Surface Modification of Diaspirin Cross-linked Hemoglobin (DCLHb

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Bioconjugate Chem. 2000, 11, 705−713

705

Surface Modification of Diaspirin Cross-linked Hemoglobin (DCLHb) with Chondroitin-4-sulfate Derivatives. Part 1 Ton T. Hai,* David E. Pereira,† Deanna J. Nelson,† Jim Catarello, and Ana Srnak Corporate Research and Technical Services, Baxter Healthcare Corporation, 25212 West State Route 120, Round Lake, Illinois 60073-9799. Received February 21, 2000; Revised Manuscript Received June 25, 2000

Synthetic methodology was developed for the preparation of chondrotin-4-sulfate reagents that could be specifically attached to the surface of diaspirin cross-linked hemoglobin (DCLHb), a chemically stabilized human hemoglobin. The surface-modified hemoglobin solutions had a significantly higher colloidal osmotic pressures (COP) than DCLHb. The P50 of the modified DCLHb was dependent upon the reactive end group of the chondrotin-4-sulfate reagents that was used for the protein modification. Modification of DCLHb with the chondroitin-4-sulfate derivatives containing the maleimide end group 23 provided a hemoglobin with a P50 value of 23 mmHg, while the P50 of hemoglobins prepared from chondroitin-4-sulfate derivatives containing the aldehyde end group 13 and 18 remained unchanged from that of DCLHb.

INTRODUCTION

Extracellular human hemoglobin is a protein composed of four polypeptide subunits, two R chains and two β chains, each with one heme associated with it. In the hemoglobin tetramer, each R chain is associated with a β chain to form two stable R-β dimers, which in turn associate to form the tetramer with a molecular weight of about 64 500. The subunits are noncovalently associated through van der Waals forces, hydrogen bonds and salt bridges. Human hemoglobin is not suitable for use as an oxygen delivering therapeutic agent for several reasons. First, the oxygen affinity of extracellular human hemoglobin is too high and, therefore, does not effectively deliver oxygen to tissues. Second, in the circulation, extracellular hemoglobin dissociates into R-β dimers. High concentrations of these dimers overwhelm the haptoglobin scavenging system and accumulate in the tubules of the kidney, where they are nephrotoxic. These shortcomings may be overcome by the stabilization of the tetrameric hemoglobin through intramolecular cross-linking between the dimeric subunits of the native hemoglobin. One such chemically modified hemoglobin is diaspirin crosslinked hemoglobin (DCLHb), which is intramolecularly cross-linked between the R chains (lysine 99R1-lysine 99R2) by a fumarate bridge (1, 2). Although stabilization of the tetrameric hemoglobin molecule by chemical modification was successful in addressing the above shortcomings, the full potential of extracellular hemoglobin solutions as an oxygen delivering therapeutic agent could not be met by tetrameric hemoglobin solutions. A primary drawback of chemically modified tetrameric hemoglobins, such as DCLHb, was the limited duration of the hemoglobin in the circulatory system. A hemoglobin of this size, approximately 64 500, is able to traverse cellular pores in the membrane of the circulatory system and enter interstitial spaces between endothelial cells * To whom correspondence should be addressed. Phone: (847) 270-5837. Fax: (847) 270-5897. E-mail: [email protected]. † Current address: Magellan Laboratories Inc., P.O. Box 13341, Research Triangle Park, NC 27709.

lining the lumen of the circulatory system. As a result, tetrameric hemoglobin can leave the intravascular space at a rate that is greater than that which is desired for optimal therapeutic utility. DCLHb has an elimination half-life of 2.5 h for a 25- and 50 mg/kg dose and an elimination half-life of 3.3 h for a 100-mg/kg dose in man (3). Therefore, further modifications to the hemoglobin that have the effect of increasing the molecular size of the resulting hemoglobin composition have been developed to extend the circulatory duration of the hemoglobin molecules. These additional modifications can be divided into two groups: polymerization and surface modification. DCLHb has been polymerized with PEG and polyamide cross-linking agents in this laboratory (4, 5) and human hemoglobin has been polymerized with other reagents, such as, glutaraldehyde and an o-raffinose derived dialdehyde (6). Less investigated is the surface modification of hemoglobin. Surface modification of hemoglobin is accomplished with monofunctional derivatization reagents that react with either the 44 available lysine -amino groups and terminal amino groups or the two thiol groups of the cysteines at position 93 of the β-chains (β-93-Cys). Both human and bovine hemoglobin have been surface modified with monofunctional poly(ethylene glycol) derivatives (7). This modification is reported to extend the circulating half-life of the modified hemoglobin molecules. An area of hemoglobin surface modification that has not been extensively explored is the use of hydrophilic, anionic reagents. Hemoglobin molecules surface modified with a reagent that could impart a global negative surface charge to the molecule would be expected to be repulsed from the surface of the endothelial cells lining the lumen of the circulatory system. It has been reported in the literature that macromolecules with an anionic molecular charge have a decrease rate of vascular endothelial and glomerular permeability (8-11). Thus, a surface-modified hemoglobin with a global negative charge may be less likely to cross through capillary junctions or glomerular pores, thereby, extending the circulatory half-life of the hemoglobin composition (12).

10.1021/bc000021i CCC: $19.00 © 2000 American Chemical Society Published on Web 08/25/2000

706 Bioconjugate Chem., Vol. 11, No. 5, 2000

Figure 1.

