Review Papers (Misc) - KOUMOTO Kazuya
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水和を介して酵素の活性化、安定化を引き起こす低分子双性イオン性分子の開発 Invited
甲元一也
野口研究所時報 ( 66 ) 3 - 9 2023.10
Authorship：Lead author, Corresponding author Publishing type：Article, review, commentary, editorial, etc. (other)
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「受動的な学び」から「能動的な学び」へ 〜高大連携活動の展開と可能性〜 Invited
甲元一也
月刊「兵庫教育」 74 ( 3 ) 4 - 7 2022.6
Authorship：Lead author, Corresponding author Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)
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Formation of beta-(1,3-1,6)-D-glucan-complexed [70]fullerene and its photodynamic activity towards macrophages (vol 15, pg 1990, 2017) International journal
Atsushi Ikeda, Motofusa Akiyama, Kouta Sugikawa, Kazuya Koumoto, Yuta Kagoshima, Jiawei Li, Toshio Suzuki, Takeshi Nagasaki
ORGANIC & BIOMOLECULAR CHEMISTRY 15 ( 45 ) 9734 - 9734 2017.12
Publisher：ROYAL SOC CHEMISTRY
Correction for 'Formation of β-(1,3-1,6)-d-glucan-complexed [70]fullerene and its photodynamic activity towards macrophages' by Atsushi Ikeda et al., Org. Biomol. Chem., 2017, 15, 1990-1997.
DOI： 10.1039/c7ob90179h
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化学修飾技術を利用した乳酸菌模倣粒子による免疫賦活 Invited
甲元一也、長濱宏治、松井淳
バイオインダストリー 32 ( 9 ) 42 - 47 2015.9
Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)
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代謝産物アナログによる酵素活性化・安定化
高木琴味、甲元一也
月刊バイオインダストリー 30 ( 8 ) 43 - 50 2013.8
Authorship：Lead author Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media) Publisher：CMC出版
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Cellular zwitterionic metabolite analogs simultaneously enhance reaction rate, thermostability, salt tolerance, and substrate specificity of alpha-glucosidase International journal
Eisuke Deguchi, Kazuya Koumoto
BIOORGANIC & MEDICINAL CHEMISTRY 19 ( 10 ) 3128 - 3134 2011.5
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
We investigated the structural effects of metabolite analogs derived from a naturally-occurring zwitterionic metabolite, glycine betaine, on the activity of several hydrolases. The initial velocities of the hydrolases were enhanced by the addition of the solutes into the buffer solution. Based on a detailed study using alpha-glucosidases, the acceleration efficiency of the enzymatic activity was strongly induced by solutes possessing bulky and aliphatic ammonium cations, indicating that enhancement of activity by the solutes depended on their chemical structures. Kinetic analysis revealed that the acceleration of the hydrolysis reaction was related to both the decrement of K-m and increment of V-max values. Furthermore, the addition of the metabolite analogs enhanced not only the rate constant but also the thermostability, salt tolerance, and substrate specificity of alpha-glucosidase simultaneously through the reduction of conformational perturbation of the enzyme. (C) 2011 Elsevier Ltd. All rights reserved.
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糖分子を認識する人工レセプター：ボロン酸
甲元一也、新海征治
月刊バイオインダストリー 27 ( 2 ) 18 - 23 2010.2
Authorship：Lead author Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media) Publisher：CMC出版
水中で糖のもつ水酸基と可逆的なエステル結合を形成するボロン酸基を有する糖レセプターの設計、合成に関する動向を纏めた。
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Reversible stability switching of a hairpin DNA via a photo-responsive linker unit International journal
Li Wu, Kazuya Koumoto, Naoki Sugimoto
CHEMICAL COMMUNICATIONS ( 14 ) 1915 - 1917 2009
Publisher：ROYAL SOC CHEMISTRY
We have designed and synthesized a novel photo-responsive linker unit, 4,4'-bis(hydroxymethyl)-azobenzene, which was introduced to the loop oligonucleotide in a DNA hairpin and was able to reversibly control the stability of the whole hairpin structure via UV or visible light irradiation.
DOI： 10.1039/b819643e
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Hydration is an important factor to regulate thermodynamic stability of a DNA duplex under molecular crowding conditions
Kazuya Koumoto, Hirofumi Ochiai, Naoki Sugimoto
CHEMISTRY LETTERS 37 ( 8 ) 864 - 865 2008.8
Publisher：CHEMICAL SOC JAPAN
By using a DNA duplex. (5'-GTTACTATATGA-3'/5'-TCATATAGTAAC-3') under molecular crowding, conditions, it was revealed that hydration plays a significant role in the stability of the duplex, in contrast to viscosity and dielectric constant which have little effect.
DOI： 10.1246/cl.2008.864
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NanoBioNow(10)周辺の分子環境に応答するDNAナノスイッチの構築
狩俣寿枝, 三好大輔, 藤本健史, 甲元一也, WANG Zhong‐Ming, 杉本直己
日本化学会講演予稿集 88th ( 2 ) 823 2008.3
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Structural effect of synthetic zwitterionic cosolutes on the stability of DNA duplexes
Kazuya KouMoto, Hirofumi Ochiai, Naoki Sugimoto
TETRAHEDRON 64 ( 1 ) 168 - 174 2008.1
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
The molecular design of useful cosolutes for polymerase chain reaction (PCR), which is one of the most important techniques in molecular biology, plays a significant role in amplification of highly stable genome sequences because during PCR, strand dissociation sometimes fails due to high melting temperature. Here, we designed and synthesized eight new zwitterionic cosolutes derived from glycine betaine, a destabilizing reagent for GC-rich DNA duplexes, and systematically compared their ability to destabilize DNA duplexes and to amplify genome DNA by PCR. We found that introduction of n-butyl groups rather than methyl groups into the ammonium group reduced the melting temperature of DNA duplexes 11-fold more than what was observed for the scaffold cosolute, glycine betaine, and furthermore, the cosolute can amplify the stable genome sequence by PCR. (c) 2007 Elsevier Ltd. All rights reserved.
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Enhanced amplification of polymerase chain reaction by addition of cosolutes derived from a cellular compatible solute. International journal
Kazuya Koumoto, Hirofumi Ochiai, Naoki Sugimoto
Nucleic acids symposium series (2004) 52 ( 52 ) 257 - 8 2008
We designed and synthesized artificial compatible solutes which can not only decrease the melting temperature of DNA duplexes dependent of their chemical structures but also improve the amplification of highly stable genome DNA sequence.
