United States Patent
`
`[193
`
`Rowley et al.
`
`[1 ll
`
`[45]
`
`4,220,722
`
`Sen. 2, 1980
`
`[54]
`
`METHOD FOR CONJUGATING TO
`POLYAMINO COMPOUNDS EMPLOYING
`HALOACYI. GROUPS AND COMPOSITIONS
`PREPARED THIEREBY
`
`Primary Exami'r:er—Lionel M. Shapiro
`Attorney, Agent, or Ft'rm—Bertra.rn I. Rowland
`
`[57]
`
`ABSTRACT
`
`Inventors:
`
`I75]
`
`Gerald L. Rnwley, San Jose; Danton
`Leung, Campbell; Prithiphal Singh,
`Santa Clara, all of Calif.
`
`[731
`
`[21]
`
`[221
`
`[51]
`I52]
`
`[53]
`
`[56]
`
`Assign.-ae:
`
`Syva Company. Palo Alto, Calif.
`
`Appl. No; 876.772
`Filed:
`Feb. 10, 1978
`
`Int. C13 ............................................. .. C12N 9/95
`us. Cl.
`435/188; 435/177;
`435/7; 260/112 R; 424/12; 260/112 B;
`260/112.5 R; 260/112.7
`Field of Search ................. .. 195x63. 63, DIG. 11;
`260/112 R; 424/12; 435/7, 177. 174, 133
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4-,tl6'l',T'F4
`4.100.263
`4.l-14,131
`
`195/63
`Rubenstein
`l/19TH
`.... .. 424-/I2
`Scherr
`T/19'.-'3
`
`3/l9'?9 Richardson .......................... "195/68
`
`Methods and compositions are provided for conjugat-
`ing 3 wide variety of compounds, particularly polyfnnc-
`tional compounds. having :1 mercapto group, either
`naturally present or synthetically introduced, to a poly-
`amino compound, particularly a polypeptide (including
`proteins). The method employs £1 haloalkylcarbonyl
`compound, which is conjugated to one or more of the
`amino groups under mild acylating conditions. This is
`followed by combining the acyiated polyamino com-
`pound with a mercapto containing compound, whereby
`the halogen is displaced by the sulfur of the mercapto
`group to form a stable thioether linkage. The resulting
`conjugates, depending on the compounds involved, can
`find uses in immunoassays. as hapten-antigen conjugates
`for the production of antibodies, and as ligand analog
`enzyme conjugates for use as reagents in controlling the
`distribution of substitution of a mercapto compound to
`a polynmino compound.
`
`14 Claims, No Drawings
`
`SANOFI V. GENENTECH
`SANOFI v. GENE(cid:49)TECH(cid:3)
`IPR2015-01624
`IPR2015-01624
`EXHIBIT 2055
`EXHIBIT 2055
`
`
`

`
`1
`
`4,220,722
`
`METHOD FOR CONJUGATING TO POLYAMINO
`COMPOUNDS EMPLOYING IIALOACYL
`GROUPS AND COMPOSITIONS PREPARED
`' THEREBY
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`There is a continuously expanding interest in being
`able to conjugate, frequently selectively, a compound to
`another compound which is polyfunctional. Where
`both compounds are polyfunctional,
`the problem of
`conjugation is exacerbated, if one does not wish all of
`the functional groups to participate in the reaction.
`Also, the need to functionalize a polyfunctiona] com-
`pound for conjugating to a polyamino compound will
`frequently require the introduction ofprotective groups
`for alcohols and amines, so that the reactive functional-
`ity does not polymerize the compound to be conju-
`gated.
`One area of particular interest is the conjugation of a
`wide variety of haptens and antigens to polypeptides
`{including proteins), particularly where conjugation is
`to occur at available amino groups. In preparing anti-
`bodies for use in competitive protein binding assays.
`where the analyte of interest is haptenic. it is generally
`necessary to conjugate the hapten to an antigen, nor-
`mally a protein. Where the analyte has a plurality of
`functionalities which can react with the active function-
`ality to be used for conjugating to the polypeptide, it
`becomes necessary to introduce removable protective
`groups to prevent polymerization of the analyte- After
`conjugation, it is usually diflicult to efficiently remove
`the protective groups.
`Where the conjugate is to be used for the preparation
`of antibodies. the resulting antibodies not only recog-
`nize the analyte of interest, but the analyte having the
`protective groups. This may result in substantially re-
`ducing the specificity of the antibody composition for
`the analyte of interest.
`One class of competitive protein binding assays in-
`volves the use of enzymes as a label. It is necessary to
`conjugate the analyte of interest to the enzyme. It is
`desirable that certain reactive site positions on the en-
`zyme be preferentially conjugated as compared to other
`reactive site positions. A method which would provide
`the ability to discriminate to even a partial degree is
`desirable.
