Synthesis and Target Identification of Benzoxepane Derivatives as Potential Anti‐Neuroinflammatory Agents for Ischemic Stroke

Benzoxepane derivatives were designed and synthesized, and one hit compound emerged as being effective in vitro with low toxicity. In vivo, this hit compound ameliorated both sickness behavior through anti‐inflammation in LPS‐induced neuroinflammatory mice model and cerebral ischemic injury through anti‐neuroinflammation in rats subjected to transient middle cerebral artery occlusion. Target fishing for the hit compound using photoaffinity probes led to identification of PKM2 as the target protein responsible for anti‐inflammatory effect of the hit compound. Furthermore, the hit exhibited an anti‐neuroinflammatory effect in vitro and in vivo by inhibiting PKM2‐mediated glycolysis and NLRP3 activation, indicating PKM2 as a novel target for neuroinflammation and its related brain disorders. This hit compound has a better safety profile compared to shikonin, a reported PKM2 inhibitor, identifying it as a lead compound in targeting PKM2 for the treatment of inflammation‐related diseases.     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

Conformational Analysis,Thermal Rearrangement,and EI‐MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)‐Germacradien‐6‐ol by 13C‐Labeling Experiments

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)‐(1(10)E,4E,6S,7R)‐germacradien‐6‐ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen (¹³C₁)FPP isotopomers (FPP=farnesyl diphosphate) and (¹³C₁₅)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including ¹³C, ¹³C COSY experiments. The (¹³C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.     

Photoinduced Electron Transfer‐Regulated Protein Labeling With a Coumarin‐Based Multifunctional Photocrosslinker

We developed a novel diazirine‐based photolabeling agent having a (coumarin‐4‐yl)methyl ester scaffold, which exhibited multiple photochemical properties of crosslinking, fluorogenicity and cleavage. These properties can be kinetically regulated via photoinduced electron transfer between diazirine and coumarin moieties. The C?O bond of (coumarin‐4‐yl)methyl ester can be cleaved via photochemical excitation of coumarin moiety, that function has been initially quenched by the diazirine moiety. Upon diazirine photolysis with 365‐nm light, interacting protein was stably captured with photoactivatable ligand probe. Then, the unlocked cleavage function was activated with 313 nm light, and the reaction was accelerated in a weakly‐basic solution. The crosslinked protein could be selectively isolated with attachment of a small coumarin tag on the surface. This multi‐functional labeling agent has a great potential to facilitate LC‐MS/MS‐based protein identification.     

Isotope‐Filtered 4D NMR Spectroscopy for Structure Determination of Humic Substances

Humic substances, the main component of soil organic matter, could form an integral part of green and sustainable solutions to the soil fertility problem. However, their global‐scale application is hindered from both scientific and regulatory perspectives by the lack of understanding of the molecular make‐up of these chromatographically inseparable mixtures containing thousands of molecules. Here we show how multidimensional NMR spectroscopy of isotopically tagged molecules enables structure characterization of humic compounds. We illustrate this approach by identifying major substitution patterns of phenolic aromatic moieties of a peat soil fulvic acid, an operational fraction of humic substances. Our methodology represents a paradigm shift in the use of NMR active tags in structure determination of small molecules in complex mixtures. Unlike previous tagging methodologies that focused on the signals of the tags, we utilize tags to directly probe the identity of the molecules they are attached to.     

Tag‐Convertible Photocrosslinker with Click‐On/Off N‐Acylsulfonamide Linkage for Protein Identification

The N‐acylsulfonamide group, known as a safety‐catch linker, has been applied to photoaffinity labeling (PAL) using a cinnamate‐type photocrosslinker to improve the efficiency of PAL‐based target identification. A bioorthogonal sulfo‐click reaction was used to stably link a photocrosslinker unit with N‐acylsulfonamide linkage to produce a photoactivatable probe without any protection. In addition, the crosslinked protein was selectively isolated with a small cinnamate tag via linkage disruption upon N‐alkylation. Furthermore, the tag moiety was photochemically converted to a stable coumarin derivative by losing a water molecule, which is a useful property in MS‐based identification.     

High‐Throughput Proteomics Enabled by a Photocleavable Surfactant

Mass spectrometry (MS)‐based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high‐throughput proteomics. Herein, we report a high‐throughput bottom‐up proteomic method enabled by a newly developed MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS‐analysis following UV degradation. Moreover, we developed an Azo‐aided bottom‐up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all‐in‐one” MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications.