Peptides and proteins are involved in almost all biological events. In this review, three chemical biology tools, which were developed for peptide/protein sciences from a viewpoint of peptide/amide bond cleavage, are overviewed. First, study on an artificial amino acid that enables stimulus-responsive functional control of peptides/proteins is briefly described. Two N–S acyl transfer reaction-based tools, one a linker molecule for facile identification of target proteins of bioactive compounds and the other a reagent for selective labeling of proteins of interest, are then discussed.

Herein, the deprotonative functionalization of pyridine derivatives with aldehydes under ambient conditions has been demonstrated using an amide base generated in situ from a catalytic amount of CsF and a stoichiometric amount of tris(trimethylsilyl)amine (N(TMS)₃). Pyridine substrates bearing two electron-withdrawing substituents (i.e., fluoro, chloro, bromo, and trifluoromethyl moieties) at the 3- and 5-positions efficiently react at the 4-position with various aldehydes including arylaldehydes, pivalaldehyde, and cyclohexanecarboxaldehyde.

For rational drug design, it is essential to predict the binding mode of protein–ligand complexes. Although various machine learning-based models have been reported that use convolutional neural networks (deep learning) to predict binding modes from three-dimensional structures, there are few detailed reports on how best to construct and use datasets. Here, we examined how different datasets affected the prediction of the binding mode of CYP3A4 by a three-dimensional neural network when the number of crystal structures for the target protein was limited. We used four different training datasets: one large, general dataset containing various protein complexes and three smaller, more specific datasets containing complexes with CYP3A4-like pockets, complexes with CYP3A4-binding ligands, and complexes with CYP protein family members. We then trained models with different combinations of datasets with or without subsequent fine-tuning and evaluated the binding mode prediction performance of each model. The best receiver operating characteristic (ROC) area under the curve (AUC) model with respect to area under the receiver operating characteristic curve was obtained by training with a combination of the general protein and CYP family datasets. However, the ROC AUC—recall balanced model was obtained by training with this combination of datasets followed by fine-tuning with the CYP3A4-binding ligands dataset. Our results suggest that datasets that balance protein functionality and data size are important for optimizing binding mode prediction performance. In addition, datasets with large median binding pocket sizes may be important for the binding mode prediction specifically of CYP3A4.

Polygalaxanthone III, a xanthone glycoside that is a major constituent of “Polygala Root” (Polygala tenuifolia roots, Onji in the Japanese Pharmacopoeia), has been used as a standard in the quality control of crude drugs. However, we previously noted differences in the chromatographic properties of one of three samples of polygalaxanthone III. Therefore, standardization of the standard itself is extremely important. The structures of three standard samples commercially available as polygalaxanthone III were characterized by LC/MS and NMR. LC/MS analysis revealed that two molecular types exist. Both types are chromatographically separable but have an identical mass number with distinguishable MS/MS spectra. One dimensional (1D)-NMR analyses demonstrated that both had the same xanthone moiety and heteronuclear multiple bond correlation (HMBC) analyses revealed that they are structural isomers at the connecting position of glucose to apiose 1-position. Consequently, the isomers were identified as polygalaxanthone III and its regioisomer, polygalaxanthone XI. Based on the findings, we recommend using the LC-MS/MS detection method, which discriminates polygalaxanthone III and XI, to confirm the quality of the standard.

A new catalytic system comprising chiral Ag complex and Li aryloxide/bisphosphine oxide is developed for the synthesis of β^2,2-amino acids via direct asymmetric Mannich-type reaction of 4-subsituted isoxazolidin-5-ones. The Mannich adduct is a direct precursor of β-peptidic compounds otherwise difficult to obtain.

Membrane curvature formation is important for various biological processes such as cell motility, intracellular signal transmission, and cellular uptake of foreign substances. However, it remains still a challenging topic to visualize the membrane curvature formation on the cell membranes in real-time imaging. To develop and design membrane curvature-sensors, we focused on amphipathic helical peptides of proteins belonging to the Bin/Amphiphysin/Rvs (BAR) family as the starting point. BAR proteins individually have various characteristic structures that recognize different curvatures, and the derived peptides possess the potential to function as curvature sensors with a variety of recognition abilities. Peptide-based curvature sensors can have wide applications in biological research fields due to their small size, easy modification, and large production capability in comparison to protein-based sensors. In the present study, we found that an amphipathic peptide derived from sorting nexin1 (SNX1) has a curvature-recognition ability. The mutation studies of the initial peptide revealed a close correlation between the α-helicity and lipid binding ability of the peptides. In particular, the amino acids located on the hydrophobic face played a vital role in curvature recognition. The α-helix formation of the peptides was thought to serve to accommodate lipid-packing defects on the membrane surface and to maintain their binding to lipid vesicles. The structure–activity correlation found in this study have the potential to contribute to the design of peptide-based curvature sensors that will enable the capture of various life phenomena in cells.

