Olefin formation in palladium-catalyzed reactions is commonly rationalized by β-hydride elimination. However, under photoexcited conditions, palladium intermediates can differ substantially from those formed under thermal conditions, raising questions about the operative hydrogen removal pathway. In this study, we performed a computational mechanistic analysis of olefin formation in a ketyl radical-mediated photoexcited palladium-catalyzed reaction. Density functional theory calculations were carried out starting from a common intermediate to compare β-hydride elimination with hydrogen atom transfer (HAT). The lower-energy β-hydride elimination pathway requires ligand dissociation and the formation of an agostic intermediate before Cβ-H bond cleavage, whereas the HAT pathway proceeds more directly while retaining the phosphine coordination environment. Although the HAT pathway was consistently calculated to be energetically favored in the present system, β-hydride elimination can still become competitive when the preceding elementary steps are taken into account. These findings indicate that the operative hydrogen removal pathway in photoexcited palladium catalysis cannot be evaluated solely from the Cβ-H bond-cleavage step but must be considered together with the elementary steps required to reach each transition state.
The structure determination of destruxin F was achieved by total synthesis. Four isomers of the α,γ-dihydroxycarboxylic acid derivatives were comprehensively prepared from chiral glycidols in an asymmetric manner, enabling the successful synthesis of all structural candidates of destruxin F. The spectral data of the (1αR,1γR)-isomer were in good agreement with those of natural destruxin F, leading to the determination of the absolute configuration of the undetermined chiral centers as 1αR and 1γR, respectively. Furthermore, biological evaluation of the synthetic compounds revealed that the proper conformation of the cyclic peptide scaffold is required to exhibit the cytotoxicity and that the structure of α-hydroxycarboxylic acid moiety regulates the potency of biological activity.
Prostate cancer remains a leading cause of cancer-related mortality. Targeted alpha therapy using 211At is a promising treatment, although the low tumor-to-kidney ratios of 211At-labeled agents pose a persistent challenge. In this study, we designed prostate-specific membrane antigen (PSMA)-targeted radioligands incorporating 125I-labeled p-iodophenyl (IP) or 211At-labeled p-astatophenyl (AP) derivatives with different carbon-chain lengths and evaluated their impact on pharmacokinetics. In biodistribution studies, while [125I]IP-Cn-PSMA (n = 1, 2, or 4) exhibited low blood retention or high renal uptake, [125I]IP-C3-PSMA showed marked blood retention and low renal uptake, resulting in a higher tumor-to-kidney ratio compared with [125I]IP-C1-PSMA. [211At]AP-C1-PSMA and [211At]AP-C3-PSMA showed pharmacokinetic trends in the blood and kidneys comparable to those of the corresponding 125I-labeled compounds, despite partial deastatination. These results suggest that C3-chain-based IP and AP derivatives are effective for improving the tumor-to-kidney ratio, although the C1-chain-based derivatives retain advantages in early pharmacokinetics. Further comprehensive evaluations in tumor-bearing mice are required to determine the optimal candidate for PSMA-targeted 211At-labeled agents, potentially involving additional pharmacokinetic fine-tuning.
An encrusting, algae-associated octocoral, Briareum stechei, afforded three 8,17-epoxybriarane diterpenoids, including the known compound briaexcavatolide B (1) and two previously undescribed analogues, briastecholides Q (2) and R (3). The absolute configuration of 1 was established for the first time by single-crystal X-ray diffraction analysis. The planar structures and relative configurations of 2 and 3 were determined through extensive spectroscopic analyses. In bioactivity evaluation, compound 2 exhibited cytotoxic effects toward the human acute lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM.
