2
`
`TEACHING EDITORIAL
`
`Bioconjugate Chem. 1992, 3, 2-13
`
`A Brief Survey of Methods for Preparing Protein Conjugates with Dyes,
`Haptens, and Cross-Linking Reagents
`
`Michael Brinkley
`
`Molecular Probes, Inc., 4849 Pitchford Avenue, Eugene, Oregon 97402. Received July 24, 1991
`
`I. INTRODUCTION
`Modification of proteins, DNA, and other biopolymers
`by labeling them with reporter molecules has become a
`very powerful research tool in immunology, histochem-
`istry, and cell biology. A number of excellent reviews of
`this subject have been published (1-6). In addition, there
`are a growing number of commercial applications of these
`modified biomolecules, including clinical immunoassays,
`DNA hybridization tests, and gene fusion detection
`systems. In these techniques, a small molecule with special
`properties, such as fluorescence or binding specificity, is
`covalently bound to a protein, a DNA strand, or other
`biomolecule. Specific examples include fluorescent-
`labeled antibodies for detection and localization of cell-
`surface antigens, biotin-labeled single-stranded DNA
`probes for detection of DNA hybridization, and hapten-
`labeled proteins that, when introduced into a suitable host
`animal, generate hapten-specific antibodies.
`This review will focus on the experimental design and
`procedures for preparing protein conjugates with dyes,
`biotin, and haptens such as drugs and hormones. Methods
`for covalently linking two unlike biopolymers through the
`judicious choice of cross-linking reagents will also be
`discussed. The following specific topics will be addressed:
`(a) reactive groups of proteins that are available for
`modification, including their naturally occurring amino
`acids, and reactive groups introduced by chemical mod-
`ification, (b) reagents that can be used to couple molecules
`to these reactive sites, (c) experimental procedures for
`preparing conjugates, (d) purification and isolation of
`conjugates, and (e) techniques for determining the degree
`of labeling.
`
`11. GENERAL DISCUSSION OF METHODS
`A. Reactive Groups of Proteins. Proteins and pep-
`tides are amino acid polymers containing a number of
`reactive side chains. In addition to, or as an alternative
`to, these intrinsic reactive groups, specific reactive moieties
`can be introduced into the polymer chain by chemical
`modification. These groups, whether or not they are
`naturally a part of the protein or are artificially introduced,
`serve as "handles" for attaching a wide variety of molecules,
`including other proteins. The intrinsic reactive groups of
`proteins are described in the following section.
`(1) Amines (Lysines, a-Amino Groups). One of the most
`common reactive groups of proteins is the aliphatic eamine
`of the amino acid lysine. Lysines are usually present to
`some extent and are often quite abundant. For example,
`the protein bovine insulin contains only a single lysine
`amine, while avidin, a protein found in egg whites, contains
`36 lysines (7). Lysine amines are reasonably good nu-
`cleophiles above pH 8.0 (pKa = 9.18) (8) and therefore
`react easily and cleanly with a variety of reagents to form
`
`stable bonds (eq 1). Other reactive amines that are found
`
`m,t.i,,w, + RX -->
`
`Rot.*r-NnR + xn
`
`(1)
`
`in proteins are the a-mino groups of the N-terminal amino
`acids. The e-amino groups are less basic than lysines and
`are reactive at around pH 7.0. Sometimes they can be
`selectively modified in the presence of lysines. There is
`usually at least one a-amino acid in a protein, and in the
`case of proteins that have multiple peptide chains or several
`subunits, there can be more (one for each peptide chain
`or subunit). Bovine insulin has one N-terminal glycine
`residue and one N-terminal phenylalanine (9). There are
`proteins that do not possess free a-amino groups, such as
`cytochrome C and ovalbumin. In these molecules, the
`N-terminal amino group is N-acylated, and therefore not
`reactive toward the usual modification reagents. Since
`either N-terminal amines or lysines are almost always
`present in any given protein or peptide, and since they are
`easily reacted, the most commonly used method of protein
`modification is through these aliphatic amine groups.
