Hermanson G. Bioconjugate Techniques, Second Edition

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260 4. Homobifunctional Crosslinkers

contains two reactive ﬂ uorine atoms that can couple to amine-containing molecules, yielding

stable arylamine bonds ( Figure 4.19 ). However, the reactivity of aryl halides is not totally spe-

ciﬁ c for amines. Thiol, imidazolyl, and phenolate groups of amino acid side chains also can

react (Zahn and Meinhoffer, 1958). Conjugates formed with sulfhydryl groups, however, are

reversible by cleaving with an excess of thiol (such as DTT) (Shaltiel, 1967). The compound

is especially useful in crosslinking cellular membrane proteins, since it is able to penetrate the

hydrophobic regions of the lipid bilayer.

Diﬂ uorobenzene reagents have been used for crosslinking phospholipids in human eryth-

rocyte membranes (Berg et al., 1965; Marfey and Tsai, 1975), conjugation of small peptides

to the carrier protein albumin (Tager, 1976), studying the interaction of proteins in the myelin

membrane (Golds and Braun, 1978), crosslinking of cytochrome oxidase subunits (Kornblatt

and Lake, 1980), and studying the conformational effects of calcium on troponin C (Kareva

et al., 1986). DFDNB also has been used to investigate the conformational changes in

Chlamydomonas outer arm dynein in response to calcium ions (Sakato et al., 2007), to study

the dimerization properties of villin (George et al., 2007), and to characterize the intraﬂ agellar

transport complex B core (Lucker et al ., 2005).

4.2. DFDNPS

DFDNPS (4,4-diﬂ uoro-3,3-dinitrophenylsulfone) is a di-aryl halide reagent containing a cen-

tral sulfone group ( Figure 4.20 ). The aromatic ﬂ uorines are reactive with amines, sulfhydryls,

phenolates, and imidazolyl groups of proteins (see previous section). The reaction with amines

forms stable arylamine linkages. The reaction with sulfhydryl groups, however, is reversible

by treatment with an excess of thiol. The central sulfone group provides cleavability through

hydrolysis with base at pH 11–12 at 37 °C (Wold, 1961, 1972; Zarling et al ., 1980).

Figure 4.19 DFDNB is a small crosslinker able to form covalent bonds between amine-containing molecules.

The aromatic ﬂ uorine atoms are readily displaced by nucleophiles.

5. Homobifunctional Photoreactive Crosslinkers

Although there are a number of photo-sensitive coupling chemistries that have been used in

modiﬁ cation and conjugation reactions (Chapter 2, Section 7), it has been primarily aryl azides

Figure 4.20 DFDNPS reacts with amine-containing molecules to form arylamine crosslinks. The central sulfone

group in the cross-bridge can be cleaved under alkaline conditions.

5. Homobifunctional Photoreactive Crosslinkers 261

262 4. Homobifunctional Crosslinkers

that have found application in homobifunctional crosslinkers. The photolysis reaction requires

exposure of the phenyl azide to a bright light source at a wavelength of 265–275 nm (Ji, 1979).

If the aromatic ring contains a nitro group meta to the azide functionality, then photolysis can

occur at higher wavelengths (300–460 nm). The photolysis process initially forms a highly reac-

tive aryl nitrene, but these quickly undergo ring expansion to create a dehydroazepine. This

active species principally reacts with nucleophiles, rather than inserting in C  H or N  H

bonds or adding to double bonds. Thus, instead of nonselective coupling into nearly any part

of a molecular structure, aryl azides ultimately react with primary amines more than any other

functionality (Schnapp et al ., 1993).

Reported structures for homobifunctional aryl azides include a biphenyl derivative and

a naphthalene derivative (Mikkelsen and Wallach, 1976), a biphenyl derivative contain-

ing a central, cleavable disulﬁ de group (Guire, 1976), and a compound containing a central

1,3-diamino-2-propanol bridge between phenyl azide rings that are nitrated (Guire, 1976). The

only commercially available homobifunctional photoreactive crosslinker is BASED.

5.1. BASED

Bis-[-(4-azidosalicylamido)ethyl]disulﬁ de (BASED) is a homobifunctional photoreactive cross-

linking agent-containing phenyl azide groups at both ends (Thermo Fisher). Its central bridge

contains a cleavable disulﬁ de bond that may be broken after conjugation with the appropriate

reducing agent (Chapter 1, Section 4.1). The aryl azides are salicylate derivatives that contain

hydroxylic functions that activate the ring toward electrophilic reactions. Thus, the phenolic rings

are modiﬁ able with

125

I using traditional oxidative radioiodination reagents. Prior to the photo-

reactive conjugation step, the crosslinker may be iodinated with IODO-GEN or IODO-BEADS

(Chapter 12, Sections 2 and 3). After two proteins are crosslinked, cleavage of the conjugate with

DTT releases the link but maintains a radiolabel on each of the molecules ( Figure 4.21 ).

