JPH0147472B2 - - Google Patents

Description

[Detailed description of the invention]

fluorescing compounds
compounds) have a wide range of applications because of their ability to emit light when excited by energy within a certain energy range. Because of this ability, fluorescers are used in the advertising of new products and in chemical or biological processes.
For example, it has found use as a label in assays. That is, various compounds can be conjugated to a fluorogenic compound, the conjugate is subjected to some type of distribution, and the fate of the conjugate is to illuminate the sample and release the conjugate. Determined by detecting the zone where the body is located. This method can be used in immunoassays involving specific binding pairs such as antigens and antibodies. The amount of antigen in an unknown sample is determined between the solid and liquid phases by conjugating a fluorogenic agent to one of the members of the specific binding pair and using various protocols. The fluorophore complex can be dispensed. By measuring the fluorescence of either of the phases, one can relate the level of fluorescence observed to the concentration of antigen in the sample. Alternatively, partitioning of the fluorescent label can be avoided by providing a mechanism that changes the fluorescence of the label depending on the label environment in the liquid medium. For example, in addition to labeling one of the members of a specific binding pair with a fluorophore,
Other members can be labeled with quenchers, molecules capable of absorbing the excitation energy of the fluorescence generator molecule, preventing the emission of photons. Quenching then occurs only when the two members of the specific binding pair are associated such that the fluorogen and quencher have the necessary spatial proximity for quenching. There are many pressing needs in producing fluorophores. As a fluorescence generator, a high extinction coefficient, preferably a high quantum efficiency close to or equal to 1, chemical stability, a large Stokes shift are desired, and the fluorescence cannot be influenced by other reagents. When used, an effective response to such reagents is desired. Furthermore, if the fluorogenic agent is to be used in the presence of serum or other compositions that are themselves fluorogenic, the fluorogenic agent will be substantially free from the energy absorbed by other compounds in the medium. It is desirable to absorb energy in different ranges. In the case of serum, it is desirable to have a fluorogenic agent that absorbs light substantially above 450 nm, preferably above 500 nm. For quencher molecules, the quencher effectively quenches the fluorophore molecule, i.e., there is substantial overlap between the wavelength range of emission by the fluorophore and the wavelength range of absorption by the quencher. This is desirable. Furthermore, the quencher should be chemically stable, preferably non-fluorescent, and should provide a high quenching efficiency for the phosphorescent-quencher pair. Furthermore, advantageous compounds should be amenable to a rational mode of synthesis so as to provide the desired product in substantially pure form. U.S. Pat. No. 3,998,943 uses photochromic hindrance of the simultaneous binding of an antibody to a ligand and an antibody to a fluorophore to generate a ligand, where the antibody to the fluorophore substantially quenches the fluorescence. - Discloses an immunoassay involving a fluorophore complex. U.S. Pat. No. 3,996,345 discloses a fluorophore-quencher in which the fluorogen-quencher binds to one member of a specific binding pair and the quencher binds to the same or a different member of the specific binding pair. An immunoassay involving the pair is described. Assay depends on the degree to which the quencher and fluorophore are brought into close proximity to the extinction range based on the amount of analyte in the medium. There is an extensive list of compounds that include derivatives of fluorogenic agents. Known compounds are 4',5'-dihydroxyfluorescein and 4',5'-dihydroxy2',
7′-dibromofluorescein (CA 61 , 7407d)
including. Isocyanate derivatives of fluorescein are commercially available, while isocyanate derivatives are described in CA 59 , 563b and derivatives containing sulfonic groups are described in CA 58 , 9012a. Di(chalcokene ether) symmetrically substituted fluorescein [Di
(chalcogen ether) symetrically substituted
fluoresceins] are provided. The compound is coupled to other substances for immunoassays, particularly reagents in immunoassays containing serum samples. The fluorescein compound may be halogenated. Fluorescence generators have large extinction coefficients, high quantum yields, maximum absorption higher than 500 nm, and are commonly
It has a Stokes shift greater than 10 nm and is stable on its own and when bound to other compounds. The quencher has an absorption maximum above 500 nm, has little or no observable fluorescence, and effectively quenches a broad spectrum of fluorescent compounds. The invention is particularly useful when conjugated to other compounds, particularly polypeptides, or soluble or insoluble supports for use as reagents in immunoassays, capable of accepting or donating electronic energy. Chromogenic di(chalcogen ether)
The present invention relates to symmetrically substituted fluorescein compounds. Fluorescein compounds are unsubstituted at 1', 8' and symmetrically disubstituted on the xanthene ring at either the 4', 5'- or 2', 7'-positions. The compounds are usually 2,7-di(aliphatic ether substituted) or 4,5-di(aliphatic ether substituted)-9-phenyl-6-hydroxy-3H-xanthen-3-ones. be. The molecule has at least 15 carbon atoms, usually at least 16 carbon atoms, and up to about 45, usually up to 35 carbon atoms. There are at least 5 chalcogen atoms (atomic number 8 to 16, oxygen and sulfur), at least 3 of which are oxygen. In addition to the chalcogen atoms, 0 to 8, usually 0 to 6 heteroatoms, such as nitrogen, halogens with atomic numbers 9 to 53, especially 17 to 53, i.e.
There may be fluorine, chlorine, bromine and iodine or other hetero functionalities which may be present to provide specific effects. Usually at least one anionic group, usually carboxylates and sulfonates, and one linking group, especially non-oxocarbonyl groups, including isothiocyanates and isocyanates.
carbonyl); sulfonamide, mercapto and amino, which may or may not be directly bonded to a ring carbon atom. Often the linking group is on a group substituted at the 9-position of the xanthene, usually the phenyl group, but the linking group may also be present as a substituent on the ether group. These compounds are conjugated to haptens and antigens to provide complexes that can fluoresce or quench a fluorescer when the quencher is in close spatial distance to the fluorescer. The compositions of the invention have a wavelength above 500nm, typically 510nm.
It has a relatively narrow band absorption maximum above, and typically at least 50% of the area of longest wavelength absorption exceeds a wavelength range of about 50 nm. The fluorogenic compounds are characterized by good chemical stability, a large Stokes shift and an extinction coefficient of more than 65,000, usually more than 75,000. Stokes shift is at least 10 nm preferably at least about
It is 20nm. The quenching compound fluoresces with a quantum efficiency of less than 10%, preferably less than 5%, in 0.05 molar phosphate when irradiated with light of maximum absorption. The compounds of the present invention provide novel compounds with important spectroscopic and physical properties. The compound is
It has an absorption maximum above 500 nm. By choosing the position for the oxy substituent, it is possible to provide highly fluorescent compounds or compounds that are substantially fluorescent and can be used as quenchers. Compounds with ether substituents in the 2',7'-position (fluorescein position number) give fluorescent compounds with high quantum efficiency. 4′, 5′―
Compounds with ether substituents in position provide compounds that do not fluoresce and absorb at long wavelengths to act as effective quenchers. Often, the compounds of the invention will have the following formula: In the formula, each of α and each of δ may be the same or different, and either α or δ is bonded to a carbon atom of the ring via a chalcogen (atomic number 8-16, oxygen and sulfur). α can be a substituent other than chalcogen and δ can be any convenient functionality, including chalcogen, if it is not a chalcogen bonding pair; or ω can be taken together with Λ to provide the active functionality for binding, or Λ can be the binding functionality for Λ if not taken together; ω can be a non-interfering functionality or hydrogen; Λ can be a ligand or receptor if not taken together to form an active functionality for binding; γ can be a non-interfering functionality or hydrogen; Spacer arms of about 1 to 20 atoms, usually 1 to 16 carbon atoms, usually 6 to 16
aliphatic (including cycloaliphatic) groups of 1 to 7 carbon atoms with more than 4 ring carbon atoms in the case of cycloaliphatic or aromatic groups of usually 6 to 10 ring atoms; is a water- or base-soluble compound having the following: and one or more groups in bonded to Λ are present when Λ is a ligand or receptor. Regarding the quencher molecule, 4,5-diether-6-hydroxy-3H-xanthene-3-one,
The presence or absence of substituents at the 2 and 7 positions does not affect extinction, but can be used to modify the absorption properties of the molecule. Therefore, if α is an ether, then
δ can be hydrogen or any convenient substituent such as alkyl of 1 to 6 carbon atoms, oxy (hydroxy and 1
thio (alkoxy of ~6 carbon atoms), thio (mercapto, alkylthio and sulfonic acids, esters and amides of 1 to 6 carbon atoms), non-oxocarbonyl (acids, esters and amides of 1 to 6 carbon atoms) (including), cyano, nitro, halo, 1-6
or a combination thereof. The choice of substitution is governed by the maximum absorbance obtained, the convenience of synthesis, and the effect on the physical and chemical properties of the molecule, such as water solubility, chemical reactivity, oxidative susceptibility, and the like. Regarding the fluorescence generator molecule, the substituents at the 4,5-positions can vary widely so long as the fluorescence efficiency is not significantly adversely affected. Therefore, although this can vary widely, the 4,5-position should not be substituted by chalcogen, which has the effect of substantially reducing the fluorescence of the molecule. Therefore, the range of substituents for the 4,5-position of the fluorescence generator is more limited than the range of substituents for the 2,7-position of the quencher. The compounds of the present invention having γ as an aromatic group mainly have the following formula; where the two A's are the same or different, usually the same except for the functionality for bonding, the two D's are the same or different, usually the same except for the functionality for bonding, and A or D is a chalcogen ether (chalcogen with atomic number 8 to 16), usually an oxyether of -JMX, where J is oxygen or sulfur; when other than -JMX, A is hydrogen or a halogen of atomic number 9-53, i.e. fluorine, chlorine, bromine or iodine, in particular chlorine and iodine, while D is hydrogen or, in particular carbon and hydrogen and as heteroatoms oxygen, sulfur, nitrogen and halogen. and can be any substituent which is usually chemically inert under the conditions of use, and M is a divalent hydrocarbon group, usually 1 to 8, usually 1 to 6. , preferably a normally straight chain saturated aliphatic of 1-3 carbon atoms, one of the X's having an active functionality for binding to the ligand, receptor or support.
or a functionality attached to the ligand or support, where X together with W provides the active functionality for attachment, and XW is a non-oxocarbonyl functionality, including its sulfur and nitrogen analogues. , for example carboxylic acid,
Carboxylic acid esters, such as lower alkyl (1
-3 carbon atoms) or active esters capable of forming amide bonds in aqueous media, such as N-
oxysuccinimide and p-nitrophenyl,
Isocyanates, isothiocyanates, imidate lower alkyl esters, activated olefins, e.g. maleimides, mercaptans (-
SH), formyl (--CHO), sulfonyl chloride, amino, active halo such as haloacetyl or halotriazine, provided that XW, when attached to M, is non-oxocarbonyl or sulfonyl; When X is not combined with W, one of the and sulfur analogs), carbamyl, thiocarbamyl, substituted ethylene from activated olefins,
thio, methylene (by reductive amination from formyl), amide nitrogen or secondary amino, sulfonyl,
or oxocarbonylmethyl from an active halo, and if X is a bonding functionality attached to M, then X is a non-oxocarbonyl; if not a bonding group, then X is hydrogen or a non-oxocarbonyl, e.g. acid, ester or amide, sulfonamide, sulfonic acid or halo, especially when attached to a ring carbon atom, p is 1 if W together with X, otherwise On average, the molecular weight is between 1 and W.
