bioconjugate techniques || modification with synthetic polymers

15 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 cross-linking 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 poly-urethanes, 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 etal., 1993).

The two polymers most often used in these applications are dextran and 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 Hnear strands (PEG or dextran) or branched constructs (dextran). 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 methods to allow

605

606 15. Modification with Synthetic Polymers

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 linear strand. Derivatives of both polymers are commercially available.

The foUow ing sections discuss the major properties and conjugation chemistries associated w ith the use of these polymers in modifying or conjugating proteins and other molecules.

1. Protein Modification with Activated Polyethylene Glycols

Since the first reports by Abuchow^ski and co-wôrkers in 1977 (Abuchow^ski et ai, 1977b) concerning the alteration of immunological properties towârd BSA that had been modified w ith PEG, the interest in polymer modification of biological molecules has grow^n almost exponentially. PEG coupled to other molecules can be used for altering solubiUty 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-fife of substances in vivo (Knauf et ai, 1988), to aid in penetrating cell membranes, to alter pharmacological properties (Dunn and Ottenbrite, 1991), to increase biocompatibility, especially towârd implanted foreign substances, and to prevent protein adsorption to surfaces.

PEG consists of repeating units of ethylene oxide that terminate in hydroxyl groups on either end of a linear chain. It is made from the anionic polymerization of ethylene oxide, resulting in the formation of polymer strands of various potential molecular wêights, depending on the polymerization conditions. 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.

H X .

PEG mPEG Poly(ethylene glycol) Monomethoxy-

Poly(ethylene glycol)

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 to which it is coupled—the "pegylated" molecule being protected from immediate breakdown or from being complexed and inactivated by immunoglobulins in the bloodstream.

The properties of PEG in solution are especially unusual, frequently displaying amphiphilic tendencies, having the ability to solubilize both 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

1. Protein Modification with Activated Polyethylene Glycols 607

create the ability to use it in aqueous two-phase systems for the purification of biological molecules (Johansson, 1992; Tjerneld, 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, 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 cross-linking when using a bifunctional polymer, monofunctional PEG polymers can be used that contain one end of each chain blocked with a methyl ether group. Monomethoxypolyethylene glycol (mPEG) contains only one hydroxyl per chain, thus limiting activation and coupling to one site and preventing the cross-linking and polymerization of modified molecules.

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., 1977a,b). 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 amine-containing 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 (Fig. 375).

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 (Mumtaz and Bachhawat, 1991; Abuchowski et al., 1977a). Once the first chlorine is coupled, the second one requires at least room temperature conditions at the same pH to efficiently react. If two chlorines are conjugated to nucleophilic groups, the third is even more difficult to couple, requiring at least 80°C. After activation of mPEG with TsT, it is therefore, for all practical purposes, only possible to couple one additional component to the triazine ring.


H.C. OH +

CI

C I ^ N ^ C I

- • H3C. O " N ^Cl

mPEG Trichloro-s-triazine TsT-Activated mPEG

H,C

R—NH2

Amine Containing Molecule

Pegylated Molecule

Figure 375 mPEG polymers may be activated by trichloro-s-triazine for the modification of amine-containing molecules.

TsT activation provides a simple route to an amine-reactive PEG derivative and has been used extensively as an activation method for modifying proteins (Wieder et ai, 1979; Zalipsky and Lee, 1992; Gotoh et ai, 1993). The modification of primary amine-containing molecules such as proteins is pH dependent. At physiological pH values, the reaction w ill proceed slov^er than in a more alkaline pH environment. Optimal derivatization efficiency is reached at conditions equal to or above pH 9. 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 ofmPEG 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 that 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.

