Biotinylated cyclooligosaccharides for paclitaxel solubilization

Article abstract of DOI:10.3390/molecules23010090

The poor water solubility of paclitaxel causes significant problems in producing cancer therapeutic formulations. Here, we aimed to solubilize paclitaxel using biocompatible cyclic carbohydrates. Generally recognized as safe, labeled β-cyclodextrin (β-CD), a cyclic α-1,4-glucan consisting of seven glucoses, was prepared, and bio-sourced cyclosophoraoses (CyS), which are unbranched cyclic β-1,2-glucans with 17–23 glucose units, were purified using various chromatographic methods from Rhizobium leguminosarum cultural broth. For effective targeting, CyS and β-CD were modified with a biotinyl moiety in a reaction of mono-6-amino CyS and mono-6-amino-β-CD with N-hydroxysuccinimide ester of biotinamidohexanoic acid. Interestingly, the aqueous solubility of paclitaxel was enhanced 10.3- and 3.7-fold in the presence of biotinyl CyS and biotinyl β-CD, respectively. These findings suggest that biotin-appended cyclooligosaccharides can be applied to improve the delivery of paclitaxel.

Full text of DOI:10.3390/molecules23010090

molecules
Article
Biotinylated Cyclooligosaccharides for
Paclitaxel Solubilization
Eunae Cho 1 and Seunho Jung 2,
*
1
Institute for Ubiquitous Information Technology and Applications (UBITA) & Center for Biotechnology
Research in UBITA (CBRU), Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea;
echo@konkuk.ac.kr
Department of Bioscience and Biotechnology, Microbial Carbohydrate Resource Bank (MBRC) & Center for
Biotechnology Research in UBITA (CBRU), Konkuk University, 120 Neungdong-ro, Gwangjin-gu,
Seoul 05029, Korea
2
*
Correspondence: shjung@konkuk.ac.kr; Tel.: +82-2-450-3520
Received: 17 November 2017; Accepted: 28 December 2017; Published: 2 January 2018
Abstract: The poor water solubility of paclitaxel causes significant problems in producing cancer
therapeutic formulations. Here, we aimed to solubilize paclitaxel using biocompatible cyclic
carbohydrates. Generally recognized as safe, labeled
consisting of seven glucoses, was prepared, and bio-sourced cyclosophoraoses (CyS), which are
unbranched cyclic -1,2-glucans with 17–23 glucose units, were purified using various chromatographic
methods from Rhizobium leguminosarum cultural broth. For effective targeting, CyS and -CD were
modified with a biotinyl moiety in a reaction of mono-6-amino CyS and mono-6-amino- -CD with
N-hydroxysuccinimide ester of biotinamidohexanoic acid. Interestingly, the aqueous solubility of
paclitaxel was enhanced 10.3- and 3.7-fold in the presence of biotinyl CyS and biotinyl -CD, respectively.
β-cyclodextrin (β-CD), a cyclic α-1,4-glucan
β
β
β
β
These findings suggest that biotin-appended cyclooligosaccharides can be applied to improve the
delivery of paclitaxel.
Keywords: solubilization; cyclooligosaccharide; paclitaxel; biotin
1. Introduction
Paclitaxel (PTX) is a complex diterpene natural product isolated from the stem bark of
Taxus brevifolia [
dynamics [ ], PTX has shown excellent antitumor activity for treating metastatic breast, advanced ovarian,
and non-small cell lung cancer [ ]. However, its clinical usage has been limited by its poor water
solubility, making the development of injectable formulations difficult [ ]. Poor aqueous solubility is
also a serious problem associated with drug discovery and combinatorial screening systems, and at
least 40% of identified active substances are poorly soluble in water [ ]. To increase the water solubility
of hydrophobic drugs, nanosizing, liposomes, and synthesized water-soluble derivatives have been
utilized [ ]. Although co-solvent systems including ethanol and Cremophore have also been employed,
significant side-effects such as neurotoxicity, nephrotoxicity, and anaphylactoid hypersensitivity reactions
occur [ ]. Therefore, there is a growing need for alternative biocompatible strategies to solubilize
hydrophobic drugs.
For drug solubilization, supramolecular complexation technology has been introduced and the
1]. Because it induces cell death by binding to β-tubulin and affecting microtubule
2
3
4
5
6–8
9
recognition ability of carbohydrates is attractive for intermolecular interactions [10]. Supramolecular
complexation with appropriate host molecules can solubilize hydrophobic guest molecules, separate
chiral compounds, and catalyze organic transformation. Particularly, macrocyclic carbohydrate hosts,
such as cyclodextrins (CD) and cyclosophoraose (CyS), have received attention because of their
characteristic structural features, including hydrophobic cavities, hydrophilic exteriors, and selective
Molecules 2018, 23, 90; doi:10.3390/molecules23010090
www.mdpi.com/journal/molecules

Molecules 2018, 23, 90
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molecular recognition abilities [11
,12]. CDs are
α
-1,4-linked glucans composed of 6, 7, or 8 glucose
units for α-, β-, and γ-CD, and are produced by cyclodextrin glycosyl transferase treatment of starch.
Using -cyclodextrin ( -CD) as a complexing agent, the stability and solubility of various drugs
containing ibuprofen, itraconazole, and atenolol have been enhanced [13 15]. With the availability
β
β
–
of various guest molecules due to the large size and different glycosidic linkages, CyS are produced
via the metabolic processes of many fast-growing soil bacteria such as Rhizobium species in their
periplasmic space as intra- and extra-oligosaccharides [16,17]. CyS, which are cyclic β-1,2 glucans
with 17–23 glucose residues, have been reported to exhibit induced fit complexation with hydrophobic
bioactive molecules [12,18,19].
