Terminal Groups in Starburst Dendrimers: Activation and Reactions with Proteins †

Terminal Groups in Starburst Dendrimers: Activation andReactions 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 primaryamino-terminal groups. The presence of these groups on any macromolecule limits its usefulnessespecially 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 anumber of reactive electrophilic and nucleophilic groups and utilization of these reactive groups information of covalent bonds between dendrimers and alkaline phosphatase. The protein-dendrimercomplexes have been reacted further with the Fab′ fragment of an anti-creatine kinase MB isoenzymeantibody to form multifunctional dendrimer reagents. The enzymatic and immunochemical propertiesof these protein-dendrimer reagents have been evaluated by an immunoassay system. Nucleophilicthiols and electrophilic phenyliodoacetamido, iodoacetamido, and epoxy groups have been incorporatedinto amino-terminal dendrimers by their reactions with appropriate heterobifunctional reagents. Twoindependent sets of reactions have been used to prepare the reactive N-hydroxysuccinimidyl estersfrom dendrimers containing the terminal carboxyl groups. Quantitation of the reactive groups hasbeen carried out by direct titration of these activated dendrimers and the products obtained by reactionsof these dendrimers with small molecules and proteins.

INTRODUCTION

In recent years, dendrimers (1-6) have attractedattention as structurally unique synthetic macromol-ecules with a very broad range of applications in physical,chemical, and biological processes (7-12). Dendrimersare synthesized by either a divergent or a convergentapproach (1). The method of choice depends on thedesired generation (4) of a specific structure, which inturn dictates the molecular mass, the terminal functionalgroups, and the physical dimensions of the molecule.To modulate some specific properties of biologically

active molecules, a number of these molecules that showspecific activity such as drugs, enzymes, antibodies, andnucleotides have been covalently coupled to macromol-ecules [e.g. poly(ethylene glycol) or polysaccharides]. Verylimited chemical manipulations are generally possible onmany of the biologically active molecules, of eithernatural or synthetic origin, due to the labile nature orspecific structural requirements for the maintenance oftheir biological activity.Functional groups on the surface of dendrimers show

remarkably higher chemical reactivity (13) in comparisonto their activity when present in other macromolecules.This unusual reactivity is expected to allow couplingreactions to occur under mild reaction conditions. Thesecharacteristics make a dendrimer, with its controlledinterior, the macromolecule of choice for covalent coupling

reactions. A biologically active molecule is likely to retainits maximum possible activity when present in a den-drimer-biologically active molecule complex prepared viathe surface groups of dendrimers. The feasibility of sucha concept has been demonstrated by preparation ofdendrimer-peptide (14), dendrimer-antibody (11, 15),dendrimer-fullerene (16), and dendrimer-antibody-porphyrin (17) complexes. However, full exploitation ofthese molecules for a number of applications is limiteddue to the nature of the terminal functional groupsavailable in the presently known dendrimer structures.A limited number of dendrimers are known (18) tocontain reactive terminal groups. However, these reac-tive dendrimers have been synthesized by the convergentmethod, where it is often difficult to prepare largequantities of higher-generation macromolecules, thuslimiting the choice of available molecules. Kilogramquantities of highly pure dendrimers, with broad molec-ular mass ranges, that have been synthesized by thedivergent method are available (19, 20). One category

† Presented as a poster at the Fourth Pacific Polymer Confer-ence held at Koloa, Kauai, HI, on December 12-16, 1995.* Author to whom correspondence should be addressed. Tele-

phone: (305) 591-5556. Fax: (305) 597-5176.X Abstract published in Advance ACS Abstracts, December

15, 1997.

1 Abbreviations: ALP, calf intestine alkaline phosphatase;ALP-dendrimer (Dn), ALP-dendrimer complex prepared byreaction of ALP with the activated dendrimer of the nthgeneration; ALP-PDH, 6-[3-(2-pyridyldithio)propionamide]hex-anoate derivative of ALP; BAHA, [6-(bromoacetamido)hexyl]-amine; CKMB, creatine kinase MB isoenzyme; dendrimer-PDH, 6-[3-(2-pyridyldithio)propionamide]hexanoate derivativeof dendrimer; dendrimer-X, derivatized dendrimer containingthe surface group X; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride; NIA, N-hydoxysuccinimidyl iodo-acetate; sulfo NHS LC SPDP, sulfosuccinimidyl 6-[3-(2-py-ridyldithio)propionamide]hexanoate; sulfoSIAB, sulfosuccinim-idyl (4-iodoacetyl)aminobenzoate; TSTU,N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate.

54 Bioconjugate Chem. 1998, 9, 54−63

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

of dendrimers available in large quantities is the full andhalf-generations of Starburst dendrimers (1).This paper describes methods for modifying the ter-

minal amino and carboxyl groups of Starburst dendrim-ers (1) to prepare dendrimer derivatives containing adefined number of reactive nucleophilic and electrophilicgroups. These reactive groups have been utilized tocouple two proteins in sequence, namely calf intestinealkaline phosphatase (ALP)1 and the Fab′ fragment ofan anti-creatine kinase MB isoenzyme (CKMB) antibody,to form ALP-dendrimer and ALP-dendrimer-Fab′ com-plexes. The performance characteristics of these protein-dendrimer complexes have been studied on Stratus II,an automated enzyme immunoassay system.

