[19]
`United States Patent
`4,351,760
`[11]
`
`Khannn et al.
`[45]
`Sep. 28, 1982
`
`[56]
`
`References Cited
`
`“'5' PATENT DOCUMENTS
`4,1:-¢,3s4 1|/1979 Ullman et a1.
`1:133:33 33$ }é'§2;§2f.T.f.‘;..:::
`4.230.305 10/1930 Singh et al.
`4.318.846
`3/1932 Khnnna et al.
`
`
`
`260/112 R
`:::..’.f§.{}}§,”;
`zen/112 R
`260/121 7:
`
`[54] NOVEL ALKYL SUBSTITUTED
`FLUORESCENT COMPOUNDS AND
`POLYAMINO ACID CONJUGATES
`
`F51
`
`Im="°rs= gig; ghglgv» Meg-in Vigwh F
`.
`'1
`-
`"'3":
`‘
`'="‘’“-
`'5“
`‘3
`C3115
`
`.
`_
`.
`U3] Asslgnea Syn Company’ Pam Aha’ Calif’
`
`Primary Exam:'r1er—Howard E. Schsin
`Attorney, Agent. or F:'rm—Bertram I. Rowland
`
`[21] Appl. No.: 73,153
`
`_
`[22] Filed!
`
`Sell 7; 1979
`
`[51]
`
`Int. Cl.3 .. ............... .. A61K 39/385- A6II{ 39/44;
`’
`co7G 1/on
`..... .. 260/112 R; 23/230 B;
`....
`[52] U.S. CI. ......
`260/I12 B; 260/112.5 R; 260/112.7; 260/121;
`424/8; 424/12; 424/35; 424/38; 435/7;
`435/188; 525/420; 549/388
`[53] Field of Search ............. .. 260/112 R, 112 B. 121;
`424/85. 88; 435/188; 525/420
`
`[571
`
`ABSTRACT
`
`_Fluore§cent antigen conjugates are provided compris-
`mg antlgens covalently bonded to at least one 2,7-diaIi-
`phatic
`substituted-9-phenyl-6-hydroxy-3H-xanthen-
`3-one. wherein the 1- and 8-positions are unsubstituted.
`Also provided are novel fluorescent compounds ab-
`Snrbing at wavelengths in excess of 500 nm, having
`active funclionalities for linking to the antigen. Finally,
`methods are provided for analyzing antigens in serum,
`whereby serum interference is avoided.
`
`7 (Iinims, No Drawings
`
`SANOFI V. GENENTECH
`SANOFI v. GENENTECH
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2036
`EXHIBIT 2036
`
`

`
`1
`
`4.351360
`
`NOVEL ALKYL SUBS'ITI'U'l'ED FLUORESCENT
`COMPOUNDS AND POLYAMINO ACID
`CONJUGATES
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`
`Fluorescent compounds find a wide variety of appli-
`cations. They find use in fluorescent
`immunoassays.
`histochemica] staining. displays, inks, and the like. Of
`particular interest for the subject invention is the use of
`antigenic conjugates (includes receptor conjugates)
`with fluorescent compounds to be used in the determi-
`nation of a variety of ligands. both antigens and recep-
`tors. A substantial proportion of the ligands are assayed
`in physiological fluids, such as serum, where the serum
`can provide substantial background fluorescence. One
`way to diminish the background fluorescence resulting
`from naturally present fluorescers is to provide a fluo-
`rescent compound which absorbs at relatively long
`wavelengths. The compound should desirably have a
`large Stokes shift. be stable under conditions of the
`assay. be relatively free of non-specific interference.
`both from materials in solution and the compound to
`which the fluorescer is conjugated and to provide high
`quantum yields. In addition. for certain applications. it
`is desirable that
`the fluorescer be coupled with a
`quencher molecule. that is a molecule which is capable
`of absorbing the energy of the fluorescer in the excited
`state when within a predetermined distance. so that the
`Iluorescer does not tluoresce.
`2. Description of the Prior Art
`A large number of fluorescein derivatives have been
`reported in the literature. The following are believed to
`be the most exemplary in relation to the subject inven-
`tion and are reported in conjunction with the Chemical
`Abstracts citation. The numbering is based on the par-
`ent molecule
`3',6'-dihydroxyspiro
`[isobenzofuram
`l(3H).9'-(91-I)xanthen]-3-one.
`2'.7'-di(n-hexyl) or di(n-heptyl)-4'. 5'-dibromo-1.'l5
`dich|oro- are reported as being prepared. C.A. 31. I621;
`2'.'l’-di(n-berry!)-, C.A. 31, 1621; 2'.T-di(allryl)-; C.A. 31,
`1388; 2‘.7'-diethyl or 2'.'l”-dibutyl—. CA. 27. 5056; 2'.7'-
`tlimethyl-. C.A. 83.
`l8972s; 2’.4’,5'.7'-tetrabromo-5 or
`6-carboity, CA. 63, l32l0h.
