Molecular Necklaces. Cross-Linking Hemoglobin with Reagents

The activated material is then combined with a reagent (3-aminoisophthalic acid) that ... Timothy A. Roach, Victor W. Macdonald, and Ramachandra S. Ho...
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1058

Bioconjugate Chem. 1999, 10, 1058−1067

Molecular Necklaces. Cross-Linking Hemoglobin with Reagents Containing Covalently Attached Ligands Sanda Crapatureanu, Ruxandra Serbanescu, Sharon Bisley Brevitt, and Ronald Kluger* Lash Miller Laboratories, Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6. Received May 27, 1999; Revised Manuscript Received August 13, 1999

Hemoglobin can be cross-linked and converted to a bioconjugate in one step by a “molecular necklace”, a reagent that contains two reacting sites and a pendant ligand. The compound to be conjugated is activated as an electrophile. The activated material is then combined with a reagent (3-aminoisophthalic acid) that contains a nucleophilic (amino) site and two latent (carboxyl) sites. The latent sites of the product are activated as 3,5-dibromosalicylates to produce the cross-linker. Illustrative examples of cross-linking are presented with pendant biotin [bis(3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate] and pendant N-trifluoroacetyl-L-isoleucylglycine [bis(3,5-dibromosalicyl) N-(N-trifluoroacetylL-isoleucylglycyl)-5-aminoisophthalate)]. The resulting modified hemoglobins contain two principal types of cross-link: (β-Lys-82-β′-Lys-82) and (R-Lys-99-R′-Lys-99). The functional properties of the modified hemoglobin containing biotin in a (β-Lys-82-β′-Lys-82) cross-link are (pH 7.4, 55 µM heme, 25 °C, 0.1 M chloride, and 50 mM Bis-Tris) P50 ) 4.9 Torr, n50 ) 3.0, values which are approximately the same as for native hemoglobin. The results of affinity chromatography of the biotinylated crosslinked hemoglobin using a column of immobilized avidin indicate that the pendant biotin is much less accessible than free biotin. We suggest that the results are consistent with the pendant species being strongly attracted into the hemoglobin environment.

INTRODUCTION

Human hemoglobin contains four subunits (RRββ) that dissociate spontaneously into Rβ dimers (1). The hemoglobin contained in red cell substitutes (for use in transfusions) must be altered to prevent its dissociation into dimers in order to avoid excretion via the kidney (2, 3). Appropriate chemical cross-linking can prevent dissociation of the tetramer and has been used to create a wide variety of red cell substitutes. Site-directed reagents introduce cross-links and produce altered hemoglobins with defined structures (4-7). The chemical alteration of hemoglobin that introduces a cross-link has potential utility beyond producing red cell substitutes: the entity created in the cross-link can serve as a site for tethering species with useful functions. In one potential application, the conjugated species can combine the properties of a red cell substitute with those of a drug-delivery agent (8-10). This complements an approach in which conjugation is achieved by de novo synthesis of mutants of hemoglobin containing additional cysteines that serve as linking points (11). The existing method for producing conjugates on a cross-link (9) uses a trifunctional reagent [trimesyl tris(3,5-dibromosalicylate), TTDS], in which only two sites react with hemoglobin. The third site is available for addition to the resulting cross-linked hemoglobin. A schematic diagram of the cross-linked hemoglobin resulting from the reaction with TTDS is shown below. Substitution of the dibromosalicylate ester creates a novel adduct with a nucleophile. Hydrolysis of the ester derived from TTDS in the crosslinked protein will compete with the desired substitution reaction that is used to produce a bioconjugate, reducing the potential yield of conjugated protein. Since hydrolysis * To whom correspondence should be addressed.

and the desired ester substitutions compete in the necessarily aqueous medium, it is not likely that hydrolysis can be suppressed once the cross-linked ester has formed. An alternative approach can avoid the competition: form the bioconjugate with the reagent prior to cross-linking the protein. Such a reagent can be visualized as a “molecular necklace” with the bioconjugate materials contained in the unreactive portion of the crosslinking reagent. In the schematic diagram below, molec-

ular necklace “A” has a pendant species “X” and two sites “Y” that react with the protein. We have developed an efficient general route to produce compounds with the properties of “A”. First, the compound to be conjugated (X) is activated as an elec-

10.1021/bc990067f CCC: $18.00 © 1999 American Chemical Society Published on Web 10/21/1999

Molecular Necklaces Scheme 1

trophile. The activated material is then combined with a reagent that contains a nucleophilic site and two latent sites. The latent sites of the product are activated to produce the molecular necklace cross-linker. The general strategy is exemplified in Scheme 1, where the pendant ligand is added as an acid chloride and the carboxyls are activated as “OY-”, which is an anionic ester group. We illustrate the method by creating a cross-linking reagent with two examples of pendants, biotin (an affinity group) and a peptide, N-trifluoroacetyl-L-isoleucylglycine (for drug delivery or receptor activation). These react directly with hemoglobin, converting it into a cross-linked bioconjugated species. EXPERIMENTAL PROCEDURES

Materials. D-Biotin, N-t-Boc-Ile-Gly, N-t-Boc-L-Phe, avidin, 3,5-dibromosalicylic acid, and 5-aminoisophthalic acid were obtained from commercial sources and used Scheme 2

