A new inosine disaccharide from the crustacean Ligia exotica: Isolation and structure elucidation by total synthesis

Article abstract of DOI:10.1021/np0000724

A novel nucleoside has been isolated from the crustacean Ligia exotica, and the structure was elucidated as 3'-O-(α-D-glucosyl)inosine, 1, by analysis of spectroscopic data and by total synthesis.

Full text of DOI:10.1021/np0000724

1188
J . Nat. Prod. 2000, 63, 1188-1191
Sch em e 1
A New In osin e Disa cch a r id e fr om th e
Cr u sta cea n Ligia exotica : Isola tion a n d
Str u ctu r e Elu cid a tion by Tota l Syn th esis
Seong Ho Kim, Su-Mi Yoo, Il Sung Park, and
Yong Hae Kim*
Center for Molecular Design and Synthesis, Department of
Chemistry, Korea Advanced Institute of Science and
Technology, Taejon 305-701, Korea
Received February 14, 2000
Abstr a ct: A novel nucleoside has been isolated from the
crustacean Ligia exotica, and the structure was elucidated as
3′-O-(R-D-glucosyl)inosine, 1, by analysis of spectroscopic data
and by total synthesis.
showing two singlets at low-field resonances of δH 8.04 (s,
H-2) and 8.28 (s, H-8) due to the hypoxanthine ring. The
comparison of the NMR spectra of inosine with those of 1
suggests that compound 1 is an inosine derivative that
contains a hexopyranose sugar unit, which was identified
Numerous bioactive nucleoside analogues have been
isolated from marine organisms such as nudibranches,1
sponges,2 a tunicate,3 and a seaweed.4 Tubercidin 5′-R-D-
glucopyranose was isolated from a Hawaiian blue green
algae.5 Suzuki and his co-worker reported a mixture of 2′-
O- and 3′-O-substituted inosine, and their structures were
thought to be 2′-O- and 3′-O-(â-D-galactosyl)inosine but
without any detailed experimental evidence.6 During our
studies on bioactive substances from marine organisms, we
recently isolated a novel nucleoside from the crustacean
Ligia exotica (Ligiidae). In this paper we describe the
isolation, structure elucidation, and synthesis of 3′-O-(R-
D-glucosyl)inosine (1).
1
as a glucopyranose ring. The H NMR signals for the two
1
sugar units were firmly assigned with the aid of H-1H
COSY and HMBC, and the correlation of 13C NMR signals
1
with H NMR signals was accomplished by performing an
HMQC experiment. The 13C NMR chemical shifts for the
ribose unit of 1 were coincident with those from the sugar
moiety of inosine except for that of C-3′ (1, δC 77.5; inosine,
δC 71.2), implying that the second sugar unit is attached
to the C-3′ position.9 The 13C NMR chemical shift for the
anomeric carbon C-1′′ (δC 100.0, lit.;10a δC-1′′ ) 100-101 in
R-form, δC-1′′ ) 103-104 in â-form) and the coupling
constant for the anomeric proton of the glucose unit H-1′′
The water-soluble extract of the crustacean subjected to
gel filtration followed by repeated reversed-phase HPLC
yielded 1. Compound 1 was obtained as a white amorphous
solid.
(J 1′′-2′′ ) 3.4 Hz, lit.;10b J 1′′-2′′ ) 3.5 Hz in R-form, J 1′′-2′′
)
8.0 Hz in â-form) indicate that the stereochemistry of
glucoside is via an R-linkage. J udging from the results of
all the spectral data, it is suggested that the structure of
1 is 3′-O-(R-D-glucosyl)inosine.
3′-O-(R-D-Glucosyl)inosine has been neither isolated nor
synthesized. Only a mixture of 2′-O- and 3′-O-(â-D-galac-
tosyl)inosine was isolated from the reaction of O-nitrophen-
yl-â-galactoside and inosine mediated by â-galactosidase
prepared from E. coli.6 Thus, it is a challenge to ste-
reospecically synthesize four isomeric 2′-O-R- and -â-, and
3′-O-R- and -â-disaccharide nucleosides.
To confirm the correct structure of the new inosine
disaccharide, the four stereoisomers 2′-O-(R-D-glucosyl)-
and 2′-O-(â-D-glucosyl)inosine and 3′-O-(R-D-glucosyl)- and
3′-O-(â-D-glucosyl)inosine were synthesized. The synthetic
approach to these compounds must include an O-glycosy-
lation step to form the disaccharide. Although there are
plenty of examples of efficient nucleoside formation from
simple, elaborated, or disaccharidal glycosyl donors, there
are few cases of successful O-glycosylation of a nucleoside.11
Attachment of a glucopyranosyl subunit at O-3′ of inosine
would represent a rare example of O-glycosylation of a
purine nucleoside because it requires overcoming the
double obstacle of steric hindrance and competing depuri-
nation. Glycosyl acceptor 4a was prepared through two
steps (Scheme 1).
