Umted States Patent
`
`[191
`
`m]
`
`4,318,846
`
`Khanna et a].
`
`[45]
`
`Mar. 9, 1982
`
`[54] NOVEL ETHER SUBSTITUTED
`FLUORESCEIN POLYAMINO ACID
`COMPOUNDS AS FLUORESCERS AND
`omega“
`
`[TS]
`
`Inventors: Pym Khannl, Mountain View;
`F' m Athena" both or
`
`' '
`MERGE: Sm COMM}, P110 A110.
`[2!] Appl. No.: 13.163
`.
`‘
`In] F'kd'
`[51]
`Int. CL!
`
`5‘" 7' my
`A611! 39/385; A611: 39/“;
`(1076 7/11)
`260/112 3: 23/230 B;
`[52] US. Cl.
`260/112 R; 260/1115 11; 260/112.7; 260/121;
`424/8; 424/12; 424/85; 424/88; 435/7;
`435/188; 525/420; 260/335
`260/112 R. 112 B. 12!;
`424/85, 88; 525/420; 435/188
`
`[58] Field ofSeIrch
`
`
`
`[56]
`
`ReferencesCited
`“'5' PATENT DOCUMENTS
`4.114.384 12/1979 Ullmen c191.
`260/112 3
`4,220,450 9/1930 Maggie
`..... .. 435/? X
`
`4.220.722
`9/1980 Rowley et a].
`................... .. 435/7 :1
`Primary Examiner—Howard E. Schain
`Amman Agent, or fi'mmflertmm 1. Rowland
`
`Diether symmetrically substituted fluoresceins are pro-
`vidcd having at least one enicnic group and a linking
`functionality. Depending upon the site of substitution.
`the compounds can be used as fluoresoers absorbing at
`wavelengths in excess of 500 um or as quenchcrs. ab-
`sorbing at wavelengths in excess 01'500 nm and exhibit-
`in; annoyingly no fluorescence- “: oojnpmmds find
`application. Dame-"1311' as label! In fluorescent
`unmunoassays.
`.
`
`12 (Helms, No Drum
`
`SANOFI V. GENENTECH
`SANOFI v. GENE(cid:49)TECH(cid:3)
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2049
`EXHIBIT 2049
`
`
`
`

`

`1
`
`4,318,846
`
`2
`2. Description of the Prior Art
`[5.8. Pat. No. 3.993.943 discloses an immunoassay
`involving a ligand-fluorescer conjugate employing ste-
`ric inhibitiou of simultaneous binding of antibody for
`ligand and antibody for fluorescer. where the antibody
`for lluorescer 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 pmximity 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','l'-
`dibromofluorescein (CA. 61. 1401a). Isothiocyanate
`derivatives of fluorescein are commercially available.
`while isocyanate derivates are described in CA. 59.
`563b and sull'onic derivatives are described in (LA. 58.
`90123.
`
`NOVEL ETHER 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
`rate of the conjugate determined by irradiating the sam-
`ple with light and detecting the zone in which the cen-
`jugate exists.
`This technique can be employed in immunoassays.
`involving specific binding pairs. such as antigens and
`antibodies By conjugating a fluo'rescer' to one of the
`members of the specific binding pair and employing
`various protocols. one can previde 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 partitiomng 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 witha 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 fluorescers. there are many desiderata.
`For a fluorescer. one desires it high extinction co-effi.
`cient. a high quantum elliciency. preferably approach-
`ing or equal to one. chemical stability. a 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 at»
`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 ol'450 mm 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.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`
`
`sulfonamide. mercapto. and
`
`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
`immunoassays. 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 shifis. normally in excess of 10 nm
`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.
`
`DESCRIPTION OF SPECIFIC EMBODIMENTS
`
`The subject invention concerns chromogertic di(chal-
`cogen ether)
`symmetrically substituted fluorescein
`compounds capable of accepting or donating electronic
`energy. which lind 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
`L',ll' and are symmetrically disubstituted on the san-
`thene ring at- either the 4'.5‘- or the 2’,‘l'-positions. The
`compounds are normally 2.7-di(aliphatic ether substi-
`tuted) or 4,5-di(aliphatic ether substitutedy9-phenyl-6-
`hydroxy-3H—xanthene-3-ones.
`The molecules will have at least 15 carbon atoms.
`usually at least 16 carbon'atoms. 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 B to l6. oxygen and sulfur). of which
`at least 3 will be oxygen. In addition to the chalcogert
`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 1'." 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-
`anate and isocyanate;
`
`