Bioconjugate Chem., Vol. 10, No. 6, 1999 1059

directly. Solutions of trypsin and endoproteinase Glu-C were freshly prepared. Human hemoglobin A was obtained in the carbonmonoxy form from Hemosol, Inc. All other materials were of the highest grade commercially available. The identity and purity of newly synthesized materials was assessed by NMR spectroscopy, mass spectrometry, infrared spectroscopy and thin-layer chromatography. Proton NMR spectra were recorded at 200 and 300 MHz. Carbon NMR and fluorine NMR spectra were recorded on multinuclear spectrometers whose proton resonance is 500 and 400 MHz. Melting points are uncorrected. Oxygen-binding analysis followed the automated method described originally by Imai (12-14). We developed a computer interface and program that enabled the data to be collected in an MS-DOS computer environment. The structures of compounds in the descriptions of syntheses are numbered in Scheme 2. The synthesis of bis (3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate (3) involves addition of amino-isophthalic acid to the acid chloride of biotin (1) to form the dicarboxylic acid (2) followed by activation of the diacid to the bis-dibromosalicylate (3). Biotin (1.07 g, 4.38 mmol) was dissolved in 20 mL of distilled thionyl chloride at room temperature and stirred until it dissolved. After 20 min, excess thionyl chloride was distilled away. The residue, the acid chloride of biotin (1), was dried in vacuo. 5-Aminoisopthalic acid (0.99 g, 5.5 mmol) was dissolved in 25 mL of anhydrous N,N-dimethylacetamide and stirred for 20 min under nitrogen. 4-(Dimethylamino)pyridine (7.1 × 10-3 g, 0.58 mmol) was added to this solution and stirred until all material dissolved. The solution was cooled to 0 °C and then added to a solution of the biotin acid chloride. The reaction mixture was stirred at room temperature for 14 h. A light brown precipitate formed and was collected on a filter. The solid was dissolved in DMF and precipitated with water. This was collected by filtration, washed with hot methanol, and dried under vacuum (0.700 g, 1.72 mmol) to give bis(3,5-dibromosalicyl)-N-biotinyl-5-aminoisophthalate: mp 220-221 °C (decomposed); IR (KBr, cm-1) 3391 (m), 2922 (m), 1696 (s), 1452 (m), 758 (m), 697 (s); 1NMR (DMSO-d6) δ 10.23(s, 1H, NH), 8.42(s, 2H, ArH), 8.12 (s, 1H, ArH), 6.43 (s, 1H, NH), 6.35 (s, 1H, NH), 4.25-4.29 (m, 1H), 4.11-4.15 (m, 1H), 3.10-3.12 (m, 1H), 2.77-2.80 (m, 1H), 2.58 (s, 1H), 2.32 (s, 2H), 1.34-1.61 (m, 6H); 13C NMR (DMS0-d6) δ 25.01, 28.11, 28.21, 36.26, 40.10, 55. 41, 59.20, 61.05, 123.4, 124.3,

1060 Bioconjugate Chem., Vol. 10, No. 6, 1999

131.6, 139.9, 162.7, 166.5, 171.7; MS parent peak (CI) calcd, 407.4; found, 408 (MH+). Bis((1-tert-butoxycarbonyl)-3,5-dibromosalicyl) N-Biotinyl-5-amino-isophthalate (3). N-Biotinyl-5aminoisophthalic acid (0.18 g, 0.43 mmol), tert-butyl 3,5dibromosalicylate (0.340 g, 0.97 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.334 g, 1.74 mmol), and 4-(dimethylamino)pyridine (0.0250 g, 0.200 mmol) were dissolved in 15 mL of anhydrous DMF and stirred at 4 °C for 20 h. The precipitated byproduct was removed by filtration. The filtrate was diluted with water and ethyl acetate. The aqueous layer was extracted several times with ethyl acetate. The organic layers were pooled, washed with water, saturated sodium bicarbonate, and water. The pooled organic extracts were dried over anhydrous magnesium sulfate and evaporated under vacuum (off-white solid). The product was purified by flash chromatography (silica gel 60; eluent, acetone). The product, bis((1-tert-butoxycarbonyl)-3,5-dibromosalicyl) N-biotinyl-5-amino-isophthalate was isolated, 0.19 g, 0.17 mmol: 1H NMR (DMSO-d6) δ 10.6 (s, 1H, NH), 8.83 (d, J ) 1.5, 2H, ArH), 8.48 (t, J ) 1.5, 1H, ArH), 8.35 (d, J ) 2.4, 2H, ArH), 8.03 (d, J ) 2.4, 2H, ArH), 6.46 (s, 1H, NH), 6.38 (s, 1H, NH), 4.31-4.27 (m, 1H), 4.15-4.12 (m, 1H), 3.13-3.10 (m, 1H), 2.79 (dd, J ) 5, 1H), 2.58 (d, J ) 12.3, 1H), 2.38 (t, J ) 7.35, 1H), 1.66-1.59 (m, 1H), 1.31 (s, 18 H); 13C (DMSO-d6) δ 172.2, 162.7, 162.1, 161.5, 145.6, 141.1, 138.8, 133.0, 129.4, 128.6, 125.0, 124.9, 119.6, 119.1, 83.1, 61.0, 59.2,55.4, 40.0, 36.3, 28.2, 28.1, 24.8. Bis(3,5-dibromosalicyl) N-Biotinyl-5-aminoisophthalate (4). Bis((1-tert-butoxycarbonyl)-3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate (0.18 g, 0.17 mmol) was dissolved in 15 mL of anhydrous trifluoroacetic acid (0 °C) and stirred 10 min at 0 °C, then for 30 min at room temperature. Diethyl ether was added to induce crystallization. The solution was left for 1 h at 4 °C. The resulting solid was collected by filtration, washed with diethyl ether, and dried under vacuum (0.16 g, 0.17 mmol): mp 235-236 °C (dec); IR (KBr, cm-1) 3368 (m), 3025 (m), 2921 (s), 1708 (s), 1452 (s), 1198 (s), 697 (s); 1H NMR (DMSO-d ) δ 10.56 (s, 1H, NH), 8.77 (s, 2H, 6 ArH), 8.41 (s, 1H, ArH), 8.34 (d, J ) 2.1, 2H, ArH), 8.10 (d, J ) 2.1, 2H, ArH), 6.47 (s, 1H, NH), 6.38 (s, 1H, NH), 4.31-4.27 (m, 1H), 4.15-4.10 (m,1H), 3.24-3.11 (m, 1H), 2.80 (dd, J ) 5,1H), 2.58 (d, J ) 12.6, 1H), 2.37 (t, J ) 7.2, 2H), 1.65-1.40 (m, 6H); 13 C NMR (DMS0-d6) δ 24.92, 28.15, 28.25, 36.34, 55.43, 59.26, 61.10, 119.36, 119.54, 124.8, 127.6, 129.5, 133.5, 139.05, 139.70, 140.90, 162.4, 162.8, 163.53. MS(ESI) calcd, 963.29; found: 963.4. Bis(3,5-dibromosalicyl) N-(N-trifluoroacetyl-Lisoleucylglycyl)-5-aminoisophthalate (8). N-(N-tertbutyloxycarbonyl-L-isoleucylglycyl)-5-aminoisophthalic Acid Dimethyl Ester (5). N-t-Boc-Ile-Gly (2.88 g, 9.97 mmol), dimethyl 5-amino-isophthalate (2.11 g, 10.1 mmol) and 4-(dimethylamino)pyridine (0.125 g, 1.03 mmol) were dissolved in 60 mL of anhydrous DMF under nitrogen. DCC (2.05 g, 9.94 mmol) was dissolved in 30 mL of anhydrous DMF and added dropwise to the first solution. The reaction mixture was stirred for 3 days at room temperature under dry nitrogen. The precipitated N,N-dicyclohexylurea was removed by filtration. Ethyl acetate and water were added to the filtrate and the layers were separated. The aqueous layer was extracted three times with ethyl acetate. The organic layers were pooled, filtered to remove additional precipitate, and washed with 2 M hydrochloric acid (1.2 L), saturated salt solution (500 mL), saturated sodium bicarbonate solution (700 mL), and again with saturated salt solution (250

