Capturing Fleeting Intermediates in a Claisen Rearrangement Using Nonequilibrium Droplet Imbibition Reaction Conditions.

The Claisen rearrangement of aromatic allyl phenyl ether to 2-allyl phenol is known to be induced by heat, acid, and air-water interfacial (on-water) effects. In this work, we show that the combination of acid and interfacial effects in an "on-droplet" experiment accelerates this reaction even further (by a factor >10×). The reaction acceleration was achieved through a droplet imbibition mass spectrometry (MS) experiment that allows reactants to be deposited on rapidly moving (100 m/s), charged microdroplets while avoiding turbulent mixing. In this case, reactants are concentrated mainly at the surface of the short-lived microdroplets (microseconds), enabling enhanced interfacial effects. By doping n-butylamine in the spray solvent and subsequently exposing the resultant electrosprayed microdroplets to formic acid vapor, the ketone intermediate, 6-allylcyclohexa-2,4-dien-1-one, involved in this Claisen rearrangement was captured and characterized by tandem MS, successfully differentiating it from the corresponding isobaric reactant (allyl phenyl ether) and product (2-allyl phenol). Similar results showing rate acceleration and subsequent capture of the ketone intermediate via an instantaneous reaction with n-butylamine were demonstrated for p-methyl and p-nitro substituted allyl phenyl ether. Density functional theory calculations confirmed that the on-droplet reaction condition, with a high abundance of proton sources, is different from the neutral rearrangement. With a calculated free energy of activation of 5.2 kcal mol-1 for the protonated reactant, the on-droplet experimental condition provides a unique mechanism for catalyzing the Claisen rearrangement on the microsecond lifetime of the droplets. This experiment marks the first direct capture and detection of a short-lived ketone intermediate in the Claisen rearrangement, a task that is challenged by a thermodynamically favorable tautomerization step to give a more stabilized product (by 20 kcal/mol).

Molecular Docking as a Tool to Examine Organic Cation Sorption to Organic Matter.

Molecular docking simulations were performed to examine the structural effects of organic cations on their sorption to organic matter. A set of benzylamine compounds was used to assess the sorption trends arising from the systematic structural differences between ring or nitrogen substituents. Binding simulations were performed using AutoDock 4.2 with Schulten's proposed soil organic matter as a representative organic matter structure. The calculated binding energies for the sorbate compounds correlated strongly with the measured sorption energies for Pahokee peat, indicating that the simulated binding energies and their associated sorbate orientations were representative of the experimental conditions. Graphical docking orientations showed primary, secondary, and tertiary aminium compounds to form hydrogen-bond interactions with deprotonated carboxylic acid groups in a pocket of the organic matter structure. Quaternary ammonium compounds formed pi-pi or cation-pi interactions with the aromatic groups elsewhere in the same organic matter pocket. Ring substituents showed no clear trends in sorption energies with the substituent group type for primary aminium compounds. Rather, substituent groups altered the simulated van der Waals, electrostatic, hydrogen-bond, and desolvation energy contributions to the overall sorption energies, in part because of the variations in docking orientations between compounds. Increasing methyl substitution of the aminium nitrogen group was associated with an increase in van der Waals energy contributions and a decrease in electrostatic energy contributions to the overall compound sorption energies because of aminium charge delocalization into methyl substituents and steric hindrance from methyl substituents to form specific interactions. The findings illustrate how molecular docking can be used to explore the effects of organic cation structure on sorption interactions with organic matter.

Alkynyl-Based Covalent Organic Frameworks as High-Performance Anode Materials for Potassium-Ion Batteries.

