Terminal Groups in Starburst Dendrimers: Activation and Reactions

54

Bioconjugate Chem. 1998, 9, 54−63

Terminal Groups in Starburst Dendrimers: Activation and Reactions with Proteins† Pratap Singh* Dade Behring Inc., P.O. Box 520672, Miami, Florida 33152-0672. Received April 2, 1997; Revised Manuscript Received August 21, 1997X

Starburst dendrimers are novel nanoscopic synthetic polymers of defined molecular mass and geometry. These macromolecules, available in commercial quantities, contain methyl carboxylate and primary amino-terminal groups. The presence of these groups on any macromolecule limits its usefulness especially in cases where, for specific modulation of the properties of biologically active molecules, covalent bond formation is desirable between the biologically active molecules and the macromolecule. This paper describes activation of the surface groups of Starburst dendrimers for incorporation of a number of reactive electrophilic and nucleophilic groups and utilization of these reactive groups in formation of covalent bonds between dendrimers and alkaline phosphatase. The protein-dendrimer complexes have been reacted further with the Fab′ fragment of an anti-creatine kinase MB isoenzyme antibody to form multifunctional dendrimer reagents. The enzymatic and immunochemical properties of these protein-dendrimer reagents have been evaluated by an immunoassay system. Nucleophilic thiols and electrophilic phenyliodoacetamido, iodoacetamido, and epoxy groups have been incorporated into amino-terminal dendrimers by their reactions with appropriate heterobifunctional reagents. Two independent sets of reactions have been used to prepare the reactive N-hydroxysuccinimidyl esters from dendrimers containing the terminal carboxyl groups. Quantitation of the reactive groups has been carried out by direct titration of these activated dendrimers and the products obtained by reactions of these dendrimers with small molecules and proteins.

INTRODUCTION

In recent years, dendrimers (1-6) have attracted attention as structurally unique synthetic macromolecules with a very broad range of applications in physical, chemical, and biological processes (7-12). Dendrimers are synthesized by either a divergent or a convergent approach (1). The method of choice depends on the desired generation (4) of a specific structure, which in turn dictates the molecular mass, the terminal functional groups, and the physical dimensions of the molecule. To modulate some specific properties of biologically active molecules, a number of these molecules that show specific activity such as drugs, enzymes, antibodies, and nucleotides have been covalently coupled to macromolecules [e.g. poly(ethylene glycol) or polysaccharides]. Very limited chemical manipulations are generally possible on many of the biologically active molecules, of either natural or synthetic origin, due to the labile nature or specific structural requirements for the maintenance of their biological activity. Functional groups on the surface of dendrimers show remarkably higher chemical reactivity (13) in comparison to their activity when present in other macromolecules. This unusual reactivity is expected to allow coupling reactions to occur under mild reaction conditions. These characteristics make a dendrimer, with its controlled interior, the macromolecule of choice for covalent coupling † Presented as a poster at the Fourth Pacific Polymer Conference held at Koloa, Kauai, HI, on December 12-16, 1995. * Author to whom correspondence should be addressed. Telephone: (305) 591-5556. Fax: (305) 597-5176. X Abstract published in Advance ACS Abstracts, December 15, 1997.

reactions. A biologically active molecule is likely to retain its maximum possible activity when present in a dendrimer-biologically active molecule complex prepared via the surface groups of dendrimers. The feasibility of such a concept has been demonstrated by preparation of dendrimer-peptide (14), dendrimer-antibody (11, 15), dendrimer-fullerene (16), and dendrimer-antibodyporphyrin (17) complexes. However, full exploitation of these molecules for a number of applications is limited due to the nature of the terminal functional groups available in the presently known dendrimer structures. A limited number of dendrimers are known (18) to contain reactive terminal groups. However, these reactive dendrimers have been synthesized by the convergent method, where it is often difficult to prepare large quantities of higher-generation macromolecules, thus limiting the choice of available molecules. Kilogram quantities of highly pure dendrimers, with broad molecular mass ranges, that have been synthesized by the divergent method are available (19, 20). One category 1 Abbreviations: ALP, calf intestine alkaline phosphatase; ALP-dendrimer (Dn), ALP-dendrimer complex prepared by reaction of ALP with the activated dendrimer of the nth generation; ALP-PDH, 6-[3-(2-pyridyldithio)propionamide]hexanoate derivative of ALP; BAHA, [6-(bromoacetamido)hexyl]amine; CKMB, creatine kinase MB isoenzyme; dendrimerPDH, 6-[3-(2-pyridyldithio)propionamide]hexanoate derivative of dendrimer; dendrimer-X, derivatized dendrimer containing the surface group X; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; NIA, N-hydoxysuccinimidyl iodoacetate; sulfo NHS LC SPDP, sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide]hexanoate; sulfoSIAB, sulfosuccinimidyl (4-iodoacetyl)aminobenzoate; TSTU, N,N,N′,N′-tetramethylO-(N-succinimidyl)uronium tetrafluoroborate.

S1043-1802(97)00048-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/12/1998

Starburst Dendrimers: Terminal Group Activation

of dendrimers available in large quantities is the full and half-generations of Starburst dendrimers (1). This paper describes methods for modifying the terminal amino and carboxyl groups of Starburst dendrimers (1) to prepare dendrimer derivatives containing a defined number of reactive nucleophilic and electrophilic groups. These reactive groups have been utilized to couple two proteins in sequence, namely calf intestine alkaline phosphatase (ALP)1 and the Fab′ fragment of an anti-creatine kinase MB isoenzyme (CKMB) antibody, to form ALP-dendrimer and ALP-dendrimer-Fab′ complexes. The performance characteristics of these proteindendrimer complexes have been studied on Stratus II, an automated enzyme immunoassay system. MATERIALS AND METHODS

