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In this paper, the structural and dynamic properties of the peptide with the sequence CAGQW have been extensively investigated using molecular dynamics simulations. Several simulations for a total simulation time of > 1.5μs were performed using the GROMOS96 and OPLS-AA/L force fields. The effects of simulation protocols and force fields on the structural and dynamic properties of the peptide have been analyzed. Accounting for long range electrostatic interactions using the particle mesh Ewald method resulted in a tendency to slow the end-to-end contact formations. In contrast to the relatively modest effect of the simulation protocol, the force field had a larger influence on the properties of the peptide. Use of GROMOS96 resulted in a faster end-to-end contact formation kinetics than using OPLS-AA/L. The ratio of maximum lifetime values, obtained from GROMOS96 (16 ns) and OPLS-AA/L (24 ns) force fields (accounting for the difference between the experimental and calculated solvent viscosity) vs the experimental lifetime (73 ns) resulted in 4.6 and 3.0 times faster kinetics, respectively. This faster encounter kinetics can be related to the free energy profile versus end-to-end distance which indicates a stronger preference for small end-to-end distance states in the case of GROMOS96 compared to other force fields. Furthermore, the comparison with results obtained on the same peptide by Yeh and Hummer (J. Am. Chem. Soc. 2002, 124, 6563-6568) using AMBER94 and CHARMM22 force fields indicates the following order for the end-to-end contact formation kinetics: GROMOS96 > OPLS-AA/L > AMBER94 ≥ CHARMM22.

The factors affecting host-guest complexation between the molecular container compound cucurbit[6]uril (CB6) and various guests in aqueous solution are studied, and a detailed complexation mechanism in the presence of cations is derived. The formation of the supramolecular complex is studied in detail for cyclohexylmethylammonium ion as guest. The kinetics and thermodynamics of complexation is monitored by NMR as a function of temperature, salt concentration, and cation size. The binding constants and the ingression rate constants decrease with increasing salt concentration and cation-binding constant, in agreement with a competitive binding of the ammonium site of the guest and the metal cation with the ureido carbonyl portals of CB6. Studies as a function of guest size indicate that the effective container volume of the CB6 cavity is approximately 105 A(3). It is suggested that larger guests are excluded for two reasons: a high activation barrier for ingression imposed by the tight CB6 portals and a destabilization of the complex due to steric repulsion inside. For example, in the case of the nearly spherical azoalkane homologues 2,3-diazabicyclo[2.2.1]hept-2-ene (DBH, volume ca. 96 A(3)) and 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO, volume ca. 110 A(3)), the former forms the CB6 complex promptly with a sizable binding constant (1300 M(-1)), while the latter does not form a complex even after several months at optimized complexation conditions. Molecular mechanics calculations are performed for several CB6/guest complexes. A qualitative agreement is found between experimental and calculated activation energies for ingression as a function of both guest size and state of protonation. The potential role of constrictive binding by CB6 is discussed.

A new fluorescent host-guest system based on the inclusion of the fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) into the cavity of the molecular container compound cucurbit[7]uril (CB7) has been designed which possesses an exceedingly long-lived emission (690 ns in aerated water). The large binding constant of (4/spl plusmn/1)/spl times/10/sup 5/ M/sup -1/ along with the resistance of the CB7/spl middot/DBO complex toward external fluorescence quenchers allow the use of CB7 as an enhancer in time-resolved fluorescence-based assays, e.g., to screen enzyme activity or inhibition by using DBO-labeled peptides as substrates. The response of CB7/spl middot/DBO to different environmental conditions and possible quenchers are described.

Two novel azoalkane bichromophores and related model compounds have been synthesised and photophysically characterised. Dimethylphenylsiloxy (DPSO) or dimethylnaphthylsiloxy (DNSO) serve as aromatic donor groups (antenna) and the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as the acceptor. The UV spectral window of DBO (250–300 nm) allows selective excitation of the donor. Intramolecular singlet–singlet energy transfer to DBO is highly efficient and proceeds with quantum yields of 0.76 with DPSO and 0.99 with DNSO. The photophysical and spectral properties of the bichromophoric systems suggest that energy transfer occurs through diffusional approach of the donor and acceptor within a van der Waals contact at which the exchange mechanism is presumed to dominate. Furthermore, akin to the behaviour of electron-transfer systems in the Marcus inverted region, a rate of energy transfer 2.5 times slower was observed for the system with the more favourable energetics, i.e. singlet–singlet energy transfer from DPSO proceeded slower than from DNSO, although the process is more exergonic for DPSO (−142 kJ mol−1 for DPSO versus −67 kJ mol−1 for DNSO).

