Chemical and enzymatic synthesis of neoglycolipids in the presence of cyclodextrins

Article abstract of DOI:10.1016/j.tetlet.2008.03.103

The efficiency of cyclodextrins (CDs), α-CD, β-CD and γ-CD, for the blotting of hydrophobic substrates to water-soluble polymers and for the synthesis of neoglycolipids using glycosyltransferase is described. CDs did not disturb HPLC purification and did not bring lather. These merits are superior to surfactants commonly used in such a case. The utility of CDs as a novel and efficient supporting material for enzymatic glycosylation is also discussed.

Full text of DOI:10.1016/j.tetlet.2008.03.103

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3413–3418
Chemical and enzymatic synthesis of neoglycolipids
in the presence of cyclodextrins
a
a,
a
a,b,
*
*
Izuru Nagashima , Hiroki Shimizu , Takahiko Matsushita , Shin-Ichiro Nishimura
a Drug-Seeds Discovery Research Laboratory, Hokkaido Center, National Institute of Advanced Industrial Science and Technology (AIST),
Sapporo 062-8517, Japan
b Division of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genomic Science and Technology,
Hokkaido University, Sapporo 011-0021, Japan
Received 13 February 2008; revised 18 March 2008; accepted 24 March 2008
Available online 28 March 2008
Abstract
The efficiency of cyclodextrins (CDs), a-CD, b-CD and c-CD, for the blotting of hydrophobic substrates to water-soluble polymers
and for the synthesis of neoglycolipids using glycosyltransferase is described. CDs did not disturb HPLC purification and did not bring
lather. These merits are superior to surfactants commonly used in such a case. The utility of CDs as a novel and efficient supporting
material for enzymatic glycosylation is also discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Enzyme; Glycosyltransferase; Cyclodextrin; Polymer-supported synthesis; Carbohydrate
The development of carbohydrate analysis has revealed
the biological importance of oligosaccharides,1–3 and car-
bohydrate chemistry is expected to contribute to the phar-
maceutical and industrial sectors. From this point of view,
we have developed new methods to prepare oligosaccha-
rides easily using glycosyltransferases, which can control
glycosylation with precise regio- and stereoselectivities.
As a part of our researches, we have reported the parallel
synthesis of glycopeptides in lmol scale to produce an
oligopeptide-focused library by the molecular transporter
between solid-phase and water-soluble polymers.4,5 Our
next topics of interest have been focused on preparing
glycolipid derivatives. Although we have reported the
utility of the water-soluble polymer in enzymatic reactions,
the key point should still be the solubility of neoglycolipids,
which are generally more hydrophobic than glycopeptides.
To increase the solubility of substrates in enzymatic
reactions, it is common to use a small amount of a polar
solvent, like DMSO, or to use surfactants like cholic acid
sodium salt and sodium dodecyl sulfate (SDS) as ionic sur-
factants and octylphenyl ethoxylates (Triton-X) as a non-
ionic surfactant. Although surfactants are helpful for
enzymatic reactions, only a limited concentration of sub-
strates becomes soluble and using a large quantity of sur-
factants resulted in other problems during purification
stages.
In this Letter, we studied the utility of simple cyclodex-
trins (CDs) for the synthesis of neoglycolipids using glycos-
yltransferase. CDs have the ability to include the
hydrophobic area of a compound, and the formed complex
becomes soluble in water due to hydroxyl groups orien-
tated on the surface of CDs. Thus, we thought CDs would
have similar properties with surfactants in enzymatic reac-
tions. While there are reports of the use of CDs for the
enzymatic glycosylations,6–8 there are no systematic studies
of the effect for the enzymatic synthesis of neoglycolipids,
which have large hydrophobic parts; namely the relation-
ship between concentrations of CD and substrates; or the
*
Corresponding authors. Fax: +81 11 857 8435 (H.S.); fax: +81 11 706
9042 (S.N.).
E-mail addresses: hiroki.shimizu@aist.go.jp (H. Shimizu), shin@
glyco.sci.hokudai.ac.jp, tiger.nishimura@aist.go.jp (S.-I. Nishimura).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.103

3414
I. Nagashima et al. / Tetrahedron Letters 49 (2008) 3413–3418
differences between a-, b- and c-CD. Thus, here, we studied
the details of the efficiency of cyclodextrins for galactosyl-
transferase in vitro to synthesize neoglycolipids in a large
scale.
could conjugate it with the reported water-soluble poly-
mer4,5 through a suitable linker, we could modify more
complicated oligosaccharides by glycosyltransferase
in vitro.
