Leptocarposide: A new triterpenoid glycoside from Ludwigia leptocarpa (Onagraceae)

Article abstract of DOI:10.1002/mrc.4023

A new triterpenoid bidesmoside (leptocarposide) possessing an acyl group in their glycosidic moiety (1), together with the known luteolin-8-C-glucoside (2) and 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(R)-2-hydroxypalmitoylamino]-8- octadecen-1,3- diol (3) was isolated from the n-butanol-soluble fraction of whole plant of Ludwigia leptocarpa (Nutt) Hara (Onagraceae). Structure of compound 1 has been assigned on the basis of spectroscopic data (¹H and ¹³C NMR, ¹H-¹H COSY, HSQC, HMBC, and ROESY), mass spectrometry, and by comparison with the literature. This compound was further screened for its potential antioxidant properties by using the radical scavenging assay model 2,2-diphenyl-1-picrylhydrazyl and reveals non-potent antioxidant activities, while compound 2 shows SC₅₀ of 0,038 mM.

Full text of DOI:10.1002/mrc.4023

MRC letters
Received: 24 July 2013
Revised: 3 October 2013
Accepted: 4 October 2013
Published online in Wiley Online Library: 30 October 2013
(wileyonlinelibrary.com) DOI 10.1002/mrc.4023
Leptocarposide: a new triterpenoid glycoside
from Ludwigia leptocarpa (Onagraceae)
Florence Déclaire Mabou,a Perrin Lanversin Foning Tebou,a David Ngnokam,a*
Dominique Harakatb and Laurence Voutquenne-Nazabadiokoc
A new triterpenoid bidesmoside (leptocarposide) possessing an acyl group in their glycosidic moiety (1), together with the
known luteolin-8-C-glucoside (2) and 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2′R)-2-hydroxypalmitoylamino]-8-octadecen-1,3-
diol (3) was isolated from the n-butanol-soluble fraction of whole plant of Ludwigia leptocarpa (Nutt) Hara (Onagraceae).
1
Structure of compound 1 has been assigned on the basis of spectroscopic data (1H and 13C NMR, H-1H COSY, HSQC, HMBC,
and ROESY), mass spectrometry, and by comparison with the literature. This compound was further screened for its potential
antioxidant properties by using the radical scavenging assay model 2,2-diphenyl-1-picrylhydrazyl and reveals non-potent
antioxidant activities, while compound 2 shows SC50 of 0,038 mM. Copyright © 2013 John Wiley & Sons, Ltd.
1
Keywords: NMR; H NMR; 13C NMR; 2D NMR; Ludwigia leptocarpa; Onagraceae; triterpenoid glycoside; bidesmoside; leptocarposide;
NMR complete assignments
and 323.2 suggesting the elimination of 3-hydroxybutyric acid,
one pentosyl, 3-hydroxybutyric + one pentosyl, one pentosyl +
Introduction
Ludwigia leptocarpa (Nutt) Hara (Onagraceae) is a pantropical
genus that is also well represented in North America and in
tropical Africa.[1] It is used in Nigerian folk medicine for the treat-
ment of rheumatism and dysentery.[2] Previous work on this
species has revealed the presence of flavonoids.[3]
In the course of our continuing search for secondary metabolites
of biological importance from Cameroonian medicinal plants, we
investigate the MeOH extract of the whole plant of L. leptocarpa.
In the present paper, we report the isolation and structural elucida-
tion of new leptocarposide isolated from this plant, using chemical
and spectroscopic methods. 2D NMR techniques, including 1H-1H
COSY, TOCSY, HSQC-Jmod, HMBC, and ROESY, were utilized in the
structure elucidation and complete assignments of 1H and 13C
NMR spectra. The antioxidant activities of compounds 1 and 2 have
been tested through DPPH (2,2-diphenyl-1-picryhydrazyl) radical
scavenging model.
one desoxyhexosyl, 3-hydroxybutyric acid + one pentosyl + one
desoxyhexosyl, and two pentosyl + one desoxyhexosyl moieties,
respectively.
