Note: Descriptions are shown in the official language in which they were submitted.
PATENT
701 7J39D-267
CHEMILUMINESCENT COMPOUNDS
by
M. Paramesw'ara Reddy,~'Maged Aziz Michael, Chan S. Oh,
Thomas S. Dobashi, & Nabih S. Girgis
~IELD OF THE INVENTION
The present invention is directed to
chemiluminescent compounds for use as labels in analytic
reactions such as immunochemical reactions.
BACKGROUND OF THE I~VENTION
In many assays for biological molecules,
particularly immunoassays, enzyme assays, and nucleic
acid hybridization assays, it is necessary to detect
small quantities o~ specifically labeled molecules. In
most cases, when particularly high sensitivity is
required, three types of labels have been used:
radioactive labels, fluorescent labels, and enzyme
labels. While all of these labels are used extensively,
each type o~ label has unique disadvantages.
Radioactive labels, particularly high-energy
radioactive labels such as 12sI '(the most commonly
employed radioisotope in immunochemistry), have several
disadvantages. For example, the radioisotopes have a
short half~ e, i.e., l25I decays with a hal~ e o~
approximately sixty days. Moreover, when a radioactive
atom incorporated in a molecule decays, it destroys the
I'C2~P~'~7017-2.-pp 11.20.~0
.
--2-- PATENT
;701 7/39D-267
molecule in which it is incorporated and can cause damage
to other molecules in the preparation. As such,
radioactively labeled preparations have extremely short
shelf-lives. Moreover, because of the radiation they
emit, radioactive labels require special safety
precautions, such as the use of lead shielding. Their
disposal is also subject to special restrictions imposed
by the Nuclear Regulatory Commission, state licensing
bodies, and even local agencies. Radiation emitted also
poses a potential health hazard to workers using
radioactive labels.
Fluorescent labels avoid the disadvantages of
radioactive labels but have other disadvantages of their
own. Notably, they are less sensitive than radioactive
labels, and the use thereof requires activation by an
extrinsic light source. This requirement of activation
by an extrinsic light source makes their detection more
di~icult, as two wavelengths of light are involved, an
emission wavelength and an excitation wavelength.
Accordingly, more complex apparatuses are required to be
utilized ln conjunction with these labels.
Enzyme labels can be extremely sensitive, but
these too have disadvantages. Their detection requires
at least one additional step, a development step, to
allow the enzyme to carry out its reaction so that a
detectable product can be produced. This step requires
the use of additional reagents to the reaction, including
O bu~fers, substrates and/or coenzymes. Moreover, it is
not possible to use enzyme labels in all assays. If the
assay requires a step that inactivates or denatures the
enzyme or results in ionic, pH, or other conditions
PC2\~PP\7017-2.~pp 1 1.2D.~0
- 3-- PATENT
701 7/39D-267
incompatible with the activity of the particular enzyme
used as the label, enzyme labels cannot be used.
Because of the above deficiencies, increased
attention has focused on chemiluminescent labels as
alternative labels for these types of assays.
Chemiluminescence is a direct generation of light from a
chemical reaction. The mechanism of most
chemiluminescent reactions is not known in detail, but a0 generalized mechanism can be outlined:
A ~ B ~ B + h~.
Compound A undergoes a chemical reaction, usually
oxidation, to yield a product in an electronically
excited state ("B "). As this product returns to its
ground state ("B"), it gives off energy in the form of
light (~Ihv~). Typically, the light is in the visible
range.
Generally speaking, chemiluminescence occurs
when the vibronically excited product of an exogenic
chemical reaction reverts to its ground state with the
emission of protons, with the reactions invariably being
both oxidatlve and biphasic. Because the excitation
energy is obtained from the chemical energy of reaction,
the process is chemiluminescence. The characteristics
and behavior of several dif~erent chemiluminescent
compounds can be found in Gundermann & McCapra,
Chemiluminescence in Or~anic Chemistry (Springer-Verlag
1987).
Chemiluminescent labels are preferred over the
previously noted labels ~or several reasons.
Chemiluminescent labels have high sensitivity -- in many
cases, sensitivity down to the femtomole (1015 mole) to
PC2\APP\~Olj 2.-pp ~1,20.1\0
~4~ ~ 7017/39D-267
attomole (10-18 mole) range has been recorded. In
immunoassays, chemiluminescent labels can thus match or
exceed the sensitivity of radioactive labels or enzyme
labels.
Luminol and isoluminol derivatives are the most
widely used chemiluminescent reagents for immunoassays.
The light-yielding reaction is initiated by oxidation
with alkaline hydrogen peroxide in the presence of
catalysts such as horseradish peroxidase,
microperoxidase, or transition metal ions. Light
emission occurs at about 465 nm, which corresponds to the
~luorescence emission of the product, aminophthalic acid.
Aminobutylethyl isoluminol ("ABEI") can be used as a
label in immunoassays and is commercially available.
A second group of chemiluminescent reagents is
aryl oxalates. These reagents have been used as
commercial cold light sources and in high-performance
liquid chromatography ("HPLC") detectors. It is thought
that aryl oxalates react with hydrogen peroxide in
bu~ered or unbu~fered solvents to give a dioxetane-dione
that decomposes quickly to give Co2 in an exclted state.
Enerqy is then transferred by electron transfer to a
fluorescent molecule that emits light. In some
applications, bis-N-alkyl-N-trifluoromethyl sulfonyl
oxalamides have been substituted for the aryl oxalate
esters.
A third group o~ reagents,
10-methyl-acridinium-9-carboxylic acid aryl esters, are
chemiluminescent in the presence of alkaline hydrogen
peroxide and in the absence o~ a catalyst. The mechanism
is believed to involve initial attack by a hydroperoxide
PC2\~PP\7017-2.. pp ~1.20.~0
--5 - ~ , ~ PATENT
70 17/3 9D-267
anion, followed by intramolecular displacement of the
phenolate (the "leaving group") to give a strained
dioxetane-one. The strained dioxetane-one decomposes to
Co2 and excited N-methyl-acridone, which emits light at
430 nm. Carboxy-substituted acridinium salts have been
used as labels in immunoassays. Also, 5-methyl-
phenanthridinium-6-carboxylic acid aryl esters, which are
isomeric with the acridinium aryl esters, have been used
as labels in immunoassays.
These previously used types of chemiluminescent
labels have several disadvantages, including relatively
low quantum yield and undue sensitivity to hydrolysis,
especially under conditions necessary to preserve the
stability of the labile biological molecules such as
antibodies to which they are attached. For example, it
has been reported that antibody-conjugated phenyl
10-methyl-9-acridinium carboxylates lose more than 10% of
their activity within three days at about pH 4Ø These
2~ labels are only stable below pH 4.0, a degree of acidity
to which many antibodies and other proteins are
sensitive.
Because o~ the ongoing need for
25 chemiluminescent labels due to the disadvantages of ~ ~`
radioactive, fluorescent, and enzyme labels, acrldinium,
phenanthridinium, or other chemlluminescent compounds
usea~le under conditions compatible with labeling o~
biological molecules would be useful and highly
desirable.
PC2\.~PP\7017 2.-pp 1~.20.ao
--6 ~ PATENT
701 7/39D-267
SUMMARY OF THE INVENTION
In order to meet these needs, we disclose novel
chemiluminescent compounds. The labels are represented
generically by the structuré
(~ ~F)
where U represents a chemical group that can produce
light by chemiluminescence, F represents a leaving group,
and n has a value of at least one.
One class of these chemiluminescent compounds
comprises salts in which the leaving group contains a
carboxyl carbon atom or its isoelectronic equivalent and
a ~ive-membered unsaturated ring, including at least one
heteroatom. For this c~ass, the moiety represented by
"U" contains a polycyclic aromatic moiety containing a
quaternary nitrogen atom, covalently linked to a moiety
C=Z' .
Th~s class o~ molecules comprises a cation and
an anion wherein:
(1) the cation is represented by the following
schematic: .
