Synthesis and biological activities of analogs of d-glucosyl-l-tyrosine, a humoral factor that stimulates transcription of the acyl-CoA binding protein in the pheromone gland of the Silkmoth, Bombyx mori

Article abstract of DOI:10.1016/j.bmc.2006.10.008

β-d-Glucosyl-O-l-tyrosine (1) is a humoral factor that stimulates transcription of the acyl-CoA binding protein (ACBP) in the pheromone gland of the Silkmoth, Bombyx mori. This paper describes stereoselective synthesis of five analogs that changed the sug

Full text of DOI:10.1016/j.bmc.2006.10.008

Bioorganic & Medicinal Chemistry 15 (2007) 97–103
Synthesis and biological activities of analogs
of D-glucosyl-L-tyrosine, a humoral factor
that stimulates transcription of the acyl-CoA binding
protein in the pheromone gland of the Silkmoth, Bombyx mori
Shunya Takahashi,* Keiko Hasumi, Atsushi Ohnishi,
Hiroyuki Koshino and Shogo Matsumoto*
RIKEN (The Institute of Physical and Chemical Research), Wako-shi, Saitama 351-0198, Japan
Received 16 August 2006; revised 4 October 2006; accepted 6 October 2006
Available online 10 October 2006
Abstract—b-D-Glucosyl-O-L-tyrosine (1) is a humoral factor that stimulates transcription of the acyl-CoA binding protein (ACBP)
in the pheromone gland of the Silkmoth, Bombyx mori. This paper describes stereoselective synthesis of five analogs that changed
the sugar and/or amino acid part in 1 and their stimulatory activities on the ACBP transcription in the pheromone gland of B. mori.
Among the analogs tested, b-D-galactosyl-O-L-tyrosine showed a 1/5 potency compared to the activity of 1.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
kol precursor and eventually releasing it for bombykol
production after eclosion in response to the neurohor-
mone termed pheromone-biosynthesis-activating neuro-
peptide (PBAN).5–7 We applied RNAi methodologies
and demonstrated that targeted disruption of the
pgACBP gene prevents TG accumulation within the cyto-
plasmic lipid droplets and consequently the availability of
the bombykol precursors.8 These results indicate the in vi-
vo biological relevance of pgACBP in the biosynthesis of
the TGs that constitute the cytoplasmic lipid droplets.
Acyl-CoA binding protein (ACBP) is a highly conserved
10-kDa intracellular lipid-binding protein that specifical-
ly binds medium- and long-chain fatty acyl-CoA esters
with high affinity and is structurally highly conserved
from yeast to mammals as well as insects.1 While ACBP
can functionally act as an acyl-CoA carrier or as an
acyl-CoA pool maker within the cell,2,3 the regulatory
mechanisms underlying ACBP gene expression are
unclear.
By exploiting this unique sex pheromone production
system in the moth PG, we have also discovered that
transcription of pgACBP is triggered by a hemolymph
based humoral factor and provided evidence that tran-
scription of some ACBPs can be triggered by specific
humoral factors. Following purification and structure
elucidation by means of high-resolution electrospray-
ionization mass spectrometry (HR-ESIMS) and NMR
analyses, in conjunction with stereochemical analyses
using acid hydrolysates, we have identified the humoral
factor to be b-D-glucosyl-O-L-tyrosine (1) (Fig. 1).9
In the Silkmoth, Bombyx mori, a distinct ACBP designat-
ed as pgACBP, is specifically expressed in the pheromone
gland (PG) during pheromonogenesis.4 In addition, dur-
ing pheromonogenesis, the PG cells accumulate a large
number of lipid droplets within the cytoplasm just before
eclosion.5 Because these lipid droplets contain triacylgly-
cerols (TGs) composed of unsaturated C16 and C18 fatty
acids with the pheromone (bombykol, (E,Z)-10,12-hexa-
decadien-1-ol) precursor, D10,12-hexadecadienoic acid as
a major component, they play a role in storing the bomby-
b-D-Glucosyl-O-L-tyrosine (1) has been shown to be a transient
metabolite of some insects and it appears to be the major tyrosine-
storage metabolite for the production of tanning diphenol substrates
in lepidoptera.10–12
Keywords: D-Glucosyl-O-L-tyrosine; Acyl-CoA binding protein;
D-Galactosyl-L-tyrosine.
*
Corresponding authors. Tel.: +81 48 467 9223; fax: +81 48 462 4627
(S.T.); e-mail: shunyat@riken.go.jp
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.10.008

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S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
OH
O
OH
O
HO
HO
HO
O
O
HO
NH2
CO2H
NH2
CO2H
OH
OH
D-glucosyl-D-tyrosine (2)
D-glucosyl-L-tyrosine (1)
HO
HO
HO
OH
OH
O
HO
HO
O
O
O
NH2
CO2H
NH2
CO2H
OH
L-glucosyl-L-tyrosine (3): α-NH2
L-glucosyl-D-tyrosine (4): β-NH2
D-galactosyl-L-tyrosine (5): α-NH2
D-galactosyl-D-tyrosine (6): β-NH2
Figure 1. Structures of b-D-glucosyl-O-L-tyrosine (1) and its analogs.
