Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-04-20
2000-08-22
Lambkin, Deborah C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 19, 540 31, 548216, 549214, 549221, 549334, C12Q 168, C12Q 144, C07J 2100, C07D26352, C07D30500
Patent
active
06107036&
DESCRIPTION:
BRIEF SUMMARY
The invention concerns heterocyclic dioxetane substrates, processes for their production and their use in enzymatic analytical methods.
It has been known for a long time that oxiluciferin is formed by the reaction of luciferin with luciferase, oxygen and ATP. In this reaction light (wavelength maximum at 562 nm) is emitted as chemiluminescence. In this process a dioxetane is presumably formed as an energy-rich intermediate product (F. McCapra, Chem. Commun. 155 (1968)). Numerous chemiluminescent 1,2-dioxetane compounds have been developed based on this postulate.
The adamantyl residue has been described for the stabilization of the unstable 1,2-dioxetanes (EP-A 0 254 051, EP-A 0 352 713, WO 91/03479, WO 90/07511, WO 92/04341 as well as publications cited therein). The chemiluminescence yield then develops with the decay of the dioxetanes and is dependent among others on the fluorescent properties of the emitter formed. Although the phenolates that are formed on decay of the dioxetane derivatives that are usually used (3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetan e or corresponding dioxetanes halogenated on the adamantyl residue) have a good fluorescence quantum yield, the substrates have an overall low chemiluminescence. Dioxetanes with improved chemiluminescence yields would be particularly desirable in the triggered decay of dioxetanes.
Stabilized dioxetane derivatives are known from WO 93/20083 which decay into oxyluciferin and phenylthiazoline derivatives as emitters. Although these dioxetanes decay with a higher light yield than other known dioxetanes, they are very difficult to prepare synthetically.
Hence the object of the present invention was to provide new 1,2-dioxetanes which are stable and which only decay by reaction with an activating agent with the formation of a high light yield and which can be prepared by relatively simple chemical synthesis. This object is achieved by a compound of the general formula Ia or Ib ##STR2## in which R.sup.1 and R.sup.2 are each the same or different and represent hydrogen, straight-chain or branched C.sub.1 to C.sub.6 lower alkyl, individually or together a 3-6 carbon atom cycloalkyl or an aryl group (preferably phenyl or naphthly with optionally one or several substituted electron-withdrawing groups provided that only one of the residues R.sup.1 or R.sup.2 is hydrogen, agent, residues and is preferably in the ortho position relative to the O-R.sup.3 group, dioxetane structure and at most one of the groups R.sup.4 or R.sup.5 represents hydrogen, meanings for R.sup.1 and R.sup.2 or it is a double bond in a mesomeric system or it represents a carbonyl group and n denotes the number 0 or 1.
Acids, bases, salts, enzymes, inorganic or organic catalysts and electron donors are preferably used as activating agents.
The group O-R.sup.3 can be located at any position of the phenyl ring. If X is oxygen, the group O-R.sup.3 is preferably located in the 5-position in the case of compounds of the general formula Ia and where n is 0, in the case of Ib and where m equals 1 it is preferably in the 3-position and in the case of Ib and where m equals 2 it is preferably in the 4-position.
The group O-R.sup.3 is preferably a hydroxy salt or an oxy acid, phosphate, arylcarboxyl ester or alkylcarboxyl ester, alkyloxy, alkylsilyloxy or arylsiloxy, sulfate, oxypyranoside or a glyceridyl or phosphoryl residue or a steroid derivative. Phosphate and a dimethyl-tertiary butyl-silyloxy group are particularly preferred for O-R.sup.3.
The composition of the aryl and alkyl residues is not critical. Preferred aryl in this application are phenyl and naphthyl, and preferred alkyl residues are C.sub.1 -C.sub.6, unless otherwise noted. Any person skilled in the art can select suitable residues for R.sup.3 without difficulty. The only requirements are solubility and cleavability of R.sup.3 by the activating agents. Any reagent which is capable of removing the R.sup.3 group, and therefore provide an activated phenoxide ion which decomposes with light emission can be us
REFERENCES:
patent: 5770743 (1998-06-01), Schaap et al.
patent: 5773628 (1998-06-01), Akhavan-Tafti et al.
patent: 5929254 (1999-07-01), Matsumoto et al.
Adam Waldemar
Beckert Rainer
Heindl Dieter
Herrmann Rupert
Holtke Hans-Joachim
Lambkin Deborah C.
Roche Diagnostics GmbH
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