United States Patent
`
`[19]
`
`[11]
`
`4,313,345
`
`Khanna et el.
`[45]
`Mar. 9, 1982
`
`
`[54] NOVEL ETHER SUB
`FLUORESCEIN POLYAM
`-ACID
`COMPOUNDS AS nuonnscnns AND
`QUENCHERS
`
`[75]
`
`Inventors: Pyare Khanm, Mountain View;
`Edwin F‘ mlmu‘ Atheflom both of
`C""f'
`S9178 Company, Palo Alto. Calif.
`
`[73] Assignee:
`
`[56]
`
`References Cited
`_
`_
`NT DOCUM
`U 5 “TE
`4,114,384 12/19'.-'9 Ullmatl et al.
`4,220,450 9/1980 Maggto
`4,220,721
`9/1980 Rowley et el.
`Primary Examt'ner—Howard E. Schain
`Anomey, Agent, or Firm-——Be11rarn 1. Rowland
`[57]
`ABSTRACT
`
`Ems
`................ .. 260/112 3
`
`435/‘! X
`
`Int. Cl.3
`
`[21] APPL N05 735153
`rm
`sen mm
`{S1}
`AGIK 39/335; A6lK 39/44;
`.
`cox} 1/no
`260/112 B; 23/23:: 13;
`[52] vs. C].
`250/112 11; 250/1125 11; 250/1123; 250/121;
`424/3; 424/13; 434/35; 424/33; 435/7;
`435/188; 525/420, 260/335
`260/112 R. 112 B. 121;
`424/85, 88; 525/420; 435/188
`
`[58] Field ofseu-ch
`
`Diether symmetrically substituted fluoresceins are pro-
`:*.::.‘:‘;i.,‘:.*::;::',=.3,1,::.1‘:,';°:,,‘::":,:e*:i::*;:::..:.:::::;2.€
`the compountls can be used as fluoresoers absorbing at
`wavelengths m excess of 500 nm or as quenchers,_a_b-
`gorbms at w_aw=lensths In excess of 500 nm and exhibit-
`mg substm_mal_ly no fluorescence. The compounds find
`tvrde eppltcatton, parttcularly as labels In fluorescent
`unrnunoassays.
`
`22 Claims, No Drawings
`
`Mylan v. Genentech
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2049
`
`Genentech Exhibit 2049
`
`IPR2016-00710
`
`

`
`1
`
`4,318,846
`
`2
`2. Description of the Prior Art
`U.S. Pat. No. 3.993.943 discloses an immunoassay
`involving a ligand-fluorescer conjugate employing ste-
`ric inhibition of simultaneous binding of antibody for
`ligand and antibody for fluorescer, where the antibody
`for fluorescer substantially quenches the fluorescence.
`U.S. Pat. No. 3,996,345 describes an immunoassay in-
`volving fluorescer-quencher pairs, where a fluorescer is
`bonded to one member of a specific binding pair and a
`quencher bonded to the same or different member of a
`specific binding pair. The assay is dependent upon the
`degree to which the quencher and‘ fluorescer are
`brought within quenching proximity based on the
`amount of analyte in the medium.
`There is an extensive list of compounds involving
`derivatives of fluorescers. Known compounds include __
`4',5'-dihydroxyfluorescein
`and
`4',5’-dihydroxy-2','lT-
`dibrornofluorescein (CA. 61,
`'l'4(}'ld). Isothiocyanate
`derivatives of lluorescein are commercially available,
`while isocyanate derivates aredescribed in CA. 59,
`S63b and sulfonic derivatives are described in CA. 58,
`9012a.
`
`SUMMARY OF THE INVENTION
`
`Di(chalcogen ether) symetrically substituted fluores-
`ceins are provided having at least one anionic group and
`one functionality for linking to another molecule. The
`compounds are linked to other materials for reagents in
`irnmunoassays, particularly immunoassays involving
`serum samples. The fluorescein compounds may also be
`halogenated.
`The fluorescers have large extinction coefficients,
`high quantum yields. have absorption maxima above
`500 nm, have Stokes shifts, normally in excess of 10 rim
`and are stable by themselves and when bonded to other
`, compounds.-The quenchers have absorption-maxima
`above 500 nm, have little or no observable fluorescence
`and efficiently quench a broad spectrum of fluorescent
`compounds.
`
`10
`
`I5
`
`20
`
`25
`
`30
`
`35
`
`NOVEL ETI-[ER SUBSTITUTED FLUORESCEIN
`POLYAMINO ACID COMPOUNDS AS
`FLUORESCERS AND QUENCHERS
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`Fluorescing compounds find wide application, be-
`cause of their ability to. emit light upon excitation with
`energy within certain energy ranges. By virtue of this
`ability, fluorescers have found employment in advertis-
`ing, novelty items, and as labels in chemical or biologi-
`cal processes, e.g. assays. That is, various compounds
`can be conjugated to a fluorescing compound, the con-
`jugate subjected to some type of partitioning. and the
`fate of the conjugate determined by irradiating the sam-
`ple with light and detecting the zone in which the con-
`jugate exists.