Furthermore, by using an in vitro system with trimmed
PG, we have demonstrated that synthetic 1 directly acts
on the PG cells, although the molecular mechanisms
underlying how the signal from 1 stimulates the tran-
scription of pgACBP within the PG cells remain to be
clarified.9 As the first step in attempting to identify the
mechanisms, here we describe the stereoselective synthe-
ses of the diastereomers and galactosyl analogs 2–6
which could be used as ligand controls, and their stimu-
latory activities on the pgACBP transcription in the PG
of B. mori.
cause compounds 10 showed a single spot on TLC, a
fractional crystallization from ethanol afforded 11 as a
pure form. In the H NMR spectrum of 11, the proton
1
at C-10 was observed at d 4.82 as a doublet with
0
0
J1 ;2 ¼ 8:8 Hz, showing the presence of a b-glycosidic
bond. Finally, all benzyl protecting groups in 11 were re-
moved by hydrogenolysis in the presence of 10% Pd/C in
ethyl acetate/aq acetic acid to give analog 2.
Synthesis of the L-glucose series was also conducted
according to the procedure described above. Treatment
of 2,3,4,6-tetra-O-benzyl-L-glucopyranose (12)17 with
DCC-CuCl followed by the addition of 8 afforded ano-
meric mixture 14 as a white solid in 40% yield (a/b = ca.
1/6) (Scheme 2). Different from the case of 10, fractional
crystallization failed. Consequently, the anomeric
mixture 14 was employed in a reversed-phase HPLC
to provide the desired isomer 16 as a pure form. Hydro-
genation of 16 gave analog 4. The use of the correspond-
ing L-amino acid derivative 1318 instead of 8 gave the
diastereomers 15 (a/b = 1/7) in 59% yield. Fractional
crystallization followed by hydrogenation of the result-
ing compound 16 provided analog 3.
2. Results and discussion
For preparation of analogs of 1, formation of a 1,2-trans
glycosidic bond between the tyrosine residue and the su-
gar part is a major problem.13–15 Usually, 1,2-trans gly-
cosidation is achieved by employing acylated sugars as a
glycosyl donor. The use of acylated sugars, however, re-
quires deprotection under alkaline conditions at the final
stage, hiding the possibility that causes a partial epimer-
ization of amino acid residue. Horvat et al.13 reported
that glycosidation of a benzylated D-glucosyl isourea16
with amino acids afforded b-D-glucopyranosyl ethers
stereoselectively. We planned to utilize Horvat’s meth-
od13 using a benzylated glycosyl isourea as a glycosyl
donor. This strategy is expected to enable us to make
analogs without technical problems arising from the
handling of final products.
Having completed the stereoselective synthesis of the
diastereomers of b-D-glucosyl-O-L-tyrosine (1), we
turned our attention to the synthesis of D-galactosyl
analogs.à There are no reports on this type of reaction
of a galactose derivative. Therefore we initially searched
the stereoselectivity of model compounds (Scheme 3).
2,3,4,6-Tetra-O-benzyl-D-galactopyranose (18) was
treated with DCC and CuCl at 90 ꢁC and then the
resulting galactosyl isourea reacted with p-cresol (19)
or methyl 4-hydroxyphenylacetate (20), providing the
corresponding aryl glycosides 21 (47%) or 22 (51%),
respectively. The 1,2-trans/cis selectivity in both cases
was shown to be >4–5/1 by the 1H NMR analyses.
Encouraged by these results, we conducted a coupling
Synthesis began from the preparation of b-D-glucosyl-
O-D-tyrosine (2). A commercially available N-benzyl-
oxycarbonyl-D-tyrosine (7) reacted with benzyl
bromide-sodium hydrogencarbonate to give N-benzyl-
oxycarbonyl-D-tyrosine benzyl ester (8) in 94% yield
(Scheme 1). This compound was treated with a benzylat-
ed glycosyl isourea, which was prepared in situ from
2,3,4,6-tetra-O-benzyl-D-glucopyranose (9) and N,N-
dicyclohexylcarbodiimide (DCC) in the presence of cop-
per chloride (CuCl) at 90 ꢁC, giving glycosides 10 (73%;
1
reaction of 18 with 8 (Scheme 4). H NMR spectra of
23 obtained in 67% yield revealed that this reaction also
1
a-anomer/b-anomer = ca. 1/7 judged by H NMR anal-
à
In the surface layer glycoprotein of Thermoanaeobacter thermohy-
drosulfuricus strains, b-D-galactosyl-L-tyrosine (5) was found as the
core component along with 1.19
ysis) as a white solid. Although separation of the desired
b-anomer 11 by chromatography on silica gel failed be-

S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
99
BnO
HO
HO
HO
OBn
NHZ
CO2Bn
NHZ
CO2H
O
a
BnO
+
R
OH
OBn
18
OBn
O
19: R = Me
20: R = CH2CO2Me
a
BnO
BnO
BnO
b
OBn
O
OH
OBn
9
O
BnO
OBn
O
OBn
BnO
BnO
O
NHZ
CO2Bn
R
OBn
21: R = Me
22: R = CH2CO2Me
10
Scheme 3. Reagents and conditions: (a) 18, DCC, CuCl, 90 ꢁC, and
then 19 or 20, 90 ꢁC, 47% for 21, 51% for 22.
