Synthesis and Reactivity of 6,7‐dihydrogeranylazides: Reagents for Primary Azide Incorporation into Peptides and Subsequent Staudinger Ligation

Abstract

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphinebased reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azidecontaining reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.

Covalent protein–oligonucleotide conjugates by copper-free click reaction

“…DIBO-phosphoramidite 1 , 50 10-undecyn-1-yl-(2-cyanoethyl)-( N,N -diisopropyl)phosphoramidite 2 , 10 1-azido-6,7-dihydrogeranyl diphosphate 3(C 10 ) , 51 and 1-azido-10,11-dihydrofarnesyl diphosphate 3(C 15 ) 26 were synthesized as described previously. Analytical and preparative size-exclusion HPLC was carried out using a Beckman model 125/168 instrument equipped with a UV-vis detector, ABI Analytical Spectroflow 980 fluorescence detector, and a Superdex 200 10/300 GL (GE Healthcare Life Sciences, Piscataway, NJ).…”

Understanding and Exploiting Protein Prenyltransferases

“…The CAAX box represents the shortest specific transferase recognition sequence known so far, and the phosphoisoprenoids were shown to cross the cell membrane, both as pyrophosphates and as alcohols 95. The proof‐of‐principle studies used substrates with functionalized isoprenoids that were augmented with fluorophores, azides, alkynes, and dienes 96–103. The protein‐conjugated isoprenoids were then used for further protein derivatization, imaging, or protein immobilization.…”

Probing protein function by chemical modification

“…Interestingly, PPTases can tolerate diverse modifications of their lipid substrates. Therefore, bioorthogonal groups or probes can be incorporated into proteins containing a CaaX tag via prenylation71–73 (Figure 5). This strategy has been employed for proteomic analysis of prenylated substrates in cells71, 74, for identification of PPTases inhibitors75, 76, for protein immobilization77, 78, and for selective protein labeling79, 80.…”