Chemical synthesis and tyrosinase-inhibitory activity of isotachioside and its related glycosides

Article abstract of DOI:10.1016/j.carres.2018.06.004

Isotachioside (1) and its related natural product 2 are isolated from Isotachis japonica and Protea neriifolia, respectively, and are categorized as analogs of arbutin (3), a tyrosinase inhibitor for practical use. Both of the natural products and several derivatives such as glucoside 4, xyloside 5, cellobioside 6, and maltoside 7 were synthesized via Schmidt glycosylation as a key step, and their tyrosinase inhibitory activity was evaluated. The half maximal inhibitory concentration (IC₅₀) of 1–3 could not be determined even when the concentration was increased to 1000 μM. Contrastingly, glycosides 4–7, missing methyl and benzoyl groups, acted as tyrosinase inhibitors with IC₅₀s of 417 μM, 852 μM, 623 μM, and 657 μM, respectively. Among these novel inhibitors, derivative 4 was the most potent, indicating that the structural combination of resorcinol and glucose was significant for inducing the inhibitory effect.

Full text of DOI:10.1016/j.carres.2018.06.004

CarbohydrateResearch465(2018)22–28
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemical synthesis and tyrosinase-inhibitory activity of isotachioside and its
related glycosides
Takashi Matsumotoa, Takuya Nakajimaa, Takehiro Iwadateb, Ken-ichi Niheia,b,∗
a
Department of Applied Biological Chemistry, School of Agriculture, Utsunomiya University, Tochigi, 321-0943, Japan
Department of Applied Life Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, 183-8509, Japan
b
A R T I C L E I N F O
A B S T R A C T
Dedicated to the memory of Professor
Toshikatsu Okuno (1940–2017)
Isotachioside (1) and its related natural product 2 are isolated from Isotachis japonica and Protea neriifolia, re-
spectively, and are categorized as analogs of arbutin (3), a tyrosinase inhibitor for practical use. Both of the
natural products and several derivatives such as glucoside 4, xyloside 5, cellobioside 6, and maltoside 7 were
synthesized via Schmidt glycosylation as a key step, and their tyrosinase inhibitory activity was evaluated. The
half maximal inhibitory concentration (IC50) of 1–3 could not be determined even when the concentration was
increased to 1000 μM. Contrastingly, glycosides 4–7, missing methyl and benzoyl groups, acted as tyrosinase
inhibitors with IC50s of 417 μM, 852 μM, 623 μM, and 657 μM, respectively. Among these novel inhibitors, de-
rivative 4 was the most potent, indicating that the structural combination of resorcinol and glucose was sig-
nificant for inducing the inhibitory effect.
Keywords:
Isotachioside
Arbutin analog
Total synthesis
Glycosylation
Tyrosinase inhibitor
1. Introduction
activity of tyrosinase as well as the total synthesis of the natural arbutin
analogs 1 and 2 has not been achieved to date. Therefore, in this study,
Hyperpigmentation of mammalian skin is responsible for the excess
deposition of melanin [1,2]. Melanin biosynthesis results from the en-
zymatic action of tyrosinase (EC,1.14.18.1), a copper-containing oxi-
doreductase, which catalyzes two successive reactions including the o-
hydroxylation of tyrosine and the o-quinone formation of DOPA [3,4].
Thus, the inhibition of tyrosinase can lead to the regulation of abnormal
melanogenesis caused by several types of stress such as sunlight-in-
duced irritation and scratching [5]. In addition, enzymatic oxidation is
a key step in food browning and insectile development [6–8], because
various monophenols and o-diphenols in plants and insects are re-
cognized as substrates of the tyrosinase family [9–13]. Therefore, tyr-
osinase inhibitors are promising substances for the development of
novel antiaging, food antibrowning, antifungal, and insecticidal agents
[14,15].
Isotachioside (2-methoxy-4-hydroxyphenyl-β-D-glucoside; 1) and
2,4-dihydroxyphenyl-(6′-O-benzoyl)-O-β-D-glucoside (2) were first iso-
lated from Isotachis japonica (Hepaticae) and Protea neriifolia
(Proteaceae), respectively (Fig. 1) [16,17]. Both structures possess a
high similarity to arbutin (3), which has previously been used as a
whitening agent in cosmetics [18,19]. The essential whitening me-
chanism of the glycoside 3 is considered to be the inhibition of the
oxidation by tyrosinase and, in particular, the water-soluble property
can expand its applications. However, evaluation of the inhibitory
we conducted the concise chemical synthesis of 1, 2, and 4–7 to de-
velop a novel water-soluble tyrosinase inhibitor and evaluated their
tyrosinase-inhibitory activity.
2. Results and discussion
2.1. Chemical synthesis of the natural glucoside 1
The starting material, 2,4-dihydroxybenzaldehyde, was transformed
into phenol 8 through benzyl etherification and Baeyer–Villiger oxi-
dation (Scheme 1) [20,21]. A glucose donor, 9, was synthesized in
several steps including peracetylation of glucose, removal of the 1-
acetyl moiety, and imino esterification [22,23]. In the presence of a
catalytic amount of boron trifluoride diethyl etherate (BF3·OEt2), 8 was
glycosylated with 9 under low-temperature conditions to obtain the
glucoside 10 in an excellent yield (94%). In contrast, glucoside 10 was
obtained in only a moderate yield (55%) by using a phase transfer
catalyst under basic condition in a previous work [17]. Trimethylsilyl
trifluoromethanesulfonate could be useful as a Lewis acid because the
glycosylation proceeded to yield 10 with 82% [24].
Diphenol 11 was obtained from 10 with 94% yield by catalytic
hydrogenolysis at room temperature (rt). The selective protection of the
phenolic hydroxyl at the C-4 of 11 was achieved by the treatment of
∗
Corresponding author. Department of Applied Biological Chemistry, School of Agriculture, Utsunomiya University, Tochigi, 321-0943, Japan.
E-mail address: nihei98@cc.utsunomiya-u.ac.jp (K.-i. Nihei).
https://doi.org/10.1016/j.carres.2018.06.004
Received 21 May 2018; Received in revised form 7 June 2018; Accepted 7 June 2018
Availableonline08June2018
0008-6215/©2018ElsevierLtd.Allrightsreserved.

T. Matsumoto et al.
CarbohydrateResearch465(2018)22–28
Fig. 1. Structure of 1–7.
Scheme 1. Synthesis of 1.
tert-butyldimethylchlorosilane (TBSCl) and N,N-diisopropylethylamine
(DIPEA) in CH2Cl2 (87% yield). When the solvent was replaced by N,N-
dimethylformamide (DMF), the excess production of a disilyl derivative
could not be avoided. In addition, the silylation did not proceed
smoothly when using imidazole as a base.