`

`
`
`4,318,846
`
`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 in fluorescer when the
`quencher is in close spatial proximity to the fluorescer.
`The subject compositions have absorption maxima
`above 500 not. 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 75.030. 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 TIP-positions (fluorescein
`numbering) provide fluorescent compounds with high
`quantum efficiencies. Compounds with ether substitu-
`ents at the 4',5'—positiorls 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:
`
`5
`
`10
`
`IS
`
`20
`
`25
`
`35
`
`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-dietheo6-
`hydroxy-SH-xanthen-S-oncs. 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 others. the 6s may be hydrogen or any convenient
`substituent such as alkyl of l to 6 carbon atoms. oxy
`(hydroxy and alkony of 1 to 6 carbcm atoms). thio {mer-
`capto.‘ alkylthio of l to 6 and sulfonic acid. ester and
`amide). non-oxo-carbonyl of I
`to 6 carbon atoms (in—
`cludes acid, esters and amides), cyano. nitro. halo. oxo-
`carbonyl of l
`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 andthe like.
`With the fluorescer molecule. the substituents at the
`Ales—positions may be varied widelyéso 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 of substantially reduc-
`ing the fluorescence of the molecule. Therefore, the
`range of substituents for the 4.53poaition of the fluo-
`rmcer is more restricted than thefigihg'e of substituents
`for the 2.7-poaition ol' the qaencher.
`For the most part. the compounds of this invention
`having 1 as aromatic will have the following formula:
`
`
`
`H0
`
`T'hfl
`
`wherein:
`each of the 11's and each of the 8's may be the same or
`different. either the o‘s or the 8’s being bonded to
`an annular carbon atom through a chalcogen
`(atomic number 8 to 16. oxygen and sulfur); when
`not the chaloogen bonded pair the 11‘s may be any
`substituent other than chalcogeu and the 8's may be
`any convenient functionality including chalcogen;
`one of the a’s or 5's bonded through an ether or a.)
`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. a: may be any
`non-interfering functionality or hydrogen;
`It is a ligand or receptor when not taken together to
`fonn an active functiOnaIity for linking;
`7 is a bond. or a spacer arm of from about 1 to 20
`atoms. usually 1
`to 16 carbon atoms; usually an
`aliphatic (includes cycloaliphatic) group of from 1
`to 7 carbon atoms having more than 4 annular
`Carbon atoms when cycloaliphatic or an aromatic
`
`55
`
`65
`
`wherein:
`the two A5 are the same or different, normally being
`the same. when other than the functionality for
`linking;
`the two D5 are the same or different. normally being
`the same, when other than the functionality for
`linking;
`either the As or the D5 are chaicogen ethers (chalco-
`gen of atomic number 8 to l6). usually oxyethers.
`of the fonnula —JMX. where J is oxygen or sulfur;
`when other than —JMX. the As are hydrogen or halo
`of atomic number 9 to 53 i.e. fluorine. chlorine.
`bromine. or iodine, particularly chloro and iodo.
`while the US may be hydrogen or any substituent.
`particularly having carbon and hydrogen and as
`heteroatoms. oxygen. sulfur, nitrogen and halogen.
`normally chemically inert under couditions of us-
`338;
`M is a divalent hydrocarbon group. normally satu-
`rated aliphatic. of from 1 to 8, usually I to 6 and
`
`
`
`