`In addition, to have an enzyme which has been modi-
`fied. whereby the same sites will be conjugated to ana-
`lytes. regardless of the particular analyte, can provide a
`number of advantages. For example. in one of the assays
`which employs an enzyme as a label, it is desirable that
`the enzyme retain a substantial proportion of its initial
`activity after conjugation. but when antibody or other
`receptor is bound to the analytes conjugated to the
`enzyme. the enzymatic activity is substantially reduced.
`The fewer the analytes necessary to conjugate to the
`antibody to obtain the desired degree of reduction is
`enzymatic activity upon the binding of antibody or
`other receptor to the conjugated analyte.
`the more
`sensitive will be the assay response.
`In addition. where a universal reagent can be em-
`ployed for conjugation, greatly increased experience
`can be obtained in the handling of the compounds. the
`reacting of the compounds, as well as the subsequent
`handling and treatment after conjugation. This can
`
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`2
`provide for great efficiencies in synthesizing and subse-
`quenr formulation.
`DESCRIPTION OF THE PRIOR ART
`
`Kato, et al. Eur. J. Biochem. 62, "285 ([976). discloses
`the use of maleic anhydride with a polyamino com-
`pound to provide one or more maleimide groups. fol-
`lowed by the addition of a compound with a mencapto
`group to add to the double bond of the maleimide. See.
`also, Lee and Kenny, Clinical Chem. 2I,967{l9'.-'5).
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for combin-
`ing a polyfunctional compound having a plurality of
`reactive primary and/or secondary amino functionali-
`ties with a second compound having a mercapto func-
`tionality, usually polyfunctional, having functionalities
`reactive to acyl groups e.g. hydroxylic and amino. The
`polyarnino compound is initially reacted with a linking
`compound having an active halogen or pseudohalogen
`and a non-oxo carbonyl functionality for reacting with
`at least one of the amino functionalities. The rnercapto
`compound is then added to the halo or pseudohalo
`containing polyarnino compound for substitution of the
`halo groups to provide a thioether linked conjugate of
`the rnercapto compound with the polyamino com-
`pound.
`The method finds particular use in the preparation of
`polypeptide and protein conjugates for preparing anti-
`gens. enzyme conjugates for immunoas-says, fluorescent
`labeling of polypeptides and proteins and the like. By
`employing the subject method, one can obtain a consis-
`tent pattern of substitution, the conjugation can be car-
`ried out under extremely mild conditions and some
`control of the positions of substitution can be achieved.
`
`DESCRIPTION OF THE‘. SPECIFIC
`EMBODIMENTS
`
`The method of the subject invention involves the
`labeling as a First step of a poly(amino acid) compound,
`normally a polypeptide or protein, with a compound
`having an ct-halo or a-pseudohaloalkylcarbonyl func-
`tionality and a non-oxo carbonyl functionality (includ-
`ing the nitrogen analog,
`imido. and sulfur analog,
`thiono), which may be the same or different from the
`carbonyl of the whale or orpseudohaloalkylcarbonyl
`Functionality. The reaction is carried out in a normally
`aqueous medium under mild pH conditions, generally at
`9.5 or below. so as to form an amide (including the
`nitrogen and sulfur analogs. imidine and thioamide re-
`spectively). The product may then be purified under
`conventional conditions and the halo or pseudohalo
`substituted by a Iuercaptan under mild conditions in an
`aqueous solution at moderate pH, normally basic pH.
`The product may then be worked up and isolated.
`The compounds prepared in accordance with this
`invention have many uses, for example haptens or anti-
`gens may be conjugated to labels, such as fluorescers
`and enzymes. and the resulting compounds employed in
`immunoassays for the determination of such haptens
`and antigens. In addition, haptens may be conjugated to
`antigens to be used for the production of antibodies,
`which may also serve as reagents in irnmunoassays.
`The subject method provides a means for preparing
`derivatives of polyamino compounds, which have a
`limited number of active sites. The distribution of these '
`active sites may be retained substantially constant. so
`that when conjugating haptens and antigens to the poly-
`
`
`
`

`
`3
`amino compounds, substitution will be relatively uni-
`form, regardless of the particular compound which is
`conjugated.
`In addition. it will normally be found that due to the
`position of the halo substituent on the polyamino com-
`pound, the halo compounds may have varying activi-
`ties. One can then distinguish between the varying ac-
`tivities. by employing two different meroaptan reagents.
`the first reagent being added in a suilicient amount to
`react with all or substantially all of the more reactive
`halogen. In this manner, the rnercaptan compound of
`interest may be directed either to the more or less reac-
`tive sites. Also. the subject method provides for syn-
`thetic convenience, for so far as the polyamino com-
`pound. the same compound may be repetitively pre-
`pared, regardless of the compound to which it is to be
`conjugated.