DOI： 10.1093/nass/nrn130
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分子クラウディング環境下においてDNA二重鎖の安定性に及ぼす水和の影響
甲元一也, 落合洋文, 狩俣寿枝, 中野修一, 杉本直己
高分子学会予稿集(CD-ROM) 56 ( 2 Disk1 ) 2X15 2007.9
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Delivery of antisense oligonucleotides to nuclear telomere RNA by use of a complex between polysaccharide and polynucleotide
Jusaku Minari, Takanori Kubo, Hideki Ohba, Naohiko Shimada, Yoich Takeda, Ryouji Karinaga, Takahisa Anada, Kazuya Koumoto, Takeshi Kawazu, Takeshi Nagasaki, Seiji Shinkai, Kazuo Sakurai
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 80 ( 6 ) 1091 - 1098 2007.6
Publisher：CHEMICAL SOC JAPAN
Telomerase, which is highly activated in neoplastic cells, can be a target for antisense therapy, and for that purpose, antisense oligonucleotides (AS ODNs) have to be effectively delivered into cellular nucleus where the target telomerase is present. The present work shows a new strategy to deliver AS ODNs to nucleus by use of a novel complex made from a natural polysaccharide schizophyllan (SPG) and AS ODNs. Nuclear transport is strictly regulated by the nuclear pore size and the related proteins. If the molecular weight of SPG is decreased, the SPG/AS ODN complex should be easily transported, although the stability of the complex decreases with a decrease in the molecular weight. We optimized the molecular weight of SPG to be 25 K. Furthermore, we attached importin-beta (a nuclear transport protein) to the side chain of SPG by use of a streptavidin-biotin interaction. When this complex was added to Jurkat cells, the telomerase activity was more suppressed than the naked dose, indicating that the importin-P in the complex induced the nuclear transport of the complexed AS ODN and the AS ODN inhibited the telomerase. The present work shows a new methodology for nuclear anti-sense therapy that should be important in future anti-cancer therapies.
DOI： 10.1246/bcsj.80.1091
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Artificial G-wire switch with 2,2 '-bipyridine units responsive to divalent metal ions International journal
Daisuke Miyoshi, Hisae Karimata, Zhong-Ming Wang, Kazuya Koumoto, Naoki Sugimoto
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 129 ( 18 ) 5919 - 5925 2007.5
Publisher：AMER CHEMICAL SOC
Development of a guanine nanowire (G-wire) that is controllable and can be switched by external signals is important for the creation of molecular electronic technologies. Here, we constructed a G-wire in which the thymines of the main chain of d(G(4)T(4)G(4)) were replaced with 2,2'-bipyridine units, which have two aromatic rings that rotate arbitrarily upon coordination with metal ions. Circular dichroism of the DNA oligonucleotides with or without the 2,2'-bipyridine unit showed that divalent metal ions induce the bipyridine-containing oligonucleotide to switch from an antiparallel to a parallel G-quadruplex. Native polyacrylamide gel electrophoresis showed that the parallel-stranded G-quadruplex DNA had a high-order structure. Circular dichroism and native gel electrophoresis analyses suggested that adding Na(2)EDTA causes a reverse structural transition from a parallel-stranded high-order structure to an antiparallel G-quadruplex. Moreover, atomic force microscopy showed a long nanowire (similar to 200 nm) in the presence of Ni2+ but no significant image in the absence of Ni2+ or in the presence of both Ni2+ and Na(2)EDTA. These observations revealed that the parallel-stranded high-order structure is a G-wire containing numerous DNA oligonucleotide strands bound together via divalent metal ion-2,2'-bipyridine complexes. Finally, we found that alternating addition of Ni2+ and Na(2)EDTA can cycle the G-wire between the high-order and disorganized structures, with an average cycling efficiency of 0.95 (i.e., 5% loss per cycle). These results demonstrate that a DNA oligonucleotide incorporating the 2,2'-bipyridine unit acts as a G-wire switch that can be controlled by chemical input signals, namely, divalent metal ions.
DOI： 10.1021/ja068707u
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Conformational switch of a functional nanowire based on the DNA G-quadruplex. International journal
Hisae Karimata, Daisuke Miyoshi, Takeshi Fujimoto, Kazuya Koumoto, Zhong-Ming Wang, Naoki Sugimoto
Nucleic acids symposium series (2004) 51 ( 51 ) 251 - 2 2007
A G-wire composing of multiple G-rich sequences in parallel orientations via G-quartet formations is promising as a structurally well-defined nanowire. Here, in order to control the G-wire by the metal ion, we incorporated 2,2'-bipyridine unit, which has two aromatic rings rotating arbitrarily by coordination with metal ions, into the main chain of d(G4T4G4) instead of thymine (G1). Circular dichroism (CD) spectra showed that G1 formed an antiparallel G-quadruplex. Interestingly, Ni(2+) induces G1 to switch from an antiparallel G-quadruplex to a parallel. On the other hand, the parallel G-quadruplex reversed the structural transition to an antiparallel G-quadruplex by the addition of Na2EDTA. Moreover, the study of the atomic force microscopy (AFM) showed that G1 formed a long nanowire ( approximately 200 nm) in the presence of Ni(2+), whereas no significant image was observed in the absence of Ni(2+) or in the presence of both Ni(2+) and Na2EDTA. From these results, it can be concluded that Ni(2+) induces a structural transition of G1 from an antiparallel G-quadruplex to a G-wire and that metal ion chelator induces the reverse structural transition from the G-wire to the antiparallel G-quadruplex. The G-wire responsive to metal ions would be useful for development of functional nanomaterials.
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CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect
T Anada, N Okada, J Minari, R Karinaga, M Mizu, K Koumoto, S Shinkai, K Sakurai
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 ( 5 ) 1301 - 1304 2006.3
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
Zymosan, classified among beta-(1-->3)-D-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the 'cocktail effect' of the two immunostimulants. (C) 2005 Elsevier Ltd. All rights reserved.
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Galactose-PEG dual conjugation of beta-(1 -> 3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake
R Karinaga, T Anada, J Minari, M Mizu, K Koumoto, J Fukuda, K Nakazawa, T Hasegawa, M Numata, S Shinkai, K Sakurai
BIOMATERIALS 27 ( 8 ) 1626 - 1635 2006.3
Publisher：ELSEVIER SCI LTD
Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery. (c) 2005 Elsevier Ltd. All rights reserved.
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Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection
T Anada, R Karinaga, K Koumoto, M Mizu, T Nagasaki, Y Kato, K Taira, S Shinkai, K Sakurai
JOURNAL OF CONTROLLED RELEASE 108 ( 2-3 ) 529 - 539 2005.11
Publisher：ELSEVIER SCIENCE BV
Our previous work showed that a natural beta-(1 -> 3)-D-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system. (c) 2005 Elsevier B.V. All rights reserved.