All reagents used were of analytical grade or better and were purchased from either Aldrich Chemical Co. (Milwaukee, WI), Sigma Chemical Co. (St. Louis, MO), or Fluka Chemical Corp. (Ronkonkoma, NY). The heterobifunctional reagents were purchased from BioAffinity Systems Inc. (Rosco, IL). Ultrogels AcA 44 and AcA 34 were obtained from BioSepra (Marlborough, MA). BCA protein assay reagent was obtained from Pierce (Rockford, IL). Alkaline phosphatase was obtained from Boehringer Mannheim Corp. (Indianapolis, IN). Mass spectra were run by the Mass Spectrometry Laboratory of the University of California at Riverside. The performance of the protein-dendrimer complexes was evaluated on Stratus II, an automated fluorometric enzyme immunoassay system available from Dade International Inc. (Miami, FL). The ALP-Fab′ conjugate, sold commercially in the CKMB assay kit for Stratus II, was used to compare the performance of the ALP-dendrimer-Fab′ complexes. Unless otherwise indicated, all reactions were carried out at room temperature. All full and half-generation poly(amidoamine) dendrimers, with ethylenediamine as the initiator core molecule (1), were obtained from Dendritech, Inc. (Midland, MI). The half-generation dendrimers were obtained as the sodium salts prepared by hydrolysis of the corresponding methyl esters with sodium hydroxide. Partially hydroxylated dendrimers were prepared at Dendritech, Inc., by using a mixture of ethylenediamine and ethanolamine in place of ethylenediamine at the final stage of synthesis. This modified synthetic procedure was used to prepare dendrimers containing a mixture of terminal hydroxyl (77%) and primary amino (23%) groups. Partially carboxymethylated dendrimer was prepared by treating a methanol solution of the dendrimer with 0.85 mol of bromoacetic acid at 37 °C for 4 h. The methanol solution was evaporated under reduced pressure, and the number of amino groups remaining in the treated dendrimer was determined by reaction with fluorescamine (21). Derivatized dendrimers of generations 1-3 were purified by repeated precipitation of a methanol solution on addition of ethyl acetate, benzene, or dioxane. The 4.0-, 4.5-, and 5th generation dendrimers were purified by ultrafiltration in an Amicon cell using a YM 10 membrane (molecular mass cutoff of 10 kDa). The Amicon concentrate, containing the derivatized dendrimer solution, was either used immediately to react with a protein or evaporated under reduced pressure to isolate the derivative. The ALP-dendrimer complexes were separated from the low-molecular mass contaminants, including the derivatized dendrimers by ultrafiltration (Amicon cell with a YM 10 membrane) or by gel filtration. A

Bioconjugate Chem., Vol. 9, No. 1, 1998 55

column of either Sephadex G-25 or Ultrogel AcA 44 (fractionation range of 10-130 kDa; exclusion limit of 200 kDa) equilibrated with a buffer containing 100 mM phosphate/1.0 mM magnesium chloride (phosphate buffer) at pH 7.0-7.4 was used for gel filtration. Free thiols in the derivatized dendrimer and the protein-dendrimer-SH complex were determined by measuring the increase in absorption at 324 nm on addition of a solution of 4,4′-dithiodipyridine (22). Titration of electrophilic epoxy, phenyliodoacetamido, and iodoacetamido groups in the derivatized dendrimers and the dendrimer-X and protein-dendrimer-X complex (X ) epoxy, phenyliodoacetamido, and iodoacetamido) was carried out by reaction with an excess of dithiothreitol followed by reaction of the remaining dithiothreitol with 4,4′-dithiodipyridine essentially as previously described (23). The N-hydroxysuccinimidyl esters were quantitated as described by Miron and Wilchek (24). The periodate-oxidized ALP was prepared by a procedure similar to that described by Husain and Bieniarz (25). ALP-SH was prepared by the reaction of ALP with sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide]hexanoate (sulfo NHS LC SPDP; 26) followed by reaction with dithiothreitol. Excess dithiothreitol was separated from the protein solution by passage over a Sephadex G-25 column equilibrated with the phosphate buffer at pH 7.0. The poly(amidoamine) dendrimers and many of the derivatives prepared in this study do not contribute to absorptions between 260 and 280 nm. Protein concentrations (mg/mL) of the solutions containing ALP and ALP-dendrimer complexes were therefore calculated from absorptions measured at 280 and 260 nm and using the formula A280/0.689 - A260/1.351. In this formula, 0.689 and 1.351 are the extinction coefficients (milliliters per milligram per centimeter) at 280 and 260 nm, respectively. BCA protein assay reagent was used to determine protein concentrations for the ALP-dendrimer complexes prepared from a dendrimer derivative containing an aromatic or another chromophoric group, e.g. sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfoSIAB)-activated dendrimer. Preparation and Reactions of Dendrimer Derivatives Containing Phenyliodoacetamido 1 and Iodoacetamido Groups 3. The carboxymethylated 5th generation dendrimer (16 mg, 0.5 µmol) was dissolved in 1 mL of the phosphate buffer at pH 7.4. A 277 µL solution of sulfoSIAB (10 mg/mL in water, 5.5 µmol) was then added. After 1 h at 30 °C, the reaction mixture was diluted with 50 mL of the phosphate buffer at pH 7.6 and concentrated to about 2 mL in an Amicon ultrafiltration cell. This dilution-concentration process was repeated two more times. The dendrimer derivative 1 present in about 1 mL of concentrate was combined with a 1 mL solution of the periodate-oxidized ALP (8.1 mg, 58 nmol) in the phosphate buffer at pH 7.4 and the combined mixture concentrated to 1.2 mL. After 16 h at 4 °C, the reaction mixture was adjusted to pH 6.3 by a careful addition of 1 N HCl and then incubated for 1 h with a 200 µL aqueous solution of sodium cyanoborohydride (30 mg/mL, 95 µmol). The ALP-dendrimer complex 2 formed by this sequence of reactions was purified by passage over an Ultrogel AcA 44 column equilibrated and eluted with the phosphate buffer at pH 7.4. A solution of N-hydroxysuccinimidyl iodoacetate (NIA, 60.4 mg, 213.4 µmol) in 1 mL of tetrahydrofuran was added dropwise to a solution of the 1st generation aminoterminal dendrimer (32.7 mg, 20.4 µmol) in 2 mL of 50% alcohol. After 1 h, excess solvents were removed under reduced pressure, and the residue was washed with