`
`MATERIALS
`
`The materials which are employed in the subject
`invention are the active halogen or pseudohalogen com-
`pound,
`the polyamino polyfunctional compound to
`which the halo or pseudohalo compound is conjugated.
`and the mercaptan which is employed for substitution
`on the halogen or pseudohalogen.
`The first compounds to be considered will be the halo
`or pseuclohalo compounds. These compounds will nor-
`mally be of from 2 to 20, more usually of from 2 to 16
`carbon atoms, and preferably of from about 2 to 12
`carbon atoms. Other than the halo or pseudohalo group.
`the compound will normally have at least two heteroat-
`orns. and may have as many as 20 heteroatoms, more
`usually having from about 2 to 12 heteroatoms, and
`preferably from about 2 to 8 heteroaton-is. The heteroat-
`oms will normally be oxygen, nitrogen and sulfur or any
`appropriate counterion for a charged species. Oxygen
`will normally be present as in nitro, oxo or ether (an
`ester includes oxo and ether oxygens); nitrogen will be
`present as in nitro, amido, or bonded solely to carbon,
`e. g. tertiary amine; and sulfur will be present as thiono
`or thioether. The compounds will of necessity include
`aliphatic groups. but may also include alicyclic, aro-
`matic. and heterocyclic groups.
`For the most part, the compounds used for conjuga-
`tion to the amino functionalized compounds will have
`the following formula:
`
`in"
`ii
`-xCH.‘.C((A iii Dlmlcl.-n)m(Z).p
`
`wherein:
`X—Cl,Br,CH3SO3 (mesylate). preferably Br;
`Y and Y1—0. NH, 5, preferably 0;
`A—--NH, 0, preferably NH;
`D—-chain of from I to 9, usually I to 6 atoms in the
`chain, having a total number of atoms other than hydro-
`gen of from I to 12, usually 1 to 10, preferably 1 to 6,
`which may be-C, 0. N and S. usually C. O and N,
`wherein: O is present as once or ether, particularly non-
`oxo carbonyl; N is present as arnido or bonded solely to
`carbon and may be present as terminal nitrogen doubly
`bonded to (CYI) where Y‘ is S to form isothiocyanate;
`and S is present as thiono or thioether; preferably hy-
`drocarbon to form a hydrocarbylene group which may
`be
`aliphatic.
`alicyclic.
`aromatic or
`combinations
`thereof, preferably aliphatic, which may be aliphatically
`saturated or unsaturated having from 0 to 1 site of unsat-
`uration i.e. ethylenic and acetylenic. preferably satu-
`
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`4,220,722
`
`4
`rated and may be straight or branched chain, preferably
`straight chain;
`Z - 0V or OCG)-alkyl, wherein alkyl is of from 1 to 6,
`usually 1 to 4 carbon atoms and V is hydrogen. p-
`nitrophenyl. N-oxy succinimide. or when Y is NH and
`m is zero, or Y‘ is NH and m is 1, alkyl of from 1 to 6
`carbon atoms.
`k, m and p—zero or 1. wherein p is zero when D and
`(CYI) form an isothiocyanate group
`The preferred halo compounds of this invention will
`have the following formula:
`
`1‘.I.l
`VI
`II
`II
`l3rCH1CNHD'CZi
`
`wherein:
`Y3 and Y3—0. NH. preferably 0
`Dl—alkylene of from 1 to 8. usually 1 ‘to 4 carbon
`atoms
`
`Zl—t_l1e_same as Z, usually OH or N-oxy snccinimide
`Illustrative compounds include
`N-bromoacetyl glycine, N-bromoacetyl valine, N-
`bromoacetyl 4-arninobutyric acid, N-bromoacetyl 3-
`atninopropionic acid, p-chloroacetylbenzoic acid, p-
`bromoacetylphenylacetic
`acid. N-bromoacetyl
`4-
`aminocrotonic acid,
`their p-nitrophenyl esters.
`their
`N-succinirnidyl esters, p-chloroaoetylphenyl isothiocy-
`anate, and methyl N-bromoacetyl glycinirnidate.
`The next group of compounds to be considered, are
`the polyamino functionalized compounds. which are
`primarily polypeptides and proteins, but may also in-
`clude polyglucosarnines and nucleic acids. These com-
`pounds may be included in combinations or assemblages
`which include bacteria, viruses, chromosomes, genes,
`mitochondria, nuclei. cell membranes and the like.
`_
`For the most part. the compounds will have a molec-
`ular weight of at least about 5,000, more usually at least
`about 10,000. In the poly(amino acid) category (in-
`cludes polypeptides and proteins),
`the poly(arnino
`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. In this category.