PC2\~PP\7017 2.~pp 11.20.D0
--7-- PATENT
2 ~
C-Z/ "
F
where:
(a) n is at least one;
tb) A~ is a positively charged moiety
capable of producing light by chemiluminescence, such as
an acridinium, substituted acridinium, phenanthridinium,
substituted phenanthridinium, quinolinium, substituted
quinolinium, benzacridinium, or substituted
benzacridinium moiety; and
(c) Z' is selected from the group
consisting of O, S, NH, NOH, NR' and NOR' where R' is C1-
C5 alkyl;
(d) F is selected from the group
consisting of moieties of the structure O-CH-CH-W, and
moieties represented by the following schematic:
I
Q
where:
(i) W is selected from the group
consisting of hydrogen and c1-C5 alkyl; and
(ii) X is selected ~rom the group
consisting of O, S, Se, Te and NR" where R" is C,-C5 alkyl
20. or arylsulfonyl;
(iii) Q is selected from the group
consisting of the following structures:
PC2~PP~70~7 2.~pp : 11.20.~0
--8-- PATENT
Q ~ ~ 701 7/39D-267
A~
where:
(A) Y is selected from the
group consisting of O, S, S=O, Se, SO2, Se=O, SeOz, Te,
Te~O, TeO2 and N-R"', where R"' i9 selected from the group
consisting of hydrogen and c1-Cs alkyl;
(B) A2, A3 and A4 are each
independently selected ~rom the group consisting of a
valence bond, C1-Clo alkyl, C2-C10 alkenyl, C2-C10 alkynyl,
c3-c12 cycloalkyl, C5-C~2 cycloalkenyl and aryl; and
(C) Z2, Z3 and Z4 are each
independently selected from the group conslsting of
hydrogen, carboxyl, carboxyl halide, sulfonyl halide,
carboalkoxy, carboxyl acylate, carboxamldo, cyano,
carboxime, isocyanate, sul~o, N-succinimidylcarboxyl and
N-maleimido, except that where all of A2, A3, and A4 are
valence bonds, all f Z21 Z3/ and Z~ are not hydrogen
unless X is NR" where R" i9 arylsulfonyl; and
(2) the anion is selected from the group
consisting of sul~ate, methosulfate, perhalomethosulfate,
haloborate, haloacetate, halophosphate, phosphate,
halide, phosphite, nitrate, nitrite, carbonate, and
bicarbonate.
PC2~PP~70 ~ 7 2.-pp 1 1 .20.~0
--9 PATENT
~ ~ ~f ~ 7 0 1 713 9 D 2 6 7
In this chemiluminescent salt, A~ can be an
acridinium moiety represented by the following schematic:
lo ~ ~ ~R3
where:
(a) Al is selected from the group consisting of
a valence bond, C1-c1o alkyl, c2-c10 alkenyl, Cz-C10 alkynyl,
C3-Cl2 cycloalkyl, C5-C12 cycloalkenyl, and aryl;
(b) Z1 is selected ~rom the group consisting o~
hydrogen, methyl, carboxyl, carboxyl halide, sul~onyl
~o halide, carboalkoxy, carboxyl acylate, carboxamido,
cyano, carboxime, lsocyanato, sulfo, N-
succinlmidylcarboxyl, and N-maleimido groups, with the
condition that where A1 is a valence bond, Z1 is not
hydrogen;
(c) Rl, R3, Rs/ R7 and R9 are each lndependently
selected ~rom the group consisting of a valence bond,
hydrogen, and a moiety A-Z where A is de~ined as A1 above
an`d Z is de~ined as Z1 above, with the conditlons that
only one of R1, R3, Rs~ R7 and R9 is a valence bond; and
(d) R2, R4, R6 and R8 are each independently
selected from the group consisting o~ hydrogen and a
moiety A-Z where A is de~ined as Al above and Z is defined
as Zl above.
PC2~APP\7017~2,~pp 11.20.~0
-10-- PATENT
7 01 7 /3 9 D-2 6 7
In such an acridinium moiety, at least one of
the carbon atoms of A1 other than the carbon atom located
furthest from the acridinium moiety can be substituted
with a substituent selected from the group consisting of
hydroxy, halo, alkoxy, amino, alkylamino, arylamino,
carboxyl, carboxyester, carboxythioester,-
thiocarboxyester, sulfonyl, nitro, sulfonic acid,
sulfoester, sulfinyl, cyano, isothiocyano, ureido, oxo,
imino, mercapto, carboxamide, alkylthio, mercaptoester,
phosphoryl, and phosphorylester.
When A1 is selected from the group consisting of
substituted and unsubstituted straight chain aliphatic
groups, at least one o~ the carbon atoms o~ Al other than
the carbon atom located furthest from the acridinium
moiety can be replaced with a replacement moiety selected
from the group consisting of -0-, -NH-, and -NL-, wherein
L is selected ~rom the group consisting of alkyl groups,
cycloalkyl groups, oxo groups, hydroxy groups, sulfo
groups, sulfoester groups, carboxyester groups,
phosphoryl groups, and phosphorylester groups.
Alternatively, Al can selected from the group
consisting o~ benzyl and aryl groups, in which case at
least one of the aromatic carbon atoms of Al can be
replaced with a replacement moiety selected from the
group consisting of -N= and -~L'=, wherein L' is selected
from the group consisting of C1-Cs alkyl, C3-C12
cycloalkyl, oxo, and hydroxyalkyl.
In the acridinium moiety, A1 can be a valence
bond, in which case Z1 can be selected from the group
consisting of carboxyl, carboxyl halide, sulfonyl halide,
carboalkoxy, carboxyl acylate, carboxamido, cyano,
PC2\~PP~7017-2,-pp 11.20.80
- l l - PATENT
~ t~ - ~J !1 lt 7017~39D-267
carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and
N-maleimido. These reactive groups can be used to couple
the chemiluminescent compound to a biomolecule.
Preferably, R5 is a valence bond and the leaving
group moiety is linked to R5; R1, R2, R3, R4, R6, R7, R~,
and R~ are each hydrogen.
Preferably, in these chemiluminescent salts,
Z' is O and X is S. Q is
~ ~ ~4
~ ~
Pre~erably, Y is S. In one pre~erred
20 embodiment, A1 is a valence bond and Z1 is CH3; A2, A3 and
A4 are each valence bonds; and the anion is CF3SO3-.
The molety A~ can al~o be a phenanthridlnlum
moiety repreeented by the structure
~5
PC2\~PP\7017-2.~p~ 11.20.~0
--12-- PATENT
701 7/39D-267
in which:
(l) A1 is selected from the group consisting of
a valence bond, C1-C10 alkyl, Cz-C10 alkenyl, C2-C10 alkynyl,
C3-C12 cycloal~yl, Cs-C~2 cycloalkenyl, and aryl;
(2) Z1 is selected from the group consisting of
hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl
halide, carboalkoxy, carboxyl acylate, carboxamido,
cyano, carboxime, isocyanato, sulfo, N-
succlnimidylcarboxyl, and N-maleimido groups, except that
where A1 is a valence bond, 21 is not hydrogen;
(3) each of R14, R17, and R18 is selected from
the group consisting of a valence bond, hydrogen, and a
moiety A-Z in which A is one of the groups defined as A
above and in which Z is one of the groups defined as Z1
ab~ove, in which the A and Z can be selected independently
for each o~ R14, R17, and R18 with the condition that only
one of R14, R17, and R1a is a valence bond; and
(4) each of R1o, R11, R12, R13~ R1s~ and R16 is
selected from the group consistir.g of hydrogen and the
moiety A-Z, in which the A and Z can be selected
independently for each of R1~, R11, R12, R13, R15, and R16.
The phenanthridinium moiety can be substltuted
in a manner analogous to that for the acridinium moiety
previously described.
