6-Hydroxymethyltriazolyl-6-deoxy-β-cyclodextrin: A highly water soluble and structurally well-defined β-cyclodextrin click cluster

Article abstract of DOI:10.1016/j.tetlet.2012.08.073

The structural, physical, and biological properties of heptakis{6-(4- hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-β-cyclodextrin (HTβCD) were investigated by a variety of methods, including NMR, UV/vis, circular dichroism spectroscopy, computer modeling, turbidity testing, K_a measurements, and the MTT assay. The experimental results suggest that HTβCD is structurally well-defined, highly water-soluble, and has low cytotoxicity. These advantages of HTβCD versus β-CD indicate that β-cyclodextrin click clusters may function both as host molecules and as potential, alternative excipients to β-CD.

Full text of DOI:10.1016/j.tetlet.2012.08.073

Tetrahedron Letters 53 (2012) 5791–5795
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
6-Hydroxymethyltriazolyl-6-deoxy-b-cyclodextrin: a highly water soluble
and structurally well-defined b-cyclodextrin click cluster
⇑
⇑
Dong-Hwan Kim , Jae Gyu Jang , Hoa Thi Le, Jin Young Kim, Choon Woo Lim , Tae Woo Kim
Graduate School of East–West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The structural, physical, and biological properties of heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-
yl)-6-deoxy}-b-cyclodextrin (HTbCD) were investigated by a variety of methods, including NMR, UV/
vis, circular dichroism spectroscopy, computer modeling, turbidity testing, Ka measurements, and the
MTT assay. The experimental results suggest that HTbCD is structurally well-defined, highly water-
soluble, and has low cytotoxicity. These advantages of HTbCD versus b-CD indicate that b-cyclodextrin
click clusters may function both as host molecules and as potential, alternative excipients to b-CD.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 14 May 2012
Revised 8 August 2012
Accepted 17 August 2012
Available online 24 August 2012
Keywords:
Cyclodextrin
Copper(I)-catalyzed azide–alkyne
cycloaddition
Cyclodextrin click cluster
Cyclodextrin host guest chemistry
b-Cyclodextrin (b-CD) is a cyclic oligosaccharide that consists of
seven glucose units. Several members of b-CD are used industrially
in pharmaceutical and allied applications.1 The ability to use the
hydrophobic cavity of b-CD to encapsulate bioactive molecules in
water has drawn tremendous interest from the pharmaceutical
industry because encapsulation improves the stability and bio-
availability of drug molecules.2 b-CD itself is the most commonly
used CD although it is the least soluble (solubility in water at
20 °C, 1.85 g/100 mL). When parenterally administered, b-CD is
not metabolized but accumulates in the kidneys as insoluble cho-
lesterol complexes, resulting in severe nephrotoxicity.3 To over-
come these disadvantages of b-CD, chemically modified b-CD
derivatives have been prepared with a view to extending the phys-
icochemical properties and inclusion capacity of parent b-CD.
For instance 2-hydroxypropyl-b-cyclodextrins (HPbCDs), which
are chemically modified b-CDs, have been shown to have better
aqueous solubility4 and a better pharmacokinetic profile for intra-
venous administration of lipophiles than b-CD.5 However, HPbCDs
are not single components; they are mixtures of compounds with
different substitution degrees (e.g. the average degree of substitu-
tion of commercially available HPbCD is 0.6–0.8 units of
2-hydroxypropyl per glucose unit). This heterogeneity in conven-
tional, modified b-CD derivatives makes it difficult for researchers
to precisely investigate the inclusion phenomena at the molecular
level. It is therefore a challenging task to synthesize novel b-CD
derivatives that are well-defined, single component molecules
with high aqueous solubility.
Several synthetic approaches to use copper(I)-catalyzed
azide–alkyne cycloaddition6 (described as the ‘click’ reaction in
this paper) to generate cyclodextrin click clusters (CCCs) have been
explored.25 CCCs have unique characteristics, namely well-defined
structures, precise molecular weights, and multivalent functional-
ization sites. Researchers have utilized CCCs as polymers,7 nucleic
acid carriers,8 sensors,9 glycoconjugates,10 and magnetic resonance
imaging probes.11 These previous studies have focused on the
development of new applications, but the systematic studies on
CCC’s physical and biological properties are rare. If you consider
the industrial needs to develop new chemically modified b-CDs
in the field of drug, food, and cosmetic formulation,26 the synthesis
and characterization of CCCs as potential, alternative excipients to
b-CD are really challenging. Before pharmaceutical studies on new
chemically modified CCCs, the basic properties of the CCCs should
be investigated by diverse techniques such as NMR, UV/vis, circular
dichroism, turbidity testing, binding constant studies, and cell
viability assay.
As a model scaffold for CCCs, we designed HTbCD (heptakis{6-(4-
hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-b-cyclodextrin)12
and prepared it using a microwave-assisted, protection-free, click
reaction, and Diaion™ HP-20 chromatography (Scheme 1). Reaction
of azido-cyclodextrin 2 with propargyl alcohol (1.5 equiv per N3)
under the optimized condition (CuSO4/THPTA (tris(3-hydrox-
ypropyltriazolylmethyl)amine) (1:5), THF/phosphate buffer
(0.1 mM, pH 7.0), microwave) followed by chromatography on
⇑
Corresponding authors.
E-mail addresses: choonwoo.lim@khu.ac.kr (C.W. Lim), tw1275@khu.ac.kr
(T.W. Kim).
These authors contributed equally to this work.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.08.073

