Isoluminol Derivatives Research Articles

Synthesis and Chemiluminescent Properties of Amino-Acylated luminol Derivatives Bearing Phosphonium Cations.

The monitoring of reactive oxygen species in living cells provides valuable information on cell function and performance. Lately, the development of chemiluminescence-based reactive oxygen species monitoring has gained increased attention due to the advantages posed by chemiluminescence, including its rapid measurement and high sensitivity. In this respect, specific organelle-targeting trackers with strong chemiluminescence performance are of high importance. We herein report the synthesis and chemiluminescence properties of eight novel phosphonium-functionalized amino-acylated luminol and isoluminol derivatives, designed as mitochondriotropic chemiluminescence reactive oxygen species trackers. Three different phosphonium cationic moieties were employed (phenyl, p-tolyl, and cyclohexyl), as well as two alkanoyl chains (hexanoyl and undecanoyl) as bridges/linkers. Synthesis is accomplished via the acylation of the corresponding phthalimides, as phthalhydrazide precursors, followed by hydrazinolysis. This method was chosen because the direct acylation of (iso)luminol was discouraging. The new derivatives’ chemiluminescence was evaluated and compared with that of the parent molecules. A relatively poor chemiluminescence performance was observed for all derivatives, with the isoluminol-based ones being the poorest. This result is mainly attributed to the low yield of the fluorescence species formation during the chemiluminescence oxidation reaction.

Chemiluminescence properties of isoluminol related mono azo disperse dyes: Experimental and DFT based approach to photophysical properties

A six novel isoluminol derivatives containing azo unit were synthesized by coupling different N,N-disubstituted anilines with the diazotized 5-nitro and 7-nitro-6-amino-2,3-dihydrophthalazine-1,4-dione. The chemiluminescent properties were studied in hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide solution. The chemiluminescence properties were compared with the standard isoluminol systems it was observed that the chemiluminescence properties of the novel derivatives were nearly same to isoluminol. The geometries of the synthesized disperse dyes for their azo and hydrazone tautomeric forms were optimized using density functional theory and their electronic excitation properties were evaluated using density functional theory and time dependent density functional theory.

Chemiluminescence properties of luminol related o-hydroxybenzimidazole analogues: Experimental and DFT based approach to photophysical properties

Novel luminol–isoluminol derivatives containing o-hydroxyphenyl benzimidazole unit were synthesized from aromatic aldehydes and diaminophthalates followed by heating under reflux with hydrazine hydrate. The chemiluminescent properties were studied in hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide solution. The chemiluminescence properties were compared with the standard luminol and isoluminol systems it was observed that the chemiluminescence properties of the novel derivatives were superior to luminol and isoluminol. Density Functional Theory computations have been used in order to have a greater understanding of the structural, molecular, electronic and photophysical properties. The experimental absorption and emission wavelength values were in good agreement with the computed vertical excitation and emission values.

Enhancement of the Chemiluminescence of Two Isoluminol Derivatives by Nanoencapsulation with Natural Cyclodextrins

The chemiluminescence (CL) yield of two isoluminol derivatives, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and N-(6-aminohexyl)-N-ethylisoluminol (AHEI), is remarkably increased in the presence of natural cyclodextrins (CDs). The most notable effect is produced by the addition of gamma-CD that produces enhancements up to 15-fold in the light emission of both compounds. Although proton nuclear magnetic resonance ((1)H NMR) measurements prove the encapsulation of these luminescent reagents in the CDs, with more stable associations by decreasing the width of the CD cavity, the improvement in the light emission of ABEI and AHEI is mainly due to the topology of the complexes. Evaluation of the rotating frame Overhauser effect spectroscopy (ROESY) spectra cross-peaks combined with semirigid-docking simulations has been used to gather information about the spatial conformation of the guest molecules into the CDs. These calculations have shown that a deeper inclusion in the CD cavity of the heterocyclic moiety of the luminescent molecules is directly related with a higher enhancement in the CL. The augment of the CL by natural CDs is of interest for increasing the detection limit in biochemical assays or liquid chromatography, for example, in which the CL of these compounds serves to quantify other molecular species that may take part, direct or indirectly, in the luminescent reaction.

Selectivity and sensitivity in the measurement of reactive oxygen species (ROS) using chemiluminescent microspheres prepared by the binding of acridinium ester or ABEI to polymer microspheres

