Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

A highly chemoselective and practical alkynylation of thiols.

A thiol-alkynylation procedure utilizing the hypervalent iodine alkyne transfer reagent TIPS-ethynyl-benziodoxolone has been developed. This scalable reaction proceeds in five minutes at room temperature in an open flask using commercially available reagents. The scope of the reaction is broad, with a variety of phenolic, benzylic, heterocyclic, and aliphatic thiols undergoing alkynylation in excellent yield. The method is highly chemoselective as a vast array of functional groups are tolerated. The utility of the thiol-alkynylation in postsynthetic elaboration has been demonstrated through the facile installment of a fluorophore tag on a cysteine-containing peptide.

Comparison of double dynamic compression plating versus two configurations of an internal veterinary fixation device: Results of in vitro mechanical testing using a bone substitute.

OBJECTIVE: To compare the mechanical properties of 2 configurations of a veterinary fixation system (VFS) for large animal long bones with dynamic compression plating (DCP). SAMPLE POPULATION: Eighteen pairs of Canevasit tubes (Canevasit; Amsler und Frei, Schinznach Dorf, Switzerland) (length, 170 mm; diameter, 47.5 mm; cortex thickness, 10 mm), aligned with a 10-mm gap, and stabilized with 2 DCP or 2 VFS implants. METHODS: Three groups (n = 6) were compared. Group 1 Canevasit tubes were stabilized with two 10-hole, broad 4.5-mm stainless steel DCP applied with both plates centered over the gap, in orthogonal planes parallel to the long axis of the tubes and staggered to allow bicortical fixation with ten 4.5-mm, 52-mm-long cortex screws each. Group 2 tubes were stabilized similarly with 2 VFS implants, each composed of a stainless steel rod (length, 167 mm; diameter, 8 mm), and 10 clamps were applied in alternating fashion left and right on the rod and fixed bicortically with ten 4.5-mm, 52-mm-long, cortex screws. Group 3 tubes were stabilized similarly, but using only 6 clamps/rod. All groups were tested initially in torsion within elastic limits and subsequently in 4-point bending, with 1 implant on the tension side, until gap closure occurred. RESULTS: None of the constructs failed, but all had plastic deformation after 4-point bending. No statistically significant differences were found among the 3 groups in torsional stiffness. Double DCP fixation was significantly stiffer and stronger in 4-point bending, compared with both configurations of double VFS fixation. CONCLUSIONS: The plate design was favored in this study. The VFS system may have to be adapted before further tests are conducted. Test modalities have to be chosen closer to clinical conditions (real bone, cyclic loading, closed gap). CLINICAL RELEVANCE: The veterinary fixation system has not yet proven its advantages for large animal long bone fracture repair. From the pure mechanical point of view, double DCP is the favored method for the treatment mentioned.