Studies of cyclodextrin effect for glycosylation by galactosyltransferase

Article abstract of DOI:10.1016/j.tet.2014.03.058

Cyclodextrins (CDs) do not disturb HPLC purification and do not form lather, which makes them superior to commonly used surfactants. In this paper, the authors describe the utility of CDs, specifically α-CD, β-CD, and γ-CD, for the synthesis of neoglycolipids from insoluble peptide-linked substrates. γ-CD was found to be well matched to the bulky benzyl group and accelerated the reaction for the substrate having Phe (F), while β-CD was well matched to the methyl group and accelerated the reaction for the substrate having Ala (A). Galactosylation to water-insoluble peptide-linked glucosaminide substrate by galactosyltransferase proceeded 63% at the most, but using either β-CD or γ-CD gave quantitative yield within 10 h.

Full text of DOI:10.1016/j.tet.2014.03.058

Tetrahedron 70 (2014) 3146e3154
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Studies of cyclodextrin effect for glycosylation by
galactosyltransferase
*
Izuru Nagashima, Hiroki Shimizu
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-higashi, Toyohira,
Sapporo 062-8517, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclodextrins (CDs) do not disturb HPLC purification and do not form lather, which makes them superior
Received 24 January 2014
Received in revised form 13 March 2014
Accepted 17 March 2014
to commonly used surfactants. In this paper, the authors describe the utility of CDs, specifically
-CD, and -CD, for the synthesis of neoglycolipids from insoluble peptide-linked substrates. -CD was
found to be well matched to the bulky benzyl group and accelerated the reaction for the substrate having
Phe (F), while -CD was well matched to the methyl group and accelerated the reaction for the substrate
having Ala (A). Galactosylation to water-insoluble peptide-linked glucosaminide substrate by gal-
a-CD,
b
g
g
Available online 22 March 2014
b
Keywords:
Glycosylation
Enzymatic synthesis
Glycosyltransferase
Cyclodextrin
actosyltransferase proceeded 63% at the most, but using either
b
-CD or
g-CD gave quantitative yield
within 10 h.
Ó 2014 Elsevier Ltd. All rights reserved.
Oligosaccharide
1. Introduction
water. In addition, some enzymes (e.g. glycosyltransferases) are not
stable, which results that their activities are lost during progression
of reactions and the reactions are not completed in some cases. We
have already reported on approaches to overcome these disad-
vantages. In particular, applying water-soluble polymer and addi-
tive cyclodextrins were found to effectively increase the solubility
It is well known that carbohydrates play key roles in the fields of
medicine and biology,1e3 and carbohydrate-based drug discovery is
a subject of current interest and challenge.4 For example, oselta-
mivir (Tamiflu from Roche) is a derivative of neuraminic acid and
has been developed by computational drug design and synthetic
chemistries.5e7 In addition, a number of biopharmaceuticals are
glycoproteins.8 Carbohydrate moieties of therapeutic glycoproteins
are diverse mixtures because they are produced in human or ani-
mal cell lines. The degree of diversity is varied, depending on the
cell line, the culture conditions and so on. Despite its subtle dif-
ferences, glycosylation status could influence efficacy, safety, and
immunogenicity. Biobetters are improved recombinant protein
drugs that are unlike any existing biopharmaceuticals. It is com-
monly thought that the standardization of carbohydrates on ther-
apeutic glycoproteins could be a key to developing biobetters.
Therefore, carbohydrate chemists are expected to contribute to
these research fields.
of the neoglycolipid. This allowed galactosylation by
b1,4-
galactosyltransferase9 to proceed, even though the substrate itself
was not soluble in water.10
Cyclodextrin (CD) is a family of compounds made up of
copyranoside units 1e4 linked in a ring and widely used in food,
cosmetic, and pharmaceutical areas. Naturally occurring -CD,
CD, and -CD are composed of six, seven, and eight -glucopyr-
D-glu-
a
a
b-
g
D
anoside units, respectively, and all of them are commercially
available. Although these cyclic oligosaccharides are similar forms,
their solubilities are different as 145 g/L, 18.5 g/L, and 232 g/L at
25 ꢀC for
a-CD, b-CD, and g-CD, respectively. They are topologically
represented as toroids, and size and volume of cavities are reported
as 0.57e1.37 nm and 173 A3, 0.78e1.53 nm and 262 A3, and
Chemical, enzymatic, and cellular syntheses are the three main
methods for obtaining oligosaccharides artificially, and each
method has advantages and disadvantages. Enzymatic synthesis
can control glycosylation in specific regions as well as controlling
stereoselectivities with ease, but substrates must be soluble in
0.95e1.69 nm and 427 A3 for -CD, respectively.11
a-CD, b-CD, and g
Exterior of the toroids shows hydrophilicity and interior possesses
relatively hydrophobic character, therefore CDs could form hoste-
guest complexes with other hydrophobic molecules and the com-
plexes become to soluble in water. Thermodynamical studies of
CDeguest complex were well studied.12
Cyclodextrins do not disturb HPLC purification and do not form
lather, which makes them superior to commonly used surfactant. In
* Corresponding author. Tel.: þ81 11 857 8497; fax: þ81 11 857 8980; e-mail
address: hiroki.shimizu@aist.go.jp (H. Shimizu).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.03.058

I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
3147
previous paper,10 we have reported relationship between concen-
trations of -CD and compound 9. Here, we report further details
g
about the effects of cyclodextrins on a variety of peptide-linked
neoglycolipids.
2. Results and discussion
We first considered peptide linkers, which can conjugate with
neoglycolipids. To take into account the use of BLase protease,13
which releases synthesized oligosaccharide from the water-
soluble polymer (as shown in the previous report10), Glu (E) was
essential and hydrophobic amino acids situated next to Glu were
preferred. To study the effectiveness of cyclodextrins on reactions
for peptide linkers employing
b
1,4-galactosyltransferase,9 we
designed and synthesized seven kinds of ‘oxo-XEYG’ peptide
linkers (1e7), where oxo-¼CH3C(]O)CH2CH2CH2C(]O)e and X,
Y¼Gly (G), Ala (A) or Phe (F). These peptide linkers were synthe-
sized by microwave-applied SPPS (solid-phase peptide synthesis)
Fmoc strategy (Scheme 1). When the synthesized peptides were
released from the supporting resin, the protecting group of side
chain of Glu (E) was maintained, in order to account for the cou-
pling with N-terminals of the peptides and 12-azidododecyl glu-
cosaminide (8).14e16 Therefore, we chose a benzyl ester protecting
group for the side chain of Glu (E). Deprotection of the Fmoc group
on Glu (E) by piperidine and successive amino acid coupling stages
were performed then at room temperature for 2 min and 1 h,
successively. If those reactions have been performed under mi-
crowave irradiation at 50 ꢀC, deprotection of the benzyl ester group
and pyroglutamization would have occurred. Finally, 110e7 were
successfully synthesized in 47e85% overall yields.
TFE
Conjugation of 12-azidododecyl glucosaminide (8) with the
linkers followed by deprotection of benzyl group proceeded in
43e87% yields (Scheme 2). Although cyclodextrins are able to form
hosteguest complexes, Gly (G) has no side chain, and thus cyclo-
dextrins would not work well in a hosteguest system for Gly (G).
Therefore, we judged that oxo-GEAG and oxo-AEGG linkers would
not need to be tested.
Scheme 1. Syntheses of peptide linkers. Reagents. Coupling: HBTU, HOBt, DIEA in
DMF, deprotection: 20% piperidine in DMF, cleavage: AcOH/CF3CH2OH/CH2Cl2 (2/2/6,
v/v/v).
We then studied galactosylation of these substrates by
b1,4-
galactosyltransferase (Scheme 3). When the reactions of 2 mM of
substrate were performed with no additives, 14 (oxo-GEFG-gl)
glycosylated in 63% for 10 h, but others proceeded only 36% at the
most. Even though the reactions continued for 24 h and 48 h, only
14 (oxo-GEFG-gl) and 10 (oxo-AEAG-gl) were completed, succes-
sively, and 9 (oxo-FEFG-gl) was not galactosylated at all.
When 0.1% of Triton X-100 was added to the reactions, reactivity
of 10 (oxo-AEAG-gl) and 11 (oxo-GEGG-gl) improved dramatically;
the substrates with a single Phe (F) improved only to a small degree.
9 (oxo-FEFG-gl) did not become an enzyme substrate at all (data
was not shown), although 1% of Triton X-100 was somewhat
helpful.10 Triton X-100 is a nonionic surfactant, and from these
experiments, 0.1% Triton X-100 was found to be effective for in-
creasing the solubility of the substrates with peptide linkers con-
sisting of less hydrophobic amino acids, i.e., Gly (G) and Ala (A), but
not for those with more hydrophobic amino acids, i.e., Phe (F).
In order to clarify the effects of adding cyclodextrins, we first
added 70 mM of
improved on those with no additives, but the reactions with 0.1% of
Triton X-100 added gave better results. However, when 10 mM of
a-CD. The subsequent reactions were somewhat
Scheme 2. Conjugation between 12-azidododecyl 2-acetoamido-2-deoxy-
pyranoside and the peptide linkers (1e7).
b-D-gluco-
b
-
CD was added to the reactions, all of the reactions improved. Cy-
clodextrins are able to form hosteguest complexes with hydro-
Therefore,
substrates like a phase transfer catalyst. Because of the solubility of
-CD itself, we could not test this by adding a higher concentration
of -CD.
Next, we tested the effects of -CD. The addition of 70 mM of g-
b-CD may have an ability to increase the solubility of
phobic molecules. From these experiments,
b
-CD was found to
b
involve side chains of Ala (A) and Phe (F), namely CH3e and
C6H5CH2e, which then increased the solubility of the substrates,
b
g
resulting
in
accelerated
galactosylation
by
b1,4-
CD resulted in all seven reactions completing within 12 h. It was
galactosyltransferase. We also observed that the reaction for 11
especially effective for 9 (oxo-FEFG-gl), but it was less effective for
(oxo-GEGG-gl) was improved, but Gly (G) has no side chain.
