Structure and Absolute Stereochemistry of Syphonoside, a Unique Macrocyclic Glycoterpenoid from Marine Organisms Margherita Gavagnin,*,† Marianna Carbone,† Pietro Amodeo,† Ernesto Mollo,† Rosa Maria Vitale,‡ Vassilios Roussis,§ and Guido Cimino† Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, 80078 Pozzuoli (Naples), Italy, Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Naples, Italy, and Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, UniVersity of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece
[email protected] ReceiVed March 8, 2007
The glycoterpenoid syphonoside (1) is the main secondary metabolite of both the marine mollusk Syphonota geographica and the sea-grass Halophila stipulacea, two Indo-Pacific species migrated to the Mediterranean Sea through the Suez Canal. The structure and the absolute stereochemistry of 1, which displays unique structural features, has been accomplished by using a combination of spectroscopic techniques, degradation reactions, and conformational analysis methods. Compound 1 was able to inhibit high density induced apoptosis in a number of human and murine carcinoma cell lines.
Introduction Marine organisms show a great variety in the structure and biological activity of their secondary metabolites,1 many of which play a chemical mediation role in defense, communication, and reproduction.2 Among marine organisms, mollusks and, in particular, gastropods of the subclass Opisthobranchia have been recognized as a very promising sources of new bioactive molecules.3-6 Most of the natural products from opisthobranchs * Author to whom correspondence should be addressed. Fax + 39 0818041770; Tel +39 0818675094. † Istituto di Chimica Biomolecolare - CNR. ‡ Istituto di Biostrutture e Bioimmagini - CNR. § University of Athens.
(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2007, 24, 31-86; and earlier articles in this series. (2) Paul, V. J. Ecological Roles of Marine Natural Products; Comstock Publishing Associates: Ithaca, NY, 1992. (3) Cimino, G., Gavagnin, M., Eds. Molluscs, From Chemo-ecological Study to Biotechnological Application; Springer: Berlin, 2006; Vol. 43. (4) Karuso, P. In Bioorganic Marine Chemistry; Scheuer P. J., Ed.; Springer-Verlag: Berlin, 1987; Vol. 1, pp 31-60. (5) Cimino, G.; Fontana, A.; Gavagnin, M. Curr. Org. Chem. 1999, 3, 327-372.
have a dietary origin, whereas a significantly smaller number of compounds are biosynthesized de noVo.4-7 The “sea hares” (family Aplysiidae) are a group of opisthobranchs frequently found in shallow waters that generally feed on algae from which they sequester secondary metabolites. Consequently, most of chemicals reported from sea hares are typical algal metabolites or derivatives of them.6,8 In the course of our investigations on natural products from opisthobranchs,5-7 we have recently studied the chemical constituents of the skin9 of a Mediterranean population of the aplysiid Syphonota geographica, which is a circumtropical species10 first recorded in the Indo-Pacific Ocean. In this article it is suggested for the first time that there is a (6) Cimino, G.; Ciavatta, M. L.; Fontana, A.; Gavagnin, M. In BioactiVe Natural Products: Isolation, Structure Elucidation and Biology Properties; Tringali, C., Ed.; Taylor and Francis: London, UK, 2001; pp 577-637. (7) Cimino, G.; Fontana, A.; Cutignano, A.; Gavagnin, M. Phytochem. ReV. 2004, 3, 285-307. (8) Carefoot, T. H. Oceanogr. Mar. Biol. Annu. ReV. 1987, 25, 167284. (9) Gavagnin, M.; Carbone, M.; Nappo, M.; Mollo, E.; Roussis, V.; Cimino, G. Tetrahedron 2005, 61, 617-621. (10) Eales, N. B. Bull. Br. Mus. (Nat. Hist.) Zool. 1960, 5, 267-404.
