Chloride- and Hydrosulfide-Bound 2Fe Complexes as Models of the Oxygen-Stable State of [FeFe] Hydrogenase.

[FeFe] hydrogenases demonstrate remarkable catalytic efficiency in hydrogen evolution and oxidation processes. However, susceptibility of these enzymes to oxygen-induced degradation impedes their practical deployment in hydrogen-production devices and fuel cells. Recent investigations into the oxygen-stable (Hinact) state of the H-cluster revealed its inherent capacity to resist oxygen degradation. Herein, we present findings on Cl- and SH-bound [2Fe-2S] complexes, bearing relevance to the oxygen-stable state within a biological context. A characteristic attribute of these complexes is the terminal Cl-/SH- ligation to the iron center bearing the CO bridge. Structural analysis of the t-Cl demonstrates a striking resemblance to the Hinact state of DdHydAB and CbA5H. The t-Cl/t-SH exhibit reversible oxidation, with both redox species, electronically, being the first biomimetic analogs to the Htrans and Hinact states. These complexes exhibit notable resistance against oxygen-induced decomposition, supporting the potential oxygen-resistant nature of the Htrans and Hinact states. The swift reductive release of the Cl-/SH-group demonstrates its labile and kinetically controlled binding. The findings garnered from these investigations offer valuable insights into properties of the enzymatic O2-stable state, and key factors governing deactivation and reactivation conversion. This work contributes to the advancement of bio-inspired molecular catalysts and the integration of enzymes and artificial catalysts into H2-evolution devices and fuel-cell applications.

Palladium-Mediated C(sp3)-H Bond Activation of N-Methyl-N-(pyridin-2-yl)benzamide: Direct Arylation/Alkylation and Mechanistic Investigation.

Herein, we present a facile synthetic methodology to produce a range of N-(CH2-aryl/alkyl)-substituted N-(pyridin-2-yl)benzamides via palladium-mediated C(sp3)-H bond activation. The N-methyl-N-(pyridin-2-yl)benzamide precursor was first reacted with palladium(II) acetate in a stoichiometric manner to obtain the key dinuclear palladacycle intermediates, whose structures were elucidated by mass spectrometric, NMR spectroscopic, and X-ray crystallographic studies in detail. The subsequent C(sp3)-H bond functionalizations on the N-methyl group of the starting substrate show facile productions of the corresponding N-(CH2-aryl/alkyl)-substituted N-(pyridin-2-yl)benzamides with good functional group tolerance. A plausible mechanism was proposed based on density functional theory calculations in conjunction with kinetic isotope effect experiments. Finally, the synthetic transformation from the prepared N-(CH2-aryl)-N-(pyridin-2-yl)benzamides through debenzoylation to N-(CH2-aryl)-2-aminopyridine was successfully demonstrated.

A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins.

Using a chemical approach to crosslink functionally versatile bioeffectors (such as peptides) to native proteins of interest (POI) directly inside a living cell is a useful toolbox for chemical biologists. However, this goal has not been reached due to unsatisfactory chemoselectivity, regioselectivity, and protein selectivity in protein labeling within living cells. Herein, we report the proof of concept of a cytocompatible and highly selective photolabeling strategy using a tryptophan-specific Ru-TAP complex as a photocrosslinker. Aside from the high selectivity, the photolabeling is blue light-driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light-photocrosslinking a UV-sensitive peptide to POI. Our visible light photolabeling can generate photocaged proteins for subsequent activity manipulation by UV light. Cytoskeletal dynamics regulation is demonstrated in living cells via the unprecedented POI photomanipulation and proves that our methodology opens a new avenue to endogenous protein modification.

One-Pot Glycosylation Strategy Assisted by Ion Mobility-Mass Spectrometry Analysis toward the Synthesis of N-Linked Oligosaccharides.