Crapatureanu et al.

mL). The ethyl acetate solution was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The mixture was kept at 4 °C for 24 h. The resulting yellow precipitate was collected by filtration, washed with hexane, and dried under vaccuum (1.75 g, 3.65 mmol): mp 124-125 °C; IR (KBr) 3307 (m), 2967 (m), 1730 (s), 1555 (s), 1515 (s), 14 440 (s), 1343 (s), 1250 (s), 1168 (s), 1009 (m), 758 (s); 1H NMR (DMSO-d6) δ 10.2 (s, 1H, NH), 8.48 (d, J ) 1.2, 2H, ArH), 8.34 (t, J ) 5.4, 1H, NH), 8.14 (t, J ) 1.5, 1H, ArH), 6.94 (d, J ) 8.1, 1H, NH), 3.91-3.83 (m, 3H, masked), 3.87 (s, 6H), 1.71-1.69 (m, 2H), 1.35 (s, 9H), 1.16-1.11 (m, 1H), 0.86-0.78 (m, 6H); 13C NMR (DMSO-d6) δ 172.00 (s), 168.33 (s), 165.18 (s), 155.70 (s), 139.62 (s), 130.70 (s), 124.1 (s), 123.5 (s), 78.20 (s), 58.99 (s), 52.50 (s), 42.73 (s), 36.24 (s), 28.11 (s), 24.50 (s), 15.34 (s), 11.00 (s); MS (ESI) calcd, 479.5; found, 480.4 (MH+). N-[N-tert-butyloxycarbonyl-L-isoleucylglycyl]-5aminoisophthalic Acid (6). N-[N-tert-butyloxycarbonylL-isoleucylglycyl]-5-aminoisophthalic acid dimethyl ester (1.75 g, 3.65 mmol) was dissolved in 100 mL of methanol, 20 mL of 6 N sodium hydroxide was added, and the reaction mixture stirred under nitrogen for 2 h. After addition of water and 50 mL of 2 N HCl to the reaction mixture, a white precipitate formed, which was filtered, washed with water, and dried under vacuum (1.58 g, 3.50 mmol): mp 215-216 °C; IR (KBr, cm-1) 3329 (m, br), 2970 (m), 1699 (s), 1558 (m), 1252 (m), 667 (w); 1H NMR (DMSO-d6) δ 10.15 (s, NH), 8.43 (d, J ) 1.5, 2H, ArH), 8.33 (t, J ) 5.7, 1H), 8.15 (t, J ) 1.5, 1H, ArH), 6.90 (d, J ) 8.1,1H, NH), 3.90-3.80 (m, 3H), 1.70-1.68 (m, 2H), 1.35 (s, 9H), 1.14-1.09 (m, 1H), 0.86-0.78 (m, 6H); 13C NMR (DMSO-d6) δ 172.04 (s), 168.24 (s), 166.45 (s), 155.74 (s), 139.35 (s), 131.87 (s), 124.69 (s), 123.56 (s), 78.24 (s), 59.04 (s), 42.78 (s), 36.31 (s), 28.20 (s), 24.57 (s), 15.40 (s), 11.07 (s); MS(ESI) calcd, 451.45; found, 452.4 (MH+). Bis((1-tert-butoxycarbonyl)-3,5-dibromosalicyl) N-[N-tert-butyloxycarbonyl-L-isoleucylglycyl]-5-aminoisophthalate (7). N-[N-tert-butyloxycarbonyl-L-isoleucyl-glycyl]-5-aminoisophthalic acid (1.58 g, 3.50 mmol), 4-(dimethylamino)pyridine (0.085 g, 0.7 mmol), and tertbutyl-3,5-dibromosalycilate (2.46 g, 7.00 mmol) were dissolved in 300 mL of anhydrous THF and cooled in an ice bath. 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (2.4 equiv, 1.61 g, 8.40 mmol) was dissolved in dry dichloromethane and added via a syringe, under nitrogen, dropwise to the solution. After stirring the mixture at 0 °C for 5 min and at room temperature for 48 h, the solvent was evaporated and the solid dissolved in ethyl acetate. The solution was washed successively with saturated sodium bicarbonate and water, evaporated under reduced pressure. The white solid product was used directly (1.40 g, 1.25 mmol). Bis(3,5-dibromosalicyl) N-(N-trifluoroacetyl-L-isoleucylglycyl)-5-aminoisophthalate (8). Bis((1-tertertbutoxycarbonyl)-3,5-dibromosalicyl) N-[N-tert-butyloxycarbonyl-L-isoleucylglycyl]-5-aminoisophthalate (1.40 g, 1.25 mmol) was dissolved in a mixture of trifluoroacetic acid and trifluoroacetic acid anhydride and stirred under nitrogen at room temperature for 2 h. The solvent was removed under vacuum, leaving a clear oil as a residue. Addition of ether resulted in the formation of a white precipitate, which was filtered and identified as a decomposition product. Adding hexane to the filtrate precipitated the desired compound. The solid was collected by filtration and dried under vacuum (0.411 g, 0.410 mmol): mp 170-172 °C, IR (KBr, cm-1) 3350 (m, br), 2923 (m), 1718 (s), 1558 (s), 1452 (s), 1199 (s), 697 (s);