10 (oxo-AEAG-gl) than for the addition of b-CD. This may be

3148
I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
Scheme 3. Syntheses of lactosamine derivatives by
b
1,4-galactosyltransferase. Graphs 1: no additives, 2: with 0.1% Triton X-100, 3: with 70 mM a-CD, 4: with 10 mM b-CD, 5: with
10 mM g-CD, 6: with 70 mM g-CD. Substrates 9: square, 10: white circle, 11: triangle, 12: cross, 13: plus, 14: gray circle, 15: white triangle.
because
C6H5CH2e, but not to the side chain of Ala (A), CH3e, while it was
the inverse for -CD. All reactions became slower after the addition
of 10 mM of -CD than after 70 mM -CD, and 9 (oxo-FEFG-gl) did
not react at all. This indicated that -CD worked concentration-
dependently. In addition, comparing the addition of 10 mM
to 10 mM -CD for 11 (oxo-GEGG-gl) led to the conclusion that
g
-CD was well matched to the bulky side chain of Phe (F),
functional groups. With the results of the previous report,10 the
higher concentration of 9 required the more -CD to perform this
enzymatic galactosylation, we concluded that cyclodextrins should
g
b
g
g
work mainly in a hosteguest system in this enzymatic gal-
g
actosylation. From the result of 10 mM
CD worked well for methyl group. From comparison of reactions of
12 and 13, methyl group of sugar side might well coordinate with
CD. In addition, -CD could work also to benzyl groups as reactions
b-CD (Scheme 3, graph 4), b-
b
b
-CD
-CD
g
b-
had a stronger ability to solve the substrates than
g-CD.
b
of 14, 15, and 9 have been accelerated. Same as the case of methyl
3. Conclusions
group, benzyl group of sugar side might well recognized that of N-
terminal side. Regarding
and 15 with 10 mM -CD were similar and 14 had been more ac-
celerated than 15 by 70 mM -CD indicated that benzyl groups of
sugar side might be well involved with -CD. The reactions for 9
and 13 were similar in either with 10 mM or with 70 mM -CD, thus
-CD to Ala (A) and Phe (F) on N-terminal side would be
g-CD, the result that the reactivities for 14
One way to study how cyclodextrins affect substrates at the
molecular level is to use NMR. Observation of chemical shift
changes of the substrates with or without cyclodextrins could
clearly show whether cyclodextrins form hosteguest complexes
with peptide side chains. These experiments have been difficult,
however, due to the poor solubility of the substrates.
g
g
g
g
affinity of
g
same.
Nevertheless this background, we successfully showed that the
effectiveness of cyclodextrins is highly dependent upon side chains
From a synthetic chemistry perspective, discussing the enzy-
matic reaction by V0 and Km in this paper is not appropriate,

I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
3149
because we aim to use enzymes as reaction catalyses and to have
products in higher yields. We faced many cases in which despite
two reaction conditions having the same V0, one could give
a quantitative yield while another had the reaction stop. As seen in
Scheme 3, using either b-CD or g-CD gave quantitative yield within
10 h for these galactosylations, which was desirable from a syn-
thetic chemistry standpoint.
2.55 mmol), and HOBt (345 mg, 2.55 mmol) in DMF (5 ml) was
added DIEA (888 l, 5.10 mmol) at rt. The reaction mixture was
shaken for 1 h at rt, and the resin was washed with DMF and CH2Cl2.
The resulting resin in 20% piperidine/DMF (5 ml) was shaken for
3 min at 50 ꢀC under microwave irradiation and washed with DMF
and CH2Cl2. To a mixture of the resulting resin, 5-oxohexanoic acid
m
(304
ml, 2.55 mmol), HBTU (967 mg, 2.55 mmol), and HOBt (345 mg,
2.55 mmol) in DMF (5 ml) was added DIEA (888
ml, 5.10 mmol) at rt.
4. Experiments
4.1. General
The reaction mixture was shaken for 10 min at 50 ꢀC under mi-
crowave irradiation and the resulting resin was washed with DMF
and CH2Cl2, and dried. A part of the resulting resin was treated with
AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/v, 10 ml) at rt, filtered, and
washed with the same cocktail in three times. The combined fil-
trates were concentrated by streaming of nitrogen gas and the
resulting residue was purified by RP-HPLC [column: Inertsil ODS-3
Polymer supported peptide syntheses with microwave irradia-
tion were performed in the EYELA MWS-1000, and reaction tem-
peratures were monitored by an optional equipped fiber optic
probe, which was directly inserted into the reaction medium. All
reagents were used as purchased without further purification and
were purchased from Wako Pure Chemical Industries, Ltd. (Japan),
Nacalai tesque, Inc. (Japan), Dojindo Laboratories (Japan) or Sig-
maeAldrich Chemical Co. (USA). Reagents for peptide derivatives
were also purchased from Watanabe Chimical Industries, Ltd. (Ja-
pan), Peptide Institute, Inc. (Japan) or Novabiochem, Merck group
(f
20ꢁ250 mm); column oven temperature: 40 ꢀC; flow rate: 5 ml/
min, detection: 220 nm UV; eluent A: 25 mM ammonium acetate
buffer (pH 5.8), eluent B: acetonitrile containing 10%, eluent C: H2O
containing 0.1% TFA and eluent D: acetonitrile containing 0.1% TFA]
Elution conditions were shown individually.
4.2.1. N-(5-Oxohexanoyl)-L-alanyl-L-(g-benzyl)glutamyl-L-alanyl
(Germany).
UDP-galactose
(UDP-Gal)
and
b
1,4-
glycine (2). Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-Ala-OH
(802 mg, 2.58 mmol) and Fmoc-Ala-OH (816 mg, 2.62 mmol),
successively. A part (657 mg) of the dried resulting resin (1.415 g)
was treated with AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/v, 10 ml) for
1.5 h at rt. RP-HPLC [eluent B in eluent A, gradient increase from 10%
to 40% over 50 min] gave 2 (168 mg, 78%) as an amorphous. 1H NMR
galactosyltransferase (GalT) were purchased from Yamasa Co. (Ja-
pan) and SigmaeAldrich Chemical Co. (USA), respectively. Re-
actions were monitored by TLC, which was performed with
0.25 mm precoated silica gel 60F254 on glass from Merck (Darm-
stadt, Germany). Compounds were detected by dipping the TLC
plates in an ethanolic solution of phosphomolybdic acid (5% v/v) or
spraying ninhydrin and heating. Silica gel N60 (40e50 nm) from
Kanto Chemical (Tokyo, Japan) was used for silica gel chromatog-
raphy. All NMR data are reported in parts per million downfield
shift from tetramethylsilane. 1H NMR spectra and 13C NMR spectra
were routinely recorded at 400 MHz and 100 MHz on a BRUKER
AVANCE 400 spectrometer at 300 K, respectively, and chemical
shifts were expressed relative to methyl proton of tetramethylsi-
(400 MHz, (CD3)2S]O): d
8.04e7.99 (3H, m, Ala1-NH, Glu2-NH, and
Ala3-NH), 7.73 (1H, t, J¼4.4 Hz, Gly4-NH), 7.39e7.29 (5H, m, aro-
matic-H), 5.08 (2H, s, PhCH2Oe), 4.30e4.20 (3H, m, Ala1-H , Glu2-
a
H
a
, and Ala3-H
a
), 3.51 (1H, dd, J¼16.6, 4.4 Hz, Gly4-H
a
), 3.48 (1H,
dd, J¼16.6, 4.4 Hz, Gly4-H
a g and
), 2.41e2.38 (4H, m, Glu2-H
eCH2C(]O)CH3), 2.08 (2H, t, J¼7.2 Hz, eNHC(]O)CH2e), 2.04 (3H,
s, eCH2C(]O)CH3), 1.97 (1H, m, Glu2-H ), 1.81 (1H, m, Glu2-H
b b),
1.64 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e), 1.20 (3H, d,
lane (
d
0.00 ppm). High-resolution ESI-mass data were recorded by
J¼7.2 Hz, Ala1-H
b
or Ala3-H
b
), 1.18 (3H, d, J¼7.2 Hz, Ala1-H
b
or Ala3-
mass spectrometry services at the Center for Instrumental Analysis,
Hokkaido University. In lactosamine disaccharide cases, proton
positions of glucose and galactose parts were shown by simple
positional numbers and numbers with single prime (0), respectively.
In peptide linkers, amino acid moieties were numbered from N-
terminal.
Hb d 208.17, 172.46, 172.29,
). 13C NMR (100 MHz, (CD3)2S]O):
171.69, 171.66, 170.75, 170.37, 136.20, 128.40, 127.92, 127.85, 65.41,
51.52, 48.17, 48.06, 41.92, 34.07, 29.95, 29.69, 27.25, 19.30, 18.24,
17.84. ESI-HRMS: C26H35N4O9 [MꢂH]ꢂ calcd (m/z) 547.24095, found
(m/z) 547.24259.
4.2.2. N-(5-Oxohexanoyl)-glycinyl-L-(g-benzyl)glutamyl-glycinyl
4.2. Syntheses peptide linkers
glycine (3). Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-Gly-OH
(754 mg, 2.54 mmol) and Fmoc-Gly-OH (783 mg, 2.63 mmol),
successively. A part (784 mg) of the dried resulting resin (1.304 g)
was treated with AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/v, 10 ml) for
3 h at rt. RP-HPLC [eluent D in eluent C, gradient increase from 10%
to 30% over 60 min] gave 3 (125 mg, 47%) as an amorphous. 1H NMR
All the reactions were carried out in a 20 ml LibraTubeÒ. To
a mixture of swelled H-Gly-Trt(2-Cl) resin (0.85 mmol/g, 1.00 g,
0.85 mmol), which was pretreated by DMF for a period of 2 h,
Fmoc-Xxx-OH, HBTU (O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethy-
luronium hexafluorophosphate) (967 mg, 2.55 mmol), and HOBt
(1-hydroxybenzotriazole) (345 mg, 2.55 mmol) in DMF (5.5 ml) was
(400 MHz, (CD3)2S]O):
d 12.56 (1H, br, eC(]O)OH), 8.24 (1H, t,
J¼5.6 Hz, Gly1-NH, Gly3-NH or Gly4-NH), 8.11 (1H, t, J¼5.6 Hz, Gly1-
NH, Gly3-NH or Gly4-NH), 8.05e8.02 (2H, m, Glu2-NH and Gly1-NH,
Gly3-NH or Gly4-NH), 7.40e7.31 (5H, m, aromatic-H), 5.09 (2H, s,
added DIEA (N,N-diisopropylethylamine) (888 ml, 5.10 mmol) at rt.
The reaction mixture was shaken for 10 min at 50 ꢀC under mi-
crowave irradiation, and the resin was washed with DMF and
CH2Cl2. The resulting resin in 20% piperidine/DMF (5 ml) and
shaken for 3 min at 50 ꢀC under microwave irradiation and washed
with DMF and CH2Cl2.
PhCH2Oe), 4.32 (1H, dt, J¼7.6, 6.0 Hz, Glu2-H
a
), 3.82e3.71 (6H, m,
Gly1-H , Gly3-H , and Gly4-H ), 2.44e2.39 (4H, m, Glu2-H
a a a g and
eCH2C(]O)CH3), 2.11 (2H, t, J¼7.2 Hz, eNHC(]O)CH2e), 2.05 (3H,
s, eCH2C(]O)CH3), 1.99 (1H, m, Glu2-H ), 1.82 (1H, m, Glu2-H
b b),
To a mixture of the resulting resin, Fmoc-Glu(OBn)-OH (1.18 g,
2.55 mmol), HBTU (967 mg, 2.55 mmol), and HOBt (345 mg,
1.67 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e). 13C NMR
(100 MHz, (CD3)2S]O):
d 208.16, 172.27, 172.25, 171.15, 171.06,
2.55 mmol) in DMF (5 ml) was added DIEA (888
m
l, 5.10 mmol) at rt.