`SUMMARY OF THE INVENTION
`
`The subject compounds include novel fluorescent
`conjugates with members of specific binding pairs, li-
`gands and receptors. as well as the fluorescent precur-
`sors to the conjugates. The conjugates find a wide vari-
`ety of uses. particularly as reagents in in-imunoassays.
`The compounds are 2.7-dialiphatic-6-hydroxy-3H-xarb
`then-3-ones, normally having at least two chloro sub-
`stituents. with the precursors having a linking group or
`functionality on a group, either aliphatic or aromatic.
`bonded to the 2- or 9-position of the xanthene.
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`
`invention concerns fluorescent com-
`The subject
`pounds, which are analogs of fluorescein. being particu-
`larly
`2,7-dialiphatic
`subsututed-9-substituted-&
`hydroxy-3H-xanthen-3-ones. usually having at least two
`chloro substituents at other than the 1.8-positions and
`having a functional group for linking to a member of a
`specific binding pair bonded to a hydrocarbon group
`substituted at the 2- or 9-position. particularly 9-posi-
`
`IO
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`2
`tion. as well as the conjugates of the fluorescent com-
`pound to the member of the specific binding pair. The
`conjugates find particular use as reagents in assays for
`members of‘ specific binding pairs.
`The fluorescent precursors will have at least about 15
`carbon atoms. usually 21 carbon atoms. and usually not
`more than about 40 carbon atoms. usually having from
`about 22 to 36 carbon atoms. There will preferably be at
`least two chlorine groups at other than the 1,8-positions
`and may be as many as 6 chlorines. In addition to chlo-
`rine. the only other heteroatoms are bromine. chalce-
`gen. particularly oxygen and sulfur. and nitrogen. there
`being at least 4 heteroatoms and usually not more than
`20 heteroatorns. more usually not more than about 16
`heteroatoms and preferably not more than about 12
`heteroatoms. Of the heteroatorns other than chlorine,
`there will be at least 3 oxygens, more usually at least 5
`oxygens. and other than the oxygens which are part of
`the xanthene chromophore, are oxygerts as non-oxo-
`carbonyl or oxy, particularly acid, ester or ether (nor-
`mally bonded solely to carbon and hydrogen); sulfur is
`normally present as sulfony], thioether or rnercapto;
`while nitrogen is normally present as amino or arnido
`(bonded solely to carbon and hydrogen).
`The fluorescent compounds are further characterized
`by having absorption maxima in 0.05 M phosphate
`buffer pH8 of at least about 500 run. an extinction coeffi-
`cient in the same medium of at least about 65,000. more
`usually at least 70.000 and a Stokes shift in the same
`medium of at least about 10 run. more usually at least
`about 12 nm.
`The 9-substituted-2,7-dialltylsubstituted xanthenes of
`this invention will for the most part have the following
`fonnula:
`
`0
`
`éo
`
`H
`
`Lil:
`
`H
`
`..
`
`‘
`
`mt
`
`(«)3
`
`no
`
`IV»
`
`wherein:
`
`pis an aliphatic group, normally aliphatic hydrocar-
`byieue (composed solely of carbon and hydrogen), satu-
`rated or unsaturated. branched or straight chain. partic-
`ularly alkylene. more particularly (CH;)¢. wherein a is
`of from 1 to 12, usually I to 6. more usually 1 to 4; p is
`normally of from I to 12. usually 1 to 6, more usually 1
`to 4 carbon atoms;
`the two ur’s are the same or different. normally being
`the same, except when linking to ct. and are hydrogen.
`a non-onto-carbonyl functionality or one of the life may
`be a non-oxo-carbonyl linking functionality;
`L is a bond or divalent radical, usually an organic
`radical. of at least one carbon atom and not more than
`20. usually not more than 16, more usually not more
`than 10 carbon atoms, normally having an aliphatic or
`aromatic hydrocarbon chain. or combination thereof.
`wherein the aliphatic chain is usually of from about '2 to
`6 carbon atoms and the aromatic chain is of from about
`6 to 12, usually 6 to 10 carbon atoms; L normally has
`from 0 to 4. when aromatic. usually I to 4. more usually
`2 to 4 substituents. wherein the substituents may be
`halo. particularly chloro; non-oxo-carbonyl;
`thio,
`in-
`cluding inert sulfur acids, esters and amides; amino.
`particularly tert-amino or arnido; and oxy, wherein the
`
`

`
`4,351,760
`
`3
`substituents are normally of from 0 to 4 carbon atoms,
`there being at least two carbon atoms between heteroat-
`oms bonded to saturated carbon atoms;
`ct is an organic compound, a member of a specific
`binding pair. either a ligand or receptor;
`B is I, when at is covalently bonded to Ill or Ill’, and is
`otherwise 0; the covalent bond normally involves an
`amide. methylene sec-amino, ether, thioether or am
`link;
`up is a group terminating in a heteroatom containing
`functionality when not bonded to 11, wherein the tenni-
`nal heteroatorn containing functionality may be bonded
`directly to a carbon atom of L or through an oligomcr
`of from 1 to 4 units. each unit of l to 4, usually 2 to 4
`carbon atoms. which units are amino acids,
`al-
`kyleneamino. or alkyleneoxy groups; the terminal func-
`tionality is normally oxo, including oxo-carbonyl and
`non-oxo-carbonyl; amino; oxy; thio; or active halogen;
`particularly non—oxo-carbonyl; and
`1: is one when ,8 is 0 and is otherwise on the average
`at least one and not more than the molecular weight of
`ct divided by 500, usually divided by l,000.