500, usually 1000, more commonly 1500, most commonly 2000, and generally p is about 1 to
200, usually in the range 1 to 100; if W is not combined with
or a ligand encompassing an antigenic receptor, generally having a molecular weight of about 125 to 2000 if haptenic and about 5000 x 1 x 10 ⁷ if antigenic. In Although,
Combinations of antigens with other substances can have much higher composite molecular weights, and the ligand is usually linked to X via amino, hydroxy, mercapto or activated ethylene, although other linkages can be used. to form an amide, amidine, thioamide, ether or thioether, or W is a naturally occurring or synthetic, modified or unmodified polysaccharide, a naturally occurring or synthetic polymer, a glass,
A soluble or insoluble support, which can be a liposome or the like, where Q is a bond or spacer arm, usually aliphatic, aromatic, heterocyclic or a combination thereof, generally aliphatic. aliphatically, saturated, wherein the arms are 1 to 16, more usually 1 to 12, preferably 1 to
It has 8 atoms in the chain, including carbon, nitrogen,
oxygen and sulfur, where nitrogen is an amide or is bonded only to carbon and hydrogen, such as tertiary amino, oxygen is oxy, and sulfur is a thioether, and chalcogen is a carbon and the heteroatoms are separated by at least two carbon atoms when bonded to a saturated carbon atom; the total number of carbon atoms is generally 1 to 20, usually 1 to 12; The total number of atoms is from 0 to 10, usually from 0 to 8, and oxygen may be present as non-oxocarbonyl or oxy;
0-9, usually 0-4 heterofunctionality is present;
Q is usually a bond when X is not a bond functionality or group, Y is a halogen with atomic number 9 to 53, especially chlorine, n is an integer from 0 to 4, where m+n is from 4 to Z is an acidic anionic group, such as a carboxylic acid or a sulfonic acid, and m is an integer from 0 to 3, usually from 1 to 3. Quite obviously, the compounds of the invention can be modified outside the scope of the above formula without significantly affecting the properties of the compounds. for example,
One or more acidic anionic groups can be esterified or amidated, or alkyl groups and other groups such as cyano, nitro, etc. can be substituted on the phenyl group. However, these changes often require additional synthetic steps, which are not justified by the degree of enhancement, if any, in the spectroscopic or chemical properties of the resulting products. The compounds of this invention have many desirable properties. The product can be conjugated to a wide variety of polypeptides without significantly adversely affecting the aqueous solubility of the polypeptide and without adversely affecting the spectroscopic properties of the compounds of the invention. It has considerable water solubility that allows for Regarding the spectroscopic properties of the compounds of the present invention, they generally have wavelengths greater than 500 nm and
Absorbs at relatively long wavelengths exceeding 510 nm. Thus, naturally occurring fluorescence that may be encountered when working with physiological fluids is substantially avoided by using excitation light in a wavelength range that does not significantly excite naturally occurring fluorogens. Furthermore, the compounds have relatively sharp absorption peaks and the fluorogens have relatively sharp emission peaks. Therefore, effective overlap is obtained between the fluorescence generator and the quencher, which allows effective quenching up to a distance of about 70 Å. Fluorescent compounds also have large Stokes shifts such that the absorption and emission band peaks are at least 10 nm, often at least 15 nm apart. A large Stokes shift minimizes background interference due to observed fluorescence. Quenchers have little or no fluorescence so that they do not contribute to background interference with the observed signal. By providing a fluorogen-quencher couple, where the absorption band of the quencher substantially overlaps the emission band of the fluorogen, the quencher is phosphorized by binding of an immunologically relevant substance. Provides an effective system for performing immunoassays that rely on quenching of fluorescence when brought into close proximity to a light generating agent. In describing the present invention, we will first consider the simple spectroscopically active monomer compounds used in conjugation, followed by a discussion of the various conjugates.
The compounds are chemically stable even at basic PH so that they retain their spectroscopic properties during use. Compounds used for conjugation to other compounds are characterized by having active functionality that forms stable covalent bonds, usually amide or thioether bonds, with other compounds. Often, the bonding functionality will include non-oxocarbonyl, including its nitrogen and sulfur analogs, and will be bonded directly to the ring carbon atom of the phenyl group of the fluorescein, or via a bonding group; It can be attached directly to the oxy- or thioether functionality or via a linking group. A variety of functionalities can be used that are comparable to other functionalities in the molecule. Functionality includes carboxylic acids, which can be activated with carbodiimides or activated alcohols to give active ester groups, isocyanates, isothiocyanates, imidates, etc., which can react with amino functionality to give amides, thioamides, etc. or form an amidine. Alternatively, it can have an amino group as functionality, which can be combined with a carboxylic acid or derivative to provide an amide linkage. Finally, mercapto functionality can be used, which can be combined or vice versa with ethylenic groups, especially activated ethylenic groups, such as maleic acid derivatives, to give thioethers. A preferred group of compounds mainly has the following formula: where the two A ^1s are the same or different, usually the same except when one is functional for bonding, and the two D ^1s are the same or different, usually one is functional for bonding. and either A ¹ or D ¹ is an oxyether of the formula -OM ¹ X ^b , where M ¹ is 1 to 6, usually 1 to
Saturated aliphatic hydrocarbylene groups of 3 carbon atoms, preferably straight-chain and saturated aliphatic hydrocarbylene groups of 1 to 2 carbon atoms, i.e. methylene or ethylene (hydrocarbylene consists only of carbon and hydrogen) ) and when not an oxyether, A ¹ is preferably hydrogen or especially halo of atomic number 9 to 53, more particularly chloro or iodo, and D ¹ is preferably hydrogen, halo or alkyl of up to 6 carbon atoms, and one of X ^a or X ^b , usually X ^a , together with W ¹ forms an active functionality, which functionality is - May have the same definition as XW, but usually with functionalities such as mixed anhydrides (its sulfur-thiono-analogs), for example with butyl chloroformates, carboxylic acids, activated esters, isocyanates or isothiocyanates. provided that X ^b is a carboxylic acid or a derivative thereof when combined with W ¹ , and when not combined with W ¹ , one of X ^a or X ^b is a non-oxocarbonyl containing W ¹ . A bonding group to W ¹ which is carbonyl to form an amide or ester, carbamyl to form urea with W ¹ or thiocarbamyl to form thiourea with W ¹ , and if it is not an active or bonding functionality, X ^a is hydrogen or non-oxocarbonyl, for example carboxy, X ^b is hydrogen or carboxyl, usually hydrogen, Q ¹ can be the same as Q, can be a bond or a spacer arm, but usually there is one in the chain. A bond or spacer arm having ~12, usually 2 to 12 atoms, the atoms being carbon, nitrogen and oxygen, typically 1 to 10, usually 1 to 8
carbon atoms and 0 to 8 heteroatoms,
The heteroatoms are nitrogen, oxygen and sulfur, where oxygen is present only bonded to carbon, such as a non-oxocarbonyl or oxyether, sulfur is analogous to oxygen, and nitrogen is amide or bonded only to carbon. For example, it is a tertiary amino,
Q ¹ is usually a bond if X ^a is a reactive functionality or other than a bonding functionality, and if W ¹ does not go together with X ^a or X ^b ,
W ¹ is usually an amino or hydroxyl, in particular a ligand, receptor or support with an amino functionality for binding, p ¹ may be the same as p and one of X ^a or X ^b is W If it is combined with ¹ , it is 1; otherwise, the molecular weight of 1 to W ¹ is 500, usually
It is the value divided by 1500, generally in the range of 1 to 200,
Usually in the range of 1 to 100, more usually in the range of 1 to 50, Z ¹ is an acidic anionic group such as carboxylic acid or sulfonic acid, m ¹ is an integer of 1 to 3, Y ¹ is a halogen with atomic number 9 to 53, especially chloro, n ¹ is an integer from 0 to 3, where m ¹ + n ¹ is 4
not greater than, has . The compound has 0 to 6, usually 0 to 5 halogens with atomic number 9 to 53, preferably chlorine or iodine, usually 0 to 4 chlorines, often 2 to 4
of chlorine. The compounds usually have at least 2 and up to 5 carboxylic acid groups, preferably 2 to 3 carboxylic acid groups. The non-oxocarbonyl bond functionality may or may not be attached to a carbon atom, but is preferably attached to a carbon atom. Preferred compounds having active functionality are mainly of the following formula: where either A ¹ or D ¹ is alkoxy of 1 to 3, usually 1 to 2 carbon atoms; if not alkoxy, they are as described above for A and D, and Y ¹ ′, Z ¹ ′, m ¹ ′ and n ¹ ′ have the same range as the dashed symbol, W ¹ ′ is an active functionality with a non-oxocarbonyl group or a sulfur analog (thiono), and including acyl halides, mixed anhydrides and activated esters, and isocyanates and isothiocyanates, where Q ¹ ' is a bond or spacer arm of 1 to 12, usually 1 to 10 atoms, and the atoms are carbon, nitrogen and oxygen, usually carbon and nitrogen;
and about 1 to 12, usually 1 to 10 carbon atoms,
0 to 8, usually 0 to 6 heteroatoms;
The atoms are nitrogen, oxygen and sulfur, especially nitrogen and oxygen, where nitrogen exists as an amide or is bonded only to carbon, chalcogens (oxygen and sulfur) are bonded only to carbon, and double bonds (oxo and thiono) or single bonded (oxy or thio), and Q ¹ ' is 1 to 8 as a spacer arm.