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Cholesterol-appended beta-(1 -> 3)-D-glucan schizophylllan for antisense oligonucleotides delivery to enhance the cellular uptake
K Koumoto, M Mizu, T Anada, T Nagasaki, S Shinkai, K Sakurai
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 78 ( 10 ) 1821 - 1830 2005.10
Publisher：CHEMICAL SOC JAPAN
Schizophyllan is a natural beta-(1 -> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide is added to a schizophyllan solution. the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enables us to introduce a cholesterol group only to the side chain of schizophyllan. We prepared four cholesterol-appended schizophyllans with different modification levels. Using these compounds, we made complexes and carried out an in vitro antisense assay, administrating a phosphorothioate AS ODN to the several cell lines to depress their c-myb mRNA. When we used 2.2-2.3 mol % modified schizophyllan as the carrier, the antisense effect was most enhanced among others. The addition of beta-cyclodextrin improved the complexation ability as well as the up-take for highly modified samples. Furthermore, the cytotoxicity for these modified schizophyllan samples was negligibly as small as the natural (unmodified) schizophyllan. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.
DOI： 10.1246/bcsj.78.1821
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PEG-appended beta-(1 -> 3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation
R Karinaga, K Koumoto, M Mizu, T Anada, S Shinkai, K Sakurai
BIOMATERIALS 26 ( 23 ) 4866 - 4873 2005.8
Publisher：ELSEVIER SCI LTD
Schizophyllan is a natural beta-(1 -> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier. (C) 2004 Elsevier Ltd. All rights reserved.
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Parallel vs. anti-parallel orientation in a curdlan/oligo(dA) complex as estimated by a FRET technique
M Numata, K Koumoto, M Mizu, K Sakurai, S Shinkai
ORGANIC & BIOMOLECULAR CHEMISTRY 3 ( 12 ) 2255 - 2261 2005.6
Publisher：ROYAL SOC CHEMISTRY
We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3' (or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.
DOI： 10.1039/b500156k
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Gene delivery system using polysaccharide schizophyllan and poly(DA) tailed DNA.
T Anada, R Karinaga, K Koumoto, M Mizu, S Shinkai, K Sakurai
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 229 U1160 - U1160 2005.3
Publishing type：Research paper, summary (international conference) Publisher：AMER CHEMICAL SOC
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In situ monitoring of polysaccharide-polynucleotide interaction using a schizophyllan-immobilized QCM device
DH Yang, AH Bae, K Koumoto, SW Lee, K Sakurai, S Shinkai
SENSORS AND ACTUATORS B-CHEMICAL 105 ( 2 ) 490 - 494 2005.3
Publisher：ELSEVIER SCIENCE SA
Schizophyllan belongs to the beta-(1 -> 3)-D-glucan family and is well known as a triple helix polysaccharide. It is already known that the single chain of schizophyllan (s-SPG) can specifically form a complex with particular polynucleotides. As an application of this recognition capability, a novel schizophyllan derivative connected with a disulfide bond through along methylene spacer was synthesized, and its interaction with polynucleoticles was directly monitored with a self-assembled monolayer on QCM. The gold-coated QCM electrode was covered in ca. 60% as a monolayer of s-SPG. The QCM resonator showed sensitive mass increases for poly(C) and poly(U) in the presence of K+ ion, which are consistent with our previous results. The present results indicate that the schizophyllan-immobilized QCM resonator can be a new sensing device for biomolecules. (c) 2004 Elsevier B.V. All rights reserved.
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Competition between polysaccharide/polynucleotide complexation vs., polynucleotide hybridization; salt concentration dependence of the reaction direction
Ryouji Karinaga, Kazuya Koumoto, Jusaku Minari, Masami Mizu, Takahisa Anada, Kazuo Sakurai, Seiji Shinkai
e-Journal of Surface Science and Nanotechnology 3 38 - 45 2005.2
Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide [ex. poly (A) or poly(dA)] was added to the schizophyllan/DMSO solution and subsequently DMSO was exchanged for water, the single chain of schizophyllan formed a complex with the polynucleotide. In this paper, using an adenine-thymine (or uracil) double strand as a model system, we explored whether s-SPG can unzip the polynucleotide duplexes, examining the competition between the schizophyllan/polynucleotide complexation and the polynucleotide hybridization with circular dichroism and fluorescence spectroscopy at various NaCl concentrations. The unzipping reaction happened for the hetero-duplexes (i.e., RNA-DNA duplexes) at low salt concentrations, on the other hand, it was not observed for the homo-duplexes at all the salt concentrations. When we compared dissociation temperatures, the unzipping reaction of the homo-duplexes was expected at low salt concentrations. This discrepancy between the experimental results and the expectation, as well as the different behavior between the hetero- and homo- duplexes, can be ascribed to the difference in the activation energy of intermediate states. © 2005 The Surface Science Society of Japan.
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Poly(N-substituted-acrylamide)-branched schizophyllans are useful for selective recovery of homopolynucleotides through convenient and quick precipitation procedures
T Matsumoto, M Umeda, M Numata, T Hasegawa, K Sakurai, K Koumoto, S Shinkai
POLYMER JOURNAL 37 ( 3 ) 177 - 185 2005
Publisher：SOC POLYMER SCIENCE JAPAN
Schizophyllans carrying poly(N-substituted-acrylamide)-branches were synthesized from native schizophyllan through Ce(IV)-initiated radical homo- or co-polymerization of N-iso-propyl- and N-sec-butyl-acrylamide. A series of structural analyses including elemental analysis, gel permeation chromatography, H-1 NMR, and enzymatic degradation using β-1,3-glucanase revealed that schizophyllans (150kDa) bearing long poly (N-substituted-acrylamide)-branches (ca. 290 kDa) are obtained. These SPG-conjugates form macromolecular complexes with certain homopolynucleotides such as poly(C), poly(dA), and poly(A) and then efficiently co-precipitate them on heating. For example, schizophyllan carrying poly(N-iso-propylacrylamide)-branches can selectively recover poly(C) from a mixture of poly(C) and poly(U) by simple heating (36° C) followed by centrifugation. HPLC analysis of the recovered polynucleotide revealed the high poly(C)-selectivity (poly(C):poly(U) = 95:5). Similarly, schizophyllan carrying more hydrophobic poly(N-iso-propylacrylamide-co-N-sec-butylacrylamide) -branches precipitates at lower temperature (ca. 20° C) and is applicable to isolation of poly(A) that forms less stable macromolecular complexes with SPG and SPG-derivatives (the dissociation temperatures are around 30° C).
