Hermanson G. Bioconjugate Techniques, Second Edition

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330 5. Heterobifunctional Crosslinkers

4.4. Benzophenone-4-maleimide

Benzophenone-4-maleimide is a heterobifunctional photoreactive crosslinker that has sulf-

hydryl reactivity similar to benzophenone-4-iodoacetamide discussed in the previous section

(Invitrogen). In this case, the sulfhydryl-reactive portion is provided by the presence of a male-

imide group that couples to thiols by addition to the double bond (Chapter 2, Section 2.2).

The maleimide group is speciﬁ c for sulfhydryls under physiological conditions, and the reac-

tion results in a thioether linkage that is quite stable. Sulfhydryl-containing proteins or other

molecules modiﬁ ed with this reagent may be used in photoafﬁ nity labeling studies to investi-

gate the speciﬁ c interactions between two molecules. After mixing a modiﬁ ed protein with a

sample, the solution may be photolyzed to create a covalent crosslink between any interacting

species. UV photolysis of the benzophenone group results in a highly reactive triplet-state inter-

mediate which can rapidly insert or add to organic components within van der Waals distance

(Figure 5.34 ). Decay of the active-state intermediate returns the photosensitive group to its origi-

nal chemical form, thus allowing repeated photoactivations without losing the potential for cou-

pling to its intended target.

Benzophenone-4-maleimide is water-insoluble and should be pre-dissolved in DMF or

another organic solvent prior to adding an aliquot to an aqueous reaction mixture. Stock solu-

tions may be prepared and stored successfully if protected from light. The hydrophobicity and

bulkiness of the benzophenone group may cause insolubility problems in the initial protein that

is modiﬁ ed if the derivatization is done at too high a level. Fortunately, the use of a sulfhydryl-

reactive reagent can limit the degree of derivatization, since thiol groups usually are present in

lower quantities and in more discrete locations than amines.

5. Carbonyl-Reactive and Photoreactive Crosslinkers

Crosslinking reagents containing a photoreactive group on one end and a carbonyl-

reactive group on the other end are rare. The use of an amine group on one end of a pho-

tosensitive heterobifunctional reagent has been described (Draﬂ er and Marinetti, 1977; Das

and Fox, 1979; Gorman and Folk, 1980), but the presence of a hydrazide is required for

spontaneous reactivity toward carbonyls. The following compound is the only commercially

available reagent containing a phenyl azide photoreactive group and a hydrazide functional

group.

5.1. ABH

ABH, p-azidobenzoyl hydrazide, is a small, hetero bifunctional crosslinker containing a photoreac-

tive phenyl azide group on one end and a hydrazide functionality on the other end (Thermo Fisher).

The hydrazide can react with carbohydrate containing molecules after oxidation with sodium peri-

odate (Chapter 1, Section 4.4) to create aldehyde residues. The reaction forms a hydrazone linkage.

Thus, glycoproteins may be speciﬁ cally labeled on their polysaccharide chains for subsequent inves-

tigation of their interaction with receptor molecules ( Figure 5.35 ). In this sense, lectin–carbohydrate

interactions may be studied through direct modiﬁ cation of the sugar groups at or adjacent to the

binding site. Other amine- or sulfhydryl-reactive probes may not be suitable for such studies due to

the lack of amine or sulfhydryl groups near enough to a polysaccharide structure.

Figure 5.34 Benzophenone-4-maleimide can couple to thiol-containing molecules to form stable thioether

bonds. Exposure of the benzophenone group to UV light causes transition to a triplet-state ketone of high reac-

tivity for insertion into C  H or N  H bonds.

5. Carbonyl-Reactive and Photoreactive Crosslinkers 331

332 5. Heterobifunctional Crosslinkers

The cross-bridge of ABH consists of a benzoic acid derivative and thus provides a short

spacer between conjugated molecules. After ABH modiﬁ cation of a glycoprotein and incuba-

tion with a potential target molecule, the solution may be photolyzed with UV light to initiate

the ﬁ nal crosslink. Prior to photolysis, the reagent and all modiﬁ ed species should be protected

from light to prevent degradation of the phenyl azide group.

ABH is relatively insoluble when directly added to water or buffer, and therefore it should

be pre-dissolved in DMSO prior to addition of an aliquot to an aqueous reaction medium.