, usually an alkylene of 1 to 4 carbon atoms, glycine or a polyglycyl of 1 to 4 glycyl units, where the final carboxy is W ¹ ', etc. Below is a list of compounds of the invention used for conjugation. Table 1 The compound is a substituted 9-phenyl-6-hydroxy-3H-xanthen-3-one, or if the compound is a fluorescein with a 2-carboxy group, the compound is a 3′,6 ′-dihydroxyspiro [isobenzofuran-(3H),
9′-[9H]xanthene]-3-one. The numbers in this list are based on the former name. 2,7-dimethoxy-9-(2',4'-dicarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 2,7-diethoxy-9-(2',3',4' -tricarboxyphenyl)-6-hydroxy-3H-
Xanthene-3-one, 2,7-diethoxy-9-(2',4',5'-tricarboxy-3',6'-dichlorophenyl)-6-
Hydroxy-3H-xanthene-3-one, 2,7-dipropoxy-4,5-dichloro-9
-(2',4'-dicarboxy-6-sulfonatophenyl)-6-hydroxy-3H-xanthene-3
-one, 2,7-dimethoxy-4,5-dibromo-9-
(2′,4′,5′-tricarboxy-phenyl)-6-
Hydroxy-3H-xanthene-3-one, 2,7-dimethoxy-4,5-dichloro-9-
(2′,4′,5′-tricarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 2,7-dimethoxy-9-(3′,4′-dicarboxyphenyl)-6 -Hydroxy-3H-xanthene-3-one, 2,7-dimethoxy-9-(3',4'-dicarboxy-2',5',6-trichlorophenyl)-6-
Hydroxy-3H-xanthene-3-one, 2,7-(2″-carboxyethoxy)-9-
(2′-carboxyphenyl)-6-hydroxy-
3H-xanthene-3-one, 2,7-diethoxy-4,5-dibromo-9-
(2′,4′,5′-tricarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 2,7-dimethoxy-9-[2′-carboxy-
4′-Fluoro-5′-(N-carboxymethylcarboxamide)phenyl]-6-hydroxy-3H
-xanthene-3-one, 2,7-dimethoxy-9-(2',4'-dicarboxy-5'-aminophenyl)-6-hydroxy-
3H-xanthene-3-one, 2,7-dimethoxy-9-(2'-carboxy-
4'-isothiocyanatophenyl)-6-hydroxy-3H-xanthene-3-one, 2,7-diethoxy-9-(2'-carboxy-
4'-mercaptophenyl)-6-hydroxy-
3H-xanthene-3-one, 2,7-dimethoxy-4,5-dibromo-9-
(2′-carboxy-4′-mercaptomethylphenyl)-6-hydroxy-3H-xanthene-3-
on, 2,7-dimethoxy-4,5-dichloro-9-
(2′-carboxy-4′-cyanophenyl)-6-
Hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-9-(2',4',5'-tricarboxyphenyl)-6-hydroxy-3H-
Xanthene-3-one, 4,5-dimethoxy-9-(2',5'-dicarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-2,7-diiodo ―9―
(2′,4′,5′-tricarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-2,7-dichloro-9-
(2′,4′,5′-tricarboxyphenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-2,7-dichloro-9-
(2′,4′,5′-tricarboxy-3′,6′-dichlorophenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-di(2″-carboxyethoxy)-2 ，
7-diiodo-9-(2'-carboxy-4'-sulfonatophenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-dipropoxy-2,7-dibromo-9
-(2',4'-dicarboxy-3,5',6'-trichlorophenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-diethoxy-2,7-dichloro-9-
(2′-carboxy-4′-amino-5′-sulfonatophenyl)-6-hydroxy-3H-xanthene-
3-one, 4,5-dimethoxy-2,7-diiodo-9-
(2′-carboxy-4′-isothiocyanatophenyl)-6-hydroxy-3H-xanthene-3-
on, 4,5-diethoxy-2,7-diiodo-9-
[2',4'-dicarboxy-5'-(N-carboxymethylformamido)phenyl]-6-hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-2,7-dichloro-9-
(2′,4′-dicarboxy-3′,5′,6′-trichlorophenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-diethoxy-2,7-dichloro-9-
(4′,5′-dicarboxy-2′,3′,6′-trichlorophenyl)-6-hydroxy-3H-xanthene-3-one, 4,5-dimethoxy-2,7-diiodo-9-
(2′-carboxy-4′-mercaptomethylphenyl)-6-hydroxy-3H-xanthene-3-
on. The above list is intended to be illustrative only and does not represent all of the compounds included within the scope of the invention. Compounds of the present invention having active functionality may have complementary heterofunctionality.
can be conjugated to a ligand or support with functionalities). The table below shows exemplary ligand functionalities, active functionalities for coupling compounds of the invention to ligands, and the resulting linking units.

Table: In most cases, the ligand functionality and spectroscopic compound functionality can be switched. Primarily, the ligand complex has the following formula: where A ² are the same or different, usually the same except for the bonding site, D ² are the same or different, usually the same except for the bonding site, and either A ² or D ² is an ether, usually of ^the formula - ^{HM 2} is an alkylene group, and X ^d
is an oxyether of carboxy or hydrogen, usually hydrogen, and if not an ether as above for A and D, and Y ² , Z ² , m ² and n ² are Y, Z respectively , m and n, Q ² may be the same as Q but is usually the same as Q ¹ , and X ^c can be included in the same definition as L by considering W ² as W. is usually a non-oxocarbonyl containing functionality including nitrogen and sulfur analogs, such as carbonyl, imide, carbamyl and thiocarbamyl to form an amide, respectively, amidine, urea and thiourea when combined with the ligand amino. , p ² is the molecular weight of 1 to W ² divided by 500, usually 1000; for haptenic ligands with a molecular weight of about 125 to 2000, p ² is usually 1, greater than 2000; For antigenic ligands with molecular weights typically greater than 5000, ^p2 generally averages about 1
200 to 200, usually 1 to 100, more commonly 2 to 50
and W ² is a ligand, receptor or support, W ² as a ligand is haptenic or antigenic, and haptenic ligands generally have a molecular weight of about 125 to 2000
and has at least one polar functionality, which may or may not be a site of binding, and the antigenic ligand is polyepitopic, usually a poly(amino acid), Nucleic acids, polysaccharides and combinations thereof, generally have a molecular weight of at least 2,000, usually at least 5,000, and can have a molecular weight of 10,000,000 or more; has a molecular weight of at least 20,000, and can be more than 10,000,000,
Soluble supports generally have a molecular weight of 10 million or less, and the supports may be naturally occurring or synthetic, may be particulate or in tangible form, and are swellable by an aqueous medium. or may be non-swellable; the support may be cross-linked or non-crosslinked; it may be a single material or a mixture of materials; naturally occurring supports include polysaccharides, nucleic acids,
poly(amino acids), such as polypeptides and proteins, gums, lignin, vesicles,
Synthetic supports include addition and condensation polymers such as polystyrene, polyacrylic resins, vinyl compounds, polyesters, polyethers and polyamides, charcols, metal chalcogenides, glasses, liposomes, etc. A preferred group of ligand complexes has the following formula: where, for a quencher molecule, A ³ is alkoxy of 1 to 2 carbon atoms and D ³ is hydrogen or halo, especially chloro, bromo or iodo or 1 to 2 carbon atoms.
is alkyl of 6 carbon atoms; for the fluorescence generator molecule, D ³ is alkoxy of 1 to 2 carbon atoms; A ³ is hydrogen or halo, especially chloro or bromo; and Z ³ is carboxyl, Y ³ is halo, especially chloro, Q ³ is 1 to 9 in the bond or chain, usually 2 to 9
a spacer arm of 3 atoms, the atoms being carbon and nitrogen, amide nitrogen, the spacer arm consisting only of carbon, oxygen, nitrogen and hydrogen, oxygen bonded only to carbon, and oxy or oxo, Particularly non-oxocarbonyl, useful spacer arms are alkylenes of 1 to 6, usually 2 to 4 carbon atoms, mono- or poly-amidomethylene (-CONHCH ₂ -) aminomono- or aminopoly-amidomethylene (- NH(CONHCH ₂
―) _x ), or aminothionomono- or poly(amiminethylene carbonyl) amino-methylene
_{NHCS (} ^NHCH ₂ ^CO ) and W ³ is usually a haptenic receptor ligand with a molecular weight of about 125 to 1000, or about 20 to 10 million,
Usually 5000 to 2 million, usually 5000 to 100
W ² is particularly a poly(amino acid) or polysaccharide antigen, and may be a hapten, and p ³ is between 1 and the molecular weight of W ³ divided by 500, usually about 2000. p 3 is generally in the range of about 1 to 200, more commonly in the range of about 2 to 150, and often in the range of about 1 to 75; for ligands with molecular weights below 500,000, p ³ is generally about 2 to 50, m ³ is an integer from 1 to 2, n ³ is an integer from 0 to 3, and m ³ +n ³ is not greater than 4. In some instances, it may be desirable for the ligand and fluorescence generating agent complex to be covalently or non-covalently attached to the support.
