Recently, oligonucleotide-based drug discovery has attracted considerable amounts of attention. As oligonucleotide therapeutics have evolved into practical use, research into the development of functional artificial nucleic acids has been vigorously conducted worldwide. However, the synthesis of artificial nucleic acids generally requires long sequences from starting materials; hence, structurally optimizing oligonucleotide therapeutics is extremely difficult. In response to this challenge, we have been developing reactions that use oligonucleotides as starting materials. As a part of this work, we focused on ligand–oligonucleotide conjugates because the conjugates of functional ligands and oligonucleotides have attracted attention as drug-delivery systems that improve the efficacies of oligonucleotide therapeutics; they are also DNA-encoded-library-based drug-discovery tools. In this review, we broadly introduce our research into ligand–oligonucleotide conjugates.

Paracoccidioidomycosis (PCM) is an infectious disease caused by dimorphic fungi of the Paracoccidioides genus and causes a series of discomforts in affected patients. This work aimed to evaluate the antifungal potential of synthetic chalcones against Paracoccidioides brasiliensis (Pb) and to determine in silico possible therapeutic targets. An in silico evaluation of a database of 21 synthesized chalcones was carried out based on pharmacokinetic parameters, enzymatic inhibition, Tanimoto similarity, and the prediction of the spectrum of activity by PASS (prediction of activity spectra of substances). The most viable chalcones from the previous evaluation were selected for the measurement of minimum inhibitory concentration (MIC) against Pb and for cytotoxicity assays pre- and post-metabolization using HEPG2 cells. After in silico evaluation, the compounds 4, 11, 12, 20, and 21 were selected to carry out the molecular docking and in vitro tests. In the docking studies, multiple hydrophobic and polar intermolecular interactions were observed, such as hydrogen bonds, with emphasis on compound 20 in the active site of thioredoxin, where it made 4 hydrogen bonds with the residues Gln43, Ala36, and Thr38. In vitro testing revealed antifungal activity, with the MICs ranging from 32 to 128 μg/mL. In cytotoxicity assays, the 5 compounds exhibited reduced IC₅₀ values (5.51–14.85 μg/mL pre-metabolization and 10.48–35.4 μg/mL post-metabolization). The compounds 4, 11, 12, 20, and 21 have shown favorable predictions of pharmacokinetic characteristics and distinct actions compared to conventional medications, as well as antifungal activity with less toxicity after metabolization, making them the best candidates for further studies.

In this study, a palladium-catalyzed, amide-directed C–H acetoxylation of cubanes has been developed. The ortho positions of the amide directing group on cubanes were selectively acetoxylated without any stoichiometric strong bases. The number (1–3) of acetoxy groups introduced was determined by the amount of PhI(OAc)₂ used. Substitution at the C4 position of cubane dramatically affected the outcome of the acetoxylation reaction.

Conventional peptide synthesis involves multiple protection and deprotection steps, and typically relies on stoichiometric amounts of coupling reagents and additives. This makes the process cumbersome, and results in poor atom economy and hazardous waste generation. Therefore, direct peptide bond formation using unprotected amino acids is a promising alternative. However, this approach presents some challenges: 1) Solubility of unprotected amino acids in organic solvents; 2) Control of undesired side reactions; 3) Chemo-selective activation of the carboxylic acid group in the presence of an amine functionality; and 4) Epimerization. To address these challenges, we developed tris(2,2,2-trifluoroethoxy)silane [H-Si(OCH₂CF₃)₃], a cost-effective and accessible coupling reagent. This single reagent efficiently synthesizes N-terminal free peptides from unprotected amino acids and amino acid tert-butyl esters, without the need for any additives. H-Si(OCH₂CF₃)₃ enhances amino acid solubility through coordinating with both termini and plays a dual role, serving as a transient amine-protecting group and as a carboxylic acid activating or promoting reagent for peptide bond formation. This method is operationally simple and versatile, enabling the efficient synthesis of N-terminal free peptides from unprotected amino acids and amino acid tert-butyl esters, with good yields, high optical purity, and broad side-chain compatibility.

