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Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.


ABSTRACT: Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

SUBMITTER: Hovlid ML 

PROVIDER: S-EPMC2836812 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.

Hovlid Marisa L ML   Edelstein Rebecca L RL   Henry Olivier O   Ochocki Joshua J   DeGraw Amanda A   Lenevich Stepan S   Talbot Trista T   Young Victor G VG   Hruza Alan W AW   Lopez-Gallego Fernando F   Labello Nicholas P NP   Strickland Corey L CL   Schmidt-Dannert Claudia C   Distefano Mark D MD  

Chemical biology & drug design 20100101 1


Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3  ...[more]

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