`(2) Thiols (Cystine, Cysteine, Methionine). Another
`common reactive group in proteins is the thiol residue
`from the sulfur-containing amino acid cystine and ita
`reduction product cysteine (or half-cystine), which are
`counted together as one of the 20 amino acids. Cysteine
`contains a free thiol group, which is more nucleophilic
`than amines and is generally the most reactive functional
`group in a protein. It reacts with some of the same
`modification reagents as do the amines discussed in the
`previous section and in addition can react with reagents
`that are not very reactive toward amines. Thiols, unlike
`most amines, are reactive at neutral pH, and therefore
`they can be coupled to other molecules selectively in the
`presence of amines (eq 2). This selectivity makes the thiol
`NH*-Rot.h-sH + Rx ->
`NH,-Rot.irrsA + XH
`group the linker of choice for coupling two proteins
`together, since methods which only couple amines (e.g.,
`glutaraldehyde, dimethyl adipimidate coupling) can result
`in formation of homodimers, oligomers, and other un-
`wanted products (10). Since free sulfhydryl groups are
`relatively reactive, proteins with these groups often exist
`in their oxidized form as disulfide-linked oligomers or have
`internally bridged disulfide groups. Immunoglobulin M
`is an example of a disulfide-linked pentamer, while im-
`munoglobulin G is an example of a protein with internal
`disulfide bridges bonding the subunits together.
`In
`proteins such as this, reduction of the disulfide bonds with
`a reagent such as dithiothreitol (DTT) is required to
`generate the reactive free thiol (11). In addition to cys-
`tine and cysteine, some proteins also have the amino acid
`methionine, which contains sulfur in a thioether linkage.
`When cysteine is absent, methionine can sometimes react
`
`(2)
`
`0 1992 American Chemical Society
`
`IMMUNOGEN 2079, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Teaching Edfforial
`with thiol-reactive reagents such as iodoacetamides (12).
`However, selective modification of methionine is difficult
`to achieve and therefore is seldom used as a method of
`attaching small molecules to proteins.
`(3) Phenols (Tyrosine). The phenolic substituent of
`the amino acid tyrosine can react in two ways. The
`phenolic hydroxyl group can form esters and ether bonds,
`and the aromatic ring can undergo nitration or coupling
`reactions with reagents such as diazonium salts at the
`position adjacent to the hydroxyl group. There is con-
`siderable literature describing the reaction of tyrosyl
`residues with diazonium compounds (13). For example,
`a p-aminobenzoyl biocytin derivative has been diazotized
`and reacted with protein tyrosine groups (14). Modifi-
`cation of tyrosines has primarily been used in structural
`studies, rather than as ameans for attaching specific labels,
`since acetylation and nitration can give useful information
`concerning the participation of tyrosine in the binding
`properties of proteins. Often, the reactivity of tyrosines
`with amine-selective modification reagents to form un-
`stable carboxylic acid esters or sulfate esters is an unwanted
`side reaction resulting in conjugates that slowly hydrolyze
`during storage. Methods for preventing this problem are
`discussed in a later part of this teaching editorial (section
`V.B.l).
`( 4 ) Carboxylic Acids (Aspartic Acid, Glutamic Acid).
`Proteins contain carboxylic acid groups at the carboxy-
`terminal position and within the side chains of the di-
`carboxylic amino acids aspartic acid and glutamic acid.
`The low reactivity of carboxylic acids in water usually
`makes it difficult to use these groups to selectively modify
`proteins and other biopolymers. In the cases where this
`is done, the carboxylic acid group is usually converted to
`a reactive ester by use of a water-soluble carbodiimide
`
`0
`
`0
`II
`Rot- - II
`> RotA-CoX + RNHNH, -
`>
`ii
`
`Rotein- CNHNHR (3)
`
`and then reacted with a nucleophilic reagent such as an
`amine or a hydrazide (15,16). The amine reagent should
`be weakly basic in order to react specifically with the
`activated carboxylic acid in the presence of the other
`amines on the protein. This is because protein cross-
`linking can occur when the pH is raised to above 8.0, the
`range where the protein amines are partially unproto-
`nated and reactive. For this reason, hydrazides, which
`are weakly basic, are useful in coupling reactions with a
`carboxylic acid (17). This reaction can also be used
`effectively to modify the carboxy terminal group of small
`peptides.