In another class of chemiluminescent compounds
according to the present invention, a second ring of at
least five atoms can be formed in the moiety Q by
eliminating two of the terminal groups Z2~ Z3, and Z4, and
linking the corresponding groups of A2, A3, and A4 on
which the terminal groups have been eliminated. In this
class of compounds, Q is selected from the group
consisting of the following structures:
PC2\APP\70 t 7 2 . ~pp 1 1 .20.~0
--13-- PATENT
, J ~ 701 7/39D-267
~ ~ Y~^~ 'q"~ (~A~-~
4J
~4
a second ring containing at least five atoms in addition
to the five-membered unsaturated heterocyclic ring being
formed by covalent linkage o~ two of A2, A3, and A4,
where:
(1) Y is selected from the
group consisting of 0, S, S=0, SO2, Se, Se=0, Seo2, Te,
Te=0, TeO2, and N-R"', wherein R"' is selected from the
group conslsting of hydrogen and C1-Cs alkyl;
t2) the moiety A2, A3, or A4 not
invo.lved in ~ormation o~ the second rlng i9 selected from
the group consisting of a valence bond, C1-C10 alkyl, C2-C10
alkenyl, C2-C10 alkynyl, C3-C12 cycloalkyl, C5-C12
cycloalkenyl, and aryl;
(3) the moiety Z2 ~ Z3, or Z4 not
involved in the formation of the second ring is selected
from the group consisting of hydrogen, carboxyl, carboxyl
halide, sulfonyl halide, carboalkoxy, carboxyl acylate,
PC2\~PP\70 1 7-2.-pp 1 1 .20,~10
--14 - PATENT
, ,, ,; 7 0 1 7 / 3 9 D 2 6 7
carboxamido, cyano, carboxime, isocyanato, sulfo, N-
succinimidylcarboxyl, and N-maleimido;
~ 4) of the two of A2, A3, and A4
involved in formation o~ the second ring, one is a
valence bond and the other is selected from the possible
groups A1 as defined above, the valence bond being linked
to the terminal carbon of the other group to form the
second ring.
The carbon atoms of Q, except for those carbon
atoms involved in formation of the second ring, can be
substituted as described above.
Another class of chemiluminescent compounds
accordlng to the present invention comprises arylsulfonyl
esters. This class of compounds comprises a cation and
an anion wherein:
(1) the cation is represented by the following
schematic:
~ t
C=o O
~ --S~~
where
(a) A~ is a positively charged moiety
capable of producing light by chemiluminescence, and
(b) Y is selected from the group
consisting of O, S, S=O, Se, SO2, Se=O, SeO2, Te, Te=O,
PC2WP~7 0 1 7 2.~pp 1 1 .20.~0
--15 - PATENT
70 17/3 9D-267
TeO2 and N-R"', where ~"' is selected from the group
consisting of hydrogen and c1-cS alkyl; and
(2) the anion is selected from the group
consisting of sul~ate, methosulfate, perhalomethosulfate,
haloborate, haloacetate, halophosphate, phosphate,
halide, phosphite, nitrate, nitrite, carbonate, and
bicarbonate.
In this class of compounds, the positively
charged moiety capable of producing light by
chemiluminescence can be selected from the group
consisting of acridinium, substituted acridinium,
phenanthridinium, substituted phenanthridinium,
quinolinium, .".~fbenzacridinium, and substituted ben~acridinium.
Another class of chemiluminescent compounds
according to the present invention is chemiluminescent
salts comprising an anion and a cation, the cation
comprising at least one chemical group capable of
producing light covalently linked to a leaving group
selected ~rom the group consisting of the following
chemical structures:
pC2WP\701 7 2.. pp ' 1 .20.~0
--16 - PATENT
701 7/~9D-26 7
l2 l2 ll D 11
f -R3; -O- f -R3; -S-Rl; -O-S-Rl; - ll -Rl;
R, Rl O
., ,
o R3 R3 R3
Il 11 ~ 11
~~ ll ~~1; - C-Rl; -O- C-Rl; and -C~I2- C-R
where:
(1) the chemical group that can produce light
by chemiluminescence is a heterocyclic ring or ring
~ystem selected from the group consisting of acridinium,
phenanthridinium, quinolinium, and benzacridinium;
(2) R1 is selected from the group consisting of
alkoxy groups, aryloxy groups, thioderivatives of alkoxy
groups, thioderivatives of aryloxy groups, pyrrole and
. substituted derivatives thereof, lmidazole and
substituted derivatives thereof, pyrazole and substituted
derivatives thereof, triazole and substituted derivatives
thereof, oxazole and substituted derivatives thereof,
thiazole and substituted derivatives thereof, tetrazole
and substituted derivatives thereof, indole and
15 substituted derivatives thereof, primary amino groups,
secondary amino groups, tertlary amlno groups, quaternary
amino groups, anilino derivatives, and morphollne
derivatives;
( 3 ) R2 i8 selected irom the group consisting of
hydrogen, alkyl groups and thloderlvatives thereof, aryl
groups and thioderivatives thereof, alkoxy groups and
thioderivatives thereo~, aryloxy groups and
thioderivatives thereo~, and derivatives of alkyl, aryl,
alkoxy, aryl, and aryloxy groups substituted wlth at
least one of nitro, cyano, halo, and sulfonyl;
PC2\~PP\7017-2.-pp 11.20.aO
--17 - PATENT
701 7/39D-267
(4) R3 is selected from the group consisting of
O, S, N~, NR1' NR2, CH2, C(R1)2, C(R2)2, and CR1R2 where R
and R2 are as defined above; and
(5) the anion is selected from the group
consisting of sulfate, methosulfate, perhalomethosulfate,
halohorate, haloacetate, halophosphate, phosphate,
halide, phosphite, nitrate, nitrite, carbonate, and
bicarbonate.
In this class of compounds, the chemical group
producing light is preferably N-methylacridinium, and the
leaving group is preferably
R3
-C-R
in which R1 is selected ~rom the group consisting of
pyrrole, pyrazole, 2-methylindole, and isatin and ~3 is 0.
Another aspect of the present invention is a
method ~or determining the quantity o~ a blomolecule in
solution by using any o~ the chemiluminescent compounds
o~ the present invention. The method comprises:
(l) reacting a covalent conjugate comprising
the cation of a chemiluminescent salt of the present
invention covalently lin~ed to the biomolecule with an
oXidizer selected ~rom the group consisting o~ hydrogen
peroxide, molecular oxygen, and organic peroxide to
generate light by chemiluminescence; and
(2) determining the quantity of light
generated to determine the quantity of the biomolecule
present.
PC2~PP~7017-2.-pp t~.20.~0
.
-18- PATENT
701 7/39D-267
t~J '~,' S ~ f .~_ ~
DESCRIPTION
We have developed novel chemiluminescent
compounds suitable for attachment to biological molecules
for use as labels. In general, these chemiluminescent
compounds comprise a conjugated heterocyclic ring or ring
system covalently linked to a stable leaving group. The
present invention encompasses both a number of possible
conjugated heterocyclic rings or ring systems and a
number of different leaving groups. In general, the
leaving groups ali include a polar moiety containing
phosphorus, sulfur, or carbon bonded to a different atom,
which can be carbon, nitrogen, oxygen, or sulfur. This
bond can be a double bond. For example, the leaving
group can include a carbonyl, thiocarbonyl, sulfone,
sul~oxide, or imide moiety.
As used herein, "leaving group" is de~ined as
that portion o~ the chemiluminescent compound susceptible
to attack by molecular oxygen, hydrogen peroxide, or
organic peroxides to form an intermediate that decays to
produce chemiluminescence. Typically, the compound
includes an ester, thioester, amide, or comparable
functional group derived from condensation o~ an acid
function, although other compounds are intended to be
within the scope of the present invention. When the
chemiluminescent compound includes a ~unctional group
derived from condensation o~ an acid functionj the bond
that is broken is the single bond between the carbon and
the substituted oxygen ~in an ester) or nitrogen (in an
amide); i.e., the C-O bond in a -COOH group. The
carbonyl group remains with the con~ugated aromatic ring;
it is electronic transitions within the portion o~ the
molecule bearing the conjugated aromatic ring that
P~:2\~P\7017-2.~pp 11.20.~0
.