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D.-H. Kim et al. / Tetrahedron Letters 53 (2012) 5791–5795
Scheme 1. Reactions and reagents. (a) Methanesulfonyl chloride, DMF, 65 °C; (b) NaN3, DMSO, MW; (c) propargyl alcohol, CuSO4/THPTA, sodium ascorbate, THF/phosphate
buffer (0.1 mM, pH 7.0), MW. Abbrev.: THPTA = tris(3-hydroxypropyltriazolylmethyl)amine. MW = microwave irradiation.
Figure 1. Partial 1H NMR spectrum (600 MHz) of HTbCD in D2O.
Diaion™ HP-20 afforded the desired product at a moderate yield
(52%).13 Copper ligands are often employed both to enhance the rate
of the reaction and to protect Cu(I) from oxidation in the presence of
adventurous oxygen.6a,14 THPTA developed by the Finn group has
proven to be invaluable for bioconjugation in water.15 Diaion™
HP-20 is a kind of porous-type synthetic adsorbent that is made
up of a styrene and divinyl benzene copolymer.16 Because of its
large porous size, Diaion™ HP-20 is suitable for the purification of
relatively larger small-molecules (MW P1500 Da). Diaion™ HP-
20 has been successfully utilized for CD derivative purification.17
Complete triazole formation was evident by the presence of ax-
ial symmetry in the NMR spectra of HTbCD. Only a single doublet
for all H1 protons appeared in the 1H NMR spectra (d = 5.12 ppm,
JH1–H2 = 3.2 Hz), and the corresponding C1 atoms also appeared as
one singlet (d = 103.2 ppm) in the H1 decoupled 13C NMR spec-
trum.18 All protons and carbons were fully assigned by HH COSY
(homonuclear correlation spectroscopy) and HSQC (heteronuclear
single quantum coherence) (Supplementary data A).
Figure 2. (a) Ultraviolet (solid, 4.0 ꢂ 10ꢁ5 M) and (b) circular dichroism (dot,
5.0 ꢂ 10ꢁ4 M) spectra of HTbCD in water.
The 1H NMR spectrum of HTbCD in D2O shows the unique spin–
spin coupling pattern of the two H9 protons (Fig. 1). If we consider
the long distance between the methylene carbon (C9) and the
nearest chiral carbon center (C5), the germinal coupling
(J = 13.3 Hz) of two diastereomic H9–H90 experimentally supports
the structural rigidity of HTbCD. In other words the triazole rings
on the crowded primary rim of b-CD are conformationally fixed
and the diastereomeric environment around C5 carbon propagates
to the H9 protons.
The circular dichroism spectrum of HTbCD in water, together
with the absorption spectrum, are shown in Figure 2 (Supplemen-
tary data B). Because the b-CD backbone is chiral, the excitonic
coupling between the individual triazoles borne on b-CD is circular
dichroism active. HTbCD shows a positive Cotton effect in which
the zero crossover point between the peak and the trough corre-
sponds closely to the normal UV kmax HTbCD has
Figure 3. Molecular modeling (DFT/B3LYP calculation with 6-31g(d) basis set in
vacuum, GAUSSIAN 09’ rev. 03) of HTbCD. (a) Top view and (b) side view.
emax ꢀ 23,000 Mꢁ1 cmꢁ1 and
D
e
(L–R)(max) ꢀ 350 Mꢁ1 cmꢁ1. As a
bearing
a
pendent chromophore (1-[5-(4-dimethylamino-
precedent, Blanchard-Desce and co-workers reported the circular
dichroism of their multichromophoric cyclodextrin derivative
benzyliden)-4-oxo-2-thioxo-thiazolidin-3-yl]-acetic acid): emax
ꢀ
200,000 Mꢁ1 cmꢁ1 and
D
e
(L–R)(max) ꢀ 1000 Mꢁ1 cmꢁ1 19
.
If we