c, d
OR
O
RO
RO
O
NHR'
CO2R
BnO
OR
HO
OBn
O
NHZ
CO2Bn
BnO
+
11: R = Bn, R' = Z
2: R = R' = H
OH
OBn
18
8: β-NHZ
a
13: α-NHZ
Scheme 1. Reagents and conditions: (a) BnBr, NaHCO3, DMF, rt,
94%; (b) 9, DCC, CuCl, 90 ꢁC, and then 8, 90 ꢁC, 73%; (c) fractional
crystallization from ethanol, 51%; (d) 10% Pd/C, H2, ethyl acetate/aq
acetic acid, rt, 75%.
BnO
OBn
O
O
BnO
NHZ
OBn
23: β-NHZ
24: α-NHZ
CO2Bn
HO
BnO
NHZ
CO2Bn
b, c
OBn
OH
BnO
BnO
RO
O
+
OR
O
O
RO
12
8: β-NHZ
NHR'
a
OR
13: α-NHZ
CO2R
BnO
OBn
25: β-NHR', R = Bn, R' = Z
6: β-NHR', R = R' = H
BnO
BnO
O
O
NHZ
26: α-NHR', R = Bn, R' = Z
5: α-NHR', R = R' = H
14: β-NHZ
15: α-NHZ
CO2Bn
b, c
Scheme 4. Reagents and conditions: (a) 18, DCC, CuCl, 90 ꢁC, and
then
or 13, 90 ꢁC, 67% for 23, 64% for 24; (b) fractional
crystallization from ethanol, 67% for 25 or HPLC separation, 55%
for 26; (c) 10% Pd/C, H2, ethyl acetate/aq acetic acid, rt, 63% for 6,
66% for 5.
RO
O
8
OR
O
RO
RO
NHR'
CO2R
16: β-NHR', R = Bn, R' = Z
4: β-NHR', R = R' = H
hydrogenation of 26 afforded the diastereomer 5.15 All
analogs synthesized were submitted to the bioassay.
17: α-NHR', R = Bn, R' = Z
3: α-NHR', R = R' = H
3. Biological activity9 of synthetic analogs
Scheme 2. Reagents and conditions: (a) 12, DCC, CuCl, 90 ꢁC, and
then
or 13, 90 ꢁC, 40% for 14, 59% for 15; (b) fractional
8
crystallization from ethanol, 74% for 16 or HPLC separation, 75%
for 17; (c) 10% Pd/C, H2, ethyl acetate/aq acetic acid, rt, 89% for 4,
59% for 3.
To examine the biological activity that stimulates tran-
scription of pgACBP, the synthetic analogs were inject-
ed into female P50 pupae three days before eclosion. For
measurement of pgACBP transcript levels, RT-PCR
analyses were performed by using total RNA prepared
from the trimmed PGs 18 h after injection. We previous-
ly demonstrated that the b-D-glucosyl-O-L-tyrosine titer
in the hemolymph of B. mori during pupal–adult molt
reaches a maximum of as much as 5 mg/mL, the concen-
tration of which is responsible for the up-regulation in
pgACBP transcription in the PG during pheromonogen-
proceeded in high b-selectivity (a/b = 1/8). Chromato-
graphy of 23 on silica gel followed by recrystallization
from ethanol provided the b-anomer 25, which under-
went hydrogenolysis to give D-galacto analog 6. Similar-
ly, glycosidation between 18 and 13 afforded glycosides
24 (a/b = 1/5), from which b-glycoside 26 was separated
by HPLC separation and recrystallization. Finally,

100
S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
NMR spectra were recorded at 400 or 600 MHz with
JEOL a 400 or JNM-ECA 600 spectrometers, using tet-
ramethylsilane as the internal standard. HPLC was per-
formed with a Shiseido Capcell-pak C18 (20 · 250 mm)
column in a Senshu Scientific Co. SSC-6300 F liquid
chromatograph equipped with an SSC-5200 UV detec-
tor. Column chromatography was performed on Kanto
silica gel 60 N (spherical, neutral; 40–100 lm). Merck
precoated silica gel 60 F254 plates, 0.25 mm thickness,
were used for analytical thin-layer chromatography.
The solvent extracts were dried with magnesium sulfate,
and the solutions were evaporated under diminished
pressure at 40–42 ꢁC.
4.2. N-Benzyloxycarbonyl-D-tyrosine benzyl ester (8)
To a vigorously stirred suspension of 7 (1.0 g, 3.2 mmol)
and sodium hydrogencarbonate (530 mg, 6.3 mmol) in
N,N-dimethylformamide (50 ml) was added dropwise
benzyl bromide (1.9 ml, 15.9 mmol) at rt, and the mix-
ture was stirred at rt for 21 h. After addition of ice-
water, the resulting mixture was extracted with ethyl
acetate. The extracts were washed with water, brine,
dried, and concentrated. The residue was chromato-
graphed on silica gel (n-hexane/ethyl acetate = 4:1) to
give 8 (1.2 g, 94%) as a white solid.
