3 4,931,223
synthetic polymers, proteinsý nucleic acids and the like, straight chain alkyl group having I to 20 carbon atoms,
by ut-recogwed direct chemical/physical probe tech- inclusive, e.g., methyl, n-butyl or decyl; a branched or
niques. straight chain betercalkyl group having 11 to 7 carbon
These and other objects, as well as the nature, scope atoms, inclusive, e.g., methoxy, hydroxyethyl or by
and utilization of this invention, will become readily 5 droxypropyk an aryl group having I or 2 inigs, e.g.,
apparent to those skilled in the art from the following phenyl;- a heteroaryl group having 1 or 2 rings, e.g.,
description and the appended claim& pyrrolyl or pyrazolyl;- a cycloalkyl group having 3 to 7
DETAILED DESCRIPTION OF THE carbon atoms, inclusive, in the ring, e.g., cyclohexyl; a
DiVENTION licterocycloalkyl group having 3 to 6 carbon atoms,
10 inclusive, in the ring, eg., dioxanc; an aralkyl group
'Me enzymatically cleavable chemiluminescent 1,2- having I or 2 rings, eg., benzyl; an alkaryl group having
dioxetaxte compounds disclosed and claimed in the I or 2 rings, e.g., tolyl; an electron-withdrawing group,
abovementioned BronsteiE6 Bronstein et al and Ed- such as a perfluoroalkyl group having between I and 7
wards applications can be represented by the general carbon atoms, inclusive, e.g., trifluorumethyl;- a hdo
formula. 15 gels; C02H, ZC02H, S03FL N02, ZN02, C-N, or
ZC-N, where Z is a branched or straight chain alkyl
0-0 R M group having I to 7 carbon atoms, inclusiv% eýg.,
methyl, or in aryl group having I or 2 rings, e.g.,
phenyl; an electmis-domaing group, eg., a branched or
T. R2 20 straight Chain CI-C7 alkoxy group, e.g., methoxy or
ithoxy: - aralluixy group having I or 2 rings, e.g,
In this lornmila the symbol R1 represents hydrogen, or phenoxy; a branched or straight chain CI-C7 alkoxy
a bond when R2 represents a subsutuent bound to the group, e.g., xethoxy or ethoxy-, an aralkoxy group hav
dm"eume ring through a Spiro linkage, Or an Organic mg I or 2 rings, e.g., phenoxy; a branched or straight
substmient that does not interfere with the production 25 Chain CI-C7 hydroxyalkyl group, eg., hydroxymethyl
of light and that satiffies the valence of the dioxetane or hydroxyethyl;- a hydroxyaryl group having I or 2
ring carbon atom to which it is attached to result in a
tetravalent dlosessine ring Onzbon aton4 such as an a,_ rmgsv g., hydroxyphenyl; a branched or straight chain
kyl. arYL aralkyl, aficaryl, heteroalkyl, hetmaryL cy_ C,1, alkyl ester group, e.g., acetate; an aryl ester
cloalkyl or cyclohcteroalkyl group, e.g., a straight or 30 group having I or 2 rings, eg., benzDate; or a hetero"
branched chdin alkyl group having from I to 7 carbon . group having I or 2 rings, e.g., benzonzole, benz
atoms, inclusive; a straight or branched chain hydroxy- thiazOle, bmirimidazole or bertzmazole.
aIkYI group having from I to 7 carbon atoms, inclusive, The symbols R1 and R2, taken together, can be a
Or Ml -OR group in which R is a C1-C2o unbratiched fused fluorescent chromophore group bonded to the
Or branched, unsUbStifilted Or substituted, saturated or 35 dic'setalle ring through a spiro linkage, e.g, one having
=saturated alkYI, cYcloalkyl, cycloalkenyl, aryl, aral- the general formula:
kYl Or aralkanYl group, any of which my additionally
be fused to R2 such that the emitting fragment contains R3 (11)
a lactotte ring, a fused ring ycloadkyj, cycloalkenyl,
aryl, aralkyl Or aVaIkC31YI group, or an N, 0 or S 4o R3
heteroatoin-containing group; or a light-emitting
fluorophoreforniing fluorescent chromcphore group R,: R3
bonded to the dioxetfine ring through a single bond or a
Vim linkage, i.e., a group capable of absorbing energy R3 R3
to form za ezcdtd energy state from which it eunts 4s
optically detectable energy to return to its original en
ergy state, to which an enzyme-cleavable group is In this formula X is
bonded by a bond cleavable by an enzyme to yield an
electron-rich moiety bonded to the diozetaim ring, e.g., R4 R5
a bond which, when cleaved, Yields an Oxygen anion. a 50 C
sulfur anion or a nitrogen anion, and particularly an
WaidO, anion such as a suffammudo anion. prelerbly,
R1 is a methoxy group.
The symbol R2 represents hydrogen, or a bond when -0-, S Or -NR6 where each of R4, R5 and R6,
R1 represents a SUbstituent bound to the dioxetane ring 55 independently, is hydrogen, a branched or straight
through a sPirO linkage, or a light-emitting fluorophor,. chain alkyl group having I to 20 carbon atoms, iWlu_
forming fluorescent chromophore group bonded to the sive, e.g., methyl, n-butyl or decyl, a branched or
CUOXCMEC ring through a single bond or a spiro linkage. straight chain heteroalkyl group having I to 7 carbon
to which an enzYme-cleavable group is bonded by a atoms, inclusive, e.g., metboxy, hydroxyethyl or by.
bond cleavable by an enzyme to yield an electron-rich 6o droxypropyl; an Ml group having I or 2 rinA e.g.,
moiety bonded to the dioxetane ring, a.&, one of the phenyl; a heterriaryl group having I or 2 rings, e.g.,
aforementioned bonds which, when cleaved, yields an PY=lY1 Or PYrazolYl; a cYcloalkyl group having 3 to 7
Oxygen, Sulfitr or nitrogen aniom carbon atoms, inclusive, in the ring, e.g., cyclohexyl; a
The fight-emitting fluorophore-forming fluorescent heterocycloalkyl group having 3 to 6 carbon atom,
chrOmOPhOre groups which can be symbolized by Ri or 65 inclusive, in the ring, e.g., dioxane; an aralkyl group
R2 G811 be the residues of the aUXili,,y fluoophrS having I or 2 rings, e.g., benzyl; an alkaryl group hav
listed below, unSubstituted or substituted with one or ing 1 or 2 rings, eag., tolYl. or an enzynic-cleavable
more non-labile substituents such as a branched or group as defined above; and each R6, independently,
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Last modified: November 3, 2007