`(5) Other Amino Acid Side Chains (Arginine, Histi-
`dine, Tryptophan). Chemical modification of other amino
`acid side chains in proteins has not been extensive,
`compared to the groups discussed above. The high pK,
`of the guanidine functional group of arginine (pK, = 12-
`13) necessitates more drastic reaction conditions than most
`proteins can survive. Arginine modification has been
`accomplished primarily with glyoxals and a-diketone
`reagents (18). Tryptophan modification requires harsh
`conditions and is seldom carried out except as a method
`of analysis in structural or activity studies. Histidines
`have been subjected to photooxidation (19) and reaction
`with iodoacetates (20).
`B. Protein Modification Reagents. This section will
`survey the extensive selection of reagents that are available
`
`Bioconjugate Chem., Vol. 3, No. 1, 1992 3
`
`for the purpose of protein modification. The fundamental
`principles for understanding how to use these reagents
`are (1) recognition of the reactive group(s) on the protein
`or peptide that can be modified and (2) knowledge of the
`type of chemical reactions these reactive groups will
`participate in and the nature of the chemical bonds that
`will result from these reactions.
`(1) Amine-Reactive Reagents. These reagents are those
`which will react primarily with lysines and the a-amino
`groups of proteins and peptides under both aqueous and
`nonaqueous conditions. Some amine-reactive reagents are
`more reactive, and therefore less selective, than others,
`and it will be necessary to understand this property in
`order to choose the best reagent for modification of a
`specific protein. The following amine-reactive reagents
`are available.
`(a) Reactive Esters (Formation of an Amide Bond).
`Reactive esters, especially N-hydroxysuccinimide (NHS)
`esters, are among the most commonly used reagents for
`modification of amine groups (21). These reagents have
`intermediate reactivity toward amines, with high selec-
`tivity toward aliphatic amines. Their reaction rate with
`aromatic amines, alcohols, phenols (tyrosine), and histi-
`dine is relatively low. Reaction of NHS esters with amines
`under nonaqueous conditions is facile, so they are useful
`for derivatization of small peptides and other low mo-
`lecular weight biomolecules. The optimum pH for reaction
`in aqueous systems is 8.0-9.0. The aliphatic amide
`products which are formed are very stable (eq 4). The
`
`0
`0
`II + H O - N b (4)
`> Rotein-NHCR
`
`P
`
`0
`
`0 )rl
`
`NHS esters are slowly hydrolyzed by water (221, but are
`stable to storage if kept well desiccated. Virtually any
`molecule that contains a carboxylic acid or that can be
`chemically modified to contain a carboxylic acid can be
`converted into its NHS ester (eq 51, making these reagents
`
`among the most powerful protein-modification reagents
`available. Newly developed NHS esters are available with
`sulfonate groups that have improved water solubility (23).
`A shortlist of reactive NHS ester derivatives of fluorescent
`probes, biotin, and other molecules is given in Table I.
`(b) Isothiocyanates (Formation of a Thiourea Bond).
`Isothiocyanates, like NHS esters, are amine-modification
`reagents of intermediate reactivity and form thiourea
`bonds with proteins and peptides (eq 6). They are
`
`II S
`Rot4nfdH,+ RN=C=S - > Protein-NHC-NHR
`
`(6)
`
`somewhat more stable in water than the NHS esters and
`react with protein amines in aqueous solution optimally
`at pH 9.0-9.5. Since this is a higher pH than the optimal
`pH for NHS esters (which undergo competing hydrolysis
`at pH 9.0-9.51, isothiocyanates may not be as suitable as
`NHS esters when modifying proteins that are sensitive to
`alkaline pH conditions. One of the most commonly used
`fluorescent derivatization reagents for proteins is fluo-
`rescein isothiocyanate (FITC). A number of other fluo-
`
`IMMUNOGEN 2079, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`4 Bioconjugate Chem., Vol. 3, No. 1, 1992
`Table 1. Succinimidyl Ester Probes
`probes
`
`succinimidyl fluorescein-5-(and -6-)carboxylate
`
`Brinkley
`
`structure
`
`function
`
`ref
`
`( .-pCooH
`
`fluorescent label
`
`75,76
`
`N - 0 - C
`
`f
`
`0
`
`+
`
`succinimidyl N,N,”,”-tetramethylrhodamine-5-
`(and -6-)carboxylate
`
`+o
`
`.-@coo”
`
`fluorescent label
`
`76
`
`succinimidyl7-amino-4-methylcoumarin-3-acetate
`
`N - 0 - C
`
`f
`
`0
`
`HzNq:H2
`;- 0 -?-$
`
`-
`
`fluorescent label
`
`77
`
`CH3
`
`0
`
`succinimidyl X-rhodamine-5-(and -6-)carboxylate
`
`fluorescent label
`
`75,78
`
`succinimidyl D-biotin
`
`0=C
`I
`0
`I
`
`0
`
`0
`
`°
`
`
`
`ligand, affinity label
`
`79
`
`radioiodination label
`
`80
`
`0
`
`rescent dyes (coumarins and rhodamines) have been
`coupled to proteins via their reactive isothiocyanates (24).
