Papers - Hanaya, Kengo

One-Step Maleimide-Based Dual Functionalization of Protein N-Termini.

Kengo Hanaya, Kazuaki Taguchi, Yuki Wada, Masaki Kawano

Angewandte Chemie International Edition (Wiley)     e202417134 2024.11

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted,  ISSN  14337851

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Maleimide derivatives are privileged reagents for chemically modifying proteins through the Michael addition reaction with cysteine due to their selectivity, operational simplicity, and commercial availability. However, since accessible free cysteine is rarely found in natural proteins, it is highly desirable to find alternative targets to enable direct bioconjugation of proteins with maleimides. In this study, we have developed an operationally simple and straightforward method for the N-terminal modification of proteins without the need for mutagenesis via a copper(II)-mediated [3+2] cycloaddition reaction with maleimides and 2-pyridinecarboxaldehyde (2-PCA) derivatives under non-denaturing conditions at pH 6 and 37 °C in aqueous media. Our method utilizes commercially available maleimides to attach diverse functionalities to various N-terminal amino acids. We demonstrate the preparation of a ternary protein complex cross-linked at the N-termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE-Cy5-trastuzumab). MMAE-Cy5-trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2-positive cells. Furthermore, MMAE-Cy5-trastuzumab allowed successful visualization of HER2-positive cancer cells in mouse tumors. This straightforward method will expand the accessibility of protein conjugates with well-defined structures in a wide range of research fields.

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Dual delivery of carbon monoxide and doxorubicin using haemoglobin-albumin cluster: proof of concept for well-tolerated cancer therapy

Ito C., Taguchi K., Yamada T., Hanaya K., Enoki Y., Sugai T., Komatsu T., Matsumoto K.

Journal of Materials Chemistry B 12 ( 23 ) 5600 - 5608 2024.05

ISSN  2050750X

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A serious concern of doxorubicin (DOX) therapy is that it causes severe adverse effects, particularly cardiotoxicity. Carbon monoxide (CO) possesses powerful cytoprotective effects against drug-induced organ injury and is expected to ameliorate DOX-induced cardiotoxicity. In this study, a dual carrier of DOX and CO (CO-HemoAct-DOX) was fabricated based on a haemoglobin-albumin cluster (HemoAct), which is a protein cluster with a haemoglobin core structure wrapped by serum albumin. CO-HemoAct-DOX was synthesised by binding CO to a haemoglobin core and covalently conjugating (6-maleimidocaproyl)hydrazone derivative of DOX to an albumin shell. The average DOX/cluster ratio was about 2.6. In the in vitro cytotoxicity assay against cancer cells, the anti-tumour activity of CO-HemoAct-DOX was 10-fold lower than that of DOX in a 2D-cultured model, whereas CO-HemoAct-DOX suppressed the growth of tumour spheroids to the same extent as DOX in the 3D-cultured model. In colon-26 tumour-bearing mice, CO-HemoAct-DOX achieved DOX delivery to the tumour site and alleviated tumour growth more effectively than DOX. Furthermore, CO-HemoAct attenuated DOX-induced cardiomyocyte atrophy in H9c2 cells and elevated the levels of cardiac biomarkers in mice exposed to DOX. These results suggest that the dual delivery of CO and DOX using HemoAct is a promising strategy as an anti-tumour agent to realise well-tolerated cancer therapy with minimal cardiotoxicity.

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Synthesis and property of 2,2-difluoro-1,4-diazaborole

Nakahara M., Wada Y., Kawano M., Hanaya K., Sugai T., Higashibayashi S.

Chemistry Letters 53 ( 5 )  2024.05

ISSN  03667022

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The synthesis of 2,2-difluoro-1,4-diazaboroles was achieved through the condensation of aliphatic acyltrifluoroborates and amino-N-heteroarenes under acidic conditions. Various N-heteroarenes, including pyridine, pyrimidine, pyridazine, thiazole, and isoquinoline, were successfully employed in the reaction. Among the synthesized 2,2-difluoro-1,4-diazaboroles, the one with an isoquinoline structure exhibited the most intense light blue emission.

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Utilization of Lipase-Catalyzed Acetylation and Deacetylation in the Synthesis of a Natural Product with a Hydroquinone Skeleton

Sugai T., Honda K., Hashimoto R., Hanaya K., Higashibayashi S.

