Affinity purification and characterization of a key enzyme responsible for circadian rhythmic control of nyctinasty in Lespedeza cuneata L

Article abstract of DOI:10.1016/j.bmc.2008.02.035

The synthesis of an affinity gel aimed at leaf-opening factor β-glucosidase (LOFG) and affinity purification of LOFG is presented. A gluconamidine-based β-glucosidase inhibitor was used as the ligand of the affinity gel. β-Glucosidase exhibiting an activi

Full text of DOI:10.1016/j.bmc.2008.02.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4600–4616
Affinity purification and characterization of a key enzyme
responsible for circadian rhythmic control of nyctinasty
in Lespedeza cuneata L.
Eisuke Kato, Takehiko Sasaki and Minoru Ueda*
Department of Chemistry, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 4 January 2008; revised 11 February 2008; accepted 11 February 2008
Available online 15 February 2008
Abstract—The synthesis of an affinity gel aimed at leaf-opening factor b-glucosidase (LOFG) and affinity purification of LOFG is
presented. A gluconamidine-based b-glucosidase inhibitor was used as the ligand of the affinity gel. b-Glucosidase exhibiting an
activity shift throughout the day was selectively purified from Lespedeza cuneata Don by the affinity gel. The resulting LOFG exhib-
ited high substrate specificity toward the leaf-opening factor.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
enzyme, leaf-opening factor b-glucosidase (LOFG),
whereas that of 2 remains nearly constant during the
day (Fig. 1).6 This implies that LOFG plays a key role
in the diurnal control of nyctinasty. In this paper, we de-
scribe affinity purification and some biochemical proper-
ties of LOFG.
A b-glucosidase is responsible for the storage and release
of bioactive substances such as flavonoids, steroid sapo-
nins, and plant hormones in plants.1,2 Glucosylation is
thus important in controlling the concentrations of
numerous bioactive substances in vivo. These com-
pounds are converted to their corresponding b-gluco-
sides and stored in the vacuole where their internal
concentration is strongly affected by the activity of b-
glucosidase.
2. Results and discussion
2.1. Syntheses of affinity gels with glucosidase inhibitor
ligands
Recently, the glucosylation–deglucosylation cycle has
been confirmed as being involved in the control of the
circadian rhythms in plant leaf-movement.3,4 Legumi-
nous plants close their leaves at night and open them
in the morning according to the circadian rhythm. This
rhythmic movement of the leaves is referred to as nycti-
nasty, which is controlled by a pair of leaf-movement
factors, leaf-opening factor (LOF) and leaf-closing fac-
tor (LCF) (Fig. 1).3,4 In Lespedeza cuneata, potassium
lespedezate (1)5 and potassium D-idarate (2)6 have been
identified as the LOF and LCF, respectively. Diurnal
variations in the relative endogenous concentrations of
LOF and LCF produce the rhythm in nyctinasty: the le-
vel of 1 in the plant body is controlled by the metabolic
Affinity chromatography7,8 has been used extensively
for the purification of enzymes. In some instances, inhib-
itors of the corresponding enzymes have been used effec-
tively as affinity ligands. For example, the amidine-type
b-glucosidase inhibitor9 b-glycosidase, which is also
found in nature,10–12 has been developed by Ganem,13–15
Tatsuta,16 Wong,17 Heck et al.,18 and Hiratake co-work-
ers19,20 In particular, Ganem’s glyconamidine (3) and
Hiratake’s glycosylamidine (4) are both strong inhibi-
tors of b-glucosidase at micromolar levels. Glycosidases
are classified into 90 families21 and each family contains
a number of glycosidases with different substrate speci-
ficities; several of the b-glucosidases exhibit strong sub-
strate specificity due to the structure of the aglycon
moiety. Thus, for affinity purification of substrate-spe-
cific glycosidase, a glycosidase inhibitor with a specific
aglycon structure is required as a ligand. We designed
Keywords: b-Glucosidase; Inhibitor; Gluconamidine; Affinity purifica-
tion; Nyctinasty.
*
Corresponding author. E-mail: ueda@mail.tains.tohoku.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.035

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4601
responding bromide or chloride instead of 15, the cou-
pling reaction gave a complex mixture because of the
instability of these substrates under basic conditions.
Importantly, our method gave only gluconamidine with-
out the by-production of mannoamidine. This was be-
cause the free hydroxyl group at the 20-position of 17,
which exists as an alkoxide-form under the coupling
condition, prevents the abstraction of the 20-H from
the resulting 17 and subsequent formation of undesir-
able mannoamidine. Deprotection of 17 with trifluoro-
acetic acid (TFA) resulted in good yields of 18. The
geometry of the olefin and the stereochemistry of the
N1-alkyl amidine moiety in 18 were confirmed to be
0
anti-(Z) by NOE correlations between H2 and H2 , and
0
H2 and H3 (Scheme 1). Then, 18 was photoisomerized
into a 1:1 mixture of (Z)-18 and (E)-5, with the desired
(E)-5 was isolated by HPLC. The small NOE between
Figure 1. Leaf-opening factor b-glucosidase (LOFG) controlling the
balance between the concentration of two leaf-movement factors (1
and 2) in the plant body.
0
H2 and olefinic H demonstrated that the geometry of
olefin in 5 is (E) and the stereochemistry of the amidine
moiety is anti (Scheme 1).
b-glucosidase inhibitors 5 and 6 based on the structure
of leaf-opening factor 1. These two amidines are ex-
pected to function well as ligands for affinity purification
of LOFG.
We also synthesized an N1-alkyl glycosylamidine analog
of 1 (6a and 6b) according to a modification of Hira-
take’s method.19 A thioamide aglycon (24) was synthe-
sized from 19 as shown in Scheme 2. Then, formation
of an N1-alkyl glycosylamidine moiety was conducted
using one-pot synthesis: methylation of 24 by Meerw-
ein’s salt and subsequent addition of protected b-gluco-
sylamine (25) with NBS gave 26 as an anti-isomer. After
deprotection, the N1-alkyl glycosylamidine analog of 1
(6a and 6b) was obtained as a mixture of syn- and
anti-isomers, which isomerize easily and could not be
isolated (Scheme 2). Our one-pot synthesis of the N1-al-
kyl glycosylamidine using Meerwein’s salt would be
widely applicable to the syntheses of other glycosylami-
dine-type inhibitors.
Synthesis of N1-alkyl gluconamidine 5 was carried out
as shown in Scheme 1. Syntheses of N1-alkyl glyconam-
idine were reported by Ganem15 and Vasella.22–24 How-
ever, these known methods produced a mixture of
gluconamidine and mannoamidine. The mannoamidine,
an epimer of gluconamidine, was produced under basic
coupling conditions through epimerization of the acidic
20-H in the resulting gluconamidine. We developed a
concise method for the synthesis of N1-alkyl glyconam-
idine25 using a non-protected thionolactam (7) prepared
according to Vasella’s method (Scheme 1).22–24
p-Hydroxycinnamic acid (8) was fully protected26 and
diol was introduced to the olefin moiety by osmium
tetroxide to give 10. After deprotection, the a- and b-hy-
droxyl groups in 10 were substituted with azide and fluo-
ride, respectively, to give 14. Reduction of 14 gave 15,
which was coupled with thionolactam (7) using N-bro-
mosuccinimide (NBS) to give a mixture of 16 and 17.
Fluoride 16 can be converted to 17 with triethylamine
(TEA). Coupling product 17 was obtained in total yields
of 94% with formation of olefin and deprotection of
TBS group. An excess amount of 15 (ca. 10 equiv) was
essential for a good yield. When the methyl ester of
the corresponding amine was used, the complex oligo-
mer of the amine produced by self-condensation was ob-
tained. The use of fluoride (15) was essential for
obtaining the coupling product. When we used the cor-
We examined the inhibitory activities of synthetic ami-
dine analogs (5, 6, and 18) against various glycosidases
(Table 1). The Ki values were determined against several
a-glucosidases and b-galactosidases using a Dixon plot.27
Interestingly, a distinct difference was observed in the Ki
values of (E)-5 and (Z)-18 against b-glucosidase from
Aspergillus niger. The Ki value of (Z)-18 was
5.8 · 10ꢀ5 M, whereas that of (E)-5 was 1.6 · 10ꢀ6 M
(Table 1). The inhibitory activity of 5 was 40-fold stron-
ger than that of 18. Additionally, the Ki value of 6 against
b-glucosidase from A. niger was determined to be
1.3 · 10ꢀ6 M, which is as strong as (E)-5. Weak or no
inhibition was observed against a-glucosidase for both
5 and 6. Extremely high-specific inhibitory activity
against b-glucosidase from A. niger was observed for 5
and 6, whereas 18 inhibited b-glucosidase as strongly as
a-glucosidase (Table 1); none of the amidines inhibited
b-galactosidase. We developed micromolar b-glucosidase
inhibitors (5 and 6) which are expected to be effective
affinity ligands for the purification of LOFG. The inhib-
itory activity of each amidine against b-glucosidase varies
with pH, with the optimum pH for 5 and 6 being 5.5 and
5.0, respectively (Table 2).
Stabilities of synthetic amidines are also important when
applied as affinity ligands. We therefore examined the

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E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
Scheme 1. Synthesis of affinity gel with gluconamidine-type LOF 5.
Scheme 2. Synthesis of affinity gel with glucosylamidine-type LOF 6.
stabilities of 5 and 6 in aqueous solutions at a variety of
pH values. Both 5 and 6 were extremely stable up to pH
6.5. However, 5 was unstable in an aqueous solution
above pH 7, while 6 was stable over a wide range of
pH from 4.5 to 9.0. These results suggest that com-
pounds 5 and 6 are sufficiently effective as inhibitors
and stable for practical application as useful affinity li-
gands under neutral or weakly acidic pH.

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4603
Table 1. Inhibitory activities of 5, 6, and 18 against glycosidases. Ki values were measured in 50 mM acetate buffer at optimal pH of each enzyme (pH
5.0 for b-glucosidases and b-galactosidase, pH 6.8 for a-glucosidase)
Enzyme
Ki (lM)
5
6
18
b-Glucosidase (from Almond)
255
1.6
Less than 90% inhibition at 1 mM
1.3
540
58
b-Glucosidase (from Aspergillus niger)
a-Glucosidase (from Bacillus stearothermophilus)
b-Galactosidase (from Aspergillus oryzae)
436
No inhibition at 1 mM
No inhibition at 1 mM
No inhibition at 1 mM
52
No inhibition at 1 mM
Next, we synthesized two affinity gels (27 and 46) with
LOF-based inhibitors 5 and 6 as ligands (Schemes 3
and 4). Synthesis of gluconamidine-type affinity gel 27
is shown in Scheme 3. Affinity gel 27 has a linker moiety
on the phenolic hydroxyl group which connects gluco-
namidine ligand 5 to the gel. Direct introduction of lin-
ker unit (28) to 5 gave a complex mixture because of the
instability of the amidine moiety under coupling condi-
tions. Therefore, the linker must be introduced before
the formation of the amidine moiety. Compound 10
was coupled with linker 28 containing the protected
thiol group. Then, the a- and b-hydroxyl groups were
substituted with azide and fluoride, respectively, to give
32. Reduction of the azide in 32 gave 33, which was cou-
pled with thionolactam (7) using NBS to give 34. Succes-
sive deprotection and photoisomerization with UV light
(312 nm) gave a 1:1 mixture of 35 and 36, with the de-
sired 36 obtained by HPLC purification. Stereochemis-
try of the olefin moiety of 36 was defined by
comparison of spectral data with 5. The synthetic ligand
36 was then coupled with gel 37 containing the malei-
mide moiety, which was prepared from the commer-
cially available Toyopearl AF-Amino-650M, to give
affinity gel 27. Similarly, glucosylamidine-type affinity
gel 46 was synthesized using a slight modification of
the method used for 27 (Scheme 4) using Toyopearl
AF-Carboxyl-650M.
Table 2. Inhibitory activity of 5 and 6 against b-glucosidase from
Aspergillus niger under various pHs
pH
Ki (lM)
2.2. Affinity purification of LOFG
5
6
4.5
5.0
5.5
6.0
1.64
1.60
1.51
3.50
1.30
1.33
4.12
We examined the affinity purification of LOFG from
L. cuneata using affinity gels 27 and 46. Preliminary
purification of LOFG using 27 was partially communi-
cated in Ref. 28.
12.9
Scheme 3. Synthesis of affinity gel with gluconamidine-ligand (27).

