6-Triazolyl-6-deoxy-β-cyclodextrin derivatives: synthesis, cellular toxicity, and phase-solubility study

Article abstract of DOI:10.1016/j.carres.2014.03.020

Heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy} -β-cyclodextrin (HTβCD) and heptakis{6-(4-sulfonylmethyl-1H-[1,2,3] triazol-1-yl)-6-deoxy}-β-cyclodextrin (STβCD) were prepared using copper(I)-catalyzed azide-alkyne cycloaddition between 6-azido-6-deoxy-β-CD and one of two alkynes, propargyl alcohol, and sodium propargyl sulfonate, respectively. The structures of HTβCD and STβCD were characterized by NMR techniques. NMR interpretations and computer modeling suggested that the limited freedom of rotation of the triazole moieties keeps HTβCD and STβCD rigid and compact. Water solubility tests of HTβCD and STβCD showed that the minimum water solubility of HTβCD and STβCD is at least 20 times higher than that of β-CD. MTT assay showed that HTβCD and STβCD did not influence the cell viability under 1 mM. A phase-solubility study of prednisolone with the CD derivatives showed increased solubility of prednisolone in the presence of increasing concentrations of HTβCD and STβCD.

Full text of DOI:10.1016/j.carres.2014.03.020

Carbohydrate Research 391 (2014) 22–28
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
6-Triazolyl-6-deoxy-b-cyclodextrin derivatives: synthesis, cellular
toxicity, and phase-solubility study
a,
Hoa Thi Le a, Hyun Mi Jeon a, Choon Woo Lim b, , Tae Woo Kim
⇑
⇑
a Graduate School of East-West Medical Science, Kyung Hee University, Gyeonggi-do 449-701, Republic of Korea
b Department of Chemistry, College of Life Science and Nano-technology, Hannam University, Daejeon 305-811, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-b-cyclodextrin (HTbCD) and heptakis{6-
(4-sulfonylmethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-b-cyclodextrin (STbCD) were prepared using cop-
per(I)-catalyzed azide–alkyne cycloaddition between 6-azido-6-deoxy-b-CD and one of two alkynes,
propargyl alcohol, and sodium propargyl sulfonate, respectively. The structures of HTbCD and STbCD
were characterized by NMR techniques. NMR interpretations and computer modeling suggested that
the limited freedom of rotation of the triazole moieties keeps HTbCD and STbCD rigid and compact. Water
solubility tests of HTbCD and STbCD showed that the minimum water solubility of HTbCD and STbCD is at
least 20 times higher than that of b-CD. MTT assay showed that HTbCD and STbCD did not influence the
cell viability under 1 mM. A phase-solubility study of prednisolone with the CD derivatives showed
increased solubility of prednisolone in the presence of increasing concentrations of HTbCD and STbCD.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 29 October 2013
Received in revised form 23 February 2014
Accepted 18 March 2014
Available online 28 March 2014
Keywords:
Cyclodextrin
Cyclodextrin click cluster
Copper(I)-catalyzed azide–alkyne
cycloaddition
6-Triazolyl-6-deoxy-b-cyclodextrin
derivatives
Phase-solubility study
1. Introduction
as a means for solving the solubility problem seen with parenteral
b-CD administration.5
Several cyclodextrins (CDs) are used industrially in pharmaceu-
tical and allied applications.1 The ability to utilize 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 bioavailability
of drug molecules.2 The host guest chemistry between CDs and a
variety of guest molecules, like aliphatic or aromatic compounds,
has been intensively explored and well reviewed.3
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 accumu-
lates in the kidneys as insoluble cholesterol complexes, resulting
in severe nephrotoxicity.4 To overcome these disadvantages of
b-CD, chemically modified b-CD derivatives have been prepared
For example, 2-hydroxypropyl-b-CD (HPbCD) and sulfobutyl
ether-b-CD (SBEbCD), which are chemically modified b-CDs, have
been shown to have increased aqueous solubility6 and a better
pharmacokinetic profile for intravenous administration of
lipophiles than b-CD.7 These modified b-CDs have been used in
the market as pharmaceutical excipients.8 However, these
modified b-CDs are not single components: they are mixtures of
compounds with different degrees of substitution. For instant,
the average degree of substitution of commercially available
HPbCD is 0.6–0.8 units of 2-hydroxypropyl per glucose unit. This
heterogeneity in conventional modified b-CD derivatives makes it
difficult for researchers to precisely investigate the inclusion phe-
nomena at a molecular level. Therefore, the need exists to identify
and synthesize novel b-CD derivatives that are well-defined, single
component molecules with high aqueous solubility and improved
pharmacokinetic properties.
