X-ray crystallography remains a powerful technique for determining protein three-dimensional structures with high spatial resolution and high throughput, even though cryo-electron microscopy has become widely used. In conventional X-ray crystallography, most obtained structures were static. However, serial femtosecond crystallography enables the visualization of dynamic structures with high temporal and spatial resolution by combining reaction triggers such as light excitation and substrate mixing. In addition, we have recently established and begun operating an X-ray crystallography experimental station at the fourth-generation synchrotron radiation facility NanoTerasu. In this article, we introduce recent topics in X-ray protein crystallography, including not only experimental advances but also widely used protein structure prediction and molecular design tools.
Per-O-methylated beta -cyclodextrin (TMe-beta -CD) forms a very stable 2:1 inclusion complex with water-soluble 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) in water. The supramolecular complex provides strong hydrophobic cavity to the porphyrin scaffold, which is similar to the environment of heme in heme proteins. In our laboratory, per-O-methylated beta -CD dimer having pyridine linker (Py3CD) was synthesized to make a biomimetic model compound of oxygen-binding heme protein like hemoglobin (Hb) and myoglobin (Mb). The inclusion complex of Py3CD with iron complex of TPPS (FeTPPS) is the first and only-one biomimetic complex that functions in water. The complex, named hemoCD, showed a very high CO binding affinity. When hemoCD was injected to rodent animals after exposure to CO gas, hemoCD captured CO during circulation and was excreted in urine without showing any toxic effect. These properties are quite suitable to the use of an antidote against CO poisoning. Our laboratory has started the drug development to implement hemoCD as a CO antidote for clinical use.
In recent years, manufacturing technologies for peptides and oligonucleotides have advanced considerably; however, research and development efforts have remained predominantly focused on upstream processes, particularly synthetic methodologies. In industrial practice, downstream operations, including purification and lyophilization, often constitute major bottlenecks to productivity, and meaningful, sustainable improvements at scale are unlikely without technological innovation in these areas. To address this challenge, we have focused our efforts on developing downstream processes beyond purification, with an emphasis on continuous purification and mixer-type lyophilization technologies. In this study, we describe the key features of these two technologies and evaluate their implementation in the large-scale manufacturing of a cyclic peptide, in comparison with conventional approaches. Application of these technologies resulted in an approximately 1.5-fold increase in overall yield and improvements in product quality. Additionally, the time required for downstream processing was reduced to nearly one-quarter of that associated with the traditional workflow. Collectively, these findings demonstrate that innovation in downstream operations can substantially enhance both productivity and product quality in peptide and oligonucleotide manufacturing.
Radiotheranostics integrates diagnostic imaging and radionuclide therapy using radiopharmaceuticals and is a promising approach for personalized medicine. Central to this paradigm is the development of theranostic pairs diagnostic and therapeutic radiolabeled analogs that exhibit comparable biodistribution. However, constructing halogen based pairs such as F-18 /(211) At remains challenging because of their divergent chemistry and the pronounced in vivo lability of astatine carbon bonds. Here, we describe a neopentyl labeling strategy designed to address these limitations. The neopentyl group leverages steric shielding to suppress nucleophilic dehalogenation and incorporates hydrophilic functionality to mitigate metabolic degradation. Using stable precursors, including carbamoyl difluoromethanesulfonate (CDf) esters, we established an efficient synthetic route enabling high yield radiolabeling. The resulting( 211 )At labeled compounds demonstrated high in vivo stability and biodistribution closely matching their radioiodinated counterparts, outperforming conventional aromatic labeling approaches. Application of this platform to a PSMA targeting ligand achieved robust tumor uptake and significant therapeutic efficacy in a prostate cancer model. In parallel, an efficient synthesis of the corresponding F-18 labeled analog was developed, permitting automated production without HPLC purification. Collectively, the neopentyl labeling platform provides a versatile and stable scaffold for generating (18) F/(211) At theranostic pairs and may facilitate clinical translation of targeted alpha therapy
Clinical development of antisense oligonucleotides (ASOs) is frequently constrained by the efficacy-safety dilemma, in which increased target affinity is accompanied by enhanced off-target toxicity. We propose that this apparent trade-off arises from an equilibrium specificity limit inherent to conventional thermodynamically controlled ASO-RNA recognition. To address this limitation, we developed BROTHERS (TM) technology, a non-equilibrium-inspired molecular design framework that mitigates the efficacy-toxicity coupling by integrating two orthogonal principles. 1) Static shielding suppresses non-specific interactions of single-stranded ASOs, while 2) dynamic selection enables kinetically driven target RNA recognition via a toehold-mediated strand displacement (TMSD) reaction. In vivo studies demonstrate that BROTHERS (TM)-based ASOs (BROs) achieve potent and selective gene silencing while markedly reducing toxicities observed with conventional single-stranded ASOs. These results indicate that non-equilibrium control of molecular recognition can effectively alleviate the efficacy-safety dilemma, establishing BROTHERS (TM) technology as a robust platform for next-generation antisense therapeutics.