Figure 2. Stimulatory activities of b-D-glucosyl-O-L-tyrosine analogs
on pgACBP transcription in the PG of Bombyx mori. (A) pgACBP
transcript levels after injection of synthetic b-D-glucosyl-O-L-tyrosine
analogs. Synthetic tyrosine glycosides (0.2 mg) were injected into day
ꢁ3 female pupae. Trimmed PGs were prepared from pupae 18 h after
injection and subjected to RT-PCR. Lane 1, b-D-glucosyl-O-L-tyrosine
(1); lane 2, b-D-galactosyl-O-L-tyrosine (5); lane 3, b-D-galactosyl-O-D-
tyrosine (6); lane 4, b-D-glucosyl-O-D-tyrosine (2); lane 5, b-L-glucosyl-
O-L-tyrosine (3); lane 6, b-L-glucosyl-O-D-tyrosine (4). (B) The
percentage of the pgACBP transcript level induced by D-glucosyl-L-
tyrosine analogs injection. Results are expressed as percentage of
control. Bars represent mean values SD (n P 3).
21
D
Compound 8; mp 119–120ꢁC (lit.18 118–119 ꢁC); ½aꢀ
22
ꢁ5.7 (c 1.06, CHCl3); ½aꢀ +13.8 (c 0.48, methanol);
D
{for antipode: lit.18 [a]D ꢁ13.9 (methanol)}. IR (neat)
3312, 3032, 2953, 1720, 1692, 1514, 1250, 1213, 1174,
1055, 1028 cmꢁ1
.
1H NMR (400 MHz, CDCl3): d
7.36–7.23 (10H, m), 6.84 (2H, d, J = 8.8 Hz), 6.67 (2H,
d, J = 8.8 Hz), 6.17 (1H, s), 5.24 (1H, brd, J = 8.3 Hz),
5.16, 5.11 (2H, each d, J = 12.2 Hz), 5.09, 5.07 (2H, each
d, J = 12.2 Hz), 4.65 (1H, m), 3.07–3.01 (2H, m). 13C
NMR (100 MHz, CDCl3): d 171.6, 155.8, 154.9, 136.0,
134.9, 130.4, 128.6, 128.5, 128.2, 127.1, 115.5, 67.3,
67.1, 54.9, 37.3. HRMS calcd for C24H24NO5 [M+H]+
406.1654, found 406.1691.
esis.9 We, therefore, injected 0.2 mg of each synthetic
analog into P50 pupae (final concentration, 5.0 mg/mL
hemolymph). The results indicate that b-D-galactosyl-
O-L-tyrosine (5) revealed the stimulatory activity albeit
the potency was about 1/5 compared to that of 1
(Fig. 2; lanes 1–2), while the other synthetic analogs 2–
4, and 6 had no effect on the transcription of pgACBP
(Fig. 2; lanes 3–6). These results show that both of the
absolute configurations of the sugar part and the amino
acid residue are important for stimulatory activity. Fur-
thermore, the configurational change at the C-4 position
in 1 influenced the activity to a lesser extent, suggesting
that the possibility of artificial modifications around the
position. This information would be useful for designing
a bioprobe for understanding the mechanism of the
transcription of pgACBP within the PG cells.
4.3. 4-O-(2,3,4,6-Tetra-O-benzyl-b-D-glucopyranosyl)-N-
benzyloxycarbonyl-D-tyrosine benzylester (11)
To a stirred mixture of 9 (1.0 g, 1.85 mmol) and N,N-
dicyclohexylcarbodiimide (384 mg, 1.86 mmol) was add-
ed copper chloride (32.0 mg, 0.32 mmol) and the mix-
ture was stirred at 90 ꢁC for 2 h. Then, 8 (500 mg,
1.23 mmol) was added and the resulting mixture was
stirred at the same temperature for 1.5 h, cooled, diluted
with dichloromethane, and then filtered through a pad
of Celite. The filtrate was concentrated to give a syrup,
which was chromatographed on silica gel (dichloro-
methane/ether = 30:1) to give 10 {830 mg, 73% (a/
b = 1/7)} as a solid. Fractional crystallization from
ethanol gave 11 (584 mg, 51%) as a white solid.
4. Experimental
4.1. General procedures
21
Compound 11. Mp 104–105 ꢁC; ½aꢀ ꢁ5.2 (c 1.03,
.
