Hong-Xue Chen’s research while affiliated with Tsinghua University and other places

We report a facile synthetic strategy toward CH2-substituted phosphothreonine mimetic. Herein, inexpensive valine with directing group was converted into homothreonine via palladium-catalyzed γ-methyl C(sp³)-H bond activation, followed by construction of phosphorus-carbon bond via 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 chances to prepare not only phosphopeptides but also phosphoproteins resistant to phosphatases.

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 β-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.

Phosphatase-inert peptidomimetics containing phosphonate pSer analogue have been developed as valuable biological tools for probing and regulating pSer-dependent protein-protein interactions (PPIs) in cellular context. Herein, we report a facile and efficient synthesis route of Fmoc-protected phosphonate pSer mimetic and also present the application of this building block in the solid-phase synthesis of a phosphatase-resistant substrate peptide of 14-3-3 ζ, retaining 14-3-3 ζ binding efficacy similar to the parent pSer-containing peptide.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancers. 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 non-hydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with non-phosphorylable 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.