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Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3',5'-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.

Generally, cage-shaped hosts for saccharides can bind strongly to guest molecules because of the three-dimensional preorganized hydrogen-bonding sites. However, the preparation of cage molecules is often difficult because of the low yield of the macrocyclization step. Here, we report a three-arm-shaped molecule possessing pyridine-acetylene-phenol units as a new kind of host having a preorganized three-dimensional hydrogen-bonding site. This three-arm-shaped host was readily prepared compared to a cage-shaped analogue. This host associated with lipophilic glycosides to form chiral complexes, and the association constants were sufficiently high as to be comparable to those of the cage-shaped analogue. Furthermore, this host extracted native monosaccharides into a lipophilic solvent.

In order to create new functions of foldamer-type hosts, various kinds of recognition arrays are expected to be developed. Here, a pyridine-acetylene-aniline unit is presented as a new class of a saccharide recognition array. The conformational stabilities of this array were analyzed by DFT calculation, and suggested that a pyridine-acetylene-aniline oligomer tends to form a helical structure. An oligomer of this array was synthesized, and its association for octyl β-D-glucopyranoside was confirmed by 1 H NMR measurements. UV/Vis, circular dichroism, and fluorescence titration experiments revealed its high affinity for octyl glycosides in apolar solvents (Ka =104 to 105 M-1 ). This oligomer was relatively stable under basic conditions, and therefore this array was expected to be applied to the derivatization of saccharides. A 4-(dialkylamino)pyridine attached pyridine-acetylene-aniline oligomer proved to catalyze the acylation of the octyl glucoside.

A macrocycle consisting of six ethynylphenol units was developed as a host architecture for saccharides. The rigid framework of the macrocycle suppressed the intramolecular hydrogen-bonding between adjacent phenolic hydroxy groups and recognized saccharides by intermolecular hydrogen-bonding within the hole. The well-defined hydrogen-bonding sites enabled the size-selective guest recognition and showed preference to pentoses over hexoses.

Halogenated 2-aminopyridine was attached to the acetylene terminal of ethynyl C-2-deoxy-β-d-ribofuranoside as a nucleobase substitute, and then, the C-nucleoside was incorporated into natural DNAs. The resulting chimeric DNA constructed double helical structures with the complementary chimeric DNA. In the duplex, 2-aminopyridine functioned as an adenine analogue that formed a base pair with a non-natural thymine isostere. Artificial homooligomers were also prepared only from the adenine-type C-nucleoside and proven to form completely artificial double helices with the corresponding artificial thymine-type homooligomers.

A chiral 1,1'-bi-2-naphthol (BINOL)-containing pyridine-acetylene-phenol macrocycle and a pyridine-BINOL alternating macrocycle were developed for enantioselective recognition of saccharides. In solid-liquid extraction of native saccharides, these macrocycles selectively extracted one enantiomer from each of a racemic mixture of d/l-fructose, d/l-glucose, and d/l-mannose into a lipophilic solvent. This is the first example in which native saccharides, especially such important hexoses, were enantioselectively extracted with artificial host molecules.

We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding-from a viewpoint of hydrogen-bonding patterns-to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5'-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5'-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3'-natural⋅5'-artificial, 3'-artificial⋅5'-natural, and 3'-artificial⋅5'-artificial terminal combination at the nicks.

Halogenated 2-aminopyridine was attached to the acetylene terminal of ethynyl

The blood-to-retina supply of cyanocobalamin (vitamin B12) across the blood–retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine, and chloroquine and was also significantly reduced under Ca2+-free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluore...

A nonplanar macrocycle consisting of four pyridine–acetylene–phenol units was developed as a host for saccharide guest molecules. The macrocycle was found to strongly associate with a lipophilic maltose derivative, with an association constant of 107 M–1, over monosaccharide derivatives, for which much smaller association constants were determined, ranging from 103 M–1 to 104 M–1. The macrocycle was found to adopt a boat-like conformation, encapsulating β-d-maltoside in a twisted manner through approximately seven intermolecular hydrogen bonds.

