Publications by authors named "Yuichi Yoshimura"

63 Publications

Synthesis and evaluation of trypanocidal activity of derivatives of naturally occurring 2,5-diphenyloxazoles.

Bioorg Med Chem 2021 Jul 12;42:116253. Epub 2021 Jun 12.

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan.

African trypanosomiasis is a zoonotic protozoan disease affecting the nervous system. Various natural products reportedly exhibit trypanocidal activity. Naturally occurring 2,5-diphenyloxazoles present in Oxytropis lanata, and their derivatives, were synthesized. The trypanocidal activities of the synthesized compounds were evaluated against Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, and T. evansi. Natural product 1 exhibited trypanocidal activity against all the species/subspecies of trypanosomes, exhibiting half-maximal inhibitory concentrations (IC) of 1.1-13.5 μM. Modification of the oxazole core improved the trypanocidal activity. The 1,3,4-oxadiazole (7) and 2,4-diphenyloxazole (9) analogs exhibited potency superior to that of 1. However, these compounds exhibited cytotoxicity in Madin-Darby bovine kidney cells. The O-methylated analog of 1 (12) was non-cytotoxic and exhibited selective trypanocidal activity against T. congolense (IC = 0.78 µM). Structure-activity relationship studies of the 2,5-diphenyloxazole analogs revealed aspects of the molecular structure critical for maintaining selective trypanocidal activity against T. congolense.
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http://dx.doi.org/10.1016/j.bmc.2021.116253DOI Listing
July 2021

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein.

J Biol Chem 2021 Jan-Jun;296:100788. Epub 2021 May 18.

Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C, Denmark; Department of Chemistry, Aarhus University, Aarhus C, Denmark. Electronic address:

The intrinsically disordered human protein α-synuclein (αSN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric αSN is cytotoxic, making it important to devise strategies to either prevent oligomer formation and/or inhibit the ensuing toxicity. (-)-epigallocatechin gallate (EGCG) has emerged as a molecular modulator of αSN self-assembly, as it reduces the flexibility of the C-terminal region of αSN in the oligomer and inhibits the oligomer's ability to perturb phospholipid membranes and induce cell death. However, a detailed structural and kinetic characterization of this interaction is still lacking. Here, we use liquid-state NMR spectroscopy to investigate how EGCG interacts with monomeric and oligomeric forms of αSN. We find that EGCG can bind to all parts of monomeric αSN but exhibits highest affinity for the N-terminal region. Monomeric αSN binds ∼54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the αSN association reaction. In contrast, preformed oligomers only bind ∼7 EGCG molecules per protomer, in agreement with the more compact nature of the oligomer compared with the natively unfolded monomer. In previously conducted cell assays, as little as 0.36 EGCG per αSN reduce oligomer toxicity by 50%. Our study thus demonstrates that αSN cytotoxicity can be inhibited by small molecules at concentrations at least an order of magnitude below full binding capacity. We speculate this is due to cooperative binding of protein-stabilized EGCG dimers, which in turn implies synergy between protein association and EGCG dimerization.
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http://dx.doi.org/10.1016/j.jbc.2021.100788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191297PMC
May 2021

Synthesis and Properties of 4'-ThioLNA/BNA.

Org Lett 2021 05 3;23(10):4062-4066. Epub 2021 May 3.

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Komatsushima 4-4-1, Aoba-ku, Sendai 981-8558, Japan.

To develop a new nucleoside analogue applicable to oligonucleotide therapeutics, we designed a 4'-thio analogue of an LNA/BNA monomer. Synthesis of 4'-hydroxymethyl-4'-thioribonucleoside was achieved by a tandem ring-contraction-aldol reaction of a 5-thiopyranose derivative and the subsequent Pummerer-type thioglycosylation reaction of the corresponding sulfoxide. Treatment of 4'-hydroxymethyl-4'-thiopyrimidine nucleosides with diphenyl carbonate in the presence of catalytic NaHCO gave the desired 4'-thioLNA/BNA monomers, which were introduced into oligonucleotides.
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http://dx.doi.org/10.1021/acs.orglett.1c01306DOI Listing
May 2021

Carbonyl C-detect solution-state protein NMR experiments to circumvent amide-solvent exchange broadening: Application to β-microglobulin.

