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Marine Drugs 2003, 1(4), 54-65;
Abstract: 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.
The pseudopterosins are a class of diterpene glycosides isolated from the sea whip Pseudopterogorgia elisabethae [1–3]. As with many marine natural products, diverse congeners of the pseudopterosins are found in different locations and there are presently fifteen known pseudopterosin derivatives (A–O). All of the known pseudopterosins contain the amphilectane skeleton with a glycosidic linkage at either C-9 or C-10.
The identity of the sugar and the degree of acetylation account for the additional structural variation of this family of diterpenes. Pseudopterosins A–D (1–4), from Sweetings Cay in the Bahamas, possess the amphilectane skeleton with an attached xylose sugar which is acetylated at different locations (Figure 1).
The seco-pseudopterosins A–D are a related group of compounds (5–8) belonging to the serrulatane class of diterpenes initially isolated from Pseudopterogorgia kallos in the Florida Keys . More recently, novel seco-pseudopterosins were reported to co-occur with pseudopterosins in P. elisabethae . The pseudopterosin and seco-pseudopterosin classes of diterpenes exhibit potent anti-inflammatory and analgesic activity [3, 5]. The pseudopterosins are pharmacologically distinct from typical NSAIDs and they appear to act by a novel mechanism of action [6, 7]. The commercial market for the pseudopterosins, presently as ingredients in a skin cream, indicates a need for the development of a sustainable supply of these compounds. Consequently, a continuing goal in our laboratory is to elucidate all steps in the biosynthetic pathway leading to the pseudopterosins.
We recently confirmed the identity of the diterpene cyclase product leading to the pseudopterosins as elisabethatriene (9) (Figure 2).
This hydrocarbon, with the serrulatane skeleton, was isolated from extracts of P. elisabethae collected in the Florida Keys and in Sweetings Cay, Bahamas. The utilization of 9 in pseudopterosin biosynthesis was confirmed through biosynthetic experiments . Erogorgiaene (11) was recently reported from a collection of P. elisabethae off Colombia . Given the structure of the pseudopterosin class of diterpenes and our report of the transformation of 9 to 1–4, it seems reasonable to suggest that 11 is an intermediate in this biosynthetic pathway. Further, it seems plausible that an endocyclic isomer of 9 such as 10 could be an intermediate in the conversion of 9 to 11.
This report describes the results of experiments directed at testing the hypothesis that isoelisabethatriene (10) and erogorgiaene (11) are early intermediates in pseudopterosin biosynthesis. Our approach was to identify these in our P. elisabethae extracts through the synthesis of standard samples of 10 and 11 from 9 and if present, test the compounds as metabolic intermediates.
Results and Discussion
Identification of Plausible Biosynthetic Intermediates
We have observed that the Bahamian populations (e.g. Sweetings Cay) of P. elisabethae contain much higher concentrations of pseudopterosins than P. elisabethae from the Florida Keys; the latter, however, exhibits a greater diversity of diterpene chemistry . A preliminary survey of the non-polar fraction of an extract of P. elisabethae from Sweetings Cay indicated a lack of hydrocarbons that were structurally related to the pseudopterosins and therefore, not likely involved in pseudopterosin biosynthesis. Given the diverse diterpene chemistry of Floridian specimens of P. elisabethae, we examined these collections for the presence of hydrocarbons such as 10 and 11.
To aid in the search for erogorgiaene (11), we synthesized a standard sample of 11 from 9 using Pd/C under nitrogen  (Scheme 1). We utilized this sample of 11 as a standard to screen for the presence of this compound in a hexane extract. This analysis revealed the presence of compound 11 in 0.2% of the 100 g extract. NMR analysis of this HPLC fraction confirmed the purity and identity of erogorgiaene.
Similarly, a standard sample of 10 was synthesized from 9, as previously described , and this was used to monitor its presence in Floridian P. elisabethae extracts. We began the search for naturally occurring compound 10 in P. elisabethae by obtaining a crude organic extract and partially purifying the extract using a silica flash column. After eluting the silica column with 100% hexanes, analysis of this non-polar fraction by HPLC indicated the presence of a compound with the same retention time and UV absorption profile (λmax = 245) as synthetic 10. Subsequent NMR analysis confirmed the purity and identity of naturally occurring 10. Interestingly, neither 10 nor 11 were present in isolable quantities in extracts of Bahamian samples of P. elisabethae.
