New isocopalane diterpene diester from a sub-Antarctic marine nudibranch

Two known isocopalane diterpene diacylglycerides 1 and 2 and one new isocopalane diterpene diacetate, (12 S , 13 R, 14 S )-isocopalan-13-ol-12,14-diacetate, 3 were isolated from an unidentified sub-Antarctic nudibranch. The structure and stereochemistry of the new minor metabolite 3 was established from spectroscopic data and recourse to biosynthetic arguments.


Introduction
Dorid nudibranchs, or sea slugs, (Order Nudibranchia, Suborder Doridina) are soft-bodied, often brightly coloured, shell-less marine opisthobranch molluscs with surprisingly few predators. 1he dorid nudibranch species occurring in the Southern Ocean surrounding Antarctic are no exception and, in common with their warmer water counterparts, are able to defend themselves against predators by exuding unpalatable secondary metabolites from specialized glands strategically located in their mantle tissue. 2,3However, while most warmer water nudibranch species sequester their toxic chemical defence metabolites from their diet of other marine invertebrates e.g.sponges and soft corals, the majority of Antarctic species are not restricted to a dietary source for their defensive metabolites and instead biosynthesize these metabolites de novo. 2 Clerodane, ent-labdane and isocopalane diterpenoid glyceryl esters clearly dominate the structural classes of chemical defence metabolites reported thus far from Antarctic dorid nudibranchs. 3,4As part of our ongoing investigation of the secondary metabolites either sequestered or biosynthesized de novo by southern African and Antarctic nudibranchs [5][6][7][8] we have extended our studies to include those nudibranchs occurring off Marion Island in the Southern Ocean (approximately 1800 km south east of South Africa).