Several approaches to b-CD derivative synthesis have used
copper(I)-catalyzed azide–alkyne cycloaddition9 (the ‘click’ reac-
tion) to generate cyclodextrin click clusters (CCCs). Azides and
alkynes are inert to most biological and organic conditions, includ-
ing molecular oxygen, water, and the majority of common reaction
conditions in organic synthesis.10 The click reaction is an efficient
way to make a connection between azide and alkyne and has
Abbreviations: CCC, cyclodextrin click cluster; CD, cyclodextrin; HPbCD, 2-
hydroxypropyl-b-CD; HTbCD, heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-
yl)-6-deoxy}-b-cyclodextrin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide; SBEbCD, sulfobutyl ether-b-CD; CCC, cyclodextrin click clusters;
STbCD, heptakis{6-(4-sulfonylmethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-b-cyclodex-
trin; THPTA, tris(3-hydroxypropyltriazolylmethyl)amine.
⇑
Corresponding authors. Tel.: +82 (31)2013704; fax: +82 312048119.
E-mail addresses: cwlim@hnu.kr (C.W. Lim), tw1275@khu.ac.kr (T.W. Kim).
http://dx.doi.org/10.1016/j.carres.2014.03.020
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
23
Table 1
CD-sulfated sodium salt failed because the compounds had phar-
macological activity, were metabolically unstable, or had limited
ability to complex with the drugs to be delivered. These issues
were overcome with the development of sulfoalkyl-modified CDs.
For instance, SBEbCD, sold under the name CAPTISOLÒ (degree of
substitution ꢀ7), has been a component of six FDA-approved
parenteral products and is a component of a number of other
parenteral products in late stage development.5 The development
history of SBEbCD shows that, while CD sulfate is metabolically
unstable and pharmacologically active, CD sulfonate is metaboli-
cally stable and pharmacologically inactive.
Binding constants for inclusion complexation of prednisolone with various b-CDs
Prednisolone
Cyclodextrin K1:1
Solubility
mg/mL in
50 mM CD
solution
(Mꢁ1
)
ND*
5.1
4.7
1.9
O
b-CD
HPbCD
2100
530
HO
H
OH
HO
HTbCD
STbCD
350
120
H
H
Copper(I)-catalyzed azide–alkyne cycloaddition reactions are
reliable and efficient, but their efficiency in the generation of
multiple triazoles on CD is still subject to debate.24,25 Copper
ligands are often employed both to enhance the rate of reaction
and to protect Cu(I) from oxidation in the presence of oxygen.26,27
Thus, we used a water-soluble ligand, THPTA (tris(3-hydrox-
ypropyltriazolylmethyl)amine, Supplementary data S1.1) to
maximize our click efficiency in aqueous medium. THPTA,
developed by the Finn group, has proven to be invaluable for
bioconjugation in water.28
6-Azido-6-deoxy-b-CD (Supplementary data S1.2–3) was pre-
pared from b-CD by a two-step reaction: (1) chlorination of the pri-
mary alcohol and (2) azide substitution at a gram scale without
column chromatography (Scheme 1). In this study, we modified
our original procedure for scale-up and obtained purified HTbCD
without column chromatography in moderate yield (55%). Using
propargyl sulfonate (Supplementary data S1.4), b-CD with seven
sulfonate groups at its primary face (STbCD) was prepared without
column chromatography in high yield (90%).
