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.

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.

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.

A method for chemoselective acylation of the vacant amide of diketopiperazines (DKPs) using di-tert-butyl dicarbonate (Boc₂O) was developed. Nucleophilic tributylphosphine reacts immediately with acid anhydride to form an active quaternary phosphonium cation. This intermediate induces acylation of the amide group on the empty side, resulting in chemoselectivity that could not be controlled by using N,N-dimethyl-4-aminopyridine (DMAP). This method achieved high yields of mono-acylated DKPs, avoiding the acylation of amino acids with bulky substituents. This reaction system also provides chemoselectivity based on the bulkiness of the protecting groups of active functional groups at the side chain, and enhances the range of substrate applicability by achieving acylation of the amido groups of amino acids, except for glycine. Furthermore, using the substrates prepared here, elongation reactions induced by introducing amino acid esters associated with ring opening were also successfully demonstrated under reaction conditions without any additives, highlighting the possibility to form peptides with a broad range of sequences.

Furanosteroids are known to exhibit inhibitory activity against phosphatidylinositol-3-kinase and are expected to serve as a basis for the development of therapeutic drugs for various diseases. In this study, a novel protocol is presented for preparation of the furanosteroid A-ring moiety. More specifically, the lipase-catalyzed kinetic resolution of racemic 1-(3-bromofuran-2-yl)-2-chloroethanol with β-substituted (Z)-acrylates and the subsequent intramolecular Diels–Alder reaction of the generated enantiomerically enriched esters were performed to obtain multi-functionalized fused cyclohexenes in excellent enantiomeric ratios (99 : 1 or 98% enantiomeric excess (ee)) and diastereomeric ratios (≥98 : 2). The obtained products possess the appropriate stereochemical structures and absolute configuration for use in the asymmetric synthesis of the A-ring moieties of naturally occurring furanosteroids, including viridin and viridol.

Oxyluciferin is the key photon emitter in the firefly/beetle luciferase-catalyzed bioluminescence reaction, and elucidating its chemical form within the active site of luciferase is essential for understanding and modulating emission wavelength. In this study, we focused on aminoluciferin (AL), a d-luciferin (d-LH₂) analog in which the hydroxy group is substituted with an amino group. AL has been widely used as an alternative substrate to d-LH₂ and in the development of bioluminescence probes. To identify the emissive form of AL in bioluminescence, we synthesized OxyAL (the emitter derived from AL) and 5-methyl-OxyAL, and measured their fluorescence spectra under various conditions. We assigned the range of emission wavelengths of keto-, enol- and enolate-OxyAL using 5,5-dimethyl-OxyAL, fixed in the keto form, as a reference. Based on spectral assignments and comparison with the bioluminescence spectra of AL, we conclude that enolate-OxyAL is the most probable light-emitting species in the active site of luciferase. These findings provide new insight into AL-based bioluminescence.

Bisenarsan is an organoarsenic natural product identified from actinomycetes and a derivative of (2-hydroxyethyl)arsonic acid (2-HEA) esterified with 2,4,6-trimethyl-2-nonenoic acid (2,4,6-TMNA). Our previous study suggested that bisenarsan is biosynthesized from arsenate [As(V)] via arsonoacetaldehyde (AnAA). In contrast, the late-stage biosynthetic steps from AnAA to bisenarsan and the roles of transporter genes within the biosynthetic gene clusters (BGCs) of bisenarsan remain unclear. In this study, through in-frame deletions and heterologous expression targeting the bisenarsan BGC in Streptomyces lividans 1326 (bsn cluster), we identified bsnF (nicotinamide adenine dinucleotide phosphate-dependent oxidoreductase), bsnPKS (iterative type I polyketide synthase), and bsnFB (3-ketoacyl-acyl carrier protein synthase III family protein) as genes encoding enzymes likely responsible for the late-stage biosynthesis of bisenarsan. BsnF, BsnPKS, and BsnFB are presumed to catalyze the reduction of AnAA to 2-HEA, the formation of the 2,4,6-TMNA moiety, and the ester bond formation, respectively. Furthermore, based on the functional analysis of the transporter genes in the bsn cluster, BsnT2 (major facilitator superfamily transporter) appears to be involved in the efflux of bisenarsan. Although the roles of other transporters in bisenarsan biosynthesis remain unclear, they may contribute to the uptake and efflux of inorganic arsenic, presumably to ensure a consistent substrate supply and mitigate toxicity caused by its overaccumulation. Our study provides valuable insights into the biosynthesis of a rare class of organoarsenic natural products, with arsonopyruvate as an intermediate.