~ .
-19- PATENT
70 1 7/39D-267
eventually produce light. The remai~nde~ o~'the ester,
amide, or comparable function constitutes the leaving
group. The stability of the leaving group is important
in obtaining efficient chemiluminescence, i.e., a
relatively high ~uantum yie'ld, because a stable leaving
group means that the C-O bond or its equivalent i5 more
readily broken. The present invention encompasses a
number of leaving groups not previously known to be used
in chemiluminescent molecules.
I. CHEMILUMINESCENT COMPOUNDS
A. Chemiluminescent Compounds in Which the Leaving
Group Contains a Carboxyl Carbon Atom or Its
Isoelectronic Equivalent and a Five-membered
Ring. Including at Least One ~eteroatom
It has been found that chemiluminescent
compounds in which the leaving group contains a carboxyl
carbon atom or its isoelectronic equivalent and a
~ive-membered ring, including at least one heteroatom,
exhibit chemiluminescent quantum yields than which are
equivalent to or higher than previously described
chemiluminescent compounds. This class o~ molecules is
represented generically by the structure:
~C~Z/~= (I)
wherein the bracketed portion o~ the molecule includes A~,
a positively charged'moiety capable of producing light by
chemiluminescence attached by a valence bond to a leaving
group F. Pre~erably, A~ is a polycyclic aromatic moiety
containing a quaternary nitrogen atom.
The anion associated with the quaternary
nitrogen atom is typically sul~ate; methosul~ate;
PC2~PP\7017-2.~pp 1 ~.20,110
--2 0-- PATENT
~ ~ 7017/3 9D 267
perhalomethosulfate; haloborate; halophosphate;
halosulfonate; haloacetate; phosphate; halide; phosphite;
nitrate; carbonate; or bicarbonate. Preferably, the
anion is CF3SO3 or FSO3-.
l. The Leavinq Group
The leaving group F can be: (a) a vinyl ester;
tb) a vinyl ester in which the terminal vinylic carbon is
substituted with C1-C5 alkyl; or, preferably (c) a moiety
having the following structure (Structure II):
(~r)
Q ,
where X is attached to the group A+ by a moiety C=Z',
where Z' can be O, S, NH, NOH, or NOR', where R' is C1-Cs
alkyl. Preferably, Z' is O. For the preferred F
represented by Structure II:
(l) X is o, S, Se, Te, or NR", where R" is
C1-C5 alkyl or arylsulfonyl; preferably, X i6 O; and
(2) Q is a five-membered unsaturated ring
containing a heteroatom, the ~ive-membered unsaturated
ring being o~ Structure III or IV:
~3 ~)
In Structure III or IV:
(a) Y is O, S, S=O, SO2, Se, Se-O, SeO2, Te,
Te=O, TeO2, or N-R"'. R"' is hydrogen or C1-C5 alkyl.
Pre~erably, Y is S.
rc2wP\70l7-2.~pp ' 11.20.aO
--21-- PATENT
701 7/39D-267
(b) Each of Az~ A3, and A4 can be independently
chosen from the following: a valence bond, C1-Clo alXyl,
C2-C10 alkenyl, C2-C10 al~ynyl, C3-C12 cycloalkyl, C5-C12
cycloalkenyl, and aryl. As used herein in the
specification and claims, the term "aryl~' refers to
unsubstituted or substituted aromatic moieties contalning
a single un~used benzene ring, and the terms "alkyl,"
alkenyl," alkynyl," "cycloalkyl," and "cycloalkenyl"
refer to unsubstituted or substituted groups. The terms
"alkyl,""alkenyl," alkynyl," "cycloalkyl,"
"cycloalkenyl," and "aryl" as used herein ~urther
encompass groups in which one or more carbon atoms are
optionally replaced by a replacement moiety as described
in the speci~lcation.
The carbon-containing groups can themselves be
substituted. Thus, at least one of the carbon atoms o~
any o~ A2, A3, or A4 (other than the carbon atom directly
attached to Z2~ Z3, or Z4 and most distant ~rom the
~ive-membered ring) can be substituted with a
substituent. The carbon atom most distant ~rom the five-
membered ring is the carbon atom separated ~rom the ring
by the greatest possible number o~ carbon atoms o~ A2, A3,
or A4. Thus, i~ A2 is a propyl (C3) group, the carbon
atom most distant ~rom the five-membered ring is
separated from the ring by the other two carbon atoms o~
the propyl group. The substituents can be any o~ the
~ollowing: hydroxy, halo, alkoxy, amino, alkylamino,
arylamino, carboxyl, carboxyester, carboxythioester,
thiocarboxyester, sul~onyl, nitro, sul~onic acid,
sul~oester,.sul~inyl, cyano, isothiocyano, ureido, oxo,
imino, mercapto, carboxamide, alkylthio, mercaptoester,
phosphoryl, or phosphorylester.
PC~PP\~017 2.-pp 11.20.aO
- 2 2-- ~ 1 PAT~NT
7017/:~9D-267
When A2, A3, or A4 is a substituted or
unsubstituted straight-chain aliphatic group, such as,
for example, an alXyl, alkenyl or alkynyl group, at least
one of the saturated carbon atoms of A2, A3, or A4 (other
than the carbon atom directly attached to ~z, Z3, or Z4
and most distant from the five-membered ring as defined
aboYe) can be replaced with a replacement moiety. The
replacement moiety can be -O-, -NH-, or -NL-. L can be
alkyl, cycloalkyl, oxo, hydroxy, sulfo, sulfoester,
carboxyester, phosphoryl, or phosphorylester.
~ Alternatively, when A2, A3, or A4 is a benzyl or
aryl group, at least one of the aromatic carbon atoms of
A2, A3, or A4 can be replaced with a replacement moiety.
The replacement moiety can be -N= or -~L'. L' can be C~-
C5 alkyl, C3-C12 cycloalkyl, oxo, or hydroxyalkyl.
(c) Each f Z2~ Z31 or Z4 can independently be
chosen from any o~ the following: hydrogen, carboxyl,
carboxyl hallde, sulfonyl halide, carboalkoxy, carboxyl
acylate, carboxamido, cyano, carboxime, isocyanato,
sulfo, N-succinimidylcarboxyl, or N-maleimido. With the
exception o~ hydrogen, these groups are reactive and can
be utilized to couple the chemiluminescent label to the
molecule to be labeled, such as, for example, an antigen
or antibody. See, for example: (a) EPO No. 0 273 115A,
which describes the conjugation of N-sulfonyl acridinium
carboxamide to antigens, haptens, and antibodies; (b) EP0
No. 0 322 926A, describing the coupling o~ ~2,6-dimethyl-
4-substituted) phenyl-N-methyl-acridinium-9-carboxylate
to haptens and proteins; ~c) Weeks, et al. "Acridinium
Esters As High-Speci~ic-Activity Labels in Immunoassay,"
Clin. Chem. 29, 1424-1479 (1983), describing the reaction
of 4-(2-succinimidyloxy carboxylethyl) phenyl-lo-
PC2~PP\7017~2.. Pp I 1.20.~0
- 2 3-- PATENT
r~ ~ S ': iJ ~ ~ 7017J~9D-267
methylacridinium-9-carboxylate with proteins. Other
conjugation reactions and methods are considered to be
well known to those in the art.
