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 Autónoma de México, 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.
S
which represent ester-type dimers of acylated tetra-4 and pentasaccharides.5,6 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-drugresistant phenotype in Gram-positive8 and -negative9 bacteria as well as in human cancer cells.10 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, anemia, hypertension, diabetes, and hemorrhages.11 It is noted 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 essential for optimal health.12 In this context, the white-skinned 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 nhexane-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.
weet potato (Ipomoea batatas L.) belongs to the Convolvulaceae (morning glory family) and is native to 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 pre-Christian times in Europe.1 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 the Convolvulaceae.1,2 They illustrate how nature creates structural diversity by using simple metabolic building blocks to generate complex mixtures of bioactive oligosaccharides of hydroxylated fatty acids.1 The sugars are D-glucose and four methylpentoses: D-fucose, L-rhamnose, D-quinovose, and Dxylose. 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 with reversed-phase columns.3 The most complex resin glycoside structures that have been isolated are those found in the Mexican varieties of sweet potato and the jalap root, © XXXX American Chemical Society and American Society of Pharmacognosy
Received: June 26, 2014
A
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION The crude n-hexane-soluble extract from the white-skinned variety of sweet potato was subjected to column chromatography 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. 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 to isolate the resin glycoside as intact constituents.3−10 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 III and IV) was saponified to liberate the same H2O-soluble glycosidic acid that was methylated and further acetylated to yield the peracetylated derivative of simonic acid B methyl ester. This product was used to generate a 13C NMR profile7
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. Comparison of the melting point, optical rotation, and 13C NMR data with published values for the peracetylated derivative of simonic acid B methyl ester14c 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-rhamnopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)]-O-α-Lrhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-β-Dfucopyranoside, which has been previously found in I. batatas13 and other members of the genus Ipomoea.14 The liberated organic acids were found to be 2-methylpropanoic (iba), 2methylbutanoic (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 identified by GC-MS coelution with authentic samples.7 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 VIII (1; C124H216O48); m/z 2448.3403 for batatin IX (2; C123H204O48); m/z 2444.3975 for batatin X (3; C122H212O48); and m/z 2500.4503 for batatin XI (4; C126H220O48). FABMS of compounds 1−4 provided readily detectable ions resulting from the characteristic elimination of the esterifying groups.15 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, B
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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Supporting Information).13 The characteristic high-mass ion fragment [M/2 − H]− was observed in both ionization techniques, which indicated the rupture of the ester bond in the dimeric structure:4−6 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 1221. As an example of the fragmentation pattern of this type of ester dimers, the FABMS of batatins IX (2) and XI (4) could be used since they were similar to that reported for batatin VII,5b and the following peaks were observed (Figures S15 and S31, Supporting Information) at m/z 1251 [unit B, C63H111O24]− and 1249 [unit A, C63H109O24]−, both produced from the rupture of the ester bond forming the dimeric structure; 1165 [1249 − C5H8O]−, indicating the loss of 84 amu from the methylbutanoyl residue; and 1067 [1249 − C12H22O]−, indicating the loss of 182 amu from the dodecanoyl residue. Structure elucidation of batatins VIII−XI (1−4) was accomplished by comparison of their NMR data with those reported for the batatins I−II and VII.5 In order to identify each constitutive monosaccharide,1 COSY (Figures S3, S11, S19, 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 differentiating each of the 1H NMR signals (Tables 1−4). Furthermore, 2D 1H−13C 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 the following common features: (a) diagnostic anomeric signals7 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 macrocyclic lactone-type4,5b structures; and (c) signals for four short-chain fatty acid residues esterifying the oligosaccharide core: H-2 of these moieties4,5b was used as a diagnostic resonance centered at δ 2.62 (1H, sept) for the methylpropanoyl group, 2.3−2.5 (1H, tq) for the methylbutanoyl groups, and 2.3−2.5 (2H, triplet-like signal) for the n-decanoyl (deca) and n-dodecanoyl residues. These postulations were 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 (2,3JCH) 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 (Figure 1): between H-2 (δ 5.94, Rha) and the carbonyl resonance at δc 172.9, assigned to the lactone functionality4,5b due to its 2JCH coupling with the diastereotopic C-2 methylene protons (δH 2.24−2.39 and 2.22−2.38); H-2 of the terminal branched rhamnose (δH 5.94, Rha‴) and the carbonyl group of the ester for unit B (δC 173.1; unit B); H-2 (δ 6.00, Rha′) and 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 one of the 2-methylpropanoyl residues due to its 2JCH with the
Table 1. 1H (400 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin VIII (1) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Iba-1 2 3 3′ Dodeca-1 2 12
δC
unit A 4.73 4.16 4.07 3.99 3.77 1.51 5.47 5.94 5.01 4.22 4.44 1.61 6.15 6.00 4.59 4.29 4.32 1.65 5.92 4.68 4.48 5.82 4.37 1.39 5.48 5.94 5.01 4.24 4.29 1.60