56 Bioconjugate Chem., Vol. 9, No. 1, 1998

tetrahydrofuran. The tetrahydrofuran-insoluble semisolid product, the iodoacetamido-dendrimer 3, was dried. The solid residue dissolved in 1 mL of alcohol and 0.5 mL of the phosphate buffer at pH 7.0 was mixed with a solution of ALP-SH (11.9 mg, 85 nmol) in 12 mL of the phosphate buffer at pH 7.0. ALP-SH, used in these reactions, contained an average of 1.4 free thiols per mole of protein. After incubation for 16 h at 4 °C, the proteindendrimer complex 4 was purified over a column of Ultrogel AcA 44 in the phosphate buffer at pH 7.0. Preparation of Dendrimer Derivatives 6 Containing Epoxy Groups. A solution of the 5th generation dendrimer (165.8 mg, 5.7 µmol), in 1 mL of 50% alcohol, was mixed with sodium carbonate (200 mg, 1.9 mmol) and epibromohydrin (0.96 g, 7.1 mmol). After stirring for 4 h, the reaction mixture was diluted with 10 mL of 50% alcohol. The solution was concentrated to about 1 mL in an Amicon ultrafiltration cell. The concentrate was diluted again to 10 mL with 50% alcohol. This concentration-dilution process was continued till the effluent was neutral (pH paper). The dendrimer derivative 6 present in the concentrate was stored at 4 °C until it was used for reaction with ALP. A 0.4 mL alcohol solution of the 2nd generation dendrimer (41.5 mg, 13 µmol) was mixed with epibromohydrin (0.5 g, 3.7 mmol) and triethylamine (36 mg, 0.36 mmol). After 4 h at 37 °C, the reaction mixture was evaporated under vacuum to a semisolid residue. The product 6 was purified by repeated precipitation (alcohol/ benzene). A solution of this product in 0.2 mL of alcohol and 0.2 mL of the phosphate buffer at pH 7.2 was mixed with a 1.5 mL solution of ALP-SH (1.9 mg, 13.6 nmol) in the phosphate buffer at pH 7.2. The ALP-SH used for this reaction contained 2.6 free thiols per mole of the protein. After 16 h at 4 °C, the ALP-dendrimer complex 7, formed in the reaction mixture, was then separated from other contaminants by passage over a Sephadex G-25 column in the phosphate buffer at pH 7.2. The succinyl-dendrimer-epoxide was prepared by stirring a reaction mixture containing the 5th generation dendrimer (222 mg, 7.7 µmol) and succinic anhydride (148 mg, 1.5 mmol) in 5 mL of 50% alcohol. After 2 h, epibromohydrin (2 mL, 23.5 mmol) was added and the mixture stirred for an additional 16 h. The derivatized dendrimer was purified by ultrafiltration in an Amicon cell (YM 10 membrane) using 50% alcohol as described above. Reaction of Dendrimers To Incorporate Thiol Groups. A 3.0 mL methanolic solution of the 5th generation dendrimer (330 mg, 11.4 µmol) was flushed with nitrogen. The dendrimer solution was then allowed to react for 1 h with a solution of sulfo NHS LC SPDP (50 mg, 95 µmol) in 0.5 mL of water. This reaction produced the 6-[3-(2-pyridyldithio)propionamide]hexanoate derivative of dendrimer 8, i.e. NH2-dendrimerNHCO(CH2)5NHCO(CH2)2S-S-Py (dendrimer-PDH). The methanol solution was evaporated under vacuum and the semisolid residue stored at -10 °C. To block the residual amino groups in the dendrimerPDH derivative prepared above, a 1.8 mL methanol solution of this derivative (148 mg, 5 µmol) was mixed with succinic anhydride (550 mg, 5.5 mmol) and 2 mL of tetrahydrofuran. The trinitrobenzenesulfonic acid color test (27) was used to monitor reaction of the primary amino groups in the dendrimer-PDH derivative. After 24 h, the organic solvents were removed by evaporation under reduced pressure and the dendrimer derivative was separated from the side products by Amicon ultrafiltration of the mixture using water. The aqueous

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solution of the succinyl-dendrimer-PDH, present in the Amicon concentrate, was evaporated under vacuum to provide 173 mg of the succinyl-dendrimer-PDH derivative as an oil. The 2nd generation dendrimer (75 mg, 23.4 µmol) dissolved in 4 mL of methanol was reacted for 1 h with a solution of sulfo NHS LC SPDP (55 mg, 104.4 µmol) in 0.5 mL of water. The reaction mixture containing this dendrimer-PDH derivative 8 was concentrated to 1.0 mL by evaporation under reduced pressure and then mixed with a 9 mL solution of the periodate-oxidized ALP (18 mg, 128.6 nmol) in 50 mM sodium phosphate/50 mM sodium carbonate at pH 9.0. After 1 h, the reaction mixture was adjusted to pH 6.5 by a careful addition of 1 N HCl. An aqueous solution (0.7 mL) of sodium cyanoborohydride (27 mg, 0.44 mmol) was then added. After 30 min, the resulting ALP-dendrimer-PDH complex was buffer exchanged with the phosphate buffer at pH 7.0 in an Amicon ultrafiltration cell (YM 10). The dithiothreitol/4,4′-dithiodipyridine titration of this ALPdendrimer-PDH complex showed the presence of 25 free thiols (as pyridyl disulfide linkage) per mole of the protein. The thiol content was calculated from the increase in absorption at 343 nm resulting from the formation of pyridine-4-thione. The dendrimer derivative succinyl-dendrimer-PDH (43 mg, 1.5 µmol) dissolved in 1.0 mL of 100 mM sodium phosphate/5 mM EDTA at pH 6.0 (phosphate/EDTA buffer) was mixed with a 0.2 mL solution of dithiothreitol (3.1 mg, 20 µmol) in the phosphate/EDTA buffer. After 1 h at 30 °C, the reaction mixture was passed through a Sephadex G-25 column equilibrated and eluted with the phosphate/EDTA buffer. The column fractions, containing the dendrimer derivative with free thiols, were monitored by measuring the increased absorption at 324 nm on addition of a solution of 4,4′-dithiodipyridine (22) to aliquots of the individual fractions. The dendrimer derivatives dendrimer-PDH and ALP-dendrimer-PDH were similarly treated with dithiothreitol to provide the derivatives NH2-dendrimer-SH 9 and ALP-dendrimerSH 10, respectively. The derivative ALP-dendrimerSH 10 was separated from excess dithiothreitol either by a Sephadex G-25 column or by an exchange with the phosphate/EDTA buffer in an Amicon ultrafiltration cell till the effluent showed a negative reaction on addition of a solution of 4,4′-dithiodipyridine. A titration of the NH2-dendrimer (D5)-SH 9 and succinyl-dendrimer (D5)-SH derivatives with 4,4′-dithiodipyridine (22) showed the presence of 3.8 and 3.7 thiols per mole of the dendrimer, respectively. Synthesis of [6-(Bromoacetamido)hexyl]amine (BAHA). BAHA 14 was synthesized (Scheme 2) by a procedure similar to that described by Heindel et al. (28). In brief, 2 mL of a methanol solution of N-BOC-1,6diaminohexane 11 (0.5 g, 2.3 mmol) was added to a solution of N-hydroxysuccinimidyl bromoacetate 12 (0.5 g, 2.1 mmol) in 5.0 mL of tetrahydrofuran. The ethyl acetate solution (20 mL) of the residue, obtained on evaporation of the organic solvents, was washed with 10 mM HCl (3 × 5 mL). The dichloromethane solution (5.0 mL) of the dry intermediate 13, obtained after removal of ethyl acetate, was mixed with trifluoroacetic acid (5 mL, 65 mmol). The trifluoroacetate salt of BAHA 14 was obtained in 50% yield as a white hygroscopic solid on crystallization from 2-propanol/hexane: mp 114-115 °C (corrected). The purity of the material was determined to be >90% by quantitation of the bromoacetamido groups as described before (23). MS m/z (relative intensity): 236, 238 (M+ , due to two isotopes of bromine of



