Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Aminopeptidases (APs) in the renin-angiotensin system (RAS) and their activity balance play a crucial role in regulating vascular functions. Multiplexed analysis of RAS-related AP activities is useful for diagnosing diseases, including cancer. Dynamic nuclear polarization coupled magnetic resonance imaging (DNP-MRI) enables simultaneous detection of multiple enzymatic activities in vivo. However, developing practical DNP-MRI probes, especially for multiplexed detection, remains challenging. Here, we report the design of DNP-MRI probes for in vivo multiplexed analysis of AP activities. By integrating quantum mechanical calculations, organic synthesis, and physicochemical/biochemical evaluations, we developed a series of AP-responsive DNP-MRI probes with high enzymatic reactivities and distinguishable chemical shifts. Using these probes, we successfully detected and visualized multiple AP activities in vivo. Furthermore, we performed in vivo multiplex analysis of RAS-related AP activities in tumor-bearing mice, demonstrating the potential of this approach for monitoring the efficacy of anti-angiogenic cancer therapy and for accurate discrimination of tumor types.

DOI： 10.1021/jacs.5c16910

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Polymer conjugation is a common strategy to improve the pharmacokinetics of aptamers, yet its effects on aptamer properties are incompletely understood.

DOI： 10.1039/d5bm01078k

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/d5ra04836b

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Aminopeptidase N (APN) is a key biomarker associated with various diseases, including cancers and cardiovascular diseases. Nuclear magnetic resonance imaging (MRI) coupled with dynamic nuclear polarization (DNP) represents a powerful technique for in vivo detection of enzymatic activity. We previously developed Ala-[1-13C]Gly-d2-NMe2 as a DMP-MRI molecular probe capable of visualizing and mapping APN activity in vivo. However, the broader application of this probe to disease animal models is hindered by significant signal attenuation during sample transfer through low magnetic fields, such as the Earth’s magnetic field, primarily due to scalar relaxation via the 13C–14N bond. Here, we present Ala-[1-13C]Gly-d2-15NMe2 as an alternative, incorporating a 15N nucleus in place of 14N. This modification produces markedly enhanced MR signal intensity without the need for stringent sample handling to avoid exposure to low magnetic fields. Consequently, this developed probe can potentially offer significant advantages for cellular studies and applications in complex organisms, where elaborate pre-injection procedures are often required.

DOI： 10.1093/chemle/upaf150

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Abstract

Enzymes play a crucial role in regulating physiological functions, and abnormal enzyme activity is associated with various pathological conditions. Precise imaging of enzyme activity in tissues, providing detailed spatial and quantitative information, advances our understanding of physiological and pathological processes. Despite their importance, there is still a lack of methods for high‐resolution 3D imaging of enzyme activity across entire tissues. In this research, we report a methodology for high‐resolution, whole‐organ 3D mapping of enzyme activity, which combines tissue clearing with an activity‐based covalent chemical probe. Focusing on aminopeptidase N (APN) as a representative target of peptidase, we developed ANA‐o‐BODIPY, an activity‐based covalent fluorescent probe compatible with tissue clearing for imaging APN activity. Upon activation by APN, ANA‐o‐BODIPY produces a reactive intermediate, aza‐quinone methide, which covalently binds to proximal proteins. This covalent probe is successfully utilized to record the location of APN activity during the tissue‐clearing process. By combining the probe with tissue clearing, we have achieved high‐resolution 3D mapping of APN activity across whole organs for the first time. Moreover, this advancement allowed us to visualize the heterogeneity of APN activity in individual tubular structures and to uncover the inhibitory effects of different APN inhibitors.

DOI： 10.1002/anie.202504668

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Dissolution dynamic nuclear polarization (DNP) is a technique that enhances the detection sensitivity of molecular probes in magnetic resonance imaging (MRI). DNP-MRI enables the detection of various biological parameters such as pH, perfusion, and metabolic reactions. However, the range of its applications is limited by the short lifetime of hyperpolarized probes. The detectable duration of in vivo hyperpolarized signal is ~10 min, i.e., within a minute timescale, which is insufficient to track in vivo events occurring on an hour timescale. Here, we report [15N]betaine-d11 as a DNP-MRI probe exhibiting extremely long hyperpolarization retention in vivo. The [15N]betaine-d11 effectively suppresses nuclear-spin relaxations and showed a very long spin-lattice relaxation time (T1 = 1,584 s, deuterated PBS, 1.4 T). Using this probe, we successfully monitored in vivo hyperpolarized 15N MR signals from a mouse body for approximately one hour. Furthermore, we detected the distribu-tion and accumulation of [15N]betaine-d11 for over 50 min in the tumor, where betaine plays a critical role.

DOI： 10.26434/chemrxiv-2025-0qxks

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Zwitterionic polymer PMPC was conjugated to a DNA aptamer to enhance its therapeutic efficacy in vivo, while minimizing the recognition by anti-PEG antibodies.

DOI： 10.1039/d4bm01541j

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Language：English   Publishing type：Research paper (scientific journal)  

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 (T1). Because of the limitation in T1, 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 13C-oligopeptides. Structure-based T1 relaxation analysis suggests that the C-terminal [1-13C]Gly-d2 residue affords sufficient T1 for biological uses, even in relatively large oligopeptides, and allowed us to develop 13C-β-casomorphin-5 and 13C-glutathione. It was found that the metabolic response and perfusion of the hyperpolarized 13C-glutathione in the mouse kidney were significantly altered in a model of cisplatin-induced acute kidney injury.

DOI： 10.1126/sciadv.adp2533

PubMed

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Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

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.

DOI： 10.1126/sciadv.ado8471

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

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.

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DOI： 10.1007/s44211-024-00520-y

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Other Link： https://link.springer.com/article/10.1007/s44211-024-00520-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/acsnano.3c12716

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Oxidized thioether-closed macrocyclic peptides are site-selectively linearized upon collision-induced fragmentation, which enhances sequence deconvolution of cyclic peptides.

DOI： 10.1039/d4cc03179b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

We developed a label-free lipid bilayer permeability assay, which realized the measurement of the penetration speed of cyclosporin A.

DOI： 10.1039/d2sc05785a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Society of Synthetic Organic Chemistry, Japan  

DOI： 10.5059/yukigoseikyokaishi.80.994

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Language：English   Publishing type：Research paper (scientific journal)  

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-13C]glycine (γ-Glu-[1-13C]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-[1-13C]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 13C-enriched γ-Glu-Gly (γ-Glu-[1-13C]Gly and γ-[5-13C]Glu-Gly) to support the validity of γ-Glu-[1-13C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T 1) of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]Gly-d 2 under various conditions (D2O, PBS, and serum) and confirmed that the T 1 of γ-Glu-[1-13C]Gly and γ-Glu-[1-13C]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-[1-13C]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-[1-13C]Gly-d 2 as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

DOI： 10.1039/d1ra07343e

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s42003-021-02575-3

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.isci.2021.102036

PubMed

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-020-63160-y

Web of Science

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/anie.202001211

Web of Science

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/anie.202001211

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-019-08833-7

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Other Link： https://www.nature.com/articles/s41467-019-08833-7

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126/science.aap9801

Web of Science

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Authorship：Lead author   Publisher：John Wiley & Sons, Ltd  

DOI： 10.1002/047084289x.rn02052

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acscatal.6b02317

Web of Science

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J-GLOBAL

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Symposium, workshop panel (public)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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