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E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
Scheme 4. Synthesis of affinity gel with glucosylamidine-ligand (46).
and HPLC analysis of 49 can be used to reliably mea-
sure b-glucosidase activity using 1. All of the subse-
quent purifications are based on this analytical
method.
Our previous study showed that LOFG activity in the
plant body increased in the evening.6 Therefore, it is
important to determine when LOFG is activated in L.
cuneata before attempting purification thereof. We pre-
pared a series of acetone powders from the leaves of
L. cuneata as a crude enzyme containing LOFG by col-
lecting leaves every 2 h from 10:00 to 20:00 h. Each ace-
tone powder was extracted with 0.1 M Bis–Tris buffer
(pH 7.0), and LOFG activity of each extract was mea-
sured using 1 as a substrate. Analysis of 49, which is
formed by the in situ coupling of 47 and 48 in aqueous
solution,30 revealed a remarkable increase in LOFG
activity between 14:00 and 16:00 h (Fig. 2). Thus, fur-
ther purification was conducted using acetone powder
prepared from the plants that were sampled between
14:00 and 16:00 h.
Figure 2. Diurnal fluctuation in b-glucosidase activity in the leaves of
Lespedeza cuneata L.
Before initiating affinity purification, we established a
reliable method for quantitative analysis of LOFG
activity using 1 as a substrate (Scheme 5). a-Keto acid
47, a hydrolytic product of 1, was unstable and easily
decomposed in aqueous solution, which meant that
reproducible measurement of LOFG activity using 1
has so far been difficult. Therefore, we developed a no-
vel method for the quantitative analysis of unstable a-
keto acids. a-Keto acid 47 can be converted quantita-
tively into stable semicarbazone (49) (Scheme 5),28,29
The acetone powder collected between 14:00 and 16:00 h
was extracted with Bis–Tris buffer (pH 7.0), and the pro-
teins were precipitated with acetone. The resulting pre-
cipitate was dissolved in Bis–Tris buffer (pH 7.0) and
then partially purified by anion exchange chromatogra-
phy. All fractions with b-glucosidase activity were col-
lected and subsequently applied to an affinity column
with gel 27 or 46. After washing the gel with sodium ace-
tate buffer (pH 5.0), sodium acetate buffer (pH 5.0) con-
taining 10 mM 1 was applied to the gel to elute the
adsorbed LOFG. No LOFG activity was eluted by D-
glucose because of the strong affinity of the ligand with
LOFG. Next, the fraction containing LOFG was ap-
plied to gel-filtration chromatography to remove 1.
The overall purification of LOFG is summarized in
Scheme 6.
Purified LOFG using gel 27 showed a glucosidase activ-
ity of 4.8 U/mg protein against 1; a 240-fold enhance-
Scheme 5. In situ formation of semicarbazone (49) from a-keto acid
(47) for quantitative analysis of LOFG activity.

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4605
Figure 3. Purification of LOFG (A) SDS–PAGE analysis of LOFG [1: MW marker, 2: purified LOFG], (B) HPLC analysis of LOFG [column: TSK-
Gel SuperSW3000; eluent: 50 mM phosphate, 150 mM sodium chloride buffer (pH 7.0)], (C) RP-HPLC analysis of LOFG [column: Sephasil protein
C4; eluent: linear gradient from 2% CH3CN–0.065% TFA to 65% CH3CN–0.065% TFA in 50 min; flow rate: 1.0 mL/min; detection: 215 nm, temp:
40 ꢁC].
ment of activity compared with the first extract.28 One
enzyme unit of LOFG was defined as the quantity of en-
zyme required to hydrolyze one micro mole of 1 in 1 min
under optimum pH conditions at 30 ꢁC. LOFG of sim-
ilar purity was also obtained using affinity gel 46. SDS–
PAGE analysis of the resulting LOFG gave a smear
band at approximately 150 kDa (Fig. 3A), whereas this
LOFG produced a single sharp peak in gel-filtration
HPLC analysis using TSK-Gel SuperSW3000 (Fig. 3B)
and RP HPLC analysis (Fig. 3C). The molecular weight
of LOFG estimated from the elution volume in gel-fil-
tration HPLC corresponded to that observed using
SDS–PAGE.
The MW of normal b-glucosidase is approximately
60 kDa.20,31–36 Since LOFG is considerably larger than
normal b-glucosidase, we assumed that LOFG under-
goes some type of post-translational modification. We
examined the presence of the sugar chain on LOFG be-
cause many of the glycoproteins are known to exhibit a
smear band on SDS–PAGE.37–40 A modification of
O’Shannessy’s method41 was employed for the detec-
tion of the sugar chain, and LOFG was subjected to
SDS–PAGE with Western blotting on a polyvinylidene
difluoride (PVDF) membrane. The membrane was first
treated with 10 mM NaIO4 and then with 0.1 mM biot-
inhydrazide to biotinylate the sugar chain of the glyco-
protein. While chemiluminescence detection with
streptavidin-horseradish peroxidase (HRP) conjugate
clearly demonstrated the presence of a sugar chain on
LOFG (Fig. 4), treatment of LOFG with N-glycopepti-
dase F,42 which is commonly used for the deglycosyla-
tion of glycoproteins, did not produce any successful
results (Fig. 5). We therefore examined the chemical
dissociation of the sugar chain using trifluoromethane
sulfonic acid (TfOH).43,44 Purified LOFG was treated
with TfOH and analyzed by SDS–PAGE. The smear
Scheme 6. Summary of purification of LOFG from the leaves of
Lespedeza cuneata L. G. Don.

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E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
Figure 4. Detection of the sugar chain in LOFG. (Left) membrane
stained by colloidal gold total protein stain (Bio-Rad Laboratories).
(Right) biotinylated sugar chain detected by chemiluminescence (1,
marker protein; 2, LOFG; 3, BSA as negative control; 4, ovalbumin as
a positive control).
band produced by LOFG of approximately 150 kDa on
SDS–PAGE shifted to a smaller, sharper band which at
approximately 60 kDa. In addition, gel-filtration HPLC
analysis of the TfOH-treated LOFG also produced a
single, sharp peak at approximately 60 kDa (Fig. 5).
Therefore, it was concluded that LOFG is a 60 kDa
protein which was subjected to post-translational glyco-
sylation by plural sugar chains whose total mass would
be about 90 kDa.
We then sought to undertake the de novo sequence anal-
ysis of LOFG. In general, de novo sequence analysis by
MALDI-TOF MS produces reliable results when a pro-
tein can be supplied in sufficient quantities and is detect-
able by Coomassie brilliant blue (CBB) staining.
However, the amount of LOFG obtained using 27 was
so limited that de novo sequence analysis was unable.
In addition, affinity gel 27 gradually decomposed during
use, and affinity to LOFG decreased abruptly due to re-
peated use. Although a large amount of gel 27 is re-
quired for large-scale purification of LOFG, the
supply is limited because of the low total yield associated
with the synthesis of 27.
Figure 5. Gel-filtration analysis of LOFG by using TSK-gel Super
SW3000 (A). Molecular weight standards [IgG: 150 kDa, BSA:
68 kDa, carbonic anhydrase (CA): 34 kDa, cytochrome C (CytC):
12 kDa], (B) intact LOFG, (C) LOFG treated with N-glycopeptidase
F, (D) LOFG treated with TfOH.
We compared the total synthetic yields of two affinity
gels: ligand 36 was synthesized in 13 steps with a total
yield of 0.4% from commercially available 8 (Schemes
1 and 3), while ligand 44 required only seven steps and
had a total yield of 18% from commercially available
19 (Schemes 2 and 4). Therefore, for the large-scale
purification of LOFG, we conducted large-scale syn-
thesis of affinity gel 46 and used it instead of 27 be-
cause it was highly stable, sufficiently durable for
repeated use, and could be supplied in sufficiently large
quantities because of the high synthetic yield. We ob-
tained 1.1 · 10ꢀ1 unit of purified LOFG using 46,
which was sufficient for further biochemical examina-
tion and de novo sequence analysis by MALDI-TOF
MS.
Purified LOFG was hydrolyzed using trypsin in H2O
and H218O, respectively, and the two hydrolyzates
were analyzed by MS/MS. Digestion in H218O labels
the C-terminal of the fragment peptide and enables
differentiation between fragments based on the N-
and C-terminals in the MS/MS experiment. Compari-
son of obtained data gave partial sequences from
which the de novo sequence was elucidated (Table
3). The obtained partial sequences showed that the se-
quence of LOFG is highly similar to b-glucosidase of
Family III.21

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4607
Table 3. Partial amino acid sequence of LOFG by de novo sequence analysis by MS/MS experiment
Entry
m/z
(N-Terminus) partial sequence from MS/MS (C-terminus)
1
2
1626
1827
1727
1856
1568
1206
1462
1485
1184
1018
(511) (I or L) DEV (I or L) SA (I or L) (Q or K) K C
(370) (RD)V (I or L) DCPV (Q or K) (I or L) SR
(155) GFSY (I or L) SS PE VAF (I or L) R
3
4
(574) Y (I or L) SS [PE or (I or L) (I or L)] VAF (I or L) R
[(379)N or (396)P] VA (I or L) (83) (Q or K) GVYSR
(Q or K) [D or (44)A] (72) AYYYVGR
5
6
7
(271) V (Q or K) (I or L) G (196) V (74) (193) GR
[(543)P or (526)N] E (Q or K) EHFR
8
9
10
(214) (168) (I or L) (I or L) NPFAK or (214) (168) (I or L) (I or L) NDEAK
(562) (I or L) VPK
Table 4. Relative activity of LOFG against various substrates
Entry Substrate
Relative activity
1
2
3
4
5
6
Potassium lespedezate (1)
Potassium isolespedezate (47)
48
100
0
26
0
Potassium galactolespedezate (49)
Potassium galactoisolespedezate (50)
51
0
113
parable to other substrate-specific b-glucosidases.32–35,45
As shown in the comparison with potassium isolespe-
dezate, LOFG recognized the olefin moiety of 1 (47)5
and 485 (Table 4). Potassium galactolespedezate (49)46
and potassium galactoisolespedezate (50),46 which have
galactose instead of glucose, could not be hydrolyzed.
On the other hand, the hydroxymethyl analog of 1
(51) was a good substrate for LOFG. These results
showed that LOFG recognizes an aglycon moiety to-
gether with a glycon moiety, and that it showed high
substrate specificity toward 1.
We also examined the substrate specificity of the LOFG
(Fig. 6 and Table 4). The Km value of LOFG against 1
was determined to be 548 lM at pH 4.5, which is com-
In conclusion, we have succeeded in the affinity purifica-
tion of a candidate of LOFG, a key enzyme involved in
Figure 6. Substrate specificity of LOFG ((a) analogs containing the olefine moiety, (b) analogs containing the sugar moiety, (c) analog containing the
carboxylate).

