bioconjugate techniques || zero-length cross-linkers

3 Zero-Length Cross-linkers

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 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 cross-Unked. 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 cross-linking agent used in the conjugation procedure. Zero-length cross-linking agents eliminate the potential for this type of cross-reactivity by mediating a direct linkage between two substances.

The reagents described in this chapter can initiate the formation of three types of bonds: an amide linkage made by the condensation of a primary amine with a car-boxylic acid, a phosphoramidate linkage made by the reaction of a 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 cross-linked 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 a car-boxylate and an amine or phosphoramidate linkages between a phosphate and an amine (Hoare and Koshland, 1966; Chu et al, 1986; Ghosh et al., 1990). They are probably the most popular type of zero-length cross-linker in use, being efficient in forming conjugates between two protein molecules, between a peptide and a protein, between oligonucleotides and proteins, or any combination of these with small mole-

169

170 3. Zero-Length Cross-linkers

cules. There are two basic types of carbodiimides: water-soluble and water-insoluble. The water-soluble ones are 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; l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] is perhaps the most popular carbodiimide for use in conjugating biological substances. Its water solubility allows for direct addition to a reaction without prior organic solvent dissolution. Excess reagent and the isourea formed as the by-product of the cross-linking reaction are both water-soluble and may be easily removed by dialysis or gel filtration (Sheehan et ai, 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 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.

EDC 1-Ethyl-3-(3-dimethylaminopropyl)

Carbodiimide Hydrochloride MW 191.7

A variety of chemical conjugates may be formed using EDC (Yamada et al., 1981; Chase et al, 1983; Chu et al, 1976, 1982; Chu and Ueno, 1977), provided one of the molecules contains a primary amine and the other a carboxylate group. N-substituted carbodiimides can react with carboxylic acids to form a highly reactive, O-acylisourea intermediates (Fig. 106). This active species can then react with a nucleophile such as a primary amine to form an amide bond (Williams and Ibrahim, 1981). Other nucleo-philes 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 etal, 1990). The potential for hydrolysis of the active ester is reflected in the rate constant at pH 4.7 for the water-soluble carbodiimide, EDC, of only 2 - 3 s"^.

1. Carbodiimides 171

OH

Carboxylic Acid

o-Acylisourea Active Intermediate

N N"

Isourea By-product

I CH,

Figure 106 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.

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 can then 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 9, 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 cross-linking 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 between 4.7 and 6. Flowever, the carbodiimide reaction occurs effectively up to at least pH 7.5 without significant loss of yield. See Chapter 9, Section 3 for additional information on the properties of EDC conjugation using small peptides coupled to carrier proteins.

Some procedures utilize 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 low 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 that do not interfere with the reaction may be used 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). Finally, EDC may promote unwanted polymerization due to the usual abundance of both amines and carboxylates on protein molecules.

The following protocol is a generaUzed 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, coup-Hng 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 the small molecule added to the reaction, thus resulting in a lower density of substitution.

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-|JL1 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-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, 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 ^.1.

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 jxl of peptide solution to the 200 |xl of protein solution.

4. Add EDC (Pierce) 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 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 rapidly. 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 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 h 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 fihration. When using EDC to prepare immu-nogen 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 functional groups with carboxylate groups using the water-soluble compound N-Hydroxysulfo-succinimide (sulfo-NHS) (Pierce). Sulfo-NHS esters are hydrophilic active groups that react rapidly with amines on target molecules (Staros, 1982; Anjaneyulu and Staros, 1987;^GthetaL, 1986; Kotite i /., 1984; Donovan and Jennings, 1986; Denney and Blobel, 1984; Jennings and Nicknish, 1985; Ludwig and Jay, 1985). Unlike nonsulfo-nated 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 and long-lived and hydrolyze relatively slowly in water. However, in the presence of amine nucleophiles that can attack at the carbonyl group of the ester, the N-hydroxysulfosuccinimide group rapidly leaves, creating a stable amide Unkage 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.

The advantage of adding sulfo-NHS to EDC reactions is to increase the 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 subject to rapid hydrolysis 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. Forming a sulfo-NHS ester intermediate from the reaction of the hydroxyl group on sulfo-NHS with the EDC active-ester complex extends the half-life of the activated carboxylate to hours. Since the concentration of added sulfo-NHS is usually much greater than the concentration of target molecule, the reaction preferentially proceeds through the longer-lived 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 (Fig. 107).

