Chemoenzymatic syntheses of naturally occurring β-glucosides

Article abstract of DOI:10.1016/S0957-4166(99)00228-1

Enzymatic glycosidation of the various kinds of primary alcohols 5, 7, 9, 11, 13 and 15 and 4-nitrophenyl-β-D-glucopyranoside 4 using β- glucosidase from almonds gave stereoselectively β-D-glucosides 6, 8, 10, 12, 14 and 16 including the naturally occurring β-glucosides in moderate yield. Among them, the β-glucosides 6, 8 and 10 were converted to the cyanoglycosides, rhodiocyanoside A 20a, osmaronin 24a and sutherlandin 29, respectively.

Full text of DOI:10.1016/S0957-4166(99)00228-1

Pergamon
Tetrahedron: Asymmetry 10 (1999) 2429–2439
Chemoenzymatic syntheses of naturally occurring β-glucosides
Hiroyuki Akita,a,∗ Katsumi Kurashima,b Takuya Nakamura a and Keisuke Kato a
aSchool of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba 274-8510, Japan
bCentral Research Laboratory, Godo Shusei Co. Ltd, 250, Nakahara, Kamihongo, Matsudo, Chiba 271-0064, Japan
Received 21 April 1999; revised 24 May 1999; accepted 27 May 1999
Abstract
Enzymatic glycosidation of the various kinds of primary alcohols 5, 7, 9, 11, 13 and 15 and 4-nitrophenyl-β-D-
glucopyranoside 4 using β-glucosidase from almonds gave stereoselectively β-D-glucosides 6, 8, 10, 12, 14 and
16 including the naturally occurring β-glucosides in moderate yield. Among them, the β-glucosides 6, 8 and 10
were converted to the cyanoglycosides, rhodiocyanoside A 20a, osmaronin 24a and sutherlandin 29, respectively.
© 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The development of stereoselective methods for the synthesis of glycosidic linkages offers a consider-
able challenge to synthetic chemists.1 Although the well described chemical synthesis of glycosidic
structures is becoming increasingly established, several steps of selective protection, activation and
coupling are necessary. This problem in chemical synthesis has enhanced the development of enzymatic
approaches because a large number of the enzymes involved in carbohydrate biosynthesis are well
described and some enzymes are commercially available. For example, β-glucosidase catalyzes the
stereospecific hydrolytic cleavage of the β-glucosidic bond in the substrate 1 to give glucose 2 (Scheme 1,
path a). Meanwhile, the reaction of the β-glucoside 1 and a nucleophile such as an alcohol are reported
to afford a new glucoside 3 exclusively with the β-configuration (Scheme 1, path b).2 In the case where
the alcohol is cheap and readily available, the use of high concentrations of the alcohol acceptor in order
to favor formation of 3 over 2 is reported to provide good yields of the β-glucosides. However, the need
for using high concentrations of the alcohol clearly limits the scope and application of this reaction. We
are attracted by this transglycosylation reaction since if a second nucleophile was a suitably selected
functionalized alcohol used in limited amounts it might provide an effective method of glycosidation
under aqueous conditions without the need for any protection of the hydroxyl group. We now describe
∗
Corresponding author. E-mail: akita@phar.toho-u.ac.jp
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00228-1

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the syntheses of β-glucosides using β-glucosidase from almonds and its application to the short path
synthesis of naturally occurring β-glucosides.
Scheme 1.
2. Results and discussion
In order to determine the effective enzyme and glycosyl donor, the synthesis of benzyl-β-D-glucoside
or n-hexyl-β-D-glucoside was selected as a model transglycosylation reaction. From a screening experi-
ment using either glycosyl transferases or glucosidases as catalysts, β-D-glucosidase (EC 3.2.1.21) from
almonds was effective for transglycosylation. This enzyme was purchased from the Sigma Chemical Co.
(G-0395, 2.5–3.4 U/mg). Meanwhile, 4-nitrophenyl-β-D-glucopyranoside 43 as a glycosyl donor was
chosen from either several kinds of phenyl-β-D-glucopyranoside congeners or cellobiose. The following
functionalized alcohols such as 3-methyl-2-buten-1-ol 5, 2-methyl-2-propen-1-ol 7, 2-benzyloxy-1,3-diol
9, 4-methoxybenzyl alcohol 11, 4-hydroxyphenethyl alcohol 13, and cinnamyl alcohol 15 were selected
as nucleophiles. Addition of the glycosyl donor 4 to a solution of the acceptor substrate dissolved in
phosphate buffer containing β-glucosidase was carried out over a period of 3–26 h. The reaction can be
easily monitored by reverse phase HPLC and terminated when the formation of the desired product is at
a maximum. The results are summarized in Table 1. The structures of all products were determined by
either conversion to the corresponding acetates or direct comparison with the corresponding natural β-
glucosides. Identification of the β-configuration of the anomeric center was easily achieved via analysis
of the C1-H/C2-H coupling constant. The spectral data of the synthetic β-D-glucosides (6: mp 78–81°C,
[α]D −40.8 (c=0.54, MeOH); 12: mp 139–140°C, [α]D −57.5 (c=0.55, MeOH); 14: mp 159–160°C,
[α]D −28.4 (c=0.5, MeOH); 16: mp 114–116°C, [α]D −48.8 (c=0.3, MeOH)) were identical with
those of the reported naturally occurring β-D-glucosides (3-methyl-2-buten O-β-glucopyranoside 6:4 mp
1
68–70°C, [α]D −23.6 (c=0.2, MeOH), H and 13C NMR; 4-methoxybenzyl O-β-glucopyranoside 12:5
1
mp 137–138°C, [α]D −51.1 (MeOH), H and 13C NMR; salidroside (rhodioside) 14:6 mp 161–164°C,
[α]D −28.3 (MeOH), 1H and 13C NMR; cinnamyl O-β-glucopyranoside 16:7 mp 114–116°C, [α]D −48.1
1
(c=1.0, MeOH), H and 13C NMR), respectively. The β-glucoside 16 was reported to exhibit potential
anti-tumor-promoting activity which corresponds with the inhibitory effects on Epstein–Barr virus (EBV)
activation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA).7b In spite of the moderate chemical
yield, in all cases only the β-glucoside was obtained. Prolonged reaction times (>24 h) generally resulted
in decreased yields of the glucosides presumably due to competing hydrolysis of the product by β-
glucosidase. In the case of the transglucosylation of an alcohol with 2-nitrophenyglucopyranoside or 4-
nitrophenyglucopyranoside 4 in the presence of β-glucosidase in phosphate buffer, the chemical yield of
the β-glucoside is reported to be low and less than 30%.2 In order to avoid the cleavage of the glycosidic
bond of the produced β-glucoside, transglucosylation of 5-phenyl-1-pentanol with 4 using lipid-coated
β-glucosidase in dry isopropyl ether is reported to give the corresponding β-glucoside in 23% yield.8

H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
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Table 1
Enzymatic formation of the glycosidic bond is thought to be mechanistically similar to the acid-
catalyzed formation of glycosides. By the use of appropriate glycosyl donor 1, the enzyme-bound
glycosyl cation (Scheme 1) can be captured by an alcohol to yield a glycoside. A proximal carboxylate
moiety appears as a common structural feature among β-glucosidases and presumably acts to stabilize
this glycosyl cation with an α-configuration at the anomeric carbon.9 Nucleophilic alcohol presumably
attacks at the anomeric carbon from the β-side to afford exclusively β-glucoside.