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E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
nyctinasty. The glucosylamidine ligand 6, designed
based on the structure of 1 and synthesized easily in
short steps, showed strong inhibitory activity toward
LOFG and was relatively effective for the selective puri-
fication of LOFG. It was also shown that the obtained
LOFG strongly recognized an aglycon part of the sub-
strate and that it exhibited high substrate specificity to-
ward 1. Research currently being undertaken is directed
at cloning LOFG. It is important for the identification
of LOFG to observe the circadian rhythm of the protein
expression of this enzyme. A molecular genetic ap-
proach will reveal the mechanisms involved in the circa-
dian rhythmic control of LOFG activity and how it was
controlled by a biological clock.
1H NMR (300 MHz, CDCl3, rt): 7.55 (1H, d,
J = 15.6 Hz), 7.51 (2H, d, J = 8.4 Hz), 7.10 (2H, d,
J = 8.4 Hz), 6.32 (1H, d, J = 15.6 Hz), 2.30 (3H, s),
1.53 (9H, s) ppm; 13C NMR (75 MHz, CDCl3, rt):
169.1, 166.2, 151.8, 142.4, 132.4, 129.0, 122.0, 120.3,
80.6, 28.2, 21.1 ppm; HR ESI-MS (positive):
[M+Na]+. Found m/z 285.1098, C15H18NaO4 requires
þ
m/z 285.1097; IR (film) m: 2978, 2934, 1769, 1709,
1638, 1508, 1369 cmꢀ1
.
3.1.2. Synthesis of tert-butyl 2,3-dihydroxy-3-(p-hydroxy-
phenyl)propionate (10). To a solution of 9 (13.05 g,
49.8 mmol) and N-methylmorpholine N-oxide (12.82 g,
109.6 mmol) in H2O/acetonitrile = 1:2 (450 mL), OsO4
dissolved in tert-butanol (125 mg/25 mL) was added at
0 ꢁC under argon atmosphere. After stirring for 18 h,
saturated Na2SO3 aq (30 mL) was added. The organic
layer was separated and the aqueous layer was extracted
with EtOAc. The combined organic layer was washed
with brine, dried over sodium sulfate and concentrated
under vacuum. The residue was dissolved in MeOH
(270 mL) and sodium methoxide (1.10 g, 20.4 mmol)
was added and stirred for 11 h under argon atmosphere.
The reaction mixture was neutralized by Amberlite-
IR120B (H+) and filtered. Evaporation of the filtrate
gave 10 (13.1 g, quant.) as a colorless oil.
3. Experimental
3.1. General procedures
NMR spectra were recorded on a Jeol JNM-AL300 [1H
(300 MHz) and 13C (75 MHz)] using TMS in CDCl3,
CD2HOD in CD3OH (1H; 3.33 ppm, 13C; 49.8 ppm),
or t-BuOH (1H; 1.23 ppm, 13C; 32.1 ppm) in D2O as
internal standards at various temperatures. The ESI-
MS and HR ESI-MS spectra were recorded on a Bruker
Esquire 3000 plus and a Bruker APEX-III, respectively.
The IR spectra were recorded on a JASCO FT/IR-410.
The specific rotations were measured by JASCO DIP-
360 polarimeter. The HPLC purification was carried
out with a Shimadzu LC-6A pump equipped with
SPD-6A detector using COSMOSIL 5C18-AR column
(/ 20 · 250 mm) (Nakalai Tesque Co. Ltd). The sol-
vents used for HPLC were available from Kanto Chem-
ical Co. and were filtered through a Toyo Roshi
membrane filter (cellulose acetate of 0.45 lm pore size,
47 mm dia.) before use. Silica gel column chromatogra-
phy was performed on Silica Gel 60 K070 (Katayama
Chemical Co. Ltd) or Silica Gel 60N (Kanto Chemical
Co. Ltd). Reversed-phase open-column chromatogra-
phy was performed on Cosmosil 75C18-OPN (Nakalai
Tesque Co. Ltd). TLC was performed on Silica gel
F254 (0.25 mm or 0.5 mm, Merck) or RP-18F254S
(0.25 mm, Merck). Purification of the protein was per-
1H NMR (300 MHz, CD3OD, rt): 7.19 (2H, d,
J = 8.4 Hz), 7.74 (2H, d, J = 8.4 Hz), 4.68 (1H, d,
J = 5.7 Hz), 4.08 (1H, d, J = 5.7 Hz), 1.31 (9H, s) ppm;
13C NMR (75 MHz, CD3OD, rt): 173.2, 158.2, 132.8,
129.5, 115.8, 82.6, 75.6, 76.5, 28.1 ppm; HR ESI-MS
(positive): þ[M+Na]+.
Found
m/z
277.1047,
C13H18NaO5 requires m/z 277.1046; IR (film) m: 3410,
2979, 2935, 1720, 1616, 1599, 1518, 1371 cmꢀ1
.
3.1.3. Synthesis of tert-butyl 2,3-dihydroxy-3-(p-(tert-
butyldimethylsilyloxy)phenyl)-propionate (11). Com-
pound 10 (64.7 mg, 0.255 mmol) was dissolved in THF
(2.5 mL) and TEA (71 lL) was added. To this solution,
TBSCl (43.8 mg) was added at 0 ꢁC under argon atmo-
sphere. After stirring for 24 h, the mixture was evapo-
rated to dryness. The residue was purified by silica gel
column chromatography with n-hexane/EtOAc = 1:1 to
give 11 (63.1 mg, 67%) as a colorless oil.
¨
formed on an AKTA S-100 FPLC system (GE Health-
care Co.) equipped with various columns.
1H NMR (300 MHz, CD3OD, rt): 7.26 (2H, d,
J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.71 (1H, d,
J = 5.7 Hz), 4.09 (1H, d, J = 5.7 Hz), 1.32 (9H, s), 0.98
(9H, s), 0.18 (6H, s) ppm; 13C NMR (75 MHz, CD3OD,
rt): 165.9, 156.5, 135.0, 129.5, 120.7, 82.6, 77.4, 76.3, 28.1,
26.1, 19.0, ꢀ4.3 ppm; HR ESI-MS (positive): [M+Na]+.
Found m/z 391.1913, C19H32NaO5Si+ requires m/z
3.1.1. Synthesis of tert-butyl trans-p-O-acetyl coumarate
(9). trans-p-Coumaric acid (8) (5.30 g, 32.3 mmol) was
dissolved in pyridine (200 mL) and Ac2O (100 mL) was
added at 0 ꢁC under argon atmosphere. After stirring
overnight at room temperature, water was added and
the resulting solution was evaporated to dryness. To
the residue tert-butyl trichloroacetimidate (24.68 g,
112.9 mmol) and THF (150 mL) was added. To the
solution, BF3ÆEt2O (0.8 mL, 6.31 mmol) was added at
0 ꢁC under argon atmosphere. After stirring for
30 min, sodium hydrogen carbonate (0.73 g) was added
and stirred for another 10 min. The reaction mixture
was filtered and concentrated under vacuum. The resi-
due was purified by silica gel column chromatography
with n-hexane/EtOAc = 5:1 to give 9 (6.01 g, 71%) as a
colorless powder.
391.1911; IR (film) m: 3369, 1695, 1608, 1508 cmꢀ1
.
3.1.4. Synthesis of tert-butyl 3-(p-(tert-butyldimethylsi-
lyloxy)phenyl)-3-hydroxy-2-(p-nitrobenzenesulfonyl)pro-
pionate (12). To a stirred solution of 11 (520.5 mg,
1.41 mmol) and TEA (0.4 mL, 2.87 mmol) in dichloro-
methane (15 mL), p-nitrobenzenesulfonyl chloride
(347.3 mg, 1.57 mmol) was added at 0 ꢁC under argon
atmosphere. After stirring for 3 h, the reaction mixture
was concentrated and the residue was purified by silica

