Bioconjugate Chem. 1998, 9, 645−654
645
ARTICLES Synthesis of Water-Soluble, Nonimmunogenic Polyamide Cross-Linking Agents† Ton That Hai, David E. Pereira,* and Deanna J. Nelson Hemoglobin Therapeutics Division, Baxter Healthcare Corp., 25212 West State Route 120, Round Lake, Illinois 60073. Received November 17, 1997; Revised Manuscript Received August 15, 1998
Novel polyamides were developed that can be used as cross-linking agents for proteins such as hemoglobin. Water-soluble, nonimmunogenic polyamides containing oxygen and sulfur atoms in the backbone were prepared by the polycondensation of the diacids bis(carboxymethyloxyacetyl)-1,4diaminobutane (1a) or 3,3′-thiodipropionic acid (1b) with diethylene glycol bis(3-aminopropyl) ether (2). The resulting R,ω-diacids were converted to the corresponding activated esters using any of a variety of carboxylic acid activating reagents including the novel reagent diphenyl(1-methylimidazol2-thiyl)phosphonate (9). The resulting polyamides could be activated with a broad spectrum of groups that allow for the cross-linking and surface modification of proteins.
INTRODUCTION
Cross-linking reagents that react with the amino or sulfhydryl groups of proteins have a broad range of applications (1-3). While some applications, such as the identification of binding sites (receptors) for ligands, the introduction of fluorescent or spin labels, or intramolecular cross-linking of neighboring functional groups, have few associated spatial requirements, many other applications require that the cross-linking agent have substantial length to prevent steric hindrance to cross-linking. To reduce the resulting heterogeneity of the crosslinked proteins and to maximize the cross-linking efficiency, it is important that the size of the cross-linking agent be defined and restricted to as narrow a range as can be achieved. Concomitantly, it is important the size of the cross-linker be controlled and limited, so that water solubility of the product is maintained. Similarly, the ability to activate the termini of the cross-linker using a spectrum of approaches is an important feature of useful cross-linking agents, since there may be some variability from a general pattern in the effectiveness of modification of a specific protein. One important application of cross-linking agents is to decrease immunogenicity and to increase the lifetime of the cross-linked product in the blood stream. Therefore, it is key that the cross-linker be nontoxic and nonimmunogenic. Finally, most protein modification reactions are completed in aqueous media. Thus, it is key that the crosslinking agent be water soluble. Likewise, to reduce the opportunistic denaturation of the protein during reaction, it is desirable that the cross-linking agent not behave like a detergent or chaotropic agent. † This article is dedicated to Nelson J. Leonard, Faculty Associate in Chemistry, California Institute of Technology. * Author to whom correspondence should be addressed. Phone: (847) 270-5838. Fax: (847) 270-5897. E-mail: pereird@ baxter.com.
The purpose of our current work was to synthesize novel polyamides that could act as cross-linking agents for hemoglobin in particular and proteins in general. The resulting modified hemoglobins or proteins could be candidates as blood substitutes or therapeutic agents, respectively. In addition to meeting the requirements defined above, we anticipated that cross-linking agents having molecular masses in the range 1000-7600 Da would provide the most useful tether length while minimizing undesirable and unnecessary additions to overall molecular mass or viscosity of the resulting crosslinked products. EXPERIMENTAL PROCEDURES
General. NMR spectra were recorded on a Bruker 270 MHz or a Bruker 300 MHz spectrometer. 1H and 13C chemical shifts were measured relative to partially deuterated solvent peaks but are reported relative to tetramethylsilane. 31P chemical shifts are reported relative to H3PO4 (85%). (NMR chemical shifts for the compounds and polymers described herein are included in the Supporting Information to this paper.) Thin-layer chromatography (TLC) of the polyamides was completed using Whatman 250 µm silica gel TLC plates and 2-propanol/NH4OH/water, 10:1:2, by volume, as the eluent. Melting points were determined on a Fisher-Johns melting point apparatus and are uncorrected. Elemental analyses were performed by Oneida Research Services, Whitesboro, NY, or Quantitative Technologies, Inc., Whitehouse, NJ. Nomenclature. For simplicity, the following acronyms are employed as designating nomenclature. The acronym descriptions are presented in Table 1. The term polyamide is abbreviated “PA” (or “PAT” if one component of the polyamide is 3,3′-thiodipropionate). If each end of the polyamide terminates in a succinate residue, the letter “S” is added to the acronym. Finally, if the activating group is N-oxysuccinimide, a second letter “S”
10.1021/bc970199d CCC: $15.00 © 1998 American Chemical Society Published on Web 10/24/1998
646 Bioconjugate Chem., Vol. 9, No. 6, 1998
Hai et al.
