Review Papers (Misc) - KOUMOTO Kazuya
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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