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4609
gel column chromatography with n-hexane/EtOAc = 3:1
to give 12 (621.8 mg, 80%) as a yellow solid.
14 (232.5 mg, 0.587 mmol) in MeOH (6 mL) 10% Pd–
C was added and stirred for 12 h under hydrogen atmo-
sphere. The reaction mixture was filtered on Celite and
the filtrate was concentrated to dryness to give crude
15. Crude 15 was then dissolved in DMF (5 mL) and 7
(12.6 mg, 0.065 mmol) was added to this solution. To
this mixture, NBS (36.5 mg, 0.21 mmol) was added at
0 ꢁC under argon atmosphere. After 1 h the reaction
mixture was dried and the residue was purified by silica
gel column chromatography with CHCl3/MeOH = 9:1
followed by elution with CHCl3/MeOH = 3:1 to give
16 (13.2 mg, 40%) and 17 (13.9 mg, 54%) as a colorless
oil. The obtained 16 (13.2 mg, 0.025 mmol) was then
stirred with TEA (5 lL, 0.034 mmol) in DMF (0.2 mL)
for 1 h to give additional 17 (9.9 mg, total 94%).
1H NMR (300 MHz, CDCl3, rt): 8.30 (2H, d,
J = 9.0 Hz), 7.92 (2H, d, J = 9.0 Hz), 7.13 (2H, d,
J = 8.4 Hz), 6.71 (2H, d, J = 8.4 Hz), 5.10 (1H, dd,
J = 4.8, 5.4), 4.91 (1H, d, J = 4.8 Hz), 2.81 (1H, d,
J = 5.4 Hz, –OH), 1.38 (9H, s), 1.01 (9H, s), 0.20 (6H,
s) ppm; 13C NMR (75 MHz, CDCl3, rt): 165.3, 156.1,
150.4, 141.6, 130.0, 129.1, 127.7, 124.1, 120.0, 83.9,
82.9, 73.2, 27.6, 25.5, 18.1, ꢀ4.57, ꢀ4.60 ppm; HR
ESI-MS (positive): [M+Na]+. Found m/z 576.1691,
C25H35NNaO9SSi+ requires m/z 576.1694; IR (film) m:
3449, 1736, 1608, 1535 cmꢀ1
.
3.1.5. Synthesis of tert-butyl 2-azido-3-(p-(tert-butyldi-
methylsilyloxy)phenyl)-3-hydroxypropionate (13). To a
solution of 12 (507.6 mg, 0.918 mmol) in DMF (6 mL),
sodium azide (313.8 mg, 4.83 mmol) was added and stir-
red at 50 ꢁC under argon atmosphere. After 16.5 h,
water was added to this solution and the mixture was ex-
tracted by n-hexane/EtOAc = 1:1. The organic layer was
washed with brine and dried over absolute sodium sul-
fate. After evaporation, the residue was purified by silica
gel column chromatography with n-hexane/EtOAc = 6:1
to give 13 (146.3 g, 41%) as a colorless oil.
1
Compound 16: H NMR (300 MHz, CD3OD, rt): 7.39
(2H, d, J = 7.6 Hz), 6.92 (2H, d, J = 7.6 Hz), 5.85 (1H,
dd, J = 6.0, 45.6 Hz), 6.14 (1H, dd, J = 6.0, 18.6 Hz),
4.22 (1H, d, J = 9.0 Hz), 3.79 (1H, dd, J = 3.3,
11.1 Hz), 3.68 (1H, dd, J = 7.2, 8.7 Hz), 3.62 (1H, dd,
J = 8.7, 9.0 Hz), 3.60 (1H, dd, J = 4.2, 11.1 Hz), 3.44
(1H, ddd, J = 3.3, 4.2, 7.2 Hz), 1.34 (9H, s), 1.00 (9H,
s), 0.22 (6H, s) ppm; 13C NMR (75 MHz, CD3OD, rt):
166.9, 166.2 (d, J = 7.9 Hz), 158.4 (d, J = 2.4 Hz),
129.7 (d, J = 6.1 Hz), 128.8 (d, J = 20.0 Hz), 121.4,
94.4 (d, J = 178.1 Hz), 85.6, 74.0, 70.1, 69.5, 62.6, 61.7,
61.0 (d, J = 25.4 Hz), 27.9, 26.1, 19.1, ꢀ4.3 ppm; HR
ESI-MS (positive): [M+H]+. Found m/z 529.2740,
1H NMR (300 MHz, CDCl3, rt): 7.25 (2H, d, J = 8.4 Hz),
6.83 (2H, d, J = 8.4 Hz), 4.92 (1H, dd, J = 4.8, 6.6 Hz),
3.97 (2H, d, J = 6.6 Hz), 3.01 (1H, d, J = 4.8 Hz, –OH),
1.45 (9H, s), 0.98 (9H, s), 0.19 (6H, s) ppm; 13C NMR
(75 MHz, CDCl3, rt): 168.0, 155.9, 131.8, 127.9, 120.1,
83.6, 73.7, 67.1, 27.9, 25.6, 18.2, ꢀ4.5 ppm; HR ESI-
MS (positive): [M+Na]+. Found m/z 516.1975,
C19H31N3NaO4Si+ requires m/z 416.1976; IR (film) m:
C25H42FN2O7Si requires m/z 529.2740; IR (film) m:
26
3319, 1735, 1676, 1610, 1514 cmꢀ1; ½aꢁ ꢀ15.7 (c 1.00,
D
CH3OH).
1
Compound 17: H NMR (600 MHz, CD3OD, rt): 7.55
(1H, s), 7.51 (2H, d, J = 8.4 Hz), 6.81 (2H, d,
J = 8.4 Hz), 4.30 (1H, d, J = 8.4 Hz), 3.71 (1H, dd,
J = 7.2, 8.0 Hz), 3.69 (1H, dd, J = 3.0, 11.4 Hz), 3.62
(1H, dd, J = 8.0, 8.4 Hz), 3.55 (1H, dd, J = 4.2,
11.4 Hz), 3.24 (1H, ddd, J = 3.0, 4.2, 7.2 Hz), 1.54
(9H, s) ppm; 13C NMR (75 MHz, CD3OD, rt): 166.6,
163.9, 162.0, 140.4, 133.9, 124.5, 120.9, 117.1, 83.7,
74.4, 70.2, 69.8, 62.4, 61.7, 28.3 ppm; HR ESI-MS (po-
sitive): [M+H]+. Found m/z 395.1814, C19H27N2O7 re-
3449, 2931, 2960, 2110, 1735, 1608, 1512 cmꢀ1
.
3.1.6. Synthesis of tert-butyl 2-azido-3-(p-(tert-butyldi-
methylsilyloxy)phenyl)-3-fluoropropionate (14). To a stir-
red solution of 13 (106.8 mg, 0.27 mmol) in
dichloromethane (5 mL), DAST (72 lL, 0.55 mmol)
was added at 0 ꢁC. After stirring for 5 min, water was
added and the organic layer was separated. The aqueous
layer was extracted by CHCl3. The combined organic
layer was washed with brine and dried over absolute so-
dium sulfate. After evaporation, the residue was purified
by silica gel column chromatography with n-hexane/
EtOAc = 10:1 to give colorless oil 14 (56.7 mg, 53%).
quires m/z 395.1813; IR (film) m: 3186, 1655, 1603,
26
1508 cmꢀ1; ½aꢁ +11.5ꢁ (c 1.00, CH3OH).
D
3.1.8. Synthesis of (Z)-2-(N,N-glucoamidine)-3-(p-
hydroxyphenyl)acrylic acid (18). Compound 17 (1.6 mg,
4.1 lmol) was dissolved in TFA (0.5 mL) and stirred
for 30 min under argon atmosphere. The reaction mix-
ture was dried and purified by preparative TLC (RP-
18, 1% AcOH, 50% MeOH aq) to give 18 (1.2 mg,
87%) as a colorless solid.
1H NMR (300 MHz, CDCl3, rt): 7.26 (2H, d,
J = 8.4 Hz), 6.86 (2H, d, J = 8.4 Hz), 5.75 (1H, dd,
J = 5.1, 45.9 Hz), 3.94 (1H, dd, J = 5.1, 22.5 Hz), 1.41
(9H, s), 0.98 (9H, s), 0.20 (6H, s) ppm; 13C NMR
(75 MHz, CDCl3, rt): 166.0, 156.7, 127.8 (d,
J = 6.2 Hz, C-F), 127.5 (d, J = 19.7 Hz, C-F), 120.2,
93.5 (d, J = 178.8 Hz, C-F), 83.7, 66.1 (d, J = 66.6 Hz,
C-F), 27.8, 25.6, 18.2, ꢀ4.5 ppm; HR ESI-MS (positive):
[M+Na]+. Found m/z 418.1935, C19H30FN3NaO3Si+ re-
quires m/z 418.1933; IR (film) m: 2932, 2860, 2114, 1744,
1H NMR (300 MHz, CD3OD, rt): 7.59 (1H, s), 7.43
(2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.32 (1H,
d, J = 9.6 Hz), 3.75 (1H, dd, J = 7.2, 9.3 Hz), 3.69 (1H,
dd, J = 3.0, 11.7 Hz), 3.61 (1H, dd, J = 9.3, 9.6 Hz),
3.52 (1H, dd, J = 3.6, 11.7 Hz), 3.21 (1H, ddd, J = 3.0,
3.6, 7.2 Hz) ppm; 13C NMR (75 MHz, CD3OD, rt):
170.0, 165.3, 160.6, 136.4, 132.9, 125.9, 125.5, 116.8,
74.2, 70.4, 69.7, 62.3, 61.9 ppm; HR ESI-MS (positive):
[M+H]+. Found m/z 339.1189, C15H19N2O7 requires m/z
1610, 1514 cmꢀ1
.
3.1.7. Synthesis of tert-butyl (Z)-2-(N,N-glucoamidine)-3-
(p-hydroxyphenyl)acrylate (17). To a stirred solution of

4610
E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
339.1187; IR (film) m: 3207, 1668, 1606, 1558, 1514, 1379
umn chromatography with n-hexane/EtOAc = 10:1 to
give 23 (954.4 mg, quant.) as a colorless oil.
26
cmꢀ1; ½aꢁ ꢀ21.6ꢁ (c 0.50, CH3OH).
D
3.1.9. Synthesis of (E)-2-(N,N-glucoamidine)-3-(p-
hydroxyphenyl)acrylic acid (5). Compound 18 (17.2 mg,
50.9 lmol) was dissolved in MeOH (2 mL) and irradi-
ated with UV lamp (365 nm) for 1 h. The reaction mix-
ture was dried and purified by HPLC [Develosil-ODS-
MG-5, 1% AcOH/2% MeOH aq] to give 5 (7.0 mg,
30%) and 18 (4.8 mg, 18%) as colorless solids.
1H NMR (300 MHz, CDCl3, rt): 8.34 (1H, s, NH), 7.39
(2H, d, J = 9.0 Hz), 6.79 (2H, d, J = 9.0 Hz), 4.15 (2H,
s), 0.950 (9H, s), 0.947 (9H, s), 0.15 (6H, s), 0.13 (6H,
s) ppm; 13C NMR (75 MHz, CDCl3, 20 ꢁC): 168.7,
152.4, 130.8, 120.9, 120.4, 63.2, 25.8, 25.6, 18.14,
18.09, ꢀ4.52, ꢀ5.57 ppm; HR ESI-MS (positive):
[M+Na]+. Found m/z 418.2204, C20H37NNaO3Si2 re-
þ
quires m/z 418.2204; IR (film) m: 3395, 2932, 1638,
Compound 5: 1H NMR (300 MHz, D2O, rt): 7.40 (2H, d,
J = 8.4 Hz), 6.88 (2H, d, J = 8.4 Hz), 6.73 (1H, s), 4.48
(1H, d, J = 8.8 Hz), 3.891 (1H, dd, J = 8.8, 9.5 Hz),
3.893 (1H, dd, J = 3.0, 12.4 Hz), 3.84 (1H, dd, J = 9.0,
9.5 Hz), 3.75 (1H, dd, J = 4.4, 12.4 Hz), 3.59 (1H, ddd,
J = 3.0, 4.4, 9.0 Hz) ppm; 13C NMR (75 MHz, D2O,
rt): 172.0, 166.0, 158.2, 133.0, 132.5, 128.0, 126.6, 117.0,
73.5, 70.3, 68.9, 61.7, 61.6 ppm; HR ESI-MS (positive):
[M+H]+. Found m/z 339.1188, C15H19N2O7 requires
1508, 1236 cmꢀ1
.
3.1.12. Synthesis of 2-(tert-butyldimethylsilyloxy)-N-(p-
(tert-butyldimethylsilyloxy) phenyl)thioacetamide (24).
Compound 23 (620.1 mg, 1.57 mmol) and Lawesson’s
reagent (0.50 g, 1.23 mmol) were dissolved in toluene
(30 mL). This solution was stirred at 70 ꢁC for 2 h under
argon atmosphere. The reaction mixture was evaporated
to 10 mL and purified by silica gel column chromatogra-
phy with n-hexane/EtOAc = 10:1 to give 24 (559.8 mg,
87%) as a yellow solid.
m/z 339.1187; IR (film) m: 3246, 1664, 1609, 1560, 1514,
26
1411 cmꢀ1; ½aꢁ ꢀ26.1 (c 0.40, H2O).
D
3.1.10. Synthesis of 2-benzyloxy-N-(p-hydroxyphenyl)
acetamide (21). To the solution of 19 (2.5 g, 22.9 mmol)
and TEA (15 mL) in THF (150 mL), benzyloxyacetyl
chloride (10.5 g, 56.9 mmol) was added at 0 ꢁC under ar-
gon atmosphere. After stirring for 10 h, the resulting
solution was evaporated to dryness to give crude 20.
The crude 20 was then dissolved in MeOH (150 mL),
and NaOMe (3.10 g, 57.4 mmol) was added to this solu-
tion. This mixture was then stirred for 13 h at room tem-
perature under argon atmosphere. The mixture was
concentrated to 20 mL and mixed with water. This solu-
tion was then extracted by EtOAc. Organic layer was
washed with brine, dried over absolute sodium sulfate.
After evaporation, the residue was purified by silica
gel column chromatography with n-hexane/EtOAc = 2:1
to give 21 (5.90 g, quant.) as a colorless oil.
1H NMR (300 MHz, CDCl3, rt): 9.97 (1H, s, NH), 7.75
(2H, d, J = 9.0 Hz), 6.85 (2H, d, J = 9.0 Hz), 4.51 (2H,
s), 0.96 (9H, s), 0.95 (9H, s), 0.18 (6H, s), 0.14 (6H, s)
ppm; 13C NMR (75 MHz, CDCl3, 19 ꢁC): 195.7, 154.1,
131.4, 123.5 (2C), 120.1 (2C), 70.3, 25.7 (3C), 25.6
(3C), 18.1 (2C), ꢀ4.52 (2C), ꢀ5.47 (2C) ppm; HR ESI-
MS (positive): þ[M+Na]+. Found m/z434.1977,
C20H37NNaO2SSi2 requires m/z 434.1976; IR (film) m:
3300, 2954, 2530, 2858, 1508, 1400, 1261 cmꢀ1
.
3.1.13. Synthesis of N-(p-(tert-butyldimethylsilyloxy)
phenyl)-N0-(2,3,4,6-tetraacetyl-1-b-D-glucopyranosyl)-(tert-
butyldimethylsilyloxy)methylamidine (26). Compound 24
(22.8 mg, 55.5 lmol) and Me3OÆBF4 (13.5 mg,
91.2 lmol) were dissolved in dried CH2Cl2. The mixture
was stirred for 14 h at room temperature under argon
atmosphere. To the reaction mixture, molecular sieves
4A (153.5 mg) and 25 (75.3 mg, 217 lmol) were added
and the solution was stirred for 15 min. Then, mixture
was cooled to 0 ꢁC and NBS (21.4 mg, 120.2 lmol)
was added. After stirring for 2 h at 0 ꢁC, the reaction
mixture was evaporated to dryness and purified by silica
gel column chromatography with n-hexane/EtOAc = 2:1
to give 26 (35.4 mg, 89%) as a yellow oil.
1H NMR (300 MHz, CDCl3, rt): 8.19 (1H, s), 7.46 (1H,
s), 7.26 (5H, m), 7.20 (2H, d, J = 8.7 Hz), 6.68 (2H, d,
J = 8.7 Hz), 4.52 (2H, s), 4.00 (2H, s) ppm; 13C NMR
(75 MHz, CDCl3, rt): 168.6, 154.2, 136.7, 129.1, 129.0,
128.7, 128.4, 122.8, 116.1, 74.0, 69.6 ppm; HR ESI-MS
(positive): [M+Na]+. Found m/z 280.0944, C15H15NO3-
Na requires m/z 280.0942; IR (film) m: 3292, 3030,
2883, 1653, 1516, 1456, 1103 cmꢀ1
.
1H NMR (300 MHz, CDCl3, rt): 6.71 (2H, d, J = 9.0 Hz),
6.57 (2H, d, J = 9.0 Hz), 6.26 (1H, d, J = 9.0 Hz, NH),
5.43 (1H, dd, J = 9.0, 9.0 Hz), 5.31 (1H, dd, J = 9.6,
9.6 Hz), 5.11 (1H, dd, J = 9.6, 9.9 Hz), 5.05 (1H, dd,
J = 9.0, 9.6 Hz), 4.31 (1H, dd, J = 3.9, 12.6 Hz), 4.10
(1H, dd, J = 1.8, 12.6 Hz), 4.07 (1H, d, J = 15.0 Hz),
4.01 (1H, d, J = 15.0 Hz), 3.85 (1H, ddd, J = 1.8, 3.9,
9.9 Hz), 2.07 (3H, s), 2.04 (3H, s), 2.01 (3H, s), 2.00
(3H, s), 0.95 (9H, s), 0.84 (9H, s), 0.14 (6H, s), ꢀ0.02
(6H, s) ppm; 13C NMR (75 MHz, CDCl3, 19 ꢁC): 170.7,
170.2, 170.0, 169.6, 156.1, 151.1, 142.7, 121.8 (2C),
120.6 (2C), 79.2, 73.3, 73.1, 70.7, 68.5, 62.0, 59.6, 25.7
(6C), 20.8, 20.7, 20.62, 20.60, 18.23, 18.18, ꢀ4.47 (2C),
ꢀ5.71 (2C) ppm; HR ESI-MS (positive): [M+Na]+.
3.1.11. Synthesis of 2-(tert-butyldimethylsilyloxy)-N-(p-
(tert-butyldimethylsilyloxy) phenyl)acetamide (23). Com-
pound 21 (642.5 mg 2.5 mmol) were dissolved in THF
(25 mL) and 5% Pd–C was added under argon atmo-
sphere. The mixture was stirred overnight under hydro-
gen atmosphere and filtered on Celite pad. The filtrate
was evaporated to give 22. Resulting 22, imidazole
(0.62 g, 9.11 mmol), and DMAP (11.0 mg, 0.09 mmol)
was dissolved in DMF (10 mL). TBSCl (1.00 g,
6.62 mmol) was added at 0 ꢁC and stirred overnight.
Water was added to the resulting mixture and extracted
by n-hexane/EtOAc = 1:1. The organic layer was washed
with brine and dried over absolute sodium sulfate. After
evaporation, the residue was purified by silica gel col-
þ
Found m/z 747.3311, C34H56N2NaO11Si2 requires m/z