10.1021/jo0704917 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/20/2007
J. Org. Chem. 2007, 72, 5625-5630
5625
Gavagnin et al. TABLE 1. NMR Dataa,b for Syphonoside (1) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
δ 1H
m, J, Hz
δ 13C
mc
1.68 2.23 6.44 1.16 2.16 1.36 1.98 1.55 4.12 2.35 2.76 5.46 4.64 1.86 1.03 1.33 0.99
m m t,3 ddd, 3, 13, 14 bd, 13 m m m dd, 1, 8 bd, 15 dd, 9, 15 bt, 7 m s d, 7 s s
19.8 27.7 138. 5 144. 6 38.7 36.5
t t d s s t
29.5 35.8 45.7 46.6 81.1 42.4 141.1 123.4 62.4 16.4 19.2 169.2 21.7 13.8
long-range correlationsd
long-range correlationsd
position
δ 1H
m, J, Hz
δ 13C
mc
H3-19 H-3, H3-19 -
1′ 2′ 3′ 4′ 5′ 6′
5.51 3.37 3.43 3.37 3.59 4.27 4.54
d, 8 m t,9 m m m dd, 2, 12
95.5 72.0e 78.1 71.5e 76.3 63.9
d d d d d t
-
t d s d d t
H3-17 H3-17, H3-20 H-12a, H3-17 H3-19, H3-20 H-12a, H3-20, H-1′′′ H3-16
1′′ 2′′ 3′′ 4′′ 5′′
2.61 2.64 -
ABq, 14 s -
172.8 46.3 71.0 49.6 171.7
s t s t s
H2-15, H2-2′′ H2-4′′, H3-6′′ H2-2′′, H2-4′′, H3-6′′ H3-6′′ H2-6′, H2-4′′
s d t q q s q q
H-11, H2-12, H2-15, H3-16 H-12a, H-15, H3-16 H-12a, H-14 H-3, H-1′ H-10 H-10
6′′
1.46
s
26.9
q
H2-2′′, H2-4′′
1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′
4.30 3.20 3.40 3.40 3.28 3.71 3.91
d, 8 m m m m dd, 2, 12 dd, 6, 12
102.3 75.2 78.6 74.0 77.8 63.1
d d d d d t
H-11, H-2′′′ -
a Bruker DPX-300, DPX-600, and AVANCE 400 MHz spectrometers, CD OD, chemical shifts (ppm) referred to CH OH (δ 3.34) and to CD OD (δ 3 3 3 49.0). b Assignments made by 1H-1H COSY, 2D-TOCSY, HSQC, and HMBC. c By DEPT sequence. d HMBC experiments (J ) 10 Hz). e Assignments may be reversed.
trophic relationship between the mollusk and the invasive IndoPacific sea-grass Halophila stipulacea,11,12 since specimens of the sea-grass were identified in the stomach of the mollusks.9 The presence of both species along Mediterranean coasts is due to the phenomenon known as Lessepsian migration from the Indo-Pacific Ocean to the Mediterranean Sea through the Suez Canal. Here, we report on our investigation of the chemical constituents of the sea-grass and the digestive gland of the mollusk, both containing a main secondary metabolite, syphonoside (1). The structure determination and the assignment of the absolute stereochemistry of this unique compound possessing a novel macrocyclic glycoterpenoid skeleton were accomplished through a combination of spectroscopic techniques, degradation experiments, and conformational analysis methods.
a trisubstituted isolated double bond and an R,β-unsaturated double bond, respectively. Finally, a vinyl methyl signal at δ 1.86 (H3-16) and four methyl signals at δ 0.99 (H3-20), 1.03 (H3-17), 1.33 (H3-19), and 1.46 (H3-6′′) accounted for the proton spectrum. Detailed analysis of the 2D NMR spectra led to the recognition of four distinct partial structures in the carbon skeleton: a terpenoid acyl moiety, two glycopyranosyl units, and a dicarboxylic acid residue. The spectral data in the terpenoid part indicated the presence of a clerodane skeleton oxidized at C-11, C-15, and C-18, exhibiting a glycosylated secondary hydroxyl, an esterified allylic primary hydroxyl, and an R,β-unsaturated ester function, respectively.