The identification of compound 10 in the Florida Keys’ P. elisabethae represents the isolation of a novel marine metabolite which may be involved in pseudopterosin biosynthesis. Additionally, the natural occurrence of compounds 10 and 11 in the Florida Keys’ P. elisabethae indicates that this specific population of the coral may be more useful for the identification of putative biosynthetic intermediates than the previously analyzed Bahamian populations. While we confirmed the presence of compounds 10 and 11 in P. elisabethae, the presence alone is, of course, not sufficient to confirm their involvement in pseudopterosin biosynthesis. This question was addressed by conducting various incubation experiments and using derivatizations to rigorously confirm radiochemical purity.
Purification of 3H-elisabethatriene
Our approach for the biosynthetic experiments was to utilize a cell-free extract of P. elisabethae to produce 3H-labeled 9 from [1-3H]-geranylgeranyl diphosphate (GGPP) and then use this to test for the conversion of 9 to 10 and 9 to 11. Thus, a protein preparation of P. elisabethae was incubated with 50 μCi of [1-3H]-GGPP. After extracting with hexanes, the organic fraction was partially purified through a small silica column and elisabethatriene (9) was rigorously purified by reversed phase HPLC and a portion subjected to scintillation counting. Compound 9 was generated in a radiochemical yield of 0.6% (300,000 dpm) and shown to be radiochemically pure as previously described . This low yield is expected for a reactive intermediate and greater radioactivity was observed for more polar metabolites including the pseudopterosins.
Purified 3H-labeled 9 (300,000 DPM) was reincubated with a cell-free extract of P. elisabethae (Florida Keys) for 1 hour. The quenched incubation mixture was partially purified by elution through a silica column with 100% hexanes and the resulting non-polar fraction subjected to repeated HPLC fractionation. The radioactivity of fractions corresponding to compounds 9–11 was measured using a scintillation counter (Table 1).
The isolated 10 was not radioactive and consequently was deemed not to be involved in the pseudopterosin biosynthetic pathway. The lack of radioactivity in 10 could be explained by the fact that there could be two different diterpene cyclases in the Floridian populations of P. elisabethae yielding two different cyclase products. This is consistent with the wide variety of diterpenes present in the Florida samples of this gorgonian .
As described in Table 1, 9 was transformed to 11 with a radiochemical yield of 0.7% (2,060 dpm). Radiochemical purity was established for erogorgiaene by conversion of the recovered 11 to an epoxide and monitoring the specific activity of the substrate 11 and the epoxide product 12 (Scheme 2).
Conditions for this derivitization were established with non-radioactive erogorgiaene (11). The reaction of 11 with meta-chloroperoxybenzoic acid (m-CPBA) proceeded in excellent yield. Importantly, compound 12 eluted more than 10 min. earlier than 11 on reversed phase HPLC, thus ensuring a simple purification of the product. The HREIMS of 12 established a molecular formula of C20H30O. The 1H-NMR spectrum of 12 was similar to that of 11. One variation between the NMR spectra of the two compounds was that the olefinic proton in 11 [δ5.27 (1 H, br t)] was replaced with a signal at δ2.57 (1 H, dd) for 12. Additionally, the two signals for the olefinic methyl groups in 11 [δ1.60 (3 H, br s) and 1.71 (3 H, br s)] were shifted upfield to δ1.14 (3 H, br d) and δ1.17 (3 H, br s) for 12.
Following the purification of radioactive 11 from the incubation with 3H-labeled 9, 3H-labeled 11 (4.27 × 106 dpm/mmol) was treated with m-CPBA in CHCl3 and the peak corresponding to the epoxide (12) was purified by reversed phase HPLC. Compound 12 was found to have the same specific activity (4.94 × 106 dpm/mmol) as 11 (4.27 × 106 dpm/mmol). The lack of change of specific activity in this derivatization reaction establishes the radiochemical purity of compound 11 and thus confirms that erogorgiaene is derived from elisabethatriene (9).
