Abstract

Tuning the electronic and steric environment of olefin metathesis catalysts with specialized ligands can adapt them to broader applications, including metathesis in aqueous solvents. Bidentate salicylaldimine ligands are known to stabilize ruthenium alkylidene complexes, as well as allow ring-closing metathesis in protic media. Here, we report the synthesis and characterization of exceptionally robust olefin metathesis catalysts bearing both bidentate salicylaldimine and N-heterocyclic carbene ligands, including a trimethylammonium-functionalized complex adapted for polar solvents. NMR spectroscopy and X-ray crystallographic analysis confirm the structures of the complexes. Although the N-heterocyclic carbene–salicylaldimine ligand combination limits the activity of these catalysts in non-polar solvents, this pairing enables efficient ring-closing metathesis of both dienes and enynes in methanol and methanol/water mixtures under air.

The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the α-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an α-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide–alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site—the C terminus—of virtually any protein.

Unlocking fluorescence. The “trimethyl lock” is an effective way to mask structurally diverse fluorescent molecules for the preparation of fluorogenic enzyme substrates. These novel probes exhibit remarkable stability in water, and this makes them useful for a variety of biochemical and biological applications.