O
*
The prednisolone solubility in 50 mM of b-CD is not determined because of the
low solubility of b-CD (solubility, 1.85 mg/100 lL; saturation concentration
16 mM).
tolerance for a wide ranges of solvents, temperature, and pH. The
resulting 1,2,3-triazole ring is structurally rigid and chemically
stable.
In viewpoint of synthesis, CCCs have a couple of merits. CCCs
can be easily prepared from 6-azido-6-deoxy CDs in gram scale.
They have unique characteristics, namely well-defined structures
and precise molecular weights. Because of the bioorthogonal char-
acteristics, the click reaction can modify CDs with highly function-
alized biological molecules without any time-consuming
protection/deprotection steps.11 Thus researchers have utilized
CCCs as nucleic acid carriers,12,13 glycoconjugates,14,15 magnetic
resonance imaging probes,16,17 etc.
To investigate the CCCs’ potential as new modified CD deriva-
tives in the field of pharmaceutical application, we should reply
to three questions. The first one is about CCC synthesis: are CCCs
are easy to prepare? The second one is about safety: are CCCs are
non-toxic in cellular level? The last one is on guest encapsulation:
do CCCs make a stable inclusion complex with drugs in water?
However, CCC studies on pharmaceutical field are rare.
In order to answer these questions, we decided to test cellular tox-
icity and drug inclusion property of two 6-triazolyl-6-deoxy-b-CDs,
heptakis{6-(4-hydroxymethyl-1H-[1,2,3]triazol-1-yl)-6-deoxy}-b-
cyclodextrin (HTbCD) and heptakis{6-(4-sulfonylmethyl-1H-[1,
2,3]triazol-1-yl)-6-deoxy}-b-cyclodextrin (STbCD). 3-(4,5-Dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay gives
a guideline for cellular toxicity.18 A phase-solubility study provides
evidence for CCC application as a drug solubilizer. Prednisolone (a
poorly water-soluble glucocorticoid, Table 1) was selected as a
model compound for the phase-solubility study. CDs, especially
2.2. Structural characterization by NMR and computer
modeling
Complete triazole formation was evident by the presence of
axial symmetry in the NMR spectra. Only a single doublet for all
H1 protons appeared in the 1H NMR spectra (d = 5.12 ppm, JH1–H2
= 3.2 Hz for HTbCD; d = 5.15 ppm, JH1–H2 = 3.4 Hz for STbCD), and
the corresponding C1 atoms also appeared as one singlet
(d = 103.2 ppm for HTbCD; d = 102.1 ppm for STbCD) in the H1
decoupled 13C NMR spectrum. All protons and carbons were fully
assigned by HH COSY (homonuclear correlation spectroscopy)
and CH HSQC (heteronuclear single quantum coherence) spectros-
copy (Supplementary data S2).
The 1HNMR spectrum of HTbCD and STbCD 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 cou-
pling (J = 13.3 Hz for HTbCD; J = 14.7 Hz for STbCD) of the diaste-
b- and
c-CD, have been shown to form complexes with many
steroids, leading to improved solubility and dissolution rate
compared with the pure steroids.19 Improved bioavailability of
CD-based steroid formulations has been demonstrated.20,21 Except
conventional CD application for drug formulation, cyclodextrin
clusters were applied to nanochemistry, like quantum dot
cappingorsupramolecularglycocluster.22,23 Consideredthesynthetic
advantage of CCCs, CCC synthesis will be adopted as a basic toolbox in
emerging cyclodextrin–nanochemistry.
0
reomic protons (H9–H9 ) supports the structural rigidity of HTbCD
and STbCD. Accordingly, the triazole rings on the crowded primary
rim of b-CD are conformationally fixed, and the diastereomeric
environment around the C5 carbon propagates to the H9 protons.
Computer modeling of HTbCD and STbCD provided insight into
the details of their molecular structure (Fig. 2). 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. In
addition the computer modeling implies that triazolyl pendants lit-
tle influence on the hydrophobic cavity size of CD.