`(c) Aldehydes (Formation oflmine, Reduction to Alkyl-
`amine Bond). Aldehyde groups react under mild aqueous
`conditions with aliphatic and aromatic amines to form an
`intermediate known as a Schiff base (an imine), which can
`be selectively reduced by the mild reducing agent sodium
`cyanoborohydride to give a stable alkylamine bond (eq 7)
`(44,53). This method of amine modification is not used
`
`N.BH,CN
`R o t W H , + RCH=O - > Rotein-N=CHR ->
`Roteicl-NHCHP (7)
`in protein conjugations as frequently as the activated ester
`method, but when the molecule to be attached has an
`aldehyde group, or can be easily converted to an alde-
`
`hyde, the method is mild, simple, and very effective. Al-
`dehydes (glyoxals) can also react with protein arginine
`groups (25,26) and the nucleic acid base guanosine, making
`them of some use in nucleic acid modification (27).
`( d ) Sulfonyl Halides (Formation of a Sulfonamide
`Bond). Sulfonyl halides are highly reactive amine-
`modifying reagents. They are unstable in water, especially
`at the pH required for reaction with aliphatic amines, but
`they form extremely stable sulfonamide bonds which can
`survive even amino acid hydrolysis (eq 8). It is for this
`
`0
`0
`II
`I1
`> Rotsin-NH-S-R + HCI Roteicl-NH, + R-S-CI -
`
`II
`II
`0
`0
`reason that sulfonamide conjugates are useful for amine-
`terminus derivatization (Dansyl-Edman degradation) and
`
`(8)
`
`IMMUNOGEN 2079, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Teachlng Edttorlal
`
`as tracers (28). In addition to amines, sulfonyl halides
`also react with phenols (tyrosine), thiols (cysteine), and
`imidazoles (histidine) on proteins (29); therefore, they are
`less selective than either NHS esters or isothiocyanates.
`The conjugates formed with thiols, imidazoles, and phe-
`nols are all unstable and, if not removed during purifi-
`cation, can lead to loss of the label from the protein during
`long-term storage (see section V.B.1). One of the most
`widely used long-wavelength fluorescent probes, Texas
`Red, is a sulfonyl chloride. It has the longest wavelength
`spectral properties of any of the common amine-reactive
`fluorescent labeling reagents (30).
`(e) Miscellaneous Amine Reactive Reagents (Dichlo-
`rotriazines, Alkyl Halides, Anhydrides). The dichloro-
`triazine derivative of fluorescein, known as DTAF (I), has
`
`I
`
`I
`
`high reactivity with protein amines and has been used to
`prepare fluorescein tubulin with minimal loss of activity
`(31). In addition to amines, dichlorotriazines will react
`with alcohols a t elevated temperatures (60-90 "C) and are
`used to prepare polysaccharide conjugates (32). Some alkyl
`halides, including iodoacetamides commonly used to
`modify thiols, will react with amines of proteins if the pH
`is in the range 9.0-9.5 (33). Other reagents that have been
`used to modify amines of proteins are acid anhydrides.
`Succinic anhydride is commonly used to succinylate amine
`groups of basic proteins for the purpose of changing their
`isoelectric point and other charge-related properties (34).
