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Peptides play essential roles in biological phenomena, and, thus, there is a growing interest in detecting in vivo dynamics of peptide metabolisms. Dissolution-dynamic nuclear polarization (d-DNP) is a state-of-the-art technology that can markedly enhance the sensitivity of nuclear magnetic resonance (NMR), providing metabolic and physiological information in vivo. However, the hyperpolarized state exponentially decays back to the thermal equilibrium, depending on the spin-lattice relaxation time ( T 1 ). Because of the limitation in T 1 , peptide-based DNP NMR molecular probes applicable in vivo have been limited to amino acids or dipeptides. Here, we report the direct detection of in vivo metabolic conversions of hyperpolarized 13 C-oligopeptides. Structure-based T 1 relaxation analysis suggests that the C-terminal [1- 13 C]Gly- d 2 residue affords sufficient T 1 for biological uses, even in relatively large oligopeptides, and allowed us to develop 13 C-β-casomorphin-5 and 13 C-glutathione. It was found that the metabolic response and perfusion of the hyperpolarized 13 C-glutathione in the mouse kidney were significantly altered in a model of cisplatin-induced acute kidney injury.

Because of the extracellular acidic microenvironment of cancer cells, many pH-responsive molecules have become indispensable materials for bioanalysis and targeted therapy development. pH-Responsive DNA aptamers, which selectively bind to target proteins in cancer cells, have become a key research target in the therapeutic field. However, conventional pH-responsive aptamers have fatal drawbacks, such as complex structures, sequence limitation, and difficulties in mass production, as they require special nucleic acid structures, including the i-motif and DNA triplex. To address these issues, we utilized An C , which is an unnatural nucleobase with a p K a H of 5.9, to construct a simple pH-responsive DNA aptamer (CSL1-II) for selective binding to the c-Met protein expressed in cancer cells. CSL1-II in a weakly acidic environment had a stronger inhibitory effect on the HGF/c-Met pathway and exerted a strong controlling effect on the spreading and migration of cancer cells. Our strategy provides a simple and versatile method to develop pH-responsive DNA aptamers and represents the first example of a cancer-selective c-Met antagonist that inhibits cell migration., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

DNA aptamers have attracted attention as an alternative modality for biomolecules due to their excellent target binding specificity and thermal stability, and they are also expected to be applied as artificial agonists for receptor proteins. DNA aptamer agonist TD0 targeting the receptor of fibroblast growth factor (FGFR), which plays an important role in the fields of wound healing and regenerative medicine, has been reported to induce cellular responses as well as its native ligands. However, it was also noted that there were some different responses upon long-term stimulation, suggesting that the intracellular signals induced by DNA aptamer agonist TD0 are different from those of natural ligands. In this paper, we comprehensively analyzed the intracellular signals induced by DNA aptamer agonist TD0 targeting FGFR1, and compared them with those by natural protein ligand FGF2. It was found that the intracellular signals were highly similar for short-term stimulation. On the other hand, the receptor and the downstream cellular signals showed different activation behaviors for long-time stimulation. Evaluating the stability and sustained activity of DNA aptamer agonist TD0 and FGF2 in the medium suggested that ligand stability may be important in properly regulating cellular responses., (© 2024. The Author(s).)

Oxidation of a thioether linkage in thioether-closed macrocyclic peptides led to collision-induced site-selective linearization of the peptides. This method has allowed for de novo sequencing of thioether macrocyclic peptides. The utility of the sequencing method was demonstrated by identifying the correct peptide sequences from a virtually randomized thioether macrocyclic peptide library.

Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.

Postoperative peritoneal adhesion (PPA) is a prevalent complication of abdominal surgery, posing a significant hindrance to postsurgical recovery. Although several strategies have been developed to alleviate and prevent adhesions, their efficacy remains unsatisfactory. For the first time, we studied the therapeutic effect and mechanism of our recently developed thermally stable oligonucleotide-based mimetics of hepatocyte growth factor (HGF DNA aptamer) to prevent PPA. The HGF DNA aptamer effectively inhibited canonical TGF-β1 signaling transduction, partially suppressing mesothelial mesenchymal transition. Additionally, the aptamer, respectively, upregulated and downregulated the expression of tissue plasminogen activator and plasminogen activator inhibitor 1, thereby enhancing fibrinolytic activity. As a pleiotropic factor, the HGF DNA aptamer also enhanced the migratory and proliferative capacities of mesothelial cells. Finally, the aptamer demonstrated a higher level of effectiveness in preventing PPAs than the commercially available antiperitoneal adhesion barrier, Seprafilm. Due to its therapeutic benefits, excellent stability, biosafety, cost-effectiveness, and versatility, the HGF DNA aptamer demonstrates promise for preventing PPA in future clinical settings.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye., (© 2024. The Author(s).)

