Bioconjugate Reagents

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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 ...
PART II

Bioconjugate Reagents

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 fluorescent, labeling molecules with biospecific ligands for subsequent affinity 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, regardless of the particular need, a workable reagent system that will yield a useful derivative almost always can be found. The best and most effective of the reported reagent systems usually are available from commercial sources, and thus do not even have to be synthesized. In Part II, the reagents of modification 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 ligands, peptides, proteins, carbohydrates, nucleic acids, oligonucleotides,

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lipids, and a host of other compounds may be accomplished by the judicious choice of the appropriate reagent system. The following reagents have been used in everything from benchscale experiments in research laboratories to process-optimized applications in the diagnostic and therapeutic industries. Conjugated or modified molecules have been applied in procedures designed to visualize target substances, as key components in clinical assay systems, and in the latest affinity-directed therapeutics, such as anti-tumor immunotoxins. Some of the reagent systems described in Part II have formed the basis for literally a multi-billion dollar biotechnology industry.

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3 Zero-Length Crosslinkers

The smallest available reagent systems for bioconjugation are the so-called zero-length crosslinkers. These compounds mediate the conjugation of two molecules by forming a bond containing 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 final complex is bound together by virtue of chemical components that add foreign structures to the substances 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 specificity 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 phosphoramidate 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 secondary amine with an aldehyde group. Therefore, using these reagent systems, substances containing 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 efficient, 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 zerolength crosslinker in use, being efficient in forming conjugates between two protein molecules, between a peptide and a protein, between an oligonucleotide and a protein, between a biomolecule 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 215

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the most common choice for biochemical conjugations, because most macromolecules of biological 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 purification. 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 bioconjugation 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 filtration (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 prepared 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 contains 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 competing reaction, cleaving off the activated ester intermediate, forming an isourea, and regenerating the carboxylate group (Gilles et al., 1990).

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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.

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 benzylamine as the amine functional groups to be conjugated. Their results indicate that carboxylate 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 peptides 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 significant 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,

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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 phenolate 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 modified by changing the pH, buffer salts, and ratios of reactants to obtain the desired product. Specific 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 modified to retain solubility or activity of the resulting conjugate. For instance, coupling hydrophobic molecules to the surface of proteins often causes partial or complete precipitation. This problem may be somewhat alleviated by decreasing 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 modified at a concentration of 10 mg/ml in one of the following 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 concentrations 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 molecules, add them to the reaction in at least a 10-fold molar excess to the amount of protein 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 concentration, 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.

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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 immediately. 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 filtration or dialysis using the buffer of choice (for many conjugates 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 is appropriate). If some turbidity has formed during the conjugation procedure, it may be removed by centrifugation or filtration. When using EDC to prepare immunogen conjugates, the presence of some precipitated 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 carboxylate groups using the water-soluble compound, NHS (sulfo-NHS) (Thermo Fisher). SulfoNHS 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 first dissolved in organic solvent before being added to aqueous solutions, sulfo-NHS esters typically 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 stability 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, having a rate constant measured in seconds (Hoare and Koshland, 1967). If the target amine does not find 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 intermediate 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 efficient sulfo-NHS ester

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Figure 3.2 The efficiency of an EDC-mediated reaction may be increased through the formation of a sulfoNHS ester intermediate. The sulfo-NHS ester is more effective at reacting with amine-containing molecules. Thus, higher yields of amide bond formation may be realized using this two-step process as opposed to using a single-step EDC reaction.

intermediate. However, the final product of this two-step reaction is identical to that obtained using EDC alone: the activated carboxylate reacts with an amine to give a stable amide linkage (Figure 3.2). EDC/sulfo-NHS coupled reactions are highly efficient and usually increase the yield of conjugation significantly over that obtainable solely with EDC. Staros et al. (1986) shows that the addition of just 5 mM sulfo-NHS to the EDC coupling of glycine to keyhole limpet hemocyanin increased the yield of derivatization about 20-fold as compared to using EDC alone. This technique also can be used to create activated proteins containing sulfo-NHS esters (Grabarek and Gergely, 1990). A protein can be incubated in the presence of EDC/sulfo-NHS, the active ester form isolated and then mixed with a second protein or other amine-containing molecule for conjugation. This two-step process allows the active species to form only on one protein, thus gaining greater control over the conjugation (Figure 3.3). In addition to the potential side reactions of EDC as mentioned previously (Section 1.1, this chapter), the additional efficiency obtained by the use of a sulfo-NHS intermediate in the process may cause other problems. In some cases, the conjugation actually may be too efficient to result in a soluble or active complex. Particularly when coupling some peptides to carrier proteins, the use of EDC/sulfo-NHS often causes severe precipitation of the conjugate. Scaling back the amount of EDC/sulfo-NHS added to the reaction may be done to solve this problem. However, eliminating the addition of sulfo-NHS altogether may have to be done in some instances to preserve the solubility of the final product. The following protocol is a generalized description of how to incorporate sulfo-NHS ester intermediates in EDC conjugation procedures. For specific applications of this technology, the amount of each reagent and unconjugated species may have to be adjusted to obtain an optimal conjugate. See also Chapter 14 and Chapter 9, Section 10 for protocols using EDC/sulfo-NHS in the coupling of proteins to particles and quantum dots, respectively.