Scheme 1. Activation and Reactions of Dendrimers Containing Terminal Amino Groupsa

a (a) sulfoSIAB; (b) ALP-CHO, sodium cyanoborohydride; (c) NIA; (d) ALP-SH; (e) Fab′; (f) epibromohydrin; (g) ALP-SH; (h) sulfo NHS LC SPDP; (i) dithiothreitol; (j) ALP-CHO, sodium cyanoborohydride, dithiothreitol.

Scheme 2. Synthesis of [6-(Bromoacetamido)hexyl]amine

atomic weights 79 and 81; 28%), 121, 123 (the bromoacetyl residue; 58%), and 116 [the protonated form of the residue NH2(CH2)6NH; 100%]. Activation of the Half-Generation Carboxy-Terminal Dendrimers. The sodium salts of the halfgeneration dendrimers, dissolved in water, were converted to the acid form by acidification with 5 N hydrochloric acid to pH 3.0. The acidified solutions were evaporated to dryness under high vacuum. Activation reaction of the free acid form of these dendrimers was carried out with N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) essentially as described by Bannwarth and Knorr (29). A solution containing the 2.5th generation dendrimer (30 mg, 7.2 µmol) in 0.2 mL of water and 0.8 mL of DMF was mixed with N,N-diisopropylethylamine (90 mg, 0.7 mmol) and TSTU (74 mg, 0.24 mmol). After 1 h, solvents were removed from a red reaction mixture under reduced pressure. The residue was washed with ethyl acetate (3 × 10 mL), and the red residue obtained on removal of all the solvents was dissolved in 0.4 mL of water. The aqueous solution of the dendrimer-active ester 15 (Scheme 3) was stored in an ice bath while the active ester concentration was being determined by titration (24). A 100 µL aliquot of the aqueous solution, containing 4.4 µmol of the active ester as determined by titration, was mixed with 1 mL of a refrigerated solution of ALP (3 mg, 21 nmol) in the phosphate buffer at pH 7.0. After

incubation for 30 min in an ice bath, the reaction mixture was combined with 0.4 mL of a 2-propanol solution of BAHA (20 mg, 62.5 µmol) and stirred gently at 4 °C for 16 h. The ALP-dendrimer complex 17, containing reactive bromoacetamido groups, was purified over an Ultrogel AcA 44 column in the phosphate buffer at pH 7.0. To incorporate other functional groups, the intermediate 16, obtained after reaction of ALP with the activated dendrimer, was combined separately with either a 1 mL solution of 100 mM ethylenediamine or 2-aminoethanethiol in the phosphate buffer to react with a homo- or heterobifunctional nucleophile. These reactions produced the complexes ALP-dendrimer-NH2 18 and ALP-dendrimer-SH 19, respectively. A solution of the free acid form of the 1.5th generation carboxyl-terminal dendrimer (33.4 mg, 15.2 µmol) in 1 mL of 80% DMF was mixed with N-hydroxysuccinimide (115 mg, 1 mmol) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC, 200 mg, 1 mmol). The reaction mixture was stirred for 16 h followed by washing the reaction mixture with ethyl acetate (2 × 5 mL) and 2-propanol (2 × 5 mL). The active ester 15 obtained on removal of the combined organic solution was dissolved in 0.5 mL of water. The aqueous solution containing 32 µmol of the active ester was reacted with a 1.8 mL solution of ALP (6.2 mg, 44.3 nmol) after titration of its active ester content as described above.


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Scheme 3. Activation and Reactions of Dendrimers Containing Terminal Carboxyl Groupsa

a

(a) TSTU or EDC/NHS; (b) ALP-NH2; (c) BAHA; (d) ethylenediamine; (e) 2-aminoethanethiol; (f) Fab′.