N-Glycans are major constituents of several cellular glycoproteins. One-pot strategies for the synthesis of N-glycans are crucial for the rapid generation of pure samples to determine their biological functions. Herein, we describe a double one-pot strategy for the synthesis of N-glycans assisted by an IM-MS analysis approach for rapid screening of optimized glycosylation reaction conditions. This research includes triflate-mediated direct ß-mannosylation and tandem glycosylation in a one-pot strategy for the synthesis of the challenging N-linked trisaccharide core ß-5. Furthermore, a one-pot sequential glycosylation of the N-linked trisaccharide core 7 furnishes diverse high-mannose type N-glycans with excellent stereo- and regioselectivities. In particular, ion mobility-mass spectrometry-based quantitative analysis is applied to identify the stereo- and regioselective outcomes of the crude reaction mixtures to develop a highly efficient one-pot protocol.

Synergistic Catalysis by Brønsted Acid/Carbodicarbene Mimicking Frustrated Lewis Pair-Like Reactivity.

Carbodicarbene (CDC), unique carbenic entities bearing two lone pairs of electrons are well-known for their strong Lewis basicity. We demonstrate herein, upon introducing a weak Brønsted acid benzyl alcohol (BnOH) as a co-modulator, CDC is remolded into a Frustrated Lewis Pair (FLP)-like reactivity. DFT calculation and experimental evidence show BnOH loosely interacting with the binding pocket of CDC via H-bonding and π-π stacking. Four distinct reactions in nature were deployed to demonstrate the viability of proof-of-concept as synergistic FLP/Modulator (CDC/BnOH), demonstrating enhanced catalytic reactivity in cyclotrimerization of isocyanate, polymerization process for L-lactide (LA), methyl methacrylate (MMA) and dehydrosilylation of alcohols. Importantly, the catalytic reactivity of carbodicarbene is uniquely distinct from conventional NHC which relies on only single chemical feature of nucleophilicity. This finding also provides a new spin in diversifying FLP reactivity with co-modulator or co-catalyst.

Direct Oligosaccharide Profiling Using Thin-Layer Chromatography Coupled with Ionic Liquid-Stabilized Nanomatrix-Assisted Laser Desorption-Ionization Mass Spectrometry.

The in-depth characterization of glycan structures is crucial to understanding their structure-function relationships and their effects on health and various diseases. Despite advances in rapid analysis, the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is limited for complex mixtures of carbohydrates due to their low ionization efficiency and the difficulty in separating oligosaccharides because of their high structural similarity. In this study, we developed an ionic liquid (IL)-stabilized, nanomatrix-decorated, thin-layer chromatography (TLC)-MALDI MS method for simultaneous and rapid separation, detection, and identification of oligosaccharides to achieve efficient profiling. The IL demonstrated good dispersion and stabilization for the spin coating of dihydroxybenzoic acid-functionalized magnetic nanoparticles (DHB@MNPs) on the TLC plate with spot homogeneity, which contributed to the observed high reproducibility (<20% CV) and 12- and 28-fold signal enhancement. Although the TLC was not able to separate isomeric glycans, the DHB@MNPs generate diagnostic glycosidic and cross-ring cleavage ions, enabling on-spot structural elucidation of composition, sequence, branching, and linkage of glycans in each separated spot. Without chemical derivatization of glycan samples, glycan visualization by TLC and tandem MS, our integrated platform, allowed the identification of 25 oligosaccharides from human milk, and heatmap analysis revealed the variability in the oligosaccharide abundance in samples from individual donors at different lactation times, which may provide insight into the microbiota and immunity of infants. With the demonstrated simplicity of our sample preparation method along with the achieved separation and in-depth structural characterization, our approach can be used for the rapid screening of other oligosaccharide-rich samples.

Rapid fabrication of functionalized plates for peptides, glycopeptides and protein purification and mass spectrometry analysis.