Biochim Biophys Acta Proteins Proteom 2021 03 25;1869(3):140593. Epub 2020 Dec 25.

Institute for Protein Research, Osaka University, Yamada-oka 3-2, Suita, 565-0871 Osaka, Japan.

The N-H heteronuclear single-quantum correlation (HSQC) technique in protein NMR spectroscopy suffers from line-broadening effects, such as chemical exchange of labile protons with solvent, and exchange broadening for residues undergoing conformational dynamics. The amide resonance of β-microglobulin residue S88 is not observed in the HSQC spectrum but can be obtained through C-detect experiments that circumvent the problem of amide-solvent exchange broadening. Line broadening of S88 resonance beyond detection in the HSQC spectrum is not attributed to conformational exchange but rather to solvent exchange occurring on the order of ~10 s.
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http://dx.doi.org/10.1016/j.bbapap.2020.140593DOI Listing
March 2021

Concise Syntheses of Violaceoids A and C.

Chem Pharm Bull (Tokyo) 2021 Feb 26;69(2):232-235. Epub 2020 Nov 26.

Laboratory of Synthetic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.

The concise syntheses of two alkylated hydroquinone natural products, violaceoids A and C, were accomplished by a protecting-group-free method employing the commercially available 2,5-dihydroxybenzaldehyde as the starting material. The key strategy of the syntheses is the utilization of alkenylboronic acid as both the coupling and temporary protective reagents to efficiently introduce the requisite alkenyl side chain of violaceoid A. Moreover, the synthesis of violaceoid C is reported here for the first time.
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http://dx.doi.org/10.1248/cpb.c20-00816DOI Listing
February 2021

Sensitive and simplified: a combinatorial acquisition of five distinct 2D constant-time C-H NMR protein correlation spectra.

J Biomol NMR 2020 Dec 17;74(12):695-706. Epub 2020 Aug 17.

Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.

A procedure is presented for the substantial simplification of 2D constant-time C-H heteronuclear single-quantum correlation (HSQC) spectra of C-enriched proteins. In this approach, a single pulse sequence simultaneously records eight sub-spectra wherein the phases of the NMR signals depend on spin topology. Signals from different chemical groups are then stratified into different sub-spectra through linear combination based on Hadamard encoding of CH multiplicity (n = 1, 2, and 3) and the chemical nature of neighboring C nuclei (aliphatic, carbonyl/carboxyl, aromatic). This results in five sets of 2D NMR spectra containing mutually exclusive signals from: (i) C-H correlations of asparagine and aspartic acid, C-H correlations of glutamine and glutamic acid, and C-H correlations of glycine, (ii) C-H correlations of all residues but glycine, and (iii) C-H correlations of phenylalanine, tyrosine, histidine, and tryptophan, and the remaining (iv) aliphatic CH and (v) aliphatic CH/CH resonances. As HSQC is a common element of many NMR experiments, the spectral simplification proposed in this article can be straightforwardly implemented in experiments for resonance assignment and structure determination and should be of widespread utility.
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http://dx.doi.org/10.1007/s10858-020-00341-xDOI Listing
December 2020

Palladium-Catalyzed Three-Component Coupling of Ynamides.

Org Lett 2020 07 26;22(14):5299-5303. Epub 2020 Jun 26.

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Komatsushima 4-4-1, Aoba-ku, Sendai 981-8558, Japan.

A palladium-catalyzed regioselective three-component coupling of ynamides was developed. The reaction proceeded smoothly to furnish the desired products when carried out at 70 °C in acetonitrile/water with potassium carbonate in the presence of 2.5 mol % Pd(dba)·CHCl without a ligand. Various iodides and boronic acids were used in this reaction, and a carbon-carbon bond was formed with satisfactory regioselectivity from the ynamides.
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http://dx.doi.org/10.1021/acs.orglett.0c01426DOI Listing
July 2020

Strategy for Designing Selective Lysosomal Acid α-Glucosidase Inhibitors: Binding Orientation and Influence on Selectivity.

