Enamine N-oxides: Design, Synthesis, and Function in Bioorthogonal Reactions.

Enamine N-oxides act as a chemical linchpin bridging two bioorthogonal associative and dissociative reactions. This article describes the design of enamine N-oxides; their synthesis through the retro-Cope elimination reaction; the use of solvent, hyperconjugation, strain, and rehybridization effects to achieve bioorthogonal reactivity; and their rapid reductive cleavage with diboron reagents. The coordinated assembly and disassembly of the enamine N-oxide motif constitutes a powerful chemical operation that enables the attachment and detachment of small molecules from biomacromolecules in a biological setting.

Quantitative interferon-gamma releasing assay in predicting tuberculosis in South Korean military: a retrospective cohort study.

OBJECTIVE: The interferon-gamma releasing assay (IGRA) has been widely used to diagnose latent tuberculosis infection (TBI). However, there are limited data on the association between performance in the IGRA and risk of tuberculosis disease (TBD), as well as on the appropriate IGRA threshold for initiating TBI treatment. METHODS: The analysis was performed using the IGRA results in the Korean Military Manpower Administration database (January 2017 to December 2021), and TBD cases reported to the Korean Military Medical Command (January 2017 to June 2023). All Korean candidates for 18-month military service underwent the IGRA in the pre-enlistment examination, and enlistees who tested positive (≥0.35 IU/mL) were advised to receive TBI treatment before enlistment. RESULTS: From 2017 to 2021, 1 647 941 individuals were screened, with 29 574 testing positive for IGRA. Excluding nonenlistees namely individuals with TBD before enlistment, 19 387 individuals were IGRA positive and 1 356 324 IGRA negative. Of the positives, 4351 were excluded due to discontinued or ongoing TBI treatment at or after enlistment. During follow-up of 9219 untreated and 5818 treated positive individuals and 1 356 324 negatives, TBD occurred in 22 of the IGRA-positive individuals (97.5/100 000 person-years [95% CI, 61.1-147.7]), predominantly in the untreated group (18 cases, 130.1/100 000 person-years [95% CI, 77.1-205.7]) compared to the treated group (4 cases, 45.9/100 000 person-years [95% CI 12.5 - 117.4]), whereas 57 cases occurred in the IGRA-negative group (2.8/100 000 person-years [95% CI, 2.2-3.6]). Elevating the cutoff of IGRA from 0.35 IU/mL to 1.33 IU/mL increased positive predictive value (0.2% vs. 0.4%, p 0.03), with insignificant loss of sensitivity (24% vs. 20%, p 0.69) and decreased numbers needing treatment from 790.5 to 415.3. DISCUSSION: Elevated IGRA levels before enlistment are associated with risk of TBD during military service. It is worth considering raising the IGRA threshold for treatment of TBI in cohorts of healthy, young military individuals.

Discovery of a Potent Deubiquitinase (DUB) Small-Molecule Activity-Based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates, often leading to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

A Single-Center, Randomized, Double-Blind, and Placebo-Controlled Comparative Human Study to Verify the Functionality and Safety of the Lespedeza cuneata G. Don Extract for the Improvement of Aging Male Syndrome.

OBJECTIVE: Aging male syndrome is a clinical biochemical syndrome characterized by typical aging symptoms and serum testosterone deficiency. Although it is accompanied by various health problems, directly affects life satisfaction, and requires proper management, no clear prevention or treatment other than hormone replacement therapy is currently available for this syndrome. Here, we aimed to determine the efficacy and safety of the Lespedeza cuneata extract in the management of the aging male syndrome. METHODS: Males aged 43-70 years who provided consent for participation and had a total Aging Males' Symptom questionnaire score ≥ 37 and testosterone level ≤ 500 ng/dL were enrolled in this study. This study was conducted in a randomized, double-blind manner. Participants were randomly assigned to either the experimental or control groups and orally administered the assigned product twice a day. Efficacy was evaluated by measuring changes in Aging Males' Symptom score, Androgen Deficiency in the Aging Male questionnaire score, International Index of Erectile Function score, International Prostatic Symptom Score, blood test results, and body mass index at 8 weeks. RESULTS: After 8 weeks, the experimental group had significantly improved symptom scores compared to the control group on the Aging Males' Symptom and Androgen Deficiency in the Aging Male questionnaires. However, no significant differences in the International Index of Erectile Function score, International Prostatic Symptom Score score, blood test results, and body mass index were observed between the experimental and control groups. CONCLUSION: Lespedeza cuneata extract safely alleviates andropause symptoms without any significant side effects, suggesting its potential for the treatment of the aging male syndrome.