Asian Journal of Organic Chemistry 13 ( 12 )  2024

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Elaborated conditions for lipase-catalyzed preferential monoacetylation enabled the large-scale preparation of hydroquinone monoacetate. This product was then applied to the synthesis of tournefolin B, a highly oxygenated prenylated hydroquinone of plant origin (Tournefortia sarmentosa Lam.). During the course of the synthetic transformations, lipase-catalyzed deacetylation under mild conditions was demonstrated.

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Unified short syntheses of oxygenated tricyclic aromatic diterpenes by radical cyclization with a photoredox catalyst

Hashimoto R., Hanaya K., Sugai T., Higashibayashi S.

Communications Chemistry (Communications Chemistry)  6 ( 1 )  2023.12

Research paper (scientific journal), Joint Work, Accepted

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The biomimetic two-phase strategy employing polyene cyclization and subsequent oxidation/substitution is an effective approach for divergent syntheses of [6-6-6]-tricyclic diterpenes. However, this strategy requires lengthy sequences for syntheses of oxygenated tricyclic aromatic abietane/podocarpane diterpenes owing to the many linear oxidation/substitution steps after cyclization. Here, we present a new synthetic route based on a convergent reverse two-phase strategy employing a reverse radical cyclization approach, which enabled the unified short syntheses of four aromatic abietane/podocarpane diterpenes and the divergent short syntheses of other related diterpenes. Oxygenated and substituted precursors for cyclization were convergently prepared through Friedel-Crafts acylation and rhodium-catalyzed site-selective iodination. Radical redox cyclization using an iridium photoredox catalyst involving neophyl rearrangement furnished the thermodynamically favored 6-membered ring preferentially. (±)-5,6-Dehydrosugiol, salvinolone, crossogumerin A, and Δ5-nimbidiol were synthesized in only 8 steps. An oxygenated cyclized intermediate was also useful for divergent derivatization to sugiol, ferruginol, saprorthoquinone, cryptomeriololide, and salvinolone.

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Synthesis and luminescence properties of substituted benzils

Yasui M., Fujihara T., Ohtsu H., Wada Y., Shimada T., Zhu Y., Kawano M., Hanaya K., Sugai T., Higashibayashi S.

Communications Chemistry (Communications Chemistry)  6 ( 1 )  2023.12

Research paper (scientific journal), Accepted

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Photophysical properties of benzil (1,2-diphenylethane-1,2-dione) and its derivatives in the crystal state have recently attracted much attention. However, the study of substituted benzils has mostly been limited to para-substituted derivatives, which did not induce a significant effect on the emission wavelength compared to pristine benzil. The effects of ortho- and meta-substituents on the photophysical properties in the crystal state have not been investigated so far. Our recently developed organocatalytic pinacol coupling of substituted benzaldehydes allowed us to prepare various ortho-, meta-, and para-substituted benzil derivatives and to investigate their luminescence properties. Ortho- and meta-substituents affected the electronic states of benzils in the crystal state, resulting in differences in their luminescence properties. The luminescence wavelength and type, i.e., phosphorescence or fluorescence, were altered by these substituents. Fast self-recovering phosphorescence-to-phosphorescence mechanochromism by the para-CF3 substituent at room temperature was also discovered.

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Selective modification of tryptophan in polypeptides via C-N coupling with azoles using in situ-generated iodine-based oxidants in aqueous media

Watanabe S., Wada Y., Kawano M., Higashibayashi S., Sugai T., Hanaya K.

Chemical Communications (Chemical Communications)  59 ( 87 ) 13026 - 13029 2023.10

Research paper (scientific journal), Last author, Corresponding author, Accepted,  ISSN  13597345

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This study demonstrates the C-N coupling of tryptophan with azoles, promoted by an in situ-generated iodine-based oxidant. The protocol was successfully applied to the selective modification of tryptophan in nonprotected polypeptide bearing oxidatively sensitive residues in acidic aqueous media. The present method allows the attachment of reactive handles to polypeptides and the peptide stapling.

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Inversion of Diaza[5]Helicenes Through an N−N Bond Breaking Pathway

Kobayashi T., Ishiwari F., Fukushima T., Nojima Y., Hasegawa M., Mazaki Y., Hanaya K., Sugai T., Higashibayashi S.

