Modification with Synthetic Polymers

25 Modification with Synthetic Polymers

Modification or attachment of proteins or other molecules with synthetic polymers can provide many benefits for both in vivo and in vitro applications. Covalent coupling of polymers to large macromolecules can alter their surface and solubility properties, creating increased water solubility or even organic solvent solubility for molecules normally sparingly miscible in such environments. Polymer modification of foreign molecules can provide increased biocompatibility, reducing the immune response, increasing in vivo stability, and delaying clearance by the reticuloendothelial system. Modification of enzymes with polymers can dramatically enhance their stability in solution. Polymer attachment can provide cryoprotection for proteins sensitive to freezing. Polymers with multivalent reactive sites can be used to couple numerous small molecules for creating pharmacologically active agents that possess long half-lives in biological systems. Similar complexes can be formed to create highly potent immunogens consisting of hapten–polymer conjugates for induction of an antibody response toward the hapten. Polymer modification of surfaces can effectively mask the intrinsic character of the surface and thus prevent nonspecific protein adsorption. Finally, multifunctional polymers can serve as extended crosslinking agents for the conjugation of more than one molecule of one protein to multiple numbers of a second molecule, creating large complexes with increased sensitivity or activity in detecting or acting upon target analytes. Many polymers have been studied for their usefulness in producing pharmacologically active complexes with proteins or drugs. Synthetic and natural polymers such as polysaccharides, poly(L-lysine) and other poly(amino acids), poly(vinyl alcohols), polyvinylpyrrolidinones, poly(acrylic acid) derivatives, various polyurethanes, and polyphosphazenes have been coupled to with a diversity of substances to explore their properties (Duncan and Kopecek, 1984; Braatz et al., 1993). Copolymer preparations of two monomers also have been tried (Nathan et al., 1993). The two polymers most often used in these applications are dextran and polyethylene glycol (PEG). Both polymers consist of repeating units of a single monomer—glucose in the case of dextran and an ethylene oxide basic unit in the case of PEG. The polymers may be composed of linear strands or branched constructs. An additional similarity is that both of them possess hydroxyl and ether linkages, lending hydrophilicity and water-solubility to the molecules. Dextran and PEG can be activated through their hydroxyl groups by a number of chemical 936

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methods to allow efficient coupling of other molecules. Dextran can be activated at multiple sites throughout its chain, since each monomer contains hydroxyl resides. PEG, by contrast, only has hydroxyls at the termini of each polymer strand. Derivatives of both polymers are commercially available. The following sections discuss the major properties and conjugation chemistries associated with the use of these polymers in modifying or conjugating proteins and other molecules.

1. Protein Modification with Activated Polyethylene Glycols Since the first report by Abuchowski et al. (1977a, b) concerning the alteration of immunological properties toward bovine serum albumin (BSA) that had been modified with PEG, the interest in polymer modification of biological molecules has grown incessantly. PEG coupled to other molecules can be used for altering solubility characteristics in aqueous or organic solvents (Inada et al., 1986), for modulation of the immune response (Delgado et al., 1992), to increase the stability of proteins in solution (Berger and Pizzo, 1988), to enhance the half-life of substances in vivo (Knauf et al., 1988), to aid in penetrating cell membranes, to alter pharmacological properties (Dunn and Ottenbrite, 1991), to increase biocompatibility, especially toward implanted foreign substances, and to prevent protein adsorption to surfaces. PEG consists of repeating units of ethylene oxide which terminate in hydroxyl groups on either end of a linear chain. Some constructs have branches that have multiple linear strands emanating from these points. PEG is made from the anionic polymerization of ethylene oxide, resulting in the formation of polymer strands of various potential molecular weights, depending on the polymerization conditions. Thus, all polymeric PEGs are polydisperse and exist as distribution of multiple lengths and molecular weights. Most forms of PEG useful in bioconjugate applications have molecular weights less than 20,000 and are soluble both in aqueous solution and in many organic solvents.

Unlike the PEG molecules formed from anionic polymerization techniques, there now exist highly discrete forms of the polymer made by controlled addition of small PEG units to create chains of exacting molecular size. These discrete PEGs have a single molecular weight and do not display the polydispersity of the traditional PEG polymers. See Chapter 18 for a complete discussion of discrete PEG-based reagents and their applications. Since the polymer backbone of PEG is not of biological origin, it is not readily degraded by mammalian enzymes (although some bacterial enzymes will break it down). This property results in only slow degradation of the polymer when used in vivo, thus extending the half-life of modified substances. PEG modification serves to mask any molecule that it is coupled to—the “PEGylated” molecule being protected from immediate breakdown or from being complexed and inactivated by immunoglobulins in the bloodstream.

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25. Modification with Synthetic Polymers

PEG’s properties in solution are especially unusual, frequently displaying amphiphilic tendencies, having the ability to both solubilize in aqueous layers and in hydrophobic membranes or organic phases. The partitioning quality of PEG across membranes is important in aiding the formation of hybridomas in the production monoclonal antibodies (Goding, 1986b). The partitioning characteristics of PEG also create the ability to use it in aqueous two-phase systems for the purification of biological molecules (Johansson, 1992). PEG in solution is a highly mobile molecule that creates a large exclusion volume for its molecular weight, much larger in fact than proteins of comparable size. Whether in solution or attached to other insoluble supports or surfaces, PEG has a tendency to exclude other polymers. This property forms a protein-rejecting region that is effective in preventing nonspecific protein binding (Bergstrom et al., 1992). Conjugation with PEG can create the same exclusion effects surrounding a macromolecule, even preventing interaction between a ligand and its target (Klibanov et al., 1991), an enzyme and its substrate (Berger and Pizzo, 1988), or the immune system and a foreign substance (Davis et al., 1979). Thus, PEG-modified molecules display low immunogenicity, have good resistance to proteolytic digestion, and survive in the bloodstream for extended periods (Abuchowski et al., 1977a; Dreborg and Akerblom, 1990). PEG can be conjugated to other molecules through its two hydroxyl groups at the ends of each linear chain. This process is typically done by the creation of a reactive electrophilic intermediate that is capable of spontaneously coupling to nucleophilic residues on a second molecule. To prevent the potential for crosslinking when using a bifunctional polymer, monofunctional PEG polymers can be used which contain one end of each chain blocked with a methyl ether group. Monomethoxypolyethylene glycol (mPEG) contains only one hydroxyl group per linear chain, thus limiting activation and coupling to one site and preventing the crosslinking and polymerization of modified molecules. The mPEG derivative also stabilizes the blocked end to degradation in solution.