`hormones of interest will generally range‘ from about
`5,000 to 60.000 molecular weight. Enzymes of interest
`will generally range from about 10.00010 300,000 mo-
`lecular weight. Immunoglobulins and portions thereof
`e.g. Fab fragments and Bence-Iones proteins, will gen-
`erally range from about 23,000 to 1.000.000, with the
`immunoglobulins generally ranging from 150,000 to
`1,000,000.
`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:
`
`protamines
`histories
`albumins
`globulins
`scleroproteins
`Phosphoproteins.
`mucoproteins
`chromoproteins
`lipoproteins
`
`
`
`

`
`4,220,722
`
`at; D
`U4
`C5
`C6
`C’?
`C8
`C9
`Important blood clotting factors include:
`
`BLOOD CLCITTING FACTORS
`
` lnlemational designation Name
`I
`Fibrinogen
`II
`Pmthnombin
`Ila
`Thrornbin
`Ill
`Tissue thromboplastin
`V and Vi
`Proaccelerin. accelerator
`globulin
`Froconwsrtin
`Antihemophilic globulin (Al-IGJ
`Christmas faeiar.
`plasma thrornboplastin
`component (FTC)
`Stum-r~Pruwer factor,
`autnprothrombin III
`Plasma Ihromboplestin
`antecedent (PTA)
`Hagemann factor
`XII
`
`XIII Fibrin-stabilizing Factor
`
`VII
`VIII
`Ix
`
`X
`
`X1
`
`15
`
`20
`
`25
`
`30
`
`35
`
`nucleoproteins
`glycoproteins
`unclassified proteins, e.g. somatotropin, prolactin.
`insulin, pepsin
`A number of proteins found in the human plasma are 5
`important clinically and include:
`Prealbumin
`Albumin
`a;~Lipoprotein
`(11-Acid glycoprotein
`an-Antitrypsin
`a;Glycoprotein
`Transcortin
`4.65-Postalbumin
`Tryptophan-poor
`at-glycoprotein
`aux-Glycoprotein
`Thyroxin-binding globulin
`Inter-ct-trypsin-inhibitor
`Go-globulin
`(Gt: 1-!)
`(Go 2-!)
`(Go 2-2)
`I-Iaptoglobin
`(Hp 1-1)
`(HP 2-1)
`[Hp 2-2)
`Ceruloplasmin
`Cholinesterase
`rt;-I..ipoprotein(s)
`a1~Macroglobt11in
`:11-HS-glycoprotein
`Zn-oz-glycoprotein
`ct;-Neuraniino-glycoprotein
`Erythropoietln
`,3-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`B2-glycoprotein I
`B3-glycoprotein II
`Immunoglobulin G
`(IgG) or 'yG-globulin
`Mol. formula:
`'
`72K: 01“ ‘Jul:
`Irnmunoglobulin A (IgA) or 7A-globulin
`Mol. formula.-
`iazlczl” Dr (tI27l2)"
`Irnmunoglobulin M
`(IgM) or 7M-globulin
`Mol. formula:
`
`Important protein hormones include:
`Peptide and Protein Hormones
`Parathyroid hormone (parathromone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`Erythropoietin
`Melanotropin (melanocyte-stimulating hormone; in-
`termedin)
`Somatotropin (growth hormone)
`Corticotropin (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone (interstitial cell-stimulating hor-
`mone)
`Luteomammotropic hormone (luteotropin, proiactin)
`Gonadotropin (chorionic gonadotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogon
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF. Somatotropin-RF, GRF, FSH-
`RF, PIF. MIF
`The microorganisms which are assayed may be in-
`tact, lysed. ground or otherwise fragmented, and the
`60 resulting composition or portion, e.g. by extraction,
`assayed. Microorganisms of interest include:
`Corynebacteria
`Corynebacterium diptheriae
`Pneumococci
`Diplococcus pneumoniae
`Streptococci
`Streptococcus pyogenes
`Streptococcus salivarus
`
`45
`
`SD
`
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`
`(ltzlczls 01' (,u27\2)5
`Immuuoglohulin D(IgD) or 'yD-Globulin ('yD)
`Mol. formula:
`(52K2) or (5212)
`Immunoglobulin E (IgE) or 7E-Globulin (yE)
`Moi. formula:
`(£2Kz) 0? (E232)
`Free K and ‘)1 light chains
`Complement factors:
`C'l
`C’lq
`C1:
`C15
`C2