Oligonucleotide therapeutics have recently emerged as an important modality in drug discovery. The use of artificial nucleic acids is essential for improving both nuclease stability and duplex-forming ability. We previously demonstrated that oligonucleotides incorporating 2'-O,4'-C-methylene-bridged nucleic acid (2',4'-BNA) exhibit high duplex-forming ability toward complementary RNA. In recent years, we have developed several novel bridged nucleic acids, including 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA), 2'-O,4'-C-spirocyclopentylene-bridged nucleic acid (scpBNA2), and alkyl-substituted guanidine-bridged nucleic acids (GuNA [R]). These analogs provide RNA-binding affinity comparable to or greater than that of 2',4'-BNA, while dramatically enhancing resistance to nuclease degradation. In particular, scpBNA and scpBNA2 were found to reduce the hepatotoxicity of antisense oligonucleotides. These artificial nucleic acids are expected to contribute to the development of highly potent and safer oligonucleotide therapeutics.
Cyclic disulfide peptides are attracting significant attention as a new modality in drug development because their rigid structures enable selective binding to target molecules and confer high resistance to metabolic enzymes. Therefore, new methods for constructing disulfide bonds can contribute to more efficient preparation of these peptides. In this paper, we report the development of disulfide bond-forming methods using 3-nitro-2-pyridinesulfenyl (Npys) compounds and their application to the synthesis of disulfide peptides. The paper covers two topics. First, we developed a one-pot solid-phase disulfide ligation (SPDSL) method, which readily affords disulfide-linked products composed of two thiol-containing components. Based on this SPDSL strategy, we further demonstrate a disulfide-driven synthesis of cyclic peptides from two different peptide fragments. Second, we identified methyl 3-nitro-2-pyridinesulfenate (Npys-OMe) as a mild oxidative reagent that promotes intramolecular disulfide bond formation between two thiols within a peptide. As an application of Npys-OMe, we show that disulfide bond formation can be achieved on thiol-containing peptidyl resins. In addition, a water-soluble Npys derivative functions as a disulfide-forming reagent in aqueous buffer, enabling the direct oxidation of thiol-containing peptides prepared by native chemical ligation.
Mirror-image proteins (D-proteins) comprise D-amino acids and achiral glycine, which are capable of assembling into mirror-image architectures of native L-proteins. Although D-proteins cannot be obtained through recombinant technology, synthetic proteins with more than 100 residues have been prepared using advanced peptide chemistry techniques, including solid-phase peptide synthesis and native chemical ligation. To date, a number of mirror-image versions of target proteins have been synthesized and used to screen the therapeutic potential of mirror-image peptides (D-peptides), nucleic acids (L-nucleic acids), and natural products (mirror-image enantiomers). We have been exploring the looking-glass world to expand the repertoire of protein-based therapeutics through mirror-image screening. To this end, we have established processes for synthesizing nanobodies (VHH antibodies) and monobodies, which contain three variable regions on stable scaffolds to bind epitopes on target proteins. Despite their identical sequences, the mirror-image forms of nanobodies and monobodies are significantly less immunogenic in mice than their L-form counterparts. Scaffolds of mirror-image proteins have also been chemically modified to study their pharmacokinetic properties and potential for bioconjugation.