The methylation at the 2-OH of silyl ether 12 failed with the use of
MeI and several bases such as K2CO3, DBU, or DIPEA. One reason for
this failure arose from the steric hindrance of the peracetylated gluco-
side. Thus, all the acetyl groups of 12 were removed by transester-
ification (67% yield). When polyol 13 was treated with MeI and K2CO3,
the ether 14 was synthesized with 68% yield. Finally, the removal of
the TBS group under acidic conditions yielded the natural glucoside 1
with 85%. The obtained spectral data were consistent with those that
were previously reported [16,25–27]. The total yield of 1 from 8 was
30% over six steps.
Scheme 2. Synthesis of 2 and 4.
2.2. Chemical synthesis of natural glucoside 2
hydrogenolysis of 16 resulted in the natural glucoside 2 with an ex-
cellent yield (98%). The spectral data obtained was in accordance with
those that had been reported previously [17]. The total yield of 2 from
8 was 67% over five steps.
Transesterification of 10 using NaOMe yielded the polyol 15 with
92% (Scheme 2). The monoester 16 could not be prepared by the direct
benzoylation of 15 with benzoyl chloride (BzCl), when pyridine, 4-di-
methylaminopyridine, or trimethylamine were used as bases and when
the reaction temperatures were controlled within −40 °C to 80 °C. The
hydroxyl group at C-6 in 3 was selectively benzoylated with benzoic
acid by using bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride as a
coupling reagent [17]. Although this method was applied, 16 could not
be isolated by the chromatographic purification using a silica gel
column.
Dibutyltin oxide (Bu2SnO) forms a nucleophilic O–Sn linkage, which
has been used as an effective additive for the regioselective mono-
acylation of several polyols [28]. In particular, methyl β-D-glucopyr-
anoside can be transformed into the 6-benzoyl derivative with over
80% yield through the preparation of the stannylidene complex fol-
lowed by treatment with BzCl [29]. As a result of the application of this
procedure, 16 was synthesized from 15 with 79% yield. Finally, the
2.3. Tyrosinase-inhibitory activity of 1 and 2
The inhibitory activity of the natural glucosides 1 and 2 was eval-
uated by using a commercially available tyrosinase. Unfortunately, the
half maximal inhibitory concentrations (IC50s) could not be identified,
even when the concentration of both synthetic compounds was in-
creased up to 1000 μM (Table 1). It has been reported previously that
the standard substance 3 can act as a weak tyrosinase inhibitor
(IC50 = 8.4 mM) [30]. This low efficacy is partially due to 3 having a
monophenolic structure that acts as alternative substrate [10,12,13].
However, several resorcinol glycosides represented potent tyrosinase-
inhibitory activity without oxidation of the substrate [31].
23

T. Matsumoto et al.
CarbohydrateResearch465(2018)22–28
converted 21 into 7 with 81% yield (two steps). Consequently, analogs
4–7 were synthesized from 8 in three steps and the overall yields ob-
tained were 85%, 96%, 47%, and 70%, respectively.
Table 1
Tyrosinase inhibitory activity of 1–7.
a
Compounds tested
IC50 (μM)
1
2
3
4
5
6
7
> 1000
> 1000
> 1000b
2.5. Tyrosinase-inhibitory activity of 4–7
In contrast to the natural glycosides 1 and 2, the IC50 of glucoside 4
could be performed, and it was found to be 417 μM. Thus, it was de-
termined that uncovering the resorcinol and sugar moieties of the ar-
butin analogs was significant in eliciting an inhibitory activity. In the
case of the bibenzyl glycosides [33], the activity of the relatively small
xyloside was higher than that of the other glycosides. However, the
IC50s of 5, 6, and 7 were measured as 852 μM, 623 μM, and 657 μM,
respectively, which were lower than that of 4. Therefore, the hydro-
philic glucose moiety might have interacted well with the enzymic key
structure.
417
852
623
657
40
74
61
79
a
The IC50s represent means
SE of three different
experiments.
b
The IC50 was 8.4 mM according to the previous re-
port [30].
The methyl modification at the 2-OH of 1 could lead to the dimin-
ished advantage of resorcinol structure that is essential to a potent
tyrosinase inhibitor. Benzoyl modification of 2 might prevent access to
the active site due to the increased steric demand of the molecule.
Furthermore, the tyrosinase-inhibitory efficacy of several resorcinol
glycosides has been found to replace glucoside in xyloside, cellobioside,
or maltoside [32,33]. Along with those hypotheses, derivatives 4–7,
which have a resorcinol moiety and an unmodified glycoside, were
synthesized and evaluated for their activity.
3. Conclusions
The total synthesis of the natural products 1 and 2 was accom-
plished for the first time by Schmidt glycosylation and selective alky-
lation or acylation as the key steps. The tyrosinase-inhibitory activity of
1 and 2 was not estimated because the IC50 values could not be de-
termined in concentrations lower than 1000 μM. In contrast, the syn-
thetic arbutin analogs 4–7 acted as weak but novel hydrophilic tyr-
osinase inhibitors. In particular, the efficacy of 4 suggested that the
glucoside structure was a requisite to increasing the tyrosinase-in-
hibitory activity.
2.4. Chemical synthesis of derivatives 4–7
Glucoside 4 was prepared from 15 b y hydrogenolysis with 98%
yield (Scheme 2) [17]. To apply a procedure similar to that mentioned
above, the glycosylation of 8 with a xylose donor, 17 [31], was un-
dertaken and proceeded smoothly to yield xyloside 18, quantitatively
(Scheme 3). Removal of the acetyl and benzyl groups by transester-
ification and hydrogenolysis converted 18 into 5 with 96% yield (two
steps). Cellobioside 19 was synthesized from phenol 8 and a cellobiose
donor 20 with 61% yield [33]. Deprotection of 19 was performed in
two steps and yielded 6 with 77%. Similarly, 8 was transformed into
maltoside 21 b y Schmidt glycosylation with a maltose donor 22 with
86% yield [33]. Transesterification followed by hydrogenation
4. Experimental section
4.1. General
NMR spectra were recorded in CD3OD, CDCl3, C5D5N, or (CD3)2SO
on a JEOL EX-400 spectrometer (1H at 400 MHz and 13C at 100 MHz).
Chemical shifts were recorded as ppm relative to the solvent signal for
CD3OD (3.30 ppm for 1H NMR, 49.0 ppm for 13C NMR), CDCl3
(7.24 ppm for 1H NMR, 77.0 ppm for 13C NMR), C5D5N (8.71 ppm for
Scheme 3. Synthesis of 5–7.