To endow cyclooligosaccharides with advanced functionality, they can be modified with various
substrates including methyl, carboxy-methyl, and hydroxypropyl groups [20]. Additionally, original
β-CD shows a limitation: a rigid and stable cavity size (7 Å) [21]. When breaking ordered hydrogen
bonds of secondary hydroxyl groups, they show increased water solubility and expanded guest
ranges [22]. At the point of site-specific delivery, specific CD derivatives, including biphenylylacetic acid
or methicilline-β-CD conjugates, have been reported [23,24]. Here, we modified cyclooligosaccharides
with biotin, whose receptor is overexpressed on the surface of cancer cells [25], to achieve cancer cell
specificity. Biotin is a growth promoter known as vitamin B7 and is composed of a valeric acid and ureido
ring fused with a tetrahydrothiophenene ring. Although the paclitaxel solubilization is important for its
clinical effectiveness in anticancer therapy, and thus researchers have consistently studied the topic so
far [26–28], more advanced paclitaxel delivery systems are still challengeable [29]. Here, our own strategy
is the use of cell-derived cyclooligosaccharides and their biotinyl derivatives.
In the present study, we isolated and purified bio-sourced CyS using ethanol precipitation and
chromatography. The mono-biotinylation of the primary hydroxyl groups of cyclooligosacchrides
was successfully performed through a mono-tosylation, azidation, amination, and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) coupling reaction, and the resulting structure was analyzed by
nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption/ionization (MALDI)
time-of-flight (TOF) mass spectrometry. Based on the flexible and distorted rings of various sizes, CyS and
biotinyl derivatives were evaluated for their ability to solubilize paclitaxel in a phase solubility study,
and the representative cyclic host β-CD and derivatives were also compared.
2. Results and Discussion
2.1. Synthesis and Structural Analyses of Biotinyl Cyclooligosaccharides
Rhizobial CyS, which is a cyclic beta 1,2 glucan with 17–23 glucose units, was purified using
chromatographic methods and the structure was confirmed by MALDI-TOF mass spectrometry,
NMR analysis, and Fourier-transform infrared spectroscopy [19]. From the initial unbranched cyclic
structure, mono-6-amino CyS was prepared via monotosylation, azidation, and amination, as described
in the Materials and Methods (Scheme 1). Next, in addition to the peaks between 3.40 and 5.10 ppm
assigned to the H1–H6 protons of glucose units, signals for Hs6 and Hs6’ protons with an amino group
appeared at 0.77 and 0.83 ppm lower in the field than those of H6 and H6’ of unsubstituted glucoses
(Figure 1). The mono-6-amino CyS was tethered by biotin with an amidocaproyl linker in an EDC
coupling reaction with biotinamidohexanoic acid N-hydroxysuccinimide ester. The characteristic Hs6
peaks at 3.21 and 2.97 ppm disappeared, and 14 proton signals appeared at 3.35 and 3.21 ppm because
of the next amide bond. The presence of the ureido and tetrahydrothiophenene-fused ring caused
proton peaks (1–4) at approximately 4.40–4.63, 3.26, and 2.70–3.03 ppm. Other 5–13 proton peaks were
observed from aliphatic chains from the amidocaproyl linker and valeic acid part. In the same manner,
mono-6-amino-β-CD was prepared, and identical Hs6 peaks appeared ranging from 2.90 to 3.20 ppm
as a ddd signal due to an AB pattern (Figure 2) [30]. Hs4 protons were also clearly observed in the
upfield shifted region compared with original H4 protons of glucoses. After biotinyl functionalization,
the patterns of other signals were similar to that of biotinyl CyS.

Molecules 2018, 23, 90
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OH
OH
OH
HO
HO
OH
OH
OH
OH
O
H
OH
HO
O
O
OH
OH
OH
N
O
HO
HO
O
O
O
HN
O
O
O
OH
OH
OH
OH
OH
S
O
O
O
HO
HO
O
O
O
O
O
HO
HO
O
CyS
OH
OH
OH
O
O
HO
O
NH
O
HO
O
O
OH
OH
OH
O
O
O
HO
O
O
O
O
HO
S
HO
O
O
O
OH
N3
iii
HO
O
HO
O
NH2
iv
NH
O
OH
O
O
OH
OH
OH
OH
OH
HO
HO
OH
OH
HO
OH
i
ii
Cyclo-
oligosaccharide
Cyclo-
oligosaccharide
Cyclo-
oligosaccharide
Cyclo-
oligosaccharide
Cyclo-
OH
oligosaccharide
O
HO
O
HO
O
OH
HO
O
OH
OH
HO
O
OH
HO
OH
HO
HO
-CD
O
O
O
OH
HO
HO
O
OH
O
OH
HO
OH
O
O
OH
OH
O
Scheme 1. Synthetic scheme for biotinyl cyclooligosaccharides. (i) Monotosylation step; (ii) azidation
step; (iii) amination step; and (iv) biotinylation step.
