Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
Title of the Invention:
1,2 CHEMILUMINESCENT DIOXETANES OF IMPROVED PERFORMANCE
BACKGROUND OF TFiE INVENTION
Field of the Invention:
This invention pertains to 1,2 dioxetanes which are stable
under ambient conditions, and can be caused to chemiluminesce by
removal of a protecting group to leave an oxyanion, which
decomposes with the release of light. The dioxetanes are
typically stabilized by a tricycloalkyl moiety, which may be
spiro-bound, and bear an aryl group to which is bound the
protected oxygen atom and an electron active substituent. The
dioxetanes of this invention also include a halogenated oxy
substituent on the 2-carbon. The dioxetanes show improved
performance, enhanced sensitivity, and are suitable for use in
a wide variety of assays and other detection applications.
Backcrround of the Prior Art:
The assignee of the invention addressed herein, Tropix,
Inc., has pioneered and commercialized chemiluminescent
dioxetanes for use in a wide variety of applications, including
immunoassays, nucleic acid assays, artificial lighting materials
and the like. Additionally, Tropix has developed dioxetanes
which can be used to detect the presence of enzymes, including
proteases and other endogenous enzymes, and a variety of
exogenous enzymes, such as alkaline phosphatase, widely used as
a marker or label.
Such compounds, and their preparation in purified form, are
the subject of U.S. Patent 4,931,569. An early commercial
compound of this type is 3- (2' -spiroadamantane) -4-methoxy-4- (3" -
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
2
phosphoryloxy)-phenyl-1,2-dioxetane disodium salt, generally
identified as AMPPD, and available from Tropix, Inc. of Bedford,
Massachusetts. A variety of assays have been identified for
compounds of this type, including the mufti-analyte assay of U.S. ,
Patent 4,931,223, also assigned to Tropix, Inc. Use of these
compounds to generate a chemiluminescent signal which is easily
detected, and/or quantif ied, can be improved by the incorporation
in the assay of "enhancer" compositions, as is specifically
addressed in U.S. Patent 4,978,614 and extensively disclosed in
U.S. Patent 5,330,900, also commonly assigned herewith.
Typically, these enhancement agents have a quaternary onium salt
structure, such as poly(vinylbenzyltributylammonium chloride) and
poly(vinylbenzyl tributylphosphonium chloride) as well as the
corresponding phosphonium and sulfonium salts and can form
hydrophobic regions or areas within an aqueous environment, to
enhance chemiluminescence.
Commercially developed dioxetanes can be generally
represented by the structural Formula:
O O
Z I
As noted, among the "first generation" dioxetanes commercially
OX
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
3
developed, Y'-, Y2, and Z are hydrogen, and R is a methyl group.
In AMPPD, X is a phosphate group, while other "first generation"
dioxetanes have also been developed and disclosed, wherein X is
a different group which can. be cleaved by an enzyme. Potential
identities for X are well known, and include as well as
phosphate, acetate, various galactosides and glucuronides and in
general, any group susceptible to cleavage by an enzyme.
Representative identities are set forth in Table 1 of U. S . Patent
4,978,614, identified as Group Z. "Second generation" dioxetanes
have been developed, disclosed and patented, wherein one or more
of Y1 and/or Y2 of the above general Formula I have identities
other than hydrogen, so as to improve chemiluminescence
intensity, chemiluminescence kinetics, or both. Compounds of
this type bear an active substituent on the spiroadamantyl group,
that is, at least one of Y1 or YZ is a group other than hydrogen.
In an embodiment characteristic of this "second generation"
either bridgehead carbon bears a chlorine substituent (CSPD).
A wide variety of other active substituents are set forth in U. S .
Patent 5,112,960 and other patents assigned to Tropix, Inc.
Instead of a chlorine substituent, the adamantyl ring may bear
a methylene substituent, as recited in Claim 1 of U. S . Patent
5,326,882, to Tropix, Inc. The importance of control over enzyme
kinetics (including T1/2), light intensity and detection
sensitivity are stressed in U.S. Patent 5,112,960.
U.S. Patent 5,326,882 also discloses and claims "third
generation" tri-substituted phenyl compounds, that is, dioxetanes
of the structure set forth above, wherein each of Y1 and YZ may
be either hydrogen or an active group, and the phenyl ring bears
in addition to the enzyme cleavable group linked to the phenyl
through an oxygen atom, an electron' active substituent which
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
4
influences enzyme kinetics and/or chemiluminescence intensity.
This electron active.group, Z in the above Formula, can either
retard or accelerate the chemiluminescence obtained.
Chemiluminescence is produced after the cleavage of the enzyme-
cleavable X group of general Formula I by admixing or combining
a suitable dioxetane with a corresponding enzyme specif is for the
X moiety. This can be accomplished in an aqueous sample, as
discussed above, or on a membrane or other solid support.
Membranes and similar solid supports can be optimized for
increased chemiluminescent signal intensity and sensitivity of
detection, by providing a polymeric membrane as disclosed in U. S .
Patent 5,336,596 to Tropix, Inc.
The dioxetanes described above are specifically prepared for
use in connection with enzymatic assays. Thus, the X
substituent, whose removal induces decomposition and
chemiluminescence, is specifically designed to be removed by an
enzyme. The enzyme may be the target analyte in the sample
inspected, or it may be a reporter molecule attached to a probe,
antigen or antibody, or any member of a specific binding pair,
to detect the presence of the other member of the specific
binding pair. Assay formats of this type are well known, the
dioxetane chemiluminescence allowing the assay to be improved
such that highly efficient, precise and sensitive detection of
specific targets can be achieved.
It is also possible to select X such that it is not
susceptible to removal by an enzyme, but can be removed by a
specific family of chemicals. U.S. Patent 4,956,477 describes ,
various synthesis methods to prepare a wide family of dioxetanes
of general Formula 1, wherein X can either be an enzyme-cleavable
group, or a chemically cleavable group, such as a hydrogen atom,
CA 02231199 2004-12-16
WO 97/14692 5 PCT/US96/14390
an alkanoyl or aroylester, an alkyl or aryl silyloxy or similar
groups. Compounds of this type are also described in U.S. Patent
4,962,192, chaa , wherein the moiety X of general Formula I can
be either cleavable by an enzyme or removed by a chemical. In
its simplest form, X ~s hydrogen, whose departure can be
triggered by a wide variety of "activating agents", among the
simplest of which is sodium hydroxide. Because the decomposition
reaction produced by the removal of the cleaving group X produces
light through the decomposition of O-O bond of the dioxetane
ring, to produce two carbonyl-based compounds, where the
activating group is a chemical, only one photon of light can be
produced per molecule c~ activating agent. This should be
contrasted with the enzyme-triggerable dioxetanes discussed
above, wherein the enzyme, as a catalyst, triggers the
decomposition of many dioxetane molecules present as substrates.
This catalytic multiplying effect has led to the commercial
development and acceptance of enzyme-triggerable dioxetanes.
Advanced chemically (non-enzymatically) triggerable dioxetanes
are the subject of U.S. Patent No. 5,783,381, filed on October
19, 1995.
Accordingly, it remains an object of those of skill in the
art to obtain dioxetanes which give adequate chemiluminescence,
with appropriate emission kinetics, and which are triggerable by
activating agents including enzymes and non-enzymatic chemicals,
such that they can be used in assays wherein high light
intensity, improved enzyme kinetics, and high sensitivity are
requirements.
SUI~iARY OF T8E INVENTION
The above objects, and others made clear by the discussion
set forth below, is met by a new family of dioxetanes which can
CA 02231199 2004-12-16
WO 97/14692 PCT/US96/14390
6
be triggered to decompose and chemiluminesce by either enzymatic
triggering agents or chemical triggering agents, depending on the
identity of the protective group, which dioxetanes, by virtue of
the substituents selected on the stabilizing and aryl groups,
together with the provision of a halo-substituted alkoxy moiety
on the 2-carbon of the dioxetane, exhibit enhanced sensitivity,
enzyme kinetics and light intensity.
The dioxetanes are of the general formula II set forth
below.
OR
T- X-OZ
T is a polycycloalkyl dioxetane, preferably adamantyl dioxetane
and most preferably spiroadamantane. X is phenyl or other aryl.
The aryl moiety bears, in addition to the group OZ, 1-3 electron
active groups, such as chlorine or methoxy, as described in U.S.
Patent No. 5,582,980, filed April 25, 1994, which is a
continuation-in-part of U.S. Patent No. 5,538,847, filed May 7,
1993. By selecting the identity(s) of these electron-active
substituents, particular aspects of the chemiluminescent
properties of the dioxetane, including half-life, quantum
yield, S/N ratio, etc. can be altered.
Z is a protecting group which can be removed either by an
enzyme, such as a phosphate group removed by a phosphatase, or
O O
CA 02231199 2004-12-16
WO 97!14692 PCT/US96/14390
7
a protecting group which may be removed by a chemical triggering
agent, such as hydrogen, or substituted silyl moieties, which can
be removed by the addition of a base. Moiety R is of 1-20 carbon
atoms, and an alkyl, aryl, aralkyl or cycloalkyl, each of which
may include 1-2 heteroatoms which may be P, N, O or S which group
R is halogenated. Most preferably, R is a trihaloalkyl moiety.
When X is naphthyl, OZ is at a point of attachment,
preferably, to the naphthyl ring, in relation to the ring's point
of attachment to the dioxetane ring, such that the total number
of ring atoms separating these points of attachment, including
the ring atoms at the points of attachment, is an odd whole
number, in a fashion analogous to the substitution pattern
disclosed in U.S. Patent 4,952,707.
The polycyclic group T can be substituted with an electron
active group, including electron donating and electron
withdrawing groups, or may be unsubstituted. Preferred
substituents include hydroxyl, halo (preferably F and C1) and
alkyl. Preferred identities for R include fluorinated alkyls,
aryls, cycloalkyls or cycloaryls, including heteroalkyls, wherein
the carbons are partially or fully substituted with fluorine or
chlorine atoms.
Dioxetanes of this structure can be used to detect the
presence of the triggering agent, such as an enzyme or a base,
used in immunoassays in the fashion disclosed in U.S. Patent
5,112,960, in nucleic acid probes, in the fashion disclosed in
U.S. Patent 5,326,882, either alone, or in conjunction with
enhancement agents, such as those disclosed in U.S. Patent
4,978,614.
CA 02231199 2004-12-16
WO 97/14692 PC'T/US96l14390
8
These enhancement agents, which are generally water -soluble
macromolecules, both natural and synthetic, are further described
in U.S. Patent 5,145,772. Preferred enhancement agents include
Water-soluble polymeric quaternary opium salts, such as the
ammonium salts poly(vinylbenzyltrimethylammonium chloride) (TMQ),
poly(vinlybenzyltributylammonium chloride) (TBQ) and
poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ) as well
as their phosphonium and sulfonium counterparts.
DETAILED DESCRIPTION OF THE INVENTION:
The' dioxetanes of this invention, having the general formula
II
OR
'1' X-UG
show improved light intensity, enzyme kinetics, and/or
sensitivity, depending on the particular identities of the
substituent selected.
Substituent T is principally selected as a stabilizing
moiety. Dioxetanes without such stabilizing moieties
spontaneously decompose, or decompose under mildly elevated
thermal conditions. A polycyclic moiety, such as an adamantyl
O O
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
9
group, lends stability to the overall dioxetane. In preferred
embodiments, T is spiroadamantane. Either bridgehead carbon, or
both, may bear electroactive substituents, which may affect
enzyme kinetics and light intensity. Each bridgehead carbon (5
and 7 positions on the adamant-2-ylidene substituent) can be,
independently, hydrogen, a hydroxyl group, a halo substituent,
particularly fluoro or chloro, an unsubstituted straight or
branched chain lower alkyl group of 1-6 carbon atoms, a lower
alkyl group mono- or di-substituted with a hydroxy, 1-3 halogens
or similar substituent, a phenyl group, unsubstituted or
substituted with halogen or lower alkoxy substituents, a cyano
group, an amide group, and other substituents which are either
electron donating or electron withdrawing.
X in general formula II is either phenyl or naphthyl,
substituted with 1-3 groups A, which are independently selected
from a wide variety of identities which are electron active
substituents. The essential characteristic of the A substituents
on the phenyl or naphthyl moiety is that it does not suppress the
chemiluminescent behavior of the dioxetane, although it may alter
the emission kinetics. Preferred electron-active substituents
include halogen, such as chloro or fluoro; alkoxy, such as
methoxy, o-nitro-benzyloxy, or polythyleneoxy; aryloxy, such as
fluorophenoxy or 4-hydroxy-3-methyl-naphth-1-oxy;
trialkylammonium, such as trimethylammonium or trihexylammonium;
alkylamido, such as N-methyl-acetamido or N-phenyl-acetamido;
arylamido, such as benzoylamido or N-methylbenzolamido;
arylcabamoyl, such as phenylcarbamoyl, or nitrphenylcarbamoyl;
alkylcarbamoyl such as N-methylbenzylcarbamoyl or t-
butylcarbamoyl; cyano; nitro; ester, such as t-butoxycarbonyl;
alkkysulfonamido, such as N-methylmethanesulfonamido;
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
i0
arylsufonamido, such as N-methyltoluenesulfonamido;
trifluoromethyl; aryl or heteroaryl, such as phenyl,
benzothiazol-2-yl, or quinazolin-4-one-2-yl; alkyl, such as
methyl, butyl, phenethyl; trialkyl-; triaryl- or alkylarylsilyl-; -
such as trimethylsilyl; t-butyldimethylsilyl or t-butyl
diphenylsilyl; alkyl or arylamidosulfonyl, such as
dibutylamidosulfonyl or diphenylamidosulfonyl; alkyl or
arylsulfonyl, such as methanesulfonyl, trifluoromethanesulfonyl,
or benzenesulfonyl; alkyl or arylthioether, such as methylthio,
butylthio, or phenylthio. The size of the A substituent is
generally limited only by solubility concerns. Where reference
is made to alkyl or R, R', etc., the alkyl moiety should have 1-
12 carbon atoms. Suitable aryl moieties include phenyl and
naphthyl as exemplary moieties. Particularly preferred species
include chloro and alkoxy. Substitution at the four and five
positions is especially preferred.
