A trinorsesterterpene glycoside from the North American fern Woodwardia virginica (L.) Smith

Article abstract of DOI:10.1016/S0031-9422(03)00290-5

A new trinorsesterterpene glycoside was isolated from the ethanol extract of the American fern Woodwardia virginica having a 3-[6-(4,8-dimethyl-nona-1,3,7-trienyl)-4-hydroxy-2,6-dimethyl-cyclohex-1-enyl] -3-hydroxypropionic acid, as the aglycone and a saccharide moiety linked at C-4 to glucoses, xylose or arabinofuranose. The structure was elucidated using extensive spectroscopic analysis (1D and 2D NMR, MS, IR and UV) including determination of absolute stereochemistry by means of the MTPA and PGME derivatives and also by chemical methods.

Full text of DOI:10.1016/S0031-9422(03)00290-5

Phytochemistry 63 (2003) 869–875
www.elsevier.com/locate/phytochem
A trinorsesterterpene glycoside from the North American fern
Woodwardia virginica (L.) Smith
Lumır O. Hanusa,*, Tomas Rezankab, Valery M. Dembitskya
aDepartment of Medicinal Chemistry and Natural Products, School of Pharmacy, PO Box 12065,
The Hebrew University of Jerusalem, Jerusalem 91120, Israel
bInstitute of Microbiology, Czech Academy of Sciences, Videnska 1083, 142 20 Prague, Czech Republic
Received 13 January 2003; received in revised form 22 April 2003
Abstract
A new trinorsesterterpene glycoside was isolated from the ethanol extract of the American fern Woodwardia virginica having a
3-[6-(4,8-dimethyl-nona-1,3,7-trienyl)-4-hydroxy-2,6-dimethyl-cyclohex-1-enyl]-3-hydroxypropionic acid, as the aglycone and a
saccharide moiety linked at C-4 to glucoses, xylose or arabinofuranose. The structure was elucidated using extensive spectroscopic
analysis (1D and 2D NMR, MS, IR and UV) including determination of absolute stereochemistry by means of the MTPA and
PGME derivatives and also by chemical methods.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Trinorsesterterpene glycoside; American fern; Woodwardia virginica
1. Introduction
(Wynne et al., 1998), L. flexuosum and L. circinnatum
(Takeno et al., 1989). The protoilludane sesquiterpene
glycoside, pteridanoside, was isolated from fern Pter-
idium aquilinum var. caudatum (Castillio et al., 1999).
In the present paper, we describe the identification of
Ferns are a very ancient family of plants: early fern
fossils predate the beginning of the Mesozoic era, 360
million years ago. They thrived on Earth for 200 million
years before the flowering plants evolved (Hallowell and
Hallowell, 2001). Many types of natural compounds
have been discovered from different fern species during
the last decade. Some ferns contain bioactive flavonoids
such as chalcones, dihydrochalcones, avanones, dihy-
dro-flavonols, flavones, flavonols, and bisflavonoids
which have recently been reviewed (Wollenweber and
Schneider, 2000, and references cited). Five lipophilic
components, hopane type triterpenes, have been iso-
lated from fronds of the fern Lophosoria quadripinnata
(Lophosoriaceae, Pteridophyta) (Tanaka et al., 1991).
Unusual betaine ether-linked lipids, phospholipids, and
fatty acids have also been studied in some fern species
(Rozentsvet et al., 2001; Dembitsky, 1996). Biological
active terpenoids, such as gibberellins, were identified
from ferns belonging to genus Lygodium: L. japonicum
a new trinorsesterterpene glycoside isolated from
American fern Woodwardia virginica.
2. Results and discussion
The leaves of fern W. virginica were extracted by ethyl
alcohol and the extract was subsequently separated on
Sephadex LH-20. The fractions were further purified by
RP-HPLC to give glycoside (1)—see Fig. 1, which was
1
identified by IR, UV, MS and H- and 13C NMR spec-
tral data and chemical degradation.
Woodwardinoside (1) was obtained as a major com-
ponent from W. virginica. The molecular formula was
deduced as C50H80O27 from a quasi-molecular ion
observed at m/z 1113.4969 (M+H)+ (ꢀ 4 mmu) in the
FABMS and from the 13C NMR spectrum. The IR
spectrum showed absorbance at 3450 and 1681 cmꢀ1
,
that indicated the presence of a hydroxyl and carbonyl
groups in the molecule, and the UV absorption band at
252 nm (conjugated double bonds).