24

T. Matsumoto et al.
CarbohydrateResearch465(2018)22–28
1H NMR, 149.2 ppm for 13C NMR), or (CD3)2SO (2.49 ppm for 1H NMR,
39.7 ppm for 13C NMR). HRMS spectra were measured on an AB SCIEX
TripleTOF 5600 fitted with an electrospray ion source in positive io-
nization mode. Optical rotations were recorded on a Jasco P-1020 po-
larimeter. Preparative RP-HPLC was performed on a Hitachi LaChrome
Elite instrument and a detection wavelength of 254 nm were used for
separations. For the purification of 1 and 2, an Inertsil ODS-3 column
(7.6 mm × 250 mm) was used with a flow rate of 2.0 mL/min. For the
4.5. 2-Hydoxy-4-(tert-butyldimethylsiloxy)phenyl-β-D-glucoside (13)
NaOMe (163 mg, 3.02 mmol) was slowly added to a solution of 12
(434 mg, 761 μmol) in MeOH (20 mL) at 0 °C and the resultant solution
was stirred for 1 h at 0 °C. After being stirred for 3 h at rt, the solution
was neutralized with Amberlite IR-120H. Filtration and concentration
followed by silica gel chromatography (20% MeOH in CHCl3) gave the
title compound 13 (204 mg, 507 μmol, 67% yield) as a white solid.
[α]D25 − 34.1 (c 0.48, MeOH). 1H NMR (400 MHz, CD3OD) δ 7.06 (d,
J = 8.7 Hz, 1H, H-6), 6.35 (d, J = 2.8 Hz, 1H, H-3), 6.25 (dd, J = 2.8,
8.7 Hz, 1H, H-5), 4.62 (d, J = 7.2 Hz, 1H, H-1′), 3.88 (dd, J = 1.4,
12.0 Hz, 1H, H-6′), 3.71 (dd, J = 4.8, 12.0 Hz, 1H, H-6′), 3.40 (m, 4H,
H-2′, H-3′, H-4′, H-5′), 0.97 (s, 9H, TBS), 0.16 (s, 6H, TBS). 13C NMR
(100 MHz, CD3OD) δ 153.4 (C-4), 149.4 (C-2), 141.7 (C-1), 120.3 (C-6),
120.0 (C-5), 109.2 (C-3), 105.5 (C-1′), 78.3 (C-3′), 77.6 (C-5′), 74.9 (C-
2′), 71.2 (C-4′), 62.4 (C-6′), 26.2 (TBS), 19.0 (TBS), −4.4 (TBS).
ESIHRMS m/z 425.1615 [M+Na]+ (calcd for C18H30NaO8Si,
425.1608).
purification of 4–7,
a
Wakosil-II 5C18 AR Prep column
(4.6 mm × 250 mm) was used with a flow rate of 1.0 mL/min.
4.2. 2,4-Dibenzyloxyphenyl-tetra-O-acetyl-β-D-glucoside (10)
Under Ar atmosphere, BF3·OEt2 (22 μL, 176 μmol) was slowly added
to a stirred solution of phenol 8 (682 mg, 2.23 mmol) [20,21] and
2,3,4,6-tetra-O-acetyl-α-D-glucosyl trichloroacetimidate (9) (1.22 g,
2.48 mmol) [22,23] in anhydrous CH2Cl2 (20 mL) at −40 °C. After
being stirred for 5 min at −40 °C, the reaction was quenched with TEA
(30 μL) and EtOAc (150 mL) was poured into the solution. The organic
layer was washed with 1% hydrochloric acid (20 mL × 3), deionized
water (20 mL × 3), and brine (20 mL × 3). The resultant aqueous
layers were extracted with EtOAc (50 mL). The combined organic layers
were dried over Na2SO4. Filtration and concentration followed by silica
gel chromatography (30–40% EtOAc in hexane) gave the title com-
pound 10 (1.33 g, 2.09 mmol, 94% yield from 8) as a white solid.
Spectral data were fully consistent with those that were previously re-
ported [17].
4.6. 2-Methoxy-4-(tert-butyldimethylsiloxy)phenyl-β-D-glucoside (14)
K2CO3 (81 mg, 586 μmol) and MeI (40 μL, 643 μmol) were added to
a solution of 13 (187 mg, 465 μmol) in DMF (15 mL) at rt. The resultant
mixture was stirred for 24 h, poured into deionized water (40 mL), and
extracted with EtOAc (100 mL). The organic layer was washed with
deionized water (10 mL × 3) and brine (20 mL × 2). The resultant
aqueous layers were extracted with EtOAc (50 mL). The combined or-
ganic layers were dried over Na2SO4. Filtration and concentration fol-
lowed by silica gel chromatography (20% MeOH in CHCl3) gave the
title compound 14 (131 mg, 315 μmol, 68% yield) as a white solid.
[α]D25 − 67.2 (c 0.35, MeOH). 1H NMR (400 MHz, CD3OD) δ 7.05 (d,
J = 8.8 Hz, 1H, H-6), 6.47 (d, J = 2.7 Hz, 1H, H-3), 6.36 (dd, J = 2.7,
8.8 Hz, 1H, H-5), 4.75 (d, J = 7.7, 1H, H-1′), 3.86 (dd, J = 2.0, 12.2 Hz,
1H, H-6′), 3.80 (s, 3H, Me), 3.68 (dd, J = 5.1, 12.2 Hz, 1H, H-6′), 3.40
4.3. 2,4-Dihydoxyphenyl-tetra-O-acetyl-β-D-glucoside (11)
To a solution of 10 (100 mg, 157 μmol) in EtOAc (20 mL) was added
20% Pd(OH)2/C (10 mg). The reaction mixture was stirred for 6 h at rt
under a balloon of H2 and filtered through a pad of Celite. The filtrate
was concentrated and the residue was purified by silica gel chromato-
graphy (5% MeOH in CHCl3) to give the title compound 11 (67 mg,
147 μmol, 94% yield) as a white solid. Spectral data were fully con-
sistent with those that were previously reported [17].
(m, 4H, H-2′, H-3′, H-4′, H-5′), 0.98 (s, 9H, TBS), 0.18 (s, 6H, TBS). 13
C
NMR (100 MHz, CD3OD) δ 152.9 (C-4), 151.7 (C-2), 142.7 (C-1), 119.5
(C-6), 112.5 (C-5), 106.5 (C-3), 103.8 (C-1′), 78.1 (C-3′), 77.8 (C-5′),
75.0 (C-2′), 71.3 (C-4′), 62.5 (C-6′), 56.6 (Me), 26.2 (TBS), 19.0 (TBS),
−4.4 (TBS). ESIHRMS m/z 455.1507 [M+K]+ (calcd for C19H32KO8Si,
455.1504).
