Synthesis of mono-glucose-branched cyclodextrins with a high inclusion ability for doxorubicin and their efficient glycosylation using Mucor hiemalis endo-β-N-acetylglucosaminidase

Article abstract of DOI:10.1016/j.bmcl.2004.12.040

The mono-glucose-branched cyclodextrins having an appropriate spacer between the β-cyclodextrin and a glucose moiety were synthesized from β-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anticancer agent, in the range of 10 ⁵-10⁶ M^-1, and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by endo-β-N- acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the high yields of 65-67%.

Full text of DOI:10.1016/j.bmcl.2004.12.040

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1009–1013
Synthesis of mono-glucose-branched cyclodextrins with a
high inclusion ability for doxorubicin and their
efficient glycosylation using Mucor hiemalis
endo-b-N-acetylglucosaminidase
Takashi Yamanoi,a,* Naomichi Yoshida,a Yoshiki Oda,a,b Eri Akaike,a
Maki Tsutsumida,a,b Natsumi Kobayashi,a,b Kenji Osumi,a Kenji Yamamoto,c
Kiyotaka Fujita,c,d Keiko Takahashib and Kenjiro Hattorib
aThe Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan
bDepartment of Nanochemistry, Faculty of Engineering, Tokyo Polytechnic University, Atsugi 243-0297, Japan
cGraduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
dDepartment of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
1-21-24, Korimoto, Kagoshima 890-0065, Japan
Received 27 October 2004; revised 13 December 2004; accepted 14 December 2004
Available online 18 January 2005
Abstract—The mono-glucose-branched cyclodextrins having an appropriate spacer between the b-cyclodextrin and a glucose moiety
were synthesized from b-cyclodextrin and arbutin. They had the significantly high association constants for doxorubicin, the anti-
cancer agent, in the range of 105–106 MÀ1, and worked as highly reactive glycosyl acceptors for the transglycosylation reaction by
endo-b-N-acetylglucosaminidase of Mucor hiemalis to produce sialo-complex type oligosaccharide-branched cyclodextrins in the
high yields of 65–67%.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Saccharides are known to be involved in a number of
significant biological recognition phenomena on the sur-
faces of cell membranes. Cyclodextrins (CDs) have the
ability to carry drugs in their cavities. Therefore, the
CD derivatives conjugated with saccharide(s) are ex-
pected to be drug carrier molecules capable of specific
cell recognition, and might be useful as targeting drug-
delivery systems.1 However, no targeting drug carrier
using the CDs has yet been reported. In order to obtain
effective drug carrier molecules based on the CD–sac-
charide(s), efficient methods for binding saccharide(s)
to the CDs and for increasing the drug inclusion ability
of the CD cavities are required.
been utilized for synthesizing them.1p,q The endo-b-N-
acetylglucosaminidase of Mucor hiemalis (Endo-M)
has proved to be a practical tool to synthesize glycocon-
jugates by transferring the natural oligosaccharide
blocks from asparagine-linked glycopeptides to accep-
tors.2 In our previous study,3 we found that the enzyme
had the transglycosylation ability of transferring the oli-
gosaccharides to the CD derivative prepared from N-a-
Fmoc-N-x-(2-acetamide-2-deoxy-b-D-glucopyranosyl)-
asparagine and 6-mono-amino-b-CD. Three kinds of
natural (sialo-complex type, asialo-complex type, and
high-mannose type)4oligosaccharide-branched CDs
were successfully obtained although the transglycosyla-
tion yields were only 6–12%.
To synthesize the CD–saccharide(s) conjugates is a chal-
lenging objective. Enzymatic glycosylations have often
This letter describes an efficient method for preparing
targeting drug carriers based on the CD–saccharide(s)
molecules using Endo-M. We designed novel CD deriva-
tives which were expected to work as highly reactive
glycosyl acceptors of the Endo-M transglycosylation
and to have a high drug inclusion ability. The molecular
Keywords: DDS; Cyclodextrin; Endo-M; DXR.
*
Corresponding author. Tel./fax: +81
tyama@noguchi.or.jp
3
5944 3213; e-mail:
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.040

1010
T. Yamanoi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1009–1013
interaction between the CD derivatives and the antican-
cer agent, doxorubicin (DXR), was evaluated using the
SPR optical biosensor, and the transglycosylation reac-
tion by recombinant Endo-M to the CD derivatives was
examined.
of the arbutin linked with CD through an appropriate
spacer would exhibit a structure like the cap of the
CD, and this Ôpseudo-capping structureÕ and the hydro-
phobicity of the phenyl group would increase the hydro-
phobicity of the CD cavity. It was also speculated that
when DXR was included into the CD cavities, the stack-
ing complex by the p–p interaction between the phenyl
group and DXR would be formed as shown in Figure
1. These effects were expected to enhance the inclusion
association between these CD derivatives and DXR.