1456, 1356, 1287, 1233, 1196, 1069 cmꢁ1 1H NMR
(400 MHz, CDCl3): d 7.36–7.17 (30H, m), 6.90 (4H, s),
5.22 (1H, d, J = 7.8 Hz), 5.16–4.49 (12H, m), 4.82 (1H,
d, J = 8.8 Hz), 4.67 (1H, m), 3.79–3.57 (6H, m), 3.07
All reactions were carried out under an argon atmo-
sphere, unless otherwise noted. Melting points were
determined using a Yanaco MP-500 apparatus and are
uncorrected. Optical rotations were measured with a
JASCO DIP-370 polarimeter. IR spectra were recorded
with a JASCO VALOR-III spectrophotometer. 1H
D
CHCl3). IR (neat) 3325, 1736, 1717, 1696, 1559, 1509,

S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
101
(2H, d, J = 5.4 Hz). 13C NMR (100 MHz, CDCl3): d
171.3, 156.5, 155.6, 138.4, 138.2, 138.1, 138.0, 136.1,
135.0, 130.4, 129.5, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 127.9, 127.8, 127.7, 127.6, 117.0, 101.6, 84.6,
81.9, 77.6, 75.7, 75.1, 75.0, 73.5, 68.7, 67.3, 67.0, 54.8,
37.3. HRMS calcd for C58H58NO10 [M+H]+ 928.4061,
found 928.4076.
(2H, m), 3.56 (1H, t, J = 8.8 Hz), 3.49 (1H, t,
J = 8.7 Hz), 3.23 (1H, m), 3.08 (1H, m). 13C NMR
(100 MHz, D2O): d 174.8, 156.8, 131.6, 130.7, 117.8,
101.0, 76.9, 76.4, 73.8, 70.2, 61.4, 56.9, 36.5. HRMS calcd
for C15H22NO8 [M+H]+ 344.1345, found 344.1306.
4.7. 4-O-(2,3,4,6-Tetra-O-benzyl-b-L-glucopyranosyl)-N-
benzyloxycarbonyl-L-tyrosine benzylester (17)
4.4. 4-O-(b-D-Glucopyranosyl)-D-tyrosine (2)
Treatment of 12 (814 mg, 1.51 mmol) and 13 (500 mg,
1.23 mmol) as described for preparation of 10 from 8
and 9 yielded 15 {578 mg, 59% (a/b = 1/7)}. Fractional
crystallization of 15 (381 mg) from ethanol gave 17
(287 mg, 75%) as a white solid.
A mixture of 11 (103 mg, 0.11 mol) and 10% Pd/C
(33 mg) in acetic acid/ethyl acetate/water (8:3:1; 3.2 ml)
was vigorously stirred at rt under hydrogen for 12 h
and then filtered through a pad of Celite. The filtrate
was concentrated to give a syrup, which was treated with
methanol to give 2 (29 mg, 75%) as a white powder.
27
Compound 17. Mp 104–105 ꢁC; ½aꢀ +7.8 (c 0.92,
D
CHCl3). The 1H and 13C NMR data were in good agree-
ment with those of 11. HRMS calcd for C58H58NO10
[M+H]+ 928.4061, found 928.4050.
22
Compound 2. ½aꢀ ꢁ25.4 (c 0.11, H2O). IR (neat) 3325,
D
1610, 1585, 1509, 1406, 1337, 1316, 1231, 1076, 1044,
1
988 cmꢁ1. H NMR (600 MHz, D2O): d 7.28 (2H, d,
J = 7.6 Hz), 7.12 (2H, d, J = 7.6 Hz), 5.12 (1H, d, J =
7.6 Hz), 3.94 (1H, br s), 3.92 (1H, dd, J = 12.6,
2.0 Hz), 3.74 (1H, dd, J = 12.6, 5.5 Hz), 3.62 (1H, m),
3.60 (1H, dd, J = 9.1, 8.6 Hz), 3.56 (1H, dd, J = 9.1,
7.6 Hz), 3.49 (1H, t, J = 9.1 Hz), 3.23 (1H, brd,
J = 11.6 Hz), 3.08 (1H, m). 13C NMR (100 MHz,
D2O): d 176.9, 158.9, 133.7, 132.7, 120.0, 103.1, 79.1,
78.5, 75.9, 72.4, 63.5, 59.0, 38.5. HRMS calcd for
C15H22NO8 [M+H]+ 344.1345, found 344.1364.
4.8. 4-O-(b-L-Glucopyranosyl)-L-tyrosine (3)
Treatment of 17 (100 mg, 0.11 mmol) as described for
preparation of 2 yielded 3 (22 mg, 59%) as a white
powder.
23
D
Compound 3. ½aꢀ +27.0 (c 0.11, H2O). The 1H and 13
C
NMR data were in good agreement with those of 2.
HRMS calcd for C15H22NO8 [M+H]+ 344.1345, found
344.1371.
4.5. 4-O-(2,3,4,6-Tetra-O-benzyl-b-L-glucopyranosyl)-N-
benzyloxycarbonyl-D-tyrosine benzylester (16)
4.9. p-Tolyl 2,3,4,6-tetra-O-benzyl-D-galactopyranosides
(21)
Treatment of 12 (835 mg, 1.54 mmol) and 8 (501 mg,
1.24 mmol) as described for preparation of 10 from 8
and 9 yielded 14 {456 mg, 40% (a/b = 1/6)}. A reversed-
phase HPLC separation (acetonitrile/water = 10:1) of 14
(369 mg) gave 16 (273 mg, 74%) as a white solid.