Evaluation of 3H-erogorgiaene in Pseudopterosin Biosynthesis
The data presented above demonstrated that elisabethatriene (9) is transformed to its aromatic derivative 11 in P. elisabethae. To test for the conversion of 11 to the pseudopterosins, and thus confirm the involvement of 11 in the pseudopterosin biosynthetic pathway, 3H-labeled 11 (5,130 DPM) was incubated with a Bahamian P. elisabethae cell-free extract . Radioactive 11 was obtained from the incubation of [1-3H]-GGPP and also by oxidation of 9. Following incubation, pseudopterosins A-D (1–4) were rigorously purified by HPLC. Liquid scintillation counting of the pseudopterosins from the HPLC separation indicated transformation of 3H-11 to 1–4. The overall radiochemical yield of the four pseudopterosins was 5.7% (290 DPM). The radiochemical yield of the pseudopterosins obtained in this experiment is an order of magnitude higher than that reported for the conversion of GGPP to 9.
These data suggest that in pseudopterosin biosynthesis, erogorgiaene is produced from elisabethatriene by a dehydrogenation and spontaneous aromatization (Scheme 3). It is conceivable that isomers of elisabethatriene, other than 10, could be involved in this pathway. However, lack of such radioactive fractions in the HPLC analysis suggests that this is not likely.
The lack of transformation of elisabethatriene to the isomeric 10 suggests that the conversion of 9 to 11 involves a dehydrogenation which is then presumably followed by a spontaneous aromatization. In addition, utilization of the chemical variance of different populations of P. elisabethae for the isolation of biosynthetic intermediates and subsequent radiolabeling experiments proved to be invaluable for this investigation.
[1-3H]-Geranylgeranyl diphosphate (60 Ci/mol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All other chemicals and reagents were purchased from Fisher Scientific, Sigma Chemical Co., or Aldrich Chemical Co. The 1H-NMR spectra (one- and two-dimensional) were recorded in C6D6 on a Varian 500 NMR spectrometer at 500 MHz. Mass spectral measurements were conducted at the Midwest Center for Mass Spectrometry at University of Nebraska-Lincoln. TLC was performed using silica gel, GF254 pre-coated plates and HPLC was performed using either on an HP 1090 or using a Perkin Elmer Series 410 pump with a Perkin Elmer LC-30 RI detector.
Collection and Identification of Pseudopterogorgia elisabethae
P. elisabethae from Sweetings Cay, Bahamas was collected by SCUBA in the month of May in 1998–2002 at depths of 10–15 m. Specimens of the gorgonian were identified by standard morphological analysis and pseudopterosins A–D identified by analyzing the crude extract by analytical TLC. Samples were flash frozen with liquid nitrogen and stored at −80°C. P. elisabethae from the Florida Keys was collected by SCUBA in 1999–2001 from Tennessee reef at a depth of 25 m. Organisms were flash frozen with liquid nitrogen and stored at −80°C. Specimens collected from Florida were identified as P. elisabethae by Fredrick M. Bayer, Department of Invertebrate Zoology, Natural Museum of Natural History, Smithsonian Institution, Washington, D.C. A voucher specimen (USNM100430) has been deposited in this institute.
Cell-Free Extract Preparation
Flash frozen P. elisabethae (100 g) was homogenized with buffer [20 mM Tris-HCl (pH 7.7) containing 3 mM EDTA, 5 mM β-mercaptoethanol, and 5 mM MgCl2] in a commercial blender. The buffer was added to the blender and was the coral blended in the presence of liquid nitrogen. After homogenizing, the cell-free extract was centrifuged to remove insoluble debris. Initially, centrifugation was conducted at 9,700 g for 10 min. The pellet was discarded and the supernatant was centrifuged again at 39,000 g for 20 min. The pellet was discarded and the supernatant centrifuged one last time at 39,000 for 30 min. A portion of the cell-free extract (40 mL) was incubated with radiolabelled compounds.