The analysis of its 1H NMR spectrum (Table 1) indicated the
presence of six tertiary methyl protons at δH 0.80, 0.90, 0.98,
1.31, 1.39, and 1.41, one olefinic proton at δH 5.36, and three
O-bearing methine protons at δH 4.15, 4.33, and 4.49. Its DEPT
spectrum exhibited the methyl signals at δC 13.2, 16.1, 16.5,
23.6, 25.8, and 32.0, the ethylenic carbons at δC 121.9 (CH) and
143.5 (C), attributable to an olean-12-ene skeleton[8–10] (Table 1),
in which three hydroxy groups were located at δC 69.9, 85.3, and
73.3 together with two carboxylic groups at 183.5 and 176.0.
C
The 1H-1H COSY showed cross-peak correlations between H-2
(δH 4.33) and H-3 (δH 4.15) and between H-15α (δH 1.67), H-15β
(δH 1.47), and H-16 (δH 4.49) indicating the hydroxylation in
positions 2 and 16 of the genine. The cross peak correlations
observed in HMBC spectrum (Figure 2) between the signal of
the carbonyl at δC 183.5 (C-23) and the protons at δH 4.15 (H-3),
δH 1.64 (H-5), and δH 1.39 (CH3-24), and between the signal of
ester carbonyl at C-28 (δC 176.0) and the oxymethine proton
H-16 at δH 4.49 confirm the place of the substituent.[8–10]
The configuration of the 2β, 3β, 16α-trihydroxy groups, and
Results and Discussion
The purification of the n-BuOH soluble extract from the methanolic
extract gave a new compound; namely, leptocarposide (i) in
addition to the known compounds, luteolin-8-C-glucoside (ii),[4,5]
and 1-O-β-D-glucopyranosyl-(2S,3R,8E)-2-[(2’R)-2-hydroxypalmitoylamino]-
8-octadecen-1,3-diol (iii)[6,7] (Figure 1).
The high resolution time of flight electrospray ionisation mass
spectrometry (HR-TOF-ESI-MS) of compound 1 exhibited a
pseudo-molecular ion peak at m/z 1431.6395 [M + Na]+ consis-
tent with a molecular formula C66H104O32 (calcd. 1431.6408)
and indicating 15° of unsaturation and an ion fragment at m/z
703.3499 due to the loss a saccharidic ester chain. In ESI-MS, an
ion fragment was observed at m/z 751.3 corresponding to the
saccharidic ester chain. The MS/MS of this ion gave other
fragment-ion peaks at m/z 647.3, 619.3, 515.3, 473.2, 369.2,
*
Correspondence to: David Ngnokam, Faculty of Science, Department of Chem-
istry, University of Dschang, P.O. Box 67. Dschang Cameroon. E-mail:
dngnokam@yahoo.fr
a Faculty of Science, Department of Chemistry, University of Dschang, P.O.
Box 67, Dschang, Cameroon
b Service Commun d’Analyses, Institut de Chimie Moléculaire de Reims (ICMR),
CNRS UMR 7312, Bat. 18 B.P.1039, 51687, Reims Cedex 2, France
c
Groupe Isolement et Structure, Institut de Chimie Moléculaire de Reims (ICMR),
CNRS UMR 7312, Bat. 18 B.P.1039, 51687, Reims Cedex 2, France
Magn. Reson. Chem. 2014, 52, 32–36
Copyright © 2013 John Wiley & Sons, Ltd.

New oleanane-type saponin
Figure 1. Structure of the isolated compounds.