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4611
747.3315; IR (film) m: 2932, 2858, 1753, 1655, 1499, 1366,
1H NMR (300 MHz, CDCl3, 20 ꢁC): 8.22 (2H, d,
J = 9.0 Hz), 7.85 (2H, d, J = 9.0 Hz), 7.12 (2H, d,
J = 8.7 Hz), 6.73 (2H, d, J = 8.7 Hz), 5.09 (1H, m),
4.86 (1H, d, J = 4.2 Hz), 4.04 (2H, t, J = 4.5 Hz), 3.83
(2H, t, J = 4.5 Hz), 3.73 (2H, t, J = 6.6 Hz), 3.03 (2H,
t, J = 6.6 Hz), 2.63 (1H, d, J = 4.2 Hz, –OH), 1.49 (9H,
s), 1.38 (9H, s) ppm; 13C NMR (75 MHz, CDCl3,
20 ꢁC): 169.0, 165.3, 159.0, 150.4, 141.7, 129.7, 129.1,
127.6, 124.0, 114.4, 84.9, 84.0, 82.9, 73.1, 70.3, 69.2,
67.4, 30.4, 28.2, 27.7 ppm; HR ESI-MS (positive):
21
1227 cmꢀ1; ½aꢁ +14.3 (c 0.20, CHCl3).
D
3.1.14. Synthesis of N-(p-hydroxyphenyl)-N0-(1-b-D-glu-
copyranosyl)hydroxylmethyl-amidine (6). Compound 26
(8.6 mg, 11.9 lmol) was dissolved in methanol (1 mL)
and acetyl chloride (50 lL) was added at 0 ꢁC under ar-
gon atmosphere. The reaction mixture was stirred for
24 h at room temperature and evaporated to dryness.
The residue was purified by HPLC equipped with Cos-
mosil 5C18AR column (Nacalai Co.) and 0.1% TFA/
5% MeOHaq. as an eluent to give 6 (4.3 mg, quant.)
as a mixture of 6a and 6b.
[M+Na]+. Found m/z 666.1650, C28H37NNaO12S2 re-
þ
quires m/z 666.1649; IR (film) m: 3508, 2982, 2936,
2876, 1757, 1716, 1699, 1610, 1533, 1514, 1369,
1350 cmꢀ1
.
Major isomer in D2O: 1H NMR (300 MHz, D2O,
40 ꢁC): 7.34 (2H, d, J = 8.7 Hz), 7.04 (2H, d,
J = 8.7 Hz), 4.86 (2H, s), 4.76 (1H, d, J = 8.7 Hz), 3.89
(1H, dd, J = 1.8, 12.6 Hz), 3.71 (1H, dd, J = 4.8,
12.6 Hz), 3.55 (1H, ddd, J = 1.8, 4.8, 9.0 Hz), 3.54
(1H, dd, J = 9.0, 9.0 Hz), 3.41 (1H, dd, J = 8.7,
9.0 Hz), 3.39 (1H, dd, J = 9.0, 9.0 Hz) ppm; 13C NMR
(75 MHz, D2O, 40 ꢁC) ppm; HR ESI-MS (positive):
3.1.17. Synthesis of tert-butyl 2-azido-3-(p-(2-(2-(tert-
butoxycarbonylsulfanyl)ethoxy)-ethoxy)phenyl)-3-hydroxy-
propionate (31). To a stirred solution of 30 (5.09 g,
7.92 mmol) in DMF (80 mL), sodium azide (2.60 g,
40.0mmol) was added and heated to 60 ꢁC. After 15 h,
water (550 mL) was added and extracted with EtOAc.
The organic layer was washed with brine and dried over
absolute sodium sulfate. After evaporation, the residue
was purified by silica gel column chromatography with
n-hexane/EtOAc = 4:1 to give 31 (2.37 g, 61%) as a yel-
low solid.
[M+H]+. Found m/z 329.1341, C14H21N2O7 requires
þ
m/z 329.1343; IR (film) m: 3261, 1654, 1516, 1439,
1202 cmꢀ1
.
1H NMR (300 MHz, CDCl3, rt): 7.30 (2H, d,
J = 9.0 Hz), 6.91 (2H, d, J = 9.0 Hz), 4.92 (1H, dd,
J = 4.8, 6.6 Hz), 4.11 (2H, t, J = 4.8 Hz), 3.97 (1H, d,
J = 6.6 Hz), 3.83 (2H, t, J = 4.8 Hz), 3.71 (2H, t,
J = 6.6 Hz), 3.04 (1H, d, J = 4.8 Hz, –OH), 3.01 (2H, t,
J = 6.6 Hz), 1.49 (9H, s), 1.46 (9H, s) ppm; 13C NMR
(75 MHz, CDCl3, 20 ꢁC): 169.0, 167.9, 158.8, 131.5,
127.9, 114.5, 84.8, 83.6, 73.5, 70.3, 69.3, 67.3, 67.0,
30.3, 28.1, 27.9 ppm; HR ESI-MS (positive): [M+Na]+.
Found m/z 506.1932, C22H33N3NaO7S+ requires m/z
506.1931; IR (film) m: 3447, 2979, 2936, 2876, 2110,
3.1.15. Synthesis of tert-butyl 3-(p-(2-(2-(tert-but-
oxycarbonylsulfanyl)ethoxy)ethoxy)-phenyl)-2,3-dihydroxy-
propionate (29). To a stirred solution of 10 (4.91 g,
19.3 mmol) and 2-(2-(tert-butoxycarbonylsulfanyl)eth-
oxy)ethyl iodide (7.04 g, 21.2 mmol) in DMF, sodium
hydride in oil (1.07 g) was added at 0 ꢁC under argon
atmosphere. After stirring for 15 h, water (1200 mL)
was added and the mixture was extracted with n-hex-
ane/EtOAc = 1:1. The organic layer was washed with
brine and dried over absolute sodium sulfate. After
evaporation, the residue was purified by silica gel col-
umn chromatography with n-hexane/EtOAc = 1:1 to
give 29 (3.88 g, 44%) as an orange oil.
1719, 1701, 1612, 1514, 1369 cmꢀ1
.
3.1.18. Synthesis of tert-butyl 2-azido-3-(p-(2-(2-(tert-
butoxycarbonylsulfanyl)ethoxy)-ethoxy)phenyl)-3-fluoro-
propionate (32). To a stirred solution of 31 (3.10 g,
6.42 mmol) in dichloromethane (120 mL), DAST
(1.7 mL, 128.8 mmol) was added at 0 ꢁC. After 10 min,
methanol (10 mL) was added. After evaporation, the
residue was purified by silica gel column chromatogra-
phy with n-hexane/EtOAc = 6:1 to give 32 (1.88 g,
60%) as a yellow solid. Two diastereomers (32a and
32b) can be separated by column chromatography.
1H NMR (300 MHz, CDCl3, rt): 7.31 (2H, d,
J = 8.7 Hz), 6.90 (2H, d, J = 8.7 Hz), 4.84 (1H, d,
J = 3.6 Hz), 4.20 (1H, d, J = 3.6 Hz), 4.10 (2H, t,
J = 4.8 Hz), 3.83 (2H, t, J = 4.8 Hz), 3.71 (2H, t,
J = 6.6 Hz), 3.01 (2H, t, J = 6.6 Hz), 1.49 (9H, s), 1.43
(9H, s) ppm; 13C NMR (75 MHz, CDCl3, rt): 171.9,
168.9, 158.4, 132.5, 127.7, 114.4, 84.8, 83.1, 74.7, 74.4,
70.2, 69.3, 67.3, 30.3, 28.1, 27.8 ppm; HR ESI-MS (po-
sitive): [M+H]+. Found m/z 481.1867, C22H35O8S+ re-
quires m/z 481.1868; IR (film) m: 3473, 2979, 2933,
2875, 1716, 1703, 1612, 1514, 1369 cmꢀ1
.
1
Compound 32a: H NMR (300 MHz, CDCl3, rt): 7.32
(2H, d, J = 8.4 Hz), 6.94 (2H, d, J = 8.4 Hz), 5.70 (1H,
dd, J = 6.0, 44.4 Hz), 4.18 (1H, dd, J = 6.0, 10.8 Hz),
4.13 (2H, t, J = 4.8 Hz), 3.85 (2H, t, J = 4.8 Hz), 3.72
(2H, t, J = 6.6 Hz), 3.02 (2H, t, J = 6.6 Hz), 1.49 (9H,
s), 1.47 (9H, s) ppm; 13C NMR (75 MHz, CDCl3, rt):
169.0, 166.0 (d, J = 5.4 Hz, C-F), 159.6 (d, J = 1.9 Hz,
C-F), 128.0 (d, J = 6.7 Hz, C-F), 127.1 (d, J = 20.6 Hz,
C-F), 114.6, 92.3 (d, J = 177.5 Hz, C-F), 84.9, 83.8,
70.3, 69.2, 67.4, 65.8 (d, J = 29.0 Hz, C-F), 30.4, 28.2,
27.9 ppm; HR ESI MS (positive): [M+Na]+. Found
m/z 508.1890, C22H32FN3NaO6S+ requires m/z
3.1.16. Synthesis of tert-butyl 3-(p-(2-(2-(tert-but-
oxycarbonylsulfanyl)ethoxy)ethoxy)-phenyl)-3-hydroxy-2-
(p-nitrobenzenesulfonyl)propionate (30). To a stirred
solution of 29 (3.88 g, 8.47 mmol) and TEA (2.4 mL,
17.2 mmol) in dichloromethane, p-nitrobenzenesulfonyl
chloride (1.88 g, 8.47 mmol) was added at 0 ꢁC under ar-
gon atmosphere. After stirring for an hour, the reaction
mixture was concentrated and the residue was purified
by silica gel column chromatography with CHCl3/
EtOAc = 7:1 to give 30 (5.09 g, 93%) as a yellow solid.