OH
(a)
OH
OH
HO
HO
OH
OH
OH
O
OH
OH
HO
O
HO
O
O
HO
HO
OH
OH
O
NH
2
O
HN
O
O
O
OH
O
OH
3
5
O
H
O
1
O
O
7
8
HO
HO
O
N
12
11
S
4
O
14
O
10
9
OH
6
OH13
O
O
O
NH
HO
HO
O
OH
OH
OH
O
O
HO
O
O
HO
O
O
OH
OH
OH
HO
O
O
O
O
HO
HO
O
O
O
1
O
HO
HO
OH
O
OH
O
OH
13
2,3
14
9
OH
OH
5,7,10,12,6,11
HO
OH
OH
OH
HO
HO
OH
8
OH
4
8.9
8.8
8.7
8.6
8.5
8.4
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
6.1
6.0
5.9
5.8
5.7
5.6
5.5
5.4
5.3
5.2
5.1
5.0
4.9
4.8
4.7
4.6
4.5
4.4
4.3
4.2
4.1
4.0
3.9
3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
3.0
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
C
h
e
m
i
c
a
l
S
h
i
f
t
(
p
p
m
)
HO
HO
OH
(b)
OH
OH
H6
HO
HO
OH
OH
OH
OH
O
OH
HO
O
H5,H4
H5,H4
HO
OH
H6'
O
HO
HO
OH
OH
OH
O
OH
H4
O
O
OH
O
O
O
O
OH
H3
H6
O
O
O
O
HO
HO
O
NH2
OH
H3,H6’
O
Hs6, Hs6'
H2
OH
O
O
O
H5
O
HO
NH2
O
OH
OH
OH
H1
Hs6, Hs6’
O
O
HO
HO
HO
O
O
O
O
O
OH
OH
OH
O
HO
HO
O
O
O
O
HO
O
O
O
O
HO
3.2 3.1 3.0 2.9
Chemical Shift (pp...
H2
HO
O
OH
O
OH
H1
OH
OH
HO
OH
OH
HO
OH
OH
HO
OH
OH
8
7
6
5
4
3
2
1
Chemical Shift (ppm)
Figure 1. 1H NMR spectra of (
a) biotinyl cyclosophoraose (CyS); and (
b) amino CyS. Inset shows the
corresponding chemical structures (NMR solvent: D2O).
(a)
O
O
HO
O
HO
O
NH
2
HN
OH
HO
3
O
5
HO
O
OH
O
1
7
8
12
11
OH
S
O
4
HN
14
10
9
6
HO
HO
13
OH
HO
NH
O
O
8,13
O
OH
OH
HO
OH
O
1
OH
9
14
HO
OH
2,3
5,7,10,12,6,11
HO
O
O
O
4
OH
O
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
(b)8.0
Chemical Shift (ppm)
O
HO
H6,H6'
O
H3,H6’
HO
H1
O
OH
H6
O
O
O
HO
OH
HO
OH
H5,H4
H2
HO
OH
H4 NH2
HO
H3
HO
H2
Hs6, Hs6'
H5
O
H1
OH
O
O
OH
HO
HO
O
OH
OH
O
Hs6, Hs6’
HO
O
OH
Hs4
O
OH
8
7
6
5
4
3
2
1
Chemical Shift (ppm)
Figure 2. 1H NMR spectra of (
a) biotinyl
β-cyclodextrin (
β-CD); and (
b) amino
β-CD. Inset shows the
corresponding chemical structures (NMR solvent: D2O).
The molecular weights (MW) of synthesized biotinyl CyS and biotinyl β-CD were determined
by mass spectrometry (Figure 3). Because CyS has a degree of polymerization (DP) from 17 to 23,
the biotinyl CyS showed increased masses according to the respective DP. The molecular ions were

Molecules 2018, 23, 90
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assigned as [DP17 + Na]+, [DP18 + Na]+, [DP19 + Na]+, [DP20 + Na]+, [DP21 + Na]+, [DP22 + Na]+,
and [DP23 + Na]+, with signals at m/z 3117.9832, 3279.9475, 3442.0567, 3604.1257, 3767.1342, 3929.2961,
and 4091.3112, respectively. For the biotinyl
β-CD, the sodium adduct peak of the product was
observed at m/z 1496.1189, which is differentiated from the molecular weight (1134.9842) of
β-CD
consisting of seven glucose residues. These results indicate that biotin tethered macrocyclic host
molecules were successfully synthesized. In addition, because of the amidocaproyl linker between
biotin and cyclooligosaccharide, the possibility of a self-inclusion state can also be avoided, and an
efficient supramolecular interaction with other compounds can be expected. Since monosubstituted
β-cyclodextrin can undergo self-inclusion [31], the reduction of competitive interaction using a long
linker is favorable for a target molecule.
Figure 3. MALDI-TOF mass spectra of biotinyl cyclooligosaccharides. (a) Biotinyl CyS; and (b) biotinyl β-CD.
2.2. Biocompatibility of Cyclooligosaccharides and the Biotinyl Derivatives
To assess the biocompatibility of the prepared cyclooligosaccharides and biotinyl derivatives,
a water soluble tetrazolium salts (WST) assay, which is a useful method for quantifying cytotoxicity [32],
was conducted using the carbohydrate host molecules. Figure 4 shows the effects of four types of
macrocyclic hosts on HEK 293 cells and HeLa cells. The normal cell viabilities compared to those of
untreated cells were
Furthermore, cancer cell viability was more than 90% in the presence of four compounds (
biotinyl -CD: 91.1%, CyS: 105.7%, and biotinyl CyS: 96.7%). Compared with the results for generally
recognized as safe-labeled -CD (GRN 000074 in October 2001) [33], no significant decreases were
observed in all samples. Because cell viability less than 70% is regarded as toxic [34], materials such as
-CD, biotinyl -CD, CyS, and biotinyl CyS are considered non-toxic to normal and cancer cell lines.
β-CD: 85.9%, biotinyl
β-CD: 93.0%, CyS: 95.6%, and biotinyl CyS: 85.3% at 500
µM.