The development of high-performance organic electrodes for potassium-ion batteries (KIBs) is attracting interest due to their sustainability and low costs. However, the electrolyte systems and moieties that generally proved to be successful in high-performance Li-ion batteries have found relatively little success in KIBs. Herein, two alkynyl-based covalent organic frameworks (COFs) containing 1,3,5-tris(arylethynyl)benzene (TAEB) and dehydrobenzoannulene (DBA) units are utilized as bulk anode materials for KIBs in a localized high-concentration electrolyte. TAEB-COF provides a high capacity value of 254.0 mAh g-1 at ∼100% efficiency after 300 cycles, and DBA-COF 3 provides a capacity of 76.3 mAh g-1 with 98.7% efficiency after 300 cycles. DFT calculations suggest that the alkynyl units of TAEB-COF facilitate the binding of K-ions through both enthalpic and geometric driving forces, leading to high reversible capacities.

Stem cell-derived polarized hepatocytes.

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Stereo- and Regioselective Synthesis of Molecular Baskets.

We describe a stereoselective method for obtaining multigram quantities of molecular basket 1 syn in overall 11% yield, using inexpensive cyclopentadiene and diethyl fumarate as starting materials. First, an asymmetric synthesis of enantioenriched bromo(trimethylstannyl)alkene (-)-8 was accomplished by the stereoselective bromination of dibromonorbornene (+)-4 guided by anchimeric assistance and subsequent syn- exo-elimination of tetrabromonorbornane (-)-5a as the key steps. Subsequent Cu(I)-catalyzed cyclotrimerization of (-)-8 was optimized to give 1 syn/ anti in 85% yield and 1:1 ratio of diastereomers. Importantly, the results of our mechanistic experiments were in line with the cyclotrimerization occurring in a chain-type fashion with racemization of a Cu(I) homochiral dimeric intermediate, reducing the stereoselectivity of the transformation. Enabled by more facile access to molecular baskets of type 1 syn, a range of recognition studies can now be completed for producing novel supramolecular catalysts, organophosphorus scavengers, and nanostructured materials.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

IKKß is a ß-catenin kinase that regulates mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) can give rise to both adipocytes and osteoblasts, but the molecular mechanisms underlying MSC fate determination remain poorly understood. IκB kinase ß (IKKß), a central coordinator of inflammation and immune responses through activation of NF-κB, has been implicated as a critical molecular link between obesity and metabolic disorders. Here, we show that IKKß can reciprocally regulate adipocyte and osteoblast differentiation of murine and human MSCs through an NF-κB-independent mechanism. IKKß is a ß-catenin kinase that phosphorylates the conserved degron motif of ß-catenin to prime it for ß-TrCP-mediated ubiquitination and degradation, thereby increasing adipogenesis and inhibiting osteogenesis in MSCs. Animal studies demonstrated that deficiency of IKKß in BM mesenchymal stromal cells increased bone mass and decreased BM adipocyte formation in adult mice. In humans, IKKß expression in adipose tissue was also positively associated with increased adiposity and elevated ß-catenin phosphorylation. These findings suggest IKKß as a key molecular switch that regulates MSC fate, and they provide potentially novel mechanistic insights into the understanding of the cross-regulation between the evolutionarily conserved IKKß and Wnt/ß-catenin signaling pathways. The IKKß-Wnt axis we uncovered may also have important implications for development, homeostasis, and disease pathogenesis.

Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress.

ADP-ribosylation is a post-translational modification that is known to be involved in cellular homeostasis and stress but has been challenging to analyze biochemically. To facilitate the detection of ADP-ribosylated proteins, we show that an alkyne-adenosine analog, N6-propargyl adenosine (N6pA), is metabolically incorporated in mammalian cells and enables fluorescence detection and proteomic analysis of ADP-ribosylated proteins. Notably, our analysis of N6pA-labeled proteins that are upregulated by oxidative stress revealed differential ADP-ribosylation of small GTPases. We discovered that oxidative stress induced ADP-ribosylation of Hras on Cys181 and Cys184 in the C-terminal hypervariable region, which are normally S-fatty-acylated. Downstream Hras signaling is impaired by ADP-ribosylation during oxidative stress, but is rescued by ADP-ribosyltransferase inhibitors. Our study demonstrates that ADP-ribosylation of small GTPases not only is mediated by bacterial toxins but is endogenously regulated in mammalian cells. N6pA provides a useful tool to characterize ADP-ribosylated proteins and their regulatory mechanisms in cells.

