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Chemical investigations of the MeOH–CH 2 Cl 2 extract of Pseudopterogorgia elisabethae specimens collected in the islands of San Andres and Providencia, Colombian Caribbean, yielded four new diterpenes ( 1 , 3 , 5 , 7 ) along with seco-pseudopterosin J ( 8 ), and amphilectosins A ( 9 ) and B ( 10 ). The structures of the new compounds were established through spectral studies as an elisabethatriene analog named elisabethatrienol ( 1 ), 10-acetoxy-9-hydroxy- and 9-acetoxy-10-hydroxy-amphilecta-8,10,12,14-tetraenes (isolated as an interconverting mixture) ( 3 ), amphilecta-8(13),11,14-triene-9,10-dione ( 5 ), and a seco -pseudopterosin 7- O -α- l -fucopyranoside named seco -pseudopterosin K ( 7 ). Elisabethatrienol can be regarded as a biosynthetic intermediate leading to erogorgiaene.

The Bahamian octocoral Pseudopterogorgia elisabethae is the source of pseudopterosins, diterpene glycosides with potent anti-inflammatory activity. The first committed step in pseudopterosin biosynthesis comprises the cyclisation of the universal diterpene precursor geranylgeranyl diphosphate to elisabethatriene. This reaction is catalysed by elisabethatriene synthase, which was purified to homogeneity from a crude coral extract. This represents the first purification to apparent homogeneity of a terpene cyclase from any marine source. The reaction kinetics of elisabethatriene synthase was examined using a steady state approach with 3 H-labelled isoprenyldiphosphates varying in carbon chain length (C 10 , C 15 , C 20 ). For the reaction of elisabethatriene synthase with its natural substrate geranylgeranyl diphosphate, values of K m (2.3 × 10 − 6 M), V max (3.4 × 10 4 nM elisabethatriene * s − 1 ) and the specificity constant ( k cat / K m = 1.8 × 10 − 10 M − 1 * s − 1 ) were comparable with diterpene cyclases from terrestrial plants. Elisabethatriene synthase also catalysed the conversion of C 15 and C 10 isoprenyldiphosphate analogues to monoterpene and sesquiterpene olefins, respectively. Kinetic parameters indicated that substrate specificity and K m of elisabethatriene synthase decreased with decreasing isoprenoid carbon chain length. Furthermore, GC–MS analysis showed increased product diversity with decreasing isoprenoid carbon chain length.

Putative precursors in pseudopterosin biosynthesis, the hydrocarbons isoelisabethatriene (10) and erogorgiaene (11), have been identified from an extract of Pseudopterogorgia elisabethae collected in the Florida Keys. Biosynthetic experiments designed to test the utilization of these compounds in pseudopterosin production revealed that erogorgiaene is transformed to pseudopterosins A-D. Together with our previous data, it is now apparent that early steps in pseudopterosin biosynthesis involve the cyclization of geranylgeranyl diphosphate to elisabethatriene followed by the dehydrogenation and aromatization to erogorgiaene.

In the past, we have questioned the accuracy of the stereochemistry of elisabethatriene, a putative biosynthetic intermediate of pseudopterosins, in light of the configuration of elisabethatrienol isolated from Pseudopterogorgia elisabethae, which was represented as 1S,4R,9S,11S. We have reinvestigated the stereochemistry of elisabethatriene. Elisabethatriene with the reported 1S,4R,9R,11S configuration was synthesized starting from (-)-isopulegol in its enantiomeric form. The (1)H- and (13)C-NMR data of the synthesized compound differed from those reported for elisabethatriene. In addition to the fact that elisabethatriene is converted into pseudopterosins, this finding has allowed us to propose that elisabethatriene should have the 1S,4R,9S,11S stereochemistry, which is identical to that of elisabethatrienol.

The pseudopterosins are a family of diterpene glycosides isolated from the gorgonian coral Pseudopterogorgia elisabethae. These metabolites exhibit potent anti-inflammatory activity, and this review describes our efforts to elucidate their biosynthetic origin. A radioactivity-guided isolation was used to identify the terpene cyclase product. In addition, a detailed NMR-guided search for potential biosynthetic intermediates identified metabolites which were tested by incubating 3H-labeled analogues with a cell-free extract of the coral. All labeled metabolites were generated biosynthetically, and radiochemical purity was established by a combination of HPLC purification and derivatization. In summary, pseudopterosins are produced by a cyclization of geranylgeranyl diphosphate to elisabethatriene, aromatization to erogorgiaene, two successive oxidations to 7,8-dihydroxyerogorgiaene and a glycosylation to afford a seco-pseudopterosin as a key intermediate. A dehydrogenation leads to amphilectosins which undergo ring closures to yield the pseudopterosins.

Analysis of the terpene metabolites of Pseudopterogorgia elisabethae collected from the Florida Keys has resulted in the identification of a novel hydroxyquinone, elisabethadione (1), as well as new pseudopterosins and seco-pseudopterosins. Anti-inflammatory assays indicate that elisabethadione is more potent than the well characterized pseudopterosin A and E. This report also describes the co-occurrence of pseudopterosins and seco-pseudopterosins, diterpenes with amphilectane and serrulatane skeletons, respectively. This together with our previously described isolation of elisabethatriene as the sole diterpene cyclase product in P. elisabethae suggests that the amphilectane and serrulatane families of diterpenes are derived from the same geranylgeranyl diphosphate cyclase product.

The pseudopterosins are a family of diterpene pentosides isolated from the marine octocoral, Pseudopterogorgia elisabethae. These compounds possess non-steroidal anti-inflammatory and analgesic properties which have been shown to be greater than the industry standard, indomethacin. In our investigations, we are interested in examining the biosynthesis and enzymology of these compounds for the development of a biotechnological production method. We have isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. This has provided us with an assay to isolate the diterpene cyclase enzyme. The amino acid sequence of the purified diterpene cyclase will facilitate cloning and expression of the gene in a suitable host. In addition, we have identified over 25 novel diterpenes from one of our collections of P. elisabethae. Several of these compounds appear to be involved in pseudopterosin biosynthesis and are presently being evaluated as potential intermediates. These compounds have also been evaluated for anti-inflammatory activity and some possess greater activity than that of the pseudopterosins. We therefore propose a production method utilizing a combination of recombinant enzyme technology and synthetic methods/biocatalysis in order to produce one or more anti-inflammatory metabolites in P. elisabethae.