`C3
`l‘3IA
`
`
`
`

`
`'7
`
`4,220,722
`
`8
`
`Staphylococci
`Staphylococcus aureus
`Staphylococcus albus
`Neisseriae
`Neisseria meningitidis
`Neisseria gonorrheae
`
`Enterobacteriaciae
`Escfierichliz rah‘
`Aerobacrer at-rogeries
`Kiebsieifa pncuinanfae
`Sufmoru-Ha 9-phom
`Safmanefla choferaeruilr
`Salmonella urphimurium
`Slrfgefla dysenfcrine
`Shigeffa 5:.‘inH'£z.*i'
`Sllifgeffa ambinorarda
`Siiilgefla flexrm-1'
`Siiijgcffu be
`if
`SJ'iI;ge."."a Sonnei‘
`Cllher enlcric bacilli
`Pmrims rulgnnk
`Pmleus m|'m£!:'.’|':
`Proteus mnrgani
`Pseu damanm rieruynaaia
`Afcaffgeiier _,"im:~a!:'s
`Fibrin cfrafeme
` 2S
`
`The mliform bacteria
`
`The Salmcnellae
`
`The Shigcllae
`
`Protc I-IE species
`
`J»
`>
`
`J
`
`10
`
`I5
`
`20
`
`Treponema pallidum
`Treponema pertenue
`Treponema carat:-um
`Borrelia recurrcntis
`I___.cpto_spira icterohemorrhagiae
`Leptospira canicola
`Spirillum minus
`Streptobacillus moniliformis
`Mycoplasmas
`Mycoplasma pneumoniae
`Other paihogens
`Listeria monocytogencs
`Erysipelothrix rhusiopathiae
`Streptobacillus moniliformis
`Dorwania granulomatis
`Bartonella bacilliformis
`Rickettsine (bacteria-like parasites)
`Rickettsia prowazekii
`Rickettsia mooseri
`Rickettsia rickettsii
`Rickettsia conori
`Rickettsia australis
`Rickettsia sibiricus
`Rickettsia akari
`Rickettsia tsutsugamushi
`Rickettsia burnelii
`Rickettsia quintana
`Chlamydia (unclassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`Fungi
`'
`Cryptococcus neoformans
`Blastomyces dermatidis
`Histoplasma cnpsulatum
`Coccidioidcs immitis
`Paracoccidioides brasiliensis
`Candida albicans
`Aspergillus fumigatus
`Mucor corymbifer (Absidia corynibifera)
`_
`
`Phyoomycelcs
`Rhizupus arr.‘u‘m.r
`}
`RMzupusaryzm
`RMZflph‘S m'gri'r.:ms
`
`Sporotrichum schenkii
`Fonsecaea pedrosoi
`Fonsecaea cornpncta
`Fonsecaea dermatitidis
`Cludosporium carrionii
`Phialophora verrucosa
`Aspergillus nidulans
`Madurella rnycctomi
`Madurclla grisea
`Allescheria boydii
`Phialosphora jeanselmei
`Microsporurn gypseum
`Trichophyton mentagrophytes
`Keratinomyces ajelloi
`Microsporum cianis
`Trichophyton rubrurn
`Microsporurn andouini
`Viruses
`Adenoviruscs
`Herpes viruses
`Herpes simplex
`Varicella (Chicken pox)
`Herpes Zoster (Shingles)
`Virus B
`
`Hemophilus-Bordetclla group
`Hemophilus influenzae.
`H. ducreyi
`H. hemophilus
`H. aegypticus
`H. paraiufluenzae
`Bordetella pertussis
`Paste-urellae
`Pnsteurella peslis
`Pasteurella tulareusis
`Brucellae
`Brucella melitensis
`Brucella abortus
`Brucella suis
`Aerobic Spore-forming Bacilli
`Bacillus anthracis
`Bacillus subtilis
`Bacillus megaterium
`Bacillus cereus
`Anaerobic Spore-forming Bacilli
`Clastridium botulinum
`Clostridium tetani
`Clostridium perfringens
`Clostridium novyi
`Clostridium septicum
`Clostridium histolyticum
`Clostridium tertinm
`Clostridium bifennentans
`Clostridiurn sporogenes
`Mycobacteria
`Mycobacterium tuberculosis hominis
`Mycobacterium bovis
`Mycobacterium avium
`Mycobacterium leprae
`Mycobacterium paratuberculosis
`Actinornycetes (fungus-like bacteria)
`Actinomyces israelii
`Actinomyces bovis
`Actinornyces naeslundii
`Noe-ardia asteroicles
`Nocardia hrasiliensis
`The Spirochetes
`
`30
`
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`
`40
`
`45
`
`50
`
`55
`
`6]]
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`65
`
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`
`

`
`4-,220.?22
`
`Cytornegalovirus
`Pox Viruses
`Variola (smallpox)
`Vaccinia.