The nonspecific binding of the ALP-dendrimer complexes was measured by using a solution of the complex in place of the ALP-Fab′ conjugate on Stratus II. The complex was diluted to 500 ng of protein/mL of solution in a buffer containing 100 mM Tris/1% BSA/ 1% Triton X-100/2 mM magnesium chloride/0.1 mM zinc sulfate at pH 7.2. Coupling of Fab′ with the ALP-Dendrimer Complex. The mouse monoclonal IgG1 was converted to its fragments F(ab′)2 and Fab′ as described by Ishikawa et al. (pages 29-30 of ref 23). The number of free thiols per mole of Fab′ was found to be 2.5 ( 0.2. A typical procedure for the coupling of Fab′ and the ALP-dendrimer complex, 4 or 17, involved mixing of a 5.5 mL solution of Fab′ (5.5 mg, 119.6 nmol) in the phosphate/EDTA buffer with 5.4 mL of a solution of the ALP-dendrimer complex (2.9 mg of protein, 20.7 nmol) in the phosphate buffer at pH 7.0. For this specific example, the ALP-dendrimer complex was prepared by a reaction of the NIA-activated 3rd generation dendrimer and ALP-SH. The mixture of Fab′ and the ALPdendrimer complex was concentrated to about 2 mL in an Amicon ultrafiltration cell (YM 10) and then buffer exchanged with the phosphate buffer at pH 7.6 till concentration of EDTA in the concentrate was e5 µM and the pH of the effluent was 7.6. The reaction mixture, 1.6 mL, was incubated at 4 °C for 16 h and then mixed with 32 µL of a 20 mg/mL solution of N-ethylmaleimide (0.64 mg, 5.1 µmol) in N,N-dimethylformamide. After 2 h, the reaction mixture containing free unconjugated Fab′ and the ALP-dendrimer-Fab′ complex, 5 or 20, was applied to an Ultrogel AcA 34 column (fractionation range of 20-350 kDa; exclusion limit of 750 kDa) equilibrated with 10 mM Tris/100 mM NaCl/0.1 mM magnesium chloride/0.01 mM zinc sulfate/0.1% azide at pH 7.0 (Tris buffer). The performance of the fractions was evaluated on Stratus II. The fractions containing the ALPdendrimer-Fab′ complex, eluting as the first peak from the AcA 34 column, were pooled on the basis of their performance being similar to that shown by the ALPFab′ conjugate. The protein concentration of the ALPdendrimer-Fab′ conjugate was determined using the BCA protein assay with alkaline phosphatase as an internal standard. The column was calibrated with protein standards from Pharmacia Biotech using blue dextran (2000 kDa), thyroglobulin (669 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67

kDa), and ovalbumin (43 kDa). The linear regression analysis of this calibration curve (fraction number vs molecular mass) was used to calculate the apparent molecular mass of the ALP-dendrimer-Fab′ complex present in the specific column fraction. RESULTS

The biochemical and immunochemical evaluations of the protein-dendrimer complexes, ALP-dendrimer and ALP-dendrimer-Fab′, were carried out on Stratus II. This heterogeneous immunoassay system utilizes a sandwich format to analyze samples containing an analyte, e.g. CKMB. Glass fiber filter paper, a negatively charged surface, is used in this system to immobilize an antiCKMB antibody called the capture antibody. The capture antibody provides one component necessary to form the sandwich. The second component of the sandwich is provided by a solution of the conjugate ALP-Fab′. These two sandwich components that utilize antibodies have distinctly different requirements for the solid phase in any heterogeneous immunoassay system. For optimum performance, the capture antibody must be able to bind strongly to the solid phase whereas the antibodyenzyme conjugate should have the least affinity for the solid support. An affinity of the antibody-enzyme conjugate for the solid support would increase nonspecific binding and thus compromise the sensitivity of the system. During the initial phase of these studies, it was clear that to minimize the solid support (glass fiber) affinity for a protein-dendrimer complex it would be necessary to block all the terminal primary amino groups of the full generation dendrimer used to prepare the complex. Two strategies were applied for this purpose. The first approach involved selection of the activation conditions in such a way so as to functionalize all the terminal groups of the full generation dendrimer. Alternatively, the amino groups that remained free during the activation procedure were blocked by reaction with a reagent so as to introduce terminal carboxyls or neutral hydroxyl groups, the groups that are expected to have the least affinity for a negatively charged solid support such as glass fiber. The reactions used to activate the terminal groups of the full and half-generation dendrimers are shown in Schemes 1 and 3, respectively. The electrophilic iodoacetamido groups are generally introduced under mild reaction conditions by treatment



Table 1. Incorporation of Electrophilic Iodoacetamido Groupsa in Dendrimers and the ALP-Dendrimer Complex Prepared from the Activated Amino-Terminal Dendrimers number of iodoacetamido groupsa incorporated ALP-dendrimer complex prepared by dendrimer generation

activation reagent

1 2 2 3 4 5 5d 5d 5e 5e

NIA NIA SIAB NIA NIA NIA NIA SIAB SIAB NIA

dendrimers (1, 3) 2.1a 4.2

Schiff baseb (2)

26c 26

thioetherb (4) 1.3 2.5

1.3 5.7 8.1 19.2

18 13 15

5.8 10.8 3.7 6.7 14.2 15.3 15.2

a Iodoacetamido groups, as determined by titration, per mole of dendrimer or protein. b See Scheme 1 and the text for details. c Percent coupling of iodoactemido groups, calculated from the experimentally determined value of the incorporated groups and the total number of terminal groups expected to be available for reaction. d Carboxymethylated dendrimers; see the text for details. e Partially hydroxylated dendrimers; see the text for details.

of molecules containing primary amines, such as a full generation poly(amidoamine) dendrimer, with a heterobifunctional reagent (e.g. the commercially available sulfoSIAB and NIA). By activation with a 10-20-fold molar excess of sulfoSIAB, it is possible to incorporate 3-5 phenyl iodoacetamido groups in the 5th generation dendrimer containing a total of 128 surface amino groups to produce 1 (NH2-dendrimer-NHCOPhNHCOCH2I). Irrespective of the generation of dendrimer used, all efforts to incorporate the maximum possible number of the reactive phenyliodoacetamido groups by reaction of all the surface amino groups with an excess of the activating reagent produced a white nonreactive precipitate. This precipitate was found to be insoluble in buffers of pH 2.0-12.0 and all common solvents such as alcohol, N,N-dimethylformamide, and dimethyl sulfoxide. A clear solution was obtained when a solution of the full generation dendrimer, in 50% alcohol, was treated with an excess of NIA dissolved in tetrahydrofuran. By this method, it was possible to activate different generations of dendrimers, including partially functionalized dendrimers such as the partially hydroxylated and carboxymethylated dendrimers. Fluorescamine titration of these NIA-activated dendrimers 3 showed the absence of any surface amino groups present originally before the activation reaction. However, the reactive iodoacetamido groups that can be titrated with dithiothreitol in these derivatized dendrimers ranged from 13 to 26% of the values expected (Table 1). To calculate these values, it is assumed that all the terminal amino groups present originally in the dendrimer would react with the activating reagent. The first two generations of dendrimers contained the highest percentage of the incorporated reactive groups. All of these dendrimer derivatives were stable to storage in 50% alcohol or pH 5.0 buffer for at least 3 days at -10 °C. The dendrimers derivatized by reaction with sulfoSIAB or NIA were reacted with ALP to form the complex ALPdendrimer-COCH2I in two different sets of reactions (Scheme 1). A Schiff base was formed between the periodate-oxidized ALP and amino groups of the dendrimer activated with sulfoSIAB to form the intermediate

