Stereoselective synthesis of a phosphonate pThr mimetic via palladium-catalyzed γ-C(sp3)-H activation for peptide preparation.

We report a facile synthetic strategy toward CH2-substituted phosphothreonine mimetics. Herein, inexpensive valine with a directing group was converted into homothreonine via palladium-catalyzed γ-methyl C(sp3)-H bond activation, followed by construction of a phosphorus-carbon bond via the well-developed Appel reaction and Michaelis-Becker reaction with a total yield of 30%. Furthermore, the derived mimetic was applied for solid-phase synthesis of two phosphopeptide inhibitors. This efficient synthesis provides a chance to prepare not only phosphopeptides but also phosphoproteins resistant to phosphatases.

Ethanol Extract of Glycyrrhiza uralensis Fisch: Antidiarrheal Activity in Mice and Contraction Effect in Isolated Rabbit Jejunum.

OBJECTIVE: To evaluate the antidiarrheal effect of ethanol extract of Glycyrrhiza uralensis Fisch root (GFR) in vivo and jejunal contraction in vitro. METHODS: In vivo, 50 mice were divided into negative control, positive control (verapamil), low-, medium- and high-dose GFR (250, 500, 1,000 mg/kg) groups by a random number table, 10 mice in each group. The antidiarrheal activity was evaluated in castor oil-induced diarrhea mice model by evacuation index (EI). In vitro, the effects of GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) on the spontaneous contraction of isolated smooth muscle of rabbit jejunum and contraction of pretreated by Acetylcholine (ACh, 10 µmol/L) and KCl (60 mmol/L) were observed for 200 s. In addition, CaCl2 was accumulated to further study its mechanism after pretreating jejunal smooth muscle with GFR (1 and 3 g/L) or verapamil (0.03 and 0.1 µmol/L) in a Ca2+-free-high-K+ solution containing ethylene diamine tetraacetic acid (EDTA). RESULTS: GFR (500 and 1,000 mg/kg) significantly reduced EI in castor oil-induced diarrhea model mice (P<0.01). Meanwhile, GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) inhibited the spontaneous contraction of rabbit jejunum (P<0.05 or P<0.01). Contraction of jejunums samples pretreated by ACh and KCl with 50% effective concentration (EC50) values was 1.05 (0.71-1.24), 0.34 (0.29-0.41) and 0.15 (0.11-0.20) g/L, respectively. In addition, GFR moved the concentration-effect curve of CaCl2 down to the right, showing a similar effect to verapamil. CONCLUSIONS: GFR can effectively against diarrhea and inhibit intestinal contraction, and these antidiarrheal effects may be based on blocking L-type Ca2+ channels and muscarinic receptors.

Synthesis of α,α-Difluorinated Phosphonate pSer/pThr Mimetics via Rhodium-Catalyzed Asymmetric Hydrogenation of ß-Difluorophosphonomethyl α-(Acylamino)acrylates.

A novel and facile synthetic strategy for α,α-difluorinated phosphonate mimetics of phosphoserine/phosphothreonine utilizing rhodium-catalyzed asymmetric hydrogenation was developed. The dehydrogenated substrate ß-difluorophosphonomethyl α-(acylamino)acrylates were first prepared from protected serine/threonine followed by asymmetric hydrogenation using the rhodium-DuPhos catalytic system to generate the chiral center(s). These important phosphonate building blocks were successfully incorporated into phosphatase-resistant peptides, which displayed similar inhibition to the 14-3-3 ζ protein as the parent pSer/pThr peptides.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.