The synthetic β-glucopyranoside 6 was converted to the cyanoglucoside rhodiocyanoside A 20a which
was isolated from the underground part of Rhodiola quadrifida (Pall.) Fisch. et Mey. (Crassulaceae) and
found to show antiallergic activity in a passive cutaneous anaphylaxis test in rat (Scheme 2).10 Acetyla-
tion of 6 gave an acetate 17 (98% yield, [α]D −27.6 (c=0.38, CHCl3)) which was subjected to ozonolysis
to afford the aldehyde 18. The Horner–Emmons reaction of 18 using diethyl (1-cyanoethyl)phosphonate
furnished (Z)-19a (32% yield from 17, [α]D −2.65 (c=0.68, MeOH)) and (E)-19b (10% yield from
17, [α]D −25.3 (c=0.43, CHCl3)). The physical data of (Z)-19a were identical with those (1H and 13C
NMR) of the reported (Z)-19a.10 Deprotection of (Z)-19a and (E)-19b provided the β-D-glucosides 20a
(77% yield, [α]D −15.3 (c=0.34, MeOH)) and unnatural 20b (87% yield, [α]D −23.5 (c=0.34, MeOH)),
respectively. The physical data ([α]D, 1H and 13C NMR) of the synthetic 20a were identical with those
([α]D −16.1 (c=0.4, MeOH), 1H and 13C NMR) of the natural rhodiocyanoside A (20a).10

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Scheme 2. (a) Ac2O/pyridine; (b) (1) O3, (2) Me2S; (c) (EtO)2POCH(CN)CH3/NaH, THF; (d) K2CO3/MeOH; (e)
(EtO)2POCH2CN/NaH, THF; (f) H2/10% Pd–C; (g) Jones reagent
The synthetic 8 was converted into the cyanoglucoside osmaronin 24a which was isolated from a
methanolic extract of the leaves of Osmaronia cerasiformis.11 Acetylation of 8 gave an acetate 21
(99% yield, [α]D −26.8 (c=0.6, CHCl3)) which was subjected to ozonolysis to afford a ketone 22.
The Horner–Emmons reaction of 22 using diethyl cyanomethylphosphonate furnished (Z)-23a (22%
yield from 21, [α]D +10.3 (c=0.6, CHCl3)) and (E)-23b (10% yield from 21, [α]D −26.2 (c=0.68,
CHCl3)). Deprotection of (Z)-23a and (E)-23b gave the β-D-glucosides 24a (83% yield, [α]D +20.0
(c=0.7, MeOH)) and 24b (94% yield, [α]D −17.5 (c=0.51, MeOH)), respectively. The spectral data of
the synthetic 24a were identical with those (1H and 13C NMR) of the natural osmaronin (24a).11
Then the synthetic 10 was converted to the cyanoglucoside sutherlandin 29 which was isolated from
leaves of Acacia sutherlandii.12 Acetylation of a diastereomeric mixture of 10 gave the corresponding
acetate 25 which was subjected to the hydrogenation (26) and the subsequent oxidation to yield the α-
acetoxyl ketone 27 (84% overall yield from 25, [α]D −7.55 (c=1.10, CHCl3)). The Horner–Emmons
reaction of 27 using diethyl cyanomethylphosphonate furnished the (Z)-28a (33% yield from 27, [α]D
−2.48 (c=1.10, CHCl3)) and (E)-28b (31% yield from 27, [α]D −23.0 (c=1.10, CHCl3)). Deprotection of

H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
2433
the presumably desired (Z)-28a afforded the (Z)-29a (76% yield, [α]D −14.6 (c=0.54, MeOH)) whose
13C NMR spectra were identical with those of the natural sutherlandin 29.12
In summary, we have demonstrated the practical syntheses of a series of β-glucosides 6, 8, 10, 12, 14,
and 16 directly from the sugar donor and an alcohol acceptor using β-glucosidase from almonds. Among
them, the first syntheses of the cyanoglucosides, rodionoside A 20a, osmaronin 24a, and sutherlandin 29
were achieved from the β-glucosides 6, 8, and 10, respectively.
3. Experimental
3.1. General
All melting points were measured on a Yanaco MP-3S micro melting point apparatus and are
1
uncorrected. H and 13C NMR spectra were recorded on a Jeol EX 400 spectrometer in CDCl3. Carbon
substitution degrees were established by DEPT pulse sequence. The fast atom bombardment mass spectra
(FABMS) were obtained with a Jeol LMS-DX 303 spectrometer. IR spectra were recorded on a Jasco
FT/IR-300 spectrometer. Optical rotations were measured with a Jasco DIP-370 digital polarimeter. All
evaporations were performed under reduced pressure. For column chromatography, silica gel (Kieselgel
60) was employed.