The stable dioxetanes of this invention are caused to
chemiluminesce by deprotection of the phenoxy or naphthyloxy
group, by removal of group Z. Z may be either a protective group
removable by addition of a base or salt, or an enzyme-cleavable
moiety. Thus, Z may be H or E3Si, wherein E is independently
hydrogen, alkyl, aryl or arylalkyl, each of 1-12 carbon atoms.
Alternatively Z may be an enzyme-cleavable moiety. Thus, upon
proper contact with a suitable enzyme, Z is cleaved from the
molecule, leaving the oxygen attached to the phenyl ring, and
thus, the phenoxy anion. Z may be phosphate, galactoside,
acetate, 1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, ,
adenosine triphosphate, adenosine diphosphate, adenosine
monophosphate, adenosine, a-D-glucoside, ~i-D-glucoside, (3-D-
glucuronide, a-D-mannoside,,Ci-D-mannoside, (3-D-fructofuranoside,
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
11
~i-glucosiduronate, P-toluenesulfonyl-L-arginine ester, P-
toluenesulfonyl-L-arginine amide, phosphoryl choline, phosphoryl
inositol, phosphoryl ethanolamine, phosphoryl serine,
diacylglycerol phosphate diester and monoacylglycerol phosphate
diester. The phosphoryl and phosphate ester moieties may be
cleaved by, e.g., a phospholipase, generating a phosphate group,
which may be cleaved by a phosphatase. Z, if an enzyme-cleavable
group, is preferably phosphate, galactoside or glucuronide, most
preferably phosphate. Preferably, when substituted on the phenyl
ring, OZ is meta with respect to the point of attachment to the
dioxetane ring, that is, it occupies the three position.
Group R is a straight or branched alkyl, aryl, cycloalkyl
or arylalkyl of 1-20 carbon atoms. R may include 1 or 2
heteroatoms which may be P, N, S or O. The substituent R is
halogenated. The degree of halogenation will vary depending on
the selection of substituents on the adamantyl group, on the aryl
group, and the desired enzyme kinetics for the particular
application envisioned. Preferred groups include trihalo lower
alkyls, including trifluoroethyl, trifluoropropyl, heptafluoro
butyryl, hexafluoro-2-propyl, a-trifluoromethyl benzyl, a-
trifluoromethyl ethyl and difluorochloro butyl moieties. The
carbon atoms of substituent R may be partially or fully
substituted with halogens. When R is aryl preferred groups
include a phenyl ring substituted with one or more chloro,
fluoro, or trifluoromethyl groups, e.g. 2,5-dichlorophenyl, 2,4-
difluorophenyl, 2,3,5,-trifluorophenyl, 2-chloro-4-fluorophenyl
or 3-trifluoromethyl phenyl. Fluorine and chlorine are
particularly preferred substituents, although bromine and iodine
may be employed in special circumstances.
The halogen atoms, being particularly powerful electron
CA 02231199 2004-12-16
WO 97/14692 PCT/US96I14390
12
withdrawing groups, are involved in several different types of
interactions on the adamantyl, aryl and R substituents.
Additionally, polyhal.oalkyl groups are well known solubilizing
agents. Thus, selection of the degree and type of halogenation
on the R group will be made based on the identities and the
presence of halogens on the adamantyl and aryl group, as well as
the desired characteristics for the dioxetane.
One particularly preferred dioxetane within the scope of
this invention is
O O
O
I I
OPO-Na+
O-Na+
C1
Other compounds, for purposes of demonstration of the invention,
bearing similar structures have been prepared. Some of these,
bearing the designation "star" fall within the scope of this
invention. Comparative compounds, including CSPD, and TFE, have
been presented for purposes of comparison. CSPD is the subject
of the U.S. Patent 5,112, 960. TFE is addressed in commonly owned
U.S. Patent No. 5,773,628. These compounds do not constitute
part of the invention, but are included for the purposes of
comparison.
CA 02231199 1998-03-OS
WO 97/14692 13 PCT/US96/14390
O
I I
OPO-Na+
TFE-ADPStar
O-Na+
Cl
O
I I
OPO-Na+
TFE-ADPStar
O-Na+
C1
O
II
OPO-Na+
ADP -STAR
O-Na+
C1
O O
O
II
OPO-Na+
CDP STAR
O-Na+
C1
O O
O
II
- OPO-Na+
CSPD
O-Na+
O O
O O
O O
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
14
TFE-ADP-Star may be made by the following synthesis. 4-chloro-3-
methoxybenzoyl chloride. 4-chloro-3-methoxybenzoic acid, 3.7 g,
was wet down with chloroform, 3 mL, and thionyl chloride, 6 mL,
was added under an argon atmosphere. The resulting paste was -
subjected to reflux. After several minutes, the light yellow
solution was cooled to room temperature and another 4.0 g of the
substituted benzoic acid was added. The mixture was again
brought to reflux for one hour. A still head was added to the
flask in order to distill off chloroform and excess thionyl
chloride at the minimum temperature possible. The still pot
residue, containing the product, then solidified. Petroleum
ether, 5 mL, was added to dissolve the solid with slight heating.
Upon cooling to room temperature, the mother liquor was pipetted
off gently, and the remaining solid was pumped in vacuo to obtain
7.3 grams of 4-chloro-3-methoxybenzoyl chloride as a light tan
solid. Another 1.03 grams of less pure material could be
obtained from the mother liquor.
2.2,2-Trifluoroethyl 4-chloro-3-methoxybenzoate. 4-chloro-3-
methoxybenzoyl chloride, 2.3 g (11.2 mmol), was weighed out into
a round-bottom flask under argon. Methylene chloride dried over
3 A molecular sieves, 20 mL, was added, followed by
trifluoroethanol, 0.9 mL. The solution was stirred under argon
in a water bath at 10°C during the dropwise addition of
triethylamine, 1.7 mL. Upon warming to room temperature, the
slurry was stirred for one hour. Water, 20 mL, and methylene
chloride, 10 mL, were added to the flask. The mixture was
transferred to a separatory funnel, rinsing the reaction flask
with methylene chloride. The lower organic layer was removed.