Our approach was to introduce the commercially avail-
able 4-hydroxyphenyl-b-D-glucopyranoside5 (natural
product called arbutin 1) into the CDs in order to syn-
thesize the mono-glucose-branched CDs, which may
function as the glycosyl acceptors of the Endo-M trans-
glycosylation. The reasons were as follows: (1) As Endo-
M showed a high transglycosylation activity on the b-
isomer of glucopyranose among the naturally occurring
hexopyranoses, the transglycosylation reaction was ex-
pected to efficiently proceed on the b-glucopyranose of
the arbutin. (2) We speculated that the phenyl group
First, we synthesized the mono-glucose-branched CDs (9
and 14) as the glycosyl acceptors of the Endo-M trans-
glycosylation. The reaction of allyl bromide (1.2equiv)
and the dry sodium salt of arbutin 1, which was ob-
tained by the treatment of 1 with NaOH (1 equiv) in
H2O, was carried out in DMF for 24 h and produced
hydrophobicity
HO
O
O
OH
OH
OH
OH
OH
OH
O
O
O
HO
spacer
O
π−π interaction
O
OMe
O
Me
NH2HCl
OH
DXR
DXR
Figure 1.
OBn
O
BnO
BnO
O
O
O
OBn
OH
OBn
f
b
O
O
HO
HO
BnO
O
OR1
O
OR1
BnO
OH
OBn
(OBn)20
8
1: R1=H
2: R1=
3: R1=
4: R1=
5: R1=
6: R1=
g
c
a
OH
d
e
OH
OTs
I
O
HO
HO
O
O
O
OH
HO
9
(OBn)20
7
Scheme 1. Reagents and conditions: (a) NaOH/H2O, then dry, AllBr/DMF, 99%; (b) NaH, BnBr/DMF, 91%; (c) 9-BBN/THF, then NaOHaq,
H2O2aq, 95%; (d) TsCl, Et3N/CH2Cl2, quant; (e) NaI/DMF, 60 ꢁC, 92%; (f) KOH, 7/DMF, 51%; (g) H2/Pd(OH)2/ether–MeOH–H2O, then gel-
filtration, 73%.

T. Yamanoi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1009–1013
1011
OBn
H
N
O
BnO
O
O
BnO
OBn
O
c
a
O
OBn
R1
BnO
BnO
3
O
O
OBn
10: R1=CHO
13
d
b
11: R1=COOH
H2N
OH
H
N
O
HO
HO
O
O
O
OH
12
14
Scheme 2. Reagents and conditions: (a) O3, Ph3P/CH2Cl2, 97%; (b) 2-methyl-2-butene, NaH2PO4, NaClO2/t-BuOH–H2O, 98%; (c) Me2P(S)Cl,
DIEA, DMF; (d) H2/Pd(OH)2/ether–MeOH–H2O, then gel-filtration, 63% from 11.
Table 1. Kinetic parameters of CD derivatives (9 and 14) for the association with the immobilized DXR
Entry
CD derivative
Ka · 103 MÀ1
kass · 103
M
À1 sÀ1
kdiss · 10À2 sÀ1
1
2
3
9
2.2 · 102
5.3 · 103
3.1
4.6 0.6
18 20.34 0.2
0.12 0.005
2.1 0.3
14
15
4.1 0.3
the allylated compound 2, followed by separation using
silica-gel column chromatography (chloroform/metha-
nol = 8:1) in 99% from 1. The benzylation of 2 using
benzyl bromide (12equiv) and NaH (16 equiv) in
DMF was carried out for 3 d, and afforded the com-
pound 3, which was purified by silica-gel column chro-
matography (hexane/ethyl acetate = 4:1) in 91%. The
hydroboration of 3 with 9-BBN (2equiv) in THF at
0 ꢁC for 24 h, followed by oxidation using aq H2O2
(10 equiv) and by hydrolysis with aq NaOH (3 equiv)
for 24 h gave the compound 4, which was purified by sil-
ica-gel column chromatography (hexane/ethyl ace-
tate = 4:1) in 95%. The reaction of 4 with TsCl
(4 equiv) in pyridine for 5 h quantitatively gave 5, fol-
lowed by separation on preparative silica-gel TLC (hex-
ane/ethyl acetate = 2:1). The treatment of 5 with NaI
(4 equiv) in DMF at 60 ꢁC for 1.5 h afforded 6, followed
by separation on preparative TLC (hexane/ethyl ace-
tate = 2:1) in 92%. The reaction of 6 (4.5 equiv) with
the benzylated b-CD 76 using KOH (120 equiv) in
DMF for 24 h gave 8, which was purified by preparative
silica-gel TLC (hexane/ethyl acetate = 2:1) in 51%. The
treatment of 8 with H2–Pd(OH)2 in ether–methanol–
H2O for 24 h provided the desired compound 9, which
was purified by gel-filtration using Sephadex G25 (5%
EtOH aq) in 73% (Scheme 1).