EDC/sulfo-NHS-coupled reactions are highly efficient and usually increase the yield of conjugation dramatically 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 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 and 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 (Fig. 108).


Carboxylic Acid

SO.Na

O Sulfo-NHS Sulfo-NHS Ester

Intermediate


Figure 107 The efficiency of an EDC-mediated reaction may be increased through the formation of a sulfo-NHS ester intermediate. The sulfo-NHS ester survives in aqueous solution longer than the active ester formed from the reaction of EDC alone with a carboxylate. Thus, higher yields of amide bond formation may be realized using this two-stage process.

In addition to the potential side reactions of EDC as mentioned previously (Chapter 3, Section 1.1), 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 v^hen coupling some peptides to carrier proteins, I have found that 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 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 product.

The following protocol is a generalized description of how to incorporate sulfo-NHS ester intermediates in EDC conjugation procedures. For specific appUcations of this technology, the amount of each reagent and unconjugated species may have to be adjusted to obtain an optimal conjugate.

Protocol

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; Pierce), 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. Dissolve the molecule to be coupled in the same buffer used in step 1. For small


Amine Containing Molecule

Conjugation Through Amide Bond Formation

Figure 108 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.

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, then add an aliquot of this stock solution to the protein solution. 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. Add EDC (Pierce) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.05-0.1 M EDC concentration in the reaction usually w^orks well. Also, add sulfo-NHS (Pierce) 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 rapidly. 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 h 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 w ith EDC/sulfo-NHS and subsequent conjugation w ith amine-containing molecules is given below . The variation in the pH of activation from that described above provides greater stability for the active ester intermediate. At pH 6, 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 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 car-bodiimide, according to Carraway and Triplett (1970). In the following protocol, sulfo-NHS is used instead of NHS so that active ester hydrolysis is slowed (Thelen and Deuticke, 1988; Anjaneyulu and Staros, 1987).

Protocol

1. Dissolve the protein to be activated in 0.05 M MES, 0.5 M NaCl, pH 6 (reaction buffer), at a concentration of 1 mg/ml.

2. Add to the solution in step 1 a quantity of EDC and sulfo-NHS (Pierce) 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 min at room temperature. 4. Add 2-mercaptoethanol to the reaction solution to obtain a final concentration

of 20 mM. Mix and incubate for 10 min 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 Sephadex G-25 or equivalent. The desalting operation should be done rapidly to minimize hydrolysis and recover as much active ester function as possible. The use of centrifugal spin columns of some sort may afford the greatest speed in purification. After purification, add the activated protein to the second molecule for conjugation. The second protein or other 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 to initiate the active ester reaction.

6. React for at least 2 h at room temperature. 7. Remove excess reactants by gel filtration or dialysis.


1.3. CMC

l-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMC) (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 the water solubility. Along with EDC (Chapter 3, Section 1.1), 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 cross-linking of two molecules, but to coupling of Hgands to insoluble support materials for use in affinity chromatography (Schmer, 1972; Lowe and Dean, 1971; Marcus and Balbinder, 1972).

CMC 1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide

MW 423.58 (as the metho-p-toluene sulfonate salt)

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 (Fig. 109). 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 cross-linking reaction. In aqueous solutions, hydrolysis of the active ester is by far the most frequent side reaction. Reaction of the group 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 cross-linking applications should be essentially the same as those given previously for EDC, except substituting a molar equivalent quantity of CMC. See Chapter 3, Sections 1.1 and 1.2 for additional information concerning carbodiimide reactions.

1.4. DCC

Dicyclohexyl carbodiimide (DCC) is one of the most frequently used coupling agents, especially in organic synthesis applications. It has been used for peptide synthesis since


OH

Carboxylic Acid CMC

H3N — R ' Primary Amine

Containing Molecule


Isourea By-product

Figure 109 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.

1955 (Sheehan and Hess, 1955) and continues to be a popular choice for creating peptide bonds (Barany and Merrifield, 1980). DCC is water-soluble, but has been used in 80% DMF for the immobilization of small molecules onto carboxylate-containing chromatography supports for use in affinity separations (Larsson and Mosbach, 1971: Lowe etal., 1973; Gutteridge and Robb, 1973). In addition to forming amide Hnkages, 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 Chapter 3, Section 1.2, active ester synthesis done with DCC is in organic solvent, and therefore does not have the hydrolysis problems of water-soluble EDC-formed esters. Thus, DCC is most often used to synthesize active ester-containing cross-linking and modifying reagents and not to perform biomolecular conjugations.