d (7.5) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) d (2.0) dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) d (2.0) dd (3.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, t (9.7) dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
unit B 7.5) 2.5) 2.0)
2.0) 3.0) 6.0)
2.0) 3.0) 6.0)
1.0) 3.0) 6.5)
1.0) 3.0) 6.0)
4.73 4.15 4.08 3.99 3.76 1.51 5.59 4.81 4.48 4.22 4.44 1.62 6.14 6.00 4.59 4.29 4.33 1.65 5.92 4.68 4.48 5.78 4.35 1.39 5.59 4.81 4.48 4.21 4.44 1.61
d (7.5) dd (8.0, dd (8.0, d (1.0) dq (6.5, d (6.5) d (1.0) bs dd (9.9, t (9.9) dq (9.9, d (6.5) d (2.0) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.0) d (1.2) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.5) d (1.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0)
7.5) 2.5) 1.0)
3.2) 6.5)
2.0) 3.0) 6.0)
1.2) 3.0) 6.5)
3.0) 6.0)
2.24 ddd (12.0, 8.0, 3.5) 2.39 ddd (12.0, 8.0, 3.5) 3.85 m 0.88 t (7.0)
2.24 ddd (12.0, 8.0, 3.5) 2.39 ddd (12.0, 8.0, 3.5) 3.85 m 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) 0.93 t (7.5)
2.31 dd (16.0, 7.5) 0.86 t (7.0)
2.25−2.40* 0.87 t (7.0)
unit A
unit B
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
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
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Iba, 2-methylpropanoyl; Dodeca, dodecanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
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 C
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H (500 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin IX (2) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Cna-1 2 3 Dodeca-1 2 12
δC
unit A 4.77 4.16 4.06 3.99 3.77 1.52 5.49 5.95 4.99 4.18 4.43 1.61 6.16 6.00 4.59 4.29 4.33 1.65 5.92 4.67 4.48 5.78 4.36 1.39 5.48 5.94 5.00 4.18 4.45 1.61
d (7.5) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) d (2.0) dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) d (2.0) dd (3.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, t (9.7) dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.5) 2.5) 2.0)
2.0) 3.0) 6.0)
2.0) 3.0) 6.0)
1.0) 3.0) 6.5)
1.0) 3.0) 6.0)
unit B 4.73 4.15 4.10 3.99 3.79 1.51 5.59 4.81 4.49 4.21 4.18 1.59 6.03 4.81 4.61 4.30 4.31 1.60 5.97 4.97 5.93 5.96 4.51 1.72 5.69 4.81 4.38 4.19 4.21 1.55
d (7.5) dd (8.0, dd (8.0, d (1.0) dq (6.5, d (6.5) d (1.0) bs dd (9.9, t (9.9) dq (9.9, d (6.5) d (2.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0) d (1.2) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.5) d (1.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0)
7.5) 2.5) 1.0)
3.2) 6.5)
3.0) 6.0)
1.2) 3.0) 6.5)
3.0) 6.0)
2.22 ddd (12.0, 8.0, 3.5) 2.38 ddd (12.0, 8.0, 3.5) 3.85 m 0.88 t (7.0)
2.25* 2.41* 3.89 m 0.88 t (7.0)
2.37 tq (7.0, 7.0) 1.06 d (7.0) 0.84 t (7.5)
2.50 tq (7.0, 7.0) 1.20 d (7.0) 0.93 t (7.5)
δH
unit A
unit B
positiona
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
82.3 14.3 175.4 41.4 16.8 11.8
82.3 14.3 176.3 41.5 17.0 11.8 166.9 118.4 128.7
Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Deca-1 2 12
6.67 d (15.9) 7.79 d (15.7) 2.31 dd (16.0, 7.5) 0.88 t (7.0)
Table 3. 1H (400 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin X (3) in Pyridine-d5 (δ in ppm, J in Hz) δC
unit A 4.83 4.50 4.18 3.94 3.77 1.52 6.36 5.33 5.63 4.64 5.02 1.61 6.18 6.03 4.59 4.29 4.33 1.66 5.93 4.67 4.48 5.81 4.36 1.40 5.95 4.75 4.39 5.81 4.29 1.60
d (7.8) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs bs dd (9.4, t (9.4) dq (9.4, d (6.0) d (1.6) dd (2.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, bs dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.8) 2.5) 2.0)
3.0) 6.0)
1.6) 3.0) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
4.75 4.16 4.18 3.94 3.77 1.52 5.95 4.71 4.51 4.22 4.31 1.61 5.67 5.98 4.50 4.29 4.36 1.66 5.65 4.67 4.39 5.81 4.36 1.40 5.50 4.75 4.48 4.20 4.29 1.60
unit B
unit A
unit B
d (7.8) dd (8.5, 7.8) dd (8.5, 2.5) d (1.0) dq (6.5, 2.0) d (6.5) bs dd (3.0) dd (9.4, 3.0) t (9.4) dq (9.4, 6.0) d (6.0) d (2.0) dd (3.0, 2.0) dd (9.0, 3.0) t (9.0) dq (9.0, 6.0) d (6.0) d (1.0) dd (3.0, 1.0) dd (9.7, 3.0) bs dq (9.8, 6.0) d (6.5) d (1.4) dd (3.0, 1.0) dd (9.3, 3.0) t (9.3) dq (9.3, 6.0) d (6.0)
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
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
2.20−2.24* 2.95 ddd (12.0, 8.0, 3.5) 3.87 m 0.88 t (7.0)
2.20−2.24*
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.26−2.35* 0.86 t (7.0)
2.26−2.35* 0.86 t (7.0)
3.87 m 0.88 t (7.0)
a
172.9 34.5 14.3
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Mba, 2-methylbutanoyl; Deca, decanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; 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 (δH 5.93, 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 carbonyl group for the ester (δC 173.4, unit B) and H-4 of the
compound 1 with the only difference involving the interaction between H-4 (δ 5.78, Rha″) and the carbonyl resonance at δ 175.4, assigned to the 2-methylbutanoyl residues due to its 2JCH 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 D
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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the signal centered at δ 2.50 identified the presence of a 2methylbutanoyl moiety, which was placed at C-4 in the terminal rhamnose unit (Rha″) of both units A and B through the 3JCH interaction of the same carbonyl carbon and the triplet-like signal at δ 5.81. 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 convolvulaceous 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 hungaiensis.16 Tricolorins H−J, ester-type dimers consisting of two trisaccharide units of tricoloric acid C, were purified from I. tricolor.17 Tyrianthins A and B, acylated ester-type heterotetraglycosides consisting of two scammonic acid A units, were isolated from I. orizabensis.18 Purgins I−III,6 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− IV4,5a were isolated from the n-hexane-soluble extract prepared from a Mexican white-skinned staple-type cultivar. Finally, three CHCl3-soluble dimers have been described: batatins V and VI4 were isolated from the extract of a purple-skinned cultivar, while batatin VII5b 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 potato.15 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.