Peptoids are a promising drug modality targeting disease-related proteins, but how a peptoid engages in protein binding is poorly understood. This is primarily due to a lack of high-resolution peptoid-protein complex structures and systematic physicochemical studies. Here, we present the first crystal structure of a peptoid bound to a protein, providing high-resolution structural information about how a peptoid binds to a protein. We previously reported a rigid peptoid, oligo( N -substituted alanine) (oligo-NSA), and developed an oligo-NSA-type peptoid that binds to MDM2. X-ray crystallographic analysis of the peptoid bound to MDM2 showed that the peptoid recognizes the MDM2 surface predominantly through the interaction of the N -substituents, while the main chain acts as a scaffold. Additionally, conformational, thermodynamic, and kinetic analysis of the peptoid and its derivatives with a less rigid main chain revealed that rigidification of the peptoid main chain contributes to improving the protein binding affinity. This improvement is thermodynamically attributed to an increased magnitude of the binding enthalpy change, and kinetically to an increased association rate and decreased dissociation rate. This study provides invaluable insights into the design of protein-targeting peptoids., Competing Interests: The authors declare the following competing financial interest: the authors (J. M., Y. F., and S. S.) have filed a patent application (PCT/JP2020/27010). All other authors declare they have no competing interests., (This journal is © The Royal Society of Chemistry.)

There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few μm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.

Elucidation of biological phenomena requires imaging of microenvironments in vivo . Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Bone marrow-derived cells (BMDCs) infiltrate hypoxic tumors at a pre-angiogenic state and differentiate into mature macrophages, thereby inducing pro-tumorigenic immunity. A critical factor regulating this differentiation is activation of SREBP2-a well-known transcription factor participating in tumorigenesis progression-through unknown cellular mechanisms. Here, we show that hypoxia-induced Golgi disassembly and Golgi-ER fusion in monocytic myeloid cells result in nuclear translocation and activation of SREBP2 in a SCAP-independent manner. Notably, hypoxia-induced SREBP2 activation was only observed in an immature lineage of bone marrow-derived cells. Single-cell RNA-seq analysis revealed that SREBP2-mediated cholesterol biosynthesis was upregulated in HSCs and monocytes but not in macrophages in the hypoxic bone marrow niche. Moreover, inhibition of cholesterol biosynthesis impaired tumor growth through suppression of pro-tumorigenic immunity and angiogenesis. Thus, our findings indicate that Golgi-ER fusion regulates SREBP2-mediated metabolic alteration in lineage-specific BMDCs under hypoxia for tumor progression., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (R F ) group adjacent to the α-carbon. R F -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. R F -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (R F -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel R F -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs)., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cell membrane receptors regulate cellular responses through sensing extracellular environmental signals and subsequently transducing them. Receptor engineering provides a means of directing cells to react to a designated external cue and exert programmed functions. However, rational design and precise modulation of receptor signaling activity remain challenging. Here, we report an aptamer-based signal transduction system and its applications in controlling and customizing the functions of engineered receptors. A previously reported membrane receptor-aptamer pair was used to design a synthetic receptor system that transduces cell signaling depending on exogenous aptamer input. To eliminate the cross-reactivity of the receptor with its native ligand, the extracellular domain of the receptor was engineered to ensure that the receptor was solely activated by the DNA aptamer. The present system features tunability in the signaling output level using aptamer ligands with different receptor dimerization propensities. In addition, the functional programmability of DNA aptamers enables the modular sensing of extracellular molecules without the need for genetic engineering of the receptor.

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides., (© 2023. The Author(s).)

