Radiolabeled mevalonate, incorporated

Statins were also instrumental in the discovery of protein prenylation. In 1984, Glomset and coworkers used mevinolin (lovastatin) and radiolabeled mevalonate to demonstrate that a product of mevalonate could become posttranslationally incorporated into select proteins [115]. The use of the statin to deplete cells of endogenous mevalonate and downstream isoprenoids enabled sufficient incorporation of the exogenous radiolabeled mevalonate and subsequent detection of the radiolabeled protein fraction. 

The incorporation of acetate into the monoterpene unit of the indole alkaloids has recently been reexamined (176). Using [l,2- C2]acetate it was established that no intact incorporation occurred, and a similar labeling pattern to that from [2- C2]acetate was observed, i.e., C-3, C-4, C-20, C-22, and C-23. Extensive scrambling of the acetate occurred via the Krebs cycle to label the 1 and 2 positions of acetate prior to incorporation. [2- C]Mevalonate was incorporated equally into C-17 and C-22 of ajmalicine (39), indicating that an equilibration occurs at some point in the pathway, as had been established previously with radiolabeled precursors 176). 

In the 1970s the biosynthesis of cannabinoids was investigated with radiolabeling experiments. 14C-labeled mevalonate and malonate were shown to be incorporated into tetrahydrocannabinolic acid and cannabichromenic acid at very low rates (< 0.02%). Until 1990 the precursors of all terpenoids, isopentenyl diphosphate and dimethyl-allyl diphosphate were believed to be biosynthesized via the mevalonate pathway. Subsequent studies, however, proved that many plant terpenoids are biosynthesized via the recently discovered deoxyxylulose phosphate pathway (Eisenreich et al., 1998 Rohmer, 1999). It was shown that the Cio-terpenoid moiety of cannabinoids is biosynthesized entirely or predominantly (>98%) via this pathway (Fellermeister et al., 2001). The phenolic moiety is generated by a polyketide-type reaction sequence. 

Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins, which had been previously treated with juvenile hormone III (JH III, 2.2 pg/beetle in acetone) and then placed in an aeration tube for 25 to 30 h. Ips paraconfusus and I. pini were each injected with 0.2 pCi of sodium [1-14C]acetate prior to placement in cut pine logs and volatile collection, while D. jeffreyi were each injected with 3.8 (male) and 3.7 (female) pCi of sodium [1-14C]acetate 6.4 (male) and 10.7 (female) h after JH application. (G) The role of the mevalonate pathway in frontalin biosynthesis is supported by the incorporation of radiolabel from [2-14C]mevalonolactone into frontalin by male D. jeffreyi (2.2 pg JH 11 l/beetle in acetone, 10 h incubation and volatile collection, 1.1 pCi of [2 14C] mevalonolactone injected, 20 h volatile collection). Figures adapted from Seybold et al. (1995b) and Barkawi (2002). 

C-Methyl-D-erythritol 4-phosphate (4), a key isoprenoid precursor in the mevalonate-independent pathway leading to isopentenyl diphosphate, has been synthesized in eight steps from 1,2-0-isopropylidene-a-D-xylofuranose in such a way as to facilitate the incorporation of C or radiolabels. Syntheses of the non-phosphorylated derivative, 5, and its L-threitol diastereomer, 6, from d-glucose and D-galactose respectively, have also been reported. 3p-(5 -D-Ribityl)cholestane (7), a putative biological precursor for fossil 3-alkylsteranes, has been synthesized from cholestanone by the stepwise and stereoselective construction and subsequent reductive opening of a 3p-(5 -deoxy-5 -yl-D-ribono-l,4-lactone) substituent/... 

Subsequently, Birch and associates reported the biosynthetic incorporation of doubly radiolabeled brevianamide F (cydo-L-tryptophyl-L-proline) into brevianamide A in Penicillium brevicompactum [13]. Thus, feeding experiments were conducted with DL-[met/zy/ene- C]-tryptophan, l-[5- H]-proline, dl-[2- C]-mevalonic acid lactone, and 9/c/o-L-[met/zy/ene- C]-tryptophyl-L-proline as shown by the resulting data in Table 2. The specific molar incorporations were determined by feeding precursors of certain specific activities and recrystallizing the brevianamide A produced under these conditions to constant activity without the addition of cold, unlabeled brevianamide A. 

Holzapfel and co-workers carried out some preliminary biosynthetic work and identified via C-labeled precursor feeding studies that this substance is fashioned from tryptophan, mevalonic acid, and acetate [77]. These workers were able to demonstrate further that )3-cyclopiazonic acid is a biosynthetic precursor to a-cyclopiazonic via biosynthetic radiolabeling of j -cyclopiazonic acid and efficient incorporation of this precursor into a-cyclopiazonic acid. In addition, it was noted that j9-cyclopiazonic acid is present in the mycelium at a stage when only traces of a-cyclopiazonic acid can be detected. The concentration of -cyclopiazonic acid then rapidly decreases as the a-cyclopiazonic acid concentration increases and seems to indicate clearly a temporal separation of the biosynthetic steps. 

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