The high-resolution FABMS data of 1 established the
molecular formula as C16H22N4O10 [m/z 431.1378 (M + H)+,
calcd for C16H23N4O10, 431.1414], which was supported by
the 13C NMR spectrum, showing signals for 16 carbon
atoms. The IR absorption bands at 3376 and 1686 cm-1
suggested the presence of hydroxyl and carbonyl groups,
respectively, and the UV spectrum showed an absorption
maximum at λmax 248 nm, which may be attributable to
the chromophore of a purine base.7 Extensive analysis of
2D NMR spectra (1H-1H COSY, HMQC, HMBC, NOESY)
suggested that 1 consisted of a purine base and two sugar
units. Since the 13C NMR chemical shifts for the purine
moiety [δC 147.0 (d, C2), 148.4 (s, C4), 124.4 (s, C5), 158.0
(s, C6), 138.4 (d, C8)] coincided well with those of the purine
base of inosine,8 the purine base of 1 was assigned as
hypoxanthine. This was confirmed by the 1H NMR spectra,
The protection of inosine (2) with tert-butyldimethylsilyl
chloride in pyridine gave a mixture of 2′,5′-di-O- and 3′,5′-
di-O-(tert-butyldimethylsilyl)inosines (3a , 3b). N-1-Benz-
ylation of the 3a and 3b mixture with benzyl bromide
yielded the glycosylation acceptor mixture of 4a and 4b,
which was separated by Si gel column chromatography as
* To whom correspondence should be addressed. Tel.: 82-42-869-2818.
Fax: 82-42-869-5818. E-mail: kimyh@sorak.kaist.ac.kr.
10.1021/np0000724 CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/02/2000

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J ournal of Natural Products, 2000, Vol. 63, No. 9 1189
Sch em e 3
Sch em e 2
described in the Experimental Section. The TBDMS moiety
is known to migrate to adjacent hydroxyl groups (from 4a
to 4b and vice versa) under basic conditions.12 Thus, the
second step was performed without separation of 3a and
3b. Benzyl-protected glucopyranosyl donor 5 was prepared
according to a literature procedure.13 The nonparticipating
benzyloxy group at C-2′′ was expected to direct R-glycosy-
lation. Scheme 2 shows the synthetic procedure used for
the synthesis of 1.
on a Bruker AVANCE 400 MHz spectrometer; signals are
reported in parts per million (δ), referenced to the solvent used.
Mass spectra were obtained on a VG Autospec Ultma mass
spectrometer.
An im a l Ma ter ia l. The crustacean Ligia exotica (Ligiidae)
was collected off the Haeundae coast, near Busan, Korea. The
animal was verified by Prof. S. H. Hur at Bukyung National
University. A voucher specimen (PK-9108YHK) was deposited
in the Department of Chemistry, Korea Advanced Institute of
Science and Technology, Taejon, Korea.
Activation of the donor 5 with BF3‚Et2O at -10 °C in
the presence of acceptor 4a led to the consumption of both
starting materials and formation of glycosylated products
as an approximate 8:1 mixture of R- and â-anomers. Silica
gel column separation of the anomers was readily effected
after desilylation to give the R-disaccharide 7 and its
â-anomer 8. Compound 7 was treated with Pd(OH)2/C in
the presence of cyclohexene and EtOH to give compound
1, for which spectral data were identical to those of the
natural product, and 8 to give 3′-O-(â-D-glucosyl)inosine
(1b). 2′-O-(R-D-Glucosyl)inosine (12) and 2′-O-(â-D-glucosyl)-
inosine (13) were respectively prepared by a method similar
to that described above using 4b as glycosyl acceptor
(Scheme 3) and identified by 1H and 13C NMR spectroscopy.
Thus the structure of 1 was concluded to be 3′-O-(R-D-
glucosyl)inosine.
Extr a ction a n d Isola tion . The crustacean L. exotica was
immediately stored in methanol (2 L). The crustacean (1 kg
wet wt) was separated by decanting the supernatant methanol
solution, and the animal tissue was crudely homogenized with
distilled water (1.5 L). This homogenate was centrifuged three
times with a superspeed centrifuge (5000 rpm, 10 min). The
supernatant was freeze-dried to give a gelatinous mixture,
which was dialyzed using a cellulose membrane, SPECTAPOR
(cylinder vol.; 0.85 mL/min, MW cut off: ca. 3500). Dialysis
was performed with water and then a 1% AcOH-water
solution. The dialysate was lyophilized to give 38 g of sticky
solid. The water extract of L. exotica was subjected to gel
filtration on Sephadex G-10 (1.9 × 110 cm column; flow rate,
3 mL/min; eluent, H2O) followed by repeated reversed-phase
HPLC [J AI GS-320, 20 × 500 mm, and ODS 20 × 600 mm;
flow rate, 3 mL/min; UV detection at 254 nm; eluent, MeOH-
H2O ) 70:30, v/v] to give 1 (3.4 mg, 0.0003%).
Com p ou n d 1: white amorphous solid; [R]20 +38° (c 0.34,
D
H2O); UV λmax (H2O) 248 nm (ꢀ 8400); IR (NaCl) νmax 3376,
Exp er im en ta l Section
2926, 1686, 1592, 1312, and 1078 cm-1; 1H and 13C NMR [Table
Gen er a l Exp er im en ta l P r oced u r es. All solvents were
redistilled. Merck Si gel 60 (230-400 mesh) and Sephadex
G-10 were used for chromatography. HPLC was conducted
using a UV detector, a J AI GS 320, and a Waters ODS column.