We compared the passive permeability of cyclosporin A (CsA) derivatives with side chain deletions across lipid bilayers. CsA maintained passive permeability after losing any one of the side chains, which suggests that the propensity of the backbone of CsA is an important component for high passive permeability., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Recently, targeted protein degradation (TPD) has attracted much attention as a powerful strategy for effective inhibition of disease-related proteins. However, development of ligands with high affinity and specificity for a target protein is still a demanding task and poses a particular challenge for designing TPD therapeutics. In this work, we report a novel TPD strategy called aptamer-mediated cleavage of extracellular antigen (Apt-clean), where oligonucleotide-based affinity agents are used for selective recruitment of proteases to target membrane proteins. Our data demonstrate that Apt-clean induces selective degradation of the target protein both in vitro and in cellulo. In addition, the potential of Apt-clean was demonstrated through the inhibition of tumor-related growth factor signaling. This novel TPD modality may serve as an efficient and flexible strategy for targeting membrane proteins.

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Spin hyperpolarization enables real-time metabolic imaging of carbon-13-labeled substrates. While hyperpolarized l-(1- 13 C)alaninamide is a probe of the cell-surface tumor marker aminopeptidase-N (APN, CD13), its activity in vivo has not been described. Scanning the kidneys of rats infused with hyperpolarized alaninamide shows both conversion to [1- 13 C]alanine and several additional spectral peaks with distinct temporal dynamics. The (1- 13 C)alaninamide chemical shift is pH-sensitive, with a p K a of 7.9 at 37 °C, and the peaks correspond to at least three different compartments of pH 7.46 ± 0.02 (1), 7.21 ± 0.02 (2), and 6.58 ± 0.05 (3). An additional peak was assigned to the carboxyamino adduct formed by reaction with dissolved CO 2 . Spectroscopic imaging showed nonuniform distribution, with the low-pH signal more concentrated in the inner medulla. Treatment with the diuretic acetazolamide resulted in significant pH shifts in compartment 1 to 7.38 ± 0.03 ( p = 0.0057) and compartment 3 to 6.80 ± 0.05 ( p = 0.0019). While the pH of compartment 1 correlates with blood pH, the pH of compartment 3 did not correspond to the pH of urine. In vitro experiments show that alaninamide readily enters blood cells and can detect intracellular pH. While carbamate formation depends on pH and pCO 2 , the carbamate-to-alaninamide ratio did not correlate with either arterial blood pH or pCO 2 , suggesting that it may reflect variations in tissue pH and pCO 2 . This study demonstrates the feasibility of using hyperpolarized sensors to simultaneously image enzyme activity, pCO 2 , and pH in vivo.

N -Substituted peptides, such as peptoids and β-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for β-peptoids; to date, no preorganized β-peptoid monomers have been reported. Here, we report the first design strategy for β-peptoids, in which all four backbone dihedral angles ( ω , ϕ , θ , ψ ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a β-peptoid with a stable twisted strand shape.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1- 13 C]Gly- d 2 -NMe 2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications., (© 2022 Wiley-VCH GmbH.)

Backbone stereochemistry of cyclic peptides has been reported to have a great influence on microsomal stability and membrane permeability, two important factors that determine oral bioavailability. Here, we comprehensively investigated the correlation between the backbone stereochemistry of cyclic hexapeptide stereoisomers and their stability in liver microsomes, as well as passive membrane permeability.

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo . We recently developed γ-glutamyl-[1- 13 C]glycine (γ-Glu-[1- 13 C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo . However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu-Gly scaffold and develop a deuterated probe, γ-Glu-[1- 13 C]Gly- d 2 , that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu-Gly and the magnetic properties of 13 C-enriched γ-Glu-Gly (γ-Glu-[1- 13 C]Gly and γ-[5- 13 C]Glu-Gly) to support the validity of γ-Glu-[1- 13 C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time ( T 1 ) of γ-Glu-[1- 13 C]Gly and γ-Glu-[1- 13 C]Gly- d 2 under various conditions (D 2 O, PBS, and serum) and confirmed that the T 1 of γ-Glu-[1- 13 C]Gly and γ-Glu-[1- 13 C]Gly- d 2 was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1- 13 C]Gly revealed a significant contribution of the dipole-dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T 1 . In fact, by using γ-Glu-[1- 13 C]Gly- d 2 as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Here, we investigated the effect of CH 3 to CF 3 substitution on the membrane permeability of peptides. We synthesized a series of peptides with CF 3 groups and corresponding nonfluorinated peptides and measured the membrane permeability of the peptides. As a result, we demonstrated that CH 3 to CF 3 substitution is useful for increasing the membrane permeability of di-/tri-peptides.

