Photochemical DNA modifications induced by 1,2-dioxetanes.

1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation. In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases. Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus. For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux. DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications. The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme. The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds. The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers. The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen. Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent. A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane.     

Improved chemiluminescent western blotting procedure.

A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.     

1,2-Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays

We have synthesized and studied two 1,2-dioxetane-based chemiluminescent enzyme substrates: 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), and, 3-(2′-spiroadamantane)-4-methoxy-4- (3″-β-D ′-galactopyrano-yloxy)phenyl-1,2-dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD -galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due to the presence of a hydrophobic microenvironment provided by the enhancer which ‘stabilizes’ the dephosphorylated AMPPD emitter. Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD we were able to detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer. AMPPD and AMPGD offer alternatives to colorimetric and fluorescent subsrates for alkaline phosphatase and β-D -galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β-hCG, LH, TSH and others).     

Unusual luminescent properties of odd- and even-substituted naphthyl-derivatized dioxetanes

With the advent of enzymatically induced chemiluminescence and improved instrumentation for luminometry, ultrasensitive detection of a wide variety of analytes is now possible using standard immunoassay and DNA probe formats. Model molecular orbital calculations and literature precedent suggest that the singlet efficiencies observed upon decomposition of dioxetanes appended with donor substituted aromatic moieties are dependent on substitution pattern. We have recently discovered, in a series of 3-(2′-spiroadamantane)-4-methoxy-4-acetoxynaphth-2′-yl-1,2-dioxetanes, that enzymatic generation of a nonconjugated, charge transfer excited state results in luminescence of markedly different properties than that observed from an isomeric, conjugated excited state. An example of the former type, 3-(2′-spiroadamantane)-4-methoxy-4-(7″-acetoxy)naphth-2′-yl-1,2-dioxetane (1) emitting at 550 nm, not only provides an increase in Φ_CL but exhibits a dramatic bathochromic shift of 80–110 nm from the 460 nm emission of the conjugated isomer 3-(2′-spiroadamantane)-4-methoxy-4-(6″-acetoxy)naphth-2′-yl-1,2-dioxetane (2). These developments, along with the attendant glow-type luminescence kinetics displayed during the enzymatic decomposition of the new ‘odd-pattern’ dioxetane, allow the design of simple protocols capable of simultaneous or ‘multichannel’ detection of several analytes.     

Imaging β-Galactosidase Activity in Human Tumor Xenografts and Transgenic Mice Using a Chemiluminescent Substrate

Background

Detection of enzyme activity or transgene expression offers potential insight into developmental biology, disease progression, and potentially personalized medicine. Historically, the lacZ gene encoding the enzyme β-galactosidase has been the most common reporter gene and many chromogenic and fluorogenic substrates are well established, but limited to histology or in vitro assays. We now present a novel approach for in vivo detection of β-galactosidase using optical imaging to detect light emission following administration of the chemiluminescent 1,2-dioxetane substrate Galacto-Light PlusTM.

Methodology and Principal Findings

B-gal activity was visualized in stably transfected human MCF7-lacZ tumors growing in mice. LacZ tumors were identified versus contralateral wild type tumors as controls, based on two- to tenfold greater light emission following direct intra tumoral or intravenous administration of reporter substrate. The 1,2-dioxetane substrate is commercially available as a kit for microplate-based assays for β-gal detection, and we have adapted it for in vivo application. Typically, 100 µl substrate mixture was administered intravenously and light emission was detected from the lacZ tumor immediately with gradual decrease over the next 20 mins. Imaging was also undertaken in transgenic ROSA26 mice following subcutaneous or intravenous injection of substrate mixture.

Conclusion and Significance

Light emission was detectable using standard instrumentation designed for more traditional bioluminescent imaging. Use of 1,2-dioxetane substrates to detect enzyme activity offers a new paradigm for non-invasive biochemistry in vivo.     