4.4. 2-Hydoxy-4-(tert-butyldimethylsiloxy)phenyl-tetra-O-acetyl-β-D-
glucoside (12)
4.7. Isotachioside (1)
A solution of 11 (400 mg, 876 μmol) in CH2Cl2 (20 mL) was treated
with TBSCl (165 mg, 1.10 mmol) and DIPEA (200 μL, 1.18 mmol) at
0 °C under Ar atmosphere. The reaction mixture was stirred for 24 h at
rt, poured into deionized water (50 mL), and extracted with EtOAc
(150 mL). The organic layer was washed with deionized water
(20 mL × 2) and brine (20 mL × 2). The resultant aqueous layers were
extracted with EtOAc (50 mL). The combined organic layers were dried
over Na2SO4. Filtration and concentration followed by silica gel chro-
matography (25% EtOAc in hexane) gave the title compound 12
To a solution of 14 (52 mg, 125 μmol) in THF (2 mL) at 0 °C, 1%
hydrochloric acid (1 mL) was added dropwise. After being stirred for
18 h at rt, the solution was neutralized with Amberlite IRA-400.
Filtration and concentration followed by solid phase extraction with
InertSep Slim C18-B cartridge (10–40% MeCN in H2O) gave the title
compound 1 (32 mg, 106 μmol, 85% yield) as a white solid. For bio-
logical evaluation, 1 was further purified by preparative RP-HPLC (12%
MeCN in H2O, tR = 8.5 min). [α]D25 − 41.9 (c 0.26, MeOH). 1H NMR
(400 MHz, C5D5N) δ 7.52 (d, J = 8.8 Hz, 1H, H-6), 6.96 (d, J = 2.7 Hz,
1H, H-3), 6.80 (dd, J = 2.7, 8.8 Hz, 1H, H-5), 5.48 (d, J = 7.6 Hz, 1H,
H-1′), 4.50 (dd, J = 2.2, 12.0 Hz, 1H, H-6′), 4.34 (m, 4H, H-2′, H-3′, H-
25
(434 mg, 761 μmol, 87% yield) as a white solid. [α]D −45.4 (c 0.22,
CHCl3). 1H NMR (400 MHz, CDCl3) δ 6.77 (d, J = 8.7 Hz, 1H, H-6),
6.43 (d, J = 2.8 Hz, 1H, H-3), 6.26 (dd, J = 2.8, 8.7 Hz, 1H, H-5), 6.07
(s, 1H, OH), 5.25 (t, J = 9.4 Hz, 1H, H-4′), 5.21 (dd, J = 7.6, 9.4 Hz,
1H, H-2′), 5.12 (t, J = 9.4 Hz, 1H, H-3′), 4.82 (d, J = 7.6 Hz, 1H, H-1′),
4.27 (dd, J = 5.4, 12.3 Hz, 1H, H-6′), 4.15 (dd, J = 2.5, 12.3 Hz, 1H, H-
6′), 3.78 (dd, J = 2.5, 5.4, 9.4 Hz, 1H, H-5′), 2.10 (s, 3H, Ac), 2.08 (s,
3H, Ac), 2.02 (s, 3H, Ac), 2.01 (s, 3H, Ac), 0.94 (s, 9H, TBS), 0.15 (s,
6H, TBS). 13C NMR (100 MHz, CDCl3) δ 170.6 (Ac), 170.2 (Ac), 169.7
(Ac), 169.4 (Ac), 153.4 (C-4), 148.4 (C-2), 138.9 (C-1), 119.3 (C-6),
111.5 (C-5), 108.3 (C-3), 102.6 (C-1′), 72.3 (C-3′), 72.2 (C-5′), 71.3 (C-
2′), 68.1 (C-4′), 61.6 (C-6′), 25.6 (TBS), 20.7 (Ac), 20.63 (Ac), 20.57
(Ac), 20.5 (Ac), 18.1 (TBS), −4.5 (TBS). ESIHRMS m/z 609.1772 [M
+K]+ (calcd for C26H38KO12Si, 609.1770).
4′, H-6′), 4.05 (dd, J = 2.2, 5.2, 9.5 Hz, 1H, H-5′), 3.74 (s, 3H, Me). 13
C
NMR (100 MHz, CD3OD) δ 154.9 (C-4), 152.0 (C-2), 141.0 (C-1), 120.5
(C-6), 107.6 (C-5), 104.3 (C-3), 101.8 (C-1′), 78.1 (C-3′), 77.8 (C-5′),
75.1 (C-2′), 71.4 (C-4′), 62.5 (C-6′), 56.5 (Me). ESIHRMS m/z 341.0642
[M+K]+ (calcd for C13H18KO8, 341.0639). The spectral data that
mentioned above were consistent with those that were previously re-
ported [16,25–27].
4.8. 2,4-Dibenzyloxyphenyl-β-D-glucoside (15)
NaOMe (400 mg, 7.41 mmol) was slowly added to a solution of 10
(1.05 g, 1.65 mmol) in MeOH (20 mL) at 0 °C and the resultant solution
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CarbohydrateResearch465(2018)22–28
was stirred for 1 h at 0 °C. After being stirred for 3 h at rt, the solution
was neutralized with Amberlite IR-120H. Filtration and concentration
followed by crystallization from EtOAc in MeOH gave the title com-
The spectral data that mentioned above were consistent with those that
were previously reported [17].
pound 15 (708 mg, 1.51 mmol, 92% yield) as
a
white solid.
