Hermanson G. Bioconjugate Techniques, Second Edition

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190 2. The Chemistry of Reactive Groups

Figure 2.2 A number of small thiol-containing molecules have proven useful for modiﬁ cation of gold or metal-

lic surfaces. The dithiol derivatives provide better dative bond stability and can ’t be displaced easily by compet-

ing thiols or oxidation. Most thiol-containing compounds used for surface modiﬁ cation also contain terminal

functional groups or reactive groups for coupling afﬁ nity ligands.

Figure 2.3 SAM surface modiﬁ cation has been done using monothiol and dithiol compounds containing PEG

linkers. Useful coatings typically contain mainly PEG-hydroxyl or PEG-monomethyl ether linkers that provide a

biocompatible lawn, which prevents nonspeciﬁ c binding of proteins to the metallic surface. About 10 percent of

the surface modiﬁ cations are done using a longer carboxylate-containing thiol-PEG linker that provides sites for

attachment of afﬁ nity ligands.

2.9. Native Chemical Ligation

Native chemical ligation involves reactions that are very similar to intein splicing and peptide

ligation found in certain protein expression systems. A peptide having a C-terminal thioester

reacts with an N-terminal cysteine residue in another peptide to undergo a transthioesteriﬁ ca-

tion reaction, which results in the formation of an intermediate thioester with the cysteine thiol.

However, due to the proximity of the neighboring -amine group on cysteine, a subsequent

nucleophilic attack of the electron-rich nitrogen on the ester carbonyl results in an S ;N shift,

this then forms a native amide (peptide) bond (Reaction 29).

(Reaction 29)

Native chemical ligation is effective at conjugating two peptides together in aqueous solu-

tion to form a longer peptide. The reaction proceeds at physiological pH under mild conditions

without any additional additives, except for the presence of the thioester-containing peptide

and the cysteine-containing peptide. In addition, other thioester compounds may be used in

this reaction to label speciﬁ cally N-terminal cysteine peptides through amide bond formation.

For instance, a biotin thioester derivative may be used to add a biotin group to a peptide or a

ﬂ uorescent label may be added by the same process. Thus, native chemical ligation is an excel-

lent way of discretely labeling peptides only at their N-terminal. See Chapter 17, Section 6 for

additional information on the use of this reaction.

2. Thiol Reactions 191

2.10. Cisplatin Modiﬁ cation of Methionine and Cysteine

Platinum complexes long have been used as tumor therapeutic agents for their ability to bind

to DNA at guanine and adenine residues and interfere with transcription (Repta and Long,

1980; Eastman, A., 1987; Reedijk et al., 1987). The reaction of Platinum II compounds with

nucleic acids has been studied in great detail (Anin et al., 1992), and the reactive group has

been applied to bioconjugation labeling reagents (van Belkum et al., 1994; Heetebrij et al .,

2003).

Cisplatin reagents also react with certain amino acid residues in proteins. Rapid coupling

occurs with methionine and cysteine residues over a broad pH range (2–9), while slower reac-

tion kinetics results in modiﬁ cation of histidine imidazole ring with an optimal pH at 8.0. The

relative rate of reaction of cisplatin derivatives for sulfur nucleophiles is over 100 times more

than their reaction with nitrogen nucleophiles (Hay and Porter, 1999). The basic structure of

cisplatin labeling reagents is shown in Figure 2.4 and Figure 2.5 illustrates the modiﬁ cation

reactions of a cisplatin derivative with amino acid or nucleic acid targets. The reactive group

is stable in aqueous conditions and covalent bonds formed with it are stable to typical condi-

tions used in biological assays and detection procedures. Reagent systems are available from

Kreatech.

3. Carboxylate Reactions

Chemical groups that speciﬁ cally react with carboxylic acids are limited in variety. In aqueous

solutions, the carboxylate functionality displays rather low nucleophilicity. For this reason, it

is unreactive with the great majority of bioconjugate reagents which couple through a nucle-

ophilic addition process.

Several important chemistries, however, have been developed that allow conjugation through

a carboxylate group. The following sections brieﬂ y describe these reactions.

192 2. The Chemistry of Reactive Groups

Figure 2.4 The general design of a cisplatin modiﬁ cation agent consists of the reactive cisplatin group and a

short linker that typically terminates in a detectable label.

3.1. Diazoalkanes and Diazoacetyl Compounds

Diazomethane and other diazoalkyl derivatives long have been used to label carboxylate

groups for analysis (Herriott, 1947; Riehm and Scheraga, 1965). A major application of such

3. Carboxylate Reactions 193

Figure 2.5 The cisplatin reactive group can covalently couple to methionine-, cysteine-, and histidine-containing

peptides or proteins. It also reacts with guanine groups to form a covalent modiﬁ cation on the N

nitrogen.

reagents has been in the HPLC analysis of low-molecular-weight compounds such as fatty

acids (DeMar et al., 1992). Several coumarin derivatives containing stable, carboxylate-

reactive diazoalkane functionalities also are available for ﬂ uorescent-labeling of target mole-

cules (Molecular Probes) (Ito and Sawanobori, 1982; Ito and Maruyama, 1983).

Diazoalkanes and diazoacetyl compounds (amides and esters) are spontaneously reactive

with carboxylate groups without addition of other reactants or catalysts. The reaction mecha-

nism involves attack of a negatively charged carboxylate oxygen atom on a protonated diazo-

alkyl group, liberating nitrogen gas, and forming a covalent linkage (Reaction 30).

(Reaction 30)

The reaction with carboxylates occurs over a range of pH values, but is optimal at pH 5.0.

Unfortunately, the diazoalkyl compounds will cross-react with sulfhydryl groups at this pH. At

higher pH conditions, the reaction is even less speciﬁ c due to reaction with other nucleophiles.

In aqueous solution, the most-likely side reaction is hydrolysis.

3.2. Carbonyldiimidazole

CDI, is a active carbonylating agent that contains two acylimidazole leaving groups (Chapter 3,

Section 3). CDI reacts with carboxylic acids under non-aqueous conditions to form N -acylimi-

dazoles of high reactivity (Reaction 31). The active intermediate forms in excellent yield due to

the driving force created by the liberation of carbon dioxide and imidazole (Anderson, 1958).

An active carboxylate then can react with amines to form amide bonds or with hydroxyl

groups to form ester linkages (Reaction 32). The reaction has been used successfully in peptide

synthesis (Paul and Anderson, 1960, 1962). In addition, activation of a styrene/4-vinylbenzoic

acid copolymer with CDI was used to immobilize the enzyme lysozyme through its available

amino groups to the carboxyl groups on the matrix (Bartling et al ., 1973).

(Reaction 31)

(Reaction 32)

194 2. The Chemistry of Reactive Groups

CDI functions as a zero-length crosslinker if the activated species is a carboxylic acid,

because the attack of another nucleophile liberates the imidazole leaving group. The conjuga-

tion reaction can be done in organic solvent or aqueous conditions, depending on the solubility

of the nucleophile. For aqueous coupling of N-acylimidazoles to amine-containing compounds,

optimal conditions include an alkaline pH environment from about pH 7–9 and in buffers con-

taining no amines (avoid Tris or imidazole).

3.3. Carbodiimides

Carbodiimides function as zero-length crosslinking agents capable of activating a carbox-

ylate group for coupling with an amine-containing compound. There are several major types

of carbodiimide reagents commonly available that can be used for organic or aqueous reac-

tions, depending on their individual solubility characteristics (Chapter 3, Section 1). The water

soluble reagents are used mainly for biological conjugations involving proteins and other mac-

romolecules. The water-insoluble carbodiimides can be used in peptide synthesis or for the syn-

thesis of other organic compounds.

Carbodiimides are used to mediate the formation of amide or phosphoramidate linkages

between a carboxylate and an amine or a phosphate and an amine, respectively (Hoare and

Koshland, 1966; Chu et al., 1986; Ghosh et al., 1990). Regardless of the type of carbodiimide,

the reaction proceeds by the formation of an intermediate o-acylisourea that is highly reactive

and short-lived in aqueous environments. The attack of an amine nucleophile on the carbonyl

group of this ester results in the loss an isourea derivative and formation of an amide bond (see

Reactions 11 and 12). The major competing reaction in water is hydrolysis.

4. Hydroxyl Reactions

Hydroxyl-reactive chemical compounds include not only those modiﬁ cation agents able to

directly form a stable linkage with an OH group, but also a broad range of reagents that

are designed to temporarily activate the group for coupling with a secondary functional group.

Many of the chemical methods for modifying hydroxyls originally were developed for use with

chromatography supports in the coupling of afﬁ nity ligands. Some of these same chemical reac-

tions have found application in bioconjugate techniques for crosslinking a hydroxyl-containing

molecule with another substance, usually containing a nucleophile. For instance, carbohydrate-

containing molecules such as polysaccharides or glycoproteins can be coupled through their

sugar residues using hydroxyl-speciﬁ c reactions. In addition, polymers and other organic com-

pounds containing hydroxyls (such as PEG) may be conjugated with another molecule using

these chemistries.

4.1. Epoxides and Oxiranes

An epoxide or oxirane group can react with nucleophiles in a ring-opening process. The reac-

tion can take place with primary amines, sulfhydryls, or hydroxyl groups to create secondary

4. Hydroxyl Reactions 195

amine, thioether, or ether bonds, respectively. See Section 1.7 (this chapter) for further informa-

tion on this reaction.

4.2. Carbonyldiimidazole

CDI, is a active carbonylating agent that contains two acylimidazole leaving groups (Chapter 3,

Section 3). The compound can react with a carboxylate to form an active acylimidazole group

capable of coupling with amine-containing molecules (Section 3.2, this chapter). However, CDI

also can react with hydroxyl groups to create a reactive intermediate. If CDI is used to acti-

vate a hydroxyl functional group, the reaction proceeds quite differently from its reaction with

carboxylates. The active intermediate formed by the reaction of CDI with an OH group is

an imidazolyl carbamate (Reaction 33). Attack by an amine releases the imidazole, but not

the carbonyl. Thus, hydroxyl-containing molecules may be coupled to amine-containing mol-

ecules with the result of a one-carbon spacer, forming stable urethane ( N-alkyl carbamate)

linkages (Reaction 34). This coupling procedure has been applied to the activation of hydroxyl-

containing chromatography supports for the immobilization of amine containing afﬁ nity lig-

ands (Bethell et al., 1979; Hearn et al., 1979, Hearn et al., 1983) and also to the activation of

PEG for the modiﬁ cation of amine-containing macromolecules (Beauchamp et al ., 1983).

(Reaction 33)

(Reaction 34)

4.3. N , N -Disuccinimidyl Carbonate or N -Hydroxysuccinimidyl Chloroformate

N , N -Disuccinimidyl carbonate (DSC) consists of a carbonyl group containing, in essence, two

NHS esters. The compound is highly reactive toward nucleophiles. In aqueous solutions, DSC

will hydrolyze to form two molecules of NHS with release of one molecule of CO

. In non-

aqueous environments, the reagent can be used to activate a hydroxyl group to a succinimi-

dyl carbonate derivative (Reaction 35). DSC activated hydroxylic compounds can be used to

conjugate with amine-containing molecules to form stable crosslinked products (Reaction 36).

The linkage created from this reaction is a urethane derivative or a carbamate bond, displaying

excellent stability.

196 2. The Chemistry of Reactive Groups

(Reaction 35)

(Reaction 36)

A related reagent, N-hydroxysuccinimidyl chloroformate also is a bifunctional carbonyl deriva-

tive containing an NHS ester and a acid chloride. In aqueous solutions, the compound is unstable

to hydrolysis, rapidly breaking down to NHS, CO

, and HCl. In non-aqueous environments, how-

ever, NHS-chloroformate may be used to activate a hydroxyl group similar to DSC. Reaction of

the chloroformate with a hydroxylic residue forms the same succinimidyl carbonate derivative as

the reaction of DSC with OH groups (Reaction 37). Subsequent conjugation with an amine-

containing compound yields a carbamate linkage. The bond is identical to that formed from the

reaction of CDI activated hydroxyls with amine-containing compounds (see previous section).

(Reaction 37)

4.4. Oxidation with Periodate

Sodium periodate can be used to oxidize hydroxyl groups on adjacent carbon atoms, forming

reactive aldehyde residues suitable for coupling with amine or hydrazide-containing molecules.

The reaction occurs with two adjacent secondary hydroxyls to cleave the carbon–carbon bond

between them and create two terminal aldehyde groups (Reaction 38). When one of the adjacent

hydroxyls is a primary hydroxyl, reaction with periodate releases one molecule of formalde-

hyde and leaves a terminal aldehyde residue on the original diol compound (Reaction 39). These

reactions can be used to generate crosslinking sites in carbohydrates or glycoproteins for subse-

quent conjugation of amine-containing molecules by reductive amination (Chapter 1, Section

4.4 and Chapter 3, Section 4). Sodium periodate also reacts with 2-aminoethanol derivatives—

compounds containing a primary amine and a secondary hydroxyl group on adjacent carbon

atoms. Oxidation cleaves the carbon–carbon bond, forming a terminal aldehyde group on the

side that had the original hydroxyl residue (Reaction 40). This reaction can be used to create

reactive aldehydes on N-terminal serine residues of peptides (Geoghegan and Stroh, 1992).

4. Hydroxyl Reactions 197

(Reaction 38)

(Reaction 39)

(Reaction 40)

4.5. Enzymatic Oxidation

Certain enzymes may be used to oxidize hydroxyl-containing carbohydrates to create aldehyde

groups (Chapter 1, Section 4.4). For example, the reaction of galactose oxidase on terminal

galactose or N -acetyl- D-galactose residues proceeds to form C-6 aldehyde groups on polysac-

charide chains (Reaction 41). These groups then can be used for conjugation reactions with

amine or hydrazide-containing molecules.

(Reaction 41)

4.6. Alkyl Halogens

Reactive alkyl halogen compounds can be used to modify speciﬁ cally hydroxyl groups in car-

bohydrates, polymers, and other molecules. Chloro- or bromo-derivatives of short alkyl chains

198 2. The Chemistry of Reactive Groups

containing an electron withdrawing second functional group on their other end (typically

a carboxylate group) can be used to form spacer arms useful for conjugation with another

substance. Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups of

dextran, forming the carboxymethyl derivative (Reaction 42). The carboxylates then may be

coupled with amine-containing molecules using a carbodiimide reaction scheme. In a some-

what similar approach, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting

6-bromohexanoic acid with a dextran polymer (Chapter 25, Section 2.2).

(Reaction 42)

Modiﬁ cation of hydroxyl groups with such compounds can be done in 3–10 M NaOH by

reacting from 25°C to 40°C for 1.5–4 hours.

4.7. Isocyanates

Isocyanates can be formed from the reaction of an aromatic amine with phosgene (Rifai and

Wong, 1986). They also can be created from acyl azides by treatment at 80°C in the presence of

an alcohol (Chapter 9, Section 5). In the transformation, the acyl azide group rearranges to form

an isocyanate that can react with hydroxyl-containing molecules to form a urethane (carbamate)

linkage (Reaction 43). The reactivity of isocyanates is excellent, but for the same reason their

stability can be a problem. In storage, moisture decomposes them, releasing CO

and leaving

an aromatic amine in its place. In aqueous environments, the aromatic amine can react with

another molecule of isocyanate to form a urea derivative (Annunziato et al., 1993).

(Reaction 43)

Isocyanate-containing reagents can be used to crosslink or label hydroxyl-containing mol-

ecules, including polysaccharides. Carbohydrate modiﬁ cation can be done without the need for

prior oxidation of sugar residues with periodate to form reactive aldehydes, as is common in

many protocols (Chapter 1, Section 4.4). The reaction occurs best at alkaline pH values (e.g.,

pH 8.5). Many coupling protocols avoid the hydrolysis problem by performing the reaction in

organic solvent (i.e., DMSO).

4. Hydroxyl Reactions 199