Stock solutions at a concentration of 50 mM ABH in DMSO work well. Since both reactive

groups on ABH are stable in aqueous environments as long as the solution is protected from

light, a secondary stock solution may be made from the initial organic preparation by adding

an aliquot to the hydrazide reaction buffer (0.1 M sodium acetate, pH 5.5; O ’Shannessy et al. ,

1984; O ’Shannessy and Quarles, 1985). Make a 1:10 dilution of the ABH/DMSO solution in

the reaction buffer. This solution may be stored in the dark at 4°C without decomposition.

6. Carboxylate-Reactive and Photoreactive Crosslinkers

A carboxylate-reactive crosslinking compound typically contains a primary amine functional

group that can be coupled to a carboxylic acid group in a protein or other molecule through

the use of a suitable activating agent, such as a carbodiimide. The carbodiimide forms an

active ester intermediate that then reacts with the amine to create an amide bond (Chapter 3,

Section 1). Reported use of diazoalkyl derivatives that spontaneously react with carboxylates

have been tried with ﬂ uorescent probes, but not yet applied to heterobifunctional crosslink-

ing agents (DeMar et al., 1992; Schneede and Ueland, 1992) (Chapter 2, Section 3.1). The

Figure 5.35 ABH reacts with aldehyde-containing compounds through its hydrazide end to form hydrazone

linkages. Glycoconjugates may be labeled by this reaction after oxidation with sodium periodate to form alde-

hyde groups. Subsequent photoactivation with UV light causes transformation of the phenyl azide to a nitrene.

The nitrene undergoes rapid ring expansion to a dehydroazepine that can couple to nucleophiles, such as amines.

following heterobifunctional reagent is the only carboxylate-reactive photosensitive crosslinker

currently available commercially.

6.1. ASBA

ASBA, 4-( p-azidosalicylamido)butylamine, is a carboxylate-reactive crosslinking agent contain-

ing a primary amine on one end and a photosensitive phenyl azide group on the other (Thermo

Fisher). The crosslinker is not spontaneously reactive with carboxylates, but must be used with

another activating agent that facilitates bond formation. For instance, it can be used in conjunc-

tion with a carbodiimide or other such reagent system that can initiate covalent bond formation

with a carboxylic acid. A water-soluble carbodiimide-like EDC (Chapter 3, Section 1.1) is able to

activate the carboxylates on a target molecule, forming active ester intermediates ( Figure 5.36 ).

In the presence of ASBA, derivatization will occur resulting in amide bond formation, and thus

leading to modiﬁ cation of the carboxylate-containing molecule with a photoreactive group.

The cross-bridge of ASBA provides a reasonably long spacer (16.3 Å). The phenyl azide por-

tion is constructed from a salicylic acid derivative and thus possesses a ring-activating hydroxyl

group. The presence of this group allows radioiodination of the ring prior to crosslinking

(Chapter 12, Section 5). Before the photolyzing step is initiated, the reagent should be handled

in the dark or protected from light to avoid decomposition of the phenyl azide group.

ASBA has been used to identify parasite adhesive proteins (Gowda et al., 2007), for active

site-directed labeling of glucosidase I (Romaniouk et al., 2004), and to study interactions with

the proteasome (Qureshi et al. , 2003).

7. Arginine-Reactive and Photoreactive Crosslinkers

The guanidinyl group on arginine ’s side chain can be speciﬁ cally targeted by the use of 1,2-

dicarbonyl reagents, such as the diketone group of glyoxal (Chapter 2, Section 5.2). Under alka-

line conditions, this type of group can condense with the guanidinyl residue to form a Schiff

base-like complex. The presence of other chemical compounds in the reaction can cause fur-

ther structural rearrangements, such as stabilization by boronate (Pathy and Smith, 1975).

Derivatives such as phenylglyoxal and p-nitrophenylglyoxal can be used to block or quanti-

tatively determine the amount of arginine in a protein (Yamasaki et al., 1981). Studies have

shown that if the reaction is done with a 2:1 ratio of glyoxal compound to arginine residues,

7. Arginine-Reactive and Photoreactive Crosslinkers 333

334 5. Heterobifunctional Crosslinkers

then the modiﬁ cation that results is reversible (Takahashi, 1968). However, if the modiﬁ cation

is done at a 1:1 stoichiometry, then it is irreversible (Konishi and Fujioka, 1987).

The ability to direct conjugation or modiﬁ cation speciﬁ cally through arginine residues using this

chemistry has been exploited in the availability of the only photoreactive glyoxal derivative, APG.

7.1. APG

APG, p-azidophenyl glyoxal, is a heterobifunctional crosslinker containing an arginine-speciﬁ c

diketone group on one end and a photosensitive phenyl azide group on the other end (Thermo

Figure 5.36 ASBA contains a primary amine group that may be conjugated to carboxylate compounds using the

carbodiimide EDC. Subsequent exposure to UV light initiates the photoreaction leading to covalent crosslinks.