We designed and synthesized two new types of crosslinked helical peptides that interact with the target DNA. One of them was crosslinked with a conventional isophthalic acid-based agent and was modified with α-lipoic acid at the N-terminus. The other peptide was crosslinked with a novel isophthalic acid-based agent containing α-lipoic acid at its own 5-position. Quartz crystal microbalance analyses showed that both the helical peptides strongly bound to their target DNAs on the Au surface.

Coordination cages were composed by self-organization of rigid C3v-symmetric heptaarene tridentates and Pd(II) precursors. The heptaarene framework involves one mesitylene, three phenol, and three pyridine moieties, which were connected by Suzuki coupling reactions. The treatment of the tridentates with Pd(dppp)(OTf)2 or Pd(en)(NO3)2 in a 2:3 molar ratio furnished coordination cages, which was ascertained by crystallography, 1H NMR and DOSY measurements, and ESI-TOFMS and UV–vis spectra. The cages have six phenolic hydroxy groups inside and were expected to incorporate hydrogen-bonding guest molecules such as saccharides. CD and DOSY measurements showed that octyl hexoside guests could be incorporated into the cage.

An amphiphilic pyridine-phenol alternating oligomer, in which oligo(ethylene glycol) chains are attached at the 4-positions of the pyridine and the phenol rings, was synthesized as a helical host to realize saccharide recognition in water. This oligomer was soluble not only in less-polar solvents but also in water at room temperature. Circular dichroism investigations revealed that the oligomer could recognize several kinds of saccharides and their derivatives including glycodrugs to form chiral higher-order structures in water. Especially, the oligomer showed high affinity for D-glucosamine, and their association constants were over 10³ M¯¹.

The helical structures of m-ethynylpyridine (2,6-pyridyleneethynylene) oligomers have been stabilized by mechanical crosslinking. A heptameric oligomer, in which the two terminal pyridine rings bear two carbaldehyde groups, has been designed. When this heptamer was treated with octyl β-d-glucopyranoside, a circular dichroism (CD) band was induced at around 330 nm, which corresponds to the formation of a chiral ethynylpyridine helix. The CD band was enhanced by the addition of a diamine and a diammonium dumbbell as a result of the formation of a [3]rotaxane structure linked to the terminal pyridines of the helical structure. A decameric oligomer containing four diformylarene units showed stronger CD enhancement in similar experiments owing to two crosslinking events.

We attached D-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The D-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-XL. The D-Ala attached helical peptides were much more stable in cells than original ones and efficiently induced apoptosis of the cells.

Hexagonal shape-persistent macrocycles (SPMs) consisting of three pyridine and three phenol rings linked with acetylene bonds were developed as a preorganized host for saccharide recognition by push-pull-type hydrogen bonding. Three tert-butyl or 2,4,6-triisopropylphenyl substituents were introduced on the host to suppress self-aggregation by steric hindrance. In spite of the simple architecture, association constants Ka of the host with alkyl glycoside guests reached the order of 106 m-1 on the basis of UV/Vis titration experiments. This glycoside recognition was much stronger than that in the cases of acyclic equivalent hosts because of the entropic advantage brought by preorganization of the hydrogen-bonding sites. Solid-liquid extraction and liquid-liquid transport through a liquid membrane were demonstrated by using native saccharides, and much preference to mannose was observed.

A shape-persistent macrocycle (SPM) consisted of three 2,6-pyridylene and three 2,6-pyrazylene rings alternately linked with acetylene bonds was prepared by Sonogashira reactions. X-ray crystallography revealed the planar macrocyclic structure, and intermolecular π-stacking was observed at the sides of the hexagon. An AFM measurement of the sample prepared by drop-casting on a mica substrate showed a reticulate fibril.

Recently, many reports have emerged about circularly polarized luminescence (CPL) based on excimer emission of pyrenes. The intense CPL was observed from various pyrene derivatives such as pyrenes having chiral side chains, chiral oligomers bearing multiple pyrene rings, and pyrenes encapsulated by γ-cyclodextrins. The luminescence dissymmetry factor was found to be obviously higher than the absorption one in those pyrenes. In addition, several pyrenes revealed reversible “on-off” CPL switching upon the complexation/decomplexation of metal ions.