Pulsed Electromagnetic Field (PEMF) Treatment Ameliorates Murine Model of Collagen-Induced Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of the joint synovial membranes. RA is difficult to prevent or treat; however, blocking proinflammatory cytokines is a general therapeutic strategy. Pulsed electromagnetic field (PEMF) is reported to alleviate RA's inflammatory response and is being studied as a non-invasive physical therapy. In this current study, PEMF decreased paw inflammation in a collagen-induced arthritis (CIA) murine model. PEMF treatment at 10 Hz was more effective in ameliorating arthritis than at 75 Hz. In the PEMF-treated CIA group, the gross inflammation score and cartilage destruction were lower than in the untreated CIA group. The CIA group treated with PEMF also showed lower serum levels of IL-1ß but not IL-6, IL-17, or TNF-α. Serum levels of total anti-type II collagen IgG and IgG subclasses (IgG1, IgG2a, and IgG2b) remained unchanged. In contrast, tissue protein levels of IL-1ß, IL-6, TNF-α, receptor activator of nuclear factor kappa-Β (RANK), RANK ligand (RANKL), IL-6 receptor (IL-6R), and TNF-α receptor1 (TNFR1) were all lower in the ankle joints of the PEMF-treated CIA group compared with the CIA group. The results of this study suggest that PEMF treatment can preserve joint morphology cartilage and delay the occurrence of CIA. PEMF has potential as an effective adjuvant therapy that can suppress the progression of RA.

Promotion of Colitis in B Cell-Deficient C57BL/6 Mice Infected with Enterotoxigenic Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) causes colitis and is implicated in inflammatory bowel diseases and colorectal cancer. The ETBF-secreted B. fragilis toxin (BFT) causes cleavage of the adherence junction, the E-cadherin, resulting in the large intestine showing IL-17A inflammation in wild-type (WT) mice. However, intestinal pathology by ETBF infection is not fully understood in B-cell-deficient mice. In this study, ETBF-mediated inflammation was characterized in B-cell-deficient mice (muMT). WT or muMT C57BL/6J mice were orally inoculated with ETBF and examined for intestinal inflammation. The indirect indicators for colitis (loss of body weight and cecum weight, as well as mortality) were increased in muMT mice compared to WT mice. Histopathology and inflammatory genes (Nos2, Il-1ß, Tnf-α, and Cxcl1) were elevated and persisted in the large intestine of muMT mice compared with WT mice during chronic ETBF infection. However, intestinal IL-17A expression was comparable between WT and muMT mice during infection. Consistently, flow cytometry analysis applied to the mesenteric lymph nodes showed a similar Th17 immune response in both WT and muMT mice. Despite elevated ETBF colonization, the ETBF-infected muMT mice showed no histopathology or inflammation in the small intestine. In conclusion, B cells play a protective role in ETBF-induced colitis, and IL-17A inflammation is not attributed to prompted colitis in B-cell-deficient mice. Our data support the fact that B cells are required to ameliorate ETBF infection-induced colitis in the host.

Synthesis of push-pull-activated ynol ethers and their evaluation in the bioorthogonal hydroamination reaction.