1.1. Trichloro-s-triazine Activation and Coupling The most common activation methods for PEG create amine-reactive derivatives that can form amide or secondary amine linkages with proteins and other amine-containing molecules. The oldest method of PEG activation is through the use of trichloro-s-triazine (TsT; cyanuric chloride) (Abuchowski et al., 1977). TsT is a symmetrical heterocyclic compound containing three reactive acyl-like chlorines. This reagent and its derivatives are extensively used in industrial applications to form strong covalent bonds between dye molecules and fabrics. The compound also has been used to activate affinity chromatography supports for the coupling of aminecontaining ligands (Finlay et al., 1978). Reaction of the TsT with PEG results in the formation of an activated derivative with an ether bond to the hydroxyl group of the polymer. If mPEG is used, TsT activation will be restricted to the one free hydroxyl, thus forming a monovalent intermediate that can be coupled to proteins without polymerization (Figure 25.1). The three reactive chlorines on TsT have dramatically different reactivities toward nucleophiles in aqueous solution. The first chlorine is reactive toward hydroxyls as well as primary and secondary amine groups at 4°C and a pH of 9.0 (Abuchowski, 1977a; Mumtaz and Bachhawat, 1991). Once the first chlorine is coupled, the second one requires at least room temperature conditions at the same pH to react efficiently. If two chlorines are conjugated to nucleophilic groups, the third is even more difficult to couple, requiring at least 80°C at alkaline

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Figure 25.1 mPEG polymers may be activated by trichloro-s-triazine for the modification of amine-containing molecules.

pH. After activation of mPEG with TsT, it is therefore, for all practical purposes, only possible to couple one additional component to the triazine ring. TsT activation provides a simple route to an amine-reactive PEG derivative and it has been used extensively as an activation method for modifying proteins (Wieder et al., 1979; Zalipsky and Lee, 1992; Gotoh et al., 1993). The modification of primary amine-containing molecules such as proteins is pH dependent. At physiological pH values, the reaction will proceed slower than in a more alkaline pH environment. Optimal derivatization efficiency is reached at conditions equal to or above pH 9.0. However, TsT reactivity is not exclusive toward amines. TsT–mPEG modification of proteins can result in modifying other nucleophilic groups such as sulfhydryls and the phenolate ring of tyrosine. In addition, there is potential for toxicity associated with TsT and its derivatives—an especially important consideration for in vivo use. The following protocol for mPEG activation using TsT and its coupling to proteins is based on the protocols of Abuchowski et al. (1977b) and Gotoh et al. (1993). Protocol for the Activation of mPEG with TsT Note: All operations should be done in a fume hood. Dispose of hazardous waste according to EPA guidelines. 1. Dissolve 5.5 g of TsT in 400 ml of anhydrous benzene which contains 10 g of anhydrous sodium carbonate. 2. Add to the TsT solution, 50 g of mPEG-5000 (monomethoxypolyethylene glycol having a molecular weight of 5000). Mix well to dissolve.

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3. React overnight at room temperature with stirring. 4. Filter the solution through a glass-fiber filter pad and slowly add, with stirring, 600 ml of petroleum ether (bp 35–60°C). 5. Collect the precipitated product by filtration and redissolve it in 400 ml of benzene. Repeat steps 4–5 several times to assure complete removal of unreacted TsT. The residual TsT may be detected by HPLC using a 250 3.2 mm LiChrosorb (5 m particle size) column from E. Merck. The separation is done using a mobile phase of hexane, and peaks are detected with a UV detector. 6. Remove excess solvents by rotary evaporation. The TsT–mPEG should be used immediately or stored in anhydrous conditions at 4°C. Protocol for Coupling of TsT–mPEG to Proteins 1. Dissolve the protein to be modified with TsT–mPEG in ice-cold 0.1 sodium borate, pH 9.4, at a concentration of 2–10 mg/ml. Other buffers at lower pH values (down to pH 7.2) can be used and still obtain modification, but the yield will be less. Avoid amine-containing buffers such as Tris or the presence of sulfhydryl-containing compounds, such as disulfide reductants. 2. Slowly add TsT–mPEG to the protein solution at a level of at least a 5-fold molar excess over the desired modification level. For example, Gotoh et al. (1993) added 100 lmg of TsT–mPEG-5000 to 19 mg of protein dissolved in 6 ml of buffer. Add the polymer over a period of about 15 minutes with stirring at 4°C. 3. React for 1 hour at 4°C. 4. Remove excess TsT–mPEG by dialysis or gel filtration using a column of Sephacryl S-300.