`This technique can be employed in immunoassays,
`involving specific binding pairs, such as antigens and
`antibodies. By conjugating a fluorescer‘ to one of the
`members of the specific binding pair and employing
`various protocols, one can provide for partitioning of
`the fluorescer conjugate between a solid phase and a
`liquid phase in relation to the amount of antigen in an
`unknown sample. By measuring the fluorescence of
`either of the phases, one can then relate the level of
`fluorescence observed to a concentration of the antigen
`in the sample.
`'
`Alternatively, one can avoid partitioning of the fluo-
`rescent label by providing for a mechanism which var-
`ies the fluorescence of the label, depending upon the
`label environment in a liquid medium. For example, in
`addition to labeling one of the members of the specific
`binding pair with the fluorescer, one may label the other
`member with.a quencher, that is, a molecule which is
`able to absorb the excitation energy of the fluorescer
`molecule, preventing the emission of a photon. The
`quenching then will occur only when the two members
`of the specific binding pair are associated, so that fluo-
`rescer and quencher have the required spatial proximity
`for quenching.
`In preparing lluorescers, there are many desiderata.
`For a fluorescer, one desires a high extinction co-effi-
`cient, a high quantum elficiency, preferably approach-
`ing or equal to one, chemical stability. 21 large Stokes
`shift. and, where the fluorescence is to be affected by
`another agent, an efficient response to such reagent.
`Furthermore, where the fluorescer is to be used in the
`~ presence of serum or other composition, which is in
`itself fluorescent, it is desirable that the fluorescer ab-
`sorb energy in a substantially different range from that
`absorbed by the other compounds in the medium. In the
`case of serum, it is desirable to have fluorescers which
`absorb light substantially in excess of 450 nm, preferably
`in excess of 500- nm.
`the
`is desirable that
`it
`For quencher molecules,
`quencher efficiently quench the fluorescer molecule,
`that is, that there be substantial overlap between the
`wavelength range of emission of the fluorescer and the
`wavelength range of absorption by the quencher. In
`addition,
`the quencher should be chemically stable,
`preferably non-fluorescent, and provide a fluorescer-
`quencher pair with a high quenching efficiency.
`In addition, any compounds of interest should be
`susceptible to reasonable modes of synthesis to provide
`the desired product in substantially pure form.
`
`DESCRIPTION OF SPECIFIC EMBODIMENTS
`
`The subject invention concerns chromogenic di{cha|-
`cogen ether)
`symmetrically substituted fluorescein
`compounds -capable of accepting or donating electronic
`energy, which find particular use when conjugated to
`other compounds, particularly polypeptides, or soluble
`or insoluble supports for use as reagents in immunoas-
`says. The fluorescein compounds are unsubstituted at
`13,8‘ and are symmetrically disubstituted on the san-
`thcne ring at either the 4',S’- or the 2’,'l’-positions. The
`compounds are normally 2,7-di(aliphatic ether substi-
`tuted) or 4,5-di(aliphatic ether substituted)-9-phenyl-E
`hydroxy-31-1-xanthene-3-ones.
`The molecules will have at least 15 carbon atoms,
`usually at least 16 carbonatoms, and not more than
`about 45 carbon atoms, usually not more than about 35
`carbon atoms. There will be at least 5 chalcogen atoms
`(atomic number of 8 to l6, oxygen and sulfur), of which
`at least 3 will be oxygen. In addition to the chalcogen
`atoms. there may be from 0 to 8, usually from O to 6
`heteroatoms. such as nitrogen, halogen of atomic num-
`ber 9 to 53. particularly of l‘.-' to 53, that is. fluorine,
`chlorine, bromine and iodine, or other heterofunc-
`tionalities which may be present
`to provide specific
`effects. There will usually be at least one anionic group,
`normally carboxylate or sulfonate, and one linking
`group. inter alia non-oxo-carbonyl, including isothiocy-
`anale and isocyanate;
`sulfonamide,
`rnercapto, and
`
`'45
`
`SS
`
`65
`
`

`
`3
`amino, which may or may not be bonded directly to an
`annular carbon atom. For the most part. the linking
`group will be on the group, usually phenyl, substituted
`at the nine position of the xanthene, although linking
`groups may also be present as substituents on the ether
`group. These compounds are conjugated to haptens and
`antigens to provide conjugates which are capable of
`fluorescing or of quenching a fluorescer when the
`quencher is in close spatial proximity to the fluorescer.