Table 4. 1H (500 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin XI (4) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Deca-1 2 12
δC
unit A 4.83 4.50 4.18 3.94 3.77 1.52 6.36 5.34 5.63 4.64 5.02 1.61 6.19 6.04 4.59 4.29 4.33 1.66 5.93 4.67 4.48 5.81 4.36 1.40 5.95 4.75 4.39 5.81 4.29 1.60
d (8.2) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) bs bs dd (9.0, t (9.0) dq (9.0, d (6.0) bs dd (3.0, dd (9.7, bs dq (9.8, d (6.5) bs dd (3.0, dd (9.3, bs dq (9.3, d (6.0)
8.2) 2.5) 2.0)
2.0) 3.0) 6.0)
2.7) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
unit B 4.76 4.16 4.18 3.94 3.77 1.52 5.95 4.71 4.51 4.22 4.31 1.61 5.68 5.98 4.50 4.29 4.36 1.66 5.65 4.67 4.39 5.81 4.36 1.40 5.50 4.75 4.48 4.20 4.29 1.60
d (7.4) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) bs bs dd (9.0, t (9.0) dq (9.0, d (6.0) bs dd (3.0, dd (9.7, bs dq (9.8, d (6.5) bs dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.4) 2.5) 2.0)
2.0) 3.0) 6.0)
2.6) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
2.20−2.24* 2.95 ddd (12.0, 8.0, 3.5) 3.87 m 0.88 t (7.0)
2.20−2.24*
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.26−2.35* 0.86 t (7.0)
2.26−2.35* 0.86 t (7.0)
3.87 m 0.88 t (7.0)
unit A
unit B
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
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
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EXPERIMENTAL SECTION
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. 1 H (500 MHz) and 13C (125.7 MHz) NMR experiments were conducted on a Bruker DMX-500 instrument, while 1H (400 MHz) NMR measurements were carried out on a Varian VXL instrument. The instrumentation used for HPLC analysis consisted of a Waters (Millipore Corp., Waters Chromatography Division, Milford, MA, 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 spectrometer. GC conditions:7 HP-5MS (5% phenyl)-methylpolysiloxane column (30 m × 0.25 mm, Agilent Technologies, Santa Clara, CA, USA), film thickness = 0.25 μm; He, linear velocity = 30 cm/s; 50 °C isothermal for 3 min, linear gradient to 300 °C at 20 °C/min; final 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. Highresolution 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
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Mba, 2-methylbutanoyl; Dodeca, dodecanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
terminal branched rhamnose (Rha‴, δH 5.81) of unit A. The carbonyl resonance at δc 173.4 was assigned to the lactone functionality at unit A, and the macrolactonization site at C-3 of the first rhamnose unit (Rha) was established by the 3JCH correlation between this carbonyl carbon and the proton at δH 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 (C1, δC 171.5), decanoyl for 3 and dodecanoyl for 4, as confirmed by FABMS data. The observed 2JCH correlation between the carbonyl resonance at δ 174.9 and the sextet-like spin pattern of E
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Figure 1. Key HMBC correlations (3JCH) for the sites of esterification of compounds 1 and 3. mode3 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 was isocratic with MeOH−CH3CN (3:2) using a flow rate of 8.16 mL/min. Eluates with a tR of 25.7 and 29.5 min were collected by the technique of heart cutting and independently reinjected in the recycling mode.3 Suitable chromatographic conditions consisted of an analytical RP-18 column (4.6 × 250 mm, 5 μM); flow 0.5 mL/min; mobile phase MeOH. This analysis and the techniques of peak shaving and heart cutting3 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 1.82 × 10−4 % on a dry weight basis, and that from fraction IV was 1.9 × 10−3 % on a dry weight basis. Batatin VIII (1): colorless microcrystals; mp 115−120 °C; [α]D −19 (c 0.1 MeOH); 1H and 13C NMR, see Table 1; negative FABMS m/z 1235 [unit A, C63H109O24]−, 1053 [1235 − C12H22O]−, 965 [1053 − C4H7O − H2O]−, 819 [965 − C6H10O4]−, 691, 545, 417; ESIMS m/z 2472.3990 [M − H]− (calcd for C124H215O48, 2472.4382). Batatin IX (2): colorless microcrystals; mp 108−112 °C; [α]D −41 (c 0.1 MeOH); 1H and 13C NMR, see Table 2; negative FABMS m/z 1249 [unit A, C63H109O24]−, 1103 [1249 − C6H10O4]−, 1067 [unit B − C9H6O]−, 1019 [1249 − C5H8O − C6H10O4]−, 921 [1067 − C6H10O4]−, 837 [921 − C5H8O]−, 691, 545, 417; ESIMS m/z 2448.3403 [M − H]− (calcd for C123H203O48, 2448.3443). Batatin X (3): colorless microcrystals; mp 125−126 °C; [α]D −42 (c 0.1 EtOH); 1H and 13C NMR, see Table 3; negative FABMS m/z 2445 [M − H]−, 1223 [unit B, C61H107O24]−, 1221 [unit A, C61H105O24]−, 1137 [1221 − C5H8O]−, 991 [1137 − C6H10O4]−, 837 [991− C10H18O]−, 545, 417, 271; ESIMS m/z 2444.3975 [M − H]− (calcd for C122H211O48, 2444.4069). Batatin XI (4): colorless microcrystals; mp 118−120 °C; [α]D −57 (c 0.1 EtOH); 1H and 13C NMR, see Table 4; negative FABMS m/z 2501 [M − H]−, 2417 [M − H − C5H8O]−, 1251 [unit B, C63H111O24]−, 1249 [unit A, C63H109O24]−, 1165 [1249 − C5H8O]−, 1067 [1249 − C12H22O]−, 921 [1137 − C6H10O4]−, 837 [921 − C5H8O]−, 545, 417, 271; ESIMS m/z 2500.4503 [M − H]− (calcd for C126H219O48, 2500.4695).
were visualized by spraying with 3% CeSO4 in 2 N H2SO4 followed by heating. Plant Material. The tuberous roots of the white-skinned cultivar of I. batatas were collected in plantations in San Nicolás, Salvatierra, 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), Instituto de Biologiá UNAM. Extraction and Isolation. The powdered dry roots of the whiteskinned 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 CH2Cl2 in n-hexane, Me2CO in CH2Cl2, and MeOH. The process was monitored by TLC (CHCl3−MeOH−H2O, 6:4:1), and a total of 36 fractions (150 mL each) were collected and combined in eight resin glycoside-containing fractions with an Rf 0.5 (I−VIII eluted with CH2Cl2−Me2CO−MeOH, 6:3:1). All fractions were partially purified by passing through activated charcoal to eliminate pigmented residues. Previoulsy isolated major resin glycosides from this plant species, batatins I−IV4,5 (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; 5 mm, 4.6 × 250 mm) with an isocratic elution of CH3CN−MeOH and a flow rate of 1 mL/min. Fractions III (1.46 g; 1.46 × 10−1 % on a dry weight basis) and IV (1.12 g; 1.12 × 10−1 % on a dry weight basis) were selected for further recycling HPLC separation of minor constituents. Recycling HPLC Separation. These analytical separations were done on a Symmetry RP-18 column (Waters; 5 mm, 4.6 × 250 mm) with an isocratic elution of various proportions of CH3CN−MeOH 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 was isocratic with MeOH−CH3CN (9:1) using a flow rate of 9 mL/ min. The eluate with a tR of 11.7 min was collected by the technique of heart cutting and reinjected in the apparatus operated in the recycle F
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μBondapak NH2 column (3.9 × 300 mm; 10 μm); elution, CH3CN− H2O (4:1); flow rate 1 mL/min. Simonic acid B was identified by coelution with an authentic sample (tR 14.6 min) previously isolated from I. pes-caprae.14d