1.2. NHS Ester and NHS Carbonate Activation and Coupling Carboxylate groups activated with N-hydroxysuccinimide (NHS) esters are highly reactive toward amine nucleophiles. In the mid-1970s, NHS esters were introduced as reactive ends of crosslinking reagents (Bragg and Hou, 1975; Lomant and Fairbanks, 1976). Their excellent reactivity at physiological pH quickly established NHS esters as the major amine-coupling chemistry in bioconjugate chemistry. NHS ester-containing compounds react with nucleophiles to release the NHS leaving group and form an acylated product (Chapter 2, Section 1.4). The reaction of such esters with sulfhydryl or hydroxyl groups is possible, but doesn’t yield stable conjugates, forming thioesters or ester linkages. Both of these bonds typically hydrolyze in aqueous environments or can undergo transesterification reactions. Histidine side-chain nitrogens of the imidazolyl ring also may be acylated with an NHS ester reagent, but they too hydrolyze rapidly (Cuatrecasas and Parikh, 1972). Reaction with primary and secondary amines, however, creates stable amide and imide linkages, respectively, that don’t readily break down. In protein molecules, NHS ester groups primarily react with the -amines at the N-terminals and the -amines of lysine side chains, due to their relative abundance. PEG contains no carboxylate groups in its native state, but can be modified to possess them by reaction with anhydride compounds. Either PEG or mPEG may be acylated with anhydrides

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Figure 25.2 mPEG may be derivatized with succinic anhydride to produce a carboxylate end. A reactive NHS ester can be formed from this derivative by use of a carbodiimide-mediated reaction under nonaqueous conditions. The succinimidyl succinate-mPEG is highly reactive toward amine nucleophiles.

to yield ester derivatives terminating in free carboxylate groups. Modification of PEG with succinic anhydride or glutaric anhydride gives bis-modified products having carboxylates at both ends. Modification of mPEG yields the mono-substituted derivative containing a single carboxylate. Creation of the succinimidyl succinate and succinimidyl glutarate derivative of PEG was described by Abuchowski et al. (1984). A method for the succinylation of mPEG can be found in Section 1.3, this chapter. Subsequent formation of the NHS ester derivatives of these acylated PEG compounds produce highly reactive polymers that can be used to modify aminecontaining molecules under mild conditions and with excellent yields (Figures 25.2 and 25.3). The main deficiency of the succinimidyl succinate or succinimidyl glutarate activation procedures is the potential for hydrolysis of the ester bond formed by acylation of the hydroxyl end groups of mPEG. A modification of the anhydride-acylation route to obtaining reactive NHS ester–PEG compounds was introduced by Zalipsky et al. (1991, 1992). In this approach, the terminal hydroxyl group of mPEG is treated with phosgene to give a reactive intermediate, an mPEGchloroformate compound. Next, the addition of N-hydroxysuccinimide (NHS) gives the succinimidyl carbonate (SC) derivative (Figure 25.4). Nucleophiles, such as the primary amino groups of proteins, can react with the SC to give stable carbamate (aliphatic urethane) bonds (Figure 25.5). The linkage is identical to that obtained through N,N-carbonyldiimidazole (CDI) activation of hydroxyl groups with subsequent coupling of amines (Chapter 3, Section 3, and this chapter, Section 1.4). However, the reactivity of the SC is much greater than that of the imidazole carbamate formed as the active species in CDI activation. Unlike the succinimidyl succinate or succinimidyl glutarate activation methods, SC chemistry does not suffer from the presence of a labile ester bond. The intermediate reactive NHS carbonate may hydrolyze in aqueous solution to release NHS and CO2, essentially regenerating

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Figure. 25.3 Succinimidyl succinate–mPEG may be used to modify amine-containing molecules to form amide bond derivatives. The ester bond of the succinylated mPEG, however, is subject to hydrolysis.

Figure 25.4 An SC derivative of mPEG was first prepared through the use of phosgene to form a chloroformate intermediate. Reaction with NHS gives the amine-reactive SC–mPEG.

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Figure 25.5 An SC–mPEG can be used to modify amine-containing molecules to form stable carbamate linkages.

the underivatized PEG hydroxyl. After coupling to amine-containing molecules, however, the resultant carbamate linkage stabilizes the chemistry to the point that a modified molecule will not lose PEG by hydrolytic cleavage. For these reasons, the SC method of PEG activation and coupling has become the chemistry of choice for attaching the polymer to amine-containing proteins and other molecules. A modification of Zalipsky’s method by Miron and Wilchek (1993) simplifies the creation of the SC-activated species. Instead of using highly toxic phosgene to form a chloroformate intermediate and then reacting with NHS, the new procedure utilizes either N-hydroxysuccinimidyl chloroformate or N,N-disuccinimidyl carbonate (DSC; Chapter 4, Section 1.7) to produce the SC–PEG in one step (Figure 25.6). Since both activation reagents are commercially available, creating an amine-reactive PEG derivative has never been easier. The following procedure is based on the Miron and Wilchek modification of Zalipsky’s method. Protocol for the Activation of PEG with N-Succinimidyl Chloroformate or N,N-Disuccinimidyl Carbonate Caution: The steps using flammable solvents, especially diethyl ether, should be done in a fume hood. 1. Dissolve 5 g PEG or mPEG (MW 5,000; 1 lmmol) in 25 ml of dry dioxane. Heating in a water bath may be necessary to solubilize fully the polymer. Cool to room temperature.

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Figure 25.6 An alternative route to an SC derivative of mPEG can be accomplished by the reaction of the

terminal hydroxyl group of the polymer with either N,N-disuccinimidyl carbonate or N-hydroxysuccinimidyl chloroformate.

