Hermanson G. Bioconjugate Techniques, Second Edition

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270 4. Homobifunctional Crosslinkers

water-soluble carbodiimide, 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride (EDC),

to yield imide linkages-containing terminal alkyl hydrazides. The hydrazide-activated protein then

can be used to conjugate with a glycoprotein that had been previously oxidized with sodium peri-

odate to generate reactive aldehyde residues. The resulting hydrazone bonds can be further stabi-

lized by reducing with sodium cyanoborohydride to give the secondary amine linkage.

These techniques have been used to target, detect, or assay glycoproteins in solution or on

cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section

5) (Bayer and Wilchek, 1990; Bayer et al., 1987a, b, 1990) and to form conjugates with

glycoproteins.

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-

stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create

numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunc-

tional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-

binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support

matrices suitable for afﬁ nity chromatography have been activated in a similar fashion to create

the hydrazide derivative (O ’Shannessy and Wilchek, 1990).

8.1. Adipic Acid Dihydrazide

The dihydrazide derivative of adipic acid (Aldrich) is perhaps the most-popular homobifunc-

tional hydrazide compound in use. The reagent provides a 10-atom bridge between crosslinked

molecules after conjugation. Adipic dihydrazide (ADH) is a solid that is soluble in aqueous

solutions, but may need to be moderately heated to create concentrated solutions. Aldehyde-

containing substances may be modiﬁ ed with this reagent to form hydrazone bonds with alkyl

hydrazide spacers suitable for reaction with other formyl-containing molecules ( Figure 4.29 ).

In this sense, afﬁ nity chromatography matrices have been activated with ADH to produce a

hydrazide derivative for coupling to aldehyde-containing ligands (O ’Shannessy and Wilchek,

1990), enzymes have been modiﬁ ed at available carboxylate groups using an EDC-facilitated

reaction to create hydrazide-activated derivatives appropriate for targeting oxidized glycopro-

teins (Bayer et al ., 1987a, b), and the biotin-binding proteins avidin and streptavidin have been

Figure 4.29 ADH spontaneously reacts with aldehydes to form hydrazone linkages.

activated with bis-hydrazides to assay glycoconjugates using biotinylated enzymes (Bayer and

Wilchek, 1990; Bayer et al., 1990). The crosslinker also has been utilized to study the carbo-

hydrate portion of yeast acid phosphatase (Kozulic et al. , 1984).

Protocols for the use of ADH in the modiﬁ cation of aldehyde or carboxylate functionalities

can be found in Chapter 1, Section 4.5 and Chapter 23, Section 5.

8.2. Carbohydrazide

Carbohydrazide (carbonic dihydrazide or 1,3-diaminourea) is a small homobifunctional rea-

gent containing reactive hydrazide groups on both ends. Its lack of an internal aliphatic bridge,

as found in ADH, gives the compound excellent solubility characteristics in aqueous solutions.

Carbohydrazide is freely soluble in water, but practically insoluble in ethanol and other organic

solvents. The two hydrazide functional groups of the molecule can react with aldehyde or

ketone groups to form hydrazone linkages. When reacted in excess with a molecule-containing

carbonyl groups, carbohydrazide modiﬁ cation results in short derivatives terminating in avail-

able hydrazides ( Figure 4.30 ). The compound has been used to modify microplate wells that

have been graft polymerized with glycidyl methacrylate to form surfaces that would couple

antibodies through their carbohydrate portions (Allmer et al., 1990; Brillhart and Ngo, 1991).

Although its use for protein modiﬁ cation has not been realized, carbohydrazide may be a supe-

rior alternative to ADH due to its hydrophilicity. Its only disadvantage may be in its shorter

bridge (5-atom spacer as opposed to ADH ’s 10-atom bridge).

A protocol for the use of carbohydrazide in the modiﬁ cation of aldehyde or carboxylate

functional groups can be found in Chapter 1, Section 4.5.

9 . Bis-diazonium Derivatives

Diazonium groups react with active hydrogens on aromatic rings to give covalent diazo bonds.

Generation of a diazonium-reactive group usually is done from an aromatic amine by reaction

Figure 4.30 Carbohydrazide can be used to transform an aldehyde residue into a hydrazide group.

9. Bis-diazonium Derivatives 271

272 4. Homobifunctional Crosslinkers

with sodium nitrite under acidic conditions at 0 °C (see Chapter 1, Section 4.3 and Chapter 2,

Section 6.1). The highly reactive and unstable diazonium is reacted immediately with an active-

hydrogen-containing compound at pH 8–10. In general, at pH 8.0 the diazonium group will

react principally with histidine residues, attacking the electron rich nitrogens of the imidazole

ring. At higher pH, the phenolic side chain of tyrosine groups can be modiﬁ ed. The reaction

proceeds by electrophilic attack of the diazonium group toward the electron rich points on the

target molecules. Phenolic compounds are modiﬁ ed at positions ortho and para to the aromatic

hydroxyl group. For tyrosine side chains, only the ortho modiﬁ cation is available.