`Poxvirus bovis
`Par-avaccinia
`Molluscum contagiosum
`Picornaviruses
`Poliovirus
`Coxssckievirus.
`Echoviruscs
`Rhinoviruses
`Myxoviruses
`Influenza (A, B. and C)
`Parainfluenza (1-4)
`Mumps Virus
`Newcastle Disease Virus
`Measles Virus
`Rinderpest Virus
`Canine Distemper Virus
`Respiratory Syncytial Virus
`Rubella Virus
`Arboviruses
`Eastern Equine Encephalitis Virus
`Western Equine Eucephalitis Virus
`Sindbis Vims
`Chikugun ya Virus
`Semliki Forest Virus
`Mayors Virus
`St. Louis Encephalitis Virus
`California Encephalitis Virus
`Colorado Tick Fever Virus
`Yellow Fever Virus
`Dengue Virus
`Reoviruses
`Reovirus Types 1-3
`Hepatitis
`Hepatitis A Virus
`Hepatitis B Virus
`Tumor Viruses
`Rauscher Leukemia Virus
`Gross Virus
`Maloney Leukemia Virus
`Enzymes of interest are clasified in accordance with
`the I.U.B. classification as follows:
`1. Oxidoreductases
`1.1 Acting on the CH—UH group of donors
`|.l.l With NAD or NADP as acceptor
`1. alcohol dehydrogenase
`6. glycerol dehydrogenase
`26. glyoxylate reductase
`2?. L-lactate dehydrogenase
`37. malate dehydrogenase
`49. glucose 6-phosphate dehydrogenasc
`I7. mannitol 1-phosphate dehydrogenase
`1.1.2 With cytochrome as an acceptor
`3. L-lactate dehydrogenase
`1.1.3 With 03 as acceptor
`4. glucose oxidase
`9. galactose oxidase
`1.2 Acting on the CH-N1-13 group of donors
`1.4.3 With 01 as acceptor
`2. L—arnino acid oxidase
`3. D-amino acid oxidase
`1.6 Acting on reduced NAD or NADP as donor
`1.6.99 With other acceptors diaphorase
`1.10 Acting on diphenols and related substances as
`donors
`
`[0
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`I55
`
`Lu-J
`
`10
`1.10.3 With 02 as acceptor
`1. polyphenol oxidase
`3. sscorbate oniidase
`1.11 Acting on H20; as acceptor
`1.11.1
`6. catalase
`'3-'. peroxidase
`. Hydrolases
`3.1 Acting on ester bonds
`3.1.1 Carboxylic ester hydrolases
`'1'. cholinesterasc
`3.1.3 Phosphoric rnonoester hydrolase.-3
`1. alkaline phosphatase
`3.1.4 Phosphoric diester hydrolases
`3. phospholipase C
`3.2 Acting on glycosyl compounds
`3.2.1 Glycoside hydrolases
`1. rt-amylase
`4. celiulase
`17. lysozyme
`23. B-galactosidase
`27. am yloglucosidase
`31. ,8-glucuronidase
`3.4 Acting on peptide bonds
`3.4.2 Peptidyl-amino acid hydrolase
`1. carboxypeptidase A
`3.4.4 Peptidyl-peptide hydrolase
`S. o.-chymotrypsin
`ll]. papain
`15 Acting on C-N bonds other than peptide bonds
`3.5.1 In linear amides
`5. urease
`3.6 Acting on acid arfltydride bonds
`3.6.1 In phosphoryl—containing anhydrides
`1. inorganic pyrophosphatase
`4. Lyases
`4.1 Carbon-carbon lyases
`4.1.2 Aldehyde lyases
`7. aldolase
`4.2 Carbowoxygen lyases
`4.2.1 Hydrolases
`l. carbonic enhyclrase
`4.3 Carbonmitrogen Iyases
`4.3.1 Ammonia. lyases
`3. histidase
`Of particular interest are the dehydrogenases, illus-
`trated by malate dehydrogenase and g1ucose-6-phos-
`pirate dehydrogenase, the hydrolases, such as ;3-galac-
`tosidase and lysozyme, and peroxidases.
`In the next group of compounds are the ruercaptan
`compounds, which may have a mercaptan group natu-
`rally or may have a merctiptan group introduced. With
`a naturally occurring compound of interest having a
`plurality of mercaptan groups.
`it will frequently be
`necessary to deactivate such groups by functionalizing
`them with permanent or removable groups. Usually. the
`functionalization will be by the formation of a thioether.
`Therefore, when the compound of interest has a plural-
`ity of mercaptan groups present, the compound ofititer-
`est will be treated with a reagent which will react with
`the mercapto groups to prevent their subsequent reac-
`tion in the conjugation and a mercaptan group then
`introduced synthetically to provide the unique site of
`conjugation.