`The subject compositions have absorption maxima
`above 500 nm, usually above 510 nm, with relatively
`narrow bands. usually at least 50% of the area of the
`longest wave length absorption being over a wave-
`length range of about 50 nm. The fluorescing com-
`pounds are characterized by having good chemical
`stability, large Stokes shifts and extinction coefficients
`in excess of 65,000, usually in excess of 'J'5,(.‘KlJ. The
`Stokes shifts will be at least 10 nm, and preferably at
`least about 20 nm. The quenching compounds, will
`fluoresce with a quantum efficiency less than 10%,
`preferably less than 5%. in 0.05 molar phosphate when
`irradiated with light at the absorption maximum.
`The compounds of the subject
`invention provide
`novel compounds having important spectroscopic and
`physical properties. The compounds have absorption
`maxima above 500 nm. By choosing the positions for
`the oxy substituents one can provide highly fluorescent
`compounds or compounds that are substantially non-
`fluorescent and can be used as quenchers. Compounds
`with ether substituents at the 2’,"."-positions (fluorcs-ccin
`numbering) provide fluorescent compounds with high
`quantum efficiencim. Compounds with ether substitu-
`ents at the -I-‘,5’-positions provide compounds with sub-
`stantially no fluorescence, while absorbing at
`long
`wavelengths so as to act as efficient quenchers.
`For the most part the compounds of this invention
`will be water or base soluble compounds having the
`following formula:
`
`H0
`
`1-»:
`
`wherein:
`each of the u.'s and each of the 8‘s may be the same or
`different. either the o.‘s or the 6's being bonded to
`an annular carbon atom through a chalcogen
`(atomic number 8 to 16, oxygen and sulfur); when
`not the chalcogen bonded pair the a‘s may be any
`substituent other than chalcogen and the 5's may be
`any convenient functionality including chalcogen;
`one of the it's or 8's bonded through an ether or to
`may be taken together with A to provide an active
`functionality for linking or, when not taken to-
`gether with A, may be a linking functionality to A;
`when not taken together with A.
`in may be any
`non-interfering functionality or hydrogen;
`A is a ligand or receptor when not_ taken together to
`form an active functionality for linking;
`-y is a bond. or a spacer arm of from about I to 20
`atoms, usually 1
`to 16 carbon atoms; usually an
`aliphatic (includes cycloaliphatic) group of from I
`to 7 carbon atoms having more than 4 annular
`carbon atoms when cycloaliphatic or an aromatic
`
`5
`
`10
`
`l5
`
`20
`
`25
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`35
`
`45
`
`55
`
`60
`
`65
`
`4,318,346
`
`4
`group of from 6 to 16, usually 6 to 10 annular
`atoms;
`there being one or more of the group in the parenthe-
`sis bonded to A when A is a ligand or receptor.
`With the quencher molecules,
`the 4.5-diether-6-
`hydroxy-3H-xanthen-3-ones. the presence or absence of
`a substituent at the 2 and 7 positions does not affect the
`quenching, but can be used to modify the absorption
`characteristics of the molecule. Therefore, when the as
`are ethers, the 65 may be hydrogen or any convenient
`substituent such as alkylof l to 6 carbon atoms, oxy
`(hydroxy and aikoxy of l to 6 carbon atoms), thio (mer-
`capto, alkylthio of l to 6 and sulfonic acid, ester and
`amide), non-oxo-carbonyl of 1 to 6 carbon atoms (in-
`cludes acid, esters and amides), cyano, nitro, halo, oxo-
`carbonyl of 1
`to 6 carbon atoms, or combinations
`thereof. The choice of substitution will be governed by
`the resulting absorption maximum. synthetic conve-
`nience and the effect on the physical and chemical prop-
`erties of the molecule, such as water solubility, chemical
`reactivity, oxidation sensitivity and. the like.
`With the fluorescer molecule, the substituents at the
`4,5-positions may be varied widel-y-Lao long as the fluo-
`rescent efficiency is not significantly adversely affected.
`Therefore, while the substituents may be widely varied.
`the 4,5-position should not be substituted with chalco-
`gen. which would have the effect ofsubstantially reduc-
`ing the fluorescence of molecule. Therefore, the
`range of substituents for the 4:5-"position of the fluo-
`rescer is more restricted than thei"r:iing‘e of substituents
`for the 2,?-position of the quencher.
`For the most part. the compounds of this invention
`having '1! as aromatic will have the following formula:
`
`
`
`wherein:
`the two A5 are the same or different, normally being
`the same, when other than the functionality for
`linking;
`the two D3 are the same or different, normally being
`the same, when other than the functionality for
`linking;
`either the As or the D5 are chalcogen ether-s (chalce-
`gen of atomic number 8 to I6). usually oxyethers.
`of the formula —JMX. where J is oxygen or sulfur;
`when other than —.lMX, the A5 are hydrogen or halo
`of atomic number 9 to 53 Le. fluorine. chlorine.
