Isolation of the elusive (−)-germacrene-a from the gorgonian, eunicea mammosa chemistry of coelenterates. XVIII.

Weinheimer, Alfred J.; Youngblood, William W.; Washecheck, Paul H.; Karns, T. K. B.; Ciereszko, Leon S.

doi:10.1016/0040-4039(70)89009-8

Cited by 104 publications

(58 citation statements)

References 11 publications

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“…Selective ion monitoring of the reaction product at m/ z 204 showed a single peak with the same retention time. A library search indicated this fragmentation pattern is compatible with that of lg-elemene rearranged from germacrene A (Weinheimer et al, 1970). The retention times and the fragmentation patterns of the product and (-)-[3-elemene in black pepper oil, as confirmed by co-injection, were identical (data not shown), an indication that the enzymatic product is (+)-germacrene A (Prosser et al, 2002).…”

Section: Bacterial Expression and Characterizationmentioning

confidence: 53%

“…Three allyl methyl groups (H-13, -14, and -15) were also evident. This assignment was based on the report of Weinheimer et al (1970) and through the aid of ACD/Structure Elucidator Version 4.5 (Advanced Chemistry Development, Canada). After standing for several days at room temperature, the aged sample exhibited spurious peaks, presumably due to rearrangement.…”

Section: Bacterial Expression and Characterizationmentioning

confidence: 99%

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cDNA isolation and characterization of (+)-germacrene a synthase fromIxerls dentata form.albiflora Hara

et al. 2005

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Ixeris dentata form. albiflora Hara, a perennial medicinal herb, accumulates various sesquiterpenes whose first committed step in biosynthesis is the cyclization of farnesyl diphosphate by terpene synthase. To isolate the synthase gene IdGAS, we amplified a 184-bp DNA fragment of sesquiterpene synthase gene from I. dentata genomic DNA, using a homology-based PCR technique. Sequence information derived from the rapid amplification of cDNA ends was used to produce a 1956-bp full-length cDNA sequence. This included a 1755-b open reading frame for 584 amino acids, with a deduced size of 67.1 kDa and a pl of 5.16. The partially purified recombinant synthase had an optimum temperature and pH at 37~ and 7.5 to 8.0, respectively, as well as a Km of 11.0 and 14.9 mM at 25 and 37~ respectively. The expressed protein was inactive with geranyl diphosphate, but did catalyze the cyclization of farnesyl diphosphate to produce a sesquiterpene that was then identified through GC-MS and NMR analyses as (+)-germacrene A. When 44 residues were deleted from its N-terminal, the mutant lost 90% of its activity, suggesting that additional residues are necessary for full enzymatic activity. Transcript levels were comparable between roots and leaves, but began to decline in leaves near the onset of flowering.

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Section: Bacterial Expression and Characterizationmentioning

confidence: 53%

Section: Bacterial Expression and Characterizationmentioning

confidence: 99%

cDNA isolation and characterization of (+)-germacrene a synthase fromIxerls dentata form.albiflora Hara

et al. 2005

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“…[13] It was partly because of the temperature induced Cope rearrangement in GC-MS experiments that 4 a was first identified as a minor product of FPP turnover by AS. [5] To further characterize compound 4 b, GC-MS analysis was performed with varying injector temperatures but under otherwise identical conditions (Figure 1).…”

Section: Thermal Rearrangement Of 4 Bmentioning

confidence: 99%

Aristolochene Synthase‐Catalyzed Cyclization of 2‐Fluorofarnesyl‐Diphosphate to 2‐Fluorogermacrene A

Miller

Allemann

2007

ChemBioChem

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The mechanism of the conversion of (E,E)-farnesyl diphosphate (FPP, 1a) to aristolochene (6) catalyzed by aristolochene synthase from Penicillium roqueforti has been proposed to proceed through the neutral intermediate germacrene A (4a). However, much of the experimental evidence is also in agreement with a mechanism in which germacrene A is not an intermediate in the predominant mechanism that leads to the formation of aristolochene, but rather an off-pathway product that is formed in a side reaction. Hence, to elucidate the mechanism of FPP cyclisation the substrate analogue 2-fluoroFPP (1b) was synthesized, and upon incubation with aristolochene synthase was converted to a single pentane extractable product according to GC-MS analysis. On the basis of NMR analyses this product was identified as 2-fluorogermacrene A (4b). Variable temperature (1)H NMR spectroscopy indicated the existence of two conformers of 4b that were in slow exchange at -60 degrees C, while at 90 degrees C the two isomers gave rise to averaged NMR signals. In the major isomer (approximately 75%) the methyl groups on C3 and C7 were most likely in the down-down orientation as had been observed for other (E,E)-germacranes. This work suggests that after an initial concerted cyclisation of FPP to germacryl cation deprotonation leads to the formation of germacrene A, and provides compelling evidence that germacrene A is indeed an on-pathway product of catalysis by aristolochene synthase.

