Supramolecular Chemistry

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Inclusion of neutral guests by water-soluble

macrocyclic hosts – a comparative thermodynamic
investigation with cyclodextrins, calixarenes and
cucurbiturils

Dong-Sheng Guo, Vanya D. Uzunova, Khaleel I. Assaf, Alexandra I. Lazar, Yu

Liu & Werner M. Nau

To cite this article: Dong-Sheng Guo, Vanya D. Uzunova, Khaleel I. Assaf, Alexandra I. Lazar, Yu
Liu & Werner M. Nau (2015): Inclusion of neutral guests by water-soluble macrocyclic hosts –
a comparative thermodynamic investigation with cyclodextrins, calixarenes and cucurbiturils,
Supramolecular Chemistry, DOI: 10.1080/10610278.2015.1105374

To link to this article: http://dx.doi.org/10.1080/10610278.2015.1105374

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 Supramolecular Chemistry, 2015
http://dx.doi.org/10.1080/10610278.2015.1105374

Inclusion of neutral guests by water-soluble macrocyclic hosts – a comparative

thermodynamic investigation with cyclodextrins, calixarenes and cucurbiturils
Dong-Sheng Guoa,b, Vanya D. Uzunovaa, Khaleel I. Assafa, Alexandra I. Lazara, Yu Liub and Werner M. Naua
a
Department of Life Sciences and Chemistry, Jacobs University Bremen, Bremen, Germany; bState Key Laboratory of Elemento-Organic Chemistry,
Department of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin, P.R. China

ABSTRACT ARTICLE HISTORY

The driving forces of association between three different families of macrocycles as hosts, namely Received 31 August 2015
cyclodextrins (α-, β-, and γ-), p-sulfonatocalix[n]arenes (n = 4–6) as well as cucurbit[n]urils (n = 6–8), Accepted 2 October 2015
Downloaded by [University of Sussex Library] at 07:44 05 December 2015

and three different bicyclic azoalkane homologues as guests, namely 2,3-diazabicyclo[2.2.1] KEYWORDS
hept-2-ene (DBH), 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as well as 2,3-diazabicyclo[2.2.3]non-2- Molecular recognition;
ene (DBN), were examined by means of calorimetric titrations, NMR spectroscopy and molecular thermodynamics;
dynamics simulation, all in aqueous solution. The small, spherical and uncharged guests preferably macrocycles; neutral guests;
bind inside the cavities of the medium sized hosts. The inclusion complexation by β-cyclodextrin hydrophobic effect
and p-sulfonatocalix[4]arene shows medium binding affinities (millimolar), while cucurbit[7]uril
macrocycle shows very strong binding (micromolar). For all types of macrocycles, the complex
formation is enthalpically driven (ΔH° < 0), accompanied by slightly unfavourable entropy changes
(ΔS° < 0). The results are discussed in terms of the flexibility of the hosts, the hydrophobic character
of their cavities and the release of high-energy water upon binding, and generalised by including
two additional guests, the ketones cyclopentanone and (+)-camphor.

Introduction guest is augmented for derivatives with protic or anionic

Cyclodextrins (α-CD, β-CD, γ-CD), p-sulfonatocalix[n]arenes
(CX4, CX5, CX6) and cucurbit[n]urils (CB6, CB7, CB8) are
water-soluble macrocycles (see structures in Chart 1)
which are well known to form host–guest inclusion com-
plexes with a large range of compounds (1–4). In many
cases, the complexation is driven, fully or to a large degree,
by the hydrophobic effect (5, 6), which allows, in particular,
non-polar organic residues to be encapsulated inside the
host cavities, concomitantly releasing high-energy water
molecules into the bulk (7). Frequently, the affinity of the
groups (8, 9), which reinforce the complexes on account
of hydrogen bonding, ion–ion or ion–dipole interactions
for the CDs rims (1, 7), CXs (3) or CBs (10), respectively.
In order to further investigate hydrophobic interactions,
neutral organic guests are attractive, since puristic, models.
Neutral guest binding by CDs (1, 11, 12), CXs (13–17) and
CBs (9, 10, 18–20) has received considerable experimental
attention, especially by means of isothermal titration cal-
orimetry (ITC), which is a powerful tool for determining
host–guest complex interactions; the technique not only
affords the binding constants (Ka), but also thermodynamic

CONTACT Dong-Sheng Guo dshguo@nankai.edu.cn; Werner M. Nau w.nau@jacobs-university.de

© 2015 Taylor & Francis
 2 D.-S. Guo et al.
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Chart 1. Water-soluble macrocycles used in this study.

