sensing ability. In-
deed, chemical sensors based on cyclodextrin derivatives
have 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 chemical
sensors use CD derivatives with an appended photoactive
moiety, with fluorescent-based sensors occupying a promi-
nent place among the optical sensors. In most cases, the
sensing ability of CD-derived chemical sensors is based on
a competition between self-inclusion of the active moiety
and 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 to
Scheme 2. Bile salts used as guests.
Herein, we also describe a cyclodextrin-based ratiometric
fluorescence sensor for the detection of NaC and NaDC.
To the best of our knowledge, this is the first ratiometric
fluorescence sensor developed for the detection of bile salts
(Scheme 2).
Results and Discussion
We have reported the one-step synthesis of mono-2-O-
the bulk aqueous media causes a fluorescence intensity de- propargyl-β-CD (13), which is a very convenient building
crease. Alternatively, so-called “turn-on” fluorescent chemi- block for the construction of β-CD compounds function-
cal sensors, having the fluorophore appended to CD alized on the secondary face. The terminal alkyne group of
through a rigid spacer, have been reported in which fluores- 13 linked to the macrocycle at O-2 offers the possibility of
cence intensity increased upon host–guest complex forma- coupling functional structures by using CuI-catalyzed alk-
tion.[10]
yne–azide cycloaddition (CuAAC)[34] or oxidative coupling
Herein, we report the preparation of a series of CD di- reactions.[55] Thus, we performed the synthesis of bis-
mers in which the two CD units are linked by rigid-rod [(azidophenyl)ethynyl]benzene derivatives 9–12 containing
phenylene-ethynylene tethers of different length and shape suitable azide groups for the coupling of CD derivative 13
through the CD secondary sides. Previously, we have inves- by CuAAC. Oxidative coupling of diethynylbenzenes 2 and
tigated the supramolecular behavior of 2–2Ј CD dimers 3 with iodoanilines 1 and 4 in piperidine at room tempera-
linked with short and rigid spacers towards multitopic ture, using [Pd(PPh3)2Cl2] and CuI as catalysts under an
guests such as bile salts and observed a distinctive behavior atmosphere of hydrogen gas diluted with nitrogen, gave
with respect to their 6–6Ј counterpart.[35] The rigid rod-like bis[(3Ј-aminophenyl)ethynyl]benzenes 5–8 in 85–93% yields.
character and conjugation of the oligo- and poly(phen- These reaction conditions led to an improved synthesis of
yleneethynylene) compounds are responsible of their inter- compound 5 that had previously been synthesized in much
esting electronic and photophysical properties,[36–38] how- lower yield.[53,54] Diamine compounds 5–8 were then trans-
ever, the strong π–π interactions between the tether chains formed through aromatic nucleophilic substitution of the
also results in a tendency to form aggregates.[39,40]
corresponding diazonium salts into diazide derivatives 9–
In addition to providing a rigid linker that prevents self- 12 in 79–93% yield. Finally, the coupling of CD 13 with
inclusion into the CD cavity, the phenylene-ethynylene bis[(azidophenyl)ethynyl]benzenes 9–12 was performed in
tether provides a fluorescent moiety and therefore potential N,N-dimethylformamide (DMF) at 105 °C with catalytic
sensing properties. We have investigated the sensing proper- amounts of (EtO)3P·CuI, generating the phenylene-eth-
ties of the newly synthesized fluorescent amphiphilic CD ynylene-tethered bis(CD) compounds 14–17. Compounds
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14–17 were very insoluble in solvents other than dimethyl compound 13 and the appearance a singlet at δ = 8.75–
sulfoxide (DMSO) and DMF. The more water-soluble com- 8.93 ppm corresponding to the 1,2,3-triazole proton.
pounds 15 and 17 were purified by column chromatog- Analysis of the HSQC spectra for compounds 14–17
raphy, isolating the bis(CD) derivatives in 65 and 63% yield, showed that the triazole methine carbon atoms resonate at
respectively. Compounds 14 and 16 were purified by soxhlet δ = 122.4–123.6 ppm. Such signals as well as those at δ =
extraction with methanol, thus removing unreacted mono- 145.0–145.6 ppm shown by the 13C NMR spectra and as-
propargyl 2I-O-β-cyclodextrin 13 and isolating the bis(CD) signed to the triazole C-4, confirm the presence of such a
derivatives in 71 and 67% yield, respectively.
ring in the molecule (Scheme 3).
The molecular weights of compounds 14–17 were veri-
Given the potential of the phenylene-ethynylene moieties
fied by MALDI-TOF mass spectrometry and they were to aggregate due to π–π interactions, we investigated the
fully characterized by NMR spectroscopic techniques em- self-aggregation properties by fluorescence spectroscopy.