The aqueous layer was extracted with methylene chloride, 10 mL.
The combined organics were washed several times with water,
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
passed through cotton and rotary-evaporated to an oil which
solidified to yield 2._55 g. of a light tan product. TLC of the
solid exhibited one spot (K5F Si02/methylene chloride:hexanes,
1:1) with an Rf value of 0.68. The infrared spectrum showed C=O
(ester) stretch at 1737 cm-1.
2-Chloro-5-(2.2,2-trifluoroethoxy-tricyclo[3.3 1 13~')dec-2-
ylidenemethyl) anisole. Titanium trichloride, 7.9 g (51.3 mmol),
was weighed into a round-bottomed flask in a glove bag under an
argon atmosphere. Freshly distilled tetrahydrofuran (from LAH),
60 mL, was added quickly under argon flow. Care should be taken,
as this addition can be exothermic. The purple suspension was
stirred vigorously so as to break up the solid adhering to the
walls of the flask. After 15 minutes, zinc dust, 5.2 g, was
added all at once under argon with continued stirring. A
moderate exotherm resulted in a reddish-brown mixture which did
not contain black suspended solids. After 15 minutes stirring,
the triethylamine, 11 mL, was added with exclusion of air. The
mixture was then refluxed for 2 hours. A solution of 2-
adamantanone, 3.0 g (20 mmol), and 2,2,2-trifluoroethyl-4-chloro-
3-methoxybenzoate, 2.6 g (10 mmol) in dry THF, 30 mL, was added
dropwise to the refluxing brown mixture over approximately 65
minutes. Reflux was continued overnight for 16 hours. The
solvents were rotary evaporated from the cooled reaction mixture
to yield a brown-black gum. This gum was triturated with
hexanes, 100 mL, and ethyl acetate, 20 mL. The yellow-orange
supernate was decanted and the trituration procedure was
repeated. After 10 minutes of agitation, triethylamine and
methanol, 5 mL each, were added.The gum began to stiffen and
eventuallybecame a lumpy solid. The solid was broken up the
and
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
Zs
supernate decanted. One final trituration was accomplished with
20~ ethyl acetate-hexanes. The combined decanted supernates were
rotary evaporated to a light yellow, semisolid paste. The
mixture was treated with hot hexanes, 50 mL, and filtered warm
to remove some insolubles. The residue obtained after rotary
evaporation of the filtrate was applied to a 2.1 X 20 cm column
of activity I aluminum oxide as a slurry in a small amount of
warm hexanes. The column was eluted with hexanes to obtain
adamantylidene adamantane. The elution was continued with 10%
dichloromethane-hexanes to obtain an oil which contained two
major mid Rf, UV-active components. The oil was taken up in a
small amount of hot hexanes. A crystalline, colorless solid,
weighing 0.718 and having a melting point of 108-112°C,
precipitated out upon cooling.
I.R. (CHaCla): 2920, 2850, 1590, 1572, 1485, 1465, 1450, 1400,
1203, 1160, 1100, 1065, 1035, 993, 965, 870, 828 cm-'-.
1H NMR (400 MHz-CDC13) : 8 1.75-1.96 (m, 14H) 2.60 (s, 1H) ; 3.27 (s,
1H) ; 3 . 70-3 .76 (m, 2H-CH.,CF3) ; 6. 80-6 . 89 (m, 2H) ; 7.31-7.33 (m,
1H) .
2-Chloro-5- (2 , 2 , 2-trifluoroethoxy tricyclo f3 3 1 13~'] dec-2-
ylidenemethyl) phenol Sodium hydride (60% in mineral oil), 0.12
g, was washed three times with hexanes under an argon atmosphere.
DMF, 6 mL, was added, and the slurry was cooled to 0°C in an ice
bath. With magnetic stirring, ethanethiol, 0.206 mL, was added
dropwise with evolution of hydrogen gas. The mixture was then
allowed to warm to room temperature over 15 minutes. 2-Chloro-5-
(2,2,2-trifluoroethoxy tricyclo[3.3.1.13~'] dec-2-ylidenemethyl)
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
17
anisole, 0.70 g, was added as a solid under argon flow. The
flask was placed in an oil bath at 120°C and stirred for one
hour. The cooled mixture was then diluted with ethyl
acetate:hexanes (1:1), 20 mL. An equal volume of aqueous 1M
NH4C1 was added with vigorous swirling. The biphase was
transferred to a separatory funnel where the organic layer was
extracted again with 20 mL 0.5 M NH4C1 solution, and then twice
with 20 mL volumes of water. The organic layer was dried over
Na2S04 and pumped in vacuo to an oily product . TLC showed a
single W-active spot at Rf 0.56 (KSF; methylene
chloride:hexanes, 1:1).
IR(neat): 3540 (OH), 3455 (OH), 2910, 2850, 1600, 1570, 1482,
1447, 1410, 1280, 1100, 1052, 996, 887, 855, 823, 800, 732 cm-'-.
Disodium 2-Chloro-5-(2.2,2-trifluoroethoxy-
tricyclo f3 .3 . 1 .13~'7 dec-2-ylidenemethvl) phenyl phosphate. 2-
Chloro-5- (2, 2, 2-trifluoroethoxy tricyclo [3 .3 . 1 . 13~'] dec-2-
ylidenemethyl) phenol, 490 mg (1.31 mmol), was dissolved in 10
mL dry THF. Triethylamine, 238 microliters, was added dropwise
by syringe under argon. The solution was cooled in an ice bath
for the dropwise addition of 2-chloro-2-oxo-1,3,2-
dioxaphospholane, 143 microliters. The slurry was warmed to room
temperature and stirred for 3 hours. The supernate was removed
from triethylamine hydrochloride using a cotton-tipped syringe
under argon flow. The solid was washed with two aliquots of dry
THF, 5.0 mL each. The solvent was stripped from the combined
filtrate to give a straw-colored gum. This was taken up in dry
DMF, 4.0 mL, and treated with dry NaCN, 0.077 g, under argon.
The mixture was allowed to stir overnight at room temperature.
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
18
The solvent was stripped in vacuo at 50-55°C. The orange gum
obtained was dissolved in anhydrous methanol, 5.0 mL, and treated
dropwise with a methanolic solution (4.3 M) of sodium methoxide,
320 microliters. After stirring for 45 minutes at room _
temperature, a sample was removed for analytical HPLC on a
Polymer Laboratories PLRS-S polystyrene, reverse-phase column.
