Heterodimerization of dye-modified cyclodextrins with native cyclodextrins

The heterodimerization behavior of dye-modified beta-cyclodextrins (1-6) with native cyclodextrins (CDs) was investigated by means of absorption and induced circular dichroism spectroscopy in an aqueous solution. Three types of azo dye-modified beta-CDs (1-3) show different association behaviors, depending on the positional difference and the electronic character of substituent connected to the CD unit in the dye moiety. p-Methyl red-modified beta-CD (1), which has a 4-(dimethylamino)azobenzene moiety connected to the CD unit at the 4' position by an amido linkage, forms an intramolecular self-complex, inserting the dye moiety in its beta-CD cavity. It also associates with the native alpha-CD by inserting the moiety of 1 into the alpha-CD cavity. The association constants for such heterodimerization are 198 M(-1) at pH 1.00 and 305 M(-1) at pH 6.59, which are larger than the association constant of 1 for beta-CD (43 M(-1) at pH 1.00). Methyl red-modified 2, which has the same dye moiety as that for 1 although its substituent position is different from that of 1, does not associate even with alpha-CD due to the stable self-intramolecular complex, in which the dye moiety is deeply included in its own cavity of beta-CD. Alizarin yellow-modified CD (3), which has an azo dye moiety different from that of 1 and 2, caused a slight spectral variation upon addition of alpha-CD, suggesting that the interaction between 3 and alpha-CD is weak. On the other hand, phenolphthalein-modified beta-CD (4), which forms an intermolecular association complex in its higher concentrations, binds with beta-CD with an association constant of 787 M(-1) at pH 10.80, where 4 exists as the dianion monomer in the absence of beta-CD. p-Nitorophenol-modified beta-CDs (5 and 6), each having p-nitorophenol moieties with a different connecting part with an amido and amidophenyl group, respectively, associated with alpha-CD with association constants of 66 and 16 M(-1) for 5 and 6, respectively. The phenyl unit in the connecting part of 6 may prevent the smooth binding with alpha-CD. All these results suggest that the dye-modified CDs, in which the dye part is not tightly included in its CD cavity, associate with the native CD to form heterodimer composed of two different CD units by inserting the dye moiety into the native CD unit. The resulting heterodimers have a cavity that can bind another appending moiety of host molecules. On this basis, more ordered molecular arrays or the supramolecular hereropolymers can be constructed.     

Complexation of tauro- and glyco-conjugated bile salts with three neutral beta-CDs studied by ACE

Complexation of the bile salts (BS) taurocholate, tauro-beta-muricholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, glycodeoxycholate, and glycochenodeoxycholate common in rat, dog, and man with natural beta-CD and the chemically modified beta-CDs 2-hydroxypropyl-beta-CD and 2-O-methyl-beta-CD was studied using mobility shift ACE. The CDs were selected due to their frequent use in preformulation and drug formulation as oral excipients for the solubilization of drug substances with low aqueous solubility. ACE was demonstrated to be a feasible and efficient technique for investigation of the interactions between BS and beta-CDs. All the investigated BS possessed affinity for the three CDs with stability constants ranging from 2x10(3) to 4x10(5) M(-) (1). The requirements and assumptions related to the use of ACE for estimating high affinity stability constants were discussed. The extent and pattern of hydroxylation significantly influenced the affinity of the glyco- and tauro-conjugated BS toward the beta-CDs (chenodeoxycholates > deoxycholates > cholates) whereas the nature of the beta-CD derivatization and BS conjugation played a minor role only. The results indicate that displacement of drug substances from beta-CD inclusion complexes is likely to occur in the small intestine where BS are present potentially influencing drug bioavailability.     