*Corresponding author. Tel.: +972-2-675-8635; fax: +972-2-675-
7076.
E-mail address: lumir@cc.huji.ac.il (L.O. Hanus).
0031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00290-5

870
L.O. Hanusˇ et al. / Phytochemistry 63 (2003) 869–875
which correlates the anomeric protons with their
respective skeleton carbons atoms (Mimaki et al., 1996).
This allowed identification of 13C NMR signals for all
the monosaccharides and the substituted positions of
the inner glycosyl moieties, with only little knowledge of
1
the sequential H NMR assignment.
The glycoside was acidic hydrolyzed and the aglycone
woodwardine (2) was isolated from the reaction mixture
as colorless needles, with HRMS establishing its mole-
cular formula as C22H34O4 (Fig. 2). The 13C NMR
spectrum showed the signals of five methyls, five
methylenes, six methines, and six quaternary carbons.
The IR absorption at 3450 and 1681 cmꢀ1 indicated the
presence of a hydroxyl and carbonyl groups in the
molecule. The 1H NMR spectrum of 2 showed the
occurrence of a methyl group at ꢀH 1.07 and a geranyl
side chain. The 1H NMR spectrum in combination with
the 1H–1H COSY NMR spectrum (Fig. 3) indicated the
presence of the partial structure –CH2CH(OH)CH2–
from the multiplet signal at ꢀH 3.54 (ddt, J=12.0, 9.2,
6.0 Hz), which coupled with two pairs of methylene
protons at ꢀH 2.31 (dd, J=17.2, 6.0 Hz), 1.94 (dd,
J=17.2, 9.2 Hz), 1.76 (dd, J=12.0, 6.0 Hz), and 1.36 (t,
J=12.0 Hz). Another partial structure, –CH(OH)CH2-
COOH, was accounted for by the observation of the
signals at ꢀC 60.7, 42.2, and 171.4 in the 13C NMR
spectrum and the signals at ꢀH 3.64 (dd, J=6.0, 5.0 Hz),
3.27 (dd, J=10.8, 5.0 Hz), and 3.50 (dd, J=10.8, 6.0 Hz)
Fig. 1. Structure of woodwardinoside 1.
The negative-ion FABMS of 1 showed fragment ion
peaks at 979 [MꢀHꢀ132]ꢀ and 949 [MꢀHꢀ162]ꢀ; the
former was assignable to the loss of a pentosyl group
from the parent ion at m/z 1111, and the latter corre-
sponded to the loss of a glucosyl group. Furthermore,
three prominent fragment ion peaks at m/z 847
[MꢀHꢀ2ꢁ132]ꢀ,
817
[MꢀHꢀ162–132]ꢀ,
685
[MꢀHꢀ162–2ꢁ132]ꢀ, were due to the elimination of
two pentosyl, glucosyl and pentosyl, and the pentosyl
and two glucosyl groups, respectively. This was sugges-
tive of a branched pentaglycoside. The severe over-
lapping of the proton signals for the pentaglycoside
moiety excluded the possibility of a complete assign-
ment in a straightforward way using a conventional
heteronuclear COSY spectrum in 1. An HSQC-TOCSY
technique was applied to solve the sugar sequence of 1,
1
in the H NMR spectrum.
Fig. 2. Structure of woodwardine 2.
Fig. 3. HMBC, COSY and NOE correlations of compound 2.

L.O. Hanusˇ et al. / Phytochemistry 63 (2003) 869–875
871
The 1H–1H COSY spectrum, enhancing the long
range couplings, allowed connection of an isoprenoid
chain. The ethylenic proton at 5.20 ppm (H-700), which
was vicinal to the methylene protons at 1.95 and 2.10
ppm, gave cross peaks with signals of methyl protons at
1.73 (H-900) and 1.57 ppm (H-1100). The ethylenic proton
at 5.78 ppm, H-300, gave correlation spots with methyl
protons at 1.63 ppm (H-1000) and methylene protons at
1.95 and 2.10 ppm. This proton H-300 is coupled
(J=10.8 Hz) with the proton at 6.32 ppm (H-200), which
in turn gave an E ethylenic coupling (J=15.3 Hz) with
the proton at 5.62 ppm (H-100). This latter proton (H-100)
ibility of the non-aromatic ring and of the isoprenoid
chain. The geometry of the trisubstituted double bond
(C-300) was established as E owing to the presence of an
NOE between H-300 and H-500 (ꢀ 1.95) observed in the
NOESY spectrum and the chemical shift of methyl car-
bon C-1000 (ꢀ 16.4) of 2 (Okubo et al., 1980; Barlow and
Pattenden, 1976) (Fig. 4). In the HMBC spectrum, the
H-100 proton provided a good starting point for the
assignment of the proton and carbon resonances of the
geranyl function. Proton H-100 exhibited correlations
with the C-50, C-80, C-600 and C-300 carbons. This per-
mitted assignment of the C-60 position of the geranyl
group.