Designing synthetic surrogates of functional proteins is an important, albeit challenging, task in the field of chemistry. A strategy toward the design of synthetic agonists for growth factor or cytokine receptors that elicit a desired signal activity has been in high demand, as such ligands hold great promise as safer and more effective therapeutics. In the present study, we used a DNA aptamer as a building block and described the strategy-guided design of a synthetic receptor agonist with fine-tuned agonism. The developed synthetic partial agonist can regulate therapeutically relevant cellular activities by eliciting fine-tuned receptor signaling., (© 2021 Wiley-VCH GmbH.)

Many cadherin family proteins are associated with diseases such as cancer. Since cell adhesion requires homodimerization of cadherin molecules, a small-molecule regulator of dimerization would have therapeutic potential. Herein, we describe identification of a P-cadherin-specific chemical fragment that inhibits P-cadherin-mediated cell adhesion. Although the identified molecule is a fragment compound, it binds to a cavity of P-cadherin that has not previously been targeted, indirectly prevents formation of hydrogen bonds necessary for formation of an intermediate called the X dimer and thus modulates the process of X dimerization. Our findings will impact on a strategy for regulation of protein-protein interactions and stepwise assembly of protein complexes using small molecules., (© 2021. The Author(s).)

We developed a photoreactive molecular glue, BP Glue-N 3 , which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BP Glue-N 3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BP Glue-N 3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu + ) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BP Glue-N 3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BP Glue-N 3 and also a nitrene species, possibly generated by the BP-to-N 3 energy transfer in BP Glue-N 3 . We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.

The development of inhibitors of intracellular protein-protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo( N -substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo( N -substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N -substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N -substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors., Competing Interests: The authors declare the following competing financial interest: J. M., Y. F., and S. S. have filed a patent application (PCT/JP2020/27010)., (This journal is © The Royal Society of Chemistry.)

Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR spectroscopy. By application of this powerful tool, small molecules with stable isotopes have been used for highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to the rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with success in vitro or in vivo., (© 2020 Wiley-VCH GmbH.)

Optochemical tools that can modulate the activity of the target protein provide an opportunity for studying and regulating the related biological processes. Here we present a DNA-based nongenetic optochemical tool that can control the dynamics of growth factor signaling. This photo-caged mimicry of growth factor can be a promising tool for elucidating a linkage between the dynamics of signaling and the resulting biological outcomes, as well as for manipulating cellular functions and the fate of living cells.

Growth factor receptors are activated through dimerization by the binding of their ligands and play pivotal roles in normal cell function. However, the aberrant activity of the receptors has been associated with cancer malignancy. One of the main causes of the aberrant receptor activation is the overexpression of receptors and the resultant formation of unliganded receptor dimers, which can be activated in the absence of external ligand molecules. Thus, the unliganded receptor dimer is a promising target to inhibit aberrant signaling in cancer. Here, we report an aptamer that specifically binds to fibroblast growth factor receptor 2b and inhibits the aberrant receptor activation and signaling. Our investigation suggests that this aptamer inhibits the formation of the receptor dimer occurring in the absence of external ligand molecules. This work presents a new inhibitory function of aptamers and the possibility of oligonucleotide-based therapeutics for cancer., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Bile acids are metabolites of cholesterol that facilitate lipid digestion and absorption in the small bowel. Bile acids work as agonists of receptors to regulate their own metabolism. Bile acids also regulate other biological systems such as sugar metabolism, intestinal multidrug resistance, and adaptive immunity. However, numerous physiological roles of bile acids remain undetermined. In this study, we solved the crystal structure of human serine hydroxymethyltransferase (hSHMT) in complex with an endogenous secondary bile acid glycine conjugate. The specific interaction between hSHMT and the ligand was demonstrated using mutational analyses, biophysical measurements, and structure-activity relationship studies, suggesting that secondary bile acid conjugates may act as modulators of SHMT activity., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Cell culture media are often supplemented with recombinant growth factors and cytokines to reproduce biological conditions in vitro. Basic fibroblast growth factor (bFGF) has been widely used to support the pluripotency and self-renewal activity of human induced pluripotent stem cells (hiPSCs). We had previously developed a synthetic surrogate for bFGF on the basis of a DNA aptamer that binds to one of the FGF receptors. Since DNA aptamers have advantages over recombinant proteins in terms of thermal stability and production cost, replacing recombinant growth factors in cell culture media with DNA aptamers would be of great interest. Herein, we describe our protocol for feeder-free hiPSC culture using a DNA aptamer-based mimic of bFGF.