parameters (enthalpy and entropy changes, ΔH°, ΔS°). N N N

Systematic comparative studies of the complexation ther- N N N
Hendo' Hsyn
Hendo Hsyn Hendo
modynamics of neutral guests with these three classes of Hendo
Hb Hanti Hb Hb Hanti
macrocycles are not at hand. However, they are particularly Hexo Hexo Hexo
Hexo'

desirable to obtain insights into the hydrophobic effect DBH DBO DBN
that drives the binding to the respective class of macro-
cycles, CDs, CXs and CBs. O H3C CH3
In contrast to the situation for neutral guests, selected H
H
comparative studies on the binding of ionic guests with
the different macrocycles have already been conducted. H
O CH3
Mutihac and co-workers made a survey on thermodynamic
properties for the complexation of some calixarene vs. CB cyclopentanone (+)-camphor
receptors towards amino acids, nucleobases and di- and
Chart 2. Structures of the investigated bicyclic azoalkanes and
tri-peptides (21). García-Río and co-workers compared the the two ketones.
complexation of cationic surfactants with β-CD and CB7
by ITC (22). Whereas the binding affinity of surfactants
with β-CD increases with alkyl chain length, the binding with the three macrocycle classes by fluorescence titra-
constants between CB7 and surfactants remain essentially tions (23). CB7 always caused enhanced fluorescence of the
unchanged, indicating that CB7 exhibits a pronounced dyes, while CX4 always resulted in quenched fluorescence.
preference towards the cationic residues while β-CD pre- Moreover, the pH values, solvent effects and salt effects
fers to bind neutral or anionic guests. Liu and co-workers exert extraordinary influence over the binding ability of
have compared the complexation of organic dye cations the three kinds of macrocycles.
 Supramolecular Chemistry  3

Table 1. Binding constants (Ka) and thermodynamic values for the complex formation between the macrocyclic hosts and the bicyclic
azoalkanes and ketone guests.

Host Guest Ka/(M–1)a ΔH°/(kJ mol–1) TΔS°/(kJ mol–1)

α-CD DBH – – –
DBO 57.8 ± 0.4 [50]b −13.4 ± 0.1 −3.4 ± 0.1
DBN – – –
β-CD DBH 241 ± 27 −14.6 ± 0.3 −1.0 ± 0.6
DBO 876 ± 10 −18.4 ± 0.1 −1.6 ± 0.1
DBN 2680 ± 10 −21.4 ± 0.1 −1.9 ± 0.1
γ-CD DBH – – –
DBO [6]b – –
DBN – – –
CX4c DBH 660 −23.3 −7.2
DBO 760 −25.4 −8.88
DBN 860 −26.4 −9.62
CX5 DBH 34.9 ± 2.1 −11.2 ± 0.6 −2.4 ± 0.9
DBO 24.8 ± 1.9 −10.3 ± 0.7 −2.4 ± 0.9
DBN 58.2 ± 2.2 −10.8 ± 0.3 −0.7 ± 0.8
CX6 DBH 55.2 ± 0.3 −15.3 ± 0.8 −5.4 ± 0.2
DBO 82.0 ± 0.1 −16.3 ± 0.4 −5.4 ± 0.4
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DBN 13.2 ± 0.6 −16.4 ± 0.6 −4.3 ± 0.1

Cyclopentanoned (3520 ± 490) [−27.4 ± 2.9] [−7.1 ± 2.5]
CB6 DBH [1300]e – –
DBO – – –
DBN – – –
(+)-Camphor – – –
CB7 Cyclopentanonef 3.30 × 105 −41 −9
DBH (3.81 ± 0.17) × 105 −38.7 ± 0.1 −6.9 ± 0.2
DBOf 1.34 × 107 −75 −34
DBN (2.08 ± 0.57) × 107 −60.2 ± 1.1 −22.7 ± 2.3
(+)-Camphor (1.94 ± 0.01) × 107 −89.9 ± 3.6 −48.2 ± 3.4
CB8 Cyclopentanonef 1100 −35 −18
DBH 2770 ± 160 −21.0 ± 1.0 −1.3 ± 1.1
DBOf 1.6 × 104 −37 −13
DBN (5.85 ± 0.20) × 105 −39.7 ± 0.3 −6.8 ± 0.3
(+)-Camphor (3.05 ± 0.27) × 107 −36.7 ± 1.9 6.0 ± 1.7
a
Mean values measured from at least three experiments at 25 °C in aqueous solution, pH 6.0; error given as standard deviation (±1σ)
b
Binding constants previously measured in D2O using 1H NMR titration: 50 M–1, cf. Ref. (25)
c
From a previous publication in aqueous solution at pH 6, cf. Ref. (16)
d
Titrations in water showed no detectable heat effect, value in square brackets measured in 200 mM Na2SO4; the Ka value was similar to the literature value of
3.4 × 103 M–1 in 200 mM Na2SO4, cf. Ref. (10)
e
In 200 mM Na2SO4 from Ref. (30)
f
At pH 6 from Ref. (20).