Chemistry - A European Journal (Chemistry - A European Journal)  29 ( 43 )  2023.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09476539

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1,1’,10,10’-Biphenothiazine and its S,S,S’,S’-tetroxide are diaza[5]helicenes with N−N linkages. Kinetic experiments on racemization together with DFT calculations revealed that they undergo inversion through the N−N bond breaking pathway rather than the general conformational pathway. In these diaza[5]helicenes with this inversion mechanism, the reduction of electronic repulsion in the N−N bond by modification of S to SO2 at the outer position of the helix led to a significantly higher inversion barrier, 35.3 kcal/mol, compared to [5]helicene. 1,1’,10,10’-Biphenothiazine S,S,S’,S’-tetroxide was highly resistant to acid-mediated N−N bond breaking and racemization under acidic conditions.

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Single-Step N-Terminal Modification of Proteins via a Bio-Inspired Copper(II)-Mediated Aldol Reaction

Hanaya K., Yamoto K., Taguchi K., Matsumoto K., Higashibayashi S., Sugai T.

Chemistry - A European Journal (Chemistry - A European Journal)  28 ( 47 )  2022.08

Research paper (scientific journal), Lead author, Corresponding author, Accepted,  ISSN  09476539

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The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The N-terminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the N-terminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.

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One-Step Synthesis of Acylborons from Acyl Chlorides through Copper-Catalyzed Borylation with Polystyrene-Supported PPh<inf>3</inf>Ligand

Nakahara M., Kurahayashi K., Hanaya K., Sugai T., Higashibayashi S.

Organic Letters (Organic Letters)  24 ( 30 ) 5596 - 5601 2022.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  15237060

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We developed a one-step synthesis of acylborons from both readily available acyl chlorides and bis(pinacolato)diboron through copper(I)-catalyzed borylation. Under the reaction conditions using tBuOLi, polystyrene-supported triphenylphosphine as a copper ligand was found to promote the borylation of acyl chlorides while suppressing alcoholysis. This method enables the facile synthesis of potassium acyltrifluoroborates.

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Synthesis of 1H-2-Benzopyran-5,8-dione Skeleton through a Cascade Reaction between Benzoquinone and β-Ketoester

Nakahara M., Ohtsu H., Kawano M., Hanaya K., Sugai T., Higashibayashi S.

Chemistry Letters (Chemistry Letters)  51 ( 4 ) 356 - 359 2022.04

Research paper (scientific journal), Joint Work, Accepted,  ISSN  03667022

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The 1H-2-benzopyran-5,8-dione structure was synthesized by a cascade reaction through Michael addition, elimination of HBr, and O-alkylation between a dibromobenzoquinone derivative and β-ketoesters under basic conditions. The reaction using potassium carbonate and 18-crown-6 in THF was the best condition, giving 1H-2-benzopyran-5,8-dione derivatives in good yields. The UV-vis absorption spectrum and cyclic voltammogram of the synthesized 1H-2-benzopyran-5,8-dione derivative were measured, clarifying the photophysical and redox properties.

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1,2-Rearrangement from o-Quinols to Multisubstituted Catechols via Retro Diels-Alder Reaction of o-Quinol Dimers

Hashimoto R., Hanaya K., Sugai T., Higashibayashi S.

Bulletin of the Chemical Society of Japan (Bulletin of the Chemical Society of Japan)  95 ( 4 ) 663 - 672 2022

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00092673

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The 1,2-rearrangement of o-quinols has been a long-standing unsolved problem since 1958. Although the rearrangement is expected to be useful for syntheses of catechol derivatives, it is hampered by many competing reactions and has not been developed as a useful methodology. Here, we succeeded in settling this problem by a first systematic thorough investigation, establishing the 1,2-rearrangement as a cascade reaction with a retro Diels-Alder reaction from o-quinol dimers. This is a useful strategy for syntheses of substituted catechols used as synthetic building blocks for bioactive compounds and material molecules. o-Quinol dimers were synthesized by improved oxidative hydroxylation of substituted phenols followed by spontaneous Diels-Alder reaction. The dimers then underwent the retro Diels-Alder reaction to regenerate the o-quinols followed by 1,2-rearrangement under neutral heating conditions at an appropriate temperature depending on the migratory substituent, furnishing substituted catechols in good yields. The competing reactions such as an elimination of a substituent or α-ketol rearrangement were minimized by controlling the reaction temperature.