Attachment of the conjugate to the support may be by covalent attachment to a functionality on the ligand or the fluorogenic agent of the invention, or typically the polyepitopic ligand is attached to the support either covalently or non-covalently. It may also be through non-covalent bonding between a receptor, usually an antibody, which may be present. Ligand bound to support
The fluorophore complex mainly has the following formula: where the two A ^4s are the same or different, usually the same unless one is the bonding functionality, and the two D _4s are the same or different, usually one is the bonding functionality. are the same except that either A ⁴ or D ⁴ is the formula - JM ⁴ X ⁴ , where J
is an ether of oxygen or sulfur, usually oxygen, - A ⁴ and D ⁴ if other than JM ⁴ X ⁴
is as described above for A and D, and M ⁴ is a divalent hydrocarbon group, usually having 1 to 8, usually 1 to 6, preferably 1 to 3 carbon atoms, and usually It is a straight chain saturated aliphatic, one of the ^X4 is the binding functionality to the ligand,
non-oxocarbonyl (including nitrogen and its sulfur analogs), carbamyl, thiocarbamyl, imide, thioether, alkylamino, thio- or oxyacetyl with one valence to carbon, provided that X ⁴ as the linking group is a non-oxocarbonyl when attached to M ⁴ , if X ⁴ is not a bonding functionality, then X ⁴ attached to M ⁴ is hydrogen or a non-oxocarbonyl, and Q ⁴
X ⁴ attached to is hydrogen, non-oxocarbonyl, or halo, where ^{Q 4 is} a bond or usually about 1 to 16, usually 1 to
an aliphatic, aromatic, multi-ring or combination thereof spacer arm having 12, more commonly 1 to 8 atoms in the chain, the atoms being carbon, nitrogen, oxygen and sulfur; nitrogen being Amide or amino, amino is usually tertiary amino, oxygen is oxy, sulfur is thioether,
Chalcogens are bonded only to carbon, and heteroatoms are separated by at least two carbon atoms when bonded to saturated carbon atoms, with a total number of carbon atoms ranging from 1 to 20, usually about 1 to 12. and the total number of heteroatoms is 0 to 10, usually 0 to 8, with oxygen as oxy or non-oxocarbonyl, and 0 to 9, usually 0 to 4 heterofunctionalities, such as amide, amino and oxy is present, Z ⁴ is an anionic group such as a carboxylic or sulfonic acid, Y ⁴ is a halogen with atomic number 9 to 53, usually chloro, m ⁴ is an integer from 0 to 3, usually 1 n ⁴ is an integer from 0 to 4, and the sum of m ⁴ + n ⁴ is 4.
not greater, p ⁴ is equal to the molecular weight of W ⁴ divided by 500, usually 1000, more commonly 1500, generally between 1 and
200, usually from 1 to 100, more usually from 1 to 50, W ⁴ generally has a molecular weight of at least about 125;
A receptor or ligand that may be haptenic or antigenic; haptenic ligands are usually about
125 to 2000, and the antigenic ligand and receptor have a molecular weight of about 5000 to 1 x 10 ⁷ , and the bond is generally through an amino, thio or oxy group of the ligand, with one of the E's being the linking group. If present and not a linking group, it has no meaning; if W ⁴ is a hapten, the E attached to the phenyl is usually a linking group, whereas if W ⁴ is antigenic, it has no meaning. , E attached to W ⁴ is usually a linking group, E has the same limitations as X ⁴ but is preferably a non-oxocarbonyl, amino or thioether, T is a soluble or insoluble support, and W
and γ ⁴ is the molecular weight of at least 1 to T divided by 500, usually 1000, more usually 1500. When the 9 positions of the xanthene ring bear aliphatic substituents, the compound primarily has the following formula; where A' and D' are as defined above, where -
OMX is the same as -OMX', G is 0 or 2 to 7 saturated hydrocarbon groups with 5 to 7 ring members, and X' falls within the definition of carbonyl, and especially carboxy when taken together with W, W and p having the same definitions as above. For use in immunoassays or other diagnostic situations, the spectroscopically active compounds of the invention are conjugated to advantageous compounds that include receptors for analytes or ligands. (By receptor is meant a compound that specifically binds to a particular spatial and polar molecular configuration, and a ligand is an organic molecule with such a configuration). Analytes are usually haptenic or antigenic. If these compounds do not have functionality available for attachment, they are modified to introduce such functionality while still retaining receptor recognition properties in the resulting product. Those compounds that are analogs of the analyte, which can also be referred to as ligands, are called ligand analogs. As described above, the compound of the present invention can be complexed with a compound that can be measured by a known immunoassay method. The resulting complex is
A reagent that competes in the assay medium with the compound or analyte of interest in the sample. Thus, the complex retains a sufficient proportion of the structure of the compound in question to be able to compete with the compound for the receptor, usually an antibody. The analyte or its analogue, receptor or ligand conjugated to the spectroscopically active compound of the invention is characterized in that it is monoepitopic or polyepitopic. Analyte, receptor or ligand, monoepitopic or polyepitopic are all used in the sense known in the art. Analytes are compounds to be identified and measured. The epitomic position of an analyte is the binding position (if any) of a complementary compound; when an analyte has only one epitomic position, the analyte is said to be monoepitomic; When it has, the analyte is said to be polyepitomic. A receptor and a ligand are two constituent compounds of an immune binding pair that are complementary to each other in order to generate immune binding. Typically the ligand is an antigen and the receptor is an antibody. Analytes Analytes of the ligands of the invention are characterized by being monoepitopic or polyepitopic. Analytes of polyepitopic ligands are usually poly(amino acids) or polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Combinations of such groups include bacteria, viruses, chromosomes, genes, thread bodies,
Includes the nucleus, cell membrane, etc. For the most part, the polyepitopic ligand analytes used in this invention will have a molecular weight of at least about 5,000, and more usually at least about 10,000. In the poly(amino acid) category, poly(amino acids) of interest are generally about
Molecular weight of 5000-5000000, more usually about 20000-
It would have a molecular weight of 1,000,000. Among the hormones of interest, the molecular weight will usually be in the molecular weight range of about 5,000 to 60,000. Broad proteins can be thought of as families of proteins with similar structural characteristics, proteins with characteristic biological functions, proteins associated with particular microorganisms, especially disease-causing microorganisms, and so on. The following are classes related by structure: protamines histones albumins globulins scleroproteins phosphoproteins mucoproteins chromoproteins riboproteins nucleoproteins glycoproteins proteoglycans unclassified proteins such as somatotropin, prolactin, insulin, pepsin human A number of proteins present in plasma are clinically important and include: prealbumin albumin alpha ₁ - lipoprotein alpha ₁ - acid glycoprotein alpha ₁ - antitrypsin alpha ₁ - glycoprotein trans Cortin 4,6S - postalbumin tryptophan - poor α ₁ - glycoprotein α ₁ x - glycoprotein thyroxy-binding globulin inter - α - tribucin - inhibitory factor Gc - globulin (Gc1-1) (Gc2- 1) (Gc2-2) Haptoglobin (Hp1-1) (Hp2-1) (Hp2-2) Ceruloplasmin cholinesterase α ₂ - Lipoprotein(s) Myoglobin C-Reactive protein α ₂ - Macroglobulin α ₂ - HS - Glycoprotein Zn - α ₂ - Glycoprotein α ₂ - Neuromino - Glycoprotein erythropoietin β - Lipoprotein transferin Hemopexin fibrinogen Plasminogen β ₂ - Glycoprotein β ₂ - Glycoprotein imunoglobulin G (IgG) or γG-globulin molecular formula: γ ₂ k ₂ or γ ₂ λ ₂ immunoglobulin A (IgG) or γA-globulin molecular formula: (α ₂ k ₂ ) ⁿ or (α ₂ λ ₂ ) ⁿ immunoglobulin M (IgG) or γM-globulin molecular formula: (μ ₂ k ₂ ) ⁵ or (μ ₂ λ ₂ ) ⁵ immunoglobulin D (IgD) or γD-globulin (γD) molecular formula: (δ ₂ k ₂ ) or (δ ₂ λ ₂ ) Immunoglobulin E (IgE) or γE-globulin (γE) Molecular formula: (ε ₂ k ₂ ) or (ε ₂ λ ₂ ) Free k and λ short chain complement factors: C′1 C′1q C′ 1γ C′1s C′2 C′3 β ₁ A α ₂ D C′4 C′5 C′6 C′7 C′8 C′9 Important coagulation factors include:

【table】

[Table] Important protein hormones include: Peptide and protein hormones Parathromone Thyrocalcitonin Glucagon Relaxin Erythropoietin Melanotropin (Melanotropin) (Megacell-stimulating hormone; Intermedin) Somatotropin (Growth hormone) Coricotropin (Adrenocorticotropic hormone) Thyrotropin (Follicle-stimulating hormone) Luteinizing hormone ( Luteinzing hormone (interstitial cell stimulating hormone) Luteomammotropic hormone (luteotropin, prolactin) Gonadotropin (choroidal gonadotropin) Tissue hormones Secretin Gastrin Angiotensin and Bradykinin Human placental lactodiene (placental lactogen) Peptide hormones from the posterior pituitary gland Oxytocin Vasopressin Releasing factors (RF) CRF, LRF, TRF, Somatotropin-RF, GRF, FSH-RF, PIF, MIF Other interests Certain polymeric substances are mucopolysaccharides and polysaccharides. Examples of antigenic polysaccharides derived from microorganisms are:

【table】

TABLE Microorganisms that can be assayed can be intact, lysed, ground or otherwise fragmented, and the resulting composition or portion can be assayed for potency, eg, by extraction. Microorganisms of interest include: Corynebacteria Corynebacterium diptheriae Pneumococci Diplococcus pheumoniae Streptococci Streptococcus Streptococcus pyogenes pyogenes) Streptococcus salivarus Staphyloccci Staphylococcus aureus Staphylococcus albus Neisseriae Neisseriae meningitidis
(Neisseria meningitidis) Neisseria gonorrheae
(Neisseria gonorrheae) Enterobacteriaceae
