Synthesis and characterization of protein and polylysine conjugates of

Maria Astrid Walter , Dietmar Pfeifer , Werner Kraus , Franziska Emmerling , Rudolf J. Schneider ... Nahla A. Abdelshafi , Ulrich Panne , Rudolf J. Sc...
1 downloads 0 Views 1MB Size
Download PDF
124

Bioconjugate Chem. 1991, 2, 124-132

Synthesis and Characterization of Protein and Polylysine Conjugates of Sulfamethoxazole and Sulfanilic Acid for Investigation of Sulfonamide Drug Allergy Jayant G. Tatake, Michele M. Knapp, and Charlotte Ressler; Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030. Received November 21,1990

Conjugates of sulfamethoxazole (SMX) with human serum albumin (HSA), transferrin (TR), and poly(L-lysine) (PL, degrees of polymerization 16 and 430) have been prepared. As a model, succinylSMXglycine methyl ester was synthesized by carbodiimide and active ester routes. The proteins and PL were acylated with succinylSMX succinimido ester, affording conjugates (succinylSMX)z-21-HSA, (succinylSMX)17,~,-TR,(succinylSMX)11-Lysl6, and (succinylSMX)71-Lys~,0in which SMX was linked by a spacer chain of four carbons. This represents substitution of up to 35,46,65, and 17% of the amino groups of HSA, TR, PL16, and PL430, respectively. HSA was also acylated with the succinimido esters of succinylSMX-glycine and succinylSMX-eaminohexanoic acid, affording conjugates (succinylSMXIn these conjugates SMX was linked by a spacer Gly)53-HSA and (succinylSMX-t-NHzhex)51-HSA. chain of 7 and 11 carbons, respectively, and almost all the amino groups of HSA were substituted. Factors apparently influencing the extent of conjugation to HSA were the stability of the active ester and the solubility of the conjugation reaction mixture. A sulfanilic acid (SA) conjugate, containing 12 mol of ligand/mol of HSA, was also prepared. The route of synthesis involved acylation of HSA with sulfanilyl fluoride. N-e-Sulfanilyl-L-lysinedihydrochloride, required for quantitation of bound SA, was synthesized by a new route starting from a-Boc-L-lysine. Conjugates (sulfanilyl)lz-HSA and (succinylSMX)13-HSA, differing in molecular weight from HSA by only 2.6 and 6.596, were distinguishable from HSA by gel-filtration HPLC, as were the more highly substituted conjugates from their respective unsubstituted materials. Conjugates (succinylSMX),-HSA (n = 2,3,6,9) were readily distinguishable from each other and from HSA on cellulose acetate electrophoresis. Mobilities were clearly dependent on epitope density.

INTRODUCTION Sulfonamides continue to have an important place as antibacterial, antiprotozoal, and antirheumatic drugs. Trimethoprim-sulfamethoxazole (Bactrim, Septra, Co-trimoxazole) in particular is valuable for the treatment of shigellosis, bronchitis, urinary tract infections, and otitis media and for the treatment and prophylaxis of Pneumocystis carinii pneumonia in AIDS (I). However, its use is limited by the development of adverse drug reactions generally attributed to the sulfonamide. Evidence suggesting an immunological basis for such drug reactions has been limited. In the absence of other information, the synthesis was undertaken of determinants that might be suitable for detecting serum antibodies to sulfamethoxazole with the idea that elevated levels of Ab might be diagnostic of sulfa drug allergy. In general analogy to the design of penicilloylpolylysine, the major determinant that can detect up to 75% of individuals sensitive to penicillin ( 2 , 3 ) ,it was desired to prepare conjugates of SMX' linked covalently to proteins and polylysine. To provide an additional determinant and to derive salient information on the structuralfeatures of the SMX molecule required for immunological recognition, sulfanilic acid representing half the molecule of SMX, was also to be linked covalently. To our knowledge, well-characterized protein and polypeptide conjugates with sulfonamide drugs have not been previously reported, although largely undefined sulfonamide-azo serum proteins, sulfonamide-azoalbumin and -azocasein have long been known (4, 5). Likewise, conjugates with sulfanilyl groups in which the p-amino group is free have not been