gave cross peaks, on the H–1H-COSY, with H-80 (1.07
1
ppm) and also with the methylene that gave the signals
at 1.76 and 1.36 ppm (H-50); these results lead to the
linkage of C-50, C-60, and C-80 to the remaining quar-
tenary carbon atom (138.5 ppm, C-10).
Finally, strong NOE were observed between: H-100
and H-3, H-300 and H-500, H-500 and H-700, H-200 and H-
1000, and H-700 and H-900, indicating that they are in a cis
relative disposition. Irradiation of H-100 enhanced the
signals of H-80 and H=50 ax. This data shows the flex-
The relative configuration at the chiral center at C-40
was determined by the coupling constants of the H-40
resonance at ꢀH 3.54 (ddt, J=12.0, 9.2, 6.0 Hz) with the
two pairs of methylene protons, which inferred an
equatorial orientation of the hydroxyl group at C-40.
The absolute stereochemistry of C-4 was confirmed by
Mosher ester derivatization (Ohtani et al., 1991a,b).
Both the (S)- and (R)-MTPA esters of 2 were prepared
(Fig. 5) and subjected to 1H NMR analysis (Table 2). In
the H NMR spectrum, the H-3 ax, H-3 eq, and Me-20
signals of 2a shifted upfield due to the phenyl group,
whereas in 2b, the H-50ax signal experienced shielding.
Therefore, the absolute configuration of 2 at C-40 was
determined as R. The reaction of aglycone (2) having a
secondary hydroxyl group on C-3 proceeded slowly and
the yield of corresponding ester was very poor, therefore
we used the method based on reaction of free acid with
(S)- and (R)-PGME (Yabuuchi and Kusumi, 2000) for
determination of absolute stereochemistry on C-3. The
principle of the PGME method is following: a chiral
b,b-disubstituted propionic acid is condensed with (R)-
and (S)-PGME. In the diastereomeric pair of PGME
amides, the protons of the (S)-isomer will have larger
upfield chemical shifts than those of the (R)-isomer
0
0
1
Fig. 4. 3D-MM2 models of compound 2.
Fig. 5. ꢀꢀ=(ꢀS–ꢀR) values (ppm) obtained for the MTPA esters.

872
L.O. Hanusˇ et al. / Phytochemistry 63 (2003) 869–875
Table 1
13C-NMR of woodwardinoside (1)
Table 2
1H- and 13C-NMR data of woodwardine (2)
C no.
C no.
No. 1H
13C
1
177.0
42.2
60.7
41
51
61
12
22
32
42
52
62
13
23
33
43
53
63
14
24
34
44
54
15
25
35
45
55
80.2
76.8
62.1
104.8
75.2
86.1
84.8
76.4
62.0
105.2
75.0
76.7
70.0
76.8
62.3
104.9
75.1
78.2
70.8
67.3
111.0
84.5
78.6
85.4
62.4
1
–
177.0
42.2
2
2
3.27 (1H, dd, J=10.8, 5.0); 3.50 (1H, dd J=10.8, 6.0)
3
3
3.64 (1H, dd, J=6.0, 5.0)
60.7
10
138.5
132.9
42.2
10
20
–
–
138.5
132.9
20
30
30 2.31 eq (1H, dd, J=17.2, 6.0); 1.94 ax (1H, dd, J=17.2, 9.2) 44.5
40
73.8
44.3
40 3.54 (1H, ddt, J=12.0, 9.2, 6.0)
50 1.76 eq (1H, dd, J=12.0, 6.0); 1.36 ax (1H, t, J=12.0)
60
67.0
48.9
50
60
24.2
17.9
–
24.2
700
700 1.71 (3H, s)
17.9
800
100
200
300
4
23.5
800 1.07 (3H, s)
23.5
132.6
127.0
122.3
140.7
40.2
100 5.62 (1H, d, J=15.3)
200 6.32 (1H, dd, J=15.3, 10.8)
300 5.78 (1H, dq, J=10.8, 1.3)
132.6
127.0
122.3
140.7
40.2
4
–
500
600
700
800
900
1000
1100
11
21
31
500 1.95 (2H, m)
25.1
600 2.10 (2H, m)
700 5.20 (1H, tq, J=6.8, 1.2)
800
900 1.73 (3H, s)
1000 1.63 (3H, d, J=1.3)
1100 1.57 (3H, d, J=1.2)
25.1
122.8
133.9
26.3
122.8
133.9
26.3
–
16.4
17.6
16.4
17.6
104.0
86.4
77.3