α-orientation of the COOH at C-4 was confirmed from the ROESY
experiments (Figure 3). The coupling constant (J= 3.2 Hz) between
H-2 and H-3 is in accordance with the literature.[9,10] On this
basis, the aglycone moiety of compound 1 was established
to be zanhic acid or 2β,3β,16α-trihydroxyolean-12-en-23,
28-dioic acid. Deshielding 13C NMR chemical shift at δC 85.3
(C-3) and shielding 13C NMR chemical shift at δC 176.0 (C-28)
suggested that this saponin was a bidesmosidic saponin with
glucosidic linkages at C-3 through an O-heterosidic bond and
at C-28 through an ester bond.[9,10] The 1H NMR spectrum
showed five anomeric protons signal at δH 4.41 (d, J = 6.7 Hz,
H-1′″′), 4.42 (d, J = 7.8 Hz, H-1′), 4.50 (d, J = 7.6 Hz, H-1″″), 5.39
(d, J = 1.7 Hz, H-1″′), and 5.41 (d, J = 8.1 Hz, H-1″). Their
corresponding anomeric carbons were detected at δC 104.4
(C-1″″′), 103.2 (C-1′), 105.6 (C-1″″), 100.1 (C-1″′), and 93.6 (C-1″) after
the analysis of HSQC spectrum indicating the presence of five sugar
residues. Two methyl carbons at δC 15.5 (C-6″) and 17.0 (C-6″′)
indicate the presence of two 6-desoxyhexoses and the three
oxymethine carbons at δC 60.9 (C-6′), 65.9 (C-5″″), and δC
65.6 (C-5″″′) suggest the presence of one hexose and two
pentoses. The analysis of COSY, TOCSY, and ROESY spectra
allowed the full indication of the spin systems of one
rhamnopyranose and one fucopyranose from the anomeric signals
at δH 5.39 (d, J = 1.7Hz, H-1″′) and δH 5.41 (d, J = 8.1 Hz, H-1″). A
glucopyranose moiety was identified starting from the anomeric
proton at δH 4.42 (d, J = 7.8 Hz, H-1′). One arabinopyranose and
one xylopyranose were assigned starting from the anomeric pro-
tons at δH 4.41 (d, J = 6.7 Hz, H-1″″′) and 4.50 (d, J = 7.6 Hz, H-1″″).
The anomeric configurations of glucose, fucose, and xylose in this
saponin were all determined to be β, and that of arabinose and
rhamnose to be α from the 3 J H1-H2 value of the anomeric proton
signals and the chemical shift of anomeric carbon.[11] The sugar
units were confirmed by thin layer chromatography (TLC) after
hydrolysis, and the D or L-configurations were proved by gas
chromatography-mass spectrometry (GC-MS) after derivatization.
The carbons of each monosaccharides were attributed by
analysis of HSQC spectrum and indicated the presence of a
terminal β-D-glucopyranose, a terminal β-D-xylopyranose, a ter-
minal α-L-arabinopyranose, a 4-substitued α-L-rhamnopyranose
(δC-4″′ 82.7), and a 2,3,4-trisubstitued β-D-fucopyranose (δC-2″ 72.8,
δC-3″ 80.8, and δC-4″ 73.6) (Table 1). The downfield shift of protons
H-4″ (δH 5.30 (d, J = 2.8 Hz) suggested an esterification of the
fucose unit in this C-4″ position.
The 1H NMR spectrum showed also two oxymethine groups at
4.18 (m, H-3″″″′) and at 5.33 (m, H-3″″″) and two methyl at 1.23
(d, J = 6.2 Hz, H-4″″″′) and at 1.36 (d, J = 6.3 Hz, H-4″″″), which
suggested the presence of two 3-hydroxybutanoic acid (HBA)[12]
In the[13]C NMR spectrum, resonances of two other ester carbonyl
at δC 170.1 and 171.1 corresponding, respectively, to HBA C-1″″″
and HBA′ C-1″″″′, two methyl carbons at δC 18.6 (HBA C-4″″″) and
21.9 (HBA′ C-4″″″′), and two oxymethine carbons at δC 64.1 (HBA
C-3″″″′) and δC 67.3 (HBA′ C-3″″″) confirmed the presence of two
HBA.[13,14] The HMBC cross peaks observed between the signal
of ester carbonyl at δC 170.1 (HBA C-1″″″) and three protons at
δH 2.81 (HBA H-2″″″a), δH 2.73 (HBA H-2″″″b) for methylene protons
and δH 5.33 (HBA H-3″″″); and between the signal of carbonyl at δC
171.1 (HBA′ C-1″″″′) and two oxymethine protons at δH 5.33 (HBA
H-3″″″) and 4.18 (HBA′ H-3″″″′), and the methylene at δH 2.45
(HBA′ H-2″″″′a) and δH 2.50 (HBA′ H-2″″″′b) suggest that the two
HBA were attached together.[13,14]
The sequences and linkage sites of the different monosaccha-
ride units were determined with the aid of key HMBC correlations
observed between the signal of ester carbonyl at C-28 (δC 176.0)
and the anomeric proton at δH 5.41 (Glc H-1″) and between the
signal of C-3 (δC 85.3) and anomeric proton at δH 4.42 (Fuc H-1″).
