CA2074014A1 - Chemiluminescent compounds - Google Patents
Chemiluminescent compoundsInfo
- Publication number
- CA2074014A1 CA2074014A1 CA002074014A CA2074014A CA2074014A1 CA 2074014 A1 CA2074014 A1 CA 2074014A1 CA 002074014 A CA002074014 A CA 002074014A CA 2074014 A CA2074014 A CA 2074014A CA 2074014 A1 CA2074014 A1 CA 2074014A1
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- Canada
- Prior art keywords
- group
- chemiluminescent
- salt
- groups
- moiety
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- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
- C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
- C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
- C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
- C07D221/02—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00 condensed with carbocyclic rings or ring systems
- C07D221/04—Ortho- or peri-condensed ring systems
- C07D221/06—Ring systems of three rings
- C07D221/10—Aza-phenanthrenes
- C07D221/12—Phenanthridines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/56—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/576—Six-membered rings
- C07F9/64—Acridine or hydrogenated acridine ring systems
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
ABSTRACT OF THE DISCLOSURE
Novel chemiluminescent compounds are suitable for use in labeling biological molecules for assays such as immunoassays. These chemiluminescent labels are characterized by the incorporation of stable leaving groups. One class of these chemiluminescent labels comprises salts in which the leaving group contains a carboxyl carbon atom or its isoelectronic equivalent and a five-membered ring, including at least one heteroatom.
The heteroatom is preferably oxygen or sulfur. This leaving group is linked to a positively charged moiety capable of producing light by chemiluminescence, which can be an acridinium, phenanthridinium, quinolinium, or benzacridinium moiety. Both the leaving group and the positively charged moiety can be substituted, such as with reactive substituents for covalently linking the label to a biological molecule. Substituents on the five-membered ring of the leaving group can form an additional ring. An additional class of these chemiluminescent labels comprises a chemical group that can produce light by chemiluminescence covalently linked to a leaving group comprising a moiety containing a sulfur, phosphorus, or carbon atom double-bonded to a more electronegative atom or atoms.
Novel chemiluminescent compounds are suitable for use in labeling biological molecules for assays such as immunoassays. These chemiluminescent labels are characterized by the incorporation of stable leaving groups. One class of these chemiluminescent labels comprises salts in which the leaving group contains a carboxyl carbon atom or its isoelectronic equivalent and a five-membered ring, including at least one heteroatom.
The heteroatom is preferably oxygen or sulfur. This leaving group is linked to a positively charged moiety capable of producing light by chemiluminescence, which can be an acridinium, phenanthridinium, quinolinium, or benzacridinium moiety. Both the leaving group and the positively charged moiety can be substituted, such as with reactive substituents for covalently linking the label to a biological molecule. Substituents on the five-membered ring of the leaving group can form an additional ring. An additional class of these chemiluminescent labels comprises a chemical group that can produce light by chemiluminescence covalently linked to a leaving group comprising a moiety containing a sulfur, phosphorus, or carbon atom double-bonded to a more electronegative atom or atoms.
Description
PATENT
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 .
;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 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 a 0 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
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
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 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 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 ~ ~ ~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 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 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;
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 .
;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 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 a 0 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
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
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 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 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 ~ ~ ~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 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 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 , J ~ 701 7/39D-267 ~ ~ Y~^~ 'q"~ (~A~-~
~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 , ,, ,; 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 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.~0 701 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
(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 -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 .
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 .
~ .
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 ~ ~ 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
(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 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
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 ,~ ~ / ,' ' 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 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;
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 , J ~ 701 7/39D-267 ~ ~ Y~^~ 'q"~ (~A~-~
~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 , ,, ,; 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 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.~0 701 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
(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 -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 .
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 .
~ .
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 ~ ~ 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
(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 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
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 ,~ ~ / ,' ' 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 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 . .
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
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 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
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
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 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
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
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 . .