Results and Discussion Spectral Analysis. The molecular formula C38H58O17, indicating 10 degrees of unsaturation, was deduced by the sodiatedmolecular ion at 809.3545 m/z in the HRESI mass spectrum. In the 13C NMR spectrum, 31 signals were observed between 13.8 and 102.3 ppm, corresponding to sp3 carbons along with 7 signals in the region of 123.4-172.8 ppm which were attributed to two double bonds and three ester carbonyl groups (Table 1). The remaining five degrees of unsaturation were thus assigned to five rings. The 1H NMR spectrum of compound 1 contained two diagnostic doublets at δ 5.51 (J ) 8 Hz) and δ 4.30 (J ) 8 Hz), which were assigned to two anomeric protons of β-glycosyl residues, along with a series of signals in the region of 3.20-4.64 ppm that could be attributed to protons linked to oxygen bearing carbons. Two olefinic signals observed at δ 5.46 (H-14) and at δ 6.44 (H-3) indicated the presence of (11) Lipkin, Y. Isr. J. Bot. 1975, 24, 198-200. (12) Boudouresque, C. F.; Verlaque, M. Mar. Pollut. Bull. 2002, 44, 32-38.
5626 J. Org. Chem., Vol. 72, No. 15, 2007
Careful proton and carbon NMR analyses (Table 1) led us to the identification of the dicarboxylic acid moiety as 3-hydroxy-3-methyl-glutaric acid and of both glycosyl units as β-glucose. The complete spin sequence for the two glucosyl residues was determined as reported in Table 1 by detailed analysis of 1H-1H COSY and 2D-TOCSY spectra that allowed the connection between the anomeric proton and the methylene group in each glucose moiety. In particular, diagnostic crosspeaks in the 2D-TOCSY experiment between the anomeric proton H-1′ (δ 5.51) and H-5′ (δ 3.59) clearly indicated that
Syphonoside, a Unique Macrocyclic Glycoterpenoid
the same glucose unit exhibits both C-1′ and C-6′ engaged in ester linkages (H-1′ and H2-6′ resonated at lower fields with respect to typical values) whereas the second glucose, which is linked to the residue through a common glycosyl linkage (H-1′′′ resonated at δ 4.30), displays the primary hydroxyl group at C-6′′′ free. Analysis of the HMBC experiment allowed the connection of the four distinct parts leading to the macrocyclic framework depicted in structure 1. Diagnostic long-range correlations were observed between C-11 (δ 81.1) and H-1′′′ (δ 4.30), C-18 (δ 169.2) and H-1′ (δ 5.51), C-5′′ (δ 171.7) and H2-6′ (δ 4.27 and 4.54), C-1′′ (δ 172.8) and H2-15 (δ 4.64). These data were in agreement with the proposed structure where the R,β-unsaturated acyl function (C-18) of the diterpene moiety esterifies the hydroxyl group at anomeric position (C-1′) of a β-glucose unit, the primary hydroxyl function (6′-OH) of which is in turn linked to the carboxyl group (C-5′′) of 3-hydroxy-3methyl-glutaric acid, further esterified at C-1′′ with a primary hydroxyl function (15-OH) of the same diterpene. An additional glucose is connected by a glycosyl linkage to a secondary hydroxyl function (11-OH) of the diterpene. The relative stereochemistry of the clerodane diterpene part was determined by analysis of the carbon chemical shifts and comparison with model clerodanes. In order to accomplish such a comparative study, compound 1 was submitted to alkaline methanolysis leading to three fragments: a) a glucosyl terpene 2; b) the dimethyl ester of 3-hydroxy-3-methylglutaric acid, and c) glucose. Compound 2 was acetylated to afford the β-pentaacetyl-derivative 3, which was fully characterized with the exception of the stereochemistry at C-11.
The trans-junction between the two rings of the clerodane skeleton was indicated by the diagnostic carbon chemical shift of the angular methyl H3-19 (δ 21.7), since the corresponding methyl in cis-clerodanes resonates at approximately 10 ppm lower fields.13 The equatorial orientation of H3-17 was inferred by the chemical shift of C-6 (δ 36.5), taking into consideration that in trans-clerodane models this carbon resonates at higher fields, when the substituent at C-8 is axial. The relative stereochemistry at C-9 was suggested by both biosynthetic considerations and the carbon value of the axially oriented C-20 (δ 13.8), which was further deshielded by the presence of the glucosylated hydroxyl group at C-11. Furthermore, a series of NOE effects were observed between H-10 and H-8, H-11 and H2-12, and between the two angular methyl groups H3-19 and H3-20. The identity of 3-hydroxy-3-methylglutaric acid, recovered as the dimethyl ester from the ether phase of the alkaline methanolysis of 1, was confirmed by LC-ESIMS comparison with an authentic sample. The glucose moiety esterified at both C-1′ and C-6′ was recovered from the aqueous phase of the (13) Nogueira, R. T.; Shepherd, G. J.; Laverde, A., Jr.; Marzaioli, A. J.; Imamura, P. M. Phytochemistry 2001, 58, 1153-1157.