`bromine. or iodine, particularly chloro and iodo,
`while the D5 may be hydrogen or any substituent,
`particularly having carbon and hydrogen and as
`heteroatoms, oxygen, sulfur, nitrogen and halogen,
`normally chemically inert under conditions of us-
`333;
`M is a divalent hydrocarbon group, normally satu-
`rated aliphatic, of from 1 to 8, usually I
`to. 6 and
`
`
`
`

`
`5
`to 3 carbon atoms, usually straight
`
`preferably 1
`chain;
`one of the X’s is an active functionality for linking to
`a ligand, receptor, or support or a functionality
`linked to said ligand or support;
`wherein when X is taken together with W to provide
`an active functionality for linking, XW can be a
`non-oxo-carbonyl functionality including-the sulfur
`and nitrogen analogs thereof, e.g. carboxylic acid,
`carboxylic acid ester, e.g. lower alkyl (1-3 carbon
`atoms) or active ester capable of forming amide
`bonds in an aqueous medium, e.g. N-oxy succini-
`mide and p-nitrophenyl,
`isocyanate,
`isothiocya-
`nate, imidate lower alkyl ester; activated olefin, e.g.
`maleirnido; rnercaptan (—Sl-I); formyl (--Cl-I0);
`sulfonyl chloride; amino; active halo e.g. halos-
`cetyl or halctriazine, with the proviso that XW is
`non-oxoncarbonyl or sulfonyl when bonded to M;
`when X is not taken together with W, one of the X3
`is a linking functionality bonded to W and depend-
`ing upon the particular active functionality will be
`non«-oxo-carbonyl (including the nitrogen and sul-
`fur analogs thereof) having one valence to carbon;
`carbamyl, thiocarbamyl; substituted ethylene from
`activated olefin; thio; methylene (from formyl by
`reductive amination); amido nitrogen or sec-amino;
`sulfonyl; or oxo-carbonyl methyl from active halo;
`when X is a linking functionality bonded to M, X is
`non-oxo-carbonyl; when not a linking group, X is
`hydrogen or non-oxo-carbonyl, e.g. carboxylic
`acid, ester or amide, sulfonamide, sulfonic acid or,
`particularly when bonded to an annular carbon
`atom. halo;
`is one when W is taken together with X and is
`otherwise on the average 1 to the molecular weight
`of W divided by 500, usually 1000. more usually
`1500 and most usually 2000, generally p ranges
`from about I to 200, usually 1 to 100;
`when W is not taken together with X, W is a ligand,
`including receptors, of at least about 125 molecular
`weight, being haptenic or antigenic, generally
`being from about 125 to 2000 molecular weight
`when haptenic and from about 5000 to IX 107
`when antigenic, although combinations of antigens
`and other materials may have a much higher com-
`posite molecular weight; the ligand will be joined
`to X, normally through amino, hydroxy, mercapto
`or active ethylene, to form amido, arnidine, thioa-
`mide, ether, or thioether, although other linkages
`may be employed, or W is a soluble or insoluble
`support which may be a polysaccharide, naturally
`occurring or synthetic, modified or unmodified, a
`naturally occurring or synthetic polymer. glass,
`inorganic solids, liposomes, or the like;
`Q is a bond or spacer arm (linking chain), usually
`aliphatic, aromatic, heterocyclic, or combination
`thereof. generally aliphatically saturated, where
`the am: will usually have from 1 to E6, more usu-
`ally I to 12, preferably 1 to 8 atoms in the chain,
`which are carbon, nitrogen, oxygen and sulfur,
`wherein the nitrogen is amido or bonded solely" to
`carbon and hydrogen.' e.g. tert-amino, oxygen is
`oxy, and sulfur is thioether, with the chalcogen
`bonded solely to carbon'and he-teroatoms being
`separated by at
`least
`two carbon atoms when
`bonded to saturated carbon atoms; the total num-
`ber of carbon atoms being generally I to 20 usually
`I to 12 and the total number of heteroatorns being
`
`10
`
`I5
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`20
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`25
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`30
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`
`4,318,846
`
`5
`
`_
`
`6
`0 to 10, usually 0 to 8; oxygen may-be present as
`' non-oxo-carbonyl or oxy, there being from O to 9,
`usually 0 to 4 heterofunctionalities; when X is not a
`linking functionality or group. Q will normally be a
`bond;
`'.
`'
`_
`'
`Y is halogen of atomic number 9 to 53, particularly
`chloro:
`n is an integer of from 0 to 4 wherein in plus 11 is not
`greater than 4;
`'
`Z. is an acidic anionic group, such" as carboxylic acid
`or sulfonic acid; and
`__
`rn is an integer of from 0 to 3, usually 1 to 3.
`Quite obviously, the compounds of the subject inven-
`tion can be modified so as-not to be within the above
`formula, without significantly affecting the properties
`of the compounds. For example, one "or more of the
`acidic anionic groups could be esterified or amidified,
`or alkyl groups can be substituted on the phenyl. as well
`as other groups, such as cyano, nitro, or the like. How-
`ever, these changes will in most cases require additional
`synthetic steps which are not warranted by the degree
`of enhancement, if any, in the spectroscopic or" chemical
`properties of the resulting product.
