Batatins VIII-XI, glycolipid ester-type dimers from ipomoea batatas

Article abstract of DOI:10.1021/np500523w

Sweet potato (Ipomoea batatas) is native to the tropics of Central and South America, where many varieties have been consumed for more that 5000 years. In developing countries, this crop is a recognized effective food for fighting malnutrition. Purification of the minor lipophilic glicolipids found in the n-hexane-soluble resin glycosides from the white-skinned variety was performed by preparative-scale recycling HPLC. Application of column overload, peak shaving, heart cutting, and recycling techniques permitted the purification of four new oligosaccharide ester-type dimer derivatives of jalapinolic acid, batatins VIII-XI (1-4). The structural characterization of these complex lipo-oligosaccharides was performed through NMR spectroscopy and MS, indicating that batatins VIII-XI (1-4) possess an oligomeric structure consisting of two pentasaccharide units of the known simonic acid B.

Full text of DOI:10.1021/np500523w

Article
pubs.acs.org/jnp
Batatins VIII−XI, Glycolipid Ester-Type Dimers from Ipomoea batatas
Daniel Rosas-Ramírez and Rogelio Pereda-Miranda*
́ ́
Facultad de Química, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Mexico City 04510 DF, Mexico
*
S Supporting Information
ABSTRACT: Sweet potato (Ipomoea batatas) is native to the tropics
of Central and South America, where many varieties have been
consumed for more that 5000 years. In developing countries, this crop
is a recognized effective food for fighting malnutrition. Purification of
the minor lipophilic glicolipids found in the n-hexane-soluble resin
glycosides from the white-skinned variety was performed by
preparative-scale recycling HPLC. Application of column overload,
peak shaving, heart cutting, and recycling techniques permitted the
purification of four new oligosaccharide ester-type dimer derivatives of
jalapinolic acid, batatins VIII−XI (1−4). The structural characterization
of these complex lipo-oligosaccharides was performed through NMR
spectroscopy and MS, indicating that batatins VIII−XI (1−4) possess
an oligomeric structure consisting of two pentasaccharide units of the
known simonic acid B.
4
weet potato (Ipomoea batatas L.) belongs to the
Convolvulaceae (morning glory family) and is native to
which represent ester-type dimers of acylated tetra- and
5
,6
S
pentasaccharides. Several biological activities evident of the
crude extracts of the Mexican Ipomoea species with purgative
activity have been attributed to their resin glycoside contents.
This class of secondary metabolites has been evaluated in
several bioassays, and the results suggest that they represent
potential efflux pump inhibitors for overcoming the multi-drug-
Central America. It is a creeping plant with edible tuberous
roots, which have been much appreciated since pre-Hispanic
times in Mexico and now play an important role as a basic diet
staple worldwide. The presence of secretory cells of resin
glycosides in foliar tissues and roots is a distinctive anatomical
characteristic of members of the morning glory family. From
the ethnomedicinal point of view, two prominent examples of
these bindweeds are I. purga (officinal jalap or jalap root) and I.
orizabensis (Mexican scammony or false jalap), which are the
New World succedanea of the scammony or Syrian purging
bindweed (Convolvulus scammonia) that had been used since
8
9
resistant phenotype in Gram-positive and -negative bacteria
10
as well as in human cancer cells.
The content of resin glycosides in sweet potato is responsible
for its purgative activity. It is safe to eat a half-cup without
cooking, but eating more than that can cause flatulence and
even a drastic purge. It has been reported that the infusions of
leaves and roots are effective for the treatment of leukemia,
1
pre-Christian times in Europe. The resin glycosides are
complex mixtures of an extensive family of secondary
metabolites known as glycolipids or lipo-oligosaccharides and
represent unique metabolites in the plant kingdom confined to
11
anemia, hypertension, diabetes, and hemorrhages. It is noted
1
,2
for its ability to adapt to adverse conditions because of its high
productivity per unit area/time as well as for being a rich source
of vitamins, minerals, antioxidant flavonoids, and dietary fiber
the Convolvulaceae.
They illustrate how nature creates
structural diversity by using simple metabolic building blocks
to generate complex mixtures of bioactive oligosaccharides of
1
12
hydroxylated fatty acids. The sugars are D-glucose and four
essential for optimal health. In this context, the white-skinned
methylpentoses: D-fucose, L-rhamnose, D-quinovose, and D-
xylose. Fatty acids with different lengths esterify the
oligosaccharide core at variable positions. The most significant
chemical feature of these resin glycosides is the macrocyclic
structure formed by the intramolecular lactonization with the
aglycone. Their amphiphilic properties have been useful to
isolate them since they are highly soluble in hexanes or
chloroform. However, successful purification has relied
exclusively on the use of improved recycling HPLC techniques
tuber variety was reinvestigated for expanding the knowledge
on the resin glycoside structural diversity. The present study
describes the procedures used by recycling HPLC, which
allowed for the isolation and purification of four minor n-
hexane-soluble glycolipid ester-type dimers, named batatins
VIII−XI (1−4). Their intact oligomeric structures were
identified by a combination of HRESIMS, FABMS, and
NMR methods.
3
with reversed-phase columns. The most complex resin
glycoside structures that have been isolated are those found
in the Mexican varieties of sweet potato and the jalap root,
Received: June 26, 2014
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
RESULTS AND DISCUSSION
■
The crude n-hexane-soluble extract from the white-skinned
variety of sweet potato was subjected to column chromatog-
raphy to eliminate pigments and nonpolar constituents in order
to concentrate the resin glycoside fraction. This procedure is
not suitable for purification of the individual constituents since
resin glycosides are always present as complex mixtures of
homologues having the same polar oligosaccharide core.