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
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 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
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
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 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
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
Claims (83)
1. A chemiluminescent salt comprising a cation and an anion wherein:
(a) the cation is represented by the following schematic:
where (i) n is at least one;
(ii) A+ is a positively charged moiety capable of producing light by chemiluminescence attached by a valence bond to a leaving group F;
(iii) Z' is selected from the group consisting of O, S, NH, NOH, NR' and NOR' where R' is C1-C5 alkyl;
(iv) F is selected from the group consisting of moieties of the structure O-CH=CH-W, and moieties represented by the following schematic:
where:
(A) W is selected from the group consisting of hydrogen and C1-C5 alkyl; and (B) X is selected from the group consisting of O, S, Se, Te and NR" where R" is C1-C5 alkyl or arylsulfonyl;
(C) Q is selected from the group consisting of the following structures:
and where:
(i) Y is selected from the group consisting of O, S, S=O, Se, SO2, Se=O, SeO2, Te, Te=O, TeO2 and N-R'", where R'" is selected from the group consisting of hydrogen and C1-C5 alkyl;
(ii) A2, A3 and A4 are each independently selected Prom the group consisting of a valence bond, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C12 cycloalkyl, C5-C12 cycloalkenyl and aryl; and (iii) Z2, Z3 and Z4 are each independently selected from the group consisting of hydrogen, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanate, sulfo, N-succinimidylcarboxyl and N-maleimido, except that where all of A2, A3, and A4 are valence bonds, all of Z2, Z3, and Z4 are not hydrogen unless X is NR" where R"
is arylsulfonyl; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
(a) the cation is represented by the following schematic:
where (i) n is at least one;
(ii) A+ is a positively charged moiety capable of producing light by chemiluminescence attached by a valence bond to a leaving group F;
(iii) Z' is selected from the group consisting of O, S, NH, NOH, NR' and NOR' where R' is C1-C5 alkyl;
(iv) F is selected from the group consisting of moieties of the structure O-CH=CH-W, and moieties represented by the following schematic:
where:
(A) W is selected from the group consisting of hydrogen and C1-C5 alkyl; and (B) X is selected from the group consisting of O, S, Se, Te and NR" where R" is C1-C5 alkyl or arylsulfonyl;
(C) Q is selected from the group consisting of the following structures:
and where:
(i) Y is selected from the group consisting of O, S, S=O, Se, SO2, Se=O, SeO2, Te, Te=O, TeO2 and N-R'", where R'" is selected from the group consisting of hydrogen and C1-C5 alkyl;
(ii) A2, A3 and A4 are each independently selected Prom the group consisting of a valence bond, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C12 cycloalkyl, C5-C12 cycloalkenyl and aryl; and (iii) Z2, Z3 and Z4 are each independently selected from the group consisting of hydrogen, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanate, sulfo, N-succinimidylcarboxyl and N-maleimido, except that where all of A2, A3, and A4 are valence bonds, all of Z2, Z3, and Z4 are not hydrogen unless X is NR" where R"
is arylsulfonyl; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
2. The chemiluminescent salt of claim 1 wherein A+ is selected Prom the group consisting of acridinium, substituted acridinium, phenanthridinium, substituted phenanthridinium, quinolinium, substituted quinolinium, benzacridinium, and substituted benzacridinium.
3. The chemiluminescent salt of claim 1 wherein F is a moiety of the structure O-CH=CH-W, wherein W is selected from the group consisting of hydrogen and C1-C5 alkyl.
4. The chemiluminescent salt of claim 3 wherein W is H.
5. The chemiluminescent salt of claim 1 wherein A+ is an acridinium moiety represented by the following structure:
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, with the condition that where A1 is a valence bond, Z1 is not hydrogen;
(c) R1, R3, R5, R7 and R9 are each independently selected from the group consisting of a valence bond, hydrogen, and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above, with the condition that only one of R1, R3, R5, R7 and R9 is a valence bond; and (d) R2, R4, R6 and R8 are each independently selected from the group consisting of hydrogen and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above.