β-CD: 92.6%,
β
β
β
β
This suggests that the cyclooligosaccharides and biotinyl derivatives can be used as biocompatible
functional materials for future biomedical applications.

Molecules 2018, 23, 90
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140
120
100
80
140
(a)
(b)
120
100
80
60
40
20
0
β-CD
β-CD
Biotinyl β-CD
CyS
Biotinyl β-CD
CyS
60
40
Biotinyl CyS
Biotinyl CyS
20
0
10
50
100
500
10
50
100
500
Concentrations (µM)
Concentrations (µM)
Figure 4. Cell viability after exposure to cyclooligosaccharides and biotinyl derivatives. (
cells; and (b) HeLa cells.
a) HEK 293
2.3. Phase Solubility Study
Here, the effect of macrocyclic carbohydrate molecules on the solubilization of anticancer
paclitaxel was investigated. In supramolecular chemistry, the phase solubility technique is generally
used to evaluate the effect of host molecules on drug solubility [35]. The UV absorption of hydrophobic
paclitaxel in water noticeably and gradually increased as the concentration of biotinyl CyS increased
(Figure 5). The linear correlation forming an AL-type curve indicated a 1:1 association of paclitaxel
and biotinyl CyS [36], and the association constant (Ka) was found to be 4703 M−1. Solubilizing
efficiency is defined as the ratio between drug solubility in the presence of a host molecule at a certain
concentration and drug intrinsic solubility in water. The solubilizing efficiency of biotinyl CyS of
2 mM was 10.3, indicating a 10.3-fold increase in solubility, which is a much higher value than those of
other cyclooligosaccharides.
Biotinyl
effect on paclitaxel was relatively low compared with that of biotinyl CyS. The association constant
of paclitaxel and biotinyl
-CD was 1409 M−1 and the solubilizing efficiency was 3.7. The result is
reasonable if the flexible and large-sized ring structure of CyS is more favorable than -CD containing
β-CD also showed a linear plot for the phase solubility diagram and the solubilization
β
β
a small cavity and rigid hole, in the case of the supramolecular association with paclitaxel. Molecular
recognition of substrates by CyS is attributed to the noncovalent interactions of their hydrophobic
entities or hydroxyl groups in CyS [37]. CyS is microbial cyclic polysaccharide with
which has both better aqueous solubility and more diverse flexible hydrophobic cavities compared
with -CD [38]. The superior efficiency of CyS may be because of differences in physicochemical and
β-1-2 glucans,
β
structural properties compared with those of the CD analogs. In addition, biotin and amidocaproyl
moieties positively affect the solubilization of paclitaxel, which could be because of the effective
noncovalent interaction with paclitaxel due to the additional hydrogen bonding sites between the
amide bond in biotinyl cyclooligosaccharides and ester linkages in paclitaxel. The initial increase
and decrease pattern of paclitaxel solubility by
between these molecules. Although the aromatic rings of paclitaxel can be incorporated in the cavity
of -CD, considering the cavity size (7 Å) and benzene ring [21], the complexed structure is not very
soluble in water because of its other bulky moiety. In actual fact, the extrapolate area of paclitaxel is
31.74 Å2 [39]. CyS may have a collapsed structure because of some intramolecular associations [40
β-CD may be related to the partial complexation
β
]
and the interaction with paclitaxel may be limited. In addition, the small cavity size of 3.7 Å and
the non-symmetric conformation inferred from NMR spectroscopic and a molecular modeling study,
support this [17]. These results demonstrate that efficient supramolecular interactions between biotinyl
CyS and paclitaxel contribute to increasing paclitaxel solubility in water. Thus, the hydrophobic
aggregation of paclitaxel in water may be noticeably reduced because of the association with biotinyl
CyS and dispersion by biotinyl CyS.

Molecules 2018, 23, 90
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90
80
70
60
50
40
30
20
10
0
Cys
Biotinyl Cys
β-CD
Biotinyl β-CD
0
0.5
1
1.5
2
2.5
Concentration of host (mM)
Figure 5. Phase solubility diagram.
1
2.4. H NMR Analysis
To confirm and compare the interaction between paclitaxel and biotinyl CyS, 1H NMR
1
spectroscopy was performed. NMR spectroscopy reveals valuable information about noncovalent
interactions at the molecular level [41]. Noncovalent interactions are indicated as protons shifted
upfield or downfield by the molecular environmental change. Figure 6 shows the noticeable chemical
shift in the aromatic ring of paclitaxel by biotinyl CyS, with the values summarized in Table 1.
In particular, protons of the A, B, and C aromatic rings were affected, indicating a local polarity change
in the moiety in the presence of biotinyl CyS. The splitting pattern of C aromatic ring protons was also
clearly altered after complexation with biotinyl CyS, with the largest chemical shift change observed
in ortho protons of the B aromatic ring. A new resonance at 7.80 ppm appeared next to the para proton
of the A aromatic ring. Although biotinyl β-CD was relatively less affected by the proton resonance of
paclitaxel, the splitting pattern of para protons of the A aromatic ring changed, and new peaks near the
para protons of B aromatic ring were observed. Thus, some interaction is suspected, which is correlated
with the small effect (2.8-fold) on the paclitaxel solubility enhancement by some association with
paclitaxel as shown in Figure 5. This molecular leveled evidence from NMR spectroscopic analysis
confirms the efficient supramolecular interaction between paclitaxel and biotinyl CyS as a key factor
affecting the solubility increase of paclitaxel.