2. Dissolve 6 mmol of either N-succinimidyl chloroformate or N,N-disuccinimidyl carbonate (Aldrich) in 10 ml of dry acetone. 3. Dissolve 6 mmol of 4-(dimethylamino)pyridine in 10 ml of dry acetone (base). 4. With stirring, add the solution prepared in step 2 to the PEG solution prepared in step 1. Next, slowly add the solution prepared in step 3. 5. React for 2 hours if activating with N-succinimidyl chloroformate or 6 hours if using N,N-disuccinimidyl carbonate. Maintain stirring with a magnetic stirring bar. 6. If N-succinimidyl chloroformate was used, filter out the white precipitate of 4-(dimethylamino) pyridine hydrochloride using a glass fiber filter pad. Collect the supernatant. 7. For either activation chemistry, precipitate the SC–PEG formed by addition of diethyl ether until no further precipitation is observed (typically 3–4 volumes of solvent). 8. Redissolve the precipitated product in acetone and precipitate again using diethyl ether. Repeat at least once more to remove completely excess reactants. 9. Dry the SC–PEG and store at 4°C. Protocol for the Coupling of SC–mPEG to Proteins 1. Dissolve the protein to be PEGylated in cold 0.1 M sodium phosphate, pH 7.5, at a concentration of 1–10 mg/ml.

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Figure 25.7 Succinylated mPEG derivative may be coupled to amine-containing molecules using a carbodiimide reaction to form an amide bond.

2. With stirring, add a quantity of SC–mPEG to the protein solution at the molar ratio of polymer-to-protein desired. The ratio of activated polymer addition typically is expressed versus the molar quantity of primary amines present on the protein being modified. Ratios of SC–PEG-to-amines between 0.3:1 and 8:1 were investigated by Miron and Wilchek (1993) for the derivatization of egg white lysozyme. The greater the ratio of activated polymer to protein, the higher the molecular weight of the resultant complex. Experiments may have to be done using a number of different reaction ratios to determine the optimal PEGylation level for a particular protein. 3. React overnight at 4°C. 4. Remove excess SC–PEG by dialysis or gel filtration.

1.3. Carbodiimide Coupling of Carboxylate–PEG Derivatives PEG contains only hydroxyl functional groups in its native state that need to be activated or modified in some manner to allow efficient conjugation to other molecules. These hydroxyls can be modified to possess carboxylates by reaction with anhydride compounds. Acylation of PEG with succinic anhydride or glutaric anhydride gives bis-modified products having carboxylates at both ends. Modification of mPEG yields the monosubstituted derivative containing a single carboxylate. Creation of these derivatives was first described by Abuchowski et al. (1984). Once the carboxylate–PEG modification is formed, it can be used to couple directly with amine-containing molecules by use of the carbodiimide reaction (Chapter 3, Section 1). A carbodiimide may be used to activate the carboxylates to highly reactive o-acylisourea intermediates. When generated in the presence of an amine-containing protein or other molecule, these active esters will react with the nucleophiles to give amide bond derivatives (Figure 25.7). Atassi and Manshouri (1991) used this technique to PEGylate various peptides. In this instance, the reaction was carried out in dimethylformamide (DMF) due to the solubility of the peptides in this solvent. The organic-soluble carbodiimides dicyclohexyl carbodiimide (DCC) (Chapter 3, Section 1.4) or diisopropyl carbodiimide (DIC) (Chapter 3, Section 1.5) were used to perform the conjugation. However, aqueous phase reactions can be done using this approach just as easily as organic-based conjugations if the water-soluble reagent EDC (1-ethyl-3-(3-dimethylam inopropyl)carbodiimide) is employed (Chapter 3, Section 1.1). The general protocols for using carbodiimides outlined in the referenced sections may be used to conjugate a carboxylatecontaining PEG derivative to an amine-containing protein or other molecule. The formation of a carboxylate-containing PEG derivative can be done according to the following protocol (adapted from Atassi and Manshouri, 1991).

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Protocol 1. Dissolve 1 g of mPEG (MW 5,000) in 5 ml of anhydrous pyridine by heating to 50°C. 2. To the stirring mPEG solution, add several 0.5 g aliquots of solid succinic anhydride over a period of several hours. 3. React for a further 2 hours at 50°C. 4. Evaporate the pyridine solvent by using a flash evaporator or a rotary evaporator under vacuum. 5. Redissolve the residue in water (the solution may have to be heated to fully dissolve) and again evaporate to dryness. Repeat until the odor of pyridine is nearly gone. 6. Remove remaining reactants by dialysis against water using a membrane having a molecular weight cutoff of 1,000.

1.4. CDI Activation and Coupling N,N-carbonyldiimidazole (CDI) is a highly reactive carbonylating compound which was first shown to be an excellent amide bond forming agent in peptide synthesis (Paul and Anderson, 1962). Later it was used to activate both carboxylic groups and hydroxyls in the immobilization of amine-containing ligands to prepare affinity chromatography supports (Battling et al., 1973; Hearn, 1987). The activation of a carboxylate group with CDI proceeds to give an intermediate imide with imidazole as the active leaving group. In the presence of a primary amine-containing compound, the nucleophile attacks the electron-deficient carbonyl, displacing the imidazole and forming a stable amide bond. For hydroxyl-containing compounds, CDI will react to form an intermediate imidazolyl carbamate, which in turn can react with N-nucleophiles to give an N-alkyl carbamate linkage. Proteins normally couple through their N-terminals (-amine) and lysine side-chain (-amine) functional groups. The final bond is an uncharged, urethane derivative having excellent chemical stability (Figure 25.8). The result of CDI activation of PEG and subsequent coupling to a protein or other amine-containing molecule is a linkage identical to that obtained using SC chemistry, described previously (Section 1.2, this chapter). CDI-activated PEG is stable for years in a dried state or in organic solvents devoid of water. The activated polymer also will have an excellent half-life to hydrolysis even in the coupling environment. Unlike some activation chemistries that degrade rapidly and have half-lives on the order of minutes, imidazole carbamates have half-lives measured in hours. For instance, an agarose chromatography support activated with CDI will take up to 30 hours at pH 8.5–9.0 for complete loss of activity. The hydrolysis of CDI–PEG derivatives causes the release of CO2 and imidazole. The hydrolyzed product thus reverts back to the original hydroxylic PEG compound, leaving no residual groups to cause potential sites for nonspecific interactions. The optimal coupling condition for a CDI–PEG or CDI–mPEG reaction is in an alkaline pH environment, typically above pH 8.5. The coupling reaction proceeds at greatest efficiency when the target molecule is reacted at about 1 pH value above its pI or pKa. The reaction can be done directly in an organic solvent environment if the molecule to be modified demonstrates poor solubility in aqueous systems. The advantage of an organic coupling reaction is that there is no competing hydrolysis of the active groups, so very high modification yields of PEG can be realized.