The blood-to-retina supply of cyanocobalamin (vitamin B

Phenol-based oligomers linked with acetylenes at their meta positions, " meta"-ethynylphenol oligomers, were developed as a synthetic helical foldamer. The architecturally simple oligomers spontaneously formed helical higher-order structures by sequential intramolecular hydrogen bonds through the multiple phenolic hydroxy groups inside the cavities. The hydrogen bonds forced C-C≡C-C bond angles to largely bend toward the inside. Addition of chiral amines caused the helices to be chiral by electrostatic interactions between the resulting chiral ammonium cations and the phenolate anions.

Coordination cages were composed by self-organization of rigid C

An amphiphilic meta-ethynylpyridine polymer with chiral amide side chains was developed. The polymer was prepared by sequential Sonogashira reactions, and the product was soluble in polar and apolar solvents. The additive effects of metal salts on the polymer were examined in water and aqueous EtOH on the basis of UV-vis and CD spectra. The enhancement of the positive Cotton effect and hypochromism around 360 nm occurred by the addition of various metal salts, indicating the coordination of the cations to the amide side chains of the polymer to stabilise the helical structure. Among them, rare-earth metal salts, especially Sc(OTf)3 showed more efficient additive effects probably because of its strong coordination ability even in water. Positive cooperativity was observed for the coordination of Sc(OTf)3 to the polymer in aqueous EtOH.

We attached d-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The d-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-X

The Sonogashira coupling of γ-CD-encapsulated alkynylpyrenes with terphenyl-type stopper molecules gave a doubly alkynylpyrene-threaded [4]rotaxane. The rotaxane showed only excimer emission, with a high fluorescence quantum yield of Φf =0.37, arising from the spatially restricted excimer within the cavity of the γ-CD. The excimer emission suffered little from self-quenching up to a concentration of 1.5×10(-5) M and was circularly polarized with a high glum value of -1.5×10(-2) . The strong circularly polarized luminescence may result from the two stacked pyrenes existing in the rotaxane in an asymmetrically twisted manner.

Macrocyclic compounds consisting of three 2,6-pyridylene and three 3,5-pyridylene units linked by acetylene bonds were synthesized by a Sonogashira reaction. The X-ray structures showed π-stacked pairs of two macrocycles, in which a 2,6-pyridylene unit of the one molecule overlaps a 3,5-pyridylene of the other molecule because of dipole-dipole interaction. Atomic force microscope (AFM) measurements revealed fibril structures indicating the stacking of the rigid planar macrocycles. Hydrogen-bonding ability of the macrocyclic inside was demonstrated by the addition of octyl β-D-glucopyranoside.

We synthesized novel photochromic non-natural amino acids based on diarylethene skeletons. The non-natural amino acids can be introduced at any positions in various types of peptides by solid/solution phase synthesis. In this study, linear and cyclic peptides including the diarylethene residues were prepared. Their peptides showed photochromism peculiar to original diarylethenes.

The chain length dependence involving a chiral memory effect of 2,6-pyridylene ethynylene oligomers ‘ meta -ethynylpyridines’ was investigated. The meta -ethynylpyridine oligomers associated with octyl β- d -glucopyranoside form a helical complex giving induced CD; the induced chirality could be memorized with the help of Cu(OTf) 2 as an additive. Contrary to our expectations, the study of the length dependence revealed that the 12-meric oligomer showed a more sustainable chiral memory effect than those observed in both of the shorter 6-meric and longer 18-meric oligomers.

Tetrameric and octameric 2,6-pyridylene ethynylene oligomers linked to a β-D-glucopyranoside template through an o-phenylene linker were prepared and studied for their higher-order structure. These oligomers formed chiral helical structures through intramolecular hydrogen bonding between the ethynylpyridine moiety and the glucoside template. The rigidity of the o-phenylene linker stabilizes the helical structure to improve its CD activity and resistance against protic surroundings. Furthermore, the helical stabilization was enhanced by the addition of Cu(OTf)2 and Zn(NO3)2 salts.