B
O
O
O
(a)
A
O
HO
O
O
NH
C
O
HO
O
OH
O
O
O
(b)
(c)
m,o-C
o-B
m-A
m-B
o-A
p-A
p-B
p-C
8.0
7.5
7.0
Chemical Shift (ppm)
Figure 6. 1H NMR spectra. (
a) Paclitaxel interacted with biotinyl CyS; (b) Paclitaxel interacted with
biotinyl
β-CD; and (c) Paclitaxel alone (NMR solvent: DMSO-d6, 99%). Inset shows the chemical
structure of paclitaxel.

Molecules 2018, 23, 90
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Table 1. Chemical shift changes in aromatic rings of paclitaxel in the presence of biotinyl CyS and
biotinyl β-CD.
Paclitaxel
Biotinyl CyS (ppm)
Biotinyl β-CD (ppm)
Protons
δ a
∆δ
δ a
∆δ
0
0
0
0
0
0
0
0
δ b
o-A
m-A
p-A
o-B
m-B
p-B
o-C
m-C
p-C
7.97
7.63
7.72
7.88
7.49
7.55
7.39
7.40
7.22
8.00
7.67
0.03
0.04
0.04
0.08
0.01
0.03
0.03
0.03
0.03
7.97
7.63
7.76 (7.80) c
7.96
7.72 d
7.88
7.50
7.58
7.49
7.55 (7.57) c
7.39
7.42 d
7.43 d
7.25 d
7.40
7.22
0
a
b
δ
= after complexation with biotinyl cyclooligosaccharides;
δ
= before complexation with biotinyl
cyclooligosaccharides; c new peak; d changed splitting pattern. ∆δ = δa − δb.
3. Materials and Methods
3.1. Materials
β-CD was obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Paclitaxel was
provided by Samyang Genex Corporation (Seoul, Korea). Sodium azide, 1-(p-toluenesulfonyl)
imidazole, and p-toluenesulfonyl chloride were purchased from Tokyo Chemical Industry (Tokyo,
Japan). Triphenylphosphine (99%) and biotinamidohexanoic acid N-hydroxysuccinimide ester were
purchased from Sigma Aldrich (St. Louis, MO, USA). N,N-Dimethylformamide (anhydrous, 99.8%)
was obtained from Alfa Aesar (Haverhill, MA, USA), and acetone was obtained from Avantor. All other
chemicals used in the study were of analytical grade.
3.2. Isolation and Purification of CyS
Rhizobium leguminosarum VF-39 was provided by the Microbial Carbohydrate Resource Bank,
◦
Konkuk University, Korea. Cells were grown in a GMS medium at 25 C for 14 days. CyS was refined
from R. leguminosarum VF-39, as described previously [42]. After removing the cells, the culture
supernatants were concentrated by five-fold. After adding two volumes of ethanol, the alcoholic
supernatant fluid was concentrated, and the concentrate was re-precipitated by adding 7 volumes of
ethanol. The precipitate was applied on a Bio-Gel P6 column, and the fractions containing desired
glucans were concentrated and desalted. Purified CyS was characterized by NMR spectroscopic and
MALDI-TOF mass spectrometric analyses.
3.3. Synthesis of Biotinyl Cyclooligosaccharides
Biotin tethering to cyclooligosaccharides starts from monotosylation on the primary hydroxyl
group, but the monotosylation method of both cyclooligosaccharides slightly differed from each other.
For monotosylation of β-CD, β-CD (10.0 g, 8.8 mmol) was dissolved in water (250 mL) and heated to
60 ◦C. After cooling to room temperature, 1-(p-toluenesulfonyl) imidazole (6 g, 27 mmol) was added
and mixed for 6 h. Sodium hydroxide (4.5 g, 112.5 mmol) solution in water (12.5 mL) was added
dropwise for 20 min. After filtration, the filtrate was quenched using ammonium chloride (12.05 g,
225 mmol), and the resulting mixture was subjected to air flow for drying. The precipitated product
was washed with water and acetone. The yield of monotosylated β-CD was 28%. For monotosylated
CyS, CyS (1 g, 0.28 mmol) was dissolved in water (50 mL), and copper sulfate (1 g, 4 mmol) in water
(100 mL), was added. NaOH (1 g, 25 mmol) solution in water (50 mL) was mixed and stirred
for 10 min. The suspension became dark blue, and p-toluenesulfonyl chloride (2.5 g, 13.2 mmol)
dissolved in 0.1 mL of acetonitrile was added in a dropwise manner for 1 h. The suspension was
stirred at room temperature for 4 h then neutralized with ammonium chloride. After removing the

Molecules 2018, 23, 90
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precipitate, the filtrate was concentrated and desalted on a Bio-Gel P2 column. The sample was
subjected to semi-preparative HPLC (Agilent Technologies 1200 series, Santa Clara, CA, USA) on a
reverse-phase column (Eclipse XDB-C18, 9.4
of monotosylated CyS was 25%. Further azidation and amination were performed, as described
×
250 mm, 5 µm) at room temperature. The yield
in our previous report [43
,
44]. The mono-6-amino-
β
-CD or mono-6-amino-CyS were dissolved◦in
DMF and reacted with biotinamidohexanoic acid N-hydroxysuccinimide ester for 24 h at 40 C.
After acetone precipitation, the samples were desalted and lyophilized. The overall synthetic yield from
original cyclooligosaccharide to biotinylated one is about 8%. The structures of the resulting biotinyl
cyclooligosaccharides were analyzed by NMR spectroscopy and MALDI-TOF mass spectrometry.