MATERIALS AND METHODS

All reagents used were of analytical grade or betterand were purchased from either Aldrich Chemical Co.(Milwaukee, WI), Sigma Chemical Co. (St. Louis, MO),or Fluka Chemical Corp. (Ronkonkoma, NY). The het-erobifunctional reagents were purchased from BioAffinitySystems Inc. (Rosco, IL). Ultrogels AcA 44 and AcA 34were obtained from BioSepra (Marlborough, MA). BCAprotein assay reagent was obtained from Pierce (Rock-ford, IL). Alkaline phosphatase was obtained fromBoehringer Mannheim Corp. (Indianapolis, IN). Massspectra were run by the Mass Spectrometry Laboratoryof the University of California at Riverside. The perfor-mance of the protein-dendrimer complexes was evalu-ated on Stratus II, an automated fluorometric enzymeimmunoassay system available from Dade InternationalInc. (Miami, FL). The ALP-Fab′ conjugate, sold com-mercially in the CKMB assay kit for Stratus II, was usedto compare the performance of the ALP-dendrimer-Fab′complexes.Unless otherwise indicated, all reactions were carried

out at room temperature. All full and half-generationpoly(amidoamine) dendrimers, with ethylenediamine asthe initiator core molecule (1), were obtained fromDendritech, Inc. (Midland, MI). The half-generationdendrimers were obtained as the sodium salts preparedby hydrolysis of the corresponding methyl esters withsodium hydroxide. Partially hydroxylated dendrimerswere prepared at Dendritech, Inc., by using a mixture ofethylenediamine and ethanolamine in place of ethylene-diamine at the final stage of synthesis. This modifiedsynthetic procedure was used to prepare dendrimerscontaining a mixture of terminal hydroxyl (77%) andprimary amino (23%) groups. Partially carboxymeth-ylated dendrimer was prepared by treating a methanolsolution of the dendrimer with 0.85 mol of bromoaceticacid at 37 °C for 4 h. The methanol solution wasevaporated under reduced pressure, and the number ofamino groups remaining in the treated dendrimer wasdetermined by reaction with fluorescamine (21).Derivatized dendrimers of generations 1-3 were puri-

fied by repeated precipitation of a methanol solution onaddition of ethyl acetate, benzene, or dioxane. The 4.0-,4.5-, and 5th generation dendrimers were purified byultrafiltration in an Amicon cell using a YM 10 mem-brane (molecular mass cutoff of 10 kDa). The Amiconconcentrate, containing the derivatized dendrimer solu-tion, was either used immediately to react with a proteinor evaporated under reduced pressure to isolate thederivative. The ALP-dendrimer complexes were sepa-rated from the low-molecular mass contaminants, includ-ing the derivatized dendrimers by ultrafiltration (Amiconcell with a YM 10 membrane) or by gel filtration. A

column of either Sephadex G-25 or Ultrogel AcA 44(fractionation range of 10-130 kDa; exclusion limit of 200kDa) equilibrated with a buffer containing 100 mMphosphate/1.0 mMmagnesium 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 bymeasuring the increase in absorption at 324 nm onaddition of a solution of 4,4′-dithiodipyridine (22). Ti-tration of electrophilic epoxy, phenyliodoacetamido, andiodoacetamido groups in the derivatized dendrimers andthe dendrimer-X and protein-dendrimer-X complex (X) epoxy, phenyliodoacetamido, and iodoacetamido) wascarried out by reaction with an excess of dithiothreitolfollowed by reaction of the remaining dithiothreitol with4,4′-dithiodipyridine essentially as previously described(23). TheN-hydroxysuccinimidyl esters were quantitatedas described by Miron and Wilchek (24).The periodate-oxidized ALP was prepared by a proce-

dure similar to that described by Husain and Bieniarz(25). ALP-SH was prepared by the reaction of ALP withsulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide]hex-anoate (sulfo NHS LC SPDP; 26) followed by reactionwith dithiothreitol. Excess dithiothreitol was separatedfrom the protein solution by passage over a SephadexG-25 column equilibrated with the phosphate buffer atpH 7.0. The poly(amidoamine) dendrimers and many ofthe derivatives prepared in this study do not contributeto absorptions between 260 and 280 nm. Protein con-centrations (mg/mL) of the solutions containing ALP andALP-dendrimer complexes were therefore calculatedfrom absorptions measured at 280 and 260 nm and usingthe formula A280/0.689 - A260/1.351. In this formula,0.689 and 1.351 are the extinction coefficients (millilitersper milligram per centimeter) at 280 and 260 nm,respectively. BCA protein assay reagent was used todetermine protein concentrations for the ALP-dendrimercomplexes prepared from a dendrimer derivative contain-ing an aromatic or another chromophoric group, e.g.sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfoS-IAB)-activated dendrimer.Preparation and Reactions of Dendrimer Deriva-

tives Containing Phenyliodoacetamido 1 and Io-doacetamido Groups 3. The carboxymethylated 5thgeneration dendrimer (16 mg, 0.5 µmol) was dissolvedin 1 mL of the phosphate buffer at pH 7.4. A 277 µLsolution of sulfoSIAB (10 mg/mL in water, 5.5 µmol) wasthen added. After 1 h at 30 °C, the reaction mixture wasdiluted with 50 mL of the phosphate buffer at pH 7.6and concentrated to about 2 mL in an Amicon ultrafil-tration cell. This dilution-concentration process wasrepeated two more times. The dendrimer derivative 1present in about 1 mL of concentrate was combined witha 1 mL solution of the periodate-oxidized ALP (8.1 mg,58 nmol) in the phosphate buffer at pH 7.4 and thecombined mixture concentrated to 1.2 mL. After 16 h at4 °C, the reaction mixture was adjusted to pH 6.3 by acareful addition of 1 N HCl and then incubated for 1 hwith a 200 µL aqueous solution of sodium cyanoborohy-dride (30 mg/mL, 95 µmol). The ALP-dendrimer com-plex 2 formed by this sequence of reactions was purifiedby passage over an Ultrogel AcA 44 column equilibratedand eluted with the phosphate buffer at pH 7.4.A solution ofN-hydroxysuccinimidyl iodoacetate (NIA,

60.4 mg, 213.4 µmol) in 1 mL of tetrahydrofuran wasadded dropwise to a solution of the 1st generation amino-terminal dendrimer (32.7 mg, 20.4 µmol) in 2 mL of 50%alcohol. After 1 h, excess solvents were removed underreduced pressure, and the residue was washed with