nol = 5:1) in 98%. The condensation of 11 (1 equiv)
with 6-mono-amino-b-CD 12 using Me2P(S)Cl (1 equiv)
was carried out in the presence of DIEA (1 equiv) in
DMF for 4 d, and gave the crude 13. The following de-
benzylation of 13 by H2–Pd(OH)2 afforded the desired
product 14, which was purified by gel-filtration using
Sephadex G25 (5% EtOH aq) in 63% (Scheme 2).
Next, we estimated the inclusion associations of 9 and
14 with the immobilized DXR by the SPR biosensor
according to the reported method.1a The association
constants (Ka), association rate constants (kass) and dis-
sociation rate constants (kdiss) of 9 and 14 are summa-
rized in Table 1 and compared with the previously
reported data of the CD derivative 15, which has no
phenyl group (Figure 2). The kass and kdiss values of 9
were 4.6 · 103 MÀ1 sÀ1 and 2.1 · 10À2 sÀ1, and those of
14 were 1.8 · 104 MÀ1 sÀ1 and 3.4 · 10À3 sÀ1. The Ka
values of 9 and 14 were calculated to be 2.2 · 105 MÀ1
HO
OH
OH
OH
O
O
O
HO
HO
OH
OH
HN
S
The ozonization of 3 in the presence of Ph3P (3 equiv)
gave 10, followed by separation on silica-gel preparative
TLC (hexane/ethyl acetate = 1:1) in 97%. The oxidation
of 10 using NaClO2 (10 equiv)–NaH2PO4 (1.5 equiv) in
the presence of 2-methyl-2-butene in t-BuOH–H2O
afforded the compound 11, which was purified by sil-
ica-gel column chromatography (chloroform/metha-
15
Figure 2.

1012
T. Yamanoi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1009–1013
and 5.3 · 106 MÀ1, respectively. The CD derivatives 9
and 14 indicated the significantly high association con-
stants for DXR in the range of 105–106 MÀ1. Particu-
larly, the Ka value of 14 was 1700 times higher (the
kass value was 150 times higher and the kdiss value was
12times lower) than that of 15. As expected, the phenyl
groups of 9 and 14 had a remarkable effect of enhancing
the inclusion interactions with the immobilized DXR.
The molecular model of 14 including DXR also sup-
ported our speculation (Fig. 3). The comparison of the
Ka values of 9 and 14 suggested that 14 would form a
more rigid Ôcapping structureÕ than 9 due to the genera-
tion of the hydrogen bonds by the existence of the amide
group of the spacer.
Finally, we investigated the synthesis of natural oligo-
saccharide-branched CDs from 9 and 14 by the transgly-
cosylation activity of the recombinant Endo-M using
Table 2. Transglycosylations using recombinant Endo-M to 1, 9, 14
Entry Glycosyl acceptor Product Transglycosylation yield/%
1
2
3
1
16
17
18
56
67
65
9
14
Figure 3. Molecular model of the inclusion association between 14 and
DXR.
Endo-M
+
Transglycosylation product
Glycosyl acceptor
Glycosyl donor
(16, 17, 18)
(1, 9, 14)
(SGP)
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
6
Manβ1-4GlcNAcβ1-4GlcNAc
3
H-Lys-Val-Ala-Asn-Lys-Thr-OH
SGP
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
6
3
Manβ1-4GlcNAc1−Glcβ1
O
O
OH
O
16
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
6
3
Manβ1-4GlcNAc1−Glcβ1
O
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
17
H
N
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1
6
Manβ1-4GlcNAc1−Glcβ1
O
O
3
O
18
Scheme 3. Reaction conditions: 0.1 lmol of glycosyl acceptor, 0.2 lmol of SGP, and 1.48 mU of Endo-M in a total volume of 10 lL of 60 mM
potassium phosphate buffer (pH 6.25) for 2 h at 37 ꢁC.