In our present investigation, we have selected a series of
bicyclic azoalkane homologues (Chart 2), namely 2,3-diaza-
bicyclo[2.2.1]hept-2-ene (DBH), 2,3-diazabicyclo[2.2.2]oct-
2-ene (DBO) and 2,3-diazabicyclo[2.2.2]non-2-ene (DBN),
and investigated their binding to three different macrocy-
cles (Chart 1) by ITC, NMR and molecular dynamics (MD)
simulation. These azoalkanes, while neutral, are sufficiently
water soluble (mM range, comparable to the investigated
macrocycles) to also allow for the assessment of weak
host–guest complexation phenomena (down to 10 M−1)
(24, 25). Additionally, they are non-protic and weakly basic,
which precludes hydrogen bonding as the predominant
interaction with the host molecules (e.g. CDs). Each of
the macrocyclic host families by itself provides a distinct
chemical environment for complexation with respect to
shape, flexibility, hydrophobicity and high-energy water
content. Therefore, these uncharged spherical guests are
ideally suited to study the inner pockets of the three host
families. To generalise some important conclusions of this
study, we have also included two ketones (cyclopentanone
and (+)-camphor, Chart 2) as guests with cucurbiturils,
which meet the same criteria with respect to solubility and
neutrality, as well as non-protic and basic character as our
bicyclic azoalkanes. The combined set of guest molecules,
while matching the macrocyclic cavities with respect to
their spherical shape, allowed us to explore the size selec-
tivity of the macrocycles families.
Results
We have structured our experimental study such that
we first examined the binding affinities and thermody-
namic origins of the analytes to the macrocyclic hosts by
ITC. Additionally, we performed 1H NMR investigations
(facilitated by the millimolar solubilities of most hosts)
to confirm the ITC findings and characterise the mode of
host–guest binding. Finally, packing coefficient (PC) cal-
culations, together with MD simulations, were carried out
 4 D.-S. Guo et al.
to obtain further information on the inclusion complex
formation between the macrocyclic hosts and the bicyclic
azoalkanes.
ITC measurements
The thermodynamic data and binding constants obtained
by ITC are summarised in Table 1, where some of the
binding constants have been taken from previous inves-
tigations done with either 1H NMR or ITC titrations. In all
cases, the binding is an enthalpically driven process. Within
each macrocyclic family (CDs, CXs and CBs), the binding
constants of the azoalkanes have comparable orders of
magnitude. There is also an obvious trend between the
differently sized guests: the enthalpy of the reaction (ΔH°),
as well as the binding constant (Ka) increases upon going
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from DBH (smallest), to DBO (intermediate), to DBN (largest
guest). Among the cyclodextrin family, the β-CD macro-
cycle is the prime candidate for azoalkane binding due
to the favourable size-fit relationship. Among the calix-
arene family, CX4 shows the highest binding affinities,
presumably due to its pronounced structural rigidity (17,
26–29). The most interesting cases, however, are those of
the cucurbituril macrocycles CB7 and CB8, which show
contrasting enthalpic and entropic terms. CB6 presents
an exception due to its small cavity size, that is it is only
capable of binding the smallest azoalkane, DBH, and the
smaller ketone, cyclopentanone. Our detailed results on
the thermodynamics of binding are first outlined sepa-
rately for each type of macrocycle.
Cyclodextrins
For cyclodextrins, only the medium sized β-CD shows sig-
nificant binding with all three bicyclic azoalkanes, while
the smaller and larger homologues, α-CD and γ-CD, show
insignificant heat effects in our calorimetric experiments,
with the exception of DBO with α-CD (Table 1). Previous 1H
NMR studies had also revealed the low binding constants
of DBO to both, α-CD and γ-CD, with association constants
of 50 and 6 M–1, respectively (25, 31). The accessible ther-
modynamic data for complex formation between the bicy-
clic azoalkanes and the cyclodextrin macrocycles indicate
that the binding is enthalpically driven (ΔH° from −13.4
to −21.4 kJ mol−1), accompanied by slightly unfavourable
entropy changes (TΔS° from −1.0 to −3.4 kJ mol−1). This
pattern is similar to the one observed for the binding of lin-
ear aliphatic guests to CDs, with the complexation energy
being proportional to the number of added methylene
groups. The enthalpic unit increment is ca. −3.3 kJ mol−1
per methylene group (1), going from −14.6 kJ mol−1 for the
DBH•β-CD complex, to −18.4 kJ mol−1 for the DBO•β-CD
complex and −21.4 kJ mol−1 for the DBN•β-CD complex.
This increment goes in line with increasing dispersion
interactions and the release of more high-energy water
molecules (6, 32). The comparably small entropic contribu-
tions indicate that the resulting inclusion complexes are
not strongly co-conformationally restricted, as expected
from the spherical shape of the guests (31, 33, 34).
Calixarenes
Calixarene macrocycles are very versatile hosts, since they
not only provide a variety of conformations in solution (26,
27, 35, 36), but also change their charge status depend-
ing on the working pH (37–39). In this respect, although
structurally similar, calixarene macrocycles may provide
different environments for complexation, which can com-
plicate the interpretation of thermodynamic data. As can
be seen from Table 1, the enthalpies of complexation in
the case of calixarene hosts do not follow any obvious
trend, CX5 < CX6 < CX4, which can be attributed to the
above-mentioned conformational diversity. The rigid cone
conformation of the CX4 macrocycle (27, 28) provides the
best shape-fit relationship for the bicyclic azoalkanes,
with the highest complex stability for the DBN•CX4 case
(ΔH° = –26.4 kJ mol–1). The pentameric homologue (CX5)
has a larger and more flexible cone structure compared
to the smaller CX4 one (37, 40), and therefore, its com-
plexation enthalpies with bicyclic azoalkanes are lower
(ca. –10 kJ mol–1). Surprisingly, the largest CX6 homologue
shows slightly higher enthalpies of binding compared to
CX5. One probable explanation is the ‘alternate’ confor-
mation composed of two trimeric partial cones, which
is commonly assumed for the CX6 macrocycle (38, 39).
The ‘alternate’ conformation of the CX6 macrocycle can
possibly lead to 1:2 binding stoichiometries (41), which,
however, could not be experimentally differentiated in
this study due to the low absolute binding affinities (from
55 to 132 M–1, extracted by assuming a 1:1 binding pat-
tern). An additional complication lies in the competitive
binding of Na+ as counterions, which modifies not only
the absolute binding constants of the organic guests but
also the measured thermodynamic parameters (42, 43). It
can furthermore be projected that the binding affinities of
Na+ with CX4, CX5 and CX6 will be significantly different.1
As a result, the present thermodynamic data for CXs are
more difficult to rationalise due to conformational flex-
ibility and also due to the competitive Na+ binding. The
latter applies particularly for guests with lower binding
constants, where higher concentrations of CXs need to
be used in the ITC reaction cell, resulting in a higher frac-
tion of CX•Na+ complex and directly related effects on the
apparent binding constant, as documented in detail by
García-Río and co-workers (43, 44, 45).
Keeping the complications in mind, the enthalpic con-
tributions in calixarenes are mainly due to dispersion and
C–H⋯π interactions; the release of high-energy water