D.-H. Kim et al. / Tetrahedron Letters 53 (2012) 5791–5795
5793
Figure 4. (a) The turbidity changes b-CD (
D
)and HTbCD (h) at 595 nm, (b) a photograph of 40 mg of b-CD and 40 mg of HTbCD in 100 lL water.
Figure 5. UV–visible absorption spectra of MO at various concentrations of HTbCD, upper-right box: the expanded spectra around 495 nm.
consider that the emax of HTbCD is 10 times lower than that
of Blanchard-Desce and co-workers’ multichromophoric CD, the
form (35 times) compared with b-CD.27 If you consider the polarity
difference between 4-hyroxymethyl and 4,5-dicarboxyl group, the
6-hydroxymethyltriazolyl modification of b-CD dramatically im-
proved its water solubility.
D
D
e
e
(L–R)(max) of HTbCD is unexpectedly high. The high
(L–R)(max) of HTbCD may result from the structural rigidity
and directionality of the triazole moieties borne on b-CD.
Computer modeling of HTbCD provided insight into the details
of the molecular structure (Fig. 3). The seven triazole rings have
a C7 symmetrical and directional orientation along the axis that
penetrates the cavity (top view). The limited freedom of rotation
of the triazole rings keeps them rigid and compact. This interpreta-
tion is consistent with the NMR and circular dichroism
observations.
To investigate the influence of the 6-hydroxymethyltriazolyl
moiety on the inclusion phenomenon, we determined binding
constants by two different methods: UV/vis spectrophotometry
and isothermal titration calorimetry (ITC) measurement. Binding
constants of inclusion complexes comprising methyl orange (MO)
and b-CD/HTbCD were measured by a UV/vis spectrometer follow-
ing Nakagaki’s report (Fig. 5 and Supplementary data D).21 The
binding constant of HTbCD to MO (390 Mꢁ1) was smaller than that
of b-CD to MO (2900 Mꢁ1). Because the UV/vis absorbance changes
The water solubility of HTbCD was determined by the turbidity
measurement.20 Compared with the relatively low water solubility
were relatively small (DA = 0.058) for the CD/MO system, we
of b-CD (solubility in water, 1.85 mg/100
l
L), HTbCD has higher
decided to double-check the binding constant with ITC. We per-
formed ITC experiments with two reported guests: 1-adamantane
carboxylate and decanoate (Supplementary data D). The binding
constants between the guests and HTbCD (log K 4.2 for 1-adaman-
tanecarboxylate and log K 3.0 for decanoate) were also smaller
than those reported for the guests and b-CD (log K 4.60 for 1-ada-
mantanecarboxylate22 and log K 3.82 for decanoate23) (Table 1).
The increased water solubility of HTbCD may influence the
water solubility and the solution containing HTbCD looked clean
at the full experimental concentration (Fig. 4 and Supplementary
data C). Figure 4 suggests that the minimum water solubility of
HTbCD is higher than 40 mg/100 lL; at least 20 times higher than
that of b-CD. C. Roehri-Stoeckel et al. synthesized 6-deoxy-6-(1-
(4,5-dicarboxyl)-l,2,3-triazolyl)-b-CD and reported the enhanced
water solubility of it under its neutral (8.5 times) and carboxylate