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 ensure complete removal of unre-acted TsT. The residual TsT may be detected by HPLC using a 250 x 3.2-mm LiChrosorb (5-(jLm particle size) column from E. Merck. The separation is done using a mobile phase of hexane, and peaks are detected w ith 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 ofTsT-mPEG to Proteins

1. Dissolve the protein to be modified with TsT-mPEG in ice-cold 0.1 M 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 fivefold molar excess over the desired modification level. For example, Gotoh et at. (1993) added 100 mg of TsT-mPEG-5000 to 19 mg of protein dissolved in 6 ml of buffer. Add the polymer over a period of about 15 min with stirring at 4°C.

3. React for 1 h at 4°C. 4. Remove excess TsT-mPEG by dialysis or gel filtration using a column of Seph-

acryl S-300.

1.2. NHS Ester and NHS Carbonate Activation and Coupling

Carboxylate groups activated with NHS esters are highly reactive toward amine nu-cleophiles. In the mid-1970s, NHS esters were introduced as reactive ends of cross-linking 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 does not yield stable conjugates, forming thioesters or ester linkages. Both of these bonds typically hydrolyze in aqueous environments. 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 do not readily break down. In protein molecules NHS ester groups primarily react with the a-amines at the N-termi-nals and the e-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 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 monosubstituted derivative containing a single carboxylate. Creation of the suc-cinimidyl succinate and succinimidyl glutarate derivative of PEG was described by


H 3 C . . "OH + H3C. OH

mPEG

H,C.

Succinic Anhydride

Carbodiimide

Nonaqueous

'"^rx)

Succinylated mPEG

N-OH

N-Hydroxy-succinimide

(NHS)

Succinimidyl Succinate (SS)-mPEG

Figure 376 mPEG may be derivatized with succinic anhydride to produce a carboxylate end. A reactive NHS ester may 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.

H.C^ ,A^-A Succinimidyl Succinate (SS)-mPEG

H: H

N-OH

NHS

- N K


H.C.

Amide Bond Formation

Figure 377 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.


Abuchowski et al. (1984). A method for the succinylation of mPEG can be found in Section 1.3. Subsequent formation of the NHS ester derivatives of these acylated PEG compounds produce a highly reactive polymer that can be used to modify amine-containing molecules under mild conditions and with excellent yields (Figs. 376 and 377). 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 groups of PEG.

A modification of the anhydride-acylation route to obtaining reactive NHS ester-PEG compounds was introduced by Zalipsky etal. (1991,1992). In this approach, the terminal hydroxyl group of mPEG is treated with phosgene to give a reactive intermediate, an mPEG-chloroformate compound. Next, the addition of NHS gives the succinimidyl carbonate derivative (Fig. 378). Nucleophiles, such as the primary amino groups of proteins, can react with the succinimidyl carbonate functional groups to give stable carbamate (aliphatic urethane) bonds (Fig. 379). The linkage is identical to that obtained through GDI activation of hydroxyl groups with subsequent coupling of amines (Chapter 3, Section 3, and this chapter. Section 1.4). However, the reactivity of the succinimidyl carbonate is much greater than that of the imidazole carbamate formed as the active species in GDI activation.

Unlike the succinimidyl succinate or succinimidyl glutarate activation methods, succinimidyl carbonate chemistry does not suffer from the presence of a labile ester bond. The intermediate carbonate may hydrolyze in aqueous solution to release NHS and GO2, essentially regenerating 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 succinimidyl carbonate method of PEG activation and

H,C.

mPEG

H X .

O CI

N-OH

N-Hydroxy-succinimide

(NHS)

Succinimidyl Carbonate (SC)-mPEG

Figure 378 A succinimidyl carbonate derivative of mPEG was first prepared through the use of phosgene to form a chloroformate intermediate. Reaction with NHS gives the amine-reactive succinimidyl carbonate-mPEG.



H NHS

N-OH

R —NH2


Carbamate Bond Formation

Figure 379 Succinimidyl carbonate-mPEG can be used to modify amine-containing molecules to form stable carbamate linkages.

coupling has become the chemical reaction of choice for attaching the polymer to amine-containing proteins and other molecules.