Nucleic acid aptamers are single-stranded oligonucleotides that recognize diverse molecular targets with high affinity and specificity. Owing to their small size, amenability to chemical synthesis, and facile functionalization, aptamers are emerging as versatile alternatives or complements to antibodies in pharmaceutical sciences. This review summarizes recent advances from 2020 to 2025 in the pharmaceutical applications of aptamers, focusing on four areas: therapeutics, biosensing, bioanalysis, and process analysis. In therapeutics, the U.S. Food and Drug Administration (FDA) approval of Izervay (avacincaptad pegol) in 2023 marked a significant milestone, renewing interest in aptamer-based drug development. Aptamers are being explored for cancer therapy, immunotherapy, and neurodegenerative disease treatment, with several candidates in clinical trials. In diagnostics, aptamer-based biosensors (aptasensors) enable sensitive detection of tumor markers, disease biomarkers, and infectious disease agents, demonstrating particular utility in point-of-care applications. For bioanalysis of biopharmaceuticals, anti-idiotype aptamers serve as capture molecules for antibody drug quantification, offering advantages in batch-to-batch consistency and stability. In process analytical technology, aptamers are being applied to real-time monitoring of cell culture conditions and quality control of antibody drug manufacturing. These advances position nucleic acid aptamers as increasingly important tools in pharmaceutical sciences.
Bioanalysis of the immune checkpoint inhibitor nivolumab is crucial for pharmacokinetic studies. However, conventional ligand-binding assays (LBAs) often suffer from the lot-to-lot variability of capture antibodies. Here, we developed a high-affinity anti-nivolumab DNA aptamer as a robust alternative reagent. An integrated strategy combining Fast Protein Liquid Chromatography-based screening, next-generation sequencing, aptamer search algorithm, and computational modeling enabled rapid aptamer identification and optimization. The anti-nivolumab aptamer, mutant 2-3, discovered through in silico modification exhibited a dissociation constant (KD) of 33 nM. Using this aptamer, an enzyme-linked aptamer assay (ELAA) successfully quantified nivolumab in 400-fold diluted plasma samples over a range of 0.02-5.0 µg/mL, demonstrating good precision with a coefficient of variation less than 11.9%. This novel analytical method provides a sufficient quantitative range for the bioanalysis of nivolumab, thus offering a robust, specific, and cost-effective alternative for future pharmacokinetic studies.
DNA aptamers are attractive alternatives to antibodies and advances in selection technologies have yielded several candidate sequences, creating a strong need for efficient methods to evaluate their binding specificity. We previously established a photoaffinity electrophoretic mobility shift assay (EMSA) for analyzing DNA-protein interactions. In this study, we investigated whether this method can be applied to assess the binding specificities of α-thrombin-binding DNA aptamers. The binding specificities of a 15-mer and a 29-mer aptamer were evaluated via photoaffinity EMSA using reaction mixtures enriched in zymogen prethrombin-2, whose structure closely resembles α-thrombin but lacks fibrinogen-clotting activity. Photoaffinity EMSA enabled quantification of the relative binding strengths of α-thrombin and prethrombin-2 resolved in the same sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) lane, revealing that the specificity for α-thrombin was approximately 2.5-3.5-fold higher than that for prethrombin-2 for both aptamers. In the case of the 15-mer aptamer, introducing the photoreactive group at the 9th nucleotide reduced its binding specificity for α-thrombin. This finding indicates that the position of the modification affects aptamer-protein recognition and offers clues about the structural basis of binding. Photoaffinity EMSA also enabled us to distinguish the binding sites of the 15-mer and 29-mer aptamers on α-thrombin using competition experiments with dabigatran and heparin, while allowing selective detection of α-thrombin in human plasma. Together, these results demonstrate that photoaffinity EMSA is a simple and informative approach that can evaluate the binding specificities of multiple DNA aptamers for a target protein in a single SDS-PAGE-based assay.
Controlling indoor humidity is crucial for preventing health concerns, such as heatstroke and atopic dermatitis. Sugarcane bagasse (BG), a byproduct of sugar production primarily composed of cellulose, is a promising, sustainable, humidity-control material owing to its charcoal- and bamboo-like characteristics. This study systematically investigates the humidity-control ability of BG-derived porous carbons prepared over a wide range of calcination temperatures. BG calcined at 900°C exhibited excellent humidity-control ability (158.7 mg/g between water activity levels of 0.98 and 0.59) and retained sorbed water across a wide range of water activities. With increasing calcination temperature, the specific surface area increased, whereas the amount of acidic functional groups decreased above 300°C, indicating significant changes in the surface chemistry. Humidity-control ability was strongly correlated with specific surface area and mesopore volume. Differential heat analysis indicated that physisorption dominated in all samples, while chemisorption was present in raw BG and BG calcined at 200-700°C. Entropy analysis further suggested that BG calcined at 800-1000°C contained abundant free water and bottleneck-shaped pores, resulting in water retention over a broad range of water activities. These findings demonstrate the potential of BG calcined at 900°C as a sustainable humidity-control material for indoor environmental applications.