6. Homobifunctional Aldehydes

Numerous bis-aldehyde reagents have been used for the conjugation of biomolecules. Nearly

every small organic compound-containing two aldehyde groups has been at least tried in

crosslinking reactions. The repertoire of available homobifunctional aldehydes ranges from

the single-carbon formaldehyde (Section 6.1, this chapter; yes, it behaves as if it were bifunc-

tional) through the 2-carbon atom glyoxal (Brooks and Klamerth, 1968), the 3-carbon malond-

ialdehyde (Cater, 1963), the 4-carbon succinaldehyde (Cater, 1963), the popular 5-carbon

glutaraldehyde (Section 6.2, this chapter), the 6-carbon adipaldehyde as well as its -hydroxy

derivative (Fein and Filachione, 1957; Seligsberger and Sadlier, 1957; Cater, 1963; Richard

and Knowles, 1968; Hopwood, 1969), and several pyridoxal-polyphosphate derivatives that

are internally cleavable with acid or base (Shimomura and Fukui, 1978; Benesch and Kwong,

1988).

By far, the two most popular bis -aldehyde reagents are formaldehyde and glutaraldehyde.

6.1. Formaldehyde

Formaldehyde is the smallest crosslinking reagent available that does not create a zero-length

bridge between two molecules (Chapter 3). Although technically not a homo-bifunctional rea-

gent, it undergoes crosslinking reactions as though it possessed two functional groups. In con-

centrated aqueous solutions, it can form the low-molecular-weight polymers typically observed

Figure 4.21 BASED can react with molecules after photoactivation to form crosslinks with nucleophilic groups,

primarily amines. Exposure of its phenyl azide groups to UV light causes nitrene formation and ring expansion

to the dehydroazepine intermediate. This group is highly reactive with amines. The cross-bridge of BASED is

cleavable using a disulﬁ de reducing agent.

6. Homobifunctional Aldehydes 263

264 4. Homobifunctional Crosslinkers

in formalin preparations. In dilute solutions, it exists mainly in its monomeric state. Older

solutions of formaldehyde may contain precipitated polymer that often can be resolubilized by

heating. Commercial preparations of formaldehyde may be obtained as a 37 percent solution

stabilized against polymerization by the addition of methanol.

Conjugation reactions using formaldehyde may proceed by one of two routes: the Mannich

reaction or via an immonium cation intermediate. The Mannich reaction consists of the con-

densation of formaldehyde (or sometimes another aldehyde) with ammonia (in the form of

its salt) and another compound containing an active hydrogen. For a review of this reaction

mechanism, see Adams et al. (1942). Instead of ammonia, however, this reaction can be done

with primary or secondary amines, or even with amides. An example of this is illustrated in

Figure 4.22 by the condensation of phenol, formaldehyde, and a primary amine salt (the active

hydrogens are shown underlined). Figure 4.23 shows some active-hydrogen-containing func-

tional groups that can participate in the Mannich reaction.

The Mannich reaction can be used for the immobilization of certain drugs, steroidal com-

pounds, dyes, or other organic molecules that do not possess the typical nucleophilic groups

able to participate in traditional coupling reactions (Hermanson et al., 1992). It also can be

used to conjugate hapten molecules to carrier proteins when the hapten contains no convenient

nucleophile for conjugation (Chapter 19, Section 6.2). In this case, the carrier protein contains

the primary amines and the hapten contains at least one sufﬁ ciently active hydrogen to partici-

pate in the condensation reaction.

To obtain acceptable yields, the Mannich reaction must be done at elevated temperatures.

Incubation at 37–57 °C for at least 2–24 hours usually is required to complete the reaction.

Addition of formaldehyde is done by adding an aliquot of a 37 percent solution to the reaction

to obtain about a 10- to 100-fold molar excess over t he amount of active-hydrogen-containing

Figure 4.22 The Mannich reaction occurs between an active-hydrogen-containing compound (phenol) and an

amine-containing molecule in the presence of an aldehyde (formaldehyde). The condensation reaction forms

stable crosslinks.

Figure 4.23 Examples of active-hydrogen-containing compounds that can participate in the Mannich reaction.