A modification of the Zalipsky method by Miron and Wilchek (1993) simplifies the creation of the succinimidyl carbonate-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 succinimidyl carbonate—PEG in one step (Fig. 380). 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 (1993) modification of the Zalipsky 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 5000; 1 mmol) in 25 ml of dry dioxane. Heating in a water bath may be necessary to solubilize fully the polymer. Cool to room temperature.

2. Dissolve 6 mmol of either N-succinimidyl chloroformate or N,N'-disuccini-midyl carbonate (Aldrich) in 10 ml of dry acetone.


'!xxx> N,N'-Disuccinimidyl Carbonate (DSC)

O

N-OH

NHS

mPEG

N-Hydroxysuccinimidyl Chloroformate


Figure 380 An alternative route to an succinimidyl carbonate 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.

3. Dissolve 6 mmol of 4-(dimethylamino)pyridine in 10 ml of dry acetone (catalyst).

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 h if activating with N-succinimidyl chloroformate or 6 h 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 succinimidyl carbonate (SC)-PEG formed by addition of diethyl ether until no further precipitation is observed (typically 3-4 vol 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 ofSC-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.

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 peg-ylation 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 to 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 (Fig. 381). Atassi and Manshouri (1991) used this technique to pegylate various peptides. In this instance, the reaction was carried out in DMF due to the solubility of the peptides in this solvent. The organic-soluble carbodiimides DCC (Chapter 3, Section 1.4) or DlC (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 is employed (Chapter 3, Section 1.1). The general protocols for using carbodiimides outUned in the referenced sections may be used to conjugate a carboxylate-containing 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).

Protocol

1. Dissolve 1 g of mPEG (MW 5000) in 5 ml of anhydrous pyridine by heating to 50°C.

o

Succinylated mPEG Amide Bond Formation

Figure 381 A succinylated mPEG derivative may be coupled to amine-containing molecules using a carbodiimide reaction to form an amide bond.


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 h at 50°C. 4. Evaporate the pyridine solvent by using a flash evaporator or a rotary evapora

tor 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 1000 cut-off.

1.4. CDI Activation and Coupling

N,N'-Carbonyldiimidazole is a highly reactive carbonylating compound that 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 (Bartling 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 im-idazolyl carbamate that in turn can react with N-nucleophiles to give an N-alkyl carbamate linkage. Proteins normally couple through their N-terminals (a-amine) and lysine side chain (e-amine) functional groups. The final bond is an uncharged, urethane-like derivative having excellent chemical stability (Fig. 382). 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 succinimidyl carbonate chemistry, described previously (Section 1.2).

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 chemical reactions 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 h at pH 8.5-9 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 with the potential to cause 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 pK^. 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 substitution yields of PEG can be realized.

There are a few precautions that should be noted when doing a CDI activation and


H,C,

J n

Carbamate Linkage

Imidazole Carbamate Active Intermediate

Figure 382 N,N'-Carbonyldiimidazole (GDI) 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.

coupling experiment. First, GDI itself is extremely unstable to aqueous environments, much more so than the active imidazolyl carbamate that is formed after PEG activation. Therefore, the activation step must be done in a solvent that is free of w^ater. If unacceptable amounts of w^ater are present, GDI will be immediately broken dovv n to GO2 and imidazole. The evolution of bubbles upon addition of GDI 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% in the solvent is usually all right for a GDI activation.

A second precaution is to carry out the activation step in a fume hood away from sources of ignition. Most GDI activation protocols use flammable or toxic solvents and care should be taken in handling and disposing of them.

The coupling reaction using GDI-mPEG or GDI-PEG derivatives is slower than that obtained using NHS ester or succinimidyl carbonate coupHng methods. Therefore, the reaction times used with GDI chemistry are typically on the order of 1-2 days at 4°G 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 ofmPEG with GDI

1. Dissolve mPEG (MW 5000) in dioxane at a concentration of 50 mM (0.25 gm/ml) by heating to 37°G.

2. Add solid GDI to a final concentration of 0.5 M (81 mg/ml).


3. React for 2 h at 37°C with stirring. 4. To remove excess CDI and reaction by-products, Beauchamp et al. (1983) dia-

lyzed 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 for succinimidyl carbonate activation (Section 1.2).