Starburst Dendrimers: Terminal Group Activation Bioconjugate Chem., Vol. 9, No. 1, 1998 55

tetrahydrofuran. The tetrahydrofuran-insoluble semi-solid product, the iodoacetamido-dendrimer 3, was dried.The solid residue dissolved in 1 mL of alcohol and 0.5mL of the phosphate buffer at pH 7.0 was mixed with asolution of ALP-SH (11.9 mg, 85 nmol) in 12 mL of thephosphate buffer at pH 7.0. ALP-SH, used in thesereactions, contained an average of 1.4 free thiols per moleof protein. After incubation for 16 h at 4 °C, the protein-dendrimer complex 4 was purified over a column ofUltrogel AcA 44 in the phosphate buffer at pH 7.0.Preparation of Dendrimer Derivatives 6 Contain-

ing Epoxy Groups. A solution of the 5th generationdendrimer (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 stirringfor 4 h, the reaction mixture was diluted with 10 mL of50% alcohol. The solution was concentrated to about 1mL in an Amicon ultrafiltration cell. The concentratewas diluted again to 10 mL with 50% alcohol. Thisconcentration-dilution process was continued till theeffluent was neutral (pH paper). The dendrimer deriva-tive 6 present in the concentrate was stored at 4 °C untilit 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 epibro-mohydrin (0.5 g, 3.7 mmol) and triethylamine (36 mg,0.36 mmol). After 4 h at 37 °C, the reaction mixture wasevaporated under vacuum to a semisolid residue. Theproduct 6 was purified by repeated precipitation (alcohol/benzene). A solution of this product in 0.2 mL of alcoholand 0.2 mL of the phosphate buffer at pH 7.2 was mixedwith 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 usedfor this reaction contained 2.6 free thiols per mole of theprotein. After 16 h at 4 °C, the ALP-dendrimer complex7, formed in the reaction mixture, was then separatedfrom other contaminants by passage over a SephadexG-25 column in the phosphate buffer at pH 7.2.The succinyl-dendrimer-epoxide was prepared by

stirring a reaction mixture containing the 5th generationdendrimer (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 themixture stirred for an additional 16 h. The derivatizeddendrimer was purified by ultrafiltration in an Amiconcell (YM 10 membrane) using 50% alcohol as describedabove.Reaction of Dendrimers To Incorporate Thiol

Groups. A 3.0 mL methanolic solution of the 5thgeneration dendrimer (330 mg, 11.4 µmol) was flushedwith nitrogen. The dendrimer solution was then allowedto react for 1 h with a solution of sulfo NHS LC SPDP(50 mg, 95 µmol) in 0.5 mL of water. This reactionproduced the 6-[3-(2-pyridyldithio)propionamide]hex-anoate derivative of dendrimer 8, i.e. NH2-dendrimer-NHCO(CH2)5NHCO(CH2)2S-S-Py (dendrimer-PDH).The methanol solution was evaporated under vacuumand the semisolid residue stored at -10 °C.To block the residual amino groups in the dendrimer-

PDH derivative prepared above, a 1.8 mL methanolsolution of this derivative (148 mg, 5 µmol) was mixedwith succinic anhydride (550 mg, 5.5 mmol) and 2 mL oftetrahydrofuran. The trinitrobenzenesulfonic acid colortest (27) was used to monitor reaction of the primaryamino groups in the dendrimer-PDH derivative. After24 h, the organic solvents were removed by evaporationunder reduced pressure and the dendrimer derivativewas separated from the side products by Amicon ultra-filtration of the mixture using water. The aqueous

solution of the succinyl-dendrimer-PDH, present in theAmicon concentrate, was evaporated under vacuum toprovide 173 mg of the succinyl-dendrimer-PDH deriva-tive as an oil.The 2nd generation dendrimer (75 mg, 23.4 µmol)

dissolved in 4 mL of methanol was reacted for 1 h witha solution of sulfo NHS LC SPDP (55 mg, 104.4 µmol) in0.5 mL of water. The reaction mixture containing thisdendrimer-PDH derivative 8was concentrated to 1.0 mLby evaporation under reduced pressure and then mixedwith a 9 mL solution of the periodate-oxidized ALP (18mg, 128.6 nmol) in 50 mM sodium phosphate/50 mMsodium carbonate at pH 9.0. After 1 h, the reactionmixture was adjusted to pH 6.5 by a careful addition of1 N HCl. An aqueous solution (0.7 mL) of sodiumcyanoborohydride (27 mg, 0.44 mmol) was then added.After 30 min, the resulting ALP-dendrimer-PDH com-plex was buffer exchanged with the phosphate buffer atpH 7.0 in an Amicon ultrafiltration cell (YM 10). Thedithiothreitol/4,4′-dithiodipyridine titration of this ALP-dendrimer-PDH complex showed the presence of 25 freethiols (as pyridyl disulfide linkage) per mole of theprotein. The thiol content was calculated from theincrease in absorption at 343 nm resulting from theformation of pyridine-4-thione.The dendrimer derivative succinyl-dendrimer-PDH

(43 mg, 1.5 µmol) dissolved in 1.0 mL of 100 mM sodiumphosphate/5 mM EDTA at pH 6.0 (phosphate/EDTAbuffer) was mixed with a 0.2 mL solution of dithiothreitol(3.1 mg, 20 µmol) in the phosphate/EDTA buffer. After1 h at 30 °C, the reaction mixture was passed through aSephadex G-25 column equilibrated and eluted with thephosphate/EDTA buffer. The column fractions, contain-ing the dendrimer derivative with free thiols, weremonitored by measuring the increased absorption at 324nm on addition of a solution of 4,4′-dithiodipyridine (22)to aliquots of the individual fractions. The dendrimerderivatives dendrimer-PDH and ALP-dendrimer-PDHwere similarly treated with dithiothreitol to provide thederivatives NH2-dendrimer-SH 9 and ALP-dendrimer-SH 10, respectively. The derivative ALP-dendrimer-SH 10 was separated from excess dithiothreitol eitherby a Sephadex G-25 column or by an exchange with thephosphate/EDTA buffer in an Amicon ultrafiltration celltill the effluent showed a negative reaction on additionof a solution of 4,4′-dithiodipyridine. A titration of theNH2-dendrimer (D5)-SH 9 and succinyl-dendrimer(D5)-SH derivatives with 4,4′-dithiodipyridine (22) showedthe presence of 3.8 and 3.7 thiols per mole of thedendrimer, respectively.Synthesis of [6-(Bromoacetamido)hexyl]amine