Alkaline Hydrolisis of Resin Glycoside Fraction. A solution of fractions III and IV (10 mg, each) in 5% KOH−H2O (2 mL) was refluxed at 95 °C for 2 h. The reaction mixture was acidified to pH 4.0 and extracted with CHCl3 (10 mL). The organic layer was washed with H2O, dried over anhydrous Na2SO4, evaporated under reduced pressure, and directly analyzed by GC-MS and comparison of their spectra and retention times with those of authentic samples.7 All analytical standards were purchased with a purity of >98%: 2methylpropanoic acid (240168, Aldrich); 2-methylbutanoic acid (193070, Aldrich); cinnamic acid (C80857, Sigma); n-decanoic acid (C1875, Sigma); n-dodecanoic acid (L4250, Sigma). Four peaks were detected for fraction III: 2-methylpropanoic acid (tR 4.0 min), m/z [M]+ 88 (10), 73 (27), 60 (3), 55 (5), 45 (7), 43 (100), 41 (40), 39 (10), 29 (6), 27 (24); 2-methylbutanoic acid (tR 7 min), m/z [M]+ 102 (3), 87 (33), 74 (100), 57 (50), 41 (28), 39 (8); cinnamic acid (tR 16.5 min), m/z [M]+ 148 (100), 147 (96), 131 (25), 103 (40), 102 (20), 77 (25), 74 (8), 51 (20), 50 (8), 39 (5), 38 (4); and ndodecanoic acid (tR 18 min), m/z [M]+ 200 (15), 183 (2), 171 (18), 157 (40), 143 (20), 129 (48), 115 (20), 101 (15), 85 (33), 73 (100), 60 (80), 57 (30), 55 (47), 43 (30). Three peaks were detected for fraction IV: 2-methylbutanoic acid (tR 7.2 min), m/z [M]+ 102 (3), 87 (33), 74 (100), 57 (50), 41 (28), 39 (8); n-decanoic acid (tR 14.6 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), 39 (6); and n-dodecanoic acid (tR 17.8 min), m/z [M]+ 200 (15), 183 (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 follows: a solution of benzyl alcohol (10.5 mg) in CH2Cl2 (1 mL), containing dicyclohexylcarbodiimide (3 mg) and 4-dimethylaminopyridine (1 mg), was added to the mixture of carboxylic acids.19 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 spectroscopic constants registered for the eluate with tR 8.39 min were identical in all aspects to those previously reported19,20 for (S)(+)-benzyl α-methylbutyrate: oil, [α]D +10. The n-butanol-soluble residue (6 mg) extracted from the saponification aqueous phase was subjected to preparative HPLC on a Waters μBondapak NH2 column (7.8 × 300 mm; 10 μm). The elution was isocratic with CH3CN−H2O (4:1), using a flow rate of 1 mL/min and a sample injection of 500 μL (5 mg/mL). This procedure yielded the same glycosidic acid for the two fractions, which was identified as simonic acid B by comparison of its physical constants and NMR data with published values.13,14 Both residues were methylated with CH2N2 to yield 9.5 mg of a white powder: mp 113−115 °C; [α]D −83 (c 1.0, MeOH) HRFABMS m/z 1015.5322 [M − H]− (calcd for C47H83O23 requires 1015.5325). The glycosidic acid methyl ester was further acetylated (Ac2O−pyridine, 2:1) to give a residue (9.3 mg), which was purified on RP-18 HPLC (7 μm, 19 × 300 mm; CH3CN−MeOH, 4:1; flow rate 8 mL/min). Eluates across the peak with tR values of 10.8 min afforded the peracetylated derivative of simonic acid B methyl ester, which was identified by coelution with an authentic sample and comparison of its physical constants and NMR data with published values:14c mp 81−85 °C; [α]D −37 (c 0.2, MeOH); 13C NMR (pyridine-d5, 125 MHz) 99.8 (CH, Fuc-1), 99.7 (CH, Rha‴-1), 99.2 (CH, Rha″-1), 99.1 (CH, Rha1), 97.4 (CH, Rha′-1), 13.9 (CH3, Jla-16), 33.9 (CH2CO2), 50.8 (OCH3), 78.0 (CH, Jla-11), 173.6 (C, Jla-1). Alkaline Hydrolisis of 1−4. Each individual compound 1−4 (3 mg) was submitted to an alkaline hydrolysis following the same procedures described above. The organic layer from extraction was directly analyzed by GC-MS. For compound 1, two peaks were detected, 2-methylpropanoic acid (tR 4.0 min) and n-dodecanoic acid (tR 17.8 min); for 2, the peaks for 2-methylbutanoic acid (tR 7 min) and cinnamic acid (tR 16.5 min) were identified; and compounds 3 and 4 afforded methylbutanoic acid (tR 7.2 min) as well as n-decanoic acid (tR 14.6 min) and n-dodecanoic acid (tR 17.8 min), respectively. The aqueous phases were individually dried and analyzed by HPLC:
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ASSOCIATED CONTENT
* Supporting Information S
Spectra for batatins VIII−XI (1−4): negative FABMS, negative ESIMS, 1H and 13C NMR, COSY, TOCSY, HSQC, HMBC. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +5255 56225288. Fax: +5255 56225329. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by Dirección General de Asuntos del Personal Académico, UNAM (IN212813), and Consejo Nacional de Ciencia y Tecnologiá (220535). D.R.-R. received graduate scholarships from CONACyT. Thanks are due to G. ́ Duarte (USAI, Facultad de Quimica, UNAM) for the recording of mass spectra and to Dr. M. Fragoso-Serrano (Departamento ́ de Farmacia, Facultad de Quimica, UNAM) for HPLC technical assistance.
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REFERENCES
(1) Pereda-Miranda, R.; Rosas-Ramírez, D.; Castañeda-Gómez, J. Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D., Falk, H., Kobayashi, J., Eds.; Springer-Verlag: New York, 2010; Vol. 92, Chapter 2, pp 77−152. (2) Eich, E. Solanaceae and Convolvulaceae: Secondary Metabolites. Biosynthesis, Chemotaxonomy, Biological and Economic Significance; Springer: Heidelberg, 2008; p 532. (3) Pereda-Miranda, R.; Hernández-Carlos, B. Tetrahedron 2002, 58, 3145−3154. (4) Rosas-Ramírez, D.; Escalante-Sánchez, E.; Pereda-Miranda, R. Phytochemistry 2011, 72, 773−780. (5) (a) Escalante-Sánchez, E.; Pereda-Miranda, R. J. Nat. Prod. 2007, 70, 1029−1034. (b) Rosas-Ramírez, D.; Pereda-Miranda, R. J. Agric. Food Chem. 2013, 61, 9488−9494. (6) (a) Castañeda-Gómez, J.; Pereda-Miranda, R. J. Nat. Prod. 2011, 74, 1148−1153. (b) Castañeda-Gómez, J.; Figueroa-González, G.; Jacobo, N.; Pereda-Miranda, R. J. Nat. Prod. 2013, 76, 64−71. (7) Pereda-Miranda, R.; Fragoso-Serrano, M.; Escalante-Sánchez, E.; Hernández-Carlos, B.; Linares, E.; Bye, R. J. Nat. Prod. 2006, 69, 1460−1466. (8) (a) Pereda-Miranda, R.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod. 2006, 69, 406−409. (b) Chérigo, L.; Pereda-Miranda, R.; FragosoSerrano, M.; Jacobo-Herrera, N.; Kaatz, G. W.; Gibbons, S. J. Nat. Prod. 2008, 71, 1037−1045. (c) Chérigo, L.; Pereda-Miranda, R.; Gibbons, S. Phytochemistry 2009, 70, 222−227. (d) EscobedoMartínez, C.; Cruz-Morales, S.; Fragoso-Serrano, M.; Rahman, M. M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2010, 71, 1796− 1801. (9) (a) Corona-Castañeda, B.; Pereda-Miranda, R. Planta Med. 2012, 78, 128−131. (b) Corona-Castañeda, B.; Chérigo, L.; Fragoso-Serrano, M.; Gibbons, S.; Pereda-Miranda, R. Phytochemistry 2013, 95, 277− 283. (10) (a) Figueroa-González, G.; Jacobo-Herrera, N.; ZentellaDehesa, A.; Pereda-Miranda, R. J. Nat. Prod. 2012, 75, 93−97. (b) Cruz Morales, S.; Castañeda-Gómez, J.; Figueroa-González, G.; G
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Batatins VIII−XI, Glycolipid Ester-Type Dimers from Ipomoea batatas Daniel Rosas-Ramírez and Rogelio Pereda-Miranda* Facultad de Química, Universidad Nacional Autónoma de México, 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.