`Desirably, there are from 2 to 6 chloro substituents on
`the fluorescent group (in the brackets), bonded at other
`than the 1,8-positions of the xanthenone. Also, the 4.5-
`positions may be unsubstituted or one or both, usually
`both, substituted with bromo, chloro, or allcyl of from I
`to 6, usually 1 to 3 carbon atoms.
`The lluorescer compound or conjugate with the or-
`ganic compound (u) may be linked, covalently or non-
`covalently to a support. The conjugate may be bound
`either through the lluorescer or organic compound.
`The support will be described in greater detail subse-
`quently.
`For the most part, the compounds of this invention
`having a 9-phenyl will have the following formula:
`
`
`
`wherein:
`R is an aliphatic group of from 1 to 3, usually I to 6,
`more usually I to 4, and preferably 1 to 3 carbon atoms,
`which may be substituted or unsubstituted, aliphatically
`saturated or unsaturated, particularly alkyl or carbony-
`alkyl of from I to 6, usually 1 to 4 carbon atoms;
`Z is carboxy;
`W is a bond or divalent radical having from 0 to 16.
`either 0 or usually I to 16 carbon atoms, more usually 1
`to 8 carbon atoms and from 0 to It], usually 2 to 8 het-
`eroatoms, which are chalcogen (oxygen and sulfur) or
`nitrogen, wherein chalcogen is present bonded solely to
`carbon (may or em) and nitrogen is present bonded
`-solely to carbon and hydrogen (amino and amido); car-
`bon is normally aromatic or aliphatic, particularly free
`of aliphatic unsaturation. having from 0 to 2 sites of
`ethylenic unsaturation; W is conveniently a monomer
`or oligomer of units of from 1 to 4 carbon atoms e.g.
`alkylene, aminoacid. oxyalkylene, aminoalkylene, etc.;
`
`4
`Y may be taken together with A to form an active
`functionality capable of forming a covalent bond with a
`heterofunctionality. such as amino, hydroxy, mercapto;
`that is with those functioualities present on A, when A
`is not taken together with ‘i’; such heterofunctionality
`as, oxo- and non-oxo-carbonyl, oxy, thio, amino, active
`halo, active olefin, inorganic acyl group e.g. sulfonyl.
`etc. or acts as a linking functionality. being either meth-
`ylene or heteroatom containing;
`A, when not taken together with Y. is a member of a
`specific binding pair, which is ligand or receptor,
`wherein the ligand may be haptenic or antigenic, nor-
`mally being of from about 125 molecular weight to an
`indefinite upper limit, although for the most part. most
`ligands will be under 10 million molecular weight, more
`usually under 2 million molecular weight, with varying
`ranges depending upon the nature of the ligand or re-
`ceptor;
`m will be 0 to 3, more usually 0 to 2; and
`n will be I when Y and A are taken together and will
`otherwise be on the average 1 to the molecular weight
`of A divided by 500, more usually divided by 1,000, and
`more frequently divided by 2,000, wherein with specific
`binding pair members over 600,000 molecular weight A
`will normally be not greater than A divided by 5,000. In
`addition, there will usually be at least two chloro sub-
`stituents bonded on any ofthe available positions where
`no specific atom is indicated. Also, the 4,5-positions
`may be substituted as described previously. Further-
`more, either the conjugate or the fluorescer precursor
`may be bonded to a support of at least about 10,000
`molecular weight and up to an indefinite molecular
`weight.
`A preferred group of compounds will for the most
`part have the following formula:
`
`HO
`
` DR‘
`
`W’Y’
`
`TD
`
`Ar
`
`wherein:
`
`R’ is alkylene of from 1 to 6, usually 1 to 4, and prefer-
`ably I to 3 carbon atoms;
`D is hydrogen or earboay; ‘
`Z‘ is carboxy;
`m‘ is 0 to 3, usually 0 to 2;
`Y’ may be taken together with A’ to form an active
`functionality which may be non-oxo-carbonyl, includ-
`ing the sulfur analog thereof, arnino bonded to at least
`one hydrogen atom, mercapto, active ethylene. usually
`having an oz-carbonyl, halomethylcarbonyl, wherein
`halo is of atomic number 17 to 53, sulfonyl, or the like;
`when not taken together with A’, Y’ will be a linking
`functionality, either methylene or a heteroatorn contain-
`ing linking functionality, usually being an amide, ester.