Aim: To study the antioxidant activity, UV absorption, concentration and stability of ascorbic acid (AA) in porcine and bovine aqueous humour (AH). Methods: Porcine and bovine AH was taken within 5 min after death and frozen at –70°C. The characteristic UV absorption band of AA and the concentration of AA in AH was determined by UV spectrophotometry. The antioxidant activity of AA to serve as a free radical scavenger in AH has been determined by using a novel fluorescent probe for antioxidants, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO). The fluorescence lifetime and intensity of this probe reflect the concentration of dissolved antioxidants. The time-resolved fluorescence of DBO (laser excitation at 351 nm) in AH and in a neutral phosphate-buffered saline (PBS) solution containing only the natural amount of AA as an additive were measured. Results: The characteristic UV absorption band of AA has its maximum at 266 nm in AH. The concentration of AA in porcine and bovine AH was found to be 0.547 ± 0.044 and 1.09 ± 0.16 mM, respectively, by spectrophotometry. The fluorescence lifetime of the probe DBO was reduced from 320 ± 5 ns in pure aerated PBS to 205 ± 5 ns in porcine AH and 165 ± 3 ns in bovine AH. A detailed kinetic analysis of the lifetime shortening suggests that AA contributes approximately 75 and 85% to the antioxidant activity of porcine and bovine AH, respectively. Conclusions: Our experiments suggest that AA is the major contributor to the antioxidant activity of porcine and bovine AH. The role of AA to serve as an antioxidant in AH is discussed. In addition, UV spectrophotometry is established as an alternative method to determine the concentration of AA in AH.

A novel fluorescence-based method, which entails contact quenching of the long-lived fluorescent state of 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO), was employed to measure the kinetics of end-to-end collision in short single-stranded oligodeoxyribonucleotides of the type 5'-DBO-(X)n-dG with X = dA, dC, dT, or dU and n = 2 or 4. The fluorophore was covalently attached to the 5' end and dG was introduced as an efficient intrinsic quencher at the 3' terminus. The end-to-end collision rates, which can be directly related to the efficiency of intramolecular fluorescence quenching, ranged from 0.1 to 9.0 x 10(6) s(-1). They were strongly dependent on the strand length, the base sequence, as well as the temperature. Oligonucleotides containing dA in the backbone displayed much slower collision rates and significantly higher positive activation energies than strands composed of pyrimidine bases, suggesting a higher intrinsic rigidity of oligoadenylate. Comparison of the measured collision rates in short single-stranded oligodeoxyribonucleotides with the previously reported kinetics of hairpin formation indicates that the intramolecular collision is significantly faster than the nucleation step of hairpin closing. This is consistent with the configurational diffusion model suggested by Ansari et al. (Ansari, A.; Kuznetsov, S. V.; Shen, Y. Proc.Natl. Acad. Sci. USA 2001, 98, 7771-7776), in which the formation of misfolded loops is thought to slow hairpin formation.

Monte Carlo simulations of chain conformations and the diffusion equation were used to analyze the fluorescence kinetics of short polymer chains labeled with a probe and a quencher at opposite ends. In simulations, three chain models were considered: an ideal chain (without volume interactions); a self-avoiding chain taking into account the exclusive volume effect; and a self-avoiding chain with limited flexibility between nearest segments. For each model, end-to-end distance distribution functions were obtained, which were different from Gaussian ones. The distribution functions were used in a diffusion equation to simulate the fluorescence kinetics of the probe affected by intramolecular end-to-end collisions of short chains. The kinetics has been numerically calculated for a representative experimental system in a nonviscous solution. The simulated time-resolved fluorescence decays were monoexponential except at very short times (

[reaction: see text] The fluorescence quenching of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) by nucleotides has been studied. The quenching mechanism was analyzed on the basis of deuterium isotope effects, tendencies for exciplex formation, and the quenching efficiency in the presence of a molecular container (cucurbit[7]uril). Exciplex-induced quenching appears to prevail for adenosine, cytidine, and uridine, while hydrogen abstraction becomes competitive for thymidine and guanosine. Compared to other fluorescent probes, DBO responds very selectively to the type of nucleotide.

We now report a novel scale for the flexibility of amino acids, which provides an absolute measure for the time scale of conformational changes in short structureless peptides as a function of the amino acid type. This experimental scale is derived from kinetic measurements of the collision frequency between the two ends of short random-coil polypeptides. The present experiments provide the first application of our recently introduced fluorazophore method for measuring the kinetics of end-to-end collision in polypeptides. [16, 17] In

UVA-irradiation of the photo-Fenton reagents N-isopropoxypyridone 2b and N-isopropoxythiazole-2(3H)-thione 3b releases radicals which induce strand breaks. Transient spectroscopy establishes N-O bond scission [Phi(N)(-)(O) = (75 +/- 8)% for 2b and (65 +/- 7)% 3b] as the dominating primary photochemical process to afford the DNA-damaging radicals. Product studies and laser-flash experiments reveal that the thiazolethione 3b leads primarily to the disulfide 5, from which through C-S bond breakage, the bithiazyl 6, the thiazole 7, and the isothiocyanate 8 are derived. Upon irradiation of pyridone 2b (300 nm) in aqueous media, a mixture of isopropoxyl and 2-hydroxyprop-2-yl radicals is formed, as confirmed by trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and EPR spectroscopy. In contrast, the photolysis of the thiazolethione 3b (350 nm) affords exclusively the DMPO adducts of the isopropoxyl radicals. Control experiments disclose that the thiazolethione-derived photoproduct disulfide 5, or the intermediary thiyl radicals B, scavenge the carbon-centered 2-hydroxyprop-2-yl radicals, which are generated from the isopropoxyl radicals by hydrogen shift. With supercoiled pBR 322 DNA in a 60:40 mixture of H(2)O-MeCN, the pyridone 2b and the thiazolethione 3b display moderate strand-break activity (17% open-circular DNA for 2b and 12% for 3b). In pure water, however, the pyridone 2b photoinduces substantially more DNA cleavage (32% open-circular DNA), which is attributed to the peroxyl radicals generated from the 2-hydroxyprop-2-yl radicals by oxygen trapping. The lower strand-break activity of the thiazolethione 3b derives presumably from isopropoxyl radicals, because only these are detected in the photolysis of this photo-Fenton reagent.