Until now, there have been many reports of glycosyl-
ation studies, namely chemical coupling, enzymatic synthe-
ses in vitro, and syntheses using cell function in vivo. The
combination of both chemical and enzymatic methods
has become a common strategy nowadays and has been
called chemoenzymatic synthesis. As shown in Figure 1,
we have attempted to also add cell-media synthesis, which
we named cellular-chemoenzymatic synthesis, as a triple-
factor combination method, to make oligosaccharide
synthesis easier.
Based on this strategy, the linker should be designed to
have the following: (i) a functional group to couple with
the azido group onto oligosaccharides synthesized by cells;
(ii) a functional group to conjugate with the water-soluble
polymer; (iii) a part to be released from the polymer; and
(iv) leaving a UV tag on the synthesized oligosaccharides
was favourable to apply further researches, like the devel-
opment of glycan array.11 According to (i), the carboxyl
group would be suitable for application in the Staudinger
reaction.12–14 In the case of amino acids’ carboxyl groups,
glycine should be used because conjugation with azido
would lead to racemization at a-position. According to
(ii), conjugation with ketone and aminooxy groups onto
the linker and the water-soluble polymer, respectively,
has already been reported.4,5 According to (iii), the reac-
tion to release synthesized oligosaccharides from the
polymer is preferred under mild conditions because the
target oligosaccharides have multi-functional groups.
Thus, our first option was an enzymatic reaction. BLase
protease15 can hydrolyze a bond between amino acids
of Glu and Phe, and hydrophobic amino acids situated
next to Glu could accelerate the reaction. In fact, we
already showed the utility of the BLase as a releasing
reagent from the polymer.5 In summary, we designed
the linker as CH3C(@O)CH2CH2CH2C(@O)-Phe-Glu-
Phe-Gly-OH (oxo-FEFG),16 which would result in leaving
a UV tag, phenylalanine, on the side of the synthesized
neoglycolipid when released from the polymer.
Yamagata, Sato, Hatanaka et al. have reported 12-azi-
dododecyl lactose as a primer, a starting material for the
synthesis of GM3 analogue by cells.9–11 We thought if we
Sugar
N3
Oligosaccharide synthesis
by cells in vivo
Sugar
N3
Sugar
Sugar
Chemical conversion
O
NC
H
Water-soluble
Polymer
Sugar
E
Linker
Sugar
Sugar
Trimming & further elongation
by enzyme in vitro
Sugar
Sugar
Sugar
O
NC
H
Sugar
Linker
To test along the above strategy, we chose a glucosa-
mine derivative as a model material. 12-azidododecyl
2-acetoamido-2-deoxy-b-D-glucopyranoside (3)17 was syn-
thesized from bromide (1)18 in 45% yield in three steps.
Instead of applying the Staudinger reaction, in this case,
reduction with triphenylphosphine followed by coupling
with the peptide linker, oxo-FEFG,16 and deprotection
gave 4,17 which the linker was successfully introduced to
the azidoalkyl group (Scheme 1).
Cleaveage from polymer
Sugar
Sugar
O
NC
H
Sugar
Linker
Fig. 1. Scheme of the triple-factor combination method for oligosaccha-
ride synthesis.
OAc
O
OAc
O
OH
b), c)
a)
O
AcO
AcO
AcO
AcO
HO
HO
O(CH2)12N3
O(CH2)12N3
Br
NDCPhth
NDCPhth
NHAc
1
2
3
O
OH
Cl
Cl
d), e)
O
HO
HO
NDCPhth =
N
O(CH2)12NH-GFEF-oxo
NHAc
O
4
Scheme 1. Preparation of model primer, N-acetyl-glucosamine derivative and chemical modification. Reagents and conditions: (a) N3(CH2)12OH
(1.0 equiv and 1.4 equiv of 1 was used), AgOTf (1.4 equiv), s-collidine (1.0 equiv), CH2Cl2, (3 ml/1g of 1), ꢀ20 °C, 2 h (59% from N3(CH2)12OH); (b)18
H2NCH2CH2NH2 (1 ml/1 g of 2),17 MeOH (3 ml/1 g of 2), rt, 18 h, then Ac2O (2 ml/1 g of 2), Py (8 ml/1 g of 2), rt, 5 h (91%): (c) NaOMe (cat.), MeOH,
rt, 1 h (97%); (d) PPh3 (1.0 equiv), EtOH (10 ml/1 g of 3), 50 °C, 18 h (90%); (e) oxo-FE(OBn)FG16 (1.1 equiv), WSC (1.2 equiv), HOBt (1.2 equiv), Et3N
(1.0 equiv), DMF (5 ml/100 mg of 3), THF (5 ml/100 mg of 3), rt, 1 h, then Pd–C (cat.), dioxane (1.5 ml/100 mg of 3), water (1.5 ml/100 mg of 3), 50 °C,
1 h, (87%).