3.4. MALDI-TOF MS and NMR Spectroscopic Analysis
NMR spectroscopic analysis was performed on a Bruker (AMX, Billerica, MA, USA) spectrometer
(500 MHz) with deuterium water (D2O; 99.9 at. % D or DMSO-d6). All NMR measurements were
performed with 0.6 mL samples in 5 mm NMR tubes. MALDI-TOF experiments were essentially
performed using a Voyager-DETM STR BioSpectrometry (PerSeptive Biosystems, Framingham, MA,
USA) in positive ion reflector mode. Samples were prepared according to the dried droplet method by
using 2,5-dihydroxybenzoic acid as a matrix.
3.5. Cytotoxicity Assay
The cytotoxicity of cyclooligosaccharides and biotinyl derivatives was assessed by a WST assay
on HEK293 and HeLa cells [45]. The assays were performed by incubating 5
×
103 cells/well with
increasing concentrations (0, 10, 50, 100, and 500
µM) in a 96-well plate (Corning Costar, Corning,
◦
NY, USA) at 37 C in a humidified incubator containing 5% carbon dioxide for 24 h. After treatment of
cyclooligosaccharides between 0 and 500 µM, the cells were incubated for 24 h. Next, 10 µL of WST-1
was added to each well, and the cells were incubated at 37 ◦C for 4 h. The absorbance of each well
at 450 nm was measured using a SpectraMax 190 microplate reader (Molecular Devices Corporation,
Sunnyvale, CA, USA). Cell viability was calculated as follows.
V (%) = A/A0 × 100
(1)
where A and A0 are the absorbance values after and before addition of cyclooligosaccharides,
respectively. Experiments were performed in triplicate.
3.6. Phase Solubility Studies
Paclitaxel (2 mM) was dissolved in methanol and this solution (0.25 mL) was added to aqueous
solutions containing β-CD, biotinyl β-CD, CyS, and biotinyl CyS (0.0–4.0 mM), in vials. The mixed
solution was stirred at 26 ◦C for 24 h then the methanol was evaporated using nitrogen gas.
After lyophilization, the samples were dissolved in water (0.5 mL) and the precipitate was removed
by centrifugation. After dilution, the solubilized paclitaxel was analyzed at 260 nm using a UV visible
spectrometer (UV 2450, Shimadzu Corp., Kyoto, Japan) [46]. The apparent association constants (Ka) of
paclitaxel/cyclooligosaccharides were calculated from the phase solubility diagram according to Higuchi
and Connor’s method [35].
Ka (M−1) = slope/Si(1-slope)
(2)
where Si is the initial solubility of paclitaxel equilibrated in water.
4. Conclusions
The poor water solubility of paclitaxel limits its clinical usage because of injection difficulties.
To overcome this problem, supramolecular complexation technology using carbohydrate host molecules
was attempted. In the present study, bio-sourced CyS, cyclic β-1,2 glucans with DP 17–23, was purified

Molecules 2018, 23, 90
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and modified with a biotin group via mono-tosylation, azidation, amination, and EDC coupling reaction.
GRAS -CD, a cyclic -1,4-glucan with DP 7, was also prepared and tethered with a biotin group in
β
α
the same manner. The results of the WST cytotoxicity assay on normal and cancer cell lines suggested
that the modified and original macrocyclic carbohydrates can be used as drug solubilizers to improve
biocompatibility. Accordingly, using those molecules, the effect on paclitaxel solubility in water was
investigated. In particular, biotinyl CyS and β-CD showed solubilization efficiencies of 10.3 and 3.7,
with apparent association constants (Ka) of 4703 and 1409 M−1, respectively. The supramolecular
interaction between paclitaxel and biotinyl cyclooligosaccharides was also determined at the molecular
level by NMR spectroscopy. The present results may be important for biomedical applications, to resolve
solubility problems and target oriented advantages as novel strategies for anticancer drug delivery.
Acknowledgments: This study was supported by the KU Research Professor Program of Konkuk University.
It was also supported by the Basic Science Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01058686), the Bio & Medical Technology
Development Program of the NRF funded by the Korean government, MSIP (2015M3A9B8031831), and Basic
Science Research Program through the NRF funded by the Ministry of Science, ICT, & Future Planning
(NRF-2015R1C1A1A02037375). SDG.
Author Contributions: E.C. and S.J. conceived and designed the experiments; E.C. performed the experiments
and analyzed the data; E.C. and S.J. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
Nicolaou, K.C.; Dai, W.M.; Guy, R.K. Chemistry and biology of taxol. Angew. Chem. Int. Ed. 1994, 33, 15–44.
[CrossRef]
Jordan, M. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr. Med.
Chem. AntiCancer Agents 2002, 2, 1–17. [CrossRef] [PubMed]
Suffness, M. Overview of Paclitaxel Research: Progress on Many Fronts; ACS Publications: Washington, DC,
USA, 1995.
Goldspiel, B.R. Clinical overview of the taxanes. Pharmacother. J. Hum. Pharmacol. Drug Ther. 1997, 17,
110S–125S.
Lipinski, C. Poor aqueous solubility—An industry wide problem in drug discovery. Am. Pharm. Rev. 2002, 5,
82–85.
Merisko-Liversidge, E.; Liversidge, G.G.; Cooper, E.R. Nanosizing: A formulation approach for poorly-water-
soluble compounds. Eur. J. Pharm. Sci. 2003, 18, 113–120. [CrossRef]
Zhang, J.A.; Anyarambhatla, G.; Ma, L.; Ugwu, S.; Xuan, T.; Sardone, T.; Ahmad, I. Development and
characterization of a novel cremophor® el free liposome-based paclitaxel (lep-etu) formulation. Eur. J.