Identification tests based on gas chromatography (GC) with a flame ionization detector (GC-FID) are specified for sage oil (SO) and sage water (SW) in the Japanese Standards of Quasi-Drug Ingredients. In the current methods, a packed column (PC) is used for both products, causing prolonged analysis and limited separation efficiency. In addition, sage extract (SE) is designated as the reference standard for the SW identification test; however, its major constituents remain uncharacterized. The capillary column (CC) has become the standard in recent GC procedures, making PC replacement with CC desirable for SO and SW. In this study, we aimed to improve CC-based GC-FID identification tests for SO and SW. The separation behavior of the SO marker components, including α-pinene, thujone, and camphor, was first examined using GC-MS, which demonstrated good separation using CC. Rapid GC conditions were subsequently evaluated, achieving a substantial reduction in analysis time while maintaining sufficient separation and detection sensitivity. The analysis of SW and SE revealed that α-thujone, camphor, and 1,8-cineole were the common major components. However, dilution with ethanol occasionally hindered the visual recognition of marker components. The application of hexane liquid-liquid extraction (Hex-LLE) as a sample pretreatment effectively removed these interfering components, enabling the clear identification of marker compounds even in aqueous samples. These results demonstrate that the combination of CC-based GC-FID and Hex-LLE provides an effective and practical approach for the identification of SO and SW. The method may be applicable to other plant-derived essential oils and aromatic distilled waters.
Nitroxyl radicals have long been recognized as powerful catalysts for alcohol oxidation. However, challenges remain regarding their efficiency, selectivity, and substrate scope. This review summarizes the author's efforts to advance alcohol oxidation catalysis through two complementary approaches: the discovery of new nitroxyl radical-based catalysts and the development of cooperative catalytic systems to address chemoselectivity challenges. Studies on 2-azaadamantane N-oxyl derivatives have led to the unexpected identification of alkoxyamine catalysts, whose unique structures and mechanistic features provide new insights into catalyst design. Further studies have established cooperative systems combining nitroxyl radicals with copper salts that exhibit distinctive chemoselectivity and enable transformations previously inaccessible to purely organocatalytic methods. Collectively, these studies demonstrate how mechanistic understanding and catalyst development can expand the utility of alcohol oxidation in synthetic chemistry, thereby offering new tools for the selective oxidation of complex molecules.
Focused beam reflectance measurement (FBRM) has been used for investigating tablet dissolution behavior. However, few studies have used FBRM to analyze sustained-release formulations. In this study, we prepared tablets containing hydroxypropyl methylcellulose (HPMC), a widely used sustained-release excipient, and evaluated dissolution behavior using both conventional dissolution testing and FBRM. To assess the effect of drug solubility on FBRM parameters, we also compared acetaminophen (readily soluble in water) with ethenzamide (poorly soluble in water). Conventional dissolution testing revealed a difference of approximately 360 min among the different HPMC grades in the time required to reach 60% drug release, while FBRM detected an approximate threefold difference in the final particle counts. These findings suggest that FBRM is a useful analytical tool for elucidating the dissolution behavior of sustained-release formulations.
Aster yomena (Kitam.) Honda, a perennial herb belonging to the family Asteraceae, is widely used in traditional Japanese cuisine and folk medicine. However, the bioactive constituents of A. yomena have not been sufficiently characterized, limiting its practical applications. In this study, the phytochemicals and bioactivities of A. yomena and related Aster species collected from multiple locations across Kyushu, Japan were analyzed. Total phenolic, flavonoid, and saponin contents, antioxidant capacity, and antiproliferative activity against gastric cancer cells (AGS) were evaluated. Among the different plant parts, the leaves of A. yomena exhibited the highest phytochemical content and bioactivity. Regional comparisons revealed that leaf samples from coastal regions showed significantly higher flavonoid and saponin accumulation, as well as stronger antioxidant and antiproliferative activities, than those from inland populations. HPLC and LC/MS analyses identified chlorogenic acid, rutin, isoquercetin, nicotiflorin, and triterpenoid saponins as the major bioactive constituents. Regression analyses demonstrated that available phosphorus, exchangeable magnesium, and certain climatic factors (e.g., temperature range) were positively correlated with metabolite levels and bioactivities. These findings are expected to provide essential foundational data for future applications of the bioactive constituents of A. yomena in functional foods and pharmaceuticals.