`Similar considerations are involved with the poly-
`atnino functionalized compound. Where the polyamino
`functionali-ted compound has one or more active mer-
`
`
`
`

`
`4,220, 722
`
`11
`capto groups, these may be deactivated prior to intro-
`duction ol' the o.—haloalkyl carbonyl compound.
`The compounds of interest will generally have either
`hydroxyl or amino functionalities or both as sites for
`conjugation to a disulfide linkage. The linking Function-
`ality will normally be an ester, amide or ether. Where a
`plurality of functionalities are present in die molecule of
`interest, which functionalitics may react with the disul-
`lidc compound, it will frequently be necessary to pro-
`tect
`the other functionalities with removable groups
`prior to conjugation of the disulftde containing com-
`pound. In some instances, a mercapto group will be
`naturally present, and this can serve as the site for link-
`ing without synthetic introduction of :1 mercaptan.
`For the most part. the mercapto compounds and their
`precursors will have the following formula:
`
`ENIIQLJTSU
`
`wherein:
`E——tl1.e compound of interest, which will be discussed
`in more detail subsequently, which may be modified by
`introduction of an hydroxyl or amino functionality, by
`protection of one or more reactive functionalities with
`protective groups, desirably removable, or in any other
`manner appropriate to the purpose of its intended use;
`M—0. NH
`Q—-C=W. wherein W is 0. NH or S. particulary O,
`T—a linking group having at least one carbon atom
`and not more than 9 atoms, other than hydrogen, usu-
`ally one to four atoms, which are carbon, oxygen. nitro-
`gen and Sulfur. preferably carbon, the terminal atoms
`being carbon atoms, any oxygen is present as oxy ether
`or oxo. particularly non-oxo carbonyl; nitrogen is pres-
`ent as amido or bonded solely to carbon and hydrogen
`e.g. amino. primary, secondary or tertiary; and sulfur is
`present as thiono or thioether; the number of heteroat-
`oms being in the range of zero to 4. usually zero to 2, T
`is preferably hydrocarbon, more preferably alkylene,
`there being not more than one site ofaliphatlc unsatura-
`tion, either branched or straight chained. preferably
`straight chained, particularly methylene or polymethyl-
`ene (CHz);., where b is from I to 4;
`S-——sulf'ur
`U—H, alkylthio of from one to four carbon atoms.
`particularly methyl, or an alkali metal cation of atomic
`number 3 to 19;
`a—zero or 1.
`Where E has a natural occurring meroaptan group. M
`and T are taken together to form a single bond. at is zero
`and U is hydrogen.
`Depending upon the purpose of the conjugate, the
`rnercaptan containing compound may vary widely. Of
`particular interest are conjugates to enzymes which are
`intended for use in imrnunoassays. Of greater interest,
`are those enzyme conjugates which are employed in a
`homogeneous enzyme immunoassays as described in
`US. Pat. No. 3.817.337. In these conjugates, when anti-
`body binds to the rnercapto compound conjugated to
`the enzyme. there is a substantial reduction in the enzy-
`matic activity. Therefore. it is quite advantageous once
`one has developed a particular distribution of active
`sites for a particular enzyme. that the mercapto com-
`pounds will bond substantially to the same sites, regard-
`less of the nature of the mercapto compound.
`Another use for the conjugates is for forming anti-
`genic products for haptens. so as to be able to produce
`antibodies.
`In this situation. any non-antigenic com-
`pound may be bonded to a convenient polypeptide or
`
`12
`protein. desirably a relatively large number of haptenic
`compounds being conjugated to the polypeptide or
`protein.
`A third possibility is the conjugation oi‘ a label to a
`polypeptide or protein for use in an immunoassay for
`determination of‘ the polypeptide or protein. Labels can
`take many forms. One class of labels is fluorescent com-
`pounds. Another class of labels is labels having enzy-
`matically labile bonds. In this instance, the labile bond
`may be bonded distant from the tliio ether linkage or
`may be part of the linkage between the compound of
`interest and the polypeptide or protein. Thus. the link
`between the compound of interest and the polypeptide
`or protein may be cleaved by an enzyme. These types of
`compounds
`include
`coenzymcs,
`fluorescent
`com-
`pounds, chemiluminescent compounds. chemical cata-
`lysts. electron transfer agents. dyes, and the like.
`The first group of compounds are monoepitopic li-
`gands. which are generally involved in assays for their
`determination. These include drugs which are used for
`therapeutic purposes, naturally occurring physiological
`compounds. metabolites, pesticides, pollutants, and the
`like.
`Included among drugs of interest are the alkaloids.
`Among the alkaloids are morphine alkaloids. which
`includes morphine, codeine. heroin, dextrornethorphan.