Bis-diazonium compounds are useful in crosslinking molecules containing no other conve-

nient functional groups such as amines, carboxylates, or sulfhydryls. Conjugations done using

these compounds usually create deeply colored products characteristic of the diazo bonds.

Occasionally, the conjugated molecules may turn dark brown or even black. The diazo link-

ages are reversible by addition of 0.1 M sodium dithionite in 0.2 M sodium borate, pH 9.0.

Upon cleavage, the color of the complex is lost.

9.1. o -Tolidine, Diazotized

o-Tolidine, or 3,3-dimethylbenzidine, is a bis-aromatic-amine-containing compound that can

be readily diazotized to a homobifunctional diazonium crosslinker by reaction with sodium

nitrite ( Figure 4.31 ). The reagent is typically used in a one-step conjugation reaction wherein

two active-hydrogen-containing molecules are crosslinked through the addition of o -tolidine

immediately after it has been diazotized under acidic conditions by reacting with sodium nitrite

(Figure 4.32 ). pH adjustment to alkaline conditions after diazonium formation rapidly causes

crosslinking to occur. The diazotized form of o-tolidine must be used quickly due to its insta-

bility in aqueous solutions. The reagent has been used to couple active-hydrogen-containing

haptens to carrier proteins to form immunogens suitable for the production of antibodies

(Chapter 19, Section 6.1).

o-Tolidine is a benzidine derivative that should be considered a potential carcinogen.

Handling should be done with proper safety precautions and with the use of a fume hood to

Figure 4.31 Reaction of o-tolidine with sodium nitrite in the presence of HCl yields a highly reactive diazo

derivative.

avoid breathing in any dust particles. The reagent is sparingly soluble in water, but is more

soluble under the dilute acidic conditions necessary for activation to a diazonium derivative.

9.2. Bis-diazotized Benzidine

Benzidine, or p-diaminodiphenyl, may be diazotized with sodium nitrite to form a homo-

bifunctional diazonium crosslinking agent useful in conjugating active-hydrogen-containing

molecules ( Figure 4.33 ). The coupling reaction proceeds via electrophilic attack on atoms con-

taining extractable hydrogens. Particularly reactive are the phenolic side chains of tyrosine resi-

dues and the imidazole rings of histidine groups.

Benzidine is a known carcinogen and should be handled with extreme caution (Fourth

Annual Report on Carcinogens; NTP 85-002, 1985, p. 37). The solid and its vapors

may be rapidly absorbed through skin. Protective clothing and the use of a fume hood are

Figure 4.32 Bis-diazotized tolidine can form crosslinks with proteins through available tyrosine, histidine, or

lysine residues.

9. Bis-diazonium Derivatives 273

274 4. Homobifunctional Crosslinkers

Figure 4.33 Benzidine can be diazotized with sodium nitrite and HCl for reaction with proteins through their

tyrosine, histidine, or lysine side-chain groups.

recommended. The compound is only sparingly soluble in water as the free base. The dihydro-

chloride form, however, is soluble in water and ethanol.

Bis-diazotized benzidine has been used to create active-hydrogen-reactive spacer arms on

chromatographic matrices (Silman et al., 1966; Lowe and Dean, 1971). The compound may

be used similarly to o-tolidine for the conjugation of active-hydrogen-containing molecules (see

Section 9.1, this chapter, and Chapter 2, Section 6.1).

10. Bis-alkyl Halides

Homobifunctional reagents containing reactive halogen groups on both ends are capable of

crosslinking sulfhydryl, amine, or histidine-containing molecules by nucleophilic substitution.

Three forms of activated halogen functionalities can be used to create these reagents: haloacetyl

derivatives (see Chapter 1, Section 5.2), benzyl halides that react through a resonance activa-

tion process with the neighboring benzene ring, and alkyl halides that possess the halogen  to

a nitrogen or sulfur atom, as in N- and S-mustards. Haloacetyl compounds typically are iodo

or bromo derivatives, the simplest of which are 1,3-dibromoacetone (Husain and Lowe, 1968)

and various iodoacetyl derivatives of short diamine alkyl spacers (Ozawa, 1967) ( Figure 4.34 ).

Benzyl halides also are usually iodo or bromo derivatives, whereas the halo-mustards mainly

employ chloro and bromo forms ( Figure 4.35 ).

Reactive halogen crosslinkers are mainly speciﬁ c for sulfhydryl groups at physiological pH,

however at more alkaline pH values they can readily cross-react with amines and the imidazole

nitrogens of histidine residues. Some reactivity with hydroxyl-containing compounds also may

be realized, particularly with dichloro- s -triazine derivatives under alkaline conditions.