I. Nagashima et al. / Tetrahedron Letters 49 (2008) 3413–3418
3415
Next, we attempted to introduce 4 onto the water-solu-
ble polymer, but we only obtained 3% of 617 in the simple
steps of blotting to and cleavage from the polymer. We
thought this low yield was due to the solubility of 4 in
water buffer, and we found that c-CD could improve the
yield dramatically, blotting with 60 mM c-CD in sodium
acetate buffer and cleavage steps resulted in 76% yield
(Scheme 2).
As we found an efficiency of c-CD in this simple blotting
stage, it would be natural to think that additional CDs for
4 without blotting would be enough for the reactions using
galactosyltransferase (GalT). However, without any addi-
tional reagents, N-acetyl-lactosamine disaccharide (7) was
not produced at all even in 0.1 mM concentration condi-
tion, suggesting that the CDs turned the impossible into
possible.19
units, respectively. Although all glucose units have a1-4
linkage, only b-CD’s solubility is low. Therefore, we have
mixed only 10 mM of b-CD for the study although a-CD
and c-CD were tested up to 100 mM (Scheme 3).
Table 1 shows the glycosylation speeds of the beginning
of the reactions (V0, mM/h) and are marked if the reactions
were completed. Although a-CD was not as effective even
at high concentrations of a-CD (100 mM) against low con-
centrations of 4 (2 mM), 10 mM b-CD worked well for the
glycosylation until 4 mM of 4, but unfortunately we could
not increase the concentration of b-CD further due to its
solubility. Regarding c-CD, 30 mM, 50 mM, 70 mM and
90 mM c-CD could lead to 7 quantitatively by the condi-
tions of 2 mM, 4 mM, 6 mM and even 8 mM of 4, respec-
tively. We would like to note that the reaction mixtures of
control and 10 mM c-CD became viscous, and 90 mM led
to a white suspension although the reaction continued to
proceed; 30–70 mM resulted in clear solutions (Fig. 2).
Three types of CD are very common, a-CD, b-CD and
c-CD, which are composed of six, seven and eight glucose
OH
O
O
O
O
O
H
N
H
N
HO
O
HO
N
H
N
H
N
H
NHAc
O
O
4
COOH
O
O
O NH2
6.4 mM AcOH-NaOAc buffer (pH 5.5)
N
H
1
Cyclodextrins
NH2
20
O NH2
OH
O
O
O
O
O
N O
H
N
H
N
HO
HO
O
N
H
N
H
N
H
NHAc
O
O
5
COOH
BLase
25 mM AcOH-AcONH4 buffer (pH 6.5)
OH
O
H
N
HO
HO
O
N
H
NH2
NHAc
O
6
CD effect for the blotting
80
60
40
20
0
γ-CD
%
α-CD
20
40
60
80
100
mM
Scheme 2. Preliminary experiment for a model primer blotting to and releasing from the water-soluble polymer. Cyclodextrins, which were added in the
blotting stage only, improved the results dramatically.

3416
I. Nagashima et al. / Tetrahedron Letters 49 (2008) 3413–3418
OH
O
O
O
O
O
H
N
H
N
HO
HO
O
N
H
N
H
N
H
NHAc
O
O
4
UDP-Gal (2.5 eq.), β1,4-GalT
MnCl2, HEPES buffer (pH 7.0)
Cyclodextrins
COOH
25 oC
HO
HO
OH
O
OH
O
O
O
O
O
H
N
H
N
O
O
HO
N
N
H
N
OH
H
H
NHAc
O
O
7
COOH
Scheme 3. Study of cyclodextrins’ effect for the synthesis of N-acetyl-lactosamine derivative (7) by b1,4-galactosyltransferase.