Our group has been developing organic electrochemical reactions (since 1994) and liquid-phase peptide synthesis (since 2002) mostly independently of each other. The merger of electrochemistry and peptides had been a long-cherished goal of our group, yet apart from the limited example of electrochemical disulfide bond formation, the two had never encountered. Herein, we describe electrochemical peptide synthesis using triarylphophines as recyclable "coupling reagents," where electrochemistry and peptides are merged. In the field of synthetic organic chemistry, the standard approach to reducing reagent consumption is to develop new catalysts that promote the desired reaction. This is also true for amide bond formation and various catalysts have been reported so far, achieving remarkable outcomes. Another fascinating-yet-challenging approach is to make coupling reagents catalytic. In situ (one-pot) regenerable coupling reagents would be ideal, yet ex situ (two-pot) recyclable ones would also contribute to reducing the consumption. When it comes to recycling coupling reagents, it is crucial to avoid applying thermal energy and/or "second" coupling reagent; otherwise, it does not constitute a fundamental solution. In this context, we focused on oxidative (electrochemical) an amide bond formation using triphenylphosphine as a coupling reagent precursor. During amide bond formation, triphenylphosphine oxide is accumulated as waste, which can be recovered and reduced back (recycled) to triphenylphosphine. Tris(4-methoxyphenyl)phosphine was found to be a superior coupling reagent precursor to non-substituted triphenylphosphine in electrochemical amide bond formation, which was further enhanced by using iodide mediator. We have successfully synthesized three bioactive peptides, including leuprorelin (9-mer), bradykinin (9-mer), and icatibant (10-mer), without the use of typical coupling reagent.
Fluorescence imaging is a powerful technique for visualizing and quantifying dynamically changing biological events with high spatial resolution and sensitivity, and it has been widely applied in both fundamental biological research and clinical practice. However, conventional "always-on" fluorescent probes emit signals irrespective of their localization, often resulting in high background fluorescence and low imaging contrast in biological environments. To address these limitations, activatable fluorescent probes, which switch from a non-emissive "off" state to an emissive "on" state only in response to specific biological stimuli, have attracted increasing attention owing to their high signalto-noise ratios and improved detection sensitivity. A variety of activation mechanisms have been developed, including the removal of quenching groups, changes in molecular planarity, and modulation of jr -conjugation length through elimination reactions. Among these strategies, probes activated by recovery of the jr -conjugated system provide unique opportunities for rational molecular design. In particular, cyanine dyes incorporating nucleophilic functional groups represent a promising yet relatively underexplored platform for activatable probe development. In this account, we summarize our recent efforts on the design and synthesis of nucleophile-containing cyanine-based activatable fluorescent probes. We focus on enzyme-responsive systems, including probes activated by esterases and aldehyde dehydrogenase, and discuss their photophysical properties, activation mechanisms, and applications in biological imaging.
In contrast to the considerable progress in the development of methodologies for amide bond formation in amines (i.e., primary amines, secondary amines, anilines), the development of direct N-acylation of less nucleophilic N-heterocycles and amides with carboxylic acids is still challenging. In this article, we describe the direct N-acylation of less nucleophilic heterocycles and amides with carboxylic acids promoted by the 4-(N,N-dimethylamino)pyridine N-oxide (DMAPO)/di-tert-butyl dicarbonate (Boc(2)O) system. The new one-pot method, which does not involve pre-activation of substrates, enables the direct N-acylation of a wide variety of nitrogen nucleophiles such as indole, carbazole, pyrrole, pyrazole, lactam, and anilide with carboxylic acids in high yield. This method also enables one-pot direct synthesis of bulky N-acyl heterocycles starting from a wide variety of less nucleophilic N-heterocycles and sterically hindered alpha-fully substituted carboxylic acids. Recently, we have also successfully developed a direct and selective N1-acylation of indazole by utilizing this method. In addition, a new synthesis of N1-functionalized alkyl indazoles utilizing N1-acyl indazoles as starting materials was achieved. The new protocol is useful for the selective synthesis of structurally diverse N1-functionalized alkyl indazoles, which are difficult to synthesize by other methods such as the Mitsunobu reaction and classical S(N)2 alkylation of indazole. Our protocol is also amenable to onepot direct N-acylation of sulfoximines and can be carried out under mild reaction conditions. Furthermore, we demonstrated a simple and practical synthesis of N-acyl oxazolidinones which can then be used in various asymmetric transformations. The new method exhibits excellent functional group tolerance and broad substrate scope. As the present method is practical, operationally simple, and scalable, it should find wide applications in both academic and industrial laboratories.