2,3-Diazabicyclo[2.2.2]oct-2-ene (DBO) was used as a long-lived fluorescent probe in zeolites (NaY, Na-mordenite, Na-ZSM-5, H-ZSM-5) and related oxide materials (all silica MCM-41, silica, silica–alumina, γ-alumina). The photophysical properties are dominated by a hydroxylic environment, caused by the inorganic framework and co-adsorbed water. The quenching of DBO by oxygen was strongly dependent on the type of zeolite. In ZSM-5 zeolites, the fluorescence decays were not monoexponential in the presence of oxygen (air).

[reaction: see text] Photolysis of hydroxyacetophenone and thermolysis of the corresponding dioxetane afford spiroiminodihydantoin rather than 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (4-HO-8-oxodG) through the oxidation of 2'-deoxyguanosine (dG) by triplet-excited hydroxyacetophenone and the peroxyl radicals derived thereof by alpha cleavage and subsequent oxygen trapping. The structure of the spiroiminodihydantoin is assigned by the SELINQUATE NMR technique, which unequivocally establishes the spirocyclic connectivity.

The reaction enthalpies for the photoreduction of n,pi*-excited states (acetone, benzophenone, 2.3-diazabicyclo[2.2.2]oct-2-ene, and a 2,3-diazabicyclo[2.2.1]hept-2-ene derivative) by model hydrogen donors (methanol and dimethylamine) were calculated on the basis of a critically evaluated data set of bond dissociation energies for donors and reduced acceptors. These were compared with the observed photoreactivity, which can be assessed through quenching rate constants of the excited states by hydrogen donors. The intriguing observation of a preferential attack at electrophilic hydrogen atoms, i.e., N-H or O-H, by n,pi*-excited azoalkanes is rationalised on the basis of the calculated thermochemical data, differences in electrophilicity, and varying contributions of antibonding character in the transition state. Singlet-excited azoalkanes act as nucleophilic species, while excited ketones display an electrophilic reactivity. This is in line with the pictorial description of the electron distribution in these excited states.

The macrocycle p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) form a stable host-guest complex, in which the dye fluorescence is quenched. Incubation of live V79 and CHO cells with the CX4/LCG chemosensing ensemble resulted in its spontaneous uptake. Subsequent addition of choline, acetylcholine, or protamine, which have a high affinity for CX4 and are capable of entering cells, resulted in a fluorescence switch-on response. This can be traced to the displacement of LCG from CX4 by the analytes. The results establish the principal functionality of indicator displacement assays with synthetic receptors for the detection of the uptake of bioorganic analytes by live cells.

A comprehensive investigation of the photophysics of a broad set of fluorenones substituted with methoxy groups at different positions brings out the importance of the location of substituents on the fluorenone core in modulating fluorescence and radiationless deactivation by way of modification of the singlet-excited state energy and its character. While the substituents at para positions are found to affect neither the fluorescence quantum yield nor the lifetime, those at meta positions are found to significantly modify the latter. A cumulative effect is observed for the substituents in that the nonradiative decay (knr) becomes progressively dominant with an increasing number of meta-methoxy substituents. For example, the trimethoxy substitution in 2,4,7-trimethoxyfluorenone (8) is found to increase knr by ca. 30 fold relative to that of the parent fluorenone (1) in a polar aprotic solvent such as acetonitrile. The predominance of nonradiative decay (knr) is rationalized from stabilization of the singlet-excited state via partial charge transfer from meta-methoxy substituents to the carbonyl group. Accordingly, a nice correlation is observed for the nonradiative (knr) rate constants versus singlet-excitation energies derived from fluorescence emission maxima of all fluorenones in acetonitrile. The macrocyclic host cucurbit[7]uril (CB7) is found to not only enhance the fluorescence of the parent fluorenone (1) significantly, but also increase the singlet lifetime considerably. Based on the changes observed in the absorption spectra and the lifetimes determined, a 1:1 host–guest complex has been proposed with CB7. The fluorescence lifetime observed in the presence of CB7 suggests that the hydrophobic fluorenone (1) can be employed as a probe to report on a polar microenvironment shielded from hydrogen bonding interactions in a polar protic solvent.