4.11. 2,4-Dihydoxyphenyl-β-D-glucoside (4)
[α]D25 − 83.0 (c 0.28, MeOH). 1H NMR (400 MHz, CD3OD) δ 7.36 (m,
10H, Bn), 7.11 (d, J = 8.9 Hz, 1H, H-6), 6.68 (d, J = 2.8 Hz, 1H, H-3),
6.52 (dd, J = 2.8, 8.9 Hz, 1H, H-5), 5.10 (s, 2H, Bn), 4.98 (s, 2H, Bn),
4.79 (d, J = 7.4 Hz, 1H, H-1′), 3.84 (dd, J = 2.2, 12.1 Hz, 1H, H-6′),
3.68 (dd, J = 5.3, 12.1 Hz, 1H, H-6′), 3.40 (m, 4H, H-2′, H-3′, H-4′, H-
5′). 13C NMR (100 MHz, CD3OD) δ 156.3 (C-4), 150.9 (C-2), 142.8 (C-
1), 138.8 (Bn), 138.4 (Bn), 129.6 (Bn), 129.5 (Bn), 129.0 (Bn), 128.91
(Bn), 128.85 (Bn), 128.6 (Bn), 120.2 (C-6), 107.7 (C-5), 104.7 (C-3),
103.9 (C-1′), 78.2 (C-3′), 77.9 (C-5′), 75.1 (C-2′), 72.4 (C-4′), 71.5 (Bn),
71.4 (Bn), 62.5 (C-6′). ESIHRMS m/z 491.1685 [M+Na]+ (calcd for
To a solution of 15 (200 mg, 427 μmol) in MeOH (15 mL) was added
20% Pd(OH)2/C (30 mg). The reaction mixture was stirred for 3 h at rt
under a balloon of H2 and filtered through a pad of Celite. The filtrate
was concentrated and the residue was purified by solid phase extraction
with InertSep Slim C18-B cartridge (5–20% MeCN in H2O) gave the title
compound 4 (121 mg, 420 μmol, 98% yield) as a white solid. For bio-
logical evaluation, 4 was further purified by preparative RP-HPLC (10%
MeCN in H2O, tR = 4.1 min). [α]D25 − 42.1 (c 0.46, MeOH). 1H NMR
(400 MHz, CD3OD) δ 7.01 (d, J = 8.7 Hz, 1H, H-6), 6.31 (d, J = 2.9 Hz,
1H, H-3), 6.19 (dd, J = 2.9, 8.7 Hz, 1H, H-5), 4.56 (d, J = 7.7 Hz, 1H,
H-1′), 3.87 (dd, J = 2.2, 12.1 Hz, 1H, H-6′), 3.71 (dd, J = 5.1, 12.1 Hz,
1H, H-6′), 3.38 (m, 4H, H-2′, H-3′, H-4′, H-5′). 13C NMR (100 MHz,
CD3OD) δ 155.3 (C-4), 149.8 (C-2), 140.2 (C-1), 121.0 (C-6), 107.1 (C-
5), 106.0 (C-3), 104.4 (C-1′), 78.2 (C-3′), 77.7 (C-5′), 75.0 (C-2′), 71.2
(C-4′), 62.4 (C-6′). ESIHRMS m/z 311.0741 [M+Na]+ (calcd for
C
26H28NaO8, 491.1682).
4.9. 2,4-Dibenzyloxyphenyl-(6′-O-benzoyl)-O-β-D-glucoside (16)
Under Ar atmosphere, Bu2SnO (420 mg, 1.69 mmol) was added to a
solution of 15 (680 mg, 1.45 mmol) in MeOH (30 mL) and the mixture
was refluxed for 2 h. After being cooled to rt, the solvent was evapo-
rated to leave a white solid, which was dissolved in dioxane (30 mL). A
solution of BzCl (193 μL, 1.66 mmol) in dioxane (5 mL) was added
dropwise and the resultant solution was stirred for 36 h at rt. The sol-
vent was removed in vacuo and deionized water (20 mL) was added to
the residue. The aqueous mixture was extracted with EtOAc (150 mL)
and the organic layer was washed with deionized water (20 mL × 3)
and brine (20 mL × 2). The resultant aqueous layers were extracted
with EtOAc (50 mL). The combined organic layers were dried over
Na2SO4. Filtration and concentration followed by silica gel chromato-
graphy (20–40% EtOAc in hexane) gave the title compound 16
C
12H16NaO8, 311.0743). The spectral data that mentioned above were
consistent with those that were previously reported [17].
4.12. 2,4-Dibenzyloxyphenyl-tri-O-acetyl-β-D-xyloside (18)
Under Ar atmosphere, BF3·OEt2 (22 μL, 176 μmol) was slowly added
to a stirred solution of phenol 8 (601 mg, 1.96 mmol) [20,21] and 2,3,4-
tri-O-acetyl-α-D-xylosyl trichloroacetimidate (17) (1.21 g, 2.88 mmol)
[22,23] in anhydrous CH2Cl2 (20 mL) at −40 °C. After being stirred for
5 min at −40 °C, the reaction was quenched with TEA (30 μL) and
EtOAc (150 mL) was poured into the solution. The organic layer was
washed with 1% hydrochloric acid (20 mL × 3), deionized water
(20 mL × 3), and brine (20 mL × 3). The resultant aqueous layers were
extracted with EtOAc (50 mL). The combined organic layers were dried
over Na2SO4. Filtration and concentration followed by silica gel chro-
matography (40% EtOAc in hexane) gave the title compound 18
(1.10 g, 1.95 mmol, 100% yield from 8) as a white solid. [α]D25 − 34.2
(c 0.51, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.34 (m, 10H, Bn), 7.01
(d, J = 8.8 Hz, 1H, H-6), 6.60 (d, J = 2.8 Hz, 1H, H-3), 6.45 (dd,
J = 2.8, 8.8 Hz, 1H, H-5), 5.16 (m, 2H, H-1′, H-3′), 5.01 (s, 2H, Bn),
5.00 (m, 2H, H-2′, H-4′), 4.96 (s, 2H, Bn), 4.20 (dd, J = 4.8, 12.0 Hz,
1H, H-5′), 3.38 (dd, J = 8.0, 12.0 Hz, 1H, H-5′), 2.04 (s, 3H, Ac), 1.98
(s, 3H, Ac), 1.81 (s, 3H, Ac). 13C NMR (100 MHz, CDCl3) δ 170.1 (Ac),
169.9 (Ac), 169.5 (Ac), 155.8 (C-4), 150.7 (C-2), 140.0 (C-1), 136.8
(Bn), 136.6 (Bn), 128.60 (Bn), 128.58 (Bn), 128.0 (Bn × 2), 127.53
(Bn), 127.48 (Bn), 121.4 (C-6), 105.5 (C-5), 103.0 (C-3), 100.5 (C-1′),
70.9 (C-3′), 70.8 (Bn), 70.44 (Bn), 70.36 (C-2′), 68.8 (C-4′), 61.9 (C-5′),
20.8 (Ac), 20.7 (Ac), 20.4 (Ac). ESIHRMS m/z 587.1886 [M+Na]+
(calcd for C31H32O10Na, 587.1893).
(652 mg, 1.14 mmol, 79% yield in two steps) as
a white solid.