4612
E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
508.1888; IR (film) m: 2979, 2936, 2876, 2114, 1718, 1701,
After 2 h, the reaction mixture was evaporated to dry-
ness and the residue was dissolved in THF (0.2 mL).
To this solution, TEA (10 lL) was added and the mix-
ture was stirred for 10 min. After evaporation, the resi-
due was purified by preparative TLC with CHCl3/
MeOH/AcOH = 60:10:1 to give 34 (4.3 mg, 23%) as a
yellow solid.
1614, 1516, 1369 cmꢀ1
.
1
Compound 32b: H NMR (300 MHz, CDCl3, rt): 7.31
(2H, d, J = 8.4 Hz), 6.95 (2H, d, J = 8.4 Hz), 5.79 (1H,
dd, J = 4.8, 45.3 Hz), 4.13 (2H, t, J = 4.8 Hz), 3.93
(1H, dd, J = 4.8, 23.4 Hz), 3.85 (2H, t, J = 4.8 Hz),
3.72 (2H, t, J = 6.6 Hz), 3.02 (2H, t, J = 6.6 Hz), 1.49
(9H, s), 1.43 (9H, s) ppm; 13C NMR (75 MHz, CDCl3,
rt): 169.0, 166.1 (d, J = 6.7 Hz, C-F), 159.5 (d,
J = 1.8 Hz, C-F), 127.7 (d, J = 6.6 Hz, C-F), 127.5 (d,
J = 20.6 Hz, C-F), 114.7, 93.5 (d, J = 178.7 Hz, C-F),
84.9, 83.9, 70.4, 69.3, 67.4, 66.6 (d, J = 23.6 Hz, C-F),
30.4, 28.2, 27.8 ppm; HR ESI-MS (positive): [M+Na]+.
Found m/z 508.1888, C22H32FN3NaO6S+ requires m/z
508.1888; IR (film) m: 2979, 2936, 2876, 2116, 1717,
1H NMR (300 MHz, CD3OD, rt): 7.78 (1H, s), 7.62
(2H, d, J = 8.7 Hz), 7.02 (2H, d, J = 8.7 Hz), 4.41 (1H,
d, J = 9.6 Hz), 4.17 (2H, t, J = 4.5 Hz), 3.86-3.80 (3H,
m), 3.66 (2H, t, J = 6.6 Hz), 3.66–3.60 (3H, m), 3.31–
3.29 (1H, m), 2.99 (2H, t, J = 6.6 Hz), 1.56 (9H, s),
1.47 (9H, s) ppm; 13C NMR (75 MHz, CD3OD,
18 ꢁC): 170.5, 166.9, 163.6, 162.7, 140.7, 133.7, 125.8,
121.3, 116.2, 85.8, 83.9, 74.3, 71.3, 70.3, 70.0, 69.8,
68.8, 62.6, 61.6, 31.2, 28.4, 28.3 ppm; HR ESI-MS (po-
sitive): [M+H]+. Found m/z 599.2632, C28H43N2O10S+
1699, 1614, 1516, 1369 cmꢀ1
.
3.1.19. Synthesis of tert-butyl 2-amino-3-(p-(2-(2-(tert-
butoxycarbonylsulfanyl)ethoxy)-ethoxy)phenyl)-3-fluoro-
propionate (33). To a solution of 32 (1.06 g, 2.18 mmol)
in MeOH (44 mL), 10% Pd–C was added and stirred for
12 h under hydrogen atmosphere. The reaction mixture
was filtered on Celite and the filtrate was concentrated
to give 33 (1.06 g, quant.) as a slightly yellow oil. Two
diastereomers (33a and 33b) were separated by column
chromatography with n-hexane/EtOAc = 6:1.
requires m/z 599.2633; IR (film) m: 3307, 1701, 1661,
19
1601, 1510, 1369 cmꢀ1; ½aꢁ +7.1 (c 0.90, CH3OH).
D
3.1.21. Synthesis of (Z)-2-(N,N-glucoamidine)-3-(p-(2-(2-
mercaptoethoxy)ethoxy)-phenyl)acrylic acid (35). Com-
pound 34 (285.9 mg, 0.463 mmol) was dissolved in
TFA (6.0 mL) and stirred for an hour at 0 ꢁC under ar-
gon atmosphere. The reaction mixture was dried and
purified by column chromatography using Cosmosil
75C18-OPN (Nakalai Tesque Co., Ltd) with 0.5%
TFA/20% MeOH aq. to give 35 (119.5 mg, 58%) as a
yellow solid.
1
Compond 33a: H NMR (300 MHz, CDCl3, rt): 7.28
(2H, d, J = 8.4 Hz), 6.93 (2H, d, J = 8.4 Hz), 5.56 (1H,
dd, J = 6.0, 45.0 Hz), 4.12 (2H, t, J = 4.8 Hz), 3.85
(1H, dd, J = 4.8, 11.3 Hz), 3.84 (2H, t, J = 4.8 Hz),
3.72 (2H, t, J = 6.6 Hz), 3.02 (2H, t, J = 6.6 Hz), 1.49
(9H, s), 1.45 (9H, s) ppm; 13C NMR (75 MHz, CDCl3,
rt): 171.0 (d, J = 6.7 Hz, C-F), 169.0, 159.2 (d,
J = 1.8 Hz, C-F), 128.1 (d, J = 20.6 Hz, C-F), 127.9 (d,
J = 6.6 Hz, C-F), 114.5, 94.4 (d, J = 175.7 Hz, C-F),
84.9, 82.0, 70.3, 69.3, 67.4, 59.7 (d, J = 26.6 Hz, C-F),
30.4, 28.2, 28.0 ppm; HR ESI-MS (positive): [M+Na]+.
Found m/z 482.1982, C22H34FNNaO6S+ requires m/z
482.1983; IR (film) m: 3393, 2979, 2933, 2874, 1719,
1H NMR (300 MHz, CD3OD, rt): 7.65 (1H, s), 7.52
(2H, d, J = 8.7 Hz), 6.98 (2H, d, J = 8.7 Hz), 4.36 (1H,
d, J = 9.6 Hz), 4.16 (2H, t, J = 4.8 Hz), 3.82 (2H, t,
J = 4.8 Hz), 3.76 (1H, dd, J = 7.2, 9.6 Hz), 3.70 (1H,
dd, J = 3.0, 12.0 Hz), 3.65 (2H, t, J = 6.6 Hz), 3.62
(1H, dd, J = 9.6, 9.6 Hz), 3.53 (1H, dd, J = 4.2,
12.0 Hz), 3.22 (1H, m), 2.67 (2H, t, J = 6.6 Hz) ppm;
13C NMR (75 MHz, D2O, 23 ꢁC): 171.0, 164.9, 161.3,
138.1, 133.0, 127.0, 125.8, 116.8, 73.9, 73.4, 70.2, 69.1,
68.8, 61.6, 61.5, 24.7 ppm; HR ESI MS (positive):
[M + H]+. Found m/z 443.1481, C19H27N2O8S+ requires
1701, 1614, 1516, 1369 cmꢀ1
.
m/z 443.1483; IR (film) m: 3234, 1666, 1603, 1510,
19
1379 cmꢀ1; ½aꢁ +1.1 (c 0.50, H2O).
1
Compound 33b: H NMR (300 MHz, CDCl3, rt): 7.28
(2H, d, J = 8.4 Hz), 6.94 (2H, d, J = 8.4 Hz), 5.63 (1H,
dd, J = 4.5, 46.2 Hz), 4.12 (2H, t, J = 4.8 Hz), 3.84
(2H, t, J = 4.8 Hz), 3.72 (2H, t, J = 6.6 Hz), 3.67 (1H,
dd, J = 4.5, 23.1 Hz), 3.02 (2H, t, J = 6.6 Hz), 1.49
(9H, s), 1.40 (9H, s) ppm; 13C NMR (75 MHz, CDCl3,
rt): 170.8 (d, J = 5.4 Hz, C-F), 169.0, 159.0 (d,
J = 1.8 Hz, C-F), 129.0 (d, J = 21.2 Hz, C-F), 127.4 (d,
J = 7.3 Hz, C-F), 114.5, 94.9 (d, J = 175.1 Hz, C-F),
84.9, 82.0, 70.3, 69.3, 67.4, 60.5 (d, J = 24.2 Hz, C-F),
30.4, 28.2, 27.9 ppm; HR ESI-MS (positive): [M+Na]+.
Found m/z 482.1982, C22H34FNNaO6S+ requires m/z
482.1983; IR (film) m: 3396, 2979, 2933, 2876, 1720,
D
3.1.22. Synthesis of (E)-2-(N,N-glucoamidine)-3-(p-(2-(2-
mercaptoethoxy)ethoxy)-phenyl)acrylic acid (36). Com-
pound 35 (17.2 mg, 50.9 lmol) was dissolved in H2O
(2 mL) and irradiated with UV lamp (312 nm) for 1 h.
The reaction mixture was dried and purified by HPLC
[Develosil-ODS-MG-5, 0.5% TFA/35% MeOH aq] to
give 35 (3.5 mg, 20%) and 36 (4.8 mg, 28%) as yellow
solids.
1
Compound 36: H NMR (300 MHz, CD3OD, rt): 7.54
(2H, d, J = 8.7 Hz), 6.90 (2H, d, J = 8.7 Hz), 7.22 (1H,
s), 4.29 (1H, d, J = 8.4 Hz), 4.13 (2H, t, J = 4.5 Hz),
3.86 (1H, dd, J = 2.4, 11.4 Hz), 3.81 (2H, t,
J = 4.5 Hz), 3.71–3.65 (3H, m), 3.65 (2H, t,
J = 6.6 Hz), 3.44 (1H, m), 2.66 (2H, t, J = 6.6 Hz) ppm;
13C NMR (75 MHz, CD3OD, 25 ꢁC): 167.4, 165.4,
162.1, 144.4, 134.3, 130.9, 126.3, 115.1, 74.24, 74.17,
70.3, 70.1, 69.6, 68.7, 62.6, 61.5, 24.7 ppm; HR ESI-
1703, 1614, 1516, 1369 cmꢀ1
.
3.1.20. Synthesis of tert-butyl (Z)-2-(N,N-glucoamidine)-
3-(p-(2-(2-mercaptoethoxy)-ethoxy)phenyl)acrylate (34).
To a stirred solution of 33 (14.2 mg, 31.0 lmol) and 7
(1.2 mg, 6.2 lmol) in MeOH (150 lL), NBS (3.5 mg,
20.0 lmol) was added at 0 ꢁC under argon atmosphere.