2. Results and discussion
2.1. Synthesis of 6-triazolyl-6-deoxy-b-CD derivatives
The introduction of an anion charge to CDs is an important
strategy to improve the parenteral delivery of poorly-soluble
drugs. This concept was developed based on the excretion profile
of sulfate ions: sulfate ions remain ionized after glomerular
filtration, do not undergo significant tubular reabsorption, and
are excreted in the urine. However, early attempts at using
2.3. Water solubility of 6-triazolyl-6-deoxy-b-CD derivatives
The water solubility of the CDs was assessed by turbidity mea-
surement.29 Compared with the relatively low water solubility of
b-CD, solutions containing HPbCD, HTbCD, or STbCD appeared clear

24
H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
R
HTβCD, R = OH
STβCD, R = SO3Na
N
N
N
OH
Cl
N3
O
O
O
O
O
O
O
O
c or d)
a)
b)
O
H
OH
OH
OH
HO
HO
HO
HO
7
7
7
7
Scheme 1. Reactions and reagents. (a) Methanesulfonyl chloride, DMF, 65 °C; (b) NaN3, DMSO, and MW; (c) for HTbCD/propargyl alcohol, CuSO4/THPTA, sodium ascorbate,
THF/phosphate buffer, MW; (d) for STbCD/propargyl sulfonate, CuSO4/THPTA, sodium ascorbate, THF/ phosphate buffer/ethanol, 80 °C, 20 h. THPTA = tris(3-hydrox-
ypropyltriazolylmethyl)amine, MW = microwave irradiation.
Figure 1. Partial 1H NMR spectra (600 MHz in D2O). (a) HTbCD and (b) STbCD.
at the full experimental concentration (Fig. 3 & Supplementary
data S3). These results suggest that the minimum water solubility
applications. We measured the effect of the 6-hydroxymethyltriaz-
olyl modification of b -CD on cell viability using MTT assay (Fig. 4).
As a reference CD, we selected HPbCD (average Mw ꢀ 1540).
HPbCD is commercially available and considered as a safe CD (An
active ingredient of Febreze, P&G). HPbCD, HTbCD, and STbCD did
not adversely impact HeLa cell viability at concentrations under
1 mM. Only STbCD showed doze-dependent cell viability decrease
at high concentrations (2, 5 mM).
of HTbCD or STbCD is higher than 33 mg/100
higher than that of b-CD (solubility in water at 20 °C, 1.85 mg/
100 L).
lL; at least 20 times
l
Intramolecular hydrogen bonding of b-CD can result in rela-
tively unfavorable enthalpies of solution and low aqueous solubil-
ity. Substitution of any of the hydrogen-bond forming hydroxyl
groups, even with hydrophobic moieties such as methoxy and eth-
oxy functions, will result in a dramatic increase in water solubil-
ity.30 Actually HTbCD and STbCD are freely miscible in 100 mg/
2.5. Phase solubility studies
100
l
L (CD/water) and result in viscous media-a 50-fold improve-
A phase-solubility profile of prednisolone with various CDs at
25 °C was obtained according to the methods of Hirayama & Uek-
ama.31 Establishment of equilibrium was verified by the compari-
son of HPbCD phase solubility after 24, 48, and 72 h. The results
showed no significant differences. Hence, the equilibration time
was set to 24 h (Supplementary data S4). A linear standard curve
was obtained in the concentration range from 2.4 ꢂ 10ꢁ4 to
1.5 ꢂ 10ꢁ2 M for prednisolone. The standard curve showed a good
linearity in the concentration range (Supplementary data S5). The
ment compared with the solubility of b-CD. From the turbidity test
of various CD derivatives, we confirm that the 6-triazolyl modifica-
tion of b-CD dramatically improves its water solubility.
2.4. MTT Assay
The cytotoxicity of
critical for future drug delivery and food/cosmetic formulation
a new chemically modified b-CD is

H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
25
Figure 2. Molecular modeling (DFT/B3LYP calculation with 6–31g(d) basis set in vacuum, Gaussian 09’ rev. 03). (a) Top view and (b) side view of HTbCD; (c) top view and (d)
side view of STbCD (sulfonic acid instead of sulfonate).