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 ofCDI-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 h at 4°C. If higher pH or room temperature conditions are used, the reaction time can be decreased to 24 h.

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, Bergstrom et al. (1992) created an epoxy derivative of the polymer by reaction with epichlorohydrin under alkaline conditions. The reactive alkyl halogen end of epichlorohydrin is first coupled to the hydroxyls of PEG to give the terminal glycidyl ether derivative (Fig. 383). The epoxy-functionalized polymer could then be used to modify covalently 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).

PEG-Amine Derivative (Jeffamine Series from Texaco;

Various Polymer Lengths Available)

Creation of a sulfhydryl-reactive PEG derivative was done by Goodson and Katre (1990) by reacting a active ester-maleimide heterobifunctional cross-linker with the


OH + H,C.

mPEG

CI

Epichlorohydrin Epoxy-Activated mPEG

- N H ,


HX

Secondary Amine Bond Formation

Figure 383 Epichlorohydrin may be used to activate the hydroxyl group of mPEG, creating an epoxy derivative. Reaction with amine-containing molecules yields secondary amine bonds.

amino groups of a PEG-amine polymer (Pillai and Mutter, 1980). An amine-terminal derivative of a PEG-type polymer is the Jeffamine series from Texaco Chemical. Reaction w ith the N-maleimido-6-aminocaproyl ester of l-hydroxy-2-nitro-4-benzene sulfonic acid resulted in an amide bond derivative (Fig. 384). This created terminal maleimide groups on each PEG-amine molecule. Maleimide compounds can be used in a site-directed coupling procedure to pegylate specifically at the sulfhydryl groups of proteins and other molecules (Chapter 2, Section 2.2).

In another approach, Wirth etal. (1991) and Chamow etal. (1994) transformed the terminal hydroxyl group of mPEG into an aldehyde residue by the Moffatt oxidation procedure (Harris et ai, 1984). In this reaction, the hydroxyl is treated with acetic anhydride in DMSO containing triethylamine, converting it to the aldehyde (Fig. 385). After stirring at room temperature for 48 h, 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 cya-noborohydride (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 that is well tolerated by most proteins.

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

2. Protein Modification with Activated Dextrans 619

H,N NH2 + 2

PEG-Amine Derivative (Jeffamine"^^)

N-Maleimido-6-anninocaproyl ester of 1-Hydroxy-2-nitro-4-benzene sulfonic acid

2 > ^ HO

w ^O

I 0-

O " " O -

Bifunctional Maleimide Derivative

Figure 384 A PEG-amine compound, such as the Jeffamine polymers from Texaco, may be reacted with this heterobifunctional cross-Hnker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of cross-linking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

H,C, Acetic Anhydride DMSO

Triethylamine

48 hours

Room Temp. HX^

n O

S ^ H

mPEG Oxidation to Terminal Aldehyde

NaCNBH

H X .

-NH,


Secondary Amine Bond Formation

Figure 385 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.


D-glucose linked together in glycosidic bonds (Chapter 1, Section 2), wherein the carbon-1 of one monomer is attached to the hydroxy 1 group at the carbon-6 of the next residue. This configuration is the same as that found in the a-l,6-Unked disaccharide isomahose. 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 a-1,2, a-1,3, or a-1,4 glycosidic linkages. The branch type and degree of branching vary by species.

