Synthesis of Farnesyl Diphosphate Analogues Containing Ether-Linked Photoactive Benzophenones and Their Application in Studies of Protein Prenyltransferases

Abstract

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 Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer 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. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal 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 Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.

The Chaperone Protein SmgGDS Interacts with Small GTPases Entering the Prenylation Pathway by Recognizing the Last Amino Acid in the CAAX Motif

“…Fractions were analyzed using an analytical C 18 reverse phase HPLC column employing a linear gradient (0 -100% Solvent B over 60 min at a flow rate of 1 ml/min) and detected at 214 nm. Fractions containing peptide product of at least 90% purity were pooled and concentrated by lyophilization to yield 35 Synthesis of Rap1B (C10-m-Bp) Peptide-Synthesis followed the same prenylation conditions as described above using C10-meta-Bp-Br (22.8 mg, 53.5 mol, 5 eq) that was prepared as previously described (34 -37). This reaction yielded 3.4 mg (14%) of the desired alkylated peptide.…”

Lipidated Ras and Rab Peptides and Proteins—Synthesis, Structure, and Function

“…Fluorescent or photoactive groups have been incorporated into the prenyl moiety, but smaller steric constraints have also been investigated with regard to the binding and subsequent functionality of the prenyltransferases. [87,[93][94][95][96][97][98][99][100][101][102][103][104][105][106][107] Azide-carrying farnesyl groups are an especially promising approach for proteomics strategies. [108] A second approach that incorporates synthetic lipidated peptides is based on the use of the maleimidocaproyl (MIC)-controlled ligation (Scheme 11 B).…”

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

“…1. Photoaffinity labeling studies have been used to identify binding regions in specific enzymes and to isolate a number of previously unidentified proteins, (Yokoyama et al, 1995;Gaon et al, 1996a;Turek et al, 1997Turek et al, , 2001Zhang et al, 1988Zhang et al, , 2004Webb et al, 1999) including protein prenyltransferases (Omer et al, 1993;Bukhtiyarov et al, 1995;Edelstein and Distefano, 1997) with remarkable specificity (Dorman and Prestwich, 1994). FPP binding to the rubber transferase active site occurs in a similar manner to the FPP-requiring enzymes mentioned above (Mau et al, 2003).…”

“…In studies with farnesyltransferase, all of these analogues ( Fig. 2) could inactivate and covalently label the enzyme upon photolysis (Gaon et al, 1996b;Turek et al, 1996;Yokoyama et al, 1995;Turek et al, 2001) suggesting they would be good probes for studying the rubber transferase.…”