A rapid and simple approach for fabricating a disposable functionalized membrane on matrix-assisted laser desorption ionization (MALDI) targets, glass, or plastic substrates, without using complex mechanical protocols or chemical reactions, was developed for sample enrichment and mass spectrometry analysis. By coating functionalized-silica particles on a polydimethylsiloxane (PDMS)-coated plate, these particles can form a monolayer of materials on the PDMS membrane for sample handling without peeling off. An octadecyl(C18)-functionalized plate was fabricated by coating porous C18-silica particles on a PDMS-coated plate. The C18 particle-coated PDMS plate (CP plate) has better sensitivity than C18 tips and magnetic nanoparticles, along with a higher sample recovery (64.3 ± 4.9%) compared to the C18 tip method, when analyzing trace amounts of 5 fm BSA digest samples. The CP plate shows significantly higher urea/SDS removal efficiency on the cell lysate proteome compared to C18 tips. The capacity of the C18 spot (∼2.8 mm in diameter) on the CP plate was ∼10 µg of BSA digests. A hydrophilic particle-coated PDMS plate was also fabricated and successfully used for glycopeptide enrichment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis.

Functionalized HgTe nanoparticles promote laser-induced solid phase ionization/dissociation for comprehensive glycan sequencing.

Glycoconjugates are ubiquitously present and play a critical role in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycan generally requires chemical derivatization and sophisticated instrumentation. Herein, we report a method for complicated glycan characterization in a single assay by employing 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in matrix-assisted laser desorption/ionization mass spectrometry. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive and intense dissociation of the glycan, superior to HgTe microparticles and other inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ions) ions permits unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated by the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. This phenomenon was delineated further by investigating the NP's physico-chemical characteristics, revealing that their nanoscale-dependent thermodynamic properties, including UV absorption, photoelectron release dynamics and thermal energy, were the key to levitate temperature synergistically, thus inducing spontaneous glycan decomposition during the nanoparticle-assisted laser desorption-ionization process. Our results show that this "pseudo-MS/MS" obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated carbohydrates, without the need for chemical pre-derivatization and conventional tandem mass spectrometry.

Redox communication within multinuclear iron-sulfur complexes related to electronic interplay in the active site of [FeFe]hydrogenase.

The one-electron oxidations of a Fe2 complex lead to the formation of a persistent metal-stabilized thiyl radical Fe2 species, mixed-valent Fe4, and Fe8 complexes. The unpaired spin in the Fe2 radical species delocalizes over the Fe2 and the aromatic dithiolate, mostly on the terminal sulfur. The subsequent dimerization of the singly oxidized Fe2 to the Fe4 retains the partial thiyl radical character. For an analogue with less steric hindrance, the π-π stacking interaction between the dithiolato aromatic rings induces generation of the Fe8, in which process electronic structures of the species are modulated through reducing the thiyl radical to the thiolate. Electronic reorganization repeats when the Fe8 is converted to Fe4. Electronic interplay in the complexes decreases the energy gap of frontier MOs and buffers electronic impacts upon redox events. Easier accessible redox potentials and increased stability of the species are facilitated. The results demonstrate that electronic versatility of the benzenedithiolate exerts pronounced influences on electronic and coordination structure of the metal complexes.

Lithiation of a silyl ether: formation of an ortho-Fries hydroxyketone.

A hydroxy-directed alkylation of an N,N-diethylarylamide using CIPE-assisted α-silyl carbanions (CIPE = complex-induced proximity effect) has been developed using a simple reagent combination of LDA (lithium diisopropylamide) and chlorosilane. A study of the mechanism, and the application of the procedure to an anionic Snieckus-Fries rearrangement for a highly efficient synthesis of the potent phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, are reported.

Lithium-Ion Dynamic and Storage of Atomically Precise Halogenated Nanographene Assemblies via Bottom-Up Chemical Synthesis.