Increasing the concentration of c-CD led to a raise in the
Table 1
Reaction speed (V0, mM/h) by galactosyltransferase from
calculated from the reaction ratio at 1 h, 2 h and 3 h18
4
to 7,
limitation of the concentration of substrate 4, which sug-
gested that CDs did not work as surfactants nor as phase
transfer systems but as molecular recognition systems.
Thus, although c-CD worked well, a-CD was not as effi-
cient even at high concentrations, because the number of
recognition parts was different, namely c-CD supposed to
involve phenyl groups on the linker, while a-CD might
not. In practicality, CDs did not prevent HPLC purifica-
tion. CDs eluted completely in early fractions and we could
not observe any CD with purified 7 by NMR, although we
had planned to use a-amylase to convert c-CD to glucose if
the product was contaminated. Finally, regarding the scale
of the reactions, c-CD could allow for the synthesis of 7 in
the 8 mM condition, that is, 8 mg of 4 in 1 ml buffer could
be converted. This is a rather high concentration of neo-
glycolipid prepared by glycosyltransferase in vitro. In addi-
tion, modified CDs, like methylated CDs with higher
solubility, are also available, which suggest the possibility
to further increase the reaction scale.
Concentration of 4
2 mM
4 mM
6 mM
8 mM
10 mM
a-CD
b-CD
c-CD
100 mM
10 mM
10 mM
20 mM
30 mM
50 mM
70 mM
90 mM
0.02
NR
0.14c
NR
0.32b
0.06
NR
0.03
NR
0.28a
0.22a
0.26a
0.26a
0.16b
0.03
0.20c
0.32b
0.28b
0.20
NR
0.05
0.24b
0.24b
0.36
NR
0.06
0.36b
NR
0.07
Triton X-100 1%
NR means no reaction. Blank areas indicate reactions that were not
performed.
a, b,
c
Indicate that the reactions were completed within 12 h, 24 h and
48 h, respectively.
In contrast, the reaction mixture with 1% of Triton X-100
became a gel phase (photo not shown), so the reaction was
not concluded in either case of 4 mM and 6 mM substrates
even though the V0 were similar to those with CDs.
In conclusion, we have demonstrated the efficiency of
cyclodextrins for the enzymatic synthesis of neoglycolipids
that are barely water soluble, and based on this study we
could make a bridge between oligosaccharide syntheses
using cell function in vivo and enzymatic synthesis
in vitro and established cellular-chemoenzymatic synthesis.
Acknowledgements
This research was supported by a grant for the ‘Develop-
ment of Novel Diagnostic and Medical Applications
through Elucidation of Sugar Chain Functions’ from the
New Energy and Industrial Technology Development
Organization (NEDO). The authors thank Professor K.
Hatanaka at the University of Tokyo and Professor T. Sato
at Keio University for valuable suggestions and discussion
through the NEDO project, Mr. T. Ito, one of our col-
leagues, for the guidance of enzymatic research and the
members of the Center for Instrumental Analysis, Hokkai-
do University, for measuring high-resolution FAB-MS.
non
10
30
50
70
90
References and notes
(mM)
Fig. 2. Photos of reaction flasks for 2 mM substrates. Concentration of
the present c-CD is shown. The control and 10 mM c-CD formed a gel
and the 90 mM became a white suspension, while the 30 mM, 50 mM and
70 mM led to clear solutions.
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16. Oxo-FEFG was synthesized on Fmoc-Gly-OH preloaded with
53.51, 51.80, 41.65, 40.62, 37.51, 37.17, 34.12, 29.86, 29.65, 27.34,
19.22. MALDI-TOF-MASS m/z for C38H44N4O9 [M+Na]+ calcd
723.30, found 723.58; [M+K]+ calcd 739.27, found 739.60. ESI HR
MS m/z for C38H44N4O9 [M+H+] calcd 701.31865, found 701.32016.