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, with the condition that where A1 is a valence bond, Z1 is not hydrogen;
(c) R1, R3, R5, R7 and R9 are each independently selected from the group consisting of a valence bond, hydrogen, and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above, with the condition that only one of R1, R3, R5, R7 and R9 is a valence bond; and (d) R2, R4, R6 and R8 are each independently selected from the group consisting of hydrogen and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above.
6. The chemiluminescent salt of claim 5 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is 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.
7. The chemiluminescent salt of claim 5 wherein A1 is selected from the group consisting of substituted and unsubstituted straight chain aliphatic groups.
8. The chemiluminescent salt of claim 7 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is replaced with a replacement moiety selected from the group consisting of -O-, -NH-, and -NL-, wherein L is selected from the group consisting of alkyl groups, cycloalkyl groups, oxo groups, hydroxy groups, sulfo groups, sulfoester groups, carboxyester groups, phosphoryl groups, and phosphorylester groups.
9. The chemiluminescent salt of claim 6 wherein A1 is selected from the group consisting of benzyl and aryl groups.
10. The chemiluminescent salt of claim 7 wherein at least one of the aromatic carbon atoms of A1 is replaced with a replacement moiety selected from the group consisting of -N= and -?L'=, wherein L' is selected from the group consisting of C1-C5 alkyl, C3-C12 cycloalkyl, oxo, and hydroxyalkyl.
11. The chemiluminescent salt of claim 5 wherein A1 is a valence bond.
12. The chemiluminescent salt of claim 11 wherein Z1 is selected from the group consisting of carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido.
13. The chemiluminescent salt of claim 5 wherein R5 is a valence bond and the leaving group moiety is linked to R5.
14. The chemiluminescent salt of claim 13 wherein R1, R2, R3, R4, R6, R7, R8 and R9 are each hydrogen.
15. The chemiluminescent salt of claim 14 wherein Z' is O and X is S.
16. The chemiluminescent salt of claim 15 wherein Q is
17. The chemiluminescent salt of claim 16 wherein Y is O.
18. The chemiluminescent salt of claim 16 wherein Y is S.
19. The chemiluminescent salt of claim 18 wherein A1 is a valence bond and Z1 is CH3.
20. The chemiluminescent salt of claim 19 wherein A2, A3 and A4 are each valence bonds.
21. The chemiluminescent salt of claim 20 where the anion is CF3SO3.
22. The chemiluminescent salt of claim 21 wherein Z2, Z3 and 24 are each H.
23. The chemiluminescent salt of claim 21 wherein Z2 is COOC2H5, and Z3 and Z4 are H.
24. The chemiluminescent salt of claim 21 wherein Z2 is COOCH3, Z3 is COOC2H5, and Z4 is H.
25. The chemiluminescent salt of claim 21 wherein Z2 and Z3 are COOCH3, and Z4 is H.
26. The chemiluminescent salt of claim 21 wherein Z2 is COOCH3, Z3 is COOH, and Z4 is CF3.
27. The chemiluminescent salt of claim 21 wherein Z2 is CH3, and Z3 and Z4 are H.
28. The chemiluminescent salt of claim 21 wherein Z2 and Z4 are each H, and Z3 is CH3.
29. The chemiluminescent salt of claim 21 wherein Z2 and Z4 are each H, and Z3 is C6H5.
30. The chemiluminescent salt of claim 21 wherein Z2 and Z3 are each COOCH3, and Z4 is Br.
31. The chemiluminescent salt of claim 1 wherein A+ is a phenanthridinium moiety represented by the structure:
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, except that where A1 is a valence bond, Z1 is not hydrogen;
(c) 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 A1 above and in which Z is one of the groups defined as Z1 above, in which the A and Z can be selected independently for each of R14, R17, and R18 with the condition that only one of R14, R17, and R18 is a valence bond; and (d) each of R10, R11, R12, R13, R15, and R16 is selected from the group consisting of hydrogen and the moiety A-Z, in which the A and Z can be selected independently for each of R10, R11, R12, R13, R15, and R16.