Molecules 2020 Jun 19;25(12). Epub 2020 Jun 19.

School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan.

Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1--heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1--alkyl chain. It is noteworthy that α-1--heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.
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http://dx.doi.org/10.3390/molecules25122843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357040PMC
June 2020

Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity.

EMBO J 2020 06 7;39(12):e101732. Epub 2020 May 7.

Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.
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http://dx.doi.org/10.15252/embj.2019101732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298289PMC
June 2020

Learning Self-Triggered Controllers With Gaussian Processes.

IEEE Trans Cybern 2020 Apr 1. Epub 2020 Apr 1.

This article investigates the design of self-triggered controllers for networked control systems (NCSs), where the dynamics of the plant are unknown a priori. To deal with the unknown transition dynamics, we employ the Gaussian process (GP) regression in order to learn the dynamics of the plant. To design the self-triggered controller, we formulate an optimal control problem, such that the optimal control and communication policies can be jointly designed based on the GP model of the plant. Moreover, we provide an overall implementation algorithm that jointly learns the dynamics of the plant and the self-triggered controller based on a reinforcement learning framework. Finally, a numerical simulation illustrates the effectiveness of the proposed approach.
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http://dx.doi.org/10.1109/TCYB.2020.2980048DOI Listing
April 2020

How internal cavities destabilize a protein.

Proc Natl Acad Sci U S A 2019 10 30;116(42):21031-21036. Epub 2019 Sep 30.

Interdisciplinary Nanoscience Center, University of Aarhus, 8000 Aarhus C, Denmark;

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å.
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http://dx.doi.org/10.1073/pnas.1911181116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800337PMC
October 2019

Synthesis of 2'-aminouridine derivatives as an organocatalyst for Diels-Alder reaction.

Nucleosides Nucleotides Nucleic Acids 2020 30;39(1-3):365-383. Epub 2019 Jul 30.

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, 981-8558, Japan.

To develop a novel asymmetric organocatalyst based on a ribonucleoside skeleton, we designed and synthesized 2'-aminouridine derivatives. The synthesized 2'-aminouridines having bulky substituents at both base and sugar moieties could catalyze the Diels-Alder reaction between cinnamaldehyde and cyclopentadiene. However, the optical purities of the resulting products were unexpectedly low.
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http://dx.doi.org/10.1080/15257770.2019.1646917DOI Listing
September 2020

Glycosylation reactions mediated by hypervalent iodine: application to the synthesis of nucleosides and carbohydrates.

Beilstein J Org Chem 2018 28;14:1595-1618. Epub 2018 Jun 28.

Graduate School of Pharmaceutical Science, Tokushima University, Shomachi 1-78-1, Tokushima, 770-8505, Japan.

To synthesize nucleoside and oligosaccharide derivatives, we often use a glycosylation reaction to form a glycoside bond. Coupling reactions between a nucleobase and a sugar donor in the former case, and the reaction between an acceptor and a sugar donor of in the latter are carried out in the presence of an appropriate activator. As an activator of the glycosylation, a combination of a Lewis acid catalyst and a hypervalent iodine was developed for synthesizing 4'-thionucleosides, which could be applied for the synthesis of 4'-selenonucleosides as well. The extension of hypervalent iodine-mediated glycosylation allowed us to couple a nucleobase with cyclic allylsilanes and glycal derivatives to yield carbocyclic nucleosides and 2',3'-unsaturated nucleosides, respectively. In addition, the combination of hypervalent iodine and Lewis acid could be used for the glycosylation of glycals and thioglycosides to produce disaccharides. In this paper, we review the use of hypervalent iodine-mediated glycosylation reactions for the synthesis of nucleosides and oligosaccharide derivatives.
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http://dx.doi.org/10.3762/bjoc.14.137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6037013PMC
June 2018

Synthesis of 4'-Thionucleosides as Antitumor and Antiviral Agents.

Chem Pharm Bull (Tokyo) 2018 ;66(2):139-146

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University.