Evaluation of norbornene- beta-cyclodextrin-based monomers and oligomers as chiral selectors by means of nonaqueous capillary electrophoresis

Norbornen-5-yl carboxylic acid and norbornen-5-ylmethylsilyl ether-based beta-cyclodextrins (beta-CDs) containing up to three norbornene ester and up to five norbornene silyl ether units have been prepared from beta-CD and norbornen-5-carboxylic chloride and norbornen-5-ylmethyldichlorosilane, respectively. Oligomers (n = 2-4) were prepared therefrom using ring-opening metathesis polymerization (ROMP). Monomeric and oligomeric substituted beta-CDs were evaluated as chiral selectors in nonaqueous capillary zone electrophoresis using 35 mM sodium bicarbonate in N-methylformamide (NMF) as background electrolyte. Both monomeric and oligomeric norbornene ester- and norbornene silyl ether-type selectors showed good enantioresolution for dansylated (DNS-) amino acids using concentrations of the chiral selector of up to 4% w/v. A significant improvement in resolution was observed upon the introduction of up to five norbornene silyl ether units into a beta-CD molecule, whereas higher degrees of substitution with norbornen-5-yl-carboxyl groups lead to a reduction in enantioresolution of DNS-amino acids. Thus, pentakis(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD turned out to be superior to mono(norbornen-5-ylmethylhydroxysiloxyl)-beta-CD in terms of enantioresolution. Moreover, norbornene silyl ether-type selectors were found to be more efficient than norbornene ester-type selectors. Finally, oligomeric selectors were found to possess superior or at least comparable enantioselectivity in the separation of DNS-amino acids compared to the parent monomers. A maximum in enantioresolution was obtained with oligo(pentakis(norbornen-5-ylmethylhydroxysiloxyl)beta-CD).     

Separation and migration behavior of structurally related phenothiazines in cyclodextrin-modified capillary zone electrophoresis

The influences of buffer pH and the concentration of beta-cyclodextrins (beta-CDs) on the separation and migration behavior of 13 structurally related phenothiazines in CD-modified capillary zone electrophoresis (CD-CZE) using a phosphate background electrolyte at low pH were investigated. We focused on the separation of these phenothiazines, including the enantiomers of chiral analytes, with the use of beta-CD and hydroxypropyl-beta-CD (HP-beta-CD) as electrolyte modifiers or chiral selectors at concentrations less than 8 mM. The results indicate that the interactions of phenothiazines with beta-CDs are very strong and that effective separations of 13 analytes can be achieved with addition of 0.3 mM beta-CD or 0.5 mM HP-beta-CD in a phosphate buffer at pH 3.0. Binding constants of phenothiazines to beta-CDs were evaluated for a better understanding of the interactions of phenothiazines with beta-CDs.     

Synthesis of bridged and metallobridged bis(beta-cyclodextrin)s containing fluorescent oxamidobisbenzoyl linkers and their selective binding towards bile salts

A series of beta-cyclodextrin (beta-CD) dimers containing fluorescent 2,2'-oxamidobisbenzoyl and 4,4'-oxamidobisbenzoyl linkers--that is, 6,6'-[2,2'-oxamidobis(benzoylamino)]ethyleneamino-6,6'-deoxy-bis(beta-CD) (2), 6,6'-[2,2'-oxamidobis(benzoylamino)]diethylenediamino-6,6'-deoxy-bis(beta-CD) (3), 6,6'-[4,4'-oxamidobis(benzoylamino)]ethyleneamino-6,6'-deoxy-bis(beta-CD) (4), and 6,6'-[4,4'-oxamidobis(benzoylamino)]diethylenediamino-6,6'-deoxy- bis(beta-CD) (5)--were synthesized from the corresponding oxamidobis(benzoic acid)s through treatment with mono[6-aminoethyleneamino-6-deoxy]-beta-CD or mono[6-diethylenetriamino-6-deoxy]-beta-CD. Further treatment of 2-5 with copper perchlorate gave their Cu(II) complexes 6-9 in satisfactory yields. The conformation and binding behavior of 2-9 towards two bile salt guests--sodium cholate (CA) and sodium deoxycholate (DCA)--was comprehensively investigated by circular dichroism, 2D NMR spectroscopy, and fluorescence spectroscopy in Tris-HCl buffer solution (pH 7.2) at 25 degrees C. Thanks to the cooperative host-linker-guest binding mode, the stoichiometric 1:1 complexes formed by bis(beta-CD)s 2-5 with bile salts gave high stability constants (KS values) of up to 10(3)-10(4) M(-1). Significantly, benefiting from the intramolecular 1:2 or 2:4 binding stoichiometry, the resulting complexes of metallobis(beta-CD)s 6-9 with bile salts gave much higher KS values of up to 10(6)-10(7) M(-2). The enhanced binding abilities of bis(beta-CD)s and metallobridged bis(beta-CD)s are discussed from the viewpoints of induced-fit interactions and multiple recognition between host and guest.     