S
which represent ester-type dimers of acylated tetra-4 and pentasaccharides.5,6 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-drugresistant phenotype in Gram-positive8 and -negative9 bacteria as well as in human cancer cells.10 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, anemia, hypertension, diabetes, and hemorrhages.11 It is noted 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 essential for optimal health.12 In this context, the white-skinned 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 nhexane-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.
weet potato (Ipomoea batatas L.) belongs to the Convolvulaceae (morning glory family) and is native to 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 pre-Christian times in Europe.1 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 the Convolvulaceae.1,2 They illustrate how nature creates structural diversity by using simple metabolic building blocks to generate complex mixtures of bioactive oligosaccharides of hydroxylated fatty acids.1 The sugars are D-glucose and four methylpentoses: D-fucose, L-rhamnose, D-quinovose, and Dxylose. 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 with reversed-phase columns.3 The most complex resin glycoside structures that have been isolated are those found in the Mexican varieties of sweet potato and the jalap root, © XXXX American Chemical Society and American Society of Pharmacognosy
Received: June 26, 2014
A
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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■
RESULTS AND DISCUSSION The crude n-hexane-soluble extract from the white-skinned variety of sweet potato was subjected to column chromatography 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. 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 to isolate the resin glycoside as intact constituents.3−10 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 III and IV) was saponified to liberate the same H2O-soluble glycosidic acid that was methylated and further acetylated to yield the peracetylated derivative of simonic acid B methyl ester. This product was used to generate a 13C NMR profile7
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. Comparison of the melting point, optical rotation, and 13C NMR data with published values for the peracetylated derivative of simonic acid B methyl ester14c 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-rhamnopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4)]-O-α-Lrhamnopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2)-β-Dfucopyranoside, which has been previously found in I. batatas13 and other members of the genus Ipomoea.14 The liberated organic acids were found to be 2-methylpropanoic (iba), 2methylbutanoic (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 identified by GC-MS coelution with authentic samples.7 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 VIII (1; C124H216O48); m/z 2448.3403 for batatin IX (2; C123H204O48); m/z 2444.3975 for batatin X (3; C122H212O48); and m/z 2500.4503 for batatin XI (4; C126H220O48). FABMS of compounds 1−4 provided readily detectable ions resulting from the characteristic elimination of the esterifying groups.15 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, B
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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Supporting Information).13 The characteristic high-mass ion fragment [M/2 − H]− was observed in both ionization techniques, which indicated the rupture of the ester bond in the dimeric structure:4−6 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 1221. As an example of the fragmentation pattern of this type of ester dimers, the FABMS of batatins IX (2) and XI (4) could be used since they were similar to that reported for batatin VII,5b and the following peaks were observed (Figures S15 and S31, Supporting Information) at m/z 1251 [unit B, C63H111O24]− and 1249 [unit A, C63H109O24]−, both produced from the rupture of the ester bond forming the dimeric structure; 1165 [1249 − C5H8O]−, indicating the loss of 84 amu from the methylbutanoyl residue; and 1067 [1249 − C12H22O]−, indicating the loss of 182 amu from the dodecanoyl residue. Structure elucidation of batatins VIII−XI (1−4) was accomplished by comparison of their NMR data with those reported for the batatins I−II and VII.5 In order to identify each constitutive monosaccharide,1 COSY (Figures S3, S11, S19, 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 differentiating each of the 1H NMR signals (Tables 1−4). Furthermore, 2D 1H−13C 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 the following common features: (a) diagnostic anomeric signals7 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 macrocyclic lactone-type4,5b structures; and (c) signals for four short-chain fatty acid residues esterifying the oligosaccharide core: H-2 of these moieties4,5b was used as a diagnostic resonance centered at δ 2.62 (1H, sept) for the methylpropanoyl group, 2.3−2.5 (1H, tq) for the methylbutanoyl groups, and 2.3−2.5 (2H, triplet-like signal) for the n-decanoyl (deca) and n-dodecanoyl residues. These postulations were 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 (2,3JCH) 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 (Figure 1): between H-2 (δ 5.94, Rha) and the carbonyl resonance at δc 172.9, assigned to the lactone functionality4,5b due to its 2JCH coupling with the diastereotopic C-2 methylene protons (δH 2.24−2.39 and 2.22−2.38); H-2 of the terminal branched rhamnose (δH 5.94, Rha‴) and the carbonyl group of the ester for unit B (δC 173.1; unit B); H-2 (δ 6.00, Rha′) and 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 one of the 2-methylpropanoyl residues due to its 2JCH with the
Table 1. 1H (400 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin VIII (1) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Iba-1 2 3 3′ Dodeca-1 2 12
δC
unit A 4.73 4.16 4.07 3.99 3.77 1.51 5.47 5.94 5.01 4.22 4.44 1.61 6.15 6.00 4.59 4.29 4.32 1.65 5.92 4.68 4.48 5.82 4.37 1.39 5.48 5.94 5.01 4.24 4.29 1.60