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, except that where A1 is a valence bond, Z1 is not hydrogen;
(c) 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 A1 above and in which Z is one of the groups defined as Z1 above, in which the A and Z can be selected independently for each of R14, R17, and R18 with the condition that only one of R14, R17, and R18 is a valence bond; and (d) each of R10, R11, R12, R13, R15, and R16 is selected from the group consisting of hydrogen and the moiety A-Z, in which the A and Z can be selected independently for each of R10, R11, R12, R13, R15, and R16.
32. The chemiluminescent salt of claim 31 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the phenanthridinium moiety is 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.
33. The chemiluminescent salt of claim 31 wherein A1 is selected from the group consisting of substituted and unsubstituted straight chain aliphatic groups.
34. The chemiluminescent salt of claim 33 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is replaced with a replacement moiety selected from the group consisting of -O-, -NH-, and -NL-, wherein L is selected from the group consisting of alkyl groups, cycloalkyl groups, oxo groups, hydroxy groups, sulfo groups, sulfoester groups, carboxyester groups, phosphoryl groups, and phosphorylester groups.
35. The chemiluminescent salt of claim 31 wherein A1 is selected from the group consisting of benzyl and aryl groups.
36. The chemiluminescent salt of claim 35 wherein at least one of the aromatic carbon atoms of A1 is replaced with a replacement moiety selected from the group consisting of -N= and -?L'-, wherein L' is selected from the group consisting of C1-C5 alkyl, C3-C12 cycloalkyl, oxo, and hydroxyalkyl.
37. The chemiluminescent salt of claim 31 wherein Z' is O and X is S.
38. The chemiluminescent salt of claim 37 wherein Q is
39. The chemiluminescent salt of claim 38 wherein Y is O.
40. The chemiluminescent salt of claim 38 wherein Y is S.
41. The chemiluminescent salt of claim 40 wherein A1 is a valence bond and Z1 is CH3.
42. The chemiluminescent salt of claim 41 wherein A2, A3 and A4 are each valence bonds.
43. The chemiluminescent salt of claim 42 wherein the anion is CF3SO3-.
44. A chemiluminescent salt comprising a cation and an anion wherein:
(a) the cation is represented by the following schematic:
where (i) n is at least one;
(ii) A+ is a positively charged moiety capable of producing light by chemiluminescence attached by a valence bond to a leaving group F;
(iii) Z' is selected from the group consisting of O, S, NH, NOH, NR' and NOR' where R' is C1-C5 alkyl;
(iv) F is selected from the group consisting of moieties represented by the following schematic:
where:
(A) X is selected from the group consisting of O, S, Se, Te and NR" where R" is selected from the group consisting of C1-C5 alkyl and arylsulfonyl;
and (B) Q is selected from the group consisting of the following structures:
, , , , , and , a second ring containing at least five atoms in addition to the five-membered unsaturated heterocyclic ring being formed by covalent linkage of two of A2, A3, and A4, where:
(1) Y is selected from the group consisting of O, S, S=O, SO2, Se, Se=O, SeO2, Te, Te=O, TeO2, and N-R"', wherein R"' is selected from the group consisting of hydrogen and C1-C5 alkyl;
(2) the moiety A2, A3, or A4 not involved in formation of the second ring is 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, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido; and (4) of the two of A2, A3, and A4 involved in formation of 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; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, or bicarbonate.