Many attempts have been made to synthesize structurally novel nucleoside derivatives in order to identify effective compounds for the treatment of tumors and virus-caused disease. At our laboratories, as part of our efforts to synthesize 4'-thionucleosides, we have identified and characterized biologically active nucleosides. During the course of our synthetic study, we developed the Pummerer-type thioglycosylation reaction. As a result, we synthesized a potent antineoplastic nucleoside, 1-(2-deoxy-2-fluoro-β-D-4-thio-arabino-furanosyl)cytosine (4'-thioFAC), and several novel 4'-thionucleosides that possess antiherpes virus activities.
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http://dx.doi.org/10.1248/cpb.c17-00636DOI Listing
November 2018

Practical Synthesis of 4'-Thioribonucleosides from L-Arabinose via Novel Reductive Ring-Contraction Reaction and Pummerer-Type Thioglycosylation.

Curr Protoc Nucleic Acid Chem 2017 12 24;71:1.43.1-1.43.12. Epub 2017 Dec 24.

Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai, Japan.

The detailed practical synthesis of 4'-thionucleosides starting from L-arabinose is described here. 1,4-Anhydro-2,3-O-isopropylidene-4-thioribitol, which is the key intermediate for the synthesis of 4'-thionucleosides, is obtained from L-arabinose in several steps, including a novel reductive ring-contraction reaction. After oxidation of the key intermediate, the sulfoxide is subjected to Pummerer-type thioglycosylation in the presence of persilylated nucleobases to obtain the 4'-thioribonucleosides in good yield and β-selectively. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpnc.45DOI Listing
December 2017

Active-Site pKa Determination for Photoactive Yellow Protein Rationalizes Slow Ground-State Recovery.

Biophys J 2017 May;112(10):2109-2116

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh, Groningen, the Netherlands; Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Aarhus, Denmark. Electronic address:

The ability to avoid blue-light radiation is crucial for bacteria to survive. In Halorhodospira halophila, the putative receptor for this response is known as photoactive yellow protein (PYP). Its response to blue light is mediated by changes in the optical properties of the chromophore para-coumaric acid (pCA) in the protein active site. PYP displays photocycle kinetics with a strong pH dependence for ground-state recovery, which has remained enigmatic. To resolve this problem, a comprehensive pK determination of the active-site residues of PYP is required. Herein, we show that Glu-46 stays protonated from pH 3.4 to pH 11.4 in the ground (pG) state. This conclusion is supported by the observed hydrogen-bonded protons between Glu-46 and pCA and Tyr-42 and pCA, which are persistent over the entire pH range. Our experimental results show that none of the active-site residues of PYP undergo pH-induced changes in the pG state. Ineluctably, the pH dependence of pG recovery is linked to conformational change that is dependent upon the population of the relevant protonation state of Glu-46 and the pCA chromophore in the excited state, collaterally explaining why pG recovery is slow.
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http://dx.doi.org/10.1016/j.bpj.2017.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443972PMC
May 2017

MOAG-4 promotes the aggregation of α-synuclein by competing with self-protective electrostatic interactions.

J Biol Chem 2017 05 23;292(20):8269-8278. Epub 2017 Mar 23.

Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark. Electronic address:

Aberrant protein aggregation underlies a variety of age-related neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Little is known, however, about the molecular mechanisms that modulate the aggregation process in the cellular environment. Recently, MOAG-4/SERF has been identified as a class of evolutionarily conserved proteins that positively regulates aggregate formation. Here, by using nuclear magnetic resonance (NMR) spectroscopy, we examine the mechanism of action of MOAG-4 by characterizing its interaction with α-synuclein (α-Syn). NMR chemical shift perturbations demonstrate that a positively charged segment of MOAG-4 forms a transiently populated α-helix that interacts with the negatively charged C terminus of α-Syn. This process interferes with the intramolecular interactions between the N- and C-terminal regions of α-Syn, resulting in the protein populating less compact forms and aggregating more readily. These results provide a compelling example of the complex competition between molecular and cellular factors that protect against protein aggregation and those that promote it.
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http://dx.doi.org/10.1074/jbc.M116.764886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437234PMC
May 2017

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy.

Angew Chem Int Ed Engl 2017 01 29;56(1):239-242. Epub 2016 Nov 29.

Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.

Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that a protein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a C-detected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects. In the experiment, the N and N chemical shifts of an arginine head group are correlated with that of the directly attached C . In the resulting spectrum, the number of protons in the arginine head group can be obtained directly from the N- H scalar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three H atoms were previously identified by neutron crystallography, we show that R52 is predominantly protonated in solution.
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http://dx.doi.org/10.1002/anie.201609605DOI Listing
January 2017

Aberrant increase of NMR signal in hydrogen exchange experiments. Observation and explanation.

Biochem Biophys Res Commun 2016 09 18;478(3):1185-8. Epub 2016 Aug 18.

Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, University of Aarhus, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark. Electronic address:

Hydrogen exchange (HX) NMR spectroscopy is widely used for monitoring structure, stability and dynamics of proteins at the level of individual residues. The stochastic replacement of protons by deuterons typically leads to an exponential decrease of the NMR signals. However, an unusual signal increase was observed in HX of several amides for T4 lysozyme L99A. This effect can be attributed to peak sharpening as a result of reduced dipolar relaxation from proximal amide protons that experience more rapid hydrogen/deuterium (H/D) exchange. The behavior was specifically observed at the termini of secondary structure elements, where large differences in protection against H/D exchange are observed. This effect is expected to be more widespread in NMR HX studies, and is important for the accurate determination of protection factors.
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http://dx.doi.org/10.1016/j.bbrc.2016.08.092DOI Listing
September 2016

Detecting O2 binding sites in protein cavities.

Sci Rep 2016 Feb 2;6:20534. Epub 2016 Feb 2.

Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), University of Aarhus, Gustav Wieds Vej 14, DK-8000 Aarhus C, Denmark.

Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in (1)H, (15)N, and (13)C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.
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http://dx.doi.org/10.1038/srep20534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735865PMC
February 2016

Docking study and biological evaluation of pyrrolidine-based iminosugars as pharmacological chaperones for Gaucher disease.

Org Biomol Chem 2016 Jan 3;14(3):1039-48. Epub 2015 Dec 3.

Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

We report on the synthesis and biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives as pharmacological chaperones for Gaucher disease. The parent compound, DAB, did not show inhibition of human β-glucocerebrosidase but showed moderate intestinal α-glucosidase inhibition; in contrast, extension of α-1-C-alkyl chain length gave a series of highly potent and selective inhibitors of the β-glucocerebrosidase. Our design of α-1-C-tridecyl-DAB (5j) produced a potent inhibitor of the β-glucocerebrosidase, with IC50 value of 0.77 μM. A molecular docking study revealed that the α-1-C-tridecyl group has a favorable interaction with the hydrophobic pocket and the sugar analogue part (DAB) interacted with essential hydrogen bonds formed to Asp127, Glu235 and Glu340. Furthermore, α-1-C-tridecyl-DAB (5j) displayed enhancement of activity at an effective concentration 10-times lower than isofagomine. α-1-C-Tridecyl-DAB therefore provides the first example of a pyrrolidine iminosugar as a new class of promising pharmacological chaperones with the potential for treatment of Gaucher disease.
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http://dx.doi.org/10.1039/c5ob02223aDOI Listing
January 2016

Structural basis for RNA-genome recognition during bacteriophage Qβ replication.

Nucleic Acids Res 2015 Dec 17;43(22):10893-906. Epub 2015 Nov 17.

Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.
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http://dx.doi.org/10.1093/nar/gkv1212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678825PMC
December 2015

Near-complete 1H, 13C, 15N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T.

Biomol NMR Assign 2016 Apr 15;10(1):25-9. Epub 2015 Aug 15.

Department of Chemistry, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D (1)H-(15)N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain (1)H, (13)C and (15)N resonances for unfolded FAS1-4 A546T at 25 °C.
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http://dx.doi.org/10.1007/s12104-015-9630-2DOI Listing
April 2016

Construction of an isonucleoside on a 2,6-dioxobicyclo[3.2.0]-heptane skeleton.

Molecules 2015 Mar 12;20(3):4623-34. Epub 2015 Mar 12.