17. Characterization of compounds. Compound 2: 1H NMR (400 MHz,
CDCl3): d 7.96 (2H, s, aromatic-H), 5.74 (1H, dd, J = 10.7, 9.2 Hz, H-
3), 5.34 (1H, d, J = 8.5 Hz, H-1), 5.19 (1H, dd, J = 9.9, 9.2 Hz, H-4),
4.34 (1H, dd, J = 12.3, 4.6 Hz, H-6), 4.29 (1H, dd, J = 10.7, 8.5 Hz,
H-2), 4.19 (1H, dd, J = 12.3, 2.3 Hz, H-6), 3.28 (2H, t, J = 7.0 Hz,
N3CH2–), 2.13 (3H, s, CH3C(@O)–), 2.06 (3H, s, CH3C(@O)–), 1.90
(3H, s, CH3C(@O)–). 13C NMR (100 MHz, CDCl3): d 170.72, 170.32,
169.43, 139.39, 130.52, 125.69, 97.97, 77.23, 71.89, 70.84, 70.21, 68.90,
62.05, 55.15, 51.51, 29.59, 29.52, 29.48, 29.45, 29.21, 29.16, 28.85,
26.72, 25.87, 20.77, 20.63, 20.47. ESI HR MS m/z for
C32H42Cl2N4O10 [M+Na+] calcd 735.21757, found 735.22036. 3: 1H
NMR (400 MHz, MeOD): d 4.41 (1H, d, J = 8.4 Hz, H-1), 3.70 (1H,
dd, J = 11.6, 5.6 Hz, H-6), 3.64 (1H, dd, J = 10.3, 8.4 Hz, H-2), 3.29
(2H, t, J = 6.7 Hz, N3CH2–), 1.99 (3H, s, CH3C(@O)–). 13C NMR
(100 MHz, MeOD): d 173.63, 102.75, 77.97, 76.11, 72.22, 70.62, 62.87,
57.49, 52.49, 30.82, 30.73, 30.71, 30.70, 30.65, 30.56, 30.29, 29.94,
27.85, 27.17, 23.04. ESI HR MS m/z for C20H38N4O6 [M+Na+] calcd
453.26890, found 453.26837. Compound 4: 1H NMR (400 MHz,
MeOD): d 7.29–7.18 (10H, m, aromatic-H), 4.56 (1H, dd, J = 9.9,
5.0 Hz, Phe-Ha), 4.46 (1H, dd, J = 9.2, 6.0 Hz, Phe-Ha), 4.38 (1H, d,
J = 8.4 Hz, H-1), 4.20 (1H, dd, J = 8.3, 5.3 Hz, Glu-Ha), 3.91 (1H, d,
J = 16.8 Hz, Gly-Ha), 3.67 (1H, dd, J = 11.9, 5.6 Hz, H-6), 3.65 (1H,
d, J = 16.8 Hz, Gly-Ha), 3.62 (1H, dd, J = 10.3, 8.4 Hz, H-2), 3.10
(1H, dd, J = 13.9, 5.0 Hz, Phe-Hb), 3.01 (1H, dd, J = 13.9, 9.2 Hz,
Phe-Hb), 2.84 (1H, dd, J = 13.9, 9.9 Hz, Phe-Hb), 2.33 (2H, t,
J = 7.3, –CH2CH2C(@O)–), 2.06 (3H, s, CH3C(@O)–), 1.96 (3H, s,
CH3C(@O)–), 1.72 (2H, qui, J = 7.3 Hz, –C(@O)CH2CH2-
CH2C(@O)–). 13C NMR (100 MHz, MeOD): d 211.11, 176.90,
176.01, 174.56, 173.90, 173.87, 173.67, 171.38, 138.50, 130.33,
130.30, 129.67, 129.58, 127.97, 127.91, 102.78, 78.02, 76.16, 72.27,
70.65, 62.91, 57.54, 56.97, 56.34, 54.86, 43.70, 43.11, 40.58, 38.46,
37.95, 35.72, 31.22, 30.85, 30.78, 30.77, 30.74, 30.58, 30.49, 30.43,
29.94, 28.00, 27.66, 27.19, 23.08, 20.75. ESI HR MS m/z for
2-chlorotrityl
chloride
resin
(l-glycine-2-chlorotrityl
resin,
0.80 mmol/g), DIEA (840 ll, 4.8 mmol) was added to the mixture
of the resin (1 g) pre-washed by CH2Cl2, Fmoc-Phe-OH (930 mg,
2.4 mmol), HBTU (910 mg, 2.4 mmol) and HOBt (320 mg, 2.4 mmol)
in DMF (5 ml), and the mixture was stirred for 10 min under
occasional microwave irradiation at 50 °C. This coupling process was
performed twice. After washing the resin with CH2Cl2 and DMF, 20%
piperidine in DMF (4 ml) was added and the mixture was stirred for