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Transferrin-appended β-(1→3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake
Takahisa Anada, Ryouji Karinaga, Masami Mizu, Kazuya Koumoto, Takahiro Matsumoto, Munenori Numata, Seiji Shinkai, Kazuo Sakurai
e-J. Surface Sci. Nanotech. 3 195 - 202 2005
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Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions
K Koumoto, R Karinaga, M Mizu, T Anada, K Sakurai, T Kunitake, S Shinkai
BIOPOLYMERS 75 ( 5 ) 403 - 411 2004.12
Publisher：JOHN WILEY & SONS INC
Thermal stabilization of the complex between polycytidylic acid [poly(Q] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T. value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14degreesC In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions. (C) 2004 Wiley Periodicals, Inc.
DOI： 10.1002/bip.20148
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Proposal of new modification technique for linear double-stranded DNAs using the polysaccharide schizopyllan
T Anada, H Matsunaga, R Karinaga, K Koumoto, M Mizu, K Nakano, S Shinkai, K Sakurai
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 14 ( 22 ) 5655 - 5659 2004.11
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
A natural polysaccharide schizophyllan (SPG) has been known to form a stable complex with poly(dA). We attached a poly(dA)(80) tail to the both ends of a linear double-stranded DNA, which had been prepared from a plasmid DNA vector. The poly(dA) tailed DNA verified to form complex with SPG by gel electrophoresis and atomic force microscopy (AFM). AFM images indicated that the complexes exhibit a dumbbell-like architecture, that is, quite similar to that of adenovirus genome. The complex demonstrated excellent exonuclease resistance, probably because of the protection effect by SPG complexation. (C) 2004 Elsevier Ltd. All rights reserved.
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A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion
M Mizu, K Koumoto, T Anada, T Matsumoto, M Numata, S Shinkai, T Nagasaki, K Sakurai
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 126 ( 27 ) 8372 - 8373 2004.7
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Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis
M Mizu, K Koumoto, T Anada, K Sakurai, S Shinkai
BIOMATERIALS 25 ( 15 ) 3117 - 3123 2004.7
Publisher：ELSEVIER SCI LTD
Schizophyllan is a beta-(1--> 3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cellfree) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect. (C) 2003 Elsevier Ltd. All rights reserved.
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Protection of polynucleotides against nuclease-mediated hydrolysis by complexation with schizophyllan
M Mizu, K Koumoto, T Kimura, K Sakurai, S Shinkai
BIOMATERIALS 25 ( 15 ) 3109 - 3116 2004.7
Publisher：ELSEVIER SCI LTD
Schizophyllan is a beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan (s-SPG) forms a complex with the polynucleotide. The present work demonstrates that the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide), using high-performance liquid chromatography and ultraviolet absorbance technique. A kinetic study for the hydrolysis clarified that the simple Michaelis-Menten relation is held and the maximum velocity for the complex is one-sixth as small as that of the naked polynucleotide. This low hydrolysis rate for the complex suggests that s-SPG is applicable to a carrier for antisense oligonucleotides. (C) 2003 Elsevier Ltd. All rights reserved.
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Long-term expression with a cationic polymer derived from a natural polysaccharide: Schizophyllan
T Nagasaki, M Hojo, A Uno, T Satoh, K Koumoto, M Mizu, K Sakurai, S Shinkai
BIOCONJUGATE CHEMISTRY 15 ( 2 ) 249 - 259 2004.3
Publisher：AMER CHEMICAL SOC
Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.
DOI： 10.1021/bc034178x
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Lactose-appended schizophyllan is a potential candidate as a hepatocyte-targeted antisense carrier
T Hasegawa, M Umeda, T Matsumoto, M Numata, M Mizu, K Koumoto, K Sakurai, S Shinkai
CHEMICAL COMMUNICATIONS ( 4 ) 382 - 383 2004.2
Publisher：ROYAL SOC CHEMISTRY
A schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination can form stable macromolecular complexes with polynucleotides, shows excellent affinity with a lactose-binding lectin, and effectively mediates gene transfection into hepatocytes.
DOI： 10.1039/b313426a
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Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency
T Matsumoto, M Numata, T Anada, M Mizu, K Koumoto, K Sakurai, T Nagasaki, S Shinkai
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1670 ( 2 ) 91 - 104 2004.1
Publisher：ELSEVIER SCIENCE BV
Schizophyllan is a natural beta-(1 --> 3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1 --> 3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier + AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers. (C) 2004 Elsevier B.V. All rights reserved.
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Low M-W sulfated curdlan with improved water solubility forms macromolecular complexes with polycytidylic acid
K Koumoto, M Umeda, M Numata, T Matsumoto, K Sakurai, T Kunitake, S Shinkai
CARBOHYDRATE RESEARCH 339 ( 1 ) 161 - + 2004.1
Publisher：ELSEVIER SCI LTD
The water solubility of curdlan was enhanced by partial sulfation at O-6. Circular dichroism measurements suggest that the sulfated curdlan with the degree of substitution (DS) from 0 to 8.7 mol % forms macromolecular complexes with polycytidylic acid (poly(C)). Although the thermal stability of the complexes decreased with increase in DS, this could be overlapped by addition of NaCl in the concentration above that of serum. The results clearly indicate that the drawback arising from the electrostatic repulsion between the anionic charges can be partially compensated by the presence of salt. Furthermore, the polynucleotide chain complexed with the sulfated curdlan was protected from the enzymatic hydrolysis, corroborating the assumption that the sulfated curdlan has an ability to bind poly(C). (C) 2003 Elsevier Ltd. All rights reserved.
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Enhancement of antisense effect by reducing interactions between proteins and ODNs
Masami Mizu, Kazuya Koumoto, Takahisa Anada, Ryouji Karinaga, Taro Kimura, Kazuo Sakurai, Takeshi Nagasaki, Seiji Shinkai
Bull. Chem. Soc. Jpn. 77 1101 - 1110 2004
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Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier
T Hasegawa, T Fujisawa, M Numata, T Matsumoto, M Umeda, R Karinaga, M Mizu, K Koumoto, T Kimura, S Okumura, K Sakurai, S Shinkai
ORGANIC & BIOMOLECULAR CHEMISTRY 2 ( 21 ) 3091 - 3098 2004
Publisher：ROYAL SOC CHEMISTRY
Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-Lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.