For these reasons, we chose to modify the surface of DCLHb with an anionic polysaccharide that would not only increase the size of the hemoglobin but also impart a global negative charge to the protein. In addition to imparting a negative charge, the polysaccharide needs to be nonimmunogenic and biocompatible. A polysaccharide that met these criteria was chondroitin-4-sulfate, one of the most abundant mucopolysacharides in the human body (13). Chondroitin-4-sulfate (C4S) is a long, unbranched polysaccharide chain composed of a repeating disaccharide structure containing uronic acid and hexosamine moieties. Chondroitin-4-sulfate molecules are strongly polyanionic due to the carboxylic acid and sulfate groups that are regularly spaced along the polymer backbone. The structure of chondroitin-4-sulfate (1) is shown in Figure 1. EXPERIMENTAL PROCEDURES

General. Diaspirin cross-linked hemoglobin (DCLHb), Plasma Lyte A, and Lactated Ringer’s irrigation solution were obtained from Baxter Healthcare Corporation, Deerfield, IL. Chondroitin-4-sulfate (injection grade, average MW of 50 000) was purchased from Maruha Corporation, Japan. All other commercial reagents were of the highest purity available. All solvents used were of HPLC grade. NMR spectrometry was performed on either a Bruker 270 MHz or a 300 MHz spectrometer. Analytical Methods. Thin-layer chromatography (TLC) was performed with Whatman MK6F silica gel 60 Å TLC plates. The products were eluted with 2-propanol: ammonium hydroxide: water (6:1:3, v/v/v). The plates were analyzed with UV light or stained with either ninhydrin or 2,3,5-triphenylphenyltetrazolium chloride (TTC) (25). The TTC spray solution was freshly prepared before use by combining one part 4% methanolic TTC solution with 1 part 1 N NaOH solution. The sprayed plate was heated for 5-10 min at 100 °C. Reducing sugars produce a red color upon development. Analytical RP-HPLC was performed on a Vydac Protein C4 column (5 µm, 4.6 × 250 mm). The mobile phase was delivered at 1 mL/min. as a linear gradient of B (CH3CN:H2O:TFA, 60:40:0.1, by volume) in A (CH3CN:H2O: TFA, 20:80:0.1, by volume) as follows: (1) 50% B to 55% B over 20 min, (2) 55% B to 75% B over 10 min; and (3) 75% B to 85% B over 10 min. Analytes were detected by measuring the absorbance at 220 nm. Size exclusion chromatography (SEC) was performed with a Superdex 200 (Pharmacia Biotech) SEC Column. A mobile phase of 50 mM phosphate buffer pH 7.0 containing 0.15 M NaCl at delivered a flow rate of 0.7 mL/min was used. SEC was also performed with a TSK

Hai et al.

G4000SW and G3000 SW columns (7.5 × 300 mm, Toso Haas) connected in series. Analytes were eluted with a binary mobile phase (50 mM phosphate, pH 6.5/2propanol, 9:1 by volume) delivered at a flow rate of 1 mL/ min. In both cases, analytes were detected by measuring the absorbance at 220 nm for chodroitin-4-sulfate and at 280 nm for hemoglobin products. Sample preparation for chondroitin sulfate analysis: a 5 mg of the C4S sample was dissolved in 0.5 mL of Nanopure water, and the solution was filtered through a 0.2 µm filter. A 20 µL aliquot of the solution was loaded on the column. Sample preparation for hemoglobin analysis: a 7 µL aliquot of the hemoglobin solution was added to 0.5 mL of Nanopure water and the solution was filtered through a 0.2 µm filter. A 20 µL aliquot of the solution was loaded on the column. Oxygen equilibrium curves were recorded by reoxygenation of nitrogen equilibrated deoxyhemoglobin in 0.1 M Bis-Tris buffer (pH 7.4 at 37 °C) in the spectral cuvette of a Hemox Analyzer (TCS Medical Products Co., Huntington Valley, PA). The oxygen pressure at which hemoglobin is half saturated (P50) and the subunit cooperativity (n40-60) were calculated from the oxygen equilibrium curves. The concentrations of oxy-, deoxy-, and methemoglobin were calculated from their absorbance at three wavelengths (630, 576, and 560 nm) using the reported molar extinction coefficients (14). Total hemoglobin concentration was calculated by summation of the concentrations of these components, and percent methemoglobin (MetHb) was calculated as its portion of the total hemoglobin concentration. The number of reactive thiols on the polyDCLHb molecules was determined by the method of Neis et al. (15). Red Cell Aggregation Testing. A simple in vivo test in which red cell aggregation was monitored was used for assessing the biocompatibilty with blood of the chemically modified DCLHb. In this test, human blood was diluted with physiological electrolyte solution (negative control) or chemically modified DCLHb solution (test solution) and then incubated. Solutions containing 10, 30, and 50% (by volume) of the chemically modified DCLHb solution were prepared and tested in this way. If there was no interaction between the test solution and the red cells or platelets, the red cells were dispersed randomly through the sample and a score of “none” was recorded. However, if there were interactions between the test sample and red cells platelets, aggregates and/ or platelet “clumps” were observed. A score of “very few”, “few”, “1”, and “2” served to define increasing degrees of red cell aggregation of the chemically modified DCLHb solution. Chondroitin-4-sulfate-4K (2). Chondroitin-4-sulfate (400.8 g) was dissolved in 5 L of 0.5 N HCl. The solution was heated at 65 °C for about 24 h. The solution was cooled in an ice bath to RT. The pH was adjusted to 7.6 with 5 N NaOH. To the solution was added 12 L of ethanol. The mixture was stirred for 3 h under ice bath cooling. The solvent was decanted and 4 L of ethanol was added. The mixture was stirred to obtain a solid. The solid was collected and washed with ethanol (2 × 500 mL) followed by ethyl ether (1 × 500 mL). The solid was vacuum-dried to give 343.5 g of product having MW of 4000 (16). The compound had a SEC a retention time of 26.3 min obtained on a Superdex 200 (Pharmacia Biotech) SEC column. Diethylene Glycol Bis(3-aminopropyl)ether Monotert-butyl Carbonate (4). Diethylene glycol bis(3-aminopropyl)ether (3) (528.0 g, 2.4 mol) was dissolved in 2 L of dichloromethane. A solution of di-tert-butyl dicar-