To a stirred mixture of 18 (158 mg, 0.29 mmol) and DCC
(60 mg, 0,29 mmol) was added copper chloride (4.7 mg,
0.05 mmol) and the mixture was stirred at 90 ꢁC for 2 h.
Then, 19 (24 ll, 0.23 mmol) was added and the resulting
mixture was stirred at the same temperature for 1.0 h,
cooled, diluted with dichloromethane, and then filtered
through a pad of Celite. The filtrate was concentrated
and then chromatographed on silica gel (n-hexane/ethyl
acetate = 30:1 ! 20:1 ! 10:1 ! 4:1) to give 21 {71 mg,
47% (a/b = 1/5)} as a syrup.
21
Compound 16. Mp 83–84 ꢁC (methanol); ½aꢀ +3.1 (c
D
0.58, CHCl3). IR (neat) IR 3350, 1717, 1541, 1522,
1456, 1235, 1206, 1070, 1059, 1013 cmꢁ1 1H NMR
.
(400 MHz, CDCl3): d 7.34–7.17 (30H, m), 6.91 (4H, s),
5.24 (1H, d, J = 7.8 Hz), 5.13–4.49 (12H, m), 4.84 (1H,
d, J = 8.3 Hz), 4.69 (1H, m), 3.79–3.67 (5H, m), 3.66–
3.59 (1H, m), 3.07 (2H, d, J = 5.9 Hz). 13C NMR
(100 MHz, CDCl3): d 171.3, 156.5, 155.6, 138.4, 138.2,
138.0, 137.9, 136.2, 135.0, 130.4, 129.6, 128.6, 128.5,
128.4, 128.3, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6,
127.5, 117.0, 101.7, 84.6, 81.9, 77.6, 75.7, 75.0, 74.9,
73.4, 68.7, 67.2, 67.0, 54.8, 37.3. HRMS calcd for
C58H58NO10 [M+H]+ 928.4061, found 928.4080.
Compound 21. IR (neat) 3032, 2919, 1508, 1496, 1226,
1
1088 cmꢁ1. H NMR (400 MHz, CDCl3) of anomeric
mixture (a/b = ca. 1/1): d 7.43–6.96 (24H, m), 5.47
(0.5H, d, J = 2.9 Hz, a-H1), 5.03–4.35 (8H, m), 4.93
(0.5H, d, J = 7.8 Hz, b-H1), 4.19 (0.5H, dd, J = 9.7,
2.9 Hz, a-H2), 4.15 (0.5H, dd, J = 9.7, 2.4 Hz, a-H3),
4.16–4.05 (1H, m, a-H5, b-H2), 4.06 (0.5H, d,
J = 2.4 Hz, a-H4), 3.94 (0.5H, d, J = 2.9 Hz, b-H4),
0
4.6. 4-O-(b-L-Glucopyranosyl)-D-tyrosine (4)
3.69–3.54 (2.5H, m, a-H6, b-H3, H5, H6, H6 ), 3.49
0
(0.5H, dd, J = 9.3, 5.9 Hz, a ꢁ H6 ), 2.30 (3H, s). HRMS
Treatment of 16 (100 mg, 0.11 mmol) as described for
preparation of 2 yielded 4 (33 mg, 89%) as a white powder.
calcd for C41H43O6 [M+H]+ 631.3060, found 631.3070.
4.10. 4-(Methoxycarbonylmethyl)phenyl 2,3,4,6-tetra-O-
benzyl-D-galactopyranosides (22)
22
Compound 4. ½aꢀ +70.0 (c 0.15, H2O). IR (neat) 3350,
D
1635, 1558, 1508, 1458, 1240, 1190, 1072 cmꢁ1
.
1H
NMR (400 MHz, D2O): d 7.27 (2H, d, J = 6.8 Hz), 7.11
(2H, d, J = 6.8 Hz), 5.12 (1H, d, J = 7.4 Hz), 3.94–3.88
(2H, brd), 3.74 (1H, dd, J = 12.2, 5.2, Hz), 3.64–3.57
Treatment of 18 (173 mg, 0.32 mmol) and DCC (66 mg,
0.32 mmol) as described for preparation of 21 from 18
and 19 yielded 22 {92 mg, 51% (a/b = 1/4)} as a syrup.

102
S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
25
Compound 26. Mp 113–115 ꢁC (ethanol); ½aꢀ ꢁ9.7 (c
Compound 22. IR (neat) 3032, 2920, 1735, 1509, 1453,
D
0.96, CHCl3). IR (neat) 3300, 1686, 1512, 1455, 1238,
1
1233, 1087 cmꢁ1. H NMR (400 MHz, CDCl3) of ano-
meric mixture (a/b = 1/4): d 7.44–7.17 (24H, m), 5.47
(0.2H, d, J = 3.4 Hz, a-H1), 5.02–4.34 (8H, m), 4.96
(0.8H, d, J = 7.8 Hz, b-H1), 4.19 (0.2H, dd, J = 10.2,
3.4 Hz, a-H2), 4.15 (0.2H, dd, J = 10.2, 2.9 Hz, a-H3),
4.14-4.05 (1H, m, a-H5), 4.11 (0.4H, dd, J = 9.8,
7.8 Hz, b-H2), 4.08 (0.2H, br s, a-H4), 3.95 (0.8H, d,
J = 2.9 Hz, b-H4), 3.72–3.52 (1.6H, m, a-H6, b-H3, H5,
1211, 1171, 1098, 1060, 1028 cmꢁ1
.