alkaline methanolysis of 1 and was directly treated with benzoyl chloride to give the mixture of the corresponding R- and β-penta-benzoyl derivatives, which were subsequently separated. The circular dicroism profiles of both epimers were compared with those of authentic R- and β-penta-benzoyl derivatives prepared from D-glucose,14 indicating the D absolute stereochemistry of the glucose linked at C-18 of the terpene moiety in syphonoside (1). In order to establish the stereochemistry of the glucose unit attached at C-11 of the clerodane moiety, glucosyl terpene 2 was further treated with HCl/MeOH to give a mixture of R- and β-methyl glucopyranoses, that was further benzoylated. The CD curves of the purified benzoyl derivatives were identical with those of the corresponding compounds obtained from benzoylation of commercial R- and β-methylD-glucopyranoses,14 thus indicating the D absolute stereochemistry of the sugar unit linked at C-11 of compound 1. Conformational Analysis. Once the absolute stereochemistry of both glucose units and the relative stereochemistry of the bicyclic clerodane substructure were assigned, a conformational study was undertaken to establish the absolute stereochemistry at C-11, C-3′′, and of the clerodane rings of syphonoside (1) which were not possible to be determined by direct spectroscopic methods. The eight possible diastereomers, obtained by permutation of absolute chiralities of C-11 and C-3′′and of the overall clerodane skeleton, were labeled with a three-letter code, based on R/S chirality of C-8, C-11, and C-3′′ atoms, respectively. Structural determination by simulated annealing followed by energy minimization (SA/EM),15 with restraints derived from NOESY spectra, was performed on all eight possible diastereomers, followed by a compared analysis of restraint violations. Sixteen strong- and six medium-intensity peaks were translated into 1.7-3.0 Å and 1.7-4.0 Å allowed distance ranges, respectively. The restraints involving the D-glucosyl residues either are directly violated in some diastereomers or induce violations in other restraints involving their partner protons, thus allowing an unambiguous identification of the absolute stereochemistry of syphonoside (Table 2): (a) Systematic violations of two strong distance restraints, corresponding to H-11/H2-1 and H-11/H3-16 NOE cross-peaks, were observed in both RS(R/S) and SR(R/S) diastereomers bundles, leaving us with only four out of eight possible diastereomers: RR(R/S) and SS(R/S). (b) Observed strong cross-peaks between methyl hydrogens of C-6′′ and both C-2′ and C-4′ protons of the sugar moiety helped us to assign the absolute chirality of the C-3′′ atom as R. In fact only in this case C-6′′ methyl hydrogen atoms satisfy their distance restraints with H-2′ and H-4′ sugar hydrogens. (c) The SSR diastereomer can be excluded after analysis of overall interproton distances within the final energetically selected 10 structure bundles. In fact, several short distances in the SSR bundle (e.g., H-3/H-2′, H-2′′′/H2-20) do not exhibit a counterpart in NOE cross-peaks, while applied distance restraints are violated either in their upper or lower limits (e.g., H-6′′/H3/H-6′′ distance in models is systematically 4.0 Å). The latter were used for analysis, but not imposed as restraints. Lowercase is used in the “violating diastereomers” column when violation is only detected in 60% of the calculated structures.