Modification of Hemoglobin with Chondroitin-4-Sulfate Derivatives

bonate (261.4 g, 1.2 mol) in 1 L of dichloromethane was added dropwise over 4.5 h. The solution was stirred for 1 h and then extracted with 1 L of water. The organic phase was evaporated to an oil. The oil was dissolved in 2 L of water and the pH was adjusted to 5 with 5 and 1 N HCl. The solution was extracted with 1 L of dichloromethane. The pH of the aqueous phase was adjusted to 13 with 1 N NaOH. The solution was extracted with dichloromethane (4 × 750 mL). The extracts were combined and dried with MgSO4. The solution was evaporated to give 241.3 g (31%) of viscous oil product. 1H NMR (CDCl ): δ 1.39 (s, 9-H); 1.67 (m, 4-H); 2.75 3 (t, 2-H, J ) 6.75 Hz); 3.17 (q, 2-H, J ) 6.2 Hz); 3.51 (m, 14-H) 5.19 (s, 1-H). 13C NMR (CDCl3): δ 28.3; 33.2; 39.5; 69.3; 69.5; 70.1; 70.4; 70.5; 78.7; 156.0; impurity resonances at 29.5; 38.4; 70.0. Anal. calcd for C15H32N2O5: C, 56.23; H, 10.07; N, 8.74. Found: C, 56.87; H, 9.86; N, 8.36. Chondroitin-4-sulfate Derivative 5. Compound 2 (222.0 g, 55.0 mmol) and 4 (150.1 g, 470.5 mmol) were dissolved in 1.2 L of sterile water for irrigation, USP, and the solution pH was adjusted to 8.3 with 1 N HCl. Ethanol (600 mL) was added to the solution followed by borane-pyridine complex (57 mL). The solution was heated at 40 °C for 4 days. The solution was cooled to RT and the pH was adjusted to 10 using 5 N NaOH followed by 1 N NaOH. Ethanol (12 L) was added to solution with stirring. After stirring 3 h, the mixture was allowed to stand for 1 h. The solvent was decanted and the solid was collected and washed with ethanol (2 × 500 mL) followed by ethyl ether (1 × 500 mL). The solid was vacuum-dried to give 210.7 g (89%) of product. 1H NMR (D O): δ 1.38 (s); 1.70 (t, J ) 6.48 Hz); 1.98 2 (s); 3.09 (t, J ) 6.48 Hz); 3.29-3.75 (m); 3.95 (m); 4.04 (m); 4.12 (m); 4.17 (m); 4.45 (m). 13C NMR (D2O): δ 25.4; 30.6; 31.6; 39.9; 53.6; 53.8; 54.4; 63.8; 70.3; 70.5; 71.2; 71.1; 72.4; 74.6; 75.3; 75.4; 75.5; 76.5; 77.4; 77.8; 77.9; 78.1; 78.4; 79.0; 79.2; 82.5; 82.8; 83.1; 83.7; 103.6; 104.2; 106.3; 106.6; 106.9; 107.1; 161.1; 177.2; 177.4; 177.5; 177.8; 178.8; 178.9. Chondroitin-4-sulfate Derivative 6. Compound 5 (184.2 g) was dissolved in 750 mL of water and 750 mL of 1 N HCl. The pH of the solution was adjusted to 0.85 with 1 N HCl. The solution was stirred at RT for 24 h. The solution was evaporated from about 1.8 L to a volume of about 1 L. The solution was diluted with ethanol (8 L) and the mixture was stirred for 2 h. The precipitate was collected and washed with ethanol (250 mL) and then with ethyl ether (250 mL). The solid was vacuum-dried to give 143.9 g (79%) of 6. 1H NMR (D O): δ 1.89 (m); 3.09 (t, J ) 6.48 Hz); 3.292 3.75 (m); 3.93 (m); 4.06 (m); 4.14 (m); 4.51 (m); 4.69 (m). 13C NMR (D O): δ 23.1; 23.5; 26.4; 27.6; 38.7; 47.4; 48.4; 2 52.0; 52.2; 52.7; 62.0; 63.7; 68.5; 68.7; 69.4; 70.5; 70.6; 72.3; 72.4; 73.1; 73.3; 73.5; 73.7; 74.5; 74.8; 75.5; 75.6; 76.0; 76.1; 81.0; 99.2; 102.4; 104.8; 105.0; 105.3; 105.4; 172.4; 172.6; 173.6; 173.8; 173.7; 176.0; 176.2. Methyl 4-Thia-7-diethoxy-heptanoate (10). Methyl 3-mercaptopropionate (8) (120.0 g, 1.0 mol) was added to a mixture of potassium carbonate (250.0 g, 1.8 mol) in 1 L of DMF. 3-Chloropropionaldehyde diethyl acetal (9) (194.6 g, 1.17 mol) was added to the mixture. The mixture was heated to 85 °C for 5 h. An additional 10.0 g portion of the acetal and 10.1 g of potassium carbonate was added. The mixture was heated for an additional 1.5 h at 85 °C. The mixture was cooled to about 20 °C and ethyl ether (1 L) was added to the mixture. Water (1 L) was added to the mixture while the mixture was well stirred. The phases were separated and the aqueous phase was