1
ploying H and 13C NMR, COSY and HSQC experiments. The fluorescence spectra of compounds 14–17 measured in
1
Inspection of H NMR spectra showed the absence of a water solutions (see Figure S1 in the Supporting Infor-
signal at δ = 3.52 ppm, corresponding to alkyne proton of mation) show a band at 365 nm corresponding to emission
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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A Cyclodextrin-Based Ratiometric Sensor for Bile Salts
from the phenylene-ethynylene core. Compound 16 also has dilution. ITC dilution experiments with 15 (450 μm) and 17
an emission at 463 nm that may be assigned to the emission (520 μm) gave rise to a series of endothermic heat pulses
of phenylene-ethynylene excimer due to π–π interactions be- that decreased in a nonlinear fashion as the concentration
tween adjacent phenylene-ethynylene groups as a result of of such compounds increased in the calorimetric cell (Fig-
the CD dimer aggregation. Careful observation of the emis- ure 2). These results agree with the fluorescence experi-
sion spectrum for 14 reveals a very slight shoulder at ments described above and suggest that both CD dimers
around 460 nm that could be due to an excimer emission. are involved in a self-association equilibrium. The resulting
Both compounds 14 and 16 share a linear phenylene-ethyn- thermal dilution profile is consistent with the simplest
ylene tether but the latter has the two CD units attached at n2 Ǟ2n dissociation model.[34] Therefore, analysis of the
the meta position of the phenyl ring through a triazole moi- ITC data by nonlinear regression procedures provided ag-
ety, instead of the para position as in compound 14. Such gregation constant (Kagg
)
values of 1.9ϫ104 and
emission at around 460 nm is not observed for 15 and 17, 4.4ϫ103 m–1 for 15 and 17, respectively. However, when
both of which share a nonlinear tether. Compounds 14 and similar ITC experiments were performed for these com-
16 showed low water solubility, which limited the type of pounds at lower concentration (20 μm), similar to that used
experiments that could be performed (see below). The fluo- for the titrations of the CD dimers with NaC and NaDC
rescence spectra of compounds 15–17 measured in water
solutions at different concentrations (2 and 50 μm) showed
both a change of the emission intensity and a redshift with
increasing concentration.
As shown in Figure 1, from 1 to 100 μm, the intensity of
the 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 of
the solute to water. This behavior was also observed in the
case 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 interactions
between the aromatic systems.[36,39,40] The capacity of CD
dimers 15–17 to undergo self-aggregation is consistent with
the reported tendency for self-aggregation of other amphi-
philic cyclodextrins.[56–58]
Figure 1. Fluorescence spectra of compound 17 (λexc = 320 nm) in
water at concentrations from 1 to 100 μm. The intensity of the
fluorescence emission (I) increases with concentration and the
emission band is bathochromically shifted.
We also studied the self-aggregation of the bis(CD) by
isothermal titration calorimetry (ITC). Thus, dilution ex-
periments were carried out to measure the heat change
when a concentrated solution of CD dimer was injected
into the calorimeter cell initially containing phosphate
buffer. The heat arises from dimers or oligomers present
Figure 2. Thermograms for dilution experiments conducted with
compounds 15 (a) and 17 (b). The studies involved injecting 25
aliquots (10 μL each) of these compounds (stock concentration of
0.45 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 of
in the higher concentration solution that dissociate upon the experimental data to a n2 Ǟ2n dissociation model.
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(see below), no heat exchange was observed, indicating that (Figure 3, A and B) is indicative of an increase in the po-
at such concentrations the size of the aggregate does not larity of the environment around the spacer moiety.
depend on the concentration.
The presence of bile salt thus appears to induce a higher
exposure of the spacer to the solvent. Given the rigidity of
the spacers, which prevents self-inclusion of the fluorescent
moiety, 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 from
the 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 presence
of bile salt did not cause any appreciable change in the size
distribution of the aggregates (see Table 1).
We carried out ITC experiments in 10 mm phosphate
buffer (pH 7.2) to gain a deeper insight into the binding
abilities of the CD dimers. Stepwise addition of aliquots of
a solution of bile salt (NaC or NaDC) to an aqueous solu-
tion containing CD dimers 14–17 led to a decrease in the
extent of released heat (see Figure 4 and the Supporting
Information).
To confirm the self-aggregation of CD dimers 14–17,
DLS measurements were performed in 10 mm phosphate
buffer at pH 7.2. It was found that these CD dimers form
a polydisperse solution of aggregates with an average size
of 218Ϯ42 nm for 14, 205Ϯ41 nm for 15, 157Ϯ9 nm for
16, and 184Ϯ14 nm for 17 (Table 1) at a concentration (2
μm) 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 the
absence and the presence of NaDC and NaC (20 μm) in 10 mm
phosphate buffer (pH 7.2).
Bis(CD)
Rh [nm]
+ NaDC
+ NaC
14
15
16
17
218Ϯ42
205Ϯ41
157Ϯ9
196Ϯ35
181Ϯ26
151Ϯ19
142Ϯ21
163Ϯ28
150Ϯ22
141Ϯ15
216Ϯ34
184Ϯ14
ITC data can be fitted using a nonlinear least square al-
gorithm based on different models, the simplest of which
The sensing properties of CD dimers 14–17 towards bile considers n equal and independent binding sites. We as-
salts sodium cholate (NaC) and sodium deoxycholate sumed that the distance between the CD units in CD dimers
(NaDC) were studied using fluorescence spectroscopy. As 14–17 is long enough that guest binding to one of the CD
illustrated in Figure 3 (see also Supporting Information), units does not affect the guest binding to the other CD
the fluorescence intensity of CD dimers 14–17 decreased moiety. The experimental data provided a good fit to this
with stepwise addition of NaC and NaDC. The decrease in model, affording the desired thermodynamic parameters (n,
fluorescence intensity as concentration of bile salt increased ΔH, and K) (Figure 5 and Table 2). ITC data for the bind-
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 fluorescence
intensities of compounds 14 (♦), 15 (᭿), 16 (᭡), and 17 (᭹) at 2 μm with the concentration of NaC (c) and NaDC (d). The smooth solid
lines represent the trend of the variation.
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A Cyclodextrin-Based Ratiometric Sensor for Bile Salts
unfavorable entropic contribution. The apparent affinity (K
values) varies as 16Ͼ15Ͼ17Ͼ14. Such K values are for
the NaDC binding to CD dimers 15 and 16 one order of
magnitude higher than those for the binding to CD dimers
14 and 17. As shown in Table 2, n values were 0.5 for the
three bis(CD) compounds, which means that the NaC·CD
dimer complexes with 15–17 are formed with a 1:2 stoichi-
ometry. Furthermore, the complexation of NaDC with CD
dimers 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. This
suggests that, in some cases, supramolecular structures for
hosts 14–17 either contain unbound CD units or CD cavi-
ties that are for some reason unavailable, perhaps due to
steric hindrance caused by aggregation. By comparing the
K values for each CD dimer, those for NaC are lower than
those for NaDC, which is consistent with the behavior of
other reported CD dimers.[34,52]
Figure 4. Titration of compound 17 with NaDC in 10 mm phos-
phate buffer (pH 7.2) at 25 °C. The smooth solid lines represent
the best fit of the experimental data to a model of n equal and
independent binding sites.
To analyze the sensing abilities of compounds 14–17 in
water, 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. This
parameter would enable the variation of the fluorescence
ing of NaC to CD dimers 15–17 showed that it was exother- intensity induced by the interaction of the bile salt with the
mic with a unfavorable entropic contribution and afforded CD dimers to be evaluated. Unlike the stability constant
stable complexes with apparent K values of 3.9–9.0ϫ104 value, which indicates the stability of the complex, the Ps
m–1.
value reflects the nature of the environment in which the
The ITC experiments performed with bis(CD) 14 did not fluorophore group remains upon interaction with the guest.
provide reliable data, most likely due to its low water solu- Thus, if the complex is very stable but the polarity of the
bility. ITC data showed that the complexation of CD di- environment in which the phenylene-ethynylene group re-
mers 14–17 with NaDC are enthalpy-driven, along with an mains unchanged, then Ps will be very low. In contrast, the
Figure 5. Free energy (–ΔG0, black), enthalpy (–ΔH, grey), and entropy changes (TΔS0, white) for the binding of conjugates 14–17 to
NaC (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
NaC
Bis(CD)
ϫ10–4 [m–1]
–ΔG0 [kJ/mol]
–ΔH [kJ/mol]
–TΔS0 [kJ/mol]
n
14
15
16
17
14
15
16
17
–
–
–
–
–
9.0Ϯ0.5
4.9Ϯ0.5
3.9Ϯ0.4
6.1Ϯ0.6
16.3Ϯ1.1
18.8Ϯ0.8
8.4Ϯ0.7
28.0Ϯ0.4
26.8Ϯ0.4
25.9Ϯ0.3
27.2Ϯ0.4
29.7Ϯ0.3
30.1Ϯ0.4
28.0Ϯ0.4
30.6Ϯ4.6
37.7Ϯ5.9
33.9Ϯ7.9
84.1Ϯ20.1
39.7Ϯ1.3
54.8Ϯ1.3
43.1Ϯ2.1
2.1Ϯ0.8
10.9Ϯ5.9
7.5Ϯ7.9
56.9Ϯ18.1
10.0Ϯ2.3
24.7Ϯ5.3
15.1Ϯ4.1
0.5Ϯ0.1
0.5Ϯ0.1
0.5Ϯ0.1
1.1Ϯ0.2
0.8Ϯ0.1
0.6Ϯ0.1
1.0Ϯ0.1
NaDC
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higher the exposure of the phenylene-ethynylene moiety to particularly in the case of CD dimer 16.[52,60,61] Further-
water upon bile salt binding interaction, the higher the Ps more, the required concentrations of the host for providing
value will be.
reliable sensing parameters are lower than in most reported
The sensitivity parameters Ps of CD dimers 14–17 for cases.[52,59–61]
bile salts NaC and NaDC are illustrated in Figure 6. The
Ps values are negative in all cases because the I0 values for
the CD dimers are higher than I values in the presence of
the bile salts. This behavior is consistent with the above
mentioned higher exposure of the fluorophore moiety to
the bulk water. As seen in Figure 6, Ps values for all CD
dimers 14–17 increase with the addition of bile salts NaC
(Figure 6, A) and NaDC (Figure 6, B). The sequence of the
sensing factors of the four CD dimers 14–17 for guest
NaDC is 16Ͼ14Ͼ15Ͼ17 over the range of concentrations
of bile salts used. The sensitivity of CD dimer 16 towards
low to moderate concentrations of NaDC was notable. The
When the obtained sensitivity parameter values for
NaDC and NaC were compared, we observed that whereas
CD dimer 17 showed a similar sensitivity towards the two
bile salts, CD dimers 14–16 were more selective towards
NaDC (Figure 6). Such selectivity increases with the con-
centration of bile salts, which indicates that NaDC induces
a larger change in the fluorophore environment, and most
likely a larger structural change in the mode of aggregation
of compounds 14–16. These observations are consistent
with results obtained from ITC measurements discussed
above.