Table 1. Polyamide Acronym Definitions and Potential Reactivity With Proteins acronym PA PAT PAS PATS PAD PATD PASS PATSS PADSC PATDSC PADMP PATDMP PADA PATDA
acronym definition
reactivity with proteins
polyamide polyamide containing 3,3′-thiodipropionate residues functional end group is a succinate residue
not activated not activated not activated
functional end group is an amine residue
not activated
polyamide activated as bis(N-oxysuccinimidyl ester)
cross-linking achieved by reaction with amines to form amides
polyamide activated as bis(N-oxysuccinimidyl urethane)
cross-linking achieved by reaction with amines to form ureas
polyamide activated as bis(maleimide)
cross-linking achieved by reaction with amines and/or thiols
polyamide activated as bis(aldehyde)
cross-linking achieved by reaction with amines and reduction of the imine intermediates
is added. The approximate molecular mass of the polyamide follows a hyphen. Thus, the acronym PASS-7600 describes the bis(N-oxysuccinimidyl) activated ester of a polyamide having a molecular mass of about 7600 Da. If the polyamide end groups are amino groups, these are designated by the acronym PAD (or PATD for thioether-containing analogues). The polyamide bis(amines) were converted to reactive urethane, maleimide, or aldehyde end groups. The acronyms for urethane, maleimide, or aldehyde end groups are SC, MP, or A, respectively. Thus, the acronym PATDSC-5700 denotes a urethane-activated, thiodipropionate-containing polyamide having a molecular mass of about 5700 Da. Size-Exclusion Chromatography (SEC). Analytes were eluted from a Superose-12 column (Pharmacia) with 50 mM phosphate buffer, pH 6.5, delivered at a flow rate of 0.4 mL/min, with analyte detection at 220 nm. Mr Determination of PAS Polymers by 1H NMR. An interval of approximately 6.3 s between excitation pulses was used; this interval was sufficient to realize a complete return of the spin system to the equilibrium magnetization state. The average number of repeating units per molecule of polymer was determined by comparing the area of the resonances attributable to protons attached to the repeating unit with the area of the resonances attributable to the protons attached to the succinic acid terminal group. The areas were normalized, as appropriate, for the relative number of equivalent protons generating each resonance. A total of six resonances attributable to the repeating unit was resolved and identified. Hence, it was possible to make six estimates of the number of repeating units per polymer molecule. It should be stressed that the six estimates were not completely independent: all were derived from a single estimate of the area of the resonance of the protons attached to the terminal unit. The n value was obtained from the average of the six estimates of the number of repeating units per polymer molecule. This method was used to determine the average Mr of PAS7600, PAS-5600, PAS-4000, and PAS-2800 (Supporting Information, Table S-2). Mr Determination of PATS Polymers by 13C NMR. Composite-pulse (WALTZ-16) decoupling was employed. Artificial enhancements of resonance areas due to the nuclear Overhauser mechanism were suppressed by the use of an inverse gating technique. An interval of approximately 22 s between excitation pulses was used; this interval was sufficient to realize a complete return of the spin system to the equilibrium magnetization state. The average number of repeating units per molecule of polymer was determined by comparing the area of the resonances attributable to carbons attached to the repeating unit with the area of the resonances attributable