Bioconjugate Chem., Vol. 11, No. 5, 2000 707

extracted with ethyl ether (1 × 1 L; 1 × 500 mL). The combined extracts were extracted with a 5% potassium carbonate solution (800 mL). The organic phase was dried with MgSO4 and filtered, and the filtrate was evaporated to an liquid. The product was collected by distillation from 115 to 120 °C at 0.2 mm Hg to give 186.1 g (74%) of product. 1H NMR (CHCl ): δ 1.19 (t, J ) 7.1 Hz, 6-H); 1.86 (q, 3 J ) 7.7 Hz, 2-H); 2.59 (m, 4-H), 2.76 (m, 2-H); 3.45 (m, 2-H); 3.64 (m, 5-H); 4.57 (t, J ) 5.6 Hz, 1-H). 13C NMR (CHCl3): δ 14.7; 26.3; 26.5; 33.0; 33.9; 51.0; 60.8; 100.9; 171.5. Anal. calcd for C11H22O4S: C, 52.77; H, 8.86; S, 12.81. Found: C, 52.37; H, 8.66; S,12.71. 4-Thia-7-diethoxy-heptanoic Acid (11). Methyl 4-thia-7-diethoxy-heptanoate (10) (250.3 g, 1.0 mol) was dissolved in ethanol (600 mL). Potassium hydroxide (61.8 g, 1.1 mol) in water (200 mL) was added in one portion. After stirring for 45 min, the solvent was evaporated to two-thirds of the starting volume. To the remaining solution was added 500 mL of water. The solution was extracted with ethyl ether (2 × 300 mL). The aqueous phase was collected, and ethyl ether (500 mL) was added. The mixture was stirred vigorously as 5 N HCl (182 mL) was slowly added. The phases were separated and the aqueous phase was extracted with ethyl ether (2 × 300 mL). The extracts were combined and extracted with 1.00 N NaOH (1 × 72 mL) to remove impurities. The organic phase was dried with MgSO4 and evaporated to provide 183.6 g (78%) of 11 as viscous oil. 1H NMR (CDCl ): δ 1.15 (t, J ) 7.0 Hz, 6-H); 1.86 (q, 3 J ) 6.8 Hz, 2-H); 2.54 (t, J ) 7.5 Hz, 2-H); 2.60 (t, J ) 6.8 Hz, 2-H); 2.73 (q, J ) 7.0 Hz, 2-H); 3.48 (m, 2-H); 3.61 (m, 2-H); 4.57 (t, J ) 5.6 Hz, 1-H). 13C NMR (CDCl3): δ 15.3; 25.5; 26.3; 27.3; 31.8; 33.5; 61.6; 101.4; 167.1; 168.9. Anal. calcd for C10H20O4S: C, 50.77; H, 8.30; S, 13.57; Found: C, 50.89; H, 8.30; S, 13.41. N-Succinimidyl 4-Thia-7-diethoxy-heptanoate (12). 4-Thia-7-diethoxy-heptanoic acid (11) (153.9 g, 0.65 mol) and triethylamine (72.9 g, 0.72 mol) were combined in 1 L of acetonitrile. N,N′-Disuccinimidyl carbonate (184.6 g, 0.72 mol) was added as a solid in portions. The resulting solution was stirred at RT overnight. The solution was evaporated to an oil, and the oil was dissolved in 1 L of chloroform. The solution was extracted with 5% NaHCO3 (2 × 500 mL). The organic phase was treated with activated carbon, and the mixture was dried with MgSO4. The mixture was filtered through a pad of MgSO4, and the filtrate was evaporated to give 186.5 g (95%) of 12 as an oil. The oil was used without further purification. 1H NMR (CDCl ): δ 1.19 (t, J ) 7.0 Hz, 6-H); 1.89 (q, 3 J ) 6.7 Hz, 2-H); 2.60 (t, J ) 7.5 Hz, 2-H); 2.85 (m, 12H); 3.49 (m, 2-H); 3.62 (m, 2-H); 4.58 (t, J ) 5.6 Hz, 1-H). 13 C NMR (CDCl3): δ 15.2; 25.5; 26.2; 27.3; 31.8; 33.5; 61.5; 101.4; 167.1; 168.9. Anal. calcd for C14H23NO6S: C, 50.43; H, 6.95; N, 4.20. Found: C,49.89; H, 6.61; N, 3.77. Chondroitin-4-sulfate Derivative 7. Compound 6 (140.0 g, 35.0 mmol) was dissolved in 1.2 L of water. The pH of the solution was adjusted to 9 with 5 N NaOH. N-Succimidyl 4-thia-7-diethoxy-heptanoate 12 (58.8 g, 170.0 mmol) in 500 mL of DMF was added dropwise. After the addition was complete, the solution was stirred 4 h at RT. The solution was concentrated to 500 mL. Ethanol (4 L) was added to the solution. The solvent was decanted from the resulting oil. Ethanol (4 L) was added to the oil and the mixture was kept at 5 °C. The solvent was decanted. The remaining mixture was centrifuged to give a gellike pellet. The pellet was diluted with acetone and evaporated to a semisolid. Ethyl ether was

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added, and the mixture was allowed to stand for 1 h. The mixture was filtered, and the gellike solid was dried under vacuum at room temperature. The solid was collected to give 104.7 g (71%) of 7. 1 H NMR (D2O): δ 1.18 (m); 1.76 (m); 1.91 (m); 2.05 (s); 2.57 (m); 2.61 (m); 2.79 (m); 2.99 (s); 3.29-3.75 (m); 3.95 (m); 4.09(m); 4.15 (m); 4.21 (m); 4.49 (m); 7.91 (s). 13 C NMR (D2O): δ 15.5; 17.9; 22.9; 23.1; 23.6; 26.2; 26.4; 27.3; 27.8; 29.3; 32.5; 34.2; 36.7; 37.4; 38.0; 51.9; 52.1; 52.6; 53.1; 58.5; 62.1; 63.9; 64.0; 68.6; 68.7; 69.4; 69.5; 70.4; 70.6; 72.9; 73.5; 73.6; 73.8; 74.6; 74.7; 75.7; 76.0; 76.3; 76.4; 77.2; 77.4; 77.7; 80.6; 81.1; 81.3; 82.2; 101.9; 102.4; 103.0; 104.3; 104.5; 104.8; 105.2; 105.3; 166.0; 175.4; 175.6; 175.7; 176.1; 176.3; 176.5; 176.9; 177.0; 178.0. Chondroitin-4-sulfate Aldehyde 13. Compound 7 (104.3 g) was dissolved in a solution of 750 mL of sterile water and 750 mL of 1 N HCl. The pH of the solution was adjusted to 2 with 1 N HCl. The solution was stirred for 3.5 h. The reaction solution was concentrated to about 100 mL. Acetone (250 mL) was added to precipitate the product as an oil. The solvent was decanted and an additional 250 mL of acetone was added. The mixture was agitated, and the solvent was decanted. To the resulting semisolid was added an additional 250 mL of acetone. The mixture was allowed to stand for 1 h. The resulting solid was collected, washed with 100 mL of acetone, and dried under vacuum to give 113.6 g of 13. 1H NMR (D O): δ 1.75 (m); 2.02 (s); 2.51 (m); 2.58 (m); 2 2.79 (m); 3.29-3.75 (m); 3.95 (m); 4.04 (m); 4.12 (m); 4.17 (m); 4.54 (m); 5.08 (s); 9.64(s). 13C NMR (D2O): δ 22.9; 23.1; 23.5; 36.7; 37.4; 51.9; 52.1; 52.7; 62.0; 68.4; 68.68.7; 69.4; 70.4; 70.6; 72.3; 73.1; 73.3; 73.5; 74.7; 75.5; 75.7; 76.0; 76.8; 77.0; 77.2; 80.9; 81.1; 90.7; 102.3; 102.5; 104.4; 104.7; 104.9; 105.3; 172.6; 172.9; 173.8; 173.9; 174.0; 175.5; 175.7; 176.0; 176.2; 207.4. Compound exists in the aldehyde and hydrated form (90.8 and 207.4 resonances). N-(4-Diethoxybutyryl)succinamic Acid (15). 4-Aminobutyraldehyde diethylacetal (14) (5.3 g, 32.9 mmol) was dissolved in 10 mL of chloroform. A solution of succinic anhydride (3.3 g, 33.0 mmol) in 20 mL of chloroform was added dropwise. The oil was used without further purification. 1H NMR (CDCl3): δ 1.15 (m, 12-H); 2.00 (m, 8-H); 2.63 (m, 8-H); 3.45 (m, 2-H); 3.56 (m, 2-H); 3.69 (m, 4-H); 5.07 (d, J ) 4.05 Hz, 1-H); 5.45 (d, J ) 5.19 Hz, 1-H); 7.25 (s, 2-H). 13C NMR (CDCl3): δ 15.1; 15.2; 21.0; 22.8; 28.7; 28.8; 29.1; 29.3; 31.4; 31.6; 45.7; 46.0; 62.2; 64.3; 85.8; 87.6; 171.9; 172.0; 176.5. Impurity resonances: 176.4; 58.1; 28.6; 18.0. N-Succinimidyl N-(4-Diethoxybutyryl)succinamate (16). Disuccinimidyl carbonate (6.5 g, 25.3 mmol) was added as a solid to a solution of N-(4-diethoxy)butyrylsuccinamic acid (15) (6.0 g, 23.0 mmol) and triethylamine (2.6 g, 25.3 mmol) in acetonitrile. The solution was stirred overnight at room temperature. The solution was evaporated to an oil. The oil was dissolved in chloroform (75 mL) and extracted with water (2 × 25 mL). The organic phase was extracted with 5% NaHCO3 (2 × 25 mL) and then dried over anhydrous MgSO4. The mixture was filtered, and the filtrate was concentrated under vacuum to give 16 as an oil. The oil was used without further purification. 1 H NMR (CDCl3): δ 1.11 (m, 6-H); 1.53 (m, 4-H); 2.62 (s, 4-H); 3.42 (m, 6-H); 3.56 (m, 2-H); 4.35 (dd, 1-H); 4.40 (t, 1-H). 13C NMR (CDCl3): δ 15.1; 22.9; 25.4; 27.9; 30.7; 61.4; 102.1; 168.7; 168.9; 177.1. Anal. calcd for C16H26N2O7: C, 53.62; H, 7.31; N, 7.82. Found: C, 53.07; H, 7.01; N, 8.00. Chondroitin-4-sulfate Derivative 17. Compound 6 (10.0 g, 2.5 mmol) was dissolved in 100 mL of water, and