However, chromatographic homogeneity was achieved for the
minor resin glycoside dimers (1−4) through recycling HPLC
in combination with the techniques of column overload, peak
shaving, and heart cutting, which provided maximal resolution
in a short-term analysis through the use of RP-18 stationary
phases. This process of purification was monitored by using a
refractive index detector. The major technical challenge was
finding the most favorable analytical factors (mobile phases and
maximum sample loading) and scaling them into preparative
conditions while retaining the resolution for the recycling
process.
since its anomeric signals were readily distinguishable and used
as a fingerprint for pattern recognition and structural
dereplication, so as to identify the known glycosidic acids.
1
3
Comparison of the melting point, optical rotation, and
C
NMR data with published values for the peracetylated
14c
derivative of simonic acid B methyl ester
confirmed its
structure, which was also identified by HPLC comparison with
an authentic sample, and, therefore, both aglycone and sugar
configurations were confirmed. Consequently, the structure for
the pentasaccharide core of batatins VIII−XI (1−4) was
simonic acid B, (11S)-hydroxyhexadecanoate 11-O-α-L-rham-
nopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)]-O-α-L-
rhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-β-D-
1
3
fucopyranoside, which has been previously found in I. batatas
14
and other members of the genus Ipomoea. The liberated
organic acids were found to be 2-methylpropanoic (iba), 2-
methylbutanoic (mba), n-dodecanoic (dodeca), and cinnamic
(cna) acids for fraction III, and 2-methylbutanoic, n-decanoic
(deca), and n-dodecanoic acids for fraction IV, which were
The main approach for the structure elucidation of the resin
glycosides involves the use of degradative chemical reactions in
combination with spectroscopic and spectrometric methods.
Breaking up the large complex compounds by simple chemical
reactions into smaller, more manageable molecules has been
developed as a result of the difficulties encountered in attempts
7
identified by GC-MS coelution with authentic samples.
The spectrometric analyses of compounds 1−4 were
conducted by HRESIMS and FABMS in the negative ion
detection mode. HRESIMS allowed the identification of the
−
quasi-molecular ion [M − H] at m/z 2472.3990 for batatin
3
−10
to isolate the resin glycoside as intact constituents.
VIII (1; C1 H O ); m/z 2448.3403 for batatin IX (2;
24 216 48
Saponification of the crude material fragments the macrocyclic
lactone and liberates the fatty acids that esterify the
oligosaccharide core, which is then subjected to acid hydrolysis.
Thus, a small portion of the isolated resin glycosides (fractions
C123H204O ); m/z 2444.3975 for batatin X (3; C122H212O );
48 48
and m/z 2500.4503 for batatin XI (4; C126H220O ). FABMS of
48
compounds 1−4 provided readily detectable ions resulting
15
from the characteristic elimination of the esterifying groups.
III and IV) was saponified to liberate the same H O-soluble
Other fragments were produced by the glycosidic cleavage of
the pentasaccharide core with observed peaks at m/z 271, 417,
545, 837, and 983, which are common to resin glycosides
containing simonic acid B (Figures S7, S15, S23, and S31,
2
glycosidic acid that was methylated and further acetylated to
yield the peracetylated derivative of simonic acid B methyl
7
1
3
ester. This product was used to generate a C NMR profile
B
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
3
1
1
1
Supporting Information). The characteristic high-mass ion
Table 1. H (400 MHz, Assignments Based on H− H COSY
−
13
fragment [M/2 − H] was observed in both ionization
techniques, which indicated the rupture of the ester bond in the
FABMS of batatin VIII (1) showed the
M/2 − H] peak at m/z 1235, that of batatins IX (2) and
batatin XI (4) at m/z 1249, and that of batatin X (3) at m/z
221. As an example of the fragmentation pattern of this type of
and TOCSY Experiments) and C (125 MHz, Assignments
Based on HSQC and HMBC Experiments) NMR Data of
Batatin VIII (1) in Pyridine-d (δ in ppm, J in Hz)
4
−6
dimeric structure:
[
5
−
δH
δC
a
position
unit A
unit B
unit A unit B
1
ester dimers, the FABMS of batatins IX (2) and XI (4) could
Fuc-1
4.73 d (7.5)
4.73 d (7.5)
104.3
80.2
73.3
72.9
70.8
17.4
98.7
73.8
69.7
80.0
68.6
19.4
99.1
73.1
79.8
79.3
68.4
18.8
103.6
72.7
73.3
74.8
68.2
17.9
98.7
73.8
69.7
73.6
70.7
19.4
172.9
34.2
104.3
80.2
73.3
72.9
70.8
17.4
104.6
72.5
72.6
80.6
68.6
18.6
99.5
73.2
79.8
79.5
70.8
18.7
103.8
70.5
76.5
74.8
71.0
18.4
104.6
72.5
72.6
73.5
70.7
18.5
173.1
34.3
be used since they were similar to that reported for batatin
2
4.16 dd (8.5, 7.5)
4.07 dd (8.5, 2.5)
3.99 d (1.0)
4.15 dd (8.0, 7.5)
4.08 dd (8.0, 2.5)
3.99 d (1.0)
5
b
VII, and the following peaks were observed (Figures S15 and
3
S31, Supporting Information) at m/z 1251 [unit B,
4
−
−
C H O ] and 1249 [unit A, C H O ] , both produced
5
3.77 dq (6.5, 2.0)
1.51 d (6.5)
3.76 dq (6.5, 1.0)
1.51 d (6.5)
6
3
111 24
63 109 24
from the rupture of the ester bond forming the dimeric
6
−
structure; 1165 [1249 − C H O] , indicating the loss of 84
Rha-1
5.47 d (2.0)
5.59 d (1.0)
5
8
amu from the methylbutanoyl residue; and 1067 [1249 −
C H O] , indicating the loss of 182 amu from the dodecanoyl
2
5.94 dd (3.0, 2.0)
5.01 dd (9.4, 3.0)
4.22 t (9.4)
4.81 bs
−
3
4.48 dd (9.9, 3.2)
4.22 t (9.9)
1
2
22
residue.