A large-scale synthesis was carried out for alkynyl C-nucleotide oligomers in order to clarify the structural details of the artificial DNA. A liquid-phase synthesis by means of phosphoramidite methodology enabled us to handle ∼0.1 g of the DNA oligomers possessing a pseudouracil derivative (T*) as a non-natural nucleobase. 1H NMR measurements in aqueous media were carried out for the oligomers, succeeded in the accurate assignments of the protons accompanying with determination of the coupling constants (J values). These J values revealed that average conformation of the alkynylribose rings in the DNA was substantially biased toward the S-type conformers (2T1/E1/OT1). X-ray crystal analysis of the non-natural nucleoside also supported the S-type conformation (2E/2T1) as seen in natural B-DNA.

Hexagonal shape-persistent macrocycles (SPMs) consisting of three pyridine and three phenol rings linked with acetylene bonds were developed as a preorganized host for saccharide recognition by push-pull-type hydrogen bonding. Three tert-butyl or 2,4,6-triisopropylphenyl substituents were introduced on the host to suppress self-aggregation by steric hindrance. In spite of the simple architecture, association constants K

Encapsulation of highly emissive alkynylpyrenes with permethylated α-cyclodextrin (PM-α-CD) followed by capping reaction yielded alkynylpyrene-based [3]rotaxanes. The [3]rotaxane emitted only blue light of monomeric pyrene under various circumstances such as lipophilic, hydrophilic, and even condensed states and exhibited extremely high stability for UV irradiation. These properties would result because PM-α-CD, like bulletproof glass, protected the alkynylpyrene core from the attack of another excited alkynylpyrene and singlet oxygen generated by the energy transfer from the excited alkynylpyrene.

A tricationic shape-persistent macrocycle was obtained by methylation on the nitrogen atoms of the three 3,5-pyridylene groups of an alternating 2,6-/3,5-substituted ethynylpyridine macrocycle. The tricationic macrocycle recognized melamine in polar solvents such as DMSO and water, and the host–guest association in water induced a higher-order aggregate confirmed by UV–vis titration and dynamic light scattering experiments. Scanning electron microscopy, transmission electron microscopy, and atomic force microscopy indicated that fibrous network structures resulted from the stacking of the macrocycle and melamine complex.

Diarylethene-bridged pep- tides were developed to photoregulate biomolecular interactions. The peptides are made up of diarylethene-bridged and DNA-binding regions at their N- and C termini, respectively. The two re- gions could be independently designed and combined as desired. The a-helici- ties of the peptides were photoregulat- ed in on/off or off/on manners, and the manner depended on the positions of two ornithine (Orn) residues for cross- linking reaction at the diarylethene- bridged region. In the case of the on/ off manner, when the diarylethene structure adopted the open form on the peptides, the peptides folded into stable a-helices. Upon UV irradiation, the diarylethene moiety isomerized to its closed form to destabilize the helical structures. Quartz crystal microbalance (QCM) analysis showed that the open isomer strongly associated with a target DNA, as compared with the closed one. When the closed-form peptide ex- isting in the DNA complex was irradi- ated with a fluorescent lamp in the middle of the QCM monitoring, the frequency change (DF) was enhanced by the diarylethene photoisomeriza- tion.

A variety of fluorophores were introduced at the N-termini of short peptides for use as biological-probes. The fluorescent peptides were cross-linked with a diacetylenic cross-linking agent between the amino acid side chains of ornithine (Orn) residues to produce peptides with high helix content.

We describe syntheses and electrochemical properties of a new class of aminopyrimidinone derivatives: 2-amino-4-(3′,4′-dimethoxyphenyl)-6-pyrimidinone, 2-amino-4-(3′,4′-dihydroxyphenyl)-6-pyrimidinone, 2,4-diamino-5-(3′,4′-dimethoxyphenyl)-6-pyrimidinone, 2,4-diamino-5-(3′,4′-dihydroxyphenyl)-6-pyrimidinone, and 4-amino-5-(3′,4′-dihydroxyphenyl)-2,6-pyrimidinione, three of which possess a catechol unit as an oxidation-active moiety for developing electrochemically detectable abasic-site binders. These compounds were synthesized via a pyrimidine-ring forming reaction with guanidine. Cyclic voltammetry measurements revealed that the catechol-bearing derivatives showed oxidation potentials lower than that of DNA, indicating that they satisfied the requirements for the purpose.