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4613
MS (positive): [M+H]+. Found m/z 443.1481,
2 h at room temperature under argon atmosphere. To
the reaction mixture, molecular sieves 4A (409 mg) and
41 (214.1 mg, 0.617 mmol) were added and stirred for
15 min. Then, the mixture was cooled to 0 ꢁC and then
NBS (58.9 mg, 0.331 mmol) was added. After stirring
for 10 min at 0 ꢁC, the reaction mixture was evaporated
to dryness and purified by silica gel column chromatog-
raphy with toluene/acetone = 4:1 to give 42 (61.6 mg,
56%).
C19H27N2O8S+ requires m/z 443.1483; IR (film) m:
19
3250, 1655, 1574, 1512, 1423 cmꢀ1; ½aꢁ ꢀ30.4 (c 0.10,
D
MeOH).
3.1.23. Preparation of gluconoamidine-type affinity gel
(27). To a gel 37 (2 mL) which was prepared from Toyo-
pearl AF-Amino-650M (Tosoh Co. Ltd), 36 (488.9 mg,
1.11 mmol) dissolved in 0.1 M sodium acetate buffer
(pH 5.5) was added and shaken overnight at 4 ꢁC. The
resulting gel was washed with 0.1 M acetate buffer
(pH 5.5) to give gluconoamidine-type affinity gel (27).
1H NMR (300 MHz, CDCl3, rt): 7.35–7.27 (5H, m), 6.81
(2H, d, J = 8.7 Hz), 6.63 (2H, d, J = 8.7 Hz), 6.21 (1H,
d, J = 8.3 Hz, NH), 5.49 (1H, dd, J = 8.3, 8.7 Hz), 5.34
(1H, dd, J = 9.6, 9.6 Hz), 5.12 (1H, dd, J = 9.6,
9.6 Hz), 5.07 (1H, dd, J = 8.7, 9.6 Hz), 4.42 (2H, s),
4.32 (1H, dd, J = 3.9, 12.3 Hz), 4.13 (1H, dd, J = 1.8,
12.3 Hz), 4.10 (2H, t, J = 4.5 Hz), 4.01 (1H, d,
J = 14.4 Hz), 3.95 (1H, d, J = 14.4 Hz), 3.86 (1H, ddd,
J = 1.8, 3.9, 9.6 Hz), 3.85 (2H, t, J = 4.5 Hz), 3.75 (2H,
t, J = 4.8 Hz), 3.42 (2H, t, J = 4.8 Hz), 2.08 (3H, s),
2.05 (3H, s), 2.032 (3H, s), 2.026 (3H, s) ppm; 13C
NMR (75 MHz, acetone-d6, 19 ꢁC): 170.7, 170.6,
170.2, 170.0, 155.3, 155.1, 143.6, 138.4, 129.1, 128.6,
128.5, 122.7, 115.6, 79.1, 73.8, 73.7, 73.3, 71.8, 70.8,
70.3, 69.5, 68.4, 66.1, 62.8, 51.3, 20.634, 20.626, 20.59,
20.53 ppm; HR ESI-MS (positive): [M+Na]+. Found
3.1.24. Synthesis of p-(2-azidoethoxyethoxy)-N-(2-ben-
zyloxyacetyl)aniline (39). Compound 21 (2.05 g,
7.98 mmol) and 38 (2.74 g, 9.61 mmol) were dissolved
in DMF and cooled to 0 ꢁC. To this solution NaH in
oil (389.4 mg) was added under argon atmosphere. After
stirring for 3 h, the reaction mixture was heated to 30 ꢁC
and stirred for another 3 h. To the resulting solution,
water was added and the mixture was extracted by n-
hexane/EtOAc = 1:1. The organic layer was washed with
brine and dried over sodium sulfate. After evaporation,
the residue was purified by silica gel column chromatog-
raphy with n-hexane/Acetone = 3:1 to give 39 (2.02 g,
68%) as a yellow oil.
þ
m/z 722.2646, C33H41N5NaO12 requires m/z 722.2644;
;
1H NMR (300 MHz, CDCl3, rt): 8.23 (1H, s), 7.45 (2H,
d, J = 8.9 Hz), 7.38 (5H, m), 6.88 (2H, d, J = 8.9 Hz),
4.64 (2H, s), 4.11 (2H, t, J = 4.6 Hz), 4.09 (2H, s), 3.85
(2H, t, J = 4.6 Hz), 3.74 (2H, t, J = 5.0 Hz), 3.41 (2H,
t, J = 5.0 Hz) ppm; 13C NMR (75 MHz, CDCl3, rt):
167.6, 155.9, 136.9, 130.8, 129.0, 128.7, 128.4, 121.8,
115.3, 74.0, 70.5, 70.0, 69.9, 68.0, 51.0 ppm; HR ESI-
MS (positive): [M+Na]+. Found m/z 393.1533,
C19H22N4O4Na requires m/z 393.1534; IR (film) m:
IR (film) m: 2110, 1751, 1655, 1502, 1367, 1228 cmꢀ1
21
D
½aꢁ +9.8 (c 1.00, CHCl3).
3.1.27. Synthesis of N-(p-(2-azidoethoxyethoxy)phenyl)-
N0-(1-b-D-glucopyranosyl)-benzyloxymethylamidine (44).
Compound 42 (64.9 mg, 92.9 lmol) was dissolved in
methanol (4 mL) and acetyl chloride (200 lL) was added
at 0 ꢁC under argon atmosphere. The reaction mixture
was stirred for 24 h at room temperature and evapo-
rated to give crude 43. The crude 43 and Pd(OH)2
(10 mg) were dissolved in THF/MeOH = 1:1 (5 mL)
and stirred for 3 h under hydrogen atmosphere. Suspen-
sion was then filtered on Celite pad and the filtrate was
evaporated to dryness. The residue was purified by
HPLC equipped with Cosmosil 5C18AR column (Nac-
alai Co.) with 0.1% TFA/5% methanol aq as an eluent
to give 44 (29.3 mg, 81%).
3385, 3036, 2870, 2112, 1676, 1508, 1454, 1111 cmꢀ1
.
3.1.25. Synthesis of p-(2-azidoethoxyethoxy)-N-(2-ben-
zyloxythioacetyl)aniline (40). Compound 39 (2.02 g,
5.46 mmol) and Lawesson’s reagent (1.65 g, 4.08 mmol)
were dissolved in toluene (100 mL) and stirred at 70 ꢁC
for an hour under argon atmosphere. The reaction mix-
ture was concentrated to 30 mL and then purified by sil-
ica gel column chromatography with n-hexane/
acetone = 4:1 to give 40 (1.22 g, 58%) as a yellow oil.
1
Compound 44: H NMR (300 MHz, D2O, 40 ꢁC): 7.34
(2H, d, J = 8.7 Hz), 7.15 (2H, d, J = 8.7 Hz), 4.85 (2H,
s), 4.77 (1H, d, J = 8.4 Hz), 4.27 (2H, t, J = 4.5 Hz),
3.94 (2H, t, J = 4.5 Hz), 3.87 (1H, dd, J = 2.4, 12.3),
3.83 (2H, t, J = 4.8 Hz), 3.70 (1H, dd, J = 5.1,
12.3 Hz), 3.55 (1H, ddd, J = 2.4, 5.1, 9.0 Hz), 3.53
(1H, dd, J = 9.0, 9.0 Hz), 3.39 (1H, dd, J = 8.4,
9.0 Hz), 3.37 (1H, dd, J = 9.0, 9.0 Hz), 3.23 (2H, t,
J = 4.5 Hz) ppm; 13C NMR (75 MHz, D2O, 40 ꢁC):
170.3, 160.4, 129.4, 127.0, 117.9, 83.5, 79.0, 77.5, 73.3,
70.7, 70.5, 69.1, 68.2, 62.0, 58.9, 40.7 ppm; HR ESI-
MS (positiþve): [M+H]+. Found m/z 416.2028,
C18H31N3O8 requires m/z 416.2027; IR (film) m: 3254,
1H NMR (300 MHz, CDCl3, rt): 9.79 (1H, s), 7.68 (2H,
d, J = 8.7 Hz), 7.38 (5H, m), 6.94 (2H, d, J = 8.7 Hz),
4.68 (2H, s), 4.48 (2H, s), 4.15 (2H, t, J = 4.8 Hz), 3.87
(2H, t, J = 4.8 Hz), 3.75 (2H, t, J = 4.9 Hz), 3.42 (2H,
t, J = 4.9 Hz) ppm; 13C NMR (75 MHz, CDCl3, rt):
195.9, 157.6, 136.8, 131.2, 129.1, 128.8, 128.5, 125.0,
115.1, 76.9, 73.8, 70.6, 70.0, 68.0, 51.0 ppm; HR ESI-
MS(positive): [M+Na]+. Found m/z 409.1305,
C19H22N4O3SNa requires m/z 409.1307; IR (film) m:
3308, 2924, 2106, 1516, 1251, 1128, 1067 cmꢀ1
.
3.1.26. Synthesis of N-(p-(2-azidoethoxyethoxy)phenyl)-
N0-(2,3,4,6-tetraacetyl-1-b-D-glucopyranosyl)benzyloxyme-
thylamidine (42). Compound 40 (60.8 mg, 0.158 mmol)
and Me3OÆBF4 (36.2 mg, 0.245 mmol) were dissolved
in dried CH2Cl2 (5 mL). The mixture was stirred for
2891, 1682, 1512, 1202 cmꢀ1
.
3.1.28. Preparation of glucosylamidine-type affinity gel
(46). To gel 45 (2 mL) prepared from Toyopearl AF-
Carboxyl-650M (Tosoh Co. Ltd), 44 (104.8 mg,

