United States Patent
`
`[193
`
`Rowley et al.
`
`[1 1]
`
`[45]
`
`4,229,722
`
`Sep. 2, 1980
`
`[54]
`
`METHOD FOR CONJUGATING TO
`POLYAMINO COMPOUNDS EMPLOYING
`I-IALOACYL GROUPS AND COMPOSITIONS
`PREPARED THEREBY
`
`Primary Exarm'ner—Lionel M. Shapiro
`Attorney, Agent, or Ffrm~—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Inventors:
`
`I75]
`
`Gerald L. Ruwiey, San Jose; Danton
`Lenng, Campbell; Prithiphal Singh,
`Santa Clara, all of Calif.
`
`[731
`
`[211
`
`[231
`
`[51]
`I52]
`
`[53]
`
`E55]
`
`Syva Company, Palo Alto, Calif.
`Assignee:
`Appl. No; 876,772
`Filed:
`Feb. 10, 1978
`
`Int. Cl} ............................................. .. Cl2N 9/96
`U.S. 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 ................. .. 195/63, 68, DIG. 11;
`260/112 R; 424/12; 435/7, 17?’, 174, 138
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4.05'r,'n4
`4,100,263
`4,144,131
`
`Rubenstein ........................... .. 195/63
`l/ I9'i'8
`Scherr ........ ..
`T./19'!l:’a
`
`3/ 1979 Richardson .......................... .. 195/68
`
`Methods and compositions are provided for conjugat-
`ing a wide variety of compounds, particularly polyfunc-
`tional compounds, having a niercapto group, either
`naturally present or synthetically introduced, to a poly-
`amino compound, particularly a polypeptide (including
`proteins). The method employs a haloalkylcarbonyl
`compound, which is conjugated to one or more of the
`amino groups under mild acylating conditions. This is
`followed by combining the acylated 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 irntnunoassays, 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 polyamino compound.
`
`16 Claims, No Drawings
`
`Mylan V. Genentech
`Mylan v. Genentech
`IPR2016-00710
`Genentech Exhibit 2055
`Genentech Exhibit 2055
`
`
`IPR2016-00710
`
`

`
`1.
`
`4,220,722
`
`METHOD FOR CONJUGATING TO POLYAMINO
`COMPOUNDS EMPLOYING I-IALOACYL
`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 poiyfunctional com-
`pound for conjugating to a polyamino compound will
`frequently require the introduction of protective 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 difficult 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 anaiyte 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 ofadvantages. 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
`
`10
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`2
`provide for great efficiencies in synthesizing and subse-
`quent formulation.
`DESCRIPTION OF THE PRIOR ART
`
`Kato, et al, Eur. J. Biochem. 62, "285 (1976), 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 mercapto
`group to add to the double bond of the maleimide. See.
`also, Lee and Kenny, Clinical Chem. 2l,967(l975).
`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
`polyamino 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 mercapto
`compound is then added to the halo or pseudohalo
`containing polyamino compound for substitution of the
`halo groups to provide a thioether linked conjugate of
`the mercapto 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 immunoassays, 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 ct-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 <1-halo or a-pseudohaloaikylcarbonyl
`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 Inercaptan 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 immunoassays.