αIIbß3 binding to a fibrinogen fragment lacking the γ-chain dodecapeptide is activation dependent and EDTA inducible.

Platelet integrin receptor αIIbß3 supports platelet aggregation by binding fibrinogen. The interaction between the fibrinogen C-terminal γ-chain peptide composed of residues γ-404-411 (GAKQAGDV) and the Arg-Gly-Asp (RGD) binding pocket on αIIbß3 is required for fibrinogen-mediated platelet aggregation, but data suggest that other ancillary binding sites on both fibrinogen and αIIbß3 may lead to higher-affinity fibrinogen binding and clot retraction. To identify additional sites, we analyzed the ability of platelets and cells expressing normal and mutant αIIbß3 to adhere to an immobilized fibrinogen plasmin fragment that lacks intact γ-404-411 ('D98'). We found the following: (1) Activated, but not unactivated, platelets adhere well to immobilized 'D98.' (2) Cells expressing constitutively active αIIbß3 mutants, but not cells expressing normal αIIbß3 or αVß3, adhere well to 'D98.' (3) Monoclonal antibodies 10E5 and 7E3 inhibit the adhesion to 'D98' of activated platelets and cells expressing constitutively active αIIbß3, as do small-molecule inhibitors that bind to the RGD pocket. (4) EDTA paradoxically induces normal αIIbß3 to interact with 'D98.' Because molecular modeling and molecular dynamics simulations suggested that the αIIb L151-D159 helix may contribute to the interaction with 'D98,' we studied an αIIbß3 mutant in which the αIIb 148-166 loop was swapped with the corresponding αV loop; it failed to bind to fibrinogen or 'D98.' Our data support a model in which conformational changes in αIIbß3 and/or fibrinogen after platelet activation and the interaction between γ-404-411 and the RGD binding pocket make new ancillary sites available that support higher-affinity fibrinogen binding and clot retraction.

SET1 and p300 act synergistically, through coupled histone modifications, in transcriptional activation by p53.

The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.

Histone modifications in Trypanosoma brucei.

Several biological processes in Trypanosoma brucei are affected by chromatin structure, including gene expression, cell cycle regulation, and life-cycle stage differentiation. In Saccharomyces cerevisiae and other organisms, chromatin structure is dependent upon posttranslational modifications of histones, which have been mapped in detail. The tails of the four core histones of T. brucei are highly diverged from those of mammals and yeasts, so sites of potential modification cannot be reliably inferred, and no cross-species antibodies are available to map the modifications. We therefore undertook an extensive survey to identify posttranslational modifications by Edman degradation and mass spectrometry. Edman analysis showed that the N-terminal alanine of H2A, H2B, and H4 could be monomethylated. We found that the histone H4 N-terminus is heavily modified, while, in contrast to other organisms, the histone H2A and H2B N-termini have relatively few modifications. Histone H3 appears to have a number of modifications at the N-terminus, but we were unable to assign many of these to a specific amino acid. Therefore, we focused our efforts on uncovering modification states of H4. We discuss the potential relevance of these modifications.

Unusual histone modifications in Trypanosoma brucei.

To start to understand the role of chromatin structure in regulating transcription in trypanosomes, we analyzed covalent modifications on the four core histones of Trypanosoma brucei. We found unusually few modifications in the N-terminal tails, which are abundantly modified in other organisms and whose sequences, but not composition, are highly divergent in trypanosomes. In contrast, the C-terminal region of H2A appears to be hyper-acetylated. Surprisingly, the N-terminal alanines of H2A, H2B, and H4, were mono-methylated, a modification that has not been described previously for histones. Possible functions and evolutionary explanations for these unusual histone modifications are discussed.