3.2. 3-Methyl-2-buten-1-ol β-D-glucopyranoside 6
A mixture of 4 (1 g, 3.32 mmol), 3-methyl-2-buten-1-ol 5 (287 mg, 3.32 mmol), and β-glucosidase
(50 mg, 170 units) in phosphate buffer (pH 5, 34 ml) was incubated for 24 h at 30°C. After the reaction
mixture was boiled for 5 min and evaporated under reduced pressure, the residue was chromatographed
on silica gel (50 g, CHCl3:MeOH (30:1)) to afford 6 (205 mg, 25%) as a colorless amorphous powder.
26
1
6: Mp 78–81°C; [α]D −40.8 (c=0.54, MeOH); IR (KBr): 3346, 2882, 1073, 1025 cm−1; H NMR
(pyridine-d5): δ 1.54 (3H, s), 1.60 (3H, s), 3.94–3.97 (1H, m), 4.06 (1H, t, J=8 Hz), 4.21–4.29 (2H, m),
4.38 (1H, d, J=11.7 Hz), 4.36–4.40 (1H, m), 4.59 (1H, d, J=11.7 Hz), 4.54–4.55 (1H, m), 4.90 (1H, d,
J=7.3 Hz), 5.52 (1H, t, J=6.3 Hz); 13C NMR (pyridine-d5): δ 135.9, 121.9, 103.5, 78.6, 78.5, 75.2, 71.7,
65.7, 62.8, 25.6, 17.9; FABMS m/z: 249 (M+1)+. Anal. found: C, 52.78; H, 8.24. Calcd for C11H20O6:
C, 53.21; H, 8.12%.
3.3. Acetylation of 6
A solution of 6 (200 mg, 0.81 mmol) in pyridine (2 ml, 24.8 mmol), 4-dimethylaminopyridine (10
mg, 0.08 mmol) was treated with Ac2O (0.5 ml, 5.3 mmol) and the reaction mixture was stirred for 15
min at room temperature. The reaction mixture was diluted with H2O (50 ml) and extracted with AcOEt
(50 ml×3). The AcOEt extract was washed with sat. brine (100 ml), dried over MgSO4, and filtered.
Removal of the solvent gave a residue which was chromatographed on silica gel (20 g, n-hexane:AcOEt
27
(2:1)) to give 17 (329 mg, 98%) as colorless needles. 17: Mp 81–83°C; [α]D −27.6 (c=0.38, CHCl3);
IR (KBr): 2969, 1754, 1384, 1209 cm−1; 1H NMR: δ 1.67 (3H, s), 1.76 (3H, s), 2.00 (3H, s), 2.02 (3H, s),
2.04 (3H, s), 2.08 (3H, s), 3.67 (1H, ddd, J=2.4, 4.9, 9.8 Hz), 4.14–4.27 (4H, m), 4.53 (1H, d, J=7.8 Hz),
4.99 (1H, dd, J=7.8, 9.8 Hz), 5.08 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.8 Hz), 5.24–5.28 (1H, m); FABMS
m/z: 455 (M+K)+. Anal. found: C, 54.69; H, 6.74. Calcd for C19H28O10: C, 54.79; H, 6.78%.

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H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
3.4. Rhodiocyanoside A tetraacetate 19a and its isomer 19b
(i) A solution of 17 (837 mg, 2.01 mmol) in CH2Cl2 (15 ml) was subjected to ozonolysis at −78°C for 2
h and treated with Me2S (1 ml, 13.6 mmol). The whole reaction mixture was stirred at room temperature
for 30 min and evaporated under reduced pressure to provide a crude aldehyde 18. (ii) NaH (60%, 77
mg, 2.01 mmol) was washed with dry n-hexane (5 ml×3) and added to dry THF (8 ml) under an argon
atmosphere at 0°C. A solution of diethyl (1-cyanoethyl)phosphonate (384 mg, 2.01 mmol) in dry THF
(2 ml) was added to the above mixture and the whole mixture was stirred for 15 min at 0°C. A solution
of the crude 18 in dry THF (6 ml) was added to the above reaction mixture and the whole mixture was
stirred for 30 min at 0°C. The reaction mixture was diluted with H2O (50 ml) and extracted with AcOEt
(30 ml×3). The organic layer was washed with sat. brine and dried over MgSO4. Evaporation of the
solvent gave a residue which was chromatographed on silica gel (30 g, n-hexane:AcOEt (3:2)) to afford
19a (288 mg, 33% from 17) as a colorless oil and 19b (85 mg, 10% from 17) as colorless needles in
elution order. 19a: [α]D −2.65 (c=0.68, MeOH); IR (neat): 2956, 2220, 1752, 1046 cm−1; H NMR:
δ 2.00 (3H, d, J=1.5 Hz), 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.10 (3H, s), 3.70–3.74 (1H, m), 4.17
(1H, dd, J=2.4, 12.2 Hz), 4.26 (1H, dd, J=4.9, 12.2 Hz), 4.40–4.53 (2H, m), 4.55 (1H, d, J=7.8 Hz), 5.00
(1H, dd, J=7.8, 9.8 Hz), 5.09 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.3 Hz), 6.22–6.26 (1H, m); 13C NMR
(MeOH-d4): δ 172.3 (s), 171.6 (s), 171.2 (s), 171.1 (s), 143.7 (s), 118.0 (s), 113.8 (s), 101.1 (d), 74.2 (d),
73.0 (d), 72.7 (d), 69.8 (d), 68.2 (t), 63.0 (t), 20.7 (q), 20.6 (q), 20.6 (q), 20.6 (q), 20.3 (q); FABMS m/z:
428 (M+1)+. Anal. found: C, 52.49; H, 5.92; N, 3.37. Calcd for C19H25NO10·1/2H2O: C, 52.28; H, 6.01;
24
1
23
N, 3.21%. 19b: Mp 86–87°C; [α]D −25.3 (c=0.43, CHCl3); IR (KBr): 2229, 1756, 1647, 1212, 1068
cm−1; 1H NMR: δ 1.90 (3H, d, J=1.5 Hz), 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.10 (3H, s), 3.71 (1H,
ddd, J=2.4, 4.9, 9.8 Hz), 4.16 (1H, dd, J=2.4, 12.2 Hz), 4.25 (1H, dd, J=4.9, 12.2 Hz), 4.28 (1H, ddd,
J=1.0, 6.3, 14.2 Hz), 4.44 (1H, ddd, J=1.0, 6.3, 14.2 Hz), 4.54 (1H, d, J=7.8 Hz), 5.01 (1H, dd, J=7.8,
9.8 Hz), 5.09 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.8 Hz), 6.38 (1H, dt, J=1.5, 6.3 Hz); FABMS m/z: 428
(M+1)+. Anal. found: C, 53.23; H, 5.76; N, 3.14. Calcd for C19H25NO10: C, 53.38; H, 5.90; N, 3.28%.