`The subject compounds have many desirable proper- I
`ties. The products have significant water solubility
`which allow them to be conjugated to a wide variety of
`polypeptides, without significantly adversly affecting
`the water solubility of the polypeptide, not having the
`polypeptide adversly affect the spectroscopic proper-
`ties of the subject compounds.
`As for the spectroscopic properties of the com-
`pounds, the compounds absorb at relatively long wave-
`lengths, generally in excess of 500 nm, more usually in
`excess of 510 nm. Thus, naturally occurring fluores-
`cence which may be encountered when working with
`_ physiological fluids is substantially avoided by employ-
`ing exciting light at a wavelength range which does not
`significantly excite the naturally occurring fluorescers.
`In addition, the compounds have relatively sharp ab-
`sorption peaks, and the fluorescers relatively sharp
`emission peaks. Because of this, eflicient overlap can be
`obtained between fluorescers and quenchers which
`allow for efficient quenching up to distances of about 70
`A. The fluorescing compounds also have _large Stokes
`shifts, so that the absorption band and emission band
`peaks are separated by at least 10 nm, frequently by at
`least
`I5 nm. The large Stokes shifts minimize back-
`ground interference with the observed fluorescence.
`, The quenchers have little or no fluorescence. so they
`do not contribute to background-interference with the
`observed signal. By providing for fluorescer-quencher
`couples, where the absorption band of the quencher
`substantially overlaps the emission bands of the fluo-
`rescer, efficient systems are provided for performing
`imrnunoassays, which rely on quenching of fluores-
`cence, when a quencher is brought into close proximity
`to the lluorescer due to binding of immunologically
`related materials.
`_
`In describing the subject invention, the simple mono-
`meric spectroscopically active compounds used for
`conjugation will be considered first, followed by con-
`sideration of the various conjugates. The compounds
`are chemically stable, even at basic pHs, so that they
`maintain their spectroscopic properties during use.
`The compounds employed for conjugation to other
`"compounds will be characterized by having an active
`functionality which forms a stable covalent bond with
`another compound. usuallyan amide bond or thioether
`
`

`
`4,318,846
`
`7
`bond. For the most part. the linking functionality will
`involve a non-oxo-carbonyl, including the nitrogen and
`sulfur analogues thereof. and may be bonded directly to
`an annular carbon atom of the phenyl group of the
`fluorescein, bonded through a linking group, or bonded
`directly or through a linking group to the oxy- or tl1ioe-
`ther functionality. Various functionalities may be em-
`ployed which are compatible with the other functionali-
`ties in the molecule. The functionalities include carbox-
`ylic acid, which may be activated with carbodiimide or
`activating alcohols to provide active ester groups, iso-
`cyanates, isothiocyanates, imidates or the like which
`groups react with amino functionalities to form amides.
`thioamides or amidines. Alternatively, one can have
`amino groups as the functionality, which can be com-
`bined with carboxylic acids or derivatives to provide
`amide links. Finally, one can employ mercapto func-
`tionalities. which can be combined with ethylenic
`groups, particularly activated ethylenic groups, such as
`rnaleic acid derivatives, or vice versa, to provide thioe-
`thers.
`For the most part, a preferred group of compounds
`will have the following formula:
`
`
`
`wherein:
`the two A15 are the same or different, usually being
`the same except when one is the functionality for
`linking;
`the two D1s are the same or different, usually being
`the same except when one is the functionality for
`linking;
`either the A13 or the D's are oxyethers of the formula
`—OM1X5, wherein M1 is a saturated aliphatic hy-
`drocarbylene group of from I to 6, usually I to 3
`carbon atoms, preferably straight chain and of from
`I
`to 2 carbon atoms,
`i.e. methylene or ethylene
`{hydrocarbylene intends a divalent organic radical
`composed solely of carbon and hydrogen); when
`not oxyethers, the A15 are preferably hydrogen or
`halo particularly of atomic number 9 to 53, more
`particularly chloro or iodo, and the D1s are prefer-
`ably hydrogen. halo, or alkyl of up to six carbon
`atom;
`is taken together
`one of the X9 or X55, usually X“,
`with W1 to form an active functionality. which
`may have the same definition as —XW. but will
`usually be a non-oxo-carbonyl containing function-
`ality (including sulfur-thiono-analogs
`thereofi,
`such as mixed anhydride. eg. with butyl chlorofor-
`mate, carboxylic acid, activated ester, isocyanate
`or isothiocyanate, with the proviso that Xi-' when
`taken together with W1 is a carboxylic acid or
`derivative thereof;
`when not taken together with W1, one of the X“ or
`Xbs is a linking group to W3 which is carbonyl.