We have discovered that β-amino acid homooligomers with cis- or trans-amide conformation can fold themselves into highly ordered helices. Moreover, unlike α-amino acid peptides, which are significantly stabilized by intramolecular hydrogen bonding, these helical structures are autogenous conformations that are stable without the aid of hydrogen bonding and irrespective of solvent (protic/aprotic/halogenated) or temperature. A structural overlap comparison of helical cis/trans bicyclic β-proline homooligomers with typical α-helix structure of α-amino acid peptides reveals clear differences of pitch and diameter per turn. Bridgehead substituents of the present homooligomers point outwards from the helical surface. We were interested to know whether such non-naturally occurring divergent helical molecules could mimic α-helix structures. In this study, we show that bicyclic β-proline oligomer derivatives inhibit p53–MDM2 and p53–MDMX protein–protein interactions, exhibiting MDM2-antagonistic and MDMX-antagonistic activities.

Melt adsorption is a manufacturing method that offers precise control of particle size distribution of granules and circumvents the disadvantages of conventional melt granulation. However, drug release from particles adsorbed with hydrophobic materials has not been fully investigated, and there are missing details as to whether particles manufactured by this technique can be applied to orally disintegrating tablets (ODT). In this report, we aimed to optimize process parameters and formulation to manufacture ODT containing melt adsorption-particles with the specific characteristic of sustained release. Melt adsorption particles containing Neusilin US2 as the adsorbent were prepared by using various waxes to determine the most suitable material for controlled release formulation. Glycerol fatty acid ester (Poem TR-FB: TR-FB) was the optimal wax examined because of its drug release pattern and tabletability. We then optimized manufacturing conditions by examining granulation time, disintegrant amount per tablet and compression force on the tablet for ODT that meet the criteria of controlled drug release, tensile strength and disintegration of the tablet. Multiple regression analysis revealed the effect of process parameters on tablet properties and drug release with increasing the granulation time affording sustained release of the drug. The analysis also showed that a high compression force crushed the granules coated by TR-FB, which impaired sustained drug release. From the regression model the optimal manufacturing conditions were determined, and the tablet prepared under these conditions concurred with the predicted values and met all criteria. This new technique should contribute to the development of ODT to improve medication adherence.

The word “theranostics,” a portmanteau word made by combining “therapeutics” and “diagnostics,” refers to a personalized medicine concept. Recently, the word, “radiotheranostics,” has also been used in nuclear medicine as a term that refer to the use of radioisotopes for combined imaging and therapy. For radiotheranostics, a diagnostic probe and a corresponding therapeutic probe can be prepared by introducing diagnostic and therapeutic radioisotopes into the same precursor. These diagnostic and therapeutic probes can be designed to show equivalent pharmacokinetics, which is important for radiotheranostics. As imaging can predict the absorbed radiation dose and thus the therapeutic and side effects, radiotheranostics can help achieve the goal of personalized medicine. In this review, I discuss the use of radiolabeled probes targeting bone metastases, sigma-1 receptor, and α_Vβ₃ integrin for radiotheranostics.

Asymmetric total syntheses of dihydropyran containing natural products, (+)-eurotiumide F and (+)-eurotiumide G have been described. These total syntheses revealed the absolute configuration of eurotiumide F and G, and confirmed the reported structure of eurotiumide F and revised the reported structure of eurotiumide G. Highlight of these syntheses is thermal rearrangement with 4-methoxyisochroman-1-one derivative having propargyl ether on phenolic ether under thermal condition to construct dihydropyran ring. X-Ray crystallographic analysis of (+)-eurotiumide G clarified the stereochemistry at the C1-position.

Transition-metal nanoparticles (NPs) catalysts supported on solid material represent one of the most important subjects in organic synthesis due to their reliable carbon–carbon or carbon–heteroatom bond-forming cross-coupling reactions. Therefore methodologically and conceptually novel immobilization methods for nonprecious transition-metal NPs are currently required for the development of organic, inorganic, green, materials, and medicinal chemistry. We discovered a self-assembled Au-supported Pd NPs catalyst (SAPd(0)) and applied it as a catalyst to Suzuki–Miyaura coupling, Buchwald–Hartwig reaction, Carbon(sp² and sp³)–Hydrogen bond functionalization, double carbonylation, removal of the allyl protecting groups of allyl esters, and redox switching. SAPd(0) comprises approximately 10 layers of self-assembled Pd(0) NPs, whose size is less than 5 nm on the surface of a sulfur-modified Au. The Pd NPs are wrapped in a sulfated p-xylene polymer matrix. We thought that the self-assembled Au-supported Pd NPs could be made by in situ metal NP and nanospace simultaneous organization (PSSO). This methodology involves 4 kinds of simultaneous procedures: i) reduction of a higher valence metal salt, ii) growth of metal NPs with appropriate size, iii) growth of a matrix with appropriate pores, and iv) wrapping of the metal NPs by matrix nanopores. This methodology is different from previously reported metal NPs-immobilizing methods, which use solid supports with preformed pores or coordination sites. We also applied the in situ PSSO method to prepare various immobilized transition-metal NPs, including base metals. For example, the in situ PSSO method can be applicable to easily prepare Ni, Ru, and Fe NPs with good recyclability and low metal leaching for use in organic synthesis.