3.5. Rhodiocyanoside A 20a
A mixture of 19a (249 mg, 0.58 mmol) and K2CO3 (166 mg, 1.2 mmol) in MeOH (10 ml) was stirred
for 30 min at room temperature. The reaction mixture was evaporated under reduced pressure and the
residue was chromatographed on silica gel (5 g, CHCl3:MeOH (4:1)) to give 20a (117 mg, 77%) as a
colorless syrup. 20a: [α]D −15.3 (c=0.34, MeOH); IR (KBr): 3397, 2224, 1646, 1075 cm−1; 1H NMR
26
(MeOH-d4): δ 1.98 (3H, s), 3.19 (1H, dd, J=7.8, 8.9 Hz), 3.27–3.37 (3H, m), 3.68 (1H, dd, J=4.9, 11.7
Hz), 3.87 (1H, br. d, J=11.7 Hz), 4.30 (1H, d, J=7.8 Hz), 4.43 (1H, dd, J=6.4, 13.7 Hz), 4.55 (1H, dd,
J=6.4, 13.7 Hz), 6.46 (1H, br. t, J=6.4 Hz); 13C NMR (MeOH-d4): δ 145.0, 118.2, 112.6, 104.0, 77.9,
77.9, 74.9, 71.4, 68.4, 62.5, 20.2; FAB MS m/z: 260 (M+1)+.
3.6. Rhodiocyanoside A isomer 20b
A mixture of 19b (90 mg, 0.21 mmol) and K2CO3 (58 mg, 0.42 mmol) in MeOH (4 ml) was stirred
for 30 min at room tempertaure. The reaction mixture was evaporated under reduced pressure and the
residue was chromatographed on silica gel (5 g, CHCl3:MeOH (4:1)) to give 20b (48 mg, 87%) as a
colorless syrup. 20b: [α]D −23.5 (c=0.34, MeOH); IR (KBr): 3394, 2224, 1646, 1073 cm−1; 1H NMR
25
(MeOH-d4): δ 1.91 (3H, d, J=1.5 Hz), 3.19 (1H, dd, J=7.8, 9.3 Hz), 3.27–3.38 (3H, m), 3.64–3.68 (1H,
m), 3.88 (1H, br. d, J=11.7 Hz), 4.29 (1H, d, J=7.8 Hz), 4.38 (1H, ddd, J=1.0, 6.4, 14.7 Hz), 4.54 (1H,

H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
2435
ddd, J=1.0, 5.9, 14.7 Hz), 6.53 (1H, ddd, J=1.5, 5.9, 6.3 Hz); 13C NMR (MeOH-d4): δ 145.4 (d), 120.8
(s), 112.5 (s), 103.9 (d), 78.1 (d), 78.0 (d), 75.0 (d), 71.6 (d), 65.8 (t), 62.7 (t), 15.6 (t); FABMS m/z: 298
(M+K)+.
3.7. 2-Methyl-2-propen-1-ol β-D-glucopyranoside 8
A mixture of 4 (250 mg, 0.83 mmol), 2-methyl-2-propen-1-ol 7 (116 mg, 1.61 mmol), β-glucosidase
17 mg (42.5 unit) in phosphate buffer (pH 5, 8.5 ml) was incubated for 4 h at 30°C. After the reaction
mixture was boiled for 5 min and evaporated under reduced pressure, the residue was chromatographed
on silica gel (6 g, CHCl3:MeOH (7:1)) to afford 8 (28 mg, 14%) as colorless needles. 8: Mp 123–125°C;
25
[α]D −51.7 (c=0.24, MeOH); IR (KBr): 3434, 1648, 1455, 1380, 1082 cm−1; 1H NMR (D2O): δ 1.63
(3H, s), 3.14–3.37 (4H, m), 3.58 (1H, dd, J=5.9, 12.2 Hz), 3.77 (1H, dd, J=1.0, 12.2 Hz), 4.08 (1H, d,
J=12.7 Hz), 4.15 (1H, d, J=12.7 Hz), 4.33 (1H, d, J=7.8 Hz), 4.88 (1H, s), 4.93 (1H, s); 13C NMR (D2O):
δ 142.3 (s), 114.4 (t), 101.7 (d), 76.6 (d), 76.6 (d), 73.9 (d), 73.9 (t), 70.5 (d), 61.5 (t), 19.5 (q); FABMS
m/z: 273 (M+K)+. Anal. found: C, 51.12; H, 7.79. Calcd for C10H18O6: C, 51.27; H, 7.75%.
3.8. Acetylation of 8
A solution of 8 (55 mg, 0.24 mmol) in pyridine (1 ml, 12.4 mmol), 4-dimethylaminopyridine (10
mg, 0.08 mmol) was treated with Ac2O (240 mg, 2.4 mmol) and the reaction mixture was stirred for 1
h at room temperature. The reaction mixture was diluted with H2O (50 ml) and extracted with AcOEt
(20 ml×3). The AcOEt extract was washed with sat. brine (50 ml) and dried over MgSO4, and filtered.