Figure 3. Turbidity measurement. (a) Absorbance by concentration of b-CD (
D
), HPbCD (s), HTbCD (h), STbCD (—) at 595 nm; (b) visual appearance of ca. 50 mg of CDs in
150 L water.
l
intrinsic solubility in water of prednisolone (S0) was determined
from four individual samples. The intrinsic aqueous solubility of
prednisolone was determined to be 250 48 mg/L (Supplementary
data S6). This was slightly lower than a previously reported value
of 390 mg/L.32
solubility. The increased solubility of prednisolone in the presence
of increasing concentrations of HPbCD, HTbCD, and STbCD is
shown in Figure 5. All CDs exhibited AL-type profiles in the chosen
concentration range. AL-type phase-solubility profiles are obtained
when the solubility of prednisolone increases with CD concentra-
tion. In general, the water-soluble CD derivatives formed A-type
phase solubility profiles. In the AL case, assuming that the complex
Phase solubility analysis provides valuable data on the effect of
various CDs on the solubility of drugs with poor or limited aqueous

26
H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
Figure 4. Effect of HTbCD, STbCD, and HPbCD on cell viability in HeLa cells. After 24 h treatment of the b-CDs, the visible absorption of formazan crystals was measured at
570 nm and the cell viability was normalized by the control value ([HTbCD, STbCD, and HPbCD] = 0). Results represent the mean ( SEM) of five independent experiments
Figure 5. Phase-solubility diagram of prednisolone-CD systems at 25 °C after 24 h equilibration. b-CD (D), HPbCD (s), HTbCD (h), and STbCD (—). The mean of two
independent measurements is plotted, and error bars = |measurement1 ꢁ measurement2|.
is of 1:1 binding mode, the stability constant of the complex can be
calculated from the slope of the isotherm by Eq. (1):
simplify the logP calculation, we utilized 1-methoxy-2-propanol
(S1), 1-methyl-1H-1,2,3-triazole-4-methanol (S2), 1-methyl-1H-
1,2,3-triazole-4-methane sulfonic acid (S3) instead of HPbCD,
HTbCD, and STbCD themselves.
slope
S0ð1 ꢁ slopeÞ
K1:1
¼
ð1Þ
S2 (logP = ꢁ1.57) is hydrophilic as compared to S1
(logP = ꢁ0.45), and S3 (logP = ꢁ6.06) is more hydrophilic than S2.
Supposing the substituent degree and position of HPbCD used in
this experiment (0.6–0.8 units of 2-hydroxypropyl per glucose unit
and 2, 3, and 6-position random substitution), the practical logP
between HPbCD and HTbCD may be larger than the logP between
S1 and S2. Figure 6 shows good correlation between stability
constants and logP values. Hydrophilic triazole substituents at
the primary face enhance the water solubility of 6-triazolyl-
6-deoxy-b-CD derivatives. Conversely, the enhanced solubility
increases the aqueous solvation shell around the primary face
and reduces guest molecule accessibility.
The solubility of the prednisolone complex with CD derivatives
is summarized in Table 1. The solubility of native b-CD/predniso-
lone complexes is relatively limited because of the low solubility
of b-CD. A large linear increase in prednisolone solubility was
observed with HPbCD, HTbCD, and STbCD. The stability constant
of HTbCD and STbCD (350, 120 Mꢁ1) are relatively lower than the
one of HPbCD (530 Mꢁ1) (Table 1). The prednisolone solubility
(mg/mL) in 50 mM CD solution was 5.1, 4.7, and 1.9 for HPbCD,
HTbCD, and STbCD, respectively.
Just-physical blocking of the primary face of the CD cannot
explain the diminished binding of prednisolone to two triazolyl-
b-CDs. Computer modeling in Figure 2 shows that the inflexible tri-
azole moieties do not block the hydrophobic cavity. The calculation
seems to suggest just-physical blocking is not the case. However,
we cannot completely exclude the possibility since our calculation
did not take account of water solvation and ionization of the triaz-
olyl-b-CDs.