Organic anion recognition of naphthalenesulfonates by steroid-modified beta-cyclodextrins: enhanced molecular binding ability and molecular selectivity

Two beta-cyclodextrin (beta-CD) derivatives bearing steroid groups (1 and 2) were synthesized by the condensation of mono(6-aminoethylamino-6-deoxy)-beta-CD with cholic acid and deoxycholic acid, respectively, and their original conformations and binding behavior to the organic anion of naphthalenesulfonate derivatives were investigated by using 1H NMR spectroscopy and spectrofluorometric titration in combination with computational methods. The 2D NMR experiments reveal that the steroid groups attached to the beta-CD rim could be deeply embedded in the beta-CD cavity to form the intramolecular (for 1) or intermolecular (for 2) inclusion complexes in aqueous solution. Upon complexation with naphthalenesulfonate derivatives, modified beta-CDs display two obviously different binding modes, that is, the competitive inclusion mode and the induced-fit inclusion mode, which is consistent with the results of molecular modeling study. The two modes and the strict size/shape fitting relationship between the hosts and guests reasonably explain the different binding behaviors and molecular selectivity of host beta-CDs 1 and 2 toward the naphthalenesulfonate guests. Therefore, the cholic acid- or deoxycholic acid-modified beta-CDs could effectively recognize the size/shape of guest molecules as compared with the parent beta-CD, giving good molecular selectivity up to 24.9 for the disodium 2,6-naphthalenedisulfonate/disodium 1,5-naphthalenedisulfonate pair by the host 1.     

Chiral beta-cyclodextrin-based polymer supports prepared via ring-opening metathesis graft-polymerization

A series of norborn-2-ene-derivatized beta-cyclodextrins (beta-CDs), 6-O-(norborn-2-ene-5-carboxyl)-beta-CD (1), tetrakis(6-O-norborn-2-ene-5-carboxyl)-beta-CD (2), (3), 6-O-(6-norborn-2-ene-5-car-6-O-(7-oxanorborn-2-ene-5-carboxyl)-beta-CD bonylaminohexoyl)-beta-CD (4), 6-O-(norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (5), tris(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (6), tetrakis(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (7) and hexakis(6-O-norborn-2-ene-5-ylmethoxymethylsilyl)-beta-CD (8), have been synthesized. Compounds 1-3 were prepared via reaction of beta-CD with norborn-2-ene-5-carboxylic chloride and 7-oxanorborn-2-ene-5-carboxylic chloride, respectively; compounds 5-8 were synthesized from norborn-2-ene-5-yl-methyldichlorosilane and beta-CD, respectively. Compound 4 was accessible by reaction of norbom-2-ene-5-carboxylaminohexoyl chloride with beta-CD. Compounds 1-8 were surface grafted onto norborn-2-ene-derivatized silica-based supports using ring-opening metathesis polymerization employing the ruthenium-based initiator bis(tricyclohexylphosphino)benzylideneruthenium dichloride [Cl2Ru(CHC6H5)(PCy3)2, Cy=cyclohexyl, 9]. Generally speaking, the resulting chiral stationary phases (CSPs) I-VIII may be prepared with high reproducibility and may be used within a pH of 2-10. Thus, relative standard deviations (sigman-1) of the mean resolution (Rs) are <7%. The CSPs were used for the enantioselective separation of beta-blockers, N-dansyl-, N-3,5-dinitrobenzoyl- and Fmoc-protected amino acids and were characterized in terms of chemical stability, selectivity (alpha') and resolution (Rs). Additionally, the role of the spacer as well as influences of capacity and the degree of substitution of the beta-CD moiety on the separation characteristics were determined.     

