4,931,223 6
can, be hydrogen; an electron-witlidrawring group, such cally cleavable. Enzymatically cleavable substituents
as a perfluoroalkyl group having between I and 7 car- itclude phosphate ester groups representetl by the gen
bon atoms, inclusive, eg., trifluaromethyl; a halogen; eral formula:
C02K ZC02H, S03H, NOZ. ZN021 CýN, or ZCýN,
where Z is a branched or straight chain alkyl group 5 0 (IV)
having I to 7 carbon atom, inclusive, e.g., methyl, or an 11
aryl group having I or 2 rings, ttg-, ph=yl;- an electmn. -O-F-O-M+
donating group, e.g, a branched or straight chain I
O-M+
CI-C7 aftlY group, e-g-, methoxy or effioxy-, in aral
koxy group having I or 2 rings, e-g-, phenoxr, a 10 wherein M+ represents a cation such as alkali metal,
branched or straight chain CI-C7 hydroxyalkyl group, e.g., sodium or potassium; ammonium, or a CI-C7 alkyl,
e-g-, hydinixymethyl or hydroxyethyl; a hydroxyaryl aralkyl or aromatic quatervary ammonium cation,
group having I or 2 rings, eg., hydroityphenyl; a N(R7)4+ in which each R7 can be aikyl, eg., methyl or
branched or straight chain CI-C7 alkyl caff group, eg., ethyl, aralkyl, e.&, besuryl, or form part of a heterocy
acetate; or an aryl ester group having I or 2 rings6 eg., 15 clic ring systm r-g., pyridinium. The disodium salt is
benzoate; a heteroaryl group having 1 or 2 rings. eg.. particularly preferred. Such quaternary ammanium
beamoxazole, benzduazole, benzimidamole or benz- cations can also be connected through one of their
triazole; or hydrogen or an enzyme-cleavable or chemi- quaternizing groups to a polymeric backbotuý viz.
cally cleavable group Z as defined hereirt, with at least
one of R3 being an enzyme-cleavable group if no other 20 (In)
substiment on the diosetanc ring is a flutorophore group
having an enzyme-cleavable substituent. Furthermom
all of the R3 9MUPS together nan form a ring which cam
be substituted or unsubstatited. F T
The symbol T represents a stablizing group that pre- 25 _CH'j
CHN(R,),l
vents the diametane compound from decomposing be-
fore the bond in the labile ring substituent, e.g, the R3
enzyme-cleavable bond in an enzymLcleavable group,
on the 1whil-emittiag fluorophore-furmang fluorescent where n is greater than 1, or can be part of a p inter
chromophore group is intentionally cleaved, such as an 30 m1rY ammonium salt.
unsubstituted or substituted cycloalkyl, aryl, including Enzymatically cleavable substituents also include
fused aryl, or heteroaryl group, e.g., an unsubstituted CNIZZYme-cleavable alkimoyloxy groupsý e.g., an acetate
cycloalkyl group having from 6 to 12 ring carbon ester group. or an enzyme-cleavable oxacarboxylate
atoms, mclmve; a substituted cycloalkyl group having group, 1-phospho-2,3dincylglyceride group, 1-thio-D
from 6 to 12 ring carbon atoms, inclusive, and having 35 glucOsidc FOUP, adenosim triphosphate analog SMUP,
one or more substituents which can be an, alkyl group adenosine diphosphate analog group, adenosine mono
having from I to 7 carbon atoms, inclusive, or a hetero- phosphate analog group, adenosine analog group, a-D
atom group which can be an alkoxy group having from galactoside group, fl-D-galacloside group, a-D-glucu
I to 12 carbon atoms, inclusive, such as methoxy or side group, fi-D-glucoside group, tt-Dýmammside
ethoxy, a substituted or unsubstituted aryloxy group, 40 group, P-d-mannoside group, j8-D6firactofaranoside
such as phenoxy or carbox"henoxy, or an group, iS-D-glucosidaronate group, p-toluenesulfortyl
yloxy group, such as methoxycthoxy or polyethy- L-argaime ester group or p-toluenesulfonyl-Largunne
leneoxY, or a cYcloalkYlideris group bonded to the di- aridde group.
owtana ring through a spiro linkage and having from 6 Ile improved methods of using chemiluminescent
to 12 carbon atoms, hichistive, or a fused polycycloalk- 45 1,2-dioxetames of this invention are particularly usefud
yfidene group bonded to the dioxetime ring through a when the dicitetanes are employed as the meam ofiden.
spirri linkage and having two or more fused rings, each tifying or quantifying several analytes using otherwise
having from 5 to 12 carbon atoms, inclusivcý e.g., an art-recograzed mummoassays, such as those hitherto
adamant-2-ylidene group. employed to detect an enzyme or a member ofa specific
One or more of the substituents RI, R2atid T can also 50 binding pair, cg., an amigenýtibody pair or a nucleic
include a substituent which enhances the water solubil. acid paired with a probe capable of binding to all or a
ity of the 1,2-dioxetane, such as; a carboxylic acid, e.g., portion of the nucletc acid. Such assays include immit.
acetic acid, sWorde acid, tg., methamesulfonic acid or atiassays used to detect a hormone such as P-chorionic
ethimesufforac acid; or quaternary amino salt group, gonadotropin (j3-HCG), thyroid stimulating hormone
e.g., ammonium bronfidcý and at least one of Rt and R2, 55 MM follicle stimulating hormone (FSM, lutmmzmg
and preferably Rz, is one of the above-described light- hormone (HLR) or the like, call sturface receptor as,
enatting fluorophore-forming chromophore group con- says, and nucleic acid probe assays used to detect vi
raining an enzyme-cleavable group, and preferably an ruses, e.g., HIV or HTLV 111, herpes simplex virus
enzymaýclmvable phosphate ester group. (HSVý human papiloma. vim MM, Apd cytomegalo
Included among the labile ring substituents which 6o vims (CMV), or bacteria, e.g., E CANL, and histocom.
can be positioned on a fluorophore group to make up patibility assays; for typical assay protocols see working
the fluorophore moieties of this invention arvý substitu. exarnples I and Hjnfra, as well as the abovementioned
ents which, as indicated above, we cleaved to yield an copending Bronstein and Bronstein et al applications.
anion, e.g., an oxygen anion, a suffur anion, or a nitro- The improved methods of this invention can also be
gen anion such as a sulfonamido anion. Such substitu. 65 used in assays for chemical analytes, such as, potassium
ents can be chemically cleavable; a hydroxyl group, an or sodium inns, or in assays for substances such as inter
allumayl or aroyl ester group, or an alkyl or ary] sily- alia. cholesterol and glucose in which the analytes are
loxy group, for example, but preferably are enzymati- caused to decompose, for example using an enzyme
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