Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1999-01-07
2000-02-29
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 4, 435968, 435975, 546348, 546351, 560 1, 560 16, 560 20, C12Q 128, C12Q 100, G01N 3353
Patent
active
060308036
DESCRIPTION:
BRIEF SUMMARY
The invention relates to novel dibenzodihydropyridinecarboxylic esters, in particular acridanecarboxylic esters, and to their use as a chemiluminescent substrate in peroxidase assay methods.
Aromatic acridane esters have been known for more than 20 years (McCapra, Acc. Chem. Res., 1976; 9:201). The chemiluminescence of acridane esters proceeds in dipolar aprotic solvents upon addition of strong base. The mechanism is reported to involve a reaction with oxygen of the corresponding carbanion formed by the addition of the base. EP-A-625510 discloses certain aromatic acridane esters, in particular p-hydroxyphenyl 10-methylacridan-9-carboxylate, which can be used as a part of a signal reagent for the enzyme horseradish peroxidase (HRP). The signal reagent also contained an enhancer e.g. para-iodophenol, a chelating agent (EDTA), a nonionic surfactant, and hydrogen peroxide. According to EP-A-625510, HRP can be detected with great sensitivity at an optimal pH range of 8-9. More potent chemiluminescent polyfluoro-phenyl esters of acridane-9-carboxylic acid are disclosed in WO 95/23971 and phenyl thioesters are described in WO 95/28495.
The optimum pH for HRP is around pH 5. Therefore, it seems unsatisfactory that the optimum pH for chemiluminescence detection of HRP is at pH 8-9. This pH is presumably a compromise between the best pH for the for the enzyme and the optimal pH for intermediate acridinium ester chemiluminescence, which occurs at basic pH's (Akhavan-Tafti et al. in: Bioluminescence and Chemiluminescence. Fundamentals and Applied Aspect. Proceedings of the 8th International Symposium on Bioluminescence and Chemiluminescence. Cambridge, September 1994. Ed. Campbell, Kricka, Stanley. John Wiley and Sons 1994:199).
The present invention aims at an enzyme assay and at reagents to be used therein, which allow a higher sensitivity than the assay and reagents of the prior art. The present invention is based on the surprising discovery that certain aliphatic acridane esters and phenanthridane esters are capable of very intense and prolonged chemiluminescence upon reaction with hydrogen peroxide and certain enhancers. This reaction proceeds over a wide pH range (optimum pH range 6-8), and without the necessity of adding chelating compounds (EDTA) or surfactants. Moreover, these aliphatic esters produce a very low background luminescence, i.e. without enzyme.
Thus the invention is concerned with dibenzodihydropyridinecarboxylic ester having formula 1 or 2: ##STR1## wherein: R.sup.1 is substituted alkyl or cyano: substituted alkyl, aryl and substituted aryl; substituted aryl; and other substituents which are relatively resistant to oxidation: groups R.sup.5 or R.sup.6 may be bound together; and
The dibenzodihydropyridinecarboxylic esters according to the invention can be acridane-9-carboxylic esters having formula 1, or phenanthridane-6-carboxylic esters having formula 2.
A key feature of the compounds according to the invention is that the .alpha.-carbon atom of the ester alkyl group is an aliphatic carbon atom. It may be substituted with various aliphatic or other groups, designated in the above formulae 1 and 2 by R.sup.1, R.sup.2 and R.sup.3.
In the specification of the compounds according to the present invention, "alkyl" means straight or branched, saturated or unsaturated C.sub.1 -C.sub.24, preferably C.sub.1 -C.sub.12, most preferably C.sub.1 -C.sub.4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl and decyl. "Substituted alkyl" means alkyl as defined above containing one or more substituents such as halogen, hydroxy, alkoxy, alkanoyl, carboxyl, alkoxycarbonyl, alkanoyloxy, aryl, aryloxy, aroyl, aroyloxy, cyano, nitro and the like, especially halogen, hydroxy, alkoxy and aryl; the substituents themselves may also be substituted, e.g. with halogen, alkyl or alkoxy. "Aryl" means phenyl, naphthyl and higher carbocyclic aryl, as well as hetero-aryl such as furyl, thienyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidyl, quinolyl and the like. "Substituted aryl" means aryl as defined abov
REFERENCES:
patent: 5336772 (1994-08-01), Saiki et al.
R.R. Burtner, "Antispasmodics. II. Basic Esters of Some Polynuclear Carboxylic Acids", Journal of the American Chemical Society, vol. 65, 1943, pp. 1582-1585. Month not available.
F. McCapra et al., "Chemiluminescence Involving Peroxide Decompositions", Photochemistry and Photobiology, vol. 4, 1965, pp.1111-1121. Month not available.
Jacquemijns Marjorie
Zomer Gijsbert
De Staat der Nederlanden, vertegenwoordigd door de Minister van
Leary Louise N.
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