A quantum sequencer offers a scalable electrical platform for single-molecule analysis of genomic events. A thymidine (dT) analog exhibiting uniquely high single-molecule conductance is a key element in capturing DNA synthesis dynamics by serving as a decodable marker for enzymatic labeling of nascent strands. However, the current design strategies of dT analogs that focus on their molecular orbital energy levels require bulky chemical modifications to extend the π-conjugation, which hinders polymerase recognition. We report herein a polymerase-compatible dT analog that is highly identifiable in quantum sequencing. An ethynyl group is introduced as a small gold-binding motif to differentiate the nucleobase-gold electronic coupling, which has been an overlooked factor in modifying nucleobase conductance. The resulting C5-ethynyl-2'-deoxyuridine exhibits characteristic signal profiles that allowed its correct identification at a 93% rate while maintaining polymerase compatibility. This study would expand the applicability of quantum sequencing by demonstrating a robust nucleoside marker with high identifiability., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and 13 C DNP measurements were performed on samples containing 8.4 M [ 13 C]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual 13 C DNP properties of SA-BDPA.

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1- 13 C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1- 13 C]glycine and acquiring dynamic 13 C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1- 13 C]glycine and its product [1- 13 C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1- 13 C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1- 13 C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1- 13 C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1- 13 C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.

Although growth factors have great therapeutic potential because of their regenerative functions, they often have intrinsic drawbacks, such as low thermal stability and high production cost. Oligonucleotides have recently emerged as promising chemical entities for designing synthetic alternatives to growth factors. However, their applications in vivo have been recognized as a challenge because of their susceptibility to nucleases and limited distribution to a target tissue. Here, we present the first example of oligonucleotide-based growth factor mimetics that exerts therapeutic effects at a target tissue after systemic injection. The aptamer was designed to dimerize a growth factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the successful application of aptamer-based receptor agonists., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Unique folded structures of natural and synthetic oligomers are the most fundamental basis for their unique functions. N -Substituted β-peptides, or β-peptoids, are synthetic oligomers with great potential to fold into diverse three-dimensional structures because of the existence of four rotatable bonds in a monomer with highly modular synthetic accessibility. However, the existence of the four rotatable bonds poses a challenge for conformational control of β-peptoids. Here, we report a strategy for per-residue programming of two dihedral angles of β-peptoids, which is useful for restricting the conformational space of the oligomers. The oligomer was found to form a unique loop conformation that is stabilized by the backbone rotational restrictions. Circular dichroism and NMR spectroscopic analyses and X-ray crystallographic analysis of the oligomer are presented. The strategy would significantly facilitate the discovery of many more unique folded structures of β-peptoids.

Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo( N -substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo( N -substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N -substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

Dynamic nuclear polarization with photo-excited triplet electrons (Triplet-DNP) is demonstrated using 6,13-diphenylpentacene (DPPentacene). DPPentacene is soluble in various organic solvents, while pentacene, which is used in most of the triplet-DNP experiments, has limited solubility. An enhancement factor of 81 is obtained for 1H spins in the glass of ethanol-d6 : water = 80 : 20 (w/w) doped with 0.1 mM DPPentacene at 90 K in 0.67 T.

Parahydrogen-induced polarization (PHIP) is a rapid and cost-effective hyperpolarization technique using transition metal-catalysed hydrogenation with parahydrogen. We examined rhodium catalysts and their kinetic studies, rarely considered in the research of current PHIP. It emerged that rhodium complexes with electron-donating bisphosphine ligands, with a dicyclohexylphosphino group, appear to be more effective than conventional rhodium catalysts., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Quantum-tunneling-based DNA sequencing is a single molecular technology that has great potential for achieving facile and high-throughput DNA sequencing. In principle, the sequence of DNA could be read out by the time trace of the tunnel current that can be changed according to molecular conductance of nucleobases passing through nanosized gap electrodes. However, efficient base-calling of four genetic alphabets has been seriously impeded due to the similarity of molecular conductance among canonical nucleotides. In this article, we demonstrate that replacement of canonical 2'-deoxyadenosine (dA) with a highly conductive dA analogue, 7-deaza dA, could expand the difference of molecular conductance between four genetic alphabets. Additionally, systematic evaluation of molecular conductance using a series of dA and dG analogues revealed that molecular conductance of the nucleotide is highly dependent on the HOMO level. Thus, the present study demonstrating that signal characteristics of the nucleotide can be modulated based on the HOMO level provides a widely applicable chemical approach and insight for facilitation of single molecular sensing as well as DNA sequencing based on quantum tunneling.