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Figure 25.8 N,N-carbonyldiimidazole (CDI) may be used to activate the terminal hydroxyl of mPEG to an imidazole carbamate. Reaction of this intermediate with an amine-containing compound results in the formation of a stable carbamate linkage.

There are a few precautions that should be noted when doing a CDI activation and coupling experiment. First, CDI itself is extremely unstable to aqueous environments, much more so than the active imidazolyl carbamate that’s formed after PEG activation. Therefore, the activation step must be done in a solvent that is free of water. If unacceptable amounts of water are present, CDI will be immediately broken down to CO2 and imidazole. The evolution of bubbles upon addition of CDI to a PEG solution is the telltale sign of high water content. Only freshly obtained solvents analyzed to be extremely low in moisture or those dried over a molecular sieve should be used. A water content of less than 0.1 percent in the solvent is usually all right for a CDI activation procedure. A second precaution is to carry out the activation step in a fume hood away from sources of ignition. Most CDI activation protocols use flammable or toxic solvents and care should be taken in handling and disposing of them. The coupling reaction using CDI–mPEG or CDI–PEG derivatives is slower than that obtained using NHS ester or SC-coupling methods. Therefore, the reaction times used with CDI chemistry are typically on the order of 1–2 days at 4°C at a pH of about 8.5. Increasing the pH of the reaction to pH 9 or 10 will speed up the coupling. In addition, doing the reaction at room temperature also helps in this regard. If the molecule to be modified is stable at alkaline pH values and room temperature, then these conditions may be used to decrease the time of the suggested protocol. The following method is adapted from Beauchamp et al. (1983). Protocol for the Activation of mPEG with CDI 1. Dissolve mPEG (MW 5,000) in dry dioxane at a concentration of 50 mM (0.25 g/ml) by heating to 37°C.

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2. Add solid CDI to a final concentration of 0.5 M (81 mg/ml). 3. React for 2 hours at 37°C with stirring. 4. To remove excess CDI and reaction by-products, Beauchamp et al. (1983) dialyzed against water at 4°C. However, the imidazole carbamate groups on mPEG formed during the activation process are subject to hydrolysis in aqueous environments. A better method may be to precipitate the activated mPEG with diethyl ether as in the protocol described previously for SC activation (Section 1.2, this chapter). 5. Finally, dry the isolated product by lyophilization (if the water dialysis method is used) or by use of a rotary evaporator (if the ether precipitation method is used).

Protocol for the Coupling of CDI–mPEG to Proteins 1. Dissolve the protein to be PEGylated in 10 mM sodium borate, pH 8.5, at a concentration of 1–10 mg/ml. Higher pH values may be used to increase the reaction rate, for instance, 0.1 M sodium carbonate, pH 9–10. 2. Add CDI–mPEG to this solution with stirring to bring the final concentration of the activated polymer to 180 mM. Note: Other ratios of polymer-to-protein may be used, depending on the modification level desired. Some optimization of the derivatization level may have to be done to obtain conjugates having the best amount of polymer substitution with retention of protein activity. 3. React for 48 hours at 4°C. If higher pH or room temperature conditions are used, the reaction time can be decreased to 24 hours. 4. Remove unconjugated mPEG and reaction by-products by dialysis or gel filtration.

1.5. Miscellaneous Coupling Reactions PEG or mPEG may be conjugated to proteins or other molecules using other coupling chemistries in addition to the ones mentioned in the previous sections. Almost any activation method that can be built off of the terminal hydroxyl(s) of PEG may be employed to PEGylate target molecules. For instance, Bergström et al. (1992) created an epoxy derivative of the polymer by reaction with epichlorohydrin under alkaline conditions. The reactive alkyl halogen end of epichlorohydrin first is coupled to the hydroxyls of PEG to give the terminal glycidyl ether derivative (Figure 25.9). The epoxy-functionalized polymer then could be used to covalently modify a poly(ethylene imine)-coated polystyrene surface to prevent nonspecific protein adsorption. This type of derivative also could be used to modify other amine-, hydroxyl-, or sulfhydryl-containing molecules (Chapter 2, Section 4.1). Creation of a sulfhydryl-reactive PEG derivative was done by Goodson and Katre (1990) by reacting an active ester–maleimide heterobifunctional crosslinker with the amino groups of a PEG-amine polymer (Pillai and Mutter, 1980). An amine-terminal derivative of a discrete PEG-type polymer is available from Thermo Fisher or Quanta BioDesign (Chapter 18). Reaction with the N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitro-4-benzene sulfonic acid results in an amide bond derivative (Figure 25.10). This creates terminal maleimide groups on each PEG-amine molecule. Maleimide compounds can be used in a site-directed coupling

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Figure 25.9 Epichlorohydrin can be used to activate the hydroxyl group of mPEG, creating an epoxy derivative. Reaction with amine-containing molecules yields secondary amine bonds.

Figure 25.10 PEG-amine compounds may be reacted with this heterobifunctional crosslinker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of crosslinking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

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procedure to specifically PEGylate at the sulfhydryl groups of proteins and other molecules (Chapter 2, Section 2.2).