Graphene has received much scientific attention as an electrode material for lithium-ion batteries because of its extraordinary physical and electrical properties. However, the lack of structural control and restacking issues have hindered its application as carbon-based anode materials for next generation lithium-ion batteries. To improve its performance, several modification approaches such as edge-functionalization and electron-donating/withdrawing substitution have been considered as promising strategies. In addition, group 7A elements have been recognized as critical elements due to their electronegativity and electron-withdrawing character, which are able to further improve the electronic and structural properties of materials. Herein, we elucidated the chemistry of nanographenes with edge-substituted group 7A elements as lithium-ion battery anodes. The halogenated nanographenes were synthesized via bottom-up organic synthesis to ensure the structural control. Our study reveals that the presence of halogens on the edge of nanographenes not only tunes the structural and electronic properties but also impacts the material stability, reactivity, and Li+ storage capability. Further systematic spectroscopic studies indicate that the charge polarization caused by halogen atoms could regulate the Li+ transport, charge transfer energy, and charge storage behavior in nanographenes. Overall, this study provides a new molecular design for nanographene anodes aiming for next-generation lithium-ion batteries.

Tunnel frit: a nonmetallic in-capillary frit for nanoflow ultra high-performance liquid chromatography-mass spectrometryapplications.

In this study, an easy method to fabricate a durable in-capillary frit was developed for use in nanoflow liquid chromatography (nanoLC). A small orifice was tunneled into the sol-gel frit during the polymerization process resulting in the simple fabrication of a tunnel frit. A short packing tunnel frit column (2 cm, C(18) particles) was able to sustain over 10,000 psi continuous liquid flow for 10 days without observation of particle loss, and back pressure variation was less than 5%. The tunnel frit was successfully applied to the fabrication of nanoflow ultra high-performance liquid chromatography (nano-UHPLC) trap and analytical columns. In the analysis of tryptic peptides, the tunnel frit trap and analytical columns were demonstrated to have high separation efficiency and sensitivity. In analysis of phosphopeptides, the use of the nonmetallic tunnel frit column showed better sensitivity than the metallic frit column. This design can facilitate the preparation of nano-HPLC and nano-UHPLC columns and the packing material can easily be refilled when the column is severely contaminated or clogged.

Dibenzocyclooctendiones (DBCDOs): Arginine-Selective Chemical Labeling Reagents Obtained through Benzilic Acid Rearrangement.

We demonstrate that dibenzocyclooctendiones (DBCDOs) are efficient chemical reagents for the site-specific labeling of arginine-containing biomolecules. Unlike the commonly used probes, DBCDOs undergo an irreversible ring-contracted rearrangement with the guanidinium group on arginine residues under mild reaction conditions. The regioselective dual-labeled arginine residues were obtained in a one-pot reaction with our tested substrates. The efficiency of DBCDOs reactions and their ease of synthesis make DBCDOs an attractive choice for the site-selective bioconjugation of arginine.

An extractive nanoelectrospray ionization-mass spectrometry method for Chinese herbal medicine authentication.

Herein, we describe a rapid, sensitive, and nondestructive method-extractive nanoelectrospray ionization-mass spectrometry (EnESI-MS)-for traditional Chinese medicine (TCM) authentication. The mass-spectral fingerprints of volatile compounds released from various TCMs can be rapidly acquired using EnESI-MS without sample pretreatment. EnESI-MS was applied to successfully differentiate between two commonly used medicinal herbs, Schisandra chinensis and Schisandra sphenanthera, which are morphologically similar but exhibit different therapeutic effects. Specific volatile compounds of each herb in a ten-component Chinese herbal product, Jia Wei Xiao Yao San, were also identified, and the method was applied to discriminate between the commercial product and a substandard version.

Enhanced Production of Formic Acid in Electrochemical CO2 Reduction over Pd-Doped BiOCl Nanosheets.

Bismuth oxyhalides (BiOX, X = F, Cl, Br, I) are emerging energy materials because of their remarkable catalytic activity. The BiOX compounds usually have a tetragonal type crystal structure with unique layered morphology consisting of [X-Bi-O-Bi-X] sheets. Although the BiOX nanosheets exposed with {001} facets perform superior photoactivity, there is lack of understanding about their capability in the electrochemical CO2 reduction reaction (CO2RR). Herein, we adopt wet-chemical syntheses to make 2D BiOCl and Pd-doped BiOCl nanosheets for CO2RR. In the results, formic acid is the only one kind of product converted from CO2 along with H2 gas from water reduction over both BiOCl and Pd-doped BiOCl nanosheets. By thorough analyses with ex situ and in situ spectroscopy, the results reflect that (1) metallic Bi0 atoms generated by the applied negative potentials serve as the catalytic sites for the hydrogen evolution reaction (HER) and CO2RR and (2) the existence of doped Pd ions in the BiOCl structure reduces the barrier of charge transfer over the nanosheets, which enhances HER and CO2RR activities. We believe that the observations are important references for making catalysts toward CO2RR performance.