When all of A2, A3, and A4 are valence bonds
(i.e., when Zz, Z3, and Z4 are all attached directly to
the five-membered ring), all f Z2' Z3~ or Z4 cannot be
hydrogen unless Z' ls O and X is NR", where R" is
arylsulfonyl. In other words, when all of A2, A3, and A4
are valence bonds, all f Z2~ Z3, and Z4are hydrogen, Z'
ls oxygen, and n is 1, the cation is of Structure V:
~ - 5
where A~ and Y are as described above. The sulfonamide
group is relatively resistant to hydrolysis and does not
requlre the protection of additional bulky groups such as
A2, A3, and A4. Chemiluminescent sulfonamide~ are
desaribed ~urther below in ~ection I(B) of the
disclosure.
Alternatively, a second ring structure in
addition to the unsaturated heterocyclic five-membered
ring can be formed and two of the terminal groups Z2~ Z3
and Z4can be eliminated, forming one of the structures
depicted below as Structures VI-XI. This second ring
structure contains at least five atoms. The additional
ring structure can be formed where one of A2, A3, or A4is
PC2UPP\70 1 7 2. .pp 1 1 .20.~0
--2 4-- , PATENT
701 7/39D-267
a valence bond and the other of A2, A3, or A4 involved in
the formation of the second ring is one of the following
groups:
(1) an alkyl group;
~2) an alkenyl group;
(3) an alkynyl group;
(4) an alkyl, alkenyl or alkynyl group in
which at least one of the carbon atoms other than the
carbon atom located furthest from the original
unsaturated heterocyclic five-membered ring is
substituted with any one of the following substituents:
(a) hydroxy;
(b) halo;
(c) alkoxy;
(d) amino;
(e) alkylamino;
(f) arylamino;
(g) carboxyl;
(h) carboxylester;
(i) carboxylthioester;
(;) thiocarboxylester;
(k) sulfonyl;
(l) nitro;
(m) sulfonic acid;
(n) sulfoester;
(o) sul~inyl;
(p) cyano;
(q) isothiocyano;
(r) ureido;
(s) oxo;
(t) imino;
(u) mercapto;
(v) alkylthio;
(w) mercaptoester;
PC2WP~017.2.~pp 11.20.aO
--2 5 ~ PATENT
.~J. ',,' ~ L ~J .'_ -~ 701 7139D-267
(x) phosphoryl;
(y) phosphorylester; or
(5) an alkyl, alkenyl or alkynyl group in
which at least one of the saturated carbon atoms other
than the carbon atom located furthest from the original
unsaturated heterocyclic five-membered ring is replaced
with any of -O-, -NH-, or -NL-, in which L is C1-C5 alkyl;
C3- CB cycloalkyl; oxo; hydroxy; sulfo; sulfoester;
carboxylester; phosphoryl; or phosphorylester.
In defining the possible structures formed by
substitution, the carbon atom located furthest from the
original unsaturated heterocyclic five-membered ring is
the carbon atom separated from the ring by the greatest
possible number of carbon atoms, as explained above for
the moiety Q.
~ .
The additional ring is formed in this
alternative when the term.inal carbon of the group A2, A3
or A4 is linked by the valence bond to the five-membered
unsaturated ring of Q.
4~ ~ ~-AL P~4~\~2
~ CQ~ J C~) C ~
p';L~ 2,~
30 ~p5~
A~ C'~ J~ Y C~)
~4
PC2~AYP~7017 2.-pp 11.20.~0
--2 6-- PATENT
701 7/39D-267
,~ ~ / ,' ' J 5 !~
2. The Polycvclic Aromatic Moiety
The polycyclic aromatic moiety, A', is
preferably either an acridinium moiety or substituted
acridinium moiety of the structure (Structure XII)
t~q
0 ~ ~ R~
or a phenanthridinium moiety or substituted
phenanthridinium moiety of the structure (Structure XIII)
I~\s
R~
R~ C~)
Io
As used herein, the terms "substituted
acridinium moiety" and "substituted phenanthridinium
moiety" encompass the full range of possible
substitutions and replacements describea herein unless
otherwise limited. Alternati~ely, the polycyclic
aromatic moiety can be a quinolinium or benzacridinium
moiety. The quinolinium or benzacridinium moiety can be
PC2\~PP\~01~ 2.-pp 11.20.~10
--2 7-- PATENT
7~1 7/33D-267
substituted analogously to the acridlnium or
phenanthridinium moieties.
Preferably, A+ is an acridinium moiety o~
Structure XII.
a. The Acridinium Moiety
In the acridinium moiety, A1 can be:
(l) a valence bond;
(2) C1-C10 alkyl;
(3) C2-C10 alkenyl;
(4) C2-C10 alkynyl;
(5) C3~C12 cycloalkyl;
(6) C5-C12 cycloalkenyl;or
(7) aryl.
When A1 is other than a valence bond, at least one of the
carbon atoms (other than the carbon atom furthest from
the fused ring structure, as defined above) can be
substituted with a substituent. The substituent can be
hydroxy, halo, alkoxy, amino, alkylamino,- arylamino,
carboxyl, carboxylester, carboxylthioester,
thiocarboxylester, sulfonyl, nitro, sul~onic acid,
sul~oester, sulfinyl, cyano, isothiocyano, ureido, oxo,
imino, mercapto, carboxamide, alkylthlo, mercaptoester,
phosphoryl, or phosphorylester.
Alternatively, when A1 i9 a substituted or
unsubstituted straight-chain aliphatlc group, at least
one o~ the saturated carbon atoms o~ A1 (other than the
carbon atom ~urthest ~rom the ring nitrogen atom) can be
replaced with a replacement moiety. The replacement
moiety can be -0-; -NH-; or -NL-, where L ls alkyl,
cycloalkyl, oxo, hydroxy, sul~o, sul~oester,
carboxyester, phosphoryl, or phosphorylester.
PC2\.~PP~701~-2.-pp 11.20.110
. .
--2 8-- PATENT
701 7/39D-267
When A~ is a benzyl or aryl group, at least one
of the aromatic carbon atoms of A1 can be replaced with a
replacement moiety. The replacement moiety can be -N= or
- ~', wherein L' is C,-Cs alkyl, C3-C~2 cycloalkyl, oxo, or
hydroxyalkyl.
With further reference to acridinium structure
XII, Z1 can be a hydrogen, methyl, or chemically reactive
group. Typically, this chemically reactive group is
lb carboxyl, carboxylhalide, sulfonylhalide, carboalkoxy,
carboxy acylate, carboxamido, cyano, carboxime,
isocyanato, sulfo, N-succinimidylcarboxy, or N-maleimido.
When A1 is a valence bond, Z1 is not hydrogen. In one
preferred embodiment, A1 is a valence bond and Z1 is
lS methyl.
Further referencing the above acridinium
structure, one of R~, R3, R5, R7, and R9 is a valence bond
for attachment to the remainder o~ the compound. The
remainder of R~, R3, R5, R7, and R9 can be independently
either hydrogen or a moiety A-Z where both A and Z in the
A-Z moiety are defined as A1 and Z1 as above,
respectively. Furthermore, each A and Z in the A-Z
moiety can be selected independently for each of R~, R3,
R5, R7, and R9. Preferably, R5 is a valence bond, with R1,
R3, R7, and R9 being hydrogen.
Each of R2, R4, R6, and RB can be either hydrogen
or~a moiety A-Z as defined in the preceding paragraph.
The moiety A-Z can be selected independently for each of
R2, R4, R6, and R8. Preferably, all of R2, R4, R6, and R8
are hydrogen.
b. The Phenanthridinium Moiety
PC2\APP\70~72.~pp 11.20.iO
--2 9-- PATENT
70 17/3 9D-267
In phenanthridinium Structure XIII depicted
above, one of R14, R17, and R18 is a valence bond for
attachment to the remainder of the compound. The
remainder of R14, R17, and R18 can be independently hydrogen
or a moiety A-Z, defined as above. The A and Z can be
selected independently for each of R14, R17, and R18.
- Preferably the two of R14, R17, and R18 that are not valence
bonds are hydrogen.
Each of R10, R11~ R12~ R~3, R~5, and R16 is either a
hydrogen or a moiety A-Z as defined above. Preferably,
each of R10~ R~, R~2, R~3, R~5, and R16 is hydrogen.