Most of the bis-alkyl halides referenced in the literature are unavailable commercially, and

therefore must be synthesized. Some key references to the preparation and use of these com-

pounds for the crosslinking of sulfhydryl-containing proteins and other molecules include

Goodlad (1957), Segal and Hurwitz (1976), Ewig and Kohn (1977), Wilchek and Givol (1977),

Prestayko et al . (1981), Luduena et al . (1982), Hiratsuka (1988), and Aliosman et al . (1989).

Figure 4.34 Several varieties of iodoacetylated diamine compounds have been investigated for crosslinking pro-

teins through sulfhydryl groups.

Figure 4.35 Benzyl halides and halomustards can be used as crosslinking agents reactive toward sulfhydryl

groups.

10. Bis-alkyl Halides 275

Heterobifunctional conjugation reagents contain two different reactive groups that can couple

to two different functional targets on proteins and other macromolecules ( Figure 5.1 ). For exam-

ple, one part of a crosslinker may contain an amine-reactive group, while another portion may

consist of a sulfhydryl-reactive group. The result is the ability to direct the crosslinking reaction

to selected parts of target molecules, thus garnering better control over the conjugation process.

Heterobifunctional reagents can be used to crosslink proteins and other molecules in a two- or

three-step process that limits the degree of polymerization often obtained using homobifunctional

crosslinkers (Chapter 1, Section 1.2 and Chapter 4, Section 2.2). In a typical conjugation scheme,

one protein is modiﬁ ed with a heterobifunctional compound using the crosslinker ’s most reactive

or most labile end. The modiﬁ ed protein then is puriﬁ ed from excess reagent by gel ﬁ ltration or

rapid dialysis. Most heterobifunctionals contain at least one reactive group that displays extended

stability in aqueous environments, therefore allowing puriﬁ cation of an activated intermediate

before adding the second molecule to be conjugated. For instance, an N-hydroxysuccinimide

(NHS) ester–maleimide heterobifunctional (e.g., see Section 1.3, this chapter) can be used to react

with the amine groups of one protein through its NHS ester end (the most labile functionality),

while preserving the activity of its maleimide functionality. Since the maleimide group has greater

stability in aqueous solution than the NHS ester group, a maleimide-activated intermediate may

be created. After a quick puriﬁ cation step, the maleimide end of the crosslinker then can be used

to conjugate to a sulfhydryl-containing molecule.

Heterobifunctional Crosslinkers

Figure 5.1 The general design of a heterobifunctional crosslinking agent includes two different reactive groups

at either end and an organic cross-bridge of various length and composition. The cross-bridge may be con-

structed of chemically cleavable components for selective disruption of conjugates.

276

Such multi-step protocols offer greater control over the resultant size of the conjugate and the

molar ratio of components within the crosslinked product. The conﬁ guration or structure of the

conjugate can be regulated by the degree of initial modiﬁ cation of the ﬁ rst protein and by adjusting

the amount of second protein added to the ﬁ nal conjugation reaction. Thus, low- or high-molecule-

weight conjugates may be obtained to better fashion the product toward its intended use.

Heterobifunctional crosslinking reagents also may be used to site-direct a conjugation reac-

tion toward particular parts of target molecules. Amines may be coupled on one molecule

while sulfhydryls or carbohydrates are targeted on another molecule. Directed coupling often

is important in preserving critical epitopes or active sites within macromolecules. For instance,

antibodies may be coupled to other proteins while directing the crosslinking reaction away

from the antigen binding sites, thus maximizing antibody activity in the conjugate.

Heterobifunctional reagents containing one photoreactive end may be used to insert non-

selectively into target molecules by UV irradiation. Ligands having speciﬁ c afﬁ nity toward a

receptor may be labeled with a photoreactive crosslinker, allowed to interact with its target,

and then photolyzed to permanently label the receptor at its binding site. The photoreactive

group is stable until exposed to high-intensity light at UV wavelengths. Photoafﬁ nity labeling

techniques are an important investigative tool for determining binding site characteristics.

The third component of all heterobifunctional reagents is the cross-bridge or spacer that ties

the two reactive ends together. Crosslinkers may be selected based not only on their reactivities,

but on the length and type of cross-bridge they possess. Some heterobifunctional families differ

solely in the length of their spacer. The nature of the cross-bridge also may govern the overall

hydrophilicity of the reagent. For instance, polyethylene glycol (PEG)-based cross-bridges create

hydrophilic reagents that provide water solubility to the entire heterobifunctional compound (see

Chapter 18). A number of heterobifunctionals contain cleavable groups within their cross-bridge,

lending greater ﬂ exibility to the experimental design. A few crosslinkers contain peculiar cross-

bridge constituents that actually affect the reactivity of their functional groups. For instance, it

is known that a maleimide group that has an aromatic ring immediately next to it is less stable

to ring opening and loss of activity than a maleimide that has an aliphatic ring adjacent to it.