[α]D24 − 77.3 (c 1.0, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.03 (dd,
J = 1.2, 8.4 Hz, 2H, Bz), 7.70 (tt, J = 1.2, 8.4 Hz, 1H, Bz), 7.40 (m,
12H, Bn, Bz), 7.10 (d, J = 8.8 Hz, 1H, H-6), 6.60 (d, J = 2.8 Hz, 1H, H-
3), 6.32 (dd, J = 2.8, 8.8 Hz, 1H, H-5), 5.01 (s, 2H, Bn), 4.92 (s, 2H,
Bn), 4.64 (dd, J = 5.0, 12.0 Hz, 1H, H-6′), 4.59 (dd, J = 2.4, 12.0 Hz,
1H, H-6′), 4.58 (d, J = 7.5 Hz, 1H, H-1′), 3.57 (m, 4H, H-2′, H-3′, H-4′,
H-5′). 13C NMR (100 MHz, CDCl3) δ 166.9 (Bz), 156.0 (C-4), 150.5 (C-
2), 140.4 (C-1), 136.8 (Bn), 136.0 (Bn), 133.2 (Bz), 129.9 (Bz), 129.7
(Bz), 128.8 (Bn), 128.6 (Bn), 128.4 (Bz), 128.1 (Bn × 2), 127.7 (Bn),
127.5 (Bn), 122.2 (C-6), 105.7 (C-5), 104.2 (C-3), 102.6 (C-1′), 76.0 (C-
3′), 74.3 (C-5′), 73.3 (C-2′), 71.2 (Bn), 70.4 (Bn), 69.8 (C-4′), 63.6 (C-
6′). ESIHRMS m/z 595.1943 [M+Na]+ (calcd for C33H32NaO9,
595.1944).
4.10. 2,4-Dihydroxyphenyl-(6′-O-benzoyl)-O-β-D-glucoside (2)
To a solution of 16 (302 mg, 527 μmol) in MeOH (10 mL) was added
20% Pd(OH)2/C (30 mg). The reaction mixture was stirred for 3 h at rt
under a balloon of H2 and filtered through a pad of Celite. The filtrate
was concentrated and the residue was purified by solid phase extraction
with InertSep Slim C18-B cartridge (20–40% MeCN in H2O) gave the
title compound 2 (203 mg, 517 μmol, 98% yield) as a white solid. For
biological evaluation, 2 was further purified by preparative RP-HPLC
(32% MeCN in H2O, tR = 7.0 min). [α]D25 − 34.1 (c 0.22, MeOH). 1H
NMR (400 MHz, (CD3)2SO) δ 7.98 (d, J = 7.4 Hz, 2H, Bz), 7.69 (t,
J = 7.4 Hz, 1H, Bz), 7.56 (t, J = 7.4 Hz, 2H, Bz), 6.86 (d, J = 8.7 Hz,
1H, H-6), 6.21 (d, J = 2.8 Hz, 1H, H-3), 5.93 (dd, J = 2.8, 8.7 Hz, 1H,
H-5), 4.62 (dd, J = 2.0, 12.0 Hz, 1H, H-6′), 4.55 (d, J = 7.3 Hz, 1H, H-
1′), 4.29 (dd, J = 7.6, 12.0 Hz, 1H, H-6′), 3.67 (m, 1H, H-5′), 3.28 (m,
3H, H-2′, H-3′, H-4′). 13C NMR (100 MHz, (CD3)2SO) δ 165.7 (Bz),
153.7 (C-4), 148.1 (C-2), 138.4 (C-1), 133.6 (Bz), 129.9 (Bz), 129.4
(Bz × 2), 129.0 (Bz × 2), 118.4 (C-6), 105.3 (C-5), 103.6 (C-3), 103.5
(C-1′), 75.8 (C-3′), 74.0 (C-5′), 73.6 (C-2′), 70.5 (C-4′), 64.5 (C-6′).
ESIHRMS m/z 431.0743 [M+K]+ (calcd for C19H20KO9, 431.0744).
4.13. 2,4-Dihydoxyphenyl-β-D-xyloside (5)
NaOMe (150 mg, 2.78 mmol) was slowly added to a solution of 18
(502 mg, 889 μmol) in MeOH (20 mL) at 0 °C and the resultant solution
was stirred for 1 h at 0 °C. After being stirred for 3 h at rt, the solution
was neutralized with Amberlite IR-120H. Filtration and concentration
followed by silica gel chromatography (5% MeOH in CHCl3) gave the
polyol as a white solid, which was used in the next step without further
purification.
To a solution of the polyol in MeOH (15 mL) was added 20% Pd
(OH)2/C (45 mg). The reaction mixture was stirred for 3 h at rt under a
balloon of H2 and filtered through a pad of Celite. The filtrate was
concentrated and the residue was purified by solid phase extraction
with InertSep Slim C18-B cartridge (5–20% MeCN in H2O) gave the title
compound 5 (221 mg, 856 μmol, 96% yield in two steps) as a white
solid. For biological evaluation, 5 was further purified by preparative
26

T. Matsumoto et al.
CarbohydrateResearch465(2018)22–28
RP-HPLC (5% MeCN in H2O, tR = 5.5 min). [α]D25 − 44.8 (c 0.44,
MeOH). 1H NMR (400 MHz, CD3OD) δ 6.90 (d, J = 8.7 Hz, 1H, H-6),
6.31 (d, J = 2.8 Hz, 1H, H-3), 6.19 (dd, J = 2.8, 8.7 Hz, 1H, H-5), 4.51
(d, J = 7.2 Hz, 1H, H-1′), 3.91 (dd, J = 5.4, 11.5 Hz, 1H, H-5′), 3.55
(dd, J = 5.4, 9.0, 11.5 Hz, 1H, H-4′), 3.39 (m, 2H, H-2′, H-3′), 3.25 (t,
J = 11.5 Hz, 1H, H-5′). 13C NMR (100 MHz, CD3OD) δ 155.4 (C-4),
149.9 (C-2), 139.9 (C-1), 121.1 (C-6), 107.1 (C-5), 106.5 (C-3), 104.4
(C-1′), 77.5 (C-3′), 74.8 (C-2′), 71.0 (C-4′), 67.1 (C-5′). ESIHRMS m/z
281.0645 [M+Na]+ (calcd for C11H14NaO7, 281.0637).
H-6′, H-6″), 3.68–3.31 (m, 8H, H-2′, H-2″, H-3′, H-3″, H-4″, H-5″, H-5′,
H-6′), 3.22 (t, J = 9.2 Hz, 1H, H-4′). 13C NMR (100 MHz, CD3OD) δ
155.4 (C-4), 149.9 (C-2), 140.1 (C-1), 121.1 (C-6), 107.1 (C-5), 105.8
(C-3), 104.6 (C-1′), 104.4 (C-1″), 80.1 (C-4′), 78.1 (C-3′), 77.8 (C-3″),
76.8 (C-5′), 76.0 (C-5″), 74.9 (C-2′), 74.7 (C-2″), 71.4 (C-4″), 62.4 (C-
6′), 61.5 (C-6″). ESIHRMS m/z 489.1012 [M+K]+ (calcd for
C
18H26KO13, 489.1011).