DOI： 10.1039/b412124b
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Polysaccharide/polynucleotide complexes. Part 6. Complementary-strand-induced release of single-stranded DNA bound in the schizophyllan complex
K Koumoto, M Mizu, K Sakurai, T Kunitake, S Shinkai
CHEMISTRY & BIODIVERSITY 1 ( 3 ) 520 - 529 2004
Publisher：VERLAG HELVETICA CHIMICA ACTA AG
Spectroscopic properties of single-stranded DNA/schizophyllan ternary complexes (ss-DNA - 2s-SPG), induced by addition of either complementary or noncomplementary strands, have been investigated. The addition of the complementary strands to ss-DNA - 2s-SPG induced the quick release of the bound ss-DNA to the complementary strands (both DNA and RNA), whereas the ternary complex was unaffected upon addition of noncomplementary strands. Our experiments imply that SPG has complexation properties indispensable to the gene carriers. As far as we know, there is no report on exploitation of such nonviral gene carriers that can accomplish an intelligent release of the bound ss-DNA toward the complementary strands. We believe, therefore, that SPG, a natural and neutral polysaccharide, has a great potential to become a new ss-DNA carrier.
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Molecular weight control of curdlan (beta-1,3-glucan polysaccharide) provides unique polynucleotide binding properties
K Koumoto, H Kobayashi, M Mizu, T Kimura, K Sakurai, T Kunitake, S Shinkai
POLYMER JOURNAL 36 ( 5 ) 380 - 385 2004
Publisher：SOC POLYMER SCIENCE JAPAN
Recently, we found that schizophyllan belonging to a beta-1,3-glucan polysaccharide family forms macromolecular complexes with certain single-stranded polynucleotides. The specific complex formation is originated from the beta-1,3-glucan skeleton in the main chain. In this paper, we have reported that curdlan, which is a beta-1,3-glucan polysaccharide without a side chain glucose unit, shows unique poly(C) binding properties different from those of schizophyllan; in the experiments using curdlan samples with the controlled molecular weight, the complexation ability appeared only in a limited "M-w range window". Stoichiometric analysis revealed that the "M-w range window" appears as a result of the competition between curdlan-curdlan self-association and curdlan-polynucleotide complexation. Furthermore, when the experiments were extended to other polynucleotides such as poly(A), poly(dA) and poly(dT), the "M-w range window" also appeared at their characteristic molecular weights. We believe, therefore, that curdlan can be applicable, in spite of its cheapness, as a precious functional material to gene technology.
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First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan
R Karinaga, M Mizu, K Koumoto, T Anada, S Shinkai, T Kimura, K Sakurai
CHEMISTRY & BIODIVERSITY 1 ( 4 ) 634 - 639 2004
Publisher：VERLAG HELVETICA CHIMICA ACTA AG
Schizophyllan is a natural beta-(1 --> 3)-D-glucan that exists as a triple helix in H2O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH > 13). As we have already reported, when a homopolynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophylian/DMSO solution, and, subsequently, DMSO is exchanged for H2O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H2O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.
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Enhancement of the Antisense Effect of Polysaccharide-Polynucleotide Complexes by Preventing the Antisense Oligonucleotide from Binding to Proteins in the Culture Medium
Masami MIZU, Kazuya KOUMOTO, Takahisa ANADA, Ryouji KARINAGA, Taro KIMURA, Takeshi NAGASAKI, Seiji SHINKAI, Kazuo SAKURAI
Bull. Chem. Soc. Jpn. 77 ( 6 ) 1101 - 1110 2004
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多糖・核酸複合体を用いた機能性高分子コンジュゲート −核酸医薬デリバリーへの挑戦−
狩長亮二, 甲元一也, 櫻井和朗, 新海征治
高分子加工 53 561 - 568 2004
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Microanalysis of nucleic acids using the limulus G test
T Anada, K Koumoto, M Mizu, R Karinaga, K Sakurai, S Shinkai
ANALYTICAL SCIENCES 19 ( 12 ) 1567 - 1568 2003.12
Publisher：JAPAN SOC ANALYTICAL CHEMISTRY
The limulus G test has been used as a quantitative analysis of (1-->3)-beta-D-glucans, including schizophyllan (SPG) and curdlan. The present work extended the limulus G test to detect polynucleotide/SPG complexes. The complex showed an extremely sensitive response to the test, compared with SPG itself. The minimum concentration of the complex to show the response is almost 10-times as small as that of SPG itself, indicating the possibility to detect (1-->3)-beta-D-glucans or/and polynucleotides on the pico gram/ml scale.
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Supramolecular structure of a sugar-appended organogelator explored with synchrotron X-ray small-angle scattering
K Sakurai, Y Jeong, K Koumoto, A Friggeri, O Gronwald, S Sakurai, S Okamoto, K Inoue, S Shinkai
LANGMUIR 19 ( 20 ) 8211 - 8217 2003.9
Publisher：AMER CHEMICAL SOC
Small-angle X-ray scattering (SAXS) from an organogel system prepared from methyl 4,6-O-benzylidene-alpha-D-mannopyranoside and p-xylene was carried out with a synchrotron X-ray source at SPring-8, which revealed that hexagonally packed fibrils are formed in the gel state. The spacing between the fibrils can be evaluated to be about 60 Angstrom, and this value was almost independent of both the gelator concentration and the temperature. Furthermore, the spacing is larger than the gelator molecular size. Upon heating, this supramolecular structure completely disappeared. Time-resolved SAXS revealed that phase separation takes place initially and subsequently the hexagonal structure is formed. Wide-angle X-ray diffraction (WARD) showed that there is no crystalline peak at all and the diffraction pattern is consistent with being amorphous. H-1 NMR spectral data show that the gelator molecules still maintain thermal motion in the gel state. The present SAXS, WARD, and NMR results contrast with those of "dry gels" in which the gel fibers consist of the crystal of the gelators. Our results suggest that the solvent molecules are incorporated into the gel fiber and the present gel can be classified as a "wet gel".
DOI： 10.1021/la0346752
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Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs
K Sakurai, R Iguchi, M Mizu, K Koumoto, S Shinkai
BIOORGANIC CHEMISTRY 31 ( 3 ) 216 - 226 2003.6
Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE
Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH*) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH* range 6.5-10, and dissociated in the pH* range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH* range 4-6 and a single strand in the pH* range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species. (C) 2003 Elsevier Science (USA). All rights reserved.
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Polysaccharide-polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification
M Numata, T Matsumoto, M Umeda, K Koumoto, K Sakurai, S Shinkai
BIOORGANIC CHEMISTRY 31 ( 2 ) 163 - 171 2003.4
Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE
Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino, acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T-m was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T-m shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T-m values attained were 64 degreesC for SPG-Arg conjugate and 62 degreesC for SPG-Lys conjugate, which are higher by 13 and 11 degreesC, respectively, than those of the unmodified SPG + poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T-m values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T-m values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature. (C) 2003 Elsevier Science (USA). All rights reserved.