* To whom reprint requests should be addressed.

described, although N4-acetylsulfanilyl residues have been substituted on gelatin and polylysine (6, 7), as have (N4acetysulfanily1)glycyl residues on polylysine (7). Albumin and transferrin, the two chief components of human serum, were selected for conjugation as possible carrier proteins in vivo. They are similar in size (69 000 and 80 000 Da) and in lysine content (60 and 59 residues per mol, respectively) (8,9), but they differ otherwise in structure and function and could afford information on accessibility to conjugation and on the immunological recognition of their conjugates. The less immunogenic polylysines of 3000 and 90000 Da were selected for conjugation also for projected studies in vivo. The unprotected sulfanilyl residue was covalently coupled to HSA through linkage of the sulfonyl group without difficulty. However, before SMX could be linked to HSA, it was necessary to derivatize its weakly reactive aromatic amino group. This afforded a conjugate of SMX separated from the protein by a spacer chain of four carbons. Conjugates of SMX and HSA separated by spacer chains of 9 and 11 carbons were also synthesized to gain information on the optimum length of the spacer chain Abbreviations used: DMF, dimethylformamide;THF, tetrahydrofuran; DCC, dicyclohexylcarbodiimide; EDC, l-ethyl-3[3-(diethylamino)propyl]carbodiimidehydrochloride; SMX, sulfamethoxazole; SA, sulfanilic acid; SA-Lys,N-t-sulfanilylysine; NHzHex, t-aminohexanoic acid; HONSu, N-hydroxysuccinimide; sucSMX, N4-(3-carboxypropanoyl)sulfamethoxazole;HSA, human serum albumin; OVA, ovalbumin;TR, human transferrin; PL, polylysine; (sucSMX),-HSA, human serum albumin conjugated with n N4-(3-carboxypropanoyl)sulfamethoxazole groups; AAA, amino acid analysis; Ab, antibodies; ELISA, enzyme-linked immunosorbent assay.

l043-l002/91/29Q2-OI24$O2.5O/Q 0 1991 American Chemical Society

Bioconjugate Chem., Vol. 2, No. 2, 1991

Sulfonamide Drug-Protein Conjugates

125

Table I. Synthesis of (SucSMX),,-HSA (9f): Conjugation of Drug to HSA under Various Conditions of Temperature, Reaction Time and Mode of Addition and Molar Ratio of Reactants expt 1

2 3 4 5 6 7

HSA, mg 100 50 50 50 50 49 50

7, mg 77 50 54 79 157 253 160

7/HSA NH2 molar ratio 2

2.6 2.8

4 8

13.3 8.1

condition pHb otherC 6.8 A 9.5 B 9.5 A 6.8 A 6.8 A, C 6.8 A, c e A, C

purifcn methodd D E F F G E E

conjugate' mg SMX/HSA molar ratio 93 6.4 16 2 20 2.9 25 9 32 13 28 13 39 21

Dialyzed products established by HPLC gel filtration to be free of low molecular weight contaminants and analyzed for bound SMX as described under "General Chemical Procedures". pH 6.8 adjusted by addition of 1equiv of NaHCO,; pH 9.5, by 2 mL of 0.2 M NaHCO3Na2C03 buffer. A, solution of 7 in 0.5 mL of DMF added and reaction allowed to proceed for 4 h at room temperature; B, 7 added as a solid over 2 h and reaction allowed to proceed for 16 h at 4 "C; C, solid separated out of the reaction mixture. Low molecular weight reagents/ byproducts were removed by the following methods: D, concentration dialysis; E, dialysis; F, gel filtration on Sephadex G-25 with water; G, gel filtration on Sephadex G-15 with 0.05 M sodium phosphate followed by dialysis as described under "General Chemical Procedures". e Sodium bicarbonate limited to 4.7 mg.