UV spectra were obtained on a Beckmann DU-64 instrument
and IR spectra on a Bomem MB-100 FT spectrophotometer.
Optical rotations were obtained on an Autopoll III automatic
polarimeter at room temperature. NMR spectra were obtained
1]; HRFABMS m/z 431.1378 (M + H)+, calcd for C16H23N4O10
431.1414.
,
2′,5′-Di-O-(ter t-bu tyldim eth ylsilyl)in osin e (3a) an d 3′,5′-
Di-O-(ter t-bu tyld im eth ylsilyl)in osin e (3b). To a stirred
dispersion of inosine (5.36 g, 20 mmol) in pyridine was added
TBDMSCl (4.50 g, 30 mmol). The mixture was stirred at room
temperature for 48 h. After evaporation under reduced pres-
sure, the residue was dissolved in CH2Cl2 (100 mL). The

1190 J ournal of Natural Products, 2000, Vol. 63, No. 9
Ta ble 1. 1H and 13C NMR Data and Heteronuclear Multiple
Rapid Communications
CH3), -0.05 (3H, s, CH3), -0.15 (3H, s, CH3); 13C NMR (100
MHz, CDCl3) δ 156.5 (C-6), 147.3 (C-4), 147.0 (C-2), 138.2 (C-
8), 135.9 (Cq, Ph), 128.9,128.2,127.9 (CH, Ph), 124.6 (C-5), 87.8
(C-1′), 85.4 (C-4′), 77.4 (C-2′), 71.3 (C-3′), 63.1 (C-5′), 48.9 (N-
CH2Ph), 25.9, 25.5, 18.4, 17.8, -5.1, -5.2, -5.4, -5.5; HREIMS
m/z 586.2999 (M)+, calcd for C29H46N2O5Si2, 586.3007.
4b: mp 194-196 °C; IR (NaCl) νmax 3369, 1698, 1139 and
836 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.04 (1H, s, H-8), 7.98
(1H, s, H-2), 7.28∼7.31 (5H, m, Ph), 5.93 (1H, d, J ) 4.6 Hz,
H-1′), 5.16-5.31 (2H, d, J ) 29.4 Hz, N-CH2Ph), 4.49 (1H,
dd, J ) 4.4, 4.5 Hz, H-3′), 4.41 (1H, ddd, J ) 4.4, 4.5, 4.9 Hz,
H-2′), 4.05 (1H, m, H-4′), 3.87 (1H, dd, J ) 3.4, 11.6, Ha-5′),
3.76 (1H, dd, J ) 3.4, 11.6, Hb-5′), 3.02 (1H, d, J ) 4.9 Hz,
2′-OH), 0.92 (9H, s, C(CH3)3), 0.86 (9H, s, C(CH3)3), 0.14 (3H,
s, CH3), 0.13 (3H, s, CH3), 0.05 (3H, s, CH3), 0.04 (3H, s, CH3);
13C NMR (100 MHz, CDCl3) δ 156.5 (C-6), 147.1 (C-4), 147.0
(C-2), 138.4 (C-8), 136.2 (Cq, Ph), 128.9, 128.2, 128.1 (CH, Ph),
124.9 (C-5), 88.7 (C-1′), 85.3 (C-4′), 75.4 (C-2′), 71.3 (C-3′), 61.1
(C-5′), 49.0 (N-CH2, Ph), 25.9, 25.7, 18.3, 18.0, -4.6, -4.8, -5.4,
-5.2; HREIMS m/z 586.3007 (M)+, calcd for C29H46N2O5Si2,
586.3007.
a
Bond Correlations (HMBC) of 1 in DMSO-d6
position
1H
J (Hz) 13C (mult.)b HMBC (1H)
2
8.04s
147.0 (d)
4
5
6
8
148.4 (s)
124.4 (s)
158.0 (s)
138.4 (d)
87.5
73.2 (d)
77.5 (d)
84.2 (d)
61.3 (t)
100.0 (d)
72.0 (d)
73.8 (d)
70.0 (d)
73.6 (d)
60.7 (t)
2, 1′
8
2
8.28 (s)
5.87 (d)
1′
1′
2′
3′
4′
5′
1′′
2′′
3′′
4′′
5′′
6′′
6.1
2′, 3′
1′′
1′′
4.63 (dd)
4.26 (dd)
4.11 (ddd)
3.62 (m)
4.93 (d)
3.23 (dd)
3.47 (dd)
3.11 (t)
5.4, 6.1
3.0, 5.4
3.0
3.4
3′
3.4, 9.4
8.7, 9.4
8.7
1′′, 3′′, 5′′, 6′′
4′′
3′′, 5′′, 6′′
4′′, 6′′
3.47 (m)
3.41-3.63 (m)
4′′
a
Taken in 400 MHz for 1H and 100 MHz for 13C at 298 K.
b
Multiplicities inferred from a DEPT experiment.