CONSPECTUS: Host-guest chemistry commenced to a large degree with the work of Pedersen, who in 1967 first reported the synthesis of crown ethers. The past 45 years have witnessed a substantial progress in the field, from the design of highly selective host molecules as receptors to their application in drug delivery and, particularly, analyte sensing. Much effort has been expended on designing receptors and signaling mechanism for detecting compounds of biological and environmental relevance. Traditionally, the design of a chemosensor comprises one component for molecular recognition, frequently macrocycles of the cyclodextrin, cucurbituril, cyclophane, or calixarene type. The second component, used for signaling, is typically an indicator dye which changes its photophysical properties, preferably its fluorescence, upon analyte binding. A variety of signal transduction mechanisms are available, of which displacement of the dye from the macrocyclic binding site is one of the simplest and most popular ones. This constitutes the working principle of indicator displacement assays. However, indicator displacement assays have been predominantly exploited in a static fashion, namely, to determine absolute analyte concentrations, or, by using combinations of several reporter pairs, to achieve a differential sensing and, thus, identification of specific food products or brands. In contrast, their use in biological systems, for example, with membranes, cells, or with enzymes has been comparably less explored, which led us to the design of the so-called tandem assays, that is, dynamically analyte-responsive host-dye systems, in which the change in analyte concentrations is induced by a biological reaction or process. This methodological variation has practical application potential, because the ability to monitor these biochemical pathways or to follow specific molecules in real time is of paramount interest for both biochemical laboratories and the pharmaceutical industry. We will begin by describing the underlying principles that govern the use of macrocycle-fluorescent dye complexes to monitor time-dependent changes in analyte concentrations. Suitable chemosensing ensembles are introduced, along with their fluorescence responses (switch-on or switch-off). This includes supramolecular tandem assays in their product- and substrate-selective variants, and in their domino and enzyme-coupled modifications, with assays for amino acid decarboxylases, diamine, and choline oxidase, proteases, methyl transferases, acetylcholineesterase (including an unpublished direct tandem assay), choline oxidase, and potato apyrase as examples. It also includes the very recently introduced tandem membrane assays in their published influx and unpublished efflux variants, with the outer membrane protein F as channel protein and protamine as bidirectionally translocated analyte. As proof-of-principle for environmental monitoring applications, we describe sensing ensembles for volatile hydrocarbons.

Ternary complexes between the macrocyclic host cucurbit[8]uril, dicationic dyes, and chiral aromatic analytes afford strong induced circular dichroism (ICD) signals in the near-UV and visible regions. This allows for chirality sensing and peptide-sequence recognition in water at low micromolar analyte concentrations. The reversible and noncovalent mode of binding ensures an immediate response to concentration changes, which allows the real-time monitoring of chemical reactions. The introduced supramolecular method is likely to find applications in bioanalytical chemistry, especially enzyme assays, for drug-related analytical applications, and for continuous monitoring of enantioselective reactions, particularly asymmetric catalysis.

We report the formation of strong inclusion complexes between the macrocyclic host cucurbit[7]uril (CB7) and more than 20 perfluorinated compounds (PFCs) in aqueous solution. The binding constants and structures of the complexes were determined through a combination of dye displacement titrations, 19F as well as 1H NMR spectroscopy, and quantum chemical calculations. The high affinities, for example 1.2 × 107 M− 1 for perfluorohexane and 1.1 × 108 M− 1 for perfluoro(methylcylohexane), are driven, among others, by the low polarisability of the cucurbituril cavity, which favours the binding of non-polarisable guests such as PFCs. The complexation-induced chemical shifts of the 19F resonances are downfield in cases where a conformational flexibility allows the (partial) interconversion of gauche conformations (favoured in water) to anti conformations (favoured inside the cavity of CB7). Only for conformationally rigid substrates, for example SF6 and (CF3)3COH, upfield shifts prevail. These are attributed to the low polarisability of the inner cavity, similar to the upfield shifts observed for 19F resonances upon going from highly polarisable to less polarisable solvents. Cucurbituril homologues (CB5, CB6 and CB8) were also studied and, among those, evidence for the formation of inclusion complexes was observed for CB6 as host and CF4, SF6 and CF3CH2OH as guests. Besides contributing to the fundamental understanding of fluorophilic interactions, the results have implications for PFC separation and for the efficient removal of PFCs from and the sensing of PFCs in aqueous solution.

Derivatives of the azoalkane 2,3-diazabicyclo[2,2,2]oct-2-ene (1a) with bridgehead 1,4-dialkyl (1b), 1,4-dichloro (1c), 1-hydroxymethyl (1d), 1-aminomethyl (1e), and 1-ammoniummethyl (1f) substituents form host-guest inclusion complexes with beta-cyclodextrin. They were employed as probes to assess substituent effects on the kinetics and thermodynamics of this complexation by using time-resolved and steady-state fluorimetry, UV spectrophotometry, induced circular dichroism (ICD) measurements, and (1)H NMR spectroscopy. The kinetic analysis based on quenching of the long-lived fluorescence of the azoalkanes by addition of host provided excited-state association rate constants between 2.6 x 10(8) and 7.0 x 10(8) M(-)(1) s(-)(1). The binding constants for 1a (1100 M(-1)), 1b (900 M(-1)), 1c (1900 M(-1)), 1d (180 M(-1)), 1e (250 M(-1)), and 1f (ca. 20 M(-1)) were obtained by UV, NMR, and ICD titrations. A positive ICD signal of the azo absorption around 370 nm was observed for the beta-cyclodextrin complexes of 1a, 1d, and 1f with the intensity order 1a1d approximately 1f, and a negative signal was measured for those of 1b, 1c, and 1e with the intensity order 1c1b approximately 1e. The ICD was employed for the assignment of the solution structures of the complexes, in particular the relative orientation of the guest in the host (co-conformation).