molecules plays no major role, contributing to the compa-
rably small overall affinities (6, 16, 46). The negative entropy
can be attributed to the loss of degrees of freedom upon
complexation. The CX macrocycles show more unfavour-
able entropic changes (from –0.7 to –9.62 kJ mol–1) upon
complexation than the cyclodextrin macrocycles, point-
ing to more pronounced conformational restrictions as
imposed, for example, by the specific C–H⋯π interactions
with the bicyclic azoalkanes (15).
Cucurbiturils
Within the cucurbituril series, the CB6 homologue is rela-
tively small and it can only encapsulate cyclopentanone
and DBH. It is interesting to note that the smaller cyclo-
pentanone guest undergoes fast exchange with CB6 on
the NMR time scale, while the complexation with DBH
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undergoes slow exchange due to its large size (30). Both
CB7 and CB8, the larger homologues, display very strong
binding affinities (from 103 to 107 M–1), but with distinct
enthalpic and entropic effects: guest binding with CB7
is more strongly enthalpically driven, and there is a less
pronounced entropic loss in the case of CB8. The stronger
enthalpic response of CB7 can be traced back to the release
of high-energy water, which is more pronounced than for
CB8 (20, 30, 47, 48); in short, although the CB8 cavity hosts
more water molecules, they can form a better hydrogen
bonding network inside the larger cavity, which reduces
their net high-energy water content. The less pronounced
entropy loss for the formation of the CB8 complexes can,
in turn, be intuitively interpreted in terms of the better
mobility of the guests inside the larger cavity. It is interest-
ing to note that the enthalpic contribution of CB8 binding
shows similar values for all guests (ca. –30 kJ mol−1); as a
consequence, the entropic contributions govern the com-
plexation strength. The complexation of (+)-camphor with
CB8 constitutes an exception, because its complexation is
also facilitated by an entropy gain, presumably due to the
entropy gained from desolvation of this putatively more
hydrophobic guest is not entirely counterbalanced by the
entropic loss due to conformational restrictions accompa-
nying host–guest inclusion. Be this as it may, in terms of
the macrocyclic environments, the CB hosts, led by CB7,
provide the strongest binding.
NMR measurements
The interaction of the azoalkanes with the CBn macrocy-
cles has been additionally confirmed by NMR measure-
ments, whereas NMR studies for the CD and CX families
are already reported with same homologous guests (15,
49). For the smallest host, CB6, complexation with the large
guests (DBO, DBN, (+)-camphor) was unobservable either
by ITC measurements or by NMR experiments. Since CB8 is
Supramolecular Chemistry  5
Table 2. Complexation-induced 1H NMR chemical shifts of the
azoalkanes with 2 equiv. CB7, at pD 6.4.
Δδ/ppma
Hendo Hexo
Azoalkanes [Hendo] [Hexo] Hsyn/Hanti
DBH −0.82 −0.90 −0.84/−0.96
DBO −0.78 −0.81 –
DBN −0.75 −0.78 –
[−0.75] [−0.75]
a
NMR assignments according to Ref. (15); signals of the Hb protons overlap
with the CB7 resonances.
furthermore too poorly water soluble for systematic NMR
studies, we have concentrated our NMR investigations on
the water-soluble CB7 macrocycle. The inclusion compl-
exation with CB7 is strong, but in fast exchange with the
free host and guests on the NMR time scale. Addition of
3 mM CB7 to 1.5 mM azoalkane solution at pD 6.4 (adjusted
with NaOD) resulted in large 1H NMR upfield shifts for the
azoalkane protons (up to 0.96 ppm, see Chart 2 and Table
2), which are characteristics for the formation of inclu-
sion complexes (50). The situation for the symmetric bar-
rel-shaped CBs is different from that for the asymmetric
cone-shaped hosts CDs and CXs (15), where the penetra-
tion depth varies with size of the host, reflected in pro-
nounced changes of the complexation-induced chemical
shifts. Accordingly, the averaged complexation-induced
shifts of all three azoalkanes are rather constant, due to
their central co-conformation, with only a small size-­
dependent decrease from DBH (ca. –0.87 ppm) to DBO
(ca. –0.80 ppm), and DBN (ca. –0.76 ppm). In the case of the
CB7 NMR signals, the azoalkanes do not cause any split-
ting of the CB7 methylene signals, with the exception of
DBN, which induces a small but significant splitting of one
doublet (data not shown). This suggests that the smaller
azoalkanes, DBH and DBO, do not assume any preferen-
tial conformation on the NMR timescale, while the largest
and most tightly bound DBN (Ka = 2.08 × 107 M–1) has a
preferred co-conformation inside CB7. The conformational
fixation is also consistent with the entropy loss being larg-
est for DBN (Table 1).
Theoretical calculations
MD simulations with Amber force field were performed
to obtain detailed information on the inclusion complex
formation between the different macrocyclic hosts and
bicyclic azoalkane guests; they complement earlier calcu-
lations and allow for a direct comparison (15, 34, 51). The
size selectivity of the azoalkanes towards the chosen hosts
can be examined by evaluating the PC of their complexes,
as it was previously successfully applied to predict the sta-
bility of other CBn complexes (10). This analysis is possible
 6 D.-S. Guo et al.

Figure 1. (Colour online) Side views of the distinct cavities of β-CD, CX4 and CB7; cavity volumes were calculated in analogy to Ref. (10).
Downloaded by [University of Sussex Library] at 07:44 05 December 2015

Figure 2. (Colour online) Top and side views of the MD-average structures of the studied complexes, (A) DBO•β-CD, (B) DBO•CX4 and (C)
DBO•CB7.

for the CD and CB macrocycles, which have geometrically
well-defined cavities, see Figure 1. For the CX macrocy-
cles, even for the CX4 homologue with the best defined
cavity, no definitive cavity volume, and consequently no
PC values, could be defined due to the wide-open upper
rim (see Figure 1). In general, PCs in the range of 50–60%
provide the best size-fit relationship (9, 10, 52). As can be
seen from Table 3, the PCs of the host–guest complexes
scale nicely with the experimental binding constants
(Table 1). For example, they rationalise the strong binding
 Supramolecular Chemistry  7

Table 3. Guest volumes, inner host cavity volumes and associated consistent with the 1H NMR shifts of the guest protons,
PCs with CBsa, β-CD and CX4. e.g. for CB7 (see above). The computed and experimental
PC/% structures reveal the favourable hydrophobic interactions
Guest/host CB6 CB7 CB8 β-CD CX4 between the studied guest molecules and the hydropho-
[volume/Å3] [142] [242] [367] [277] [>102]b bic cavities of both, CB7 and β-CD. The average structure
Cyclopentanone [87] 61 36 24 of the CX4 complex (Figure 2) shows that the guest is
DBH [96] 68 40 26 35 94
DBO [111] 78 46 30 40 109 also included within the cavity, with the azo group (N=N)
DBN [126] 89 52 34 46 124 exposed to the aqueous bulk and the C–H bonds suitably
(+)-Camphor [160] 113 66 44
a
positioned for C–H⋯π interactions; this co-conformation
The data of CBs with azoalkanes are from a previous publication, cf. Ref. (53).
b
The selected cavities are shown in Figure 1; for CX4, only a lower limit could is consistent with previous 2D NMR results (15).
be estimated. The guests’ dynamics inside the host cavity, monitored
by the superposition of six snapshots extracted from the
constant for DBN•CB7 (52%, virtually ideal PC value), and MD trajectories for each complex, are shown in Figure 3.
also account for the lower binding of (+)-camphor to CB7 Rotational motion of the guest molecules inside the cavity
(66%, PC above ideal value). was observed in all complexes, while a restricted transla-
tional motion was most pronounced for the β-CD com-
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The ‘preferred capacity’ of a concave host can be cal-

culated as 55% of its cavity volume. Accordingly, the pre-
ferred capacity of CB7 is 133 Å3 and that of β-CD is 152 Å3,
while that of CX4 has been previously estimated to be
130 Å3 (15). Accordingly, these three hosts are comparable
in terms of their capacity – as are the smaller and larger
homologues – which justifies the direct comparison con-
ducted in this study.
The average structures of the 1:1 complexes of DBO
with the different hosts were obtained from 5-ns MD tra-
jectories, see Figure 2. For β-CD and CB7, the guest mol-
ecule is included within the hydrophobic cavity, which is
plexes. To point to one peculiarity, for DBH and DBO, but
not for the larger DBN, a flip-flop motion was observed
inside CB7, facilitated by the looser packing.
We have also looked at the dynamics of the free macro-
cycles (without guests) in explicit water molecules, which
in fact increases in the sequence of CB7 < β-CD ≈ CX4
(Figures S3 and S4). It was also observed that, among the
macrocyclic hosts, β-CD showed the highest flexibility after
binding with the neutral azoalkane guests, followed by
CX4, while CB7 formed, as expected, the most rigid host–
guest complex.