Identification of erogorgiaene (11) in P. elisabethae collected in the Florida Keys
Elisabethatriene (9) (0.5 mg) was reacted with a spatula tip of Pd/C in 500 μL of triethylene glycol dimethyl ether (triglyme), and the mixture refluxed under nitrogen for 3 hours. Following the reaction, the sample was filtered and the solvent evaporated under nitrogen. Analysis was then performed by reversed phase HPLC using a diode array detector (λ = 215 nm) with 100% methanol (2 mL/min.). To identify the compound in the Florida Keys’ P. elisabethae extract, the hexane layer (~6 g) obtained from partitioning a crude methylene chloride/ethyl acetate extract with hexanes and methanol/water (9:1) was passed through a small silica column with 100% hexanes. The solvent was evaporated using a rotary evaporator and fractionated by HPLC using a diode array detector (λ = 215 nm) with 100% methanol (2 mL/min.). The peak corresponding to a retention time of 22 min. was subjected to NMR analysis.
Identification of compound 10 in P. elisabethae collected in the Florida Keys
A crude methylene chloride/methanol extract of P. elisabethae (Florida Keys) was partially purified through a silica column eluted with 100% hexanes. The sample was further purified using preparative TLC with hexanes as the eluent and the band corresponding to an Rf of approximately 0.6 was isolated. Further fractionation was achieved by reversed phase HPLC using a refractive index detector and 100% methanol (2 mL/min.). The peak corresponding to a retention time of 27 min. was subjected to NMR analysis. Compound 10: UV (MeOH) λmax 245 nm; 1H-NMR (500 MHz, C6D6) δ 6.44 (1 H, s), 5.23 (1 H, t), 3.02 (1 H, m), 1.72 (3 H, s), 1.68 (3 H, s), 1.57 (3 H, s), 1.02 (3 H, d, J = 6 Hz), 0.90 (3 H, d, J = 6 Hz).
Purification of 3H-labeled 9 from incubation of [1-3H]-GGPP
Two separate reactions were conducted by incubating 20 μCi of [1-3H]-GGPP with each of two 500 μL aliquots of a Sweetings Cay P. elisabethae cell-free extract for 1 hour at 29°C and 200 rpm. A Sweetings Cay P. elisabethae cell-free extract that had been partially purified by DEAE-cellulose anion-exchange chromatography was also incubated with 10 μCi of [1-3H]-GGPP for 1 hour at 29°C and 200 rpm. The samples were extracted with hexanes and passed through a small silica pasteur pipet column (5 cm). Elisabethatriene (300,000 DPM) was rigorously purified by reversed phase HPLC using a refractive index detector and 100% methanol (2 mL/min.). The radioactivity was measured using a liquid scintillation counter.
Incubation of Florida Keys’ P. elisabethae Cell-free Extract with 3H-9 and Purification of 3H-11 and 3H-10
3H-Elisabethatriene (300,000 DPM) was collected from HPLC purification and the solvent was evaporated under a stream of N2. Following the addition of 1 mL of assay buffer and 0.05% Tween 20, the sample was sonicated for 10 min. and cell-free extract was added to a total volume of 40 mL. The sample was then incubated at 29°C and 200 rpm for 1 hour. The sample was lyophilized and partitioned between hexanes and methanol/H20 (9:1). After partial purification through a silica column (7 cm) with 100% hexanes, the sample was purified using reversed phase HPLC with 100% methanol (2 mL/min.). Compounds 9–11 were collected in separate vials and the solvent evaporated under N2. Ten percent of each sample was then reinjected and fractions were collected before and after each peak. The solvent was evaporated from the fractions and they were subjected to liquid scintillation counting. For compound 10, another 40% was added to the sample and the liquid scintillation counting repeated. The remaining amount (90%) of 9 (48,740 DPM) and 11 (1,860 DPM) was then reinjected to purify further.
