Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.
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.
Hovlid
Marisa L
ML
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
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
D001665:Binding Sites; D051231:Farnesyltranstransferase / Q000378:metabolism; D007700:Kinetics; D019920:Photoaffinity Labels; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D012717:Sesquiterpenes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D019920:Photoaffinity Labels; D011106:Polyisoprenyl Phosphates; D029701:Saccharomyces cerevisiae Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D051231:Farnesyltranstransferase