Antigen-binding proteins, such as nanobodies, modified with functional small molecules hold great potential for applications including imaging probes, drug conjugates, and localized catalysts. However, traditional chemical labeling methods that randomly target lysine or cysteine residues often produce heterogeneous conjugates with limited reproducibility. Conventional site-specific conjugation approaches, which typically modify only the N- or C-terminus, may also be insufficient to achieve the desired functionalities. Genetic code expansion offers a powerful alternative by enabling the site-specific incorporation of noncanonical amino acids bearing reactive handles—such as trans-cyclooctene (TCO)—thereby allowing precise bioorthogonal conjugation via click chemistry. Nevertheless, identifying suitable incorporation sites that tolerate such modifications without disrupting antigen binding remains a time- and cost-intensive process, as this process typically requires labor-intensive screening involving the expression and purification of each candidate variant. Here, using HER2 and an anti-HER2 nanobody as a model antigen–binder pair, we present a convenient mammalian cell-based screening platform for rapid, purification-free evaluation of site-specifically labeled nanobodies. The nanobody is fused to blue fluorescent protein (BFP), secreted by HEK293T cells, and labeled in situ with a tetrazine–fluorescein probe. The resulting supernatant is then applied directly to HEK293T cells stably expressing HER2–mCherry. Labeling efficiency and retention of antigen-binding activity are simultaneously assessed by fluorescence imaging in the BFP, fluorescein, and mCherry channels. This approach enables efficient identification of labeling sites that support productive click conjugation while preserving binding function. It should be broadly applicable to other antigens and binders, streamlining early-stage screening of engineered antigen–binder conjugates for diverse applications.

This study evaluated the cadmium (Cd) adsorption characteristics of sugarcane bagasse (BG) calcined at different temperatures (200–1000°C). The point of zero charge (pH_pzc) of the BGs ranged from 4.3 to 6.5. The amount of Cd adsorbed was higher at pH 4–8 than at pH 2. These results indicate that the negative charge on the BG surface and the presence of Cd(II) cations are important for Cd adsorption. Meanwhile, the amount of Cd adsorbed and the specific surface area (SSA) of BG increased with increasing calcination temperature of BG. Furthermore, a partial correlation analysis revealed that acidic surface functional groups (SFGs) were also significantly associated with Cd adsorption, independently of SSA. These results suggest that both SSA and acidic SFGs jointly contribute to Cd adsorption.

Several approaches for synthesizing [1-¹³C]2-oxoglutaric acid were attempted, and the synthesis was successfully achieved in 4 steps from trimethylsilyl ¹³C-cyanide. The ¹³C-breath tests on rats were conducted by orally administering the newly synthesized [1-¹³C]2-oxoglutaric acid, the previously prepared [1′-¹³C]citric acid, and [1-¹³C]acetic acid as a control drug, and the results were compared. The results indicate that [1-¹³C]2-oxoglutaric acid and [1′-¹³C]citric acid may serve as potential substrates for assessing the TCA cycle flux.

The development of analytical techniques applicable to powdered pharmaceutical co-crystals, including those containing excipients, represents a comprehensive strategy for quality control in both drug development and regulatory settings. This study investigates the structural characterization of indomethacin–nicotinamide co-crystals using a combination of microcrystal electron diffraction (microED), solid-state NMR (SSNMR), Raman spectroscopy, and powder X-ray diffraction (PXRD). MicroED analysis revealed the crystal structure of the co-crystal, while SSNMR measurements provided insights into the molecular interactions within the structure. The combination of solid-state NMR and microED with general techniques such as PXRD and Raman spectroscopy is considered useful for confirming crystal properties that cannot be identified by a single method and is applicable to both active pharmaceutical ingredients and final formulations.