Hai et al.

the solution pH was adjusted to 9.3 with 1 N NaOH. A solution of N-oxysuccinimidyl N-(4-diethoxy)butyrylsuccinamate (16) (4.5 g, 12.5 mmol) in 10 mL of DMF was added to the reaction solution. The solution was stirred for 3 h at room temperature. The solution was concentrated under vacuum to a volume of about 25 mL. To the solution was added 1 L of ethanol. The mixture was cooled to 5 °C and allowed to stand overnight. The solvent was decanted, and the solid was collected by filtration. The solid was washed with ethanol (25 mL) and then with ethyl ether (2 × 25 mL). The solid was dried under vacuum to give 6.8 g (64%) of 17. 1H NMR (D O): δ 1.24 (m); 1.76 (m); 2.06 (br. s); 2.49 2 (m); 2.72 (s); 3.2-4.20 (br. s); 4.49. 13C NMR (D2O): 14.3; 14.9; 15.4; 23.6; 26.2; 29.3; 31.0; 31.4; 32.5; 33.0; 37.4; 39.0; 52.0; 52.6; 62.1; 63.6; 64.1; 68.5; 68.8; 69.2; 69.4; 70.4; 70.6; 72.6; 72.9; 73.8; 74.7; 75.7; 76.0; 76.4; 76.6; 77.2; 77.4; 80.7; 81.0; 81.3; 101.9; 104.5; 104.8; 105.1; 105.3; 108.7; 175.3; 175.6; 175.7; 176.0; 177.0; 177.1; 178.7. Chondroitin-4-sulfate Aldehyde 18. Compound 17 (5.5 g, 1.4 mmol) was dissolved in 25 mL of water, and the solution pH was adjusted to 1.5 with 1 N HCl. The solution was stirred at RT for 4 h. The solution was concentrated under vacuum to a volume of about 10 mL, and then acetone (50 mL) was added. The solvent was decanted, and an additional 50 mL of acetone was added. The solvent was again decanted, and 50 mL of acetone was added. The mixture was stirred and the solid was collected. The solid was dried under vacuum to give 5.0 g (83%) of 18. 1H NMR (D O): δ 1.14 (m); 1.74; (br. s); 1.80 (br. s); 2 2.47 (br. s); 2.61 (br. s); 2.75 (br. s); 3.20-4.16 (m); 4.56 (br. s); 5.63 (s). 13C NMR (D2O): δ 14.5; 15.0; 22.9; 23.1; 23.5; 26.3; 28.4; 31.4; 37.5; 46.8; 47.7; 52.2; 52.8; 62.0; 68.5; 68.7; 69.5; 69.9; 70.4; 70.7; 72.3; 73.5; 74.5; 74.6; 74.8; 75.4; 75.6; 75.7; 76.0; 76.1; 76.4; 77.0; 77.3; 80.4; 81.1; 8Å.9; 82.7; 102.5; 104.5; 104.8; 105.0; 172.3; 172.5; 173.4; 173.6; 175.8; 176.0; 176.2; 183.0. N-Succinimidyl 2-Formylphenoxyacetate (20). 2-Formylphenoxyacetic acid (19) (18.0 g, 0.1 mol) and N-hydroxysuccinimide (13.0 g, 0.11 mol) were combined in 300 mL of acetonitrile. 1,3-Dicyclohexylcarbodiimide (24.1 g, 0.12 mol) in 100 mL of acetonitrile was added. The resulting mixture was stirred for 3 h and then filtered. The filtrate was concentrated to dryness under vacuum, and tetrahydrofuran (THF; 50 mL) was added. The solid was collected by filtration, and the filtrate was reduced in volume to about 15 mL. The precipitated solid was collected, combined with the first crop, and dried to give 22.8 g (82%) of 20. 1H NMR (DMSO-d ): δ 2.82 (s, 4-h); 5.54 (s, 2-H); 7.16 6 (t, J ) 7.43 Hz, 1-H); 7.25 (d, J ) 8.42 Hz, 1-H); 7.71 (m, 2-H); 10.41 (s, 1-H). 13C NMR (D2O): δ 25.2, 63.7, 122.2, 124.8, 136.2, 159.3, 165.3, 169.9, 188.8. Anal. calcd for C11H22O4: C, 56.32; H, 4.00; N, 5.05. Found: C, 56.44; H, 4.00; N, 4.92. Chondroitin-4-sulfate Aldehyde 21. Compound 6 (8.0 g, 2.0 mmol) in water (60 mL) was added dropwise to a solution of N-oxysuccimidyl 2-formylphenoxyacetate (20) (3.3 g, 12 mmol) in DMF (100 mL) over 1.5 h. The solution was stirred at RT for 1 h, and then the solution was concentrated to dryness under vacuum. The residue was dissolved in 60 mL of water, and the solution was filtered. Ethanol (500 mL) was added to the filtrate to precipitate the product. The product was washed with ethanol (25 mL) and dried to give 3.6 g (43%) of product. 1 H NMR (D2O): δ 1.78; 1.99; 3.32; 3.47; 3.59; 3.66; 3.75; 3.97; 4.06; 4.18; 4.45; 7.046 (d); 7.19 (t); 7.69 (t); 7.84 (d).