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D.-H. Kim et al. / Tetrahedron Letters 53 (2012) 5791–5795
Table 1
funded by the Ministry of Education, Science and Technology
(2011-0002609). This work was supported by a Grant from the
Kyung Hee University in 2011 (KHU-20110242).
Estimated thermodynamic parameters for guest-HTbCD complexation from ITC
measurements (T = 303 K, 1:1 binding mode)
Guesta
log K
D
H° (kJ molꢁ1
)
TD )
S° (kJ molꢁ1
Adamantane carboxylate
Decanoate
4.2
3.0
ꢁ15
ꢁ11
8.8
5.8
Supplementary data
a
Countercation: Na+.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.08.073.
References and notes
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12. Literature survey shows that Thatcher G. R. J. et al. reported the HTbCD
synthesis (Bioorg. Med. Chem. 2010, 18, 809). Their synthesis used the
conventional, organic click condition (CuI, DIEA) without a chromatographic
purification and the yield was low (15%). They did not report any structural,
physical properties of HTbCD.
Figure 6. Effect of HTbCD on cell viability in Hep-G2 cells. After 24 h of HTbCD
treatment, the visible absorption of formazan crystals was measured at 570 nm and
the cell viability was normalized by the control value ([HTbCD] = 0). Results
represent the mean ( SEM) of five independent experiments.
solvation of the cyclodextrin cavity, thereby decreasing the Kas. For
practical applications, which are currently limited by the low solu-
bility of b-CD, the reduced Ka of HTbCD can be compensated for by
the enhanced water solubility of HTbCD.
The cytotoxicity of a new chemically modified form of b-CD is
also of critical importance for future drug delivery and food/cos-
metic formulation applications. We measured the effect of the 6-
hydroxymethyltriazolyl modification of b-CD on cell viability using
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay24 (Fig. 6 and Supplementary data E). HTbCD did
not adversely impact Hep-G2 cell viability at concentrations under
13. 6-Azido-6-deoxy-b-cyclodextrin 2 (30 mg) was dissolved in a 4 mL (1:1, v/v)
cosolvent of THF/phosphate buffer (0.1 M, pH 7.0) and propargyl alcohol
(1.5 equiv per N3) was added. CuSO4/THPTA (1:5 ratio) and sodium ascorbate
were added to reach the each final concentrations of 1 mM for Cu2+ and 5 mM
for sodium ascorbate. After a short mixing period, the reaction mixture in a
closed vessel was irradiated at 70 W and 130 °C in microwave reactor (CEM
Discover, dynamic mode, no cooling air) for 10 min. The reaction progress was
roughly checked by TLC (1-propanol/ethyl acetate/water/28% ammonia = 6/1/
3/1, Rf = 0.79 for the starting, Rf = 0.35 for the desired product). THF was
evaporated in vacuo and the residue was purified by column chromatography
on a Diaion™ HP-20 (water/methanol = 100/0 to 60/40). The 80/20 (water/
methanol, v/v) fraction afforded the desired product (yield, 52%). Up to
ꢃ200 mg of HTbCD was successfully obtained by open-vessel MW irradiation
at 75 °C for 1 h.
100
centration of HTbCD (1000
In conclusion, we synthesized HTbCD, a chemically modified
b-cyclodextrin through microwave-assisted, protection-free,
lM. The cell viability decreased to 86% only at a very high con-
lM) compared with the control.
a
copper(I)-catalyzed azide–alkyne cycloaddition reaction, and inves-
tigated its properties. HTbCD has unique structural and physical
characteristics. NMR, circular dichroism, and computer modeling
showed that HTbCD has a well-defined, rigid structure. In addition
the 6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl) modification of b-
CD dramatically improved its water solubility. The binding con-
stants measured by UV/vis and ITC titration revealed that HTbCD
has a lower Ka than b-CD. An MTT assay revealed that the 6-hydrox-
ymethyltriazolyl modification of b-CD did not adversely affect cell
1H NMR (D2O, 600 MHz): d 7.89 (s, 7H, H7), 5.12 (d, 7H, J = 3.2 Hz, H1), 4.46 (d,
7H, J = 13.3 Hz, H9), 4.41 (d, 7H, J = 13.3 Hz, H9), 4.30 (app d, 7H, H6), 4.24 (app
t, 7H, H5), 4.17 (m, 7H, H6), 4.00 (app t, 7H, H3), 3.61 (dd, 7H, J = 9.8, 3.1 Hz,
H2), 3.40 (app t, 7H, H4); 13C NMR (D2O, 150 MHz): d 148.2 (C8), 127.2 (C7),
103.2 (C1), 84.1 (C4), 73.8 (C3), 73.1 (C2), 71.5 (C5), 55.8 (C9), 51.7 (C6); MALDI-
TOF (CHCA, positive): calcd 1701.5 for C63H91N21O35, observed 1724.2 for
[M+Na]+ (relative intensity 100%).
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viability for HTbCD concentrations of less than 100 lM. These exper-
imental data indicate that HTbCD meets the following basic require-
ments for further applications: (1) facile synthesis, (2) well-defined
structure, (3) high water solubility, and (4) low cytotoxicity. Consid-
ering these material properties of HTbCD, structurally well-defined,
highly water-soluble cyclodextrin click clusters appear to have po-
tential as novel excipients in pharmaceutical formulations.
19. Rekaï, E. D.; Baudin, J. B.; Jullien, L.; Ledoux, I.; Zyss, J.; Blanchard-Desce, M.
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This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)

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5795
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