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Copper-Catalyzed Stereoselective Borylation and Palladium-Catalyzed Stereospecific Cross-Coupling to Give Aryl C-Glycosides

Kurahayashi K., Hanaya K., Sugai T., Hirai G., Higashibayashi S.

Chemistry - A European Journal (Chemistry - A European Journal)  29 ( 6 )  2022

Research paper (scientific journal), Joint Work, Accepted,  ISSN  09476539

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Metabolically stable C-glycosides are an essential family of compounds in bioactive natural products, therapeutic agents, and biological probes. For their application, development of synthetic methods by connecting glycosides and aglycons with strict stereocontrol at the anomeric carbon, as well as with high functional-group compatibility and environmental compatibility is a pivotal issue. Although Suzuki–Miyaura-type C(sp3)−C(sp2) cross-coupling using glycosyl boronates is a potential candidate for the construction of C-glycosides, neither the cross-coupling itself nor the facile synthesis of the coupling precursor, glycosyl boronates, have been achieved to date. Herein, it was succeeded to develop a copper-catalyzed stereoselective one-step borylation of glycosyl bromides to glycosyl boronates and palladium-catalyzed stereospecific cross-coupling of β-glycosyl borates with aryl bromides to give aryl β-C-glycosides, in which the β-configuration of the anomeric carbon of the glycosyl trifluoroborates is stereoretentively transferred to that of the resulting aryl C-glycosides.

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Intramolecular cyclization of m-homoprenylphenols through oxidative nucleophilic aromatic substitution

Deguchi H., Hanaya K., Sugai T., Higashibayashi S.

Chemical Communications (Chemical Communications)  59 ( 6 ) 748 - 751 2022

Research paper (scientific journal), Accepted,  ISSN  13597345

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We developed an intramolecular cyclization of m-homoprenylphenols and related m-prenylphenols to bicyclic skeletons by hypervalent iodine reagents through an oxidative nucleophilic aromatic substitution using the prenyl group as a carbon nucleophile. The reaction was applicable for the syntheses of 5/6-, 6/6-, and 7/6-fused ring systems.

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Chemoselectivity-independent Cu-mediated coupling to construct the hydroquinoline skeleton of symbioimine

Fujita R., Hanaya K., Sugai T., Higashibayashi S.

Scientific Reports (Scientific Reports)  11 ( 1 )  2021.12

Research paper (scientific journal), Accepted

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Construction of the hydroquinoline skeleton of symbioimine by Cu-mediated N-alkenylation or O-alkenylation of an allyl aminoalcohol, in which either chemoselectivity could lead to the target compound, was investigated. O-alkenylation followed by Claisen rearrangement was favored with high selectivity under a ligand-free condition. Subsequent intramolecular condensation furnished the hydroquinoline skeleton of symbioimine.

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Metal-free thermal organocatalytic pinacol coupling of arylaldehydes using an isonicotinate catalyst with bis(pinacolato)diboron

Yasui M., Hanaya K., Sugai T., Higashibayashi S.

RSC Advances (RSC Advances)  11 ( 40 ) 24652 - 24655 2021.07

Research paper (scientific journal), Accepted

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The metal-free thermal organocatalytic pinacol coupling of arylaldehydes has been developed. The intermolecular coupling of arylaldehydes catalyzed byt-butyl isonicotinate with bis(pinacolato)diboron as the co-reducing agent afforded 1,2-diphenylethane-1,2-diols. This reaction was also applicable to the intramolecular coupling of 1,1′-biphenyl-2,2′-dicarbaldehydes to afford 9,10-dihydrophenanthrene-9,10-diols. Various functional groups were tolerated under this coupling condition.

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Chemoenzymatic semisynthesis of caffeic acid β-phenethyl ester, an antioxidative component in propolis, from raw coffee bean extract

Hashimoto R., Iai H., Fujita R., Hanaya K., Higashibayashi S., Inoue H., Sugai T.

Bioscience, biotechnology, and biochemistry (Bioscience, biotechnology, and biochemistry)  85 ( 3 ) 476 - 480 2021.02

Research paper (scientific journal), Accepted,  ISSN  09168451

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Caffeic acid β-phenethyl ester (CAPE), an antioxidative bioactive catechol isolated from propolis, was semisynthesized from chlorogenic acid and related compounds in an extract of raw (unroasted) Robusta coffee (Coffea canephora) beans in 5 steps and a total yield of 31%. Oxidative degradation of the intermediates and target molecule was prevented by alkaline hydrolysis of the chlorogenic acids in the presence of sodium dithionite (Na2S2O4) and deprotection of the catecholic diacetate precursor by Candida antarctica lipase B-mediated transesterification as the final step.

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Selective capture and non-invasive release of cells using a thermoresponsive polymer brush with affinity peptides

Nagase K., Shimura M., Shimane R., Hanaya K., Yamada S., Akimoto A.M., Sugai T., Kanazawa H.

Biomaterials Science (Biomaterials Science)  9 ( 3 ) 663 - 674 2021.02

Research paper (scientific journal), Accepted,  ISSN  20474830

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Tissue engineering and cell transplantation therapy have become promising therapies for intractable diseases. These approaches require cell separation technology without cell modification. Accordingly, in this study, we developed a novel cell separation method using a thermoresponsive block copolymer brush with an affinity peptide. A block copolymer brush with bottom poly(2-hydroxyethyl methacrylate [HEMA]-co-propargyl acrylate) and top poly(N-isopropylacrylamide-co-HEMA) segments was prepared through two steps of atom transfer radical polymerization. Then, cell affinity peptides were conjugated to the bottom segment of the copolymer brush through a click reaction. Using cRGD as a cell-Affinity peptide, enhancement of cell adhesion with rapid adhesion on the copolymer brush was observed at 37 °C, whereas the copolymer brush without cRGD did not exhibit cell adhesion. Temperature-modulated cell adhesion and detachment were performed with a relatively long upper segment because the affinity between peptides and cells was modulated by the swelling and shrinking of the upper thermoresponsive segment. Selective endothelial cell adhesion was performed at 37 °C using GGGREDV as an affinity peptide. Smooth muscle cells and fibroblasts did not adhere to the copolymer brush. Adhered human umbilical vein endothelial cells (HUVECs) were successfully recovered by reducing the temperature to 20 °C. Based on the properties of the copolymer brush, HUVECs could be purified using a mixture of cells simply by changing the temperature. These results demonstrated that the prepared copolymer brush with cell affinity peptides could be a useful cell separation tool because the cells could be separated with specificity and without cell modification using a simple procedure.

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Semisynthesis of prunetin, a bioactive O-methylated isoflavone from naringenin, by the sequential deacetylation of chalcone intermediates and oxidative rearrangement

Sugai T., Hanaya K., Higashibayashi S.

Bioscience, biotechnology, and biochemistry (Bioscience, biotechnology, and biochemistry)  85 ( 1 ) 143 - 147 2021.01

Research paper (scientific journal), Accepted,  ISSN  09168451

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Prunetin (4',5-dihydroxy-7-methoxyisoflavone) was semisynthesized in 8 steps from readily available naringenin in 26% total yield. The key reaction was chemoenzymatic sequential deacetylation to 6'-acetoxy-2',4″-dihydroxy-4'-methoxychalcone, the in situ-formed precursor for thallium(III) nitrate-mediated oxidative rearrangement.

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Theoretical Analysis of the Heterocyclic [4+2] Cycloaddition Between Pyridinium Ion and Enol Ether

Nakahara M., Hanaya K., Sugai T., Higashibayashi S.

ChemistryOpen (ChemistryOpen)  10 ( 6 ) 627 - 629 2021

Research paper (scientific journal), Accepted

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© 2021 The Authors. Published by Wiley-VCH GmbH Dearomative heterocyclic [4+2] cycloaddition between the N-(2,4-dinitrophenyl)pyridinium ion of nicotinamide and an enol ether was analyzed by Density Functional Theory (DFT) calculations. The calculation revealed that the reaction undergoes stepwise bond formation rather than occurring in a concerted manner. The experimental products were found to be both kinetically and thermodynamically favored. The calculated transition states and intermediate suggested that the high diastereoselectivity is derived from the electrostatic interaction between the 2-nitro group of the pyridinium ion and the hydrogen of the enol ether.

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