d (7.5) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) d (2.0) dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) d (2.0) dd (3.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, t (9.7) dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
unit B 7.5) 2.5) 2.0)
2.0) 3.0) 6.0)
2.0) 3.0) 6.0)
1.0) 3.0) 6.5)
1.0) 3.0) 6.0)
4.73 4.15 4.08 3.99 3.76 1.51 5.59 4.81 4.48 4.22 4.44 1.62 6.14 6.00 4.59 4.29 4.33 1.65 5.92 4.68 4.48 5.78 4.35 1.39 5.59 4.81 4.48 4.21 4.44 1.61
d (7.5) dd (8.0, dd (8.0, d (1.0) dq (6.5, d (6.5) d (1.0) bs dd (9.9, t (9.9) dq (9.9, d (6.5) d (2.0) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.0) d (1.2) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.5) d (1.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0)
7.5) 2.5) 1.0)
3.2) 6.5)
2.0) 3.0) 6.0)
1.2) 3.0) 6.5)
3.0) 6.0)
2.24 ddd (12.0, 8.0, 3.5) 2.39 ddd (12.0, 8.0, 3.5) 3.85 m 0.88 t (7.0)
2.24 ddd (12.0, 8.0, 3.5) 2.39 ddd (12.0, 8.0, 3.5) 3.85 m 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) 0.93 t (7.5)
2.31 dd (16.0, 7.5) 0.86 t (7.0)
2.25−2.40* 0.87 t (7.0)
unit A
unit B
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
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
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Iba, 2-methylpropanoyl; Dodeca, dodecanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
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 C
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. 1H (500 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin IX (2) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Cna-1 2 3 Dodeca-1 2 12
δC
unit A 4.77 4.16 4.06 3.99 3.77 1.52 5.49 5.95 4.99 4.18 4.43 1.61 6.16 6.00 4.59 4.29 4.33 1.65 5.92 4.67 4.48 5.78 4.36 1.39 5.48 5.94 5.00 4.18 4.45 1.61
d (7.5) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) d (2.0) dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) d (2.0) dd (3.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, t (9.7) dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.5) 2.5) 2.0)
2.0) 3.0) 6.0)
2.0) 3.0) 6.0)
1.0) 3.0) 6.5)
1.0) 3.0) 6.0)
unit B 4.73 4.15 4.10 3.99 3.79 1.51 5.59 4.81 4.49 4.21 4.18 1.59 6.03 4.81 4.61 4.30 4.31 1.60 5.97 4.97 5.93 5.96 4.51 1.72 5.69 4.81 4.38 4.19 4.21 1.55
d (7.5) dd (8.0, dd (8.0, d (1.0) dq (6.5, d (6.5) d (1.0) bs dd (9.9, t (9.9) dq (9.9, d (6.5) d (2.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0) d (1.2) dd (3.0, dd (9.5, t (9.5) dq (9.5, d (6.5) d (1.0) bs dd (9.5, t (9.5) dq (9.5, d (6.0)
7.5) 2.5) 1.0)
3.2) 6.5)
3.0) 6.0)
1.2) 3.0) 6.5)
3.0) 6.0)
2.22 ddd (12.0, 8.0, 3.5) 2.38 ddd (12.0, 8.0, 3.5) 3.85 m 0.88 t (7.0)
2.25* 2.41* 3.89 m 0.88 t (7.0)
2.37 tq (7.0, 7.0) 1.06 d (7.0) 0.84 t (7.5)
2.50 tq (7.0, 7.0) 1.20 d (7.0) 0.93 t (7.5)
δH
unit A
unit B
positiona
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
82.3 14.3 175.4 41.4 16.8 11.8
82.3 14.3 176.3 41.5 17.0 11.8 166.9 118.4 128.7
Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Deca-1 2 12
6.67 d (15.9) 7.79 d (15.7) 2.31 dd (16.0, 7.5) 0.88 t (7.0)
Table 3. 1H (400 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin X (3) in Pyridine-d5 (δ in ppm, J in Hz) δC
unit A 4.83 4.50 4.18 3.94 3.77 1.52 6.36 5.33 5.63 4.64 5.02 1.61 6.18 6.03 4.59 4.29 4.33 1.66 5.93 4.67 4.48 5.81 4.36 1.40 5.95 4.75 4.39 5.81 4.29 1.60
d (7.8) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs bs dd (9.4, t (9.4) dq (9.4, d (6.0) d (1.6) dd (2.0, dd (9.0, t (9.0) dq (9.0, d (6.0) d (1.0) dd (3.0, dd (9.7, bs dq (9.8, d (6.5) d (1.0) dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.8) 2.5) 2.0)
3.0) 6.0)
1.6) 3.0) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
4.75 4.16 4.18 3.94 3.77 1.52 5.95 4.71 4.51 4.22 4.31 1.61 5.67 5.98 4.50 4.29 4.36 1.66 5.65 4.67 4.39 5.81 4.36 1.40 5.50 4.75 4.48 4.20 4.29 1.60
unit B
unit A
unit B
d (7.8) dd (8.5, 7.8) dd (8.5, 2.5) d (1.0) dq (6.5, 2.0) d (6.5) bs dd (3.0) dd (9.4, 3.0) t (9.4) dq (9.4, 6.0) d (6.0) d (2.0) dd (3.0, 2.0) dd (9.0, 3.0) t (9.0) dq (9.0, 6.0) d (6.0) d (1.0) dd (3.0, 1.0) dd (9.7, 3.0) bs dq (9.8, 6.0) d (6.5) d (1.4) dd (3.0, 1.0) dd (9.3, 3.0) t (9.3) dq (9.3, 6.0) d (6.0)
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
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
2.20−2.24* 2.95 ddd (12.0, 8.0, 3.5) 3.87 m 0.88 t (7.0)
2.20−2.24*
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.26−2.35* 0.86 t (7.0)
2.26−2.35* 0.86 t (7.0)
3.87 m 0.88 t (7.0)
a
172.9 34.5 14.3
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Mba, 2-methylbutanoyl; Deca, decanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; 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 (δH 5.93, 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 carbonyl group for the ester (δC 173.4, unit B) and H-4 of the
compound 1 with the only difference involving the interaction between H-4 (δ 5.78, Rha″) and the carbonyl resonance at δ 175.4, assigned to the 2-methylbutanoyl residues due to its 2JCH 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 D
DOI: 10.1021/np500523w J. Nat. Prod. XXXX, XXX, XXX−XXX
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the signal centered at δ 2.50 identified the presence of a 2methylbutanoyl moiety, which was placed at C-4 in the terminal rhamnose unit (Rha″) of both units A and B through the 3JCH interaction of the same carbonyl carbon and the triplet-like signal at δ 5.81. 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 convolvulaceous 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 hungaiensis.16 Tricolorins H−J, ester-type dimers consisting of two trisaccharide units of tricoloric acid C, were purified from I. tricolor.17 Tyrianthins A and B, acylated ester-type heterotetraglycosides consisting of two scammonic acid A units, were isolated from I. orizabensis.18 Purgins I−III,6 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− IV4,5a were isolated from the n-hexane-soluble extract prepared from a Mexican white-skinned staple-type cultivar. Finally, three CHCl3-soluble dimers have been described: batatins V and VI4 were isolated from the extract of a purple-skinned cultivar, while batatin VII5b 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 potato.15 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.
Table 4. 1H (500 MHz, Assignments Based on 1H−1H COSY and TOCSY Experiments) and 13C (125 MHz, Assignments Based on HSQC and HMBC Experiments) NMR Data of Batatin XI (4) in Pyridine-d5 (δ in ppm, J in Hz) δH positiona Fuc-1 2 3 4 5 6 Rha-1 2 3 4 5 6 Rha′-1 2 3 4 5 6 Rha″-1 2 3 4 5 6 Rha‴-1 2 3 4 5 6 Jal-1 2a 2b 11 16 Mba-1 2 2-Me 3-Me Deca-1 2 12
δC
unit A 4.83 4.50 4.18 3.94 3.77 1.52 6.36 5.34 5.63 4.64 5.02 1.61 6.19 6.04 4.59 4.29 4.33 1.66 5.93 4.67 4.48 5.81 4.36 1.40 5.95 4.75 4.39 5.81 4.29 1.60
d (8.2) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) bs bs dd (9.0, t (9.0) dq (9.0, d (6.0) bs dd (3.0, dd (9.7, bs dq (9.8, d (6.5) bs dd (3.0, dd (9.3, bs dq (9.3, d (6.0)
8.2) 2.5) 2.0)
2.0) 3.0) 6.0)
2.7) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
unit B 4.76 4.16 4.18 3.94 3.77 1.52 5.95 4.71 4.51 4.22 4.31 1.61 5.68 5.98 4.50 4.29 4.36 1.66 5.65 4.67 4.39 5.81 4.36 1.40 5.50 4.75 4.48 4.20 4.29 1.60
d (7.4) dd (8.5, dd (8.5, d (1.0) dq (6.5, d (6.5) bs dd (3.0, dd (9.4, t (9.4) dq (9.4, d (6.0) bs bs dd (9.0, t (9.0) dq (9.0, d (6.0) bs dd (3.0, dd (9.7, bs dq (9.8, d (6.5) bs dd (3.0, dd (9.3, t (9.3) dq (9.3, d (6.0)
7.4) 2.5) 2.0)
2.0) 3.0) 6.0)
2.6) 6.0)
1.0) 3.0) 6.0)
1.0) 3.0) 6.0)
2.20−2.24* 2.95 ddd (12.0, 8.0, 3.5) 3.87 m 0.88 t (7.0)
2.20−2.24*
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.50 tq (7.0, 7.0) 1.21 d (7.0) 0.96 t (7.5)
2.26−2.35* 0.86 t (7.0)
2.26−2.35* 0.86 t (7.0)
3.87 m 0.88 t (7.0)
unit A
unit B
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
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
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EXPERIMENTAL SECTION
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. 1 H (500 MHz) and 13C (125.7 MHz) NMR experiments were conducted on a Bruker DMX-500 instrument, while 1H (400 MHz) NMR measurements were carried out on a Varian VXL instrument. The instrumentation used for HPLC analysis consisted of a Waters (Millipore Corp., Waters Chromatography Division, Milford, MA, 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 spectrometer. GC conditions:7 HP-5MS (5% phenyl)-methylpolysiloxane column (30 m × 0.25 mm, Agilent Technologies, Santa Clara, CA, USA), film thickness = 0.25 μm; He, linear velocity = 30 cm/s; 50 °C isothermal for 3 min, linear gradient to 300 °C at 20 °C/min; final 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. Highresolution 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
a
Abbreviations; Fuc, fucose; Rha, rhamnose; Jal, 11-hydroxyhexadecanoyl; Mba, 2-methylbutanoyl; Dodeca, dodecanoyl. Chemical shifts marked with an asterisk (*) indicate overlapped signals.
terminal branched rhamnose (Rha‴, δH 5.81) of unit A. The carbonyl resonance at δc 173.4 was assigned to the lactone functionality at unit A, and the macrolactonization site at C-3 of the first rhamnose unit (Rha) was established by the 3JCH correlation between this carbonyl carbon and the proton at δH 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 (C1, δC 171.5), decanoyl for 3 and dodecanoyl for 4, as confirmed by FABMS data. The observed 2JCH correlation between the carbonyl resonance at δ 174.9 and the sextet-like spin pattern of E
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Figure 1. Key HMBC correlations (3JCH) for the sites of esterification of compounds 1 and 3. mode3 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 was isocratic with MeOH−CH3CN (3:2) using a flow rate of 8.16 mL/min. Eluates with a tR of 25.7 and 29.5 min were collected by the technique of heart cutting and independently reinjected in the recycling mode.3 Suitable chromatographic conditions consisted of an analytical RP-18 column (4.6 × 250 mm, 5 μM); flow 0.5 mL/min; mobile phase MeOH. This analysis and the techniques of peak shaving and heart cutting3 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 1.82 × 10−4 % on a dry weight basis, and that from fraction IV was 1.9 × 10−3 % on a dry weight basis. Batatin VIII (1): colorless microcrystals; mp 115−120 °C; [α]D −19 (c 0.1 MeOH); 1H and 13C NMR, see Table 1; negative FABMS m/z 1235 [unit A, C63H109O24]−, 1053 [1235 − C12H22O]−, 965 [1053 − C4H7O − H2O]−, 819 [965 − C6H10O4]−, 691, 545, 417; ESIMS m/z 2472.3990 [M − H]− (calcd for C124H215O48, 2472.4382). Batatin IX (2): colorless microcrystals; mp 108−112 °C; [α]D −41 (c 0.1 MeOH); 1H and 13C NMR, see Table 2; negative FABMS m/z 1249 [unit A, C63H109O24]−, 1103 [1249 − C6H10O4]−, 1067 [unit B − C9H6O]−, 1019 [1249 − C5H8O − C6H10O4]−, 921 [1067 − C6H10O4]−, 837 [921 − C5H8O]−, 691, 545, 417; ESIMS m/z 2448.3403 [M − H]− (calcd for C123H203O48, 2448.3443). Batatin X (3): colorless microcrystals; mp 125−126 °C; [α]D −42 (c 0.1 EtOH); 1H and 13C NMR, see Table 3; negative FABMS m/z 2445 [M − H]−, 1223 [unit B, C61H107O24]−, 1221 [unit A, C61H105O24]−, 1137 [1221 − C5H8O]−, 991 [1137 − C6H10O4]−, 837 [991− C10H18O]−, 545, 417, 271; ESIMS m/z 2444.3975 [M − H]− (calcd for C122H211O48, 2444.4069). Batatin XI (4): colorless microcrystals; mp 118−120 °C; [α]D −57 (c 0.1 EtOH); 1H and 13C NMR, see Table 4; negative FABMS m/z 2501 [M − H]−, 2417 [M − H − C5H8O]−, 1251 [unit B, C63H111O24]−, 1249 [unit A, C63H109O24]−, 1165 [1249 − C5H8O]−, 1067 [1249 − C12H22O]−, 921 [1137 − C6H10O4]−, 837 [921 − C5H8O]−, 545, 417, 271; ESIMS m/z 2500.4503 [M − H]− (calcd for C126H219O48, 2500.4695).