A new class of push-pull-activated alkynes featuring di- and trifluorinated ynol ethers was synthesized. The difluorinated ynol ether exhibited an optimal balance of stability and reactivity, displaying a substantially improved half-life in the presence of aqueous thiols over the previously reported 1-haloalkyne analogs while reacting just as fast in the hydroamination reaction with N,N-diethylhydroxylamine. The trifluorinated ynol ether reacted significantly faster, exhibiting a second order rate constant of 0.56 M-1 s-1 in methanol, but it proved too unstable toward thiols. These fluorinated ynol ethers further demonstrate the importance of the hyperconjugation-rehybridization effect in activating alkynes and demonstrate how substituent effects can both activate and stabilize alkynes for bioorthogonal reactivity.

Bioorthogonal Click and Release: A General, Rapid, Chemically Revertible Bioconjugation Strategy Employing Enamine N-oxides.

A chemically revertible bioconjugation strategy featuring a new bioorthogonal dissociative reaction employing enamine N-oxides is described. The reaction is rapid, complete, directional, traceless, and displays a broad substrate scope. Reaction rates for cleavage of fluorophores from proteins are on the order of 82 M-1s-1, and the reaction is relatively insensitive to common aqueous buffers and pHs between 4 and 10. Diboron reagents with bidentate and tridentate ligands also effectively reduce the enamine N-oxide to induce dissociation and compound release. This reaction can be paired with the corresponding bioorthogonal hydroamination reaction to afford an integrated system of bioorthogonal click and release via an enamine N-oxide linchpin with a minimal footprint. The tandem associative and dissociative reactions are useful for the transient attachment of proteins and small molecules with access to a discrete, isolable intermediate. We demonstrate the effectiveness of this revertible transformation on cells using chemically cleavable antibody-drug conjugates.

Enamine N-Oxides: Synthesis and Application to Hypoxia-Responsive Prodrugs and Imaging Agents.

Tumor hypoxia induces the large-scale adaptive reprogramming of cancer cells, promoting their transformation into highly invasive and metastatic species that lead to highly negative prognoses for cancer patients. We describe the synthesis and application of a hypoxia-responsive trigger derived from previously inaccessible enamine N-oxide structures. Hypoxia-dependent reduction of this motif by hemeproteins results in the concomitant activation of a caged molecule and a latent electrophile. We exploit the former in a hypoxia-activated prodrug application using a caged staurosporine molecule as a proof-of-principle. We demonstrate the latter in in vivo tumor labeling applications with enamine-N-oxide-modified near-infrared probes. Hypoxia-activated prodrug development has long been complicated by the heterogeneity of tumor hypoxia in patients. The dual drug release and imaging modalities of the highly versatile enamine N-oxide motif present an attractive opportunity for theranostic development that can address the need not only for new therapeutics but paired methods for patient stratification.

Bioorthogonal Hydroamination of Push-Pull-Activated Linear Alkynes.

A bioorthogonal reaction between N,N-dialkylhydroxylamines and push-pull-activated halogenated alkynes is described. We explore the use of rehybridization effects in activating alkynes, and we show that electronic effects, when competing stereoelectronic and inductive factors are properly balanced, sufficiently activate a linear alkyne in the uncatalyzed conjugative retro-Cope elimination reaction while adequately protecting it against cellular nucleophiles. This design preserves the low steric profile of an alkyne and pairs it with a comparably unobtrusive hydroxylamine. The kinetics are on par with those of the fastest strain-promoted azide-alkyne cycloaddition reactions, the products regioselectively formed, the components sufficiently stable and easily installed, and the reaction suitable for cellular labeling.

Bioorthogonal Retro-Cope Elimination Reaction of N,N-Dialkylhydroxylamines and Strained Alkynes.

A bioorthogonal reaction between N,N-dialkylhydroxylamines and cyclooctynes is described. This reaction features a highly regioselective transformation between small, easily functionalizable reaction components with second-order rate constants reaching 84 M-1 s-1. The reaction is orthogonal to the inverse-electron demand Diels-Alder reactions between tetrazine and strained alkenes, and its components exhibit exquisite stability and chemoselectivity in cell lysate. This retro-Cope elimination reaction introduces a new member to the bioorthogonal reaction compendium outside the prolific class of cycloaddition reactions.