In addition to the high sensitivity of 16 towards guests
order of sensitivity of the CD dimers 14–17 for NaC was NaC and NaDC, as Figure 7 illustrates, upon gradual ad-
16Ͼ 17Ͼ 14Ͼ 15. Again, CD dimer 16 was the most sensi- dition of NaC and NaDC to CD dimer 16, the intensity of
tive host, although the sensitivity differences were higher at the emission bands at 348 and 364 nm significantly de-
higher concentrations of bile salt. These facts indicate that creased but that of the emission band at 463 nm slightly
the geometry of the phenylene-ethynylene moiety, which increased. The ratio of the emission intensities (I463/I348) in-
most likely influence the mode of self-aggregation, and the creased as the concentration of NaC and NaDC was in-
group at C-7 of the steroidal framework, affect the sensitiv- creased, rendering the supramolecular complex a sensitive
ity parameters.
ratiometric fluorescent sensor for both bile salts. Most fluo-
In all cases, the CD dimers allow detection of NaC and rescent chemosensors are based on fluorescence measure-
NaDC at [bile salt]/[host] ratios lower than 1. When com- ments at a single wavelength, which may be influenced by
paring the obtained values with those reported for other variations of the sample environment. However, wave-
fluorescent β-cyclodextrin-based sensors, the sensing fac- length-ratiometric probes are desirable because the ratios
tors of CD dimers 14–17 for NaC and NaDC are higher, are independent of the probe concentration. Thus, ratiomet-
Figure 6. Sensitivity parameters (Ps) of CD dimers 14–17 towards bile salts NaC and NaDC. Top: Variation of sensitivity parameter
values [Ps, (I – I0)/I0] of compounds 14 (♦), 15 (᭿), 16 (᭡), and 17 (᭹) at 2 μm with the addition of increasing amounts of NaC (A) and
NaDC (B) in 10 mm phosphate buffer (pH 7.2). The smooth solid lines represent the trend of the variation. Bottom: Ps values of
compounds 14–17 (2 μm) in the presence of different concentrations of NaC and NaDC [1 (black), 7 (grey) and 20 (white) μm].
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A Cyclodextrin-Based Ratiometric Sensor for Bile Salts
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 the
peak 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 lines
represent the trend of the variation and do not correspond to a fit to a model.
ric fluorescent probes allow the measurement of emission be used as a cyclodextrin-based ratiometric fluorescence
intensities at two wavelengths, which should provide a built- sensor for the detection of sodium cholate and deoxychol-
in correction for environmental effects. Therefore, the de- ate.
sign of ratiometric fluorescent sensors is of great current
interest.[62] There are very few examples of cyclodextrin-
based ratiometric fluorescence chemosensors, mostly for the Experimental Section
detection 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.
General Methods: TLC analysis was performed on Merck Silica
Gel 60 F254 aluminium sheets and visualized by UV light, and by
developing with either ethanolic sulfuric acid (5% v/v) or ethanolic
phosphomolybdic acid (7% w/v). Flash column chromatography
was performed on Merck Silica Gel (230–400 mesh, ASTM). Melt-
Conclusions
ing points were measured with a Büchi B-450 melting point appara-
tus and are uncorrected. Optical rotations were recorded with a
We have reported the synthesis of a series of CD dimers
in which the two CD units are linked by rigid-rod phenyl-
ene-ethynylene tethers of different length and shape through
Jasco P-1030 polarimeter at room temperature. [α]D values are
given in 10–1 deg cm2 g–1. IR spectra were recorded with a Mattson
Genesis II FTIR spectrometer. 1H, 13C and 2D NMR spectra
the CD secondary sides. The synthesis involves oxidative (gCOSY and gHSQC) were recorded with a Bruker Avance
DPX300 spectrometer equipped with a QNP 1H/13C/19F/31P probe.
Standard Bruker software was used for acquisition and processing
routines. Chemical shifts are given in ppm and referenced to in-
ternal TMS (δH and δC = 0.00 ppm); J values are given in Hz.