Pharm. Biopharm. 2005, 59, 177–187. [CrossRef] [PubMed]
8.
9.
Kingston, D.G.; Zhao, Z.-Y. Water Soluble Derivatives of Taxol. U.S. Patent 5,352,805 A, 4 October 1994.
Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Cremophor El: The drawbacks and advantages of
vehicle selection for drug formulation. Eur. J. Cancer 2001, 37, 1590–1598. [CrossRef]
10. Cho, E.; Jung, S. Supramolecular complexation of carbohydrates for the bioavailability enhancement of
poorly soluble drugs. Molecules 2015, 20, 19620–19646. [CrossRef] [PubMed]
11. Davis, M.E.; Brewster, M.E. Cyclodextrin-based pharmaceutics: Past, present and future. Nat. Rev. Drug Discov.
2004, 3, 1023–1035. [CrossRef] [PubMed]
12. Lee, S.; Seo, D.-H.; Park, H.-L.; Choi, Y.; Jung, S. Solubility enhancement of a hydrophobic flavonoid, luteolin
by the complexation with cyclosophoraoses isolated from rhizobium meliloti. Antonie Van Leeuwenhoek
2003, 84, 201–207. [CrossRef] [PubMed]
13. Salústio, P.; Cabral-Marques, H.; Costa, P.; Pinto, J. Comparison of ibuprofen release from minitablets and
capsules containing ibuprofen: B-cyclodextrin complex. Eur. J. Pharm. Biopharm. 2011, 78, 58–66. [CrossRef]
[PubMed]
14. Cartledge, J.; Midgley, J.; Youle, M.; Gazzard, B. Itraconazole cyclodextrin solution—Effective treatment for
hiv-related candidosis unresponsive to other azole therapy. J. Antimicrob. Chemother. 1994, 33, 1071–1073.
[CrossRef] [PubMed]

Molecules 2018, 23, 90
10 of 11
15. Ficarra, R.; Ficarra, P.; Di Bella, M.; Raneri, D.; Tommasini, S.; Calabro, M.; Villari, A.; Coppolino, S. Study of
the inclusion complex of atenolol with -cyclodextrins. J. Pharm. Biomed. Anal. 2000, 23, 231–236. [CrossRef]
16. Amemura, A. Synthesis of (1 2)- -D-glucan by cell-free extracts of agrobacterium radiobacter ifo 12665b 1
β
→
β
and rhizobium phaseoli ahu 1133. Agric. Biol. Chem. 1984, 48, 1809–1817.
17. André, I.; Mazeau, K.; Taravel, F.R.; Tvaroska, I. Conformation and dynamics of a cyclic (1→2)-β-D-glucan.
Int. J. Biol. Macromol. 1995, 17, 189–198. [CrossRef]
18. Koizumi, K.; Okada, Y.; Horiyama, S.; Utamura, T.; Higashiura, T.; Ikeda, M. Preparation of cyclosophoraose-a
and its complex-forming ability. In Clathrate Compounds, Molecular Inclusion Phenomena, and Cyclodextrins;
Springer: Dordrecht, The Netherlands, 1984; pp. 891–899.
19. Kwon, C.; Choi, Y.-H.; Kim, N.; Yoo, J.S.; Yang, C.-H.; Kim, H.-W.; Jung, S. Complex forming ability of a
family of isolated cyclosophoraoses with ergosterol and its monte carlo docking computational analysis.
J. Incl. Phenom. Macrocycl. Chem. 2000, 36, 55–64. [CrossRef]
20. Szente, L.; Szejtli, J. Highly soluble cyclodextrin derivatives: Chemistry, properties, and trends in development.
Adv. Drug Deliv. Rev. 1999, 36, 17–28. [CrossRef]
21. Bender, M.L.; Komiyama, M. Cyclodextrin Chemistry; Springer: Heidelberg/Berlin, Germany, 2012; Volume 6.
22. Casu, B.; Reggiani, M.; Gallo, G.; Vigevani, A. Conformation of O-methylated amylose and cyclodextrins.
Tetrahedron 1968, 24, 803–821. [CrossRef]
α-,-β-, and-γ
23. Uekama, K.; Minami, K.; Hirayama, F. 6A-O-[(4-biphenylyl) acetyl]- -cyclodextrins and 6A-deoxy-6A-
[[(4-biphenylyl) acetyl] amino]- -,- -, and- -cyclodextrins: Potential prodrugs for colon-specific delivery.
α
β
γ
J. Med. Chem. 1997, 40, 2755–2761. [CrossRef] [PubMed]
24. Karunaratne, D.N.; Farmer, S.; Hancock, R.E. Synthesis of bulky. Beta.-lactams for inhibition of cell surface.
Beta.-lactamase activity. Bioconjug. Chem. 1993, 4, 434–439. [CrossRef] [PubMed]
25. Russell-Jones, G.; McTavish, K.; McEwan, J.; Rice, J.; Nowotnik, D. Vitamin-mediated targeting as a potential
mechanism to increase drug uptake by tumours. J. Inorg. Biochem. 2004, 98, 1625–1633. [CrossRef] [PubMed]
26. Dordunoo, S.K.; Burt, H.M. Solubility and stability of taxol: Effects of buffers and cyclodextrins. Int. J. Pharm.
1996, 133, 191–201. [CrossRef]
27. Hamada, H.; Ishihara, K.; Masuoka, N.; Mikuni, K.; Nakajima, N. Enhancement of water-solubility and
bioactivity of paclitaxel using modified cyclodextrins. J. Biosci. Bioeng. 2006, 102, 369–371. [CrossRef]
[PubMed]
28. Surapaneni, M.S.; Das, S.K.; Das, N.G. Designing paclitaxel drug delivery systems aimed at improved patient
outcomes: Current status and challenges. ISRN Pharmacol. 2012, 2012. [CrossRef] [PubMed]
29. Jing, J.; Szarpak-Jankowska, A.; Guillot, R.; Pignot-Paintrand, I.; Picart, C.; Auzély-Velty, R. Cyclodextrin/
paclitaxel complex in biodegradable capsules for breast cancer treatment. Chem. Mater. 2013, 25, 3867–3873.