The points of reactivity are shown by the hydrogen atoms.

compound to be conjugated. Thermo Fisher has designed a kit for the conjugation of haptens

to carrier proteins using the Mannich reaction mechanism.

A secondary reaction pathway also is possible in formaldehyde-facilitated conjugations.

Formaldehyde may react with a primary amine to form a quaternary ammonium salt. This

intermediate spontaneously reacts to create a highly active immonium cation with loss of one

molecule of water (Blass et al., 1965; Ji, 1983). The immonium cation is reactive toward nucle-

ophiles in proteins and other molecules, including amines, sulfhydryls, phenolic groups, and

imidazole nitrogens. The reaction yields methylene bridges between two nucleophiles, binding

macromolecules with a one-carbon linker ( Figure 4.24 ).

It is obvious that the Mannich reaction pathway and the immonium ion mechanism may

occur simultaneously, especially at conditions of room temperature or greater. Formaldehyde-

facilitated crosslinking reactions between molecules that both contain nucleophiles prob-

ably occur primarily by the immonium ion pathway, since the Mannich reaction proceeds

at a slower rate. In addition, the Mannich reaction will cause nondescript polymerization

between molecules that possess both active hydrogens and amine groups. It is best to utilize the

Mannich reaction only when one of the molecules contains no nucleophilic groups but at least

one active hydrogen, and the other molecule contains a primary or secondary amine.

Formaldehyde also can be used to study protein interactions in cells or tissue sections by

crosslinking and capturing protein complexes. Chapter 28, Section 1.3 describes this method

and contains a protocol for use.

6.2. Glutaraldehyde

Glutaraldehyde is the most popular bis-aldehyde homobifunctional crosslinker in use today.

However, a glance at glutaraldehyde ’s structure is not indicative of the complexity of its possi-

ble reaction mechanisms. Reactions with proteins and other amine-containing molecules would

be expected to proceed through the formation of Schiff bases. Subsequent reduction with

sodium cyanoborohydride or another suitable reductant would yield stable secondary amine

6. Homobifunctional Aldehydes 265

266 4. Homobifunctional Crosslinkers

linkages (Chapter 2, Section 5.3 and Chapter 3, Section 4). This reaction sequence certainly is

possible, but other crosslinking reactions also occur.

Glutaraldehyde in aqueous solutions can form polymers-containing points of unsaturation

(Hardy et al., 1969, 1976; Monsan et al., 1975) ( Figure 4.25 ). Such ,-unsaturated glutaral-

dehyde polymers are highly reactive toward nucleophiles, especially primary amines. Reaction

with a protein results in alkylation of available amines, forming stable secondary amine link-

ages. These glutaraldehyde-modiﬁ ed proteins still may react with other amine-containing mole-

cules either through the Schiff base pathway or through addition at other points of unsaturation

(Figure 4.26 ). The proposed reaction mechanism of conjugation using these polymer conjugates

explains the stability of proteins crosslinked by glutaraldehyde that have not been reduced.

Schiff base formation alone would not yield stable crosslinked products without reduction.

Crosslinking using glutaraldehyde polymers is difﬁ cult to reproduce. Since the glutaralde-

hyde polymer size and structure is unknown, the exact nature of the conjugates formed by this

method is indeterminable, as well. The age of a glutaraldehyde solution is another variable,

because the older the solution the more polymer will be formed. Fresh glutaraldehyde often

will not yield the same results as aged solutions.

Figure 4.24 Two amine-containing molecules can be crosslinked by formaldehyde through formation of a qua-

ternary ammonium salt with subsequent dehydration to an immonium cation intermediate. This active species

then can react with a second amine compound to form stable secondary amine bonds.

A third method of using glutaraldehyde in conjugation reactions is through its ability to

react rapidly with hydrazide groups. A molecule-containing hydrazide functionalities or modi-

ﬁ ed to contain them (Chapter 1, Section 4.5) can be conjugated with another molecule con-

taining either amines or hydrazides. Glutaraldehyde will react with the hydrazide groups to

form hydrazone linkages. When crosslinking two macromolecules containing multiple sites of

conjugation, the multivalent hydrazone bonds are stable enough to create a stable conjugate.

Figure 4.26 Glutaraldehyde may react by several routes to form covalent crosslinks with amine-containing molecules.

6. Homobifunctional Aldehydes 267

Figure 4.25 Glutaraldehyde in aqueous solution may polymerize at either acid or basic pH.

268 4. Homobifunctional Crosslinkers

If a small molecule is involved, however, reduction of the hydrazone with sodium cyanoboro-

hydride is recommended to produce a leak-resistant bond.