for immunological recognition. In both the sulfanilyland spacer-linked SMX conjugates, the linkage of the ligand to the protein, as to polylysine, was expected to involve the t-amino group of lysyl residues. As a model reaction for the introduction of SMX into proteins and polylysines, the new sucSMX-glycine methyl ester was synthesized. Conjugation of SA and SMX to HSA, transferrin, and polylysine was judged qualitatively by enhanced UV absorption a t 254 nm relative to the 280-nm protein peak on gel-filtration HPLC. This technique was particularly informative since it also was capable of distinguishing by retention volume conjugates from parent proteins or PL differing with only small changes in molecular weight. Quantitative determination of the degree of conjugation to HSA or transferrin was accomplished by reverse-phase HPLC analysis of hydrolysates for SMX or N-t-sulfanilyllysine. Conjugation to PL was determined quantitatively by the ratio SMX: Lys as given by HPLC and amino acid analysis, respectively, of hydrolysates. When tested as a capture antigen in a new ELISA (IO), the conjugate (sucSMX)13-HSA was found capable of detecting serum IgM antibodies to sulfamethoxazole in an individual with an adverse cutaneous reaction to Bactrim (11). The ELISA activity was proportional to the degree of substitution for at least 13 residues of the ligand. By contrast, conjugate (SA)lz-HSA was inactive, suggesting the need in antibody recognition for a spacer in the conjugate or for additional structural features residing in the SMX molecule.2 The development and validation of the ELISA and an evaluation of its clinical applicability to detect hypersensitivity and cross-reactivity (12)to SMX, as well as comparison of the various conjugates, are to be presented in detail separately. EXPERIMENTAL PROCEDURES

Materials. Sulfamethoxazole, succinicanhydride, poly(L-lysine)hydrobromide (n= 430), human transferrin, and crystalline human albumin were obtained from Sigma Chemical Co., St. Louis, MO. The latter was confirmed to be free of 7-globulins and macroglobulins by HPLC on Waters Protein-Pak 1-125column. Poly(L-lysine) hydrobromide (n = 16) was from Miles-Yeda LTD, Miles Scientific, Naperville, IL. N-Hydroxysuccinimide, sulfanilyl fluoride, and acetylsulfanilyl chloride were from Aldrich Chemical Co., Milwaukee, WI; pyridine, dicyclohexylcarbodiimide, ethyl chloroformate, 40 % dimethylThese findings were presented a t the 43rd Annual Meeting of the American Academy of Allergy and Immunology, February 19-25, 1987, Washington, DC. (11).