Figure 3. (Colour online) Dynamics of the host–guest complexes shown as clustered molecular display for (A) β-CD, (B) CX4 and (C) CB7
with the homologous guests DBH, DBO and DBN (from left to right). For the hosts, only the average structure is shown for clarity. The
drawings include six representative structures that refer to the time interval of simulation from 0 to 5 ns in 1-ns step.
 8 D.-S. Guo et al.
Discussion
CDs, CXs and CBs are three classical water-soluble macro-
cycles widely studied with respect to their ability to form
host–guest complexes in aqueous media (23, 54–56). CDs
are cyclic oligomers of anhydroglucopyranose linked by
α-1,4 glycosidic bonds. This geometry gives CDs the overall
shape of a truncated cone or torus rather than that of a
perfect cylinder, with the wider side formed by the second-
ary 2- and 3-hydroxyl groups and the narrower side by the
primary 6-hydroxyl groups (1, 57). The calixarene skeleton
is built up by the base-catalysed condensation of 4-substi-
tuted phenols with formaldehyde, with a conformational
variability much larger than CDs (58). While the smaller
homologues CX4 and CX5 are typically represented as a
(flexible) truncated cone or cup, with the upper rim formed
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by the sulfonate groups and the lower rim by the phenolic
hydroxyl groups, the larger CX6 usually adopts an ‘alter-
nate’ conformation (41, 59–61). CBs are cyclic oligomers
composed of glycoluril units joined by dual methylene
bridges, possessing a rigid symmetric barrel or pumpkin
shape (2, 62). All three classes of macrocycles have demon-
strated significant biocompatibility and show therefore for
several biological applications involving molecular recog-
nition events (56, 63–68). Although the formation of host–
guest inclusion complexes is invariably facilitated by their
hydrophobic inner cavities, the intrinsic characteristics and
inclusion properties of the three hosts differ significantly,
which forms the basis for our present comparative study.
In particular, the following aspects require consideration:
(1) The CB cavity is equatorially symmetric, while
the CD and CX ones are asymmetric. Both por-
tals/rim of CBs are identical, whereas CDs and
CXs are distinguished by a wider side (upper
rim) and a narrower one (lower rim).
(2) Among the three macrocycle families investi-
gated, the CXs are the most conformationally
variable, with conformations other but the
desirable cone shape possible, including par-
tial-cone, 1,2-alternate, 1,3-alternate and more
complicated ones for larger analogues. On the
contrary, CDs and CBs have robust conforma-
tions with a better defined inner cavity (3, 10).
While maintaining an overall cone shape, CDs
are, however, considerably more flexible than
CBs (Figures S3 and S4).
(3) The hydrophobic effect is consistently a driv-
ing force for all of these three water-soluble
macrocycle families. In particular, the release
of high-energy water molecules from the
different macrocyclic cavities (non-classical
hydrophobic effect) plays an important role,
most pronounced for the medium sized CB7
and least pronounced for the smallest CX4 (6).
Additionally, the portals of CDs are capable
of forming hydrogen bonds, particularly with
anionic guest molecules, while the CXs donate
synergistically negative charge and π-stacking
interactions for cation binding; CBs offer ion–
dipole interactions with their carbonyl portals,
which also promote cation binding (23, 54, 56).
In our study, we selected neutral guests with
poorly basic and hydrogen bonding properties,
precisely with the aim to bypass the differen-
tial specific interactions provided by the rims/
portals, and to focus on their hydrophobic cavi-
ty-driven binding.
(4) The size/shape-fit always plays a crucial role in
governing the binding selectivity for each mac-
rocycle, and it depends on its geometry. CBs
have tighter portals (their diameter is smaller
than that of the equator (30)), which leads to a
more stringent size/shape selectivity. CXs have
a wide and loose opening, which allows a vari-
ety of guest molecules to be encapsulated with
lower selectivity. Taking the example of CX4
and CB7, with comparable preferred capacity
(ca. 130 Å3, see Results) (15), CB7 shows a high
affinity to methyl viologen (Ka = ~105 M–1) but
no appreciable binding to lucigenin (69–71),
whereas CX4 shows strong binding to both
methyl viologen (Ka = ~106 M–1) and lucigenin
(Ka = ~107 M–1) (16, 40, 72, 73).
(5) CDs and CBs can form deep inclusion com-
plexes, forming threaded structures in most
cases, while CXs favour a more shallow inclu-
sion (64, 74–77). This is because the hydrogen
bonding network formed by the lower rim phe-
nolic hydroxyls leaves the bottom of CX cavity
effectively closed, preventing guest threading.
The shape of CXs is therefore better described
as that of a bowl or cup, with a wide opening
and closed bottom, than that of a truncated
cone.
To turn to the comparative binding of the three macr-
ocycles with neutral guests, as can be seen from Table 1,
CXs exhibit the lowest binding selectivity for the neutral
guests employed, and also a low host selectivity when the
same guest is compared. With respect to guest selectivity,
the Ka values are at best double upon going from DBH to
DBN, and with respect to host selectivity, the Ka values
of the calixarene hosts do not follow a systematic trend,
i.e. CX5 < CX6 < CX4, a fact that can be attributed to the
previously mentioned competitive Na+ binding and con-
formational diversity of CXs in general, and CX6 in particu-
lar. Even when CX4 and CX5 are compared, the two hosts
with a cup-shaped conformation, the best host selectiv-
ity is only a factor of 30, for DBO as guest, one order of
magnitude less than that of CBs. Moreover, although CX5
possesses a larger cavity size, it does not provide a higher