`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 mercaptan reagents,
`the first reagent being added in a sufficient amount to
`react with all or substantially all of the more reactive
`halogen. In this manner, the mercaptan 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 polyarnino 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 pseudohalo 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-
`oms, and may have as many as 20 heteroatoms, more
`usually having from about 2 to 12 heteroatoms, and
`preferably from about 2 to 8 heteroatoms. The heteroat-
`orns 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 onto 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:
`
`if’
`ii
`XCH-2C((Alk(D}m(C}m)m(Z)p
`
`wherein:
`X—C1,Br,CH3SO3 (mesylate), preferably Br;
`Y and Y'—0, NH, 5, preferably 0;
`A—NH, 0, preferably NH;
`D—cl-lain of from 1 to 9, usually 1 to 6 atoms in the
`chain, having a total number of atoms other than hydro-
`gen of from 1 to 12, usually 1 to 10, preferably 1 to 6,
`which may be-C, 0, N and 5. usually C, O and N,
`wherein: C) is present as once or ether, particularly non-
`oxo carbonyl; N is present as amido or bonded solely to
`carbon and may be present as terminal nitrogen doubly
`bonded to (CY3) 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-
`
`l0
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`4,220,722
`
`4
`rated and may be straight or branched chain, preferably
`straight chain;
`Z - UV or 0C0-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
`rn is zero, or Y1 is NH and m is 1, alkyl of from 1 to 6
`carbon atoms.
`k, m and p—zero or I, wherein p is zero when D and
`(CYI) form an isothioeyanate group
`The preferred halo compounds of this invention will
`have the following formula:
`
`Y3
`Y2
`II
`II
`DrC.‘H’;CNHl'J'CZ'
`
`wherein:
`Y3 and Y3—O, NI-I, preferably 0
`Di—a1kylene of from 1 to 8, usually 1 "to 4 carbon
`atoms
`
`Z'—-the same as Z, usually OH or N-oxy succinimide
`Illustrative compounds include
`N-bromoacetyl glycine, N-bromoacetyl valine, N-
`bromoacetyl 4-aminobutyric acid, N-brotnoacetyl 3-
`aminopropionic acid, p-chloroacetylbenzoic acid, p-
`bromoacetylphenylacetic
`acid. N-bromoacetyl
`4-
`aminocrotonic acid,
`their p-nitrophenyl esters,
`their
`N-succinimidyl esters, p-chloroacetylphenyl isothiocy-
`anate, and rnethyl N-bromoacetyl glycinimidate.
`The next group of compounds to be considered, are
`the polyamino functionalized compounds, which are
`primarily polypeptides and proteins, but may also in-
`clude polyglucosamines 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(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. 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 10,000-to 300,000 mo-
`lecular weight. Immunoglobulins and portions thereof
`e.g. Fab fragments and Bence-Jones proteins, will gen-
`erally range from about 23,000 to 1,000,000, with the
`immunoglobulins generally ranging from l50,000 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
`histones
`albumins
`globulins
`scleroproteins
`Phosphoproteins
`mucoproteins
`chromoproteins
`lipoproteins
`
`
`
`

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`4,220,722
`
`(12 D
`C'4
`C'5
`C’6
`C7
`C3
`C9
`Important blood ciotting factors include;
`
`'
`
`BLOOD CLOTTING FACTORS
`
` International designation Name
`I
`Fibrinogen
`H
`Prothrornbin
`Ha
`Thrombin
`Ill
`Tissue thromboplastin
`V and VI
`Proaocelerin. accelerator
`globuiin
`Proconvertin
`Autihernophilic globulin (AHG)
`Christmas factor.
`plasma thrcrrnboplastin
`component (PTC)
`Stuart-Prower factor.
`autoprcthrombin III
`Plasma thrornboplastin
`antecedent (PTA)
`Hagemaun factor
`XII
`
`XIII Fibrin-stabilizing factor
`
`X
`
`XI
`
`VII
`VIII
`IX
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`15
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`20
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`30
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`35
`
`nucleoproteins
`glycoproteius
`unclassified proteins, e.g. somatotropin, prolactin,
`insulin, pepsin
`A number of proteins found in the human plasma are 5
`important clinically and include:
`Prealburnin
`Albumin
`a1~Lipoprotein
`at-Acid glycoprotein
`at-Antitrypsin
`a|Glycoprotein
`Transcortin
`4.68-Postalbumin
`Tryptophan-poor
`at-glycoprotein
`aux-Glycoprotein
`Thyroxin-binding globulin
`Inter-ct-trypsin-inhibitor
`Gc-globulin
`(Gc 1-l)
`(Ge 2-!)