(Enterobacteriaciae) {Escherichia coli Aerobacter aerogenes Klebsiella pneumoniae} Escherichia coli bacteria {Salmonella typhosa Salmonella choleraesuis Typhimurium
(Salmonella typhimurium)} Salmonellae (Salmonellae) {Shigella sicenteriae
(Shigella dysenteriae) Shigella schmitzii Shigella arabinotarda Shigella flexneri Shigella boydii Shigella sonnei} Shigellae Other intestinal bacteria { Proteus vulgaris Proteus mirabilis Proteus morgani
(Proteus morgani) Proteus species Pseudomonas aeruginosa Alcaligenes faecalis Vibrio cholerae Haemophilus voltella
(Hemophilus-Bordetella) group Hemophilus influenzae, H. ducreyi H. hemophilus H. aegypticus H. parainfluenzae Bordetella pertussis
(Bordetella pertussis) Pasteurellae Pasteurellae pestis
(Pasteurella pestis) Pasteurella tulareusis
(Pasteurella tulareusis) Brucellae Brucella melitensis
Brucella melittensis Brucella abortus Brucella suis Aerobic spore-forming fungus Becillus anthracis
(Bacillus anthracis) Bacillus subtilis (Bacillus subtilis) Bacillus megaterium
(Bacillus megaterium) Bacillus cereus Anaerobic spore-forming fungus Clostridium botulinum
(Clostridium botulinum) Clostridium tenani
(Clostridium tetani) Clostridium perfringens (Clostridium perfringens) Clostridium novii
(Clostridium Septicum) Crostridium Historyticum (Clostridium Historyticum) Crostridium Teltium (Clostridium Tertium) Crostridium Tertium Stridium Bifermentans (Clostridium Sporogens) Micobacteria・Mycobacterium tuberculosis hominis Mycobacterium bovis
(Mycobacterium bovis) Mycobacterium avium
(Mycobacterium avium) Mycobacterium leprae
(Mycobacterium leprae) Mycobacterium paratuberculosis Actinomycetes (bacteria-like bacteria) Actinomyces israelii
(Actinomyces israelii) Actinomyces bovis
(Actinomyces bovis) Actinomyces naeslundii (Actinomyces naeslundii) Nocardia asteroides
(Nocardia asteroides) Nocardia brasiliensis (Spirochetes) Treponema baridium
(Treponema pallidum) Spirillum minus (Treponema pallidum) Treponema pertenue
(Treponema pertenue) Streptobacillus moniliformis Treponema caratenum
(Treponema carateum) Borrelia reculentis
(Borrelia recurrentis) Leptospira icterohemorrhagiae Leptospira canicola
(Leptospira canicola) Mycoplasmas Mycoplasma neomoniae
(Mycoplasma pneumoniae) Other pathogens Listeria monocytogenes Erysipelothrix rhusiopathiae Streptobacillus moniliformis Donvania granulomatis Bartonella bacilliformis Rickettsiae ( Rickettsiae) (parasite-like bacteria) Rickettsia prowazekii Rickettsia mooseri Rickettsia mooseri
(Rickettsia rickettsii) Rickettsia conori Rickettsia australis Rickettsia sibiricus
(Rickettsia sibiricus) Rickettsia akari (Rickettsia akari) Rickettsia tsutsugamushi
(Rickettsia tsutsugamushi) Rickettsia tsutsugamushi
(Rickettsia burnetii) Rickettsia quintana
(Rickettsia quintana) Chlamydia (unclassifiable parasitic bacteria/virus) Chlamydia agents (unspecified name) Fungi (Rickettsia quintana) Cryptococcus neoformans Blastomyces dermatidis ) Histoplasma capsulatum (Histoplasma capsulatum) Kosidioides immitis
(Coccidioides immitis) Paracoccidioides brasiliensis Candida albicans
(Candida albicans) Aspergillus fumigatus
(Aspergillus fumigatus) Mucor corymbifer (Absidia
corymbifera) {Rhizopus oryzae (Rhizopus oryzae) Rhizopus archizus
(Rhizopus arrhizus) Rhizopus nigcans
(Rhizopus・nigricans)} Phycomycetes Sporotrichum schenkii Huonsecaea pedrosoi
(Fonsecaea pedrosoi) Fonsecaea compactor
(Fonsecaea compacta) Fonsecaea dermatigis
(Fonsecaea dermatidis) Cladosporium carrionii (Cladosporium carrionii) Huialohora vercosa
(Phialophora verrucosa) Aspergillus nidulans (Madurella mycetomi) Madurella grisea (Madurella grisea) Areskeria boisii
(Allescheria boydii) Phialosphora jeanselmei Microsporum gypseum Trichophyton mentagrophytes Keratinomyces ajelloi Microsporum canis
(Microsporum canis) Trichophyton rubrum
(Trichophyton rubrum) Microsporum andui
(Microsporum andouini) Viruses Adenoviruses Herpes Viruses Herpes Simplex
(Herpes simplex) Varicella (Chicken pox) Herpes soster
(Herpes Zoster) (Shingles) Virus (Virus) B Cytomegalovirus Pox Viruses Variola (smallpox) Vaccinia Poxvirus bovis Paravaccinia Molscum contagiomus (Molluscum contagiosum) Picornaviruses Poliovirus Coxsackievirus Echoviruses Rhinoviruses Myxoviruses Influenza
(Influenza) (A, B, and C) Parainfluenza
(Parinfluenza) (1-4) Mumps Virus Newcastle Disease Virus Measles Virus Reinderpest Virus Canine Distemper Virus Respiratori・Respiratory Syncytial Virus Rubella Virus Arboviruses Eastern Equine Eucephalitis Virus Western Equine Eucephalitis Virus Sidbis Virus (Sindbis Virus) Chikugunya Virus Semliki Forest Virus Mayora Virus St. Louis Encephalitis Virus California Encephalitis Virus) Colorado Tick Fever Virus Yellow Fever Virus
(Yellow Fever Virus) Dengue Virus Reoviruses Reovirus Types 1-3 Hepatitis Hepatitis A virus Hepatitis B virus Tumor Viruses Lauskel - Rauscher Leukemia Virus Gross Virus Maloney Leukemia Virus Monoepitopic ligand analytes generally have a molecular weight of about 100-2000, more usually 125-1000. . Analytes of interest include drugs, metabolites, pesticides, pollutants, and the like. Drugs of interest include alkaloids. Examples of alkaloids are: morphine alkaloids, e.g. morphine, codeine, heroin, dextromeltofan, derivatives and metabolites thereof; cocaine alkaloids, e.g. cocaine and benzoylecgonine, derivatives and metabolites thereof. ; ergot alkaloids, such as diethylamide of lysergic acid;
Steroid alkaloids; iminazoyl alkaloids; quinazoline alkaloids, isoquinoline alkaloids; quinoline alkaloids, e.g.
Quinine and quinidine; diterbene alkaloids; derivatives and metabolites thereof. The next group of drugs includes: steroids, such as estrogens, gestogens,
Androgens, andrenocortical steroids, bile
cardiotonic glycosides and aglycones, such as digoxin and digoxigenin, saponins and sapogenins, their derivatives and metabolites. Also, steroid mimetic substances, such as diethylstilbestrol. The next group of drugs are lactams with 5-6 ring members, such as the barbiturates, such as phenobarbital and secobarbital, diphenylhydantonin, primidone, ethoximide, and metabolites. The next group of drugs are aminoalkylbenzenes with alkyl of 2 to 3 carbon atoms, such as amphetamine, catecholamines, eg ephedrine, L-dova, epinephrine, narcein, papaverine, their metabolites. The next group of drugs are aminoalkylbenzenes with alkyl of 2 to 3 carbon atoms, such as efuedrine, L-dova, epinephrine, narcein, papverine, their metabolites and derivatives. A group of drugs include benzheterocyclics, e.g.
Oxazepam, chlorpromazine, tegretol, imipramine, their derivatives and metabolites, and the heterocycles are azepines, diazepines and phenothiazines. The next group of drugs are purines, such as theophylline, caffein, their metabolites and derivatives. The next group of drugs is marijuana
eg, cannabinol and tetrahydrocannabinol. The next group of drugs are vitamins, such as A,
B, examples are B ₁₂ , C, D, E and K, folic acid, thiamine. The next group of drugs are prostaglandins, which differ in the degree and site of hydroxylation and unsaturation. The next group of drugs is antibiotics, for example, penicillin, chloromycetin, actinomycetin,
Tetracyclines, terramycin, their metabolites and derivatives. The next group of drugs are nucleosides and nucleotides, such as ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine, with their appropriate sugar and phosphate substituents. The next group of drugs includes various individual drugs, e.g.
Methadone, meprobamate, serotonin, meperidine, amitriftyline, nortributyline, lidocaine, procainamide, acetylprocainamide, propanol, griseofulvin, valproic acid, butyrophenone,
Antihistamines, anticholinergic agents, such as atropine, their metabolites and derivatives. The next group of compounds are amino acids and small peptides, such as polyiodothyronine, oxytocin, ACTH, angiotensin, methoenkephalin, leuenkephalin, their metabolites and derivatives. Metabolites implicated in disease states include spermine, galactose, phenylpyruvate and polyphyllin type 1. The next group of drugs are aminoglycosides, such as gentamicin, kanamycin, tobramycin and amikacin. Examples of insecticides of interest are polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenated sulfenamides, metabolites and derivatives thereof. Acceptors are compounds that can bind to spatial and polar organizations of molecules. For the most part, the receptors are for antibiotics, especially IgG, IgA, and IgE.
and IgM. The receptor can be an analyte or used as a reagent in an immunoassay. In either case, the receptor is capable of binding a spectroscopically active compound of the invention. For receptor analytes, the molecular weight is generally
It will range from 10,000 to 2×10 ⁶ , more usually from 10,000 to 10 ⁶ . For immunoglobulin, IgA, IgG, IgE and IgM, the molecular weight generally ranges from about 160,000 to about
It will change in 10 ⁶ . Enzymes usually have a molecular weight of approximately
It would be 10,000 to 600,000. Natural receptors vary widely in molecular weight and are generally at least about 25,000 and can be greater than ¹⁰⁶ ; examples of such receptors are apidin, thyroxine-binding globulin,
These include syroxine-bound prealbumin and transcortin. The compounds of the invention can bind to a wide variety of ligands, haptens and antigens, and receptors to form complexes, and these complexes can be used in a variety of assays,
Especially used in immunoassays. The specific ligand is
Analytes are of interest because, for example, they have great importance as drugs and are easy to quantify using the compounds of the present invention. The following is the formula for a particularly important complex. The first complex of interest is the lactam-chromophore complex of the formula: where a is 1, with the proviso that when R and R ^a are phenyl, a is 0 or 1; Z is chalcogen, oxygen or sulfur;
Usually oxygen, R and R ^a are hydrocarbons of 1 to 7 carbon atoms, usually 1 to 6 carbon atoms, and when non-aromatic have 0 to 1 sites of ethylenic unsaturation, e.g.