Modification of Hemoglobin with Chondroitin-4-Sulfate Derivatives

Bioconjugate Chem., Vol. 11, No. 5, 2000 709

Table 1. Surface Modification of DCLHb with 23 molar ratio peak maximaa P50 n of 23/DCLHb (retention time, min) (mm Hg) (40-60) C4S/Hbb DCLHb 5/1 10/1 15/1 20/1 25/1 30/1 35/1 40/1

19.6 17.6 16.9 16.3 15.8 15.4 15.1 14.9 14.7

31 21 21 23 22 22 22 23 23

2.7 2.1 1.9 1.9 1.8 1.9 1.8 1.8 1.9

NA NA NA NA 7.0/1 7.8/1 9.8/1 10.7/1 NA

a SEC column: TSKG3000 and TSKG4000 in series. b Determined by NMR.

13C NMR (D O): δ 23.6; 27.6; 29.3; 37.5; 38.7; 52.1; 52.7; 2 62.2; 68.6; 69.4; 70.5; 70.6; 72.9; 73.5; 73.8; 74.8; 75.7; 76.1; 76.4; 76.4; 77.3; 77.5; 81.1; 81.4; 101.9; 105.2; 105.4; 114.4; 123.3; 125.4; 132.0; 138.5; 160.5; 171.7; 175.4; 175.7; 176.1; 177.1; 194.7. Chondroitin-4-sulfate Maleimide 23. A solution of 6 (7.0 g, 1.75 mmol) in a solution of 46 mL of water and the solution pH were adjusted to 9 with 5 N NaOH. DMF (23 mL) was added to the solution of 6, and the solution was added to a stirred solution of N-succinimidyl maleimidopropionate (22) (2.78 g, 10.5 mmol) in solution of 140 mL of DMF and 10 mL of water at a rate of 4-5 mL/min. After the addition, the solution was stirred for an additional 1 h. The reaction mixture was concentrated to dryness. The residue was dissolved in water and filtered to remove an insoluble solid. The filtrate was diluted with ethanol to precipitate the product. The solid was dried to give 5.83 g (80%) of 23. 1H NMR (D O): δ 1.78 (br. s); 1.80 (br. s); 2.14; 2.56 2 (br. s) 3.24-4.17 (m); 4.07; 4.28; 4.55; 4.57; 6.95 (s). 13C NMR (D2O): δ 23.1; 23.7; 26.3; 26.5; 29.3; 35.7; 36.0; 37.5; 47.1; 47.4; 50.6; 52.1; 62.2; 64.0; 68.7; 69.5; 70.5; 70.7; 72.0; 72.9; 73.6; 73.8; 74.8; 75.6; 76.1; 76.5; 76.8; 77.5; 80.1; 81.1; 81.4; 82.3; 102.0; 102.5; 103.9; 104.6; 104.9; 105.2; 105.4; 135.7; 173.7; 173.7; 173.8; 174.5; 175.9; 175.8; 176.1; 176.4; 17.1; 178.6. Surface Modification of DCLHb with Chondroitin4-sulfate Containing Aldehyde End Groups. A solu-

Figure 2.

tion of DCLHb in 0.1 M borate, pH 9.0, was deoxygenated by vacuum/nitrogen cycles for 1.5 h at room temperature. A solution of 13, 18, or 21 in water and borane-pyridine complex (borane-pyridine/2 in a molar ratio of 5/1) were added so that the final concentration of DCLHb was 6 g/dL in borate buffer, pH 9.0. The reaction mixture was stirred at room temperature under nitrogen for 24 h and dialyzed against Plasma-Lyte A to give the final product. The results are summarized in Tables 2 and 3 and Figure 2. Surface Modification of DCLHb with Chondroitin4-sulfate Containing Maleimide End Groups. A solution of DCLHb in 0.1 M HEPES, pH 8.0, was deoxygenated by vacuum/nitrogen cycles for 1.5 h at room

Table 2. Effect on P50 and Coopertivity by the Surface Modification of DCLHb by 13 and 18 DCLHb-13 conjugate

a

DCLHb-18 conjugate

molar ratio of 13 or 18/DCLHb

peak maximaa (retention time, min)

P50 (mmHg)

n (40-60)

peak maximaa (retention time, min)

DCLHb 20/1 25/1 30/1 35/1 40/1 45/1

22.0 20.3 20.0 19.5 19.4 19.2 19.5

30 31 32 33 33 32 31

2.7 2.3 2.4 2.3 2.4 2.2 2.2

22.0 20.7 20.5 20.3 20.1 19.7 19.7

SEC column: Superdex 200.