Molecular Necklaces

NMR (DMSO-d6) δ 10.68 (s, 1H, NH), 9.59 (d, J ) 8.4, 1H, NH), 8.75 (d, J ) 1.5, 2H, ArH), 8.68 (t, J ) 5.7, 1H, NH), 8.44 (t, J ) 1.5, 1H, ArH), 8.36 (d, J ) 2.4, 2H, ArH), 8.11 (d, J ) 2.4, 2H, ArH), 4.08-3.89 (m, 3H), 1.92-1.88 (m, 2H), 1.46-1.40 (m, 1H), 0.83-0.78 (m, 6H); 13C NMR (DMSO-d ) δ 170.69 (s), 168.78 (s), 163.89 (s), 6 162.79 (s), 156.61 (q, J ) 145), 146.95 (s), 140.94 (s), 139.42 (s), 133.90 (s), 130.10 (s), 128.04 (s), 125.51(s), 125.27 (s), 119.93 (s), 119.72 (s), 58.13 (s), 43.21 (s), 35.77 (s), 24.98 (s), 15.56 (s), 10.84 (s); 19F NMR (DMSO-d6) δ -74.07 (s, CF3CO); MS (ESI) calcd, 1003.45; found, 1004.4 (MH+). Cross-Linking Procedures. Reactions were conducted in solutions of deoxyhemoglobin, which was prepared from carbonmonoxyhemoglobin. Stock solutions of carbonmonoxyhemoglobin (1.0 mL, 1.23 mM) in 50 mM bis-tris (pH 6.9) were passed through a Sephadex G-25 column at 4 °C equilibrated with 0.1 M sodium borate buffer (pH 9). Carbonmonoxyhemoglobin was converted to oxyhemoglobin by photoirradiation in a rotating flask under a stream of humidified oxygen for 3 h at 0 °C (14). Oxyhemoglobin was converted to deoxyhemoglobin by continuously flowing wet nitrogen through the rotating flask for 3 h at 37 °C. The cross-linking reagent (2 mol/ mol of hemoglobin) was dissolved in 0.2-0.3 mL of peroxide-free dioxane and 0.3 mL of 0.1 M sodium borate buffer (pH 9). The reaction solution was deoxygenated under a stream of nitrogen for 1 h and then added to the hemoglobin solution via a syringe. The reaction mixture was kept rotating at 37° under slowly flowing nitrogen to maintain the hemoglobin in the deoxy state. The reaction was monitored by C-4 reversed-phase HPLC. The reaction was stopped when no more R- or β-chain modification was observed. At the end of the reaction period, the flask was cooled on ice, flushed with carbon monoxide, and passed through a Sephadex G-25 column equilibrated with 0.1 M borate buffer (pH 9) in order to remove unreacted cross-linking reagent. Structural Characterization of Modified Hemoglobins. Chromatographic Analysis. Modified globin chains were identified by analytical reversed-phase HPLC (15) using a 330 Å pore size C-4 Vydac column (250 × 4.6 mm). The materials were then separated by preparative reversed-phase HPLC using the same column packing (250 × 12 mm). Developers contained 0.1% trifluoroacetic acid and gradients of acetonitrile in water from 20 to 60%. The effluent was monitored for absorbance at 220 nm. Analysis of the reaction mixture and isolation of modified hemoglobins was accomplished by analytical and semipreparative anion-exchange HPLC using a Synchropak AX-300 column (analytical, 250 × 4.5 mm; semipreparative, 250 × 10 mm) and a POROS column (from Perseptive Biosystems: 7000 Å pore, 10 µm particle). Developers for the AX-300 column contained 15 mM Tris, (pH 8) and gradients of sodium acetate in water starting at 10 mM and ending at 150 mM. The flow rate was 1 mL/min for the analytical analysis and 4 mL/min for the semipreparative separation. For the analysis using the POROS column, a pH gradient from pH 8.5 to 6.5 was used. The effluent was monitored at 420 nm. Peptide fragments were separated by reversed-phase HPLC using a C-18 Vydac column (93 × 4.7 mm; 330 Å pore, 5 µm particle), with developers containing 0.1% trifluoroacetic acid. Gradients of acetonitrile in water ranged from 0 to 100%, pumped at 1 mL/min. The effluent was monitored at 220 and 280 nm (14). The presence of cross-links between globin chains can be established by molecular mass bands that are multiples those of the 16 kDa monomers in SDS-PAGE (16). 1H