The photooxidative damage of DNA, specifically guanine oxidation and strand-break formation, by sidechain-oxyfunctionalized acetophenones (hydroxy, methoxy, tert-butoxy and acetoxy derivatives), has been examined. The involvement of triplet-excited ketones and their reactivity towards DNA has been determined by time-resolved laser-flash spectroscopy. The generation of carbon-centered radical species upon Norrish-type I cleavage has been assessed by spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide, coupled with electron paramagnetic resonance spectroscopy. The observed DNA-base oxidation and strand-break formation is discussed in terms of the peroxyl radicals derived from the triplet-excited ketones by alpha cleavage and molecular oxygen trapping, as well as direct interaction of the excited states by electron transfer and hydrogen-atom abstraction. It is concluded that acetophenone derivatives, which produce radicals upon photolysis, in particular the hydroxy (AP-OH) and tert-butoxy (AP-O(t)Bu) derivatives, are more effective in oxidizing DNA.

Radical-pair disproportionation through sequential electron-proton transfer (Polanyi's harpoon) is one efficient mechanism leading to quenching fluorescence of n,π*-excited states by hydrogen donors. This mechanism is demonstrated by ab initio MS-CASPT2 calculations and experimental investigations (photoproduct studies and EPR spin-trapping experiments) on the quenching of singlet-excited azoalkanes by chlorinated hydrocarbons.

The solution structures of the beta-cyclodextrin complexes between 2,3-diazabicyclo[2.2.2]oct-2-ene (1) and its 1-isopropyl-4-methyl derivative 2 have been investigated by means of induced circular dichroism (ICD) and MM3-92 force-field calculations, which considered the effect of solvation within a continuum approximation. Of primary interest was the so-called co-conformation of the host-guest complex, i.e., the relative orientation of the guest within the host. A pool of low-energy complex structures, which were located by means of a Monte Carlo simulated annealing routine, was generated to evaluate the dynamic co-conformational variability of the complexes. The ICD effects were calculated for the computed low-energy structures by applying a semiempirical method. The experimental and theoretical ICD as well as the calculated low-energy complex geometries suggest solution co-conformations in which the parent compound 1 adapts a lateral arrangement with the ethano bridge of the guest penetrating deepest into the cavity and the azo group aligning parallel to the plane of the upper rim. In contrast, the alkyl derivative 2 prefers a frontal co-conformation with the isopropyl group penetrating deepest into the cavity and the azo group aligning perpendicular to the plane of the upper rim. The validity of the predictions of the Harata rule regarding the sign and the intensity of the ICD signals for the n(-)pi, n(+)pi, and pipi transition of the azo chromophore in dependence on the complex co-conformation are discussed. With respect to the co-conformational variability of the complexes of the two azoalkanes, it was observed that the nearly spherical guest 1 forms a geometrically better defined complex than the sterically biased, alkyl-substituted derivative 2. This dichotomy is attributed to the largely different modes of binding for azoalkanes 1 and 2. It is concluded that the goodness-of-fit in a host-guest complex cannot be directly related to the "tightness-of-fit".

Pigmentation of the iris is caused by varying amounts of melanin pigment granula in a constant number of melanocytes in the superficial stroma. Melanin has been shown to act as antioxidant. We have now investigated lipid peroxidation in dependence on stromal pigmentation in isolated porcine irises.The same number of lightly and heavily pigmented porcine irises (visual selection) were homogenized (1 : 20 w/v) in buffer (50 mmol/l phosphate buffer and 4 mmol/l sodium azide). 500 microl homogenate were incubated at 37 degrees C in duplicate for 5, 10, 20 and 40 min in absence and presence of Fe2+ as inducer of lipid peroxidation. The amount of lipid peroxidation was assayed by the thiobarbituric acid (TBA) test. The results are expressed as nmol of TBA reactive material (TBAR) produced/mg protein. Fe2+ concentration of the supernatant was determined spectrophotometrically with 1,10 orthophenanthroline. Concentrations of D-glucose and D -fructose in iris tissue homogenates were determined spectrophotometrically by enzymatic bioanalysis.70, 180 and 360 micromol/l Fe2+ induced lipid peroxidation. A plateau region was reached after 20 min. The amount of lipid peroxidation differed in dependence on stromal pigmentation in porcine irises. The effect was most significant at 180 micromol/l Fe2+, which induced 1.373 +/- 0.138 nmol TBAR/mg protein in lightly compared to 0.491 +/- 0.125 nmol TBAR/mg protein in heavily pigmented irises after 10 min incubation (p0.0001, n = 4). Similar effects (factor 2-3) were also measured after 20 and 40 min incubation. On the other hand, the content of Fe2+ in the supernatant was the same within error. Sugar concentrations (D-glucose and D-fructose) did not differ significantly for the two differently pigmented iris tissues.There is a stronger induction of lipid peroxidation in lightly compared to heavily pigmented porcine irises. This effect may be related to the difference in stromal melanin content and its antioxidant activity.