3. Preferred Embodiments of Acridinium Salts
With reference to the general structure
~A~ 1
l I--Z .I n
A~ is an acridinium moiety represented by Structure XII
where A1 is a valence bond; Z1 is methyl; Rs is a valence
bond; each of R~, R2, R3, R4, R5, R6, R7, R8, and R9 is
hydrogen; Z' is O; F is represented by Structure II (X-Q)
where X is O; and Q is represented by the five-membered
ring of Structure III
PC2\~PP\70 1 ~ 2 . ~pp 1 1 ,20.~0
-3 0- PATENT
I'i r ~ ~ , 3 ~ 7017/3 9D-267
' f~
where Y is -S-, each of A2, A3, and A4 are valence bonds,
and Z2~ Z3~ and Z4 can be one of the following set of
alternatives: -
(1) each of Z2~ Z3~ and Z4 is hydrogen;
(2) Z2 is COOC2H5 and each of Z3 and Z4 is
15 hydrogen;
(3) Z2 is COOCH3, Z3 is COOC2Hs, and Z4 is
hydrogen;
(4) each o~ zz and Z3 is COOCH3 and Z4 is
hydrogen;
(5) each of Z2 and Z3 is COOCH3 and Z4 is CH3;
( 6 ) Z2 is COOC2Hs, Z3 is carboxyl, and Z4 is
methyl;
(7) one f Z2~ Z3~ and Z4 is methyl, the others
being hydrogen;
~8) Z2 and Z4 are each hydrogen and Z3 is
phenyl; and
( 9 ) Z2 and Z3 are each COOCH3 and Z4 is ~r.
Most preferablY, each f Z2~ Z3~ and Z4 i9
hydrogen.
Preferred chemiluminescent acridinium saltsaccording to the present invention accordingly have the
following structure (Structure XIV):
PC2~APP\70~7 2.~pp
1 1.20.00
--31~ PATENT
C~3 !~ C701 7/39D-267
~' ~ C)<~)
~S~
~3 o
B. Chemi-luminescent Sulfonamide Derivatives
The present invention also encompasses
chemiluminescent sulfonamide derivatives comprislng a
cation and an anion. The cation has the general
structure shown as Structure XV
I~--s-~ C~)
~ 0
In this structure, the increased resistance of
the sulfonamide to hydrolysis means that bulky protecting
groups in the five-membered unsaturated heteroayclic ring
are not required.
In compounds of Structure XV, A~ is a positlvely
charged moiety capable of producing light by
chemiluminescence, which can be acridinium, substituted
acridinium, phenanthridinium, subfitituted
phenanthridlnium, qulnolinium, or benzacridinium.
` Pre~erably, A~ is N-methylacridinium. Y is O, S, S=O,
S02, Se, Se=0, SeO2, Te, Te=O, TeO2, or N-R"', where R"' is
hydrogen or C1-Cs alkyl. Preferably, Y is S.
PC2\~PP\70~ 2 .~pp~1.20.~0
-32- ; s1sf~ PATENT
'' -' -' - 7017/39D-267
The anion i5 as described above. Preferably,
the anion is CF3SO3 or FSO3-.
A preferred chemiluminescent sulfonamide
derivative accbrding to thé present invention has the
structure shown below as Structure XVI.
u ~ C ~
C. Othe~ Chemlluminescent Compounds Capable o~
Covalent Attachment to_a BioloqicallY Active
~o~e_ule
Other chemiluminescent compounds capable of
covalent attachment to a biologically active molecule are
also within the scope of the invention. These comprise a
chemlcal group that can produce light by chemi-
luminescence coupled to a leaving group containing
phosphorus, sulfur, or carbon double-bonded to C, N or O.
If phosphorus or sul~ur is part o~ the leaving group, it
is in a relatively polar moiety in which the phosphorus
or sul~ur is bonded to more electronegative atoms. The
leaving groups include the following structures
(Structures XVII-XXV):
PC2~PP\701 7 1.-pp 1 1 ,20.~0
--3 3-- ~ PATENT
", 701 7139D-267
l2 l2 11 1111
- P-R3; -O- P-R3; -S-R; -O-S-Rl; -S-Rl;
, C~) Cx~m~ C,~) C ~) C~)
Il 113113 Rll3
_O_Il_Rl; - C-Rl; -O- C-Rl; and -CH2- C-Rl .
C~) C~)C)~) Cgiz~
In the leaving groups depicted above, R1 can be any of:
(l) an alkoxy group, an aryloxy group, or a
thioderivative of an alkoxy or an aryloxy group;
(2) pyrrole, imidazole, pyrazole, triazole,
oxazole, thiazole, tetrazole, or a substituted derivative
o~ any o~ these heterocycles;
~ 3) a primary, a secondary, a tertiary, or a
quaternary amino group; or
(4) an aniline derivative or a morpholine
derivative. The term "derivative" as used herein
encompasses all derivatives that do not af~ect the
reactivity o~ the leaving group.
Rz can be any o~:
(l) hydrogen;
(2) an alkyl group, an aryl group, an alkoxy
group, an aryloxy group, or a thioderivative of any o~
these groups; or
(3) a derivative o~ an alkyl, an aryl, an
alkoxy, or an aryloxy group substituted with at least one
of nitro, cyano, halo, or sulfonyl.
R3 can be O, S, NH, NR1, NR2, CH2, C(R1)2, C(R2)2,
or CR1R2, where R1 and R2 are defined as in the preceding
two paragraphs.
PC2\APP\7017-2,~pp 11,20.aO
_ 3 4 _ ~ S ~ PATENT
701 7/39D-267
Wi~h respect to chemiluminescent labels having
Structures XVII-XXV, inclusive, the chemical group that
can produce light by chemiluminescence is a heterocyclic
ring or ring system. The ring or ring system can be
acrldinium, phenanthridinium, quinolinium, or
benzacridinium.
With chemiluminescent labels of the type
disclosed in this section, the preferred chemical group
that can produce light by chemiluminescence is an
a~ridinium group. The leaving group is preferably either
-l~, in which R1 is pyrrole and R3 is pyrazole, or
in which R1 is pyrrole or pyrazole.
II. PREPARATION OF CHEMILUMINESCENT COMPOUNDS
The chemiluminescent compounds as disclosed in
Section I, above, can be prepared by reacting an acyl
chloride derivative, or other comparably reactive
derivative of the acridinium, phenanthridinium, or other
light-producing cyclic nitrogen-containing moiety
direatly with the leaving group. The reaction is a
condensation between the activate~ carboxyl function and
a hydroxyl, mercapto, or simllar function of the leaving
group. The reaction is preferably performed in a
chlorinated methane, such as dichloromethane or
chloro~orm, as solvent, in the presence o~ triethylamine
at room temperature. The resulting acridine derivative
i9 then quaternized by reacting the derivative with a
methylating agent such as, ~or example, methyl
fluorosulfonate. See Examples 1-6, in~ra, for
preparation of several chemiluminescent compounds
according to the present invention.
PC2~PP~70~-2.-pp 1 1.20.90
.
-35~ ,.. PATENT
~ J r i ''~ ~~ 7017/39D-267
III. REACTION OF CHEMI~UMINESCENT COMPOUNDS WI~H
BIOLOGICAL MOLECULES
In order for the chemiluminescent compounds to
be used as labels in immunoassays, as well as other
analytlcal assays, it is necessary to attach the compound
covalently to the biological molecule to be measured or
to a biological molecule reacting specifically with the
biological molecule to be measured. Typical biological
molecules or biomolecules to which the chemiluminescent
compounds of the present invention can be attached
include, ~or example, peptides, haptens, antigens,
antibodies, enzymes, receptor proteins, hormones,
carbohydrates, phospholipids, glycolipids,
oligonucleotides, nucleic acids, therapeutic drugs, and
drugs of abuse.