Bioconjugate Chem., Vol. 10, No. 6, 1999 1061

Polyacrylamide gels (12%) were run in a dual-slab cell apparatus at 200 mV. Prior to electrophoresis, the protein sample was dissolved in 0.5 M tris-HCl, pH 6.8, which contained 0.05% bromophenol blue, 4% v/v β-mercaptoethanol, 2% SDS, and 10% v/v glycerol. The sample was kept for 5 min in boiling water to denature it. Approximately 10 µg of protein was applied to each lane of the gel and subjected to electrophoresis for 35 min. Unmodified hemoglobin and molecular weight standards were used to estimate the molecular weight of the resulting bands after fixation followed by staining with Coomassie Brilliant Blue R. The molecular masses of cross-linked hemoglobins were determined precisely by electrospray ionization mass spectrometry, coupled to reversed-phase HPLC, as described previously (17). Standard masses were taken from literature compilations (18). Separation of peptides from tryptic digestion of partially acylated globin chains reveals the site of amide modification by comparison with the intensity of peaks of tryptic peptides from native globin chains (14). Native and cross-linked R and β chains were separated and collected using the reversed-phase HPLC procedure outlined above. Organic developers were evaporated under reduced pressure and water removed by lyophilization. The globin chains were then dissolved in 8 M urea to help denature the protein and promote complete hydrolysis. They were kept for 2 h at room temperature. The solution was diluted to 2 M urea with, pH 8.5, 0.8 M bicarbonate. Freshly prepared TPCKtreated trypsin solution (2 mg/mL, 4% of mass of total protein) was added and the digestion continued for 24 h at room temperature. The tryptic hydrolysate was heated in boiling water for 2 min and diluted to 1 M urea with 0.8 M bicarbonate buffer (pH 8.5). A solution of endoproteinase Glu-C (1 mg/mL, 2% of mass of total protein) was added and the mixture digested for 72 h at room temperature. The sample was clarified by filtration through a 0.45-µm filter before injection onto the C-18 reversed-phase HPLC column. Peptide fragments were separated according to the procedure described above. The modified peaks were identified by peptide pattern analysis. The tryptic-Glu-C peptide maps of modified R and β subunits were analyzed by comparison with the peptide maps of unmodified R and β subunits and by comparison with published standards (14). Quantitative Affinity Chromatography. 4-Nitrophenyl chloroformate was use to activate Sepharose CL-4B. All steps in the activation procedure were performed in an ice bath according to the reported procedure (19). Sepharose Cl-4B (50 mL) was washed in batches through a sintered glass funnel with increasing concentrations of acetone in water, starting at 0% and ending at 100% absolute acetone. To the washed Sepharose gel, an acetone solution (50 mL) containing 4-nitrophenyl chloroformate (0.3 mmol/mL) was added. To this suspension, 50 mL of an acetone solution containing 4-dimethylaminopyridine (0.35 mmol/mL) was added dropwise with stirring, and the resin was stirred for 90 min. The activated Sepharose was then washed exhaustively with acetone (500 mL), acetone-2-propanol (1:1) (200 mL), 2-propanol (200 mL), 2-propanol-water (1:1) (200 mL), and distilled water until the solution remained clear in the presence of 0.2 N sodium hydroxide. The activated resin (0.737 mmol 4-nitrophenol/g resin) was dried by lyophilization and stored at 4 °C. Avidin (3.8 mg) was dissolved in 6 mL of 0.1 M sodium bicarbonate (pH 8.5). To this solution, 2.73 g of activated Sepharose was added, and the reaction mixture stirred

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for 1 h at room temperature then overnight at 4 °C (20). The resin was then washed successively with water, 0.2 M acetic acid, water, and 0.01 M sodium hydroxide, followed by distilled water, 0.1 M sodium bicarbonate, and phosphate-buffered saline (PBS), pH 7.4. The immobilized avidin was stored at 4 °C in PBS. Circular dichroism (CD) spectra of the native and modified hemoglobins were recorded on a Jasco J-710 instrument between 200 and 280 nm in, pH 9, 0.003 M sodium borate buffer. Circular dichroism (CD) spectra were recorded on a Jasco J-710 system between 200 and 280 nm (hemoglobin concentration 0.3 mM in sodium borate buffer, pH 9). RESULTS AND DISCUSSION

We have been able to produce cross-linking reagents with appended molecules by a process that first adds the appendage to the amino group of amino isophthalic acid. We then activate the carboxylic acid groups by converting them to reactive esters. The resulting material has the potential to attach the pendant ligand while linking between two sites on the protein, functioning as a “molecular necklace”. Producing the cross-linking reagent in this manner avoids hydrolysis of the activated ester, which occurs if the ester group is appended to the protein. The reaction sequence is presented in Scheme 2. Bis(3,5dibromosalicyl) N-biotinyl-5-aminoisophthalate (DBBA, 4) is a conjugation agent that adds a pendant molecule of biotin in the cross-linking process. Addition of 5-aminoisophthalic acid to biotin acid chloride (1) gives N-biotinyl-5-aminoisophthalic acid (2). This is coupled with the substituted salicylate to give bis[(1-tert-butoxycarbonyl)-3,5-dibromosalicyl] N-biotinyl-5aminoisophthalate (3). Removal of the tert-butyl groups

gives the biotin-substituted cross-linker, bis(3,5-dibromosalicyl) N-biotinyl-5-aminoisophthalate (4, DBBA). We used an analogous route to prepare a cross-linker with a pendant dipeptide derivative. The preparation of bis(3,5-dibromosalicyl) N-[N-trifluoroacetyl-L-isoleucylglycyl]-5-aminoisophthalate (DBTIA, 8) is presented in Scheme 3. N-[N-tert-butyloxycarbonyl-L-isoleucylglycyl]5-amino-isophthalic acid dimethyl ester (5) was prepared by coupling of N-t-Boc-Ile-Gly with dimethyl 5-aminoisophthalate. The diester was hydrolyzed to the diacid (6). This was coupled with the substituted salicylate to give the tert-butyl 3,5-dibromosalicylate ester (7). Removal of the tert-butyl group with trifluoroacetic acid and formation of trifluoroacetyl derivative of the terminal amino group with trifluoroacetic anhydride in a single step gave DBTIA (8). In our experience, hemoglobin reacts with cross-linking reagents most readily and completely in its deoxy form (21). This is also the case with the reagents in the present study. Carbonmonoxyhemoglobin reacted very slowly with DBBA (pH 9, 37 °C). The reagent was much more reactive toward deoxyhemoglobin under the same condi-

Figure 1. Globin chain separation after reaction of deoxyhemoglobin with DBBA (pH 9, 37 °C, 3 h).