Therapeutic drug monitoring (TDM) is vital for effective optimization of pharmacological treatments. In this study, we engineered a chromatography column that is sensitive to temperature fluctuations, thereby enabling safe and straightforward TDM without relying on organic solvents. Silica beads were modified by applying poly(N-isopropylacrylamide) (PNIPAAm) hydrogels, using a condensation reaction to modify the initiator, followed by radical polymerization to integrate the PNIPAAm hydrogel. We investigated the elution behavior of the drugs at varying temperatures using a PNIPAAm-modified silica bead-packed column and an aqueous mobile phase. Model samples composed of drugs and serum proteins were used to assess the elution behavior within the column. At a column temperature of 30°C, the serum proteins and drugs were successfully separated. The drug concentrations in the samples were quantified by constructing a calibration curve. Experimental results indicate that the chromatography column has significant benefits for TDM, as it allows the determination of drug concentrations in serum without requiring organic solvents in the mobile phase or sample preparation steps.

In screening for antibacterial agents from co-cultures of Mycobacterium smegmatis and microbial resources, such as actinomycetes and fungi, the known hydroxyquinone antibiotic griseorhodin A (1) was isolated from a co-culture of actinomycete strain TMPU-20A002 and M. smegmatis. Compound 1 exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), with minimum inhibitory concentrations of 0.25 and 2.0 μg/mL, respectively. In silkworm infection models with MRSA and VRE, 1 exhibited therapeutic efficacy, with ED₅₀ values of 2.5 and 18 μg/larva·g, respectively. A pharmacokinetic analysis of silkworms revealed an elimination half-life of 4.4 h in the hemolymph, indicating a favorable metabolic profile. This is the first in vivo evaluation of griseorhodin A, including its pharmacological activity and metabolic behavior, and it highlights its potential as a candidate for the development of novel antibacterial agents.

Electrophilic azides serve as valuable reagents in visible-light-induced photocatalytic processes. While the reactivity of triplet nitrenes formed via energy transfer to electrophilic azides has been relatively well studied, the synthetic potential of nitrene radical anions generated through single electron transfer remains less explored. Herein, we report a photoinduced azidosulfonylative cyclization of 1,6-dienes with sulfonyl azides in the presence of 9,10-diphenylanthracene as a photoreductant, providing access to functionalized five-membered heterocycles. Mechanistic studies suggest that nitrene radical anions are responsible for the generation of sulfonyl radicals through hydrogen atom transfer from N,N-dimethylformamide (DMF) solvent.

Therapeutic drug monitoring (TDM) is crucial for enhancing the effectiveness of pharmacological treatments. This study developed two types of two-dimensional (2D) chromatography systems. The primary column used a poly(N-isopropylacrylamide) (PNIPAAm) hydrogel-modified bead-packed column, while the secondary column employed either an octadecyl silyl (ODS) column or a PNIPAAm-terminally-modified column. The primary PNIPAAm-hydrogel-modified column effectively separated serum proteins and drugs, while the secondary ODS column exhibited a sharp peak owing to the drug concentration at the front of the column when an aqueous mobile phase was used. However, drug elution was slow, and the mobile phase required an organic solvent. By contrast, the column packed with PNIPAAm-terminally-modified beads reduced the analysis time, and organic solvent was unnecessary. These findings indicate that the developed two-column 2D chromatography system is advantageous for TDM. This system allows for the analysis of serum-drug samples without the need for sample preparation methods, such as deproteination. Moreover, drug concentration can be determined in a short analysis timeframe without organic solvents in the mobile phase.

Rosemary oil (RO) and rosemary water (RW) are widely used ingredients in cosmetics. According to the Japanese Standards of Quasi-Drug Ingredients (JSQDI), RO is defined as an essential oil obtained by the steam distillation of fresh rosemary (Rosmarinus officinalis L.) leaves, branches, and flowers, whereas RW is the water layer obtained from the steam distillation of the leaves. Although the JSQDI outlines a specific TLC identification test for RW, this test is time-consuming (15-cm development distance) and requires the use of hazardous reagents (toluene). Additionally, no identification tests are available for RO. This study therefore aimed to establish a new TLC-based identification test for RO in JSQDI, while also improving the RW identification test. Using cyclohexane/methyl tert-butyl ether/acetonitrile (20 : 10 : 1) as the developing solvent and 4-methoxybenzaldehyde sulfuric acid as the visualization reagent, 1,8-cineole and borneol were universally detected in 19 RO market products. Thus, 1,8-cineole was selected as the primary marker compound and borneol as the secondary marker compound. Additionally, the extraction process was optimized by applying these TLC conditions to RW, significantly simplifying the process. TLC analysis of the RW market products confirmed the presence of 1,8-cineole in all samples. Thus, in addition to representing a novel method for the identification of RO, these results indicate the suitability of the new extraction conditions for use as an alternative method to identify RW.