3. Conclusion
Two 6-triazolyl-6-deoxy-b-CD derivatives, HTbCD and STbCD
were synthesized using copper(I)-catalyzed azide–alkyne cycload-
dition between 6-azido-6-deoxy-b-CD and two alkynes, propargyl
alcohol, and sodium propargyl sulfonate. Their structures were
characterized by 1H, 13C NMR, HH COSY, and CH HSQC. The NMR
interpretations and the computer modeling suggested that HTbCD
and STbCD have rigid and compact structures. The water turbidity
tests of b-CD, HPbCD, HTbCD, and STbCD showed that the mini-
mum water solubility of HTbCD and STbCD is at least 20 times
Another possible explanation for the differences observed in the
binding of CDs for prednisolone is that the aqueous salvation shell
associated with the substituents of CD may diminish the size of the
cavity opening, resulting in reduced accessibility of guest mole-
cules.28 The decrease in stability constant of the CD derivatives is
parallel with the logP decrease of their substituents (Fig. 6). To

H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
27
Figure 6. Correlation diagram: logK of b-CD derivatives/prednisolone complexes versus logP of the substituents. LogPs (partition coefficients) were calculated using
Advanced Chemistry Development (ACD/Labs) Software V12.01. LogP of S3 was assumed as [logP (S2) + (logP (methanesulfonic acid) – logP (methanol))]. LogP = log
([Solute]octanol/[Solute]water).
higher than that of b-CD. HTbCD and STbCD were found to be freely
miscible in 100 mg/100 L (CD/water) and result in viscous media.
CuSO4ꢃ5H2O/THPTA (Supplementary data 1.3) (0.5 mL of 100 mM
stock in water, 1:5 molar ratio), and freshly prepared sodium
ascorbate (0.5 mL of 500 mM stock in water) were mixed with
60 mL of THF/0.1 M phosphate buffer (pH 7) (1:1, v/v). The reaction
was carried out in a microwave reactor (100 °C, 40 min, Power
60 W, open vessel). After the reaction, the mixture was shaken
with CupriSorb™ resin overnight to remove copper ions. The reac-
tion mixture was then filtered to remove CupriSorb™ resin and
insoluble impurities. The supernatant was evaporated and precip-
itated using methanol (100 mL). The precipitate was washed with
methanol (3 ꢂ 10 mL). The solid was dissolved in water (4 mL) and
triturated with acetone (100 mL). The precipitate was dissolved in
water (15 mL) and lyophilized using a freeze dryer (Ilshin Europe
B.V, model FD8512) to yield a pale yellowish powder (320 mg,
0.188 mmol, yield 55%).
l
MTT assay showed the low cellular toxicity of HTbCD and
STbCD. In other words, they did not adversely impact HeLa cell via-
bility at concentrations less than 1 mM. The phase-solubility study
of prednisolone with the CD derivatives showed increased solubil-
ity of prednisolone in the presence of increasing concentrations of
HTbCD and STbCD. The stability constants for the complexes
between prednisolone and the CDs were 2200, 530, 350, 120 Mꢁ1
for b-CD, HPbCD, HTbCD, and STbCD, respectively. The lower stabil-
ity constants of triazolyl-b-CDs suggest that the strong solvation
around triazole rings at CD primary rim hamper the accessibility
of prednisolone. If we consider that the low solubility of b-CD lim-
its its practical applications, including pharmaceutical, cosmetic,
and food excipient applications, the reduced K of HTbCD and STbCD
can be compensated for by their enhanced water solubility. To
develop triazolyl-CDs with high stability constant, we are further
investigating the synthesis and phase-solubility study of 2,3- alkyl-
ated triazolyl-b-CDs.
1H NMR (600 MHz, D2O): 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 (150 MHz, D2O): 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): calculated 1701.5 for
4. Experimental
C
63H91N21O35, observed 1724.2 for [M+Na]+.