(BAHA). BAHA 14 was synthesized (Scheme 2) by aprocedure similar to that described by Heindel et al. (28).In brief, 2 mL of a methanol solution of N-BOC-1,6-diaminohexane 11 (0.5 g, 2.3 mmol) was added to asolution of N-hydroxysuccinimidyl bromoacetate 12 (0.5g, 2.1 mmol) in 5.0 mL of tetrahydrofuran. The ethylacetate solution (20 mL) of the residue, obtained onevaporation of the organic solvents, was washed with 10mM HCl (3 × 5 mL). The dichloromethane solution (5.0mL) of the dry intermediate 13, obtained after removalof ethyl acetate, was mixed with trifluoroacetic acid (5mL, 65 mmol). The trifluoroacetate salt of BAHA 14 wasobtained in 50% yield as a white hygroscopic solid oncrystallization from 2-propanol/hexane: mp 114-115 °C(corrected). The purity of the material was determinedto be >90% by quantitation of the bromoacetamidogroups as described before (23). MS m/z (relative inten-sity): 236, 238 (M+ , due to two isotopes of bromine of

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

atomic weights 79 and 81; 28%), 121, 123 (the bro-moacetyl residue; 58%), and 116 [the protonated form ofthe residue NH2(CH2)6NH; 100%].Activation of the Half-Generation Carboxy-Ter-

minal Dendrimers. The sodium salts of the half-generation dendrimers, dissolved in water, were con-verted to the acid form by acidification with 5 Nhydrochloric acid to pH 3.0. The acidified solutions wereevaporated to dryness under high vacuum. Activationreaction of the free acid form of these dendrimers wascarried out with N,N,N′,N′-tetramethyl-O-(N-succinim-idyl)uronium tetrafluoroborate (TSTU) essentially asdescribed by Bannwarth and Knorr (29). A solutioncontaining the 2.5th generation dendrimer (30 mg, 7.2µmol) in 0.2 mL of water and 0.8 mL of DMF was mixedwith N,N-diisopropylethylamine (90 mg, 0.7 mmol) andTSTU (74 mg, 0.24 mmol). After 1 h, solvents wereremoved from a red reaction mixture under reducedpressure. The residue was washed with ethyl acetate (3× 10 mL), and the red residue obtained on removal ofall the solvents was dissolved in 0.4 mL of water. Theaqueous solution of the dendrimer-active ester 15(Scheme 3) was stored in an ice bath while the activeester concentration was being determined by titration(24). A 100 µL aliquot of the aqueous solution, containing4.4 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 mixturewas combined with 0.4 mL of a 2-propanol solution ofBAHA (20 mg, 62.5 µmol) and stirred gently at 4 °C for16 h. The ALP-dendrimer complex 17, containingreactive bromoacetamido groups, was purified over anUltrogel AcA 44 column in the phosphate buffer at pH7.0. To incorporate other functional groups, the inter-mediate 16, obtained after reaction of ALP with theactivated dendrimer, was combined separately witheither a 1 mL solution of 100 mM ethylenediamine or2-aminoethanethiol in the phosphate buffer to react witha homo- or heterobifunctional nucleophile. These reac-tions produced the complexes ALP-dendrimer-NH2 18and 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 1mL of 80% DMF was mixed with N-hydroxysuccinimide(115 mg, 1 mmol) and 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride (EDC, 200 mg, 1mmol). The reaction mixture was stirred for 16 hfollowed by washing the reaction mixture with ethylacetate (2× 5 mL) and 2-propanol (2× 5 mL). The activeester 15 obtained on removal of the combined organicsolution was dissolved in 0.5 mL of water. The aqueoussolution containing 32 µmol of the active ester wasreacted with a 1.8 mL solution of ALP (6.2 mg, 44.3 nmol)after titration of its active ester content as describedabove.

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


The nonspecific binding of the ALP-dendrimercomplexes was measured by using a solution of thecomplex in place of the ALP-Fab′ conjugate on StratusII. The complex was diluted to 500 ng of protein/mL ofsolution in a buffer containing 100 mM Tris/1% BSA/1% Triton X-100/2 mMmagnesium chloride/0.1 mM zincsulfate at pH 7.2.Coupling of Fab′ with the ALP-Dendrimer Com-