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“…Repeated column chromatography of fraction 0 (3.40 g) on silica gel (100 g) with 10% EtOAc in hexane gave four fractions. Purification of fraction I1 (287 mg) by hplc (Zorbax C-8 ODs silica gel with 35% H20 in MeOH) afforded 13.1 mg of 8-epi-uprolide-B acetate (9). Fraction I11 (1.38 g) was purified by hplc (Partisil 10 silica gel using 10% isopropanol in hexane) to give pure uprolide-B acetate (6) (9.1 mg) and uprolide-C acetate (12) (15.9 mg).…”

Section: Discussionmentioning

confidence: 99%

Isolation and structures of the uprolides. I. Thirteen new cytotoxic cembranolides from the Caribbean gorgonian Euniceamammosa

Rodrı́guez¹,

Piña²,

Soto³

et al. 1995

Can. J. Chem.

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ate cytotoxic action against several animal and human tumor cell lines, could be envisioned as functionalized structural congeners of eupalmerin acetate (I) and eupalmerin (2), two well-known y-cembranolides isolated earlier from specimens of the gorgonian collected near Puerto Rican waters (21-24). The specimens of E. mammosa were collected off the West coast of Puerto Rico near Mona Island and were frozen after collection and then freeze-dried prior to extraction with 1:l MeOH-EtOAc. From the hexane and CHC1, extracts, diterpenes 3-15 were isolated by conventional chromatographic procedures (see Experimental). Results and discussionThe composition C22H3206 for the new terpenoid uprolide-A acetate (3) derives from high-resolution fabms and I3c nmr ~CEMBRANOUDE SKELETON HO OAcH OAc H data. The latter, obtained in CDCI,, revealed 22 resonances for 4 CH,, 6 CH2, 7 CH, and 5 C. Three of the seven methine carbons were sp3 at a heteroatom, two were sp3 not connected to a heteroatom, and two were olefinic. One of the methylene carbons was sp2 and two of the quaternary carbon atoms were those of ester C=O functionalities. This suggests a tricyclic diterpene all three cycles of which are accounted for by an epoxidized cembranolide, as in the case of eupalmerin acetate (1). Uprolide-A acetate (3) shows an ultraviolet absorption maximum at 2 12 nm (E 25 300) suggesting the existence of an a-methylene-y-butyrolactone ring. The infrared spectrum inferred the presence of a hydroxyl(3550 cm-'), a conjugated carbonyl(1766 cm-I), and isolated ester (1733 cm-') moieties. The 'H nmr spectrum (300 MHz; CDC1,) shows the signals of four olefinic protons (6 6.10 (lH, d, J = 3.3 Hz), 5.45 (lH, d, J = 15.6 Hz), 5.39 (lH, m), and 5.29 (lH, d, J = 3.3 Hz)), the presence of three 3H singlets at 6 1.89, 1.35, and 1.27 indicating an acetate ester group and methyl groups in an E-trisubstituted epoxide and tertiary carbinol, respectively, while a 3H doublet at 6 0.85 ( J = 6.9 Hz) indicated a methyl group bonded to a tertiary carbon atom. In addition, the 'H nmr spectrum shows three separate methine protons on carbon atoms bearing oxygenat65.11 (lH,d,J= 10.2Hz),4.80(1H,d,J=8.1Hz), and 2.93 (lH, dd, J = 3.6, 10.2 Hz). The proton networks were determined by H-H COSY and decoupling difference spectra, and the connectivity of each network through the quaternary carbons was deduced by INEPT and NOESY spectra, which led to the structure 3 for uprolide-A acetate. The E configuration of the h6 olefin was suspected from the high-field chemical shift value (6 73.7) of the allylic C-8 resonance line (25), the presence of a strong sharp infrared band at 968 cm-', the absence of nOe between the ethylenic protons H-6 and H-7, and the coupling constant observed for the partially overlapped doublet centered at 6 5.45 ( J = 15.6 Hz) ascribed to H-7. The

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Isolation of the elusive (−)-germacrene-a from the gorgonian, eunicea mammosa chemistry of coelenterates. XVIII.

Cited by 104 publications

References 11 publications

cDNA isolation and characterization of (+)-germacrene a synthase fromIxerls dentata form.albiflora Hara

cDNA isolation and characterization of (+)-germacrene a synthase fromIxerls dentata form.albiflora Hara

Aristolochene Synthase‐Catalyzed Cyclization of 2‐Fluorofarnesyl‐Diphosphate to 2‐Fluorogermacrene A

Isolation and structures of the uprolides. I. Thirteen new cytotoxic cembranolides from the Caribbean gorgonian Euniceamammosa

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