have systematic violations of 1 Å at level of H3-6′′/H-2′/H-4′ distance restraints, thus highlighting the existence of a coupling among distance restraints involving all the chiral centers. So, only RRR implying an ent-clerodane skeleton provided a 10-structure bundle that converges to a single conformation and exhibits both no appreciable restraint violation and a counterpart of all short distances in its NOE strong peak list. Structural features of 1 are dominated by the conformation of its 22-membered ring, including one glucose ring and a clerodane ring system and being responsible for 17 out of the 19 conformational degrees of freedom of the whole molecule (excluding smaller-ring flexibility and trivial OH and CH3 rotations). The macrocycle conformation of the main (1) conformer (Figure 1), in analogy with the “chair” conformation of six-membered rings, can be described as “sofalike”. Here, the “chairlike” shape derives from two mutually parallel twoatom segments (yellow in Figure 1) while two nine-atom almost parallel strands (green in Figure 1) in an all-trans conformation (except for a 90° bulge in O-15-C1′′-C2′′-C3′′ torsion) replace the single “tip” atoms of six-membered rings. Looking at the 3D molecular surface, the macrocycle shows a rather planar shape curved at the borders, with the external glucose ring almost perpendicular to the macrocycle plane. So, the whole molecule vaguely resembles a turtle in shape, with the “head” provided by the external glucose ring, the “shell” by the macrocycle backbone plus the clerodane unsaturated ring, and “limbs” by the saturated clerodane ring, internal glucose ring, C-16 methyl group, and nonbackbone atoms of 3-hydroxy-3methylglutaric acid (Figure 1). Polar groups are concentrated in the “head” and “hind limbs”, the remainder of the molecule being mostly apolar. The “lower-shell” region is concave with a small, deep cavity in the middle, whose bottom is provided by C-3, C-4, and O-1′ atoms (Figure 1, f). 5628 J. Org. Chem., Vol. 72, No. 15, 2007
FIGURE 1. Ball-and-stick and surface representation of syphonoside (1) best conformer. Six views of the conformer are shown, obtained by sequential rotations around either x (horizontal) or y (vertical) axis, as indicated near each corresponding arrow. Only heavy atoms are shown as spheres colored according to atom types. Corresponding bonds are represented as half-sticks colored according to atom types, except for 22-atom ring bonds, where yellow is used for “chairlike” regions and green for extended strands. The half-transparent shaded solventaccessible molecular surface is also shown, painted according to atom types.
Biological Activity of Syphonoside (1). Syphonoside was submitted to cytotoxicity evaluation16 on a number of human and murine cell lines (MCF7 derived from a mammary adenocarcinoma, PC3, derived from a prostate adenocarcinoma, A431 derived from epidermoid carcinoma, HeLa derived from cervix adenocarcinoma, HT29 colon cancer cell line, NT2, teratocarcinoma, P19 murine embryonic carcinoma stem cell line). None of the concentrations of 1 (400-0.2 µM) was shown to be cytotoxic to the cell lines used in this study, even after long-term exposure (72 h). Surprisingly, 1 was found to decrease the number of floating cells especially in A431 and P19 cells with effective concentrations being 440 ( 35 nM and 360 ( 42 nm, respectively. Therefore, although syphonoside (1) did not exhibit any cytotoxic activity, it was able to inhibit high density induced apoptosis. The suppression of apoptosis resulted in a prolonged viability and maximum cell density. Conclusions The chemical analysis of the pair S. geographica/H. stipulacea led to the isolation of the same macrocyclic glycoterpenoid, syphonoside (1), from both organisms, confirming the trophic relationship between the mollusk and the sea-grass. This compound displays unique structural features among marine natural products, where analogies could be envisaged with plant secondary metabolites, thus representing an interesting conjunction point between marine and terrestrial natural products. Syphonoside is characterized by a novel macrocyclic skeleton (16) Iliopoulou, D.; Mihopoulos, N.; Vagias, C.; Papazafiri, P.; Roussis, V. J. Org. Chem. 2003, 68, 7667-7674.
