A concise gram-scale total synthesis of (±)-makaluvamine F was accomplished. The left segment, 2-aminodihydrobenzothiophene possessing an N,S-acetal moiety, was prepared using commercially available 2-fluoro-4-methoxybenzaldehyde in 6 steps via Curtius rearrangement. Subsequent condensation of the 2-aminodihydrobenzothiophene segment with a pyrroloiminoquinone segment completed the total synthesis of makaluvamine F, which was achieved in a 23% overall yield via a longest linear sequence of 7 steps. The versatility of the synthetic route involving the Curtius rearrangement was demonstrated by applying it to synthesize several unnatural makaluvamine F derivatives.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures that have emerged as attractive therapeutic targets owing to their involvement in diverse biological processes. Additionally, peptides derived from G4-binding proteins provide promising platforms for selective G4 recognition. In this study, we explored the G4-binding capacity of arginine–glycine–glycine (RGG)-rich sequences derived from the RGG3 domain translocated in liposarcoma/fused in sarcoma (TLS/FUS), a known G4 RNA binding protein. In this study, we synthesized a library of overlapping 15-mer peptides and evaluated their G4-binding affinities. Among the 10 evaluated native sequences, several peptides demonstrated measurable affinities toward G4 RNA structures, with STK5-1 exhibiting the highest G4-binding affinity. Furthermore, to investigate the impact of conformational constraints on G4 recognition, we introduced (E)-methylalkene dipeptide isosteres (MADIs) into selected Gly–Gly motifs, generating a series of RGG peptidomimetics. Subsequent binding assays revealed that some of these MADI peptidomimetics exhibited enhanced affinity and selectivity compared with their unmodified counterparts. Our findings offer new insights into the sequence and structural features governing G4-binding, establishing a foundation for the further development of peptide-based G4 ligands.

We report the development of triarylphenol-based photocatalysts that promote C–I bond borylation via halogen bonding (XB) interactions under visible-light irradiation. Traditional phenol system reactions often suffer from phenoxyl radical instability, limiting their catalytic utility. To overcome this issue, we designed sterically and electronically tuned triarylphenols that stabilize radical intermediates while maintaining high photoreactivity. Systematic evaluation revealed that 2,4,6-triphenylphenol efficiently facilitates photoinduced electron transfer (PET) and suppresses undesired side reactions such as phenol decomposition. The optimized reaction conditions enabled a broad substrate scope, showing efficient arylboronic ester formation. Density functional theory calculations confirmed the formation of XB complexes with charge-transfer character, providing mechanistic support for the PET pathway. This work shows the potential of rationally designed phenols as XB acceptors and offers a sustainable approach for C–I bond functionalization.

Aspergillus and Penicillium species produce a variety of quinazolinobenzodiazepine (QBD) alkaloids by using different biosynthetic gene clusters (BGCs). During the course of metabolome analysis of Aspergillus fumigatiaffinis IFM55214, we discovered an atropisomeric pair of QBD natural products, benzomalvin G (1) and H (2), that are produced by this fungus. The isomer 1 was favored in organic solvents, and 2 was favored in aqueous solutions. The stereochemistry and conformation of these 2 compounds were determined unambiguously by Marfey’s method and nuclear Overhauser effect (NOE) analysis, respectively. Furthermore, a putative BGC for 1 and 2 (Afben cluster), composed of 3 genes (AfbenX, AfbenY, and AfbenZ) shared between the ben cluster in Aspergillus terreus and 4 additional genes (methyltransferases and oxygenases), was identified in the genome of A. fumigatiaffinis IFM55214.

Polyunsaturated fatty acids (PUFAs) are signaling molecules exhibiting diverse bioactivities. Due to their flexible nature, PUFAs can theoretically adopt an enormous number of conformations, and it is not easy to identify the biologically active conformation(s), so rational drug design targeting the corresponding receptors is difficult. Here, we established a method to characterize conformational similarity by combining the replica exchange molecular dynamics simulation with the 3-dimensional WHIM (Weighted Holistic Invariant Molecular) descriptor. Using this method, we explored the total conformational space of 6 PUFAs and identified their conformational preferences for binding to known proteins. We found that upon binding, a specific cluster of conformers from the solution state is populated, which can be termed as bioactive conformations. By comparing the dynamics of synthetic modulators with those of PUFAs, we found that these compounds can be regarded as rigid mimics of the bioactive conformations of PUFAs, though this perspective has not been previously described. This approach is expected to be helpful in designing new bioactive molecules.

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.