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Polysaccharide-polynucleotide complexes part 17. Solvent effects on conformational-transition of polydeoxyadenylic acid in the complexes with schizophyllan
M Mizu, K Koumoto, T Kimura, K Sakurai, S Shinkai
POLYMER JOURNAL 35 ( 9 ) 714 - 720 2003
Publisher：SOC POLYMER SCIENCE JAPAN
This paper examines the relationship between the conformation and solvent for the poly(dA)/s-SPG complex using circular dichroism in the 240-300 nm wavelength region. At the low temperature and low salt concentration, poly(dA) in the complex is estimated to take C2'-endo with a different torsional angle from the anti form. With increasing temperature, the circular dichroism (CD) spectrum starts to bear characteristics of C3'-endo and anti as a minority among the majority of C2'-endo and anti. When the salt concentration is increased, the spectrum becomes almost identical to that of the high temperature form under the non-salt condition. Therefore, addition of salt provides the identical effect to increasing temperature. DO induces the same effect as decreasing temperature.
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Polysaccharide-polynucleotide interaction (XI); Novel separation system of RNAs by using Schizophyllan-appended column
T Kimura, K Koumoto, M Mizu, K Sakurai, S Shinkai
CHEMISTRY LETTERS ( 12 ) 1240 - 1241 2002.12
Publisher：CHEMICAL SOC JAPAN
Schizophyllan belongs to the beta-1,3-glucan family and can form a complex with particular polynucleotides.(1) We applied this complexation to specifically separate polynucleotides by constructing a schizophyllan-appended column. The column trapped poly(C) and poly(A), while it did not show any affinity to poly(G), which is consistent with the experimental results obtained in the homogenous solution. The present results indicate that the s-SPG column can be a new separation device for biomolecules.
DOI： 10.1246/cl.2002.1240
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Gel formation properties of a uracil-appended cholesterol gelator and cooperative effects of the complementary nucleobases
E Snip, K Koumoto, S Shinkai
TETRAHEDRON 58 ( 43 ) 8863 - 8873 2002.10
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
We designed and synthesized a uracil-appended cholesterol gelator (1) in order to control the gel stability and the gel morphology by addition of the complementary and non-complementary nucleobase derivatives. Compound 1 forms columnar stacks in cyclohexane due to the van der Waals interaction (cholesterol-cholesterol interaction) and the intergelator hydrogen bonding between uracil moieties. Addition of a 'monomeric' adenosine (3) into the gel only decreases the stability with increasing the concentration. The destabilization is ascribed to a lack of intergelator hydrogen bonding accompanied with forming the complementary base pairs between 1 and 3. In contrast, addition of adenine-appended cholesterol (7) induces a different behavior; with increasing 7 concentration the mixed gel is initially stabilized and then destabilized, giving rise to a maximum at the ratio of 1/7= 1:1 for the T-gel plot. One may consider, therefore, that when the additive has a common, column-forming cholesterol moiety, the cholesterol-cholesterol interaction can operate cooperatively with the complementary base pairing. In addition, the gel fiber structure is clearly changed by the addition of 7. Taking the fact that there is no report for such an additive effect inducing a structural change with maintaining the gel stability into consideration, our attempt combining cholesterol columnar stacks with the nucleobase additives provides a new methodology to control the stability and the morphology of organogels. (C) 2002 Elsevier Science Ltd. All rights reserved.
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Polysaccharide-polynucleotide complexes VIII. Cation-induced complex formation between polyuridylic acid and schizophyllan
K Sakurai, R Iguchi, K Koumoto, T Kimura, M Mizu, Y Hisaeda, S Shinkai
BIOPOLYMERS 65 ( 1 ) 1 - 9 2002.10
Publisher：JOHN WILEY & SONS INC
Schizophyllan belongs to the beta-1,3-D-glucan family, and dissolves as a single chain in dimethylsulfoxide (DMSO) and as a triple helix in water, respectively. It is already known that when we prepare a mixture of poly(C) and schizophyllan in DMSO and subsequently exchange the solvent to water, poly(C) anti schizophyllan can form a complex. When we applied this procedure to the poly(U) system, we could not induce the complex formation between poly(U) and schizophyllan at all. However, we found that addition of some alkaline cations is necessary to induce the complexation between poly(U) and schizophyllan. The complex stability strongly depends on both the cation species and the salt concentration. The complexation is sensitively reflected in the CD spectrum. The magnitude of the spectral change is followed by the order; Rb+ much greater than K+ > Na+ ? Cs+. This cation
DOI： 10.1002/bip.10189
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Chemical modification of curdlan: Characteristic complexation with polynucleotides by control of the molecular weight.
K Koumoto, K Sakurai, S Shinkai, T Kunitake
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 224 ( 2 ) U429 - U429 2002.8
Publishing type：Research paper, summary (international conference) Publisher：AMER CHEMICAL SOC
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Novel DNA-polysaccharide triple helix and its application to a gene carrier.
K Sakurai, S Shinkai
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 224 ( 2 ) U476 - U477 2002.8
Publishing type：Research paper, summary (international conference) Publisher：AMER CHEMICAL SOC
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Molecular design of "super" hydrogelators: Understanding the gelation process of azobenzene-based sugar derivatives in water
H Kobayashi, A Friggeri, K Koumoto, M Amaike, S Shinkai, DN Reinhoudt
ORGANIC LETTERS 4 ( 9 ) 1423 - 1426 2002.5
Publisher：AMER CHEMICAL SOC
[GRAPHIC]
As an attempt to rationally design aqueous organogelators, a bolaamphiphilic azobenzene derivative (1) bearing two sugar groups was synthesized. Compound 1 formed a gel in water even at concentrations as low as 0.05 wt% (0.65 mM). Spectroscopic studies and electron-micrographic observations have clarified the gel structure and the origin of the gelation ability for water.DOI： 10.1021/ol025519+
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Polysaccharide-polynucleotide complexes. Part 12. Enhanced affinity for various polynucleotide chains by site-specific chemical modification of schizophyllan
K Koumoto, T Kimura, M Mizu, T Kunitake, K Sakurai, S Shinkai
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1 ( 22 ) 2477 - 2484 2002
Publisher：ROYAL SOC CHEMISTRY
Recently, we found that schizophyllan (SPG), which is a natural and neutral polysaccharide, can form macromolecular complexes with certain polynucleotides. In order to increase the stability and the affinity of the complexes, we synthesized a 2-aminoethanol-appended SPG (N-s-SPG) by utilizing periodate oxidation and reductive amination. Although the modi. cation level was only 2.4 +/- 0.3%, the melting temperature of the poly(C)-N-s-SPG complex was increased by 8 degreesC comparing with the poly(C)-s-SPG complex. In addition, the conformation of the complex was scarcely perturbed by the introduction of amino groups. When we formed complexes of N-s-SPG and other polynucleotides, the melting temperature of their complexes increased without exception. Moreover, the amino modification induced complexation with poly(U), which does not form a complex with SPG. The affinity for the short chain nucleotide was also enhanced. These results indicate that the present modi. cation method is quite useful to improve complex stability and we believe, therefore, that this strategy will make it possible to apply SPG to a novel gene carrier.