A gold-catalyzed introduction of various terminal alkynes to acetals was investigated. Extensive optimization of the reaction conditions revealed that thermally stable cationic gold catalysts bearing bulky ligands such as 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene 3-1H-benzo[d][1,2,3]triazolyl gold trifluoromethanesulfonate (IPrAu(BTZ-H)OTf) were particularly suitable for the reaction. Additionally, significant solvent effects were observed. Ether solvents such as tetrahydrofuran (THF), cyclo pentyl methyl ether (CPME), and 1,4-dioxane were effective for the reaction. Studies on the scope of substrates and alkynes indicated that various alkynes and acetals were feasible to provide a wide range of propargylic ethers.

The 4-vinylpyrimidin-2-one nucleoside (T-vinyl) forms a cross-link with the RNA containing uracil at the complementary site at a high reaction rate. To obtain the stable T-vinyl derivative so that its reactivity is protected until it access to the target site, several derivatives were investigated, and the 2-thiopyridinyl- and 2-thiopyrimidinyl T-vinyl derivatives were determined to be good candidates. The 2-thiopyrimidinyl T-vinyl derivative was found to more efficiently cross-link with mRNA albeit having a better stability than the 2-thiopyridinyl T-vinyl derivative. The investigation using the luciferase (Luc) mRNA, the synthetic mRNA and non-cellular translation system revealed that the translation is terminated at the end of the cross-linked duplex between the mRNA and the oligoribonucleotide (ORN). Thus, the 2-thiopyrimidinyl T-vinyl derivative has successfully demonstrated both a good stability and high efficiency for the cross-linking reaction, and expanded its applicability in biological applications.

We aim to attain the sustainable use of longgu and have investigated the significance of longgu in Keishikaryukotsuboreito (KRB) decoction. We have already reported that longgu alters compound profiles in KRB decoction and hypothesized that it does so by adsorbing foreign organic compounds into its superficial pores. In the present study, we focused on the adsorbability of organic materials onto longgu surface as the cause of component profile alteration. We analyzed the physical changes in longgu through the decoction process by measuring the adsorbed water on longgu surface. ¹H magic angle spinning NMR (¹H-MASNMR) spectroscopic analysis revealed that raw longgu (R-raw) as well as decocted longgu [whether single (R-r) or KRB (R-krb) decoction] adsorbed water. However, the amount of adsorbed water in R-krb was smaller than that in R-raw and R-r. The nitrogen adsorption isotherms of longgu samples indicated that longgu was macroporous. The Brunauer–Emmett–Teller (BET) surface area of R-krb was smaller than that of R-raw and R-r. Further, thermogravimetric analysis of longgu samples showed that R-krb adsorbed matter that R-raw and R-r did not adsorb. The above findings and the ¹H-MASNMR analysis of heated longgu samples suggested that longgu adsorbed organic compounds into the pores. We considered that longgu adsorbed organic compounds during KRB decoction into its superficial pores through the decoction process.

Natural products are still rich sources of clinically used medicines and lead compounds for them. This review summarizes chemical studies carried out by the author on natural products of microorganism origin, many of which were discovered at the Institute of Microbial Chemistry (BIKAKEN). Caprazamycin B is a liponucleoside antibiotic from which CPZEN-45, an antituberculosis agent with a unique mode of action, was developed. Intervenolin and leucinostatin A exert antiproliferative activity toward tumor cells in the presence of the corresponding stromal cells, which implies that the primary molecular targets of these molecules should be related to growth signals from normal (stromal) cells. Details of the endeavors to establish efficient synthetic routes to these compounds which accelerated structure–activity relationship studies and further evaluation of biological activity are described.