plex. The mouse monoclonal IgG1 was converted to itsfragments F(ab′)2 and Fab′ as described by Ishikawa etal. (pages 29-30 of ref 23). The number of free thiolsper 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 a5.5 mL solution of Fab′ (5.5 mg, 119.6 nmol) in thephosphate/EDTA buffer with 5.4 mL of a solution of theALP-dendrimer complex (2.9 mg of protein, 20.7 nmol)in the phosphate buffer at pH 7.0. For this specificexample, the ALP-dendrimer complex was prepared bya reaction of the NIA-activated 3rd generation dendrimerand ALP-SH. The mixture of Fab′ and the ALP-dendrimer complex was concentrated to about 2 mL inan Amicon ultrafiltration cell (YM 10) and then bufferexchanged with the phosphate buffer at pH 7.6 tillconcentration of EDTA in the concentrate was e5 µMand the pH of the effluent was 7.6. The reaction mixture,1.6 mL, was incubated at 4 °C for 16 h and then mixedwith 32 µL of a 20 mg/mL solution of N-ethylmaleimide(0.64 mg, 5.1 µmol) in N,N-dimethylformamide. After 2h, the reaction mixture containing free unconjugated Fab′and the ALP-dendrimer-Fab′ complex, 5 or 20, wasapplied to an Ultrogel AcA 34 column (fractionation rangeof 20-350 kDa; exclusion limit of 750 kDa) equilibratedwith 10 mM Tris/100 mM NaCl/0.1 mM magnesiumchloride/0.01 mM zinc sulfate/0.1% azide at pH 7.0 (Trisbuffer). The performance of the fractions was evaluatedon Stratus II. The fractions containing the ALP-dendrimer-Fab′ complex, eluting as the first peak fromthe AcA 34 column, were pooled on the basis of theirperformance being similar to that shown by the ALP-Fab′ conjugate. The protein concentration of the ALP-dendrimer-Fab′ conjugate was determined using theBCA protein assay with alkaline phosphatase as aninternal standard. The column was calibrated withprotein standards from Pharmacia Biotech using bluedextran (2000 kDa), thyroglobulin (669 kDa), catalase(232 kDa), aldolase (158 kDa), bovine serum albumin (67

kDa), and ovalbumin (43 kDa). The linear regressionanalysis of this calibration curve (fraction number vsmolecular mass) was used to calculate the apparentmolecular mass of the ALP-dendrimer-Fab′ complexpresent in the specific column fraction.

RESULTS

The biochemical and immunochemical evaluations ofthe protein-dendrimer complexes, ALP-dendrimer andALP-dendrimer-Fab′, were carried out on Stratus II.This heterogeneous immunoassay system utilizes a sand-wich format to analyze samples containing an analyte,e.g. CKMB. Glass fiber filter paper, a negatively chargedsurface, is used in this system to immobilize an anti-CKMB antibody called the capture antibody. The cap-ture antibody provides one component necessary to formthe sandwich. The second component of the sandwichis provided by a solution of the conjugate ALP-Fab′.These two sandwich components that utilize antibodieshave distinctly different requirements for the solid phasein any heterogeneous immunoassay system. For opti-mum performance, the capture antibody must be able tobind strongly to the solid phase whereas the antibody-enzyme conjugate should have the least affinity for thesolid support. An affinity of the antibody-enzymeconjugate for the solid support would increase nonspecificbinding and thus compromise the sensitivity of thesystem.During the initial phase of these studies, it was clear

that to minimize the solid support (glass fiber) affinityfor a protein-dendrimer complex it would be necessaryto block all the terminal primary amino groups of thefull generation dendrimer used to prepare the complex.Two strategies were applied for this purpose. The firstapproach involved selection of the activation conditionsin such a way so as to functionalize all the terminalgroups of the full generation dendrimer. Alternatively,the amino groups that remained free during the activa-tion procedure were blocked by reaction with a reagentso as to introduce terminal carboxyls or neutral hydroxylgroups, the groups that are expected to have the leastaffinity for a negatively charged solid support such asglass fiber. The reactions used to activate the terminalgroups of the full and half-generation dendrimers areshown in Schemes 1 and 3, respectively.The electrophilic iodoacetamido groups are generally

introduced under mild reaction conditions by treatment

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′.


of molecules containing primary amines, such as a fullgeneration poly(amidoamine) dendrimer, with a hetero-bifunctional reagent (e.g. the commercially availablesulfoSIAB and NIA). By activation with a 10-20-foldmolar excess of sulfoSIAB, it is possible to incorporate3-5 phenyl iodoacetamido groups in the 5th generationdendrimer containing a total of 128 surface amino groupsto produce 1 (NH2-dendrimer-NHCOPhNHCOCH2I).Irrespective of the generation of dendrimer used, allefforts to incorporate the maximum possible number ofthe reactive phenyliodoacetamido groups by reaction ofall the surface amino groups with an excess of theactivating reagent produced a white nonreactive precipi-tate. This precipitate was found to be insoluble in buffersof 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 treatedwith an excess of NIA dissolved in tetrahydrofuran. Bythis method, it was possible to activate different genera-tions of dendrimers, including partially functionalizeddendrimers such as the partially hydroxylated and car-boxymethylated dendrimers. Fluorescamine titration ofthese NIA-activated dendrimers 3 showed the absenceof any surface amino groups present originally before theactivation reaction. However, the reactive iodoacetamidogroups that can be titrated with dithiothreitol in thesederivatized dendrimers ranged from 13 to 26% of thevalues expected (Table 1). To calculate these values, itis assumed that all the terminal amino groups presentoriginally in the dendrimer would react with the activat-ing reagent. The first two generations of dendrimerscontained the highest percentage of the incorporatedreactive groups. All of these dendrimer derivatives werestable to storage in 50% alcohol or pH 5.0 buffer for atleast 3 days at -10 °C.The dendrimers derivatized by reaction with sulfoSIAB

or NIA were reacted with ALP to form the complex ALP-dendrimer-COCH2I in two different sets of reactions(Scheme 1). A Schiff base was formed between theperiodate-oxidized ALP and amino groups of the den-drimer activated with sulfoSIAB to form the intermediate

