4,931,223 2
containing fluorophoric trifluoromethyl-nr benzo
METIIODS OF USING CHEMILUMUiESCENT Wazolyllientopyranyl residue), and each such com
IZ-D10XErANkS pound being structured so as to be cleavable by a differ
cut enzymatic cleaving means [e.g., one such compound
This application is a continuation-in-part of copend- 5 can contain, as mentioned above, a phosphate ester
iag Bronstein U.S. patent application Set. No. 889,823, group cleavable by a phosphatase or an acetate ester
"Method of Detecting a Substance Using Enzymatical- group cleavable by a cuboxylesterase, the other(s) can
ly-Induced Decomposition of Dioxetanes", filed July contain an a-D- orS-D-glucoside group cleavable by a
24,1986; Bronstein at al US. patent application Ser. No. glucose oxidese or a fl-D-galactoside group cleavable
140,035,"Dioxetanes for Use in Assays", filed Dec. 31, 10 by a fl-galactosidase], light of diffierent wavelengths can
1987 and Edwards U.S. patent application Set. No. be induced simultaneously or sequentially by the de
140,197, "Synthesis of 1,2-Dioxetanes and Intermediates composition of these differently configured and differ
Tberefor", filed Dec. 31, 1987, now abandoned. ently decomposable chemilummescent compounds.
FEELD OF THE INVENTION Hence, multi-channcl assays can be designed in which
is different enzymes attached to or associated with two or
Tlus invention relates to improved methods of using more different analytes will, by cleaving different en
chernflumineseent compounds, and especially enzymati- zyme cleavable dioxetane substituents, induce the emis
cally cleavable cheraffluminescent 1,2-dioxetane com- sion of light of a different wavelength for each analyte
pounds. More particularly, this invention relates to the ban assayed.
generation and detection of electromagnetic energy 20 Further, the emission of fight of different wave
released by the decomposition of enzymatically cleav- lengths by a multiplicity of decomposable chemilumi
able and chemically cleavable chemilumbleacent 1,2- nescent compounds6 e.g., in multi-charmel assays, can
dioxetane compounds used to determine the presence, also be accomplished by using one or more enzymati
concentration or structure of sidisibuices in a simple, cally cleavable chemiluminesocut 1,2-dioxetane com
especially an aqueous sample, particularly when such 25 pounds and one or more chemically or electrocherm
chemiluminescaut compounds me used to detect the cally cleavable chemiluminesceat compounds, such a;
preseDoc or determine the concentration of chemical or the chemically cleavable analogs of the enzymatically
biological substances by art-recognized mummoessay, cleavable 1,2-dioxetanes disclosed in the abovemn
teclindques, chemical assays or nucleic acid probe as- fioned Bronstein, Bronstemi at al and Edwards applica
says6 or when they a: used as direct chemical/physical 30 tions which for example contain, instead of an enzyme
probes for studying the molecular structures or micro- cleavable group, a chemically cleavable group such as a
structures of various macromoleculese synthetic poly. hydroxyl group, an alkanoyl or aroyl ester group such
mans, proteins, miclac acids and the like. as an scatoxy group, or an alkyl or aryl silyloxy group
BACKGROUND OF THE INVENTION such as a t-butyldimethylsilyloxy or t-butyldiphenyt
35 silyloxy group. together with one or more enzymes and
The decomposition of chicatilummescent chemical one or more chemical cleaving meamý each attached to
compounds to release electromagnetic energy, and es- a different substance, e.g., an analyte, to once again
pectally optically detectable energy-usually humnes- mduce the emission of light of a different wavelength
can= in the form of visible liglit-4i; well known and from each such decomposable chernfluminescent crun
understood. The incorporation of such light emitting 40 pound, a.&, for each analyte being assayed in a multi
reactants in art recognized inummoassays, chemical channel assay.
assays. nucleu; acid probe assays and chemical/physical It is, therefore, an object of this invention to provide
probe techniques as the means by which the analytc, a improved methods of using chemilummeaccut 1,2
substance whose presence, amount or structure is being diometane compounds, and especially enzymatically
determined, is actually identified or quantified has as- 45 cleavable chemiluminescent 1.2-dioutane compounds.
mined increasing importance in recent years, partiou- A further object of this invention is to provide im
holy with the advent of enzymadcally-cleirvable 1,2, proved methods of inducing the simultaneous genera
dioxetsoes;- see6 for example, the abovernentioned co- thin of light of different wavelengths by decomposing
pending Bronstein, Bronstein at &I and Edwards appli- differently configured and differently decomposable
cationst 50 chemilumirmscent compounds, including chemilumines
SUMMARY OF THE INVENTION cent 1,2-dioxetane compounds and, in particular, enzy
matically cleavable chemilumirmsceat 1,2-dioxetane
The present invention is based upon the discovery compounds.
that by using two or more enzymatically cleavable A still further object of this invention is to provide
(decomposable) chemiluminescent 1.2-ffioxetane corri- 55 multi-channel assays carried out in the presence of at
pounds, such as those disclosed in the aboversentioned least two differently configured and differently decom
copending Bronstein, Bronstein at al and Edwards ap- posable chemiluminescent compounds, including che
plications6 such compounds being configured, by means miluminescent 1,2-dioxetane compounds and, in parric.
of the inclusion of a different light-emitting fluoraphore ular, enzymatically cleavable chemiluminment 1,2.
moiety in each molecule, to emit light of a different 60 dioxetane compounds6 as substrates, each of which
wavelength from the other(s) upon decomposition [e.g., compounds emits light of a different wavelength from
one such compound can contain a fluorophoric courna- the other(s) and each of which has a labile substiment
rin (benzopyranyl) residue unsubstituted except for a cleavable by a different means from the other(s), to
labile ring substituent such as a phosphate ester or ace- detect the presence or determine the concentration, by
tate ester group, e.g., dispiro(adamantatte-2)-3'-(I',2'- 65 artýrecognized immunoassay, chemical assay and on
dioxetane)-4',2"-(7phosphýylaxy-3"ýhmmene) so- cleic acid probe assay techniques, of chemical or biolog
dium salt; see the abovementioned copending Bronstein ical substances, and to elucidate the molecular struc
at al application, the other(s) a labile ring substitnent. tures or microstructures of various macromolecules.
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