A number of methods considered to be well known
in the art can be used to react the chemiluminescent
compound with the biological molecule. Where the
compound contains a reactive group such as, for example,
carboxyl, carboxyl halide, sul~onyl halide, carboalkoxy,
carboxamido, carboxime, or N-succinimidylcarboxy, such
groups can be coupled covalently to hydroxyl ~unctions or
amino functions using conjugation reagents such as, for
example, carbodiimides or 1,1-carbonyldiimidazole.
N-maleimido groups react directly with sul~hydryl
residues in proteins. I~ the compound contains aromatic
amino groups, these can be converted to diazonium salts
and reacted with phenol groups such as those found in
tyrosine groups o~ proteins. Either a reactive group
present in the polycyclic aromatic moiety or other light-
producing group, or one present in the leaving group, can
be used to attach the compounds o~ the present inventlon
to the biological molecule.
PC2~PP\7017~2.~pp 11.20.aO
-36- ,"~ t ,~.! PATENT
~ 7017139D-267
IV. USE OF LABELS TO PRODUCE CHEMILUMINESCENCE
In general, labels according to the present
invention produce chemiluminescence by reaction with
hydrogen peroxide, molecular oxygen or an organic
peroxide in an alkaline solution. The pH of the solution
has a range from about 7 to about 14; preferably, the pH
is at least 10; most preferably, the pH is about 13.
These reactions preferably take place at room
temperature. When hydrogen peroxide or an organic
peroxide is used to trigger the reaction, it is
pre~erably present in a stoichiometric excess. In place
of hydrogen peroxide, organic peroxide can be used,
including, for example, perbenzoic acid, benzyl peroxide,
or t-butyl hydroperoxide.
Chemiluminescence is typically measured at
425-430 nm in a commercially available luminometer, such
as a Berthold Chemiluminometer produced by Berthold
Laboratorium, Wildbat, Germany.
EXAMPLES
The following Examples are presented ~or
illustration purposes only and are not intended to limit
the scope of the invention, this disclosure, or the
claims that follow.
PC2~PP\7017 2.~pp 11.20.(~0
f ~ i ,r " ~ PATENT
701 7/39D-267
Example 1
Preparation of N-Methyl-Acridlnium 9-Carboxylic Acid 2-
Methyl-3-Furanthiol Thioester
A guantity of acridine-9-acyl chloride (1940
mg) was placed in a 100 ml round-bottom flask with a
stirring bar. Dry C~Cl3 (15 ml) was added to dissolve the
solid acid chloride. Triethylamine (1300 ~1) and
1~ 2-methyl-3-furanthiol (800 ~1) were added and the flask
was rinsed with 3x 1.5 ml CHCl3, capped and stirred
overnight at room temperature. Thin-layer chromatography
of the reaction product on silica gel in CHC13-EtOAC (9:1)
showed the thiGester at an RF of 0.68 and four additional
bands at 0.61, 0.50, 0.37, and 0.00.
The solvent was then removed by distillation at
a pot temperature of 90-100C. The flask was then cooled
and the residue was triturated with approximately 25 ml
cyclohexane. The residue, initially a dark brown oil,
became a yellow s~lid; the solid wa~ ~iltered and washed
with cyclohexane. The yellow solid was dissolved in
heated methane and dried onto 8 g of silica gel. The
silica gel was placed in a 2.5 X 60 cm column packed with
110 g silica gel ~lurried with chloro~orm. The column
was eluted sequentially with chloro~orm, 98~
chloro~orm-2% ethyl acetate, 97% chloro~orm, 3% ethyl
acetate, 95% chloro~orm-5% ethyl acetate, and 90%
chloro~orm-10~ ethyl acetate. A yellow band began
eluting in the 97%-chloroform-3% ethyl acetate, and
continued through the 95% chloroform-5% ethyl acetate,
ending at the 90% chloroform-10% ethyl acetate.
PC2~PP\70~7 2.~pp ~ ~.20.~0
.
-38~ PATENT
~J ~ J ~ 7017/39D-267
Fractions containing the yellow band were
collected and subjected to thin layer chromatography (on
silica gel). A band of RF . 73 was seen. The thioester
was crystallized ~rom ethyl acetate, yielding 400 mg of
solid with a melting point of 170-171C, which was
.designated R170TD.
A mass spectral analysis of a comparable
recrystallized fraction from another preparation yielded
a molecular ion with an M/Z of 320 consistent with a
molecular formula of Cl9H13N02S or a structural formula of:
~
~S~
An elemental analysls of the preparation
su~ected to mass spectroscopy provided results of:
71.35% C, 4.10% H, 4.33% N, 10.51% 0, and 9.71% S, in
essential agreement with the calculated values o~ 71.45%
C, 4.10% H, 4.39~ N, 10.02% 0, and 10.04% S.
Spectroscopy in the visible and ultraviolet
regions revealed a major peak at 258.5 nm, with minor
absorption peaks at 219.5 nm and 363 nm.
PC2\~PP\701~-2.~pp 1 1.20.D~
-39- _ ~ , , PATENT
~ 7017/39D-267
For quaternization of the acridinium thioester,
approximately 110 mg of R170TD was placed in a 25-ml
round ~ottom flask and dissolved in 6 ml of dry CH2Cl2.
Approximately 250 ~l of methyl fluorosulfonate was added
to the flask. The reaction vessel was ~lushed with
nitrogen gas, capped, and then placed in the dark for 2-3
days, a~ter which period of time crystals were observed
on the bottom of the flask. The solution was filtered,
and 70 mg of a solid was obtained. This solid was
designated R171TD.
A mass spectral analysis of R171TD yielded a
molecular ion with an M/Z of 334 consistent with the
formula C20Hl6N02S having a structure:
C~3
O~ , ,
0~c~
\~ '
C~
o
An elemental analysis of this preparation gave
results of: 55.34% C, 3.71% H, 3.21% N, 14.92% S, and
2.59% F. Calculated values were as followa: 55.41% C,
3.72% H, 3.23% N, 14.79% S, and 4.38% F. Spectroscopy in
PC2UPP\70 1 7 2,-pp 1 1 .20.~0
--4 0-- j .`~ PATENT
('J _ J ~ t 701 7/39D-267
the visible and ultraviolet regions revealed a major peak
at 261.5 nm, with minor peaks at 221.5 nm and 368.5 nm.
Both the acridinium thioester and the
quaternized acridinium thioester exhibited
chemiluminescence with the quaternized compound yielding
approximately 40-fold greater chemiluminescence at 10l5
moles than did the thioester at 10-1Z moles.
Example 2
Preparation of N-Methyl-Acridinium 9-(5-Ethoxycarbony1-2-
Methoxycarbon~1-3-Thienyl Ester Fluorosulfonate
Acridine-9-acyl chloride (483 mg) was placed in
a 25-ml round-bottomed flask and dissolved in 7 ml of dry
chloro~orm. To the solution was added 500 mg of ethyl
methyl 3-hydroxythiophene-2,5-dicarboxylic acid ester.
Triethylamine (242 mg, or 0.34 ml) was added dropwise
while stirring at room temperature. The mixture was
stirred for one hour, during which time a white
precipitate was formed. The precipitate was collected by
filtration and washed with chloroform to give a pure
product (250 mg). The filtrate was then evaporated to
dryness, 10 ml of water was added to the residue
remaining from the evaporation, and the residue was
digested. The solid separated was collected by
filtration, washed with water, dried, and recrystallized
from ethyl acetate to yield another 450 mg of product.
The total yield of product was 700 mg or 80%.
The product was quaternized by placing 50 mg in
a 10-ml round-bottom flask, dissolving in 3 ml of dry
PC2~PP~7017-2.~pp 1 1.20.(10
--41-- PATENT
701 7/39D 26
methylene chloride, and adding 200 ~l of methyl
fluorosulfonate. The flask was left in the dark
overnight. A few drops of hexane was added to the
solution. The crystalline material that separated out
after standing for two hours was collected by filtration,
washed with hexane, and dried to give the product (45 mg,
71% yield).