[CrossRef]
30. Brunner, H.; Hankofer, P.; Holzinger, U.; Treittinger, B.; Schönenberger, H. Synthesis and antitumor activity
of platinum (ii) complexes containing substituted ethylenediamine ligands. Eur. J. Med. Chem. 1990, 25,
35–44. [CrossRef]
31. Lukin, O.; Dolgonos, G.; Leszczynski, J. A comprehensive test of computational approaches for evaluation
of cyclodextrin complexes. Self-inclusion in monosubstituted β-cyclodextrins—A case study. Tetrahedron
2017, 73, 5302–5306. [CrossRef]
32. Zhang, X.; Scialis, R.J.; Feng, B.; Leach, K. Detection of statin cytotoxicity is increased in cells expressing the
oatp1b1 transporter. Toxicol. Sci. 2013, 134, 73–82. [CrossRef] [PubMed]
33. Samperio, C.; Boyer, R.; Eigel, W.N., III; Holland, K.W.; McKinney, J.S.; O’Keefe, S.F.; Smith, R.; Marcy, J.E.
Enhancement of plant essential oils’ aqueous solubility and stability using alpha and beta cyclodextrin.
J. Agric. Food. Chem. 2010, 58, 12950–12956. [CrossRef] [PubMed]
34. International Organization for Standardization. Biological Evaluation of Medical Devices—Part 5: Tests for In
Vitro Cytotoxicity; ISO 10993-5: 2009; International Organization for Standardization: Geneva, Sweden, 2009.
35. Higuchi, T.; Connors, A. Phase-solubility techniques. Adv. Anal. Chem. Instrum. 1965, 4, 117–212.
36. Loftsson, T.; Magnúsdóttir, A.; Másson, M.; Sigurjónsdóttir, J.F. Self-association and cyclodextrin solubilization of
drugs. J. Pharm. Sci. 2002, 91, 2307–2316. [CrossRef] [PubMed]
37. Dindulkar, S.D.; Jeong, D.; Kim, H.; Jung, S. Functionalized β-cyclodextrin as supramolecular ligand and
their pd (OAc)2 complex: Highly efficient and reusable catalyst for mizoroki-heck cross-coupling reactions
in aqueous medium. Carbohydr. Res. 2016, 430, 85–94. [CrossRef] [PubMed]

Molecules 2018, 23, 90
11 of 11
38. Liu, L.; Guo, Q.-X. The driving forces in the inclusion complexation of cyclodextrins. J. Incl. Phenom. Macrocycl. Chem.
2002, 42, 1–14. [CrossRef]
39. Zhao, L.; Feng, S.-S. Effects of cholesterol component on molecular interactions between paclitaxel and
phospholipid within the lipid monolayer at the air—Water interface. J. Colloid Interface Sci. 2006, 300, 314–326.
[CrossRef] [PubMed]
40. Hu, Y.; Kim, H.; Shinde, V.V.; Jeong, D.; Choi, Y.; Cho, E.; Jung, S. Carboxymethyl cyclosophoraoses as a
flexible ph-responsive solubilizer for pindolol. Carbohydr. Polym. 2017, 175, 493–501. [CrossRef] [PubMed]
41. Schneider, H.-J.; Hacket, F.; Rüdiger, V.; Ikeda, H. Nmr studies of cyclodextrins and cyclodextrin complexes.
Chem. Rev. 1998, 98, 1755–1786. [CrossRef] [PubMed]
42. Jeong, D.; Kim, H.K.; Jeong, J.-p.; Dindulkar, S.D.; Cho, E.; Yang, Y.-H.; Jung, S. Cyclosophoraose/cellulose
hydrogels as an efficient delivery system for galangin, a hydrophobic antibacterial drug. Cellulose 2016, 23,
2609–2625. [CrossRef]
43. Kim, H.; Dindulkar, S.D.; Jeong, D.; Park, S.; Jun, B.-H.; Cho, E.; Jung, S. A synthetic encapsulating emulsifier
using complex-forming pentacosadiynoyl cyclosophoraoses (cyclic β-(1,2)-D-glucan). J. Ind. Eng. Chem.
2016, 44, 195–203. [CrossRef]
44. Cho, E.; Kim, H.; Yang, J.E.; Jun, B.-H.; Paik, S.R.; Jung, S. Supramolecular self-assembled aggregates
formed by pentacosa-10,12-diynyl amidomethyl-
[PubMed]
β-cyclodextrin. Carbohydr. Res. 2014, 391, 37–42. [CrossRef]
45. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.;
Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in
culture using human and other tumor cell lines. Cancer Res. 1988, 48, 4827–4833. [PubMed]
46. Kathiravan, G.; Sureban, S.M.; Sree, H.N.; Bhuvaneshwari, V.; Kramony, E. Isolation of anticancer drug
taxol from pestalotiopsis breviseta with apoptosis and b-cell lymphoma protein docking studies. J. Basic
Clin. Pharm. 2012, 4, 14–19. [CrossRef] [PubMed]
Sample Availability: Not available.
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