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Figure 3.3 EDC may be used in tandem with sulfo-NHS to create an amine-reactive protein derivative containing active ester groups. The activated protein can couple with amine-containing compounds to form amide bond linkages.

Protocol 1. Dissolve the protein to be modified at a concentration of 1–10 mg/ml in 0.1 M sodium phosphate, pH 7.4. NaCl may be added to this buffer if desired. For the modification of keyhole limpet hemocyanin (KLH; Thermo Fisher) as described by Staros et al., 1986, include 0.9 M NaCl to maintain the solubility of this high-molecular-weight protein. If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants as necessary to maintain the correct molar ratios. 2. Dissolve the molecule to be coupled in the same buffer used in step 1. For small molecules, add them to the reaction in at least a 10-fold molar excess over the amount of protein 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 concentration, and then add an aliquot of this stock solution to the protein solution. 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. 4. Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar excess of EDC over the amount of protein present. Alternatively, a 0.05–0.1 M EDC concentration

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in the reaction usually works well. Also, add sulfo-NHS (Thermo Fisher) to the reaction to bring its final concentration to 5 mM. To make it easier to add the correct quantity of EDC or sulfo-NHS, higher concentration stock solutions may be prepared if they are dissolved and used immediately. Mix to dissolve. If this ratio of EDC/sulfo-NHS to peptide or protein results in precipitation, scale back the amount of addition until a soluble conjugate is obtained. 5. React for 2 hours at room temperature. 6. Purify the conjugate by gel filtration or dialysis using the buffer of choice (for many conjugates 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 is appropriate). If some turbidity has formed during the conjugation procedure, it may be removed by centrifugation or filtration. A modification of a two-step protocol (Grabarek and Gergely, 1990) for the activation of proteins with EDC/sulfo-NHS and subsequent conjugation with amine-containing molecules if given below. The variation in the pH of activation from that described above provides greater stability for the active ester intermediate. At pH 6.0, the amines on the protein will be protonated and therefore be less reactive toward the sulfo-NHS esters that form. In addition, the hydrolysis rate of the esters is dramatically slower at slightly acid pH. Thus, the active species may be isolated in a reasonable time frame without significant loss in conjugation potential. To quench the unreacted EDC, 2-mercaptoethanol is added to form a stable complex with the remaining carbodiimide, according to Carraway and Triplett (1970). In the following protocol, sulfo-NHS is used instead of NHS so that active ester is more water-soluble and ester hydrolysis is slowed (Anjaneyulu and Staros, 1987; Thelen and Deuticke, 1988).

Protocol 1. Dissolve the protein to be activated in 0.05 M MES, 0.5 M NaCl, pH 6.0 (reaction buffer), at a concentration of 1 mg/ml. 2. Add to the solution in step 1 a quantity of EDC and sulfo-NHS (Thermo Fisher) to obtain a concentration of 2 mM EDC and 5 mM sulfo-NHS. To aid in aliquoting the correct amount of these reagents, they may be quickly dissolved in the reaction buffer at a higher concentration, and then a volume immediately pipetted into the protein solution to obtain the proper molar quantities. 3. Mix and react for 15 minutes at room temperature. 4. Add 2-mercaptoethanol to the reaction solution to obtain a final concentration of 20 mM. Mix and incubate for 10 minutes at room temperature. Note: If the protein being activated is sensitive to this level of 2-mercaptoethanol, instead of quenching the reaction chemically, the activation may be terminated by desalting (step 5). 5. If the reaction was quenched by the addition of 2-mercaptoethanol, the activated protein may be added directly to a second protein or other amine-containing molecule for conjugation. Alternatively, or if no 2-mercaptoethanol was added, the activated protein may be purified from reaction by-products by gel filtration using a desalting resin. The desalting operation should be done rapidly to minimize hydrolysis and recover as much active ester functionality as possible. The use of centrifugal spin columns of some sort may afford the greatest speed in purification (Thermo Fisher). After purification, add the activated protein to the second molecule for conjugation. The second protein or other