binding affinity to the larger guest as intuitively expected.
This is because the CX5 cavity resembles more that of a
shallow dish, due to the restriction of the lower rim hydro-
gen bond network (40, 78), its cavity opening is too wide to
effectively accommodate hydrophobic cargoes. The poor
binding selectivity of CXs to the neutral azoalkane guests
further validates the aforementioned points 2 and 4: the
preorganisation of the conformationally flexible CXs is not
as well developed as for the other two macrocycles, result-
ing in a reduced size/shape selectivity towards the guests.
The differential binding affinities of β-CD towards DBH,
DBO and DBN suggest an intermediary guest selectivity.
The highest selectivity, both upon varying the guest size
and upon varying the host cavity size, is observed for
the CBs, for example Ka DBN•CB8/Ka DBH•CB8 = 211, Ka DBH•CB7/
Ka DBH•CB6 = 293 and Ka DBO•CB7/Ka DBO•CB8 = 323. Consequently,
Downloaded by [University of Sussex Library] at 07:44 05 December 2015
defying the activity-selectivity principle, the CB macrocy-
cles display not only the strongest absolute binding to the
neutral guests but also the highest selectivity towards the
guests. We therefore expanded our investigation for CBs
to two additional guests: cyclopentanone (as a smallest
guest) and (+)-camphor (as a larger guest). As expected,
the smaller cyclopentanone forms a tighter complex with
CB6 than DBH (more ideal PC, 61% vs. 68 %), and the larger
(+)-camphor forms a tighter complex with CB8 than DBN;
both results follow a simple supramolecular structure–
activity relationship, that is the binding strength increases
as packing becomes more ideal (see Results, Table 3).
β-CD, CX4 and CB7 are three analogues studied most
frequently in tandem, because of their similar preferred
capacity (see Results); generally, these three also stand
out within each class as highest affinity binders (15). β-CD
shows a moderate binding to the azoalkanes, similar to
CX4, but its complexation enthalpy and entropy terms are
distinct. The enthalpy gain of CX4 is more favourable than
that of β-CD, probably resulting from the unique C-H⋯π
interactions offered by the aromatic framework of calix-
arenes that the other hosts are lacking. Compared with
CDs, CXs have a wider opening, which leads to the rela-
tively shallower inclusion that is unfavourable for compl-
exation-induced desolvation (Figures 2 and 3). This is also
reflected in MD calculations (see hydrogen bond analysis,
Table S1), which reveal that CBs and CDs shield the guest
molecules better from the surrounding water. Moreover,
CXs, inherent to their higher flexibility, always undergo a
comparably larger loss of the conformational degrees of
freedom upon complexation of guests (17).
Our study exposes that CB7, despite its comparable
cavity size, forms 2–4 orders of magnitude more stable
complexes with neutral guests than β-CD and CX4. The
main factor that drives the encapsulation of neutral guests
into macrocyclic cavities is the release of high-energy
water, that is the non-classical hydrophobic effect. Very
Supramolecular Chemistry  9
recently, we reported water-box simulations for discrete
water molecules inside cavities and concluded that if N
is the number of water molecules in a cavity and m is the
average number of hydrogen bonds which these water
molecules form inside the cavity, then the relative driving
force can be estimated as Z = N (3.62-m), where 3.62 is
the simulated hydrogen bond number in bulk water (6).
β-CD hosts 4.4 water molecules in its cavity with m = 2.96
and, thus, Z = 3.1; CX4 has 0.8 water molecules in its cav-
ity with m = 2.15 and, thus, Z = 1.2; CB7 accommodates
7.9 water molecules in its cavity with m = 2.52 and, thus,
Z = 8.7. These estimates are in good agreement with the
experimental results. For example, the cavity of CX4, also
termed ‘the smallest cup of water’ (79), is occupied by a
single water molecule, such that the release of high-energy
water (Z = 1.2) cannot contribute much to the net driving
force for neutral guest binding. In contrast, neutral guest
binding to CB7 shows a strong high-energy water-release
effect Z = 8.7, which accounts for the strongest binding
by this host. β-CD, with Z = 3.1, lies in between in terms
of water-release contributions. The same arguments have
been previously used to assess the trends within the CB
series; for CB6, the number N is smaller than for CB7,
while for CB8, the number m is higher than for CB7, both
of which result in reduced Z values and hence binding.
This delicate interplay between the individual energetic
frustration and the absolute number of the cavity water
molecules provides an explanation for the medium sized
macrocycle CB7 displaying the highest binding constants,
not only for the presently selected series of neutral guests,
but also among synthetic macrocycles in general (20).
Conclusion
We have evaluated the binding affinities and the thermo-
dynamic parameters for the binding of three azoalkane
guests, namely DBH, DBO and DBN, with three different
prototypal macrocyclic host families, CDs, CXs and CBs. All
offer a hydrophobic inner cavity, but differ in shape, flex-
ibility and hydrophobicity, resulting in different selectivi-
ties and affinities. Neutral azoalkane guests are perfectly
suited to comparatively study these three host families,
because direct host–guest interactions, namely, hydrogen
bonding in CDs, charge interactions in CXs and ion–dipole
ones in CBs, can be neglected, in a first approximation.
The obtained results show that (1) the preorganisation of
conformational flexibility governs the binding selectivity
significantly. The well-defined CB cavity provides a much
stringent size/shape-fit than the conformationally flexible