(a) the cation is represented by the following schematic:
where (i) n is at least one;
(ii) A+ is a positively charged moiety capable of producing light by chemiluminescence attached by a valence bond to a leaving group F;
(iii) Z' is selected from the group consisting of O, S, NH, NOH, NR' and NOR' where R' is C1-C5 alkyl;
(iv) F is selected from the group consisting of moieties represented by the following schematic:
where:
(A) X is selected from the group consisting of O, S, Se, Te and NR" where R" is selected from the group consisting of C1-C5 alkyl and arylsulfonyl;
and (B) Q is selected from the group consisting of the following structures:
, , , , , and , a second ring containing at least five atoms in addition to the five-membered unsaturated heterocyclic ring being formed by covalent linkage of two of A2, A3, and A4, where:
(1) Y is selected from the group consisting of O, S, S=O, SO2, Se, Se=O, SeO2, Te, Te=O, TeO2, and N-R"', wherein R"' is selected from the group consisting of hydrogen and C1-C5 alkyl;
(2) the moiety A2, A3, or A4 not involved in formation of the second ring is 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, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido; and (4) of the two of A2, A3, and A4 involved in formation of 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; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, or bicarbonate.
45. The chemiluminescent salt of claim 44 wherein A+ is selected from the group consisting of acridinium, substituted acridinium, phenanthridinium, substituted phenanthridinium, quinolinium, substituted quinolinium, benzacridinium, and substituted benzacridinium.
46. The chemiluminescent salt of claim 45 wherein at least one of the carbon atoms of A2, A3 or A4 that is not involved in formation of the second ring, other than the carbon atom located furthest from the unsaturated heterocyclic 5-membered ring, is 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.
47. The chemiluminescent salt of claim 45 wherein the one of A2, A3 and A4 that is not involved in formation of the second ring is selected from the group consisting of substituted and unsubstituted straight-chain aliphatic groups and at least one of the carbon atoms of the one of A2, A3 and A4 not involved in formation of the second ring, other than the carbon atom located furthest from the unsaturated heterocyclic 5-membered ring, is replaced with a replacement moiety selected from the group consisting of -O-, -NH-, and -NL-, wherein L is selected from the group consisting of alkyl groups, cycloalkyl groups, oxo groups, hydroxy groups, sulfo groups, sulfoester groups, carboxyester groups, phosphoryl groups, and phosphorylester groups.
48. The chemiluminescent salt of claim 45 wherein Z' is O and X is S.
49. The chemiluminescent salt of claim 48 wherein Y is O.
50. The chemiluminescent salt of claim 48 wherein Y is S.
51. The chemiluminescent salt of claim 50 wherein the anion is CF3SO3-.
52. The chemiluminescent salt of claim 4?
wherein A+ is an acridinium moiety represented by the following structure:
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, with the condition that where A1 is a valence bond, Z1 is not hydrogen;
(c) R1, R3, R5, R7 and R9 are each independently selected from the group consisting of a valence bond, hydrogen, and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above, with the condition that only one of R1, R3, R5, R7 and R9 is a valence bond; and (d) R2, R4, R6 and R8 are each independently selected from the group consisting of hydrogen and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above.
wherein A+ is an acridinium moiety represented by the following structure:
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, with the condition that where A1 is a valence bond, Z1 is not hydrogen;
(c) R1, R3, R5, R7 and R9 are each independently selected from the group consisting of a valence bond, hydrogen, and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above, with the condition that only one of R1, R3, R5, R7 and R9 is a valence bond; and (d) R2, R4, R6 and R8 are each independently selected from the group consisting of hydrogen and a moiety A-Z where A is defined as A1 above and Z is defined as Z1 above.
53. The chemiluminescent salt of claim 52 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is 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.
54. The chemiluminescent salt of claim 52 wherein A1 is selected from the group consisting of substituted and unsubstituted straight chain aliphatic groups.
55. The chemiluminescent salt of claim 54 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is replaced with a replacement moiety selected from the group consisting of -O-, -NH-, and -NL-, wherein L is selected from the group consisting of alkyl groups, cycloalkyl groups, oxo groups, hydroxy groups, sulfo groups, sulfoester groups, carboxyester groups, phosphoryl groups, and phosphorylester groups.
