Toshihisa Mizuno

Quinoxaline-oligopyrroles: Improved pyrrole-based anion receptorsElectronic supplementary information (ESI) available: synthetic details of 3 and 4, titration studies for anion binding of 3 and 4, and crystallographic details for 3. See http://www.rsc.org/suppdata/cc/b1/b111708d/

Novel quinoxaline derivatives bearing dipyrromethane or tripyrromethane substituents act as improved anion receptors as compared to the unsubstituted dipyrrolylquinoxaline core from which they are derived.

Fluorescence and CD spectroscopic sugar sensing by a cyanine-appended diboronic acid probe

Abstract A cyanine dye ( 4 ) bearing two boronic acids was designed and synthesized, expecting the selective binding of monosaccharides through the formation of 1:1 intramolecular complexes. While it aggregates in water, it exists discretely in water/methanol 1.1 mixed solvent. In the latter solvent the fluorescence spectra were scarcely affected by the medium pH but efficiently increased when it formed intramolecular 1:1 complexes with monosaccharides. This complexation mode was also corroborated by CD spectroscopy and continuous variation plots. Hence, the saccharideinduced fluorescence increase us rationalized in terms of “rigidification” of the cyanine skeleton. The association constants ( K ) were estimated from plots of saccharide concentration vs fluorescence intensity: the largest K was observed for D-fructose (1.3 x 10 5 M −1 ) and the next for D-arabinose (1.0 x 10 4 M −1 ) This is a novel system for sensitive and selective fluorescence detection of monosaccharides

Chirality sensing of saccharides using a boronic acid-appended chiral ferrocene derivative

Abstract A diboronic acid-appended chiral ferrocene derivative ( R )-9 was designed and synthesized. This chiral ferrocene scaffold was obtained by resolution of the diastereomer with a monosaccharide derivative. One can therefore expect that ( R )-9 would show d / l selectivity for specific monosaccharides. The complex formation of ( R )-9 with various saccharides using the two boronic acid functions was conveniently monitored by a change in the circular dichroism (CD) spectra. The CD spectral change (Δ[ θ ]) induced by added monosaccharides was chiroselective: in particular, d / l -alloses and d / l -galactoses induced the 8.0- and 7.0-fold difference in the magnitude of the CD spectral change. The association constants for d - and l -saccharides ( K D and K L , respectively) were determined: among them, ( R )-9 showed a significant discrimination ability for mannose ( K L / K D =2.6) and arabinose ( K L / K D =1/2.4). The origin of d / l selectivity was discussed on the basis of computational studies on ( R )-9 ·saccharide complexes.

Sugar sensing using chiral salen-Co(II) complexes

Abstract Two chiral salen-Co(II) complexes, (R)- 7 and (R)- 8 bearing two boronic acid groups were synthesized. The saccharide-binding event was conveniently minitored by a spectral change in UV-vis absorption spectroscopy arising from saccharide-boronic acids complexation. Since the distance between the two boronic acid groups is shorter than other diboronic acid-based receptors, the saccharide selectivity is quite different from other systems: the largest association constant was observed for fructose and the next for talose. In addition, chiral recognition was achieved for certain saccharides: the largest discrimination was 2.1 observed for (R)- 7 with d/l -allose. These results indicate that the salen-metal complexes provide an excellent basic skeleton for designing the chiral sugar sensing systems.

Construction of a pH-Responsive Artificial Membrane Fusion System by Using Designed Coiled-Coil Polypeptides

In many viruses, pH-responsive coiled-coil domains in the specific fusion proteins play important roles in membrane fusion and the infection of viruses into host cells. To investigate the relationship between the conformational change of the coiled coil and the fusion process, we have introduced a de novo designed polypeptide as a model system of the coiled-coil domain. This system enables the systematic study of the dynamics of pH-responsive coiled-coil polypeptide-membrane interactions. First, we designed and synthesized pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptides. Then the relationship between the pH-induced conformational change of the polypeptide and the membranes interactive properties was studied by physicochemical methods. Structural changes in the designed polypeptides were examined by means of circular dichroism measurements. And finally, the behavior of the membrane fusion was investigated by leakage of liposomal contents, turbidity analysis, dynamic light scattering, and lipid mixing experiments. Our data show that coiled-coil formation under acidic pH conditions enhances polypeptide-induced membrane fusion. The results in this study demonstrate that an artificial membrane fusion system can be constructed on a molecular level by the use of a pH-responsive isoleucine-zipper triple-stranded coiled-coil polypeptide.