Table 2. Incorporation of Electrophilic Epoxy Groupsa in Amino Dendrimers and the ALP-Activated Dendrimer Complex on Reaction with Epibromohydrin number of epoxy groupsa incorporated dendrimer generation 2 5 5d 5e

dendrimer 6 0.7a 2.1 2.5 2.3

4.4c 1.6 8.5 8.0

ALP-dendrimer complex (7) prepared by thioetherb 5.3

a Epoxy groups incorporated, as determined by titration, per mole of dendrimer or protein. b ALP containing 2.6 mol of free SH reacted with an excess of the activated dendrimer. c Percent coupling of epoxy groups, calculated from the experimentally determined value of the incorporated groups and the total number of terminal groups expected to be available for reaction. d Partially hydroxylated dendrimers; see the text for details. e Succinylated dendrimers; see the text for details.

ALP-CHdN-dendrimer-NHCOPhNHCOCH2I. This intermediate was reduced with sodium cyanoborohydride to form the stable secondary amine derivative 2. Alternatively, ALP-SH (containing 1-3 free SHs per mole of the protein) was reacted with an excess of the NIAactivated dendrimer derivative to form the complex 4 (ALP-SCH2CONH-dendrimer-NHCOCH2I). The reactive iodoacetamido groups present in the complexes prepared by the two methods were quantitated by titration with dithiothreitol (Table 1). The product formed on reaction of the 1st generation amino-terminal dendrimer with NIA was analyzed with an electrospray ionization mass spectrum. This analysis was carried out on a methanol/water solution of the product under the positive ion mode. The three most intense peaks present in this spectrum show m/z (relative intensity) of 903.3 (100%), 780.1 (85%), and 678.1 (50%) corresponding to +3, +2, and +4 charged states of the molecules, respectively. These three peaks represent the molecular species of m/z 2708.7, 1558.2, and 2708.4, respectively. The highest peak observed in this spectrum shows m/z of 1361.6 (10% relative intensity) corresponding to a +2 charged state. This peak represents a molecular species of m/z 2721.2. The 1st generation dendrimer derivative containing eight iodoacetamido groups introduced by reaction of all eight terminal amino groups would be expected to have a molecular mass of 2780 Da. A reaction of terminal amino groups of the full generation dendrimers with an excess of epibromohydrin resulted in the formation of epoxy-dendrimers 6. A number of dendrimers were functionalized with the epoxy groups. Fluorescamine titration of the derivatized dendrimers showed that 96-99% of the terminal amino groups had been reacted during this activation procedure. Similar to the NIA activation above, all the epoxydendrimers prepared by reaction with epibromohydrin showed complete solubility in 50% aqueous alcohol and these solutions could be stored for several days at -20 °C without loss of the reactive groups. The complex ALP-S-dendrimer-epoxide 7 was prepared by reaction of an excess of the epoxy-dendrimer (prepared from the 2nd generation dendrimer) with ALP-SH containing 2.6 free SHs per mole of protein (Scheme 1). Titration of the electrophilic groups in this complex and epoxy-dendrimer was carried out with dithiothreitol (22), and results are shown in Table 2. The reactive epoxide groups in the dendrimer-epoxide 6, available for titration, were found to be 1.6-8.5% of the total terminal amino groups present in the specific generation of the dendrimer.


Singh

Table 3. Incorporation of Electrophilic N-Hydroxysuccinimidyl Ester Groupsa in Carboxyl Dendrimers and the ALP-Coupled Activated Dendrimer

dendrimer generation

activation reagent

1.5 5.5 1.5 2.5 4.5

EDC/NHS EDC/NHS TSTU TSTU TSTU

number of active ester groupsa

number of additional groups in the ALP-activated dendrimer complexb NH2 SH COCH2Br (17) (18) (19) 83

42.8

503 1.2 1.4 7.1

7.5c 4.4 5.5

3.9 4.8 5.2

a Active groups incorporated, as determined by titration, per mole of dendrimer or protein. b See the text for details. c Percent coupling of the active ester groups, calculated from the experimentally determined value of the incorporated groups and the total number of terminal groups available for reaction.

Dendrimers containing the nucleophilic sulfhydryls (dendrimer-SH) 9 have been prepared by a reaction of the terminal amino groups in the full generation dendrimers with a 4-10-fold molar excess of sulfo NHS LC SPDP (26) to form the intermediate NH2-dendrimerS-S-Py (dendrimer-PDH) 8, followed by reaction of this intermediate with a reagent such as dithiothreitol. By utilizing a limited amount of the activation reagent, it has been possible to prepare a dendrimer derivative 9 containing two surface nucleophiles with distinctly different reactivity, i.e. NH2 and SH (Scheme 1). In addition, succinic anhydride/dithiothreitol treatment of the intermediate dendrimer-PDH, containing the surface amino groups that were not involved during reaction with sulfo NHS LC SPDP, resulted in the formation of COOH-dendrimer-SH. This derivative contains the nucleophilic sulfhydryls in addition to the free carboxyl groups which show a low reactivity. A solution of the succinylated dendrimer derivative with free sulfhydryl groups was found to form an insoluble precipitate when stored at -20 °C for 48 h. Quantitative conversion of the terminal amino groups in the intermediate dendrimer-PDH, during reaction with succinic anhydride, was confirmed by titration with fluorescamine. However, under identical reaction conditions, e90% of the amino groups were involved in reaction with reagents such as gluconolactone, butyrolactone, or acetoxyacetyl chloride. An excess of the intermediate dendrimer-PDH, prepared from the 2nd generation amino-terminal dendrimer, was reacted with the periodate-oxidized ALP. A cyanoborohydride/dithiothreitol treatment of the intermediate Schiff base resulted in the formation of the complex ALP-dendrimer-SH 10 (Scheme 1). BAHA, a heterobifunctional reagent, was synthesized in three steps by a procedure similar to that described by Heindel et al. (28). The synthetic scheme is shown in Scheme 2. The electrophilic N-hydroxysuccinimidyl esters 15 (Scheme 3) were prepared by activation of the terminal carboxyl groups in the half-generation dendrimers. This activation was carried out by two different methods. An 80% dimethylformamide solution of the free acid form of the dendrimer was treated either with TSTU (29) or with EDC in the presence of NHS. A titration of the active esters incorporated in different generations of the carboxyl dendrimers was found to be 4.4-7.5% of the total carboxyl groups present in the specific generation of the dendrimer (Table 3). The purified active esters 15 were reacted with ALP followed by reaction of the intermediate