Laboratory of Human Retrovirology, Leidos Biochemical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

We have built a new isonucleoside derivative on a 2,6-dioxobicyclo[3.2.0]heptane skeleton as a potential anti-HIV agent. To synthesize the target compound, an acetal-protected dihydroxyacetone was first converted to a 2,3-epoxy-tetrahydrofuran derivative. Introduction of an azide group, followed by the formation of an oxetane ring, gave a pseudosugar derivative with a 2,6-dioxobicyclo[3.2.0]heptane skeleton. The desired isonucleoside was obtained by constructing a purine base moiety on the scaffold, followed by amination.
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http://dx.doi.org/10.3390/molecules20034623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272333PMC
March 2015

Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C' chemical shifts of multiple contiguous residues in highly resolved 3D spectra.

J Biomol NMR 2015 Feb 11;61(2):109-21. Epub 2015 Jan 11.

Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000, Aarhus C, Denmark.

Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone (1)H(N), (15)N(H), and (13)C' resonance assignments to be completed from a single pair of 3D experiments.
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http://dx.doi.org/10.1007/s10858-014-9890-7DOI Listing
February 2015

Synthesis and biological evaluation of α-1-C-4'-arylbutyl-L-arabinoiminofuranoses, a new class of α-glucosidase inhibitors.

Bioorg Med Chem Lett 2014 Aug 11;24(15):3298-301. Epub 2014 Jun 11.

Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan.

A series of α-1-C-4'-arylbutyl-L-arabinoiminofuranoses 3 with functional groups attached to the phenyl ring, which are potential α-glycosidase inhibitors, was designed and synthesized by using a Negishi cross-coupling reaction as the key reaction. Arylbutyl derivatives 3a-e showed potent inhibitory activities against intestinal maltase. Among them, difluorophenylbutyl derivative 3e showed good inhibition activities against intestinal isomaltase and sucrase as compared to those of 1 and commercial drugs.
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http://dx.doi.org/10.1016/j.bmcl.2014.06.001DOI Listing
August 2014

How epigallocatechin gallate can inhibit α-synuclein oligomer toxicity in vitro.

J Biol Chem 2014 Aug 6;289(31):21299-310. Epub 2014 Jun 6.

From the Interdisciplinary Nanoscience Center, Center for Insoluble Protein Structures,

Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCG's mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.
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http://dx.doi.org/10.1074/jbc.M114.554667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118093PMC
August 2014

Asymmetric synthesis of 2,5-disubstituted 3-hydroxypyrrolidines based on stereodivergent intramolecular iridium-catalyzed allylic aminations.

Org Biomol Chem 2014 Mar 19;12(12):1983-94. Epub 2014 Feb 19.

Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan.

Intramolecular iridium-catalyzed allylic aminations of homochiral (E)-6-N-nosylaminohept-2-en-1-yl methyl carbonates were investigated. The relative position of the 2,5-substituents of the resulting pyrrolidines was found to be controlled by using both enantiomers (4 and 5) of the appropriate chiral ligand, demonstrating a simple and highly stereodivergent synthetic protocol. Selected trans- and cis-2,5-disubstituted 3-hydroxypyrrolidines (2a and 18a) were converted to (+)-bulgecinine (6) and (+)-preussin (7), respectively.
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http://dx.doi.org/10.1039/c3ob42229aDOI Listing
March 2014

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication.

Biochim Biophys Acta 2013 Dec 1;1834(12):2640-6. Epub 2013 Oct 1.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.
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http://dx.doi.org/10.1016/j.bbapap.2013.09.016DOI Listing
December 2013

Solubility and supersaturation-dependent protein misfolding revealed by ultrasonication.

Langmuir 2014 Feb 9;30(7):1845-54. Epub 2013 Oct 9.

Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Although alcohols are useful cosolvents for producing amyloid fibrils, the underlying mechanism of alcohol-dependent fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 12-16%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase in fibrillation above 60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results indicate that although fibrillation is determined by solubility, supersaturation prevents conformational transitions and ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent protein misfolding funnel useful for examining amyloidogenicity. This misfolding funnel will apply to fibrillation under physiological conditions where biological environments play important roles in decreasing the solubility.
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http://dx.doi.org/10.1021/la403100hDOI Listing
February 2014
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