`forming an amide or ester with W1, carbamyl form-
`
`8
`ing a urea with W1, or thiocarbamyl forming a
`thiourea with W1;
`when not an active or linking functionality X" is hy-
`drogen, or non-oxo-carbonyl e.g. carboxy. and X9
`is hydrogen or carboxyl. usually hydrogen;
`Q1 may be the same as Q, a bond or spacer arm, but
`will usually be a bond or spacer arm of from 1 to
`12, usually 2 to [2 atoms in the chain which are
`carbon, nitrogen and oxygen, generally having
`from 1 to It), usually 1 to 3 carbon atoms and 0 to
`8 heteroatoms which are nitrogen, oxygen and
`sulfur, wherein oxygen is present bonded solely to
`carbon e. g. non-oxo-carbonyl or may ether, sulfur is
`analogous to oxygen and nitrogen is an-iido or
`bonded solely to carbon e.g. tertiary amino; Q1 is
`usually a bond when X" is other than a reactive
`functionality or linking functionality;
`when W1 is not taken together with X“ or X11‘, W1 is a
`ligand. receptor or support, usually having amino
`or hydroxyl, particularly amino functionalities for
`linking;
`p1 may be the same as p, being I when one of X“ or
`X55 are taken together with W1, and is otherwise 1
`to the molecular weight of W1 divided by 500,
`usually divided by 1500, generally in the range of 1
`to 200, usually in the range of 1 to I00 and more
`usually in the range of I to 50;
`Z1 is an acidic anionic group, such as a carboxylic
`acid or sulfonic acid;
`ml is an integer of from 1 to 3;
`Y1 is halogen of atomic number 9 to 53, particularly
`chloro;
`n1 is an integer of from 0 to 3, wherein m1 plus n1 is
`not greater than 4;
`The compounds will normally have from 0 to 6, usu-
`ally 0 to 5 halogen of atomic number 9 to 53, preferably
`chlorine or iodine. and usually from 0 to 4 cl-ilorines,
`frequently 2 to 4 chlorines. The compounds will nor-
`mally have at least two carboxylic acid groups and up
`to 5 carboxylic acids groups. preferably having from 2
`to 3 carboxylic acid groups. The non-oxo-carbonyl
`linking functionality may or may not be bonded to a
`carbon atom, but is preferably bonded to a carbon atom.
`The preferred compounds having the active function-
`ality will for the most part have the following formula:
`
`
`
`wherein:
`to 3.
`either the A1's or D1's are alkoxy of from 1
`usually 1 to 2 carbon atoms. when not alkoxy they
`are as previously described for As and D5;
`Y1’, Z1’. m1‘ and :11’ have the same scope as the un-
`primed symbols;
`W1’ is an active functionality having a non-oxo-can
`bonyl group or sulfur analog (thionol and includes
`
`5
`
`l0
`
`i5
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`S5
`
`65
`
`

`
`9
`
`4.3 13,846"
`
`10
`
`5
`
`25
`
`so
`
`thyIPhe"VD'6‘h3'dr°'“"3H"“‘"th¢“'3‘°"‘3
`The above list is intended to merely be illustrative
`and not‘ exhaustive of the compounds included within
`the Scope of the Subject invention
`‘
`‘ The compounds of this invention having active func.
`tionalittes may be conjugated to ligands or supports
`having complementary heterofunctionalities. The fol-
`-
`-
`-
`-
`-
`-
`-
`-
`-
`lowing table Indicates Illustrative ligand functionalities,
`35 active functionnliues for linking the compounds of this
`!lI‘l\I"EIIfiOl'l
`the
`and the resulting linking llfllt.
`
`'
`
`
`
`40
`
`Ligand
`
`Linking
`iiefififlplc
`
`Fumiommy,
`my
`Gmup
`
`NH: —C0"NH"'
`NH2
`‘-NHOONH-'
`NH;
`-NHC5NH-
`NH;
`N
`
`_
`
`SH
`
`'
`
`—\\
`
`“NH<'
`
`N
`N =<
`H
`N”-
`,
`I
`I
`5-—c—c—co—
`'
`|
`
`4,5dimethoXy_2_;r_d.1ch[om.9,(2r’4*,5'-n-ical-bo,w_3',5'.