tions: combining 1 equiv of hemoglobin with 2 equiv of the reagent led to more than 75% conversion in 3 h. The solution contained two principal products. The larger fraction was the material cross-linked in the β subunits (RRβ82-82β′), from acylation of the -amino groups of Lys-82 of each of the β subunits. The other major product is cross-linked between the -amino groups of Lys-99 of each of the R subunits (ββR99-99R′) (Figure 1). Increasing the reaction time to 14 h led to over 95% acylation of hemoglobin, with an increase in the extent of inter-R cross-linking and almost no further reaction of the β subunits. The details of the analysis follow. Product Structures. Products were analyzed as previously described (22). The C-4 reversed-phase HPLC chromatogram (Figure 1) has peaks for two cross-linked hemoglobin chains along with the peaks coincident with those of the native protein (heme, R and β subunits). The relative areas of the unreacted R and β chains compared to those from unreacted hemoglobin indicates that the chemical modification takes place predominantly in the β chains. SDS-PAGE of the globin chains indicated the presence of covalent cross-links with 32 kDa bands corresponding to linked dimers from 16 kDa monomers. The mass of each globin chain was determined by HPLC-MS. The first major modified globin (β82-82β′ in Figure 1) has a mass of 32 093 Da, consistent with an inter-β cross-linked dimer containing N-biotinyl-5-aminoisophthalate (calculated mass, 32 093 Da). The second major modified globin (R99-99R′ in Figure 1) has a mass of 32 615 Da, consistent with an R-R dimer containing N-biotinyl-5aminoisophthalate (calculated mass, 32 619 Da). Two distinct peaks eluting at 43 and 45 min could not be distinguished by their molecular weight and peptide pattern analysis. Their different chromatographic behavior suggests that these could be stereoisomers, differing in the orientation of the biotin derivative with respect to the protein. The intermediate chromatographic zones correspond to cross-linked globins having a second molecule of crosslinker attached to a third site with or without one leaving group hydrolyzed, as suggested by their HPLC-MS analysis. Since they made up only a small percentage of the other two products, they were not further analyzed. The anion-exchange HPLC chromatogram of the reaction products is shown in Figure 2. The peaks were identified by comparison with the chromatogram of native hemoglobin run under the same conditions, separation of the main fractions, purification by chromatography on a POROS column and globin chain separation

Molecular Necklaces

Bioconjugate Chem., Vol. 10, No. 6, 1999 1063

Scheme 3

on a C-4 reversed-phase column of the purified hemoglobins. Retention times of globin chains separated on C-4 reversed-phase column from the purified hemoglobins were compared with those from the reaction mixture for which HPLC-MS analysis was performed. Peak A is unmodified hemoglobin. Peak B is the inter-R crosslinked hemoglobin. Regions C and D correspond to the RRβ82-82β′ hemoglobin conjugate. The inter-β crosslinked hemoglobin fraction (peak C) was concentrated and used for functional studies. Its C-4 chromatogram (Figure 3) indicates the presence of the heme, the R monomer and the β-β′ cross-linked dimer. Region E contains a mixture of modified hemoglobins corresponding to the minor globins in the C-4 chromatogram (Figure 2). To determine the site of modification of the protein chain, the cross-linked dimers (peaks β82-82β′ and R9999R′ in 1) were separated on a C-4 column and digested with trypsin followed by endoproteinase Glu-C. Peptide

peaks were identified by comparison with the peptide pattern of the digest of native R and β globins. The patterns of tryptic peptides eluting at 43 and 45 min (Figure 1) are identical to one another (Figures 4 and 5). The peptide maps are characteristic of the β chain of human hemoglobin (14) with the exception of the absence of βT-9 and βT-10a′ peptide fragments and the presence of additional peaks at 93.8 and 94.3 min. These results are consistent with the selective acylation of the β-Lys82. The peptide pattern for the peak eluting at 75 min in the C-4 chromatogram (Figure 1) is missing the RT11 and RT-12 peak (Figure 6). A large peak elutes at 111.8 min. These results suggest the formation of an amide bond between the R-Lys-99 (on the C-terminus of RT-11) on each R subunit and the cross-linker. This newly formed amide bond prevents trypsin from hydrolyzing the peptide bond between RT-11 and RT-12 peptide fragments. These peptide pattern data along with the molecular masses determined experimentally by HPLC-MS

1064 Bioconjugate Chem., Vol. 10, No. 6, 1999

Figure 2. Anion-exchange HPLC profile on a POROS column for the reaction of DBBA with deoxyhemoglobin (37 °C, pH 9, sodium borate buffer, 3 h).

Figure 3. Globin chain separation on a C-4 column of the inter-β cross-linked Hb (peak C in Figure 2) purified on an AX300 column.

Crapatureanu et al.

Figure 5. C-18 reversed-phase HPLC chromatogram of the β82-82β′ globin dimer, after trypsin/Glu-C digestion, monitored at 200 nm.

Figure 6. Tryptic digest separation on C-18 reversed-phase HPLC from cross-linked R99-99R′ globin dimer (obtained from reversed-phase HPLC of globin chains), monitored at 200 nm..

eluted with PBS, pH 7.4 (∼4 mg avidin on the column; avidin:biotinylated hemoglobin 2:1, M:M).The column was eluted until absorbance at 420 nm was minimal. The ratio of absorption from hemoglobin (all as conjugate) in the effluent to absorption of total hemoglobin conjugate that had been applied was used to calculate the amount retained on the column:

Hbtotal - Hbeffluent/Hbtotal ) (A420applied - A420effluent/A420applied) × 100%

Figure 4. C-18 reversed-phase HPLC chromatogram of the β82-82β′ globin dimer (peak eluting at 43 min in the C-4 chromatogram), after trypsin/Glu-C digestion, monitored at 200 nm.

for the globin dimers indicate that two β and two R chains are cross-linked by a N-biotinyl-5-aminoisophthalate moiety. Affinity Chromatography of Cross-Linked Biotinylated Hemoglobin. Quantitative measurements of the accessibility to avidin on the column of biotin conjugated to hemoglobin followed the procedures described by Wilchek (23). The hemoglobin-biotin conjugate was applied to the avidin Sepharose column and

These measurements indicate that only 17% of the modified hemoglobin adheres to the avidin column (which binds free biotin quantitatively) (24). It is likely that because of the relatively short tether, only part of the biotin species extends beyond the hemoglobin to which it is attached. Reaction of hemoglobin with DBTIA. The materials, method, and analytical procedures are the same as for cross-linking hemoglobin with DBBA with a 2-fold molar excess of reagent to protein. The reaction was stopped after 3 h. C-4 HPLC analysis of the reaction mixture indicated formation of a reaction mixture in which the β-globin chains are all reacted while the R chains are mostly unreacted (Figure 7). The material in each peak was identified by comparing the chromatogram with that for native protein combined

Molecular Necklaces

Bioconjugate Chem., Vol. 10, No. 6, 1999 1065

Scheme 4

Figure 7. C-4 reversed phase HPLC chromatogram of separated subunits from the reaction of deoxyhemoglobin with DBTIA (pH 9, 37 °C, 3 h).