ALP-CHdN-dendrimer-NHCOPhNHCOCH2I. This in-termediate was reduced with sodium cyanoborohydrideto form the stable secondary amine derivative 2. Alter-natively, ALP-SH (containing 1-3 free SHs per mole ofthe protein) was reacted with an excess of the NIA-activated dendrimer derivative to form the complex 4(ALP-SCH2CONH-dendrimer-NHCOCH2I). The reac-tive iodoacetamido groups present in the complexesprepared by the two methods were quantitated by titra-tion with dithiothreitol (Table 1).The product formed on reaction of the 1st generation

amino-terminal dendrimer with NIA was analyzed withan electrospray ionization mass spectrum. This analysiswas carried out on a methanol/water solution of theproduct under the positive ion mode. The three mostintense peaks present in this spectrum showm/z (relativeintensity) of 903.3 (100%), 780.1 (85%), and 678.1 (50%)corresponding to +3, +2, and +4 charged states of themolecules, respectively. These three peaks represent themolecular species of m/z 2708.7, 1558.2, and 2708.4,respectively. The highest peak observed in this spectrumshows m/z of 1361.6 (10% relative intensity) correspond-ing to a +2 charged state. This peak represents amolecular species of m/z 2721.2. The 1st generationdendrimer derivative containing eight iodoacetamidogroups introduced by reaction of all eight terminal aminogroups would be expected to have a molecular mass of2780 Da.A reaction of terminal amino groups of the full genera-

tion dendrimers with an excess of epibromohydrin re-sulted in the formation of epoxy-dendrimers 6. Anumber of dendrimers were functionalized with the epoxygroups. Fluorescamine titration of the derivatized den-drimers showed that 96-99% of the terminal aminogroups had been reacted during this activation procedure.Similar to the NIA activation above, all the epoxy-dendrimers prepared by reaction with epibromohydrinshowed complete solubility in 50% aqueous alcohol andthese solutions could be stored for several days at -20°C without loss of the reactive groups. The complexALP-S-dendrimer-epoxide 7 was prepared by reactionof an excess of the epoxy-dendrimer (prepared from the2nd generation dendrimer) with ALP-SH containing 2.6free SHs per mole of protein (Scheme 1). Titration of theelectrophilic groups in this complex and epoxy-den-drimer was carried out with dithiothreitol (22), andresults are shown in Table 2. The reactive epoxidegroups in the dendrimer-epoxide 6, available for titra-tion, were found to be 1.6-8.5% of the total terminalamino groups present in the specific generation of thedendrimer.

Table 1. Incorporation of Electrophilic IodoacetamidoGroupsa in Dendrimers and the ALP-DendrimerComplex Prepared from the Activated Amino-TerminalDendrimers

number of iodoacetamido groupsaincorporated

ALP-dendrimercomplex prepared

by

dendrimergeneration

activationreagent dendrimers (1, 3)

Schiffbaseb(2)

thioetherb(4)

1 NIA 2.1a 26c 1.32 NIA 4.2 26 2.52 SIAB 1.33 NIA 5.7 18 5.84 NIA 8.1 13 10.85 NIA 19.2 15 3.75d NIA 6.75d SIAB 14.25e SIAB 15.35e NIA 15.2

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

Table 2. Incorporation of Electrophilic Epoxy Groupsain Amino Dendrimers and the ALP-ActivatedDendrimer Complex on Reaction with Epibromohydrin

number of epoxy groupsa incorporated

dendrimergeneration dendrimer 6

ALP-dendrimer complex (7)prepared by thioetherb

2 0.7a 4.4c 5.35 2.1 1.65d 2.5 8.55e 2.3 8.0

a Epoxy groups incorporated, as determined by titration, permole of dendrimer or protein. b ALP containing 2.6 mol of free SHreacted with an excess of the activated dendrimer. c Percentcoupling of epoxy groups, calculated from the experimentallydetermined value of the incorporated groups and the total numberof terminal groups expected to be available for reaction. d Partiallyhydroxylated dendrimers; see the text for details. e Succinylateddendrimers; see the text for details.


Dendrimers containing the nucleophilic sulfhydryls(dendrimer-SH) 9 have been prepared by a reaction ofthe terminal amino groups in the full generation den-drimers with a 4-10-fold molar excess of sulfo NHS LCSPDP (26) to form the intermediate NH2-dendrimer-S-S-Py (dendrimer-PDH) 8, followed by reaction of thisintermediate with a reagent such as dithiothreitol. Byutilizing a limited amount of the activation reagent, ithas been possible to prepare a dendrimer derivative 9containing two surface nucleophiles with distinctly dif-ferent reactivity, i.e. NH2 and SH (Scheme 1). Inaddition, succinic anhydride/dithiothreitol treatment ofthe intermediate dendrimer-PDH, containing the sur-face amino groups that were not involved during reactionwith sulfo NHS LC SPDP, resulted in the formation ofCOOH-dendrimer-SH. This derivative contains thenucleophilic sulfhydryls in addition to the free carboxylgroups which show a low reactivity. A solution of thesuccinylated dendrimer derivative with free sulfhydrylgroups was found to form an insoluble precipitate whenstored at -20 °C for 48 h.Quantitative conversion of the terminal amino groups

in the intermediate dendrimer-PDH, during reactionwith succinic anhydride, was confirmed by titration withfluorescamine. However, under identical reaction condi-tions, e90% of the amino groups were involved inreaction with reagents such as gluconolactone, butyro-lactone, or acetoxyacetyl chloride.An excess of the intermediate dendrimer-PDH, pre-

pared from the 2nd generation amino-terminal den-drimer, was reacted with the periodate-oxidized ALP. Acyanoborohydride/dithiothreitol treatment of the inter-mediate Schiff base resulted in the formation of thecomplex ALP-dendrimer-SH 10 (Scheme 1).BAHA, a heterobifunctional reagent, was synthesized

in three steps by a procedure similar to that describedby Heindel et al. (28). The synthetic scheme is shown inScheme 2.The electrophilic N-hydroxysuccinimidyl esters 15

(Scheme 3) were prepared by activation of the terminalcarboxyl groups in the half-generation dendrimers. Thisactivation was carried out by two different methods. An80% dimethylformamide solution of the free acid form ofthe dendrimer was treated either with TSTU (29) or withEDC in the presence of NHS. A titration of the activeesters incorporated in different generations of the car-boxyl dendrimers was found to be 4.4-7.5% of the totalcarboxyl groups present in the specific generation of thedendrimer (Table 3). The purified active esters 15 werereacted with ALP followed by reaction of the intermediate