4.1. Materials and characterization
(Supplementary data 2 for 1H, 13C, HH-COSY, HSQC spectra of
HTbCD).
b-CD and prednisolone were purchased from TCI (PN C0777and
P0637). HPbCD was purchased from Aldrich (PN 389145). All
purchased reagents were used without purifications. The 1H and
13CNMR, HH COSY, and CH HSQC spectroscopy of HTbCD and
4.2.2. Stbcd
6-Azido-6-deoxy-b-CD (500 mg, 0.382 mmol), sodium propar-
gyl sulfonate (494 mg, 3.47 mmol, eqv. 1.3/N3 group, Supplemen-
tary data 1.4), CuSO4ꢃ5H2O/THPTA (0.8 mL of 100 mM stock in
water, 1:5 molar ratio), and sodium ascorbate (0.8 mL of 500 mM
stock in water) were mixed with 120 mL of THF/0.1 M phosphate
buffer (pH 7)/EtOH (5:5:2). The mixture was stirred at 80 °C for 1
day. The reaction mixture was evaporated to remove solvent. The
solid was dissolved in water (100 mL) and the insoluble impurities
were filtered off. The filtrate was shaken with CupriSorb™ resin for
overnight. The supernatant was evaporated and precipitated with
cooled methanol (100 mL). The solid was dissolved in 5 mL water
and acetone (100 mL) was added. The precipitate was dissolved
in water (15 mL) and stirred overnight. The mixture was centri-
fuged at 12,000 rpm for 30 min. The supernatant was lyophilized
using a Freeze dryer (Ilshin Europe B.V, model FD8512) to give a
white powder (795 mg, 0.345 mmol, yield 90%).
STbCD were performed on
spectrometer.
a Bruker Avance III 600 NMR
The other 1H and 13CNMR spectra were measured on a JNM-
AL300 (JEOL) spectrometer. Chemical shifts were reported as d in
units of parts per million (ppm), and J-values are in Hz. MALDI
MS spectra were measured on a Voyager-DE™ STR Biospectrome-
try Workstation (Applied Biosystems). A CEM Discover™ micro-
wave reactor was utilized for the microwave-assisted organic
synthesis.
4.2. Synthesis
4.2.1. HTbCD
The synthesis of HTbCD was reported by our group,33 but we
modified the procedure to scale-up without column chromatogra-
phy: 6-azido-6-deoxy-b-CD (450 mg, 0.344 mmol; Supplementary
1H NMR (600 MHz, D2O): d 8.01 (s, 7H, H7), 5.16 (d, 7H,
J = 3.4 Hz, H1), 4.58 (app d, 7H, H6), 4.39 (dd, 7H, J = 14.9, 5.8 Hz,
data 1.2), propargyl alcohol (208 lL, 3.6 mmol, eqv. 1.5/N3 group),

28
H. T. Le et al. / Carbohydrate Research 391 (2014) 22–28
H6), 4.31 (br, 7H, H5), 4.14 (d, 7H, J = 14.7 Hz, H9), 4.10 (d, 7H,
J = 14.8 Hz, H9), 4.00 (app t, 7H, H3), 3.56 (dd, 7H, J = 10.0, 3.2 Hz,
H2), 3.33 (app t, 7H, H4); 13C NMR (150 MHz, D2O): 139.6 (C8),
127.9 (C7), 102.2 (C1), 82.6 (C4), 73.0 (C3), 72.2 (C2), 70.5 (C5), 50.9
(C6), 48.0 (C9); MALDI-TOF (CHCA, positive): Calculated 2303.2 of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2014.
03.020.
C
63H84N21O49Na7S7, observed 2325.9 of [M+Na]+and 2342.1of
[M+K]+.
References
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Acknowledgments
This research was supported by the Basic Science Research
Program through the National NRF of Korea funded by the
Ministry of Education, Science and Technology (2011-0024970).
This work was supported by the National Research Foundation of
Korea (NRF) grant funded by the Korea government (MEST)
(2013R1A2A2A04016066).
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