`(Ge 2-2)
`Haptoglobin
`(Hp 1-1)
`(HP 2-1}
`(HP 2~2)
`Ceruloplasmin
`Cholinesterase
`Q2-LipopI‘otein(S)
`oz;-Macroglobulin
`ct:-HS-glyooprotein
`Zn-az-glycoprotein
`ag»Neuramino-glycoprotein
`Erythropoietin
`B-lipoprotein
`Transferrin
`Hemopexin
`Fibrinogen
`Plasminogen
`.32-glycoprotein I
`82-glycoprotein II
`Irnmunoglobulin G
`(IgG) or 11G-globulin
`Mol. formula:
`‘YIK2 Dr ?2?t2
`Immuuoglobulin A (IgA) or -yA-globulin
`Mol. fonnula:
`(<12K2)" Or (t127k2)"
`Immunoglobtllin M
`(IgM) or 7M-globulin
`Mol. formula:
`(tL2I<2)5 01' (tt27\2)5
`Irnmunoglobulin D(IgD) or 'yD-Globulin (~yD)
`Mo]. formula:
`(figrcz) or (52?L2}
`Imrnunoglobulin E (IgE) or 7E-Globulin (YE)
`Moi. formula:
`(an) 0r (€212)
`Free K and 7 light chains
`Complement factors:
`C'l
`C’lq
`C’lr
`C’ls
`C'2
`C'3
`BIA
`
`Important protein hormones include:
`Peptide and Protein I-Ionnones
`Parathyroid hormone (parathromone)
`Thyrocalcitonin
`Insulin
`Glucagon
`Relaxin
`Erythropoietin
`Melanotropin (melanocyte—stimula.ting hormone; in~
`termedin)
`-
`Somatotropin (growth hormone
`Corticotropin (adrenocorticotropic hormone)
`Thyrotropin
`Follicle-stimulating hormone
`Luteinizing hormone (interstitial cell-stimulating hor-
`mone}
`Luteomammotropic hormone (luteotropin, prolactin)
`Gonadotropin (chorionic gonadotropin)
`Tissue Hormones
`Secretin
`Gastrin
`Angiotensin I and II
`Bradykinin
`Human placental lactogen
`Peptide Hormones from the Neurohypophysis
`Oxytocin
`Vasopressin
`Releasing factors (RF)
`CRF, LRF, TRF, Sorrtatotropin-RF, G-RF, 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
`Corynelaacterium diptheriae
`Pneurnococci
`Diplococcus pneumoniae
`Streptococci
`Streptococcus pyogenes
`Streptococcus salivarus
`
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`4,220,722
`
`The coliform bacteria
`
`10
`
`Staphylacocci
`Staphylococcus aureus
`Staphylococcus albus
`Neisseriae
`Neisseria meningitidis
`Neisseria gonorrheae
`
`Enlcrobncteriaciae
`Escherichia mu’
`)>
`.-ierobacrer as-rogcmss
`Kfebsfeffa pnet.-rnoniae
`Salmonella 9-phase
`Safmonefla ckofemetui: }
`Safmanefle typhfmurium
`Shigefla dysemeriae
`Shigeffa sciamirzfi
`Sitigeffa ambfnotarda
`Shigeifa flexmrri
`S.‘n'gel'£u baydfr
`Shfgeifa Sonnet‘
`Other enteric bacilli
`Proteus vu fgaris
`Proteus mi'rabHf$
`Proteus mwgani
`Pseudomoa-ms aemgfnom
`Afcafigeuesfaecafis
`Vibnb cholera?