is 1, methyl, ethyl, sec-butyl,
n-butyl, α-methylbutyl, isoamyl, hexyl, Δ ¹ -cyclohexenyl and phenyl,
In particular, the combination of amyl and α-methylbutyl or the combination of ethyl and phenyl, and when a is 0, there are two phenyls, one of L is a single bond, a functional group or group bonded to Ch, and usually oxy, amino, non-oxycarbonyl or mercapto end, and from 0 to
6, usually has a chain of 0 to 4 carbon atoms, when outside the bonding site, L is hydrogen, Ch has the following formula, where α, δ and γ are as previously defined, and CO' is a suitable functional group attached to L to form an amide, amidine, urea, thiourea, ester, oxyether or thioether, or A secondary-amino functionality attached to a ligand and a chromophore, the above formula encompassing all of the preceding formulas within a narrow range, where X is the attachment functionality. The next important chromogenic ligand complex is for theophylline, which may be of the formula: In the formula, one of L and L ¹ is a single bond or a functional group or group bonded to Ch, usually ending in oxy, amino, non-oxocarbonyl or mercapto, and a chain of 0 to 6, usually 0 to 4 carbon atoms. and is other than the site of attachment, L is hydrogen, L ¹ is methyl, and Ch is as defined above. The following important chromophore ligand complexes are for aminoglucoxides, especially antimicrobial aminoglucosides, having for the most part the following formula: where L can be attached to any one of the R groups, otherwise R ² is amino (--NH ₂ ), except for kanamycin and amikacin, it is hydroxyl and R ³ , R ⁷ and R ⁹ are hydroxyl, R ⁴ and R ⁵ are amino, except for amikacin, R ⁴ is 2-hydroxy-4-aminobutyramide, R ⁶ is hydrogen or methyl, and gentamicin methyl, and topramycin, kanamycin or amikacin,
hydrogen, R ⁸ is amino or methylamino, methylamino when gentamicin, amino otherwise, R ¹⁰ is hydrogen or hydroxymethyl, hydrogen when gentamicin, and is hydroxymethyl, R ¹¹ and R ¹² are hydrogen or hydroxyl, hydrogen for geltamicin, hydroxyl for kanamycin and amikacin, and for topramycin R ¹¹ is hydroxyl, and R ¹² is hydrogen. , and L and Ch are as defined above. The next important chromophore-ligand complex is for tricyclic antidepressants, which would have the following formula: where one of L ¹ is a linking group as previously defined;
Otherwise, it is hydrogen, G ¹ is N-, C= or CH-, and the remaining 2
two valencies toward adjacent ring carbon atoms, and R ^c is a single or double bond connecting L- to G or aminopropyl or -ylidene with 0 to 2 methyl groups attached to nitrogen. be. The next important chromophore-ligand complex is for benzodiazepines, which would have the formula: where L and Ch are as defined above, R〓 is a single bond or methylene when L is hydrogen, R ^e is a single bond or oxy, and O is 0 to 1 ortho It is a halogen with an atomic number of 9 to 17. The bonding group represented by L will vary depending on the nature of R to which L is bonded and the group to which L is bonded. As indicated earlier, L can be a single bond, a functional group or a linking group, which ends in a functional group that can form a covalent bond with the functional group present on Ch. When L is single bonded, it will normally bond a carbon atom to a foreign atom, especially nitrogen and oxygen atoms. When L is a functional group, it is usually bonded to carbon and is usually oxy, thio, amino or non-oxycarbonyl. L
When is a linking group, it usually has a chain of 1 to 8, especially 1 to 6, especially 1 to 4 atoms, of which 0 to 2, usually 0 to 1, are heteroatoms, i.e. oxygen, sulfur and nitrogen, chalcogen is primarily bonded to carbon, nitrogen is primarily bonded to carbon and hydrogen, the chain ends as a methylene or with a heterofunctional group (as defined above for L), and L is the sum of having 1 to 10, usually 1 to 6 carbon atoms and 0 to 4, usually 1 to 3 heteroatoms, namely oxygen, sulfur and nitrogen, the chalcogen being usually an ether or non-oxocarbonyl, and nitrogen is amino or amide. The subject conjugates can be used in a variety of ways to qualitatively, semi-quantitatively, or quantitatively determine the presence of a compound of interest in a sample. If the compound is detected in a physiological fluid, the fluid can include serum, urine, saliva, lymph, and the like. When the compound in question is related to chemical processing or environmental health;
Samples may include aqueous media, organic media, soil, inorganic mixtures, etc. Assays typically involve spectroscopic changes due to changes in the environment surrounding a spectroscopically active compound, or fluorophore-quencher pairs where the quencher interacts with the fluorophore. Including getting within close enough range. In the first assay, involving steric exclusion and using receptors or antibodies for the ligand and the fluorogen, simultaneous binding of the receptor for the ligand and the receptor for the fluorophore is blocked. Furthermore, when a fluorophore acceptor (antifluorescent agent) is coupled to a fluorogenic agent, the fluorescence of the fluorogenic agent is substantially reduced. Further reductions can be achieved by coupling a quencher to an anti-fluorescence generator, where suppression of fluorescence is not complete. This assay is described in detail in US Pat. No. 3,998,943, issued December 21, 1976. Instead of the fluorescent light used therein, the fluorescent compounds of the present invention are used instead. The test is in columns 3 to 5 of the above patent.
, which is included here by reference. In general, this method involves the preparation of a sample suspected of containing an analyte, a ligand and a fluorogen, an antifluorescent agent, and when the ligand is an analyte, an acceptor or anticoordinator for the ligand. Consists of matching children. These substances are combined in an aqueous medium at a pH ranging from 5 to 10, usually 6 to 9, and a temperature ranging from about 10 to 45°C, and the fluorescence is determined in rate or equilibrium mode, and all substances are In velocity mode, combined after-readings are taken within about 1 second to 1 hour, while in equilibrium mode, readings can be taken over longer periods of time, up to about 24 hours or more. The following immunoassay technique uses a fluorogen-quencher pair in which one member of the pair binds to one member of a specific binding pair, ie, a ligand and an antiligand. , and the other chromophore member binds to the same or different member of a particular binding pair. For example, the fluorogen can bind to the anti-ligand and the quencher can bind to different molecules of the anti-ligand, so that when the two complexed anti-ligands are combined with the antigen, the fluorophore The generator and quencher are brought within extinction distance. Alternatively, one of the chromophores can be attached to the ligand and the other chromophore to the anti-ligand. This test is based on U.S. Patent No.
It is described in detail in No. 3996345. This verification technique is explained in columns 17 to 23 and is incorporated herein by reference. The chromophore to ligand and acceptor ratios are explained in columns 4-6 and are incorporated herein by reference. This assay is carried out in substantially the same manner as above, except that in this assay, the fluorescent complex and the quenching complex are added together with the sample, and the fluorescence is compared to the assay medium with a known amount of anolyte. and decide. Other techniques can be used with the subject compounds, such techniques include heavy atom quenching as described in U.S. Patent Application No. 824,576, filed August 13, 1977, or Other assays can be used if one desires a fluorescent compound that emits light at a wavelength substantially above that emitted by fluorescent compounds naturally present in the fluid or other sample. EXPERIMENTAL The following examples are presented by way of illustration and not by way of limitation. (All temperatures are in degrees Celsius, unless otherwise noted. All parts and percentages are by weight, except where indicated, by volume for mixtures of ligands. The following abbreviations are used.
THF-tetrahydrofuran; NHS-N-hydroxysuccinimide; DMF-N,N-dimethylformamide; DCC-dicyclohexylcarbodiimide; PEG-polyethylene glycol 6000.) Example 1 Production of 2-chloro-4-methoxyresorcinol In a reaction flask 2 g of 2-chloro-3-hydroxy-4-methoxybenzaldehyde in 50 ml of methylene chloride was charged with excess m-chloroperbenzoic acid and refluxed for 2.5 hours. After removing the solvent, the solid residue was dissolved in 10 ml of methanol and stirred with approximately 10 weight percent aqueous sodium hydroxide (10 ml) for 1 hour. rare HCl
After making the solution acidic to pH 1, the solution was filtered and the precipitate was washed with 10 ml of cold water. The liquids were combined, the washed material was extracted with ether, and this ether solution was diluted with 5%
NaHCO ₃ (2 x 10 ml) and saturated NaCl solution (1 x
10 ml) and then evaporated to leave the solid,
This solid was crystallized from an ethyl acetate-hexane mixture to give the product as white silk-like needles (1.35 g). Melting point 69-70°. Example 2 3′,6′-dihydroxy-4′,5′-dichloro-2′,
7′-dimethoxyspiro [isobenzofuran-1
(3H), 9′-[9H]xanthene]-5 or 6-
Carboxy-3-one The product of Example 1 (200 mg) and 100 mg of 1,
A mixture of 2,4-benzenetricarboxylic anhydride was heated with 15 mg of zinc chloride at 180-85° for 30 minutes.
The resulting red mixture was dissolved in 5% aqueous sodium hydroxide solution and brought to pH 1 with dilute HCl and precipitated. The precipitate was then dissolved in CH ₂ Cl ₂ ;MeOH:AcOH-9:
Chromatographed using 1:0.5. The obtained product showed the following physical property values. PH8.0
In 0.05MPO ^3-4 buffer solution of λ absorption max ⁼
518-519nm, λ emission max = 540-543nm. Its extinction coefficient = 80000. Example 3 3',6'-dihydroxy-4',5'-dimethoxyspiro[isobenzofuran-1(3H),9'-
[9H] Xanthene]-5 or 6-carboxy-
A mixture of 3-one pyrogallol-2-methyl ether (4 g) and 1,2,4-benzenetricarboxylic anhydride (2.3 g) was heated at 190 to 195° (external oil bath temperature).
3 in an open test tube in an oil solution preheated to
heated for an hour. A dark red object was obtained. It was cooled, dissolved in 5% NaOH (35 ml) and diluted with concentrated HCl.
The pH was made acidic to 1. A dark red oil separated. The solution was saturated with solid NaCl and stirred in a cold room overnight. A thick black tar-like substance was isolated and this tar was macerated with saturated NaCl solution.