Fisher). The reagent is a derivative of phenylglyoxal, a compound long used as an arginine

guanidinyl modiﬁ er. Reaction of APG with proteins at pH 7–8 results in selective modiﬁ cation

of arginine, leaving photoreactive groups available for subsequent crosslinking with interacting

molecules ( Figure 5.37 ). Exposure to UV light effects the ﬁ nal crosslink. The cross-bridge of an

APG crosslink is only 9.3 Å in length, allowing proximity interactions to be studied or the irre-

versible labeling of arginine areas in proteins.

APG has been used to investigate the binding step in collagen phagocytosis (Chong et al. ,

2007), to inhibit bovine heart lactic dehydrogenase, egg white lysozyme, horse liver alcohol

dehydrogenase, and yeast alcohol dehydrogenase (Ngo et al., 1981), crosslinking RNA–protein

interactions in E. coli ribosomes (Politz et al., 1981), and identifying regions of brome mosaic

virus coat protein chemically crosslinked in situ to viral RNA (Sgro et al ., 1986).

7. Arginine-Reactive and Photoreactive Crosslinkers 335

Figure 5.37 APG can be used to label speciﬁ cally arginine residues in proteins, producing stable, cyclic Schiff

base-like bonds with the side-chain guanidino groups. Photoactivation with UV light then causes ring expansion

of the phenyl azide group, initiating covalent bond formation with amines.

Trifunctional crosslinkers represent a relatively small but important category of bioconjugation

reagents, possessing three different reactive or complexing groups per molecule. The design of

this type of reagent is more elaborate than multifunctional crosslinkers such as polyaldehyde

dextran (Chapter 25, Section 2.1) or small organic molecules like trichloro- s-triazine (Chapter

25, Section 1.1) which merely contain more than two groups of the same functionaity per

molecule. The trifunctional approach incorporates elements of the heterobifunctional concept

wherein two ends of the linker contain reactive groups able to couple with two different func-

tional groups on target molecules. A trifunctional reagent, however, has a third arm terminating

in still another group able to speciﬁ cally link to a third chemical or biological target.

A convenient molecule from which to build trifunctionals is the amino acid, L-lysine. Its

three functional groups, -carboxy, -amino, and -amino, can be derivatized independently to

contain three arms. Each arm can be designed to terminate in a complexing group able to par-

ticipate in a particular type of conjugation reaction or afﬁ nity interaction.

The initial attempts at producing trifunctional reagents used biocytin as the core compound.

Biocytin is the lysine derivative of biotin having its valeric acid side chain amide-bonded to the

-amino group of the amino acid (Chapter 11, Section 3). Thus, crosslinkers built on this com-

pound have one of their trifunctional arms ending in a biotin label which is able to speciﬁ cally

complex with avidin or streptavidin probes. Creating two additional reactive arms from the

-carboxy and -amino groups of biocytin results in the completed trifunctional. The following

two crosslinkers are examples of this approach.

1. 4-Azido-2-nitrophenylbiocytin-4-nitrophenyl ester

Wedekind et al. (1989) designed a trifunctional reagent for studying the hormone binding

site of the insulin receptor. The crosslinker, 4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester

(ABNP) contains a nitrophenyl ester group that can react with amine functions in proteins and

peptides, similar to the reaction of N-hydroxysuccinimide (NHS) esters with amines (Chapter 2,

Section 1.4). This group can be used to modify a ligand (such as insulin) prior to its binding

to a speciﬁ c receptor molecule. The second chemically reactive functional group on ABNP is a

photosensitive pheny lazide group capable of being activated by exposure to UV light. After

Trifunctional Crosslinkers

336

the labeled ligand is allowed to interact with its receptor, forming an interaction complex, the

mixture is photolyzed to effect a covalent attachment point ( Figure 6.1 ). The third arm of the

trifunctional reagent is the biotin handle (from biocytin). This component allows the complex

to be puriﬁ ed by afﬁ nity chromatography on immobilized avidin or immobilized streptavidin.

Alternatively, the biotin group can be used to visualize the binding of the ligand to its receptor

using labeled avidin or streptavidin reagents (Chapter 23).

ABNP is soluble in dimethylformamide (DMF) but insoluble directly in aqueous solution.