1-N-(Ben zyl)-2′,5′-d i-O-(ter t-b u t yld im et h ylsilyl)-3′-O-
(2′′,3′′,4′′,6′′-tetr a -O-ben zylglu cop yr a n osyl)in osin e (6). To
a cooled (-10 °C), stirred mixture of 1-N-(benzyl)-2′,5′-di-O-
(tert-buthyldimethylsilyl)inosine (4a , 1.16 g, 2.0 mmol) and
O-(2,3,4,6-tetra-O-benzyl-R-D-glucopyranosyl)trichloroacetim-
idate (5, 2.055 g, 3.0 mmol) in CH2Cl2 (20 mL) was added BF3‚
Et2O (1.23 mL, 10 mmol). Stirring was continued for an
additional 1 h at -10 °C under an argon atmosphere. The
reaction mixture was quenched with saturated aqueous so-
dium bicarbonate (5 mL) and extracted with CH2Cl2 (2 × 20
mL). The combined organic layer was washed water (2 × 20
mL) and then dried over MgSO4. Evaporation of the solvent
gave the crude product, which was chromatographed on a Si
gel column (eluent, CH2Cl2-MeOH ) 95:5, v/v; flow rate, 5
mL/min) to give a mixture of the R- and â-form of 6 (1.237 g,
56%). 6: HRFABMS m/z 1109.5482 (M + H)+, calcd for
C63H81N4O10Si2 1109.5491.
solution was washed consecutively with 1 N HCl (2 × 30 mL)
and water (2 × 30 mL) and then dried over MgSO4. Evapora-
tion of the solvent gave the crude product, which was chro-
matographed on a Si gel column (eluent, CH2Cl2-MeOH )
95:5, v/v; flow rate, 10 mL/min) to give a mixture of 3a and
3b (7.94 g, 80%). 3a : mp 216-218 °C; IR (NaCl) νmax 3411,
1697, 1123 and 836 cm-1; 1H NMR (400 MHz, CDCl3) δ 13.20
(1H, s, NH), 8.23 (1H, s, H-8), 8.22 (1H, s, H-2), 6.04 (1H, d, J
) 4.8 Hz, H-1′), 4.54 (1H, dd, J ) 4.8, 4.9 Hz, H-2′), 4.26 (1H,
ddd, J ) 4.0, 4.4, 4.4 Hz, H-3′), 4.17 (1H, m, H-4′), 3.97 (1H,
dd, J ) 2.4, 11.6, Ha-5′), 3.83 (1H, dd, J ) 2.4, 11.6, Hb-5′),
2.69 (1H, d, J ) 4.4 Hz, 3′-OH), 0.92 (9H, s, C(CH3)3), 0.81
(9H, s, C(CH3)3), 0.12 (3H, s, CH3), 0.10 (3H, s, CH3), -0.05
(3H, s, CH3), -0.12 (3H, s, CH3); 13C NMR (100 MHz, CDCl3)
δ 159.3 (C-6), 148.9 (C-4), 145.3 (C-2), 138.5 (C-8), 124.8 (C-
5), 88.2 (C-1′), 85.3 (C-4′), 77.4 (C-2′), 71.1 (C-3′), 63.0 (C-5′),
25.9, 25.5, 18.4, 17.8, -5.1, -5.2, -5.4, -5.5; HREIMS m/z
496.2546 (M)+, calcd for C22H40N2O5Si2, 496.2535.
1-N-Ben zyl-3′-O-r- a n d 1-N-Ben zyl-3′-O-â-D-(2′′,3′′,4′′,6′′-
tetr a -O-ben zylglu cop yr a n osyl)in osin es (7, 8). To a cooled
(0 °C), stirred solution of a mixture of 6 (1.105 g, 1.0 mmol) in
THF (10 mL) was added 1 M tetrabutylammonium fluoride
in THF (2.2 mL, 2.2 mmol). After being stirred for 1 h at room
temperature, the reaction mixture was evaporated to give a
residue, which was dissolved in CH2Cl2 (20 mL). The organic
layer was washed with water (10 mL) and then dried over
MgSO4. Evaporation of solvent gave the crude product, which
was chromatographed on a Si gel column (eluent CHCl3-
MeOH ) 30:1, v/v; flow rate, 0.1 mL/min) to give products [7
(704 mg) and 8 (88 mg), 90%]. 7: IR (NaCl) νmax 3419, 1702,
and 1590 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.98 (1H, s, H-8),
7.79 (1H, s, H-2), 7.12 ∼ 7.35 (25H, m, PhH), 5.61 (1H, d, J )
7.3 Hz, H-1′), 5.22 (2H, s, N-CH2Ph), 4.90 (1H, d, J ) 3.9 Hz,
H-1′′), 4.44 ∼ 4.94 (8H, m, O-CH2Ph), 4.61 (1H, t like, H-2′),
4.33 (1H, m, H-3′), 4.23 (1H, m, H-4′), 4.01 (1H, m, H-3′′), 3.78
3b: mp 208-210 °C; IR (NaCl) νmax 3369, 1698, 1139 and
837 cm-1; 1H NMR (400 MHz, CDCl3) δ 13.11 (1H, s, NH), 8.16
(1H, s, H-8), 8.10 (1H, s, H-2), 5.98 (1H, d, J ) 4.4 Hz, H-1′),
4.52 (1H, dd, J ) 4.5, 5.0 Hz, H-3′), 4.44 (1H, ddd, J ) 5.0,
5.2, 5.7 Hz, H-2′), 4.08 (1H, m, H-4′), 3.91 (1H, dd, J ) 3.4,
11.5, Ha-5′), 3.75 (1H, dd, J ) 3.4, 11.5, Hb-5′), 3.06 (1H, d, J
) 5.7, 2′-OH), 0.92 (9H, s, C(CH3)3), 0.88 (9H, s, C(CH3)3), 0.14
(3H, s, CH3), 0.13 (3H, s, CH3), 0.05 (3H, s, CH3), 0.03 (3H, s,
CH3); 13C NMR (100 MHz, CDCl3) δ 159.3 (C-6), 148.7 (C-4),
145.0 (C-2), 138.7 (C-8), 125.1 (C-5), 88.9 (C-1′), 85.3 (C-4′),
77.0 (C-3′), 71.3 (C-2′), 62.1 (C-5′), 25.9, 25.7, 18.3, 18.0, -4.6,
-4.8, -5.4, -5.2; HREIMS m/z 496.2509 (M)+, calcd for
C
22H40N2O5Si2, 496.2535.