`ether or azo link;
`W’ is a bond or linking group of from 1 to 16, usually
`I to 12, and preferably 1 to 3 atoms other than hydro-
`gen, which are carbon, nitrogen. oxygen or sulfur, pref-
`erably carhon, nitrogen and oxygen. there being from 0
`
`10
`
`15
`
`20
`
`30
`
`35
`
`45
`
`SS
`
`65
`
`

`
`5
`to 8 carbon atoms and 0 to B heteroatorns. with the
`number of carbon atoms and heteroatoms being at least
`1. wherein nitrogen will be bonded solely to hydrogen
`and carbon, and will be either amino or amido. oxygen
`and sulfur will be bonded solely to carbon as city (thio)
`or oxo (thiono) and carbon is normally aliphatic and
`usually free of aliphatic tmsaturation. generally having
`from 0 to I site of ethylenic unsaturation; W‘ may be
`alkylene or allcenylene of from 1 to 8. usually 1 to 4
`carbon atoms. oxoallcylene or oxoalltenylene of from I
`to 8, usually I to 4 carbon atoms. imino (NH), N-formyl
`amino acid or N-forniyl poly(amino acid) e.g. glycine or
`polyglycine. there being from about 1 to 4 amino acids,
`with the terminal carboxy being Y'A’, or the like;
`11' is I when Y‘ and A‘ are taken together and other-
`wise is on the average at least I to the molecular weight
`of A‘ divided by 500, usually divided by 1,000, more
`usually divided by 2.000. and when A’ is over 500.000
`molecular weight. more usually divided by 5.000:
`there generally being not more than 5 carboxyl
`groups. usually not more than 4 carbonyl groups in
`total, and there being from 0 to 6 chloro groups. prefer-
`ably 2 to 5 chloro groups bonded to available carbon
`atoms; and
`A’ is a member of a specific binding pair. a ligand or
`receptor, wherein the ligand may be haptenic or anti-
`genic. and haptenic ligands will include compounds of
`interest such as drugs. hormones. pollutants. com-
`pounds of interest in processing. agricultural chemicals,
`metabolites. and the like;
`antigens will primarily be proteins, polysaccharides
`or nucleic acids, individually or in combination with
`each other or other materials. such as in cells. viruses.
`phage, or the like. The haptens will normally be from
`about 125 to 2,000. more usually to 1.000 molecular
`weight, while the antigens will normally be from about
`2.000, more usually 5,000 molecular weight up to an
`indefinite molecular weight, usually not exceeding 10
`million, more usually not exceeding 2 million.
`The 4.5-positions are preferably unsubstituted or
`chloro-substituted.
`In addition, the above conjugate may be bonded to a
`support. Various supports may be employed, both solu-
`ble or insoluble. swellable or nonswellable. by aqueous
`or organic solvents. naturally occurring or synthetic.
`organic or inorganic, porous or nonporous. or the like.
`Various polymeric materials include vinyl polymers
`and copolymers, polysaccharides. silicones, glass, car-
`bon particles. such as graphite or charcoal. metals or
`metal compounds. po1y{amino acids), nucleic acids or
`the like.
`For the most part. the fluorescent compounds of the
`subject invention employed for conjugation will have
`the following formula:
`
`
`
`wherein:
`
`5
`
`ID
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`SD
`
`55
`
`65
`
`4.351360
`
`6
`R‘ is alkylene of from 1 to 6. usually 1 to 4. preferably
`1 to 2 carbon atoms;
`D1 is hydrogen or carboxy. preferably hydrogen;
`Z‘ is carboxy;
`E" is hydrogen, alkyl of from 1 to 6, usually 1 to 3
`carbon atoms, or chloro;
`E1 is chloro;
`W1 is a bond or linking group of from I to 12, usually
`1 to 3 atoms other than hydrogen. and generally I to 3.
`usually 1 to 6 atoms in the chain wherein the atoms are
`carbon. nitrogen, oxygen and sulfur, particularly car-
`bon. nitrogen and oxygen. wherein the carbon is ali-
`phatic, the nitrogen is present as amido or amino, partic-
`ularly amino bonded solely to carbon, and oxygen and
`sulfur are bonded solely to carbon and are may or onto or
`the sulfur analogs thereof;
`W1 will generally be aliphatic, being saturated or
`unsaturated. normally saturated, having from 0 to 1 site
`of ethylenic unsaturation. alkylene or alkenylene of
`from 1
`to 3. usually 1
`to 4 carbon atoms. N-formyl
`amino acid or N-forrnyl poly(amino acid], where the
`terminal carboxy is derived from YlA'. amino. mer-
`capto. or the like;
`YIAI are taken together to form a functionality for
`linking. wherein ‘(M1 are bonded solely to carbon or
`nitrogen. with the proviso that when Y! and A‘ are
`bonded to nitrogen, YlA1 are carbonyl. including the
`nitrogen and sulfur analogs thereof and can be doubly
`bonded to nitrogen;
`YIAI can be non-oxo-carbonyl, haloacetyl. halogen
`of atomic no. 9 to 53. particularly chloro or bromo.