`

`4.318.846
`
`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, KW can be a
`non-oxo-carbonyl functionality including the sulfur
`and nitrogen analogs thereof. cg. carboaylic 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-
`rnide and p-nitrophenyl,
`isocyanate.
`isothiocya—
`nate. imidate lower alkyl ester; activated olefin. e.g.
`maleimidc; mercaptan (—SH); formyl (—Cl-IO);
`sulfonyl chloride; amino; active halo cg. halos-
`cetyl or halotriazine, with the proviso that XW is
`non-oxo-carbonyl or sulfonyl when bonded to M;
`when X is not taken together with W, one of the X5
`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 thereon 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 Ono-carbonyl methyl from active halo;
`when X is a linking functionality bonded to M. X. is
`non-oso-carbouyl: when not a linking group. X is
`hydrogen or non-oxo—carbonyl. e.g. carboxylic
`acid, ester or amide, sull'onamide, 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 1 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 101r
`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 amide. amidine. 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 arm will usually have from I to 16. more usu-
`ally I to 12. preferably 1 to S 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. tart-amino. oxygen is
`oxy. and sulfur is thioether. with the chalcogen
`bonded solely to carbon'and heteroatoms being
`separated by at
`least
`two carbon atoms when
`bonded to saturated carbon atoms; the total num-
`ber of carbon atoms being generally 1 to 20 usually
`I to 12 and the total number of heteroatcms being
`
`
`
`6
`0 to 10, usually 0 to 8; oxygen may-be present as
`non—oxo-carbonyl or oxy. there being from 0 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 111 plus 11 is not
`greater than 4;
`Z is an acidic anionic group. such as carboxylic acid
`or sulfonic acid; and
`_.
`m is an integer of from 0 to 3. usually 1 to 3.
`Quite obviously, the compounds of the subject inven-
`tlon can be modified so as not to be within the above
`formula. without significantly affecting the preperties
`of the compounds. For example, one or more of the
`acidic anionic groups could be esterified or amidified,
`or' 'alltyl 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
`ofenhancement, if any. in the spectroscopic or chemical
`prOperties of the resulting product.
`The subject compounds have many desirable proper-
`ties. The products have significant water solubility
`which allow them to be conjugated to a wide variety of
`polypeptides. without significantly adverst affecting
`the water solubility of the polypeptide. nor having the
`polypeptide adverst affect the spectrbscopic proper-
`ties of the subject compounds.
`As for the spectroscopic properties of the com-
`pounds. the compounds absorb at relatively long Wave-
`lengths. generally ln excess of 500 no]. 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, efficient overlap can be
`obtained between fluoresoers 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 saparaied by at least 10 nm. frequently by at
`least 15 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 perfonning
`immunoassays. which rely on quenching of fluores-
`cence. when a quencher is brought into close proximity
`to the fluorescer 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. usually an amide bond or thioether
`
`5
`
`ll}
`
`[5
`
`20
`
`25
`
`3D
`
`35
`
`40
`
`45
`
`55
`
`65
`
`
`
`

`

`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 thioe-
`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 oarbodiimide 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 carbonylic 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
`maleic acid derivatives. or vice verse, to provide thioe—
`thers.
`For the most part. a preferred group of compounds
`will have the following formula:
`
`
`
`wherein:
`the two A's are the same or different. usually being
`the same except when one is the functionality for
`linking:
`the two Dis are the same or different. usually being
`the same except when one is the functionality for
`linking;
`either the A's or the D‘s are oxyethers of the formula
`—OM1X°, wherein M' is a Saturated aliphatic hy-
`drocarbyicne group of from I to 6, usually 1 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 Als are preferably hydrogen or
`halo particularly of atomic number 9 to 53, more
`particularly chloro or iodo, and the D‘s 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 Wl
`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-nnalogs
`thereon,
`such as mixed anhydride. cg. with butyl chlorofor-
`rnate, carbonylic acid, activated ester. isocyanate
`or isothiocyanate. with the proviso that X!" when
`taken together with W1 is a carboxylic acid or
`derivative thereof;
`when not taken together with W'. one of the X“ or
`Xi’s is a linking group to W1 which is carbonyl.
`forming an amide or ester with W'. carbamyl form-
`
`
`
`4,318,846
`
`8
`ing a urea with W'. or thiocarbamyl forming a
`thiourea with “1';
`when not an active or linking Functionality X“ is hy-
`drogen, or non-oxo-carbonyl e.g. carboxy. and 30"
`is hydrogen or carbonyl. usually hydrogen;
`0' may be the same as Q. a bond or spacer arm, but
`will usually be a bond or spacer arm of from I to
`12. usually 2 to 12 atoms in the chain which are
`carbon, nitrogen and oxygen, generally having
`from 1 to It], usually 1 to 8 carbon atoms and O to
`3 heteroatoms which are nitrogen, oxygen and
`sulfur, wherein oxygen is present bonded solely to
`carbon e.g. non-oxo-carbonyl or my ether, sulfur is
`analogous to oxygen and nitrogen is amido 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 WI is not taken together with X“ or X5. W1 is a
`ligand. receptor or support. usually having amino
`or hydroxyl. particularly amino functionalities for
`linking;
`pl may be the same as p. being I when one of X“ or
`X’s are taken together with W]. and is otherwise l
`to the molecular weight of W1 divided by 500,
`usually divided by 1500. generally in the range of I
`to 200. usually in the range of 1 to 100 and more
`usually in the range of t to 50;
`Z' is an acidic anionic group, such as a carhoxyhe
`acid or sulfonic acid;
`to1 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 in1 plus n' is
`not greater than 4;
`The compounds will normally have frOm l} to 6. usu-
`ally 0 to 5 halogen of atomic number 9 to 53, preferably
`chlorine or iodine. and usually from O to 4 chlorines,
`frequently 2 to 4 chlorines. The compounds will nor-
`mally have at least two carbonylic acid groups and up
`to S carboxyIic acids groups, preferably having from 2
`to 3 Carbonylic 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 Al's or Dl's are alkoxy of from I
`usually 1 to 2 carbon atoms. when not alkoxy they
`are as previously described for As and D5;
`Y". 2". ml' and ni’ have the same scope as the un-
`primed symbols;
`W“ is an active functionality having a non-oxo-car-
`bonyl group or sulfur analog (thiono) and includes
`
`5
`
`10
`
`IS
`
`‘25
`
`35
`
`45
`
`SD
`
`55
`
`65
`
`
`
`
`
`