Removal of the solvent gave a residue which was chromatographed on silica gel (10 g, n-hexane:AcOEt
24
(2:1)) to give 21 (94 mg, 99%) as colorless prisms. 21: Mp 108–109°C; [α]D −26.8 (c=0.6, CHCl3);
IR (KBr): 2950, 1761, 1656, 1226, 1042 cm−1; 1H NMR: δ 1.71 (3H, s), 2.01 (3H, s), 2.02 (3H, s), 2.04
(3H, s), 2.09 (3H, s), 3.68 (1H, ddd, J=2.2, 4.7, 9.8 Hz), 4.02 (1H, d, J=12.7 Hz), 4.19 (1H, dd, J=2.2,
12.2 Hz), 4.22 (1H, d, J=12.7 Hz), 4.26 (1H, dd, J=4.7, 12.2 Hz), 4.53 (1H, d, J=7.8 Hz), 4.93 (1H, s),
4.95 (1H, s), 5.04 (1H, dd, J=7.8, 9.3 Hz), 5.01 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.3 Hz); FABMS m/z:
441 (M+K)+. Anal. found: C, 53.52; H, 6.45. Calcd for C18H26O10: C, 53.71; H, 6.52%.
3.9. Osmaronin tetraacetate 23a and its isomer 23b
(i) A solution of 21 (137 mg, 0.34 mmol) in CH2Cl2 (8 ml) was subjected to ozonolysis at −78°C
for 20 min and treated with Me2S (1 ml, 13.6 mmol). The whole reaction mixture was stirred at room
temperature for 30 min and evaporated under reduced pressure to provide a crude ketone 22. (ii) NaH
(60%, 25 mg, 0.61 mmol) was washed with dry n-hexane (6 ml×2) and added to dry THF (8 ml) under
an argon atmosphere at 0°C. A solution of diethyl cyanomethylphosphonate (108 mg, 0.61 mmol) in
dry THF (2 ml) was added to the above mixture and the whole mixture was stirred for 15 min at 0°C. A
solution of the crude 22 in dry THF (2 ml) was added to the above reaction mixture and the whole mixture
was stirred for 30 min at 0°C. The reaction mixture was diluted with H2O (50 ml) and extracted with
AcOEt (20 ml×3). The organic layer was washed with sat. brine and dried over MgSO4. Evaporation
of the solvent gave a residue which was chromatographed on silica gel (20 g, n-hexane:AcOEt (3:2)) to
afford 23a (32 mg, 22% from 21) as a colorless oil and 23b (67 mg, 46% from 21) as colorless needles
in elution order. 23a: [α]D +10.3 (c=0.6, CHCl3); IR (neat): 2221, 1751, 1373, 1231, 1045 cm−1; 1H
28
NMR: δ 1.94 (3H, d, J=1.5 Hz), 1.99 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.08 (3H, s), 3.71 (1H, ddd, J=2.4,
4.9, 9.8 Hz), 4.15 (1H, dd, J=2.4, 12.5 Hz), 4.24 (1H, dd, J=4.9, 12.5 Hz), 4.46 (2H, s), 4.51 (1H, d, J=8.1

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H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
Hz), 5.01 (1H, dd, J=8.1, 9.3 Hz), 5.07 (1H, t, J=9.8 Hz), 5.20 (1H, t, J=9.3 Hz), 5.26 (1H, d, J=1.5 Hz);
FABMS m/z: 428 (M+1)+. Anal. found: C, 52.69; H, 5.74; N, 3.19. Calcd for C19H25NO10·1/2H2O: C,
26
52.28; H, 6.01; N, 3.21%. 23b: Mp 109–110°C; [α]D −26.2 (c=0.68, CHCl3); IR (KBr): 2228, 1752,
1376, 1217, 1041 cm−1; 1H NMR: δ 2.00 (3H, s), 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s), 2.09 (3H, s),
3.70 (1H, ddd, J=2.0, 4.4, 9.6 Hz), 4.08 (1H, d, J=15.9 Hz), 4.15 (1H, dd, J=2.0, 12.2 Hz), 4.25 (1H,
dd, J=4.4, 12.2 Hz), 4.37 (1H, d, J=15.9 Hz), 4.54 (1H, d, J=8.0 Hz), 5.03–5.12 (2H, m), 5.22 (1H, t,
J=9.6 Hz), 5.43 (1H, s); FABMS m/z: 428 (M+1)+. Anal. found: C, 53.27; H, 5.76; N, 3.15. Calcd for
C19H25NO10 C, 53.38; H, 5.90; N, 3.28%.
3.10. Osmaronin 24a
A mixture of 23a (170 mg, 0.4 mmol) and K2CO3 (55 mg, 0.4 mmol) in MeOH (4 ml) was stirred
for 10 min at room temperature. The reaction mixture was evaporated under reduced pressure and the
residue was chromatographed on silica gel (5 g, CHCl3:MeOH (6:1)) to give 24a (86 mg, 83%) as a
colorless syrup. 24a: [α]D +20.0 (c=0.7, MeOH); IR (KBr): 3388, 2224, 1645, 1383, 1073 cm−1; 1H
26
NMR (MeOH-d4): δ 2.03 (3H, d, J=1.5 Hz), 3.23 (1H, dd, J=7.8, 9.3 Hz), 3.29–3.37 (3H, m), 3.70 (1H,
dd, J=5.4, 12.2 Hz), 3.88 (1H, dd, J=2.4, 12.2 Hz), 4.29 (1H, d, J=7.8 Hz), 4.46 (1H, d, J=13.2 Hz), 4.56
(1H, d, J=13.2 Hz), 5.46 (1H, m); 13C NMR (MeOH-d4): δ 162.9 (s), 117.1 (s), 103.9 (d), 97.5 (d), 78.0
(d), 78.0 (d), 74.9 (d), 71.4 (d), 70.6 (t), 62.5 (t), 20.8 (q); FABMS m/z: 260 (M+1)+.