`maleimido. mercapto, amino. or inorganic acyl. having
`phosphorous or sulfur as the central atom;
`in‘ is 0 to 3. usually 0 to 2. there usually being not
`more than a total of 5 carboxyl groups in the molecule.
`usually not more than a total of 4 carboxyl groups. and
`preferably not more than about 2 carboxyl groups,
`other than YIAI;
`x is 0 to 4, preferably 2 to 4, there generally being not
`more than a total of 6 chloro groups in the molecule,
`usually not more than a total of 4 chloro groups.
`wherein it plus ml is not greater than 4.
`For the most part, the compositions of this invention
`when bonded to ligand or support will have the follow-
`ing formula:
`
`'
`
` 1:2
`
`wherein:
`
`E5‘ is hydrogen or chloro;
`E3 is chloro;
`Z3 is carboxy;
`R3 is alkylene of from 1 to 6. usually I to 3, preferably
`1 to 2 carbon atoms;
`D3 is hydrogen or carboxy, preferably hydrogen;
`W3 is a bond or linking chain, when a linking chain
`being of from I to 12, usually of from 1 to 10, and pref-'
`
`

`
`7
`erably of from about 1 to 8 atoms other than hydrogen.
`having from about 1 to I0, usually from about 1 to B.
`and preferably from about I to 6 atoms in the chain or
`spacer arm, wherein the atoms are carbon, oxygen,
`nitrogen and sulfur, particularly carbon. oxygen and 5
`nitrogen in the spacer arm_. wherein oxygen and sulfur
`are bonded solely to carbon, as city or oxo, and nitrogen
`is bonded solely to carbon and hydrogen, namely amino
`and arnido. wherein heteroatorns bonded to saturated
`carbon atoms are separated by at
`least two carbon
`atoms;
`W3 is particularly alkylene. carboxamidoalkylene,
`wherein alkylene is of from about I to 2 carbon atoms-
`(—C0NHC1.g—),,, wherein a is in the range of from
`about 1 to 4, usually I to 3;
`thiocarbamyl,
`Y3 is non-oxo-carbonyl, carbamyl,
`methylene, amino, or thio, particularly a functionality
`having a non-oxo-carbonyl group or sulfur analog
`thereof;
`X2 is 0 to 4;
`In3 is 0 to 3, preferably 1 to 2;
`n3 is 1 to the molecular weight of A1 divided by 500,
`usually divided by 1,000, more usually divided by 2,000,
`wherein when A3 is a ligand of between about 125 to
`2,000 molecular weight, n3 will generally be of from
`about 1 to 20, when A2 is a ligand of from about 2,000
`to 600,000 molecular weight, n1 will generally be in the
`range of about I to 100, more usually in the range of
`about 2 to St}; and
`A3 is a ligand of at least about 125 molecular weight 30
`and may be l0 million or more molecular weight, which
`is haptenic or antigenic, wherein haptens are from about
`125 to 2,000 molecular weight and antigens will gener-
`ally range from about 5,000 to 10 million molecular
`weight, more usually from about 5.000 to 2 million 35
`molecular weight and frequently from about 5,000 to
`600,000 molecular weight, the ligand being a member of
`a specific binding pair, which comprises a compound
`having at least 1 determinant or epitopic site and a re-
`ceptor which is capable of recognizing the determinant
`site or A1 is a receptor of from about 10,000 to 1 million
`molecular weight.
`Finally, in some instances it may be desirable to have
`the fluorescent compound or the conjugate of the fluo-
`rescent compound with ligand, bonded to a support.
`where the linkage may be derived from either the fluo-
`rescent compound or the ligand, normally the ligand. In
`this situation, the linking group may be any convenient
`functionality which is present on the fluorescent com-
`pound or the ligand or a functionality which may be
`introduced, particularly on the ligand. These composi-
`tions will for the most part have the following formula.
`where the symbols are derived from the previous for-
`mula for the conjugate for the most part:
`
`10
`
`I5
`
`20
`
`25
`
`45
`
`50
`
`SS
`
`4,351,760
`
`8
`all of the symbols have been defined previously. ex-
`cept for:
`113 which is at least 1 and up to the molecular weight
`of A3 divided by 500, usually 1000, more usually 1,500,
`with the proviso that when :1 is 0, n3 is l;
`C] which is 0 or 1;
`p which is at least 1 and of up to the molecular weight
`of the support divided by 500, more usually the molecu-
`lar weight of the support divided by 1,000, wherein
`when the molecular weight of the support exceeds
`500,000, p will normally be not greater than the molecu-
`lar weight of the support divided by 5,000, more usually
`divided by 10,000; and
`Support intends a macromolecular support of at least
`about 10,000 molecular weight, which may be naturally
`occurring or synthetic, having a plurality of functionali-
`ties for linking e.g. carboxy, hydroxy. or amino, usually
`being a polymer, such as a polysaceliaride or an addi-
`tion polymer; the support being bonded to the conju-
`gate by any convenient functionality remaining on A2
`or the conjugate in the brackets. the particular manner
`of linking not being a significant aspect of the subject
`invention. For example, if A3 is a poly(amino acid),
`carboxylic groups on the support can be used for amide
`formation or maleirnicle groups may be introduced and
`linked to mercapto groups.