4.16. 2,4-Dibenzyloxyphenyl-hepta-O-acetyl-β-D-maltoside (21)
4.14. 2,4-Dibenzyloxyphenyl-hepta-O-acetyl-β-D-cellobioside (19)
Under Ar atmosphere, BF3·OEt2 (22 μL, 176 μmol) was slowly added
to a stirred solution of phenol 8 (432 mg, 1.41 mmol) [20,21] and
2,2′,3,3′,4,4′,6,6′-hepta-O-acetyl-α-D-maltosyl trichloroacetimidate (22)
(1.02 g, 1.31 mmol) [22,23] in anhydrous CH2Cl2 (20 mL) at −40 °C.
After being stirred for 5 min at −40 °C, the reaction was quenched with
TEA (30 μL) and EtOAc (200 mL) was poured into the solution. The
organic layer was washed with 1% hydrochloric acid (20 mL × 3),
deionized water (20 mL × 3), and brine (20 mL × 3). The resultant
aqueous layers were extracted with EtOAc (50 mL). The combined or-
ganic layers were dried over Na2SO4. Filtration and concentration fol-
lowed by crystallization from cyclohexane gave the title compound 21
(1.12 g, 1.21 mmol, 86% yield from 8) as a white solid. [α]D25 +27.1 (c
0.38, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.34 (m, 10H, Bn), 7.02 (d,
J = 8.8 Hz, 1H, H-6), 6.59 (d, J = 2.8 Hz, 1H, H-3), 6.44 (dd, J = 2.8,
8.8 Hz, 1H, H-5), 5.40 (d, J = 4.0 Hz, 1H, H-1″), 5.34 (dd, J = 9.8,
10.5 Hz, 1H, H-3″), 5.27 (t, J = 9.1 Hz, 1H, H-3′), 5.07 (dd, J = 7.7,
9.1 Hz 1H, H-2′), 5.03 (t, J = 9.8 Hz, 1H, H-4″), 5.00 (s, 2H, Bn), 4.96
(s, 2H, Bn), 4.92 (d, J = 7.7 Hz, 1H, H-1′), 4.84 (dd, J = 4.0, 10.5 Hz,
1H, H-2″), 4.44 (dd, J = 2.6, 12.1 Hz, 1H, H-6′), 4.24 (m, 2H, H-6′, H-
6″), 4.06 (t, J = 9.1 Hz, 1H, H-4′), 4.03 (dd, J = 2.1, 11.4 Hz, 1H, H-6″),
3.94 (dd, J = 2.1, 3.6, 9.8 Hz, 1H, H-5″), 3.67 (dd, J = 2.6, 4.3, 9.1 Hz,
1H, H-5′), 2.09 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.00 (s,
3H, Ac), 1.981 (s, 3H, Ac), 1.975 (s, 3H, Ac), 1.70 (s, 3H, Ac). 13C NMR
(100 MHz, CDCl3) δ 170.53 (Ac), 170.51 (Ac), 170.4 (Ac), 170.1 (Ac),
169.9 (Ac), 169.8 (Ac), 169.4 (Ac), 155.9 (C-4), 150.5 (C-2), 140.2 (C-
1), 136.8 (Bn), 136.6 (Bn), 128.58 (Bn), 128.56 (Bn), 128.03 (Bn),
128.02 (Bn), 127.5 (Bn), 127.4 (Bn), 121.6 (C-6), 105.4 (C-5), 102.9 (C-
3), 100.4 (C-1′), 95.5 (C-1″), 75.2 (C-4′), 72.6 (C-3′), 72.1 (C-5′), 72.0
(C-2′), 70.7 (Bn), 70.4 (Bn), 70.0 (C-3″), 69.3 (C-5″), 68.5 (C-2″), 68.0
(C-4″), 62.6 (C-6″), 61.5 (C-6′), 20.9 (Ac), 20.8 (Ac), 20.7 (Ac), 20.58
(Ac), 20.55 (Ac × 2), 20.2 (Ac). ESIHRMS m/z 947.2953 [M+Na]+
(calcd for C46H52NaO20, 947.2950).
Under Ar atmosphere, BF3·OEt2 (22 μL, 176 μmol) was slowly added
to a stirred solution of phenol 8 (480 mg, 1.57 mmol) [20,21] and
2,2′,3,3′,4,4′,6,6′-hepta-O-acetyl-α-D-cellobiosyl
trichloroacetimidate
(20) (1.21 g, 1.55 mmol) [22,23] in anhydrous CH2Cl2 (20 mL) at
−40 °C. After being stirred for 5 min at −40 °C, the reaction was
quenched with TEA (30 μL) and EtOAc (200 mL) was poured into the
solution. The organic layer was washed with 1% hydrochloric acid
(20 mL × 3), deionized water (20 mL × 3), and brine (20 mL × 3). The
resultant aqueous layers were extracted with EtOAc (50 mL). The
combined organic layers were dried over Na2SO4. Filtration and con-
centration followed by silica gel chromatography (40% EtOAc in
hexane) gave the title compound 19 (890 mg, 962 μmol, 61% yield
from 8) as a white solid. [α]D25 − 18.7 (c 0.61, CHCl3). 1H NMR
(400 MHz, CDCl3) δ 7.34 (m, 10H, Bn), 7.01 (d, J = 8.8 Hz, 1H, H-6),
6.59 (d, J = 2.8 Hz, 1H, H-3), 6.42 (1H, dd, J = 2.8, 8.8 Hz, H-5), 5.20
(t, J = 9.6 Hz, 1H, H-3′), 5.14 (dd, J = 7.5, 9.6 Hz, 1H, H-2′), 5.12 (t,
J = 9.5 Hz, 1H, H-3″), 5.04 (t, J = 9.5 Hz, 1H, H-4″), 4.98 (s, 2H, Bn),
4.96 (s, 2H, Bn), 4.91 (dd, J = 8.0, 9.5 Hz, 1H, H-2″), 4.83 (d,
J = 7.5 Hz, 1H, H-1′), 4.49 (d, J = 8.0 Hz, 1H, H-1″), 4.46 (dd, J = 2.0,
12.0 Hz, 1H, H-6′), 4.35 (dd, J = 4.4, 12.0 Hz, 1H, H-6′), 4.10 (dd,
J = 5.2, 12.0 Hz, 1H, H-6″), 4.02 (dd, J = 2.2, 12.0 Hz, 1H, H-6″), 3.82
(t, J = 9.6 Hz, 1H, H-4′), 3.64 (dd, J = 2.0, 4.4, 9.6 Hz, 1H, H-5′), 3.56
(dd, J = 2.2, 5.2, 9.5 Hz, 1H, H-5″), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac),
2.00 (s, 3H, Ac), 1.96 (s, 6H, Ac), 1.96 (s, 3H, Ac), 1.70 (s, 3H, Ac). 13
C
NMR (100 MHz, CDCl3) δ 170.5 (Ac), 170.3 (Ac), 170.2 (Ac), 169.8
(Ac × 2), 169.3 (Ac), 169.0 (Ac), 155.9 (C-4), 150.6 (C-2), 140.3 (C-1),
136.8 (Bn), 136.5 (Bn), 128.6 (Bn × 2), 128.1 (Bn), 128.0 (Bn), 127.51
(Bn), 127.50 (Bn), 121.7 (C-6), 105.4 (C-5), 102.9 (C-3), 100.9 (C-1″),
100.8 (C-1′), 76.5 (C-4′), 72.9 (C-3′), 72.7 (C-3″), 72.4 (C-5′), 72.0 (C-
5″), 71.6 (C-2′), 71.4 (C-2″), 70.8 (Bn), 70.4 (Bn), 67.8 (C-4″), 61.7 (C-
6′), 61.5 (C-6″), 20.8 (Ac), 20.6 (Ac), 20.53 (Ac), 20.51 (Ac × 3), 20.3
(Ac). ESIHRMS m/z 947.2953 [M+Na]+ (calcd for C46H52NaO20
947.2950).