To develop potent ligands for the vitamin D receptor (VDR), we designed and synthesized a series of vitamin D analogues with and without 22-alkyl substituents. These analogues exhibited agonistic, partial agonistic, or antagonistic activity. To elucidate the mechanism of action of the analogues, we conducted crystal structure analyses of the ligand-binding domain (LBD) of VDR complexed with the analogues. The VDR-LBD/agonist complex exhibited precise interactions, which clearly explained VDR agonism. The VDR-LBD/partial agonist complex showed two conformers (agonist and antagonist binding conformers) in a single crystal, demonstrating that partial agonism could be explained by the sum of the agonistic and antagonistic activities. Antagonist binding to the VDR-LBD structure was elucidated using both crystal structure analysis and in-solution structural analyses with the small-angle X-ray scattering (SAXS)-molecular dynamics (MD) and hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) methods. Several antagonist-binding structures were detected. We found that the antagonist binding structures differed depending on the structure of the antagonist itself, and those structures clearly explained the VDR antagonism. Furthermore, the apo VDR-LBD structure without the ligand in the ligand-binding pocket was revealed and found to have an entrance to accommodate the ligand. Thus we elucidated the mechanisms of action of agonists, partial agonists, and antagonists based on structural changes (differences) in the receptor protein induced by ligand binding.

We report here the development of phenylamino-1,3,5-triazine derivatives as novel nonsteroidal progesterone receptor (PR) antagonists. PR plays key roles in various physiological systems, including the female reproductive system, and PR antagonists are promising candidates for clinical treatment of multiple diseases. By using the phenylamino-1,3,5-triazine scaffold as a template structure, we designed and synthesized a series of 4-cyanophenylamino-1,3,5-triazine derivatives. The synthesized compounds exhibited PR antagonistic activity, and among them, compound 12n was the most potent (IC₅₀ = 0.30 µM); it also showed significant binding affinity to the PR ligand-binding domain. Docking simulation supported the design rationale of the compounds. Our results suggest that the phenylamino-1,3,5-triazine scaffold is a versatile template for development of nonsteroidal PR antagonists and that the developed compounds are promising lead compounds for further structural development of nonsteroidal PR antagonists.

Spatiotemporally controllable nitric oxide (NO) releasers are very attractive chemical tools for investigating the biological activities of NO, which is involved in the regulation of vasodilation, neurotransmission, and immune responses. We previously developed an easily synthesized, yellowish-green-light-controllable NO releaser, NO-Rosa5, and characterized its photoredox reaction mechanism. Here, we aimed to establish the biological applicability of NO-Rosa5 for in cellullo and ex vivo experiments. We successfully demonstrated yellowish-green-light-controlled NO release in HEK293T cells in vitro, as well as photomanipulation of the rat aorta response to NO in an ex vivo system. Furthermore, NO-Rosa5 showed lower toxicity than NOBL-1, a previously reported blue-light-controllable NO releaser, as determined by tetrazolium salt cell viability assay. Overall, our results indicate that NO-Rosa5 is a biocompatible, photocontrollable NO releaser with low toxicity and potentially broad applicability.

The aim of this study was to demonstrate the usefulness of T₂ measurements conducted with a time-domain NMR (TD-NMR) for the characterization of active pharmaceutical ingredients (APIs) containing solid dosage forms. A solid dispersion (SD) and a physical mixture (PM) consisting of indomethacin (IMC) and polyvinylpyrrolidone (PVP) were prepared at different weight ratios as test samples, and then their T₂ relaxation curves were measured by TD-NMR. The T₂ relaxation curve of IMC was quite different from that of PVP by nature. T₂ values of the SD and PM samples became gradually shortened with increasing IMC content. No difference in T₂ relaxation curves was observed between SD and PM. By analyzing the T₂ relaxation curves in detail, we succeeded in precisely quantifying the IMC contents incorporated in the samples. Next, this study evaluated the T₂ relaxation curves of amorphous and crystalline states of powdered IMC. T₂ relaxation rate of crystalline IMC was slightly but significantly higher than that of amorphous IMC, proving that the T₂ measurement was sensitive enough to detect these differences. Finally, a thermal stress was imposed on SD and PM samples at 60°C for 7 d, and then an amorphous-to-crystalline transformation occurred in IMC in the PM sample and was successfully monitored by T₂ measurement. We believe that T₂ measurement by TD-NMR is a promising analysis for the characterization of APIs in solid dosage forms, including SD-based pharmaceuticals.

Formylations of fluorine-containing aromatic compounds with dichloromethyl alkyl ethers have been investigated. Dichloromethyl propyl ether and dichloromethyl butyl ether have been applied for the formylation of fluorine-containing anisoles to give the corresponding aldehydes in good yields. Application of these ethers is preferable to that of methyl ether, which is prepared from volatile methyl formate. Reaction of fluorine-containing phenols with these dichloromethyl alkyl ethers did not give salicylaldehyde derivatives, leading instead to corresponding aryl formates in high yields. A plausible mechanism is discussed.