`

`
`
`4.3 1 8,846
`
`10
`
`9
`acyl halides, mixed anhydrtdes and, activated ester,
`as well as isocyanate and isothiocyanate.
`Q" is a bond or Spacer arm of from I to 12, usually 1
`to ll] atoms in the chain which are carbon. nitrogen
`and oxygen. usually carbon and nitrogen, and has 5
`from about 1 to 12, usually 1 to 10 carbon atoms
`and {l to 8. usually 0 to 6 heteroatorns which are
`nitrogen. oxygen and sulfur. particularly nitrogen
`and oxygen, wherein nitrogen is present as amido
`or bonded solely to carbon and the ohaICOgens
`[oxygen and sulfur) are bonded solely to carbon.
`doubly bonded (oxo and thiono} or singly bonded
`(013; or thin); as a spacer arm Q" can be alkylene of
`from I to 8, usually 1 to 4 carbon atoms, glycyl or
`polyglyoyl of from 1 to 4 glycyl units where the 15
`final carboxy is W“, or the like;
`The following is a list ofcompounds in this invention
`which find use for conjugating.
`TABLE I
`
`10
`
`The compounds are substituted 9-phenyl-6—hydroxy-
`3H-xnnthen-3-ones or when the compound is fluores-
`cein having a 2-carboxy group,
`the compounds are
`3’.6'-dihydroxySpiro[isobenzofurnn~(3H),9'-[9H]xen—
`thenl-J-one. The numbering in this list is based on the
`former naming.
`2,7-dimethoxy-9- 2‘,4'-dicarboxyphenyl)-6-hydroxy-
`BH-xnnthen-Zl-one
`2.7—diethoxy-9-{2‘,3',4’-tricarboxyphenyl)-6-hydroxy-
`Bannthend—one
`
`25
`
`30
`
`4,5-dimetho:Ity.2,T-dichloro-9vt‘2',4‘,3’-tricarboxy-336‘-
`dichlorophenyl)-6-hydroity-3H—xanthend-one
`4,5-dt(2"-earboxyethony-Z,7-diiodo-9-(2'carboxy-4’-
`sulfonatophenyll-fi-hydroxy-JH-xanthen—J-one
`4,5-dipropoxy-ZJ-dibromo-9-(2'.4’-dica.rhoxy-3',5',6’-
`trichlorophenylj-G-hydroxy-SH-xenthen-B-one
`
`4.5-diethoxy-2J-diehloro-Q-(2’-earboxy-4’-amino-5'-sul-
`fonatophenyi)-6-hydroxy-JH-xanthen—S-one
`4,5-dimethoxy-Z,7-diiodo-9-(2’-carboxy4'-isothi-
`ocyanutophenyl)—6‘hydroxy-3H-xanthen—S-one
`4.S-diethoxy-Z,‘i-diiodo-9-[2’,4'-dioarboxy-5'-(N-car—
`boxymethvl
`formamichphenvlj-fi-hydroxy-SH—xan-
`then-3-one
`
`4.5~dimethoxy~2.7-dichloro-9—(2',4'~dic_arboxy-3’,S’,6'-
`trichlomphenyl)-6-hydroxy—3H~xanthen-3-one
`.4.5-diethony«2,T-dichloro-9-{4‘,S‘-dicarboxy-2',3',6‘-tri-
`chlorophenyl)~6—hydroxy~3H-xnnthen-J-one
`4,5-dimethoxy-2,7-diiOdo-9-{2'-oarboxy-4'-mercaptome-
`thy[phenyll—G-hydroxy-SH-xanthen-3-one
`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-
`tionalities may be conjugated to ligands or supports
`having complementary heterofunctionalities. The fol-