Visual indicator for surfactant abundance in MS-based membrane and general proteomics applications.

The existence of surfactants in proteomics samples can severely reduce enzymatic digestion efficiency, liquid chromatography (LC) separation efficiency, column lifetime, and mass spectrometry (MS) sensitivity. Although various techniques are able to remove surfactants, surfactants may occasionally be retained in samples due to variations in sample preparation method or personal skill. Evaluation of surfactant residue in a sample, however, usually requires an additional instrument and is time-consuming. In this study, a simple and rapid visual indicator for surfactant abundance (VISA) was developed. With the detection of a visible surfactant pellet in the solution, this assay was able to detect surfactant residue in aqueous solutions within 5 min. Without the need of additional equipment such as a mass spectrometer, every user can perform a quick test on their bench before sending the sample to the MS facility. The detection limit for the commonly used surfactants, Triton X-114 and SDS, was about 0.0005% and 0.0002%, respectively. The VISA was successfully applied to evaluate the efficiency of removal of surfactants in Triton X-114 extracted membrane proteins using tube-gel. With the combination of Triton X-114 extraction and tube-gel protocol, a study of spermatozoa membrane proteome identified about 252 proteins of which about 67.5% were classified as membrane proteins. The coexistence of protein and surfactant did not affect the VISA sensitivity, suggesting that this indicator is suitable for proteomics applications. The VISA also has potential for the detection of other surfactants and can be applied to other surfactant removing protocols.

Sensitivity improvement of CE/ESI/MS analysis of gangliosides using a liquid-junction/low-flow interface.

Methodology is presented which greatly improves the sensitivity of ganglioside analysis by CE/MS without compromising separation efficiency. In this method, non-volatile additives were removed, where possible, to reduce ion suppression. Specifically, alpha-CD, used to break up ganglioside micelle formation, was replaced with isopropyl alcohol. To reduce ion suppression from sodium ions, ammonium borate was substituted for sodium borate in the preparation of borate buffer. Because borate anions were found to be essential for CE separation, a liquid-junction/low-flow interface was employed to restrict borate anions from entering the ESI ion source. With proper control over the EOF in the connecting capillary, borate anions were controlled to migrate away from the ESI ion source. With these modifications, the sensitivity of CE/MS analysis of gangliosides was improved significantly.

Dioxygen activation by a dinuclear thiolate-ligated Fe(ii) complex.

Dioxygen activation by FeII thiolate complexes is relatively rare in biological and chemical systems because the sulfur site is at least as vulnerable as the iron site to oxidative modification. O2 activation by FeII-SR complexes with thiolate bound trans to the O2 binding site generally affords the FeIV[double bond, length as m-dash]O intermediate and oxidized thiolate. On the other hand, O2 activation by Fe(ii)-SR complexes with thiolate bound cis to the O2 binding site generates FeIII-O-FeIII or S-oxygenated complexes. The postulated FeIV[double bond, length as m-dash]O intermediate has only been identified in isopenicillin N synthase recently. We demonstrated here that O2 activation by a dinuclear FeII thiolate-rich complex produces a mononuclear FeIII complex and water with a supply of electron donors. The thiolate is bound cis to the postulated dioxygen binding site, and no FeIII-O-FeIII or S-oxygenated complex was observed. Although we have not detected the transient intermediate by spectroscopic measurements, the FeIV[double bond, length as m-dash]O intermediate is suggested to exist by theoretical calculation, and P-oxidation and hydride-transfer experiments. In addition, an unprecedented FeIII-O2-FeIII complex supported by thiolates was observed during the reaction by using a coldspray ionization time-of-flight mass (CSI-TOF MS) instrument. This is also supported by low-temperature UV-vis measurements. The intramolecular NHO[double bond, length as m-dash]FeIV hydrogen bonding, calculated by DFT, probably fine tunes the O2-activation process for intramolecular hydrogen abstraction, avoiding the S-oxygenation at cis-thiolate.