HO OH

Isomaltose (a-1,6) Repeating Unit of Dextran Polymer Chains

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 three hydroxyls (four 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 10,000-500,000 has been used extensively as a modifying or cross-linking 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 elicit an immune response against coupled molecules (Shih etal, 1991; Dintzis etal, 1989), as an inducer of B-cell proliferation by coupling anti-Ig antibodies (Brunswick et al., 1988), as a multifunctional linker to cross-link monoclonal antibody conjugates with chemotherapeutic agents (Heindel etal., 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 macro-molecules 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 active derivatives may be used to couple with proteins and other molecules containing the appropriate functional groups.

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


o—I V-0 o 1,0 o C Sodium Periodate H O i ' / V n O O

W \u,OH ^ ) = 0

HO ^OH N ^ 0 0 .. ^ HO^ OH HO^ O

Dextran Polymer Carbon-Carbon Bond ' Cleavage with Oxidation

to Aldehyde Residues

Figure 386 Dextran polymers may be oxidized with sodium periodate to create a polyaldehyde derivative.

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 etal., 1978) (Fig. 386). Polyaldehyde dextran may be conjugated with amine groups by Schiff base formation followed by reductive amina-tion to create stable secondary (or tertiary amine) linkages (section 2.1.4) (Fig. 387).

Proteins may be modified with oxidized dextran polymers under mild conditions using sodium cyanoborohydride as the reducing agent. The reaction proceeds primarily through 8-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.

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 (NaI04) (Sigma) in 500 ml of deionized water at a concentration of 0.03 M (6.42 g).

R —NH„ HO I ' / V ' l iO ?"

NaCNBH, / ^ ^*A y^^Z^ R - N H O ^~~~~^ "

HO O HO

Periodate Oxidized Dextran Secondary or Tertiary Amine Formation

Figure 387 Polyaldehyde dextran may be used as a multifunctional cross-linking agent for the coupling of amine-containing molecules. Reductive amination creates secondary amine linkages.


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 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 hy-droxylamine 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 periodate-oxidized sugars are capable of reducing Cu " to Cu"^, which can be detected using the bicinchoninic acid (BCA) reagent (Pierce Chemical) (Smith 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 p'rotein 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 later. 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 milliliter 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 h 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 milliliter of the reaction. Note: If a second molecule is to be coupled after the initial protein conjugation, do not block the remaining aldehydes until the second molecule is added.

6. React for an additional 2 h at room temperature. 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 carboxylate-containing 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 cross-linking agent (Chapter 5).

This type of modification process has been used to form sulfhydryl-reactive dextran polymers by coupling amine spacers with cross-linkers containing an amine-reactive end and a thiol-reactive end (Noguchi et al, 1992; Brunswick et al., 1988). 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, 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'-iminofc/spropylamine) can be coupled in excess to polyaldehyde dextran to create an amine-terminal derivative. Carboxyl-terminal derivatives may be prepared similarly by coupling molecules such as 6-aminocaproic acid or p-alanine to polyaldehyde

Ethylene Diamine

Carboxymethyl Dextran

Figure 388 An amine terminal derivative of dextran may be prepared through a two-step process involving the reaction of chloroacetic acid w ith the hydroxyl groups of the polymer to create carboxylates. Next, ethylene diamine is coupled using a carbodiimide-mediated reaction to give the primary amine functional groups.


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. In this manner, Brunsw^ick et al. (1988) used chloroacetic acid to modify the hydroxyl groups of dextran, forming the carboxymethyl derivative. The carboxylates w ere then aminated w ith ethylene diamine to create an amine-terminal derivative (Inman, 1985). The amine w as then modified w ith iodoacetate to form a sulfhydryl-reactive polymer (Fig. 388).

In a somewhat similar scheme, Noguchi etal. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with the dextran polymer. The carboxylate was then aminated with ethylene diamine to form an amine-terminal spacer (Fig. 389). This dextran derivative was finally reacted with SPDP (Chapter 5, Section 1.1) to create the final sulfhydryl-reactive polymer (Section 2.4). 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 of dextran also may be prepared from the periodate-oxidized polymer or from a carboxyl derivative by reaction with fc/s-hydrazide compounds (Chapter 4, Section 8). A hydrazide terminal spacer provides reactivity toward al-

Dextran Polymer X

. < wQ.,„oH HO OH ) ^

HO^ OH

H,N

o o

V ^ ViiOH O OH ) ^

6 OH

Amine Functional Dextran Derivative

NH2

Ethylene Diamine

Figure 389 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.


dehyde- 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).