Conditions for CNT - Coated Textile Sensors Applied to Wearable Platforms to Monitor Limb Joint Motion.

Despite recent research on joint motion measurement to monitor human body movement, current measurement techniques and tools have significant limitations, including requiring large space for measurement and causing discomfort in test subjects wearing motion sensors. Our study aims, first, to develop carbon nanotube (CNT)-based textile joint motion sensors. Second, ours study aims to identify the most suitable CNT-based sensor structure and attachment method for use on a wearable platform during general exercise speeds. Lastly, we used these sensors on the human body, using sleeves and legs to find the most stable location, and we used the CNT-based sensor condition to monitor joint motions. We utilized our CNT-based sensor, which has proper elasticity as well as conductivity, and applied it to the elbow and knee joints. Based on the strain gauge principle, we monitored the variance of electric resistance that occurred when the CNT-based sensor was stretched due to limb motion. Our study tested 48 types of sensors. These sensors were applied to the CNT using different base knit textiles as well as different attachment methods, layers, sensor lengths, and sensor widths. The four most successful sensor types, which showed superior efficacy over the others in joint motion measurement, were selected for further study. These four sensors were then used to measure the elbow and knee joint motions of human subjects by placing them on different locations on sleeves and legs. The CNT knit textile sensors best suited to measuring joint motions are those with a double-layered CNT knit and 5 cm long × 0.5 cm or 1 cm wide sensors attached to a polyester¬-based knit using a welding method. The best position for the sensor to more stably monitor joint motions was the "below hinge position" from the elbow or knee hinge joint. Our study suggests an alternative strategy for joint-motion measurement that could contribute to the development of more comfortable and human-friendly methods of human limb motion measurement.

A human protein hydroxylase that accepts D-residues.

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

Visible light induced alkene aminopyridylation using N-aminopyridinium salts as bifunctional reagents.

The development of intermolecular alkene aminopyridylation has great potential for quickly increasing molecular complexity with two valuable groups. Here we report a strategy for the photocatalytic aminopyridylation of alkenes using a variety of N-aminopyridinium salts as both aminating and pyridylating reagents. Using Eosin Y as a photocatalyst, amino and pyridyl groups are simultaneously incorporated into alkenes, affording synthetically useful aminoethyl pyridine derivatives under mild reaction conditions. Remarkably, the C4-regioselectivity in radical trapping with N-aminopyridinium salt can be controlled by electrostatic interaction between the pyridinium nitrogen and sulfonyl group of ß-amino radical. This transformation is characterized by a broad substrate scope, good functional group compatibility, and the utility of this transformation was further demonstrated by late-stage functionalization of complex biorelevant molecules. Combining experiments and DFT calculations on the mechanism of the reaction is investigated to propose a complete mechanism and regioselectivity.

Site-Selective Functionalization of Pyridinium Derivatives via Visible-Light-Driven Photocatalysis with Quinolinone.

The selective installation of phosphinoyl and carbamoyl moieties on the pyridine scaffold is an important transformation in synthetic and medicinal chemistry. By employing quinolinone as an efficient organic photocatalyst, we developed a catalytic system driven by visible light that forms phosphinoyl and carbamoyl radicals, which react with various heteroarenium derivatives under mild, transition-metal-free conditions. This straightforward and environmentally friendly synthetic method represents a new approach to site-divergent pyridine functionalization that offers considerable advantages in both simplicity and efficiency. Ambient temperature is sufficient for the formation of the reactive radicals, and the site-selectivity can be switched from C2 to C4 by changing the radical coupling sources. Under standard reaction conditions, phosphinoyl radicals give access to C4 products, while carbamoyl radicals selectively give C2 products. We found that the carbamoyl radical overcomes the intrinsic preference for forming the ortho-product by allowing the oxo functionality of the carbamoyl radical to electrostatically engage the nitrogen of the pyridinium substrate, which preferentially gives the ortho-product. The phosphinoyl radical cannot engage in the same interaction, because the phosphorus is too large. This novel synthetic route tolerates a broad range of substrates and provides a convenient and powerful synthetic tool for accessing the core structures of numerous privileged scaffolds.