Syphonoside, a Unique Macrocyclic Glycoterpenoid
containing an ent-clerodane diterpenoid moiety, two D-glucose units, and a 3-hydroxy-3-methylglutaric residue. This structure displays an original conformation exhibiting two long nine-atom extended strands joined by two “cyclohexane-chairlike” twoatom junctions. The resulting three-dimensional shape of this 22-membered ring is a moderately curved turtle-shell structure characterized by an inhomogeneous distribution of polar vs hydrophobic regions. In fact, a prevalently polar rim (also including the perpendicular extracyclic glucose moiety) surrounds a mostly hydrophobic central region, also including a cavity that could easily host either an all-hydrophobic group or a small hydrophobic chain ending with a H-bond donor group (pointing toward O1′ atom). This cavity, together with the two long, almost parallel eight-residue strands, could represent interaction sites with other biomolecules and, at the same time, provide potential technological applications. In a preliminary cytotoxicity test, syphonoside (1) did not exhibit any cytotoxic activity against the employed cell lines but was able to inhibit high density induced apoptosis. Therefore, this compound may play an important role in regulating cell survival and cell death under specific conditions but additional studies are required for the elucidation of this role. Experimental Section General experimental procedures were performed as previously described.9 Collection and Extraction. The collection and the extraction of S. geographica individuals (seven animals) has already been reported.9 A sample of H. stipulacea was collected from the same site as the mollusk in November 2003 and was frozen at -20 °C. This material (dry residue 103.0 g) was extracted according to the standard procedures.9 The n-BuOH layer from the acetone extract was evaporated under reduced pressure to afford 1.31 g of crude extract. Analogously, the n-BuOH layer of the digestive gland extract of the mollusk was evaporated to dryness, yielding 618.0 mg of residue. Both extracts were analyzed by TLC (chloroform/ methanol in variable ratios) and compared. Isolation of 1. The n-BuOH extract (618 mg) of the mollusk was subjected to Sephadex LH-20 chromatography using a mixture of chloroform/methanol in 1:1 ratio as eluent, to yield a fraction (115 mg) which was further purified on preparative TLC (SiO2, 7:3, CHCl3:MeOH) to give 49.2 mg of pure metabolite 1 (ca. 8% of the extract). An aliquot (500 mg) of the n-BuOH soluble part of the extract of the sea-grass was chromatographed under the abovedescribed conditions to give18.8 mg of pure 1 (ca. 4% of the extract). Syphonoside (1). Rf ) 0.50 (7:3, CHCl3:MeOH); [R]D -7.0 (c ) 0.54, MeOH); CD (EtOH) [θ]209 2754, [θ]237 -8574; IR (liquid film): 3500, 2887, 1714 cm-1; UV (MeOH): λmax 206 ( ) 11 660); 1H and 13C NMR in Table 1; HRESIMS (m/z): (M + Na)+ calcd for C38H58O17Na, 809.3572; found, 809.3545. Alkaline Methanolysis of 1. A 25 mg amount of 1 was dissolved in a 0.5 M solution of MeONa in MeOH (2.5 mL), and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was first chromatographed on a DOWEX-50W column eluted with methanol (100 mL) and then extracted with Et2O to afford 15.0 mg and 4.5 mg of residue from the ethereal and aqueous phases, respectively. An aliquot of the ethereal phase was analyzed by HPLC (Phenomenex, Kromasil 5 µm 100A C18; eluent CH3CN/H2O 4:6; flow 1 mL/min). The dimethyl ester of 3-hydroxy3-methylglutaric acid was identified by comparison of its retention time (tR 4 min) with that of an authentic sample. An additional LC-ESIMS analysis was conducted confirming the identity of 3-hydroxy-3-methylglutaric dimethyl ester. The remaining ether soluble residue was purified on preparative TLC (SiO2, 9:1, CHCl3: MeOH) to give 12.0 mg of compound 2 (Rf ) 0.4); [R]D -4.8 (c