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amine-containing molecule should be dissolved in 0.1 M sodium phosphate, pH 7.5. This will bring the pH of the coupling medium above pH 7.0 to initiate the active ester reaction. 6. React for at least 2 hours at room temperature. 7. Remove excess reactants by gel filtration or dialysis.

1.3. CMC CMC, or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (usually synthesized as the metho p-toluene sulfonate salt) (Aldrich), is a water-soluble reagent used to form amide bonds between one molecule containing a carboxylate and a second molecule containing an amine. The presence of the positively charged morpholino group creates its water solubility. Along with EDC (Section 1.1, this chapter), CMC is the only other soluble carbodiimide commonly available for biological conjugations. It was first utilized in peptide synthesis (Sheehan and Hlavka, 1956) and found to be superior to other coupling agents used at the time (Ondetti and Thomas, 1965). It also has been used for the quantitative modification and estimation of total carboxyl groups in protein molecules (Hoare and Koshland, 1967) and for investigating the secondary structure of nucleic acids (Metz and Brown, 1969). Another early application area of CMC, relates not to solution phase crosslinking of two molecules, but to coupling of ligands to insoluble support materials for use in affinity chromatography (Lowe and Dean, 1971; Marcus and Balbinder, 1972; Schmer, 1972).

CMC reacts with carboxylate groups by addition of the carboxyl across one of its diimide bonds, resulting in the characteristic active ester, o-acylisourea intermediate common to all carbodiimide mechanisms. Nucleophilic attack on this intermediate yields the acylated product—usually an amide bond, resulting from the reaction with a primary amine (Figure 3.4). However, carbodiimide chemistry does create several potential side reactions. Sulfhydryl groups may react with CMC to form a stable covalent complex unreactive toward further conjugation. The reagent also may react with phenols, alcohols, and other nucleophiles to quench the crosslinking reaction. In aqueous solutions, hydrolysis of the carbodiimide and the active ester are by far the most frequent side reactions. Reaction of the ester with water molecules regenerates the carboxylate and releases a soluble isourea by-product. CMC should be able to participate in the two-step reaction using a sulfo-NHS ester intermediate similar to EDC, however there are no reports in the literature to this effect. Protocols for the use of this reagent in biological crosslinking applications should be essentially the same as those given previously for EDC, except substituting a molar equivalent quantity of CMC. See Sections 1.1 and 1.2 in this chapter for additional information concerning carbodiimide reactions.

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Figure 3.4 The water-soluble carbodiimide CMC reacts with carboxylates to form an active-ester intermediate. In the presence of amine-containing molecules, amide bond formation can take place with release of an isourea by-product.

1.4. DCC DCC (dicyclohexyl carbodiimide) is one of the most frequently used coupling agents, especially in organic synthesis applications. It has been used for peptide synthesis since 1955 (Sheehan and Hess, 1955) and continues to be a popular choice for creating peptide bonds (Barany and Merrifield, 1980). DCC is water-insoluble, but it has been used in 80 percent DMF for the immobilization of small molecules onto carboxylate-containing chromatography supports for use in affinity separations (Larsson and Mosbach, 1971; Lowe et al., 1973). In addition to forming amide linkages, DCC has been used to prepare active esters of carboxylate-containing compounds using NHS or sulfo-NHS (Staros, 1982). Unlike the EDC/sulfo-NHS reaction described in Section 1.2 (this chapter), active ester synthesis done with DCC is in organic solvent, and therefore doesn’t have the hydrolysis problems of water-soluble EDC-formed esters. Thus, DCC is most often used to synthesize active ester containing crosslinking and modifying reagents and not to perform biomolecular conjugations.

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Figure 3.5 The organic-soluble carbodiimide DCC is often used to create amide bonds, especially between water-insoluble compounds.