169.19, 168.94, 136.16, 128.39, 127.94, 127.88, 65.44, 51.73, 42.00,
41.90, 41.69, 40.54, 34.12, 29.88, 29.68, 27.15, 19.29. ESI-HRMS:
C
The reaction mixture was shaken for 10 min at 50 ꢀC under mi-
crowave irradiation, and the resin was washed with DMF and
CH2Cl2. Then, the resulting resin in 20% piperidine/DMF (5 ml) was
shaken for 2 min at rt, and washed with DMF and CH2Cl2. To
a mixture of the resulting resin, Fmoc-Yyy-OH, HBTU (967 mg,
24H31N4O9 [MꢂH]ꢂ calcd (m/z) 519.20000, found (m/z) 519.21090.
4.2.3. N-(5-Oxohexanoyl)-
L-alanyl-L-(g-benzyl)glutamyl-L-phenyl-
alanyl glycine (4). Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-

3150
I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
Phe-OH (977 mg, 2.52 mmol) and Fmoc-Ala-OH (799 mg,
2.57 mmol), successively. A part (862 mg) of the dried resulting
resin (1.438 g) was treated with AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/
v, 10 ml) for 3 h at rt. RP-HPLC [eluent D in eluent C, gradient in-
crease from 20% to 50% over 60 min] gave 4 (199 mg, 63%) as
13C NMR (100 MHz, (CD3)2S]O):
d 208.07, 172.03, 172.12, 171.14,
170.90, 170.55, 169.19, 137.65, 136.10, 129.05, 128.33, 127.94, 127.89,
127.82, 126.14, 65.35, 53.65, 51.77, 41.98, 41.85, 40.58, 37.23, 34.02,
29.73, 29.60, 27.10, 19.20. ESI-HRMS: C31H37N4O9 [MꢂH]ꢂ calcd (m/
z) 609.25660, found (m/z) 609.25813.
an amorphous. 1H NMR (400 MHz, (CD3)2S]O):
d 12.57 (1H, br,
eC(]O)OH), 8.32 (1H, t, J¼5.8 Hz, Gly4-NH), 7.99 (1H, d, J¼6.8 Hz,
Ala1-NH), 7.89 (1H, d, J¼8.0 Hz, Glu2-NH), 7.89 (1H, d, J¼8.0 Hz,
Phe3-NH), 7.41e7.11 (10H, m, aromatic-H), 5.08 (2H, s, PhCH2Oe),
4.2.6. N-(5-Oxohexanoyl)-L-phenylalanyl-L-(g-benzyl)glutamyl-gly-
cinyl glycine (7). 7 was prepared in a half scale of other peptides
(same scale with 6). Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-
Gly-OH (398 mg, 1.34 mmol) and Fmoc-Phe-OH (523 mg,
1.35 mmol), successively. The resulting dried resulting resin
(737 mg) was treated with AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/v,
10 ml) for 1.5 h at rt. RP-HPLC [eluent D in eluent C, gradient in-
crease from 20% to 40% over 60 min] gave 7 (219 mg, 83%) as an
4.54 (1H, ddd, J¼9.6, 8.0, 4.4 Hz, Phe3-H
a
), 4.26e4.17 (2H, m, Glu2-
Ha
and Ala1-H
a
), 3.80 (1H, dd, J¼17.6, 5.8 Hz, Gly4-H
a), 3.74 (1H, dd,
J¼17.6, 5.8 Hz, Gly4-H
a
), 3.05 (1H, dd, J¼14.0, 4.4 Hz, Phe3-H
b), 2.79
(1H, dd, J¼14.0, 9.6 Hz, Phe3-H
), 2.39 (2H, t, J¼7.4 Hz, eCH2C(]O)
b
CH3), 2.34e2.19 (2H, m, Glu2-H
g), 2.07 (2H, t, J¼7.4 Hz, eNHC(]O)
CH2e), 2.03 (3H, s, eCH2C(]O)CH3), 1.86 (1H, m, Glu2-H
b), 1.74 (1H,
amorphous. 1H NMR (400 MHz, (CD3)2S]O):
d 8.15e8.13 (2H, m,
m, Glu2-H
b
), 1.64 (2H, qui, J¼7.4 Hz, eC(]O)CH2CH2CH2C(]O)e),
Gly3-NH or Gly4-NH, and Glu2-NH), 8.10e8.05 (2H, m, Gly3-NH or
Gly4-NH, and Phe1-NH), 7.40e7.16 (10H, m, aromatic-H), 5.10 (2H, s,
1.14 (3H, d, J¼6.8 Hz, Ala1-H
b
). 13C NMR (100 MHz, (CD3)2S]O):
d
208.13, 172.45, 172.22, 171.71, 171.15, 170.97, 170.54, 137.55, 136.18,
PhCH2Oe), 4.55 (1H, ddd, J¼10.0, 8.0, 4.4 Hz, Phe1-H
a), 4.32 (1H, dt,
129.14, 128.40, 127.96, 127.86, 126.20, 65.39, 53.46, 51.80, 48.11,
41.91, 40.62, 37.53, 34.06, 29.83, 29.67, 27.23, 19.30, 17.73. ESI-
HRMS: C32H39N4O9 [MꢂH]ꢂ calcd (m/z) 623.27225, found (m/z)
623.27394.
J¼6.0, 7.6 Hz, Glu2-H
a
), 3.79 (2H, d, J¼5.6 Hz, Gly3-NH or Gly4-NH),
3.75 (2H, d, J¼5.6 Hz, Gly3-NH or Gly4-NH), 3.03 (1H, dd, J¼14.0,
4.0 Hz, Phe1-H
b
), 2.75 (1H, dd, J¼14.0, 10.6 Hz, Phe1-H
b), 2.41 (2H, t,
J¼7.6 Hz, Glu2-H
), 2.21 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3),
g
2.06e1.95 (3H, m, eNHC(]O)CH2e and Glu2-H
b
), 1.99 (3H, s,
), 1.54 (2H, qui, J¼7.2 Hz,
eC(]O)CH2CH2CH2C(]O)e). 13C NMR (100 MHz, (CD3)2S]O):
208.53, 172.29, 171.87, 171.64, 171.11, 171.07, 168.97, 130.08, 136.21,
4.2.4. N-(5-Oxohexanoyl)-
L
-phenylalanyl-
L
-(
g
-benzyl)glutamyl-
L
-al-
eCH2C(]O)CH3), 1.85 (1H, m, Glu2-H
b
anyl glycine (5). Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-Ala-
OH (783 mg, 2.51 mmol) and Fmoc-Phe-OH (994 mg, 2.57 mmol),
successively. A part (900 mg) of the dried resulting resin (1.387 g)
was treated with AcOH/CF3CH2OH/CH2Cl2 (2:2:6 v/v/v, 10 ml) for
3 h at rt. RP-HPLC [eluent D in eluent C, gradient increase from 20%
to 50% over 60 min] gave 5 (195 mg, 57%) as an amorphous. 1H NMR
d
129.16, 128.44, 128.01, 127.99, 127.92, 126.20, 65.49, 53.81, 51.87,
41.76, 41.70, 40.60, 37.20, 34.17, 29.90, 29.69, 27.18,19.27. ESI-HRMS:
C
31H37N4O9 [MꢂH]ꢂ calcd (m/z) 609.25660, found (m/z) 609.25836.
(400 MHz, (CD3)2S]O):
d
12.54 (1H, br, eC(]O)OH), 8.18 (1H, t,
4.3. Syntheses of peptide-linked glucosaminide substrates
J¼5.8 Hz, Gly4-NH), 8.08 (1H, d, J¼8.0 Hz, Glu2-NH), 8.04 (1H, d,
J¼8.0 Hz, Phe1-NH), 8.02 (1H, d, J¼7.2 Hz, Ala3-NH), 7.40e7.15 (10H,
4.3.1. 12-[N-(5-Oxohexanoyl)-
L
-phenylalanyl-
L
-glutamyl-
L-phenyl-
m, aromatic-H), 5.09 (2H, s, PhCH2Oe), 4.53 (1H, m, Phe1-H
a
),
alanyl-glycinyl]-aminododecyl 2-acetamido-2-deoxy-b-D-glucopyr-
4.33e4.26 (2H, m, Glu2-H
a
and Ala3-H
a
), 3.80 (1H, dd, J¼17.6,
anoside (9).10 To the solution of 1 (59 mg, 0.13 mmol), HOBt (20 mg,
0.15 mmol), and WSC (1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide hydrochloride) (29 mg, 0.15 mmol) in DMF (3 ml) were
5.8 Hz, Gly4-H
a
), 3.71 (1H, dd, J¼17.6, 5.8 Hz, Gly4-H
a), 3.00 (1H, dd,
J¼13.6, 4.0 Hz, Phe1-H
b
), 2.72 (1H, dd, J¼13.6, 10.4 Hz, Phe1-H
b),
2.40 (2H, t, J¼8.0 Hz, Glu2-H
g
), 2.20 (2H, t, J¼7.2 Hz, eCH2C(]O)
added 12-aminododecyl 2-acetamido-2-deoxy-
b-
D-glucopyrano-
CH3), 2.02e1.91 (3H, m, eNHC(]O)CH2e and Glu2-H
b
), 1.98 (3H, s,
side acetic acid salt (59 mg, 0.13 mmol) and triethylamine (18 m
l,
eCH2C(]O)CH3), 1.83 (1H, m, Glu2-H
b
), 1.53 (2H, qui, J¼7.2 Hz,
0.13 mmol) successively. The reaction mixture was stirred for 1 h at
rt and directly purified by SiO2 column chromatography. Eluent of
EtOAc/EtOH/H2O (12:2:1) gave assumed benzyl protected 9. 10%
PdeC (30 mg) was added to a solution of the resulting material in
dioxane/water (1:1 v/v, 10 ml), and the reaction mixture was stirred
under hydrogen for 1 h at 50 ꢀC. After removal of PdeC by Celite
filtration, the filtrate was concentrated, and purified by the pre-
eC(]O)CH2CH2CH2C(]O)e), 1.26 (3H, d, J¼7.2 Hz, Ala1-H
b
). 13C
NMR (100 MHz, (CD3)2S]O): d 207.91,172.25,172.20,171.66, 171.36,
170.95, 170.22, 137.93, 136.10, 129.02, 128.32, 127.87, 127.79, 126.07,
65.35, 53.62, 51.50, 47.87, 41.59, 40.46, 37.17, 34.06, 29.86, 29.58,
27.21, 19.14, 18.13. ESI-HRMS: C32H39N4O9 [MꢂH]ꢂ calcd (m/z)
623.27225, found (m/z) 623.27410.
parative RP-HPLC [column: Inertsil ODS-3 (
f
20ꢁ250 mm); eluent
4.2.5. N-(5-Oxohexanoyl)-glycinyl-
L
-(
g
-benzyl)glutamyl-
L
-phenyl-
A: H2O containing 0.1% TFA, eluent B: acetonitrile containing 0.1%
TFA; eluate B gradient increase from 30% to 60% over 50 min; col-
umn oven temperature: 40 ꢀC; flow rate: 5 ml/min, detection:
220 nm UV] to give 910 (109 mg, 87%) as an amorphous.
alanyl glycine (6). 6 was prepared in a half scale of other peptides.