`Mixed anhydrides derived from reaction of a carboxylic
`acid with carbitol or 2-methylpropanol chloroformates (eq
`9) are excellent reagents for modification of amines under
`
`0
`0
`II
`II
`R-COH + cIcoCn,CH(CHJ, ->
`0 0
`RotObNH, + RCOCOCH,CH(CH J , - >
`II II
`
`0 0
`II II
`RCOCOCH,CH(CH,),
`
`0
`II
`Rotein-NHCR + HOCOCH,CH(CHJ,
`
`(9)
`
`mild conditions (35). Of these, the carbitol mixed anhy-
`dride is relatively water soluble and is the preferred reagent
`for modification of amines in aqueous solution.
`(2) Thiol-Reactive Reagents. Thiol-reactive reagents
`are those that will couple to thiol groups on proteins to
`give thioether-coupled products. These reagents react
`rapidly at neutral (physiological) pH and therefore can be
`reacted with thiols selectively in the presence of amine
`groups.
`(a) Haloacetyl Derivatives (Formation of a Thioether
`Bond). These reagents (usually iodoacetamides) are
`among the most frequently used reagents for thiol mod-
`ification. In most proteins, the site of reaction is at cys-
`teine groups that are either intrinsically present or that
`
`Bioconjugtte Chem., Vol. 3, No. 1, 1992 5
`
`result from reduction of cystines. The reaction of iodoac-
`etate with cysteine is approximately twice as fast as that
`with bromoacetate and 20-100 times as rapid as that with
`chloroacetate (36). As mentioned previously, in the
`absence of cysteines, methionines can sometimes react
`with haloacetamides (12). Reaction of haloacetamides
`with thiols occurs rapidly at neutral pH at room temper-
`ature or below, and under these conditions, most aliphatic
`amines are unreactive. In addition to proteins, haloac-
`etamides have been reacted with thiolated peptides and
`thiolated primers for DNA sequencing (37), and also with
`RNA (on thiouridine) (38). The thioether linkages formed
`from reaction of haloacetamides are very stable. A
`potential problem in using iodoacetamides as modification
`reagents is their instability to light, especially in solution;
`therefore, they must be protected from light in storage
`and during reaction. The fluorescein and rhodamine io-
`doacetamides are among the most intensely fluorescent
`sulfhydryl reagents available for protein and peptide
`modification.
`(b) Maleimides (Formation of a Thioether Bond). Ma-
`leimides (eq 10) are similar to iodoacetamides in their
`
`0
`
`application as reagents for thiol modification; however,
`they are more selective than iodoacetamides, since they
`do not react with histidine, methionine, or thionucleotides
`(39,40). The optimum pH for the reaction of maleimides
`is near 7.0. Above pH 8.0, hydrolysis of maleimides to
`nonreactive maleamic acids can occur (41).
`(c) Miscellaneous Thiol-Reactive Reagents. These
`reagents include bromomethyl derivatives and pyridyl di-
`sulfides. The bromomethyl derivatives are similar in
`reactivity to iodoacetamides. The haloalkyl derivatives
`monobromobimane and monochlorobimane (11) react with
`
`H3*%
`H3C
`
`X = C1, Br
`CH2X
`
`I1
`
`glutathione and other thiols in cells to give fluorescent
`adducts, thus providing a method of quantitation of thi-
`ols (42). Pyridyl disulfides react in an exchange reaction
`with protein thiols to give mixed disulfides (eq 11) (43).
`
`N
`N
`(3) Carboxylic Acid- and Aldehyde-Reactive Reagents.
`(a) Aminesand Hydrazides (FormutionofAmideorAlkyl-
`amine Bonds). Amines and hydrazides can be coupled
`to carboxylic acids of proteins via activation of the car-
`boxyl group by a water-soluble carbodiimide followed by
`reaction with the amine or hydrazide. As mentioned
`previously (section II.A.41, the amine or hydrazide reagent
`must be weakly basic so that it will react selectively with
`the carbodiimide-activated protein in the presence of the
`more highly basic protein t-amines (lysines). The reaction
`of these probes with carbodiimide-activated carboxyl
`groups leads to the formation of stable amide bonds (eq
`12).