Sonogashira coupling reactions for the construction of the
tethers and final grafting of the β-cyclodextrin units by
click chemistry. The resulting CD dimers self-aggregated in
water, forming polydisperse nanoparticles. Such CD di-
HRMS (FAB) were recorded with a Micromass Autospec spec-
meric nanoparticles formed stable complexes with sodium
trometer using glycerol as matrix. HRMS (ESI) were acquired with
cholate and deoxycholate, with the latter bile salt affording
an Agilent Technologies LC/MSD-TOF spectrometer using purine
stronger complexes. In addition, we demonstrated the high
fluorescence sensing abilities of the synthesized CD dimers
toward those bile salts through the use of sensitivity param-
as internal reference. MALDI-TOF mass spectra were recorded
with a Perspective Biosystems Voyager DR-RP spectrometer using
2,5-dihydroxybenzoic acid as matrix. Pure water (MilliQ, 18.2
eters. Finally, we have demonstrated that CD dimer 16 can MΩcm) was obtained from a Millipore MilliQ Plus system. β-
Eur. J. Org. Chem. 2012, 2560–2571
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2567

A. Vargas-Berenguel et al.
FULL PAPER
Cyclodextrin was purchased from Fluka and dried at 50 °C in
vacuo 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 at
room temperature in vacuo in the presence of P2O5 for 24 h. Other
reagents 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]
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[(4Ј-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 and
the 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]
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 were
3 nm for conjugates 14–16 and 9 nm in the case of 17. Excitation
and 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 and
363.3 nm for 17, respectively. The emission of conjugates 15–17
were studied at concentrations varying from 2 to 50–100 μm in
MilliQ water. A concentration of 2 μm was used for 14. These ex-
periments were repeated for all conjugates (2 μm) in the presence
of increasing amounts of bile salts NaC and NaDC ranging from
0 to 20 μm in 2 mm phosphate buffer (pH 7.2) by mixing stock
solutions of these compounds (4 μm for conjugates, 1 mm for bile
salts).
1,3-Bis[(4Ј-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 and
the mixture was stirred at room temp. for 3.5 h. The solvent was
removed by evaporation under vacuum and the crude material was
purified by column chromatography (CH2Cl2/hexane, 5:1) to yield
6 (105 mg, 93%) as a pale-yellow solid; m.p. 183 °C (dec.). IR
ITC Experiments: Isothermal titration calorimetry (ITC) experi-
ments were performed with a MCS isothermal titration calorimeter
from Microcal, Inc. (Northampton, MA).[68] Volumes of sample
cell and injection syringe were 1.38 mL and 250 μL, respectively.
The reference cell was filled with MilliQ water. All solutions were
prepared in 10 mm phosphate buffer (pH 7.2) and degassed for
10 min under vacuum prior to the titration experiments. During
the titrations, the reaction mixture was continuously stirred at
400 rpm. The raw experimental data are presented as the amount
of heat produced per second following each injection (corrected
for the bile salts heats of dilution in the case of the interaction
experiments) as a function of time. The amount of heat produced
per injection was calculated by integration of the area under indi-
vidual peaks by using the Origin software. The errors are provided
from the best fit of the experimental data to the most suitable
model in each case, and corresponds to the standard deviation in
the fitting of the curves.
(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,
4
3
3J4,5 = 7.7, J2,4 = 1.8 Hz, 2 H, 4-H), 7.35 (d, J2Ј,3Ј = 8.8, 4 H, 2Ј-
3
3
H), 7.29 (t, J4,5 = 7.7 Hz, 1 H, 5-H), 6.65 (d, J2Ј,3Ј = 8.8 Hz, 4 H,
3Ј-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-2Ј), 130.6 (C-5), 128.4 (C-1),
124.3 (C-2), 114.9 (C-3Ј), 112.6 (C-1Ј), 90.7, 86.9 (CϵC) ppm.
HRMS (FAB): calcd. for [C22H16N2 + H]+ 309.1392; found
309.1391.
1,4-Bis[(3Ј-aminophenyl)ethynyl]benzene (7):
A
solution of
[Pd(PPh3)2Cl2] (9 mg, 0.013 mmol) and CuI (9 mg, 0.046 mmol) in
anhydrous 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 and
the mixture was stirred at room temp. for 3.5 h. The solvent was
removed by evaporation under vacuum and the crude material was
purified by column chromatography (CH2Cl2/hexane, 5:1 Ǟ 10:1)
to yield 7 (164 mg, 89%) as a pale-yellow solid; m.p. 182 °C (dec.).
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 due
to its low water solubility. The dilution profiles, under identical
experimental conditions, were obtained by injecting each bile salt
into buffer solution. The dilution heats were similar to the heat
signals detected after the saturation was reached and were sub-
tracted from heats detected during the binding experiment. A
model of n equal and independent sites was adequate to fit the
experimental data. From measurements of K, the free energy of
binding, ΔG°, could be calculated and hence the entropy of bind-
ing, ΔS°, determined from ΔG° = ΔH – TΔS° = –RT lnK. The
calculation of thermodynamic functions implies the usual approxi-
mation of setting standard enthalpies equal to the observed values.