(2 x 15ml). TLC of this tar ( _{CH2Cl2 :}
MeOH:AcOH 85:15:1) test showed the presence of red spots and some other spots with lower R _f . 5g of this tar RP-2
Silica gel 60 silanide (E.Merck Cat.No.7719)
Absorb in _CH2Cl2 : _methanol (100:0.25)
It was purified by a conventional gravity column using RP-2 silica gel (35 g) prepared in Elution was performed using the same solvent CH ₂ Cl ₂ :methanol (100:0.25). Elution was followed by TLC. Fractions containing the same red spot were combined and the solvent was removed to obtain a red residue, which was dissolved in 20 ml of _CCl4 :
Dissolved in a mixture of CH ₂ Cl ₂ (95:5). The resulting red solid (1.1 g) was filtered off. This was the only spot on TLC. It has a λ ^absorption _nax of 512 nm, ε78200 in 0.05M PO ₄ ^3- buffer pH 8.0.
It has no luminescence. Example 4 3',6'-dihydroxy-4',5'-dimethoxy-
2′,7′-diiodospiro [isobenzofuran]
1(3H),9H′-[9H]-xanthene-5 or 6-carboxy-3-one 4′,5′-dimethoxy-6-carboxyfluorescein (Example 3) (1.05 g) was dissolved in anhydrous ethyl alcohol (15 ml). ) and add _I2 powder (1
g) and solid sodium bicarbonate (0.5g) were added. The contents were refluxed under nitrogen for 1 hour (external bath temperature 80-85°). TLC examination of the refluxing mixture (CH ₂ Cl ₂ :methanol:acetic acid 85:14:1) showed formation of monoiodinated derivative and a small amount of diiodinated derivative as well as starting material. Further I ₂ (500 mg) and NaHCO ₃ (250 mg) were added and heating continued for an additional hour. TLC is a thing.
and di-iodination products were further formed. Addition of more I ₂ (500 mg) and NaHCO ₃ (250 mg) followed by heating for 1 hour was repeated two more times. TLC of the final mixture showed the presence of a large amount of diiodinated derivative as well as a very small amount of monoiodinated derivative. Ethyl alcohol was removed on a Rotovap. 20 ml of water was added to the residue and the solution was made acidic to PH1 with dilute HCL. A colored oil separated. This was extracted with ether (3x50ml).
The dark red ether layer was diluted with 5% sodium thiosulfate solution (2 x 20 ml), water (1 x 20 ml) and brine (1 x
20ml). The ether was removed on a rotovap and the residue was dissolved in 4% NaOH (20ml) and filtered. The solution was acidified with HCl to give a dark red solid (21
g) was obtained. This red solid (2.1 g) was mixed with 4 g of RP-2 silica gel silanide (E.Merck
Cat. No. 9917) and then (prepared from 30 g of RP-2 silica gel using CH ₂ Cl ₂ )
Pour onto a gravity column of RP-2 silica gel.
Dissolved _{in CH3Cl2} . Elution was followed by TLC.
Fractions with the same R _f were combined and the solvent was removed.
This residue (1.1 g) was dissolved in 10 ml of CH ₃ Cl ₂ :MeOH.
(99:1), and n-hexane (7 ml) was added. A thick precipitate separated. After this time, the mother liquor was concentrated. Further hexane was added to this to obtain further solid. The solids were combined (0.95 g) to give 2',7'-diiodo-4',5'-dimethoxy-6-carboxyfluorescelene as very lightly colored long needles. This substance turned orange-red when left alone. This product has a λ ^absorption _nax of 533 nm and ε 81780 in 0.05M PO ₄ ^3- buffer PH 8.0. Example 5 9-(2',5',6'-trichloro-3',4'-dicarboxyphenyl)-2,7-dimethoxy-4,
5-Dichloro-6-hydroxy-3-isoxanthenone A 10 g of 4-methylphthalic anhydride and 0.5 g of powdered iodine were dissolved in 25 ml of 20% oleum.
The mixture was heated to 90-100° and chlorine gas was continuously bubbled through the solution. After heating for 24 hours, an additional 0.5 g of iodine was added and heating continued for another 24 hours. After the white solid precipitated, the solution was cooled, diluted in 100 ml of ice-cold water, and the white solid was filtered off. The solid was washed with 20ml of cold water and dried in vacuo. This product is 3,5,6-
It was believed to be a mixture of trichloro-4-methylphthalic acid and anhydride. B 20 g (0.075 mol) of the above product and 400 ml of 10% aqueous potassium carbonate solution were introduced into the reaction flask. After refluxing at 120° for about 1 hour, 20 ml of t-butanol was added, followed by the portionwise addition of 33 g (0.2 mol) of powdered potassium permanganate to prevent permanganate buildup. moreover
Washed in permanganate using 100 ml of 10% aqueous potassium carbonate solution. reaction course
When the substantial absence of starting material was observed as monitored by TLC, the t-butanol was removed by distillation and the resulting slurry was reduced to pH 1 with 6M H ₂ SO ₄ .
made acidic. Excess permanganate was destroyed using solid oxalic acid while maintaining the pH at 1 by adding sulfuric acid. After concentration in vacuo, a white precipitate forms and 6M HCl is added to bring the final volume to 300.
ml. After stirring for 30 minutes at room temperature, a fine white precipitate formed. The slurry was extracted with ether (3x400ml), the ether solution was separated and the ether was evaporated in vacuo. After azeotropic drying with benzene, 20 g of a white powder was isolated, which was recrystallized from ethyl acetate-CCl ₄ and
19.5g of product was obtained. Melting point, 238-240°. C The above product (10 g) was dissolved in 30 ml of acetic anhydride and heated at 140-145° under nitrogen for 45 minutes. The acetic anhydride was removed in vacuo at a temperature of about 35-40°. The weight of the white solid was 9.5g. The products are mixed and intramolecular anhydrides of 1,2-dicarboxylic acid groups with acetic acid. This product (9.5 g) is equivalent to 12 g of 2-chloro-
It can be mixed with 4-methoxyresorcinol and 1 g of anhydrous zinc chloride and heated to 185-190° for about 1.5 hours. This product is then processed as described above. Example 6 The product of Example 3 and anti (human IgG)
A solution of 25 mg of the product of Example 3 in 1 ml of dry THF was combined with dicyclohexylcarbodiimide (10
mg) and 10 mg of NHS and stirred overnight. After filtering the solution, the solvent was removed and the residue was dissolved in 10 ml of n-hexane for 20 minutes. After filtering the resulting solid and washing the solid with 5 ml of n-hexane, the resulting ester was used without further purification. 1ml of 0.05M phosphate buffer, 1% in PH8
In a solution of anti (human IgG) (10 mg protein), add 25μ
0.7 mg of the above NHS ester in DMF was slowly added and the mixture was stirred for 1 hour at 0-5° and then for a further hour at room temperature. This product is
Purified by chromatography through a G25 Sephadex column using 0.05M phosphate buffer, PH8. The total production volume was 2.2ml. Based on UV absorption at 519 nm, the dye/protein ratio was estimated to be approximately 5. Example 7 Conjugation of the product of Example 4 with anti-(human IgG) 4',5'- in freshly distilled dry THF (3 ml)
A solution of dimethoxy-2',7'-diiodo-6-carboxyfluorescein (Example 4) (250 mg), dicyclohexylcarbodiimide (80 mg) and N-hydroxysuccinimide (50 mg) was stirred at room temperature for 6 hours. During this time, a small amount of white solid separated, which was separated. The liquid was concentrated on a rotovap and the residue was dried in vacuo for 5 minutes to remove the last traces of solvent. The resulting deep red solid was stirred for 20 minutes using 12 ml of a benzene-hexane mixture (1:1), and the resulting deep purple solid was filtered. This _solid was almost completely converted to NHS ester ( _NHS
It was found that the ester reacts with methanol to give the corresponding methyl ester (as indicated by the higher R _f compared to the starting material).
This purple solid was used as is for conjugation to proteins. This solid is believed to be approximately 90% pure by weight. B. Human-IgG (0.5 ml of 0.05 M PO ₄ ^3- buffer PH containing 1% cholic acid) cooled to 0-5° in an ice bath.
To a solution of the NHS ester (0.4 mg) prepared above in 25μ dry DMF was slowly added over 20 minutes. Stirring was continued overnight in the cold room. The next day, the solution was centrifuged for 2 minutes and the deep red solution was transferred to _a Cephadex G- ²⁵ column (1 x 30 cm ) was purified above. Faster migrating complexes were easily separated. Calculation of hapten number (ie, dye/protein ratio) was performed as follows. The amount of dye from the UV spectrum of a particular dilution is determined by dividing the OD at 539 by the extinction coefficient of 81.780. The amount of protein was determined prior to conjugation by knowing the starting protein concentration (appropriate dilution calibration was performed for the final conjugate, assuming that no protein was lost during conjugation and sepadex chromatography). Determined by using concentration numbers. When labeling according to the above method, this quencher
The labeling efficiency of NHS esters is found to be approximately 80-90%. Example 8 4,5-dimethoxy-6-hydroxy-9-
(2′-carboxyethyl)xanthene-3H-3
- Preparation of O ² -methylpyrogallol (300 mg), succinic anhydride (100 mg) and ZnCl ₂ (20 mg) into a reaction flask.
mg) and the mixture was heated to 180-85° for 15 minutes. This product was subjected to preparative TLC (CH ₂ Cl ₂ :
MeOH:AcOH, 90:10:0.5).
The purified product had a λ ^absorption _nax of 506-7 nm in 0.05M PO ₄ ^3- buffer, PH 8.0 and no fluorescence emission. Succinic anhydride with other acid anhydrides, such as glutaric anhydride, 2,3-dimethylsuccinic anhydride,
If the above method is followed by replacing 1,2-cyclopentanedicarboxylic anhydride and adipic anhydride,
A suitably substituted 4,5-dimethoxy-6-hydroxy-9-substituted xanthene-3H-3-one is obtained. Example 9 3′,9′-dihydroxy-4′,5′-dichloro-2′,
7′-dimethoxyspiro [isobenzofuran-1
(3H),9′-[9H]xanthene]-4,7-dichloro-5 or 6-carboxy-3-one production A 2-chloro-4-methoxyresorcinol (0.4g) and 3,6-dichlorotri Mellitic anhydride (0.27 g) was heated with ZnCl ₂ (10 mg) at 185° for 15 minutes and the product was converted into CH ₂ Cl ₂ :MeOH:
Purified by TLC using AcOH, 90:10:1. This product is 0.05M PO ₄ ^3- buffer, PH
λ ^absorbance _nax 533nm and λ ^emission _nax 549-5 in 8.0
0nm
It had B 3,6-dichlorotrimellitic anhydride was produced as follows. 2,5 in dilute HCl (4N, 30 ml) maintained at 0° in a three-necked flask equipped with a mechanical stirrer.