3.11. Osmaronin isomer 24b
A mixture of 23b (370 mg, 0.87 mmol) and K2CO3 (239 mg, 1.73 mmol) in MeOH (7 ml) was stirred
for 20 min at room temperature. The reaction mixture was evaporated under reduced pressure and the
residue was chromatographed on silica gel (5 g, CHCl3:MeOH (7:1)) to give 24b (212 mg, 94%) as a
colorless syrup. 24b: [α]D −17.5 (c=0.51, MeOH); IR (KBr): 3395, 2224, 1645, 1382, 1076 cm−1; 1H
24
NMR (MeOH-d4): δ 2.01 (3H, s), 3.23 (1H, dd, J=7.8, 8.8 Hz), 3.27–3.38 (3H, m), 3.65 (1H, dd, J=5.6,
11.7 Hz), 3.87 (1H, d, J=11.7 Hz), 4.23 (1H, d, J=16.9 Hz), 4.28 (1H, d, J=7.8 Hz), 4.43 (1H, d, J=16.9
Hz), 5.75 (1H, m); 13C NMR (MeOH-d4): δ 162.5 (s), 118.0 (s), 103.7 (d), 95.1 (d), 77.9 (d), 77.8 (d),
74.9 (d), 71.7 (t), 71.4 (d), 62.6 (t), 17.9 (q); FABMS m/z: 282 (M+Na)+.
3.12. 2-Benzyloxy-3-hydroxy-propan-1-ol β-D-glucopyranoside 10
A mixture of 4 (1 g, 3.32 mmol), 2-benzyloxy-1,3-propanediol 9 (1.2 g, 6.59 mmol), β-glucosidase
(93 mg, 232 unit) in phosphate buffer (pH 5, 34 ml) was incubated for 4 h at 30°C. After the reaction
mixture was boiled for 5 min and evaporated under reduced pressure, the residue was chromatographed
26
on silica gel (20 g, CHCl3:MeOH (5:1)) to afford 10 (253 mg, 22%) as a colorless syrup. 10: [α]D
−3.0 (c=0.37, MeOH); IR (KBr): 3394, 1635, 1343, 1252, 1075 cm−1; H NMR (DMSO-d6+D2O): δ
1
2.96–3.17 (3.5H, m), 3.32–3.90 (7H, m), 4.19 (1H, t, J=7.8 Hz), 4.50–4.68 (2.5H, m), 7.22–7.53 (5H,
m); FABMS m/z: 383 (M+K)+. Anal. found: C, 53.30; H, 6.96. Calcd for C16H24O8.H2O: C, 53.03; H,
7.23%.
3.13. 3-Acetoxy-2-oxopropan-1-ol β-D-glucopyranoside 27
(i) A solution of 10 (180 mg, 0.52 mmol) in pyridine (2 g, 25.3 mmol), 4-dimethylaminopyridine (10
mg, 0.08 mmol) was treated with Ac2O (534 mg, 5.24 mmol) and the reaction mixture was stirred for 1 h

H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
2437
at room temperature. The reaction mixture was diluted with H2O (50 ml) and extracted with AcOEt (20
ml×3). The AcOEt extract was washed with sat. brine (50 ml), dried over MgSO4, and filtered. Removal
of the solvent gave a crude acetate 25. (ii) A mixture of the crude 25 and 10% Pd–C (50 mg) in MeOH
(7 ml) was subjected to catalytic hydrogenation at ordinary temperature for 2 h and the reaction mixture
was filtered with the aid of Celite. The filtrate was evaporated to give a residue 26. A mixture of the
crude 26 and PCC (552 mg, 2.1 mmol) in CH2Cl2 (10 ml) was stirred at room temperature for 48 h. The
reaction mixture was directly subjected to chromatography on Florisil (20 g, AcOEt) to provide a ketone
24
27 (203 mg, 84% from 10) as a colorless oil. 27: [α]D −7.55 (c=1.1, CHCl3); IR (KBr): 1752, 1375,
1
1241, 1044 cm−1; H NMR: δ 2.02 (3H, s), 2.03 (3H, s), 2.09 (3H, s), 2.09 (3H, s), 2.16 (3H, s), 3.72
(1H, ddd, J=2.4, 4.9, 9.8 Hz), 4.13 (1H, dd, J=2.4, 12.5 Hz), 4.26 (1H, dd, J=4.9, 12.5 Hz), 4.30 (1H, d,
J=16.9 Hz), 4.36 (1H, d, J=16.9 Hz), 4.57 (1H, d, J=7.8 Hz), 4.86 (2H, s), 5.06 (1H, dd, J=7.8, 9.3 Hz),
5.09 (1H, t, J=9.8 Hz), 5.23 (1H, dd, J=9.3, 9.8 Hz); FABMS m/z: 501 (M+K)+. Anal. found: C, 49.26;
H, 5.80. Calcd for C19H26O13: C, 49.34; H, 5.67%.
3.14. Sutherlandin pentaacetate 28a and its isomer 28b
NaH (60%, 62 mg, 1.56 mmol) was washed with dry n-hexane (6 ml×3) and added to dry THF (8 ml)
under an argon atmosphere at 0°C. A solution of diethyl cyanomethylphosphonate (287 mg, 1.62 mmol)
in dry THF (2 ml) was added to the above mixture and the whole mixture was stirred for 15 min at 0°C.