Dinuclear ruthenium(II) polypyridyl complexes containing large, redox-active, aromatic bridging ligands: synthesis, characterization, and intramolecular quenching of MLCT excited states

Two new ruthenium(II) polypyridyl dimers containing the large planar aromatic bridging ligands 9,11,20,22-tetraazatetrapyrido[3,2-a:2'3'-c:3' ',2'-l:2',3'-n]pentacene (tatpp) and 9,11,20,22-tetraazatetrapyrido[3,2-a:2'3'-c:3',2' '-l:2',3'-n]pentacene-10,21-quinone (tatpq) have been synthesized and characterized by (1)H and (13)C NMR, MALDI mass spectrometry, and elemental analyses. The electronic properties (UV-vis, redox, photophysical) of these dimers are analyzed in the context of orbital calculations (PM3 level) on the bridging ligands. A localized orbital model is proposed in which low-lying acceptor orbitals on the center portion of the ligands effectively quench the Ru(II)-based MLCT emission via a mechanism that can be viewed as intramolecular electron transfer to specific subunits of the bridges.     

Fluorometric study of the inclusion interaction of beta-cyclodextrin derivatives with tetraphenylporphyrin and its analytical application

The effects of native beta-cyclodextrin (beta-CD) and four kinds of alkylated beta-cyclodextrin (beta-CDs), i.e. heptakis (2,6-di-O-isobutyl-beta-cyclodextrin) (I), heptakis (2,6-di-O-octyl-beta-cyclodextrin) (II), heptakis (2,6-di-O-dodecyl-beta-cyclodextrin) (III), and heptakis (2,6-di-O-hexadecyl-beta-cyclodextrin) (IV), on the fluorescence behaviors of tetraphenylporphyrin (TPP) are investigated. An obvious fluorescence enhancement is observed from TPP by using alkylated derivatives compared to that obtained in beta-CD aqueous or in water. A 114-N fluorescence emission intensity enhancement is found for the complex with 2,6-di-O-octyl-beta-cyclodextrin relative to the free analyte. The exact stoichiometric ratios and the formation constants of the inclusion complexes have been examined by application of curve fitting method. The linear calibration plots between fluorescence intensity and TPP concentration are determined in the 1.14 x 10(-8)-5.06 x 10(-6) mol l(-1) range.     

Study of the complexation of fisetin with cyclodextrins

In this work, the interaction between fisetin (3,3',4',7-tetrahydroxyflavone) (Fis) and cyclodextrins (CDs) (alpha and beta) was studied through UV-vis absorption, steady-state fluorescence, induced circular dichroism, and (1)H NMR experiments with dependence on temperature and pH. Some experimental data were compared with quantum-mechanics studies based on the SAM1 (AMPAC) semiempirical model, as well as with the B3LYP and MPW1PW91 functional models from density functional theory using the 6-311G and 3-21G basis sets. The spectroscopic measurements show that Fis does not form stable complexes with alpha-CD. On the other hand, at pH 4.0 and 6.5, the complex Fis-beta-CD is formed in a Fis:beta-CD 1:1 stoichiometry and an equilibrium constant (K) of 900 +/- 100 M(-1). In basic medium (pH 11.5), K decreases to 240 +/- 90 M(-1) because Fis deprotonation leads to its better solubilization in water. Molecular modeling points out that Fis is not totally inserted into the inner cavity of beta-CD. The formation of the inclusion complex renders an environment that enhances intramolecular excited state proton transfer. The inclusion complex is formed preferentially via entry of the Fis phenyl group into beta-CD.     