4.46, 4.61, 5.01, 5.28, and 6.28 were observed, corre-
sponding to signals at ꢀC 104.0, 104.8, 104.9, 105.2, and
111.0, respectively, and indicating that 1 possesses five
sugar units. Four anomeric protons (ꢀH 4.46, 4.61, 5.01
and 5.28) showed b-glycosidic linkages according to the
coupling constants of their anomeric protons (J=7.5–
7.8 Hz). A characteristic furanosyl broad singlet signal
(ꢀH 6.28, brs) was observed. Among the five anomeric
carbons of the carbohydrate units, the chemical shift at
d 111.0 demonstrated that one of those was in the a-
furanose form (Gorin and Mazurek, 1975). The con-
figuration of all of the other sugars in the pyranose form
in 1 was fully defined from the chemical shift and the
coupling constant of each of the remaining anomeric
owing to the diamagnetic anisotropic effect of the ben-
zene ring. The stereochemistry of the side chain alcohol
was resolved by the H NMR analysis of 2c and 2d
1
(Fig. 6). The signal of Me on C-20 of 2d appeared more
downfield than that of 2c, and the H-80, H-100 and H-200
protons of 2d were shielded by a phenyl group and
shifted more upfield than that of 2c. These observations
indicated that the configuration at C-3 was S. On the
basis of the above data, the absolute stereochemistry of
woodwardine was assigned as 2, i.e. (10E,100E,3S,
300E,40R,60S)-3-[60-(400,800-dimethyl-nona-100,300,700-trienyl)-
40-hydroxy-20,60-dimethyl-cyclohex-10-enyl]-3-hydroxy-
propionic acid.
1
protons. From the H–1H COSY and TOCSY spectra,
Formation of the compounds 2c, d, which have
MTPA ester on b-hydroxyl group, was also observed.
The residue was subjected to silica gel preparative TLC
with eluent n-hexane–acetone (3:1) to yield 2a and 2c or
2b and 2d, successively. After preparative TLC the yield
of compounds 2c, d was negligible and consequently the
configuration of b-hydroxyl group was determined with
the help of PGME esters. The low yield of esters for-
mation on b-hydroxyl group can be explained due to the
fact that it is not an isolated secondary hydroxyl group,
but an b-hydroxylacid where different hydroxyl groups
have different properties.
The saccharide composition of 1 was identified by
GLC (after acidic hydrolysis), and the sugars were
1
identified as glucose, arabinose and xylose. In the H
NMR spectrum of 1, five anomeric proton signals at ꢀH
Fig. 6. ꢀꢀ=(ꢀS–ꢀR) values (ppm) obtained for the PGME amides.

L.O. Hanusˇ et al. / Phytochemistry 63 (2003) 869–875
873
all proton signals belonging to each sugar moiety in 1
were identified, starting with the anomeric protons. All
sugar connectivities were established using NOESY and
HMBC experiments. In the NOESY spectrum, cross-
peak signals were observed between Glc-H-11 and H-40,
Glc-H-12 and Glc-H-41, Glc-H-13 and Glc-H-42, Xyl-H-
14 and Glc-H-21, and Araf-H-15 and Glc-H-32. The
HMBC experiment showed long-range correlations
between H-11 and C-40, H-12 and C-41, H-13 and C-43,
H-14 and C-21, and H-15 and C-32 (Fig. 1). Thus, the
structure of 1 was assigned as (40R)-40-O-[b-d-glucopyr-
anosyl-(1-4)-b-d-arabinofuranosyl-(1-3)-b-d-glucopyr-
anosyl-(1-4)]-[b-d-xylopyranosyl-(1-2)]-b-d-glucopyranosyl-1-
4-woodwardine, i.e. woodwardinoside (1).