Derivitization of 11 to 12
meta-Chloroperoxybenzoic acid (m-CPBA, 76.2%, 3.5 mg) was dissolved in dry chloroform (5 mL). A portion of this mixture (1 mL, 4.6 μmol, 1.04 mg m-CPBA) was pipetted into a small separatory funnel and added to a stirred vial of 11 (4.6 μmol, 1.25 mg) over 10 minutes. The reaction was conducted at 0°C for 2.5 hours. The reaction mixture was concentrated under N2 and purified by reversed phase HPLC with 100% methanol (2 mL/min.) to afford 12 (1.25 mg) as a colorless oil. Compound 12: UV (MeOH) λmax 220 nm; HRMS calcd for C20H30O (M+) 286.2297, found 286.2297; 1H-NMR (500 MHz, C6D6) δ 7.12 (1 H, d), 7.06 (1 H, s), 6.95 (1 H, d, J = 7.5), 2.88 (1 H, m), 2.83 (1 H, m), 2.65 (1 H, m), 2.57 (1 H, dd), 2.21 (3 H, br s), 1.22 (3 H, d, J = 6.5), 1.17 (3 H, br s), 1.14 (3 H, br. d, J = 3.5), 0.67 (3 H, d, J = 3 Hz), 0.66 (3 H, d, J = 3 Hz). EIMS m/z [M]+ 183 (28), 175 (31), 174 (31), 173 (67), 159 (83), 157 (100), 145 (28), 69 (33).
Synthesis and Purification of Radioactive 12
3H-Labeled 9 (45.6 μg, 169 nmol, 1,420 DPM) was treated with m-CPBA (39.7 μg, 175 nmol) in dry CHCl3 at 0°C for 2.5 hours as described previously. The reaction mixture was purified by reversed phase HPLC as described (methanol, 2 mL/min.) to afford 8.8 nmol (5.2%) of 12 and the radioactivity measured. Quantities of the compounds were obtained by integration of HPLC peaks.
Synthetic Conversion of 9 to 11
H5PMo10V2O40− (60 mg) was stirred with 1,2-dichloroethane (1 mL) and tetraglyme (65 μL) in a 2 mL conical vial at 70°C for 5 min . Elisabethatriene (9) (0.46 mg) was reacted with the mixture under oxygen atmosphere for 1.5 hours. The 1,2-dichloroethane was evaporated and the mixture partially purified through a small silica pasteur pipet column with hexanes. Reversed phase HPLC of the reaction (methanol, 2 mL/min.) showed the presence of erogorgiaene in approximately 80% yield.
Synthesis and Purification of Radioactive 11
3H-9 (48,740 DPM) purified from the incubation with [1-3H] GGPP was treated with H5PMo10V2O40− (26.5 mg) in tetraglyme (55 μL) and 1,2-dichloroethane at 70°C for 2 hours as described above. The sample was purified as described in the previous section and 5% was reinjected along with a small amount of “cold” erogorgiaene (11). Fractions were collected before and after the erogorgiaene peak and subjected to liquid scintillation counting. The other 95% was reinjected separately for purification.
Incubation of Cell-free Extract with 3H-11 and Purification of 3H-[1–4]
3H-11 (5,130 DPM) was added to a plastic conical vial using hexanes and the solvent evaporated. Following the addition of glycerol (4 mL, 10% v/v) and Tween 20 (20 μL, 0.05%), the sample was sonicated for 10 min. and a Sweetings Cay P. elisabethae cell-free extract was added to a total volume of 40 mL. The sample was incubated at 200 rpm and 29°C for 24 hours. Solvent partitioning was performed between hexanes and methanol/water (9:1) and then methanol/water (1:1) and methylene chloride. The pseudopterosins A–D were subsequently purified from the methylene chloride layer by normal phase HPLC with a hexane/ethyl acetate gradient (60:40 to 100% ethyl acetate over 35 min., λ=283 nm). Radioactivity was monitored using a liquid scintillation counter.
|Table 1. Recovered Radioactivity of Compounds 9–11 after Incubation of 3H-labeled 9 with a Cell-free Extract of P. elisabethae (Florida Keys).|
We thank the National Science Foundation (MCB-0119011) and a National Institutes of Health Minority Predoctoral Fellowship (5 F31 GM64005-02, A. Kohl) for funding this research as well as the Florida Institute of Oceanography for providing ship time aboard the R/V Bellows and the Keys Marine Laboratory for use of their facilities. This is contribution number P200307 from the Center of Excellence in Biomedical and Marine Biotechnology.
- Sample availability: Samples of compounds 9, 10 and 11 are available from the authors.
- Supporting Information Available. 1H-NMR spectra for compounds 10 and 12 (12 pages).
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