A gradient of acetonitrile, running against 0.1% sodium
bicarbonate showed the product eluting at 12.24 minutes. The
methanol was removed from the reaction mixture and the residue
was dissolved in water, 50 mL. This solution was filtered and
preparatively purified with two injections onto a large, one inch
PLRP-S column. An acetonitrile/water gradient was applied which
allowed the major product to be collected in two separate
fractions. These were combined and lyophilized to yield a
slightly off-white, fluffy solid weighing 517 mg.
1H NMR (D20) : b 1.79-1.96 (m, 14H) ; 2.65 (s, 1H) ; 3.22 (s, 1H) ;
4.01-4.08 (m, 2H); 7.02-7.04 (m, 1H); 7.45-7.50 (m, 2H). This
data supports the structure.
Disodium 2-chloro-5-(4-(2.2,2-trifluoroethoxy)spiro,l 2-
dioxetane-3,2'-tricvclo 3.3.1.13~'] decan ~yl-phenyl phosphate
(TFE-ADP-Star) .
480 milligrams of chromatographed disodium 2-chloro-5-(2,2,2-
trifluoroethoxy tricycloC3.3.1.13~']dec-2-ylidenemethyl)phenyl
phosphate was placed in a tube and wet down with anhydrous
methanol, 4 mL. Chloroform, 40 mL, was added to obtain a
solution. A solution of tetraphenylporphine (TPP), 6 milligrams
in 3 mL chloroform, was then added to yield a pink-purple
reaction mixture. The solution was cooled in an ice bath while
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
7. 9
sparging with oxygen through a Pasteur pipette for 5 minutes.
The gas flow was continued while the solution was irradiated with
a 400 watt sodium vapor lamp which had been filtered with a 3.0
- mil thickness of DuPont Kapton film. After 40 minutes, a small
sample was blown down and dissolved in water which contained 0 . 1
NaHC03. Analytical HPLC, under conditions described above for
the starting material, indicated a new product which eluted at
12.1 minutes. The starting material, eluting at 12.5 minutes,
was present in just a trace amount. The reaction was stripped
of solvents and pumped to yield a dark red foam. This was
dissolved in 80 mL water which contained 5 drops of 0.5 M NaOH.
The solution was filtered to remove insolubles, and was
preparatively chromatographed on a one inch PLRP-S column
(Polymer Laboratories) using an acetonitrile/water gradient. The
product peak was shaved on the front and in the back, collecting
only the middle cut. This eluant was freeze dried to obtain 293
milligrams of the product as a white solid. That the product was
the entitled 1,2-dioxetane, was confirmed by enzymatic triggering
of the material with alkaline phosphatase to produce light.
1H NMR (400 MHz, D20) : b 0.87-0.90 (d, 1H) ; 1.17-1.20 (d, 1H) ;
1.20-1.71 (m, lOH); 2.17(x, 1H); 2.86 (s, 1H); 3.83 (br.m, 2H);
7.11 (br.m, 1 H); 7.38-7.40 (m, 1H); 7.68(br.m, 1H). This data
supports the compound structure.
' TFE-CDP-Star may be made by the following synthesis.
2 - C h 1 o r o - 5 - ( 2 , 2 , 2 - t r i f 1 a o r o a t h o x y - ( 5 ' -
chlorotricyclof3.3.1.13~'7dec-2-ylidenemethyl) anisole Titanium
trichloride, 10.1 g (65.6 mmol), was weighed into a round-
bottomed flask in a glove bag under an argon atmosphere . Freshly
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
distilled tetrahydrofuran (from LAH), 70 mL, was added quickly
under argon flow. (Care should be taken, as this addition can be
exothermic.) The purple suspension was stirred vigorously so as
to break up the solid material adhering to the walls of the _
flask. After 15 minutes, zinc dust, 7.1 g, was added all at once
under argon with continued stirring. A moderate exotherm
resulted in a reddish-brown mixture which did not contain black
suspended solids. After 20 minutes stirring, the mixture was
cooled in an ice bath and several drops of triethylamine were
added with exclusion of air. After an exotherm, an additional
12 ml N(Et)3 was added. The mixture was then refluxed for 2
hours. A solution of 5-chloro-2-adamantanone, 3.7 g (20 mmol),
and 2,2,2-trifluoroethyl 4-chloro-3-methoxybenzoate, 2.6 g (10
mmol) in dry THF, 40 mL, was added dropwise to the refluxing
brown mixture over approximately 35 minutes. Reflux was
continued for 2.5 hours whereupon TLC showed unreacted ester
starting material. An additional 1.10 g of the chloro-
adamantanone was added as a solid under argon flow. After
another 2.5 hours of reflux, the cooled reaction mixture was
stirred overnight at room temperature. The solvents were rotary
evaporated from the cooled reaction mixture to yield a brown-
black gum. This gum was triturated with hexanes, 100 mL, and
ethyl acetate, 20 mL. The yellow-orange supernate was decanted
and the trituration procedure was repeated. After 10 minutes of
agitation, triethylamine and methanol, 2 mL each, were added.
The gum began to stiffen and eventually became a lumpy solid.
The solid was broken up and the supernate decanted. One final
trituration was accomplished with 5% ethyl acetate-hexanes. The
combined decanted supernates were rotary evaporated to a light
yellow oil. The residue obtained after rotary evaporation of the
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
21
filtrate was applied to a 2.1 x 20 cm. column of silica gel. The
column was eluted with 5 to 10°s ethyl acetate in hexanes to five
fractions which contained a mid Rf UV-active spot, contaminated
by iodine-sensitive spots both above and below. These fractions
were rotary evaporated to give 2.52 g of a semisolid paste. An
I.R. spectrum showed absorptions at 1583 and 1567 cm-1. The
impure product as used directly in the next reaction step.
2 - C h 1 o r o - 5 - ( 2 , 2 , 2 - t r i f 1 a o r o a t h o x y - ( 5 ' -
chloro)tricyclof3.3.1,13~'ldec-2-ylidenemethv)phenol Sodium
hydride (60o dispersion, 240 mg.; 6.0 mmol) was washed with
hexanes three time under argon. DMF, 17 mL, was added. The ice
cooled slurry was treated dropwise with ethanethiol (450
microliters; 6 mmol) from a syringe. After warming this solution
to room temperature, the evolution of hydrogen was complete. The
solution was added by pipette (argon) to the product of the
preceding reaction. The mixture was stirred and heated under
argon at 120°C for 1.25 hours. The cooled reaction mixture was
partitioned in 70 mL 40o hexanes in ethyl acetate against 70 mL
of 1 M ammonium chloride solution. After washing again with
water, 3 times, the organic layer was dried over sodium sulfate.