New natural products are the starting point for related research areas such as drug development, biosynthesis, mode of action, and organic synthesis. However, intensive screening activity over the past decades has led to frequent reisolation of known compounds. With the first priority to obtain new chemical structures, we have employed HPLC/UV-guided chemical screening instead of activityguided screening. In this article, new compounds discovered in our laboratory are presented, focusing on structural features, structure determination, biosynthesis, and synthetic studies.
Amycolamicin, which exhibits potent antibacterial activity against drug-resistant bacteria including fF5 and Kibdelosporangium sp. MA7385 by the Igarashi and Singh groups, respectively. The unique hybrid structure of amycolamicin combined with its promising biological activity attracted significant attention from the synthetic community. Herein, we describe the details of our convergent total synthesis of amycolamicin, which features: (1) a protecting group-free intramolecular Diels-Alder reaction of a hydroxy tetraenal to construct a trans-decalin system in a highly diastereoselective manner; (2) a diastereoselective nucleophilic addition of a p-methoxybenzyloxy-substituted vinyllithium reagent to an alpha,beta -bisalkoxy ketone intermediate to provide the corresponding tertiary alcohol as a single diastereomer; (3) alpha beta-selective glycosylation of a trans-decalinol intermediate using a bicyclic N,O-acetal as a glycosyl donor; (4) a completely stereoconvergent N-acylation of an anomeric N-glycoside mixture with a beta -keto thioester; and (5) a nucleophilic ring-opening of a cyclic carbonate protecting group with 2,4-dimethoxybenzyl amine to install a beta-hydroxy carbamate structure.
Nucleophilic addition to carbonyl compounds is one of the most fundamental transformations in organic synthesis. Carbonyl carbon atoms serve as electrophilic carbinol cation synthons in reactions with nucleophiles. Umpolung of the carbonyl reactivity should permit carbonyl compounds to react as nucleophilic carbinol anions with electrophiles. We have developed photocatalytic umpolung reactions of carbonyl compounds to generate anionic carbinol synthons through multielectron reduction. Here two approaches are presented. The first approach exploits carbon dioxide-promoted electron transfer (CO2ET) process. Under photocatalytic conditions in the presence of CO2, aromatic aldehydes and ketones undergo two-electron reduction to generate carbinol anion equivalents. The resulting nucleophilic species participate in a range of C-C bond forming reactions, including carboxylation, cross-pinacol coupling with second carbonyl compounds, and 1,4-addition to electron-deficient olefins. This concept is further extended to a,/3-unsaturated carbonyl compounds, where homoenolate anion equivalents are generated through CO2ET process. The second approach relies on a newly developed diazabenzacenaphthenium photocatalyst (N & horbar;BAP) with high photoredox abilities and visible-light absorption. In combination with ammonium oxalate as a traceless reductant, N & horbar;BAP promotes unprecedented four-electron reduction of esters to generate carbinol anion equivalents. The resulting carbinol anions undergo protonation to afford alcohols and react nucleophilically with carbonyl electrophiles to form unsymmetric 1,2-diols.
Endo-, beta -N-acetylglucosaminidases (ENGases) are endoglycosidases that hydrolyze the glycosidic bond between two N-acetylglucosamine residues in asparagine-linked glycans. ENGases are crucial tools for the structural analysis and glycan remodeling of glycoproteins. However, current ENGase activity assays are often complex and unsuitable for high-throughput analysis. To address this, we developed Forster resonance energy transfer (FRET)-based glycan molecular probes for the real-time detection of ENGase activity. We synthesized di-, tri-, and pentasaccharide probes bearing a fluorophore at the non-reducing end and a quencher at the reducing end, and evaluated their quenching efficiencies for activity detection. The pentasaccharide probe, MM3D, was efficiently cleaved by EndoM, resulting in a significant increase in fluorescence. These results successfully demonstrate that our Furthermore, we constructed a library of probes with diverse glycan structures. Using this library, we evaluated the activities of six commercially available ENGases and observed their distinct substrate specificities. This FRET probe library represents a valuable tool for detecting ENGase activity and will significantly contribute to advances in glycobiology research.