amine, and sulfanilamide were from Matheson, Coleman, Bell, Norwood, OH; 3-amino-5-methylisoxazole was from Pfaltz and Bauer, Flushing, NY; methyl glycinate hydrochloride and t-aminocaproic acid were from Mann Research Labs, New York, NY; a-Boc-L-lysinefrom Bachem, Marina Del Ray, CA. N,N'-Dimethylformamide was obtained from Eastman Kodak Co., Rochester, NY, and was freed of dimethylamine as described (13). General Chemical Procedures. Melting points were determined in open glass capillary tubes with a ThomasHoover apparatus and are uncorrected. Evaporations were carried out under reduced pressure. Elemental analyses were performed by Micro-tech Laboratories, Oakton, IL, or Galbraith Laboratories, Knoxville, TN. Proton magnetic resonance spectra were recorded on a Varian EM360 spectrometer a t concentrations near 0.1 M. Chemical shifts are given as 6 values in ppm downfield from sodium 3-(trimethylsilyl)propionate-2,2,3,3-d~ as internal reference in aqueous solution and from tetramethylsilane in nonaqueous solvents. Multiplicities of resonances are described as broad (br), singlet (s), doublet (d),triplet (t), quartet (q), or multiplet (m). HPLC was carried out in the apparatus described elsewhere (14). A CISpBondapak column (Waters) was used in the reverse phase with system A (30% MeOH-l% AcOH), system B (5% MeOH-1% AcOH), and system C (25 % MeOH-1 % AcOH) a t flow rates of 1-2 mL/min. A Protein-Pak 1-125gel-filtration column (Waters) was used with 0.05 M potassium phosphate buffer, pH 6.8. Protein absorption was determined a t 280 nm, sulfonamide absorption a t 254 nm. Analytical TLC was carried out on Eastman Chromagram, polymer-backed, fluorescent indicator cellulose 13254 strips with system A (CHCl3-MeOH, 5:1), B (benzene-EtOAc, 5:1), C (benzene-EtOAc, 1O:l) or D (benzene-EtOAc, 5:2) or with silica gel 13181 strips and system E (CHCl3-MeOH, 1O:l). The Bratton-Marshall test (15) was carried out with a sensitivity in solution of 0.5 nmol and with a sensitivity on paper of 0.05 pmol. Gel filtration was carried out on an equilibrated Sephadex G-15 column (2.5 X 63 cm, Pharmacia, Piscataway, NJ) in 0.05 M sodium phosphate buffer, pH 8. The (sucSMX)ls-HSA conjugate, equiv to 25 mg of starting HSA (Table I, expt 5), was applied in 3 mL of 0.2 M phosphate buffer, pH 8. Filtration was at a flow rate of 2.5 mL/min and was monitored by absorbance a t 280 nm. Peak materials were identified by HPLC on Protein-Pak 1-125, which showed protein at 120-138 mL, well-separated from low molecular weight material a t 212-250 mL. Proteincontaining fractions were pooled and dialyzed against

126

Tatake et al.

Bioconju@?teChem., Vol. 2, No. 2, 1991

+

I

Figure 1. Effect of epitope density on the electrophoretic mobility of (sucSMX),-HSA conjugates. Composite electropherogram of HSA and conjugates of n = 2,3,6,and 9. Materials: 1Opg was applied to the center of 1X 6 in. cellulose acetate strips and electrophoresedat pH 5.6 at 27 V/cm for 20 min, and then stained with Coomassie Blue. Mobilities were 10,13,18,26, and 35 mm, respectively.

distilled water, then lyophilized to give the product as a white fluffy powder. Gel filtration on Sephadex G-25 (Pharmacia, Table I, expts 3 and 4) was carried out with water in a similar way. Dialysis was carried out with membrane tubing (retention cutoff 12 000 Da, 1 in. flat, from A.H. Thomas, Philadelphia, PA) or Spectrapor tubing (cutoff 1000 Da, from Fisher Scientific, New York, NY) that was treated before use (16). Synthetic conjugates were dialyzed a t 4 "C against three changes of 1.5 L of 0.05 M sodium phosphate buffer, pH 8, until the product was free of low molecular weight material as determined by HPLC on Protein-Pak 1-125. Dialysis was then continued against 2 X 1.5 L of distilled water until the dialysate was free of phosphate ion (17). Concentration dialysis was carried out in 8-mL collodion concentration bags (retention cutoff 25 000 Da, Schleicher & Scheull, Keene, NH), and was monitored by HPLC in a similar way. Electrophoresis. (SucSMX),-HSA conjugates were electrophoresed on Sepraphore I11 cellulose acetate strips (Gilman Instrument Co., Ann Arbor, MI) in 0.06 M sodium acetate buffer, pH 5.6, at 27 V/cm for 20 min a t room temperature. Paper electrophoresis was carried out on Whatman ill strips in 2% sodium carbonate solution, pH 8.9, or pyridine-AcOH-HzO (4.9:1.2:94), pH 5.6, at 13 V/cm for 1.5 h at room temperature. Amino acid analysis of 9a and 9b for glycine was carried out semiquantitatively by hydrolysis in 6 N HCl at 110 "C for 18 h followed by paper electrophoresis and detection with 0.2 % ninhydrin in acetone. Sulfamethoxazole-polylysine conjugates were hydrolyzed in 6 N HCl2 3'% phenol for 1 h at 150 "C and analyzed quantitatively on a Beckman 7300 amino acid analyzer. Analysis of Conjugates for Covalently Bound Sulfonamide. Before hydrolysis, final conjugate products were established to be free of low molecular weight derivatives of SMX/sulfanilic acid by HPLC on ProteinPak 1-125. Typical elution volumes (in mL) were as follows: protein conjugate, 6.2; HSA, 6.4; 6, 9.7; SA, 11. With (sucSMX),-HSA conjugates, the absence of HSA was confirmed by electrophoresis on cellulose acetate. Typical mobilities were as follows: HSA, 10 mm; (sucSMX)e-HSA, 26 mm (Figure 1). (a)Hydrolysis of Conjugates of Sulfamethoxazole and HSA (9f, l l b , c), TR (9g), a n d Polylysines (9d,e). The conjugate (1.3 mg) in 0.5 mL of N NaOH in a closed 5-mL borosilicate test tube was heated in a bath held at 65-70 "C for 16-18 h. The solution was cooled and adjusted to pH 7 with 1 N HC1 and analyzed for SMX by absorbance at 254 nm on HPLC on the CIScolumn in system