Some oxidation products of ethoxyquin including those found in autoxidising systems

2,4-Dimethyl-6-ethoxyquinoline (2), 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline nitroxide (3), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine N-oxide (4), 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (5), 1,8′-di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) (6) and 1,2-dihydro-6-hydroxy-2,2,4-trimethylquinoline (7) have been prepared from 1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline (1) (ethoxyquin) and their spectroscopic properties (UV, IR, mass and NMR) examined.     

Acetophenones and terpenoids from Senecio Gallicus

Six new compounds isolated from the aerial part of Senecio gallicus were: 7,11,15-trimethyl-3-methylene hexadecan-1,2-diol diacetate; 7,11,15-trimethyl-3-methylenehexadecan-1,2-diol; 3,5-bis(3-methyl-2-butenyl)-4-acetoxyacetophenone; 3-(2-hydroxy-3-methyl-3-butenyl)-5-(3-methyl-2-butenyl)4-hydroxacetophenone; 3-(2,3-dihydroxy-3-methyl-butyl)-5-(3-methyl-2-butenyl)-4-hydroxyacetophenone and 1,10-epoxy-8α-hydroxy eremophilenolide.     

Identification of urinary metabolites of the nephrotoxic hydrocarbon 2,2,4-trimethylpentane in male rats

The compound 2,2,4-trimethylpentane (2,2,4 TMP) is reported to be especially potent in inducing kidney lesions in male rats (1,2). Although the pathology produced by 2,2,4 TMP has been examined (1), there are no reports concerning the metabolism of 2,2,4 TMP by the male rat. The eight principal urinary metabolites of 2,2,4 TMP found in the urine of Fischer 344 male rats are: 2,2,4-trimethyl-1-pentanol, 2,4,4-trimethyl-1-pentanol, 2,4,4-trimethyl-2-pentanol, 2,2,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-2-hydroxy-1-pentanoic acid, 2,2,4-trimethyl-5-hydroxy-1-pentanoic acid and 2,4,4-trimethyl-5-hydroxy-1-pentanoic acid.     

Chemical constituents from Anthemis wiedemanniana Fisch. & Mey.

The lipophilic extract of the aerial parts of Anthemis wiedemanniana Fisch. & Mey, a Turkish endemic species, was investigated. In addition to one new natural product, namely 3,4,4-trimethyl-6-carboxy-cyclohex-2-ene-1-one, five known sesquiterpene lactones, five methylated flavonoids, one simple phenolic derivative and one nor-isoprenoid were isolated. Among the sesquiterpene lactones, canin was found to be the main compound.     

5(Z)-benzylidene-1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes as non-steroidal glucocorticoid receptor modulators

A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure-activity relationships for this series of compounds are presented.     

Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry.

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.     

Radical cations and triplet states of 1,2-disubstituted cyclopropanes: comparison of potential surfaces

Radical ion pairs generated by photo-induced electron transfer from 1,2-disubstituted cyclopropanes to various acceptors undergo return electron transfer in pairs of singlet and triplet multiplicity. The pair energies relative to the reactant ground states and to accessible triplet states, respectively, determine the competition between the recombination pathways. The potential surfaces of the radical cations and triplet states of 1,2-diphenyl-, 1, and 1,2-dimethylcyclopropane, 2, have been examined by density functional theory calculations. The radical cation surfaces have minima at geometries that retain significant bonding between C-1 and C-2, preventing geometric isomerization of the radical cations. The triplet potential surfaces are dissociative with minimal rotational differentiation at long distances between C-1 and C-2.     

DNA damage by oxygen radicals and excited state species: a comparative study using enzymatic probes in vitro

Repair enzyme-containing extracts from a variety of cell types are used to analyse and compare DNA damage induced by oxygen radicals and excited molecules. The differing potentials of these extracts for recognising DNA damage leads to characteristic DNA damage profiles after treatment with superoxide (xanthine/xanthine oxidase), gamma-rays, chemically generated singlet oxygen, photosensitizers (rose bengal, methylene blue), UV254 and a 1,2-dioxetane. Three different types of damage profiles are distinguished and assigned to the predominant action of hydroxyl radicals, singlet oxygen or to the photoexcitation of thymine residues. The method applied in this study allows the analysis of DNA damage and the identification or exclusion of the participation of different ultimate reactive species without chemical identification of the lesions.     