1H NMR
(400 MHz, CDCl3): d 7.39–7.24 (30H, m), 6.88 (4H, d,
J = 2.4 Hz), 5.19 (1H, d, J = 8.3 Hz), 5.16–4.62 (10H,
m), 4.90 (1H, d, J = 7.8 Hz), 4.65 (1H, m), 4.43, 4.39
(2H, each d, J = 11.7 Hz), 4.09 (1H, dd, J = 9.8,
7.8 Hz), 3.95 (1H, d, J = 2.9 Hz), 3.68–3.59 (4H, m),
3.05 (2H, d, J = 5.9 Hz). 13C NMR (100 MHz, CDCl3):
d 171.4, 156.6, 155.6, 138.6, 138.5, 138.4, 137.8, 136.2,
135.1, 130.3, 129.4, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 127.8, 127.6, 127.5, 117.2, 102.0, 82.1, 79.2,
75.4, 74.6, 73.8, 73.6, 73.4, 73.1, 68.8, 67.2, 67.0, 54.9,
37.4. HRMS calcd for C58H58NO10 [M+H]+ 928.4061,
found 928.4033.
0
H6, H6 ), 3.70 (0.6H, s), 3.69 (2.4H, s), 3.58 (2H, br s),
0
3.50 (0.2H, dd, J = 9.3, 5.9 Hz, a ꢁ H6 ). HRMS calcd
for C43H45O8 [M+H]+ 689.3114, found 689.3102.
4.11. 4-O-(2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl)-
N-benzyloxycarbonyl-D-tyrosine benzylester (25)
Treatment of 18 (503 mg, 0.93 mmol) and 8 (301 mg,
0.74 mmol) as described for preparation of 10 from 8
and 9 yielded 23 {405 mg, 67% (a/b = 1/8)}. Fractional
crystallization of 23 (251 mg) from ethanol gave 25
(167 mg, 67%) a white solid.
4.14. 4-O-(b-D-Galactopyranosyl)-L-tyrosine (6)
Treatment of 26 (101 mg, 0.11 mmol) as described for
preparation of 2 yielded 5 (25 mg, 66%) as a white
powder.
26
Compound 25. Mp 127–129 ꢁC (ethanol); ½aꢀ ꢁ15.3 (c
25
D
Compound 5. ½aꢀ ꢁ78.9ꢁ (c 0.11, H2O). IR (neat) 3250,
D
0.61, CHCl3). IR (neat) 3300, 1740, 1696, 1509, 1456,
1600, 1717, 1509, 1474, 1458, 1399, 1233, 1049 cmꢁ1. 1H
NMR (400 MHz, D2O): d 7.29 (2H, d, J = 8.3 Hz), 7.14
(2H, d, J = 8.3 Hz), 5.07 (1H, d, J = 7.3 Hz), 4.02–3.96
(2H, m), 3.90–3.76 (4H, m), 3.26 (1H, dd, J = 14.6,
4.9 Hz), 3.10 (1H, dd, J = 14.6, 7.8 Hz). 13C NMR
(100 MHz, D2O): d 174.8, 156.9, 131.6, 130.4, 117.8,
101.5, 76.2, 73.4, 71.4, 69.3, 61.6, 56.8, 36.4. HRMS
calcd for C15H22NO8 [M+H]+ 344.1345, found
344.1313.
1
1287, 1233, 1065 cmꢁ1. H NMR (400 MHz, CDCl3): d
7.39–7.24 (30H, m), 6.89 (4H, s), 5.20 (1H, d, J =
8.3 Hz), 5.15–4.62 (10H, m), 4.91 (1H, d, J = 7.8 Hz),
4.65 (1H, m), 4.43, 4.38 (2H, each d, J = 11.7 Hz), 4.09
(1H, dd, J = 9.3, 8.3 Hz), 3.95 (1H, d, J = 2.4 Hz), 3.68–
3.59 (4H, m), 3.05 (2H, d, J = 5.4 Hz). 13C NMR
(100 MHz, CDCl3): d 171.2, 156.5, 155.5, 138.4, 138.3,
138.2, 137.7, 136.1, 134.9, 130.2, 129.3, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.5, 127.4,
116.9, 101.8, 81.9, 79.0, 75.2, 74.4, 73.7, 73.4, 73.2, 72.9,
68.6, 67.1, 66.8, 54.8, 37.2. HRMS calcd for
C58H58NO10 [M+H]+ 928.4061, found 928.4043.
4.15. Insects
Larvae of the inbred P50 strain of B. mori, a gift from T.