A in which SMX elutes at 6 mL. Treated under these conditions SMX and 6 yielded SMX with a recovery of 100 f 7 % . (b) Hydrolysis of Sulfanilyl-HSA (2). Conjugate 2 (1.2 mg) in 1 mL of 6 N HCl was heated in a bath at 120 "C for 18h. The solution was then taken to dryness, taken up in water, and analyzed for 3 by HPLC on the CIScolumn. The major peak, detected a t 254 nm, eluted in the same position as a reference sample of SA-Lys.2HC1,and it gave a positive Bratton-Marshall reaction. On paper electrophoresis the hydrolysate showed ninhydrin- and BrattonMarshall-positive material with the same mobility as authentic 3 a t pH 9 and 5.6. When treated with acid under similar conditions, 3 was recovered as 80 ?6 3 and 20 96 SA. ( c ) Calculation of Degree of Substitution. For conjugates containing protein and SMX or SA, n was calculated from the HPLC analysis for SMX or 3 + SA, respectively, in a hydrolysate of the conjugate as follows: n = M / [ ( l / A )- R ]

where n = moles of substituent per mole of conjugate; A = micromoles of SMX or 3 + SA per milligram of conjugate as determined experimentally; M = MW of the unsubstituted protein, e.g., 69 for HSA; R = 10-3 X MW of the substituent, e.g., 0.335 for sucSMX. Such values of n are uncorrected for moisture in the conjugate and may be minimal. For the conjugates of PL and SMX (sa),the fraction, f , of lysine amino groups substituted was given by the ratio SMX:Lys,the former determined by HPLC of a basic hydrolysate, the latter by AAA of an acid hydrolysate:

n = f ( m + 1) where m is the number of residues of Lys in PL. Chemical Syntheses. N-c-Sulfanilyl-L-lysine Dihydrochloride (3). To a solution of 4 (367 mg, 1.5 mmol) in 1.2 mL of water was added 0.4 mL of 30% NaOH (3 mmol) and 1 mL of acetone. Compound 5 (353 mg, 1.5 mmol) was added in portions with magnetic stirring over 2 h. Stirring was continued for an additional 2 h. The mixture was clarified by centrifugation, and acetone was evaporated. The mixture was then acidified with 2 N HC1. The sticky gum was extracted with several portions of EtOAc. The extract was washed with H2O and taken to dryness. To the residue was added 3 mL of 6 N HC1, and the mixture was heated in a bath a t 120 "C for 1h. The hydrolysate was then taken to dryness over Pz05 and KOH: 248 mg (49%);mp 210-215 "C dec. HPLC analysis of the hydrolysate on the CIScolumn showed a single peak [elution volume 6.7 mL (solvent system B)]. Paper electrophoresis at pH 5.6 for 1.5 h a t 13 V/cm showed a single spot (ninhydrin positive, Bratton-Marshall positive) located 1 cm toward the cathode and well-separated from reference sulfanilic acid and lysine. For analysis, the product was dissolved in a minimal volume of MeOH. The solution was diluted with an equal volume of i-PrOH and was allowed to evaporate in an open beaker to half its volume: 190 mg; mp 210-215 "C dec. Recrystallization furnished 111mg of product of unchanged melting point [lit. mp 205 "C (S)]. Anal. Calcd for C12H19N30&2HCl: C, 40.6; H, 5.82; S, 9.10; N, 14.1. Found: C, 40.5; H, 5.83; S, 9.10; N, 14.1. N-t-(Sulfanilyl)l2-HSA (2). A solution of HSA (102 mg, 1.5 pmol) in 2 mL of HzO in a two-necked conical 30-mL flask was adjusted to pH 9 with 50 pL of 1N NaOH. To this a solution of 1 (91 mg, 0.52 mmol) in 1mL of Et20 was added with magnetic stirring over a period of 10 min. The mixture was maintained in a bath at 35 "C for 4 h