4614
E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
0.25 mmol) dissolved in 0.1 M Bis–Tris buffer (pH 7.0)
was added and shaken overnight at 4 ꢁC. The resulting
gel was washed with water to obtain glucosylamidine-
type affinity gel (46).
(50 g) was dissolved in 800 mL of 0.1 M Bis–Tris buffer
(pH 7.0) containing 2 mM DTT, 5 mM EDTA, 1 M
NaCl, ascorbic acid (1 g) and Polyclar-VT (8% w/v).
The mixture was stirred overnight at 4 ꢁC, and then cen-
trifuged at 14,000g for 30 min. Resulting supernatant
(500 mL) was collected and used as a crude enzyme.
3.2. Determination of Ki values of 5, 6a, 6b, and 18
against glycosidases
3.5.2. Acetone precipitation. To the crude enzyme solu-
tion (500 mL), 750 mL of chilled acetone (ꢀ20 ꢁC) was
added gradually and stirred at 0 ꢁC for 5 h. Resulting
precipitate was collected by centrifugation (14,000g 30
min.) and suspended in 100 mL of 25 mM Bis–Tris buf-
fer (pH 7.0). The suspension was stirred for 5 h and then
centrifuged (14,000g 30 min.). The obtained supernatant
was used for further purification.
Ki values of inhibitor 5, 6a, 6b, and 18 were determined
by Lineweaver–Burk plot. Each enzyme (Sigma Co.,
Ltd) was preincubated with various amounts of inhibi-
tor at 37 ꢁC for 15 min and then p-nitrophenyl glycoside
was added to the mixture. The reaction was stopped by
adding 0.5 M Na2CO3 aq Absorbance at 405 nm was
measured by microplatereader Model 680 (Bio-Rad
Co., Ltd) to determine quantity of resulting p-nitrophe-
nol. And the similar experiments were carried out at var-
ious pHs (pH 4.5–5.5: 25 mM acetate buffer, pH 6.0:
25 mM Bis–Tris buffer) to determine the effect of pH
on the inhibitory activity.
3.5.3. Purification by Hiprep 16/10 Q XL. The above-
mentioned supernatant (80 mL) was applied to Hiprep
16/10 Q XL (GE Healthcare Co.) equilibrated with
25 mM Bis–Tris buffer (pH 7.0). The column was
washed with the same buffer containing 0–0.3 M so-
dium chloride (linear gradient in 120 mL), 0.3–1.0 M
sodium chloride (linear gradient in 20 mL), and 1.0 M
sodium chloride (40 mL). The fractions were collected
in 20 mL each and concentrated to 4 mL by ultrafiltra-
tion (Amicon ultra-15, MWC 10, 000, Millipore Co.)
and desalted by Hitrap Desalting column (GE Health-
care Co.). LOFG activity and protein concentration of
each fraction were analyzed by the method mentioned
above.
3.3. Stability of 5 and 6
Compound 5 or 6 was dissolved in buffer (pH 4.5–5.5:
25 mM acetate buffer, pH 6.0–7.0: 25 mM Bis–Tris buf-
fer, pH 7.5–9.0: 25 mM Tris–HCl buffer) and incubated
for 6 h at 4 ꢁC. The remaining inhibitor was quantified
by HPLC equipped with Develosil ODS-HG-5 column
(Nomura Co.) using 0.1% TFA/2% methanol aq as an
eluent.
3.4. Measurement of b-glucosidase activity using 1
3.5.4. Affinity purification at pH 5.0. Crude LOFG solu-
tion (4 mL) purified by HiPrep 16/10 Q XL was applied
to the column (/ · 100 mm) with 2 mL of affinity gel 27
or 46. After washing the column by 50 mM acetate buf-
fer (pH 5.0), stepwise elution using the same buffer con-
taining 2 M D-glucose (10 mL), 10 mM potassium
lespedezate (1, 10 mL) and then 1 M NaCl (10 mL)
was performed. The fractions were collected 5 mL each
and desalted by Hitrap Desalting column, and concen-
trated by Amicon Ultra-4 to 2 mL.
Twenty-five millimolar acetate buffer (pH 4.5) contain-
ing 1 mM potassium lespedezate (1) and 1 mM semi-
carbazide hydrochloride (48) was preincubated at
37 ꢁC for 15 min. b-Glucosidase in 25 mM acetate buffer
(pH 4.5) was added to this solution, and the mixture was
incubated at 37 ꢁC. After 2 h, the reaction was stopped
by adding 0.1 M Tris–HCl buffer (pH 9.0) and adjusting
the pH to 8.0. Quantitative analysis of the resulting
semicarbazone (49) was performed by HPLC with Dev-
elosil ODS-HG-5 (Nomura Chemical Co.) using 0.1%
TFA, 20% MeOH aq as an eluent. One enzyme unit of
LOFG was defined as a quantity of enzyme that hydro-
lyzes one micro mole of 1 in one minute under optimum
pH condition at 30 ꢁC.
3.5.5. Superdex 200 10/300 GL gel-filtration chromatog-
raphy. Resulting LOFG solution containing 1 (0.5 mL)
was applied to Superdex 200 10/30 GL (GE Healthcare
bio.) equilibrated with 25 mM Acetate buffer (pH 4.5)
containing 100 mM sodium chloride. Elution was per-
formed by the same buffer at a flow late of 0.5 mL/min
to give pure LOFG.
3.5. Purification of LOFG from the leaves of L. cuneata
The purification of LOFG from L. cuneata was carried
out according to the following procedure. In each purifi-
cation step, amount of proteins were quantified with
Quick Starttrade Bradford Protein Assay (Bio-Rad Co.,
Ltd) using BSA (20, 10, 5, 2.5, 1.25 lg/mL) as a standard.
3.5.6. Reverse-phase HPLC analysis of LOFG. LOFG
was analyzed by HPLC equipped with Sephasil Protein
C4 5 lm ST4.6/250 (GE Healthcare Co.). The linear gra-
dient analysis between eluent A (0.065% TFA, 2%
MeCN aq.) and eluent B (0.05% TFA, MeCN) in
50 min was performed at a flow rate of 1.0 mL/min at
40 ꢁC. LOFG was detected at 215 nm.
3.5.1. Preparation of crude enzyme. Leaves of L. cuneat-
a(180 g) were collected at certain time of the day and
homogenized in cold acetone (ꢀ20 ꢁC) by Warring Blen-
der. Resulting homogenate was filtrated by miracloth
under vacuo and washed with cold acetone until the fil-
trate became nearly colorless. The residue was dried in
vacuo to give acetone powder (50 g). Acetone powder
3.5.7. Gel-filtration HPLC analysis of LOFG. LOFG
was analyzed by HPLC equipped with TSK-Gel
SuperSW3000 (Tosoh Co. Ltd). The analysis was per-
formed at a flow rate of 0.2 mL/min and 50 mM phos-
phate buffer containing 0.15 M NaCl (pH 7.0) as an

E. Kato et al. / Bioorg. Med. Chem. 16 (2008) 4600–4616
4615
eluent. The proteins were detected at 215 nm. IgG
150 kDa, BSA 68 kDa, Carbonic anhydrase (CA)
34 kDa, and cytochrome C (Cyt C) 12 kDa were used
as molecular weight standards.
7.5). Blocking was performed using 2% w/v ECL Advance
Blocking Agent (GE Healthcare Co.) in PBS-T and then
washed three times with PBS-T. Oxidized glycoproteins
were biotinylated with 0.1 mM Biotinhydrazide dissolved
in 0.1 M Acetate buffer (pH 5.5) and washed twice with
PBS-T. Membrane was then soaked in Streptavidin-
HRP conjugate (GE Healthcare Co.) diluted with PBS-
T (1/250,000) for 15 min and sufficiently washed with
PBS-T. Finally chemiluminescent detection was per-
formed using ECL advance Western Blotting Detection
Kit (GE Healthcare Co.) and ECL Mini-Camera (GE
Healthcare Co.) equipped with Instant Black & White
Film FP-3000B Super Speedy (Fujifilm Co.).
3.5.8. SDS–PAGE analysis of LOFG. SDS–PAGE was
performed according to the method of Laemmli.47 Pre-
cision Plus Protein Standards (Bio-Rad Inc.) were used
as a marker protein. Samples (30 lL) were mixed with
6 lL of SDS sample buffer (0.35 M Tris–HCl, 10%
SDS, 30% glycerol, 600 mM DTT, 0.01% BPB, pH
6.8) and heated at 95 ꢁC for 5 min. Fifteen microliters
of each mixture was applied to 10% polyacrylamide
gel and electrophoresised at 200 V for 45 min. Protein
bands were visualized by silver staining using Bio-Rad
Silver Stain kit (Bio-Rad Inc.).
Total protein on the membrane was detected using Col-
loidal Gold Total Protein Detection Kit (Bio-Rad Inc.).
3.5.9. Km value of LOFG. Km value of LOFG was esti-
mated from Liveweaver–Burk plot with various concen-
trations of 3 (250, 500, 750, 1000 lM) as substrates
described above.
3.8. Deglycosylation of LOFG
3.8.1. By N-glycopeptidase A. LOFG was denatured by
either of the following: 2% Nonidet P-40, 2% Triton
X-100, 0.1% SDS, 0.75 M NaSCN, and 6 M urea for
2 h. To the solution, N-glycopeptidase A (0.1 mU) was
added and incubated for 24 h at 37 ꢁC. The resulting
mixture was analyzed by SDS–PAGE as described
above.
3.5.10. De novo sequenece analysis of LOFG. Purified
LOFG was hydrolyzed by trypsin in H2O and H218O,
respectively, in appropriate conditions. Then the two
hydrolyzates were analyzed by MS/MS using oMALDI-
Qq-TOF MS/MS QSTAR Pulsar i (Applied Biosystems
Co., Ltd) with de novo sequence analyis software PEAKS
(Infocom Co., Ltd). Comparison of obtained data gave
partial sequences and the de novo sequence was obtained
from the agreement between them (Table 4).
3.8.2. By N-glycopeptidase F. LOFG was denatured by
0.5% SDS containing 0.1 M 2-mercaptoethanol for
5 min at 95 ꢁC. To the solution, N-glycopeptidase F
(5 U) was added and incubated for 24 h at 37 ꢁC. The
resulting mixture was analyzed by gel-filtration HPLC
as described above.
3.6. Substrate specificity of LOFG
Substrate specificity of LOFG was examined as follows:
compounds 47–51 were hydrolyzed by LOFG, respec-
tively. Twenty-five millimolars acetate buffer (pH 4.5)
containing each substrate (1 mM) were preincubated at
37 ꢁC. LOFG in 25 mM acetate buffer (pH 4.5) was
added to this solution, and the mixture was incubated
at 37 ꢁC. After two hours, the reaction was stopped by
adding 0.1 M Tris–HCl buffer (pH 9.0) and adjusting
the pH to 8.0. Quantitative analysis of resulting aglycon
was performed by HPLC using Develosil ODS-HG-5
(Nomura Chemical Co.) with 0.1% TFA, 20% MeOH aq.
3.8.3. By trifluoromethanesulfonic acid (TfOH). To the
lyophilized LOFG, trifluoromethanesulfonic acid
(100 lL) was added at 0 ꢁC. After 2 h, the mixture was
cooled to ꢀ20 ꢁC and neutralized by adding 60% pyri-
dine aq. (200 lL). The resulting solution was dialyzed
with Spectra/Por Float-A-Lyzer (MWCO = 3500, Nip-
pon Genetics Co., Ltd) and purified by Superdex 200
10/300 GL (GE Healthcare Co., Ltd). Molecular weight
of the obtained protein was measured by gel-filtration
HPLC [TSK-Gel SuperSW3000 (Tosoh Co. Ltd),
50 mM phosphate buffer (pH 7.0) with 0.15 M NaCl,
flow rate: 0.2 mL/min, detection: 215 nm].
3.7. Detection of sugar chain in LOFG
Acknowledgments
SDS–PAGE analysis of LOFG was performed as de-
scribed above. BSA (100 ng) was used as negative control,
ovalbumin (100 ng) as positive control and Biotinylated
SDS–PAGE Standards (Bio-Rad Inc.) were used as
marker protein. The proteins in the gel were transferred
to Hybond-P (GE Healthcare Co., Ltd) using semidry
blotter (100 mA, 1 h) with 25 mM Tris/192 mM glycine/
20% MeOH aq as blotting buffer. The membrane was
washed three times with TBS-T (25 mM Tris–HCl,
150 mM NaCl, 0.1% Tween 20, pH 7.5) for 20 min. and
then with milliQ water for three times. Glycoproteins on
the membrane were then oxidized with 10 mM NaIO4 in
PBS (100 mM phosphate, 100 mM NaCl, pH 7.5) for
30 min at 4 ꢁC and washed three times with PBS-T
(100 mM phosphate, 100 mM NaCl, 0.1% Tween 20, pH
This work was supported by a Grant-in-Aid for Scientific
Research on Priority Area 16073216 from the Ministry of
Education, Science, Sports, and Culture (MEXT), Asahi
Glass Foundation, and Naito Foundation. We thank As-
soc. Prof. Jun Hiratake and Prof. Kanzo Sakata (Insti-
tute for Chemical Research, Kyoto University) for the
helpful discussions and suggestions on this work.
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