1-N -(Be n zyl)-2′,5′-d i-O-(t er t-b u t yld im e t h ylsilyl)in o-
sin e (4a ) a n d 1-N-(Ben zyl)-3′,5′-d i-O-(ter t-bu tyld im eth -
ylsilyl)in osin e (4b). To a stirred solution of a mixture of 3a
and 3b (7.45 g, 15 mmol) in THF (75 mL) were added benzyl
bromide (3.57 mL, 30 mmol) and potassium carbonate (4.15
g, 30 mmol). The mixture was stirred at 60 °C for 4 h and
quenched by aqueous NH4Cl (5 mL). After evaporation under
reduced pressure, the residue was dissolved in EtOAc (200
mL). The solution was washed with water (2 × 100 mL) and
then dried over MgSO4. Evaporation of solvent gave the crude
product, which was chromatographed on a Si gel column
(eluent, EtOAc-n-hexane-CH2Cl2 ) 50:35:15, v/v/v; flow rate,
10 mL/min) to give products [4a (4.48 g) and 4b (3.00 g), 90%].
4a : mp 159-160 °C; IR (NaCl) νmax 3494, 1697, 1133 and 837
cm-1; 1H NMR (400 MHz, CDCl3) δ 8.15 (1H, s, H-8), 7.97 (1H,
s, H-2), 7.28-7.31 (5H, m, PhH), 5.99 (1H, d, J ) 5.2 Hz, H-1′),
5.25 (2H, d, J ) 3.4 Hz, N-CH2Ph), 4.52 (1H, dd, J ) 5.0, 5.2
Hz, H-2′), 4.23 (1H, ddd, J ) 4.1, 4.2, 5.0 Hz, H-3′), 4.17 (1H,
m, H-4′), 3.95 (1H, dd, J ) 2.4, 11.5, Ha-5′), 3.81 (1H, dd, J )
3.4, 11.5, Hb-5′), 2.66 (1H, d, J ) 4.2 Hz, 3′-OH), 0.92 (9H, s,
C(CH3)3), 0.80 (9H, s, C(CH3)3), 0.11 (3H, s, CH3), 0.10 (3H, s,
(1H, m, H-5′′), 3.59-3.68 (6H, m, H-5′, H-2′′, H-4′′, H-6′′); 13
C
NMR (100 MHz, CDCl3) δ 156.1 (C-6), 147.0 (C-4), 145.9 (C-
2), 140.3 (C-8), 138.3, 137.7, 137.6, 136.9, 135.4 (5 x Cq, Ph),
127.7-129.1 (CH, Ph), 124.1 (C-5), 100.1 (C-1′′), 91.7 (C-1′),
86.3, 85.9, 82.1, 80.1, 74.6, 73.6, 71.6, 68.2, 62.7 (C-2′, C-3′,
C-4′, C-5′, C-2′′, C-3′′, C-4′′, C-5′′, C-6′′), 79.0, 77.7, 75.2, 74.6
(4 × O-CH2Ph), 49.3 (N-CH2Ph); HREIMS m/z 880.3555 (M)+,
calcd for C51H52N4O10, 880.3683.
8: IR (NaCl) νmax 3411, 1701, and 1587 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.98 (1H, s, H-8), 7.87 (1H, s, H-2), 7.12-7.33
(25H, m, PhH), 5.71 (1H, d, J ) 6.7 Hz, H-1′), 5.24 (2H, s,
N-CH2Ph), 4.41-4.90 (8H, m, O-CH2Ph), 4.69 (1H, t like,
H-2′), 4.48 (1H, d, J ) 7.2 Hz, H-1"), 4.46 (1H, m, H-3′), 4.21
(1H, m, H-4′), 3.50-3.84 (8H, m, H-5′, H-2′′, H-3′′, H-4′′, H-5′′,
H-6′′); 13C NMR (100 MHz, CDCl3) δ 156.1 (C-6), 146.9 (C-4),
146.0 (C-2), 140.2 (C-8), 138.1, 137.9, 137.6, 137.4, 135.5 (5 ×
Cq, Ph), 127.7-129.1 (CH, Ph), 124.1 (C-5), 103.8 (C-1′′), 91.4
(C-1′), 85.4, 84.3, 82.1, 81.8, 74.8, 73.9, 73.4, 68.2, 62.4 (C-2′,
C-3′, C-4′, C-5′, C-2′′, C-3′′, C-4′′, C-5′′, C-6′′), 77.1, 75.7, 75.2,

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J ournal of Natural Products, 2000, Vol. 63, No. 9 1191
75.1 (4 × O-CH2Ph), 49.3 (N-CH2Ph); HREIMS m/z 880.3565
(M)+, calcd for C51H52N4O10, 880.3683.