-dichloro-3,6-dimethylaniline (5
g), add a solution of sodium nitrite (50 ml)
(1.85 g in water) was added dropwise. After the addition _is complete, the diazo solution is PH'd using _Na2CO3 .
7 and added benzene (100 ml). A freshly prepared solution of cuprous cyanide (prepared from a solution of cuprous chloride and sodium cyanide) was then added to this with vigorous stirring. The mixture was allowed to reach room temperature and then stirred overnight. The next day, the benzene layer was separated and concentrated to yield about 4.5 g of a crude, colored solid as the nitrile.
This was not purified and was directly hydrolyzed to the amide. A solution of the nitrile (4.5g) in a mixture of dioxane (30ml) and 4% aqueous NaOH (70ml) was refluxed for 8 hours. The solution was cooled and extracted with ether, and the ether solution was concentrated to give a colored oil which was soaked with chloroform to give a colored solid. Add this to chloroform.
Crystallization from petroleum ether gave the amide (2.3 g). A solution of the above amide (0.9 g) in concentrated H ₂ SO ₄ (4 ml) was added with a solution of sodium nitrite (1 ml in water).
0.37 g) was added slowly while maintaining the temperature of the solution below 30°. After the addition was complete, the mixture was diluted with ice to obtain a white precipitate, which was filtered and further dissolved in _a 10% _K2CO3 solution.
Purification by filtration and acidification with 1N HCl gave 2,5-dichloro-3,6-dimethylbenzoic acid. Add alkaline to the acid produced above (0.56g)
Add KMnO ₄ (1.81 g in 10 ml 10% K ₂ CO ₃ );
This mixture was heated at 110° for 3 hours. The resulting mixture was acidified to pH 1 with 6N H ₂ SO ₄ and the excess
KMnO ₄ was removed by treatment with oxalic acid. Extraction with ether gave the desired triacid product, mp 232-33°. Example 10 2,7-di(methylthio)-6-hydroxy-
9-(2'-carboxyphenyl)xanthene-
Preparation of 3H-3-one 4-thiomethylresorcinol (200 mg), phthalic anhydride (104 mg) and zinc chloride (10 mg) were placed in a reaction flask, and the mixture was heated at 180° for 5 minutes. This product _was converted into _CH3OH : _CH2Cl2 :
Preparative using HOAc (9:1:0.1)
Purified by TLC. The product obtained is 0.05M
It had a λ ^absorption _{nax of} 512 nm and a λ ^emission _{nax of} 540 nm in PO ₄ ^3- buffer, pH 8.0. To demonstrate the usefulness of this compound, U.S. Patent No.
This protein complex was used in immunological evaluations as described in No. 3996345. In the first evaluation, about 7
A complex of fluorescein isothiocyanate and IgG with a fluorescein/protein molar ratio of was used at a concentration of 0.03 mg/ml. The product of Example 6 was used at a concentration of 4.9 mg/ml in 0.05M phosphate buffer, PH8. Various dilutions of the product of Example 6 were added to a fixed amount of fluorescein complex and incubated for 10 minutes using 0.01M phosphate, 0.15M NaCl and 2% PEG, PH 8.0 as the evaluation buffer. In the table below, 25μ of the fluorescein conjugate in 250μ of buffer is combined with 25μ of the appropriately diluted product of Example 6 in 250μ of buffer, the mixture is incubated for 10 minutes, and 2ml of buffer is added. was added and the fluorescence signal was then determined. To obtain the background from the product of Example 6, the fluorescence signal changed from 0 to 6 for various dilutions of the product of Example 6 when the fluorescein-IgG complex was replaced with buffer. However, this indicates that there was no fluorescence, and the observed fluorescence was within the error of the instrument. The table below shows the results, ie the various dilutions and the fluorescence observed. Table 1 Dilution Fluorescence Signal of Example 6 1:40 722 1:20 598 1:10 382 1:5 198 1:25 122 ∞ 928 The above results demonstrate that the quencher conjugate with respect to the fluorescein present in the medium It is shown that, depending on the concentration, the conjugates of the invention give a large dynamic range in fluorescence changes. This data shows that a sensitive evaluation of gamma globulin can be obtained. This is because by adding gamma globulin to the medium, the amount of quencher conjugate available for binding to the fluorophore conjugate is reduced in proportion to the amount of antigen present in the evaluation medium. Furthermore, by using the conjugates of the present invention, there is essentially no background radiation from the quencher conjugates. A second evaluation was conducted by replacing the quencher complex of Example 6 with the quencher complex of Example 7. The molar ratio of quencher to protein was approximately 1.5 and the concentration of the quencher solution was approximately 4 mg/ml. The fluorescent agent used instead of fluorescein was 2,7-dimethyl-9.
It was (2',5',6'-trichloro-3',4'-dicarboxyphenyl)-6-hydroxy-3-isoxanthenone. This compound has one carboxyl group.
It bound to IgG via a glycine-binding group. The fluorescent agent/protein molar ratio is approximately 7.5;
This solution had a concentration of approximately 0.03 mg/ml. The evaluation described above was performed using the quencher and antiseptic of the present invention, Example 7.
Both commercially available rhodamine isothiocyanates conjugated to IgG were tested. The table below shows a comparison of the results observed for these two quenchers.

【table】 %
(The above fluorescein compounds are prepared from 4-methylresorcinol and 2,5,6-trichloro-1,3,4-phenitricarboxylic anhydride in the manner previously described for the preparation of fluorescein derivatives. ) The next evaluation conducted was for the hapten, morphine. 6-Carboxyfluorescein NHS ester was conjugated to ^O3- (2'-aminoethyl)morphine, and the compound of Example 3 was conjugated to antimorphine using varying dye/antibody ratios as described in Example 6. . The fluorescence signal from the morphin conjugate was plotted against the concentration of anti-morphin conjugate. This evaluation mixture contained 8.75 ng/2.55 ml of fluorophore-morphine complex. anti-morphin complex, 0
Amounts of ~125 μg were varied and varying ratios of quencher to morphine were added. Buffer for evaluation mixture
0.01M _PO43-0.15M NaCl and 2 ^% PEG6000 were used. The fluorescein was added to Perkin-Elmer 1000 (Perkin
- Measured using Elmer1000). D/P ratio ¹ fluorescence signal ² 1 715 3.4 400 7.8 110 10 80 1 D/P - quencher/protein molar ratio. 2 Signal based on 1000 in the absence of antimorphin-quencher complex. By adding morphine to the evaluation mixture,
The fluorescence was enhanced, which indicates the specificity of the quenching effect. Compared to rhodamine, which is commonly used as a quencher, this quencher does not have many of the drawbacks of rhodamine and comparable dyes used as quenchers.
It has two absorption maxima, and the ratio of these two maxima changes with the degree of labeling. For effective quenching, a high rhodamine/antibody ratio is required;
This causes insolubilization. This requires the addition of undesirable solubilizing agents such as cholic acid or glycerin. From the above effects it is clear that with the conjugate according to the invention a highly efficient quenching is achieved with much lighter labeling than rhodamine. Furthermore, the contribution to the background is much smaller than that observed for rhodamine. Therefore, efficient evaluation can be performed with a high degree of extinction and a minimal amount of interference from the light emitted by the quencher. This quencher conjugate has an additional advantage compared to rhodamine, previously taught and commonly used as a quencher for fluorescein, in that it is efficiently conjugated and completely dissolved in the evaluation medium. Furthermore, due to the excellent overlap between emission of the fluorophore and absorption of the quencher, a high efficiency of transfer is achieved. At the same time, this compound is easily synthesized and
Easily complexes with proteins and other ligands.
Finally, no precipitation is observed at the level of labeling used, eliminating this problem observed with rhodamine. From the above results it is clear that the compositions of the invention, both the parent fluorescein derivative quenchers and their complexes with proteins, are highly advantageous for use in evaluation. The compound is water-soluble and can be easily complexed with proteins. When conjugated to proteins, such as antibodies, they provide useful reagents in immunological evaluations where quenching of fluorophores is required. The quenchers of the present invention provide highly efficient quenching of the fluorophore, while the amount of light observed from the evaluation medium makes only a small or negligible contribution. Because the fluorescent compounds of the present invention absorb at longer wavelengths, they provide only a small amount of excitation light, if any, to the naturally occurring fluorescent substances found in the sample to be evaluated, such as serum. can be excited. The compounds are water soluble and do not undergo significant changes in the spectroscopic properties of interest when complexed with proteins. Furthermore, the compound exhibits a large Stokes shift (Stokes shift).
shifts), with sharp absorption and emission peaks and good chemical and thermal stability. Although the foregoing invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding,
It will be obvious that certain changes and modifications may be practiced within the scope of the claims.

Claims (1)

[Claims] 1 formula where A is the same or different, D is the same or different, and either A or D has the formula - JMX, where J is a chalcogen with an atomic number of 8 to 16 and X is a hydrogen atom. if not JMX, they are hydrogen or a halogen with atomic number 9 to 53; M is a divalent hydrocarbon radical of 1 to 8 carbon atoms; QXW is a carboxylic acid Y is a halogen with an atomic number of 9 to 53, Z is an acidic anion group, m is an integer of 0 to 3, n is an integer of 0 to 4, where m+n is 4 A compound not larger than . 2. A compound according to claim 1, wherein JMX is alkoxy of 1 to 3 carbon atoms. 3. The compound according to claim 2, wherein A is alkoxy. 4. The compound according to claim 2, wherein D is alkoxy. 5 formula The compound according to claim 1, which is represented by: 6 formula The compound according to claim 1, which is represented by: 7 formula The compound according to claim 1, which is represented by: 8 formula The compound according to claim 1, which is represented by: 9 formula The compound according to claim 1, which is represented by: