Hermanson G. Bioconjugate Techniques, Second Edition

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210 2. The Chemistry of Reactive Groups

photoreaction process. Elsner and Mouritsen (1994) used psoralen linkers for modiﬁ cation of

the surface of microplates to create sites for covalent binding of afﬁ nity molecules.

8. Cycloaddition Reactions

The following sections brieﬂ y describe three cycloaddition reactions that can be used to form

bioconjugates. These reactions represent highly speciﬁ c reactant pairs that have a chemoselec-

tive nature, meaning they mainly react with each other and not other functional groups, such

as those found on biomolecules. For a complete discussion of chemoselective ligation reactions,

see Chapter 17.

8.1. Diels–Alder Reaction

The Diels–Alder reaction consists of the covalent coupling of a diene with an alkene to form

a 6-membered ring complex. This process has been used extensively in organic synthesis, but

only recently has it been applied to bioconjugation reactions (Hill et al., 2001). This reaction

proceeds at room temperature or slightly elevated temperature conditions (30°C) to give the

2  4 cycloaddition product, a hexane ring containing a single double bond. The reaction can

be done using a maleimide group as the alkene derivative and a hexadienyl group as the diene

(Reaction 58). The reaction process can give 90–95 percent yields in 1–18 hours.

(Reaction 58)

See Chapter 17, Section 1 for additional information concerning the use of the Diels–Alder

reaction in bioconjugation applications.

8.2. Complex Formation with Boronic Acid Derivatives

Boronic acid derivatives are able to form ring structures with other molecules having neigh-

boring functional groups consisting of 1,2- or 1,3-diols, 1,2- or 1,3-hydroxy acids, 1,2- or

1,3-hydroxylamines, 1-2- or 1,3-hydroxyamide, 1,2- or 1,3-hydroxyoxime, as well as various

sugars containing these species (Weith et al., 1970; Rosenberg and Gilham, 1971; Rosenberg

et al., 1972; Pace and Pace, 1980; Singhal et al., 1980). The products of these reactions are

5- or 6-membered heterocyclic rings, which in some cases are reversible with a change in pH or

by the addition of a counter-ligand having competing functional groups.

Typically, the boronic acid group is part of an aminophenyl boronic acid derivative, and

this group has been used for bioconjugation and afﬁ nity chromatography purposes (Burnett

et al., 1980; O ’Shannessy and Quarles, 1987). A common partner for a phenyl boronic acid

group in bioconjugation is the salicylhydroxamic acid (SHA) group (Chapter 17, Section 3)

(Reaction 59).

(Reaction 59)

8.3. Click Chemistry: Cu

-promoted Azide––Alkyne [3  2] Cycloaddition

Click chemistry refers to the reaction between an azido functional group and an alkyne to

form a [3  2] cycloaddition product, a 5-membered triazole ring. This reaction has been used

for many years in organic synthesis to form heterocyclic rings. Normally, the click reaction

requires high temperatures, and this was the main reason that it was not used as a bioconjuga-

tion tool. However, it was discovered that in aqueous solutions and in the presence of Cu(I),

the reaction kinetics are dramatically accelerated to provide high yields even at room tem-

perature and ambient pressures (Rostovtsev et al., 2002; Torn øe et al., 2002; Sharpless et al .,

2005).

The advantage of the click reaction for bioconjugation is that the reactant pair is not

reactive with any other functional group encountered in biological systems. This property of

8. Cycloaddition Reactions 211

212 2. The Chemistry of Reactive Groups

(Reaction 60)

bioorthogonality provides extreme selectivity for bringing together azide and alkyne derivatives

to form triazoles even in complex biological samples.

Reaction 60 shows the reaction of an alkyne with an azide to form a triazole ring in the

presence of a Cu(I) catalyst. See Chapter 17, Section 4 for additional details on the use of this

conjugation reaction.

PART II

The reagent systems used in bioconjugate procedures are as varied

as their intended applications. Whether it is for tagging proteins to

make them chromogenic or ﬂ uorescent, labeling molecules with biospe-

ciﬁ c ligands for subsequent afﬁ nity interactions, or crosslinking two or

more substances to create uniquely active conjugates, the choice of

reagents available for use is limited only by the imagination.

Over the last 30 years, the selection of crosslinking and modifying

agents has grown not only in shear number, but in the availability of

novel reactive groups and in the variety of their design. Today, regard-

less of the particular need, a workable reagent system that will yield a

useful derivative almost always can be found. The best and most effec-

tive of the reported reagent systems usually are available from com-

mercial sources, and thus do not even have to be synthesized.

In Part II, the reagents of modiﬁ cation and conjugation have been

categorized according to structural type, reactivity, and use. Where

possible and appropriate, generalized protocols have been provided for

each reagent ’ s most likely application. The options described herein,

combined with a thorough knowledge of the basic chemical reactions

that their functional groups provide (as discussed in Part I), allow the

creation of an intelligent design and plan of attack for any desired

application. The labeling, tagging, crosslinking, or targeting of small lig-

ands, peptides, proteins, carbohydrates, nucleic acids, oligonucleotides,

Bioconjugate

Reagents

214 II. Bioconjugate Reagents

lipids, and a host of other compounds may be accomplished by the judi-

cious choice of the appropriate reagent system.

The following reagents have been used in everything from bench-

scale experiments in research laboratories to process-optimized appli-

cations in the diagnostic and therapeutic industries. Conjugated or

modiﬁ ed molecules have been applied in procedures designed to visual-

ize target substances, as key components in clinical assay systems, and

in the latest afﬁ nity-directed therapeutics, such as anti-tumor immuno-

toxins. Some of the reagent systems described in Part II have formed

the basis for literally a multi-billion dollar biotechnology industry.

215

The smallest available reagent systems for bioconjugation are the so-called zero-length cross-

linkers. These compounds mediate the conjugation of two molecules by forming a bond contain-

ing no additional atoms. Thus, one atom of a molecule is covalently attached to an atom of a

second molecule with no intervening linker or spacer. In many conjugation schemes, the ﬁ nal com-

plex is bound together by virtue of chemical components that add foreign structures to the sub-

stances being crosslinked. In some applications, the presence of these intervening linkers may be

detrimental to the intended use. For instance, in the preparation of hapten–carrier conjugates the

complex is formed with the intention of generating an immune response to the attached hapten.

Occasionally, a portion of the antibodies produced by this response will have speciﬁ city for the

crosslinking agent used in the conjugation procedure. Zero-length crosslinking agents eliminate the

potential for this type of cross-reactivity by mediating a direct linkage between two substances.

The reagents described in this section can initiate the formation of three types of bonds: an

amide linkage made by the condensation of a primary amine with a carboxylic acid, a phospho-

ramidate linkage made by the reaction of an organic phosphate group with a primary amine,

and a secondary or tertiary amine linkage made by the reductive amination of a primary or sec-

ondary amine with an aldehyde group. Therefore, using these reagent systems, substances con-

taining amines can be conjugated with other molecules containing phosphates or carboxylates.

Alternatively, substances containing amines can be crosslinked to molecules containing formyl

groups. All of the reactions are quite efﬁ cient, and depending on the reagent chosen and the

desired application, they may be performed in aqueous or nonaqueous environments.

1. Carbodiimides

Carbodiimides are used to mediate the formation of amide linkages between carboxylates and

amines or phosphoramidate linkages between phosphates and amines (Hoare and Koshland,

1966; Chu et al., 1986; Ghosh et al., 1990). They are probably the most popular type of zero-

length crosslinker in use, being efﬁ cient in forming conjugates between two protein molecules,

between a peptide and a protein, between an oligonucleotide and a protein, between a biomole-

cule and a surface or particle, or any combination of these with small molecules. There are two

basic types of carbodiimides: water-soluble and water-insoluble. The water-soluble ones are

Zero-Length Crosslinkers

the most common choice for biochemical conjugations, because most macromolecules of bio-

logical origin are soluble in aqueous buffer solutions. Not only is the carbodiimide itself able

to dissolve in the reaction medium, but the by-product of the reaction, an isourea, is also

water-soluble, facilitating easy puriﬁ cation. Water-insoluble carbodiimides, by contrast, are

used frequently in peptide synthesis and other conjugations involving molecules soluble only

in organic solvents. Both the organic-soluble carbodiimides and their isourea by-products are

insoluble in water.

1.1. EDC

EDC (or EDAC; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) is the most

popular carbodiimide used for conjugating biological substances containing carboxylates and

amines. In fact, it also may be the most frequently used crosslinking agent of all. Its application

in particle and surface conjugation procedures along with NHS ( N -hydroxysulfosuccinimide)

or sulfo-NHS is nearly universal (Chapter 14) and this fact makes it the most common bio-

conjugation reagent in use today. EDC is water-soluble, which allows for its direct addition

to a reaction without prior organic solvent dissolution. Both the reagent itself and the isourea

formed as the by-product of the crosslinking reaction are water-soluble and may be removed

easily by dialysis or gel ﬁ ltration (Sheehan et al., 1961, 1965). The reagent is, however, labile

in the presence of water. The bulk chemical should be stored desiccated at 20°C. Warm the

bottle to room temperature before opening to prevent condensation occurring that will cause

decomposition of the reagent over time. A concentrated solution of EDC in water may be pre-

pared to facilitate the addition of a small molar amount to a reaction, but the stock solution

should be dissolved rapidly and used immediately to prevent extensive loss of activity.

A variety of chemical conjugates may be formed using EDC (Chu et al., 1976, 1982; Chu

and Ueno, 1977; Yamada et al., 1981; Chase et al., 1983), provided one of the molecules con-

tains an amine and the other a carboxylate group. N-substituted carbodiimides can react with

carboxylic acids to form highly reactive, o-acylisourea intermediates ( Figure 3.1 ). This active

species then can react with a nucleophile such as a primary amine to form an amide bond

(Williams and Ibrahim, 1981). Other nucleophiles are also reactive. Sulfhydryl groups may

attack the active species and form thiol ester linkages, although these are not as stable as the

bond formed with an amine. In addition, oxygen atoms may act as the attacking nucleophile,

such as those in water molecules. In aqueous solutions, hydrolysis by water is the major com-

peting reaction, cleaving off the activated ester intermediate, forming an isourea, and regener-

ating the carboxylate group (Gilles et al. , 1990).

216 3. Zero-Length Crosslinkers

Nakajima and Ikada (1995) investigated the reactions of EDC amide bond formation in

aqueous solution using hydrogels of acrylic acid- or maleic acid-containing polymers or other

carboxylate molecules to contribute the activatable groups and ethylene diamine or ben-

zylamine as the amine functional groups to be conjugated. Their results indicate that carbox-

ylate activation occurs most effectively with EDC at pH 3.5–4.5, while amide bond formation

occurs with highest yield in the range of pH 4–6. However, EDC hydrolysis occurs maximally

at acidic pH values with increasing stability of the carbodiimide in solution at or above pH 6.5.

When working with proteins and peptides, experience indicates that EDC-mediated amide

bond formation effectively occurs between pH 4.5 and 7.5. Beyond this pH range, however,

the coupling reaction occurs more slowly with lower yields.

The presence of both carboxylates and amines on one of the molecules to be conjugated

with EDC may result in self-polymerization, because the substance then can react with another

molecule of its own kind instead of the desired target. For instance, when conjugating pep-

tides to carrier proteins using EDC, the peptide usually contains both a carboxylate and an

amine. The result typically is peptide polymerization in addition to coupling to the carrier (see

Chapter 19, Section 3). For this type of immunogen conjugation, polymerization is not usually

detrimental to its use, because polymerized peptide is also immunogenic. However, for other

crosslinking applications where it may be more desirable to avoid oligomer formation, the use

of a carbodiimide may not be the best choice of reagent, especially if one of the molecules

being conjugated contains both a carboxylate and an amine.