Target-selective vesicle fusion system with pH-selectivity and responsiveness

The present paper reports on induction of target-selective liposomal vesicle fusion triggered by molecular recognition on a vesicle surface. Phosphatidylinositol (PI) having a sugar-like cyclic cys-diol structure was selected as a recognition target. Since diol sugars are abundant on cell surfaces, vesicle fusion systems based on the recognition of diol functionalities can be relevant for liposome-based drug delivery. Here, we design and synthesize a novel phenylboronic acid derivative with a tertiary amine group adjacent to the boron atom as a pilot molecule toward PI at a physiological condition. The pilot vesicle, or EggPC liposome containing this phenylboronic acid derivative causes selective membrane fusion toward a target liposomal vesicle containing sugar-like cyclic cys-diol structure at a physiological condition. From lipid mixing and inner-leaflet mixing assays, we demonstrate that the fusion event activated by inter-vesicular complex formation occurs rapidly. Furthermore, we also construct the target selective fusion system working only at the endosomal pH by the use of weakly acidic lipid, 1,2-dipalmitoyl-sn-glycero-3-succinate (DPGS) containing the pilot vesicle. The lipid mixing and inner-leaflet mixing assays make it clear that the vesicle fusion proceeds over pH range 5.0–5.5, whose range is upper than the pKa of the boronic acid moiety on cys-diol complexation and lower than the pKa of the carboxyl group of DPGS.

Sugar ‘Chirality’ Sensing Using a ‘Prochiral’ Salen–Co(II) Complex

Abstract A prochiral salen–Co(II) complex ( 6 ) bearing two boronic acid groups was synthesized. The saccharide-binding event was conveniently monitored by a circular dichroism (CD) spectral change. The exciton-coupling-type CD spectra thus obtained were compared with those of chiral salen–Co(II) complexes, ( R )- 4 and ( R )- 5 . Very interestingly, monosaccharides which are selectively bound to ( R )- 4 and ( R )- 5 with P helicity generate the P helicity CD spectra in prochiral 6 . This means that the two boronic acid groups in ( R )- 4 and ( R )- 5 are chirally preorganized suitable to the binding of these monosaccharides. Reflecting the short distance between the two boronic acid groups, 6 shows the very high selectivity for talose which has all OH groups arranged on the same side of the pyranose ring. These results indicate that 6 is useful for sugar ‘chirality’ sensing at visible wavelength region and prediction of d / l selectivity for ( R )- 4 and ( R )- 5 .

Molecular assembly of zinc chlorophyll derivatives by using recombinant light-harvesting polypeptides with His-tag and immobilization on a gold electrode.

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

The effect of the side chain length of Asp and Glu on coordination structure of Cu2+ in a de novo designed protein

Metal ions in proteins are important not only for the formation of the proper structures but also for various biological activities. For biological functions such as hydrolysis and oxidation, metal ions often adopt unusual coordination structures. We constructed a stable scaffold for metal binding to create distorted metal coordination structures. A stable four stranded alpha-helical coiled-coil structure was used as the scaffold, and the metal binding site was in the cavity created at the center of the structure. Two His residues and one Asp or Glu residue were used to coordinate the metal ions, AM2D and AM2E, respectively. Cu(2+) bound to AM2D with an equatorial planar coordination structure with two His, one Asp, and H(2)O as detected by electron spin resonance and UV spectral analyzes. On the other hand, Cu(2+) had a slightly distorted square planar structure when it bound two His and Glu in AM2E, due to the longer side-chain of the Glu residue as compared to the Asp residue. Computational analysis also supported the distorted coordination structure of Cu(2+) in AM2E. This construct should be useful to create various coordinations of metal ions for catalytic functions.

Molecular assembly of covalently-linked mesoporphyrin dimers with light-harvesting polypeptides

Abstract Light-harvesting (LH)-α and -β polypeptides separately isolated from photosynthetic bacteria, R. rubrum , organized mesoporphyrin dimers ( Scheme 1 ) in the n -octyl-β- d -glucopyranoside (OG) micelle, depending upon the porphyrin structure and temperature. An efficient energy transfer from Zn porphyrin to Ni porphyrin in 7 due to the presence of the LH polypeptides was observed.