4
Structure elucidation of batatins VIII−XI (1−4) was
5
4.44 dq (9.4, 6.0)
1.61 d (6.0)
4.44 dq (9.9, 6.5)
1.62 d (6.5)
accomplished by comparison of their NMR data with those
6
5
reported for the batatins I−II and VII. In order to identify each
Rha′-1
2
6.15 d (2.0)
6.14 d (2.0)
1
constitutive monosaccharide, COSY (Figures S3, S11, S19,
6.00 dd (3.0, 2.0)
4.59 dd (9.0, 3.0)
4.29 t (9.0)
6.00 dd (3.0, 2.0)
4.59 dd (9.5, 3.0)
4.29 t (9.5)
3
and S27, Supporting Information) and TOCSY (Figures S4,
S12, S20, S15, and S28, Supporting Information) experiments
were used to assign chemical shift values, after identifying and
4
5
4.32 dq (9.0, 6.0)
1.65 d (6.0)
4.33 dq (9.5, 6.0)
1.65 d (6.0)
1
6
differentiating each of the H NMR signals (Tables 1−4).
1
13
Rha″-1
2
5.92 d (1.0)
5.92 d (1.2)
Furthermore, 2D H− C NMR experiments, using the HSQC
technique (Figures S5, S13, S21, and S29, Supporting
Information), were used for the assignments of the 13C NMR
signals (Tables 1−4). All four compounds showed the7
following common features: (a) diagnostic anomeric signals
for an oligomeric ester-type dimer consisting of two
pentasaccharide units of simonic acid B; (b) signals attributable
to the nonequivalent protons of the methylene group at C-2 in
the aglycone moiety of unit A, which confirmed their
4.68 dd (3.0, 1.0)
4.48 dd (9.7, 3.0)
5.82 t (9.7)
4.68 dd (3.0, 1.2)
4.48 dd (9.5, 3.0)
5.78 t (9.5)
3
4
5
4.37 dq (9.8, 6.5)
1.39 d (6.5)
4.35 dq (9.5, 6.5)
1.39 d (6.5)
6
Rha‴-1
2
5.48 d (1.0)
5.59 d (1.0)
5.94 dd (3.0, 1.0)
5.01 dd (9.3, 3.0)
4.24 t (9.3)
4.81 bs
3
4.48 dd (9.5, 3.0)
4.21 t (9.5)
4
,5b
4
macrocyclic lactone-type
structures; and (c) signals for
5
4.29 dq (9.3, 6.0)
1.60 d (6.0)
4.44 dq (9.5, 6.0)
1.61 d (6.0)
four short-chain fatty acid residues esterifying the oligosacchar-
4
,5b
6
ide core: H-2 of these moieties
was used as a diagnostic
Jal-1
resonance centered at δ 2.62 (1H, sept) for the methyl-
propanoyl group, 2.3−2.5 (1H, tq) for the methylbutanoyl
groups, and 2.3−2.5 (2H, triplet-like signal) for the n-decanoyl
2
a
2.24 ddd (12.0, 8.0, 2.24 ddd (12.0, 8.0,
.5) 3.5)
3
2
b
2.39 ddd (12.0, 8.0, 2.39 ddd (12.0, 8.0,
3.5)
(deca) and n-dodecanoyl residues. These postulations were
3.5)
verified by the usual deshielding of the proton and carbon
bearing the ester group due to acylation (Tables 1−4). The
main differences among the new batatins are (a) the position
for the macrolactonization of unit A; (b) the location for the
ester-type linkage established between both pentasaccharide
moieties; and (c) the type and position of the esterifying
residues. The observed long-range correlations ( JCH) in the
HMBC spectrum were used to support the structural
differences between 1−4 (Figures S6, S14, S22, and S30,
Supporting Information). For compound 1, the following key
correlations were observed for the pentasaccharide unit A
11
16
Iba-1
2
3.85 m
3.85 m
82.3
14.3
175.4
41.4
16.8
11.8
172.9
34.5
14.3
82.3
14.3
176.3
41.5
17.0
11.8
173.1
34.2
14.3
0.88 t (7.0)
0.89 t (7.0)
2.63 sept (7.0)
1.19 d (7.0)
1.16 d (7.0)
2.50 tq (7.0, 7.0)
1.20 d (7.0)
3
3′
0.93 t (7.5)
2
,3
Dodeca-1
2
2.31 dd (16.0, 7.5)
0.86 t (7.0)
2.25−2.40*
0.87 t (7.0)
12
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexade-
canoyl; Iba, 2-methylpropanoyl; Dodeca, dodecanoyl. Chemical shifts
(
Figure 1): between H-2 (δ 5.94, Rha) and the carbonyl
marked with an asterisk (*) indicate overlapped signals.