) 0.13, CHCl3); IR (liquid film): 3390, 2930, 1725 cm-1; UV (MeOH): λmax 206 ( ) 6700); selected 1H NMR values (300 MHz, CD3OD): δ 6.57 (1H, t, J ) 3 Hz, H-3), 5.61 (1H, app. t, J ) 7 Hz, H-14), 4.36 (1H, d, J ) 8 Hz, H-1′′), 1.77 (3H, bs, H3-16), 1.33 (3H, s, H3-19), 0.99 (3H, d, J ) 6 Hz, H3-17), 0.90 (3H, s, H3-20); HRESIMS (m/z): (M + Na)+ calcd for C27H44O9Na, 535.2883; found, 535.2859. The aqueous phase from the workup was filtered on a Sep-Pak and dried to afford pure D-glucose [6.0 mg, Rf ) 0.4 (70:20:10, EtOAc:MeOH:H2O); [R]D 45.4 (c ) 0.37, H2O)]. The absolute stereochemistry of D-glucose was determined by analysis of 1H NMR and CD spectra of the corresponding benzoate derivatives.14 Compound 3. Glycosyl terpene 2 (2.0 mg) was dissolved in dry pyridine (2 mL), acetic anhydride (0.5 mL) was added to the resulting solution, and the reaction mixture was stirred overnight at room temperature. Pyridine was removed in Vacuo, and the residue was dissolved in diethyl ether and worked up as usual to afford pure 3 (1.8 mg), Rf ) 0.7 (95:5, CHCl3:MeOH); [R]D -41.4 (c ) 0.1, CHCl3); IR (liquid film): 2940, 1758, 1232 cm-1; UV (MeOH): λmax 206 ( ) 5250); 1H NMR (400 MHz, CDCl3): δ 6.52 (1H, app. t, J ) 3 Hz, H-3), 5.41 (1H, app. t, J ) 7 Hz, H-14), 5.16 (1H, app. t, J ) 9 Hz, H-3′′′), 5.00 (1H, app. t, J ) 9 Hz, H-4′′′), 4.93 (1H, dd, J ) 8, 9 Hz, H-2′′′), 4.62 (1H, d, J ) 8 Hz, H-1′′′), 4.75 (1H, m, H-15a), 4.46 (1H, m, H-15b), 4.21 (1H, m, H-6′′′a), 4.13 (1H, m, H-6′′′b), 4.09 (1H, m, H-11), 3.68 (3H, s, OMe), 3.61 (1H, m, H-5′′′), 2.40 (1H, m, H-12a), 2.22 (1H, m, H-6a), 2.20 (1H, m, H-12b), 2.17 (1H, m, H-2a), 2.12 (3H, s, OAc), 2.10 (1H, m, H-2b), 2.07 (3H, s, OAc), 2.04 (3H, s, OAc), 2.01 (3H, s, OAc), 1.98 (3H, s, OAc), 1.75 (3H, bs, H3-16), 1.69 (1H, m, H-1a), 1.57 (1H, m, H-1b), 1.55 (1H, m, H-8), 1.38 (2H, m, H2-7), 1.31 (1H, m, H-10), 1.30 (3H, s, H3-19), 1.08 (1H, m, H-6b), 0.84 (3H, s, H3-20), 0.81 (3H, d, J ) 7 Hz, H3-17); 13C NMR (300 MHz, CDCl3): δ 171.4 (s, OAc), 170.3 (s, 2C, OAc), 169.4 (s, 2C, OAc), 167.8 (s, C-18), 142.8 (s, C-4), 139.4 (s, C-13), 136.1 (d, C-3), 121.6 (d, C-14), 98.8 (d, C-1′′′), 77.4 (d, C-11), 73.1 (d, C-3′′′), 71.9 (d, C-2′′′), 71.4 (d, C-5′′′), 69.2 (d, C-4′′′), 62.4 (t, C-6′′′), 61.3 (t, C-15), 51.2 (q, OMe), 46.6 (d, C-10), 43.5 (s, C-9), 42.5 (t, C-12), 38.0 (s, C-5), 35.7 (t, C-6), 27.9 (t, C-7), 27.2 (t, C-2), 21.1 (2C, q, C-19 and OAc), 20.6 (4C, q, OAc), 18.8 (t, C-1), 17.5 (d, C-17), 16.2 (q, C-16), 12.5 (q, C-20); HRESIMS (m/ z): (M + Na)+ calcd for C37H54O14Na, 745.3411; found, 745.3430. Acid Methanolysis of 2. Compound 2 (10 mg) was dissolved in a 1 N HCl solution in MeOH (2 mL), and the obtained solution was stirred for 12 h at 40 °C. After the usual workup, the reaction mixture was dried and partitioned between CHCl3 and H2O/MeOH, 8:2. The aqueous layer was concentrated, filtered on a Sep-Pak cartridge, and dried. The 1H NMR spectrum of this fraction indicated that it was a mixture of R-and β-methyl glucopyranose, the absolute stereochemistry of which was determined by analysis of 1H NMR and CD spectra of the corresponding tetra-benzoate derivatives.14 Conformational Analysis of 1. Syphonoside structures were obtained by restrained SA/EM.15 Experimental NOE intensities were converted into 22 proton-proton distance restraints classified into two ranges: 1.7-3.0 Å and 1.7-4.0 Å, corresponding to strong/ strong-medium and medium/medium-weak NOE peaks, respectively. Weak and very weak interactions, as well as peaks corresponding to fixed distances in glucose rings, were not included. Methylene protons in the NMR spectra of syphonoside were not resolved, but they were described using an “ambiguous restraint” approach in which, for each restraint, all the possible distances between the two methylene protons and their NOE counterpart proton(s) were calculated and weighted by the inverse of the sixth power of their values. Calculations were performed with Sander J. Org. Chem, Vol. 72, No. 15, 2007 5629