Fmoc-Xxx-OH and Fmoc-Yyy-OH were Fmoc-Phe-OH (488 mg,
1.26 mmol) and Fmoc-Gly-OH (372 mg, 1.25 mmol), successively.
The resulting dried resulting resin (851 mg) was treated with AcOH/
CF3CH2OH/CH2Cl2 (2:2:6 v/v/v, 10 ml) for 1 h at rt. RP-HPLC [eluent
D in eluent C, gradient increase from 20% to 50% over 60 min] gave 6
(208 mg, 85%) as an amorphous. 1H NMR (400 MHz, (CD3)2S]O):
4.3.2. 12-[N-(5-Oxohexanoyl)-
aminododecyl 2-acetamido-2-deoxy-
the solution of 2 (83 mg, 0.15 mmol), HOBt (24 mg, 0.17 mmol), and
WSC (33 mg, 0.17 mmol) in DMF (3.5 ml) were added 12-
aminododecyl 2-acetamido-2-deoxy-
acid salt (70 mg, 0.15 mmol) and triethylamine (24
successively. The reaction mixture was stirred for 4 h at rt and di-
rectly purified by SiO2 column chromatography. Eluent of EtOAc/
EtOH/H2O (9:2:1) gave benzyl protected 10. 10% PdeC (20 mg) was
added to a solution of the resulting material in dioxane/water (1:1
v/v,10 ml), and the reaction mixture was stirred under hydrogen for
2 h at 50 ꢀC. After removal of PdeC by Celite filtration, the filtrate
was concentrated, and purified by the preparative RP-HPLC [col-
L
-alanyl-
L
-glutamyl-
L-alanyl-glycinyl]-
b-D-glucopyranoside (10). To
d
12.55 (1H, br, eC(]O)OH), 8.23 (1H, m, Gly1-NH or Gly4-NH), 8.06
(1H, m, Gly1-NH or Gly4-NH), 8.02 (1H, d, J¼8.4 Hz, Phe3-NH), 7.97
b
-D
-glucopyranoside acetic
l, 0.17 mmol)
(1H, d, J¼7.6 Hz, Glu2-NH), 7.40e7.14 (10H, m, aromatic-H), 5.08
m
(2H, s, PhCH2Oe), 4.53 (1H, m, Phe3-H
a
), 4.22 (1H, m, Glu2-H
a
),
3.80 (1H, dd, J¼17.6, 5.8 Hz, Gly1-H
a
or Gly4-H
a), 3.75 (1H, dd,
J¼17.6, 5.8 Hz, Gly1-H
a
or Gly4-H
a
), 3.68 (2H, d, J¼5.2 Hz, Gly1-H
a
or Gly4-H
a
), 3.07 (1H, dd, J¼13.6, 4.0 Hz, Phe3-H
b), 2.81 (1H, dd,
J¼13.6, 10.0 Hz, Phe3-H
), 2.42 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3),
b
2.27 (2H, t, J¼7.8 Hz, Glu2-H
g
), 2.12 (2H, t, J¼7.2 Hz, eNHC(]O)
),1.72 (1H,
CH2e), 2.05 (3H, s, eCH2C(]O)CH3),1.86 (1H, m, Glu2-H
b
m, Glu2-H
b
), 1.67 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e).
umn: Inertsil ODS-3 (
f20ꢁ250 mm); column oven temperature:

I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
3151
40 ꢀC; flow rate: 5 ml/min, detection: 220 nm UV; eluate D in eluent
C gradient increase from 10% to 40% over 60 min] to give 10 (70 mg,
0.29 mmol), and WSC (55 mg, 0.29 mmol) in DMF (4 ml) were
added 12-aminododecyl 2-acetamido-2-deoxy- -glucopyrano-
side acetic acid salt (112 mg, 0.24 mmol) and triethylamine (40 l,
b-D
55%) as an amorphous. 1H NMR (400 MHz, (CD3)2S]O):
d
12.07
m
(1H, br, eC(]O)OH), 8.12 (1H, t, J¼6.0 Hz, Gly4-NH), 8.03 (1H, d,
J¼7.2 Hz, Ala1-NH or Ala3-NH), 8.01 (1H, d, J¼6.8 Hz, Ala1-NH or
Ala3-NH), 7.96 (1H, d, J¼8.0 Hz, Glu2-NH), 7.65e7.61 (2H, m,
eNHC(]O)CH3 and eCH2CH2NHC(]O)e), 4.94 (1H, br, OH-4), 4.86
(1H, br, OH-3), 4.48 (1H, br, OH-6), 4.25 (1H, d, J¼8.0 Hz, H-1),
0.29 mmol) successively. The reaction mixture was stirred for 1 h at
rt and directly purified by SiO2 column chromatography. Eluent of
EtOAc/EtOH/H2O (9:2:1) gave assumed benzyl protected 12. 10%
PdeC (40 mg) was added to a solution of the resulting material in
dioxane/water (1:1 v/v, 10 ml), and the reaction mixture was stirred
under hydrogen for 1 h at 50 ꢀC. After removal of PdeC by Celite
filtration, the filtrate was concentrated, and purified by the pre-
4.24e4.17 (3H, m, Ala1-H
a
, Glu2-H
a
, and Ala3-H
a
), 3.73e3.56 (4H,
m, eOCH2CH2e, H-6b and Gly4-H
a), 3.46e3.25 (4H, m, H-6a, H-2,
eOCH2CH2e, and H-3), 3.06e3.01 (4H, m, eCH2CH2NHe, H-5 and
H-4), 2.42 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3), 2.24 (2H, t, J¼7.6 Hz,
parative RP-HPLC [column: Inertsil ODS-3 (
f
20ꢁ250 mm); column
oven temperature: 40 ꢀC; flow rate: 5 ml/min, detection: 220 nm
UV; eluate D in eluent C gradient increase from 10% to 50% over
60 min] to give 12 (171 mg, 76%) as an amorphous. 1H NMR
Glu2-H
g
), 2.10 (2H, t, J¼7.2 Hz, eNHC(]O)CH2e), 2.06 (3H, s,
eCH2C(]O)CH3), 1.90 (1H, m, Glu2-H
b), 1.78 (3H, s, eNHC(]O)
CH3), 1.75 (1H, m, Glu2-H
b
), 1.66 (2H, qui, J¼7.2 Hz, eC(]O)
(400 MHz, (CD3)2S]O): d 12.05 (1H, br, eC(]O)OH), 8.22 (1H, t,
CH2CH2CH2C(]O)e), 1.43e1.38 (4H, m, eOCH2CH2e and
eCH2CH2NHe), 1.26e1.21 (16H, m, eOCH2CH2(CH2)8e), 1.22 (3H, d,
J¼5.6 Hz, Gly4-NH), 8.02 (1H, d, J¼7.2 Hz, Ala1-NH), 7.98 (1H, d,
J¼7.6 Hz, Phe3-NH), 7.94 (1H, d, J¼7.6 Hz, Glu2-NH), 7.64
(1H, d, J¼8.8 Hz, eNHC(]O)CH3), 7.57 (1H, t, J¼5.6 Hz,
eCH2CH2NHC(]O)e), 7.26e7.16 (5H, m, aromatic-H), 4.94 (1H, br,
J¼6.8 Hz, Ala1-H
b
or Ala3-H
b
), 1.19 (3H, d, J¼7.2 Hz, Ala1-H
b
or Ala3-
Hb d 208.05, 173.90, 172.53,
). 13C NMR (100 MHz, (CD3)2S]O):
172.20, 171.82, 170.92, 168.77, 168.19, 100.91, 76.86, 74.17, 70.57,
68.13, 60.97, 55.35, 51.66, 48.48, 48.25, 42.00, 41.85, 38.42, 33.97,
29.94, 29.61, 29.03, 28.96, 28.93, 28.74, 28.66, 27.00, 26.27, 25.35,
22.92, 19.19, 17.66, 17.57. ESI-HRMS: C39H67N6O14 [MꢂH]ꢂ calcd (m/
z) 843.47207, found (m/z) 843.47382.
OH-4), 4.85 (1H, br, OH-3), 4.50e4.42 (2H, m, OH-6 and Phe3-H
a
a
),
),
4.25 (1H, d, J¼8.4 Hz, H-1), 4.23e4.14 (2H, m, Ala1-H
a
and Glu2-H
3.73e3.66 (3H, m, H-6b, Gly4-H
a
and eOCH2CH2e), 3.58 (1H, dd,
J¼16.4, 5.6 Hz, Gly4-H
a), 3.44 (1H, m, H-6a), 3.40e3.24 (3H, m, H-2,
eOCH2CH2e and H-3), 3.07e3.01 (5H, m, eCH2CH2NHe, Phe3-H
b,
H-5 and H-4), 2.83 (1H, dd, J¼13.6, 9.0 Hz, Phe3-H
b), 2.41 (2H, t,
4.3.3. 12-[N-(5-Oxohexanoyl)-glycinyl-
aminododecyl 2-acetamido-2-deoxy-
L
-glutamyl-glycinyl-glycinyl]-
-glucopyranoside (11). To
J¼7.2 Hz, eCH2C(]O)CH3), 2.18e2.12 (2H, m, Glu2-H
g), 2.09 (2H, t,
b-
D
J¼7.2 Hz, eNHC(]O)CH2e), 2.05 (3H, s, eCH2C(]O)CH3), 1.82 (1H,
the solution of 3 (100 mg, 0.19 mmol), HOBt (31 mg, 0.23 mmol),
m, Glu2-H ), 1.78 (3H, s, eNHC(]O)CH3), 1.69 (1H, m, Glu2-H
b b),
and WSC (44 mg, 0.23 mmol) in DMF (4 ml) were added 12-
1.66 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e), 1.42e1.38
aminododecyl 2-acetamido-2-deoxy-b-D-glucopyranoside acetic
(4H, m, eOCH2CH2e and eCH2CH2NHe), 1.27e1.21 (16H, m,
acid salt (89 mg, 0.19 mmol) and triethylamine (32
ml, 0.23 mmol)
eOCH2CH2(CH2)8e), 1.15 (3H, d, J¼7.2 Hz, Ala1-H
b
). 13C NMR
successively. The reaction mixture was stirred for 2.5 h at rt and
directly purified by SiO2 column chromatography. Eluent of EtOAc/
EtOH/H2O (6:2:1) gave assumed benzyl protected 11. 10% PdeC
(30 mg) was added to a solution of the resulting material in di-
oxane/water (1:1 v/v, 10 ml), and the reaction mixture was stirred
under hydrogen for 1 h at 50 ꢀC. After removal of PdeC by Celite
filtration, the filtrate was concentrated, and purified by the pre-
(100 MHz, (CD3)2S]O): d 208.03, 173.86, 172.53, 171.81, 171.04,
170.94, 168.77, 168.12, 137.50, 129.00, 127.94, 126.17, 100.91, 76.86,
74.17, 70.57, 68.13, 60.97, 55.36, 54.00, 51.89, 48.16, 42.06, 41.85,
38.43, 37.02, 33.99, 29.84, 29.60, 29.03, 28.95, 28.74, 28.68, 27.01,
26.27, 25.35, 22.92, 19.21, 17.62. ESI-HRMS: C45H71N6O14 [MꢂH]ꢂ
calcd (m/z) 919.50337, found (m/z) 919.50643.