We compared the film-forming ability of a heparinoid cream (HP-C) formulation, an oil-in-water-type emulsion used as a moisturizer, and found that the original HP-C (HP-C_O) formulation formed a thick and robust film that floated easily on an acrylic plate. It is suggested that the thickness and robustness of HP-C_O contributed to the retention of its high keratin water content, while its floatability helped prevent adhesion to clothing after application. Among the generic HP-C formulations tested, only one (HP-C_G1) formed a film with properties similar to those of HP-C_O. When the HP-C formulations were mixed with white petrolatum, the floatability of the resulting film was eliminated, and the film easily disintegrated upon physical stimulation with a spatula. These results suggest that mixing the HP-C formulations with other ointment bases, such as white petrolatum, is not appropriate from the standpoint of film formation. Because peaks corresponding to glycerin (GL) were clearly observed in the near-IR spectra of both HP-C_O and HP-C_G1 after 24 h at room temperature, we explored the addition of water-soluble polyhydric alcohols, such as GL and propylene glycol (PG), to generic HP-C formulations that did not initially demonstrate good film-forming properties. The state of the film and its floatability tended to improve when these generic HP-C formulations were mixed with GL or PG. Thus, these results indicate that the film-forming ability differed depending on the HP-C formulation. Furthermore, the results suggest that GL played a key role in the successful film formation observed in both HP-C_O and HP-C_G1.

Enhancing the solubility of poorly water-soluble oral small-molecule drugs remains a critical challenge in the pharmaceutical industry, particularly during the early stages of drug development when selecting solubilized formulation technologies. This difficulty largely stems from the absence of objective indicators for comparing formulations. This study aimed to establish the selection indicators for solubilized formulation technologies based on applicable formulation preparation among amorphous solid dispersion (ASD), cyclodextrin (CD), lipid-based formulation (LBF), and nanocrystal (NC), utilizing the overall molecular properties, such as molecular weight, melting point (T_m), Log P, and polar surface area (PSA) in the marketed oral drugs employing solubilized formulation technologies. Single-property plots of the selected properties revealed the regions of higher or lower frequency in the selected molecular properties. The multiple property plot with T_m and the combined property of normalized Log P and PSA identified a specific area as the optimal range for selecting ASD, LBF, and NC, while excluding CD. These findings are expected to function as guide indicators that enable the rational selection of formulation technologies and serve as a foundational approach to accelerating development timelines based on molecular properties available during the early stages of drug development.

The present study reports on the structure–activity relationships of side-chain shortened and fluorinated vitamin D₃ analogs (5–13), with the goal of developing vitamin D analogs that are silent toward the vitamin D receptor (VDR) while retaining inhibitory activity against sterol regulatory element-binding protein (SREBP). The biological activities of the synthesized fluorinated vitamin D₃ analogs were assessed through their effects on the VDR ligand-binding domain (VDR_LBD), full-length VDR transcriptional activity, and SREBP transcriptional activity. Among the synthesized analogs, 8, 11, and 13 exhibited notable SREBP inhibitory activity, although their potency was lower than that of the natural vitamin D₃ metabolite, 25(OH)D₃ (1). Compound 11 emerged as the most effective analog, displaying selective SREBP inhibition with minimal VDR activation.

We previously reported that Ag^I- and Hg^II-mediated primer extension reactions with a dT template are highly selective for dC and dT triphosphate incorporation, forming T-Ag^I-C and T-Hg^II-T base pairs, respectively. This study investigates the impact of 5-substitution of templated thymine and pH on Klenow fragment (KF) catalyzed metal-mediated primer extension reactions, as well as the thermal stability of DNA duplexes with a 5-substituted uracil derivative. The findings reveal that the selective base recognition of Ag^I and Hg^II ions, leading to T-Ag^I-C and T-Hg^II-T formation in metal-mediated primer extension, is likely governed by the absence of net charge in the resulting base pairs, the abstraction of thymine’s N3 imino proton by Ag^I, and the stability of the Hg^II-N3 bond. These findings may be useful for designing novel regulated replicating systems utilizing metal-mediated base pairs.