were visualized by spraying with 3% CeSO4 in 2 N H2SO4 followed by heating. Plant Material. The tuberous roots of the white-skinned cultivar of I. batatas were collected in plantations in San Nicolás, Salvatierra, 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), Instituto de Biologiá UNAM. Extraction and Isolation. The powdered dry roots of the whiteskinned 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 CH2Cl2 in n-hexane, Me2CO in CH2Cl2, and MeOH. The process was monitored by TLC (CHCl3−MeOH−H2O, 6:4:1), and a total of 36 fractions (150 mL each) were collected and combined in eight resin glycoside-containing fractions with an Rf 0.5 (I−VIII eluted with CH2Cl2−Me2CO−MeOH, 6:3:1). All fractions were partially purified by passing through activated charcoal to eliminate pigmented residues. Previoulsy isolated major resin glycosides from this plant species, batatins I−IV4,5 (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; 5 mm, 4.6 × 250 mm) with an isocratic elution of CH3CN−MeOH and a flow rate of 1 mL/min. Fractions III (1.46 g; 1.46 × 10−1 % on a dry weight basis) and IV (1.12 g; 1.12 × 10−1 % on a dry weight basis) were selected for further recycling HPLC separation of minor constituents. Recycling HPLC Separation. These analytical separations were done on a Symmetry RP-18 column (Waters; 5 mm, 4.6 × 250 mm) with an isocratic elution of various proportions of CH3CN−MeOH 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 was isocratic with MeOH−CH3CN (9:1) using a flow rate of 9 mL/ min. The eluate with a tR of 11.7 min was collected by the technique of heart cutting and reinjected in the apparatus operated in the recycle F
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μBondapak NH2 column (3.9 × 300 mm; 10 μm); elution, CH3CN− H2O (4:1); flow rate 1 mL/min. Simonic acid B was identified by coelution with an authentic sample (tR 14.6 min) previously isolated from I. pes-caprae.14d
Alkaline Hydrolisis of Resin Glycoside Fraction. A solution of fractions III and IV (10 mg, each) in 5% KOH−H2O (2 mL) was refluxed at 95 °C for 2 h. The reaction mixture was acidified to pH 4.0 and extracted with CHCl3 (10 mL). The organic layer was washed with H2O, dried over anhydrous Na2SO4, evaporated under reduced pressure, and directly analyzed by GC-MS and comparison of their spectra and retention times with those of authentic samples.7 All analytical standards were purchased with a purity of >98%: 2methylpropanoic acid (240168, Aldrich); 2-methylbutanoic acid (193070, Aldrich); cinnamic acid (C80857, Sigma); n-decanoic acid (C1875, Sigma); n-dodecanoic acid (L4250, Sigma). Four peaks were detected for fraction III: 2-methylpropanoic acid (tR 4.0 min), m/z [M]+ 88 (10), 73 (27), 60 (3), 55 (5), 45 (7), 43 (100), 41 (40), 39 (10), 29 (6), 27 (24); 2-methylbutanoic acid (tR 7 min), m/z [M]+ 102 (3), 87 (33), 74 (100), 57 (50), 41 (28), 39 (8); cinnamic acid (tR 16.5 min), m/z [M]+ 148 (100), 147 (96), 131 (25), 103 (40), 102 (20), 77 (25), 74 (8), 51 (20), 50 (8), 39 (5), 38 (4); and ndodecanoic acid (tR 18 min), m/z [M]+ 200 (15), 183 (2), 171 (18), 157 (40), 143 (20), 129 (48), 115 (20), 101 (15), 85 (33), 73 (100), 60 (80), 57 (30), 55 (47), 43 (30). Three peaks were detected for fraction IV: 2-methylbutanoic acid (tR 7.2 min), m/z [M]+ 102 (3), 87 (33), 74 (100), 57 (50), 41 (28), 39 (8); n-decanoic acid (tR 14.6 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), 39 (6); and n-dodecanoic acid (tR 17.8 min), m/z [M]+ 200 (15), 183 (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 follows: a solution of benzyl alcohol (10.5 mg) in CH2Cl2 (1 mL), containing dicyclohexylcarbodiimide (3 mg) and 4-dimethylaminopyridine (1 mg), was added to the mixture of carboxylic acids.19 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 spectroscopic constants registered for the eluate with tR 8.39 min were identical in all aspects to those previously reported19,20 for (S)(+)-benzyl α-methylbutyrate: oil, [α]D +10. The n-butanol-soluble residue (6 mg) extracted from the saponification aqueous phase was subjected to preparative HPLC on a Waters μBondapak NH2 column (7.8 × 300 mm; 10 μm). The elution was isocratic with CH3CN−H2O (4:1), using a flow rate of 1 mL/min and a sample injection of 500 μL (5 mg/mL). This procedure yielded the same glycosidic acid for the two fractions, which was identified as simonic acid B by comparison of its physical constants and NMR data with published values.13,14 Both residues were methylated with CH2N2 to yield 9.5 mg of a white powder: mp 113−115 °C; [α]D −83 (c 1.0, MeOH) HRFABMS m/z 1015.5322 [M − H]− (calcd for C47H83O23 requires 1015.5325). The glycosidic acid methyl ester was further acetylated (Ac2O−pyridine, 2:1) to give a residue (9.3 mg), which was purified on RP-18 HPLC (7 μm, 19 × 300 mm; CH3CN−MeOH, 4:1; flow rate 8 mL/min). Eluates across the peak with tR values of 10.8 min afforded the peracetylated derivative of simonic acid B methyl ester, which was identified by coelution with an authentic sample and comparison of its physical constants and NMR data with published values:14c mp 81−85 °C; [α]D −37 (c 0.2, MeOH); 13C NMR (pyridine-d5, 125 MHz) 99.8 (CH, Fuc-1), 99.7 (CH, Rha‴-1), 99.2 (CH, Rha″-1), 99.1 (CH, Rha1), 97.4 (CH, Rha′-1), 13.9 (CH3, Jla-16), 33.9 (CH2CO2), 50.8 (OCH3), 78.0 (CH, Jla-11), 173.6 (C, Jla-1). Alkaline Hydrolisis of 1−4. Each individual compound 1−4 (3 mg) was submitted to an alkaline hydrolysis following the same procedures described above. The organic layer from extraction was directly analyzed by GC-MS. For compound 1, two peaks were detected, 2-methylpropanoic acid (tR 4.0 min) and n-dodecanoic acid (tR 17.8 min); for 2, the peaks for 2-methylbutanoic acid (tR 7 min) and cinnamic acid (tR 16.5 min) were identified; and compounds 3 and 4 afforded methylbutanoic acid (tR 7.2 min) as well as n-decanoic acid (tR 14.6 min) and n-dodecanoic acid (tR 17.8 min), respectively. The aqueous phases were individually dried and analyzed by HPLC:
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ASSOCIATED CONTENT
* Supporting Information S
Spectra for batatins VIII−XI (1−4): negative FABMS, negative ESIMS, 1H and 13C NMR, COSY, TOCSY, HSQC, HMBC. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +5255 56225288. Fax: +5255 56225329. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by Dirección General de Asuntos del Personal Académico, UNAM (IN212813), and Consejo Nacional de Ciencia y Tecnologiá (220535). D.R.-R. received graduate scholarships from CONACyT. Thanks are due to G. ́ Duarte (USAI, Facultad de Quimica, UNAM) for the recording of mass spectra and to Dr. M. Fragoso-Serrano (Departamento ́ de Farmacia, Facultad de Quimica, UNAM) for HPLC technical assistance.
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REFERENCES
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H
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