Characterization of the Polymerization Catalyst [(2,5-norbornadiene)Rh{C(Ph)CPh₂}(PPh₃)] and Identification of the End Structures of Poly(phenylacetylenes) Obtained by Polymerization Using This Catalyst

The structures of [(2,5-norbornadiene)­Rh­{C­(Ph)CPh₂}­(PPh₃)] (<b>1</b>) and its reaction product with CH₃CO₂H were elucidated by ¹H, ¹³C, and ³¹P NMR spectroscopy, mass spectrometry, and single-crystal X-ray analysis. The presence of two conformational isomers of <b>1</b> was verified by NMR spectroscopy, which was well-supported by DFT calculations. Phenylacetylene was polymerized using <b>1</b> as a catalyst with [M]₀/[Rh] = 10 and quenched with CH₃CO₂H and CH₃CO₂D. The incorporation of H and D at the polymer ends was confirmed by MALDI-TOF mass spectrometry and ¹H and ¹H–¹³C HSQC NMR spectroscopy. The polymerization degree was calculated to be 11 by ¹H NMR spectroscopy, which agreed well with the theoretical value

Characterization of the Polymerization Catalyst [(2,5-norbornadiene)Rh{C(Ph)CPh₂}(PPh₃)] and Identification of the End Structures of Poly(phenylacetylenes) Obtained by Polymerization Using This Catalyst

The structures of [(2,5-norbornadiene)­Rh­{C­(Ph)CPh₂}­(PPh₃)] (<b>1</b>) and its reaction product with CH₃CO₂H were elucidated by ¹H, ¹³C, and ³¹P NMR spectroscopy, mass spectrometry, and single-crystal X-ray analysis. The presence of two conformational isomers of <b>1</b> was verified by NMR spectroscopy, which was well-supported by DFT calculations. Phenylacetylene was polymerized using <b>1</b> as a catalyst with [M]₀/[Rh] = 10 and quenched with CH₃CO₂H and CH₃CO₂D. The incorporation of H and D at the polymer ends was confirmed by MALDI-TOF mass spectrometry and ¹H and ¹H–¹³C HSQC NMR spectroscopy. The polymerization degree was calculated to be 11 by ¹H NMR spectroscopy, which agreed well with the theoretical value

Characterization of the Polymerization Catalyst [(2,5-norbornadiene)Rh{C(Ph)CPh₂}(PPh₃)] and Identification of the End Structures of Poly(phenylacetylenes) Obtained by Polymerization Using This Catalyst

The structures of [(2,5-norbornadiene)­Rh­{C­(Ph)CPh₂}­(PPh₃)] (<b>1</b>) and its reaction product with CH₃CO₂H were elucidated by ¹H, ¹³C, and ³¹P NMR spectroscopy, mass spectrometry, and single-crystal X-ray analysis. The presence of two conformational isomers of <b>1</b> was verified by NMR spectroscopy, which was well-supported by DFT calculations. Phenylacetylene was polymerized using <b>1</b> as a catalyst with [M]₀/[Rh] = 10 and quenched with CH₃CO₂H and CH₃CO₂D. The incorporation of H and D at the polymer ends was confirmed by MALDI-TOF mass spectrometry and ¹H and ¹H–¹³C HSQC NMR spectroscopy. The polymerization degree was calculated to be 11 by ¹H NMR spectroscopy, which agreed well with the theoretical value

A Set of Organelle-Localizable Reactive Molecules for Mitochondrial Chemical Proteomics in Living Cells and Brain Tissues

Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography–mass spectrometry (LC–MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases

Semisynthetic Lectin–4-Dimethylaminopyridine Conjugates for Labeling and Profiling Glycoproteins on Live Cell Surfaces

Glycoproteins on cell surfaces play important roles in biological processes, including cell–cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu­(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both <i>in vitro</i> and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells

Syndecan‑4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides

Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis

Discovery of Shoot Branching Regulator Targeting Strigolactone Receptor DWARF14

DWARF14 (D14) is a strigolactone receptor that plays a central role in suppression of shoot branching, and hence is a potential target to increase crop productions and biomass. Recently, we reported a fluorescence turn-on probe, Yoshimulactone Green (YLG), which generates a strong fluorescence upon the hydrolysis by D14-type strigolactone receptors. Herein, we applied a YLG-based <i>in vitro</i> assay to a high-throughput chemical screening and identified a novel small molecule DL1 as a potent inhibitor of D14. DL1 competes with endogenous strigolactones, thereby increasing the number of shoot branching in a model plant <i>Arabidopsis</i> as well as in rice. Thus, DL1 is expected to be useful not only as a tool to understand the biological roles of D14 receptors in plant growth and development, but also as a potent agrochemical to improve the crop yield