Synthesis of farnesyl diphosphate analogues containing ether-linked photoactive benzophenones and their application in studies of protein prenyltransferases

Protein prenylation is a posttranslational lipid modification in which C-15 and C-20 isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Pas protein is farnesylated coupled with the observati on that mutant forms of Pas are implicated in a variety of human cancers. I nhibition of this posttranslational modification may serve as a possible ca ncer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP ) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Su bstrate studies, inhibition kinetics, photoinactivation studies, and photol abeling experiments are also included; these experiments were performed wit h a number of protein prenyltransferases from different sources. A X-ray cr ystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Pas-based peptid e substrates allowing the preparation of molecules with photoactive isopren oids that may serve as valuable probes for the study of prenylation functio n. Photoaffinity labeling of human protein geranylgeranyltransferase with P -32-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from th e beta -subunit of this enzyme. These results,clearly demonstrate the utili ty of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP o r GGPP as their substrates.