DCC is a waxy solid that is often difficult to remove from a bottle. Its vapors are extremely hazardous to inhalation and to the eyes. It should always be handled in a fume hood. The isourea by-product of a DCC-initiated reaction, dicyclohexyl urea (DCU) (Figure 3.5), is also water-insoluble and must be removed by organic solvent washing. For synthesis of peptides or affinity supports on insoluble matrices this is not a problem, because washing of the support material can be done without disturbing the conjugate coupled to the support. For solution phase chemistry, however, reaction products must be removed by solvent washings, precipitations, or recrystallizations. A potential undesirable effect of DCC coupling reactions is the spontaneous rearrangement of the o-acylisourea to an inactive N-acylurea (Stewart and Young, 1984) (Figure 3.6). The rate of this rearrangement is dramatically increased in aprotic organic solvents, such as DMF. The activation efficiency of DCC is extraordinarily high, especially in anhydrous solutions that don’t have competing hydrolysis problems. o-Acylisourea-activated carboxylates may undergo two-side reactions that form other active groups. If DCC is added to an excess of a carboxylate-containing molecule without the presence of an amine-containing target, then the activated carboxylate may react with another carboxylic acid to form a symmetrical anhydride (Figure 3.7). The formation of an anhydride intermediate may be a frequent mechanism in route to the creation of an amide bond with an amine, especially under anhydrous conditions (Rebek and Feitler, 1974; Nakajima and Ikada, 1995). In addition, a DCC-activated carboxylate may react with an amino acid to form an azlactone (Figure 3.8) (Coleman et al., 1990). Both the anhydride and the azlactone will react with amines to form covalent amide linkages. However, the ring-opening reaction of an azlactone will form a different product than the zerolength crosslinking result of coupling directly to an amine-containing molecule (Figure 3.9).

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Figure 3.6 The active-ester intermediate formed from the reaction of DCC with a carboxylate group may undergo rearrangement to an inactive N-acylisourea product.

Figure 3.7 The reaction of DCC with a carboxylate compound in excess may create anhydride products in the absence of nucleophiles.

Figure 3.8 A DCC-mediated reaction with a carboxylate group in the presence of a small amino acid may form azlactone rings.

1.5. DIC DIC (or diisopropyl carbodiimide) is another water-insoluble amide bond-forming agent that has advantages over DCC (Section 1.4, this chapter). It is a liquid at room temperature and

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Figure 3.9

An azlactone reacts with amine groups through a ring-opening process, creating amide bond linkages with the attacking nucleophile.

Figure 3.10 The symmetrical carbodiimide DIC reacts with carboxylates to form active-ester intermediates able to couple with amine-containing compounds to form amide bond linkages.

is therefore much easier to dispense than DCC. Its by-products, diisopropylurea and diisopropyl-N-acylurea, are more soluble in organic solvents than the DCU by-product of a DCC reaction. DIC reacts similarly to DCC, forming an active o-acylisourea intermediate with a carboxylic acid group (Figure 3.10). This active species may then react with a nucleophile such as an amine to form an amide bond. Presumably, all the possible side reactions that DCC may undergo are also possible with DIC, although it is not well documented.

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2. Woodward’s Reagent K Woodward’s reagent K is N-ethyl-3-phenylisoxazolium-3⬘-sulfonate, a zero-length crosslinking agent able to cause the condensation of carboxylates and amines to form amide bonds (Woodward and Olofson, 1961; Woodward et al., 1961). The reaction mechanism involved in activating a carboxylate includes the conversion of the reagent under alkaline conditions to a reactive ketoketenimine. This intermediate then reacts with a carboxylate to create an enol ester. The enol ester is highly susceptible to nucleophilic attack. The reaction with an amine proceeds to amide bond formation with loss of the inactive diketo derivative (Figure 3.11). In aqueous solution, the major side reaction is hydrolysis which occurs rapidly (Dunn and Affinsen, 1974). Although Woodward’s reagent K has been used successfully for conjugation applications with proteins and other molecules to form amide linkages (Boyer, 1986; Pikuleva and Turko, 1989), its mechanism of reaction was called into question by Johnson and Dekker (1996), who found that the compound reacted with cysteine and histidine groups in E. coli L-threonine dehydrogenase, not the available aspartate or glutamate groups. Woodward’s reagent K is available from Fluka.