In another approach, Wirth et al. (1991) and Chamow et al. (1994) transformed the terminal hydroxyl group of mPEG into an aldehyde residue by the Moffatt oxidation procedure (Harris et al., 1984). In this reaction, the hydroxyl is treated with acetic anhydride in dimethyl sulfoxide (DMSO)-containing triethylamine, converting it to the aldehyde (Figure 25.11). After stirring at room temperature for 48 hours, the aldehyde derivative is isolated by precipitation with ether and ethyl acetate. An aldehyde can be conjugated to proteins or other amine-containing molecules by reductive amination using sodium cyanoborohydride (Chapter 3, Section 4). The advantage of an aldehyde–PEG derivative over the other amine-reactive chemistries described previously is that the active function will not hydrolyze or readily degrade before the coupling reaction is initiated. In addition, reductive amination is a reasonably mild conjugation technique well-tolerated by most proteins. For additional information on PEG-based reagents and coupling chemistry, see Chapter 18, which discusses the unique discrete PEG compounds.

Figure 25.11 A terminal aldehyde function on mPEG may be formed through an oxidative process at elevated temperatures. This derivative may be used to modify amine-containing molecules by reductive amination.

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2. Protein Modification with Activated Dextrans

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2. Protein Modification with Activated Dextrans Dextran is a naturally occurring polymer that is synthesized in yeasts and bacteria for energy storage. It is mainly a linear polysaccharide consisting of repeating units of D-glucose linked together in glycosidic bonds (Chapter 1, Section 2), wherein the carbon-1 of one monomer is attached to the hydroxyl group at the carbon-6 of the next residue. This configuration is the same as that found in the -1,6 linked disaccharide isomaltose. The same disaccharide is found at the branch points of glycogen and amylopectin. Occasional branch points also may be present in a dextran polymer, occurring as -1,2, -1,3, or -1,4 glycosidic linkages. The branch type and degree of branching vary by species.

The hydroxylic content of the dextran sugar backbone makes the polymer very hydrophilic and easily modified for coupling to other molecules. Unlike PEG, discussed previously, which has modifiable groups only at the ends of each linear polymer, the hydroxyl functional groups of dextran are present on each monomer in the chain. The monomers contain at least 3 hydroxyls (4 on the terminal units) that may undergo derivatization reactions. This multivalent nature of dextran allows molecules to be attached at numerous sites along the polymer chain. Soluble dextran of molecular weight 10,000500,000 has been used extensively as a modifying or crosslinking agent for proteins and other molecules. It has been used as a drug carrier to transport greater concentrations of antineoplastic pharmaceuticals to tumor sites in vivo (Bernstein et al., 1978; Heindel et al., 1990), as conjugated to biotin to make a sensitive anterograde tracer for neuroatomic studies (Brandt and Apkarian, 1992), as a hapten carrier to illicit an immune response against coupled molecules (Dintzis et al., 1989; Shih et al., 1991), as an inducer of B-cell proliferation by coupling anti-Ig antibodies (Brunswick et al., 1988), as a multifunctional linker to crosslink monoclonal antibody conjugates with chemotherapeutic agents (Heindel et al., 1991), and as a stabilizer of enzymes and other proteins (Zlateva et al., 1988; Nakamura et al., 1990). As is true of PEG conjugates with proteins, dextran modification of macromolecules provides increased circulatory half-life in vivo, decreased immunogenicity, and a heat and protease protective effect when coupled at sufficient density (Mumtaz and Bachhawat, 1991). The following sections describe the major activation and coupling methods used with dextran polymers. The reactive derivatives may be used to couple with proteins and other molecules containing the appropriate functional groups.

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2.1. Polyaldehyde Activation and Coupling The dextran polymer contains adjacent hydroxyl groups on each glucose monomer. These diols may be oxidized with sodium periodate to cleave the associated carbon–carbon bonds and produce aldehydes (Chapter 1, Section 4.4). This procedure results in two aldehyde groups formed per glucose monomer, thus producing a highly reactive, multifunctional polymer able to couple with numerous amine-containing molecules (Bernstein et al., 1978) (Figure 25.12). Polyaldehyde dextran may be conjugated with amine groups by Schiff base formation followed by reductive amination to create stable secondary (or tertiary amine) linkages (Chapter 2, Section 5) (Figure 25.13). Proteins may be modified with oxidized dextran polymers under mild conditions using sodium cyanoborohydride as the reducing agent. The reaction proceeds primarily through -amino groups of lysine located at the surface of the protein molecules. The optimal pH for the reductive amination reaction is an alkaline environment between pH 7 and 10. The rate of reaction is greatest at pH 8–9 (Kobayashi and Ichishima, 1991), reflecting the efficiency of Schiff base formation at this pH. Polyaldehyde dextran can be used to couple many small molecules, such as drugs, to a targeting molecule like an antibody. The multivalent nature of the oxidized dextran backbone provides more sites for conjugation than possible using direct coupling of the drug with the antibody itself. Similarly, detection molecules such as fluorescent probes can be conjugated in greater amounts using a dextran carrier than is feasible with direct modification of a protein.

Figure 25.12 Dextran polymers can be oxidized with sodium periodate to create a polyaldehyde derivative. Note that additional oxidation may occur to cleave off another carbon atom and create an aldehyde on the adjacent COH group.

Figure 25.13 Polyaldehyde dextran may be used as a multifunctional crosslinking agent for the coupling of amine-containing molecules. Reductive amination creates secondary amine or tertiary amine linkages.