IR (KBr): ν
= 3434, 3349, 1614, 1599, 1578, 836, 786 and
˜
max
685 cm–1. 1H NMR (300 MHz, CDCl3): δ = 7.48 (s, 4 H, 2-H), 7.14
(t, 3J = 7.8 Hz, 2 H, 5Ј-H), 6.95 (d, J5Ј,6Ј = 7.7 Hz, 2 H, 6Ј-H),
6.86 (s, 2 H, 2Ј-H), 6.67 (d, J4Ј,5Ј = 7.8 Hz, 2 H, 4Ј-H), 3.70 (br. s,
3
3
4 H, NH2) ppm. 13C NMR (75 MHz, CDCl3): δ = 146.5 (C-3Ј),
131.7 (C-2), 129.5 (C-5Ј), 123.9, 123.3 (C-1,1Ј), 122.3 (C-6Ј), 117.9
(C-2Ј), 115.7 (C-4Ј), 91.6, 88.7 (CϵC) ppm. HRMS (FAB): calcd.
for [C22H16N2 + H]+ 309.1392; found 309.1391.
1,3-Bis[(3Ј-aminophenyl)ethynyl]benzene (8):
A
solution of
[Pd(PPh3)2Cl2] (6 mg, 0.008 mmol) and CuI (6 mg, 0.032 mmol) in
anhydrous 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 and
the mixture was stirred at room temp. for 6.5 h. The solvent was
removed by evaporation under vacuum and the crude material was
purified by column chromatography (CH2Cl2/hexane, 5:1) to yield
8 (105 mg, 90%) as a pale-yellow solid; m.p. 105 °C (dec.). IR
DLS Measurements: Dynamic light scattering (DLS) measurements
were conducted with a Zetasizer nanoseries Nano-ZS-ZEN3600 in-
strument equipped with a laser of 633 nm using a ZEN-112 cuvette
at 15 °C. For each measurement the number of scans was 25, the
run duration was 35 s, the equilibration time was 140 s, and the
(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, J4,5
3
2568
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 2560–2571

A Cyclodextrin-Based Ratiometric Sensor for Bile Salts
3
3
= 7.8 Hz, 2 H, 3Ј-H), 7.33 (t, J4,5 = 7.8 Hz, 1 H, 5-H), 7.17 (t, J
1,4-Bis[(4Ј-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H-1,2,3-triazol-
1-yl}phenyl)ethynyl]benzene (14): CuI·(EtO)3P (10 mg, 0.028 mmol)
3
= 7.7 Hz, 2 H, 5Ј-H), 6.99 (d, J5Ј,6Ј = 7.5 Hz, 2 H, 6Ј-H), 6.87 (s,
3
2 H, 2Ј-H), 6.68 (d, J4Ј,5Ј = 8.0 Hz, 2 H, 4Ј-H), 3.64 (br. s, 4 H, was added to a solution of 9 (25 mg, 0.069 mmol) and 13 (171 mg,
NH2) ppm. 13C NMR (75 MHz, CDCl3): δ = 146.4 (C-3Ј), 134.6 0.146 mmol) in anhydrous DMF (6 mL) under N2 atmosphere and
(C-2), 131.2 (C-4), 129.4 (C-5Ј), 128.5 (C-5), 123.7, 123.7 (C-1,1Ј), the mixture was stirred at 105 °C for 1.5 h. The solution was
120.1 (C-6Ј), 117.8 (C-2Ј), 115.6 (C-4Ј), 90.4, 88.1 (CϵC) ppm. poured into acetone (100 mL) and the resulting precipitate was fil-
HRMS (FAB): calcd. for [C22H16N2 + H]+ 309.1392; found
309.1391.
tered off, dissolved in water, and freeze dried. The solid was washed
with methanol using a Soxhlet extraction apparatus (overnight) to
yield 14 (132 mg, 71%) as a pale-yellow solid; m.p. 263 °C (dec.).
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 the
[α]D = +90 (c = 0.25 in DMF). IR (KBr): ν
= 3396, 2924, 1644,
˜
max
1522, 1156, 1080, 1028 cm–1. H NMR (300 MHz, [D6]DMSO): δ
1
3
= 8.92 (s, 2 H, 5-H-C2HN3), 8.02 (d, J2Ј,3Ј = 8.3 Hz, 4 H, 3Ј-H),
3
7.83 (d, J2Ј,3Ј = 8.3 Hz, 4 H, 2Ј-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
-
same temperature for 50 min.
A solution of NaN3 (27 mg,
H, CH2O), 4.60–4.40 (m, 16 H, OH), 3.88 (t, J = 8.8 Hz, 2 H, 3I-
H), 3.70–3.49 (m, 59 H, 2I,3II–VII,4I,5I–VII,6I–VII,6ЈI–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-4Ј), 133.1
(C-3Ј), 131.9 (C-2), 122.4 (C-5-C2HN3), 122.4, 122.1 (C-1,1Ј), 120.2
(C-2Ј), 102.0–101.7 (C-1II–VII), 100.1 (C-1I), 90.5, 90.3 (CϵC),
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.