`lowing table indicates illustrative ligand funetionalities.
`active functionalities for linking the compounds of this
`invention with the ligands and the reenlting linking unit.
`
`
`
`Ligand
`Fune-
`Specuotoopie
`Carnpound
`tinn-
`Linltlng
`
`Functionalily"
`ality
`Group
`
`—co—on. —o-rl. —OCD:T
`—NOO
`—NCS
`
`NH: —CO-NH-
`NH; —NHCONH-
`NH; —NHCSNH-
`
`'—NH<
`
`N 1
`
`N =(
`
`N
`
`Cl
`
`|
`l
`-C=C-C0—
`--CHO
`—502Cl
`—CDCH;lialo
`
`NH!
`
`N 1
`
`SH
`
`—-NH<’
`
`N
`N ={
`NH-
`H
`I
`I
`S-IIZ—If—CO—
`NH: —CH;NH—
`NH: —SO:NH—
`SH or —CU'CH1—S— or
`OH
`F0—
`
`'1' is nlkyl: T‘ 'n alkyl or e1eetrone5nlive esler activating group.
`
`In most cases. the ligand functionality and spectro-
`
`scopio compound functionality may be switched.
`For the most part the ligand conjugates will have the
`following formula:
`
`
`
`2.7-diethoxy—9-(z'.4',Sttricarboxy-a',s'nichlom-
`phenyl}6-hydroxy—BH-xanthen-B-one
`2,7-dipropoxy4.5»dichlorou9-(2',4’-dicarboxy-6-sul-
`fonatophenylJ-fi-hydroxy-ZiH-xanthen-S-one
`2.7-dimethoxv-4,5-dibromo-9-(2’.4',5’-tricarboxy-
`phenyl)-6-hydroxy—BH-xanthen-B-one
`2.7-dimethoxy-4,S-dichtaro-942',4',S'-triearboxy-
`phenyl)-6-hydroxy—3H-xanthen-3-one
`2.741imethoxy-9-(3',4‘-dicarboxypheny])-6-hydroity-
`3H~xanthen-3-one
`2,7-dimethoxy-9-(3'.4‘-clicarboxy-Z',S‘,6’-triohloro-
`phenyl)—6—hvdroxy-JH-xanthen~3—one
`2.7-(2"-carboxyethoxy)-9-(2'-earboxyphenyl)-6-
`hydroxy-JH-‘xanthen-B-one
`2,7-diethoxy-4,5-dibromo-Q-(Z',4'.5'-tricarboxyphenyl)-
`6-hydroxy~3H-xanthen-3-one
`2,7-dimethoxy~9~[2'-carboxy-4‘-lluoro-fi‘-(N-earbox-
`yrnethyl
`carboxamide}phenylj-fi-hydrony«3H+xan~
`then-S-one
`2.7-dimethoxy-9-(2‘.4’-dicarboxy-5’-aminophenyl)—6-
`hydroxy-SH-xanthen-S-one
`2,7-dimethoxy-9-{2'carboxy-4’-isothiocyanatophenyl}-
`6-hydroxy-3H-Jtanthen-3—one
`2,7-diethoity-9-(2‘-carboxy-4'-mercaptophenyl)-6-
`hydroxy-JH-xnnthen-S-one
`2,7-dimethoxy-4.5-dibromo-9-(2'-carboxy-4'-mercap-
`tomethylphenyl)-6-hydroxy-3H—xenthen-3-one
`2.7-dilnetboxy-4.5'dichloro«9-(2’-carhoxy-4'-cyano.
`phenyll-fi-hydroxy-SH-aanthen-3-one
`4,5-dimethoxy-9-{2',4',5'-tricarhoxy phenyIJ-fi-hydwxy-
`3H~xanthen-3-one
`4.5-dimethoxy-9-(2‘,5'-dioarboxy
`3H-xanthen-3-one
`4.5-dirnethoxy-2,T-diiodo-9—(2'A',5’-tricarhoxy phenyl)— 65
`6-11ydroity-JH-xanthen-J-one
`4.5-dimethoxy-ZJ-dichloro-9-(2'.4'.5'-tricarhoxy-
`phenyl)-fi-hydroay~3ll-Jtanthen~3-one
`
`35
`
`ill}
`
`45
`
`50
`
`55
`
`Hi
`
`phenyll-G-hydmxy-
`
`