4,5b
resonance at δc 172.9, assigned to the lactone functionality
due to its J coupling with the diastereotopic C-2 methylene
2
septet-like spin pattern of the proton at δ 2.62. The same
HMBC connectivities were observed for the pentassacharide
unit B (Figure 1) since its acylation was similar to unit A: H-2
(δ 6.00, Rha′) and the carbonyl carbon of one dodecanoyl
residue at δ 173.1; and H-4 (δ 5.78, Rha″) and the carbonyl
resonance at δ 176.3, assigned to a second 2-methylpropanoyl
residue. Batatin IX (2) was asymmetrically substituted as
identified by FABMS. The observed HMBC correlations for its
macrocyclic unit A were similar to those described for
CH
protons (δ 2.24−2.39 and 2.22−2.38); H-2 of the terminal
H
branched rhamnose (δ 5.94, Rha‴) and the carbonyl group of
H
the ester for unit B (δ 173.1; unit B); H-2 (δ 6.00, Rha′) and
C
the carbonyl carbon for a long-chain fatty acid at δ 172.9,
assigned to a dodecanoyl moiety by the FABMS and ESI
fragmentation pattern as described above; and finally, H-4 (δ
5
.82, Rha″) and the carbonyl resonance at δ 175.4, assigned to
2
one of the 2-methylpropanoyl residues due to its J with the
CH
C
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
1
1
1
1
Table 2. H (500 MHz, Assignments Based on H− H COSY
Table 3. H (400 MHz, Assignments Based on H− H COSY
1
3
13
and TOCSY Experiments) and C (125 MHz, Assignments
Based on HSQC and HMBC Experiments) NMR Data of
and TOCSY Experiments) and C (125 MHz, Assignments
Based on HSQC and HMBC Experiments) NMR Data of
Batatin IX (2) in Pyridine-d (δ in ppm, J in Hz)
Batatin X (3) in Pyridine-d (δ in ppm, J in Hz)
5
5
δH
δC
δH
δC
a
a
position
unit A
4.77 d (7.5)
unit B
unit A unit B
position
unit A
4.83 d (7.8)
unit B
unit A unit B
Fuc-1
4.73 d (7.5)
104.3
80.2
73.3
72.9
70.8
17.4
98.7
73.8
69.7
80.0
68.6
19.4
99.1
73.1
79.8
79.3
68.4
18.8
103.6
72.7
73.3
74.8
68.2
17.9
98.7
73.8
69.7
73.6
70.7
19.4
172.9
34.2
104.3
80.2
73.3
72.9
70.8
17.4
104.6
72.5
72.6
80.6
68.6
18.6
99.5
73.2
79.8
79.5
70.8
18.7
103.8
70.5
76.5
74.8
71.0
18.4
104.6
72.5
72.6
73.5
70.7
18.5
173.1
34.3
Fuc-1
4.75 d (7.8)
100.2
79.1
76.7
73.4
70.8
17.2
98.5
69.8
77.7
78.0
68.6
17.7
97.7
79.8
72.8
79.3
68.4
17.4
102.2
72.7
71.3
74.1
68.2
17.3
102.0
75.6
69.7
73.2
70.7
18.0
173.4
33.0
102.8
73.3
76.6
73.4
70.8
17.2
102.3
71.4
69.6
75.3
69.7
17.7
97.7
79.8
72.8
79.5
70.8
17.4
103.2
70.5
71.3
73.4
71.0
17.3
97.3
72.5
72.6
73.2
70.7
18.0
171.6
33.0
2
4.16 dd (8.5, 7.5)
4.06 dd (8.5, 2.5)
3.99 d (1.0)
4.15 dd (8.0, 7.5)
4.10 dd (8.0, 2.5)
3.99 d (1.0)
2
4.50 dd (8.5, 7.8)
4.18 dd (8.5, 2.5)
3.94 d (1.0)
4.16 dd (8.5, 7.8)
4.18 dd (8.5, 2.5)
3.94 d (1.0)
3
3
4
4
5
3.77 dq (6.5, 2.0)
1.52 d (6.5)
3.79 dq (6.5, 1.0)
1.51 d (6.5)
5
3.77 dq (6.5, 2.0)
1.52 d (6.5)
3.77 dq (6.5, 2.0)
1.52 d (6.5)
6
6
Rha-1
5.49 d (2.0)
5.59 d (1.0)
Rha-1
6.36 bs
5.95 bs
2
5.95 dd (3.0, 2.0)
4.99 dd (9.4, 3.0)
4.18 t (9.4)
4.81 bs
2
5.33 bs
4.71 dd (3.0)
4.51 dd (9.4, 3.0)
4.22 t (9.4)
3
4.49 dd (9.9, 3.2)
4.21 t (9.9)
3
5.63 dd (9.4, 3.0)
4.64 t (9.4)
4
4
5
4.43 dq (9.4, 6.0)
1.61 d (6.0)
4.18 dq (9.9, 6.5)
1.59 d (6.5)
5
5.02 dq (9.4, 6.0)
1.61 d (6.0)
4.31 dq (9.4, 6.0)
1.61 d (6.0)
6
6
Rha′-1
2
6.16 d (2.0)
6.03 d (2.0)
Rha′-1
2
6.18 d (1.6)
5.67 d (2.0)
6.00 dd (3.0, 2.0)
4.59 dd (9.0, 3.0)
4.29 t (9.0)
4.81 bs
6.03 dd (2.0, 1.6)
4.59 dd (9.0, 3.0)
4.29 t (9.0)
5.98 dd (3.0, 2.0)
4.50 dd (9.0, 3.0)
4.29 t (9.0)
3
4.61 dd (9.5, 3.0)
4.30 t (9.5)
3
4
4
5
4.33 dq (9.0, 6.0)
1.65 d (6.0)
4.31 dq (9.5, 6.0)
1.60 d (6.0)
5
4.33 dq (9.0, 6.0)
1.66 d (6.0)
4.36 dq (9.0, 6.0)
1.66 d (6.0)
6
6
Rha″-1
2
5.92 d (1.0)
5.97 d (1.2)
Rha″-1
5.93 d (1.0)
5.65 d (1.0)
4.67 dd (3.0, 1.0)
4.48 dd (9.7, 3.0)
5.78 t (9.7)
4.97 dd (3.0, 1.2)
5.93 dd (9.5, 3.0)
5.96 t (9.5)
2
3
4
5
6
4.67 dd (3.0, 1.0)
4.48 dd (9.7, 3.0)
5.81 bs
4.67 dd (3.0, 1.0)
4.39 dd (9.7, 3.0)
5.81 bs
3
4
5
4.36 dq (9.8, 6.5)
1.39 d (6.5)
4.51 dq (9.5, 6.5)
1.72 d (6.5)
4.36 dq (9.8, 6.0)
1.40 d (6.5)
4.36 dq (9.8, 6.0)
1.40 d (6.5)
6
Rha‴-1
2
5.48 d (1.0)
5.69 d (1.0)
Rha‴-1 5.95 d (1.0)
2
5.50 d (1.4)
5.94 dd (3.0, 1.0)
5.00 dd (9.3, 3.0)
4.18 t (9.3)
4.81 bs
4.75 dd (3.0, 1.0)
4.75 dd (3.0, 1.0)
4.48 dd (9.3, 3.0)
4.20 t (9.3)
3
4.38 dd (9.5, 3.0)