H-5′), 3.21-3.55 (5H, m, H-3′′, H-4′′, H-5′′, H-6′′); 13C NMR
(100 MHz, CDCl3) δ 156.1 (C-6), 146.9 (C-4), 146.0 (C-2), 140.8
(C-8), 138.1, 137.9, 137.7, 137.5, 135.5 (5 × Cq, Ph), 127.7-
129.1 (CH, Ph), 126.3 (C-5), 102.1 (C-1′′), 89.5 (C-1′), 86.7, 84.9,
81.0, 80.8, 77.4, 73.2, 70.8, 68.0, 62.7 (C-2′, C-3′, C-4′, C-5′,
C-2′′, C-3′′, C-4′′, C-5′′, C-6′′), 75.6, 75.3, 75.1, 75.0 (4 × O-CH2-
Ph), 49.2 (N-CH2Ph); HREIMS m/z 880.3565 (M)+, calcd for
C51H52N4O10, 880.3683.
3′-O-(r-D-glu cosyl)in osin e (1). To a stirred solution of 7
(438 mg, 0.5 mmol) in EtOH (20 mL) were added 20%
Pd(OH)2/C (438 mg) and cyclohexene (14 mL). The mixture
was stirred for 4 h under reflux. The catalyst was removed by
filtration, and the filtrate was concentrated to dryness. The
resulting residue was separated by reversed-phase HPLC
(ODS column; eluent, MeOH-H2O ) 70:30, v/v; flow rate, 1
mL/min; detection, UV 254 nm) to give 1 (172 mg, 80%). The
spectral data of synthetic compound 1 were identical to those
2′-O-(r-D-glu cosyl)in osin e (12). This was prepared from
10 (176 mg 0.2 mmol), 20% Pd(OH)2/C (176 mg), and cyclo-
hexene (10 mL) in EtOH by the same method as 1 (69 mg,
20
80%). [R]20 +31° (c 1.0, H2O); IR (NaCl) νmax 3395, 1686, and
of the natural product. [R]D +40° (c 1.0, H2O); IR (NaCl) ν
D
max 3376, 1682 and 1592 cm-1; 1H NMR (400 MHz, DMSO-d6)
δ 8.28 (1H, s; H-8), 8.05 (1H, s, H-2), 5.87 (1H, d, J ) 6.1 Hz,
H-1′), 4.94 (1H, d, J ) 3.4 Hz, H-1′′), 4.63 (1H, dd, J ) 5.4, 6.1
Hz, H-2′), 4.26 (1H, dd, J ) 3.0, 5.4 Hz, H-3′), 4.12 (1H, ddd,
J ) 3.0 Hz, H-4′), 3.63 (2H, m, H-5′), 3.47 (2H, m, H-3′′, H-5′′),
3.41-3.62 (2H, m, H-6′′), 3.22 (1H, dd, J ) 3.4, 9.4 Hz, H-2′′),
3.11(1H, t, J ) 3.4, H-4′′); 13C NMR (100 MHz, DMSO-d6) δ
158.1 (C-6), 148.4 (C-4), 147.0 (C-2), 138.3 (C-8), 124.4 (C-5),
100.1 (C-1′′), 87.5 (C-1′), 84.3 (C-4′), 77.5 (C-3′), 73.7 (C-3′′),
73.6 (C-5′′), 73.2 (C-2′), 72.1 (C-2′′), 70.0 (C-4′′), 61.2 (C-5′), 60.5
(C-6′′); HRFABMS m/z 453.1233 (M + Na)+, calcd for
1576 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 8.20 (1H, s, H-8),
8.05 (1H, s, H-2), 6.01 (1H, d, J ) 5.0 Hz; H-1′), 4.67 (1H, d, J
) 3.2 Hz, H-1′′), 4.57 (1H, dd, J ) 4.6, 5.0 Hz, H-2′), 4.29 (1H,
dd, J ) 3.5, 4.6 Hz, H-3′), 3.99 (1H, ddd, J ) 3.5 Hz, H-4′),
3.58-3.69 (2H, m, H-5′), 3.36-3.54 (4H, m, H-3′′, H-5′′, H-6′′),
3.12 (1H, dd, J ) 3.2, 9.5 Hz, H-2′′), 3.07 (1H, t like, H-4′′);
13C NMR (100 MHz, DMSO-d6) δ 158.3 (C-6), 148.6 (C-4), 147.0
(C-2), 138.4 (C-8), 124.6 (C-5), 98.8 (C-1′′), 86.1 (C-1′), 85.7 (C-
4′), 79.1 (C-2′), 72.9 (C-3′′), 72.8 (C-5′′), 71.7 (C-3′), 69.9 (C-
2′′), 69.6 (C-4′′), 60.9 (C-5′), 60.6 (C-6′′); HRFABMS m/z
453.1236 (M + Na)+, calcd for C16H22N4O10Na 453.1234.