(10E,100E,3S,300E,40R,60S) - 3 - [6 - (4,8 - Dimethyl - nona-
1,3,7 - trienyl)-4-hydroxy-2,6-dimethyl-cyclohex-1-enyl]-
3-hydroxy-propionic acid (woodwardine, 2), could be
synthesized (see Fig. 7). The mechanism of sesterterpene
biosynthesis was recently reviewed (Dewick, 2002).
Dimethylallyl diphosphate is the starting biogenic iso-
prene unit, along with isopentyl diphosphate. Poly-
prenyl diphosphate synthases (prenyl-transferases) is
responsible for the alkylation of dimethylallyl diphos-
phate and isopentyl diphosphate resides, reactions
which provide the polyprenyl diphosphate precursors
for the various terpenoid families. In enzymatic cycli-
zation, presence of cation cyclases is possible. At pre-
sent, the oxidation in the biosynthesis of woodwardine 2
is not yet known; it probably includes some further
biosynthetic steps.
3. Experimental
3.1. General experimental procedures
UV spectra were measured in heptane within the
range of 200–350 nm by a Cary 118 (Varian) appara-
tus. Optical rotary dispersion (ORD) measurements
were carried out under dry N2 by a Jasco-500A spec-
ꢂ
tropolarimeter at 24 C. A Perkin-Elmer Model 1310
(Perkin-Elmer, Norwalk, CT, USA) IR spectro-
photometer was used for scanning IR spectroscopy of
acids and glycosides as neat films. NMR spectra were
recorded on a Bruker AMX 500 spectrometer (Bruker
Analytik, Karlsruhe, Germany) at 500.1 MHz (1H),
125.7 MHz (13C) in mixture of deuterated pyridine
and CD3OD (v/v 1:1). High- and also low-resolution
MS were recorded using a VG 7070E-HF spectro-
meter (70 eV). HRFABMS (positive and/or negative
ion mode) were obtained with a PEG-400 matrix. RP-
HPLC was carried out using Shimadzu gradient LC
system (Shimadzu, Kyoto, Japan). Gas chromato-
graphy analysis was made on a Hewlett Packard HP
5980 gas chromatograph (Hewlett Packard sro, Czech
Republic).
Fig. 7. Proposed biosynthetic pathways of woodwardine (2) in fern Woodwardia virginica.

874
L.O. Hanusˇ et al. / Phytochemistry 63 (2003) 869–875
3.2. Plant material, extraction and isolation
a residue which was chromatographed on silica gel with
hexane–ethyl acetate (3:1, v/v) as a developing solvent
to afford the amides in 70–80% yield (Yabuuchi and
Kusumi, 2000).
The specimens of W. virginica (L.) Sm. (Blechnaceae),
Virginia chain fern, were collected in Montgomery
County, Maryland (USA) in October 2002. Fresh fern
leaves were extracted with ethanol (on the spot, imme-
diately after collecting) and after that with ethanol–water
(70:30). Both extracts were combined and evaporated to
small volume. Ethanol–water extracts were separated on
a Sephadex LH-20 column eluting with MeOH–H2O
(9:1), yielding three fractions. Fraction B was further
fractionated by RP-HPLC on a C18-Bondapack col-
umn (30 cmꢁ7.8 mm, flow rate 2.0 ml/min) with ACN–
H2O (1:2) to yield compound 1.
(40R)-40-O-[b-d-Glucopyranosyl-(1!4)-b-d-arabino-
furanosyl-(1!3)-b-d-glucopyranosyl-(1!4)]-[b-d-xylo-
pyranosyl-(1!2)]-b-d-glucopyranosyl-1-4-woodwardine,
i.e. woodwardinoside (1), colourless powder, [a]D23
ꢀ42.0ꢂ; UV lmax (EtOH) (log E) 252 nm (2.24); IR (film)
ꢁmax 3450 (OH), 1680 (COOH); HRFABMS m/z
1113.4969 (M+H)+, calculated for [C50H80O27+H]+
1113.4965; negative LRFABMS m/z 1111 [MꢀH]ꢀ, 979
[MꢀHꢀ132]ꢀ, 949 [MꢀHꢀ162]ꢀ, 847 [MꢀHꢀ2ꢁ132]ꢀ,
817 [MꢀHꢀ162–132]ꢀ, 685 [MꢀHꢀ162–2ꢁ132]ꢀ, 361
[MꢀHꢀ3ꢁ162–2ꢁ132]ꢀ; 1H-NMR spectrum: 3.71 (1H,
m, H-40), 4.46 (1H, d, J=7.6 Hz, H-11), 4.61 (1H, d,
J=7.5 Hz, H-12), 5.01 (1H, d, J=7.8 Hz, H-14),
5.28 (1H, d, J=7.6 Hz, H-13), 6.28 (1H, brs, H-15);
13C-NMR spectrum, see Table 1.