T. Yamanoi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1009–1013
1013
Gonzalez, F.; Vargas-Berenguel, A. J. Org. Chem. 1999, 64,
522–531; (g) Roy, R.; Hernandez-Mateo, F.; Santoyo-
Gonzalez, F. J. Org. Chem. 2000, 65, 8743–8746; (h)
Shinoda, T.; Maeda, A.; Kagatani, S.; Konno, Y.; Sonobe,
T.; Fukui, M.; Hashimoto, H.; Hara, K.; Fujuta, K. Int. J.
Pharm. 1998, 167, 147–154; (i) Shinoda, T.; Kagatani, S.;
Maeda, A.; Konno, Y.; Hashimoto, H.; Hara, K.; Fujuta,
K.; Sonobe, T. Drug Dev. Ind. Pharm. 1999, 25, 1185–1192;
(j) Andre, S.; Kaltner, H.; Furuike, T.; Nishimura, S.-I.;
Gabius, H.-J. Bioconjugate Chem. 2004, 15, 87–98; (k)
Yasuda, N.; Aoki, N.; Abe, H.; Hattori, K. Chem. Lett.
2000, 706; (l) Ortega-Caballero, F.; Gimenez-Martinez, J.
J.; Garcia-Fuentes, L.; Ortiz-Salmeron, E.; Santoyo-Gonz-
alez, F.; Vargas-Berenguel, A. J. Org. Chem. 2001, 66, 7786;
(m) Ichikawa, M.; Woods, A. S.; Mo, H.; Goldstein, I. J.;
Ichikawa, Y. Tetrahedron: Asymmetry 2000, 11, 289–392;
(n) Mallet, C. O.; Defaye, J.; Fernandez, J. M. G. Chem.
Eur. J. 2002, 8, 1982–1990; (o) Baussanne, I.; Benito, J. M.;
Mallet, C. O.; Fernandez, J. M. G. ChemBioChem 2002, 2,
777–783; (p) Furuike, T.; Sukegawa, T.; Nishimura, S.-I.
Macromolecules 2000, 49, E933; (q) Kitahata, S.; Tanim-
oto, T.; Okada, Y.; Ikuta, A.; Tanaka, K.; Murakami, H.;
Nakano, H.; Koizumi, K. Biosci. Biotechnol. Biochem.
2000, 64, 2406–2411, The references of their enzymatic
glycosylation study using CDs were cited therein.
the hen egg yolk glycopeptide, H-Lys-Val-Ala-Asn-
[(NeuAc-Gal-GlcNAc-Man)2-Man-GlcNAc2]-Lys-Thr-OH
(SGP), as the oligosaccharide donor. The transglycosyl-
ation reaction was carried out using 0.2 lmol of the oli-
gosaccharide donor (SGP), 0.1 lmol of the glycosyl
acceptors (1, 9 and 14), and 1.48 mU of recombinant
Endo-M according to the reported procedure.7 First,
arbutin 1 was used as the glycosyl acceptor in order to
ascertain its reactivity in the Endo-M transglycosyl-
ation. After incubation for 2h at 37 ꢁC, HPLC analysis
of the reaction mixture indicated that the transglycosyl-
ation reaction proceeded with the good yield of 56%. In
general, after the yields of the Endo-M transglycosyla-
tion reached the maximum, the transglycosylation
product was gradually degraded by the hydrolytic activ-
ity of Endo-M. In the case of 1 used as the glycosyl
acceptor, the degradation of the transglycosylation
product of 16 was hardly observed even after 5 h. When
9 and 14 were used as the glycosyl acceptors, the corre-
sponding sialo complex-type oligosaccharide-branched
CDs 17 and 18 were similarly obtained in the high yields
of 67% and 65%, respectively.8 Both yields using 9 and
14 were slightly higher than that of 1. The CD cavities
in these glycosyl acceptors might influence the transgly-
cosylation activity of the enzyme. Thus we could
successfully develop an efficient Endo-M transglycosyl-
ation system to produce oligosaccharide-branched CDs
(Table 2, Scheme 3).
2. (a) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.;
Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J.
Am. Chem. Soc. 1999, 121, 284–290; (b) Haneda, K.; Inazu,
T.; Mizuno, M.; Iguchi, R.; Yamamoto, K.; Kumagai, H.;
Aimoto, S.; Suzuki, H.; Noda, T. Bioorg. Med. Chem. Lett.