Efficient pretreatment of lignocellulosic biomass with high recovery of solid lignin and fermentable sugars using Fenton reaction in a mixed solvent.

BACKGROUND: Pretreatment of biomass to maximize the recovery of fermentable sugars as well as to minimize the amount of enzyme inhibitors formed during the pretreatment is a challenge in biofuel process. We develop a modified Fenton pretreatment in a mixed solvent (water/DMSO) to combine the advantages of organosolv and Fenton pretreatments. The hemicellulose and cellulose in corncob were effectively degraded into xylose, glucose, and soluble glucose oligomers in a few hours. This saccharide solution, separated from the solid lignin simply by filtration, can be directly applied to the subsequent enzymatic hydrolysis and ethanol fermentation. RESULTS: After the pretreatment, 94% carbohydrates were recovered as soluble monosaccharide (xylose and glucose) and glucose oligomers in the filtrates, and 87% of solid lignin was recovered as the filter residue. The filtrates were directly applied to enzymatic hydrolysis, and 92% of raw corncob glucose was recovered. The hydrolysates containing the glucose and xylose from the enzymatic hydrolysis were directly applied to ethanol fermentation with ethanol yield equals 79% of theoretical yield. The pretreatment conditions (130 °C, 1.5 bar; 30 min to 4 h) are mild, and the pretreatment reagents (H2O2, FeCl3, and solvent) had low impact to environment. Using ferrimagnetic Fe3O4 resulted in similar pretreatment efficiency and Fe3O4 could be removed by filtration. CONCLUSIONS: A modified Fenton pretreatment of corncob in DMSO/water was developed. Up to 94% of the carbohydrate content of corncob was recovered as a saccharide solution simply by filtration. Such filtrate was directly applied to the subsequent enzymatic hydrolysis and where 92% of the corncob glucose content was obtained. The hydrolysate so obtained was directly applied to ethanol fermentation with good fermentability. The pretreatment method is simple, and the additives and solvents used have a low impact to the environment. This method provides the opportunity to substantially maximize the carbohydrate and solid lignin recovery of biomass with a comparatively green process, such that the efficiency of biorefinery as well as the bioethanol production process can be improved. The pretreatment is still relatively energy intensive and expensive, and further optimization of the process is required in large-scale operation.

DNA Demethylation by DNMT3A and DNMT3B in vitro and of Methylated Episomal DNA in Transiently Transfected Cells.

The DNA methylation program in vertebrates is an essential part of the epigenetic regulatory cascade of development, cell differentiation, and progression of diseases including cancer. While the DNA methyltransferases (DNMTs) are responsible for the in vivo conversion of cytosine (C) to methylated cytosine (5mC), demethylation of 5mC on cellular DNA could be accomplished by the combined action of the ten-eleven translocation (TET) enzymes and DNA repair. Surprisingly, the mammalian DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox conditions-dependent manner, although little is known about its molecular mechanisms and occurrence in a cellular context. In this study, we have used LC-MS/MS to track down the fate of the methyl group removed from 5mC on DNA by mouse DNMT3B in vitro and found that it becomes covalently linked to the DNA methylation catalytic cysteine of the enzyme. We also show that Ca2+ homeostasis-dependent but TET1/TET2/TET3/TDG-independent demethylation of methylated episomal DNA by mouse DNMT3A or DNMT3B can occur in transfected human HEK 293 and mouse embryonic stem (ES) cells. Based on these results, we present a tentative working model of Ca2+ and redox conditions-dependent active DNA demethylation by DNMTs. Our study substantiates the potential roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation during Ca2+-dependent physiological processes.