Most references to the use of EDC describe the optimal reaction medium to be at a pH from

4.7 to 6.0. However, the carbodiimide reaction occurs effectively up to at least pH 7.5 without

signiﬁ cant loss of yield. Conjugations done under mildly alkaline pH conditions (e.g., pH 8.5)

also can be done to limit the polymerization of proteins, while still facilitating the coupling

of a carboxylate-containing molecule at a low substitution level per protein. See Chapter 19,

Figure 3.1 EDC reacts with carboxylic acids to create an active-ester intermediate. In the presence of an amine

nucleophile, an amide bond is formed with release of an isourea by-product.

1. Carbodiimides 217

Section 3 for additional information on the properties of EDC conjugation using small peptides

coupled to carrier proteins.

Some procedures recommend the use of water as the solvent in an EDC reaction, while the

pH is maintained constant by the addition of HCl. Buffered solutions are more convenient,

because the pH does not have to be monitored during the course of the reaction. For acidic pH

conjugations, MES [2-( N-morpholino)ethane sulfonic acid] buffer at 0.1 M works well. When

doing neutral pH reactions, a phosphate buffer at 0.1 M is appropriate. Any buffers may be

used that do not interfere with the reaction, but avoid amine- or carboxylate-containing buffer

salts or other components in the medium that may react with the carbodiimide.

There are some side reactions that may occur when using EDC with proteins. In addition to

reacting with carboxylates, EDC itself can form a stable complex with exposed sulfhydryl groups

(Carraway and Triplett, 1970). Tyrosine residues can react with EDC, most likely through the phe-

nolate ionized form of its side chain (Carraway and Koshland, 1968). The imidazolyl group of

histidine may react with sulfo-NHS esters, resulting in an active carbonyl imidazole group which

subsequently hydrolyzes (Cuatrecasas and Parikh, 1972). Finally, EDC may promote unwanted

polymerization due to the usual abundance of both amines and carboxylates on protein molecules.

The following protocol is a generalized description of how to conjugate a small amine- or

carboxylate-containing molecule to a protein. The protocol may be modiﬁ ed by changing the

pH, buffer salts, and ratios of reactants to obtain the desired product. Speciﬁ c protocols utilizing

EDC in selected conjugation applications may be found in Part III. In some cases, the parameters

of this generalized protocol may have to be modiﬁ ed to retain solubility or activity of the result-

ing conjugate. For instance, coupling hydrophobic molecules to the surface of proteins often

causes partial or complete precipitation. This problem may be somewhat alleviated by decreas-

ing either the amount of EDC or the amount of the hydrophobic molecule added to the reaction,

thus resulting in a lower density of substitution. Protocols on the use of EDC to couple proteins

or other molecules to particles may be found in Chapter 14 and Chapter 9, Section 10.

Protocol

1. Dissolve the protein to be modiﬁ ed at a concentration of 10 mg/ml in one of the fol-

lowing reaction media: (a) water, (b) 0.1 M MES, pH 4.7–6.0, or (c) 0.1 M sodium

phosphate, pH 7.3. NaCl may be added (i.e., 0.15 M) if desired. If lower or higher con-

centrations of the protein are used, adjust the amounts of the other reactants added as

necessary to maintain the correct molar ratios. For the preparation of a peptide–protein

immunogen conjugate, a 200 l solution of the carrier protein at a concentration of

10 mg/ml in 0.1 M MES, pH 4.7 usually works well.

2. Dissolve the molecule to be coupled in the same buffer used in step 1. For small mol-

ecules, add them to the reaction in at least a 10-fold molar excess to the amount of pro-

tein present. If possible, the molecule may be added directly to the protein solution in

the appropriate excess. Alternatively, dissolve the molecule in the buffer at a higher con-

centration, and then add an aliquot of this stock solution to the protein solution. In the

example of preparing a peptide–protein conjugate, dissolve the peptide in 0.1 M MES,

pH 4.7, at a concentration of up to 2 mg/500  l.

3. Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold

molar excess of small molecule to protein. In the case of the peptide–protein immunogen

conjugate, add the 500  l of peptide solution to the 200  l of protein solution.

218 3. Zero-Length Crosslinkers

4. Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar excess

of EDC to the protein. Alternatively, a 0.5–0.1 M EDC concentration in the reaction

mixture usually works well. To make it easier to add the correct quantity of EDC, a

higher concentration stock solution may be prepared if it is dissolved and used immedi-

ately. To prepare the peptide–protein conjugate, add the solution from step 3 to 10 mg of

EDC in a test tube. Mix to dissolve. If this ratio of EDC to peptide or protein results in

precipitation, scale back the amount of carbodiimide addition until a soluble conjugate

is obtained. For some proteins, as little as 0.1 times this amount of EDC may have to be

used to maintain solubility.

5. React for 2 hours at room temperature.

6. Purify the conjugate by gel ﬁ ltration or dialysis using the buffer of choice (for many con-

jugates 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 is appropriate). If some turbid-

ity has formed during the conjugation procedure, it may be removed by centrifugation or

ﬁ ltration. When using EDC to prepare immunogen conjugates, the presence of some pre-

cipitated material is usually not of concern, because precipitated immunogens are often

more immunogenic than soluble proteins.

1.2. EDC Plus Sulfo-NHS

The water-soluble carbodiimide EDC may be used to form active ester functionalities with car-

boxylate groups using the water-soluble compound, NHS (sulfo-NHS) (Thermo Fisher). Sulfo-

NHS esters are hydrophilic reactive groups that couple rapidly with amines on target molecules

(Staros, 1982; Denney and Blobel, 1984; Kotite et al., 1984; Beth et al., 1986; Donovan and

Jennings, 1986; Jennings and Nicknish, 1985; Ludwig and Jay, 1985; Anjaneyulu and Staros,

1987). Unlike non-sulfonated NHS esters that are relatively water-insoluble and must be ﬁ rst

dissolved in organic solvent before being added to aqueous solutions, sulfo-NHS esters typi-

cally are water-soluble, longer-lived, and don ’t hydrolyze quite as quickly in water. However,

in the presence of amine nucleophiles that can attack at the carbonyl group of the ester, the

sulfo-NHS group rapidly leaves, creating a stable amide linkage with the amine. Sulfhydryl

and hydroxyl groups also will react with such active esters, but the products of such reactions,

thioesters and esters, are relatively unstable compared to an amide bond.

The advantage of adding sulfo-NHS to EDC reactions is to increase the solubility and sta-

bility of the active intermediate, which ultimately reacts with the attacking amine. EDC reacts

with a carboxylate group to form an active ester ( o-acylisourea) leaving group. Unfortunately,

this reactive complex is slow to react with amines and can hydrolyze in aqueous solutions, hav-

ing a rate constant measured in seconds (Hoare and Koshland, 1967). If the target amine does

not ﬁ nd the active carboxylate before it hydrolyzes, the desired coupling cannot occur. This

is especially a problem when the target molecule is in low concentration compared to water,

as in the case of protein molecules. In addition, Nakajima and Ikada (1995) found that if a

carboxylate-containing compound can form an anhydride from the o-acylisourea intermedi-

ate reactive ester, then the yield of amide bond formation is increased. In a similar approach,

forming a sulfo-NHS ester intermediate from the reaction of the hydroxyl group on sulfo-NHS

with the EDC active-ester complex dramatically increases the resultant amide bond formation.

Since the concentration of added sulfo-NHS usually is much greater than the concentration of

target molecule, the reaction preferentially proceeds through the more efﬁ cient sulfo-NHS ester

1. Carbodiimides 219