Figure 1. Gel filtration elution profiles of the ALP-dendrimer complexes. The NIA-activated dendrimers were reacted with ALP-SH, and the reaction mixture was applied to an Ultrogel AcA 44 column (1.6 × 70 cm) equilibrated and eluted with the phosphate buffer at pH 7.0. The figure shows the elution profiles of products obtained on reaction of the 1st, 2nd, 4th, and 5th generations of dendrimers (D1, D2, D4, and D5, respectively). The inset shows the elution profile of ALP-PDH obtained by reaction of ALP with sulfo NHS LC SPDP.

ALP-dendrimer-active ester 16 with either BAHA, ethylenediamine, or 2-aminoethanethiol to prepare complexes 17-19, respectively. Additional amino, sulfhydryl, and bromoacetamido groups present in the complex ALP-dendrimer-X (X ) NH2, SH, and NHCOCH2Br) were quantitated by titration with fluorescamine, 4,4′dithiodipyridine, and dithiothreitol/4,4′-dithiodipyridine, respectively (Table 3). A number of the ALP-dendrimer complexes were purified on a gel filtration column (Ultrogel AcA 44; fractionation range of 10-130 kDa). The elution profiles of a few of these complexes along with that of ALP-6[3-(2-pyridyldithio)propionamide]hexanoate (ALP-PDH), prepared by activation of ALP with sulfo NHS LC SPDP, are shown in Figure 1. The peak fractions (showing maximum absorption at 280 nm) in these profiles were eluted from this column in the same position, i.e. the fraction number 23 ( 1. Activation of proteins with sulfo NHS LC SPDP does not lead to polymerizations, and since the elution profiles of the ALP-dendrimer complexes are similar to that of ALP-PDH, the predominant component in all these ALP derivatives is the monomeric form of the protein. The polymeric forms of ALP, especially a dimer when present as an impurity in the monomeric form of the protein, elute in front of the main peak containing the monomeric form of the protein when chromatographed on a column prepared with this gel. A small amount of this dimeric form of ALP is evident as a small shoulder (Figure 1) in a few of the ALP-dendrimer complexes as well as in ALP-PDH. The specific enzyme activity of all of the ALPdendrimer complexes described above has been found to be very similar to that of ALP used for the coupling reactions. The enzyme activity was determined spectrophotometrically from the rate of hydrolysis of p-nitrophenyl phosphate in 1 M Tris buffer at pH 8.0. For example, ALP-dendrimer-COCH2I, prepared from the 2nd and the hydroxylated 5th generation dendrimers, shows specific activities of 450 and 399 units/mg, respectively. Under identical conditions, ALP, used for these reactions, shows an activity of 500 units/mg.


Kinetic parameters of the ALP-dendrimer complexes were found to be comparable to those of ALP. For example, the ALP-CH2NH-dendrimer complexes prepared from the 3rd and the 5th generation dendrimers show Km values of 0.19 and 0.11 mM, respectively, as compared to 0.17 mM for that of ALP. The Kcat/Km ratios for these dendrimer complexes were found to be 8.6 × 106 and 1.20 × 107 M-1 s-1, respectively. This ratio was found to be 9.8 × 106 M-1 s-1 for ALP under identical reaction conditions. Prior to conjugation of Fab′ to an ALP-dendrimer complex, the nonspecific binding of these ALP-dendrimer complexes was evaluated on Stratus II. Nonspecific binding is the response generated, at 0 ng/mL of the analyte, by the ALP-dendrimer complex when applied to the glass fiber solid support used in Stratus II. The complexes 4 prepared by a reaction of ALP-SH with the NIA- activated dendrimers showed (15) nonspecific binding of 146-2571 mV/min at a protein concentration of 500 ng/mL. This interaction was found to increase with the generation of the dendrimers containing terminal amino groups. The reagents prepared with the halfgeneration dendrimers show very low nonspecific binding (35-83 mV/min) regardless of the generation of dendrimer used. The nonspecific binding of complexes prepared by dendrimers activated with the other methods was found to be extremely high. For example, the complexes ALP-dendrimer (D2)-epoxide 7, ALP-CH2NH-dendrimer (hydroxylated D5)-NHCOPhNHCOCH2I 2, and ALP-CH2NH-dendrimer (D5)-SH 10 showed responses of 10 668 (20 ng/mL), 2544 (0.94 ng/mL), and 2973 mV/min (100 ng/mL), respectively, as compared to a response of 108 mV/min for the ALP-Fab′ conjugate. The ALP-dendrimer complexes 4 and 17 containing terminal iodoacetamido and bromoacetamido groups were reacted separately with the Fab′ fragment of an antiCKMB antibody to form the multifunctional dendrimer reagents ALP-dendrimer-Fab′. The reaction mixtures containing the ALP-dendrimer-Fab′ complexes (5 and 20) were purified on an AcA 34 column (fractionation range of 20-350 kDa; exclusion limit of 750 kDa). The column fractions were tested on Stratus II. This testing evaluates both the enzyme activity of ALP and the antigen binding activity of the antibody in the ALPdendrimer-Fab′ complex. The first peak that elutes from the column represents the ALP-dendrimer-Fab′ complex followed by elution of the two unreacted components ALP-dendrimer and Fab′. The elution profiles of a few of these representative complexes are shown in Figure 2. The presence of fractions appearing as a shoulder and representing higher-molecular mass complexes in front of the main ALP-dendrimer-Fab′ complex peak is dependent on the generation of the dendrimer used to prepare the ALP-dendrimer complex. For example, in the complex prepared from the 1st generation dendrimer, this front shoulder is almost nonexistent, whereas this set of fractions becomes the major component when the 5th generation dendrimer was used for the coupling reactions. The presence of this shoulder (Figure 2) is quite obvious in cases where the 3rd and the 4.5th generation dendrimers were used for the reactions. The apparent molecular mass of the peak fractions (showing highest absorption at 280 nm) in these ALP-dendrimerFab′ complexes prepared from the 3rd and the 5th generation dendrimers was calculated to be 194 and 517 kDa, respectively. All complexes, except for the one prepared from the 5th generation dendrimer, showed performances (15) similar to that of the ALP-Fab′