This spectrum exhibited also cross peaks between the signals of
Fuc C-2″at δC 72.8 and anomeric proton at δH 5.39 (Rha H-1″′),
between Fuc C-3″ (δC 80.8) and anomeric proton at δH 4.41
Magn. Reson. Chem. 2014, 52, 32–36
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

F. D. Mabou et al.
Table 1. 1H (600 MHz) and 13C (150 MHz) nuclear magnetic resonance data, for compound 1, in CD3OD
δC
δH (J in Hz)
δC
δH (J in Hz)
Zanhic acid. 1
43.5
1.30 (m)
Glc 1′
103.2
4.42 (d, 7.8)
2.15 (dd, 14.9, 3.2)
4.33 (q, 3.2)
4.15 (d, 3.2)
-
2
3
4
5
6
69.9
85.3
52.5
51.8
20.5
2′
3′
4′
5′
6′
74.0
76.4
69.8
76.4
60.9
3.24 (dd, 8.7, 7.8)
3.38 (m)
3.37 (t, 8.8)
1.64 (m)
3.29 (m)
1.26 (m)
3.71 (dd, 11.8, 4.8)
3.83 (dd, 11.8, 2.3)
5.41 (d, 8.1)
1.63 (m)
7
32.6
1.41 (m)
Fuc 1″
93.6
1.61 (m)
8
39.7
47.1
36.0
23.2
—
2″
3″
4″
5″
72.8
80.8
73.6
69.9
3.95 (dd, 9.5, 8.1)
4.04 (dd, 9.5, 2.8)
5.30 (d, 2.8)
9
1.69 (m)
10
11
—
1.97 (m)
3.88 (m)
2.02 (dd, 11.9, 2.5)
5.36 (t, 3.3)
—
12
13
14
15
121.9
143.5
41.5
6″
Rha 1″′
2″′
15.5
100.1
70.4
1.08 (d, 6.4)
5.39 (d, 1.7)
3.97 (m)
—
35.0
1.47 (dd, 14.9, 2.5)
1.67 (dd, 14.9, 3.2)
4.49 (t, 2.9)
—
3″′
70.8
3.83 (m)
16
17
18
19
73.3
48.9
40.9
46.8
4″′
5″′
82.7
67.7
3.57 (t, 9.3)
3.82 (m)
2.95 (dd, 13.8, 3.9)
1.08 (dd, 13.8, 3.9)
2.32 (t, 13.8)
—
6″′
17.0
1.36 (d, 6.3)
4.50 (d, 7.6)
Xyl 1″″
105.6
20
21
29.9
35.2
2″″
3″″
74.8
76.9
3.26 (dd, 8.6, 7.6)
3.32 (t, 8.6)
1.20 (m)
1.98 (m)
22
23
30.9
1.80 (td, 14.7, 4.4)
1.98 (m)
4″″
5″″
69.5
65.9
3.52 (m)
183.5
—
3.21 (t, 10.4)
3.85 (dd, 10.4, 3.1)
4.41 (d, 6.7)
3.56 (m)
24
25
26
27
28
13.2
16.1
1.39 (s)
1.31 (s)
0.80 (s)
1.41 (s)
—
Ara 1″″′
2″″′
104.4
71.1
73.0
68.3
65.6
16.5
3″″′
3.52(m)
25.8
4″″′
3.81 (t, 2.1)
3.54 (d, 11.7)
3.84 (d, 12.7)
-
176.0
5″″′
29
30
32.0
23.6
0.90 (s)
0.98 (s)
HBA 1″″″
2″″″
170.1
40.0
2.73 (dd, 16.1, 5.6)
2.81 (dd, 16.1, 7.3)
5.33 (m)
3″″″
4″″″
67.3
18.6
1.36 (d, 6.3)
-
HBA′ 1″″″′
2″″″′
171.1
43.6
2.45 (dd, 15.0, 5.6)
2.50 (dd, 15.0, 7.5)
4.18 (m)
3″″″′
4″″″′
64.1
21.9
1.23 (d, 6.2)
Glc, β-D-glucopyranosyl; Fuc, β-D-fucopyranosyl; Xyl, β-D-xylopyranosyl; Ara, α-L-arabinopyranosyl; Rha, α-L-rhamnopyranosyl; HBA, 3-hydroxylbutanoyl.