We developed a dendritic molecular glue PC Glue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PC Glue-NBD carrying multiple guanidinium ion (Gu + ) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PC Glue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PC Glue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PC Glue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PC Glue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.

Here, we report a facile permeability assay to quantitatively evaluate the membrane permeability of multiple peptides in parallel. With a fluorogenic click reaction between azidocoumarin and a terminal alkyne tag introduced on a peptide, the peptide that crossed an artificial membrane or a cell monolayer was quantitatively detected. The method allows a rapid measurement of the permeability of multiple compounds on a plate reader even in the presence of a complex mixture of biological molecules.

Replacing expensive and thermally unstable growth factors with synthetic alternatives has been an important issue in stem cell-based regenerative medicines. Here we developed DNA aptamer-assemblies that act as functional mimics of basic fibroblast growth factor (bFGF), one of the essential factors for stem cell culture. The most potent aptamer assembly named TD0, composed solely of 76-mer single-stranded DNA, could support the self-renewal and pluripotency of induced pluripotent stem cells (iPSCs). This work presents the first application of DNA aptamer in the maintenance of iPSCs.

Serine hydroxymethyltransferase (SHMT) is an enzyme that catalyzes the reaction that converts serine to glycine. It plays an important role in one-carbon metabolism. Recently, SHMT has been shown to be associated with various diseases. Therefore, SHMT has attracted attention as a biomarker and drug target. However, the development of molecular probes responsive to SHMT has not yet been realized. This is because SHMT catalyzes an essential yet simple reaction; thus, the substrates that can be accepted into the active site of SHMT are limited. Here, we focus on the SHMT-catalyzed retro-aldol reaction rather than the canonical serine-glycine conversion and succeed in developing fluorescent and 19 F NMR molecular probes. Taking advantage of the facile and direct detection of SHMT, the developed fluorescent probe is used in the high-throughput screening for human SHMT inhibitors, and two hit compounds are obtained.

Tumor biomarkers are highly desirable for the screening of patients with a risk of tumor development and progression. Here, we report a beta-galactosidase (β-gal)-responsive acetaminophen (β-GR-APAP) as a synthetic plasma biomarker for targeted tumor detection. Tumor β-gal labeling via the recognition of tumor-related antigen enabled the detection of a tumor using β-GR-APAP.

Dynamic nuclear polarization (DNP) is a technique to polarize the nuclear spin population. As a result of the hyperpolarization, the NMR sensitivity of the nuclei in molecules can be dramatically enhanced. Recent application of the hyperpolarization technique has led to advances in biochemical and molecular studies. A major problem is the short lifetime of the polarized nuclear spin state. Generally, in solution, the polarized nuclear spin state decays to a thermal spin equilibrium, resulting in loss of the enhanced NMR signal. This decay is correlated directly with the spin-lattice relaxation time T 1 . Here we report [ 13 C,D 14 ]tert-butylbenzene as a new scaffold structure for designing hyperpolarized 13 C probes. Thanks to the minimized spin-lattice relaxation (T 1 ) pathways, its water-soluble derivative showed a remarkably long 13 C T 1 value and long retention of the hyperpolarized spin state., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

α-Helix-mediated protein-protein interactions (PPIs) are important targets in biological research and drug development. Peptides containing d-amino acid residues are attractive molecules for inhibiting α-helix-mediated PPIs because of their wide surface area and high protease resistance. In this study, a peptide library was constructed using a one-bead one-compound format designed to isolate left-handed α-helical peptides, which are promising molecules as inhibitors of α-helix-mediated PPIs. Screening of the library against an α-helix-mediated PPI between MDM2 and p53 yielded an inhibitor of the PPI. Design and screening of the library, and biochemical and spectroscopic studies of the discovered peptide are presented., (Copyright © 2018 Elsevier Ltd. All rights reserved.)