Sulfonamide Drug-Protein Conjugates

during which pH 9 was maintained by addition of 460 pL of NaOH in portions. The gelatinous mixture was transferred to a test tube and extracted three times with Et20 during which the aqueous mixture gradually clarified. Residual ether in the aqueous solution was evaporated off. The solution was subjected to concentration dialysis, analyzed on the Protein-Pak 1-125 column, and lyophilized (72 mg). N4-(3-Carboxypropanoyl)sulfamethoxazole(6). A solution of 12 (10.1 g, 40 mmol) in 140 mL of AcOH was prepared by heating to 60 "C with mechanical stirring. To it was added succinic anhydride (4.4 g, 44 mmol). The clear reaction mixture was stirred and maintained at 5560 "C. The product separated out after 14 min. The thick mixture was then diluted with 30 mL of AcOH. After a total period of 40 min, the reaction mixture was cooled to room temperature, and the product was collected on a filter and washed with AcOH followed by warm water and then dried over KOH and P205: 13.1 g (92.8%);mp 211215 "C dec; HPLC, elution volume 8.8mL (solvent system A). A second peak indicated 2 % starting SMX that was further confirmed by a quantitative Bratton-Marshall reaction. The product was considered suitable for further work. For analysis a sample was recrystallized from MeOH: mp 214-218 "C; TLC, Rf0.93 (solvent system A); HPLC, elution volume 11.8 mL (solvent system C); 'H NMR (NaOD) 6 (ppm) 2.18 (s, 3, CH3), 2.62 (m, 4, CH3, 4.8 (HOD), 5.92 (s, 1, vinyl), 7.94 (m, 4, aromatic). Anal. Calcd for C14H15N306S: C, 47.6; H, 4.28; N, 11.9. Found: C, 47.5; H, 4.33; N, 11.8. N4-[3 4(Succinimidooxy)carbonyl]propanoyl]sulfamethoxazole(7). A mixture of 6 (354 mg, 1 mmol) and N-hydroxysuccinimide (117 mg, 1 mmol) in 7 mL of dry, peroxide-free THF was warmed slightly to effect solution. To the solution at room temperature was added DCC (209 mg, 1 mmol) with magnetic stirring. Stirring was continued for 3 h. The precipitate was filtered off and washed with 6 mL of THF. Combined filtrate and washings were evaporated to a glassy solid that was slurried with 10 mL of EtOAc and then collected by filtration: 436 mg (96%); mp 173-174 "C. Crystallization from THF-i-PrOH gave a white solid (mp 183-186 "C) that traveled as a single spot on TLC [Rf0.5 (solvent system B)] and HPLC [12.7 mL (solvent system A)]; lH NMR ( D M s 0 - d ~6) 2.4 (s, 3, CH3), 2.95 (m, 8, CH2), 6.3 (s, 1, vinyl), 8.0 (s, 4, aromatic). Anal. Calcd for C18HlsN408S: C, 48.0; H, 4.03; S, 7.12. Found: C, 48.2; H, 4.21; S, 7.18. 3-(4-Succinimidobenzenesulfonamido)-5-methylisoxazole (8). To a solution of 6 (353 mg, 1 mmol) in 1 mL of DMF was added with magnetic stirring DCC (103 mg, 0.5 mmol). Stirring was continued for 1 h. The precipitate was then filtered off and washed twice with 0.5mL of DMF. The combined filtrate and washings were diluted with 5 mL of dry Et20 and the mixture was allowed to stand in the cold overnight. The white solid was collected on a filter, washed well with Et20, and dried: 113 mg (66%);mp 238-242 "C dec. A run with 2 mmol of 6 and 2 mmol of DCC in 2.5 mL of DMF furnished 8 in 44% yield. The product was crystallized from CH3CN-Et20: mp 240-242 "C; it was homogeneous on TLC [Rf0.74 (solvent system C)] and HPLC [elution volume 7.0 mL (solvent system A)]; 'H NMR ( D M s 0 - d ~ 6) 2.45 (5, 3, CH3), 2.95 (s, 4, CHd, 6.51 (5, H, vinyl), 8.17 (s, 4, aromatic). Anal. Calcd forC14H13N305S: C, 50.1;H, 3.91; S, 9.56. Found: C, 50.3; H, 3.98; S, 9.74. N4-[3-[(Methyl glycinate-N-yl )carbonyl]propanoyl]sulfamethoxazole (9a). ( a ) Carbodiimide Procedure. To a solution of methyl glycinate hydrochloride (723 mg,