Aiming to develop safe and efficient cognitive enhancers, this study reports the design, synthesis, and biological evaluation of novel α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor modulators derived from the lead compound CX717. Twenty-four derivatives were designed, synthesized, and characterized by 1H-NMR, 13C-NMR, and high resolution MS (HR-MS). Molecular docking studies indicated that, compared to CX717, the compounds exhibited lower binding energy to the AMPA receptor. Cognition-improved activity was evaluated by passive avoidance, active avoidance, novel object recognition, and novel place recognition tests in mice. The results demonstrated that nine compounds significantly enhanced cognitive function in mice, with compounds B7 and C3 showing efficacy superior to CX717 in vivo. Furthermore, B7 and C3 alleviated serum oxidative stress, enhanced hippocampal glutamate transport efficiency, and reduced serum pro-inflammatory cytokine levels in scopolamine-treated mice. Subacute toxicity studies revealed favorable safety profiles for both compounds. Molecular dynamics simulations supported stable binding of B7 and C3 to the AMPA receptor, consistent with the structural modification strategy. Surface plasmon resonance (SPR) analysis demonstrated that B7 and C3 bound directly to the GluR2 protein with moderate affinity, with C3 showing higher affinity than B7. Patch-clamp experiments further showed that B7 and C3 positively modulated AMPA receptor-mediated currents, supporting their positive allosteric modulator (PAM)-like activity. The membrane permeability assay suggested that B7 and C3 had higher passive membrane permeability than CX717 in vitro. In conclusion, B7 and C3 demonstrated superior cognition-improved activity and safety compared to CX717, highlighting their potential as candidates for treating cognitive disorders.
Nonclassical N-methyl amidation reactions represent a valuable alternative to conventional coupling methods and expand access to drug-like molecules. Herein, we report a direct N-methyl amidation using N,N-disubstituted aminophosphonium salts. Treatment of a bench-stable trimethylaminophosphonium salt with silver acetate or quaternary ammonium carboxylates efficiently affords tertiary N-methyl amides at ambient temperature without the use of external coupling reagents. The aminophosphonium salts act as bench-stable and readily accessible N-methylamine equivalents, enabling the simultaneous activation of carboxylic acids and delivery of N-methylamine without overalkylation. Notably, the reaction proceeds via a mechanistically distinct pathway that does not involve iminophosphorane intermediates. This method provides a practical and mild approach to the synthesis of tertiary N-methyl amides.
2,3,4,5-Tetrahydro-1,5-benzoxazepine derivatives constitute an important class of heterocycles frequently found in pharmaceuticals. In this study, we developed an efficient and practical synthetic method for the construction of 2,3,4,5-tetrahydro-1,5-benzoxazepine cores starting from N-aryl isoxazolidines. The key step involves Lewis acid-mediated alkoxy migration induced by N-O bond cleavage using aluminum chloride, providing the desired heterocyclic scaffolds in good yields. This method offers a new route to access 2,3,4,5-tetrahydro-1,5-benzoxazepine frameworks and expands the utility of N-aryl isoxazolidines in heterocycle synthesis.
Cell-penetrating peptides (CPPs) have attracted considerable attention as carriers that facilitate the intracellular delivery of biomacromolecules. In this study, the amphipathic antimicrobial peptide K9L9 was used as a model, and a series of peptides incorporating the non-proteinogenic amino acid α-aminoisobutyric acid (Aib) were designed and synthesized to investigate the relationship between peptide structure, membrane interaction, and cellular uptake behavior. Circular dichroism analysis revealed that K9L9 adopted a β-sheet-like conformation in an aqueous solution, whereas the Aib-containing peptide formed a stable α-helical structure. In liposome leakage assays, peptides containing two Aib residues exhibited enhanced membrane-disruptive activity, whereas the peptide containing four Aib residues exhibited reduced activity. Furthermore, intracellular delivery studies using fluorescein isothiocyanate-dextran (FITC-dextran) demonstrated that delivery efficiency was significantly enhanced when the peptides were preincubated with FITC-dextran, suggesting that peptide-cargo complex formation plays a critical role in the delivery process. In addition, K9L9 exhibited rapid cellular uptake at early time points, whereas the Aib-containing peptides showed a gradual increase in uptake over time, indicating distinct uptake kinetics. These results demonstrate that Aib-induced helix stabilization modulates the membrane interaction and cellular uptake behavior of amphipathic peptides.