Macrocyclic compounds can serve as hosts for smaller organic molecules, but precise control over the uptake and release of the guests remains challenging. Now, a host–guest system has been built that responds to the addition of metal ions, showing promise for drug-delivery applications.

The photolysis of the mono-, bis-, and trisazoalkanes 1, 2, and 3 in a toluene matrix at 77 K has been studied by EPR and UV spectroscopy. The purpose was to find the optimal conditions for the generation of the corresponding organic high-spin polyradicals (the triplet diradicals D-1, D-2, and D-3, the tetraradicals T-2 and T-3, and the hexaradical H-3) all with localized cyclopentane-1,3-diyl spin-carrying units, connected by m-phenylene (except D-1) as ferromagnetic coupler. Irradiation of these azoalkanes at 333, 351, or 364 nm gave different polyradical compositions. This observed wavelength dependence is due to the secondary photoreaction (photobleaching) of the polyradical intermediate. The photobleaching process has been examined in detail for the triplet diradical D-1, for which pi,pi excitation affords the cyclopentenes 5 instead of the housane 4 (the usual product of the diradical D-1 on warm-up of the matrix). The pi,pi-excited diradical D-1 fragments into a pair of allyl and methyl radicals (the latter was observed by EPR spectroscopy of a photobleached sample), and recombination affords the cyclopentene. Similar photochemical events are proposed for the photobleaching of the tetraradical T-2 and hexaradical H-3, derived from the respective azoalkanes 2 and 3. Thus, photobleaching of the polyradicals competes effectively with their photogeneration from the azoalkane. This unavoidable event is the consequence of spectral overlap between the cumyl-radical pi,pi chromophore of the polyradical and the n,pi chromophore of the azoalkane at the wavelength (364 nm), at which the latter is photoactive for the required extrusion of molecular nitrogen.

Five aryl-substituted phenacetyl radicals (X = p-MeO, p-Me, H, p-Cl, p-CF3) were generated by laser photolysis of the corresponding dibenzyl ketones in n-hexane and acetonitrile. The decarbonylation reaction was monitored through the rise in time-resolved absorption of the benzyl radical chromophore at 317 nm. The decarbonylation rate constants were obtained by a numerical integration procedure, where second-order radical reactions were explicitly taken into account. Values of (2–3) × 106 s−1 in acetonitrile and (6–10) × 106 s−1 in n-hexane revealed a large solvent effect for all derivatives (by a factor of ∼3). The electronic substituent effect indicates that both electron-withdrawing and electron-donating para substituents accelerate the decarbonylation slightly. The rate constants followed the order MeO > Me, Cl, CF3 > H. The substituent effects are interpreted in terms of the ability of the para substituent to stabilize the benzyl radical resulting from decarbonylation. Evidence for a polar effect was not obtained. The underlying reasons for the observed solvent effect are discussed. Copyright © 2000 John Wiley & Sons, Ltd.

The direct and benzophenone-sensitized photodenitrogenation of the diethoxy-substituted diazene 3 in nonprotic solvents, e.g. n-hexane, benzene, and acetonitrile, afforded exclusively the housane 4 through the intermediary 2,2-diethoxy-1,3-diphenylcyclopentane-1,3-diyl diradical 2b. Alternatively, in the presence of methanol, the formation of the adduct 6 competed through trapping of the allylic cation 5. The intervention of the intermediates was corroborated by laser-flash photolysis experiments of the diazene 3. The diradical intermediate was characterized by its strong transient absorption at 545 nm, which decayed with a first-order lifetime in the microsecond range. The diradical lifetimes increased in the order n-hexane (0.52 μs), benzene (0.88 μs), acetonitrile (1.01 μs), and chloroform (3.73 μs). The singlet ground state of the 2,2-diethoxy-substituted 1,3-diradical 2b is suggested by the absence of EPR signals at low temperature, the lack of quenching by molecular oxygen, and the calculated (UB3LY...

The fluorescence quenching of two azoalkanes, the weak acceptor 2,3-diazabicyclo[2.2.2]oct-2-ene and its less reactive derivative 4-methyl-1-isopropyl-2,3-diazabicyclo[2.2.2]oct-2-ene, by a series of aliphatic amine donors was examined by time-resolved spectroscopy in benzene. The fluorescence quenching rate constants ranged from 0.13 × 107 to 25 × 107 M-1 s-1, while the activation energies were found to be 1.3 and 3.0 kcal mol-1 for the best (N-ethyldicyclohexylamine) and the poorest (N,N-diisopropyl-3-pentylamine) donor. The oxidation potentials of the amines and the reduction potentials of the azoalkanes were measured. Deviations from the expected dependence of the quenching rate constants on the energetics of electron transfer were observed. Steric effects on the kinetics of quenching, namely a reduction by up to a factor of 50, became significant for amines with three secondary alkyl substituents, triisopropylamine, and N,N-diisopropyl-3-pentylamine, while a stereoelectronic effect was found for 1,4-...