Real-time detection and identification of modified cysteine sulfhydryl (Cys-SH) residues are important because supersulfidation (S-sulfhydrylation) alters protein function and thereby modulates the activity of biological systems. Although fluorescence probes for rapid, sensitive detection and biotin tag-switch methods for comprehensive labeling of modified residues at the proteome level have been developed, simultaneous detection and labeling have not been achieved in a single system. Herein, we describe dinitrobenzene-based fluorogenic probes with tunable electrophilicity, which react rapidly and selectively with highly nucleophilic supersulfides such as cysteine persulfide (Cys-SSH) and Na₂S₂ and simultaneously label Cys-SSH itself with a dinitrobenzene tag.

Efficient cytosolic delivery of functional proteins such as therapeutic antibodies remains a major challenge in drug development. In this study, we sought to optimize the cytosolic delivery peptide Mel-V8G12, a melittin derivative, through structure-guided design and functional screening of its amino acid substitutions. Among seven derivatives, VG-6, featuring A10L, T11E, and S18K substitutions demonstrated superior cytosolic delivery efficiency compared with the parental Mel-V8G12, while maintaining low cytotoxicity. Notably, VG-6 exhibited enhanced membrane-lytic activity toward neutral lipid membranes, yet did not increase cellular toxicity, suggesting a delivery mechanism distinct from conventional pH-responsive endosomolytic peptides. Mechanistic studies revealed that, in contrast to Mel-V8G12 which predominantly utilizes actin-mediated endocytosis, VG-6 additionally engages caveolae-mediated endocytosis, contributing to its enhanced cytosolic delivery. Furthermore, VG-6 enabled successful cytosolic delivery of functional Cre recombinase and immunoglobulin G (IgG), facilitating biological activity and subcellular targeting. These findings suggest that VG-6 is a promising tool for intracellular delivery of protein therapeutics via a unique membrane-interacting and endocytic pathway.

Antibody-based therapeutics have shown remarkable success in targeting extracellular molecules, yet their application to intracellular targets remains largely unexplored due to the absence of efficient delivery systems. The large molecular weight and hydrophilicity of immunoglobulin G (IgG) make cytosolic delivery particularly challenging. Previously, we developed a cytosolic delivery peptide, E3MPH16, based on a modified Mastoparan X sequence, which enabled efficient delivery of macromolecules such as dextran with minimal cytotoxicity. However, effective intracellular delivery of antibodies required high concentrations, limiting its practical utility. In this study, we aimed to enhance delivery efficiency while preserving low toxicity by introducing d-amino acid substitutions into E3MPH16. The resulting peptide, e3MPH16, incorporates d-glutamic acid residues at the N-terminus to improve serum stability and protease resistance. Functional analyses demonstrated that e3MPH16 significantly improves cytosolic delivery of Cre recombinase and antibodies compared with the original E3MPH16, without increasing membrane-lytic activity or cytotoxicity. These results underscore the potential of d-amino acid-substituted peptides such as e3MPH16 as a promising platform for the intracellular delivery of unmodified functional antibodies.

Structural transformation changes the activity of biological reactions. Neurotransmitter aralkylamines, such as phenethylamine, tyramine, dopamine, tryptamine, serotonin, and histamine, absorb aerial CO₂, and heteronuclear multiple bond connectivity (HMBC) correlations between the carbon derived from CO₂ and the α-hydrogen of the several amines were confirmed in the D₂O solution. The isolation of methyl carbamate from phenethylamine and CO₂ in water with TMSCHN₂ also supported the formation of covalently bound carbamic acid in the amine aqueous solution containing CO₂. Therefore, it is suggested that CO₂ produced in the body would react with neurotransmitter amines to form covalently bound carbamic acid, which might affect biological reactions.