Figure 2. Gel filtration elution profiles for the purification of ALP-dendrimer-Fab′ complexes. The reaction mixture obtained after reaction of the ALP-dendrimer complexes with an excess of Fab′ was applied to an Ultrogel AcA 34 column (1.6 × 100 cm) equilibrated and eluted with the Tris buffer. The figure shows the profiles of products obtained from the 1st, 3rd, 5th, and 4.5th generation dendrimers (D1, D3, D5, and D4.5, respectively).

conjugate. The ALP-dendrimer-Fab′ complex, prepared from the 5th generation dendrimer, could not match the performance of the commercial conjugate even at a very high protein concentration of 9.26 µg/mL as compared to other complexes which showed comparable performances at 0.434-0.740 µg/mL protein (15). DISCUSSION

Reaction conditions normally used to activate functional groups may adversely affect the structural integrity or specific activity of a biologically active molecule. However, a number of reaction conditions affecting the surface groups of Starburst dendrimers do not impact their structural integrity (3, 30, 31). For coupling of biologically active molecules to a natural or a synthetic macromolecule, such as a dendrimer, it is therefore desirable to activate the surface groups of these macromolecules. For optimum performance, the complex prepared by reacting biologically active material with a dendrimer, e.g. antibody-dendrimer (11) and antibody-dendrimerenzyme (15), may require either partial or essentially complete reaction of the terminal functional groups present originally in a dendrimer. The results presented here make it possible to achieve these goals by activation of a dendrimer with a specific reagent in defined molar ratios. A number of full and half-generation dendrimers have been used for these derivatizations. Respective reactions with sulfoSIAB and NIA (Scheme 1 and Table 1) allow partial or complete reaction of terminal amino groups of a full generation dendrimer to incorporate the electrophilic iodoacetamido groups 1 and 3. Similarly, incorporation of the electrophilic epoxy 6 and N-hydroxysuccinimidyl groups 15 (Tables 2 and 3) has been achieved by reaction with an excess of the appropriate reagents. By a similar choice of reactions, it has been possible to derivatize dendrimers containing either two nucleophilic groups 9 (i.e. SH and NH2; Scheme 1) with selective reactivity patterns or a nucleophilic sulfhydryl and a nonreactive carboxyl or hydroxyl. These derivatized dendrimers have been reacted with a limited amount of a model protein (ALP) to form the complex


ALP-dendrimer-active group. This complex can be reacted further with a homo- or heterobifunctional reagent to achieve specific chemical or ionic characteristics. Alternatively, active groups in the intermediate are available for further reaction with other similar or dissimilar biologically active materials for formation of dendrimer-based multifunctional reagents. The feasibility of preparation of multifunctional reagents has been demonstrated by reaction of the ALPdendrimer complexes with the Fab′ fragment of an antiCKMB antibody. The resulting bifunctional reagents have been prepared using both full and half-generations of the poly(amidoamine) dendrimers. The performance characteristics of these ALP-dendrimer-Fab′ complexes are influenced by the dendrimer generation (15), the nature of the terminal functional groups present, and the method of activation used to prepare the derivatized dendrimer. The bifunctional reagent ALP-dendrimer-Fab′ prepared from the 5th generation dendrimer did not show optimum performance on Stratus II probably due to the very large size of the complex formed. However, it may rather be beneficial to prepare a large sized complex for other applications such as that utilized for a slow and sustained release of a biologically active molecule targeted either at a specific site of action or in circulation. The results presented here clearly show that by an appropriate selection of the dendrimer it is possible to control the size (Figure 2) and thus the performance of these dendrimer-based multifunctional reagents. The number of reactive functional groups introduced in the derivation reactions described here is lower than that expected on the basis of the number of terminal groups present originally in the specific generation of the Starburst dendrimer. The reasons for this effect are not very clear. Although a standard method for titration of the electrophilic groups when present in proteins, dithiothreitol/4,4′-dithiodipyridine reaction (23) may not be optimum for quantitation of such groups in dendrimers. Furthermore, the terminal functional groups in a dendrimer have been shown (13) to possess enhanced chemical reactivity as compared to their activity when present in other molecules. This higher than usual activity may be responsible for inter- and intramolecular reactions of the incorporated groups and thereby decreasing the number of such groups actually available during titrations and for coupling with biologically active molecules. The gel filtration column profiles of ALP-dendrimer complexes (Figure 1) have shown that the molecular mass of the protein eluted was not dramatically different as compared to that of ALP; i.e. formation of dimers and other polymers of ALP was not evident. This interpretation is supported further by the calculated molecular mass (194 kDa) of the peak fraction (with maximum absorbance at 280 nm) in the complex ALP-D3-Fab′. This number is very close to what would be expected (193.7 kDa) for such a complex containing ALP-D3-Fab′ in a 1:1:1 ratio. The mass spectrum of a product formed from the activation reaction of the 1st generation amino-terminal dendrimer shows the presence of species with a molecular mass of

Terminal Groups in Starburst Dendrimers: Activation and Reactions

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