56. The chemiluminescent salt of claim 52 wherein A1 is selected from the group consisting of benzyl and aryl groups.
57. The chemiluminescent salt of claim 56 wherein at least one of the aromatic carbon atoms of A1 is replaced with a replacement moiety selected from the group consisting of -N= and -?L'=, wherein L' is selected from the group consisting of C1-C5 alkyl, C3-C12 cycloalkyl, oxo, and hydroxyalkyl.
58. The chemiluminescent salt of claim 52 wherein A1 is a valence bond.
59. The chemiluminescent salt of claim 58 wherein Z1 is selected from the group consisting of carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido.
60. The chemiluminescent salt of claim 52 wherein R5 is a valence bond and the leaving group moiety is linked to R5.
61. The chemiluminescent salt of claim 60 wherein R1, R2, R3, R4, R6, R7, R8 and R9 are each hydrogen.
62. The chemiluminescent salt of claim 61 wherein A1 is a valence bond and Z1 is CH3.
63. The chemiluminescent salt of claim 44 wherein A+ is a phenanthridinium moiety represented by the structure:
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, except that where A1 is a valence bond, Z1 is not hydrogen;
(c) 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 A1 above and in which Z is one of the groups defined as Z1 above, in which the A and Z can be selected independently for each of R14, R17, and R18 with the condition that only one of R14, R17, and R18 is a valence bond; and (d) each of R10, R11, R12, R13, R15, and R16 is selected from the group consisting of hydrogen and the moiety A-Z, in which the A and Z can be selected independently for each of R10, R11, R12, R13, R15, and R16.
where:
(a) A1 is 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;
(b) Z1 is selected from the group consisting of hydrogen, methyl, carboxyl, carboxyl halide, sulfonyl halide, carboalkoxy, carboxyl acylate, carboxamido, cyano, carboxime, isocyanato, sulfo, N-succinimidylcarboxyl, and N-maleimido groups, except that where A1 is a valence bond, Z1 is not hydrogen;
(c) 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 A1 above and in which Z is one of the groups defined as Z1 above, in which the A and Z can be selected independently for each of R14, R17, and R18 with the condition that only one of R14, R17, and R18 is a valence bond; and (d) each of R10, R11, R12, R13, R15, and R16 is selected from the group consisting of hydrogen and the moiety A-Z, in which the A and Z can be selected independently for each of R10, R11, R12, R13, R15, and R16.
64. The chemiluminescent salt of claim 63 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the phenanthridinium moiety is 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.
65. The chemiluminescent salt of claim 63 wherein A1 is selected from the group consisting of substituted and unsubstituted straight chain aliphatic groups.
66. The chemiluminescent salt of claim 65 wherein at least one of the carbon atoms of A1 other than the carbon atom located furthest from the acridinium moiety is replaced with a replacement moiety selected from the group consisting of -O-, -NH-, and -NL-, wherein L is selected from the group consisting of alkyl groups, cycloalkyl groups, oxo groups, hydroxy groups, sulfo groups, sulfoester groups, carboxyester groups, phosphoryl groups, and phosphorylester groups.
67. The chemiluminescent salt of claim 63 wherein A1 is selected from the group consisting of benzyl and aryl groups.
68. The chemiluminescent salt of claim 66 wherein at least one of the aromatic carbon atoms of A1 is replaced with a replacement moiety selected from the group consisting of -N= and -?L'=, wherein L' is selected from the group consisting of C1-C5 alkyl, C3-C12 cycloalkyl, oxo, and hydroxyalkyl.