Different inclusion modes can be realized for a bicyclic azoalkane in beta-cyclodextrin (see picture). Variations in the alignment of the azo chromophore relative to the host can be detected through NMR spectroscopy and induced circular dichroism. The latter technique produces a positive signal for the lateral conformer and a negative, significantly weaker peak for the frontal one. This is in keeping with the orientation-intensity rule.

Faster in cyclohexane than in acetonitrile is the fluorescence quenching of the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) by amines and sulfides. Although this photoreaction is induced by charge transfer (CT; see picture) and exciplexes are formed, the increase in the dipole moment of the exciplex is not large enough to offset the solvent stabilization of the excited reactants, and an "inverted" solvent effect results.

The lifetimes of 33 1,3-diaryl-1,3-cyclopentanediyl triplet diradicals were determined by laser flash photolysis of the corresponding azoalkane precursors. The first-order decay rate constants range from 0.8 to 16.7 × 105 s-1 in degassed solution at 294 K and exhibit a systematic, but nonlinear dependence on Brown's σ+ substituent constants. Analysis of spin−orbit coupling in terms of the two-electrons-in-two-orbitals model indicates that the electronic effect on the rates of intersystem crossing operates by influencing the weight of ionic contributions in the lowest singlet state wave functions of the diradicals. Counter to intuition, but in agreement with the prediction by the model, push−pull substitution does not enhance the ionic contribution. Arrhenius parameters, Ea = 2−6 kcal mol-1 and A = 107−1010 s-1, were determined from the temperature dependences of the decay rate constants. A sensitive statistical test is used to establish that the Arrhenius parameters exhibit enthalpy−entropy compensation, ...

A novel fluorescent probe with n,π* configuration, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), is responsive to complexation by supramolecular hosts. The n,π* fluorescent probe serves to provide structural and, owing to its exceedingly long fluorescence lifetime (up to 1 μs), also kinetic information on host−guest complexation. The three cyclodextrins (CDs) were selected as prototypal hosts in aqueous solution, and the complexation, in both the ground and excited states, was followed by four techniques: time-resolved and steady-state fluorescence, UV absorption spectrophotometry, and NMR spectroscopy. The fluorescence quenching rate constants (kq) of DBO by α-, β-, and γ-CD (1.9, 4.0, and 0.78 × 108 M-1 s-1) were determined from the dynamic component of the biexponential time-resolved decay traces, while the static component was assigned to the fluorescence lifetimes of the complexes (τCD) of α-CD (ca. 33 ns) and β-CD (ca. 95 ns). Time-resolved and steady-state fluorescence measurements yielded...

Photoactivable (“caged”) fluorescent dyes and probes are crucial for temporally and spatially resolved tracer experiments, e.g., in cell biology or fluid physics. The thiadiazolidinedione 1 represents a new class of caged fluorophore. Upon UV-irradiation it releases in a rapid photoreaction with high quantum yield the azoalkane 2. Longer wavelength excitation of 2 to the singlet excited state results in strong and long-lived fluorescence with maximum intensity at 425 nm. It has been demonstrated that one single uncaging laser pulse suffices for time-resolved or steady-state detection of the fluorescence.

The fluorescence lifetimes of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) were determined in a large number of solvents and solvent mixtures, displaying variations from 825 ns in CD3CN to 13 ns in CHCl3. Large deuterium isotope effects (8−11) and significant activation energies (generally 2−15 kJ mol-1) for fluorescence quenching of DBO are observed, which support a novel solvent-induced quenching mechanism via an aborted hydrogen atom transfer. In CCl4 a photoinduced electron-transfer quenching mechanism may compete. The fluorescence quenching by solvent mixtures is not proportional to the mole fraction of the two components, indicating a preferential solvation of the excited state.

The n,π* singlet-excited azoalkane 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO) is efficiently quenched by the natural antioxidants α-tocopherol (kq = 5.3 × 109 M-1 s-1), uric acid (3.4 × 109), ascorbic acid (2.05 × 109), and glutathione (0.82 × 109). This trend in reactivity is the same as that observed for alternative probes for antioxidants, e.g., triplet-excited benzophenone and the trichloromethylperoxyl radical. Since the quenching of singlet-excited DBO can be readily quantified by means of its long-lived (325 ns in aerated water) and strong fluorescence (λmax ca. 425 nm), this azoalkane serves as the first fluorescent probe for the reactivity of antioxidants. The effects of pH and deuterium substitution and kinetic solvent effects were also examined. The data suggest the involvement of hydrogen atom transfer in the fluorescence quenching of DBO by antioxidants. The reaction efficiency for radical formation in the quenching of singlet-excited DBO by α-tocopherol was found to be one order of magnitude lo...