`Quite obviously, the compounds of the subject inven-
`tion can be modified so as not to be within the above
`formulas. 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.
`Turning now to a consideration of the individual
`components of the subject compositions, the fluorescein
`derivatives will be considered first. The following is a
`list of illustrative fluorescein derivatives coming within
`the scope of the subject invention.
`TABLE I
`
`2,7-din1ethyl~4,S-dichloro-9-(2',4’,5'-tricarboxyphenyl)-
`6-hydroxy-3-xanthen-3-one
`2,7-diethy]-4,S-dichloro-9-(2’,4'.5'-tricarboxy-3’,6’-
`dichlorophenyl)-6-hydroxy-31-I-xanthen-Bone
`2,?-diheityl-9-(2',4’,5‘-tricarboxyphenyl)-6-hydroxy-3H-
`xanthen-3-one
`2,T-dimethyl-4,5-dichlorc-9—(2'-carboxy-4'-isothi-
`ccyanato-3',5'-dichlorophenyl)-6-hydroxy-3l-l-xan-
`then-3-one
`Z,7-dimethyl-9-(2’-carboxy-4'-isocyanato-3’,S’,6’-tri-
`chloropheuyl)-6-h ydroxy-3 I-I—xanthen-3-one
`2,7-dimethyl-9-(4'-carboxy-5’-carboxylphenyl)glycyl-
`glycylglycine an1ide-6-hydroxy-3H-xanthen-3-one
`2,7-di(carboxymethyl)-9-(4',5’,-dicarboxy-2',3',6'-t1'i-
`chlorophenyl)-6-h yd roxy-3 H-xanthen-3-one
`2,7-di(carboxypropy1)-4,5-dichloro-9-(3 ',4'-dicarboJty-
`phenyl)-6-hydroxy-31-I-xanthen-3-one
`2,?-diethyl-9-(2’-carbony--if-arnino-3',5‘-dichloro-
`phenyl)-6-hydroxy-3H-xanthen-3-one
`2,?-dimethyl-9-(2'-carboxy-4’-mercaptophenyl)-6-
`hydroxy-31-l-xanthen-3-one
`2,?-dimethyl-9-(2'-carbony-4'-carbonymethylphenyl)-6-
`hydroxy-3H-Iulnthen-3-one
`
`65
`
`wherein:
`
`

`
`9
`2.7rdimethyl-9-(2’-carboxy-4'-(4”-carboxybutyl)-
`phenyl)-6-hydroxy-3H-xanthen—3-one
`2,7-dimethyl-4,5¥dicl1loro-9-(2’,4‘-dicarboxy-5'-(carbox-
`arnidornethylene)pl1enyl)-6-hydroxyg3H-xanthen-
`3-one
`2,'}'—dimethyl-4,5-dichloro-9(3'-carboxypropyl)-6-
`hydroxy-3H-xanthen-3-one.
`As indicated previously, the fluorescein derivatives
`of the subject invention will be conjugated with ligands
`and/or supports, The following is a description of the
`applicable ligands.
`Analyte
`The ligand analytes of this invention are character-
`ized by being monoepitopic or polyepitopic. The
`polyepitopic
`ligand
`analytes will
`normally
`be
`poIy(amino acids) i.e. polypeptides and proteins, poly-
`saccharides, nucleic acids, and combinations thereof.
`Such combinations of assemblages include bacteria,
`viruses, chromosomes. genes, mitochondria. nuclei, cell
`membranes, and the like.
`.
`For the most part, the polyepitopic ligand analyt
`employed in the subject invention will have a molecular
`weight of at least about 5,000, more usually at least
`about 10,000. In the po1y(amino acid) category,
`the
`poly(amino acids) of interest will generally be from about
`5,000 to 5,000,000 molecular weight, more usually from
`about 20,000 to 1,000,000 molecular weight; among the
`hormones of interest.‘ the molecular weights will usually
`range from about 5,000 to 60,000 molecular weight.
`The wide variety of proteins may be considered as to
`the family ‘of proteins having ‘similar structural features.
`proteins having particular biological functions, proteins
`related to specific microorganisms, particularly disease
`causing microorganisms, etc.