to the carbons attached to the terminal units. The observed resonance areas were normalized, as appropriate, for the relative number of equivalent carbons generating each resonance. A total of three resonances attributable to eight carbon atoms on the terminal units were resolved. Because only one of the three resonances could be identified unambiguously, the total area for these eight carbon atoms was obtained by summation and treated as a single area measurement. A total of seven resonances attributable to the repeating unit were resolved. Three resonances attributable to carbon atoms adjacent to oxygen in the repeating unit of 2 were resolved but not individually identified. In addition, baseline resolution of these three resonances was not achieved. Because of these two factors, the total area for the three resonances was obtained by summation and treated as a single area. Hence, it was possible to make five estimates of the number of repeating units per polymer molecule. It should be stressed that the five estimates were not completely independent: all were derived from a single estimate of the area of the resonance of the carbons attached to the terminal unit. The average of the five estimates of the number of repeating units per polymer molecule gave the degree of polymerization n. This method was used to determined the average Mr of PATSS-5700, PATSS-5200, and PATSS2800 (Supporting Information, Table S-5). Bis(carboxymethyloxyacetyl)-1,4-diaminobutane (1a). Diglycolic anhydride (1524 g, 13 mol) was dissolved in 3 L of N,N-dimethylformamide (DMF). The solution was heated to 60 °C, and the heating mantle was replaced with an ice bath. A solution of 1,4-diaminobutane (529 g, 6 mol) in 1.2 L of DMF was added at such a rate that the temperature was kept between 70 and 75 °C. After all of the 1,4-diaminobutane had been added, an additional 100 g of the anhydride was added to the reaction solution. The mixture was then cooled in an ice bath for 1.5 h, and a solid was formed. The DMF solution was decanted from the solid, and the solution volume was reduced to about 500 mL. The residue was combined with the solid, and 3 L of chloroform (CHCl3) was added to the ice-bath-cooled mixture. The mixture was stirred, and the solid was collected by filtration, washed with 1 L of CHCl3, and dried under vacuum to give 1882 g of 1a (98%). Mp: 168-169 °C. Anal. (C12H20N2O8) C, H, N. N,N′-Bis(methyloxycarbonylmethyloxyacetyl)-1,4diaminobutane (10a). Compound 1a (250 g, 0.8 mol) was combined with methanol (1 L) and 50 mL of 4.0 M HCl in 1,4-dioxane. The mixture was stirred overnight. The solvent was removed by rotary evaporation under high vacuum to give a solid. The solid was dried over NaOH under high vacuum. The solid was dissolved in
Polyamide Cross-Linking Agents
500 mL of warm acetone (dried over anhyd K2CO3) and filtered. Ethyl ether was slowly added to the filtrate, and an oil separated. Stirring with a glass rod caused the oil to solidify. The solid was collected under a flow of nitrogen. The solid was dried under a vacuum over NaOH to give 248 g (88%) of 10a. Mp: 76-77 °C. Anal. (C14H24N2O8‚0.5H2O) C, H, N. N,N′-Bis(carboxymethyloxyacetyl)-1,4-diaminobutane Bis(p-nitrophenyl ester) (10b). p-Nitrophenyl trifluoroacetate (2491 g, 10.6 mol) was combined with a solution of DMF (2 L) and 2,6-lutidine (4 L). Compound 1a (1411 g, 4.4 mol) was added as a solid in one portion to the solution of p-nitrophenyl trifluoroacetate. After 2 h, an additional 1 L of DMF was added to the resulting mixture. After stirring an