Allylic Acetals as Acrolein Oxonium Precursors in Tandem C-H Allylation and [3+2] Dipolar Cycloaddition.

The ruthenium(II)-catalyzed C-H functionalization of (hetero)aryl azomethine imines with allylic acetals is described. The initial formation of allylidene(methyl)oxoniums from allylic acetals could trigger C(sp2 )-H allylation, and subsequent endo-type [3+2] dipolar cycloaddition of polar azomethine fragments to deliver valuable indenopyrazolopyrazolones. The utility of this method is showcased by the late-stage functionalization of bioactive molecules such as estrone and celecoxib. Combined experimental and computational investigations elucidate a plausible mechanism of this new tandem reaction. Notably, the reductive transformation of synthesized compounds into biologically relevant diazocine frameworks highlights the importance of the developed methodology.

Visible-Light Excitation of Quinolinone-Containing Substrates Enables Divergent Radical Cyclizations.

Reported herein is the photochemical activity of quinolinone-containing substrates that directly reach an excited state upon light absorption to trigger radical-based bond-forming processes. The presented transformations allow divergent construction of valuable dihydro- or tetrahydrophenanthridin-6(5 H)-ones through the generation of S- or P-centered radicals, subsequent radical addition, cyclization, and a hydrogen atom transfer/electron transfer sequence. This strategy demonstrates the potential generality of quinolinone-tethered substrates to directly participate in the photoexcitation for the development of useful synthetic methods.

Visible-Light-Induced C-O Bond Formation for the Construction of Five- and Six-Membered Cyclic Ethers and Lactones.

Visible-light-induced intramolecular C-O bond formation was developed using 2,4,6-triphenylpyrylium tetrafluoroborate (TPT), which allows the regiocontrolled construction of cyclic ethers and lactones. The reaction is likely to proceed through the single-electron oxidation of the phenyl group, followed by the formation of a benzylic radical, thus preventing a competing 1,5-hydrogen abstraction pathway. Detailed mechanistic studies suggest that molecular oxygen is used to trap the radical intermediate to form benzyl alcohol, which undergoes cyclization. This new approach serves as a powerful platform by providing efficient access to valuable five- and six-membered cyclic ethers and lactones with a unified protocol.

Regiodivergent Ring-Opening Cross-Coupling of Vinyl Aziridines with Phosphorus Nucleophiles: Access to Phosphorus-Containing Amino Acid Derivatives.

Catalytic ring-opening phosphonation and phosphatation of vinyl aziridines have been developed in a regiodivergent fashion, giving linear and branched products. Generation of P-centered radicals enables SN2'-type ring-opening reactions of vinyl aziridines to afford δ-amino alkylphosphorus products at room temperature. On the other hand, in situ generated phosphate anions via the Ag-catalyzed aerobic oxidation of phosphonyl reactants underwent SN2 reaction to provide branched phosphorus-containing amine products. Furthermore, this divergent methodology serves as a powerful tool for the stereospecific synthesis of phosphorus-containing amino acid derivatives.

Reactivity of Morita-Baylis-Hillman Adducts in C-H Functionalization of (Hetero)aryl Nitrones: Access to Bridged Cycles and Carbazoles.

The rhodium(III)-catalyzed cross-coupling of (hetero)aryl nitrones with Morita-Baylis-Hillman (MBH) adducts is described. An allylated intermediate derived from aryl nitrones and MBH adducts allows the formation of bridged cyclic compounds via an exotype [3 + 2] cycloaddition. In sharp contrast, electron-rich indolinyl or aniline substrates were found to couple with MBH adducts to generate naphthalene or carbazole derivatives, respectively.