The (ammonio)amidyl (AA) groups, with the general structure of R₃N⁺N⁻–, represent a new class of π-electron-donating groups for the benzene-based π-conjugated molecules. This study investigated AA-substituted p-nitrobenzenes to assess the effects of acyclic, monocyclic, and bicyclic ammonium structures in the AA groups on the π-electron-donating ability and thermal stability of the structure. Quantum chemical calculations were performed that supported the experimentally observed favorable properties of the (quinuclidinio)amidyl (QA) group.

Newly designed optically active cage type 2-azanorbornane-based amino amide organocatalysts were developed and employed in the asymmetric Michael addition of β-keto esters with nitroolefins to afford the chiral Michael adducts with good chemical yields (up to 99%) and stereoselectivities (up to diastereomeric ratio (dr) = 97 : 3, up to 96% enantiomeric excess (ee)).

Iodine L-edge X-ray absorption near-edge structure (XANES) measurements were performed to characterize each crystal form of the compounds containing iodine atoms. 4-Iode-l-phenylalanine (4ILP) was used as a model compound. Each crystalline form had a distinctive XANES spectral shape. Complementary single-crystal X-ray structure analyses revealed diverse atomic interactions surrounding the iodine atoms, including C···I contacts and I···I halogen bonds. These different interactions influence the electronic states of the iodine atoms of 4ILP, resulting in distinct features in the XANES spectra. Notably, the spectral changes in the L₁ absorption edge of iodine atoms were found to be sensitive to van der Waals contacts. On the other hand, those in L₂ and L₃ were sensitive to the presence or absence of halogen bonds. This research highlights the crucial impact of weak nonconventional atomic interactions on the XANES spectra, providing valuable insights for the development and evaluation of crystalline materials.

Glycans, as one of the fundamental biomolecules alongside nucleic acids and proteins, play critical roles in biological processes, including glycoprotein folding, transport, degradation, and cell–cell interactions. Despite their biological importance, the structural analysis of glycans remains challenging due to their high flexibility and complex branched structures. This study addresses these challenges by combining molecular dynamics (MD) simulations and NMR spectroscopy to obtain dynamic conformational ensembles of glycans. Nonlinear correlation analyses, specifically Hilbert–Schmidt independence criterion and maximal information coefficient, were applied to decipher the structural dynamics of glycans. The study focused on GM3 trisaccharides and high-mannose glycans (GM9, M9, and M8B), uncovering the roles of glycosidic dihedral angles and intramolecular hydrogen bonds in stabilizing specific conformations. Key correlations between glycosidic linkages and hydrogen bonds were identified, offering insights into the conformational changes that underpin glycan bioactivity. Notably, the removal of specific mannose residues disrupts hydrogen bond networks, expanding conformational space and influencing glycoprotein fate in the endoplasmic reticulum. By integrating MD simulations, NMR validation, and nonlinear multivariate analysis, this study provides a robust framework for understanding glycan structural dynamics. These findings have broad implications for glycoengineering, glycan-based drug discovery, and the design of therapeutics targeting structurally dynamic biomolecules, such as intrinsically disordered proteins.