` '25
`
`Trepcmema pallidum
`Treponema pertenue
`Treponema carateurn
`Borrelia recurrentis
`Leptospira icterohemorrhagiae
`Leptospira canicola
`Spirilium minus
`Streptobacillus moniliformis
`Mycoplasrnas
`Mycoplasma pneumoniae
`Other pathogens
`Listeria monacytogenes
`Erysipelothrix rhusiopathiae
`Streptobacillus moniliformis
`Donvania granulomatis
`Bartonella bacilliformis
`Rickettsiae (bacteria-like parasites)
`Rickettsia prowazekii
`Rickettsia mooseri
`Rickettsia rickettsii
`Rickettsia conori
`Rickettsia australis
`Rickettsia sibiricus
`Rickettsia akari
`Rickettsia tsutsugamushi
`Rickettsia burnetii
`Rickettsia quintana
`Chlamydia (unclassifiable parasites bacterial/viral)
`Chlamydia agents (naming uncertain)
`Fungi
`'
`Cryptococcus neoformans
`Blastomyces derrnatidis
`Histoplasma capsulaturn
`Coccidioicles irnmitis
`Paraeoccidioides brasiliensis
`Candida albicans
`
`Aspergillus fumigatus
`Mucor corymbifer (Absidia coryrribifera)
`._
`
`Phycomycétes
`Riuiaopus arrilizus
`Ritizopus aryzae
`)
`Rhfzopu: rrigrfmns
`
`Sporotrichum schenkii
`Fonsecaea pedrosoi
`Fonsecaea compacta
`Fonsecaea dermatitidis
`Cladosporium carrionii
`Phialophcra verrucosa
`Aspergillus nidulans
`Madurella mycetomi
`Madurella grisea
`Allescheria boydii
`Phialosphora jeanselrnei
`Microsporum gypseum
`Trichophyton mentagrophytes
`Keratinomyces ajelloi
`Microsporum csnis
`Trichophyton rubrurn
`Microsporum andouini
`Viruses
`Adenoviruses
`Herpes viruses
`Herpes simplex
`Varicella (Chicken pox)
`Herpes Zoster (Shingles)
`Virus B
`
`‘I112: Saimcnellae
`
`The Shigellae
`
`1s_
`
`20
`
`Proteus species
`
`J
`
`Hemophilus-Bordetella group
`Hernopltilus influenzae,
`H. ducreyi
`H. hemophilus
`H. aegypticus
`H. paraiufluenzae
`Bordetella pertussis
`Pasteurellae
`Pasteurella pestis
`Pasteurella tulareusis
`Bruceilae
`Brucella melitensis
`Brucella abortus
`Brucella suis
`Aerobic Spore-forming Bacilli
`Bacillus anthracis
`Bacillus subtilis
`Bacillus megateriurn
`Bacillus cereus
`Anaerobic Spore-forming Bacilli
`Clcstridium botulinum
`Clostridium tetani
`Clostridium perfringens
`Clostridium novyi
`Clostridium septicum
`Clostridium histolyticum
`Clostridium tertium
`Clostridiurn biferrnentans
`Clostridium sporogenes
`Mycc-bacteria
`Mycobacterium tuberculosis hominis
`Mycobacterium bovis
`Mycobacterium avium
`Mycobacterium leprae
`Mycobacteriurn paratuberculosis
`Actinomycetes (fungus-like bacteria) _
`Actinomyces israelii
`Actinomyces bovis
`Actinomyces naeslundii
`Nocardia asteroides
`Nocardia brasiliensis
`The Spirochetes
`
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`
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`

`
`4,220,722
`
`Cytornegalovirus
`Pox Viruses
`Variola (smallpox)
`Vaccinia
`Poxvirus bovis
`Par-avaccinia
`Molluscum contagiosum
`Picornaviruses
`Poliovirus
`Coxsaclcievirus
`Echoviruses
`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 Eucephaiitis Virus
`Western Equine Encephalitis Virus
`Sinclbis Virus
`Chikugunya Virus
`Semliki Forest Virus
`Mayora Virus
`St. Louis Encephalitis Virus
`California Encephalitis Virus
`Colorado Tick Fever Virus
`Yellow Fever Virus
`Dengue Virus
`Reoviruses
`Reovirus Types I-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. Oxiclorecluctases
`1.] Acting on the CH—0H group of donors
`1.1.] With NAD or NADP as acceptor
`1. alcohol dehydrogenase
`6. glycerol dehydrogenase
`26. glyoxylate reductase
`27. L~lactate dehydrogenase
`37. malate dehydrogenase
`49. glucose 6-phosphate dehydrogenase
`1?. mannitol 1-phosphate dehydrogenase
`1.1.2 With cytochrome as an acceptor
`3. L-lactate dehydrogenase
`1.1.3 With 02 as acceptor
`4. glucose oxidase
`9. galactose oxidase