`acyl halides, mixed anhydrides and, activated ester,
`diclilorophenyl)~6~hydroxy~3H—xanthen~3-one
`as we" as isocyanate and isethiocymam
`’
`‘
`__
`H
`_
`Q'’ is a bond or spacer arm of from I to 12, usually 1
`4*5'd‘(2 ‘°”b°"5"°th°"5’)'2=7'd"°d°‘9'(2 '°a"b°"3"4 ‘
`to 10 atoms in the chain which are carbon, nitrogen
`sulfonatopheriyl}-6-hydroxy-JH-xanthen-3-one
`and oxygen, usually carbon and nitrogen, and has
`4,5_d§pmP0xy_2,7_d§bmm,3_9_(2"4*_dica1-b0xy-3',5',6*-
`from about 1 to 12, usually 1 to 10 carbon atoms
`mchl°l_ophenyD_6_hydroxy_3H_xamhen_3_0ne
`and 0 to 8, usually 0 to 6 heteroatoms which are
`,
`_
`_
`I
`I
`_
`nitrogen, oxygen and sulfur. particularly nitrogen
`4,5-diethoxy-2,7-dichloro-9-(2 ~carboxy-4 -amino-5 ~sul-
`and oxygen wherein nmogen is presem as amide
`fonatophenyl)-6-hydroxy-3H-xanthen-3-one
`or bonded solely to carbon and the chalcogens 10
`4,5-dimeihoxy-2,T-diiodo-9-(2’-carboxy-4'-isothi-
`{oxygen and sulfur) are bonded solely to carbon.
`ocyanamphCnyn_6_hydl_oxy_3H_xamhen_3‘_0ne
`doubly bonded (oxo and tliiono} or singly bonded
`_
`_,
`_
`'
`,
`,
`(oity or thio}; as a spacer arm Qi’ can be alkylene of
`_ 4,5-diethoxy-2,7-dnodo-9-[2 ,4 -dicarboxy-5 -(N-can
`from 1 10 3, usually 1 to 4 carbon atoms’ glycyl or
`boxymetliyl
`fortnamicIe)plienyl]-6-hydroxy-3H-xan-
`polyglycyl of from 1 to 4 glycyl units where the 15
`then-3.om=_
`final carboxy is W‘', or the like;
`4 5_dimeth0x}}_2 7_dichlol_o_9_(2. 4-_dica1_b0xy_3. 5. 6._
`The following is a list ofcotnpounds in this invention
`, trichlorophertiil) 6 hydroxy 31:1 xanthen 3-one ’
`which find use for conjugafing
`. 30 4,5-diethoxy-2,‘l'-clichloro-9-(4’.S'-dicarboxy-2T,3',6'-tri-
`.
`TABLE I
`The compounds are substituted 9-phenyl-6-hydroxy-
`ch1ompheny})_6_hyd,-0xy.3H.xan¢]-,en-3-one
`31-l-xanthen-3-ones or when the compound is f}uores-
`4,5_dime-1hoxy_2’7_diiod0_9_(2._cal_b°xy_4._me1_Capt°me_
`cem having a 2-carboxy group.
`the compounds are
`3’,6'-dihydroxyspirofisobenzofuran-(31-I),9’—[9H]xan—
`then]-3-one. The numbering in this list is based on the
`f01"'“9F “3f1'1iT1B-
`'
`2.7-dimethoity-9- 3',4'~dicarboxypheny1)-6~ljzydi'oxy-
`3H-xar'.then-3-one
`2‘7_diethoxy.9.{2.'3.’4._mcarbOxyphenyn‘6‘hydmxyh
`3H.xanu1en.3.o;1e
`2.7-Cii€lh01¥'9-(2'o4'g5'-l1'iC=Ifb0XY-3's5'-diCh10l'0-
`phenyl}-6-hydroxy-3H-xanthen-3-one
`2’7_dip‘_oP°xy_4’sdichlpm_9.(2,‘4,_dicatbOxy’6_Sul_
`fonamphenyD_6_hyd1.oxy_3H_xanthcn_3_one
`2",’-dimethoxy—4,5—dib[omo-9—(2"4',5"-tficafboxy—
`pheriyl}-6-hydroxy-3H—xanthen-3-one
`2,7-diinethoxy-4,5-dichloro-9-(2‘,4’,5'-tricarboxy-
`phenyl)-6-hydroxy-3H-xanthen-3-one
`2,7-dimethoiiy-9-(3',4’-rlicarboxyphenyl)-6-hydroxy-
`3H-xanthen-3-one
`2,7-dimethoxy-9-(3‘,4’-clicarboxy-2’,5’,6’-trichloro-
`phenyl)-6—hydroxy-3H~xanthen-3-one
`2,7-(2"-carboxyethoxy)-9-(2'-carboxyphenyl)-6
`hydroity-3H-'xantl1en~3-one
`2,7-diethoxy-4,5-dibromo-9-(2',4'.5’-tricarboxyphenyl}
`6—hydroxy-3H-xanthen-3-one
`2,7-dirnethoity~9~{2'-carboxy-4‘-fluoro-5'-(N-carboiv
`yniethyl
`carboxamide)plienyl]-6-hydroxy-3H-xam
`then-3-one
`2,7-diiriethoity-9-(2',4’-dicarboxy-S’-aminophenyl)-ti
`liydroxy-3H-xanthen—3-one
`2,7-dimethoity-9-(2'-cat-boxy-4’-isothincyanatoplienyl)-
`6—hydroity-3}-I-xanthen-3-one
`2,7-diethoxy-9-(2'-carboity-4'~rnercaptophenyl)~6~
`hydroxy-31-I-xanthen-3-one
`2.7-dimethoxy—4,5-dibromo-9-(2’-carboity-4’-mercap-