In this synthetic study, a gold-catalyzed cascade cyclization reaction for the construction of the caulerpin scaffold, suitable for the preparation of unsymmetrical caulerpin derivatives, was developed. The reaction proceeds through the formation of an α-imino gold(I) carbene from an azido-alkyne, followed by nucleophilic attack of an alkenylindole moiety on the gold carbene, leading to the formation of the bis-indole-fused 8-membered ring, the caulerpin core structure. The use of ethyl enol ether and a bulky phosphine ligand is suitable for successful ring closure at the desired position.

Recently, a chromatographic quantitative method using relative molar sensitivity (RMS), the so-called RMS method, was developed for the determination of target analytes in food additives, drugs, and supplements. By determining the RMS value of the analyte to a different reference compound, this method avoids the need for an analytical standard of the analyte itself to plot calibration curves for quantification. In this collaborative study performed by 10 laboratories, we demonstrated the robustness and reliability of the RMS method in the quantitative analysis of chlorogenic acid (5-O-caffeoylquinic acid, 5CQA) in apple juice. Reagent-grade 5CQA derived from natural sources is commercially available, but its purity, stability, and hygroscopic properties still need to be clarified. Therefore, there is always a risk of bias in the quantitative results, even when the calibration curve is prepared using reagent-grade 5CQA as the analytical standard. The RMS method overcomes this problem by using caffeic acid (CA) with high purity and stability as a reference compound. Prior to the collaborative study, a laboratory documented the standard operating procedure for method validation, which was then implemented in all laboratories to determine the RMS value of 5CQA to CA and quantify 5CQA in five apple juice samples. A comparison between the results obtained using a calibration curve and the RMS method validates the RMS method using CA as a reference compound for the quantitative analysis of 5CQA without an analytical standard.

Addressing the challenging field of chemoenzymatic dynamic kinetic resolution (DKR) of tertiary alcohols, for which so far only one example exists in the literature, we combined biocatalytic esterification and oxovanadium-catalyzed racemization, operating both steps in two different compartments of one reactor. The compartmentalization of the two heterogeneous catalysts, namely, immobilized lipase A from Candida antarctica (CAL-A) or its mutant and oxovanadium species on mesoporous silica, was achieved using a polydimethylsiloxane thimble, avoiding contact of the oxovanadium with water, thus maintaining the catalyst’s activity and thereby successfully improving the efficiency of the DKR. Utilizing the immobilized double mutant CAL-A V278S + S429G, the ester was obtained in 62% yield with excellent enantiomeric excess of >99% ee.

Nitric oxide (NO) is involved in numerous physiological activities including vasodilation, neurotransmission, and immune system regulation. NO-releasing small compounds are used to investigate the physiological activity of NO and to treat circulatory diseases, such as hypertension and angina pectoris. Among them, light-controllable NO releasers (caged NOs) enable spatiotemporal control of NO’s bioactivities. We previously reported NORD-1, a photoinduced electron transfer (PeT)-driven NO releaser that responds to red light. In the PeT-driven NO releasers, the NO release is triggered by photoinduced electron transfer from the N-nitrosoaminophenol to the light-harvesting dye. However, additional functionalization of PeT-driven NO releasers is required to enable introduction of tissue targeting groups or novel release triggers. As such, structure–activity relationship studies are needed to identify a suitable site for modification so as not to affect the NO-releasing efficiency of the PeT. Here, we investigated the functional impact of introducing substituents into the linker region connecting the light-harvesting antenna and NO releasing moiety. Although introduction of various substituents elicited only minor changes in NO-releasing efficiency and vasodilation activity, dialkylamino groups induced pH-dependent changes in NO-releasing reactivity. The structure–activity relationship of the linker moiety could provide fruitful information in further functionalizing PeT-driven NO releasers for biological applications.