`1.2 Acting on the CH-NH; group of donors
`1.4.3 With 0; as acceptor
`2. L—amino 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
`
`E0
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`15
`
`2|]
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`25
`
`30
`
`35
`
`45
`
`S0
`
`55
`
`65
`
`10
`1.10.3 With 02 as acceptor
`1. polyphertol oxidase
`3. ascorbate oxidase
`1.11 Acting on H102 as acceptor
`1.11.1
`6. catalase
`7. peroxidase
`3. I-lydrolases
`3.1 Acting on ester bonds
`3.1.1 Carboxylic ester hydrolases
`'}'. cholinesterase
`3.1.3 Phosphoric monoester hydrolases
`1. alkaline phosphatase
`3.1.4 Phosphoric diester hydrolases
`3. phospholipase C
`3.2 Acting on glycosyl compounds
`3.2.] Glycoside hydrolases
`I. o:—amylase
`4. cellulase
`1?. lysozyme
`23. 13-galactosidase
`27. amyloglucosidase
`31. ,8—g1ucu:-onidase
`3.4 Acting on peptide bonds
`3.4.2 Peptidyl-amino acid hydrolase
`1. carboxypeptidase A
`3.4.4 Peptidyl-peptide hydrolase
`5. o.-chymotrypsin
`10. papain
`3.5 Acting on C-N bonds other than peptide bonds
`3.5.1 In linear amides
`5. urease
`3.6 Acting on acid anhydride 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 Carbon-oxygen lyases
`4.2.1 Hydrolases
`1. carbonic anhydrase
`4.3 Carbomnitrogen lyases
`4.3.1 Ammonia Iyases
`3. histidase
`
`Of particular interest are the dehydrogenases, illus-
`trated by malate dehydrogenase and g1ucose-6-phos-
`pliate clehydrogenase, the hydrolases, such as ,B-galac-
`tosidase and lysozyme, and peroxidases.
`In the next group of compounds are the rnercaptan
`compounds, which may have a mercaptan group natu-
`rally or may have a mercaptan 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 of inter-
`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 rnereaptan group then
`introduced synthetically to provide the unique site of
`conjugation.
`Similar considerations are involved with the poly-
`amino functionalized compound. Where the polyamino
`functionalized compound has one or more active mer-
`
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`
`11
`capto groups, these may be deactivated prior to intro-
`duction of the cvltaloalkyl 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 the molecule of
`interest, which functionalities may react with the disul-
`fide compound, it will frequently be necessary to pro-
`tect
`the other functionalities with removable groups
`prior to conjugation of the disulllde 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 a mercaptan.
`For the most part, the mercapto compounds and their
`precursors will have the following formula:
`
`EM (Q),-_,TSU
`
`wherein:
`E-—the 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 0,
`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 0x0, 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 all-tylene,
`there being not more than one site ofaliphatic unsatura-
`tion, either branched or straight chained, preferably
`straight chained, particularly methylene or polymethyl-
`ene (CH2);,, where b is from I to 4;
`S-—-sulfur
`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 mercaptan group, M
`and T are taken together to form a single bond, a 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 irnmunoassays. Of greater interest,
`are those enzyme conjugates which are employed in a
`homogeneous enzyme immunoassays as described in
`U.S. Pat. No. 3,817,837. In these conjugates, when anti-
`body binds to the mercapto 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
`
`4,220,722
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`12
`protein, desirably a relatively large number of hnptenic
`compounds being conjugated to the polypeptide or
`protein.