4.19 t (9.5)
3
4.39 dd (9.3, 3.0)
5.81 t (9.3)
4
4
5
4.45 dq (9.3, 6.0)
1.61 d (6.0)
4.21 dq (9.5, 6.0)
1.55 d (6.0)
5
4.29 dq (9.3, 6.0)
1.60 d (6.0)
4.29 dq (9.3, 6.0)
1.60 d (6.0)
6
6
Jal-1
Jal-1
2a
2
2
1
1
a
2.22 ddd (12.0, 8.0, 3.5) 2.25*
2.38 ddd (12.0, 8.0, 3.5) 2.41*
3.85 m
2.20−2.24*
2.95 ddd (12.0, 8.0, 3.5)
2.20−2.24*
b
1
6
2b
3.89 m
82.3
14.3
175.4
41.4
16.8
11.8
82.3
14.3
11
3.87 m
3.87 m
80.9
12.8
174.9
40.2
16.4
10.3
171.5
32.8
12.8
80.9
13.0
174.9
40.2
16.4
10.3
171.5
32.8
12.8
0.88 t (7.0)
0.88 t (7.0)
16
0.88 t (7.0)
0.88 t (7.0)
Mba-1
2
176.3
41.5
Mba-1
2
2.37 tq (7.0, 7.0)
1.06 d (7.0)
2.50 tq (7.0, 7.0)
1.20 d (7.0)
2.50 tq (7.0, 7.0)
1.21 d (7.0)
2.50 tq (7.0, 7.0)
1.21 d (7.0)
2
3
-Me
-Me
17.0
2-Me
3-Me
Deca-1
2
0.84 t (7.5)
0.93 t (7.5)
11.8
0.96 t (7.5)
0.96 t (7.5)
Cna-1
166.9
118.4
128.7
2
3
6.67 d (15.9)
7.79 d (15.7)
2.26−2.35*
0.86 t (7.0)
2.26−2.35*
0.86 t (7.0)
12
a
Dodeca-1
2
172.9
34.5
14.3
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexade-
2.31 dd (16.0, 7.5)
0.88 t (7.0)
canoyl; Mba, 2-methylbutanoyl; Deca, decanoyl. Chemical shifts
marked with an asterisk (*) indicate overlapped signals.
1
2
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexade-
canoyl; Mba, 2-methylbutanoyl; Cna, trans-cinnamoyl; Dodeca,
dodecanoyl. Chemical shifts marked with an asterisk (*) indicate
overlapped signals.
not esterified in unit B of 2 (δ 4.8); a trans-cinnamoyl residue
(
δC‑1 166.9) was located as the acylating group at C-3 (δ 5.93,
H
unit B) of the terminal rhamnose unit (Rha″); and the
correlation between H-4 of the terminal branched rhamnose
(δH 5.96, Rha″) and the carbonyl resonance at δ 176.3 was
assigned to a 2-methylbutanoyl residue. Batatins X (3) and XI
(4) showed the same symmetrical pattern of esterification
(Figure 1), and the ester-type linkage between the acyclic unit
B at the macrocyclic unit A was established through the
compound 1 with the only difference involving the interaction
between H-4 (δ 5.78, Rha″) and the carbonyl resonance at δ
2
1
75.4, assigned to the 2-methylbutanoyl residues due to its J
CH
with the sextet-like spin pattern of the proton at δ 2.37.
Pentasaccharide unit B displayed major differences in its
acylation pattern: C-2 of the second rhamnose unit (Rha′) was
carbonyl group for the ester (δ 173.4, unit B) and H-4 of the
C
D
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
1
Table 4. H (500 MHz, Assignments Based on H− H COSY
the signal centered at δ 2.50 identified the presence of a 2-
1
3
and TOCSY Experiments) and C (125 MHz, Assignments
Based on HSQC and HMBC Experiments) NMR Data of
Batatin XI (4) in Pyridine-d (δ in ppm, J in Hz)
methylbutanoyl moiety, which was placed at C-4 in the terminal
3
rhamnose unit (Rha″) of both units A and B through the J
CH
interaction of the same carbonyl carbon and the triplet-like
signal at δ 5.81.
5
δH
δC
A large number of glycolipid ester-type dimer congeners can
occur in the same morning glory species, as they represent
multiple variations caused by acylation with fatty acids of
different lengths. Prior to this investigation, 16 ester-type
dimers have been described from four different convolvula-
ceous species. Merremin, the first example of an ester-type
heterodimer which consists of two units of the pentasaccharide
operculinic acid A, was obtained from the roots of Merremia
a
position
unit A
4.83 d (8.2)
unit B
unit A unit B
Fuc-1
4.76 d (7.4)
100.2
79.1
76.7
73.4
70.8
17.2
98.5
69.8
77.7
78.0
68.6
17.7
97.7
79.8
72.8
79.3
68.4
17.4
102.2
72.7
71.3
74.1
68.2
17.3
102.0
75.6
69.7
73.2
70.7
18.0
173.4
33.0
102.8
73.3
76.6
73.4
70.8
17.2
102.3
71.4
69.6
75.3
69.7
17.7
97.7
79.8
72.8
79.5
70.8
17.4
103.2
70.5
71.3
73.4
71.0
17.3
97.3
72.5
72.6
73.2
70.7
18.0
171.6
33.0
2
4.50 dd (8.5, 8.2)
4.18 dd (8.5, 2.5)
3.94 d (1.0)
4.16 dd (8.5, 7.4)
4.18 dd (8.5, 2.5)
3.94 d (1.0)
3
4
5
3.77 dq (6.5, 2.0)
1.52 d (6.5)
3.77 dq (6.5, 2.0)
1.52 d (6.5)
6
16
Rha-1
6.36 bs
5.95 bs
hungaiensis. Tricolorins H−J, ester-type dimers consisting of
2
5.34 dd (3.0, 2.0)
5.63 dd (9.4, 3.0)
4.64 t (9.4)
4.71 dd (3.0, 2.0)
4.51 dd (9.4, 3.0)
4.22 t (9.4)
two trisaccharide units of tricoloric acid C, were purified from I.