Insulin labeling was done in DMF:water at a ratio of 9:1. For molecules not soluble in organic

solvent, such as proteins, the trifunctional ﬁ rst may be dissolved in DMF and a small aliquot

added to an aqueous reaction medium. The nitrophenyl ester reactive group can be coupled to

amine groups at alkaline pH (7–9) and in buffers containing no extraneous amines (avoid Tris).

Unfortunately, ABNP is not commercially available at the time of this writing.

2. Sulfo-SBED

Another trifunctional crosslinking agent is sulfo-SBED or sulfosuccinimidyl-2-[6-(biotinamido)-

2-(p -azidobenzamido)hexanoamido]ethyl-1,3 9 -dithiopropionate, developed by Ed Fujimoto at

Pierce Chemical (now Thermo Fisher). Like ABNP discussed previously, sulfo-SBED is built on

a biocytin backbone. Thus, one arm of the trifunctional compound consists of a biotin handle

that can be used for puriﬁ cation or detection purposes using avidin or streptavidin probes.

The chemically reactive groups of sulfo-SBED include a sulfo-NHS ester and a phenyl azide

group. The sulfo-NHS ester provides amine-coupling capability, forming amide bond linkages

with target molecules (Chapter 2, Section 1.4). The phenyl azide is photosensitive and may

be activated by exposure to UV light at wavelengths 300 nm. Most phenyl azides react by

ring expansion to dehydroazepines with subsequent reactivity toward nucleophiles, especially

amines (Chapter 2, Section 7.1) ( Figure 6.2 ).

2. Sulfo-SBED 337

338 6. Trifunctional Crosslinkers

The sulfo-NHS ester of sulfo-SBED is negatively charged and provides a degree of water solu-

bility (about 5 mM maximum concentration) for the entire molecule. Limited water solubility is

all that can be expected due to the large size of the trifunctional, most of it consisting of relatively

hydrophobic structures. However, the reagent is much more soluble in organic solvents such as

DMF (170 mM) and dimethyl sulfoxide (DMSO) (125 mM). Concentrated stock solutions may

be prepared in these solvents prior to addition of a small aliquot to an aqueous reaction mixture.

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20

minutes in phosphate buffer at room temperature conditions), it should be coupled to an

amine-containing protein or other molecule before the photolysis reaction is done. During the

initial coupling procedure, the solutions should be protected from light to avoid decomposition

of the phenyl azide group. The degree of derivatization should be limited to no more than a

5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible

precipitation of the modiﬁ ed molecules. For a particular protein, studies may have to be done

to determine the optimal level of modiﬁ cation.

An additional feature of sulfo-SBED is the presence of a cleavable disulﬁ de group in the

cross-bridge of the NHS ester arm of the molecule. After a conjugation reaction has taken

place, the complexes ﬁ rst may be puriﬁ ed using immobilized avidin or immobilized streptavi-

din and then the conjugates released by treatment with a disulﬁ de reducing agent. This allows

analysis of the complexed molecules, for example, after the binding of a ligand to its receptor.

Alternatively, the disulﬁ de group may be cleaved after interaction and capture of unknown

proteins, thus transferring the biotin label to the prey protein. The unknown interacting pro-

tein then may be detected or puriﬁ ed using the biotin tag. Such label transfer procedures are

important options for studying protein–protein interactions (Chapter 28).

Since sulfo-SBED has three functional arms, the length of each portion should be considered

when doing conjugation studies involving interacting proteins. The biotin handle has an effec-

tive length of 19.1 Å, including the side chain length for the lysine component. The sulfo-NHS

ester arm is approximately 13.7 Å long, measuring from the same point in the lysine group.

The phenyl azide arm is the shortest, only 9.1 Å long. The structure for sulfo-SBED shown in

this section includes molecular distance measurements somewhat different from these numbers

in that the total distances between the three arms are given, which reﬂ ects the intramolecular

distances between the terminal reactive groups or interacting group on biotin (indicated by the

arrows).

Additional information on the use of sulfo-SBED for the study of protein interactions can be

found in Chapter 28, Section 3.1.

The following suggested protocol was developed by Barb Olson at Thermo Fisher for the

labeling of soybean trypsin inhibitor (STI) with its subsequent complexation with trypsin.

Modiﬁ cations to this procedure may have to be done for other proteins.

2. Sulfo-SBED 339

Figure 6.1 The Wedekind trifunctional crosslinker can react with amine groups via its p-nitrophenyl ester to

form amide bond linkages. The phenyl azide group then can be photoactivated with UV light to generate cova-

lent bond formation with a second molecule. The biotin side chain provides binding capability with avidin or

streptavidin probes.