additional hour, 2 L of ethyl ether was added. The mixture was stirred for 30 min, and the solid was collected. The solid was then combined with 4 L of ethyl ether with mixing. The solid was collected and air-dried. The ethyl ether washing was repeated to give 1997 g (80%) of 10b. Mp: 141-142 °C. Anal. (C24H26N4O12) C, H, N. N,N′-Bis[(2,3-dihydro-2-thioxo-3-benzoxazolyl)carbonylmethyloxyacetyl]-1,4-diaminobutane (10d). Compound 8 (12.7 g, 33 mmol) was added to a stirred suspension of 1a (4.8 g, 15 mmol) and 2,6-lutidine (4 mL, 34 mmol) in DMF (25 mL). The resulting solution was stirred for 2 h. The precipitate was collected and washed with ethyl ether to give 6.30 g of 10d. The filtrate was evaporated to dryness, and the residue was triturated with methanol to precipitate 0.53 g of additional product for a total yield of 6.83 g (78%) of 10d. Mp: 140-141 °C. Anal. (C26H26N4O8S2‚2H2O) C, H, N, S. N,N′-(3,3′-Thiodipropionyl)bis(benzoxazoline-2thione) (14b). Compound 8 (843 g, 2.2 mol) was added as a solid in one portion to a stirred solution of 3,3′thiodipropionic acid (178 g, 1.0 mol) and triethylamine (224 g, 2.2 mol) in 3.4 L of CHCl3. After 1.5 h, methanol (3 L) was added, and the mixture was cooled in an ice bath. After 10 min, the solid was collected and washed with ethyl ether (1 L). The solid was dried under house vacuum to give 356 g (80% yield) of 14b. Mp: 168-170 °C. Anal. (C20H16N2O4S3‚0.25H2O) C, H, N, S. Diphenyl(1-methylimidazol-2-thiyl)phosphonate (9). A solution of diphenyl chlorophosphate (11; 76.6 g, 0.285 mol) in CHCl3 (100 mL) was added in one portion to a solution of 2-mercapto-1-methylimidazole (12; 22.0 g, 0.19 mol) and triethylamine (29.1 g, 0.285 mol) in CHCl3 (200 mL). The solution was heated at reflux overnight. The solvent was evaporated, and the residue was dissolved in toluene (250 mL). The insoluble material was removed by filtration. The filtrate was passed through a pad of silica gel. The product was eluted with a solution of chloroform:acetone (5:1, v/v) (2 × 250 mL). The eluent was evaporated to an oil. Hexane (150 mL) was added to the oil, and a solid was obtained. The solid was collected, washed with hexane (50 mL), and dried to give 48.2 g (68%) of 9. Mp: 113-114 °C. Anal. (C16H15N2O3SP) C, H, N, S. S,S′-Bis(1-methyl-2-imidazolyl)-3-thiapentane-1,5dithiocarboxylate (14c). Compound 9 (8.00 g, 22.0 mmol) was added to a solution of 3,3′-thiodipropionic acid (1.77 g, 10.0 mmol) and triethylamine (3.1 mL, 22.0 mmol) in CHCl3 (50 mL). The solution was heated at reflux overnight. The solution was cooled to ambient temperature (RT) and passed through a pad of silica gel. The silica gel was washed with a solution of chloroform: acetone (5:1, v/v) (100 mL). The filtrate was evaporated to a yellow solid. The solid was dissolved in acetone (10 mL), and ethyl ether (15 mL) was added to precipitate
Bioconjugate Chem., Vol. 9, No. 6, 1998 647 Table 2. Synthesis of Polyamide from 10b and 2a molar ratio of 2 to 10b 1.1:1
1.2:1
1.3:1
1.4:1
polyamide characteristics molecular massb SECc retention time (min) product distributiond PA bis(amine) 5a R-amino-ω-acid PA 5b PA bis(acid) 5c molecular massb SECc retention time (min) product distributiond PA bis(amine) 5a R-Amino-ω-Acid PA 5b PA bis(acid) 5c molecular massb SECc retention time (min) product distributiond PA bis(amine) 5a R-amino-ω-acid PA 5b PA bis(acid) 5c molecular massb retention time (min) product distributiond PA bis(amine) 5a R-amino-ω-acid PA 5b PA bis(acid) 5c
solvent DMF
chloroform
7.6 kDa 34.7
7.6 kDa 34.1
75% 25% none detected 5.6 kDa 35.5
>95% 95% 95% 95%