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The following protocol for creating the polyaldehyde dextran derivative is based on the method of Bernstein et al. (1978). Protocol for Oxidizing Dextran with Sodium Periodate 1. Dissolve sodium periodate (NaIO4) (Sigma) in 500 ml of deionized water at a concentration of 0.03 M (6.42 g). Protect from light. 2. Dissolve dextran (Polysciences) of molecular weight between 10,000 and 40,000 in the sodium periodate solution with stirring. 3. React overnight at room temperature in the dark. 4. Remove excess reactant by extensive dialysis against water. The purified polyaldehyde dextran may be lyophilized for long-term storage. The degree of oxidation may be assessed by measurement of the aldehydes formed. Zhao and Heindel (1991) suggest derivatizing the polyaldehyde dextran with hydroxylamine hydrochloride and measuring the amount of HCl released by titration. However, this may be tedious and time-consuming. A simpler method may be to take advantage of the fact that periodateoxidized sugars are capable of reducing Cu2 to Cu, which can be detected using the bicinchoninic acid (BCA) reagent (Thermo Fisher) (Smith, P. S. et al., 1985). The formation of Cu is in direct proportion to the amount of aldehydes present in the polymer. BCA will form a purple-colored complex with Cu which can be measured at 562 nm. Protocol for Coupling Polyaldehyde Dextran to Proteins 1. Dissolve or buffer-exchange the periodate-oxidized dextran in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 10–25 mg/ml. Other buffers having a pH range of 7–10 may be used with success, as long as they do not contain competing amines (such as Tris). A reaction environment of pH 8–9 (0.1 M sodium bicarbonate) will give the greatest yield of reductive amination coupling. 2. Add 10 mg of the protein to be coupled to the dextran solution. Other ratios of dextran-to-protein may be used as appropriate. For instance, if more than one protein or a protein plus a smaller molecule are both to be conjugated to the dextran backbone, the amount of protein added initially may have to be scaled back to allow the second molecule to be coupled latter. Many times, a small molecule such as a drug will be coupled to the dextran polymer first, and then a targeting protein such as an antibody conjugated secondarily. The optimal ratio of components forming the dextran conjugate should be determined experimentally to obtain the best combination possible. 3. In a fume hood, add 0.2 ml of 1 M sodium cyanoborohydride (Aldrich) to each ml of the protein/dextran solution. Mix well. Caution: Cyanoborohydride is extremely toxic and should be handled only in well-ventilated fume hoods. Dispose of cyanide-containing solutions according to approved guidelines. 4. React for at least 6 hours at room temperature. Overnight reactions also may be done. 5. To block excess aldehydes, add 0.2 ml of 1 M Tris, pH 8, to each ml of the reaction. Note: If a second molecule is to be coupled after the initial protein conjugation, don’t block the remaining aldehydes until the second molecule is added. 6. React for an additional 2 hours at room temperature.

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7. Purify the protein–dextran conjugate from unconjugated protein and dextran by gel filtration using a column of Sephacryl S-200 or S-300. Small molecules may be removed from a dextran conjugate by dialysis.

2.2. Carboxyl, Amine, and Hydrazide Derivatives Dextran derivatives containing carboxyl- or amine-terminal spacer arms may be prepared by a number of techniques. These derivatives are useful for coupling amine- or carboxylatecontaining molecules through a carbodiimide-mediated reaction to form an amide bond (Chapter 3, Section 1). Amine-terminal spacers also can be used to create secondary reactive groups by modification with a heterobifunctional crosslinking agent (Chapter 5). This type of modification process has been used to form sulfhydryl-reactive dextran polymers by coupling amine spacers with crosslinkers containing an amine reactive end and a thiol-reactive end (Brunswick et al., 1988; Noguchi et al., 1992). The result was a multivalent sulfhydryl-reactive dextran derivative that could couple numerous sulfhydryl-containing molecules per polymer chain. Several chemical approaches may be used to form the amine- or carboxyl-terminal dextran derivative. The simplest procedure may be to prepare polyaldehyde dextran according to the procedure of Section 2.1 (this chapter) and then make the spacer arm derivative by reductively aminating an amine-containing organic compound onto it. For instance, short diamine compounds such as ethylene diamine or diaminodipropylamine (3,3-iminobispropylamine) can be reacted in large excess with polyaldehyde dextran to create numerous modifications along the polymer having terminal primary amines. Carboxyl-terminal derivatives may be prepared similarly by coupling molecules such as 6-aminocaproic acid or -alanine to polyaldehyde dextran. Alternatively, an amine-terminal spacer may be reacted with succinic anhydride to form the carboxylate derivative (Chapter 1, Section 4.2). Another approach uses reactive alkyl halogen compounds containing a terminal carboxylate group on the other end to form spacer arms off the dextran polymer from each available hydroxyl. In this manner, Brunswick et al. (1988) used chloroacetic acid to modify the hydroxyl groups to form the carboxymethyl derivative. The carboxylates then were aminated with ethylene diamine to create an amine-terminal derivative (Inman, 1985). Finally, the amines were modified with iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.14). In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. Hydrazide derivatives also may be prepared from a periodate-oxidized dextran polymer or from a carboxyl-containing dextran derivative by reaction with bis-hydrazide compounds (Chapter 4, Section 8). A hydrazide terminal spacer provides reactivity toward aldehyde- or ketone-containing molecules. Thus, the hydrazide–dextran polymer can be used to conjugate specifically glycoproteins or other polysaccharide-containing molecules after they have been oxidized with periodate to form aldehydes (Chapter 1, Section 4.4).

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Figure 25.14 An amine derivative of dextran may be prepared through a two-step process involving the reaction of chloroacetic acid with the hydroxyl groups of the polymer to create carboxylates. Next, ethylene diamine is coupled in excess using a carbodiimide-mediated reaction to give the primary amine functional groups.

Figure 25.15 Amino-dextran derivatives may be prepared by the reaction of 6-bromohexanoic acid with the hydroxyl groups of the polymer followed by coupling of ethylene diamine using EDC.