Table 3. Physical Characteristics of Surface Modified DCLHb with 13 DCLHb-13 conjugate molar ratio of 13/DCLHb total Hb (g/dL) % MetHb pH at 37 °C COP (mmHg) viscosity (cp) osmolality (mOsm/Kg) cell aggregationa a

DCLHb

25/1

35/1

40/1

10.3 2.0 7.38 43 1.2 286 none seen

7.1 8.0 7.44 92 1.5 290 none seen

6.9 11.7 7.37 124 2.0 338 none seen

7.1 17.2 7.48 123 1.6 296 none seen

Results of 50% CS-DCLHb concentration.

710 Bioconjugate Chem., Vol. 11, No. 5, 2000

temperature. A solution of 23 in water and boranepyridine complex (borane-pyridine/2 in a molar ratio of 5/1) were added so that the final concentration of DCLHb was 6 g/dL in 0.1 M HEPES, pH 8.0. The hemoglobin solution was stirred at room temperature under nitrogen, and the reaction was monitored by SEC (column: TSK G3000SW and TSK G4000SW in series). SEC indicated that the surface modification of DCLHb was completed within 6 h. The reaction mixture was dialyzed against Plasma Lyte A to give the final product. The results are summarized in Table 1.

Hai et al. Scheme 1a

RESULTS AND DISCUSSION

Synthesis of Monofunctional Chondroitin-4-sulfate Derivatives. Chondroitin-4-sulfate has a MW of 50 000. A solution of hemoglobin surface modified with chondroitin-4-sulfate molecules with this high of MW would result in a solution too viscous to be used as a therapeutic solution. Therefore, the MW of chondroitin4-sulfate was reduced to 4000 via acid-catalyzed hydrolytic cleavage at 65 °C. The hydrolysis was stopped at the time the size exclusion chromatography (SEC) profile of the reaction product was comparable with the SEC profile of a chondroitin-4-sulfate standard with a MW of 4000 provided by Biorelease, Inc. (16). The hydrolysis method provided Chondroitin-4-sulfate (C4S) (2) with reproducible product distributions as determined by SEC and yields. It has been reported in the literature that the reducing terminus of a disaccharide or polysaccharide can be directly derviatized with a primary amine by reductive amination under mild conditions (17-19). We took advantage of this chemistry to introduce a primary amine to the reducing terminus of chondroitin-4-sulfate. The reductive amination was achieved by treatment of chondroitin-4-sulfate (2) with the monoprotected diamine diethylene glycol bis(3-aminopropyl)ether mono-tert-butyl carbonate (4) in the present of a reducing agent depicted in Scheme 1. The choice of the reducing agent for this transformation was critical. Sodium cyanoborohydride has been successfully used for the reductive amination of carbohydrates (17-21). However, for large-scale syntheses the use of poisonous sodium cyanoborohydride is unattractive as is the workup, because care must be exercised with respect to the sodium cyanide and hydrogen cyanide byproducts. The use of sodium cyanoborohydride could be avoided by the use of borane-pyridine complex. Cabacungan et al. (22) found that boranepyridine complex has a higher reducing capacity than sodium cyanoborohydride for the reductive methylation of N-R-acetyl-lysine with formaldehyde. In addition, borane-pyridine complex does not readily reduce aldehydes (23, 24). Therefore, the reduction of the reducing terminus of chondroitin-4-sulfate during the reductive amination reaction could also be avoided by the use of borane-pyridine complex. The aminated product 5 was prepared by the treatment chondroitin-4-sulfate (2) with 4 in the present of borane-pyridine complex under alkaline conditions at 40 °C over four days. The reaction was terminated when the detection with 2,3,5-triphenyltetrazolium chloride (TTC) of the reducing terminus was negative (25). The diethylene glycol bis(3-aminopropyl)ether (3) was monoprotected with a tert-butyl carbonate group using di-tert-butyl dicarbonate (Scheme 1). The BOC protecting group was removed by acid hydrolysis from 5 to give the deprotected product 6. The primary amine of 6 was coupled to both alkyl and aromatic aldehyde moieties via an amide linkage. 4-Thia7-diethoxy-heptanoyl was covalently linked to 6 as out-

a (a) Di-tert-butyl carbonate, CH Cl ; (b) borane-pyridine, 2 2 EtOH, 40 °C, 4 days; (c) HCl, 24 h.

Scheme 2a

a

12, pH 9, DMF-water; (b) HCl, water.

lined in Scheme 2. Treatment of 6 with N-succinimidyl 4-thia-7-diethoxy-heptanoate (12) gave 7 in moderate yields. N-Succinimidyl 4-thia-7-diethoxy-heptanoate (12) was synthesized by a three step method (Scheme 3). Methyl 3-mercaptopropionate (8) was allowed to react

Modification of Hemoglobin with Chondroitin-4-Sulfate Derivatives Scheme 3a

Bioconjugate Chem., Vol. 11, No. 5, 2000 711 Scheme 4a

a

(a) Succinic anhydride, CHCl4; (b) DSC, Et3N, CH3CN.

Scheme 5a

a (a) K CO , DMF, 85 °C; (b) KOH, ethanol water; (c) disuc2 3 cinimidyl carbonate, triethylamine, acetolnirile.

