Prior examples of hydroacylation to form six- and seven-membered ring ketones require either embedded chelating groups or other substrate design strategies to circumvent competitive aldehyde decarbonylation. A cooperative catalysis strategy enabled intramolecular hydroacylation of disubstituted alkenes to form seven- and six-membered rings without requiring substrate-embedded chelating groups.

No abstract

Protein prenylation involves the attachment of a farnesyl or geranylgeranyl group onto a cysteine residue located near the C-terminus of a protein, recognized via a specific prenylation motif, and results in the formation of a thioether bond. To identify putative prenylated proteins and investigate changes in their levels of expression, metabolic labeling and subsequent bioorthogonal labeling has become one of the methods of choice. In that strategy, synthetic analogues of biosynthetic precursors for post-translational modification bearing bioorthogonal functionality are added to the growth medium from which they enter cells and become incorporated into proteins. Subsequently, the cells are lysed and proteins bearing the analogues are then covalently modified using selective chemical reagents that react via bioorthogonal processes, allowing a variety of probes for visualization or enrichment to be attached for subsequent analysis. Here, we describe protocols for synthesizing several different isoprenoid analogues and describe how they are metabolically incorporated into mammalian cells, and the incorporation into prenylated proteins visualized via in-gel fluorescence analysis. © 2018 by John Wiley & Sons, Inc.

Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper (I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

The tricyclic alpha-keto hemiacetals 3a,b and 8a-d obtained from ruthenium-catalyzed oxidation of tetrahalonorbornyl derivatives possessing a pendant hydroxymethyl group were cleaved using Pb(OAc)(4) or alkaline H(2)O(2) to give gamma-lactone-fused cyclopentane derivatives 5a,b and 9a-d. The alpha-keto hemiacetal 3b has also been elaborated to spiroepoxide derivative 25. The stable hydrate 4 formed from ruthenium-catalyzed oxidation of acrolein adduct 10 furnished an intramolecular hemiacetal 11 upon cleavage with Pb(OAc)(4). The alpha-halo ester moiety in 5a was transformed smoothly in a highly regio- and stereoselective manner to alpha-hydroxy esters through a lactone-assisted intermediate to furnish 18.