Figure 8. Tryptic-Glu-C peptide map of DBTIA-cross-linked β-chains (peak 1 in Figure 7).

with MS (ESI) analysis. The peaks designated “R” and “β” in Figure 7 are those of native globin chains. Peak 1 corresponds to a β-β′ dimer cross-linked by the isoleucylglycyl-5-aminoisophthalate (calculated, 32 133; found, 32 138). The molecular weight for the species in peak 2 is in the range expected for a β-β cross-linked dimer that has a second molecule of cross-linker attached as a monoamine to the protein, with the second ester intact. This dimer is cross-linked between the same residues as 1, based on the peptide pattern analysis. The experimental mass for peak 3 is consistent with that of the R globin chain to with the reagent as a monoamide with the remaining ester intact (calculated, 15 832; found, 15 839). Peak 4 corresponds to an R-R cross-linked dimer (calculated, 30 661; found, 30 665). The reaction mixture was also analyzed by anion-exchange chromatography, which separates hemoglobin tetramers and modified tetramers, giving results consistent with the HPLC analysis. To determine the site of modification of the protein chain, the cross-linked dimers (peaks 1, 2, and 4 in Figure 7) were collected and digested with trypsin followed by endoproteinase Glu-C. The peptide maps of native R and

β chains obtained by C-18 reversed-phase HPLC were compared to those for the modified chains in order to identify the site of modification. In the C-18 reversed-phase HPLC of peaks 1 and 2, the peptide fragments βT-9 and βT-10a′ are not present and new peaks elute at approximately 85-100 min (Figures 8 and 9). These two peptide fragments result from cleavage adjacent to Lys-82. In both chromatograms, the fragment β T-1 is unmodified. These results suggest that only the -amino groups of the β-Lys-82 residues have been blocked, the β-Val-1 residue [the amino group that is also modified by other reagents (25)] remaining unmodified. This is consistent with the formation of a bis-amide cross-link between the -amino group of the Lys-82 residue of one β chain and the -amino group of the Lys-82 residue of the other β chain. The peptide pattern for peak 4 in the C-4 chromatogram (Figure 7) is missing the RT-11 and the RT-12 peaks (Figure 10). These results, combined with the mass spectral data, suggest that a bis-amide cross-link between the -amino groups of two R-Lys-99 (the C-terminus of RT-11) has formed selectively.

1066 Bioconjugate Chem., Vol. 10, No. 6, 1999

Crapatureanu et al.

Figure 9. Tryptic-Glu-C peptide map of DBTIA-cross-linked β chains (peak 2 in Figure 7).

Figure 10. Tryptic-Glu-C peptide map of DBTIA-cross-linked R chains (peak 4 in Figure 7). Table 1. Product Distributions (relative percentages ot total eluted) from Reaction of Deoxyhemoglobin with DBBA and with DBTIA (pH 9, 3 h: based on reversed phase HPLC analysis) reagent

β82-X-82β′

R99-X-99R′

unreacted β

unreacted R

DBBA DBTIA

21 14.3

10 4.9

7.4 0.7

23 13

On the basis of the structural analysis and quantitative measurement of products, we determined the yield of modified hemoglobin obtained in the reactions. The results are summarized in Table 1. The cross-linked hemoglobins are produced in sufficient yields to permit isolation of reasonable amounts of bioconjugates. The process is not efficient in terms of use of hemoglobin, but it does produce stable, modified species that are easily isolated. We made no attempt to optimize the process by varying reaction conditions. The oxygen-binding properties of the biotinylated crosslinked hemoglobin [β82-(aminoisophthalylbiotintyl)-82β′ (DBBA-Hb] were determined by a modification of the Imai method (12, 14). The oxygen affinity (P50 ) 5.0; pH 7.4, 25 °C, 0.1 M chloride and 50 mM bis-tris) is very close to that of native hemoglobin (P50 ) 4.9) (25), and full cooperativity is maintained (n50 ) 3.1). Previous work has established a linear correlation between the length of the cross-link span and the oxygen affinity of the hemoglobin conjugate (for common sites of modification)

(22, 26). The oxygen-binding properties of the DBBA-Hb conjugate fit this correlation for a span of ∼7 Å between the  amino groups of the linked lysines (β82-X-82β′), the span of the isophthalyl cross-link. The cross-link is located on the 2-fold rotation axis of hemoglobin and does not introduce asymmetric distortion into the tetramer. Physical studies have shown that this cross-linking causes symmetrical perturbations in the structure of the protein (27), and related crystal structures show the retained symmetry (28). Thus, it is likely that the β8282β′ cross-link preserves the quaternary structure of the protein. The CD spectrum of the modified protein is essentially superimposable on that of the native protein, indicating that the modification does not alter the basic structure of the protein. We have used sequential activation to produce the cross-linking reagents with pendant ligands. The ligand to be appended is activated to react with the amino group of 3-amino-isophthalic acid to form an amide. The two carboxylic acid groups are then activated as 3,5-dibromosalicylates to react with the protein. The reagents that we have produced, DBBA and DBTIA, have their reactive sites in the isophthalyl geometry that with other reagents produces a cross-link selectively between β-Lys-82 residues of hemoglobin. The data in Table 1 show that DBBA and DBTIA also give significant amounts of products with R-Lys-99-R′-Lys-99 connections as well as the expected β-Lys-82 products. Walder has shown that for reactions with fumaryl bis (3,5-dibromosalicylate), the R-Lys-99 amino groups are normally reactive if the β-Lys-82 sites are blocked (29). Thus, we propose that reaction at the R-Ly-99 probably occurs because a molecule of the reagent binds between the β subunits without reacting, directing a second reagent to the alternative site between the R subunits. A schematic representation of the primary cross-linking reaction leading to the product with biotin appended is shown in Scheme 4. CONCLUSION

The procedures we have developed establish that molecular necklaces convert hemoglobin directly into cross-linked bioconjugates. Our method involves sequential activation, avoiding competition with hydrolysis in the final products. The method is general and should be applicable to generating a wide range of bioconjugates. ACKNOWLEDGMENTS