(Ara H-1″″′), between Rha C-4″′ (δC 82.7) and anomeric proton
at δH 4.42 (Xyl H-1″″), and the signal of ester carbonyl at δC
170.1 (HBA C-1″″″) and Fuc H-4″ at δH 5.30. These correlations
suggested that the sugar moieties are 3-O-β-D-glucopyranoside
and 28-O-β-D-xylopyranosyl(1→ 4)-α-L-rhamnopyranosyl(1→ 2)-
[α-L-arabinopyranosyl(1 → 3)]-4-O-(3′-hydroxybutanoyloxy-3-
hydroxybutanoyloxy)-β-D-fucopyranoside.
The structure of the compound 1 was further supported by a
1H-1H ROESY experiment, which revealed cross peak correlations
between Rha H-1″′ (δH 5.39) and Fuc H-2″ (δH 3.95), Fuc H-3″
(δH 4.04), and Ara H-1″″′ (δH 4.41) and between Xyl H-1″″
(δH 4.50) and Rha H-4″′ (δH 3.57). On the basis of aforementioned in-
formation, the structure of this compound was elucidated as 3-O-β-D-
glucopyranosyl-28-O-β-D-xylopyranosyl(1 →4)-α-L-rhamnopyranosyl
wileyonlinelibrary.com/journal/mrc
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2014, 52, 32–36

New oleanane-type saponin
Perkin-Elmer Clarus 500 GC-MS system and gas chromatograph
interfaced to a mass spectrometer (GC-MS) instrument employing
the following conditions: capillary column EQUITYTM-1 (30 m× 0.25
mm × 0.25 μm, Supelco Dex phase, composed of 25% 2,3-di-O-
acetyl-6-0-TBDMS-β-cyclodextrin embedded in SPB-20 poly(20%
phenyl, 80% dimethylsiloxane). Column temperature, 230 °C;
injection temperature, 250 °C; carrier N2 gas; detection in Electronic
impact (EI) mode, ionization potential, 70 eV; ion-source tempera-
ture, 280 °C.
Figure 2. Selected heteronuclear multiple-bond correlation correlations
for compound 1.
Plant material
The whole plant of L. leptocarpa was collected in Foto village
(Menoua Division, Western region of Cameroon), in April 2011.
Authentication was performed by Victor Nana, a botanist of
the Cameroon National Herbarium, Yaoundé, where a voucher
specimen (N° 38782/HNC) has been deposited.
Extraction and isolation
The dried whole plant of L. leptocarpa (4 kg) was extracted with
MeOH at room temperature for 3 days, and the extract was con-
centrated to dryness under reduced pressure. Part of residue
obtained (97 g) was subjected to silica gel column chromatogra-
phy and eluted with hexane containing increasing EtOAc, then
with EtOAc containing increasing MeOH. Seven fractions were
obtained: A (hexane), B (hexane–EtOAc : 9–1), C (hexane–EtOAc :
8–2), D (hexane–EtOAc : 6–4), E (hexane–EtOAc : 4–6), F (hexane–
EtOAc: 9–1), and G (EtOAc–MeOH : 1–1). Fraction G was suspended
in water and successively extracted with ethyl acetate and n-butanol
to obtain after evaporation of solvent 5.20 g and 13.76 g, respec-
tively. Part of butanol-solute extracts (13.76 g) was eluted with EtOAc
containing increasing MeOH (10%, 20%, 30%, 40%, and 50%).
Five under fractions were obtained G1, G2, G3, G4, and G5.
Fraction G5 was purified over silica gel column eluted with
the mixture EtOAc–MeOH–H2O (7–2–1) to give the compound
1 (20 mg). Fraction G2 was purified over silica gel column
eluted with the mixture EtOAc–MeOH (85–15) to give the
compound 2 (white amorphous powder, 13 mg). Fractions G3
and G4 were combined and purified over silica gel column
eluted with the mixture EtOAc–MeOH–H2O 8–1–1) to give the
compound 3 (yellow amorphous powder, 30 mg).