5 min under occasional microwave irradiation at 50 °C. After washing
the resin with CH2Cl2 and DMF, the same coupling procedure was
carried out using Fmoc-Glu(OBn)-OH (1.1 g, 2.4 mmol). We should
note here that the deprotection of the Fmoc group by piperidine was
performed for 2 min at rt instead of for 10 min under microwave
irradiation to prevent byproducts that would cause lactamization of
the glutamic acid. After the introduction of the Glu moiety as above,
Fmoc-Phe-OH was conjugated by the same procedure for 2 min at rt
for the deprotection of Fmoc, and finally 5-oxohexanoic acid (290 ll,
2.4 mmol) was used in the same procedure as above for the
introduction of a ketone group. Treatment of the resin washed with
CH2Cl2 and DMF in 10% TFA in dichloromethane (20 ml) or AcOH/
CF3CH2OH/CH2Cl2 (2:2:6, 20 ml) for 1 h at rt followed by HPLC
purification [column: Inertsil ODS-3, £ 20 mm ꢁ 250 mm; eluate:
acetonitrile, gradient increase from 20% to 90% over 50 min; column
oven temperature: 40 °C; flow rate: 5 ml/min] gave CH3C(@O)-
CH2CH2CH2C(@O)–FE(OBn)FG in 57% and 58% yields, respec-
tively. 1H NMR (400 MHz, (CD3)2S@O): d 8.34 (1H, t, J = 5.8 Hz,
Gly-NH), 8.03 (1H, d, J = 7.7 Hz, Glu-NH), 8.01 (1H, d, J = 8.2 Hz,
Phe-NH), 7.96 (1H, d, J = 8.3 Hz, Phe-NH), 5.09 (2H, s, PhCH2O–),
4.55 (1H, m, Phe-Ha), 4.52 (1H, m, Phe-Ha), 4.25 (1H, m, Glu-Ha),
3.80 (1H, dd, J = 17.6, 5.8 Hz, Gly-Ha), 3.75 (1H, dd, J = 17.6,
5.8 Hz, Gly-Ha), 3.05 (1H, dd, J = 13.9, 4.4 Hz, Phe-Hb), 2.94 (1H,
dd, J = 13.9, 3.9 Hz, Phe-Hb), 2.80 (1H, dd, J = 13.9, 9.5 Hz,
Phe-Hb), 2.68 (1H, dd, J = 13.9, 10.7 Hz, Phe-Hb), 2.29 (2H, m,
Glu-Hc), 2.18 (2H, t, J = 7.3, –CH2CH2C(@O)–), 1.98 (2H, t, J = 7.3,
–CH2CH2C(@O)–), 1.97 (3H, s, CH3C(@O)–), 1.89 (1H, m, Glu-Hb),
1.76 (1H, m, Glu-Hb), 1.52 (2H, qui, J = 7.3, –C(@O)CH2CH2CH2-
C(@O)–). 13C NMR (100 MHz, (CD3)2S@O): d 207.97, 172.20, 171.72,
171.45, 171.16, 170.99, 170.51, 138.04, 137.56, 136.18, 129.14, 129.08,
128.39, 127.96, 127.95, 127.93, 127.88, 126.19, 126.11, 65.42, 53.59,
C
51H76N6O14 [M+Na+] calcd 1019.53172, found 1019.53166. 6: 1H
NMR (400 MHz, MeOD): d 7.39–7.28 (5H, m, aromatic-H), 4.38
(1H, d, J = 8.4 Hz, H-1), 4.07 (1H, dd, J = 8.0, 6.0 Hz, Phe-Ha), 3.94
(1H, d, J = 16.4 Hz, Gly-Ha), 3.72 (1H, d, J = 16.4 Hz, Gly-Ha), 3.68
(1H, dd, J = 11.9, 5.6 Hz, H-6), 3.62 (1H, dd, J = 10.3, 8.4 Hz, H-2),
3.24 (1H, dd, J = 14.0, 6.5 Hz, Phe-Hb), 3.18 (2H, t, J = 7.1, –OCH2–
or –NHCH2–), 3.04 (1H, dd, J = 14.0, 8.0 Hz, Phe-Hb), 1.97 (3H, s,
CH3C(@O)–). 13C NMR (100 MHz, MeOD): d 173.64, 170.70,
173.07, 135.80, 130.50, 130.17, 128.91, 102.80, 78.00, 76.13, 72.24,