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The following protocols may be used to create carboxyl-, amine-, or hydrazide-containing derivatives of dextran. Preparation of Amine or Hydrazide Derivatives by Reductive Amination 1. Prepare polyaldehyde dextran according to the method of Section 2.1 (this chapter). 2. To make an amine derivative of dextran, dissolve ethylene diamine (or another suitable diamine) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 3 M. Note: Use of the hydrochloride form of ethylene diamine is more convenient, since it avoids having to adjust the pH of the highly alkaline free-base form of the molecule. Alternatively, to prepare a hydrazide–dextran derivative, dissolve adipic acid dihydrazide (Chapter 4, Section 8.1) in the coupling buffer at a concentration of 30 mg/ml (heating under a hot water tap may be necessary to completely dissolve the hydrazide compound). Adjust the pH to 7.2 with HCl and cool to room temperature. 3. Dissolve polyaldehyde dextran in the ethylene diamine (or adipic dihydrazide) solution at a concentration of 25 mg/ml. 4. In a fume hood, add 0.2 ml of 1 M sodium cyanoborohydride to each ml of the diamine/ dextran solution. Mix well. Caution: Cyanoborohydride is extremely toxic and should be handled only in well-ventilated fume hoods. Dispose of cyanide-containing solutions according to approved guidelines. 5. React for at least 6 hours at room temperature. Overnight reactions also may be done. 6. Remove excess diamine and reaction by-products by dialysis. The ethylene diamine–dextran derivative may be used for the coupling of carboxylate-containing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide–dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. Protocol for the Modification of Dextran with Chloroacetic Acid 1. 2. 3. 4.

In a fume hood, prepare a solution consisting of 1 M chloroacetic acid in 3 N NaOH. Immediately add dextran polymer to a final concentration of 40 mg/ml. Mix well to dissolve. React for 70 minutes at room temperature with stirring. Stop the reaction by adding 4 mg/ml of solid NaH2PO4 and adjusting the pH to neutral with 6 N HCl. 5. Remove excess reactants by dialysis.

The carboxymethyl–dextran derivative may be used to couple amine-containing molecules by the carbodiimide reaction. Heindel et al. (1994) prepared the lactone derivative of carboxymethyl–dextran by refluxing for 5 hours in toluene or other anhydrous solvents. The lactone derivative is highly reactive toward amine-containing molecules, thus creating a preactivated polymer for conjugation purposes.

2.3. Epoxy Activation and Coupling Epoxy activation of hydroxylic polymers is commonly used as a means to immobilize molecules on solid phase chromatographic supports that contain hydroxyl groups (Sundberg and

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Porath, 1974). Bis-oxirane compounds also can be used to introduce epoxide groups into soluble dextran polymers in much the same manner (Böldicke et al., 1988; Böcher et al., 1992). The epoxide group reacts with nucleophiles in a ring-opening process to form a stable covalent linkage. The reaction can take place with primary amines, sulfhydryls, or hydroxyl groups to create secondary amine, thioether, or ether bonds, respectively (Chapter 2, Section 1.7). Modification of dextran polymers with 1,4-butanediol diglycidyl ether results in ether derivatives of the dextran hydroxyl groups, which then contain hydrophilic spacers with terminal epoxy functions (Figure 25.16). Protocol 1. In a fume hood, mix 1 part 1,4-butanediol diglycidyl ether with 1 part 0.6 N NaOH containing 2 mg/ml sodium borohydride. 2. With stirring, add 5 mg of dextranto each ml of the bis-epoxide solution. Mix well to dissolve.

Figure 25.16 An epoxy-functional dextran derivative may be prepared by the reaction of 1,4-butanediol diglycidyl ether with the hydroxyl groups of the polymer.

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3. React for 12 hours at 25°C or 3–4 hours at 37°C. 4. Extensively dialyze the solution against water to remove excess reactants. The activated dextran may be lyophilized for long-term storage. The epoxide-activated dextran may be used to conjugate amine-, sulfhydryl-, or hydroxylcontaining molecules. The reaction of the epoxide groups with hydroxyls requires high pH conditions, usually in the range of pH 11–12. Amine nucleophiles react at more moderate alkaline pH values, typically needing buffer environments of at least pH 9–10. Sulfhydryl groups are the most highly reactive nucleophiles with epoxides, requiring a buffered system closer to the physiological range, pH 7.5–8.5, for efficient coupling.

Figure 25.17 An amine-functionalized dextran derivative may be further reacted with SPDP to create a sulfhydrylreactive product.

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Figure 25.18 An amine-derivative of dextran may be coupled with iodoacetic acid using a carbodiimide reaction to produce a sulfhydryl-reactive iodoacetamide polymer.

Figure 25.19 Polyaldehyde dextran may be modified with the hydrazide end of M2C2H to create a thiolreactive polymer.

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2.4. Sulfhydryl-Reactive Derivatives Sulfhydryl-reactive dextran derivatives may be prepared through the use of heterobifunctional crosslinking agents (Chapter 5). In particular, crosslinkers containing pyridyl disulfide, maleimide, or iodoacetyl groups on one end are quite effective in directing a conjugation reaction to thiols. Both maleimide and iodoacetyl activation procedures will yield nonreversible bonds with sulfhydryl-containing molecules. Pyridyl disulfide compounds, by contrast, react with thiols to form cleavable disulfide bonds that can be reversed by reduction. Noguchi et al. (1992) used an amine-terminal spacer arm derivative of dextran to react with SPDP (Chapter 5, Section 1.1) in the creation of a pyridyl disulfide-activated polymer (Figure 25.17). Brunswick et al. (1988) used a different amine-terminal spacer arm derivative of dextran and subsequently coupled iodoacetate to form a sulfhydryl-reactive polymer (Figure 25.18). Heindel et al. (1991) used a unique approach. They modified polyaldehyde dextran with a heterobifunctional crosslinker containing a hydrazide group on one end and a maleimide group on the other (Chapter 5, Section 2). The hydrazides reacted with the aldehyde groups to form hydrazone linkages, leaving the maleimide ends free to result in a thiol-reactive dextran derivative (Figure 25.19).

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