Simulating Electron Transfer Reactions in Solution: Radical-Polar Crossover.

Single-electron transfer (SET) promotes a wide variety of interesting chemical transformations, but modeling of SET requires a careful treatment of electronic and solvent effects to give meaningful insight. Therefore, a combined constrained density functional theory and molecular mechanics (CDFT/MM) tool is introduced specifically for SET-initiated reactions. Mechanisms for two radical-polar crossover reactions involving the organic electron donors tetrakis(dimethylamino)ethylene (TDAE) and tetrathiafulvalene (TTF) were studied with the new tool. An unexpected tertiary radical intermediate within the TDAE system was identified, relationships between kinetics and substitution in the TTF system were explained, and the impact of the solvent environments on the TDAE and TTF reactions were examined. The results highlight the need for including solvent dynamics when quantifying SET kinetics and thermodynamics, as a free energy difference of >20 kcal/mol was observed. Overall, the new method informs mechanistic analysis of SET-initiated reactions and therefore is envisioned to be useful for studying reactions in the condensed phase.

Chemoenzymatic o-Quinone Methide Formation.

Generation of reactive intermediates and interception of these fleeting species under physiological conditions is a common strategy employed by Nature to build molecular complexity. However, selective formation of these species under mild conditions using classical synthetic techniques is an outstanding challenge. Here, we demonstrate the utility of biocatalysis in generating o-quinone methide intermediates with precise chemoselectivity under mild, aqueous conditions. Specifically, α-ketoglutarate-dependent non-heme iron enzymes, CitB and ClaD, are employed to selectively modify benzylic C-H bonds of o-cresol substrates. In this transformation, biocatalytic hydroxylation of a benzylic C-H bond affords a benzylic alcohol product which, under the aqueous reaction conditions, is in equilibrium with the corresponding o-quinone methide. o-Quinone methide interception by a nucleophile or a dienophile allows for one-pot conversion of benzylic C-H bonds into C-C, C-N, C-O, and C-S bonds in chemoenzymatic cascades on preparative scale. The chemoselectivity and mild nature of this platform is showcased here by the selective modification of peptides and chemoenzymatic synthesis of the chroman natural product (-)-xyloketal D.

A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins.

G protein-coupled receptors (GPCRs) detect a wide variety of physical and chemical signals and transmit that information across the cellular plasma membrane. Dimerization is a proposed modulator of GPCR signaling, but the structure and stability of class A GPCR dimerization have been difficult to establish. Here we investigated the dimerization affinity and binding interface of human cone opsins, which initiate and sustain daytime color vision. Using a time-resolved fluorescence approach, we found that human red cone opsin exhibits a strong propensity for dimerization, whereas the green and blue cone opsins do not. Through mutagenesis experiments, we identified a dimerization interface in the fifth transmembrane helix of human red cone opsin involving amino acids I230, A233, and M236. Insights into this dimerization interface of red cone opsin should aid ongoing investigations of the structure and function of GPCR quaternary interactions in cell signaling. Finally, we demonstrated that the same residues needed for dimerization are also partially responsible for the spectral tuning of red cone opsin. This last observation has the potential to open up new lines of inquiry regarding the functional role of dimerization for red cone opsin.

Class A Plexins Are Organized as Preformed Inactive Dimers on the Cell Surface.

Plexins are single-pass transmembrane receptors that bind the axon guidance molecules semaphorins. Single-pass transmembrane proteins are an important class of receptors that display a wide variety of activation mechanisms, often involving ligand-dependent dimerization or conformational changes. Resolving the activation mechanism and dimerization state of these receptors is extremely challenging, especially in a live-cell environment. Here, we report on the dimerization state of PlexinA4 and its response to activation by semaphorin binding. Semaphorins are dimeric molecules that activate plexin by binding two copies of plexin simultaneously and inducing formation of a specific active dimer of plexin. An open question is whether there are preexisting plexin dimers that could act as autoinhibitory complexes. We address these questions with pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is a two-color fluorescence microscopy method that is directly sensitive to protein dimerization in a live-cell environment. With PIE-FCCS, we show that inactive PlexinA4 is dimerized in the live-cell plasma membrane. By comparing the cross correlation of full-length PlexinA4 to control proteins and plexin mutants, we show that dimerization of inactive PlexinA4 requires the Sema domain, but not the cytoplasmic domain. Ligand stimulation with Sema6A does not change the degree of cross correlation, indicating that plexin activation does not lead to higher-order oligomerization. Together, the results suggest that semaphorin activates plexin by disrupting an inhibitory plexin dimer and inducing the active dimer.

Paclitaxel-loaded crosslinked hyaluronic acid films for the prevention of postsurgical adhesions.

PURPOSE: Post surgical adhesion formation results in significant morbidity for surgical patients. The purpose of this study was to investigate the use of paclitaxel (PTX) as an inhibitor of adhesion formation in rats and to design and characterize a controlled release film formulation of the drug for application to exposed surgical sites. METHODS: The rat cecal side wall abrasion model was used to investigate the anti-adhesion properties of PTX. The drug was administered by either intraperitoneal injection (i.p.), as the cremophor formulation (Taxol) or by application of carbodiimide crosslinked hyaluronic acid (HA) films containing PTX. The HA films were also characterized by measurements of elasticity, degree of swelling in water and drug release rates. RESULTS: Taxol administered by i.p. injection at 4 mg/kg on a daily basis for between 3 and 5 days resulted in a significant reduction in adhesion formation. All animals in the control group (n = 10) had some form of adhesion following abrasion whereas the percent of animals without adhesions significantly increased and the mean incidence of adhesion formation decreased in the three Taxol treated groups. The application of 5% PTX loaded HA films had a similar significant effect in increasing both the % of animals without adhesions and in reducing the mean incidence of adhesions. CONCLUSIONS: Paclitaxel is an effective inhibitor of adhesion formation in rats. HA crosslinked with 2 mM water soluble carbodiimide and containing 10% glycerol and 5% PTX are flexible, mucoadhesive, biocompatible controlled release films suitable for application to surgical sites for the prevention of adhesion formation.