The direct and benzophenone-sensitized photodenitrogenation of the azoalkane 2,3-diazabicyclo[2.2.1]hept-2-ene-7,3‘-spiro-2‘,2‘-diphenyloxirane 1 at ambient temperature (ca. 15 °C) in benzene afforded exclusively the 6-oxabicyclo[3.2.0]hept-1-ene 3. The labile oxetane 3 is proposed to be formed by the selective cleavage of the C−O bond in the epoxy ring of the initially photogenerated, spiroepoxy-substituted, localized cyclopentane-1,3-diyl diradical 1,3-DR to the 1,4-DR diradical and subsequent cyclization of the latter. Even at −100 °C, the highly strained (strain energy estimated to be ca. 94 kcal/mol) spiroepoxy housane 2 could not be observed by 1H NMR (600 MHz) spectroscopy. In MeOH, instead of the oxetane 3, the two regioisomeric MeOH adducts 6 (trapping product of the diradicals 1,3-DR and 1,4-DR) and 6‘ (methanolysis product of 3) were obtained in high yields. For the first time methanol trapping of a cyclopentane-1,3-diyl singlet diradical was achieved; the required dipolar character is presumab...

The 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane (HTMD) highly efficiently damages DNA compared to the merely alkyl-substituted derivative 3,3,4,4-tetramethyl-1,2-dioxetane (TMD). To elucidate this difference in oxidative reactivity, two additional hydroxymethyl-substituted 1,2-dioxetanes, namely cis/trans-3-(hydroxymethyl)-3,4-dimethyl-4-(phenylmethyl)- (1α/1β) and 3-(hydroxymethyl)-4,4-dimethyl-3-(phenylmethyl)-1,2-dioxetane (2), were investigated in regard to their photochemical and photobiological properties. The high genotoxic effects of the hydroxymethyl-substituted 1,2-dioxetanes, which are reflected in the significant formation of single-strand breaks in plasmid pBR 322 DNA and the efficient oxidation of guanine in calf thymus DNA and the nucleoside 2'-deoxyguanosine (dGuo), are for the first time understood in terms of radical chemistry. The reactivity order of the dioxetanes 1α/1β > HTMD > 2 ≫ TMD to damage DNA parallels the propensity of these dioxetanes to generate radicals. These reactive ...

The fluorescence quenching of the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene by protic solvents (methanol, methanol-OD, water, deuterium oxide, acetic acid) has been examined. The pseudo-unimolecular quenching rate constants (k q ) vary from 0.30-44×10 6 s -1 and decrease upon deuteration of the solvent OH bonds, e.g., the isotope effect for methanol/methanol-OD is ca. 8.5. This demonstrates that the hydroxylic hydrogens are predominantly responsible for the fluorescence quenching. The activation parameters were determined for methanol and methanol-OD. The activation enthalpies are unexpectedly low (ΔH = 1.8 kcal mol -1 for methanol) and increase upon deuteration (ΔH = 3.0 kcal mol -1 for methanol-OD), while the activation entro-pies remain the same (ΔS ca. 17.5 cal K -1 mol -1 ). This provides evidence for a fully classical isotope effect related to differences in zero-point vibrational energies. Tunneling appears to play no significant role. The quenching rate constants display no trend with the acidity of the solvent (pK a values) but with the homolytic bond dissociation energies of the OH bonds. This suggests the involvement of a hydrogen atom rather than a proton transfer. All important aspects (activation enthalpies, isotope effects, etc.) of this novel quenching mechanism of protic solvents are reproduced by MCSCF quantum-chemical calculations with a complete active space of CAS (12,10) or CAS (8.7). Most importantly, the computed data indicate the occurrence of a conical intersection, i.e., a real surface crossing, which follows the transition state and provides an efficient tngger for radiationless retum to the ground-state energy surface (fluorescence quenching).

As a result of recent experimental and theoretical investigations, new aspects of the photochemical hydrogen ion by n,π * -excited states have emerged. The absolute reactivity of singlet-excited n,π * states exceeds that of the corresponding triplet states, but the singlet reaction tums out to be chemically much less efficient than the triplet reaction. Hence, radiationless deactivation of the singlet states represents the dominant pathway for interaction with hydrogen donors. The fraction of singlet encounters undergoing deactivation appears to increase with decreasing reaction exothermicity. Hence, when relatively inert solvents like methanol or chloroform act as hydrogen donors towards singlet-excited azoalkanes, the reaction becomes entirely inefficient. This constitutes an example of a novel fluorescence quenching mechanism, which is referred to as an aborted hydrogen abstraction. The inefficiency of the singlet reaction can be rationalized, making use of the most recent theoretical findings, in terms of the occurrence of a conical intersection, i.e., a real surface crossing, rather than the previously implicated avoided crossing. The conical intersection provides an efficient channel for nonadiabatic return to the ground-state reactants and, thus, serves to account for the observed inefficiency of product formation from the singlet state. In general, the conical intersection appears to follow the transition state for hydrogen abstraction, but the consequences of a situation, where the conical intersection precedes the transition state are also discussed. The latter becomes reasonable for endothermic hydrogen ions, for which one would then expect a unit efficiency of singlet deactivation. The absolute reactivities in hydrogen abstractions, the involvement of CT interactions and tunneling, and the behavior of different n,π * chromophores (azoalkanes versus ketones) are also discussed in view of the most recent experimental data. General rules for photochemical hydrogen abstractions by n,π * -excited states are expressed.