Figure 3. Selected rotating frame nuclear overhauser effect spectros-
copy correlations observed for the aglycone moiety of compound 1.
(1 → 2)-[α-L-arabinopyranosyl(1→ 3)]-4-O-(3′-hydroxybutanoyloxy-
3- hydroxybutanoyloxy)-β-D-fucopyranosyl zanhic acid named
leptocarposide.
Very similar structure was previously isolated from Filicium
decipiens with two nilic acids linked each other in position 4 of
the fucose moiety.[9] Saponins with two 3-hydroxy butanoic acids
linked to the fucose moiety in position 4 were also previously
isolated from Solidago virgaurea but with polygalacic acid as
genin.[13,14] To our knowledge, this is the first report of zanhic
acid saponin esterified by 3-hydroxybutanoic acid. The antioxi-
dant activities of compounds 1 and 2 were studied using DPPH
radical scavenging assay, and only orientin (2) showed radical
scavenging activity (SC50 = 0.038 mM) as observed previously.[5]
Materials and Methods
General
NMR data
The melting points were recorded with a Reichert microscope
(Reichert Technologies, Depew, New York USA) and are uncorrected.
IR spectra were recorded with a Shimadzu FT-IR-8400S (Shimadzu,
The NMR spectra were recorded at 298 K using a BRUKER Avance
DRX 600 spectrometer (Bruker, Wissembourg, France) using
5 mm CPTCI 1H/13C/15 N/D Zgrd operating at 600 MHz for 1H
and 150 MHz for 13C. 1D and 2D NMR experiments (COSY, TOCSY,
ROESY, HSQC-Jmod, and HMBC) were performed using standard
Bruker pulse programs (XWinNMR version 2.1).
1
France) spectrophotometer. H (600MHz) and 13C (150MHz) NMR
spectra were recorded on a Bruker Avance III 600 spectrometer
(Bruker, Wissembourg, France) equipped with a cryoplatform using
CD3OD with TMS as the internal standard. TOF-ESIMS and HR-TOF-
ESI experiments were performed using a Micromass Q-TOF micro in-
strument (Manchester, UK) with an electrospray source. The samples
were introduced by direct infusion in a solution of MeOH at a rate of
5μl minÀ1. The optical rotations were measured on a Bellingham &
Stanley ADP 220 polarimeter (Bellingham + Stanley Ltd, United King-
dom). Column chromatography was run on Merck silica gel (VWR,
France ) 60 (70–230 mesh) and gel permeation on Sephadex LH-
20 (VWR, France ), while TLC was carried out on silica gel GF254
pre-coated plates with detection accomplished by spraying with
50% H2SO4 followed by heating at 100 °C or by visualizing with a
UV lamp at 254 and 365 nm. GC-MS analysis was carried out on a
Sample was dissolved in CD3OD. Chemical shifts were
referenced to the solvent signal (δ (CD3OD) = 3.33 ppm for 1H
NMR and [δ (CD3OD) = 49.0 ppm for 13C NMR].
1H and 13C 1D spectra were acquired with relaxation delay
d1 = 1 s, 32 K data points and 90° pulses were, respectively,
9.32 μs at 3 dB and 12.0 μs at À0.5 dB for 1H and 13C. The number
of scans is 16 for 1H and 6144 for 13C.
2D experiments were recorded with the following parameters:
1H-1H gradient COSY spectrum: relaxation delay d1 = 1 s; 90°
1
pulse, 9.32 μs for H at 3 dB; number of scans 2; 2 K data points
in t2; spectral width 6.0ppm in both dimensions; 512 experiments
Magn. Reson. Chem. 2014, 52, 32–36
Copyright © 2013 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/mrc

F. D. Mabou et al.
in t1; zero-filling up to 1 K in t1; apodization with pure sine-bell in
both dimensions prior to double Fourier transformation.
was extracted with EtOAc (2 ml × 3). Combined remaining aque-
ous layer containing monosaccharides was concentrated under
reduced pressure to dryness, to give a residue. The residue was
dissolved in pyridine (0.1 ml), to which 0.1 M L-cysteine methyl
ester hydrochloride in pyridine (0.20 ml) was added. The mixture
was heated at 60° for 2 h, dried in vacuo, and trimethylsilylated
with hexamethyldisilazane-trimethylchlorosilane (0.2 ml) at 60°
for 2 h. The mixture was partitioned between n-hexane and H2O
(0.4 ml each), and the n-hexane extract was subjected to GC-MS
analysis. Absolute configurations of monosoccharides in compound
1 were determined as D-glucose, D-fucose, D-xylose, L-arabinose
and, L-rhamnose by comparison of the retention times of their
derivatives with those of literature[17] and with those of D-glucose,
D-fucose, D-xylose, L-arabinose, and L-rhamnose derivatives prepared
in the same way, which showed retention times of 10.32, 7.76, 6.32,
6.37, and 7.53.