3. N,N⬘-Carbonyldiimidazole CDI (or N,N⬘-carbonyldiimidazole) is a highly active carbonylating agent that contains two acylimidazole leaving groups (Aldrich). The result is that CDI can activate carboxylic acids or hydroxyl groups for conjugation with other nucleophiles, creating either zero-length amide bonds or one-carbon-length N-alkyl carbamate linkages between the crosslinked molecules. Carboxylic acid groups react with CDI to form N-acylimidazoles of high reactivity. The active intermediate forms in excellent yield due to the driving force created by the liberation of carbon dioxide and imidazole (Anderson, 1958). The active carboxylate then can react with amines to form amide bonds or with hydroxyl groups to form ester linkages (Figure 3.12). Both reaction

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Figure 3.11 Woodward’s reagent K undergoes a rearrangement in alkaline solution to form a reactive ketoketenimine. This active species can react with a carboxylate group to create another active group, an enol ester derivative. In the presence of amine nucleophiles, amide bond formation takes place.

Figure 3.12 CDI reacts with carboxylate groups to form an active acylimidazole intermediate. In the presence of an amine nucleophile, amide bond formation can take place with release of imidazole.

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mechanisms have 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). 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. However, if CDI is used to activate a hydroxyl functional group, the reaction proceeds quite differently. The active intermediate formed by the reaction of CDI with an ᎏOH group is an imidazolyl carbamate (Figure 3.13). Attack by an amine releases the imidazole, but not the carbonyl. Thus, a hydroxylcontaining molecule may be coupled to an amine-containing molecule with the result of a one-carbon spacer, and forming a stable urethane (N-alkyl carbamate) linkage. This coupling rocedure has been applied to the activation of hydroxyl-containing chromatography supports for the immobilization of amine-containing affinity ligands (Bethell et al., 1979; Hearn et al., 1979, 1983) and also to the activation of polyethylene glycol for the modification of aminecontaining macromolecules (Beauchamp et al., 1983). In addition, Chapter 14 describes the use of CDI for the activation of particles to immobilize proteins or other affinity ligands. CDI-activated hydroxyls also may undergo a side reaction to form active carbonates. This occurs when an imidazolyl carbamate reacts with another hydroxyl group before the second hydroxyl has had a chance to get activated with CDI. Particularly with adjacent hydroxyls on the same molecule, this can be a problem if a defined reactive species is desired. Any carbonates formed, however, are still reactive toward amines to create carbamate linkages. Formation of the activated species, whether with a carboxylate or a hydroxyl, must take place in nonaqueous environments due to the rapid breakdown of CDI by hydrolysis. Even in solvents containing small amounts of water, CDI quickly hydrolyzes to CO2 and imidazole. It is best to use solvents with less than 0.1 percent water to prevent extensive CDI breakdown.

Figure 3.13 CDI reacts with hydroxyl groups to form an active imidazole carbamate intermediate. In the presence of amine-containing compounds, a carbamate linkage is created with loss of imidazole.

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Characteristic bubble formation is an indication of reagent hydrolysis, although CO2 also is released upon reaction with a carboxylic acid. Activation of carboxylates or hydroxyls may be done in dry organic solvents such as acetone, dioxane, DMSO, THF, or DMF. If an excess of CDI is used during the activation step, it should be removed before adding the active intermediate to an amine-containing molecule for conjugation. Alternatively, equal molar quantities of CDI and the molecule to be activated may be mixed to form the active species. After about an hour of activation, add an equivalent molar quantity of the amine-containing target molecule to be conjugated. Aqueous reaction conditions that result in the best conjugation yields using CDI usually reflect the relative pKa of the nucleophilic amine being coupled. Proteins are best coupled to CDI-activated supports or molecules in an environment at least one pH unit above their pI values. Frequently the greatest coupling yields occur in alkaline buffers within the range of pH 8.0–10.0. In aqueous solutions, CDI-activated carboxylates or hydroxyls will hydrolyze and slowly lose activity. N-Acylimidazoles hydrolyze by loss of imidazole and regenerate the original carboxylate. The imidazole carbamate active species hydrolyzes by loss of CO2 and imidazole, regenerating in this case, the original hydroxyl group. CDI-activated carboxylic acids hydrolyze faster in aqueous solutions than CDI-activated hydroxyls; however, both experience increasing hydrolysis with increasing pH. Conjugation reactions using CDI also may be done in organic solutions. This is a distinct advantage if the reactants are not very soluble in aqueous environments. In addition, organic coupling will not result in concomitant loss of activity due to hydrolysis as water-based reactions, thus nonaqueous reactions usually will provide greater yields. A protocol for the use of CDI in the activation of poly(ethylene glycol) is discussed in Chapter 25, Section 1.4, while CDI activation procedures for particles are described in Chapter 14.