The product had a melting point of >250C.
Nuclear magnetic resonance in DMS0-d6 gave the following
chemical shifts ~ (ppm): 8.96 (d, 2H, J = 9.3 Hz, C1H and
C8H); 8.82 (d, 2H, J = 8.6 Hz, C4H and C5H); 8.57 (t, 2H,
C3H and C6H); 8.54 (s, lH, thiophene-C4H); 8.17 (t, 2H, C2H
and C7H~; 4.97 (s, 3H, N'-CH3); 4.42 (q, 2H, ethyl-CH2);
3.83 (s, 3H, ester-CH3); and 1.38 (t, ethyl-CH3). Mass
spectroscopy gave a quasi-molecular ion corresponding to
the anticipated M~ at M/Z 450. Because the compound is a
quaternary nitrogen salt it has a pre-existing positive
charge and does not acquire an extra proton from the
matrix ionization. Also, the negatively charged
fluorosulfonate ion did not show as part of the molecular
ion. Elemental analysis gave C 52.36%, H 3.82%, N 2.29%,
and F 3.62%, in essential agreement with the calculated
values for C24HzoN04FS2 of C 52.45%, H 3.67%, N 2.54%, and F
3.48%.
Example 3
Pre~aration of 9-(N-pyrryl)carbo~l-N~thylacrl_inium
Fluo~_ulfonat~
A quantity of acridine-9-car~oxylic acid (22.3
g; 0.1 mol) was placed in a 250-ml round-bottom flask.
PC2~PP\7017~2.-pp ~1,20,~o
-42~ ,. 7017/39D-267
Freshly distilled thionyl chloride (70 g; 42 ml) was
added, and the resulting reaction mixture was heated
under reflux for 3 hours, yielding acridine-9-carboxylic
acid chloride hydrochloride. The excess of thionyl
chloride was removed by distillation and the traces left
were removed by washing with dry benzene. The solid acid
chloride was kept under dry benzene. It was collected by
filtration to give the acid chloride as a yellow solid.
The yield was 24.7 g, or 90%.
The acridine acid chloride (0.277 g; l mmol)
was dissolved in 20 ml of dry chloroform in a round-
bottom flask. Pyrrole (67 mg; 1 mmol) was added,
followed by addition of triethylamine (0.30 g; 0.32 ml; 3
mmol). The reaction mixture was left overnight with
stirring. The solvent was removed under reduced pre-sure
and the residue was dissolved in water and extracted with
ethylacetate. Thin-layer chromatography on sillca gel in
hexane-ethyl acetate (70:30) showed a ma;or fluorescent
spot, the product. Purification of the product, 9-(N-
pyrryl)carbonyl acridine, was achieved using silica gel
column chromatography using hexane-ethyl acetate as
eluant to yield 165 mg (61%). The 9-(N-pyrryl)carbonyl
acridine was converted to 9-(N-pyrryl)carbonyl-N-
methylacridinium fluorosulfonate by treatment with methylfluorosulfonate. A quantity of the acridine compound
(1.36 mg; 0.5 mmol) was dissolved in 20 ml of dry
methylene chloride in a 50-ml round-bottom ~lask.
Methylfluorosulfonate (0.5 ml) was added and the flask
was kept in the dark overnight. The yellow solid which
formed was collected by filtratlon and washed with CH2Cl2
and hexane, and dried to give a yield of 150 mg or 83%.
PC2~PP~7017 2.. pp 11,20.iO
_ 4 3 - . . , . PATENT
.~d ~ ,' 7()17/39D-267
Example 4
Prepara$ion of 9-(2-~yrazolyl)carbonvl-N-methylacridinium
Fluorosulfonate
Acridine 9-carboxylic acid chloride (0.83 g; 3
mmol) was dissolved in 50 ml of dry chloroform. Pyrazole
(0.3 g; 4.4 mmol) was added, followed by triethylamine
(0.91 g; 9 mmol). The reaction mixture was left
overnight with stirring. The solvent was removed under
reduced pressure and the residue was dissolved in water
and extracted with ethyl acetate. Evaporation of the
ethyl acetate af~orded a crude product. Thin-layer
chromatography on silica gel in ethyl acetate-hexane
(40:60) showed a fluorescent major spot along with some
impurities. Puri~ication of the product, 9-~2-
pyrazolyl)carbonyl acridine was achieved by using silica
gel column chromatography with ethyl acetate-hexane
(60:40) as eluant. Evaporation of the fractions
containing the product gave the substituted acrldine in a
yield of 617 mg (75% yield).
The ~ubstituted acridine was converted to 9-(2-
pyrazolyl)carbonyl-N-methylacridinium fluorosulfonate by
treatment with methyl fluorosulfonate as in Example 3.
The reaction used 0.273 g (1 mmol) of the substituted
acridine along with 20 ml of CH2Cl2 and 1 ml of methyl-
fluorosulfonate. The yield was 310 mg (80~).
PC2\APP\7017-2,~pp 11,20.~0
_ 4 4 _ PATENT
701 7/39D-267
Example 5
Preparation of 9-(N-2-methylindolyl!carbonyl-N-
methylacridinium Fluorosulfonate
The compound 9-(N-2-methylindolyl)carbonyl-N-
methylacridinium fluorosulfonate was prepared essentially
as in Examples 3 and 4, starting with acridine acid
chloride and 2-methylindole. The reaction mixture
comprised 1.385 g of acridine acid chloride (5 mmol),
0.655 g of 2-methylindole (5 mmol), and 1.515 g of
triethylamine (15 mmol), in 50 ml of dry chloroform.
The ~ubstituted acridine product was purified
by silica gel chromatography, using hexane-ethylacetate
(50:503. The yield was 0.62 g of a pale yellow semi-
solid (37~). This compound, 9-(N-2-
methylindolyl)carbonyl acridine, was converted to the N-
methyl fluorosul~onate by reaction with methyl
fluorosulfonate as in Examples 3 and 4. The end
methylfluorosulfonate was purified by dlssolving in
distilled water, filtration, and evaporation untll
dryness to yield a dark yellow semi-solid product.
Example 6
Preparation o~ 9-~N-isatinyl)carbonyl-N-methylacridinium
Fluorosul~onate
9-(N-isatinyl)carbonyl-N-methylacridinium
~luorosul~onate was prepared essentially as in Examples
3-5 starting with acridine acid chloride, isatin and
triethylamine. The reaction mixture comprised 1.4 g of
acridine acid chloride (5 mmol), 0.4 g of isatin (5
PC2WP\7017.2.~pp 1 1.20.~0
~45~ ~ 7017l39D-267
mmol), and 1.51 g of triethylamine (15 mmol) in 50 ml of
dry chloroform. The yield after column chromatography on
silica gel using ethylacetate-hexane (80:20) was 0.64 g
(36~) as a pale yellow semi-solid product.
This product, 9-(N-isatinyl)carbonyl acridine,
was converted to the N-methylfluorosulfonate by reaction
with methylfluorosulfonate as in Examples 3-5. The
reaction product was a dark brown gum. It was purified
by dissolving in distilled water and filtration from the
brown impurities. The water was evaporated under reduced
pressure. The product was dried, washed w~th n-hexane,
and dried to give a yellow solid.
ADVANTAGES OF THE INVENTION
The present invention provides chemiluminescent
compounds with quantum yield and stability equal to or
exceeding the quantum yleld and stability o~ presently
available compounds that are suitable for use in labeling
biological molecules. The compounds are particularly
3uitable for conjugation to biomolecules such as
antibodles and haptens for immunoassays and other
specific binding assays.
While the invention has been described with
respect to speci~ic embodiments and compounds, it is to
be understood that modifications and equivalents may be
apparent to those skilled in the art and are intended to
be within the scope of the invention.
PC2\,~PP~7017-2.~pp 11.20.00