The solvents were stripped and the residue was chromatographed
on aluminum oxide using 50:50 ethyl acetate/hexanes, followed by
ethyl acetate and finally by 2°s methanol in ethyl acetate. The
lower Rf product was obtained from 2 fractions as an amber gum
weighing 0.58 g. The PMR spectrum showed impurities, but the
major absorptions for the desired product were found at:
1H NMR (400 MHz-CDC13) : b 1.56-2.21 (m, 13H) ; 2.73 (s, 1H) ;
3.45(s, 1H); 3.70-3.76, (m, 2H-CH~CF3); 5.62 (br. s., 1H);
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
22
6.78-7.33(m, 3H).
The product was found to be sufficiently pure to be converted to
the cyanoethyl phosphate diester enol ether as follows:
1.2 g of the phenol (2.95 mmol) in 12 mL dry THF was treated with
535 microliters of triethylamine. The mixture was stirred at 0°C
under argon while 2-chloro-2-oxo-1,3,2-dioxaphospholane
(326 microliters, 3.54 mmol) was added dropwise by syringe. The
resulting suspension was stirred at room temperature for 3 hours
and filtered under argon. The precipitate was washed with
3 X 10 mL of 3.1 dry THF/dry ether. The filtrate was pumped to
an amber oil which was dissolved in 7 mL anhydrous DMF. The
solution was treated with 60 mg dry NaCN and stirred overnight
at room temperature. The DMF was removed in vacuo at 50°C to
provide the orange, gummy cyanoethylphosphate diester which was
used directly in the next step.
Disodium 3-(4-(2,2.2-trifluoroethoxy)spiro'1 2-dioxetane-3
~' - (5' -chloro) tricyclo 3 .3 . 1 13''1 decan,~ -4-yl) phenyl phosphate
The phosphate diester was placed in a tube and wetted down with
anhydrous methanol, 4 mL. Chloroform, 40 mL, was added to obtain
a clear solution. A solution of tetraphenylporphine (TPP),
6 milligrams in 3 mL chloroform, was then added to yield a pink-
purple reaction mixture. The solution was cooled in an ice bath
while sparging with oxygen through a Pasteur pipette for 5 '
minutes. The gas flow was continued while the solution was
irradiated with a 400 watt sodium vapor lamp which had been
filtered with a 3.0 mil thickness of DuPont Kapton film. After
1.5 hours, a small sample was blown down and dissolved in water
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
33
which contained NaOH. Analytical HPLC of this basified sample,
under conditions described above, indicated that the major
product eluted at 12.6 minutes. Collection. of this peak,
dilution into pH 10 aqueous diethanolamine buffer, and addition
of excess alkaline phosphatase gave a burst of light lasting
3 minutes. The reaction was stripped of solvents and pumped to
yield a dark red gum. This was dissolved in 10 mL methanol, and
treated with 690 microliters of 4.37 M sodium methoxide in
methanol. After 40 minutes at room temperature, half of the
methanol was removed on the rotary evaporator. Water, 60 mL, was
added and the solution was filtered through 0.45 micron nylon
filters, rinsing with another 20 mL of water. This solution was
preparatively chromatographed on a one inch PLRP-S column
(Polymer Laboratories) using an acetonitrile/water gradient. The
product peak was shaved on the front and in the back, collecting
only the middle cut. This eluant was freeze dried to obtain
605 milligrams of the product as a white solid.
1H NMR (400 MHz-D20) : a 0.82-2.22 (m, 11H) : 2.38 (br. s. , 1H) ;
3.05 (s, 1H); 3.86 (br. m., 2H); 7.10-7.71 (m, 3H)
Wittia-Horner-Emmons Synthetic Pathway
An alternate methodology for the synthesis of fluoroalkoxy 1,2-
dioxetanes of the invention involves the Wittig-Horner-Emmons
~ condensation of a-fluoroalkoxy arylmethane phosphonate esters
with 2-adamantanone or substituted 2-adamantanones.
Konenigkramer and Zimmer (J. Org. Chem., 45, 3994-3998, 1980),
describe the reaction of benzaldehyde with triethylphosphite and
chloromethylsilane to give diethyl 1-trimethylsiloxy-1-
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
24
phenylmethanephosphonate. Creary and Underiner (J. Org. Chem.,
50, 2165-2170, 1985) further detail the conversion of the related
a-trimethylsiloxy substituted-phenylmethanephosphonate esters to
the corresponding cx-fluoroalkoxy phosphonates via the a-mesyloxy _
or a-trifloxy derivatives. Using this route with
trifluoroethanol, one of the possible fluorinated alcohols, 4-
chloro-3-methoxybenzaldehyde yields diethyl 1-(2,2,2-
trifluoroethoxy)-1-(4-chloro-3-methoxyphenyl)methane phosphonate.
This is then condensed with 5-chloro-2-adamantanone to yield an
enol ether which is subsequently converted to an enzyme
triggerable dioxetane in accordance with the examples provided.
One of skill in the art would also be aware that a sodium
salt of the appropriate alcohol or phenol could be reacted with
the benzyl halide derivative of the aldehyde in the resence of
assisting silver salts to give a haloalkyl or haloaryl acetal
which could then be utilized to synthesize other arylmethyl
phosphonates and enol ethers by Wittig Horner Emmons reaction.
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
CHO (HaC)3Si0 (OEt)Z
P (OEt) z
_ / 0~3 TMS-Cl
vc:n;
CF3COOH-
MeOH
HsCSO t) s HO~~PQ (OEt) _
CHaSOaCl ( \
3 OCH~
CF3 (CF2) nCH~OIi
FsC (F2C) nHZCO PO (OEt) a
OCB;
The phosphonate ester shown is synthesized by the following
method:
Svn.thesis of 4~-Chloro-3-methoxyphenvl (2 , 2 , 2-
~ trifluoroethoxy)diethyl-phosphonate.
The 2,2,2-trifluoroethoxydiethylphosphonate (compound 3, below)
was synthesized from bis(2,2,2-trifluoroethyl)4-chloro-3-
methoxybenzaldehyde acetal (compound 2, below) which was obtained
from the 4-chloro-3-methoxybenzaldehyde dichloroacetal (compound
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
26
l, below).
4-Chloro-3-methoxybenzaldehyde dichloroacetal (1).
4-Chloro-3-methoxybenzaldehyde (1.5 g, 8.8 mmol) was added to
PCIS (2.45 g, 11.8 mmol) under argon and was stirred for 15 min.
while heat evolved from the liquefied mixture. Methylene -
chloride (3 ml) was added and the solution was stirred overnight
at room temperature; TLC analysis indicated a trace of W
activity from the starting benzaldehyde remained. Additional
CH2C12 (15 ml) and saturated NaHC03 solution (40 ml) were added
and the solution was stirred for 1 hr at room temperature to
quench the reaction. The reaction mixture was partitioned
between CH~C12 and water, the aqueous layer was washed once with
CHZC12 and the combined organic layers were dried over NaZS04.