Hydrogallation of alkynes with H-GaCl2: formation of organoelement dichlorogallium compounds potentially applicable as chelating Lewis acids

Treatment of trimethylsilylethynylbenzenes C6H6-x(C[TRIPLE BOND]C-SiMe3)x(x=1-3) with the hydridodichlorogallium compound H-GaCl2 afforded, almost quantitatively, the alkenylphenyl compounds C6H6-x[C(H)C(SiMe3)-GaCl2]x[x=1 (6), 2 (7), and 3 (8)] by hydrogallation. Only compound 6 was readily soluble in n-hexane; it formed dimers via Ga-Cl bridges. The bisalkenyl compound 7 was only sparingly soluble; its molecular structure consisted of a singular dimeric formula unit with a cyclophane-type constitution and two bridging Ga 2Cl 2 heterocycles. The overall structure may be described by a molecular box formed by a large macrocycle comprising 22 Ga, C, and Cl atoms. Compound 8 proved to be insoluble in hydrocarbon solvents. Its molecular structure could not be detected. Extraction of the solid raw products of 7 and 8 with diethyl ether yielded small quantities of the ether adducts C6H6-x[C(H)C(SiMe3)-GaCl2(OEt2)]x(x=2, 3) [7(OEt2)2 and 8(OEt2)3], both of which are monomeric because of the coordinative saturation of their gallium atoms. The tetraalkyne 1,2,4,5-tetrakis(trimethylsilylethynyl)benzene gave a different reaction course. Complete hydrogallation resulted in the release of 2 equiv of GaCl3, and neighboring alkenyl groups of the product 9 were connected by GaCl bridges to form seven-membered heterocycles and an overall tricyclic compound. Compound 9 was characterized as a diethyl ether adduct.     

Advanced mesoporous organosilica material containing microporous beta-cyclodextrins for the removal of humic acid from water

A new mesoporous organosilica material (beta-CD-Silica-4%) containing microporous beta-cyclodextrins (beta-CDs) has been prepared by the co-polymerization of a silylated beta-CD monomer with tetraethoxysilane in the presence of a structure-directing template, cetyltrimethylammonium bromide. Solid-state 13C and 29Si NMR studies provided evidence for the presence of covalently attached beta-CDs in the mesoporous material. Nitrogen adsorption experiments showed that beta-CD-Silica-4% material had a BET surface area of 460 m2/g and an average mesopore diameter of 2.52 nm. Small-angle powder X-ray diffraction pattern of beta-CD-Silica-4% material revealed the lack of highly ordered mesoporous structure. Adsorption experiments showed that beta-CD-Silica-4% material removed up to 99% of humic acid from an aqueous solution containing 50 ppm of humic acid at a solution-to-solid ratio of 100 ml/g.     

Homodimerization and heteroassociation of 6-O-(2-sulfonato-6-naphthyl)-gamma-cyclodextrin and 6-deoxy-(pyrene-1-carboxamido)-beta-cyclodextrin

6-O-(2-sulfonato-6-naphthyl)-gamma-cyclodextrin (1) and 6-deoxy-(pyrene-1-carboxamido)-beta-cyclodextrin (2) were prepared. Homodimerizations of 1 and 2 and heteroassociation between 1 and 2 were investigated by (1)H NMR, circular dichroism, and fluorescence spectroscopic methods. The compounds 1 and 2 form head-to-head dimers with dimerization constants of 140 +/- 50 and 270 +/- 70 M(-)(1), respectively. We also determined the association constants of 1 with beta-CD as 270 +/- 20 M(-)(1) and 2 with gamma-CD as 100 +/- 30 M(-)(1) from fluorescence and circular dichroism titration data, respectively. The heteroassociation between 1 and 2 was manifested in increased circular dichroism ellipticities of 2, downfield shift of the H-2 proton of the pyrene group of 2, and upfield shift of the H-5 proton of the naphthyl group of 1 upon mixing 1 and 2. The analysis of circular dichroism titration data of 2 with 1 gave the association constant as 9300 +/- 1600 M(-)(1). The NMR and circular dichroism spectra suggested that the naphthyl group of 1 is deeply included into the beta-CD cavity of 2, while the pyrene group of 2 is partially inserted in the gamma-CD cavity of 1 in the complex. The energy-minimized structure from molecular modeling of the complex supports this. We believe that the facile heteroassociation of two cyclodextrin derivatives having different sizes of cavity and pendant group could be utilized as a useful strategy for assembling functionalized CDs for various applications.     