70.65, 62.90, 57.49, 56.01, 43.12, 40.58, 38.63, 30.80, 30.72, 30.70,
30.64, 30.54, 30.41, 27.99, 27.18, 23.04. ESI HR MS m/z for
C
31H52N4O8 [M+H+] calcd 609.38634, found 609.38566. Compound
7: 1H NMR (400 MHz, (CD3)2S@O): d 8.25 (1H, t, J = 5.7 Hz,
Gly-NH), 8.06 (1H, d, J = 7.4 Hz, Phe-NH), 8.02 (1H, d, J = 8.4 Hz,
Phe-NH), 7.71 (1H, d, J = 8.6 Hz, Glu-NH), 7.59 (1H, t, J = 5.5 Hz,
–CH2NHC(@O)–), 7.26–7.14 (10H, m, aromatic-H), 5.05 (1H, br,
–OH), 4.76 (1H, br, –OH), 4.61 (1H, br, –OH), 4.52 (1H, m, Phe-Ha),
4.45 (1H, m, Phe-Ha), 4.28 (1H, d, J = 7.9 Hz, H-1 or H-10), 4.23 (1H,
d, J = 7.8 Hz, H-1 or H-10), 2.95 (1H, dd, J = 13.9, 3.8 Hz, Phe-Hb),
2.84 (1H, dd, J = 13.7, 9.1 Hz, Phe-Hb), 2.68 (1H, dd, J = 13.9,
10.7 Hz, Phe-Hb), 2.19 (2H, t, J = 7.3, –CH2CH2C(@O)–), 1.99 (3H,
s, CH3C(@O)–), 1.53 (2H, qui, J = 7.3, –C(@O)CH2CH2CH2C(@O)–).
13C NMR (100 MHz, (CD3)2S@O):
d 208.34, 174.27, 172.14,
171.92, 171.48, 171.41, 168.95, 168.56, 137.41, 137.91, 129.46,
129.43, 128.39, 128.27, 126.62,104.35, 101.23, 81.83, 75.92, 75.35,
73.57, 72.64, 70.95, 68.78, 68.53, 60.83, 55.13, 54.56, 53.96, 34.49,
30.28, 30.03, 29.47, 29.38, 29.16, 29.11, 27.61, 26.71, 25.75, 23.30,
19.58. ESI HR MS m/z for C57H86N6O19 [M+Na+] calcd 1181.58454,
found 1181.58306.
18. Shimizu, H.; Ito, Y.; Matsuzaki, Y.; Iijima, H.; Ogawa, T. Biosci.,
Biotechnol., Biochem. 1996, 60, 73–76.

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I. Nagashima et al. / Tetrahedron Letters 49 (2008) 3413–3418
19. Typical procedure of Scheme 3 for 2 mM 4 with 90 mM c-CD: 4
(1.0 mg, 1.0 mol) was galactosylated using 25 mU of b1,4-GalT
(SIGMA, G-5507, practically 1.7 mg was dissolved in 1.7 ml water as
3 U/ml, and used), 50 ll of 50 mM UDP-Gal and 300 ll of 150 mM c-
CD in a total volume of 500 ll of 50 mM HEPES buffer, pH 7.0, and
10 mM MnCl2. Incubation was carried out at 25 °C and the reaction
was monitored every hour with 5 ll sampling by HPLC [column:
Inertsil ODS-3, £ 4.6 mm ꢁ 250 mm; eluate: acetonitrile, gradient
increase from 30% to 40% over 30 min; column oven temperature:
30 °C; 220 nm UV detector; flow rate: 1 ml/min] tR = 24.5 min for 7
and 27.5 min for 4. In the case of the reaction for 4 (5.00 mg in total
volume 2.5 ml, 2 mM) with 50 mM c-CD, HPLC purification gave
5.78 mg of 7 (99%) [column: Inertsil ODS-3, £ 20 mm ꢁ 250 mm;
eluate: 10% acetonitrile for 20 min followed by gradient increase
from 10% to 30% over 10 min and 30% to 45% over 50 min; column
oven temperature: 40 °C; 220 nm UV detector; flow rate: 5 ml/min. c-
CD was eluted out between 8 and 23 min, which was detected by
MALDI-TOF MS, and 7 was collected from the fractions from 49 to
53 min].