17
3
tricolor. Tyrianthins A and B, acylated ester-type hetero-
4
tetraglycosides consisting of two scammonic acid A units, were
18
6
5
5.02 dq (9.4, 6.0)
1.61 d (6.0)
4.31 dq (9.4, 6.0)
1.61 d (6.0)
isolated from I. orizabensis. Purgins I−III, ester-type dimers
formed by two operculinic acid B moieties, were purified from
aerial parts of I. purga. From sweet potato, seven intact
glycolipid ester-type dimers have been reported: batatins I−
6
Rha′-1
2
6.19 bs
5.68 bs
6.04 bs
5.98 bs
4
,5a
3
4.59 dd (9.0, 2.7)
4.29 t (9.0)
4.50 dd (9.0, 2.6)
4.29 t (9.0)
IV were isolated from the n-hexane-soluble extract prepared
4
from a Mexican white-skinned staple-type cultivar. Finally,
5
4.33 dq (9.0, 6.0)
1.66 d (6.0)
4.36 dq (9.0, 6.0)
1.66 d (6.0)
three CHCl -soluble dimers have been described: batatins V
3
4
6
and VI were isolated from the extract of a purple-skinned
5b
Rha″-1
5.93 bs
5.65 bs
cultivar, while batatin VII was isolated from a yellow-skinned
staple-type cultivar. Simonic acid B is present in batatins I, II,
and VII, while operculinic acid C is the tetrasaccharide core in
batatins III−VI. Thus, the glycolipid ester-type dimers are
formed by the linkage of the major oligosaccharide cores
present in the lipophilic resin glycoside mixtures of sweet
2
3
4
5
6
4.67 dd (3.0, 1.0)
4.48 dd (9.7, 3.0)
5.81 bs
4.67 dd (3.0, 1.0)
4.39 dd (9.7, 3.0)
5.81 bs
4.36 dq (9.8, 6.0)
1.40 d (6.5)
4.36 dq (9.8, 6.0)
1.40 d (6.5)
15
Rha‴-1 5.95 bs
5.50 bs
potato. They were slightly different from each other in the
degree and nature of their acylating residues; for this reason,
these amphipathic compounds are difficult to isolate and purify
in sufficient quantities for structural elucidation.
2
4.75 dd (3.0, 1.0)
4.75 dd (3.0, 1.0)
4.48 dd (9.3, 3.0)
4.20 t (9.3)
3
4.39 dd (9.3, 3.0)
5.81 bs
4
5
4.29 dq (9.3, 6.0)
1.60 d (6.0)
4.29 dq (9.3, 6.0)
1.60 d (6.0)
6
EXPERIMENTAL SECTION
Jal-1
■
2
2
1
1
a
2.20−2.24*
2.95 ddd (12.0, 8.0, 3.5)
2.20−2.24*
General Experimental Procedures. Melting points were
determined on a Fisher-Johns apparatus and are uncorrected. Optical
rotations were measured with a PerkinElmer model 241 polarimeter.
b
1
6
3.87 m
3.87 m
80.9
12.8
174.9
40.2
16.4
10.3
171.5
32.8
12.8
80.9
13.0
174.9
40.2
16.4
10.3
171.5
32.8
12.8
1
13
H (500 MHz) and C (125.7 MHz) NMR experiments were
0.88 t (7.0)
0.88 t (7.0)
1
conducted on a Bruker DMX-500 instrument, while H (400 MHz)
NMR measurements were carried out on a Varian VXL instrument.
The instrumentation used for HPLC analysis consisted of a Waters
Mba-1
2
2.50 tq (7.0, 7.0)
1.21 d (7.0)
2.50 tq (7.0, 7.0)
1.21 d (7.0)
2
3
-Me
-Me
(Millipore Corp., Waters Chromatography Division, Milford, MA,
0.96 t (7.5)
0.96 t (7.5)
USA) 600 E multisolvent delivery system equipped with a Waters 410
refractive index detector. The sensitivity setting of the refractometer
was increased from 8 N to 64 N to facilitate the detection of all minor
impurities. Control of the equipment, data acquisition, processing, and
management of chromatographic information were performed by the
Waters Millennium 2000 software program. GC-MS was performed on
a Hewlett-Packard 5890-II instrument coupled to a JEOL SX-102A
Deca-1
2
2.26−2.35*
0.86 t (7.0)
2.26−2.35*
0.86 t (7.0)
1
2
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexade-
canoyl; Mba, 2-methylbutanoyl; Dodeca, dodecanoyl. Chemical shifts
marked with an asterisk (*) indicate overlapped signals.
7
spectrometer. GC conditions: HP-5MS (5% phenyl)-methylpolysi-
loxane column (30 m × 0.25 mm, Agilent Technologies, Santa Clara,
terminal branched rhamnose (Rha‴, δ 5.81) of unit A. The
CA, USA), film thickness = 0.25 μm; He, linear velocity = 30 cm/s; 50
H
°
C isothermal for 3 min, linear gradient to 300 °C at 20 °C/min; final
carbonyl resonance at δc 173.4 was assigned to the lactone
temperature hold = 10 min. MS conditions: ionization energy = 70 eV;
ion source temperature = 280 °C; interface temperature = 300 °C;
scan speed = 2 scans/s; mass range = 33−880 amu. Negative ion
FABMS experiments were performed on a JEOL SX-102A
spectrometer and recorded using a matrix of triethanolamine. High-
resolution negative ion ESIMS experiments were performed on a
Bruker MicrOTOF-Q instrument. CC was performed on silica gel 60
(0.063−0.200 mm, Merck). TLC was carried out on precoated
Macherey-Nagel silica gel/UV254 plates of 0.25 thickness, and spots
functionality at unit A, and the macrolactonization site at C-3 of
the first rhamnose unit (Rha) was established by the JCH
correlation between this carbonyl carbon and the proton at δH
3
5
.63. C-2 of the third saccharide (Rha′) in both pentasaccharide
units A (δ 6.03) and B (δ 5.98) was esterified by a fatty acid (C-
, δ 171.5), decanoyl for 3 and dodecanoyl for 4, as confirmed
1
C
2
by FABMS data. The observed J correlation between the
CH
carbonyl resonance at δ 174.9 and the sextet-like spin pattern of
E
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Key HMBC correlations (3JCH) for the sites of esterification of compounds 1 and 3.