parative RP-HPLC [column: Inertsil ODS-3 (
f
20ꢁ250 mm); column
4.3.5. 12-[N-(5-Oxohexanoyl)- -glutamyl-
glycinyl]-aminododecyl 2-acetamido-2-deoxy-b-D-glucopyranoside
(13). To the solution of 5 (150 mg, 0.24 mmol), HOBt (39 mg,
0.29 mmol), and WSC (55 mg, 0.29 mmol) in DMF (4 ml) were
L
-phenylalanyl-
L
L-alanyl-
oven temperature: 40 ꢀC; flow rate: 5 ml/min, detection: 220 nm
UV; eluate D in eluent C gradient increase from 10% to 30% over
60 min] to give 11 (80 mg, 51%) as an amorphous. 1H NMR
(400 MHz, (CD3)2S]O):
d
12.08 (1H, s, eC(]O)OH), 8.22 (1H, t,
added 12-aminododecyl 2-acetamido-2-deoxy-
side acetic acid salt (111 mg, 0.24 mmol) and triethylamine (40
b
-
D
-glucopyrano-
J¼5.6 Hz, Gly1-NH, Gly3-NH or Gly4-NH), 8.05e8.01 (3H, m, Glu2-NH
and Gly1-NH, Gly3-NH or Gly4-NH), 7.70 (1H, t, J¼5.6 Hz,
eCH2CH2NHC(]O)e), 7.64 (1H, d, J¼8.8 Hz, eNHC(]O)CH3), 4.93
(1H, d, J¼3.2 Hz, OH-4), 4.85 (1H, d, J¼4.8 Hz, OH-3), 4.48 (1H, t,
ml,
0.29 mmol) successively. The reaction mixture was stirred for 1 h at
rt and directly purified by SiO2 column chromatography. Eluent of
EtOAc/EtOH/H2O (9:2:1) containing 1% AcOH gave assumed benzyl
protected 13. 10% PdeC (40 mg) was added to a solution of the
resulting material in dioxane/water (1:1 v/v, 10 ml), and the re-
action mixture was stirred under hydrogen for 1 h at 50 ꢀC. After
removal of PdeC by Celite filtration, the filtrate was concentrated,
and purified by the preparative RP-HPLC [column: Inertsil ODS-3
J¼5.6 Hz, OH-6), 4.28 (1H, t, J¼7.6 Hz, Glu2-H
a
), 4.25 (1H, d,
J¼8.0 Hz, H-1), 3.72e3.65 (6H, m, Gly1-H
a
, Gly3-H
a
, Gly4-H
a,
eOCH2CH2e and H-6b), 3.44 (1H, m, H-6a), 3.40e3.24 (3H, m, H-2,
eOCH2CH2e and H-3), 3.05e3.01 (4H, m, eCH2CH2NHe, H-5 and H-
4), 2.43 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3), 2.25 (2H, t, J¼7.8 Hz, Glu2-
H
g
), 2.11 (2H, t, J¼7.2 Hz, eNHC(]O)CH2e), 2.06 (3H, s, eCH2C(]O)
(
f
20ꢁ250 mm); column oven temperature: 40 ꢀC; flow rate:
CH3),1.93 (1H, m, Glu2-H
Glu2-H
), 1.67 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e),
1.43e1.38 (4H, m, eOCH2CH2e and eCH2CH2NHe), 1.26e1.21 (16H,
m, eOCH2CH2(CH2)8e). 13C NMR (100 MHz, (CD3)2S]O):
208.06,
b
),1.78 (3H, s, eNHC(]O)CH3),1.76 (1H, m,
5 ml/min, detection: 220 nm UV; eluate D in eluent C gradient
b
increase from 10% to 50% over 60 min] to give 13 (96 mg, 43%) as
an amorphous. 1H NMR (400 MHz, (CD3)2S]O):
d 12.09 (1H, s,
d
eC(]O)OH), 8.16 (1H, t, J¼5.6 Hz, Gly4-NH), 8.10 (1H, d, J¼6.4 Hz,
Ala3-NH), 8.10 (1H, d, J¼6.4 Hz, Glu2-NH), 8.04 (1H, d,
J¼8.4 Hz, Phe1-NH), 7.65e7.63 (2H, m, eNHC(]O)CH3 and
eCH2CH2NHC(]O)e), 7.26e7.15 (5H, m, aromatic-H), 4.93 (1H, br,
173.84, 172.19, 171.48, 169.17, 168.79, 168.77, 168.22, 100.91, 76.86,
74.17, 70.57, 68.13, 60.97, 55.35, 51.83, 42.07, 41.93, 41.89, 41.84,
38.44, 34.04, 29.86, 29.62, 29.03, 28.97, 28.95, 28.92, 28.73, 28.65,
27.03, 26.28, 25.35, 22.92, 19.21. ESI-HRMS: C37H63N6O14 [MꢂH]ꢂ
calcd (m/z) 815.44077, found (m/z) 815.44273.
OH-4), 4.85 (1H, br, OH-3), 4.54 (1H, m, Phe1-H
6), 4.28e4.16 (2H, m, Ala3-H and Glu2-H
and eOCH2CH2e), 3.59 (1H,
), 3.46e3.43 (1H, m, H-6a), 3.40e3.25
a
), 4.48 (1H, br, OH-
a
a
), 4.25 (1H, d, J¼8.0 Hz,
H-1), 3.73e3.66 (3H, m, H-6b, Gly4-H
a
4.3.4. 12-[N-(5-Oxohexanoyl)-
glycinyl]-aminododecyl 2-acetamido-2-deoxy-
(12). To the solution of 4 (150 mg, 0.24 mmol), HOBt (39 mg,
L
-alanyl-
L
-glutamyl-
L
-phenylalanyl-
dd, J¼16.8, 5.6 Hz, Gly4-H
a
b
-D-glucopyranoside
(3H, m, H-2, eOCH2CH2e and H-3), 3.06e3.03 (5H, m,
eCH2CH2NHe, Phe1-H
b
, H-5 and H-4), 2.71 (1H, dd, J¼13.2, 11.2 Hz,

3152
I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
Phe1-H
b
), 2.27e2.19 (4H, m, eCH2C(]O)CH3 and Glu2-H
g
), 2.01
of PdeC by Celite filtration, the filtrate was concentrated, and pu-
rified by the preparative RP-HPLC [column: Inertsil ODS-3
(2H, m, eNHC(]O)CH2e), 2.00 (3H, s, eCH2C(]O)CH3), 1.91 (1H,
m, Glu2-H ), 1.79 (1H, m, Glu2-H
b
b), 1.78 (3H, s, eNHC(]O)CH3),
(f
20ꢁ250 mm); column oven temperature: 40 ꢀC; flow rate:
1.54 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e), 1.43e1.38
5 ml/min, detection: 220 nm UV; eluate D in eluent C gradient in-
(4H, m, eOCH2CH2e and eCH2CH2NHe), 1.25e1.19 (19H, m,
crease from 10% to 40% over 60 min] to give 15 (102 mg, 56%) as an
eOCH2CH2(CH2)8e and Ala3-H
b
). 13C NMR (100 MHz, (CD3)2S]O):
amorphous. 1H NMR (400 MHz, (CD3)2S]O):
d 12.08 (1H, s, eC(]
d
207.91, 173.87, 173.23, 171.71, 171.46, 170.88, 168.77, 168.20, 137.95,
O)OH), 8.15 (1H, d, J¼7.6 Hz, Glu2-NH), 8.11 (1H, t, J¼5.6 Hz, Gly3-NH
or Gly4-NH), 8.09e8.05 (2H, m, Gly3-NH or Gly4-NH and Phe1-NH),
7.69 (1H, t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 7.64 (1H, d, J¼8.8 Hz,
eNHC(]O)CH3), 7.26e7.15 (5H, m, aromatic-H), 4.93 (1H, br, OH-4),
129.02, 127.85, 126.06, 100.91, 76.86, 74.17, 70.58, 68.13, 60.97,
55.36, 53.63, 51.68, 48.54, 42.00, 41.60, 38.44, 37.20, 34.06, 29.88,
29.60, 29.03, 28.94, 28.74, 28.65, 27.12, 26.28, 25.35, 22.92, 19.15,
17.57. ESI-HRMS: C45H71N6O14 [MꢂH]ꢂ calcd (m/z) 919.50337,
found (m/z) 919.50523.