A solution of 27 (500 mg, 1.8 mmol) in dry THF (4 ml) was added to the above reaction mixture and the
whole mixture was stirred for 30 min at room temperature. The reaction mixture was diluted with H2O
(50 ml) and extracted with AcOEt (20 ml×3). The organic layer was washed with sat. brine and dried
over MgSO4. Evaporation of the solvent gave a residue which was chromatographed on silica gel (20 g,
n-hexane:AcOEt (3:2)) to afford 28a (176 mg, 33% from 27) as a colorless syrup and 28b (164 mg, 31%
from 27) as a colorless syrup in elution order. 28a: [α]D27 −2.48 (c=1.1, CHCl3); IR (KBr): 2226, 1752,
1374, 1228, 1048 cm−1; 1H NMR: δ 2.01 (3H, s), 2.03 (3H, s), 2.07 (3H, s), 2.10 (3H, s), 2.13 (3H, s),
3.73 (1H, ddd, J=2.0, 4.7, 9.8 Hz), 4.18 (1H, dd, J=2.0, 12.3 Hz), 4.25 (1H, dd, J=4.7, 12.3 Hz), 4.55
(1H, d, J=7.8 Hz), 4.58 (2H, d, J=2.9 Hz), 4.74 (2H, dd, J=2.0, 3.4 Hz), 5.01 (1H, dd, J=7.8, 9.8 Hz),
5.09 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.8 Hz), 5.54 (1H, s); FABMS m/z: 524 (M+K)+. Anal. found: C,
51.78; H, 5.56; N, 2.78. Calcd for C21H27NO12: C, 51.94; H, 5.61; N, 2.89%. 28b: [α]D28 −23.0 (c=1.1,
1
CHCl3); IR (KBr): 2226, 1752, 1228, 1044 cm−1; H NMR: δ 2.01 (3H, s), 2.03 (3H, s), 2.06 (3H, s),
2.09 (3H, s), 2.13 (3H, s), 3.72 (1H, ddd, J=2.4, 4.4, 9.8 Hz), 4.15 (1H, dd, J=2.4, 12.7 Hz), 4.20 (1H,
dd, J=2.0, 15.9 Hz), 4.26 (1H, dd, J=4.4, 12.7 Hz), 4.47 (1H, dd, J=1.8, 15.9 Hz), 4.56 (1H, d, J=7.8 Hz),
4.84 (2H, br. s), 5.04 (1H, dd, J=7.8, 9.3 Hz), 5.09 (1H, t, J=9.8 Hz), 5.22 (1H, t, J=9.8 Hz), 5.62 (1H, br.
d, J=1.0 Hz); FABMS m/z: 524 (M+K)+. Anal. found: C, 51.64; H, 5.58; N, 2.63. Calcd for C21H27NO12:
C, 51.94; H, 5.61; N, 2.89%.
3.15. Sutherlandin 29
A mixture of 28a (239 mg, 0.49 mmol) and K2CO3 (68 mg, 0.49 mmol) in MeOH (10 ml) was stirred
for 30 min at room temperature. The reaction mixture was evaporated under reduced pressure and the
residue was chromatographed on silica gel (5 g, CHCl3:MeOH (4:1)) to give 29 (103 mg, 76%) as a
22
colorless syrup. 29: [α]D −14.6 (c=0.54, MeOH); IR (KBr): 3405, 2227, 1645, 1075 cm−1; 1H NMR
(DMSO-d6): δ 2.97 (1H, dt, J=4.4, 7.8 Hz), 3.07–3.26 (3H, m), 3.46 (1H, dd, J=5.9, 12.0 Hz), 3.67 (1H,
dd, J=6.4, 12.0 Hz), 4.15 (1H, d, J=7.8 Hz), 4.22 (2H, d, J=3.4 Hz), 4.37 (1H, d, J=13.0 Hz), 4.50 (1H,
d, J=13.0 Hz), 4.51 (1H, d, J=5.9 Hz), 4.93 (1H, d, J=3.9 Hz), 4.98 (1H, d, J=4.4 Hz), 5.10 (1H, d, J=4.9

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H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
Hz), 5.38 (1H, t, J=5.4 Hz), 5.72 (1H, br. s); 13C NMR (DMSO-d6): δ 165.0 (s), 116.5 (s), 102.5 (d),
94.0 (d), 76.9 (d), 76.5 (d), 73.2 (d), 69.8 (d), 66.4 (t), 61.0 (t), 60.8 (t); FABMS m/z: 276 (M+1)+.
3.16. 4-Methoxybenzyl β-D-glucopyranoside 12
A mixture of 4 (1 g, 3.32 mmol), 4-methoxybenzyl alcohol 11 (463 mg, 3.35 mmol), β-glucosidase
(100 mg, 340 unit) in phosphate buffer (pH 5, 34 ml) was incubated for 26 h at 30°C. After the reaction
mixture was boiled for 5 min and evaporated under reduced pressure, the residue was chromatographed
on silica gel (50 g, CHCl3:MeOH (30:1)) to afford 12 (249 mg, 25%) as a colorless amorphous powder.
12: Mp 139–140°C; [α]D −57.5 (c=0.55, MeOH); IR (KBr): 3338, 2900, 1514, 1075, 1031 cm−1; 1H
26
NMR (pyridine-d5): δ 3.66 (3H, s), 3.95–4.00 (1H, m), 4.09–4.13 (1H, m), 4.24–4.29 (2H, m), 4.42 (1H,
dd, J=5.4, 11.7 Hz), 4.60 (1H, d, J=2.4, 11.7 Hz), 4.81 (1H, d, J=11.2 Hz), 4.98 (1H, d, J=7.8 Hz), 5.13
(1H, d, J=11.2 Hz), 6.95 (2H, d, J=8.8 Hz), 7.48 (2H, d, J=8.8 Hz); 13C NMR (pyridine-d5): δ 159.7,
130.8, 130.0, 114.1, 114.1, 103.7, 78.6, 78.5, 75.2, 71.7, 70.6, 62.8, 55.2; EIMS m/z: 300 M+. Anal.
found: C, 55.98; H, 6.73. Calcd for C14H20O7: C, 55.99; H, 6.71%.
3.17. 4-Hydroxyphenethyl β-D-glucopyranoside 14
A mixture of 4 (600 mg, 1.99 mmol), 4-hydroxyphenethyl alcohol 13 (1.377 g, 9.97 mmol), β-
glucosidase (47 mg, 160 unit) in phosphate buffer (pH 5, 20 ml) was incubated for 26 h at 30°C.