69. The chemiluminescent salt of claim 63 wherein A1 is a valence bond and Z1 is CH3.
70. A chemiluminescent salt comprising a cation and an anion wherein:
(a) the cation is represented by the following schematic:
where (i) A+ is a positively charged moiety capable of producing light by chemiluminescence, and (ii) Y is selected from the group consisting of O, S, S=O, Se, SO2, Se=O, SeO2, Te, Te=O, TeO2 and N-R'", where R'" is selected from the group consisting of hydrogen and C1-C5 alkyl; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
(a) the cation is represented by the following schematic:
where (i) A+ is a positively charged moiety capable of producing light by chemiluminescence, and (ii) Y is selected from the group consisting of O, S, S=O, Se, SO2, Se=O, SeO2, Te, Te=O, TeO2 and N-R'", where R'" is selected from the group consisting of hydrogen and C1-C5 alkyl; and (b) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
71. The chemiluminescent salt of claim 70 wherein Y is S.
72. The chemiluminescent salt of claim 70 wherein A+ is selected from the group consisting of acridinium, substituted acridinium, phenanthridinium, substituted phenanthridinium, quinolinium, substituted quinolinium, benzacridinium, and substituted benzacridinium.
73. The chemiluminescent salt of claim 71 wherein A+ is an acridinium moiety represented by the following chemical structure:
.
.
74. The chemiluminescent salt of claim 73 wherein the anion is SO3CF3?.
75. A chemiluminescent salt represented by the following chemical structure:
.
.
76. A chemiluminescent salt comprising an anion and a cation, the cation comprising at least one chemical group capable of producing light by chemiluminescence covalently linked to a leaving group selected from the group consisting of the following chemical structures:
; ; ; ; ;
; ; ; and where:
(a) the chemical group that can produce light by chemiluminescence is a heterocyclic ring or ring system selected from the group consisting of acridinium, phenanthridinium, quinolinium, and benzacridinium;
(b) 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, imidazole 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 substituted derivatives thereof, primary amino groups, secondary amino groups, tertiary amino groups, and quaternary amino groups, anilino derivatives, and morpholine derivatives;
(c) R2 is selected from the group consisting of hydrogen, alkyl groups and thioderivatives thereof, aryl groups and thioderivatives thereof, alkoxy groups and thioderivatives thereof, aryloxy groups and thioderivatives thereof, and derivatives of alkyl, aryl, alkoxy, aryloxy groups substituted with at least one of nitro, cyano, halo and sulfonyl;
(d) R3 is selected from the group consisting of O, S, NH, NR1, NR2, CH2, C(R1)2, C(R2)2, and CR1R2 where R1 and R2 are as defined above; and (e) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
; ; ; ; ;
; ; ; and where:
(a) the chemical group that can produce light by chemiluminescence is a heterocyclic ring or ring system selected from the group consisting of acridinium, phenanthridinium, quinolinium, and benzacridinium;
(b) 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, imidazole 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 substituted derivatives thereof, primary amino groups, secondary amino groups, tertiary amino groups, and quaternary amino groups, anilino derivatives, and morpholine derivatives;
(c) R2 is selected from the group consisting of hydrogen, alkyl groups and thioderivatives thereof, aryl groups and thioderivatives thereof, alkoxy groups and thioderivatives thereof, aryloxy groups and thioderivatives thereof, and derivatives of alkyl, aryl, alkoxy, aryloxy groups substituted with at least one of nitro, cyano, halo and sulfonyl;
(d) R3 is selected from the group consisting of O, S, NH, NR1, NR2, CH2, C(R1)2, C(R2)2, and CR1R2 where R1 and R2 are as defined above; and (e) the anion is selected from the group consisting of sulfate, methosulfate, perhalomethosulfate, haloborate, haloacetate, halophosphate, phosphate, halide, phosphite, nitrate, nitrite, carbonate, and bicarbonate.
77. The chemiluminescent salt of claim 76 wherein the chemical group producing light is N-methylacridinium.
78. The chemiluminescent salt of claim 77 wherein the leaving group is in which R1 is selected from the group consisting of pyrrole, pyrazole, 2-methylindole, and isatin and R3 is 0.