Emulsion preparation using beta-cyclodextrin and its derivatives acting as an emulsifier

The preparation and formation mechanism of n-hexadecane/water emulsions using natural beta-cyclodextrin (beta-CD) and chemically modified beta-CDs (triacylated beta-cyclodextrins) as an emulsifier were investigated. The stable water/oil (W/O) emulsion was formed using tripropanoyl-beta-CD (TP-beta-CD). From observation using the contact angle (theta(ow)) of precipitates derived from CD, it was clarified that oil/water (O/W) emulsion at theta(ow)<90 degrees and (W/O) emulsion at theta(ow)>90 degrees are formed when the composition of each oil and water was mixed with natural beta-CD or triacylated beta-CDs.     

Liquid chromatography determination of citalopram enantiomers using beta-cyclodextrin as a chiral mobile phase additive

A reliable and specific method for the determination of citalopram enantiomers was developed and validated. Chromatographic resolution of citalopram enantiomers was made on a Shim-pack (5 microm particle size) cyanopropyl column with beta-cyclodextrin (beta-CD) as an effective chiral mobile phase additive. The composition of the mobile phase was (90 + 10, v/v) aqueous 0.1% triethylammonium acetate buffer, pH 4.0 (adjusted with acetic acid), and acetonitrile, containing 12 mM beta-CD. The flow rate was 0.8 mL/min with ultraviolet detection at 240 nm. The effects of the mobile phase composition, concentration of beta-CD, and pH of the triethylammonium acetate buffer on peak shape and resolution of the enantiomers were investigated. The calibration graphs were linear (r = 0.9999, n = 8) in the range of 1-40 microg/mL for S(+) citalopram and R-(-) citalopram. The limit of detection values were 5.51 x 10(-3) and 4.35 x 10(-3) pg/mL, while the limit of quantification values were found to be 1.84 x 10(-2) and 1.45 x 10(-2) microg/mL for S-(+) citalopram and R-(-) citalopram, respectively.     

Hydrazide-based quadruply hydrogen-bonded heterodimers. Structure, assembling selectivity, and supramolecular substitution

This paper describes the synthesis, self-assembly, and characterization of a new class of highly stable hydrazide-based quadruply hydrogen-bonded heterodimers. All of the hydrazide-derived heterodimers possess the complementary ADDA-DAAD hydrogen-bonding sequences. Hydrazide derivatives 1, which has two intramolecular S(6) RO.H-N hydrogen bonds, and 2 complex to afford two fastly exchanging isomeric heterodimers 1.2 and 1.2' in chloroform, as a result of two different conformational arrangements of 2. An average binding constant K(assoc) of 4.7 x 10(4) M(-)(1) was determined for heterodimer 1.2 and 1.2' by (1)H NMR titration of 1 with changing 2 in chloroform-d. In contrast, 1 binds 11 and 12, both of which are introduced with two intramolecular S(6) hydrogen bonds, to exclusively afford heterodimers 1.11 and 1.12, with K(assoc) values of 1.8 x 10(4) and 5.0 x 10(2) M(-)(1), respectively. Fluorine-containing 19, which has a hydrazide skeleton identical to that of 1 but two intramolecular S(6) F.H-N hydrogen bonds, can also complex with 2, 11, and 12, to afford heterodimers 19.2, 19.2', 19.11, and 19.12, with K(assoc) values of of 1.2 x 10(4) (average value for 19.2 and 19.2'), 5.4 x 10(3), and 1.9 x 10(2) M(-)(1), respectively. The structures of the new heterodimers have been proven with NOESY, IR, and VPO (for some of the heterodimers) experiments. Moreover, 1 and 19 can also strongly bind 2,7-dilauroylamido-1,8-naphthyridine 23 to afford dimers 1.23 and 19.23 with K(assoc) values of 6.0 x 10(5) and 1.4 x 10(5) M(-)(1), respectively. Adding 1 to the 1:1 solution of 23 and 1-octyl-3-(4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl)urea 24 or 1-octyl-3-(4-oxo-1,4-dihydro-pyrimidin-2-yl)urea 25, which had been developed initially by Zimmerman and Meijer, respectively, induces dimers 23.24 and 23.25 to dissociate, leading to the formation of dimers 1.23 and 24.24 or 25.25, respectively. The new hydrazide-based hydrogen-bonding modules described are useful building blocks for self-assembly and open a new avenue to recognition between discrete supramolecular species.     

Characterization of drug interactions with soluble beta-cyclodextrin by high-performance affinity chromatography

This study examined the use of an immobilized human serum albumin (HSA) column to study solution-phase reactions between drugs and beta-cyclodextrin (beta-CD). Chromatographic equations were developed to characterize the binding of chemicals to a soluble ligand (beta-CD) in the presence of an independent immobilized ligand (HSA). Situations considered included the presence of both a homogeneous and heterogeneous immobilized ligand, as well as complex interactions between the chemical of interest and soluble ligand. Three drugs (warfarin, tamoxifen, and phenytoin) were examined by this approach. This method involved injecting a small amount of each drug onto an HSA column in the presence of various concentrations of beta-CD in the mobile phase. By measuring the change in the drug's retention factor as the concentration of beta-CD was varied, it was possible to determine the stability constant between the injected drug and beta-CD. With this approach, warfarin and beta-CD were found to have 1:1 interactions with a stability constant of 5.2 x 10(2) M(-1) at 37 degrees C and pH 7.4, a result in close agreement with previous literature values. Tamoxifen and phenytoin were also found to have 1:1 interactions with beta-CD and had stability constants of 0.9-1.2 x 10(4) and 6-9 x 10(2) M(-1) respectively. With these latter solutes, the effects of secondary binding to the chromatographic support had to be considered. The theory and methods described in this report are not limited to these drugs and beta-CD but can be applied to other analytes and soluble ligands.     

Charge mediation by ruthenium poly(pyridine) complexes in 'second-generation' glucose biosensors based on carboxymethylated β-cyclodextrin polymer membranes