Changes of structure and energy on the route from dioxetane to carbonyl products. A quantum chemical study

Energy diagrams, changes of geometry and bond orders were calculated semi-empirically for the thermolysis of 1,2-dioxetane. Stretching of the O–O bond, then of the C–C bond and distortion of the whole quadrangular structure make major, but different, contributions to the reaction coordinate on the path to formaldehyde. The activation barrier represents a vast region where the gaps between the ground and excited states are small, and this favours horizontal radiationless transitions leading to the excitation of a product. The results show that semi-empirical calculations may help to provide better insight into the nature and mechanism of the chemiluminescence excitation. © 1998 John Wiley & Sons, Ltd.     

The metabolism of cis- and trans-indane-1,2-diol

1. The metabolism of cis-indane-1,2-diol, trans-indane-1,2-diol, indene epoxide and 2-hydroxyindan-1-one in rats has been studied. The substances were administered to the animals by subcutaneous injection. 2. The urine of the dosed animals was examined for the presence of free and conjugated cis- and trans-dihydrodiols, and for each compound it was possible to isolate both cis and trans forms of indane-1,2-diol from the urine. 3. The urines were also examined by paper chromatography for ketones and two ketonic metabolites were detected in the urine of rats dosed separately with cis-indane-1,2-diol, trans-indane-1,2-diol, 2-hydroxyindan-1-one and indene epoxide. The ketones were provisionally identified as (1-oxoindan-2-yl glucosid)uronic acid and 1-oxoindan-2-yl sulphuric acid. 4. (1-Oxoindan-2-yl glucosid)uronic acid was isolated as the 2,4-dinitrophenylhydrazone from the urine of rats dosed separately with cis-indane-1,2-diol and trans-indane-1,2-diol. 5. Possible mechanisms for the interconversion of cis- and trans-indane-1,2-diol are discussed.     

5-(1,2-Epoxy-2,6,6-trimethyl-1-cyclohexyl)-3-methyl-cis,trans-2,4-pentadienoic acid and its esters: New plant growth inhibitors structurally related to abscisic acid

Summary 5-(1,2-Epoxy-2,6,6-trimethyl-1-cyclohexyl)-3-methyl-cis,trans-2,4-pentadienoic acid and its esters were found to be potent plant growth inhibitors. Their activities were comparable to that of abscisic acid.     

Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes.

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.     

Synthesis and cytotoxic activity of some novel polycyclic gamma-butyrolactones

Baeyer–Villiger oxidation of 5-aryl-7,11,11-trimethyltricyclo[5.4.0.0^3,6]-undec-1-en-4-ones 4a–h by H₂O₂ and formic acid in methanol yields mixtures of 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-1-ones 8a–h and 3b,7,7-trimethyl-3-phenyl-3,3a,3b,4,5,6,7,8a-octahydro-1H-indeno-[1,2-c]furan-2-ones 9a–h in high yields. The obtained butyrolactones 8a–h display cytotoxic activity against a number of human cancer cells.     

A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

Flavonoids from Merrillia caloxylon

Three chalcones and three flavones isolated from the fruit of Merrillia caloxylon (Rutaceae) have been characterised. Two of the flavones and two of the chalcones are related structurally, i.e. 3′,4′,5,7-tetramethoxyflavone with 2′- hydroxy-3,4,4′,6′-tetramethoxychalcone and 3′,4′,5,5′,7-pentamethoxyflavone with 2′,3-dihydroxy-4,4′,6′- trimethoxychalcone. A minor constituent was tentatively characterized as 5-hydroxy-3′,4′,5′,6,7-pentamethoxyflavone and this is accompanied by 2-hydroxy-3,4,4′,5,6′-pentamethoxychalcone and 5-hydroxy-3′,4′,6,7-tetramethoxyfiavone.