`
`Rotoin-Sn + RS-S<
`
`3 ->
`
`A
`
`Protein-S-Sf3 + S=(
`
`9 (11)
`
`A
`
`IMMUNOGEN 2079, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`8 Bioconjugate Chem., Vol. 3, No. 1, 1992
`
`0
`0 I I
`I1
`Rotein-COH + RN=C=NR' - > Rotein-COC = N-R - >
`HAW
`
`R"NH,
`
`0
`0
`II
`It
`Rotein-CNHR" + RNHCNHR'
`
`(12)
`
`Amines and hydrazides are also able to react with al-
`dehyde groups, which can be generated on proteins by
`periodate oxidation of carbohydrate residues on the
`protein. In this case, a Schiff base intermediate is formed
`(eq 13), which can be reduced to an alkylamine with sodium
`1) RNH,
`2) NaBH,CN
`
`Rotein-gly + N a n 4 - > Protein-CH = 0 - > Protein-CHAHR
`(13)
`cyanoborohydride, a mild and selective water-soluble
`reducing agent (44) (see also section 1I.B.l.c). Since the
`Schiff base formation is reversible, it is possible to minimize
`formation of protein-protein products by adding a large
`excess of amine or hydrazide reagent.
`(4) Bifunctional Reagents. Bifunctional, or cross-
`linking, reagents are specialized reagents having reactive
`groups that will form a bond between two different groups,
`either on the same molecule or two different molecules.
`Bifunctional reagents can be divided into two types: those
`with the same reactive group at each end of the molecule
`(homobifunctional) and those with different reactive
`groups at each end of the molecule (heterobifunctional).
`Recent trends are heavily in favor of the use of hetero-
`bifunctional cross-linkers where the bifunctional reagent
`has two reactive sites, each with selectivity toward different
`functional groups (amine reactive and thiol reactive, for
`example). These reagents, some of which are available in
`a range of chain lengths, are well-suited to the task of
`controlled coupling of unlike biomolecules, such as two
`different proteins. Table I1 lists some frequently used
`heterobifunctional cross-linkers along with their reactiv-
`ities and references describing their use.
`(a) Amine Reactive-Thiol or Protected Thiol. Because
`thiols will react selectively in the presence of amines with
`a variety of reagents, these functional groups are very useful
`for attaching two different proteins together. Thiol-
`coupling methods are frequently employed to prepare
`protein-enzyme conjugates. If the proteins to be coupled
`do not contain intrinsic thiols, the procedure is typically
`carried out by introducing a single thiol group to an amine
`of one of the proteins by means of a heterobifunctional
`reagent (eq 14). Traut's reagent (iminothiolane) has been
`
`Rotein(1 )-NH, +
`
`A i
`0
`II
`I
`:N -O-CCH,CH,SCCH, - >
`3 0
`
`0
`0
`0
`II
`I1
`I/
`Rotein(1 )-NHCCH,CH,SCCH, - > Protein11 ) NHCCH,CH,SH
`
`0
`II
`Rotein(1 I-NHCCH,CH,SH + Rotein(S)-NHCCH,I - >
`0 I1
`Protein(1) NHCCH,CH,SCH,CNH-Protein(Z)
`
`(14)
`
`extensively used for the purpose of introducing thiol groups
`selectively to proteins (45,46). Many other bifunctional
`
`Brinkley
`
`reagents contain both an amine-reactive and a protected
`thiol group, such as succinimidyl (acety1thio)acetate
`(SATA) (47, 48) or succinimidyl 3-(2-pyridyldithio)pro-
`pionate (SPDP) (43, 49). After deprotection, the thiol-
`containing protein is then reacted with a thiol-reactive
`group on the other protein, which has been introduced by
`a similar technique. Alternatively, proteins with synthetic
`thiol groups that have been introduced by modification
`can be used to couple to a number of thiol-reactive
`derivatives of dyes, biotin, haptens, or other molecules.
`(b) Amine Reactive-Iodoacetamide.
`Iodoacetamides
`are primarily thiol-reactive groups with the reaction
`occurring rapidly at physiological pH, but they can react
`with amines under more alkaline conditions (greater than
`pH 9.0) and long reaction times (section II.B.2.a). Io-
`doacetamides can be introduced into a protein or peptide
`that does not have intrinsic thiols via amine-reactive
`derivatives (eq 15) (50). The resulting modified protein
`
`Y 0
`
`n
`0
`II
`II
`Protein-NHClCH,),NHCCH,I
`
`(15)
`
`can then be coupled to any thiol-containing molecule. The
`second molecule is usually a thiol-containing protein.