Four different poly(pyridine) complexes of ruthenium, viz. Ru(II)(trpy)(phen)(OH(2))](2+) (1), trans-[Ru(III)(2,2'bpy)(2)(OH(2))(OH)](2+) (2), [(2,2'bpy)(2)(OH)Ru(III)ORu(III)(OH)(2,2'bpy)(2)](4+) (3), and [Ru(II)(4,4'bpy)(NH(3))(5)](2+) (4) (2,2'bpy=2,2'-bipyridine, 4,4'bpy=4,4'-bipyridine, trpy=2,2',2"-terpyridine, phen=1,10-phenanthroline), were tested as non-physiological charge mediators of 'second-generation' glucose biosensors. The membranes for these biosensors were prepared by casting anionic carboxymethylated beta-cyclodextrin polymer films (beta-CDPA) directly onto the Pt or glassy carbon (GC) disk electrodes. Simultaneously, glucose oxidase (GOD) was immobilized in the films by covalent bonding and the Ru complexes were incorporated both by inclusion in the beta-CD molecular cavities and by ion exchange at the fixed carboxymethyl cation-exchange sites. The leakage of the mediator from the polymer has been minimized by adopting a suitable pre-treatment procedure. The biosensors catalytic activities increased in the order 1<2<3<4, as established by linear sweep voltammetry. In case of complexes 2-4, the enzymatic glucose oxidation was mediated by the Ru complexes at their redox potentials. However, this oxidation was mediated by oxygen in case of complex 1 where H(2)O(2) was detected as the reaction product. The effectiveness of the mediators used in the presence of oxygen has been estimated using Pt and GC supports. The redox potential of the mediator does not depend on the support used, while the oxidation of H(2)O(2) proceeds on GC at much higher positive potentials than on Pt. The sensitivity and the linear concentration range of the biosensor studied varied significantly. For complex 4, which forms stable inclusion complex with beta-CD, the biosensor sensitivity was the highest and equal to 7.2 micro A mM(-1) cm(-2), detectability was as low as 1 mM, but the linear concentration range was limited only to 4 mM. In contrast, for complexes 2 and 3 the sensitivity was 0.4 and 3.2 micro A mM(-1) cm(-2), while the linear concentration range extended up to at least 24 and 14 mM glucose, respectively. Even though some common interfering substances, such as ascorbate, paracetamol or urea, are oxidized at potentials close to those of the Ru complex redox couples, their electro-oxidation currents at physiological concentrations are insignificant compared to those due to the biocatalytic oxidation of glucose. The biosensor response to glucose is reversible as demonstrated by the inhibition of GOD activity by Cu(II). That is, the Cu(II) concentration required to inhibit by half the response to glucose of the biosensor containing complex 2 was 1.0 mM. This inhibitory effect was fully reversed by addition of citrate, a ligand forming sufficiently stable complex with Cu(II).     

Enzyme-like acceleration for the hydrolysis of a DNA model promoted by a dinuclear Zn(II) catalyst in dilute aqueous ethanol

The rates and products of cleavage of methyl (2-chloro-4-nitrophenyl) phosphate (2) promoted by a dinuclear Zn(II) complex (3) of 1,3-bis-N,N'(1,5,9-triazacyclododecyl)propane along with 1 equiv of ethoxide were investigated in ethanol solution containing small amounts of water (8 mM or=1.6 x 10(17) times relative to the background hydroxide reaction, suggesting that complex 3 promotes the hydrolysis at least 1000 times more effectively than ethanolysis.     

Discrimination of A/T sequences in the minor groove of DNA within a cyclic polyamide motif

Eight-ring cyclic polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp) aromatic amino acids recognize predetermined six base pair sites in the minor groove of DNA. Two four-ring polyamide subunits linked by (R)-2,4-diaminobutyric acid [(R)H2Ngamma] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin polyamides, substitution of Hp/Py for Py/Py pairs enhances selectivity for T. A base pairs but compromises binding affinity for specific sequences. In an effort to enhance the binding properties of polyamides containing Hp/Py pairings, four eight ring cyclic polyamides were synthesized and analyzed on a DNA restriction fragment containing three 6-bp sites 5'-tAGNNCTt-3', where NN = AA, TA, or AT. Quantitative footprint titration experiments demonstrate that contiguous placement of Hp/Py pairs in cyclo-(gamma-ImPyPyPy-(R)H2Ngamma-ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5'-tAGAACTt-3' site (Ka = 7.5 x 10(7)M(-1)) relative to ImPyPyPy-(R)H2Ngamma-ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(gamma-ImHpPyPy-(R)H2Ngamma-ImHpPyPy-) (3) binds a 5'-tAGTACTt-3' site with an equilibrium association constant KA= 3.2 x 10(9)M(-1), representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)H2Ngamma-ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3'-stagger regulates specificity of cyclo-(gamma-ImPyHpPy-(R)H2Ngamma-ImPyHpPy-) (5) for the 5'-tAGATCTt-3' site (Ka = 7.5 x 10(7)M(-1)) threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)H2Ngamma-ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.