3
were visualized by spraying with 3% CeSO in 2 N H SO followed by
mode to achieve total homogeneity after 10−20 consecutive cycles
employing the same isocratic elution. These techniques affored
compounds 1 (13 mg) and 2 (5.2 mg). For fraction IV, the elution
4
2
4
heating.
Plant Material. The tuberous roots of the white-skinned cultivar of
I. batatas were collected in plantations in San Nicolas, Salvatierra,
́
was isocratic with MeOH−CH CN (3:2) using a flow rate of 8.16
3
Guanajuato, Mexico, in 2010. The plant material was identified by Dr.
Robert Bye. A voucher specimen (R. Bye FB 1815) was deposited in
the Ethnobotanical Collection of the National Herbarium (MEXUM),
mL/min. Eluates with a t of 25.7 and 29.5 min were collected by the
R
technique of heart cutting and independently reinjected in the
3
recycling mode. Suitable chromatographic conditions consisted of an
́
Instituto de Biologia UNAM.
analytical RP-18 column (4.6 × 250 mm, 5 μM); flow 0.5 mL/min;
Extraction and Isolation. The powdered dry roots of the white-
skinned cultivar (1.0 kg) were extracted by maceration at room
temperature with n-hexane (8 L) to give, after removal of the solvent, a
syrup (7.5 g, 0.75% on a dry weight basis). The crude extract was
subjected to silica gel column chromatography (150 g) using gradients
of CH Cl in n-hexane, Me CO in CH Cl , and MeOH. The process
mobile phase MeOH. This analysis and the techniques of peak shaving
3
and heart cutting affored pure compounds 3 (10.6 mg) and 4 (8.4
mg), respectively. All pure compounds 1−4 were recrystallized in
MeOH. The total yield of pure resin glycosides from fraction III was
−4
1.82 × 10 % on a dry weight basis, and that from fraction IV was 1.9
−3
× 10 % on a dry weight basis.
2
2
2
2
2
was monitored by TLC (CHCl −MeOH−H O, 6:4:1), and a total of
Batatin VIII (1): colorless microcrystals; mp 115−120 °C; [α] −19
3
2
D
1
13
3
6 fractions (150 mL each) were collected and combined in eight resin
(c 0.1 MeOH); H and C NMR, see Table 1; negative FABMS m/z
−
−
glycoside-containing fractions with an R 0.5 (I−VIII eluted with
1235 [unit A, C H O ] , 1053 [1235 − C H O] , 965 [1053 −
f
63 109 24
12 22
−
−
CH Cl −Me CO−MeOH, 6:3:1). All fractions were partially purified
C H O − H O] , 819 [965 − C H O ] , 691, 545, 417; ESIMS m/z
2
2
2
4
7
2
6
10
4
H
−
by passing through activated charcoal to eliminate pigmented residues.
Previoulsy isolated major resin glycosides from this plant species,
2472.3990 [M − H] (calcd for C
O , 2472.4382).
124 215 48
Batatin IX (2): colorless microcrystals; mp 108−112 °C; [α] −41
D
4,5
1
13
batatins I−IV (1 mg/100 μL), were used as HPLC standards to
identify fractions containing new minor constituents. Co-elution
HPLC experiments were done on a Symmetry RP-18 column (Waters;
(c 0.1 MeOH); H and C NMR, see Table 2; negative FABMS m/z
−
−
1249 [unit A, C H O ] , 1103 [1249 − C H O ] , 1067 [unit B
63
109 24
6
10
4
−
−
− C H O] , 1019 [1249 − C H O − C H O ] , 921 [1067 −
9
6
5
8
6
10
4
−
−
5
mm, 4.6 × 250 mm) with an isocratic elution of CH CN−MeOH
C H O ] , 837 [921 − C H O] , 691, 545, 417; ESIMS m/z
3
6
10
4
5
8
−1
−
and a flow rate of 1 mL/min. Fractions III (1.46 g; 1.46 × 10 % on a
2448.3403 [M − H] (calcd for C H O , 2448.3443).
123 203 48
−1
dry weight basis) and IV (1.12 g; 1.12 × 10 % on a dry weight basis)
were selected for further recycling HPLC separation of minor
constituents.
Batatin X (3): colorless microcrystals; mp 125−126 °C; [α] −42
D
1 13
(c 0.1 EtOH); H and C NMR, see Table 3; negative FABMS m/z
−
−
2445 [M − H] , 1223 [unit B, C H O ] , 1221 [unit A,
61
107 24
−
−
−
Recycling HPLC Separation. These analytical separations were
C H O ] , 1137 [1221 − C H O] , 991 [1137 − C H O ] , 837
6
1
105 24
5
8
6
10
4
−
−
done on a Symmetry RP-18 column (Waters; 5 mm, 4.6 × 250 mm)
[991− C H O] , 545, 417, 271; ESIMS m/z 2444.3975 [M − H]
10
18
with an isocratic elution of various proportions of CH CN−MeOH
(calcd for C H O , 2444.4069).