,
4.17. 2,4-Dihydroxyphenyl -β-D-maltoside (7)
NaOMe (400 mg, 7.41 mmol) was slowly added to a solution of 21
(1.20 g, 1.30 mmol) in MeOH (40 mL) at 0 °C and the resultant solution
was stirred for 1 h at 0 °C. After being stirred for 3 h at rt, the solution
was neutralized with Amberlite IR-120H. Filtration and concentration
followed by silica gel chromatography (10% MeOH in CHCl3) gave the
polyol as a white solid, which was used in the next step without further
purification.
4.15. 2,4-Dihydroxyphenyl -β-D-cellobioside (6)
NaOMe (180 mg, 3.33 mmol) was slowly added to a solution of 19
(420 mg, 454 μmol) in MeOH (20 mL) at 0 °C and the resultant solution
was stirred for 1 h at 0 °C. After being stirred for 3 h at rt, the solution
was neutralized with Amberlite IR-120H. Filtration and concentration
followed by silica gel chromatography (5% MeOH in CHCl3) gave the
polyol as a white solid, which was used in the next step without further
purification.
To a solution of the polyol in MeOH (12 mL) was added 20% Pd
(OH)2/C (25 mg). The reaction mixture was stirred for 3 h at rt under a
balloon of H2 and filtered through a pad of Celite. The filtrate was
concentrated and the residue was purified by solid phase extraction
with InertSep Slim C18-B cartridge (3–10% MeCN in H2O) gave the title
compound 6 (158 mg, 351 μmol, 77% yield in two steps) as a white
solid. For biological evaluation, 6 was further purified by preparative
RP-HPLC (5% MeCN in H2O, tR = 9.5 min). [α]D25 − 50.2 (c 0.20,
MeOH). 1H NMR (400 MHz, CD3OD) δ 6.99 (d, J = 8.8 Hz, 1H, H-6),
6.31 (d, J = 2.9 Hz, 1H, H-3), 6.19 (dd, J = 2.9, 8.8 Hz, 1H, H-5), 4.60
(d, J = 7.8 Hz, 1H, H-1″), 4.43 (d, J = 7.8 Hz, 1H, H-1′), 3.88 (m, 3H,
To a solution of the polyol in MeOH (40 mL) was added 20% Pd
(OH)2/C (80 mg). The reaction mixture was stirred for 3 h at rt under a
balloon of H2 and filtered through a pad of Celite. The filtrate was
concentrated and the residue was purified by solid phase extraction
with InertSep Slim C18-B cartridge (3–10% MeCN in H2O) gave the title
compound 7 (473 mg, 1.05 mmol, 81% yield in two steps) as a white
solid. For biological evaluation, 7 was further purified by preparative
25
RP-HPLC (5% MeCN in H2O, tR = 3.5 min). [α]D +13.8 (c 0.50,
MeOH). 1H NMR (400 MHz, CD3OD) δ 7.00 (d, J = 8.7 Hz, 1H, H-6),
6.31 (d, J = 2.8 Hz, 1H, H-3), 6.19 (dd, J = 2.8, 8.7 Hz, 1H, H-5), 5.18
(d, J = 3.8 Hz, 1H, H-1″), 4.59 (d, J = 7.9 Hz, 1H, H-1′), 3.87–3.45 (m,
11H, H-2′, H-2″, H-3′, H-3″, H-4″, H-5″, H-5′, H-6′, H-6″), 3.26 (t,
J = 9.2 Hz, 1H, H-4′). 13C NMR (100 MHz, CD3OD) δ 155.4 (C-4), 149.8
(C-2), 140.1 (C-1), 121.1 (C-6), 107.1 (C-5), 105.9 (C-3), 104.4 (C-1′),
27

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CarbohydrateResearch465(2018)22–28
102.9 (C-1″), 80.8 (C-4′), 77.4 (C-3′), 76.8 (C-5′), 75.1 (C-2′), 74.8 (C-
3″), 74.5 (C-5″), 74.2 (C-2″), 71.5 (C-4″), 62.7 (C-6″), 61.9 (C-6′).
ESIHRMS m/z 473.1276 [M+Na]+ (calcd for C18H26NaO13, 473.1271).
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4.18. Tyrosinase inhibitory assay
Tyrosinase (E.C.1.14.18.1) purchased from Sigma–Aldrich. All in-
hibitors were first dissolved in DMSO and used for the experiment by
appropriate dilution with DMSO [32–34]. First, 0.1 mL of the DMSO
solution of the inhibitor was mixed with 0.3 mL of a 5.0 mM of L-DOPA
aqueous solution, 0.6 mL of 0.25 M sodium phosphate buffer (pH 6.8),
and 1.9 mL of water in this order. The resultant solution was incubated
at 30 °C for 5 min. Then, 0.1 mL of enzyme solution (4.8 units/mL) were
added to the mixture. This solution was immediately monitored for the
formation of dopachrome by measuring the linear increase in optical
density at 475 nm. The reaction was carried out under a constant
temperature of 30 °C. Absorption measurements were recorded using a
Jasco V-630 spectrophotometer. The experimental data were analyzed
by using Sigma Plot 12.0 to estimate IC50 values.
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We are grateful to Dr. Yoichi Yamada and Dr. Michinori Karikomi
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the NMR spectra and the optical rotations values. This study was sup-
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