Two kinds of chemiluminescent microspheres were prepared as tools for measuring reactive oxygen species (ROS) released into phagosomes in phagocytizing cells, by chemically binding acridinium ester or ABEI (isoluminol derivative) to polymer microspheres, and were examined from the viewpoint of specificity and sensitivity to ROS. Acridinium ester-bound microspheres (AE-ms) were found to be a sensitive probe to superoxide anion and hydrogen peroxide under a neutral condition (pH 7.2). AE-ms emitted strong chemiluminescence (CL) by hypoxanthine (HPX)/xanthine oxidase (XOD) or hydrogen peroxide. The CL by HPX/XOD was initially inhibited by superoxide dismutase. At pH 5.6, the CL intensity from AE-ms in the presence of HPX/XOD was reduced to about one-eighth of that at pH 7.2. ABEI-bound microspheres (ABEI-ms) were found to be a selective probe for singlet oxygen although not highly sensitive. ABEI-ms emitted CL of moderate intensity with hydrogen peroxide/myeloperoxidase (MPO), but not with hydrogen peroxide alone or with hypochlorite/MPO at pH 5.6. The CL from ABEI-ms with hydrogen peroxide/MPO was completely inhibited by azide. ABEI-ms did not emit CL in the presence of HPX/XOD or by potassium superoxide at pH 5.6. The result of supplemental experiments using dissolved chemiluminescent probes and non-enzymatically generated ROS supported the above-described selectivity and sensitivity of chemiluminescent microspheres.

Mechanism and Analytical Applicability of Luminol-Specific Extrinsic Lyoluminescence of UV-Irradiated Potassium Peroxodisulfate

Luminol shows strong chemiluminescence with an emission maximum at ∼430 nm in the presence of sulfate radicals. Sulfate radicals were produced by the dissolution of UV-irradiated potassium peroxodisulfate powder in aqueous luminol solutions. The UV irradiation at 6.7 eV produces a solid solution of sulfate radical in potassium peroxodisulfate by rupturing −O−O− bonds, as in solution, but now the solid solution is stable in a time scale of years in dryness. In the present system, luminol chemiluminescence is produced via several parallel pathways having a common triggering step, one-electron oxidation of luminol monoanion by sulfate or hydroxyl radical. Present chemiluminescence allows sensitive luminol detection from picomolar to micromolar level with a linear response over 5 orders of magnitude, after which luminescence is too strong for single-photon counting. The high sensitivity of luminol detection allows us to propose extrinsic lyoluminescence of potassium peroxodisulfate as a new and simple method for detection step of bioaffinity assays using luminol or isoluminol derivatives as label compounds.

UV-irradiated potassium peroxodiphosphate and nitrate as excitation sources for luminol chemiluminescence

Luminol-specific extrinsic lyoluminescence of UV-irradiated potassium peroxodiphosphate and potassium nitrate has been studied. UV irradiation of solid peroxodiphosphate can induce rupture of the peroxide bond and produce a stable solid solution of phosphate radicals in potassium peroxodiphosphate. The dissolution of the irradiated salt can liberate phosphate radicals which initiate a luminol chemiluminescence-generating pathway in alkaline solution. UV irradiation of solid potassium nitrate induces partial photoisomerisation to potassium peroxonitrite, which has been previously reported to generate hydroxyl radicals during dissolution in aqueous solution. Support for hydroxyl radical generation was obtained and the hydroxyl radical was observed to be a key intermediate for strong chemiluminescence of luminol during dissolution of irradiated KNO3 in luminol sample solutions. Both lyoluminescence systems allow the detection of luminol down to sub-nM levels with linear calibration curves spanning five orders of magnitude of luminol concentration, suggesting that they can be used as tools in bioaffinity assays utilising luminol and isoluminol derivatives as labels.

Chemiluminescence determination of proteases by flow injection using immobilized isoluminol

A flow-injection system for protease determination is described. A tripeptide–isoluminol derivative was immobilized on an affinity support, and incorporated into the flow system. Injected enzyme sample solutions (0.025 cm3) catalysed the hydrolysis of some of the derivatives at pH 8.47, and the released isoluminol was detected by the chemiluminescence produced by its cobalt-catalysed oxidation with hydrogen peroxide. Linear calibration results were obtained for α-chymotrypsin (from 5 × 10–4 to 0.41 mg dm–3), trypsin (from 4 × 10–2 to 4 mg dm–3) and a commercial protease (from 2 × 10–3 to 4 mg dm–3). The limits of detection were 2.7 × 10–4, 4 × 10–2 and 2 × 10–3 mg dm–3, respectively.

Chemiluminescence of long-alkyl-chain N-mono- and N,N-disubstituted isoluminols in homogeneous and micellar media

The synthesis and spectral parameters of some N-mono- n-alkylated and N,N-di- n-alkylated long-chain isoluminol derivatives are reported and their chemiluminescence in homogeneous and micellar media is presented. The light reactions of the title compounds are less efficient in sodium dodecylsulphate (SDS) and polyethyleneglycol dodecylether (BRIJ-35) micelles compared with that in water, whereas higher quantum yields are observed in cetyltrimethylammonium chloride (CTAC), in all cases, by factors depending on the degree of alkylation. By measuring the chemiluminescence and fluorescence of 4- n-nonylamino-phthalic acid ( IIIa), a representative of the light reaction's primary emitter, it is found that this species forms micellar aggregates at concentrations above 4.5 × 10 −3 M.