3
0.415 mmol) in water (2.5 mL) was then added dropwise and the
mixture was stirred at 0 °C for 45 min in the dark. The resulting
precipitate 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[(4Ј-azidophenyl)ethynyl]benzene (9): Starting from 5, use of
the general procedure yielded 9 (48 mg, 82%) as a pale-orange so-
lid; m.p. 151 °C. IR (KBr): ν
= 2123, 1592, 1510, 1297, 1261,
˜
max
1
1100, 1064, 833, 799, 525 cm–1. H NMR (300 MHz, [D8]THF): δ
3
= 7.57 (d, J2Ј,3Ј = 8.7 Hz, 4 H, 2Ј-H), 7.54 (s, 4 H, 2-H), 7.14 (d,
1,3-Bis[(4Ј-{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 and
the mixture was stirred at 105 °C for 4.5 h. The solution was
poured into acetone (100 mL) and the resulting precipitate was fil-
tered off, dissolved in water, and freeze dried. The crude material
3J2Ј,3Ј = 8.7 Hz, 4 H, 3Ј-H) ppm. 13C NMR (300 MHz, [D8]THF):
δ = 138.5 (C-4Ј), 131.0, 129.4 (C-2,2Ј), 121.2, 117.6 (C-1,1Ј), 117.2
(C-3Ј), 88.6, 87.0 (CϵC) ppm. HRMS (ESI): calcd. for
[C22H12N6 – N2 + 3 H]+ 335.1297; found 335.1292.
1,3-Bis[(4Ј-azidophenyl)ethynyl]benzene (10): Starting from 6, use of
the general procedure yielded 10 (46 mg, 79%) as a pale-orange
solid; m.p. 165 °C. IR (KBr): ν
= 2131, 1586, 1503, 1309, 1284, was purified by column chromatography (MeCN/water, 2:1) to
˜
max
832, 796, 685, 532 cm–1. H NMR (300 MHz, CDCl3): δ = 7.69 (s,
yield 15 (121 mg, 65%) as a pale-yellow solid; m.p. 283 °C (dec.).
1
3
3
1 H, 2-H), 7.52 (d, J2Ј,3Ј = 8.5 Hz, 4 H, 2Ј-H), 7.48 (d, J4,5
=
[α]D = +102 (c = 0.25 in DMF). IR (KBr): νmax = 3383, 2926, 1638,
˜
3
3
1
7.8 Hz, 2 H, 4-H), 7.34 (t, J4,5 = 7.8 Hz, 1 H, 5-H), 7.02 (d, J2Ј,3Ј 1595, 1155, 1040, 1027 cm–1. H NMR (300 MHz, [D6]DMSO): δ
= 8.5 Hz, 4 H, 3Ј-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 140.3
(C-4Ј), 134.6 (C-2), 133.3 (C-2Ј), 131.4 (C-4), 128.7 (C-5), 123.6 (C-
= 8.92 (s, 2 H, 5-H-C2HN3), 8.03 (d, J2Ј,3Ј = 8.5 Hz, 4 H, 3Ј-H),
3
7.83 (d, 3J2Ј,3Ј = 8.5 Hz, 4 H, 2Ј-H), 7.84 (s, 1 H, 2-H), 7.68 (d, 3J4,5
3
1), 119.7 (C-1Ј), 119.3 (C-3Ј), 89.5, 89.0 (CϵC) ppm. HRMS (ESI): = 7.3 Hz, 2 H, 4-H), 7.56 (t, J4,5 = 7.3 Hz, 1 H, 5-H), 6.00–5.90
calcd. for [C22H12N6 – N2 + 3 H]+ 335.1297; found 335.1292.
(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, J = 8.8 Hz, 2 H, 3I-
H), 3.70–3.49 (m, 59 H, 2I,3II–VII,4I,5I–VII,6I–VII,6ЈI–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-4Ј), 134.1
(C-2), 133.1 (C-2Ј), 132.6 (C-1), 131.9 (C-4), 129.6 (C-5), 122.4 (C-
5-C2HN3), 122.1 (C-1Ј), 120.2 (C-3Ј), 102.0–101.6 (C-1II–VII), 100.1
(C-1I), 89.7, 89.2 (CϵC), 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]+ 2729.9
and [C112H156N6O70 – N4 + Na + 4 H]+ 2675.9; found 2729.5 and
2675.6.
3
1,4-Bis[(3Ј-azidophenyl)ethynyl]benzene (11): Starting from 7, use of
the general procedure yielded 11 (54 mg, 93%) as a pale-orange
solid; m.p. 168 °C. IR (KBr): ν
= 2128, 1593, 1566, 1470, 1425,
˜
max
1
1319, 1295, 889, 839, 793 cm–1. H NMR (300 MHz, CDCl3): δ =
7.52 (s, 4 H, 2-H), 7.38–7.28 (m, 4 H, 5Ј,6Ј-H), 7.21–7.18 (m, 2 H,
2Ј-H), 7.01 (dt, 3J4Ј,5Ј = 7.2, 4J = 1.9 Hz, 2 H, 4Ј-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 140.5 (C-3Ј), 131.8 (C-2), 129.9, 128.3 (C-
5Ј,6Ј), 124.8, 123.1 (C-1Ј,1), 122.0 (C-2Ј), 119.4 (C-4Ј), 90.4, 90.1
(CϵC) ppm. HRMS (ESI): calcd. for [C22H12N6 – N2 + 3 H]+
335.1297; found 335.1292.