The product was recovered after evaporating the solvent under
pressure and pumping to dryness on high vacuum. Purification of
the oil on silica gel, eluting with 4% EtOAc/hexanes, yielded
1.73 g (87%) of dichloroacetal 1 as a light yellow oil.
1H NMR (CDC13, ppm) : 3.94 (3H, s) ; 6.65 (1H, s) ; 7.03 (1H, dd,
J=2 Hz, 8 Hz) ; 7.15 (1H, d, J=2 Hz) ; 7.35 (1H, d, J=8 Hz)
Bis(2,2,2-trifluoroethyl)4-chloro-3-methoxYbenzaldehyde acetal
Dichloroacetal 1 (835 mg, 3.7 mmol) was dissolved in 2,2,2-
trifluoroethanol (4 ml) and cooled to 0°C. Sodium hydride (429
mg, 60% in oil, 10.7 mmol) was added in portions to the solution
at low temperature to generate sodium 2,2,2-trifluoroethanoate
in situ. After adding anhydrous silver carbonate (2.1 g, 7.6
mmol) , the reaction mixture was heated at 80°C for 1 hr to effect
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
27
acetylation. Upon cooling, water and EtOAc were added with
stirring and the solution was filtered through a cotton plug to
remove solids. The solids were rinsed well with EtOAc and the
resulting organic solution was partitioned between EtOAc and
water. The EtOAc solution was dried over Na2S04, decanted and
evaporated to an oil which was purified on a silica gel column
(0-1°s EtOAc/hexanes) to yield 1.13 g (860) of
trifluoroethylacetal 2 as an oil. A small amount of product (42
mg) was further purified on a prep TLC plate for spectral
analysis.
IR (CHC13, nm-1): 2940, 1590, 1580, 1482, 1457, 1405, 1267, 1164,
1064, 1029, 964, 870
1H NMR (CDC13, ppm) : 3 . 83-3 . 94 (4H, m) ; 3 . 90 (3H, s) ; 5, 82 (1H,
s); 7.00-7.02 (2H, m); 7.39 (1H, d, J=8.5 Hz)
4-Chloro-3-methoxvphenvl(2,2.2-trifluoroethoxv)diethvlphos~honate
Triethyl phosphate (530 ~.1, 3.1 mmol) and boron trifluoride
etherate (420 Er.l, 3.4 mmol) were added, at 0°C under argon, to
trifluoroacetal 2 (1.09 g, 3.1 mmol) dissolved in CH2C12 (10 ml).
The reaction mixture was allowed to warm to room temperature with
stirring over 3 hrs. During this time additional triethyl
phosphite (100 ~.1, 0.6 mmol) and boron trifluoride etherate
(250 ~,1, 2.0 mmol) were added to complete the reaction. The
solution was partitioned between CH2C1~ and dilute brine and the
aqueous layer was washed well with CHZCIz. The combined organic
layers were then washed with saturated NaHC03, dried over Na2S04,
decanted and evaporated. The colorless oil was purified on a
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
28
silica gel column flushed with hexanes and eluted with 0-50°s
EtOAc/hexanes to give 752 mg (62~) of phosphonate 3 as a clear
oil. A small sample (47 mg) of the oil was further purified on
a prep TLC plate for spectral analysis.
IR (CHC13, nm-1); 2990, 1590, 1581, 1484, 1460, 1410, 1277, 1255,
1165, 1113, 1051, 1030, 970
HCCl z HC (OCH =CFa ) Z
HaC~3aCFa. NaH. AgC03, 80°C
~OMe ~OMe
1 1
1 2
(St0) aP (O) CH (OCH aCFa)
(Et0)3P. BF=~Et=O. CHZCla
OMe
1
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
29
We performed comparisons of chemiluminescence half-lives
(tl/2) for the following dioxetanes:
Determin.atior.~ ~f the Chemiluminescence Half-Life of
Deghosphorylated Dioxetane.
A 1 mL aliquot of each dioxetane (0.004 mM) was equilibrated to
30°C in 0.1 M diethanolamine, 1 mM MgCl2, pH 10. Alkaline
phosphatase (at final concentration of 1.05 X 10'9 M) was added
to the test tube and the chemiluminescent signal kinetics was
measured in a Turner TD-20E luminometer for 10 to 20 minutes.
The half-life was calculated from the plot of log RLU versus
time. For TFE and TFE-ADP-Star, the chemiluminescent half-life
was also determined in the presence of Sapphire II enhancer (in
0.1 M diethanolamine, 1 mM MgCl2, 10% polyvinylbenzyltributyl
ammonium chloride at 1 mg/mL). The results are listed in TABLE
I:
TABLE I
DIOXETANE T1/2 (MINUTES)
TFE-ADP-Star 2.0
TFE 0.9
ADP-Star 2.~
CDP-Star 1.2
CSPD 0.6
TFE/Sapphire II 15.5
TFE-ADP-Star/Sapphire II 5.0
Determination of the Peak Light Intensity of Dephosphorylated
Dioxetanes
A 0.5 mL aliquot of dioxetane (0.004 mM) was equilibrated to 30°C
in 0.1 M diethanolamine, 1 mM MgClz, pH 10. Alkaline phosphatase
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
(final concentration of 1.05 X 10-9 M) was added to the tube and
the chemiluminescent .signal was measured in a Turner TD-20E
luminometer for 10 to 20 minutes. The peak light intensity was
recorded and is shown in TABLE II
TABLE II
DIOXETANE PEAK INTENSITY (RLU) '
TFE-ADP-Star 390
TFE 282
ADP-Star 682
CDP-Star 863
CSPD 200
Detection of Biotinylated pBR322(35 mer) on Nvlon Membrane
Serial dilutions of biotinylated pBR322 (35 mer) were spotted onto
strips of Tropilon-Plus nylon membrane. Spots correspond to 210
pg, 70 pg, 23.3 pg, 7.7 pg, 2.6 pg, 0.86 pg, 0.29 pg, 0.10 pg,
32.0 fg, 10.7 fg, 3 .6 fg and 1.2 fg of biotinylated pBR322 (35
mer). After spotting, the DNA was cross-linked to the membrane
by UV fixation, the membranes were blocked with 0.2% I-Block, 5%
sodium dodecyl sulfate (SDS) in phosphate buffered saline(PBS)
(Blocking Buffer I) , incubated with a 1-5000 dilution of Avidx-AP
in Blocking Buffer I for 30 minutes, washed twice with 5% SDS in
PBS for 5 minutes, rinsed twice with Assay Buffer, incubated for
5 minutes in 0.25 mM dioxetane phosphate in Assay Buffer and
exposed to X-ray film for 5 minutes. The membrane strips
incubated with CSPD, and TFE were rinsed with 0_1 M
diethanolamine, 1 mM MgCl2, pH 9.5 and those incubated with CDP-
Star, and TFE-ADP-Star were rinsed with the same buffer at pH
9Ø The Assay Buffer for CSPD and TFE was 0.1 M diethanolamine, ,
1 mM MgCla, pH 9.5 and for CD-Star, and TFE-ADP-Star was the same
buffer at pH 9Ø The detection sensitivities, obtained in a 5
minute exposure are listed in TABLE III.