4.85 (1H, bd, J¼4.0 Hz, OH-3), 4.55 (1H, m, Phe1-H
a), 4.48 (1H, t,
J¼5.6 Hz, OH-6), 4.27 (1H, m, Glu2-H
), 4.25 (1H, d, J¼8.4 Hz, H-1),
a
3.73e3.66 (2H, m, H-6b and eOCH2CH2e), 3.72 (2H, d, J¼5.6 Hz,
4.3.6. 12-[N-(5-Oxohexanoyl)-glycinyl-
glycinyl]-aminododecyl 2-acetamido-2-deoxy-
(14). To the solution of 6 (130 mg, 0.22 mmol), HOBt (35 mg,
0.26 mmol), and WSC (50 mg, 0.26 mmol) in DMF (4 ml) were
added 12-aminododecyl 2-acetamido-2-deoxy-
side acetic acid salt (100 mg, 0.22 mmol) and triethylamine (36
L
-glutamyl-
L
-phenylalanyl-
Gly3-H
a
or Gly4-H
a
), 3.66 (2H, d, J¼5.6 Hz, Gly3-H
a a),
or Gly4-H
b-
D-glucopyranoside
3.48e3.41 (1H, m, H-6a), 3.40e3.24 (3H, m, H-2, eOCH2CH2e and
H-3), 3.08e2.99 (5H, m, eCH2CH2NHe, Phe1-H
b
, H-4 and H-5), 2.72
(1H, dd, J¼13.6, 10.8 Hz, Phe1-H
b), 2.27e2.19 (4H, m, eCH2C(]O)
b-
D-glucopyrano-
CH3 and Glu2-H
g), 2.01 (2H, m, eNHC(]O)CH2e), 2.00 (3H, s,
ml,
eCH2C(]O)CH3), 1.94 (1H, m, Glu2-H ), 1.80 (1H, m, Glu2-H
b b), 1.78
0.26 mmol) successively. The reaction mixture was stirred for 1 h at
rt and directly purified by SiO2 column chromatography. Eluent of
EtOAc/EtOH/H2O (9:2:1) containing 5% AcOH gave assumed benzyl
protected 14.
(3H, s, eNHC(]O)CH3), 1.54 (2H, qui, J¼7.2 Hz, eC(]O)
CH2CH2CH2C(]O)e), 1.43e1.37 (4H, m, eOCH2CH2e and
eCH2CH2NHe), 1.26e1.19 (16H, m, eOCH2CH2(CH2)8e). 13C NMR
(100 MHz, (CD3)2S]O):
d 207.92, 173.85, 171.72, 171.57, 171.40,
10% PdeC (40 mg) was added to a solution of the resulting
material in dioxane/water (1:1 v/v, 10 ml), and the reaction mixture
was stirred under hydrogen for 2 h at 50 ꢀC. After removal of PdeC
by Celite filtration, the filtrate was concentrated, and purified by
168.77, 168.22, 137.94, 129.02, 127.87, 126.07, 100.91, 76.86, 74.17,
70.58, 68.13, 60.97, 55.36, 53.63, 51.90, 42.11, 41.93, 41.59, 38.45,
37.11, 34.05, 29.83, 29.60, 29.03, 28.97, 28.94, 28.91, 28.74, 28.65,
27.06, 26.28, 25.35, 22.92, 19.15. ESI-HRMS: C44H69N6O14 [MꢂH]ꢂ
calcd (m/z) 905.48772, found (m/z) 905.49107.
the preparative RP-HPLC [column: Inertsil ODS-3 (f20ꢁ250 mm);
column oven temperature: 40 ꢀC; flow rate: 5 ml/min, detection:
220 nm UV; eluate D in eluent C gradient increase from 10% to 40%
over 60 min] to give 14 (108 mg, 55%) as an amorphous. 1H
4.4. Reactions with galactosyltransferase to yield lactosami-
nide derivatives
NMR (400 MHz, (CD3)2S]O): d 12.05 (1H, br, eC(]O)OH), 8.14 (1H,
t, J¼5.6 Hz, Gly1-NH or Gly4-NH), 8.08e8.05 (2H, m, Gly1-NH or
Gly4-NH and Phe3-NH), 7.98 (1H, d, J¼7.6 Hz, Glu2-NH), 7.64
(1H, d, J¼8.8 Hz, eNHC(]O)CH3), 7.58 (1H, t, J¼5.6 Hz,
eCH2CH2NHC(]O)e), 7.27e7.16 (5H, m, aromatic-H), 4.92 (2H, br,
The reactions were performed as a 0.5 ml solution of substrate
(9e15,
2 mM), MnCl2 (10 mM), UDP-Gal (5 mM) b1,4-
galactosyltransferase9 (50 mU/ml), and additives (CDs or Triton-X
100) in 50 mM HEPES buffer (pH 7.0) at 25 ꢀC. The reactions were
OH-4 and OH-3), 4.47e4.41 (1H, m, Phe3-H
a
), 4.25 (1H, d, J¼8.4 Hz,
, eOCH2CH2e and H-
or Gly4-H
), 3.46e3.25
sampled 15
ml each at 10 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h,
H-1), 3.73e3.66 (5H, m, Gly1-H or Gly4-H
a a
10 h, 11 h, 12 h, 1 d, and 2 d (not all data were shown in Scheme 3)
6b), 3.62 (1H, dd, J¼16.4, 5.6 Hz, Gly1-H
a
a
and monitored by RP-HPLC [column: Inertsil ODS-3 (
f
4.6ꢁ250 mm);
(4H, m, H-6a, H-2, eOCH2CH2e, H-3), 3.08e3.01 (5H, m,
column oven temperature: 30 ꢀC; flow rate: 1 ml/min, detection:
eCH2CH2NHe, Phe3-H
b
, H-5 and H-4), 2.84 (1H, dd, J¼13.6, 9.6 Hz,
220 nm UV].
Phe3-H
Glu2-H
b
g
), 2.43 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3), 2.17e2.10 (4H, m,
and eNHC(]O)CH2e), 2.05 (3H, s, eCH2C(]O)CH3), 1.81
4.4.1. 12-[N-(5-Oxohexanoyl)-
alanyl-glycinyl]-aminododecyl O-(
acetamido-2-deoxy-
-glucopyranoside (16).10 To obtain physical
data of 16, a 2.5 ml solution of 9 (2 mM) was treated by UDP-Gal
(5 mM) and
1,4-galactosyltransferase9 (50 mU/ml) in 50 mM
HEPES buffer (pH 7.0) with MnCl2 (10 mM) and -CD (50 mM) at
L
-phenylalanyl-
L-glutamyl-L-phenyl-
(1H, m, Glu2-H
Glu2-H
1.42e1.38 (4H, m, eOCH2CH2e and eCH2CH2NHe), 1.27e1.20 (16H,
m, eOCH2CH2(CH2)8e). 13C NMR (100 MHz, (CD3)2S]O):
208.03,
b
), 1.78 (3H, s, eNHC(]O)CH3), 1.71e1.62 (1H, m,
b-D
-galactopyranosyl)-(1/4)-2-
b
), 1.67 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e),
b-D
d
b
173.79, 172.27, 171.08, 170.99, 169.20, 168.77, 168.12, 137.62, 129.01,
127.96, 126.18, 100.91, 76.86, 74.17, 70.57, 68.13, 60.97, 55.36, 54.16,
51.85, 42.03, 41.98, 41.85, 38.44, 36.89, 34.02, 29.80, 29.61, 29.03,
28.96, 28.94, 28.74, 28.67, 27.06, 26.27, 25.35, 22.92, 19.19. ESI-
HRMS: C44H69N6O14 [MꢂH]ꢂ calcd (m/z) 905.48772, found (m/z)
905.49089.
g
25 ꢀC for 5 h. The mixture was freeze-dried and the resulting res-
idue was purified by RP-HPLC [eluent D in eluent C gradient in-
crease from 30% to 40% over 30 min] to give 1610 (5.8 mg, quant.) as
an amorphous. tR¼25 min.
4.4.2. 12-[N-(5-Oxohexanoyl)-
aminododecyl O-( -galactopyranosyl)-(1/4)-2-acetamido-2-
deoxy- -glucopyranoside (17). To obtain physical data, six of re-
L-alanyl-L-glutamyl-L-alanyl-glycinyl]-
4.3.7. 12-[N-(5-Oxohexanoyl)-
glycinyl]-aminododecyl 2-acetamido-2-deoxy-
L
-phenylalanyl-
L
-glutamyl-glycinyl-
-glucopyranoside
b-D
b
-
D
b-D
(15). To the solution of 7 (120 mg, 0.20 mmol), HOBt (32 mg,
0.24 mmol), and WSC (46 mg, 0.24 mmol) in DMF (4 ml) were
sidual reaction mixtures performed in Scheme 3 were mixed
(1.65 ml) and freeze-dried. The resulting residue was purified by
RP-HPLC [eluent D in eluent C gradient increase from 20% to 30%
over 30 min] to give 17 (2.4 mg, 72%) as an amorphous. tR¼27 min.
added 12-aminododecyl 2-acetamido-2-deoxy-
b-D-glucopyrano-
side acetic acid salt (93 mg, 0.20 mmol) and triethylamine (34
ml,
0.24 mmol) successively. The reaction mixture was stirred for 1 h at
rt and directly purified by SiO2 column chromatography. Eluent of
EtOAc/EtOH/H2O (9:2:1) containing 5% AcOH gave assumed benzyl
protected 15. 10% PdeC (40 mg) was added to a solution of the
resulting material in dioxane/water (1:1 v/v, 8 ml), and the reaction
mixture was stirred under hydrogen for 1 h at 50 ꢀC. After removal
1H NMR (400 MHz, (CD3)2S]O):
d 12.07 (1H, br, eC(]O)OH), 8.12
(1H, t, J¼5.6 Hz, Gly4-NH), 8.04 (1H, d, J¼7.2 Hz, Ala1-NH or Ala3-
NH), 8.01 (1H, d, J¼6.8 Hz, Ala1-NH or Ala3-NH), 7.96 (1H, d,
J¼7.6 Hz, Glu2-NH), 7.73 (1H, d, J¼8.4 Hz, eNHC(]O)CH3), 7.63 (1H,
t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 5.07 (1H, br, OH-20), 4.78 (1H, br,
OH-30), 4.65 (1H, t, J¼4.8 Hz, OH-60), 4.61 (1H, s, OH-3), 4.58 (1H, t,

I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
3153
J¼6.0 Hz, OH-6), 4.50 (1H, d, J¼4.8 Hz, OH-40), 4.29 (1H, d, J¼8.0 Hz,
H-1), 4.25e4.13 (3H, m, Ala1-H
a
, H-10 and Glu2-H
a
), 3.78e3.55 (6H,
H-1), 4.26e4.15 (4H, m, H-10, Ala1-H
a
, Glu2-H
a
, and Ala3-H
a
),
m, H-6b, eOCH2CH2e, Gly4-H , H-6a and H-40), 3.53e3.41 (5H, m,
a
3.78e3.68 (2H, m, H-6, eOCH2CH2e), 3.66e3.56 (4H, m, H-6, H-40
H-60, H-50, H-4, and H-2), 3.40e3.22 (5H, m, eOCH2CH2e, H-20, H-3,
and Gly4-H
a
), 3.54e3.40 (5H, m, H-60, H-50, H-4 and H-2),
H-5, and H-30), 3.07e3.01 (3H, m, eCH2CH2NH- and Phe3-H
b
), 2.83
), 2.41 (2H, t, J¼7.2 Hz, eCH2C(]O)
), 2.09 (2H, t, J¼7.2 Hz, eNHC(]O)
),1.77 (3H,
3.38e3.22 (5H, m, eOCH2CH2e, H-20, H-3, H-5, and H-30), 3.03
(2H, dt, J¼5.6, 6.4 Hz, eCH2CH2NHe), 2.42 (2H, t, J¼7.2 Hz,
(1H, dd, J¼13.6, 9.2 Hz, Phe3-H
b
g
CH3), 2.18e2.12 (2H, m, Glu2-H
eCH2C(]O)CH3), 2.24 (2H, t, J¼8.0 Hz, Glu2-H
g), 2.10 (2H, t,
CH2e), 2.05 (3H, s, eCH2C(]O)CH3),1.81 (1H, m, Glu2-H
b
J¼7.2 Hz, eNHC(]O)CH2e), 2.06 (3H, s, eCH2C(]O)CH3), 1.90 (1H,
s, eNHC(]O)CH3), 1.71 (1H, m, Glu2-H
b
), 1.66 (2H, qui, J¼7.2 Hz,
m, Glu2-H
b
), 1.77 (3H, s, eNHC(]O)CH3), 1.75 (1H, m, Glu2-H
b
),
eC(]O)CH2CH2CH2C(]O)e), 1.44e1.41 (4H, m, eOCH2CH2e and
eCH2CH2NHe), 1.25e1.21 (16H, m, eOCH2CH2(CH2)8e), 1.15 (3H, d,
1.66 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e),1.43e1.37 (4H,
m, eOCH2CH2e and, eCH2CH2NHe), 1.25e1.21 (16H, m,
J¼6.8 Hz, Ala1-H
b d 208.04,
). 13C NMR (100 MHz, (CD3)2S]O):
eOCH2CH2(CH2)8e), 1.22 (3H, d, J¼6.8 Hz, Ala1-H
b
or Ala3-H
b
),
173.86, 172.53, 171.82, 171.05, 170.95, 168.51, 168.12, 137.50, 129.00,
127.94, 126.17, 103.92, 100.79, 81.39, 75.48, 74.87, 73.13, 72.14, 70.51,
68.33, 68.09, 60.40, 54.69, 54.00, 51.89, 48.15, 42.05, 41.85, 38.43,
37.00, 33.98, 29.84, 29.61, 29.03, 28.95, 28.73, 28.68, 27.01, 26.27,
25.31, 22.86, 19.21, 17.62. ESI-HRMS: C51H81N6O19 [MꢂH]ꢂ calcd (m/
z) 1081.55620, found (m/z) 1081.55823.