Synthetic studies toward the indole diterpene penitrem E are described. A functionalized o-alkynylaniline derivative was designed as a model substrate to construct the D/E/F/G tetracyclic core bearing an exocyclic functional group on the F ring as a precursor for C18 oxygen installation. The tetracyclic compound is furnished through a Pd-catalyzed cascade cyclization of the o-alkynylaniline derivative, which involves indole formation, an intramolecular Heck reaction, and oxidative chlorination of the Heck intermediate.
We have developed a methodology for rapid extraction under continuous-flow conditions. Basic compounds with a tert-butoxycarbonyl protecting group, which are labile under acidic conditions, were extracted into an acidic aqueous phase by a mixer-settler under continuous-flow conditions and immediately neutralized prior to their decomposition. This methodology could serve as a promising option for purifying drug substances during pharmaceutical production.
The Japanese Pharmacopoeia (JP) method describes the determination of aspirin content by alkaline hydrolysis of the ester bond and neutralization of the carboxyl group, followed by back-titration of the remaining sodium hydroxide (NaOH) with sulfuric acid. Although the monograph does not explicitly address this point, the excess NaOH is expected to deprotonate the phenolic hydroxyl group of salicylic acid, generating the corresponding dianionic species. Despite its presumed formation under the JP assay conditions, this species has not yet been experimentally verified. In this study, we spectroscopically characterized the dianionic form of salicylic acid, the hydrolysis product of aspirin, to determine whether it actually forms during this procedure. Deprotonation of the phenolic hydroxyl group enhances electron delocalization within the aromatic system, inducing marked changes in both absorbance and fluorescence. Under strongly alkaline conditions (pH > 12), the UV-visible spectra (200-400 nm) exhibited predominantly red-shifts. Furthermore, the fluorescence excitation band at 250 nm increased sharply, whereas that at 294 nm remained unchanged. These absorbance and fluorescence spectral changes are consistent with the formation of the salicylate dianion as predicted by the Henderson-Hasselbalch equation. Lastly, to determine whether the dianionic species is present after treatment with NaOH, we measured the absorbance spectra of undiluted reaction mixtures. The observed spectra were well described by combinations of the monoanionic and dianionic reference spectra, supporting the presence of the dianionic form. This work sheds light on salicylate species overlooked in the aspirin titration process and contributes to a mechanistic understanding of the JP titration.
Liquid-phase peptide synthesis (LPPS) has emerged as a powerful platform for constructing complex peptide natural products, yet its efficiency remains highly dependent on carrier design, protecting group compatibility, and late-stage functional group manipulations. Here, we report a streamlined LPPS strategy enabled by a benzoyl-type tag carrier that facilitated tert-butoxycarbonyl (Boc)-based elongation, mild carrier cleavage, and direct access to C-terminal peptide alcohols. This platform enabled the concise synthesis of kozupeptin aldehyde-an exceptionally potent antimalarial peptide aldehyde-in only 11 purification steps from commercially available methyl gallate, representing a substantial improvement over previous methodologies. The robustness of the benzoyl tag system further allowed the rapid preparation of 10 analogs by parallel one-pot LPPS via single-residue scanning across the peptidic core. Biological evaluation against Plasmodium falciparum revealed tight structure-activity relationships, identifying strict sequence and conformational requirements for potency while highlighting the crucial stereoelectronic influence of the 4-methyl-proline (Pro(4-Me)) residue and the threonine (Thr)-proline (Pro) amide bond equilibrium. Collectively, this work establishes a versatile LPPS approach for accelerated access to peptide natural products and provides new insights into the conformational determinants underlying kozupeptin's antimalarial activity.