C
16H22N4O10Na 453.1234.
2′-O-(â-D-glu cosyl)in osin e (13). This was prepared from
11 (26 mg 0.1 mmol), 20% Pd(OH)2/C (26 mg), and cyclohexene
3′-O-(â-D-glu cosyl)in osin e (1b). This was prepared from
8 (88 mg 0.1 mmol), 20% Pd(OH)2/C (88 mg), and cyclohexene
(3 mL) in EtOH by the same method as 1 (10 mg, 80%). [R]20
D
20
(3 mL) in EtOH by the same method as 1 (34 mg, 80%). [R]D
-34° (c 1.0, H2O); IR (NaCl) νmax 3386, 1703, and 1579 cm-1
;
-33° (c 1.0, H2O); IR (NaCl) ν
3380, 1698 and 1585 cm-1
;
max
1H NMR (400 MHz, DMSO-d6) δ 8.32 (1H, s, H-8), 8.05 (1H, s,
H-2), 6.07 (1H, d, J ) 4.6 Hz, H-1′), 4.70 (1H, dd, J ) 4.2, 4.6
Hz, H-2′), 4.39 (1H, dd, J ) 3.5, 4.6 Hz, H-3′), 4.30 (1H, d, J )
7.7 Hz, H-1′′), 3.93 (1H, ddd, J ) 3.5, Hz, H-4′), 3.48-3.67 (2H,
m, H-5′), 3.33-3.47 (3H, m, H-5′′, H-6′′), 2.98-3.14 (3H, m,
H-2′′, H-3′′, H-4′′); 13C NMR (100 MHz, DMSO-d6) δ 157.2 (C-
6), 147.5 (C-4), 146.2 (C-2), 138.1 (C-8), 124.3 (C-5), 103.2 (C-
1′′), 86.2 (C-1′), 85.1 (C-4′), 81.2 (C-2′), 76.8 (C-3′′), 75.9 (C-
5′′), 73.5 (C-2′′), 69.5 (C-3′), 69.4 (C-4′′), 61.1 (C-5′), 60.5 (C-
1H NMR (400 MHz, DMSO-d6) δ 8.32 (1H, s, H-8), 8.05 (1H, s,
H-2), 5.89 (1H, d, J ) 5.2 Hz, H-1′), 4.55 (1H, dd, J ) 5.1, 5.2
Hz, H-2′), 4.35 (1H, dd, J ) 3.8, 5.1 Hz, H-3′), 4.33 (1H, d, J )
7.4 Hz, H-1′′), 4.13 (1H, ddd, J ) 3.8 Hz, H-4′), 3.67-3.60 (3H,
m; H-5′, H-5′′), 3.43 (1H, dd, J ) 5.2, 5.2 Hz, H-4′′), 3.17-3.08
(4H, m, H-2′′, H-3′′, H-6′′); 13C NMR (100 MHz, DMSO-d6) δ
157.3 (C-6), 148.2 (C-4), 146.5 (C-2), 138.4 (C-8), 124.4 (C-5),
102.1 (C-1′′), 87.6 (C-1′), 83.2 (C-4′), 77.2 (C-3′), 76.9 (C-3′′),
76.5 (C-5′′), 73.5 (C-2′′), 73.1 (C-2′), 69.8 (C-4′′), 60.9 (C-5′), 60.8
(C-6′′); HRFABMS m/z 453.1227 (M + Na)+, calcd for
6′′); HRFABMS m/z 453.1233 (M + Na)+, calcd for C16H22
N4O10Na 453.1234.
-
C
16H22N4O10Na 453.1234.
1-N-(Ben zyl)-3′,5′-d i-O-(ter t-b u t yld im et h ylsilyl)-2′-O-
Refer en ces a n d Notes
(2′′,3′′,4′′,6′′-tetr a-O-ben zylglu copyr an osyl)in osin e (9). This
was prepared from 4b (1.164 g, 2.0 mmol), 5 (2055 mg, 3.0
mmol), and BF3‚Et2O (1.229 mL, 10 mmol) by the same method
as 6 (931 mg, 42%). 9: HRFABMS m/z 1109.5478 (M + H)+,
calcd for C63H81N4O10Si2 1109.5491.
(1) Kim, Y. H.; Fuhrman, F. A.; Fuhrman, G. J .; Pavelka, L. A.; Mosher,
H. S. Science 1980, 207, 193-195.
(2) (a) Quinn, R. J .; Gregson, R. P.; Cook, A. F.; Bartlett, R. T.
Tetrahedron Lett. 1980, 21, 567-568. (b) Kato Y.; Fusetani, N.;
Matsunaga, S.; Hashimoto, K. Tetrahedron Lett. 1985, 26, 3483-3486.
(c) Kondo, K.; Shigemori, H.; Ishibashi, M.; Kobayashi, J . Tetrahedron
1992, 48, 7145-7148.
