Evaluation of an Alkyne-containing Analogue of Farnesyl Diphosphate as a Dual Substrate for Protein-prenyltransferases

Alkynyl-farnesol reporters for detection ofproteinS-prenylation in cells

“…32 We utilized a synthetic scheme that would allow access to a diverse panel of alkynyl-farnesol derivatives ( alk-FOH , alk-FOH-2 and alk-FOH-3 ), as well as an azide analog ( az-FOH) for comparison purposes, from a common intermediate (Figure 2). After THP protection, commercially available farnesol can be selectively oxidized at the terminal allylic position to give functionalized farnesol derivative 1 .…”

“…23 Allylic alcohol 1 was then alkylated with propargyl bromide and deprotected in acidic ethanol yielding alk-FOH , a precursor to previously reported alkynyl-farnesol diphosphate that can be incorporated onto peptide substrates by both FTase and GGTase-I in vitro . 32 Corey-Kim like halogenation 33 of allylic alcohol 1 enabled access to allylic bromide intermediate 3 . CuI/K 2 CO 3 mediated coupling with TMS-acetylene afforded enyne 4 , which was subsequently deprotected in two steps to yield alk-FOH-2 .…”

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…This is a result of the side chain cleavage between the cysteine β-carbon and sulfur atom, indicating the farnesyl group was indeed appended to cysteine. (64) Surprisingly, 13 of the 14 remaining dns-GC(x)3X peptides (all except for dns-GCGGDD) exhibited formation of the farnesylated product when monitored by RP-HPLC, with dns-GCWGEV and dns-GCQGFL showing nearly quantitative modification (Table 1 and Figure S2). While a small number of dns-GCaaX peptides do not exhibit fluorescence enhancement upon prenylation,(16) it appears the majority of the longer dns-GC(x)3X peptides examined are not compatible with the fluorescence-based assay.…”

The Protein Lipidation and Its Analysis

“…Later, the authors used a biotin-geranylgeranyl (GerGer) pyrophosphate analog and an engineered protein prenyltransferases to analyse the in vivo effects of protein prenyltransferase inhibitors by quantifying by MS the amount of biotin-GerGer-tagged RabGTPases after treatment with inhibitors [115]. Synthesis of additional farnesyl diphosphate analogs bearing alkyne or azide groups [116,117] as well as in vitro studies to characterize them as substrates for the corresponding prenyl transferases have also been described by other groups [118,119]. Recently, Tate and co-workers reported an azido-derived GerGer pyrophosphate that could serve as a substrate for both GGTase I and RabGGTase and be subsequently modified by CuAAC to incorporate a double labeled tag [120].…”

“…Metabolic labeling with alkyne-containing fatty acids has also been used by other groups for the study the lipidated N-Ras and H-Ras, [127] for the characterization of myristoylated proteins [128] or for confirming the acylation of Histone H3 variants [129,130]. In addition, isoprenoid derivatives have also been reported and used for the analysis of the prenylome using CuAAC [116,117,131,132].…”