1.18 (3H, d, J¼7.2 Hz, Ala1-H
b b
or Ala3-H ). 13C NMR (100 MHz,
(CD3)2S]O):
d 208.06, 173.90, 172.53, 172.21, 171.82, 170.92, 168.51,
168.19, 103.92, 100.79, 81.39, 75.48, 74.87, 73.12, 72.14, 70.50, 68.33,
68.09, 60.40, 54.69, 51.65, 48.48, 48.24, 42.00, 41.85, 38.42. 33.97,
29.92, 29.61, 29.04, 28.93, 28.72, 28.66, 26.99, 26.27, 25.31, 22.86,
19.18, 17.66, 17.57. ESI-HRMS: C45H77N6O19 [MꢂH]ꢂ calcd (m/z)
1005.52490, found (m/z) 1005.52729.
4.4.5. 12-[N-(5-Oxohexanoyl)-
glycinyl]-aminododecyl O-(
acetamido-2-deoxy- -glucopyranoside (20). To obtain physical
L
-phenylalanyl-
L-glutamyl-L-alanyl-
b-D
-galactopyranosyl)-(1/4)-2-
4.4.3. 12-[N-(5-Oxohexanoyl)-glycinyl-
aminododecyl O-( -galactopyranosyl)-(1/4)-2-acetamido-2-
deoxy- -glucopyranoside (18). To obtain physical data, six of re-
L
-glutamyl-glycinyl-glycinyl]-
b-D
b
-
D
data, six of residual reaction mixtures performed in Scheme 3 were
mixed (1.65 ml) and freeze-dried. The resulting residue was puri-
fied by RP-HPLC [eluent D in eluent C gradient increase from 25% to
40% over 30 min] to give 20 (2.1 mg, 59%) as an amorphous.
b-D
sidual reaction mixtures performed in Scheme 3 were mixed
(1.65 ml) and freeze-dried. The resulting residue was purified by
RP-HPLC [eluent D in eluent C gradient increase from 20% to 30%
over 30 min] to give 18 (2.8 mg, 87%) as an amorphous. tR¼24 min.
tR¼21 min. 1H NMR (400 MHz, (CD3)2S]O):
d 12.09 (1H, s, eC(]O)
OH), 8.16 (1H, t, J¼5.8 Hz, Gly4-NH), 8.10 (1H, d, J¼6.8 Hz, Ala3-NH),
8.10 (1H, d, J¼6.8 Hz, Glu2-NH), 8.04 (1H, d, J¼8.0 Hz, Phe1-NH), 7.73
(1H, d, J¼8.4 Hz, eNHC(]O)CH3), 7.65 (1H, t, J¼5.6 Hz,
eCH2CH2NHC(]O)e), 7.26e7.15 (5H, m, aromatic-H), 5.07 (1H, br,
OH-20), 4.78 (1H, br, OH-30), 4.65 (1H, dd, J¼5.2, 4.4 Hz, OH-60), 4.61
1H NMR (400 MHz, (CD3)2S]O):
d 12.07 (1H, br, eC(]O)OH), 8.23
(1H, t, J¼5.6 Hz, Gly1-NH, Gly3-NH or Gly4-NH), 8.06e8.01 (3H, m,
Glu2-NH and Gly1-NH, Gly3-NH or Gly4-NH), 7.72 (1H, d, J¼8.8 Hz,
eNHC(]O)CH3), 7.70 (1H, t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 5.07
(1H, br, OH-20), 4.78 (1H, br, OH-30), 4.65 (1H, dd, J¼5.2, 4.0 Hz, OH-
60), 4.61 (1H, s, OH-3), 4.58 (1H, t, J¼6.0 Hz, OH-6), 4.50 (1H, d,
J¼4.4 Hz, OH-40), 4.29 (1H, d, J¼8.0 Hz, H-1), 4.29e4.24 (1H, m,
(1H, s, OH-3), 4.58 (1H, t, J¼6.0 Hz, OH-6), 4.54 (1H, m, Phe3-H
a),
4.50 (1H, d, J¼4.4 Hz, OH-40), 4.29 (1H, d, J¼8.0 Hz, H-1), 4.26e4.17
(3H, m, Ala3-H
a a), 3.78e3.56 (6H, m, H-6b,
H-10 and Glu2-H
Glu2-H
a
), 4.20 (1H, m, H-10), 3.79e3.58 (10H, m, H-6b,
eOCH2CH2e, Gly4-H
a
, H-6a and H-40), 3.53e3.41 (5H, m, H-60, H-50,
eOCH2CH2e, Gly1-H
a
,
Gly3-H
a,
Gly4-H
a,
H-6a and H-40),
H-4, and H-2), 3.40e3.22 (5H, m, eOCH2CH2e, H-20, H-3, H-5,
3.53e3.41 (5H, m, H-60, H-50, H-4, and H-2), 3.40e3.22 (5H, m,
eOCH2CH2e, H-20, H-3, H-5, and H-30), 3.03 (2H, dt, J¼5.6, 6.4 Hz,
eCH2CH2NHe), 2.43 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3), 2.25 (2H, t,
and H-30), 3.05e3.00 (3H, m, eCH2CH2NHe and Phe1-H
b), 2.71 (1H,
dd, J¼13.8, 10.6 Hz, Phe1-H
b), 2.27e2.19 (4H, m, eCH2C(]O)
CH3 and Glu2-H
g), 2.00 (2H, m, eNHC(]O)CH2e), 2.00 (3H, s,
J¼7.8 Hz, Glu2-H
g
), 2.11 (2H, t, J¼7.2 Hz, eNHC(]O)CH2e),
eCH2C(]O)CH3), 1.91 (1H, m, Glu2-H ), 1.79 (1H, m, Glu2-H
b b), 1.77
2.06 (3H, s, eCH2C(]O)CH3), 1.93 (1H, m, Glu2-H
b
), 1.77 (3H, s,
(3H, s, eNHC(]O)CH3), 1.54 (2H, qui, J¼7.2 Hz, eC(]O)
CH2CH2CH2C(]O)e), 1.44e1.36 (4H, m, eOCH2CH2e and
eCH2CH2NHe), 1.25e1.19 (19H, m, eOCH2CH2(CH2)8e and Ala3-
eNHC(]O)CH3), 1.76 (1H, m, Glu2-H
b), 1.67 (2H, qui, J¼7.2 Hz,
eC(]O)CH2CH2CH2C(]O)e), 1.44e1.36 (4H, m, eOCH2CH2e and
eCH2CH2NHe), 1.26e1.22 (16H, m, eOCH2CH2(CH2)8e). 13C NMR
Hb d 207.92, 173.88, 172.23,
). 13C NMR (100 MHz, (CD3)2S]O):
(100 MHz, (CD3)2S]O):
d
208.07, 173.84, 172.19, 171.49, 169.17,
171.71, 171.47, 170.88, 168.51, 168.20, 137.95, 129.02, 127.85, 126.07,
103.92, 100.79, 81.39, 75.48, 74.88, 73.12, 72.14, 70.50, 68.33, 68.09,
60.40, 54.69, 53.63, 51.69, 48.55, 42.00, 41.59, 38.44, 37.20, 34.06,
29.89, 29.60, 29.04, 28.94, 28.73, 28.66, 27.12, 26.28, 25.32, 22.86,
168.80, 168.51, 168.23, 103.92, 100.79, 81.39, 75.48, 74.87, 73.12,
72.14, 70.50, 68.33, 68.09, 60.40, 54.68, 51.83, 42.07, 41.91, 41.89,
41.84, 38.44, 34.04, 29.86, 29.62, 29.03, 28.95, 28.93, 28.72, 28.66,
27.02, 26.28, 25.31, 22.87, 19.21. ESI-HRMS: C43H73N6O19 [MꢂH]ꢂ
calcd (m/z) 977.49360, found (m/z) 977.49586.
19.15, 17.57. ESI-HRMS:
C
51H81N6O19 [MꢂH]ꢂ calcd (m/z)
1081.55620, found (m/z) 1081.55951.