`_
`The following are classes of proteins related by struc-
`ture:
`
`protarnines
`histones
`albumins
`globulins
`scleroproteins
`phosphoproteins
`rnucoproteins
`chromoproteins
`lipoproteins
`nucleoproteins
`glycoproteins
`proteoglycans
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are
`important clinically and include:
`Prealbumin
`Albumin
`ct]-Lipoprotein
`a1-Acid glycoprotein
`oi-Antitrypsin
`a1-Glycoprotein
`Transcortin
`4.63-Postalbumin
`Tryptophan-poor
`ct]-glycoprotein
`cc1x—Glycoprotein
`Thyroxin-binding globulin
`Inter-a-trypsin-inhibitor
`Gc-globulin
`(Gc 1-1)
`(Gc 2-1)
`
`4,351,760
`
`10
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`S5
`
`65
`
`(Ge 2-2)
`Haptoglobin
`(HP 1-1}
`(HP 1-1)
`(HP 2-2)
`Ceruloplasmin
`Cholinesterase
`:13-Lipoprotein(s)
`Myoglobin
`C-Reactive Protein
`0:1-Macroglobulin
`oz-I-IS-glycoprotein
`Zn-:1;-glycoprotein
`a2—Neuramino-glycoprotein
`Erythropoietin
`,8-lipoprotein
`Transfer:-in
`Hemopexin
`Fibrinogen
`Plasminogen
`B2-glycoprotein I
`B;-glycoprotein I1
`Immunoglobulin G
`(IgG) or 7G-globulin
`Mol. formula:
`')'21]2'0r 72%:
`Irnmunoglobulin A (IgA)
`or 'yA-globulin
`Mol. formula:
`
`(a2n2)" or (a2?L2)"
`Immunoglobulin M
`(Igli/I) or -yM-globulin
`Mo]. formula:
`
`(#2n2)5 or (n~2?t2)-"
`Immunoglobulin D(IgD)
`or 'yD-Globulin ('yD)
`Mol. formula:
`(52112) 0|‘ 5212)
`Irnmunoglobulin E (IgE)
`or 'yE—Globu]in (-yE)
`Mo]. formula:
`(62112) or (ezhzl
`Free 11 and A light chains
`Complement factors:
`C' 1
`C'1q
`C’ lr
`C’ 15
`C'2
`C'3
`31A
`0.213
`C'4
`C'5
`C'6
`C"?
`C'3
`C'9
`Important blood clotting factors include:
`
`BLOOD CLOTTING FACTORS
`
`International designation
`Name
`I
`'
`Fibrinogen
`[I
`Prothrombin
`Ila
`Throrrnbin
`Ill
`Tissue thromboplastin
`V and V1
`Prouccelerirt. accelerator
`globulin
`
`

`
`4,351,760
`
`12
`-continued
`
`
`
`Species of Microorganisms
`Hrucella nbortus
`Haemaplttlu: luflnettzuc
`Haemapltflut pertussis
`Trqeonemt: teflcrl
`Veillonella
`
`Erysipelothrts
`Litterta monaggytqgenet
`Chremobaclerium
`
`Mjtcobactcrlum tuberculosis
`
`Klebsfclln ttemgmes
`Klebefello eltxictre
`Salmanelln maltose
`
`Hemosensitin Found in
`Crude extract
`Polysaccharide
`Crude
`Polysacchnride
`Lijaopolysam
`cltaride
`Polysnccharide
`Folgtsnccharide
`Lipopolysac-
`charidc
`Saline ettltthcl of‘
`90% phenol
`extracted
`rnycobacleria
`and polysac-
`ohnlide
`fraction of
`cells and
`‘tuberculin
`Polyseocharide
`I-‘olysaocharide
`Lipopolysao
`chnride.
`Polysaeeluuide
`Polyseceltaride
`
`11
`-continued
`BLOOD Cl.JDTT'ING FACTORS
`International designation
`Name
`VII
`Prtnconvertin
`VIII
`Antihetnopl-tilic globulin
`(AHG)
`Christmas factor.
`plasma thromheplestin
`component (FTC)
`Stuart-Prower factor,
`antoprolhronihin I'll
`Plasma thrornboplastirl
`antecedent (PTA)
`Hngemenn factor
`Fibrin-stabilizing factor
`
`IX
`
`X
`
`XI
`
`XII
`XIII
`
`Important protein honnones include:
`Peptide and Protein Hormones
`Parathyroid hormone
`(parathromone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`
`Erythropoietin
`Melanotropin
`(melanocyte-stimulating hormone; intermedin)
`Somatotropin
`(growth hormone)
`Cortiootropin
`(adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing honnone
`(interstJ'tial_ cell-stimulating hormone)
`Luteomammotropic hormone
`(luteotropin, prolnctin)
`Gonadotropin
`(chorionic gonndotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`Peptide Hormones from the Neumhypophysis
`Ottytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF. Son1atotropin—RF,
`GRF. FSH-RF. PIF. MIF
`Other polymeric materials of interest are mucopo]y-
`saccharides and polysaccharides.
`Illustrative antigenic polysaccharides derived from
`microorganisms are as follows:
`
` Species of ‘Mhzroergunisms Kemesensitin Found in
`
`Streptao9cI:u.rpjqge1-tes
`Polgrseocharido
`Dloleoecrus pneumonia:
`Polymccil Iride
`Neinrerio ment‘trgt‘t't'a't‘.s
`Polyseecltaride
`Neiuwrio gertorrlu-tie
`Polyseccheride
`Cot)-neltacterlum diphtheria:
`Poly-secclmide
`Actinaboclllus J-m't.i'lct'.'