`their derivatives and metabolites; cocaine alkaloids.
`which includes cocaine and bcrlzoyl ecgonine,
`their
`derivatives are metabolites; ergot alkaloids. which in-
`cludes the diethylamide of lysergic acid; steroid alka-
`loids;
`iminazoyl alkaloids; quinazoline alkaloids;
`iso-
`quinoline alkaloids; quinoline alkaloids; which includes
`quinine and quinidine; diterpene alkaloids. their deriva-
`tives and metabolites.
`The next group of drugs includes steroids. which
`includes
`the estrogens, gestrogeus, androgens.
`an-
`drenocortical steroids, bile acids, cardiotonic glycosides
`and aglycones. which includes digoxin and digoxigenin,
`saponins and sapogenins. their derivatives and metabo-
`lites. Also included are the steroid mimetic substances,
`such as diethyl stilbestrol.
`The next group of drugs is lactams having from S to
`6 annular members. which include the barbiturates, e.g.
`phenobarbital and secobarbital. diphenylhydantoin, arid
`their metabolites.
`The neitt group of drugs is aminoall-tylbenzenes. with
`alkyl of from 2 to 3 carbon atoms. which includes the
`amphetamines, cateeholamines. which includes ephed-
`rine. L-dopa, methyldopa. epinephrine, narceinc. pa-
`paverine. their metabolites and derivatives.
`The next group of drugs if benzheterocyclics which
`include bcnzothiadiazides, oxazepam, chlorpromazine.
`tegretol, imiprarnine. their derivatives and metabolites,
`the hetcrocyclic rings being azepines. diazepines.
`thiadiazines, and phenothiazines.
`The next group of drugs is purines, which includes
`theophylline. caffeine. tlteobrontine.
`their metabolites
`and derivatives.
`
`The next group of drugs includes those derived from
`marijuana, which includes cannabinol and tetrahydro-
`cannabinol.
`The next group of drugs includes the vitamins such as
`A. B. C. D. E and K.
`The next group of drugs is prostaglandins. which
`differ by the degree and sites of hydroxylation and
`unsaturntion.
`
`l0
`
`l5
`
`20
`
`25
`
`30
`
`35
`
`4t]
`
`45
`
`St]
`
`55
`
`6!]
`
`
`
`

`
`4,220,722
`
`I4
`E1—an haptenic ligand of from about 125 to 2.000
`molecular weight. usually from about 125 to 300 molec-
`ular weight;
`to 4
`T1—a linking group of from 1 to 6, usually 1
`carbon atoms, usually aliphatic hydrocarbon having
`from 0 to l site of ethylcnic unsaturation and preferably
`saturated. either branched or straight chain, perferably
`straight e.g. (CH2)... where q is a number of from l to 4;
`Y3—the two ‘[73 are the same or different and are 0
`or NH, preferably 0;
`D1—-is a linking chain of from 1 to 9, usually 1 to 5
`and more usually l
`to 3 carbon atoms, preferably al-
`phutic hydrocarbon having from O to 1 site of ethylenic
`unsaturation. preferably saturated. either straight chain
`or branched, preferably straight chain e.g.
`(CH1),-_..
`where q is a number of from 1 to 4;
`j*—is on the average a number of at least one and not
`more than 2!]. usually from about 1 to 12. more usually
`from about 1 to B.
`Enzymes of particular interest are oxidoreductases
`e.g. dehydrogenases. and hydrolases.
`A narrower genus involves the conjugation of amino-
`glynoside antibiotics with enzymes. particularly ox-
`idoreductuses and hydrolases. many of which have been
`referred to earlier. These compounds will for the most
`part have the following formula (excluding chirality):
`
`13
`The next group of drugs is antibiotics, which includes
`macrolides. aminoglycosides. B-lactams. etc. such as
`penicillins. ethambutol. isoniazid, vancomycin. methe-
`namine madelate, chloromycetin. actinomycetin. tetra-
`cyelines.
`terramycin. cephalosporins. erythromycin.
`rifampin, clindamycin the aminoglycosides such as
`streptomycin. gcntarnicin, tobramycin. a.l']1ll(aCl.n., kam-
`rnycin. neomycin; nalidiitic acid. nitrofurantoin. enlisti-
`methale. lincornycin. amphotericin B. llucytosine, their
`metabolites and derivatives.
`The next group of drugs is the nucleosides and nucle-
`otides, which include ATP, NAB. FMN. adenosine.
`guanosine. thyrnidine, uridine and cytidine with their
`appropriate sugar and phosphate substituents.
`The next group of drugs is miscellaneous individual
`drugs which include methadone, phenoxybenzamine
`and related haloallrylamines.
`tolamol. sotalol. gunne-
`th