1998, 8, 1303–1306; (c) Haneda, K.; Inazu, T.; Mizuno, M.;
Iguchi, R.; Tanabe, H.; Fujimori, K.; Yamamoto, K.;
Kumagai, H.; Tsumori, K.; Munekata, E. Biochim. Bio-
phys. Acta 2001, 1526, 242–248; (d) Haneda, K.; Inazu, T.;
Mizuno, M.; Yamamoto, K.; Fujimori, K.; Kumagai, H..
In Peptide Chemistry 1996; Kitada, C., Ed.; Protein
Research Foundation: Osaka, Japan, 1997 pp 13–16.
3. Matsuda, K.; Inazu, T.; Haneda, K.; Mizuno, M.; Yama-
noi, T.; Hattori, K.; Yamamoto, K.; Kumagai, H. Bioorg.
Med. Chem. Lett. 1997, 7, 2353–2356.
4. Our recent study showed that recombinant Endo-M had a
sufficient transglycosylation activity for transferring a
bisecting hybrid-type oligosaccharide from an ovalbumin
glycopeptide: Osumi, K.; Makino, Y.; Akaike, E.; Yama-
noi, T.; Mizuno, M.; Noguchi, M.; Inazu, T.; Yamamoto,
K.; Fujita, K. Carbohydr. Res. 2004, 339, 2633–2635.
5. Arbutin was purchased from Tokyo Kasei Kogyo Co., Ltd.
6. Wang, W.; Pearce, A. J.; Zhang, Y.; Sinay, P. Tetrahedron:
Asymmetry 2001, 12, 517–523.
In summary, we could synthesize the mono-glucose-
branched CDs, which had an appropriate spacer be-
tween the b-cyclodextrin and a glucose moiety, from
b-CD and arbutin. The obtained mono-glucose-
branched cyclodextrins indicated significantly high asso-
ciation constants for doxorubicin in the range of 105–
106 MÀ1. In addition, they worked as highly reactive gly-
cosyl acceptors for the transglycosylation reaction by
Endo-M to produce sialo-complex type oligosaccha-
ride-branched CDs in the high yields of 65–67%. The
arbutin derivatives can be widely used as the useful tags
of various substrates for the Endo-M transglycosyl-
ation. Their introduction into CDs through an appro-
priate spacer can also enhance the CDÕs inclusion
ability for drugs having an aromatic ring like DXR.
These findings will contribute to the development of
drug carriers for targeting drug-delivery systems.
7. Yamanoi, T.; Tsutsumida, M.; Oda, Y.; Akaike, E.; Osumi,
K.; Yamamoto, K.; Fujita, K. Carbohydr. Res. 2004, 339,
1403–1406.
8. Analyses or isolation of the transglycosylation product
were done using HPLC (Shimadzu LC-10AT chromato-
graph equipped with a SPD-10A ultraviolet spectropho-
References and notes
1. (a) Abe, H.; Kenmoku, A.; Yamaguchi, N.; Hattori, K. J.
Incl. Phenom. Macrocyl. Chem. 2001, 44, 39–47; (b) Ikuta,
A.; Koizumi, K.; Tanimoto, T. J. Carbohydr. Chem. 2000,
19, 13–23; (c) Furuike, T.; Aiba, S.; Nishimura, S.-I.
Tetrahedron 2000, 56, 9909–9915; (d) Kassab, R.; Felix, C.;
Parrot-Lopez, H.; Bonaly, R. Tetrahedron Lett. 1997, 38,
7555–7558; (e) Baussanne, I.; Benito, J. M.; Mellet, C. O.;
Fernandez, J. M. G.; Law, H.; Defaye, J. Chem. Commun.
2000, 1489–1490; (f) Garcia-Lopez, J. J.; Hernandez-
Mateo, F.; Isac-Garcia, J.; Kim, J. M.; Roy, R.; Santoyo-
tometer) on
a
reversed-phase column (4.6 · 250 mm,
Inertsil ODS-3, GL Sciences, Inc.). Elution was carried
out with a linear gradient of acetonitrile (5–35%) containing
0.1% aqueous trifluoromethanesulfonic acid (TFA) in
30 min at the flow rate of 1 mL/min. The reaction products
were monitored by absorptions at 214 nm. MALDI-TOF
MS; 17: Found: m/z [MÀH]À 3450.5: calcd for
C
133H213N5O98[MÀH]À 3447.2; 18: Found: m/z [M+K]+
3484.6: calcd for C132H210N6O98 [M+K]+ 3486.1.