DOI： 10.1039/b207553a
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Sol-gel phase transition induced by fiber-vesicle structural changes in sugar-based bolaamphiphiles
H Kobayashi, K Koumoto, JH Jung, S Shinkai
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 ( 11 ) 1930 - 1936 2002
Publisher：ROYAL SOC CHEMISTRY
The research objects of this paper were to design bolaamphiphilic gelators utilizing a sugar family as a source of solvophilic groups and an azobenzene segment as a solvophobic group and to monitor the aggregation mode utilizing the spectroscopic properties of the azobenzene chromophore. The results indicated that the bolaamphiphiles act, although only for specific DMSO-water mixtures, as gelators and form a unique supramolecular helical structure in the gel phase. The UV- Vis and CD spectra showed that the azobenzene segments adopt H- type face- to- face orientation and the dipole moments are arranged in the right- handed (R)- helicity. Since the fibrils as observed by electron microscope possess the right- handed helical structure, one may consider that the microscopic azobenzene-azobenzene orientation is reflected by the macroscopic supramolecular structure. When boronic acid- appended poly( L-lysine) was added, the gel phase was changed into the sol phase in the macroscopic level and the fibrous aggregate was changed into the vesicular aggregate in the microscopic level. These changes, which are usually induced by a temperature change, are due to the specific boronic acid- sugar interaction occurring at the constant temperature. Interestingly, when -fructose which shows high affinity with the boronic acid group was added, the sol phase and the vesicular aggregate were changed back to the gel phase and the fibrous aggregate, respectively. This means that the phase and morphological changes in the sugar- integrated bolaamphiphiles can be controlled reversibly.
DOI： 10.1039/b204631h
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Chemical modification of curdlan to induce an interaction with poly(C)(1)
K Koumoto, T Kimura, H Kobayashi, K Sakurai, S Shinkai
CHEMISTRY LETTERS ( 9 ) 908 - 909 2001.9
Publisher：CHEMICAL SOC JAPAN
The molecular weight of the commercially available curdlan is over 10(6), which makes it difficult to bind to polynucleotides. This paper presents a chain-scission reaction of curdlan and the molecular weight dependence of the binding ability. We found that curdlan can bind to poly(C) when the molecular weight is less than about 10(5). However, the complex stability is still less than that of the single chain of schizophyllan.
DOI： 10.1246/cl.2001.908
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Organization of nucleosides supported by boronic-acid-appended poly(L-lysine): Creation of a novel RNA mimic
H Kobayashi, M Amaike, K Koumoto, S Shinkai
BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 74 ( 7 ) 1311 - 1317 2001.7
Publisher：CHEMICAL SOC JAPAN
Boronic-acid-appended poly(L-lysine) (1) can form anionic boronate esters with diol derivatives in aqueous solution. One can thus expect that when nucleosides are mixed with 1, the resultant complexes will satisfy the basic structural characteristics of RNA, viz., one-dimensionally organized nucleic bases and anionic charges. To obtain insights into this novel RNA mimic, the solution properties of adenosine (Ad) were estimated in the absence and the presence of 1. The large absorption and CD spectral changes were observed at pH 6-10 where the poly(L-lysine) main chain tends to adopt an a-helix-rich conformation. On the basis of the fact that the magnitude of the spectral changes is much larger than the fraction of complexed Ad molecules, the polymer-supported organization mode was proposed: that is, Ad molecules complexed by covalent bonds with I act as clusters to induce further organization of uncomplexed Ad molecules by noncovalent interactions. The proposal was further confirmed by H-1 NMR spectroscopy and dynamic light scattering, On the other hand, none of these spectral changes were observed for 2 ' -deoxyadenosine. The I-Ad complex thus formed can interact with complementary poly(U) but not with noncomplementary poly(C), indicating that it can act as a novel RNA mimic.
DOI： 10.1246/bcsj.74.1311
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Polysaccharide-polynucleotide complexes (IV): Antihydrolysis effect of the schizophyllan/poly(C) complex and the complex dissociation induced by amines
K Koumoto, T Kimura, K Sakurai, S Shinkai
BIOORGANIC CHEMISTRY 29 ( 3 ) 178 - 185 2001.6
Publisher：ACADEMIC PRESS INC
Polymer complexes formed by schizophyllan and poly(C) showed a unique antihydrolysis effect when poly(C) was subjected to hydrolysis under basic conditions. The complexation reduced the hydrolysis rate to 80% of the control (i.e., poly(C) itself). However, when we added oligoamines with the intention of catalyzing the hydrolysis, the oligoamines induced dissociation of the complex instead of acceleration of the hydrolysis. (C) 2001 Academic Press.
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TEM and SEM observations of super-structures constructed in organogel systems from a combination of boronic-acid-appended bola-amphiphiles with chiral diols
K Koumoto, T Yamashita, T Kimura, R Luboradzki, S Shinkai
NANOTECHNOLOGY 12 ( 1 ) 25 - 31 2001.3
Publisher：IOP PUBLISHING LTD
Two bola-amphiphilic alpha, omega -diboronic acids separated by a (CH(2))(11) or (CH(2))(12) group were synthesized. Complexation with chiral diols readily gave new amphiphiles end-capped with the chiral substituents. Some of these acted as good gelators of organic solvents. Transmission electron microscope and scanning electron microscope observations established that a variety of super-structures are created in the organogels, depending on the solvents and the structure of the chiral end-cap groups. In most cases, the fibrous aggregates, the network structure which is the driving-force for gelation, showed a helical higher-order structure reflecting the chirality of the end-cap groups. The results indicate that the combinatorial approach utilizing boronic acid functions and diol compounds is useful in creating a variety of new super-structures in the gel phase.
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A novel technique to specifically separate RNAs by Schizophyllan
Taro Kimira, Kazuya Koumoto, Masami Mizu, Ritsuko Kobayashi, Kazuo Sakurai, Seiji Shinkai
Nucleic Acids Res. Supplement 1 283 - 284 2001
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Application of schizophyllan as a novel gene carrier
Kazuo Sakurai, Taro Kimira, Kazuya Koumoto, Masami Mizu, Ritsuko Kobayashi, Seiji Shinkai
Nucleic Acids Res. Supplement 1 223 - 224 2001
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Thermally induced conformational-transition of polydeoxyadenosine in the complex with schizophyllan and the base-length dependence of its stability
M Mizu, T Kimura, K Koumoto, K Sakurai, S Shinkai
CHEMICAL COMMUNICATIONS ( 5 ) 429 - 430 2001
Publisher：ROYAL SOC CHEMISTRY
A single chain of schizophyllan, one of the beta -1,3-glucan family, can form a stoichiometric complex with poly(dA) and the poly(dA)'s conformation and the complex stability strongly depends on the base length.