Shimada (University of Tokyo, Japan), were reared on
mulberry leaves and maintained under a 16L:8D photo-
period at 25 ꢁC. Bioassays were performed using 5–10
female pupae 3 days before eclosion (day ꢁ3). Pupal
age was determined based on the morphological charac-
teristics as described.7
4.12. 4-O-(b-D-Galactopyranosyl)-D-tyrosine (6)
Treatment of 25 (100 mg, 0.11 mmol) as described for
preparation of 2 yielded 6 (24 mg, 63%) as a white powder.
25
Compound 6. ½aꢀ ꢁ17.6 (c 0.10, H2O). IR (neat) 3250,
D
1653, 1559, 1509, 1420, 1231, 1051 cmꢁ1
.
1H NMR
4.16. Bioassay in conjunction with RT-PCR
(400 MHz, D2O): d 7.28 (2H, d, J = 8.3 Hz), 7.16 (2H,
d, J = 8.3 Hz), 5.07 (1H, d, J = 7.3 Hz), 4.00 (1H, d,
J = 2.9 Hz), 3.97 (1H, dd, J = 7.8, 5.4 Hz), 3.88 (1H, dd,
J = 6.3, 5.9 Hz), 3.84–3.75 (4H, m), 3.25 (1H, dd,
J = 14.6, 5.4 Hz), 3.10 (1H, dd, J = 14.6, 7.8 Hz). 13C
NMR (100 MHz, D2O): d 174.8, 156.9, 131.5, 130.3,
117.7, 101.4, 76.2, 73.3, 71.3, 69.2, 61.5, 56.8, 36.3; HRMS
calcd for C15H22NO8 [M+H]+ 344.1345, found 344.1368.
To examine the activity that stimulates transcription of
pgACBP, test samples were diluted to 2 lL (50 lg/lL)
with phosphate-buffered saline (PBS: 137 mM NaCl,
2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4
(pH 7.2)) and injected into 5–10 female pupae (day
ꢁ3). After maintaining for 18 h at 25 ꢁC, the injected pu-
pae were dissected and their abdominal tips were excised
into insect Ringer’s solution (35 mM NaCl, 36 mM KCl,
12 mM CaCl2, 16 mM MgCl2, 274 mM glucose, and
5 mM Tris–HCl (pH 7.5)) for preparation of trimmed
PGs.20 For RT-PCR, total RNA was prepared from
trimmed PGs by the method of Chomoczynski and Sac-
chi.21 First-strand cDNA was synthesized from total
RNA (500 ng) by using the RNA PCR kit (Takara
Bio Inc., Japan) according to the manufacturer’s
instructions. Specific oligonucleotide primer pairs (for
pgACBP, pgACBP-F1 [sense primer]: 50-ATCAATATG
TCTCTCCAAGAAAAA-30 and pgACBP-R1 [anti-
4.13. 4-O-(2,3,4,6-Tetra-O-benzyl-b-D-galactopyranosyl)-
N-benzyloxycarbonyl-L-tyrosine benzylester (26)
Treatment of 18 (794 mg, 1.47 mmol) and 13 (476 mg,
1.17 mmol) as described for preparation of 10 from 8
and 9 yielded 24 {702 mg, 64% (a/b = 1/5)}.
A reversed-phase HPLC separation (acetonitrile/
water = 10:1) of 24 (524 mg) gave 26 (288 mg, 55%) as
a white solid.

S. Takahashi et al. / Bioorg. Med. Chem. 15 (2007) 97–103
103
sense primer]: 50-CTTTTATTCTTTGAGGCCGATG-
GA-30) were designed based on their published sequenc-
es. PCR conditions consisted of 25 cycles of 94 ꢁC for
30 s, 55 ꢁC for 30 s, and 72 ꢁC for 30 s. Following
PCR, 5 lL aliquots were run on a 1.2% agarose gel in
TAE buffer.
´
5. Fonagy, A.; Yokoyama, N.; Okano, K.; Tatsuki, S.;
Maeda, S.; Matsumoto, S. J. Insect Physiol. 2000, 46, 735–
744.
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6. Fonagy, A.; Yokoyama, N.; Matsumoto, S. Arthropod
Struct. Dev. 2001, 30, 113–123.
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7. Matsumoto, S.; Fonagy, A.; Yamamoto, M.; Wang, F.;
Yokoyama, N.; Esumi, Y.; Suzuki, Y. Insect Biochem.
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8. Ohnishi, A.; Hull, J. J.; Matsumoto, S. Proc. Natl. Acad.
Sci. U.S.A. 2006, 103, 4398–4403.
Acknowledgments
9. Ohnishi, A.; Koshino, H.; Takahashi, S.; Esumi, Y.;
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Agric. Biol. Chem. 1981, 45, 687–692.
We are grateful to Dr. Y. Hongo (RIKEN) for mass
spectral measurements. This work was supported in part
by the Chemical Biology Project (RIKEN) and Ministry
of Education, Culture, Sports, Science and Technology
of Japan Grants-in-Aid for Scientific Research (B)
(17380041).
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