`

`11
`
`4,3 18, 846
`
`12
`densation polymers e.g. polystyrene,- polyacrylics.
`vinyl compounds, polyesters. polyethers and poly-
`amides; charcoal, meta] chalcogenides, glass. lipo-
`scores. and [he like.
`A preferred group of ligand conjugates will have the
`
`following formula:
`
`H0
`
`w:
`
`p:
`
`5
`
`10
`
`I 5
`
`A}
`
`A}
`
`H0
`
`D3
`
`0
`
`03
`
`“(I
`
`p3
`
`(2’)».3
`J
`{Y M
`3
`f
`Q x
`
`
`
`30
`
`45
`
`
`
`wherein:
`the A15 are the same or different. normally being the
`same, except when the linking site:
`the Dis are the same or different. normally being the
`same. except when the linking site;
`either the A15 or D25 are ethers. usually oxyethers. of m
`wherein:
`the formula —HM3XJ. wherein H is Oxygen or
`sulfur. M2 may be the same as M, but usually is an
`for quencher molecules. the A35 are alkoxy of from I
`alkylene group of from 1 to 6. usually 1 to 3 carbon
`to 2 carbon atoms and the D35 are hydrogen or
`atoms. X6 is cat-boxy or hydrogen. usually hydro-
`halo, particularly chloro. bromo or iodo, or alkyl
`gen; and when not ethers, as defined previously for 25
`of from 1 to 6 carbon atoms;
`A
`d D ‘
`Y1, ‘szazmmz aid n; are the same as Y z m and u re-
`for fluorescer molecules. the D3: are alkoxy of from
`spechvely:
`'
`'
`l to 2 carbon atoms and the A35 are hydrogen or
`Q2 may be the same as Q, but will usually be the same
`hale" particularly cmom or homo;
`as Q';
`Z3 is carboxyl;
`Y3 is halo. particularly chloro;
`Xcmay come within the same definition as X. treating
`Q3 is a bond or spacer arm of from 1 to 9. usually 2 to
`W2 as W. but will usually be a non-oxo-carbonyl
`9 atoms in the chain. which are carbon and nitro-
`containing functionality,
`including nitrogen and
`gen. particularly amido nitrogen. wherein the
`sulfur analogs, such as carbonyl, imido, oarbamyl
`Spacer arm is composed solely of carbon, oxygen,
`and thiocarbarnyl to form respectively amide. ami-
`dine. urea and thiourea. when combined with as
`nitrogen and hydrogen, wherein oxygen is bonded
`amino on the ligand;
`solely to carbon and is city or onto, particularly
`p3 is l to the molecular weight of Wzdivided by 500.
`non-oxo-carbonyl; useful spacer arms include al-
`llsufllly 1000. more usually 2000; With haptenic
`kylene of from 1 to 6. usually 2 to 4 carbon atoms.
`ligands of from about
`l25 to 2000 molecular
`mono— or poly-amidomethylene (—CONHCH2—)
`“might: P2 Win “$3311? be 1; With afltigeflic “Emmi ‘0
`aminomono-
`or
`amino
`poly-amidomethy‘lene
`0f Swat” than 2000 “101931115? dehfi usually
`(—HN(CONHCH29-x). or aminothiono mono- or
`Steal“ than 5000 “101501