with 3-chloropropionaldehyde diethyl acetal (9) in the presence of potassium carbonate to give methyl 4-thia7-diethoxy-heptanoate (10). Methyl 4-thia-7-diethoxyheptanoate (10) was hydrolyzed to the free acid (11) with potassium hydroxide in aqueous ethanol. Treatment of the free carboxylic acid with disuccinimidyl carbonate provided N-succinimidyl 4-thia-7-diethoxy-heptanoate (12) in good yields. The target compound 13 was prepared by the deprotection of the aldehyde group of 7 by acid hydrolysis. The coupling of the aldehyde groups with amino groups on the surface of proteins is dependent upon the distance of the aldehyde to a heteroatom on the reagent chain as demonstrated by Harris and Sedaghat-Herati (26). They showed that PEG propionaldehyde (PEG-OCH2CH2CHO) was more reactive toward the protein amino groups than was PEG acetaldehyde (PEG-OCH2CHO). To test the reactivity of a reagent with a greater distance between the aldehyde group and a heteroatom, a chondroitin-4-sulfate derivative containing a butyraldehyde end group (18) was prepared. The carboxylic acid of N-(4-ethoxybutyryl)succinamic acid (15) was activated with N-hydroxysucccinimide to provide 16 that was readily coupled to the primary amino group of 6 to afford the target compound 17. The ethoxy groups of 17 were removed under acidic conditions to provide 18 (Schemes 4 and 5). In addition to alkyl aldehydes, we also wanted to investigate the utility of employing aromatic aldehydes and maleimide groups for the attachment of chondroitin4-sulfate to DCLHb. The chondroitin-4-sulfate derivative 21, terminating with an aromatic aldehyde, was prepared from N-sucinimidyl 2-formylphenaoxyacetic acid and 6 shown in Scheme 6. A maleimide moiety was conveniently coupled to 6 using N-succinimidyl maleimidopropionate (22) to provide 23 (Scheme 7).

a

(a) DMF-water; (b) HCl.

Modification of DCLHb with Chondroitin-4sulfate Derivatives. The typical surface modification of DCLHb with the maleimide derivative 23 was accomplished by the reaction of deoxyDCLHb in HEPES buffer, pH 8, with the chondrotin-4-sulfate derivative under a nitrogen atmosphere at room temperature over the course of 6 h. The results are summarized in Table 1. The data indicates that 23 surface-modified DCLHb provides a product that exhibited a single peak by SEC with a shorter retention time than that of DCLHb. The greater the 23/DCLHb molar ratio, the more extensively surface modified the products are as detected by the decrease in the product SEC retention times. The P50 and n (cooperativity) values are in the range 21-23 mm Hg

712 Bioconjugate Chem., Vol. 11, No. 5, 2000 Scheme 6a

a

(a) DCC, N-hydroxysuccinimide; CH3CN; (b) 6, DMF-water.

Scheme 7a

a

(a) DMF-water.

and 1.9-2.1, respectively. The decrease in the P50 of the surface-modified DCLHb product solutions was expected and is due to the alkylation of β-cys93 residues by 23 (27, 28). The maleimide group is known to alkylate both thiol and amino groups although the reagent will preferentially react with a thiol group and, indeed, this was the case. It was found that 30-35% of the added 23 would react with the surface of DCLHb as determined by NMR spectrometry (29). Since the N-acetyl groups of chondroitin-4-sulfate give an isolated and distinct resonance at 2 ppm in a 1H NMR spectrum, this resonance was used to quantitate the number of chondroitin-4-sulfate moieties attached to the protein. The method involved generating a standard curve of integrals of the N-acetyl groups of 6 in a DCLHb solution. The degree of alkylation of DCLHb by 23 was determined by the measurement of the resonance at 2 ppm and relating that value to the standard curve. It has been demonstrated that the reductive amination of a protein or peptide is most effective using cyanoboro-

Hai et al.

hydride or borane-pyridine under slightly alkaline conditions (pH 7-9) (18, 21, 30). Thus, the surface modification of DCLHb with aldehydes 13, 18, and 21 was performed under deoxy conditions in borate buffer, pH of 9, in the presence of borane-pyridine. The latter was selected to avoid the toxicity associated with sodium cyanoborohydride. Since borane-pyridine complex does not readily reduce aldehyde groups at neutral or alkaline pH (24), the reagent can be added along with the aldehyde allowing the Schiff bases that are formed to be reduced during the course of the reaction. The data indicate that 13 and 18 effectively surfacemodified DCLHb. The surface-modified DCLHb showed a single, broad peak by SEC with the retention time in the range 19-20 min as shown in Figure 2. The P50 and n values of the products are the range of 31-33 mm Hg and 2.2-2.6, respectively. These values indicate that the surface modification of DCLHb with 13 or 18 do not greatly perturb the conformation of the protein. The chondroitin-4-sulfate derivative that contains a butyraldehyde end group, 18, is as active as the chondroitin-4sulfate derivative with the propionaldehyde end group (13) toward the surface modification of DCLHb. The aromatic aldehyde containing derivative 21 also modified DCLHb; however, the modified product was rapidly oxidized to metHb during the reaction workup. The mechanism of this rapid oxidation is unknown. The physical properties of DCLHb modified with 13 are presented in Table 3. The two most distinguishing features of the modified DCLHb are the colloidal osmotic pressure (COP) and red blood cell aggregation data. The COP of the 13-DCLHb conjugates were 2-3-fold greater than that of DCLHb. The high COP can be attributed to the highly hydrated chondroitin-4-sulfate moieties attached to the protein surface. Red blood cell aggregation was determined by mixing known concentrations of the modified hemoglobin products with red blood cells. Red cell aggregation (31) has been observed with some polyDCLHb solutions prepared by polymerization with bifunctional polyamide reagents (5). In vitro, RBCs are also known to form red cell aggregates when they are suspended in saline solution in the presence of a wide variety of macromolecules such as serum proteins (32), dextran (33-38), polylysine (3438), poly(ethylene glycol) (34, 35), poly(vinylpyrrolidone) (37), polysaccharides (36), and polyglutamic acid (36). Several mechanisms of red cell aggregate formation have been proposed (33, 35-44), including size-dependent interaction of the macromolecule with the red cell membrane, changes in the surface charge or charge density of the red cell membrane induced by the adherent macromolecule, and the volume excluded effect of the polymer. Since DCLHb-chodroitin-4-sulfate conjugates do not cause red cell agregation even though the products’ COP are high, the volume-excluded mechanism of cell aggregation cannot be invoked. The negatively charged surface of DCLHb-chodronitin-4-sulfate conjugates may prevent red cell aggregation. In conclusion, chondrotin-4-sulfate reagents that could be readily attached to the surface of diaspirin cross-linked hemoglobin (DCLHb) were prepared. Synthetic methodology has been developed that allows for the modification of proteins, such as DCLHb, through alkylation of protein thiols or amino groups by the chondroitin-4-sulfate derivatives. The modification of proteins with chondroitin-4sulfate would add an global negative charge that has the potential to alter the in vivo properties of the proteins and, possibly, increase their therapeutic utility.

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