The following protocols may be used to create carboxyl-, amine-, or hydrazide-containing derivatives of dextran.

Protocol for the Preparation of Amine or Hydrazide Derivatives by Reductive Amination

1. Prepare polyaldehyde dextran according to the method of Section 2.1. 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 dissolve completely the hydrazide compound). Adjust the pH to 7.2 with HCl.

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 milliliter 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 h 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 to couple with carboxylate-containing molecules by the carbodiimide reaction, to couple amine-reactive probes, or to further modify using heterobifunctional cross-linkers. The hydrazide-dextran derivative may be used to cross-link aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins.

Protocol for the Modification of Dextran with Chloroacetic Acid

1. In a fume hood, prepare a solution consisting of 1 M chloroacetic acid in 3 M NaOH.

2. Immediately add dextran polymer to a final concentration of 40 mg/ml. Mix well to dissolve.

3. React for 70 min at room temperature with stirring. 4. Stop the reaction by adding 4 mg/ml of solid NaH2P04 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 h in toluene or other an-


hydrous 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 (Sundberg and Porath, 1974). B/soxirane compounds also can be used to introduce epoxide functional groups into soluble dextran polymers in much the same manner (Bocher et al., 1992; Boldicke et al., 1988). The epoxide group can react w ith 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).

HO OH

Dextran Polymer

Figure 390 An epoxy-functional dextran derivative may be prepared by the reaction of 1,4-butanediol diglycidyl ether v^ith the hydroxyl groups of the polymer.


Modification of dextran polymers with 1,4-butanediol diglycidyl ether results in ether derivatives on the dextran hydroxyl groups containing hydrophilic spacers with terminal epoxy functions (Fig. 390).

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 dextran to each ml of the fo/s-epoxide solution. Mix well to dissolve.

3. React for 12 h at 25°C or 3 -4 h at 37°C.

H,N

Figure 391 An amine-functionalized dextran derivative may be further reacted with SPDP to create a sulfhydryl-reactive product.


Amine Functional Dextran Derivative

lodoacetylated Dextran Polymer

Figure 392 An amine-derivative of dextran may be coupled with iodoacetic acid using a carbodiimide reaction to produce a sulfhydryl-reactive iodoacetamide polymer.

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 hydroxyl-containing molecules. The reaction of the epoxide functional groups with hydroxyls requires high pH conditions, usually in the range pH 11-12. Amine nucleo-philes react at more moderate alkaline pH values, typically needing buffer environments of at least pH 9. 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.

2.4. Sulfhydryl-Reactive Derivatives

Sulfhydryl-reactive dextran derivatives may be prepared through the use of heterobi-functional cross-linking agents (Chapter 5). In particular, cross-linkers containing pyridyldisulfide, 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. Py-ridyl disulfide compounds, however, 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 pyridyldisulfide-activated polymer (Fig. 391). Brunswick etal. {198^) used a different amine-terminal spacer arm derivative of dextran and subsequently coupled iodoacetate to form a sulfhydryl-reactive polymer (Fig. 392). Heindel et al. (1991) used a unique approach. They

2. Protein Modification with Activated Dextrans

X

629

HO 0

Polyaldehyde Dextran

^ ^

H,N-N 2 H

>0 O-

Viio o-

Maleimide-Activated Dextran Derivative

Figure 393 Polyaldehyde dextran may be modified with the hydrazide end of M2C2H to create a thiol-reactive polymer.

modified polyaldehyde dextran with a heterobifunctional cross-linker 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 (Fig. 393).

bioconjugate techniques || modification with synthetic polymers

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