3.2.1. Acidic hydrolysis of the glycosides
The glycoside (1) was refluxed in 2 N HCl (0.5 ml) for
2 h. The aglycone was extracted three times with EtOAc
(10 ml). After separating the organic layer, the aqueous
phase was neutralized with NaHCO3, lyophilized and
the residue was chromatographed on a column of silica
gel (10 g), using CH2Cl2–MeOH–H2O (90:10:1) to pro-
(10E,100E,3S,300E,40R,60S) - 3 - [6 - (4,8 - Dimethyl - nona-
1,3,7-trienyl)-4-hydroxy-2,6-dimethyl-cyclohex-1-enyl]-3-
hydroxy-propionicꢂacid=woodwardine (2), white crys-
tals, m.p. 125–127 C; [a]2D3 ꢀ71.0ꢂ (c 0.18, MeOH); UV
lmax (EtOH) (log E) 253 nm (2.37); IR (film) ꢁmax 3450
(OH), 2950, 2910, 1981 (COOH); HREIMS m/z
348.2305 C21H32O4 [M]+, calculated for [C22H34O4]+
1
vide an acid for H NMR analysis. The identification
and the d or l configuration of sugars was determined
using gas chromatography (a glass-capillary column
Supelco SPB-1, the [acetylated (+)-2-butyl] derivatives
were eluted as a peaks with retention times, which were
identical with standards of the appropriate tetraacetyl
(+)-2-butyl-saccharides) according to the method sta-
ted in Gerwig et al. (1978), with some modifications as
previously described (Rezanka and Guschina, 2000).
1
348.2301; H- and 13C-NMR spectra, see Table 2.
References
Barlow, L., Pattenden, G., 1976. Synthesis of poly-Z-isomers of
2,6,11,15-tetramethyl-hexadeca-2,6,8,10,14-pentaene, a C20 analog
of phytoene. Re-examination of the stereochemistry of a new isomer
of phytoene from Rhodospirillum rubrum. J. Chem. Soc., Perkin
Trans. 1, 1029–1034.
3.2.2. (S)-MTPA and (R)-MTPA esters
To a CH2Cl2 solution (100 ml) of aglycone (0.3 mg),
DMAP (1.0 mg), and Et3N (2 ml), (R)-(ꢀ)-MTPACl (2.0
mg) was added at room temperature, and was stirred for
3 h. After evaporation of solvent, the residue was pur-
ified by silica gel TLC (hexane–AcOEt, 2:1) to provide
the (S)-MTPA ester as colorless oil.
Castillio, U.F., Sakagami, Y., Alonso-Amelot, M., Ojika, M., 1999.
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toxic component of the neotropical bracken fern Pteridium aquili-
num var. caudatum. Tetrahedron 55, 12295–12300.
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and biology. Prog. Lipid Res. 35, 1–51.
3.2.3. (R)-MTPA ester of aglycone
Dewick, P.M., 2002. The biosynthesis of C5–C25 terpenoid com-
pounds. Nat. Prod. Rep. 19, 181–222.
The aglycone (0.3 mg) was treated with (S)-(+)-
MTPACl (2.0 mg) by the same procedure as described
above to provide the (R)-MTPA ester as colorless oil
(Ohtani et al. 1991a,b).
Gerwig, G.J., Kamerling, J.R., Vliegenthart, J.F.G., 1978. Determi-
nation of the D and L configuration of neutral monosaccharides by
high-resolution capillary GLC. Carbohydr. Res. 62, 349–357.
Gorin, P.A.J., Mazurek, M., 1975. Assignments of signals in carbon-
13 magnetic resonance spectra of aldoses and derived methyl glyco-
sides. Can. J. Chem. 53, 1212–1223.
3.2.4. (S)- and (R)-PGME amides of a carboxylic acid
To a stirred solution of a 0.1 mmol carboxylic acid
and 0.15 mmol (S)-PGME [or (R)-PGME] in dry 2 ml
DMF were successively added 0.15 mmol BOP, 0.15
mmol HOBT, and N-methylmorpholine (200 ml) at 0 C.
After the mixture was stirred at room temperature for 3
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NaHCO3 solution, and brine. The organic layer was
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