CX cavity. (2) CXs have a wider opening and afford rather
a shallow inclusion rather than a deep encapsulation or
even threading; the latter is observed for CDs and CBs.
(3) It is the non-classical hydrophobic effect (release of
 10 D.-S. Guo et al.
high-energy water) that gives CBs the luxury of showing
superior binding affinities towards neutral guests.
Experimental
Materials
The bicyclic azoalkane homologues (DBH, DBO and DBN)
were obtained as previously reported (31). α-, β- and
γ-cyclodextrin (>95% purity) were purchased from Fluka
(Seelze, Germany) and used without purification. The calix-
arene homologues (CX4, CX5 and CX6) were synthesised
and purified according to the literature procedures (37, 39).
The CB6 sample was purchased from Merck (Darmstadt,
Germany) and used without any further purification. In
the calculations of the cucurbit[6]uril concentration,
Downloaded by [University of Sussex Library] at 07:44 05 December 2015
20% water content was taken into account, which was
determined by 1H NMR. CB7 (30% water content) was
synthesised according to the literature procedure (80).
CB8 (20% water content) was purchased from Sigma-
Aldrich (Taufkirchen, Germany). All solutions were freshly
prepared on a daily basis in ultrapure water, and the pH/
pD was adjusted using HCl and NaOH. Care was taken to
minimise the amount of NaOH used as excessive Na+ may
also complex with CB7 and affect its binding towards the
investigated guest (30, 81).
Instruments and measurements
Isothermal titration calorimetry
The microcalorimetric measurements were performed on
an isothermal titration calorimeter (VP-ITC), purchased
from MicroCal Inc., USA. The ITC instrument was peri-
odically calibrated using the internal electric heater (82)
Titration experiments were carried out at 25 °C in aqueous
solution, pH 6; each experiment consisted of 25–30 con-
secutive injections (5–10 μL) of guest solution (0.2–75 mM)
injected into the microcalorimetric reaction cell, filled with
macromolecule solution (0.03–5.00 mM) (Figure S1). All
solutions were degassed prior to the titration. Heats of
dilution were subtracted from each data set. The data
were analysed according to a 1:1 binding model in Origin
7.0 software; this affords simultaneously the binding sto-
ichiometry (N), the complex stability constant (Ka), the
standard molar reaction enthalpy (∆H°) and the standard
deviation from the titration curve. The knowledge of com-
plex stability constant (Ka) and molar reaction enthalpy
(∆H°) enabled the calculation of standard free energy (∆G°)
and entropy changes (∆S°) according to
ΔG◦ = −RT lnKa = ΔH ◦ − T ΔS◦
where R is the gas constant and T is the absolute
temperature.
NMR measurements
NMR experiments were performed at ambient tempera-
ture in D2O (99.8%). The pD values of the solutions were
adjusted by the addition of D2SO4 or NaOD. pH readings
were taken with a WTW 330i pH meter equipped with a
combined pH glass electrode (SenTix Mic) and converted
to pD (+0.40 units) (83). 1H NMR spectra (400 MHz) were
recorded using the chemical shift of HOD in D2O preset
at 4.67 ppm as reference. Concentrations of CB7 (3.0 mM)
and azoalkane (1.5 mM) were chosen to obtain a sufficient
signal strength (Figure S2).
Computational studies
The initial molecular geometry of CB7, β-CD and CX4 was
obtained from X-ray diffraction data, whereas the starting
geometries of the guest molecules were built up from
standard lengths and bond angles. The AMBER 8 program
was used throughout this work using the param99 (for β-
CD) and the general force field parameters sets (for CB7, CX4
and the guest molecules) (84). The electrostatic potentials
of the guest molecules were taken from ab initio HF/6–31G*
calculations performed within the Gaussian 03 W package
(85); atomic charges reproducing these electrostatic poten-
tials were obtained by means of the RESP methodology. All
systems were solvated in a truncated octahedral periodic
box of TIP3P water molecules with a closeness parameter of
15 Å. Periodic boundary conditions were adopted and the
particle-mesh Ewald method was used for the treatment of
long-range electrostatic interaction. The non-bonded cut-
off was set to 10.0 Å. Energy minimisation was performed
for each solvated complex using the conjugate gradient
algorithm, heated up to 298 K for 60 ps, followed by 200 ps
for achieving equilibration at 298 K and 1 atm. Production
runs were carried out for 5 ns at 298 K and a barostat at
1.0 atm (under an NPT ensemble). A 2-fs time step with a
saving of the structure every 2 ps was used, and the non-
bonded pair list was updated every 25 steps.
Supplemental material
Supplemental data for this article can be accessed here:
http://dx.doi.org/10.1080/10610278.2015.1105374
Note
1. 
We have previously measured the binding constants
of Na+ with CX4 and CX5 by fluorescence competitive
titrations, giving the values of 85 and 173 M−1 (please
see Supporting Information of Ref. 73).
Acknowledgements
The Chinese team gratefully acknowledges the support of the
NNSFC (21172119) and 21322207). The German team thanks

the Deutsche Forschungsgemeinschaft (DFG grant NA-686/5
and SPP 1807), the COST Action CM1005 ‘Supramolecular
Chemistry in Water’, and the Fonds der Chemischen Industrie
for support. Both teams thank the Robert Bosch Foundation for
bilateral collaborative project support (Wissenschaftsbrücke
China). D.-S. Guo also appreciates the DAAD for supporting his
research visit at Jacobs University Bremen.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the NNSFC [21172119], [21322207];
Deutsche Forschungsgemeinschaft DFG [grant NA-686/5], [SPP
1807]; Fonds der Chemischen Industrie; Robert Bosch Founda-
Downloaded by [University of Sussex Library] at 07:44 05 December 2015
tion; DAAD.
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