In addition, conjugates destined for use in vivo may have different properties depending on the

type of spacer on the associated crosslinker. Some spacers may be immunogenic and cause spe-

ciﬁ c antibody production to occur against them. In other instances, the half-life of a conjugate

in vivo may be altered by choice of cross-bridge, especially when using cleavable reagents.

The following heterobifunctional reagents are organized according to their reactivities. The

majority are commercially available and well documented in the literature as to their properties.

Additional heterobifunctional compounds are described in Chapter 17 (Chemoselective Ligation;

Bioorthogonal Reagents) and Chapter 18 (Discrete PEG Reagents).

1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers

Perhaps the most popular heterobifunctional reagents are those which contain amine-reactive

and sulfhydryl-reactive ends. The amine-reactive group is usually an active ester, most often

an NHS ester, while the sulfhydryl-reactive portion may be one of several different functional

groups. The amine-reactive end of these crosslinkers is typically an acylating agent possessing a

good leaving group that can undergo nucleophilic substitution to form an amide bond with pri-

mary amines. The sulfhydryl-reactive portion, by contrast, is usually an alkylating agent that is

1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers 277

278 5. Heterobifunctional Crosslinkers

capable of creating either thioether or disulﬁ de linkages with sulfhydryl-containing molecules.

Depending on the chemistry chosen, linkages with a sulfhydryl-containing molecule may be

either permanent covalent bonds or reversible disulﬁ de bonds that can be cleaved by use of a

suitable disulﬁ de reductant.

The active ester chemistry of the amine-reactive end of these crosslinkers is characteristically

the most labile functional group, being susceptible to rapid hydrolysis under the aqueous con-

ditions of a conjugation reaction. The sulfhydryl-reactive group, however, is usually much more

stable to breakdown in aqueous environments. Therefore, these reagents typically are used in

multi-step conjugation protocols wherein one protein or molecule ﬁ rst is modiﬁ ed through its

amines to yield a sulfhydryl-reactive intermediate. After removal of excess crosslinker by gel ﬁ l-

tration, a second protein or molecule containing a sulfhydryl group is added to effect the ﬁ nal

conjugation. The stability of the sulfhydryl-reactive end of these crosslinkers allows greater

control over the crosslinking process than is possible with single-step procedures.

1.1. SPDP, LC-SPDP, and Sulfo-LC-SPDP

SPDP, N-succinimidyl-3-(2-pyridyldithio)propionate, is one of the most popular heterobifunc-

tional crosslinking agents available. The activated NHS ester end of SPDP reacts with amine

Figure 5.2 SPDP can react with amine-containing molecules through its NHS ester end to form amide bonds. The

pyridyl disulﬁ de group then can be coupled to a sulfhydryl-containing molecule to create a cleavable disulﬁ de bond.

groups in proteins and other molecules to form an amide linkage. The 2-pyridyldithiol group

at the other end reacts with sulfhydryl residues to form a disulﬁ de linkage with sulfhydryl-

containing molecules (Carlsson et al., 1978) ( Figure 5.2 ). The crosslinker is used extensively to

form enzyme conjugates for use in immunoassays or in labeled DNA probe techniques. It also

is frequently used for the preparation of immunotoxin conjugates for in vivo administra-

tion (Chapter 21, Section 2.1 and Chapter 27, Section 2.4). In addition, the reagent is effec-

tive in creating sulfhydryls on proteins and other molecules (Chapter 1, Section 4.1). Once

modiﬁ ed with SPDP, a protein can be treated with dithiothreitol (DTT) (or another disulﬁ de

reducing agent) to release the pyridine-2-thione leaving group and form a free sulfhydryl. The

terminal  SH group then can be used to conjugate with any crosslinking agents containing

sulfhydryl-reactive groups, such as maleimide or iodoacetyl (both for covalent conjugation) or

2-pyridyldithiol groups (for reversible conjugation).

There are three forms of SPDP analogs currently available commercially (Thermo Fisher):

the standard SPDP, a long-chain version designated LC-SPDP, and a water-soluble, sulfo-NHS

form also containing an extended chain, called sulfo-LC-SPDP. Both the standard SPDP and

the LC-SPDP are insoluble in aqueous solutions and must be ﬁ rst solubilized in DMSO prior

to addition to the reaction solution. The sulfo-LC-SPDP may be solubilized directly in water

or buffer. The long-chain versions extend the length of the crosslinker for those applications

that require greater accessibility to react with sterically hindered functional groups. Since many

sulfhydryl residues are found below the surface of a protein structure in more hydrophobic

domains, the longer spacer arm of the LC versions may be more effective in conjugations with

1. Amine-Reactive and Sulfhydryl-Reactive Crosslinkers 279