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
31
TABLE III
DIOXETANE DNA CONC. (PICOGRAMS)
TFE-ADP-Star 0.0107
TFE 7.7
CDP-Star 0.86
CSPD 23.3
The comparison of the experimental data points out that the
presence of the haloalkoxy substituent on AMPPD (TFE) reduces the
chemiluminescence half-life in a buffer solution (tl/2 for AMPPD
is 2.1 min, tl/2 for TFE is 0.9 min.), however, in the presence
of enhancers the tl/2 increases to 15 min. These dioxetanes are
particularly commercially useful when enhanced by either a
solution enhancer (such as TBQ, TB or in a CTAB-fluorescein as
in LumiPhos 43 0 ) or a surf ace ( such as nylon membrane ) . However,
TFE without the electron active substituent on the phenyl ring
does not provide any advantages. Furthermore, even by itself in
a buffer, the CSPD half-life is shorter than TFE. The peak
intensity data in TABLE II shows the comparison of the
chemiluminescent signal intensity or the relative emission
efficiencies. The results clearly indicate that TFE-ADP-Star
generates a higher intensity signal. Superior conventional
utilities are offered by TFE-ADP-Star.
TABLE III shows the comparison of these dioxetanes in a blotting
application for the detection of DNA on a nylon membrane. This
data shows an unexpected advantage of TFE-ADP-Star, which does
not exist in TFE or other compounds. TFE-ADP-Star detects close
to one thousand times less DNA than TFE. In this particular
application, TFE-ADP-Star is also more sensitive than CDP-Star.
As demonstrated above, the dioxetanes of this invention can
CA 02231199 1998-03-OS
WO 97/14692 PCT/US96/14390
32
be used to detect the presence of an enzyme in a sample, as well
as reporter molecule -to detect the presence of a nucleic acid
sequence. Generally, there are a wide variety of assays and
assay formats which exist which can make use of the dioxetanes, ,
all employing visually detectable chemiluminescence of the
deprotected oxyanion decomposition to indicate the presence
and/or concentration of a particular substance in a sample.
For example, when using this invention to detect an enzyme
in a sample, the sample is contacted with a dioxetane bearing a
group capable of being cleaved by the enzyme being detected. The
enzyme cleaves the dioxetane's enzyme cleavable group to form a
negatively charged substituent (e.g., an oxygen anion) bonded to
the dioxetane. This negatively charged substituent in turn
destabilizes the dioxetane, causing the dioxetane to decompose
to form a fluorescent chromophore group that emits light energy.
It is this chromophore group that is detected as an indication
of the presence of the enzyme . By measuring the intensity of
luminescence, the concentration of the enzyme in the sample can
also be determined.
The above-described dioxetanes can be used in any reporter
molecule based assay with an acceptable environment. Examples
of such assays include immunoassays to detect antibodies or
antigens, e.g., 8- or ,Ci-hCG; enzyme assays; chemical assays to
detect, e.g., potassium or sodium ions; and nucleic acid assays
to detect, e.g., viruses (e.g., HTLV III or cytomegalovirus, or
bacteria (e. g., E. Coli), and certain cell functions (e. g.,
receptor binding cites).
When the detectable substance is an antibody, antigen, or
nucleic acid, the enzyme capable of cleaving the enzyme cleavable
group of the dioxetane is preferably bonded to a substance having
CA 02231199 2004-12-16
CVO 97/14692 ~ PCTIUS96/14390
-,
3.
a specific affinity for the detectable substance (i.e., a
substance that binds specifically to the detectable substance),
e:g., an antigen, an antibody, or nucleic acid probe.
Conventional methods, e.g., carbodiimide coupling, are used to
bond the enzyme to the specific affinity substance; bonding is
preferably through an amide linkage.
In general, assays are performed as follows. A sample
suspected of containing a detectable substance is contacted with
a buffered solution containing an enzyme bonded to a substance
having a specific affinity for the detectable substance. The
resulting solution is incubated to allow the detectable substance
to bind to the specific affinity portion of the specific
affinity-enzyme complex. Excess specific affinity-enzyme complex
is washed away, and a dioxetane having a group cleavable by the
enzyme portion of the specific affinity-enzyme complex is added.
The enzyme cleaves the enzyme cleavable group, causing the
dioxetane to decompose into two carbonyl compounds (e.g., an
ester, a ketone or an aldehyde). The chromophore to which the
enzyme cleavable group had been bonded is thus excited and
luminesces. Luminescence is detected (using, e.g., a cuvette,
or light-sensitive film in a camera luminometer, or a
photoelectric cell or photomultiplier tube) , an indication of the
presence of the detectable substance in the same. Luminescence
intensity is measured to determine the concentration of the
substance.
In solid state assays , the specif is membranes of U. S . Patent
5,336,596 can be advantageously employed. The use of other
membranes, as well as other solid phases in an assay, may be
improved by blocking non-specific binding to the solid phase
matrix by pre-treatment with non-specific proteins such as BSA
CA 02231199 2004-12-16
WO 97/14692 PGT/US96/14390
34
or gelatine. Non-specific binding can also be blocked by
application, to the membranes, of a variety of polymeric
quaternary opium salts, such as are disclosed in U.S. Patent
5,326,882.
In the alternative, as fully set forth in U.S. Patent
4,978,614 and U.S. Patent 5,567,836, one may use polymeric
quaternary opium (phosphonium, sulfonium and ammonium)
quaternary slats. These enhancement agents can be used alone,
or in conjunction with surfactants such as Zelec DP. It is
believed that these enhancement agents, in an aqueous
environment, sequester the hydrophobic deprotected oxyanion,
substantially excluding water from the microenvironment in which
decomposition, and chemiluminescence occurs. As water tends to
"quench" chemiluminescence, its exclusion by the hydrophobic
enhancement agents can dramatically improve chemiluminescence
characteristics, as demonstrated in the above-experiments.
The dioxetanes of this invention, and their application and
preparation have been described both generically, and by specific
example. The examples are not intended as limiting. Other
substituent identities, characteristics and assays will occur to
those of ordinary skill in the art, without the exercise of
inventive faculty. Such modifications remain within the scope
of the invention, unless excluded by the express recitation of
the claims advanced below.