Bloconjugate Chem., Vol. 2, No. 2, 1991

127

5.8 mmol), NEt3 (583 mg, 5.8 mmol), and 6 (1.77 g, 5 mmol) in 4 mL of DMF at 10 "C was added, with magnetic stirring, DCC (1.19 g, 5.8 mmol) in portions over 10 min. The mixture was allowed to warm to room temperature and maintained for 3 h. After addition of 0.4 mL of AcOH the insoluble byproduct was filtered off and washed with 1 mL of DMF. Dilution of the filtrates with an equal volume of water led to precipitation of the product. This was collected by filtration, washed successively with dilute HCl, water, sodium bicarbonate solution, and water, and dried: 1.78 g (84%); mp 200-202 "C dec. On HPLC the product eluted as a single substance [elution volume 10.6 mL (solvent system A)]. The product was recrystallized from CH3CN: mp 206-208 "C dec; TLC, Rf0.2 (solvent system E); 'H NMR ( D M s 0 - d ~ 6) 2.41 (s, 3, CHd, 2.68 (m, 4, CHd, 3.78 (s, 3, OCH3),4.03 (t, 2, glycyl CH2),6.42 (s, 1, vinyl), 8.17 (4, aromatic), 8.77 (m, 1, glycyl amide), 10.89 (m, 1, amide), 11.88 (m, 1, amide). Anal. Calcd for C17H20N407S: C, 48.1; H, 4.76; N, 13.2. Found: C, 48.1; H, 4.76; N, 13.2. ( b ) Active Ester Procedure. To a solution of methyl glycinate hydrochloride (76 mg, 0.6 mmol) and NaHC03 (51.8 mg, 0.6 mmol) in 0.4 mL of water was added, in portions, with magnetic stirring a solution of 7 (180.3 mg, 0.4 mmol) in 2 mL of DMF. The almost clear reaction mixture remained at pH 6.8-7. Additional NaHC03 (33.7 mg) was added, and stirring was continued for 3 h. TLC revealed no starting or cyclized 7. The mixture was clarified by centrifugation, cooled, acidified with 5 N HC1 to pH 1.5, and diluted (1.5X) with water. The solid that separated on cooling was collected by filtration, washed with 1 N HC1, then with water until neutral, and dried: 116 mg (68%); mp 200-202 "C dec. Recrystallization raised the melting point to 208 "C. On HPLC, the product had the same retention volume as that prepared under procedure a. N 4-[3-(Glycinocarbonyl)propanoyl]sulfamethoxazole (9b). To a solution of glycine (43 mg, 0.58 mmol) and NaHC03 (82 mg, 0.98 mmol) in 0.5 mL of water at room temperature was added a solution of 7 (201 mg, 0.45 mmol) in 0.6 mL of DMF with magnetic stirring over 10 min. Stirring was continued for 3 h, after which the clear reaction mixture was diluted with an equal volume of water, cooled, and adjusted with 5 N HC1 to pH 3. The solid product that gradually separated was collected on a filter, washed with dilute HC1, then with water until chloridefree, and then dried over P205 and KOH: 151 mg (82%); mp 223-225 "C dec. TLC showed along with the major component two minor components corresponding to 7 and 8. Results of paper electrophoresis at pH 8.9 were in agreement. Hydrolysis followed by electrophoresisat pH 8.9 confirmed the presence of glycine (ninhydrin positive) and sulfanilic acid (Bratton-Marshall positive), when mobilities were compared with those of authentic samples. For analysis a solution of the product in boiling MeOH (16 mg/mL) was reduced in volume and cooled to room temperature. The colorless needles (mp 223-225 "C dec) were homogeneous on TLC [Rf0.71 (solvent system A)] and HPLC [elution volume 9.6 mL (solvent system C)]. Anal. Calcd for C16H18N407S: C, 46.8; H, 4.42; S,7.81. Found: C, 46.8; H, 4.35; S, 7.95. N4-[3 4 (Succinimidoglycinate-N-yl)carbonyl]propanoyl]sulfamethoxazole (lob). The reaction of 9b (152 mg, 0.37 mmol) with HONSu (44 mg, 0.38 mmol) and DCC (80.6 mg, 0.39 mmol) was carried out as described for 7, except that the solvent was 1.3 mL of DMF. The precipitate was washed twice with 0.3 mL of DMF, the combined filtrate and washings were diluted with 25 mL