DCC N,N'-Dicyclohexyl

carbodiimide MW 206.32

DCC is a waxy solid that is often difficult to remove from a bottle. Its vapors are extremely hazardous to the eyes and to inhale. It should always be handled in a fume hood. The isourea by-product of a DCC-initiated reaction, dicyclohexyl urea (DCU) (Fig. 110), is also water-insoluble and must be removed by organic solvent washing.


OH

Carboxylic Acid

o-Acylisourea Intermediate

Dicyclohexylisourea

Figure 110 The organic-soluble carbodiimide DCC is often used to create amide bonds, especially between water-insoluble compounds.

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) (Fig. 111). 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 do not have competing hydrolysis problems. O-Acylisourea-activated

o-Acylisourea Intermediate Inactive N-Acylisourea

Figure 111 The active-ester intermediate formed from the reaction of DCC with a carboxylate group may undergo rearrangement to an inactive N-acylisourea product.


OH

Carboxylic Acid


Symmetrical Anhydride

Second Carboxylic Acid

Molecule

Figure 112 The reaction of DCC with a carboxylate compound in excess may create anhydride products in the absence of nucleophiles.

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 (Fig. 112). 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). In addition, a DCC-activated carboxylate may react with a amino acid to form an azlactone (Fig. 113) (Coleman etal., 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 zero-length cross-linking result of coupling directly to an amine-containing molecule (Fig. 114).

1.5. Die

Diisopropyl carbodiimide (DIC) is another water-insoluble amide bond-forming agent that has advantages over DCC (Chapter 3, Section 1.4). It is a Uquid at room temperature and is therefore much easier to dispense than DCC. Its by-products, diisopropylurea and diisopropyl-N-acylurea, are more soluble in organic solvents than

OH

Carboxylic Acid

H3N O

O

Amino Acid

DCC

R

An Azlactone

Figure 113 A DCC-mediated reaction with a carboxylate group in the presence of a small amino acid may form azlactone rings.

2. Woodward's Reagent K 181

R-

An Aziactone

HpN — R -

O

X R'

Primary Amine Containing Compound

Ring Opening with Amide Bond Formation

Figure 114 An aziactone reacts with amine groups through a ring-opening process, creating amide bond linkages with the attacking nucleophile.

the DCU by-product of a DCC reaction. DIG reacts in a manner similar to that of DCC, forming an active O-acyUsourea intermediate with a carboxyhc acid group (Fig. 115). 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.

DIC Diisopropyl carbodiimide

MW 126.2

2. Woodward's Reagent K

Woodward's reagent K is N-ethyl-3-phenylisoxazolium-3'-sulfonate, a zero-length cross-linking agent able to cause the condensation of carboxylates and amines to form amide bonds (Woodward et ai, 1961; Woodward and Olofson, 1961). The reaction

OH

Carboxylic Acid

O

,R'

Amide Bond Formation


A„XA Diisopropylurea

Figure 115 The symmetrical carbodiimide DIC reacts with carboxylates to form active-ester intermediates able to couple with amine-containing compounds to form amide bond linkages.


OH I Reactive - • ^^'^^^ Ketoketenimine

Enol Ester R Intermediate

Figure 116 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, a enol ester derivative. In the presence of amine nucleophiles, amide bond formation takes place.

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 (Fig. 116). 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 (Pikuleva and Turko, 1989; Boyer, 1986), it is not available commercially, which severely limits its application.

Woodward's Reagent K N-Ethyl-5-phenylisoxazolium-3'-

sulfonate, sodium salt M W 1 7 6

3. /V,/V'-Carbonyldiimidazole 183

3. /V,yV'-Carbonyldiimidazole

N,N'-Carbonyldiimidazole (CDI) is a highly active carbonylating agent that contains two acyUmidazole leaving groups (Aldrich). The result is that CDI can activate car-boxylic acids or hydroxyl groups for conjugation w ith other nucleophiles, creating either zero-length amide bonds or one-carbon-length N-alkyl carbamate Unkages betw^een the cross-linked molecules. Carboxylic acid groups react w ith 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 w ith amines to form amide bonds or w ith hydroxyl groups to form ester linkages (Fig. 117). Both reaction 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).

o

X CDI

N,N'-Carbonyldiimidazole MW 162

CDI functions as a zero-length cross-linker 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 —OH group is an imidazolyl carbamate (Fig. 118). Attack by an amine releases the imidazole, but not the carbonyl. Thus, a hydroxyl-containing molecule may be coupled to an amine-containing molecule with the result of a one-carbon spacer, forming a

OH

Carboxylic Acid

N=r

Acyl Imidazole Active Intermediate

Figure 117 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.