79. A method of determining the quantity of a biomolecule present in a solution comprising:
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 1 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 1 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
80. A method of determining the quantity of a biomolecule present in a solution comprising:
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 44 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 44 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
81. A method of determining the quantity of a biomolecule present in a solution comprising:
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 70 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 70 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
82. A method of determining the quantity of a biomolecule present in a solution comprising:
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 75 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 75 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
83. A method of determining the quantity of a biomolecule present in a solution comprising:
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 76 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
(a) reacting a covalent conjugate comprising the cation of the chemiluminescent salt of claim 76 covalently linked to the biomolecule with an oxidizer selected from the group consisting of hydrogen peroxide, molecular oxygen, and organic peroxide to generate light by chemiluminescence; and (b) determining the quantity of light generated to determine the quantity of the biomolecule present.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/US1991/006861 WO1992009580A1 (en) | 1990-11-21 | 1991-09-20 | Chemiluminescent compounds |
| EP92900463A EP0511366A1 (en) | 1990-11-21 | 1991-09-20 | Chemiluminescent compounds |
| CA002074014A CA2074014A1 (en) | 1990-11-21 | 1992-07-16 | Chemiluminescent compounds |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61648690A | 1990-11-21 | 1990-11-21 | |
| CA002074014A CA2074014A1 (en) | 1990-11-21 | 1992-07-16 | Chemiluminescent compounds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2074014A1 true CA2074014A1 (en) | 1994-01-17 |
Family
ID=25675333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002074014A Abandoned CA2074014A1 (en) | 1990-11-21 | 1992-07-16 | Chemiluminescent compounds |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0511366A1 (en) |
| CA (1) | CA2074014A1 (en) |
| WO (1) | WO1992009580A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731148A (en) * | 1995-06-07 | 1998-03-24 | Gen-Probe Incorporated | Adduct protection assay |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE1008216A4 (en) * | 1994-01-25 | 1996-02-20 | Biocode Sa | HETEROCYCLIC CHEMOLUMINESCENT DERIVATIVES. |
| IT1302564B1 (en) * | 1997-05-22 | 2000-09-29 | Hoffmann La Roche | POLLIZATION IMMUNOSAGE OF PERFECTED FLUORESCENCE. |
| DE69831965T2 (en) | 1997-08-21 | 2006-07-20 | Maine Medical Center | PEROXIDE-BASED CHEMILUMINESCENCE TEST AND CHEMILUMINESCENT COMPOUNDS USED IN THIS INVENTION |
| EP1397488B1 (en) | 2001-06-01 | 2006-08-16 | Roche Diagnostics GmbH | Novel compounds for chemiluminescense procedures |
| KR102540846B1 (en) | 2014-11-25 | 2023-06-07 | 삼성전자주식회사 | Compound for organic photoelectric device and organic photoelectric device, image sensor and electronic device including the same |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1461877A (en) * | 1974-02-12 | 1977-01-19 | Wellcome Found | Assay method utilizing chemiluminescence |
| DE3279029D1 (en) * | 1981-12-11 | 1988-10-20 | Welsh Nat School Med | Luminescent labelling materials and procedures |
| DE3628573C2 (en) * | 1986-08-22 | 1994-10-13 | Hoechst Ag | Chemiluminescent acridine derivatives, processes for their preparation and their use in luminescence immunoassays |
| DE3844954C2 (en) * | 1988-02-20 | 1998-07-16 | Hoechst Ag | Special chemiluminescent acridine derivatives and their use in luminescent immunoassays |
-
1991
- 1991-09-20 WO PCT/US1991/006861 patent/WO1992009580A1/en not_active Application Discontinuation
- 1991-09-20 EP EP92900463A patent/EP0511366A1/en not_active Withdrawn
-
1992
- 1992-07-16 CA CA002074014A patent/CA2074014A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5731148A (en) * | 1995-06-07 | 1998-03-24 | Gen-Probe Incorporated | Adduct protection assay |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0511366A1 (en) | 1992-11-04 |
| WO1992009580A1 (en) | 1992-06-11 |
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