`Crude extract
`Acrlnabacfllur whlremotf
`Fmnctiella tulure.-uir
`
`Ptt:tet.trellu' pesttir
`Porteurella pt-sttk
`Pastetmt-Ha tnnlrot-t'dn
`
`Lipopelyseo
`eharide
`Pulysaccilaride
`
`Polyseccheride
`(kpeular antigen
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`S0
`
`S5
`
`65
`
`Salmonella typht'-m:m'ttM.'
`Salmonella derby
`Salmonella pullortttn
`Sltlgelln tlysentertee
`Sltlgella flexnerl
`Crude. Poly-
`Sltlgt.-Ila rennet
`sacizhnlide
`Crude extract
`Rickettsiee
`Polyseccharicle
`Chndldu .ttlbt‘mn.s
`
`Emamaetu ln'.s-tnlytlm Crud: entree:
`
`Polysxtcchm-ide
`
`The miomorganis- which are assayed may be in-
`tact. lysed. ground or otherwise fragmented, and the
`resulting composition or portion, e.g. by extraction.
`assayed. Microorganisms of interest include:
`Corynebacteria
`Cotjvnebacterium dtptherlae
`Pneumococci
`
`Dtlolacacwus pneuntonfae
`Streptococci
`Streptococcus pyagertes
`Sta-eprocacctts ealivants
`Staphylococci
`Staphylocacctts ttureus
`Staphylococcus albus
`Neisseriae
`Net'.n-aria menlnglttdtit
`Net'.t.9ert'a go.-tarrfteae
`
`§E1.E1.":.;"=!'=_'.E_'i_l_'=1.'I4=_
`fl"clterfcltI’u' colt"
`Aerulmcter fl'£\l'lJgEl'lfl
`Klchrfelln pneuntmtluc
`Salmonella rjgaltnru
`Salmonella cltolemesuir
`Salmonella I)tp.lu'tm-tfl'um
`Sltlgello ulyrenrenite
`Slttgelln sclttm'tz:':'
`Sllt]gc\llo omblnamrdn
`Sliujgcllo flexneri
`stttgetta boydlt‘
`Sltlgellu Senttel
` .
`Proteus wlguris
`Pmtett.r tnlrubflir
`humus Morgan!"
` fl uemgtnom
`Alcalt1genex_fi:ecnlt.t
`l"t'brt‘tJ rltalerue
`Heraoghllus-Bordetella gong
`
`The collform
`bacteria
`
`The Salmoncllne
`
`The Shigclltse
`
`Proteus species
`
`

`
`4,35 I,'?60
`
`14
`
`Hemopmfusl.-zflumzae.
`
`pasteurelhe
`
`H. Hum-y.-‘
`H. hemophilia
`H’ ,,,w,,m
`H. pamrnfiuenxae
`
`Bordmmw pertussis
`
`I3
`Rrckerrsfa Isursugamushf
`cominucd
`Rickerrsla burnerif
`R‘-ckensm gm-mafia
`I
`.
`I
`.
`.
`.
`.
`Chlamydia _(unclass|fiable Iparasues beaten: /vtra )
`5 F Chlamycha agents (narmng uncertam)
`ungi
`Crypraoaccus neoformans
`Blestomyr.-es dermatfdis
`’é;;.::+:::::;::,«::::::::'""'
`_
`_
`_
`_
`Pfl'fflCf|'CClO'fi_0l:dES brasilfenm
`C""d'd; “ b"""":‘
`_
`Aspergi usfure gems
`_
`I
`Mucar carymbrfirr {fibsrdm carymbyfimr)
`
`
`
`Rmppu, mm
`Rhranpu: mum
`R~’"'H-‘PHI Hi":-'1'-WM
`
`)
`
`Phyeemyeetes
`
`Fasten relic rulareusis
`Bwcenae
`Bmcefia mefirensis
`Bmceya, charm.
`Bmcelia suis
`Aerobic Spore-forming Bacilli
`Bacflius anfkracis
`Bacflfus 3lln‘5l‘l'h's
`Bacillus megarerium
`Bacillus cereus
`Anaerobic Spore-forming Bacilli
`Clam-idium baruffnum
`Closrrfdfum return’
`Cfostrfdium pedringens
`Closlridfum rwvyf
`Clostridium sepricum
`Clostridfum histabrflcum
`Clostridfum terrfum
`Clastridfum btfennenrans
`Clasrrldium spamgenes
`Mycobacteria
`Mycebacrerfum tuberculosis hamlnis
`Mycobacrerium bavis
`Mycabacrefium avfum
`Mycabacterfum Iepme
`Mycobactefium pararuberculosrls
`Actinoruycetes [fungus-like bacteria)
`Actinomyces israelif
`Actirtomyces bowie
`Actinomyces aaeslundfi
`Nocardfa anerofdes
`Nocardfa bmsfffensis
`
`
`The Spiroeheles
`.S_a.'n'Hum minus
`Trepomema peflfdum
`Trep