After the reaction mixture was boiled for 5 min and evaporated under reduced pressure, the residue
was chromatographed on silica gel (30 g, CHCl3:MeOH (30:1)) to afford 14 (217 mg, 36%) as colorless
needles. 14: Mp 159–160°C (recrystallized from AcOEt); [α]D26 −28.4 (c=0.5, MeOH); IR (KBr): 3324,
2956, 1520, 1075, 1060 cm−1; 1H NMR (pyridine-d5): δ 1.54 (3H, s), 1.60 (3H, s), 4.38 (1H, dd, J=11.7,
6.1 Hz), 4.59 (1H, d, J=11.7 Hz), 4.90 (1H, d, J=7.3 Hz), 5.52 (1H, t, J=6.3 Hz); 13C NMR (MeOH-d4):
δ 156.8, 130.9, 130.9, 130.7, 116.1, 116.1, 104.4, 78.1, 77.9, 75.1, 72.1, 71.6, 62.7, 36.4; FABMS m/z:
301 (M+1)+. Anal. found: C, 55.63; H, 6.77. Calcd for C14H20O7: C, 55.91; H, 6.71%.
3.18. Cinnamyl β-D-glucopyranoside 16
A mixture of 4 (500 mg, 1.66 mmol), cinnamyl alcohol 15 (228 mg, 1.7 mmol), β-glucosidase (30 mg,
102 unit) in phosphate buffer (pH 5, 17 ml) was incubated for 16 h at 30°C. After the reaction mixture
was boiled for 5 min and evaporated under reduced pressure, the residue was chromatographed on silica
gel (25 g, CHCl3:MeOH (30:1)) to afford 16 (97 mg, 20%) as a colorless amorphous powder. 16: Mp
114–116°C; [α]D28 −48.8 (c=0.3, MeOH); IR (KBr): 3378, 2924, 1075, 1031 cm−1; 1H NMR (pyridine-
d5): δ 3.95–3.99 (1H, m), 4.12 (1H, t, J=7.8 Hz), 4.24–4.31 (2H, m), 4.40 (1H, dd, J=5.4, 11.7 Hz), 4.48
(1H, ddd, J=1.0, 5.9, 13.0 Hz), 4.58 (1H, dd, J=2.4, 11.7 Hz), 4.75 (1H, ddd, J=1.5, 5.9, 13.0 Hz), 4.98
(1H, d, J=7.8 Hz), 6.48 (1H, dt, J=5.9, 16.1 Hz), 6.78 (1H, d, J=16.1 Hz), 7.24 (1H, t, J=6.8 Hz), 7.31
(2H, t, J=6.8 Hz), 7.41 (2H, d, J=6.8 Hz); 13C NMR (pyridine-d5): δ 137.4, 132.1, 129.0, 127.9, 126.9,
126.9, 103.9, 78.5, 78.5, 75.2, 71.7, 69.7, 62.8; EIMS m/z: 296 M+. Anal. found: C, 60.29; H, 6.79. Calcd
for C15H20O6: C, 60.80; H, 6.80%.
3.19. Acetylation of 16
A solution of 16 (100 mg, 0.34 mmol) in pyridine (1 ml, 12.4 mmol), 4-dimethylaminopyridine (10
mg, 0.08 mmol) was treated with Ac2O (276 mg, 2.7 mmol) and the reaction mixture was stirred for 1 h

H. Akita et al. / Tetrahedron: Asymmetry 10 (1999) 2429–2439
2439
at room temperature. The reaction mixture was diluted with H2O (50 ml) and extracted with AcOEt (20
ml×3). The AcOEt extract was washed with sat. brine (50 ml), dried over MgSO4, and filtered. Removal
of the solvent gave a residue which was chromatographed on silica gel (20 g, n-hexane:AcOEt (3:1)) to
give 30 (154 mg, 98%) as colorless needles. 30: Mp 81–83°C (recrystallized from n-hexane/benzene);
27
1
[α]D −27.6 (c=0.38, CHCl3); IR (KBr): 3430, 2929, 1745, 1228, 1043 cm−1; H NMR: δ 2.01 (3H,
s), 2.02 (3H, s), 2.05 (3H, s), 2.08 (3H, s), 3.71 (1H, ddd, J=2.4, 4.9, 9.8 Hz), 4.16 (1H, dd, J=2.4, 12.2
Hz), 4.25–4.31 (2H, m), 4.50 (1H, ddd, J=1.5, 5.4, 13.2 Hz), 4.63 (1H, d, J=7.8 Hz), 5.05 (1H, dd, J=7.8,
9.3 Hz), 5.11 (1H, t, J=9.8 Hz), 5.21 (1H, t, J=9.8 Hz), 6.22 (1H, ddd, J=5.4, 6.4, 16.1 Hz), 6.59 (1H, d,
J=16.1 Hz), 7.23–7.39 (5H, m); 13C NMR (CDCl3): δ 170.7 (s), 170.3 (s), 169.4 (s), 169.3 (s), 136.3 (s),
133.1 (d), 128.6 (d), 127.9 (d), 126.5 (d), 124.4 (d), 99.5 (d), 72.9 (d), 71.8 (d), 71.3 (d), 69.8 (t), 68.4
(d), 61.9 (t), 20.7 (q), 20.7 (q), 20.5 (q), 20.5 (q); FABMS m/z: 503 (M+K)+. Anal. found: C, 59.57; H,
6.14. Calcd for C23H28O10: C, 59.47; H, 6.08%.
Acknowledgements
The authors are grateful to Dr. Junichi Kitajima, Showa College of Pharmaceutical Sciences, Japan,
for generously giving the spectral data of 6, 12, to Dr. Kenichiro Inoue, Gifu Pharmaceutical University,
Japan, for generously providing the spectral data of 14 and to Dr. Takao Konoshima, Kyoto Pharma-
ceutical University, Japan, for generously donating the spectral data of 16. This work was supported by
a grant for the ‘Biodesign Research Program’ from The Institute of Physical and Chemical Research
(RIKEN, Japan) to H.A.
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