Glutaraldehyde has been used extensively as a homobifunctional crosslinking reagent, especially

for antibody–enzyme conjugations (Avrameas, 1969; Avrameas and Ternynck, 1971). To help over-

come its tendency to form large-molecular-weight polymers upon crosslinking two proteins, a two-

step protocol often is employed. In this regard, one protein ﬁ rst is reacted with glutaraldehyde and

puriﬁ ed away from excess reagent. The second protein then is added to effect the conjugate forma-

tion. See the introduction to this chapter and Chapter 1, Section 1.2 and Chapter 20, Section 1.2

for additional information on the use of glutaraldehyde and two-step crosslinking procedures.

7 . Bis-epoxides

Homobifunctional compounds-containing epoxide groups on both ends can be used to

crosslink molecules-containing nucleophiles, including amines, sulfhydryls, and hydroxyls.

The reaction proceeds with epoxide ring opening to create secondary amine, thioether, or

ether bonds with these functional groups ( Figure 4.27 ). During the ring opening process, a

-hydroxy group is created. Hydrolysis of the epoxy function without coupling to a nucle-

ophile yields adjacent hydroxyls that can be oxidized with sodium periodate to create reactive

aldehydes ( Figure 4.28 ). Epoxide groups, however, are quite stable in aqueous environments

around neutral pH or above. They are reactive at alkaline pH values toward other nucleophilic

molecules, while they can be hydrolyzed to a diol at acid pH.

Certain bis-epoxide reagents have been used to activate hydroxylic matrices for coupling

ligands-containing amine, sulfhydryl, or hydroxyl groups for afﬁ nity chromatography purposes

(Hermanson et al., 1992). Conjugation reactions involving proteins also have been done using

epoxide crosslinkers, but not to the extent of their use in immobilization.

Bis-epoxy compounds that have been used for crosslinking purposes vary mainly in their

chain length, ranging from the 4-carbon bridge of 1,2:3,4-diepoxybutane (Skold, 1983; Kohn

et al., 1966), the 6-carbon spacer of 1,2:5,6-diepoxyhexane (Fearnley and Speakman, 1950),

Figure 4.27 Epoxide groups are reactive toward sulfhydryls, amines, and hydroxyls.

Figure 4.28 Hydrolysis of epoxy groups forms 1,2-dihydroxy derivatives that can be oxidized with periodate

to create reactive aldehydes.

the 7-atom bridge of bis(2,3-epoxypropyl)ether (Kohn et al., 1966), to the 12-atom spacer of

the popular 1,4-(butanediol) diglycidyl ether (discussed below) (Sundberg and Porath, 1974;

Porath, 1976). Longer chain polymeric bis-epoxide compounds also have been utilized in col-

lagen crosslinking experiments (Murayama et al ., 1988).

7.1. 1,4-Butanediol Diglycidyl Ether

The most commonly used homobifunctional epoxide compound is 1,4-butanediol diglycidyl

ether. The reagent can react with hydroxyls, amines, or sulfhydryl groups to produce ether, sec-

ondary amine, or thioether bonds, respectively. The reaction of the epoxide functionalities with

hydroxyls requires high pH conditions, usually in the range of pH 11–12. Amine nucleophiles

react at more moderate alkaline pH values, typically needing buffer environments of at least

pH 9. Sulfhydryl groups are the most highly reactive nucleophiles with epoxides, requiring a

buffered system in the range of only pH 7.5–8.5 for efﬁ cient coupling.

1,4-Butanediol diglycidyl ether is a viscous liquid having a density of 1.45 at 20 °C. It is a

hygroscopic, corrosive compound with a displeasing odor that should be handled with care in

a fume hood. Aqueous solutions of the bis-epoxide usually possess a characteristic oily ﬁ lm on

their surfaces, indicating the limited solubility of the reagent.

An example of the use of 1,4-butanediol diglycidyl ether for the activation of soluble dex-

tran polymers is given in Chapter 25, Section 2.3. One end of the bis-epoxide reacts with the

hydroxylic sugar residues of dextran to form ether linkages, which terminate in epoxy func-

tionalities. The epoxides of the activated derivative then can be used to couple additional mol-

ecules-containing nucleophilic groups to the dextran backbone.

8. Homobifunctional Hydrazides

Homobifunctional crosslinking agents containing hydrazide groups at both ends can be used

to conjugate molecules-containing carbonyl or carboxyl groups. In one scheme, a bis-hydrazide

compound can be reacted with the carboxylate groups of a protein in the presence of the

8. Homobifunctional Hydrazides 269