1-N-Ben zyl-3′-O-r- a n d 1-N-Ben zyl-3′-O-â-D-(2′′,3′′,4′′,6′′-
tetr a -O-ben zyl glu cop yr a n osyl)in osin es (10, 11) These
were prepared from 9 (800 mg, 0.72 mmol) and 1 M tetrabu-
tylammonium fluoride in THF (1.6 mL, 1.6 mmol) by the same
method as 7 and 8 [10 (532 mg) and 11 (38 mg), 90%]. 10: IR
(3) Doi, Y.; Ishibashi, M.; Kobayashi, J . Tetrahedron 1994, 50, 8651-
8656.
(4) Kazlauskas, R.; Murphy, P. T.; Wells, R. J .; Baird-Lambert, J . A.;
J amieson, D. D. Aust. J . Chem. 1983, 36, 165-170.
(5) Stewart, J . B.; Bornemann, V.; Chen, J . L.; Moore, R. E.; Caplan, F.
R.; Karuso, H.; Larsen, L. K.; Patterson, G. M. L. J . Antibiotics 1988,
41, 1048-1056.
(6) Suzuki, Y.; Uchida K. Nippon Nogeikaku Kaishi 1976, 50, 231-236.
(7) Kobayashi, J .; Doi, Y.; Ishibashi, M. J . Org. Chem. 1995, 59, 255-
257.
(8) Kalinowski, H. O.; Berger, S.; Braun, S. Carbon-13 NMR Spectroscopy;
J ohn Wiley & Sons: Chichester, 1984; p 440. Inosine [δC 146.2 (C-2),
148.5(C-4), 124.6(C-5), 156.9(C-6), and 139.1(C-8)].
(9) Lichtenthaler, F. W.; Eberhard, W.; Braun, S. Tetrahedron Lett. 1981,
22, 4401-4404.
(10) (a) Beier, R. C.; Mundy, B. P. Can. J . Chem. 1980, 58, 2800-2804.
(b) Kraska, B.; Lichtenthaler, F. W. Chem. Ber. 1981, 114, 1636-
1648.
(11) (a) Knapp, S. Chem. Rev. 1995, 95, 1859-1876. (b) Lerner, L. M.
Synthesis of Nucleosides and Nucleotides; Townsend, L. B., Ed.;
Plenum Press: New York, 1991; Vol. 2, pp 27-79. (c) Lichtenthaler,
F. W.; Sanemitsu, Y.; Nohara, T. Angew. Chem., Int. Ed. Engl. 1978,
17, 772-774. (d) Knapp, S.; Gore, V. K. J . Org. Chem. 1996, 61, 6744-
6747.
(12) Ogilvie, K. K.; Entwistle D. W. Carbohydr. Res. 1981, 89, 203
(13) Schmidt, R. R.; Michel, J . Angew. Chem., Int. Ed. Engl. 1980, 19,
731-732.
1
(NaCl) νmax 3405, 1705, and 1585 cm-1; H NMR (400 MHz,
CDCl3) δ 7.94 (1H, s, H-8), 7.80 (1H, s, H-2), 7.14-7.30 (25H,
m, PhH), 5.90 (1H, d, J ) 7.8 Hz, H-1′), 5.19 (2H, d, J ) 5.9
Hz, N-CH2Ph), 4.46-4.80 (8H, m, O-CH2Ph), 4.70 (1H, t like,
H-2′), 4.63 (1H, d, J ) 4.0 Hz, H-1′′), 4.37 (1H, t like, H-3′),
4.20 (1H, m, H-4′), 4.12 (1H, m, H-5′′), 3.84 (2H, m, H-5′, H-3′′),
3.67(2H, m, H-5′, H-6′′), 3.31-3.45 (3H, m, H-2′′, H-4′′, H-6′′);
13C NMR (100 MHz, CDCl3) δ 156.1 (C-6), 146.9 (C-4), 145.9
(C-2), 140.7 (C-8), 138.2, 137.7, 137.5, 137.2, 135.4 (5 × Cq,
Ph), 127.7-129.1 (CH, Ph), 127.1 (C-5), 98.4 (C-1′′), 88.8 (C-
1′), 88.1, 82.4, 81.5, 79.2, 77.5, 72.8, 71.5, 68.8, 62.9 (C-2′, C-3′,
C-4′, C-5′, C-2′′, C-3′′, C-4′′, C-5′′, C-6′′), 76.8, 76.7, 75.0, 73.5
(4 × O-CH2Ph), 49.3 (N-CH2 Ph); HREIMS m/z 880.3580 (M)+,
calcd for C51H52N4O10, 880.3683.
11: IR (NaCl) νmax 3415, 1698, and 1579 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.98 (1H, s, H-8), 7.88 (1H, s, H-2), 7.12-7.34
(25H, m, PhH), 5.87 (1H, d, J ) 6.8 Hz, H-1′), 4.91-5.27 (2H,
dd, J ) Hz, N-CH2Ph), 4.87 (1H, t like, H-2′), 4.71-4.81 (8H,
m, O-CH2Ph), 4.49 (1H, m, H-3′), 4.46 (1H, d, J ) 8.5 Hz, H-1′′),
4.30 (1H, t like, H-2′′), 4.18 (1H, m, H-4′), 3.61-3.88 (2H, m,
NP0000724