`(c) Amine Reactive-Maleimide. The introduction of
`maleimides into a protein or peptide can be carried out
`with heterobifunctional reagents that have an amine-
`reactive group at one end and the thiol-specific maleim-
`ide at the other end (eq 16). The applications are very
`
`0'
`
`similar to those for the iodoacetamides discussed in the
`preceding section. Specific applications include coupling
`of ricin to monoclonal antibodies (51) and linking of oli-
`gonucleotides to enzymes (52).
`( d ) Amine Reactive-Aldehyde. Aldehydes do not
`occur naturally in proteins, but can be introduced in two
`ways. In the first method, carbohydrate groups on proteins
`are treated with an oxidizing reagent, such as sodium pe-
`riodate, or are converted via a galactose oxidaselcatalase
`enzyme method, both of which split the sugar to form
`aldehyde groups (53). Not all proteins contain carbohy-
`drate groups, and therefore a second method of introducing
`aldehydes via the reagent glutaraldehyde has been em-
`ployed (10). Glutaraldehyde has been used extensively to
`couple two proteins together via their amine groups (eq
`17); however, like other homobifunctional reagents, glu-
`Rotein(1 )-NH, + Rotein(Z)-NH, + 0 = CH(CH,),CH = 0->
`
`Rotein(l)-NH(CH,),NH-RoteinlP) (17)
`taraldehyde is being replaced with more selective heter-
`obifunctional reagents such as those discussed above.
`
`IMMUNOGEN 2079, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Teaching Editorial
`
`Bioconjugate Chem., Vol. 3, No. 1, 1992 7
`
`Table 11. Heterobifunctional Cross-Linkina Reaaents
`reagent
`
`~~
`
`succinimidyl3-(2-pyridyldithio)propionate (SPDP)
`
`succinimidyl 1-carboxylate trans-4-
`
`
`(N-maleimidylmethy1)cyclohexane-
`(SMCC)
`
`succinimidyl (acety1thio)acetate (SATA)
`
`4-[ (succinimidyloxy)carboxyl]-a-methyl-a-
`(2-pyridy1dithio)toluene (SMPT)
`
`~~
`
`reactivity
`
`ref
`
`primary amine, thiol
`
`49
`
`primary amine, thiol
`
`54,48
`
`primary amine, thiol
`
`47,48
`
`0
`
`0
`
`'HZ -
`0
`
`' 0
`Go -
`
`structure
`
`0
`
`0
`
`H,CCSCH2C0 - N
`
`0
`
`0
`
`succinimidyl4- [ [ (iodoacetyl)aminolmethyll-
`cyclohexane-1-carboxylate (SIAC)
`
`C-0-N
`
`primary amine, thiol
`
`50
`
`succinimidyl p-azidobenzoate (SAB)
`
`I
`ICH2CNHCH2
`It 0
`
`N3 0 - E- 0- N>
`
`0
`
`primary amine, nonselective 56
`
`(5) Photoactiuatable Reagents. Reagents are available
`that can be activated by light (photons) to produce a
`reactive intermediate that can couple to various functional
`groups on biomolecules. Two of the most frequently used
`photoactivatable reagents for this purpose are aromatic
`azides and benzophenones.
`(a) Aromatic Azides. Aromatic azides are efficiently
`photolyzed by illumination with an ultraviolet light at
`300-350 nm. The reactive molecule produced by this pho-
`tolysis is a nitrene, which reacts rapidly and nonspecif-
`ically with either solvent molecules or with functional
`groups on biomolecules. Almost any functional group or
`amino acid can be modified, since the nitrene is very
`reactive. Recent improvements in azide-based protein
`modification reagents have resulted in perfluorinated
`azides that generate nitrene intermediates with greater
`stability, thus giving reagents with higher efficiency (up
`to 40%) of reaction with the protein (57,581. One of the
`primary uses of these highly reactive reagents is to carry
`out photoaffinity labeling experiments. In these exper-
`iments, the aromatic azide is attached to a drug or other
`molecule which binds specifically to a protein binding site
`(an example is an enzyme inhibitor or a nucleotide
`analogue) and then photolyzed. The location and type of
`bond formed in this process provides information about
`the environment near the binding site (59). In addition
`to their role as photoaffinity labels, aryl azides are useful
`as heterobifunctional cross-linkers. Succinimidyl azido-
`benzoate (SAB), p-azidophenacyl bromide, and 4-male-
`imidobenzophenone have been employed to couple pro-
`teins through dark reaction with amines or thiols followed
`by light activation (56, 58, 60, 61).