On-line method for the generation of electrochemical reagent generation for liquid chromatography with luminol-based chemiluminescence detection

An on-line method for the generation of electrochemical reagent for liquid chromatography, with luminol-based chemiluminescence detection, has been developed. An ESA Coulochem guard cell, equipped with a porous graphite working electrode, operated at − 600 mV and inserted after the column, produces an oxidative reagent for the luminol-based reaction. This method has been compared with the conventional method with post-column addition of hydrogen peroxide as the oxidative reagent. With this novel method a detection limit of 0.15 pmol of ibuprofen (labelled with an isoluminol derivative) can be obtained, and a good alternative for post-column addition of hydrogen peroxide is presented.

A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis‐B surface antigen (Anti‐HBsAg)

A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3 U/l.

Luminescent immunoassay using isoluminol derivatives.

Isoluminol derivatives (ID) were early employed in the preparation of tracers for immunoassay. Their wide use was mainly due to their high quantum efficiency, low molecular weight, well-known chemical structure, low cost and high stability. Moreover the light efficiency of some ID may be modified by specific binding to the antibody, thus allowing the development of homogenous immunoassays requiring no bound/free separation step. Some ID are commercially available under both the amino terminal and carboxyl terminal form for conjugation to respectively carboxy derivative of steroid and amino residues of protein. The linking reaction can be commonly carried on via active esters chemistry and can be easily accomplished within one day. Steroid conjugates can then be rapidly purified by silica gel thin layer chromatography, and protein conjugates by gel filtration on a short disposable column. In the field of steroid studies, isoluminol derivative conjugates were prepared for the immunoassay of almost all compounds of clinical interest. When dealing with protein, both antigens and antibody were labelled in this way, resulting in highly specific activity tracers for competitive and non-competitive immunoassays. Recently the labelling of streptavidin with amino-buty-ethyl-isoluminol allowed the development of very sensitive immunoassay methods which take advantage of the biotin-avidin system.

Immunoassay labels based on chemiluminescence and bioluminescence

Reagents required for reactions that produce chemiluminescence (CL) or bioluminescence (BL) may be coupled to antibodies or antigens and used as labels for immunoassay. Because methods based on CL and BL have very low detection limits, they have the potential to replace assays that currently employ radioisotopes as labels. The feasibility of several BL and CL labels has been demonstrated. To date, isoluminol derivatives have been most widely studied. Several steroid assays involving isoluminol labels have been reported, and labelled compound has been detected at levels approaching 10(17) moles. Acridinium ester-labelled compounds have also been detected at this level. In addition to systems in which the label is a reactant required for light, CL and BL can be used to analyze the amount of product generated by enzyme labels. This approach has also yielded very low detection limits. Systems have been developed using enzyme labels that catalyze formation of ATP which is then assayed by the firefly reaction or that catalyze formation of peroxide which is determined by either luminol or peroxyoxalate CL.

Preparation and evaluation of steroid chemiluminescent tracers

Six different isoluminol derivatives have been synthesized and evaluated as potential labels for conjugation to steroid molecules. The resulting steroid-chemiluminescent tracers have been investigated in terms of detection limit and affinity for the homologous antibody. The conjugation to the steroid molecule did not significantly affect the quantum yield of the chemiluminescent label. Consequently the detection limit was very similar using tracers with the same chemiluminescent label and different steroids and it was always at picomolar levels. The affinity of the chemiluminescent tracer versus the homologous antibody was evaluated using tritiated steroids as reference. The bridge and the chemiluminescent molecule could influence the affinity of the chemiluminescent tracer which could be higher, similar or lower than that of the tritiated steroid. In conclusion some isoluminol derivatives (i.e. ABEI or AEEI) can be considered as universal labelling compounds for steroids and their metabolites, because they possess a high light efficiency (similar to luminal itself) and favourable reaction conditions. Moreover the conjugation to the steroid molecule does not modify their chemiluminescent properties.

Immunochemiluminometric assay for hepatitis B surface antigen.

A novel "sandwich" immunoassay for hepatitis B surface antigen monitored by chemiluminescence is described. The method involves use of an antibody-coated microtitration plate and requires 100 micro L of test specimen. The antigen binds to the antibody during the first 2-h incubation and, after an intermediate wash step, the sandwich is completed by 2-h incubation with antibody to antigen that has been labeled with an isoluminol derivative. A final wash step follows. A luminometer, built in-house, adds "microperoxidase" and peroxide, to initiate chemiluminescence, and provides automated readout at 10-s intervals. Results compare well in specificity and sensitivity with those of a comparison radioimmunoassay procedure. Within- and between-assay variability (CV) is 7 to 13% (n = 6). All reagents are stable at 4 degrees C for at least several months. Use of a non-radioisotopic label in this assay avoids the stability problems and inconvenience associated with radioactivity.

Chemiluminescence yields and detection limits of some isoluminol derivatives in various oxidation systems

Chemiluminescence yields and detection limits of some isoluminol derivatives in various oxidation systems