R'-OH

Hydroxy I Containing Molecule

o Carbamate Linkage

Imidazole Carbamate Active Intermediate

Figure 118 GDI 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.

Stable urethane (N-alkyl carbamate) linkage. This coupling procedure has been ap-pHed to the activation of hydroxyl-containing chromatography supports for the immobilization of amine-containing affinity ligands (Bethell et al, 1979; Hearn et ai, 1979, 1983; M. T. W. Hearn, 1987) and also to the activation of polyethylene glycol for the modification of amine-containing macromolecules (Beauchamp et ai, 1983).

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 GDI. 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 GDI by hydrolysis. Even in solvents containing small amounts of water, GDI quickly hydrolyzes to GO2 and imidazole. It is best to use solvents with less than 0.1% water to prevent extensive GDI breakdown. Gharacteristic bubble formation is an indication of reagent hydrolysis, although GO2 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, and DMF. If an excess of GDI is used during the activation step, it should be removed before adding the activated intermediate to an amine-containing molecule for conjugation. Alternatively, equal molar quantities of GDI 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 GDI usually reflect the relative pK^ of the nucleophilic amine being coupled. Proteins are best coupled to GDI-activated supports or molecules in an environment at least one pH unit above their pi values. Frequently the greatest coupling yields occur in alkaUne

4. Schiff Base Formation and Reductive Amination 185

buffers within the pH range 8-10. 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 experience the concomitant loss of activity due to hydrolysis as water-based reactions, thus nonaqueous reactions usually result in greater yields.

A protocol for the use of CDI in the activation of poly(ethylene glycol) is discussed in Chapter 15, Section 1.4.

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 hnkages (Fig. 119). A number of reducing agents can be used to convert specifically the Schiff base bond into an alkylamine linkage. Once reduced, the bonds are highly stable. The use of reductive amination to conjugate an aldehyde-containing molecule to an amine-containing molecule results in a zero-length cross-link where no additional spacer atoms are introduced between the molecules.

Reductive amination (or alkylation) may be used to conjugate an aldehyde- or ketone-containing molecule with an amine-containing molecule. The reduction reaction is best facilitated by the use of a reducing agent such as sodium cyanoborohydride.

O R II ^

p . ^ H + H N — R • JJ R'

Aldehyde Primary Amine Containing Containing Molecule Schiff Base Molecule / Formation

Reduction to Secondary Amine

Linkage

Figure 119 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 stabiHzed to a secondary amine bond.


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 nonreactive 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, effectively preserving the activity of even some labile monoclonal antibodies. Cyanoborohydride has been shown to be at least five times milder than borohydride in reductive amination processes with antibodies (Peng et ai, 1987). Other reducing agents that have been explored for reductive amination processes include various amine boranes and ascorbic acid (Cab-acungan et al, 1982; Hornsey et ai, 1986).

Immobilization by reductive amination of amine-containing biological molecules onto aldehyde-containing solid supports has been used for quite some time (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 reaUzed from pH 7—10. At high 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 protein and other molecules can be accomplished by a number of methods (Chapter 1, Section 4.4). Glyproteins 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 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 mol of HRP per mole of antibody.

3. Add 10 |JL1 of 5 M cyanoborohydride in 1 N NaOH (Aldrich) per milUHter of the conjugation solution volume. Caution: Highly toxic compound. Use a fume hood and be careful to avoid skin contact with this reagent.

4. React for 2 h at room temperature. 5. To block unreacted aldehyde sites, add 20 |JL1 of 3 M ethanolamine (pH adjusted

to desired value with HCl) per milliliter of the conjugation solution volume. React for 15 min at room temperature.

6. Purify the conjugate by dialysis or gel filtration using a buffer suitable for the nature of the proteins being cross-linked.

bioconjugate techniques || zero-length cross-linkers

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