3
122 211 48
and a flow rate of 0.5−0.7 mL/min. The best chromatographic elution
system found was then applied to each fraction in a preparative RP-18
column (Waters; 7 mm, 19 × 300 mm), with a sample injection of 20
μL (sample concentration: 10 mg/mL). For fraction III, the elution
Batatin XI (4): colorless microcrystals; mp 118−120 °C; [α] −57
1 13
D
(c 0.1 EtOH); H and C NMR, see Table 4; negative FABMS m/z
−
−
2501 [M − H] , 2417 [M − H − C H O] , 1251 [unit B,
5
8
−
−
−
C H O ] , 1249 [unit A, C H O ] , 1165 [1249 − C H O] ,
6
3
111 24
63 109 24
5
8
−
−
was isocratic with MeOH−CH CN (9:1) using a flow rate of 9 mL/
1067 [1249 − C H O] , 921 [1137 − C H O ] , 837 [921 −
3
12 22
6
10
4
−
−
min. The eluate with a t of 11.7 min was collected by the technique of
C H O] , 545, 417, 271; ESIMS m/z 2500.4503 [M − H] (calcd for
R
5
8
heart cutting and reinjected in the apparatus operated in the recycle
C
H
O , 2500.4695).
1
26 219 48
F
DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Alkaline Hydrolisis of Resin Glycoside Fraction. A solution of
μBondapak NH column (3.9 × 300 mm; 10 μm); elution, CH CN−
2
2
3
fractions III and IV (10 mg, each) in 5% KOH−H O (2 mL) was
H O (4:1); flow rate 1 mL/min. Simonic acid B was identified by
2
refluxed at 95 °C for 2 h. The reaction mixture was acidified to pH 4.0
coelution with an authentic sample (t 14.6 min) previously isolated
R
1
4d
and extracted with CHCl (10 mL). The organic layer was washed
from I. pes-caprae.
3
with H O, dried over anhydrous Na SO , evaporated under reduced
2
2
4
pressure, and directly analyzed by GC-MS and comparison of their
spectra and retention times with those of authentic samples. All
analytical standards were purchased with a purity of >98%: 2-
methylpropanoic acid (240168, Aldrich); 2-methylbutanoic acid
ASSOCIATED CONTENT
■
* Supporting Information
7
S
Spectra for batatins VIII−XI (1−4): negative FABMS, negative
1
13
(
(
193070, Aldrich); cinnamic acid (C80857, Sigma); n-decanoic acid
C1875, Sigma); n-dodecanoic acid (L4250, Sigma). Four peaks were
ESIMS, H and C NMR, COSY, TOCSY, HSQC, HMBC.
This material is available free of charge via the Internet at
http://pubs.acs.org.
detected for fraction III: 2-methylpropanoic acid (t 4.0 min), m/z
R
+
[
(
M] 88 (10), 73 (27), 60 (3), 55 (5), 45 (7), 43 (100), 41 (40), 39
10), 29 (6), 27 (24); 2-methylbutanoic acid (t 7 min), m/z [M]
+
R
AUTHOR INFORMATION
■
1
1
02 (3), 87 (33), 74 (100), 57 (50), 41 (28), 39 (8); cinnamic acid (tR
6.5 min), m/z [M] 148 (100), 147 (96), 131 (25), 103 (40), 102
+
Corresponding Author
(
20), 77 (25), 74 (8), 51 (20), 50 (8), 39 (5), 38 (4); and n-
*Phone: +5255 56225288. Fax: +5255 56225329. E-mail:
pereda@servidor.unam.mx.
+
dodecanoic acid (t 18 min), m/z [M] 200 (15), 183 (2), 171 (18),
R
1
57 (40), 143 (20), 129 (48), 115 (20), 101 (15), 85 (33), 73 (100),
Notes
6
0 (80), 57 (30), 55 (47), 43 (30). Three peaks were detected for
+
The authors declare no competing financial interest.
fraction IV: 2-methylbutanoic acid (t 7.2 min), m/z [M] 102 (3), 87
R
(
33), 74 (100), 57 (50), 41 (28), 39 (8); n-decanoic acid (t 14.6
R
+
min), m/z [M] 172 (2), 155 (3), 143 (12), 129 (62), 115 (15), 112
12), 87 (20), 73 (100), 60 (90), 57 (40), 55 (45), 43 (30), 41 (35),
ACKNOWLEDGMENTS
■
(
3
(
(
This research was supported by Direccion
́
General de Asuntos
+
9 (6); and n-dodecanoic acid (t 17.8 min), m/z [M] 200 (15), 183
R
del Personal Academ
Nacional de Ciencia y Tecnologia (220535). D.R.-R. received
́
ico, UNAM (IN212813), and Consejo
2), 171 (18), 157 (40), 143 (10), 129 (48), 115 (20), 101 (15), 85
33), 73 (100), 60 (80), 57 (30), 55 (47), 43 (30).
Esterification of the liberated carboxylic acids was performed as
́
graduate scholarships from CONACyT. Thanks are due to G.
Duarte (USAI, Facultad de Quimica, UNAM) for the recording
́
follows: a solution of benzyl alcohol (10.5 mg) in CH Cl (1 mL),
2
2
containing dicyclohexylcarbodiimide (3 mg) and 4-dimethylaminopyr-
of mass spectra and to Dr. M. Fragoso-Serrano (Departamento
1
9
idine (1 mg), was added to the mixture of carboxylic acids. The
reaction was stirred for 12 h at room temperature and filtered, and the
solvent evaporated. The crude mixture was purified by HPLC on a
normal-phase column (μPorasil, 10 μm, 3.9 × 300 mm; Waters) using
n-hexane−EtOAc (99:1, flow rate 0.7 mL/min): The physical and
́
de Farmacia, Facultad de Quimica, UNAM) for HPLC
technical assistance.
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■
(
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spectroscopic constants registered for the eluate with t 8.39 min were
R
1
9,20
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identical in all aspects to those previously reported
+)-benzyl α-methylbutyrate: oil, [α] +10.
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1
3,14
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́
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2
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83 23
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́
́
14c
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13
[
(
1
(
α]D −37 (c 0.2, MeOH); C NMR (pyridine-d , 125 MHz) 99.8
́
5
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DOI: 10.1021/np500523w
J. Nat. Prod. XXXX, XXX, XXX−XXX