ALP-dendrimer-active ester 16 with either BAHA,ethylenediamine, or 2-aminoethanethiol to prepare com-plexes 17-19, respectively. Additional amino, sulfhydryl,and bromoacetamido groups present in the complexALP-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 profilesof 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 (showingmaximum absorption at 280 nm) in these profiles wereeluted from this column in the same position, i.e. thefraction number 23 ( 1. Activation of proteins with sulfoNHS LC SPDP does not lead to polymerizations, andsince the elution profiles of the ALP-dendrimer com-plexes are similar to that of ALP-PDH, the predominantcomponent in all these ALP derivatives is the monomericform of the protein. The polymeric forms of ALP,especially a dimer when present as an impurity in themonomeric form of the protein, elute in front of the mainpeak containing the monomeric form of the protein whenchromatographed on a column prepared with this gel. Asmall amount of this dimeric form of ALP is evident as asmall shoulder (Figure 1) in a few of the ALP-dendrimercomplexes as well as in ALP-PDH.The specific enzyme activity of all of the ALP-

dendrimer complexes described above has been found tobe very similar to that of ALP used for the couplingreactions. The enzyme activity was determined spectro-photometrically from the rate of hydrolysis of p-nitro-phenyl phosphate in 1 M Tris buffer at pH 8.0. Forexample, ALP-dendrimer-COCH2I, prepared from the2nd and the hydroxylated 5th generation dendrimers,shows specific activities of 450 and 399 units/mg, respec-tively. Under identical conditions, ALP, used for thesereactions, shows an activity of 500 units/mg.

Table 3. Incorporation of ElectrophilicN-Hydroxysuccinimidyl Ester Groupsa in CarboxylDendrimers and the ALP-Coupled Activated Dendrimer

number of additionalgroups in theALP-activated

dendrimer complexb

dendrimergeneration

activationreagent

numberof active

ester groupsaNH2(18)

SH(19)

COCH2Br(17)

1.5 EDC/NHS 83 42.85.5 EDC/NHS 5031.5 TSTU 1.2 7.5c 3.92.5 TSTU 1.4 4.4 4.84.5 TSTU 7.1 5.5 5.2

a Active groups incorporated, as determined by titration, permole of dendrimer or protein. b See the text for details. c Percentcoupling of the active ester groups, calculated from the experi-mentally determined value of the incorporated groups and the totalnumber of terminal groups available for reaction.

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


Kinetic parameters of the ALP-dendrimer complexeswere found to be comparable to those of ALP. Forexample, the ALP-CH2NH-dendrimer complexes pre-pared from the 3rd and the 5th generation dendrimersshow Km values of 0.19 and 0.11 mM, respectively, ascompared to 0.17 mM for that of ALP. The Kcat/Km ratiosfor these dendrimer complexes were found to be 8.6 ×106 and 1.20 × 107 M-1 s-1, respectively. This ratio wasfound to be 9.8 × 106 M-1 s-1 for ALP under identicalreaction conditions.Prior to conjugation of Fab′ to an ALP-dendrimer

complex, the nonspecific binding of these ALP-den-drimer complexes was evaluated on Stratus II. Nonspe-cific binding is the response generated, at 0 ng/mL of theanalyte, by the ALP-dendrimer complex when appliedto the glass fiber solid support used in Stratus II. Thecomplexes 4 prepared by a reaction of ALP-SH with theNIA- activated dendrimers showed (15) nonspecific bind-ing of 146-2571 mV/min at a protein concentration of500 ng/mL. This interaction was found to increase withthe generation of the dendrimers containing terminalamino groups. The reagents prepared with the half-generation dendrimers show very low nonspecific binding(35-83 mV/min) regardless of the generation of den-drimer used. The nonspecific binding of complexesprepared by dendrimers activated with the other methodswas found to be extremely high. For example, thecomplexes ALP-dendrimer (D2)-epoxide 7, ALP-CH2-NH-dendrimer (hydroxylated D5)-NHCOPhNHCOCH2I2, and ALP-CH2NH-dendrimer (D5)-SH 10 showedresponses of 10 668 (20 ng/mL), 2544 (0.94 ng/mL), and2973 mV/min (100 ng/mL), respectively, as compared toa response of 108 mV/min for the ALP-Fab′ conjugate.The ALP-dendrimer complexes 4 and 17 containing

terminal iodoacetamido and bromoacetamido groups werereacted separately with the Fab′ fragment of an anti-CKMB antibody to form the multifunctional dendrimerreagents ALP-dendrimer-Fab′. The reaction mixturescontaining the ALP-dendrimer-Fab′ complexes (5 and20) were purified on an AcA 34 column (fractionationrange of 20-350 kDa; exclusion limit of 750 kDa). Thecolumn fractions were tested on Stratus II. This testingevaluates both the enzyme activity of ALP and theantigen binding activity of the antibody in the ALP-dendrimer-Fab′ complex. The first peak that elutesfrom the column represents the ALP-dendrimer-Fab′complex followed by elution of the two unreacted com-ponents ALP-dendrimer and Fab′. The elution profilesof a few of these representative complexes are shown inFigure 2.The presence of fractions appearing as a shoulder and

representing higher-molecular mass complexes in frontof the main ALP-dendrimer-Fab′ complex peak isdependent on the generation of the dendrimer used toprepare the ALP-dendrimer complex. For example, inthe complex prepared from the 1st generation dendrimer,this front shoulder is almost nonexistent, whereas thisset of fractions becomes the major component when the5th generation dendrimer was used for the couplingreactions. The presence of this shoulder (Figure 2) isquite obvious in cases where the 3rd and the 4.5thgeneration dendrimers were used for the reactions. Theapparent molecular mass of the peak fractions (showinghighest absorption at 280 nm) in these ALP-dendrimer-Fab′ complexes prepared from the 3rd and the 5thgeneration dendrimers was calculated to be 194 and 517kDa, respectively. All complexes, except for the oneprepared from the 5th generation dendrimer, showedperformances (15) similar to that of the ALP-Fab′

conjugate. The ALP-dendrimer-Fab′ complex, preparedfrom the 5th generation dendrimer, could not match theperformance of the commercial conjugate even at a veryhigh protein concentration of 9.26 µg/mL as comparedto other complexes which showed comparable perfor-mances at 0.434-0.740 µg/mL protein (15).