4.4.4. 12-[N-(5-Oxohexanoyl)-
glycinyl]-aminododecyl O-(
acetamido-2-deoxy- -glucopyranoside (19). To obtain physical
L
-alanyl-
L
-glutamyl-
L
-phenylalanyl-
4.4.6. 12-[N-(5-Oxohexanoyl)-glycinyl-L-glutamyl-L-phenylalanyl-
b
-
D
-galactopyranosyl)-(1/4)-2-
glycinyl]-aminododecyl O-(
b-D-galactopyranosyl)-(1/4)-2-
b
-D
acetamido-2-deoxy- -glucopyranoside (21). To obtain physical
b-D
data, six of residual reaction mixtures performed in Scheme 3
were mixed (1.65 ml) and freeze-dried. The resulting residue was
purified by RP-HPLC [eluent D in eluent C gradient increase from
25% to 40% over 30 min] to give 19 (2.8 mg, 79%) as an
data, six of residual reaction mixtures performed in Scheme 3 were
mixed (1.65 ml) and freeze-dried. The resulting residue was puri-
fied by RP-HPLC [eluent D in eluent C gradient increase from 25%
to 40% over 30 min] to give 21 (3.3 mg, 94%) as an
amorphous. tR¼22 min. 1H NMR (400 MHz, (CD3)2S]O):
d
12.04
amorphous. tR¼21 min. 1H NMR (400 MHz, (CD3)2S]O):
d 12.06
(1H, br, eC(]O)OH), 8.22 (1H, t, J¼5.6 Hz, Gly4-NH), 8.03 (1H, d,
J¼6.8 Hz, Ala1-NH), 7.98 (1H, d, J¼7.6 Hz, Phe3-NH), 7.94 (1H, d,
J¼7.2 Hz, Glu2-NH), 7.73 (1H, d, J¼8.0 Hz, eNHC(]O)CH3), 7.58 (1H,
t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 7.26e7.16 (5H, m, aromatic-H),
5.07 (1H, br, OH-20), 4.78 (1H, br, OH-30), 4.65 (1H, dd, J¼5.2, 4.4 Hz,
OH-60), 4.61 (1H, s, OH-3), 4.58 (1H, t, J¼6.0 Hz, OH-6), 4.50 (1H, d,
(1H, br, eC(]O)OH), 8.16 (1H, t, J¼5.8 Hz, Gly1-NH or Gly4-NH),
8.09e8.05 (2H, m, Gly1-NH or Gly4-NH and Phe3-NH), 7.99 (1H, d,
J¼7.6 Hz, Glu2-NH), 7.73 (1H, d, J¼8.4 Hz, eNHC(]O)CH3), 7.58 (1H,
t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 7.27e7.16 (5H, m, aromatic-H),
5.07 (1H, br, OH-20), 4.78 (1H, br, OH-30), 4.65 (1H, dd, J¼5.2, 4.4 Hz,
OH-60), 4.61 (1H, s, OH-3), 4.58 (1H, t, J¼6.2 Hz, OH-6), 4.50 (1H, d,
J¼4.4 Hz, OH-40), 4.47e4.42 (1H, m, Phe3-H
a
), 4.29 (1H, d, J¼8.0 Hz,
J¼4.4 Hz, OH-40), 4.44 (1H, m, Phe3-H
), 4.29 (1H, d, J¼8.0 Hz, H-1),
a

3154
I. Nagashima, H. Shimizu / Tetrahedron 70 (2014) 3146e3154
4.23e4.16 (2H, m, H-10 and Glu2-H
a
), 3.78e3.57 (8H, m, H-6b,
173.85, 171.72, 171.57, 171.40, 168.77, 168.51, 168.22, 137.94, 129.02,
127.87, 126.07, 103.92, 100.79, 81.39, 75.48, 74.87, 73.12, 72.14,
70.50, 68.33, 68.09, 60.40, 54.68, 53.63, 51.90, 42.10, 41.92, 41.59,
38.45, 37.12, 34.05, 29.82, 29.60, 29.03, 28.94, 28.72, 28.65, 27.06,
eOCH2CH2e, Gly1-H , Gly4-H , H-6a and H-40), 3.54e3.41 (5H, m,
a a
H-60, H-50, H-4, and H-2), 3.40e3.22 (5H, m, eOCH2CH2e, H-20, H-3,
H-5, and H-30), 3.08e3.01 (3H, m, eCH2CH2NHe and Phe3-H
b),
2.84 (1H, dd, J¼14.0, 9.6 Hz, Phe3-H
b
), 2.43 (2H, t, J¼7.2 Hz,
26.28, 25.31, 22.87, 19.15. ESI-HRMS:
C
50H79N6O19 [MꢂH]ꢂ
eCH2C(]O)CH3), 2.14 (2H, t, J¼8.4 Hz, Glu2-H
g
), 2.12 (2H, t,
calcd (m/z) 1067.54055, found (m/z) 1067.54355.
J¼7.2 Hz, eNHC(]O)CH2e), 2.05 (3H, s, eCH2C(]O)CH3), 1.83 (1H,
m, Glu2-H
), 1.67 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e), 1.44e1.36
(4H, m, eOCH2CH2e and eCH2CH2NHe), 1.26e1.21 (16H, m,
eOCH2CH2(CH2)8e). 13C NMR (100 MHz, (CD3)2S]O):
208.04,
b
), 1.77 (3H, s, eNHC(]O)CH3), 1.71e1.62 (1H, m, Glu2-
Acknowledgements
Hb
The authors deeply appreciate Professor S.-I. Nishimura (Hok-
kaido University) in his support of this research. This research was
supported by a grant for the ‘Development of Novel Diagnostic and
Medical Applications through Elucidation of Sugar Chain Functions,
Hatakana-group’ 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, and members of Center for Instrumental Analysis,
Hokkaido University, for measuring high-resolution FABMS.
d
173.80, 172.28, 171.08, 170.99, 169.20, 168.51, 168.12, 137.62, 129.01,
127.97,126.18,103.92,100.79, 81.39, 75.48, 74.87, 73.12, 72.14, 70.50,
68.33, 68.09, 60.39, 54.68, 54.16, 51.85, 42.30, 41.96, 41.84, 38.43,
36.89, 34.01, 29.81, 29.61, 29.03, 28.94, 28.72, 28.67, 27.06, 26.27,
25.31, 22.86, 19.19. ESI-HRMS: C50H79N6O19 [MꢂH]ꢂ calcd (m/z)
1067.54055, found (m/z) 1067.54345.
4.4.7. 12-[N-(5-Oxohexanoyl)-
glycinyl]-aminododecyl O-(
acetamido-2-deoxy- -glucopyranoside (22). To obtain physical
L
-phenylalanyl-
L-glutamyl-glycinyl-
b-D
-galactopyranosyl)-(1/4)-2-
b-D
References and notes
data, six of residual reaction mixtures performed in Scheme 3 were
mixed (1.65 ml) and freeze-dried. The resulting residue was puri-
fied by RP-HPLC [eluent D in eluent C gradient increase from 25% to
40% over 30 min] to give 22 (3.5 mg, quant.) as an amorphous.
1. Varki, A. Glycobiology 1993, 3, 97e130.
2. Feizi, T. Glycoconjugate J. 2000, 17, 553e565.
3. Le Poole, I. C.; Gerberi, M. A. T.; Kast, W. M. Curr. Opin. Oncol. 2002, 14,
641e648.
4. Carbohydrate-based Drug Discovery; Wong, C.-H., Ed.; Wiley-VCH: Weinheim,
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5. Wade, R. C. Structure 1997, 5, 1139e1145.
6. Joseph, N.; Varghese, J. N.; Smith, R. W.; Sollis, S. L.; Blick, T. J.; Sahasrabudhe, A.;
McKimm-Breschkin, J. L.; Colman, P. M. Structure 1998, 6, 736e746.
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Mendel, D. M.; Laver, W. G.; Stevens, R. C. J. Med. Chem. 1998, 41, 2451e2460.
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Biol. Pharm. Bull. 2009, 32, 796e800.
tR¼22 min. 1H NMR (400 MHz, (CD3)2S]O):
d 12.09 (1H, s, eC(]O)
OH), 8.15 (1H, d, J¼7.6 Hz, Glu2-NH), 8.11 (1H, t, J¼5.6 Hz, Gly3-NH
or Gly4-NH), 8.07 (1H, t, J¼6.0 Hz, Gly3-NH or Gly4-NH), 8.06 (1H, d,
J¼8.4 Hz, Phe1-NH), 7.73 (1H, d, J¼8.4 Hz, eNHC(]O)CH3), 7.69 (1H,
t, J¼5.6 Hz, eCH2CH2NHC(]O)e), 7.26e7.15 (5H, m, aromatic-H),
5.07 (1H, br, OH-20), 4.78 (1H, br, OH-30), 4.65 (1H, dd, J¼5.2, 4.4 Hz,
OH-60), 4.61 (1H, s, OH-3), 4.58 (1H, t, J¼6.2 Hz, OH-6), 4.55 (1H, m,
Phe1-H
a
), 4.50 (1H, d, J¼4.4 Hz, OH-40), 4.29 (1H, d, J¼7.6 Hz, H-1),
9.
b1,4-Galactosyltransferase was purchased from SIGMA (G-5507).
4.29e4.24 (1H, m, Glu2-H ), 4.20 (1H, m, H-10), 3.78e3.58 (8H, m,
a
10. Nagashima, I.; Shimizu, H.; Matsushita, T.; Nishimura, S.-I. Tetrahedron Lett.
2008, 49, 3413e3418.
11. Szejtli, J. Chem. Rev. 1998, 98, 1743e1753.
H-6b, eOCH2CH2e, Gly3-H , Gly4-H , H-6a and H-40), 3.55e3.44
a a
(5H, m, H-60, H-50, H-4, and H-2), 3.39e3.22 (5H, m, eOCH2CH2e,
12. Rekharsky, M. V.; Inoue, Y. Chem. Rev. 1998, 98, 1875e1917.
13. Kakudo, S.; Kikuchi, N.; Kitadokoro, K.; Fujiwara, T.; Nakamura, E.; Okamoto, H.;
Shin, M.; Tamaki, M.; Teraoka, H.; Tsuzuki, H.; Yoshida, N. J. Biol. Chem. 1992,
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H-20, H-3, H-5, and H-30), 3.05e3.00 (3H, m, eCH2CH2NHe and
Phe1-H
b
), 2.72 (1H, dd, J¼14.0, 10.4 Hz, Phe1-H
b), 2.25 (2H, t,
J¼8.4 Hz, Glu2-H
), 2.21 (2H, t, J¼7.2 Hz, eCH2C(]O)CH3), 2.01 (2H,
g
m, eNHC(]O)CH2e), 2.00 (3H, s, eCH2C(]O)CH3e), 1.94 (1H, m,
Glu2-H ), 1.80 (1H, m, Glu2-H
b b), 1.77 (3H, s, NHC(]O)CH3),
1.54 (2H, qui, J¼7.2 Hz, eC(]O)CH2CH2CH2C(]O)e), 1.45e1.35
(4H, m, eOCH2CH2e and eCH2CH2NHe), 1.25e1.20 (16H, m,
eOCH2CH2(CH2)8e). 13C NMR (100 MHz, (CD3)2S]O):
d 207.93,
2007, 19, 1e17.