We thank the Natural Sciences and Engineering Research Council of Canada for support. The program used to collect oxygenation data was written by Howard Feldman and revised by Bryan Keith. LITERATURE CITED (1) Guidotti, G. (1967) Studies on the chemistry of hemoglobin. II. The effect of salts on the dissociation of hemoglobin into subunits. J. Biol. Chem. 242, 3685-93. (2) Winslow, R. M. (1995) Hemoglobin-Based Red Cell Substitutes, Johns Hopkins University Press, Baltimore. (3) Keipert, P. E., Gomez, C. L., Gonzales, A., Macdonald, V. W., and Winslow, R. M. (1992) The role of the kidneys in the excretion of chemically modified hemoglobins. Biomater., Artif. Cells, Immobilization Biotechnol. 20, 737-45. (4) Walder, J. A., Zaugg, R. H., Walder, R. Y., Steele, J. M., and Klotz, I. M. (1979) Diaspirins that cross-link beta chains of hemoglobin: bis(3,5-dibromosalicyl) succinate and bis(3,5dibromosalicyl) fumarate. Biochemistry 18, 4265-70. (5) Kluger, R. (1997) Chemical cross-linking and protein function. In Protein Function a Practical Approach (T. E. Creighton, Ed.) pp 185-214, IRL Press, Oxford.

Molecular Necklaces (6) Arnone, A., Benesch, R. E., and Benesch, R. (1977) Structure of human deoxyhemoglobin specifically modified with pyridoxal compounds. J. Mol. Biol. 115, 627-642. (7) Schumacher, M. A., Dixon, M. M., Kluger, R., Jones, R. T., and Brennan, R. G. (1995) Allosteric transition intermediates modelled by cross-linked haemoglobins. Nature (London) 375, 84-7. (8) Kluger, R., Song, Y., Wodzinska, J., Head, C., Fujita, T. S., and Jones, R. T. (1992) Trimesoyl Tris(3,5-Dibromosalicylate): Specificity of reactions of a trifunctional acylating agent with hemoglobin. J. Am. Chem. Soc. 114, 9275-9. (9) Kluger, R., and Song, Y. (1994) Changing a protein into a generalized acylating reagent. J. Org. Chem. 59, 733-6. (10) Kluger, R., and Li, X. (1997) Efficient chemical introduction of a disulfide cross-link and conjugation site into human hemoglobin at beta-lysine-82 utilizing a bifunctional aminoacyl phosphate. Bioconjugate Chem. 8, 921-6. (11) Trimble, S. P., Marquardt, D., and Anderson, D. C. (1997) Use of designed peptide linkers and recombinant hemoglobin mutants for drug delivery: in vitro release of an angiotensin II analogue and kinetic modeling of delivery. Bioconjugate Chem. 8, 416-23. (12) Imai, K. (1981) Measurement of accurate oxygen equilibrium curves by an automatic oxygenation apparatus. Methods Enzymol. 76, 438-49. (13) Imai, K. (1994) Adair fitting to oxygen equilibrium curves of hemoglobin. Methods Enzymol. 232, 559-76. (14) Jones, R. T. (1994) Structural characterization of modified hemoglobins. Methods Enzymol. 231, 322-43. (15) Shelton, J. B., Shelton, J. R., and Schroeder, W. A. (1984) High performance liquid chromatographic separation of globin chains on a large-pore C4 column. J. Liq. Chromatogr. 7, 1969-77. (16) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature (London), 227, 680-5. (17) Kluger, R., Paal, K., and Adamson, J. G. (1999) An etherlinked tetrafunctional acylating reagent and its cross-linking reactions with hemoglobin. Can. J. Chem. 77, 271-80. (18) Adamczyk, M., and Gebler, J. C. (1997) Electrospray mass spectrometry of alpha and beta chains of selected hemoglobins and their TNBA and TNB conjugates. Bioconjugate Chem. 8, 400-6.

Bioconjugate Chem., Vol. 10, No. 6, 1999 1067 (19) Bayer, E. A., Ben-Hur, H., and Wilchek, M. (1990) Isolation and properties of streptavidin. Methods Enzymol. 184, 809. (20) Bayer, E. A., and Wilchek, M. (1990) Avidin- and streptavidin-containing probes. Methods Enzymol. 184, 174-87. (21) Kluger, R., Jones, R. T., and Shih, D.-T. (1994) Crosslinking hemoglobin by design: lessons from using molecular clamps. Artif. Cells, Blood Substitutes, Immobil. Biotechnol. 22, 415-28. (22) Kluger, R., Shen, L., Xiao, H., and Jones, R. T. (1996) Systematically cross-linked human hemoglobin: functional effects of 10 Å spans between beta subunits at lysine-82. J. Am. Chem. Soc. 118, 8782-6. (23) Bayer, E. A., and Wilchek, M. (1990) Protein biotinylation. Methods Enzymol. 184, 138-60. (24) Green, N. M. (1990) Avidin and streptavidin. Methods Enzymol. 184, 51-67. (25) Dickerson, R. E., and Geis, I. (1983) Hemoglobin: Structure, Function, Evolution, and Pathology, The Benjamin/ Cummings Publishing Co., Menlo Park, CA. (26) Jones, R. T., Head, C. G., Fujita, T. S., Shih, D.-T., Wodzinska, J., and Kluger, R. (1993) Modification of human hemoglobin with methyl acyl phosphates derived from dicarboxylic acids. Systematic relationships between cross-linked structure and oxygen-binding properties. Biochemistry 32, 215-23. (27) Shibayama, N., Imai, K., Hirata, H., Hiraiwa, H., Morimoto, H., and Saigo, S. (1991) Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82β1 and 82β2 lysyl residues by bis(3,5-dibromosalicyl fumarate). Biochemistry 30, 8158-65. (28) Schumacher, M. A., Zheleznova, E. E., Poundstone, K. S., Kluger, R., Jones, R. T., and Brennan, R. G. (1997) Allosteric intermediates indicate R2 is the liganded hemoglobin end state. Proc. Natl. Acad. Sci. U.S.A. 94, 7841-4. (29) Snyder, S. R., Welty, E. V., Walder, R. Y., Williams, L. A., and Walder, J. A. (1987) HbXL99 alpha: a hemoglobin derivative that is cross-linked between the alpha subunits is useful as a blood substitute. Proc. Natl. Acad. Sci. U.S.A. 84, 7280-4.

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