`
`0
`
`(b) Benzophenones. Benzophenones are like azides in
`that they are photoactivatable by ultraviolet light, but
`once they have been activated, they can either react with
`functional groups or return to the ground state. Thus,
`these molecules can sometimes be reactivated if they do
`not react on the first activation. These reagents are also
`used as photoaffinity labels in a manner similar to that of
`the aromatic azides (62).
`111. PRACTICAL CONSIDERATIONS
`Along with a thorough knowledge of protein reactivity
`and the available reagents for the desired type of protein
`modification, it is of crucial importance that the researcher
`understand the practical aspects of carrying out reactions
`between highly reactive small organic molecules and large,
`complex, conformationally sensitive, water-soluble biopoly-
`mers. The following discussion will address some of the
`general rules, problems, and pitfalls of protein-modifica-
`tion chemistry.
`A. Choosing the Right Buffer. Conjugations should
`be carried out in a well-buffered system at a pH that is
`optimal for the reaction. The ionic strength should, in
`most cases, be in the range of 25-100 mM. For modification
`of thiol groups and a-amino groups, which occurs selec-
`tively at physiological pH (7.0-7.51, phosphate buffers are
`ideally suited. The more strongly basic lysine amines
`require more alkaline pH, in the range of 8.0-9.5, where
`phosphate solutions do not buffer well. For these reactions,
`carbonate/bicarbonate (pH of 100 mM bicarbonate is 9.2)
`or borate buffers are quite satisfactory. As an example,
`conjugations with NHS esters are best carried out in pH
`8.2 bicarbonate buffer, while isothiocyanates require the
`
`IMMUNOGEN 2079, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`8 Bloconlugate Chem., Vol. 3, No. 1, 1992
`
`higher pH (9.0-9.5) provided by carbonate or borate
`buffers. The choice of buffer will in some cases be directed
`by compatibility of the protein.
`B. Cosolvents. If the reagent that is to be attached
`to the biomolecule is readily soluble at millimolar con-
`centrations in water or buffer, no cosolvent is needed, and
`the reagent can be added as a concentrated aqueous
`solution to the buffered reaction solution. Unfortunately,
`aqueous systems are very often incompatible with the
`reagent, as a result of poor solubility or high reactivity
`with water. In these cases, a water-miscible cosolvent must
`be employed that will dissolve the reagent without causing
`its decomposition. At the same time, the cosolvent must
`not cause irreversible denaturation or precipitation of the
`biomolecule. Some cosolvents that have been successfully
`utilized in protein modifications are methanol, ethanol,
`2-propanol, 2-methoxyethanol, dioxane, dimethylforma-
`mide (DMF), and dimethyl sulfoxide (DMSO).
`The most versatile of these cosolvents are DMF and
`DMSO. They are recommended because of the following
`desirable properties: (a) they are inert to many of the
`reactive reagents used in preparing conjugates, (b) they
`are miscible with water in all proportions, and (c) they are
`compatible with most aqueous protein solutions even at
`up .to 30% v/v ratios. DMF is the solvent of choice for
`reactions of sulfonyl chlorides, since these reagents will
`react with DMSO. It is usually important that cosolvents
`be carefully dried and stored over a drying agent to prevent
`competing hydrolysis of the reactive modification reagent.
`C. Reaction Conditions. As a general rule, conjugation
`reactions should be done at below room temperature, since
`the rate of reaction of most conjugation reagents is rapid
`at low temperature. Low temperatures tend to increase
`the selectivity of the reaction, resulting in fewer side
`reactions and more consistent and reproducible results. A
`convenient procedure is to add the reagent to a gently
`stirred buffered solution of the protein in an ice-bath and
`then allow the bath to warm to room temperature over a
`period of about 2 h. Very reactive reagents such as sul-
`fonyl chlorides should be reacted under more carefully
`controll