Cleavage of the Robust Silicon-Fluorine σ-Bond Allows Silicon-Carbon Bond Formation: Synthetic Strategies Toward Ortho-Silyl Aryl Phosphonates.

A straightforward, mild, and transition metal-free three-component coupling reaction involving arynes, phosphites, and silyl fluorides was developed through Si-F bond activation.  Although the Si-F bond is one of the strongest bonds, Si-C bond formation via Si-F bond cleavage with the assistance of bidentate silicon and phosphonium Lewis acids has been successfully achieved.  This unprecedented strategy provides a facile approach for synthesizing ortho-silyl-substituted aryl phosphonates.  Notably, this method allows the use of not only dialkylarylsilyl fluorides and diarylalkylsilyl fluorides but also triarylsilyl fluorides as coupling partners, which is uncommon in the field of arylsilane synthesis.  Furthermore, a variety of ortho-silyl-substituted aryl phosphonates were produced in moderate to good yields with broad functional group tolerance.  Additionally, the versatility of ortho-silyl-substituted aryl phosphonates was demonstrated by the elaboration of the products into a range of silicon-containing compounds.

Identification of transglutaminase 2 kinase substrates using a novel on-chip activity assay.

Transglutaminase 2 (TG2) is an enzyme that plays a critical role in a wide variety of cellular processes through its multifunctional activities. TG2 kinase has emerged as an important regulator of apoptosis, as well as of chromatin structure and function. However, systematic investigation of TG2 kinase substrates is limited due to a lack of a suitable TG2 kinase activity assays. Thus, we developed a novel on-chip TG2 kinase activity assay for quantitative determination of TG2 kinase activity and for screening TG2 kinase substrate proteins in a high-throughput manner. Quantitative TG2 kinase activity was determined by selective detection of substrate protein phosphorylation on the surface of well-type amine arrays. The limit of detection (LOD) of this assay was 4.34µg/ml. We successfully applied this new activity assay to the kinetic analysis of 27 TG2-related proteins for TG2 kinase activity in a high-throughput manner and determined Michaelis-Menten constants (Km) of these proteins. We used the Km values and cellular locations of the TG2-related proteins to construct a substrate affinity map for TG2 kinase. Therefore, this on-chip TG2 kinase activity assay has a strong potential for the systematic investigation of substrate proteins and will be helpful for studying new physiological functions.

Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies.