A model for sequential threading of alpha-cyclodextrin onto a guest: a complete thermodynamic and kinetic study in water.

The first variable-temperature and variable-pressure stopped-flow spectrophotometric study of the sequential threading of alpha-cyclodextrin (alpha-CD) onto the guest dye Mordant Orange 10, S, is reported. Complementary (1)H one-dimensional (1D) variable-temperature kinetic studies and two-dimensional (2D) rotating-frame nuclear Overhauser effect spectroscopy (ROESY) and EXSY NMR studies are also reported. In aqueous solution at 298.2 K, the first alpha-CD threads onto S to form a 1:1 complex S.alpha-CD with a forward rate constant k(1,f) = 15 200 +/- 200 M(-1) s(-1) and dethreads with a reverse rate constant k(1,r) = 4.4 +/- 0.3 s(-1). Subsequently, S.alpha-CD isomerizes to S.alpha-CD (k(3,f) = 0.158 +/- 0.006 s(-1), k(3,f) = 0.148 +/- 0.006 s(-1)). This process can be viewed as a thermodynamically controlled molecular shuttle. A second alpha-CD threads onto S.alpha-CD to form a 1:2 complex, S.(alpha-CD)(2), with k(2,f) = 98 +/- 2 M(-1) s(-1) and k(2,r) = 0.032 +/- 0.002 s(-1). A second alpha-CD also threads onto S.alpha-CD to form another 1:2 complex, S.(alpha-CD)(2), characterized by k(4,f) = 9640 +/- 1800 M(-1) s(-1) and k(4,r) = 61 +/- 6 s(-1). Direct interconvertion between S.(alpha-CD)(2) and S.(alpha-CD)(2) was not detected; instead, they interconvert by dethreading the second alpha-CD and through the isomerization equilibrium between S.alpha-CD and S.alpha-CD. The reaction volumes, DeltaV(0), were found to be negative for the first three equilibria and positive for the fourth equilibrium. For the first three forward and reverse reactions, the volumes of activation are substantially more negative, indicating a compression of the transition state in comparison with the ground states. These data were used in conjunction with DeltaH, DeltaH degrees, DeltaS, and DeltaS degrees data to deduce the dominant mechanistic threading processes, which appear to be largely controlled by changes in hydration and van der Waals interactions, and possibly by conformational changes in both S and alpha-CD. The structure of the four complexes were deduced from (1)H 2D ROESY NMR studies.

An hermaphrodite [2]rotaxane: preparation and analysis of structure.

[structure: see text]. The pictured rotaxane is assembled in water by capping a substituted cyclodextrin composed of the wheel and axle components. The unusual dimeric structure of the rotaxane reflects the thermodynamics of the assembly process. In N,N-dimethylformamide, the corresponding monomer is the predominant product.

The synthesis and fluorescent properties of analogues of the zinc(II) specific fluorophore zinquin ester.

The synthesis and fluorescent properties in the absence and presence of zinc(II) of a range of 2-substituted derivatives of N-(6-methoxy-2-methyl-8-quinolyl)-4-methylbenzenesulfonamide are described. These analogues formed complexes with zinc(II) as indicated by a bathochromic shift in their UV/vis spectra. Analogues with isobutenyl and isobutyl side chains at the 2-position formed fluorescent complexes whose fluorescence was stronger than that of the 2-methyl-containing parent. These derivatives were converted, via conversion to the phenol with boron tribromide and reaction with ethyl bromoacetate, to systems with ester-containing side chains analogous to zinquin ester, a specific cellular fluorophore for zinc(II). All of these ester derivatives formed complexes with zinc(II) resulting in a bathochomic shift in their UV/vis spectra. Compounds with isobutyl, isobutenyl, and styryl side chains exhibited increased fluorescence compared to that of zinquin ester in the presence of zinc(II). The compound with the 2-isobutyl side chain was more selective in its fluorescence for zinc(II) over cadmium(II) compared to zinquin ester.

Alkaline earth complexes of 1,4,7,10-tetrakis(2-hydroxyethyl)- and 1,4,7,10-tetrakis(2-methoxyethyl)-1,4,7,10-tetraazacyclododecane: an equilibrium and kinetic study.

The 1,4,7,10-tetrakis(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane complexes [M(thec12)]2+, where M2+ = Mg2+, Ca2+, Sr2+, and Ba2+, are characterized by log(K/dm3 mol-1) = 2.86 +/- 0.09, 7.41 +/- 0.04, 6.47 +/- 0.04, and 4.84 +/- 0.03 at 298.2 K in aqueous Et4NClO4 (I = 0.10 mol dm-3), where K is a potentiometrically determined stability constant. The analogous literature values for the 1,4,7,10-tetrakis(2-methoxyethyl)-1,4,7,10-tetraazacyclododecane complexes [M(tmec12)]2+ are 2.47, 5.47, 5.00, and 4.72. The enantiomerization of eight-coordinate delta- and lambda-[M(thec12)]2+ is characterized by k(298.2 K) = 2310 +/- 260, 582 +/- 17, and 445 +/- 5 s-1, delta H++ = 19.1 +/- 0.8, 33.3 +/- 0.5, and 43.9 +/- 0.4 kJ mol-1, and delta S++ = -117 +/- 4, -80.3 +/- 1.8, and -47.0 +/- 1.3 J K-1 mol-1 when M2+ = Mg2+, Ca2+, and Ba2+, respectively, in methanol-12C-d4 as shown by 13C NMR spectroscopy. For the enantiomerization of eight-coordinate delta- and lambda-[M(tmec12)]2+, k(298.2 K) = 310 +/- 1 and 688 +/- 3 s-1, delta H++ = 54.0 +/- 0.2 and 39.6 +/- 0.1 kJ mol-1, and delta S++ = -16.1 +/- 0.5 and -57.9 +/- 0.3 J K-1 mol-1 when M2+ = Ca2+ and Ba2+, respectively. However, [Mg(tmec12)]2+ has a seven-coordinate structure where one of the methoxy groups is not coordinated and exchange of the methoxy groups between the coordinated and free states is characterized by k(298.2 K) = 163,000 +/- 8000 s-1, delta H++ = 35.8 +/- 0.4 kJ mol-1, and delta S++ = -25.1 +/- 1.7 J K-1 mol-1. The intermolecular exchange of thec12 and tmec12 between the coordinated and free states is substantially slower than the enantiomerizations in the first five complexes and the intramolecular exchange process observed in [Mg(tmec12)]2+.

Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin.

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship to metallothionein and intracellular zinc.

Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetate], a new intracellular zinc fluorophore, was used to reveal and to measure Zn in cultured rat hepatocytes before and after metallothionein (MT) induction. Hepatocytes labelled with an intense extranuclear fluorescence. Culture with combinations of Zn, dexamethasone and interleukin-6, increased intracellular MT by 24-fold, Zn 3-fold, and Zinquin fluorescence by approx. 2-fold above control values. Zinquin fluorescence correlated in descending order with the total cellular Zn (r = 0.747), exchangeable Zn (r = 0.735), soluble cytosolic Zn (r = 0.669) and MT (r = 0.666). When Zinquin was incubated with a cytosolic fraction of liver proteins before Sephadex G-75 column chromatography, it fluoresced with free, MT-incorporated and protein-bound Zn. Although only a slight attenuation of fluorescence was seen with high-molecular-mass protein-bound Zn, MT was degraded by 60% in the presence of Zinquin. The undegraded Zn-MT fluoresced at about 20% of the expected intensity. Although Zinquin fluoresces with all cytosolic Zn, caution is required when comparisons are made between samples with different concentrations of MT. This limitation was demonstrated by staining liver slices from adjuvant-treated rats where MT was increased 24-fold, intracellular Zn by 77%, but Zinquin fluorescence by only 19% above controls. Nevertheless, Zinquin should prove to be a useful tool for studying the distribution of Zn in living cells.

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin.

BACKGROUND: The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS: Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS: The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.