Results and Discussion
A single large yellow nudibranch was dredged from a depth of 115m near Marion Island (46° 52′S, 37° 54′E) during early autumn, 2005.The nudibranch was initially frozen, steeped in acetone and the acetone extract concentrated in vacuo to yield a brown gum (380 mg).The brown gum was subjected to initial polymeric reversed-phase separation followed by flash chromatography using a diol solid support and finally normal phase HPLC to yield the two known isocopalane diterpene diacylglycerides 1 (29 mg, Isomers 1 and 2 have been previously reported from two nudibranch species, viz.Anisodoris fontaini (originally erroneously identified as Archidoris carvi) 9,10 collected off Patagonia (southern Argentina) and Doris verucossa obtained from the Mediterranean. 11The closely related diastereoisomers 4 and 5 were first identified in extracts of the British Columbian nudibranch Archidoris montereyensis 12 and were also later isolated from the Patagonian nudibranch Archidoris tuberculata. 9Comparison of the CD data of 1 with analogous data obtained for its stereoisomer 4, and the respective oxidation and reduction products of both compounds was originally used to unequivocally confirm that 4 was a diastereoisomer of 1, possessing an ent-isocopalane as opposed to an isocopalane diterpene skeleton and sharing the same (S-2') absolute configuration in the glyceryl side chain. 9An (S) configuration at the single chiral centre in the glyceryl side-chain is a common feature amongst the plethora of 1,3diacylglycerides isolated thus far from marine molluscs. 13tereoselective syntheses of 1, 2, 4 and 5 have clearly confirmed the positive and negative signs of the specific rotation for the isocopalane diacylglyceride and ent-isocopalane diacylglyceride series respectively (Table 1). 13,14Fontana et al. 14 attributed the discrepancy between the magnitude of the specific rotation originally reported for 2 isolated from Anisodoris fontaini and the value which they obtained for their synthetic product, to the errors often associated with measuring the optical rotation of diminishing small amounts (< 1 mg) of a marine natural product.
The molecular formula of 3 was established as C 24 H 40 O 5 from HRFABMS data ([M+H] + , m/z 409.2953, calcd.m/z 409.2954) and implied five degrees of unsaturation for this compound.A comparison of the 13 C and 1 H NMR data of 3 (Table 2) with those of 1 and 2 not only suggested that all three compounds shared the same tricyclic isocopalane skeleton but also confined the differences between 3 and the former two compounds to the substitution pattern around ring C.These differences were, first, the absence of the deshielded ∆ 12 olefin resonances in the 13 C NMR spectrum of 3 and, second, the absence of any signals in the NMR data of this compound that could be attributed to a glyceryl ester moiety.In addition, the combination of the two methyl singlets (δ H 1.65 and 1.72) in the 1 H NMR spectrum and two ester carbonyl signals (δ C 169.3 and 169.7) in the 13 C NMR spectrum of 3 corroborated the presence of two acetate moieties, thus accounting for the remaining two degrees of unsaturation and four of the five oxygen atoms required by the molecular formula.A vicinal COSY coupling between the diastereotopic, deshielded oxymethylene protons (δ H 4.25 and 4.27) and the methine proton H-14 (δ H 1.44) placed an esterified oxymethylene sidechain at C-14 (δ C 58.0).Further evidence for this assignment was provided by three bond HMBC correlations from the oxymethylene protons H 2 -15 to one of the acetate carbonyls (δ C 169.7) in addition to two quaternary carbons, C-8 and C-13 (δ C 13.7 and 74.6 respectively).Confirmation that both C-12 and C-13 were oxygenated was provided by the 13 C chemical shift of the deshielded C-13 resonance and a two bond HMBC correlation from the methyl protons (δ H 1.12, H 3 -16) to the similarly deshielded C-12 methine carbon (δ C 78.3).An HMBC correlation from ARKAT USA, Inc.
H-12 (δ H 5.01) to the ester carbonyl C-1" (δ C 169.3) placed the second acetate at C-12 and thus required the remaining oxygen substituent at C-13 to be a tertiary alcohol.The relative configurations at C-12, C-13 and C-14 were determined from NOESY data (Figure 1).Crucial NOESY correlations between H-9 (δ H 1.35) and H-12 (δ H 5.01) and between H-12 and H-14 (δ H 1.44) implied a cis relationship between these three axial methine protons and accordingly placed the substituents at C-12 and C-14 in α-equatorial positions.A further NOESY correlation between H-12 and H 3 -16 (δ 1.12) required the methyl and hydroxyl substituents at C-13 to be β-equatorial and α-axial respectively.The co-occurrence of 1, 2 and 3 in the Marion Island nudibranch suggests that all three compounds are products of a similar biosynthetic pathway and hence share the same isocopalane diterpene structure.Regrettably, the acetone solvent dehydrated and distorted the Marion Island nudibranch's radula (a chitinous ribbon of teeth used by most molluscs for feeding and by mollusc taxonomists for identification purposes) to such an extent that the nudibranch could not be positively identified.A tentative identification of the Marion Island nudibranch based on chemotaxonomic evidence was also inconclusive as isocopalane diterpenes are not confined to the genera Doris and Anisodoris.For example isocopalane 2'-monoglyceryl esters austrodorins A (6) and B (7) were isolated from Austrodoris kerguelenensis, 15 a large and ubiquitous Antarctic and sub-Antarctic nudibranch ranging in colour from white to yellow and a rich source of a wide variety of diterpene metabolites. 4The Kerguelen Islands whence A. kerguelenensis derives its name, are situated 800 km due east of Marion Island.

Conclusions
A new isocopalane diterpene diacetate, (12S, 13R, 14S)-isocopalan-13-ol-12,14-diacetate, has been isolated from a single dorid nudibranch dredged from 115m near Marion Island in the Southern Ocean.The stereochemistry assigned to this compound was implied from recourse to biosynthetic arguments.The research presented here represents the first investigation of the natural products isolated from a marine invertebrate collected off this remote sub-Antarctic island and identifies a new source of naturally occurring isocopalanes, a class of rare and apparently uniquely marine diterpenes. 16perimental Section General Procedures.NMR spectra were measured on a Brüker 600 MHz NMR spectrometer using standard pulse sequences.Chemical shifts are reported in ppm and are referenced to residual solvent resonances (C 6 D 6 δ H 7.15, δ C 128.02). 17  The nudibranch was exhaustively extracted with Me 2 CO, the extracts combined and the solvent removed under reduced pressure.

Figure 1 .
Figure 1.Key NOESY correlations observed in the NOESY spectrum of 3.

Figure 2 .
Figure 2. Photograph of nudibranch shortly after collection.