4. Schiff Base Formation and Reductive Amination Aldehydes and ketones can react with primary and secondary amines to form Schiff bases. A Schiff base is a relatively labile bond that is readily reversed by hydrolysis in aqueous solution. The formation of Schiff bases is enhanced at alkaline pH values, but they are still not completely stable unless reduced to secondary or tertiary amine linkages (Figure 3.14). A number of reducing agents can be used to convert specifically the Schiff base into an alkylamine linkage. Once reduced, the bonds are highly stable. The use of reductive amination to conjugate an aldehydecontaining molecule to an amine-containing molecule results in a zero-length crosslink where no additional spacer atoms are introduced between the molecules. Reductive amination (or alkylation) may be used to conjugate an aldehyde- or ketonecontaining molecule with an amine-containing molecule. The reduction reaction is best facilitated by the use of a reducing agent such as sodium cyanoborohydride, because the specificity of this reagent is toward the Schiff base structure and will not affect the original aldehyde groups. By contrast, sodium borohydride also is used in this reaction, but its strong reducing power rapidly converts any aldehydes not yet reacted into non-reactive hydroxyls, effectively eliminating them from further participation in the conjugation process. Borohydride also may affect the activity of some sensitive proteins, whereas cyanoborohydride is gentler, successfully

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Figure 3.14 Carbonyl groups can react with amine nucleophiles to form reversible Schiff base intermediates. In the presence of a suitable reductant, such as sodium cyanoborohydride, the Schiff base is stabilized to a secondary amine bond.

preserving the activity of even some labile monoclonal a ntibodies. Cyanoborohydride has been shown to be at least 5 times milder than borohydride in reductive amination processes with antibodies (Peng et al., 1987). Other reducing agents that have been explored for reductive amination include various amine boranes and ascorbic acid (Cabacungan et al., 1982; Hornsey et al., 1986). Immobilization by reductive amination of amine-containing biological molecules onto aldehyde-containing solid supports has been used for quite sometime (Sanderson and Wilson, 1971). The reaction proceeds with excellent efficiency (Domen et al., 1990). The optimum pH for the reaction is alkaline, although good yield can be realized from pH 7 to 10. At the high end of this range (pH 9–10), the formation of the Schiff bases is more efficient, and the yield of conjugation or immobilization reactions can be dramatically increased (Hornsey et al., 1986). The introduction of aldehyde functional groups into proteins and other molecules can be accomplished by a number of methods (Chapter 1, Section 4.4). Glycoproteins may be oxidized at their carbohydrate residues using sodium periodate or a specific sugar oxidase. Amine groups may be modified to produce a formyl group by reacting with NHS-aldehydes or p-nitrophenyl diazopyruvate. The following generalized protocol assumes that the requisite groups are present on the two molecules to be conjugated. Protocol 1. Dissolve the amine-containing protein to be conjugated at a concentration of 1–10 mg/ml in a buffer having a pH between 7 and 10. Higher pH reactions will result in greater yield of conjugate formation. Suitable buffers include 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2; 0.1 M sodium borate, pH 9.5; or 0.05 M sodium carbonate, 0.1 M sodium citrate, pH 9.5. Avoid amine-containing buffers like Tris. 2. Add a quantity of the aldehyde-containing molecule to the solution in step 1 to obtain the desired molar ratio for conjugation. For instance, if the amine-containing protein is

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3.

4. 5.

6.

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an antibody and the aldehyde-containing protein is an enzyme such as horseradish peroxidase (HRP), a typical molar ratio for the reaction might be 2–4 moles of HRP per mole of antibody. Add 10 l of 5 M sodium cyanoborohydride in 1 N NaOH (Aldrich) per ml of the conjugation solution volume. Caution: Highly toxic compound. Use a fume hood and be careful to avoid skin contact with this reagent. React for 2 hours at room temperature. To block unreacted aldehyde sites, add 20 l of 3 M ethanolamine (pH adjusted to desired value with HCl) per ml of the conjugation solution volume. React for 15 minutes at room temperature. Purify the conjugate by dialysis or gel filtration using a buffer suitable for the nature of the proteins being crosslinked.

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