`tometiiylphenyl)-6-hydroxy-3H-itanthen-3-one
`2,7-dimethoxy-4.5-dichloro-9-(2’«carboxy-4’-cyano-
`phenyl)-6-hydroxy-3!-I-itanthen-3-one
`4.5~dimethoxy-9-{2',4',5’-tricarboxy phenyl)-6-hydroxy-
`3H-xaiithen~3-one
`4,5-dirnethoxy-9-(2',5‘-dicarboxy
`3H"‘a'“h°“‘3‘°“'3
`__ 4.5-diinetlioxy-2,3'-diiodo-9-(2',4’,5'-trtcarboxy phenyl)- 65
`6_hydroxy_3H_xamhen_3_°ne
`4,5dime;ho,‘y_2,',I_dich]O,-o_9_(2*'4'_5-_;,ica,boxy_
`plienyl)-6-hydroxy-3H~xanthen-3-one
`
`45 —C0—0H.—0T‘.—OCO2T
`—NCO
`-Ncs
`
`N
`- W
`_
`59 ‘Nflé
`
`N
`
`N =<
`C1
`I
`I
`55 —c=c—-co-—
`
`'
`
`'
`
`_CHo
`-5030
`tit} _C°cH’I"°‘I°
`
`_CH’NH_
`NH’
`-503”!-
`NH‘
`SH °' _C°cH1_5_ °r
`0“
`"9""
`‘T is alkyl: T‘ is. alkyl or elecironegalivc ester activating group.
`
`pheityl)-6-hydroxy
`
`In most cases, the ligand functionality and spectro-
`.
`.
`.
`.
`.
`SCOPIC compound functionality may be switched.
`For the most part the ligand conjugates will have the
`following formula:
`-
`
`

`
`11
`
`4,318,846
`
`12
`densation polymers e.g. polystyrene, polyacrylics,
`vinyl compounds. polyesters, pol yethers and poly-
`amides; charcoal, metal chalcogenides, glass, lipo-
`somes. and the like.
`A preferred group of ligand conjugates will have the
`following formula:
`
`
`
`(Z3)m3
`
`
`
`(Y3)-is
`
`Q3)‘;-
`
`wherein:
`for quencher molecules, the A3s are alkoxy of from 1
`to 2 carbon atoms and the D35 are hydrogen or
`halo. particularly chloro, bromo or iodo, or alkyl
`of from 1 to 6 carbon atoms;
`for fluorescer molecules, the D35 are alkoxy of from
`1 to 2 carbon atoms and the A35 are hydrogen or
`halo, particularly chloro or bromo;
`Z3 is carboxyl;
`Y3 is halo, particularly chloro;
`Q3 is a bond or spacer arm of from 1 to 9, usually 2 to
`9 atoms in the chain, which are carbon and nitro-
`gen, particularly arnido nitrogen, wherein the
`spacer arm is composed solely of carbon, oxygen,
`nitrogen and hydrogen, wherein oxygen is bonded
`solely to carbon and is oity or onto. particularly
`non-oxo-carbonyl; useful spacer arms include al-
`kylene of from 1 to 6, usually 2 to 4 carbon atoms,
`mono- or poly-amidomethylene (—CONHCl-12-)
`aminornono-
`or
`amino
`poly-amidomethylene
`(—HN(CONHCHz-3-x), or aminothiono mono- or
`poly(aminornethyIenecarbonyl) amino-methylene
`1i'—N3I;lCS(NI-{Cl-i;CO),.,NHCH;—), wherein it is
`to ;
`X? is non-oxo-carbonyl, carbamyl or thiocarbamyl,
`bonded to amino of W3 to form an amide bond;
`W3 is a receptor or ligand, usually haptenic of from
`about 125 to 1000 molecular weight or antigen of
`from about 2000 to 10 million, -usually 5000 to
`2,000,000. more usually 5000 to 1,000,000 molecu-
`lar weight; W3 is particularly a poly(amino acid) or
`polysaccharide antigen and may be any hapten;
`p3 is l to the molecular weight of W3 divided by 500.
`usually divided by about 2000, generally in the
`range of about 1 to 200, more usually in the range
`of about 2 to 150, and frequently in the range of
`about I to 75. For ligands below 500,000 molecular
`weight p3 will generally be in the range of about 2
`to 50;
`m3 is an integer of from 1 to 2;
`n3 is an integer of from 0 to 3. with m3 plus n3 not
`greater than 4.
`In some instances it may be desirable to have the
`conjugate of the ligand and subject lluorescer bound,
`either covalently or non—covalently to a support. The
`binding of the conjugate to a support may be