DOI： 10.1039/b009943k
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Chemical modification of schizophyllan by introduction of a cationic charge into the side chain which enhances the thermal stability of schizophyllan–poly(C) complexes
Kazuya Koumoto, Taro Kimura, Masami Mizu, Kazuo Sakurai, Seiji Shinkai
Chemical Communications 1 ( 19 ) 1962 - 1963 2001
Combining oxidation with periodate and reductive amination, the amino group is introduced only in the side chain of schizophyllan and the product can bind to poly(C) with a higher dissociation temperature than that of the unmodified schizophyllan and poly(C) complex. © 2001 Royal Society of Chemistry.
DOI： 10.1039/b105609n
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Polysaccharide-polynucleotide complexes (III): A novel interaction between the beta-1,3-glucan family and the single-stranded RNA poly(C)
T Kimura, K Koumoto, K Sakurai, S Shinkai
CHEMISTRY LETTERS ( 11 ) 1242 - 1243 2000.11
Publisher：CHEMICAL SOC JAPAN
We measured circular dichroism spectra to examine the complex-formation capability of various kinds of polysaccharides with poly(C). The results show that schizophyllan and lentinan can interact with poly(C), on the other hand, amylose, pullulan, dextran, and curdlan cannot. This difference clearly indicates that the water soluble beta -1,3-glucan can bind with polynucleotides. The molecular weight dependence of the complex formation has established that at least 30 glucose sequence length is necessary to incorporate the most bases into the complex.
DOI： 10.1246/cl.2000.1242
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Colorimetric sugar sensing method useful in "neutral" aqueous media
K Koumoto, S Shinkai
CHEMISTRY LETTERS ( 8 ) 856 - 857 2000.8
Publisher：CHEMICAL SOC JAPAN
Azobenzene derivatives bearing one or two aminomethylphenylboronic acid groups were synthesized: they were confirmed to be useful for practical colorimetric saccharide sensing in "neutral" aqueous media.
DOI： 10.1246/cl.2000.856
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The first example of positive allosterism in an aqueous saccharide-binding system designed on a Ce(IV) bis(porphyrinate) double decker scaffold
A Sugasaki, M Ikeda, M Takeuchi, K Koumoto, S Shinkai
TETRAHEDRON 56 ( 27 ) 4717 - 4723 2000.6
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
The first example of a positive, homotropic system in aqueous saccharide-binding has been achieved using a Ce(IV) bis(porphyrinate) double decker scaffold bearing two pairs of boronic acid groups (compound 2). In this system, the binding of the first guest (1:2 saccharide/boronic acid complex) suppresses the rotational freedom of the two porphyrin planes, which facilitates the binding of the second guest. As a result, two pairs of boronic acid groups in 2 can autoacceleratively bind these guests and yield CD-active species. The analyses of CD intensity-guest concentration plots according to the Hill equation resulted in K=3.7x10(4) M-2 and n=2.0 for D-fructose and K=9.6x10(5) M-2 and n=1.6 for D-glucose. The present system which is in action even in aqueous media is widely applicable to allosteric control of drug release, catalytic reaction, information transduction, etc. of saccharide-containing guest molecules. (C) 2000 Elsevier Science Ltd. All rights reserved.
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Super-structures can be constructed in organogels from combination of a boronic-acid-appended bolaamphiphile with chiral diols
T Kimura, T Yamashita, K Koumoto, S Shinkai
TETRAHEDRON LETTERS 40 ( 36 ) 6631 - 6634 1999.9
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
A bolaamphiphile bearing two boronic acid groups at the two ends was synthesized. Complexation with chiral diols readily afforded new amphiphiles end-capped with the chiral substituents. Some of them acted as gelators of organic solvents. TEM and SEM observations established that a variety of super-structures are created in the organogels, depending on the solvents and the structure of the end-cap groups. (C) 1999 Elsevier Science Ltd. All rights reserved.
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Design of a visualized sugar sensing system utilizing a boronic acid azopyridine interaction
K Koumoto, M Takeuchi, S Shinkai
SUPRAMOLECULAR CHEMISTRY 9 ( 3 ) 203 - + 1998
Publisher：TAYLOR & FRANCIS LTD
The boronic acid-amine (B-N) interaction was applied to the molecular design of a visualized sensing system for saccharides. 3-Nitrophenyl-boronic acid (3b) interacts with the pyridine nitrogen of 4-(4-dimethylaminophenyl-azo) pyridine (2) in methanol and changes its color from yellow to orange. Added saccharides form complexes with 3b and enhance the acidity of the boronic acid group. As a result, the B-N interaction becomes stronger and the intensified intramolecular charge-transfer band changes the solution color to red. By combined (1)H-NMR and absorption spectroscopic studies, four association constants governing the present ternary complex system have been determined. In case of D-fructose, the 3b.D-fructose complex can interact with 2240 times more strongly than 3b and the 2.3b complex can associate D-fructose 241 times more strongly than 3b. Clearly, the B-N interaction not only enables the colorimetric saccharide sensing but also enhances the association ability (i.e., sensitivity). Thus, the present paper offers a novel basic concept for molecular design of visualized saccharide sensing systems.
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Efficient glucoside extraction mediated by a boronic acid with an intramolecular quaternary ammonium ion
M Takeuchi, K Koumoto, M Goto, S Shinkai
TETRAHEDRON 52 ( 40 ) 12931 - 12940 1996.9
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
To develop an efficient sugar extractant on the basis of the mechanistic view a phenylboronic acid bearing a trioctylammonium group at the ortho position (4) was synthesized. To avoid the complexity we employed a simple two-phase solvent-extraction system which corresponds to the first step in membrane transport, i.e., extraction from a donating aqueous phase to an organic liquid membrane phase. The extraction rates and equilibria were estimated using alpha-p-nitrophenyl-D-glucopyranoside as a sugar and 1,2-dichloroethane as an organic phase and compared with those of a 2-methylphenylboronic acid (5)/trioctylmethylammonium chloride (TOMAC) 1 : 1 binary system. The extraction rates for 4 were faster by 2.5 - 29 fold than those for 5 + TOMAC. The distribution coefficients were also enhanced by 5 - 8 fold. The results indicate that the intramolecular quaternary ammonium group is very effective to neutralize the anionic charge developed in the boron atom upon sugar-binding and create extractable zwitterionic sugar complexes. Copyright (C) 1996 Elsevier Science Ltd