Gavagnin et al. module of AMBER8,17 using AMBER GAFF parametrization18 and semiempirical AM1-BCC charges.19 Starting structures for all eight sampled syphonoside diastereomers were built with Ghemical 1.01.20 Conformational sampling was obtained by restrained simulated annealing (SA), including an implicit solvation model (RIWSA), based on a generalized Born21 with surface area contribution22 approach (GBSA). The starting structure of each syphonoside diastereomer underwent 50 SA cycles of 100 000 MD steps, where system temperature was linearly raised from 10 to 1200 K (step 1 to 5000) and then kept constant at 1200 K (step 5001 to 50 000), and finally linearly decreased down to 10 K (step 50 001 to 100 000). A time step of 1 fs, with no constraints or restraints on bond lengths, a nonbonded cutoff of 16 Å and a 0.05 fs time constant for heat bath coupling were used, with all other parameters set at their default values. A semiparabolic penalty function, with a force constant of 20 kcal mol-1 Å-2, was applied to interprotonic distances larger than upper-limit values. transAlkene bonds were forced into a trans orientation (ω ) 180°) by torsional constraints with a force constant of 50 kcal mol-1 operating (17) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D. A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.; Ross, W. S.; Kollman, P. A. AMBER 8, University of California, San Francisco, 2004. (18) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput. Chem. 2004, 25, 1157-1174. (19) Jakalian, A.; Bush, B. L.; Jack, D. B.; Bayly, C. I. J. Comput. Chem. 2000, 21, 132-146. (20) Rowley, C.; Hassinen, T. Ghemical, 2001-2002; licensed un GNU GPL, Copyright 2006 Tommi Hassinen, Kuopio, Finland (http:// www.bioinformatics.org/ghemical/ghemical/index.html). (21) Tsui, V.; Case, D. A. Biopolymers 2001, 56, 275-291. (22) Weiser, J.; Shenkin, P. S.; Still, W. C. J. Comput. Chem. 1999, 20, 217-230.
5630 J. Org. Chem., Vol. 72, No. 15, 2007
for deviations higher than 20° from the trans form. Final structures were energy minimized (EM) with 100 steps of steepest descent followed by a conjugate gradient method, down to a gradient norm value less than 10-3 kcal mol-1 Å-1 (thus leading to the final SA/ EM structure set), both with the same penalty function used for SA (REM) and under totally unrestrained conditions (UEM). MOLMOL23 program was used for structural analysis. Cytotoxicity Assays. Cytoxicity of 1 was evaluated according to MTT dye reduction assay.24-26
Acknowledgment. We are grateful to Mr. F. Castelluccio for his valuable technical assistance. Thanks are also due to Mr. R. Turco for drawings and to Mr. C. Iodice for spectrophotometric measurements. The NMR spectra were recorded at the ICB NMR Service, the staff of which is acknowledged. Particular thanks are due to Mrs. Dominique Melck for NMR spectra processing. The authors thank also Dr. Panagiota Papazafiri (University of Athens, Dept. of Biology) for her assistance with cytotoxicity evaluation. Supporting Information Available: MS and 1D- and 2D-NMR spectra of syphonoside (1), 1H NMR spectra of compounds 2 and 3, CD spectra of benzoyl glucose derivatives and additional experimental procedures. This material is available free of charge via the Internet at http:// pubs.acs.org. JO0704917 (23) Koradi, R.; Billeter, M.; Wu¨thrich, K. J. Mol. Graph. 1996, 14, 51-55. (24) Mossmann, T. J. Immunol. Methods 1983, 65, 55-63. (25) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589-601. (26) Experimental details are reported in Supporting Information.