Based on ‘click chemistry’, the new convergent synthesis to a variety of saccharide-linked 2,6-pyridylene ethynylene ‘ethynylpyridine’ foldamers was developed. Ethynylpyridine 6-, 9-, and 12-meric blocks were linked with 1,7-octadiyne linker block by Sonogashira reaction, and joined with azido group-introduced glucoside, galactoside, and mannoside templates by Huisgen reaction. The resulting saccharide-linked ethynylpyridine foldamers exhibited typical circular dichroism to indicate the formation of a helical structure by intramolecular hydrogen bonds.

We studied the preparation and circular dichroism (CD) behavior of 2,6-pyridylene ethynylene octameric oligomer linked to a β-d-glucoside moiety with a C20-alkylene chain. The addition of Cu(OTf)(2) salt led to a remarkable enhancement of the first CD band of the oligomer, and an unexpected concentration and time dependency were observed. For example, when 1.0 × 10(-3) M of Cu(OTf)(2) was added to a 1,2-dichloroethane solution of the oligomer (5.0 × 10(-4) M, unit concentration), the CD band appeared in the positive at first and gradually inverted into the negative with time.

We report bending flexibility of damaged duplexes possessing an apurinic/apyrimidinic (AP) site analogue, a cyclobutane pyrimidine dimer (CPD), and a pyrimidine(6-4)pyrimidone photoproduct (6-4PP). Based on the electrochemical evaluation on electrodes, the duplex flexibilities of the lesions increased in the following order: CPD < AP < 6-4PP. We discussed the possibility that the emerging local flexibility might be a good sign for UV-damaged DNA-binding proteins on duplexes.

We explored the effect of α-helical stabilization upon the binding of short peptides to DNAs. The short peptides were designed according to the binding domains of DNA-binding proteins and were cross-linked between their side chains with diacetylenic or isophthalic cross-linking agents to keep stable α-helices. The binding abilities of the peptides to DNAs were evaluated by fluorescence resonance energy transfer analysis. When a cross-linked peptide based on the homeodomain of the transcription factor was titrated with a target DNA duplex, its dissociation constant (Kd) was calculated to be ~0.5 nM. This value was the double-digit smaller than that of the corresponding non-cross-linked peptide. The cross-linked peptide showed high substrate specificity for DNAs at the same level as the original DNA-binding protein.

We prepared a series of 1,3,6,8-tetrakis(arylethynyl)pyrenes with donor or acceptor substituents and investigated their photophysical properties. Solvent polarity hardly affected on the UV/vis spectra of all of the tetrakis(arylethynyl)pyrenes used in this study. On the other hand, electron-donating groups, NMe 2 and NPh 2 groups imparted fluorescence solvatochromicity to the skeleton by intramolecular charge transfer in the excited state. Furthermore, a tetrakis(arylethynyl)pyrene showed better lightfastness upon exposure to laboratory weathering using a xenon long arc lamp than coumarin.

Co-oligomers involving (1H)-4-pyridone and 4-alkoxypyridine rings were studied, and it was found that their supramolecular transformation was caused by saccharide recognition. In the co-oligomers, pyridone and pyridine rings are alternately linked at their 2,6-position with an acetylene bond. The pyridine rings behave as a hydrogen bonding acceptor, and the pyridone rings and tautomerized 4-pyridinol work as a donor. Pyridine-pyridone-pyridine 3-mer was found to self-dimerize on the basis of vapor pressure osmometry in CHCl(3), and the association constant was obtained as 2.3 x 10(3) M(-1) by (1)H NMR titration. Longer 5-, 7-, 9-, and 11-mer oligomers showed considerable broadening and anisotropy in the (1)H NMR spectra due to self-association. These longer oligomers recognized octyl beta-D-glucopyranoside and changed their form into a chiral helical complex, showing characteristic circular dichroism.