This study describes the development of manufacturing processes for TAFIa inhibitor 1 and its prodrug 2. To establish an industrial-scale production process for 1, comprehensive screening of chiral catalysts was conducted. This investigation revealed that the Ru/BINAP catalyst system in fluorous alcohol solvents (2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)) significantly enhanced both reactivity and selectivity. Consequently, a robust and efficient process was successfully developed, achieving an 85% overall yield from intermediate 12 over 5 steps. This represents a substantial improvement compared to the early-stage process (40% overall yield in 5 steps). Concurrently, a manufacturing process was developed for prodrug 2. A novel optically active prodrug fragment, (R)-32, utilizing HFIP as a leaving group, was designed to circumvent problematic chromatographic purification, and its synthetic route was established. Enzyme screening identified Chirazyme L-2, C4 as an effective catalyst, producing (R)-32 in 37% yield with 99.8% ee optical purity. Additionally, crystallization-induced asymmetric transformation (CIAT) of a diastereomeric mixture 2c from the (R,R)-form to the desired (R,S)-form was achieved, resulting in 97% yield with 94.8% de. Based on these methodologies, a manufacturing process was established for prodrug 2, achieving an overall yield of 66% from intermediate 12 through 6 steps.
Anions are fundamental species in the environment and in living cells; therefore, the recognition of anions is important. We found that silanol derivatives, including silanediols, silanetriol, 1,3-disiloxane 1,3-diol, and 1,3-disiloxane-1,1,3,3-tetraol, form multiple hydrogen bonds with anions in organic solvents. The X-ray single crystal structure of di(1-naphthyl)silanediol and Cl- revealed that the two hydroxy groups formed cooperatively hydrogen bonds to Cl-. The association constants of the silanol derivatives for anions were comparable to those of the corresponding urea derivatives. In addition, silanediols bearing pyrenyl and substituted naphthyl groups slowly react in the presence of a base to form the corresponding cyclotri- and cyclotetrasiloxanes, respectively under mild conditions without cleaving the Si-C bonds. The structures and the photophysical properties are also presented. Notably, silanol derivatives can be utilized as organocatalyst, similar to thiourea and squaramide derivatives. The addition of indole to beta-nitrostyrene catalyzed by silanetriol was faster than that with silanediol indicating that three hydroxy groups cooperatively stabilize the transition state of the reaction.
Artificial genes are engineered DNA sequences designed to express specific RNA or proteins, with broad applications in gene therapy, diagnostics, and synthetic biology. A major challenge in this field is developing technologies that enable precise, on-demand control of gene expression, which is essential for functional studies and safe therapeutic strategies. Conventional approaches, such as light-sensitive transcription factors or photo-responsive DNA modifications, are limited by poor tissue penetration of light, restricting their applicability. To address this limitation, we developed a novel system based on host-guest chemistry to reversibly regulate DNA duplex formation. Guest-modified adenosines were designed by attaching a guest molecule to the N 6 position of adenosine via an alkyl linker. These modified nucleosides were incorporated into DNA strands using a post-synthetic approach and formed stable base pairs with complementary thymidine under normal conditions. Upon addition of cucurbit[7]uril (CB[7]), bulky host-guest complexes formed on DNA, destabilizing the duplex. Subsequent introduction of a competing guest molecule displaced CB[7], restoring duplex formation. By integrating this system into the transcription initiation region of artificial genes, we successfully demonstrated reversible control of gene expression in a cell-free expression system. This study highlights a new chemical strategy for dynamic gene regulation and its potential applications in synthetic biology and therapeutic design.
We recently discovered that acrolein is produced at high concentrations in various cancer cells. We then developed an efficient in & horbar;cell reaction with acrolein in cancer; thus, the aryl azides undergo a metal & horbar;free 1,3 & horbar;dipolar cycloaddition reaction with acrolein in a highly selective manner, resulting in the formation of alpha & horbar;diazocarbonyl derivatives. In this paper, we describe our recent trials of this in & horbar;cell reaction toward medical applications, i.e., cancer diagnosis and therapy. Our in & horbar;cell reaction strategy led to the clinical application and constitutes a new modality for cancer medicine