Total correlation spectroscopy spectrum: relaxation delay
d1 = 1 s; 90° pulse, 9.32μs for 1H at 3 dB; number of scans 8; spin lock
time, 200 ms using 90° pulse of 30 μs at 14.21 dB; 2 K data points in
t2; spectral width 6.0 ppm in both dimensions; 512 experiments
in t1; apodization with sine-bell (processing parameter SSB = 3) in
both dimensions; zero-filling with linear prediction up to 1 K.
Rotating frame NOE spectroscopy using Bruker library pulse
sequence “roesyetgp”: relaxation delay d1 = 1 s; 90° pulse,
9.32 μs for 1H at 3 dB; number of scans 8; ROESY spin lock
pulse of 500 ms at 27.21 dB; 2 K data points in t2; spectral width
6.0 ppm in both dimensions; 512 experiments in t1; apodization
with squared cosine-bell in both dimensions; zero-filling up to 1
and 4 K, respectively, in t1 and t2.
Heteronuclear single-quantum correlation-J modulated using Bruker
library pulse sequence “hsqcedetgpsisp2.2”: relaxation delay d1 = 1 s;
coupling constant 1 J(1H-13C) = 145 Hz for d4 = 1.72 ms; 90° pulse,
Leptocarposide (1)
1
9.32 μs at 3 dB for H, 12 μs at À0.5 dB for 13C with gradient ratio
White amorphous powder; 1H and 13C, see Table 1; [α]2D0À23°
(c 0.06, CH3OH) HRESIMS (positive-ion mode) m/z: 1431.6395
[M+ Na]+ (calcd. for C66H104O32Na: 1431.6408), 703.3499 [M + Na-
ester chain]+; ESIMS (positive-ion mode) m/z: 1431.8 [M + Na]+.
751.3 [ester chain + Na]+, 647.3 [ester chain+ Na-C4H8O3]+; ESIMSn
(positive-ion mode): ESIMS1 (1431.8) m/z: 751.4 [ester chain + Na]+,
647.4 [ester chain+ Na-C4H8O3]+, 473.3 [ester chain+ Na-C5H8O4-
C6H10O4]+, ESIMS2 (751.3) m/z: 647.3 [ester chain + Na-C4H8O3]+,
619.3 [ester chain+ Na-C5H8O4]+, 515.3 [ester chain+ Na-C4H8O3-
C5H8O4]+, 473.3 [ester chain + Na-C5H8O4-C6H10O4]+, 369.2 [ester
chain+ Na-C5H8O4-C6H10O4-C4H8O3]+
GPZ1:GPZ2:GPZ3:GPZ4=80 :20:11:À5; 2 K data points in t2;
spectral width 6 ppm in F2 and 160 ppm in F1; number of scans 2;
512 experiments in t1; apodization with pure cosine-bell in both
dimensions; zero-filling with linear prediction up to 1 K.
Heteronuclear multiple-bond correlation using Bruker library
pulse sequence “hmbcetgpl3nd”: relaxation delay d1= 1 s; same
pulse calibration as HSQC; delay of the low-pass J-filter
d2 = 3.44ms (corresponding to 1 J(1H-13C)= 145 Hz); delay for evo-
lution of long-range coupling d6 = 62.5 ms; gradient ratio GPZ1:
GPZ3: GPZ4: GPZ5 : GPZ6= 80:14 : À8 : À4 : À2; 2 K data points in
t2; spectral width 6.0ppm in F2 and 220 ppm in F1; number of
scans 14; 512 experiments in t1; apodization with pure sine-bell in
both dimensions; zero-filling with linear prediction up to 1 K.
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