1,3-Bis[(3Ј-azidophenyl)ethynyl]benzene (12): Starting from 8, use of
the general procedure yielded 12 (48 mg, 82%) as a pale-orange
1,4-Bis[(3Ј-{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 and
the mixture was stirred at 105 °C for 3.5 h. The solution was
poured into acetone (100 mL) and the resulting precipitate was fil-
tered off, dissolved in water, and freeze dried. The solid was washed
with methanol using a Soxhlet extraction apparatus (overnight) to
yield 16 (125 mg, 67%) as a pale-yellow solid; m.p. 251 °C (dec.).
solid; m.p. 139 °C. IR (KBr): ν
= 2127, 1598, 1565, 1480, 1424,
˜
max
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, 5,5Ј,6Ј-H), 7.22–7.19 (m, 2 H, 2Ј-H), 7.02 (dt, 2 H, J4Ј,5Ј
3
= 7.3, 4J = 2.0 Hz, 4Ј-H) ppm. 13C NMR (75 MHz, CDCl3): δ =
140.5 (C-3Ј), 134.9 (C-2), 131.8 (C-4), 129.9, 128.7, 128.3 (C-
5,5Ј,6Ј), 124.8, 123.4 (C-1,1Ј), 122.0 (C-2Ј), 119.4 (C-4Ј), 89.4, 89.1
(CϵC) ppm. HRMS (ESI): calcd. for [C22H12N6 – N2 + 3 H]+
335.1297; found 335.1292.
[α]D = +121 (c = 0.25 in DMF). IR (KBr): νmax = 3388, 2925, 1642,
˜
1
1606, 1157, 1080, 1027 cm–1. H NMR (300 MHz, [D6]DMSO): δ
Synthesis of Bis[(4Ј-{4-[(2I-O-cyclomaltoheptaosyl)methyl]-1H- = 8.75 (s, 2 H, 5-H-C2HN3), 8.09 (s, 2 H, 2Ј-H), 8.00–7.90 (m, 2
1,2,3-triazol-1-yl}phenyl)ethynyl]benzenes 14–17
H, 4Ј-H), 7.80–7.60 (m, 8 H, 2,5Ј,6Ј-H), 6.00–5.90 (m, 4 H, OH),
Eur. J. Org. Chem. 2012, 2560–2571
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2569

A. Vargas-Berenguel et al.
FULL PAPER
5.80–5.60 (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,6ЈI–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-3Ј), 132.0, 131.9,
131.5 (C-2,5Ј,6Ј), 130.6 (C-1Ј), 123.6 (C-1), 122.5, 122.4 (C-4Ј, C5-
C2HN3), 120.6 (C-2Ј), 102.0–101.6 (C-1II–VII), 100.1 (C-1I), 90.3,
90.1 (CϵC), 82.0–81.3 (C-4I–VII), 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]+ 2729.9
and [C112H156N6O70 – N4 + Na + 4 H]+ 2675.9; found 2729.5 and
2675.6.
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1,3-Bis[(3Ј-{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 and
the mixture was stirred at 105 °C for 7.5 h. The solution was
poured into acetone (100 mL) and the resulting precipitate was fil-
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was purified by column chromatography (MeCN/water, 5:2) to
yield 17 (120 mg, 63%) as a pale-yellow solid; m.p. 273 °C (dec.).
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[α]D = +98 (c = 0.25 in DMF). IR (KBr): ν
= 3379, 2928, 1639,
˜
max
1608, 1175, 1006, 1029 cm–1. H NMR (300 MHz, [D6]DMSO): δ
= 8.93 (s, 2 H, 5-H-C2HN3), 8.16 (s, 2 H, 2Ј-H), 8.04–7.97 (m, 2
H, 4Ј-H), 7.87 (s, 1 H, 2-H), 7.74–7.65 (m, 6 H, 4,5Ј,6Ј-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,
1
3
16 H, OH), 3.87 (t, J = 8.8 Hz, 2 H, 3I-H), 3.70–3.45 (m, 59 H,
2I,3II–VII,4I,5I–VII,6I–VII,6ЈI–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-3Ј), 132.1, 131.5, 130.6,
129.6, 129.6 (C-1,4,5,5Ј,6Ј), 123.6 (C-5-C2HN3), 122.6, 122.5 (C-
1Ј,2Ј), 120.6 (C-4Ј), 101.9–101.7 (C-1II–VII), 100.1 (C-1I), 89.5, 89.0
(CϵC), 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-
[24]
[25]
[26]
TOF-MS: calcd. for [C112H156N6O70
+
Na]+ 2727.9 and
[C112H156N6O70 – N4 + Na + 2 H]+ 2673.9; found 2727.2 and
2673.7.
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
Supporting Information (see footnote on the first page of this arti-
cle): Fluorescence titrations, ITC titrations, 13C NMR spectra of
compounds 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 EU
European Regional Development Fund (FQM3141 and in part
FQM06903). Financial support by the EU through a Marie Curie
ITN program (CYCLON 237962) and the Spanish Ministry of
Education (PhD fellowship to M. C. M.-M.) is also acknowledged.
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Received: November 3, 2011
Published Online: March 21, 2012
Eur. J. Org. Chem. 2012, 2560–2571
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2571