Alkaline hydrolysis of the crude resin glycoside fraction from the leaves and stems of Ipomoea lacunosa L. (Convolvulaceae) yielded organic acid and glycosidic acid fractions. The organic acid fraction included n-decanoic and n-dodecanoic acids. Acidic hydrolysis of the glycosidic acid fraction yielded 3 monosaccharides (d-glucose, d-fucose, and l-rhamnose) and 2 known hydroxyl fatty acids (11S-hydroxytetradecanoic and 11S-hydroxyhexadecanoic acids). Treatment of the glycosidic acid fraction with trimethylsilyldiazomethane (in hexane) afforded 1 new glycosidic acid methyl ester (lacunosinic acid J methyl ester) and 6 known glycosidic acid methyl esters. Eight new resin glycosides (lacunosins V–XII) were isolated from the leaves and stems, along with 2 known resin glycosides. Their structures were determined using spectroscopic and chemical analyses. Three types of resin glycosides were identified: those with 18-membered macrolactone, those with 19-membered macrolactone, and those with non-macrolactone structures. All these compounds contained n-decanoic and n-dodecanoic acids as the organic acid components. Nine of the isolated resin glycosides were tested for cytotoxic activity against HL-60 human promyelocytic leukemia cells. One compound exhibited activity with an IC₅₀ value of 44.5 μM, while 3 compounds demonstrated moderate activity, with inhibition rates ranging from 53.5 to 68.7% at a concentration of 200 μM. In contrast, the remaining 5 compounds showed negligible effects even at 200 μM.

Characterizing the complex drug release profiles of nanoparticle-based pharmaceuticals is essential to ensure their efficacy and safety. In this study, we investigated the drug release of a representative liposomal drug, Doxil, to explore the applicability of the dynamic dialysis method (DDM), which offers the advantage of simple implementation. The DDM demonstrated considerable doxorubicin release from Doxil in response to increased ammonia concentration, supporting the hypothesis of ammonia-driven drug release from Doxil in tumor environments. To analyze the drug release of liposomal doxorubicin, we developed a mathematical model that (i) does not require strict sink conditions and (ii) avoids introducing numerous kinetic parameters. This model consolidates the complexities of drug partitioning into the liposomal membrane into a single apparent permeability constant. The release profiles of Doxil at 25°C and a physiological temperature of 40°C were successfully reproduced by the kinetic model, yielding reasonable permeability coefficients of 1.4 × 10⁻¹⁰ and 2.1 × 10⁻¹⁰ cm/s, respectively. Our model described the release behavior of the generic product Lipodox, yielding a permeability coefficient of 2.1 × 10⁻¹⁰ cm/s at 40°C, thereby confirming the utility of the DDM across products. Our results demonstrate that, with optimized conditions, the DDM can assess the drug release kinetics of liposomal doxorubicin. Furthermore, we believe that our study provides a valuable framework for evaluating and optimizing drug release phenomena in liposomal formulations.

Lipids, including fatty acids and phospholipids, play crucial roles in biological systems and are widely utilized in pharmaceutical and biomedical applications. However, their inherent hydrophobicity poses significant challenges for formulation and administration. In this study, we aimed to enhance the aqueous solubility of lipidic compounds by leveraging light-responsive molecular design. We synthesized azo-lipids by incorporating azobenzene units into a fatty acid and phosphatidylcholine, hypothesizing that light-induced trans–cis isomerization would improve solubility. The synthesized compounds exhibited reversible photoisomerization upon alternating UV (365 nm) and visible light irradiation, as confirmed by UV-vis spectroscopy and reverse-phase HPLC. The solubilization of these azo-lipids was quantified under UV-unirradiated and irradiated conditions. Azobenzene-incorporated phosphatidylcholine 2 exhibited a drastic increase in solubilization from 2.030 to 1008 µM (496-fold) after UV irradiation. This significant improvement was attributed to efficient photoisomerization and molecular bending in the cis, cis conformation, reducing intermolecular interactions. Our findings suggest that this on-demand aqueous solubilization strategy offers a novel approach for improving the handling, storage, and potential therapeutic administration of lipid-based compounds.