`A third possibility is the conjugation of 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 thio 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
`coenzymes,
`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, dextromethorphan,
`their derivatives and metabolites; cocaine alkaloids,
`which includes cocaine and be.-izoyl 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, gestrogens, 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 stilbestro].
`The next group of drugs is lactams having from 5 to
`6 annular members. which include the barbiturates, e.g.
`phenobarbital and secobarbital, diphenylhydantoin, and
`their metabolites.
`The next group of drugs is aminoalkylbenzenes, with
`alkyl of from 2 to 3 carbon atoms, which includes the
`amphetamines, catecholamines. which includes ephed-
`rine. L-dopa, rnethyldopa, epinephrine, nurceine, pa-
`paverine, their metabolites and derivatives.
`The next group of drugs if benzheterocyclics which
`include benzothiadiazides, oxazepam, chlcrpromazine,
`tegretol, imipraminc, their derivatives and metabolites.
`the heterocyclic rings being azepines, diazepines.
`thiadiazines, and phenothiazines.
`The next group of drugs is purines, which includes
`theophylline, caffeine.
`theobromine,
`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
`unsaturation.
`
`ID
`
`15
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`20
`
`25
`
`30
`
`35
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`40
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`45
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`S0
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`S5
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`60
`
`65
`
`
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`

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`4,220,722
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`13
`The next group of drugs is antibiotics, which includes
`macrolides, aminoglycosides,
`,8-lactams, etc. such as
`penicillins, ethambutol, isoniazid, vancomycin, mettle-
`namine madelate. chloromycetin, actinomycetin. tetra-
`cyclines,
`terramycin, cephalosporins, erythromycin,
`rifampin, clindamycin the aminoglycosides such as
`streptomycin. gcntamicin, tobramycin. an-iikacin, kanav
`mycin. neomycin; nalidixic acid, nitrofurantoin, colisti-
`methate. lincomycin, amphotericin B. flucytosine, their
`metabolites and derivatives.
`The next group of drugs is the nucleosides and nucle-
`otides, which include ATP, NAD, FMN, adenosine.
`guanosine, thymidine, 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 haloalkylamines,
`tolamol, sotalol, guane—
`thide, meprobamate, serotonin, merperidine, chlorcy-
`clazine, chlorpheniramine. amitriptyline, nortriptyline,
`lidocaine. procaineamide, acetylprocaineamide. pro-
`panolol. griseofulvin, butyrophenones, antihistamines.
`methotrexate. aminopterin. anticholinergic drugs, such
`as atropine. their metabolites and derivatives.
`The next group of compounds is amino acids and
`small peptides which include polyiodothyronines e.g.
`thyroxine, and triiodothyronine. oxytocin, ACTH, an-
`giotensin. endorphin, met- and leu-enkephalin.
`their
`metabolites and derivatives.
`Metabolites related to diseased states include sper-
`mine, galactose. phenylpyruvic acid, and porphyrin
`type 1.
`Among pesticides of interest are polyhalogenated
`biphenyls, phosphate esters,
`thiophosphates.
`carba—
`rnates, polyhalogenated sulfenamides, their metabolities
`and derivatives.
`Electron transfer labels will for the most part be
`either metal complexes or aromatic compounds.
`Compounds of interest are ligands. particularly hap-
`tenic ligands. bonded to poly(amino acids), particularly
`antigen and enzymes. These compounds will have for
`the most part the following formula:
`
`11'
`in’
`EM(QlaTSCH2C((A)k(D}m(C)mlm ;- FAA
`
`wherein all of the symbols have been defined previously
`except:
`PAA—poly(amino acid); and
`j—a number on the av