DISCUSSION

Reaction conditions normally used to activate func-tional groups may adversely affect the structural integ-rity or specific activity of a biologically active molecule.However, a number of reaction conditions affecting thesurface groups of Starburst dendrimers do not impacttheir structural integrity (3, 30, 31). For coupling ofbiologically active molecules to a natural or a syntheticmacromolecule, such as a dendrimer, it is thereforedesirable to activate the surface groups of these macro-molecules.For optimum performance, the complex prepared by

reacting biologically active material with a dendrimer,e.g. antibody-dendrimer (11) and antibody-dendrimer-enzyme (15), may require either partial or essentiallycomplete reaction of the terminal functional groupspresent originally in a dendrimer. The results presentedhere make it possible to achieve these goals by activationof a dendrimer with a specific reagent in defined molarratios. A number of full and half-generation dendrimershave been used for these derivatizations. Respectivereactions with sulfoSIAB and NIA (Scheme 1 and Table1) allow partial or complete reaction of terminal aminogroups of a full generation dendrimer to incorporate theelectrophilic iodoacetamido groups 1 and 3. Similarly,incorporation of the electrophilic epoxy 6 andN-hydroxy-succinimidyl groups 15 (Tables 2 and 3) has beenachieved by reaction with an excess of the appropriatereagents. By a similar choice of reactions, it has beenpossible to derivatize dendrimers containing either twonucleophilic groups 9 (i.e. SH and NH2; Scheme 1) withselective reactivity patterns or a nucleophilic sulfhydryland a nonreactive carboxyl or hydroxyl. These deriva-tized dendrimers have been reacted with a limitedamount of a model protein (ALP) to form the complex

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


ALP-dendrimer-active group. This complex can bereacted further with a homo- or heterobifunctional re-agent to achieve specific chemical or ionic characteristics.Alternatively, active groups in the intermediate areavailable for further reaction with other similar ordissimilar biologically active materials for formation ofdendrimer-based multifunctional reagents.The feasibility of preparation of multifunctional re-

agents has been demonstrated by reaction of the ALP-dendrimer complexes with the Fab′ fragment of an anti-CKMB antibody. The resulting bifunctional reagentshave been prepared using both full and half-generationsof the poly(amidoamine) dendrimers. The performancecharacteristics of these ALP-dendrimer-Fab′ complexesare influenced by the dendrimer generation (15), thenature of the terminal functional groups present, and themethod of activation used to prepare the derivatizeddendrimer.The bifunctional reagent ALP-dendrimer-Fab′ pre-

pared from the 5th generation dendrimer did not showoptimum performance on Stratus II probably due to thevery large size of the complex formed. However, it mayrather be beneficial to prepare a large sized complex forother applications such as that utilized for a slow andsustained release of a biologically active molecule tar-geted either at a specific site of action or in circulation.The results presented here clearly show that by anappropriate selection of the dendrimer it is possible tocontrol the size (Figure 2) and thus the performance ofthese dendrimer-based multifunctional reagents.The number of reactive functional groups introduced

in the derivation reactions described here is lower thanthat expected on the basis of the number of terminalgroups present originally in the specific generation of theStarburst dendrimer. The reasons for this effect are notvery clear. Although a standard method for titration ofthe electrophilic groups when present in proteins, dithio-threitol/4,4′-dithiodipyridine reaction (23) may not beoptimum for quantitation of such groups in dendrimers.Furthermore, the terminal functional groups in a den-drimer have been shown (13) to possess enhanced chemi-cal reactivity as compared to their activity when presentin other molecules. This higher than usual activity maybe responsible for inter- and intramolecular reactions ofthe incorporated groups and thereby decreasing thenumber of such groups actually available during titra-tions and for coupling with biologically active molecules.The gel filtration column profiles of ALP-dendrimercomplexes (Figure 1) have shown that the molecular massof the protein eluted was not dramatically different ascompared to that of ALP; i.e. formation of dimers andother polymers of ALP was not evident. This interpreta-tion is supported further by the calculated molecularmass (194 kDa) of the peak fraction (with maximumabsorbance 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-terminaldendrimer shows the presence of species with a molecularmass of <3 kDa. This observation would rule out thepossibility of the multimeric derivatives of the dendrimerbeing the predominant component of this reaction prod-uct. The unusual reactivity of a terminal group in adendrimer, the reactivity of the introduced iodoacetamidogroups, an absence of primary amino terminal groups(fluorescamine), and a small number of the availableiodoacetamido groups (dithiothreitol titration) suggest an

intramolecular coupling of the activated derivative. Workis in progress to further characterize the nature of themajor component present in this reaction mixture. Theintermolecular coupling of appropriately activated den-drimers has been used (2) to prepare multimeric formsof dendrimers but may not be the major reaction pathunder the conditions utilized in this study.In conclusion, dendrimer derivatives of different gen-

erations have been prepared that contain nucleophilic,electrophilic, and nonreactive groups by an appropriateselection of the specifically defined reaction conditions.The methods described here allow for the preparation ofreagents with desirable characteristics such as controlledmolecular mass and a defined number of reactive groups.These reactions show that Starburst dendrimers can besubjected to a series of defined chemical reactions. Dueto the controlled architecture and geometry of dendrimersand the use of a defined sequence of chemical reactions,the methods developed here allow for reproducible prepa-ration of reagents that have the potential for very broadapplications.

ACKNOWLEDGMENT

The author is grateful to Peter Cronin and TimGoodnow for their support and Ralph Spindler for sup-plying dendrimers.

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Terminal Groups in Starburst Dendrimers: Activation and Reactions with Proteins †