128

Tatake et al.

Bioconjugate Chem., Vol. 2, No. 2, 1991

Table 11. Synthesis of Conjugates of Sulfamethoxazole and HSA, Transferrin, and Polylysines Linked via a Succinyl, Succinylglycyl, or Succinyl-e-aminohexanoyl Spacer, and Conjugates of Sulfanilic Acid Linked Directly to HSA or Lysine.

conjugate % NH2 of N nucleophile (N) mg reagent (R) mg mg substituted by Rc 3 CPBOC-L-LYS 367 5 353 1 248 2 HSA 102 1 91 6 72 20 9f n = 2-21d HSA 50-100 7 -d 2-13 -d -d 3-35 llb HSA 49 10b 178 8.2 C 65 88 llc HSA 50 1oc 199 8.1 C 74 86 9g n = 2 7 * 3 TR 50 7 139 8.4 C 47 46 n=17*l TR 100 7 278 8.2 C 102 29 9d D, E 51 65e LYs16 50 7 119 1 9e D,E 56 17 LYS430 47 7 109 1.1 Reactions with activated esters were at pH 6.8 except where specified for 9f in Table I. Reaction with 1 was at pH 9. A, recrystallization; B, concentration dialysis; C, dialysis; D, insoluble product centrifuged off and washed; E, succinylation followed by dialysis and lyophilization. Value uncorrected for moisture in the Drotein coniuaate. Data of Table I. e Determined by the ratio SMX:Lys in hydrolysates; in protein conjugates determined by SMX or 3 + S A in hydrolysates.

conjugate

of dry EtzO, and the mixture was kept a t -20 "C overnight under anhydrous conditions. The oily material was separated by decantation, washed well with EtzO, and then was crystallized from THF-Et20: 138 mg (74%);mp 179181 "C dec. The product was recrystallized from THFi-PrOH without change in melting point. It showed a single spot on TLC [Rf0.14 (solvent system B)] and HPLC [elution volume 9.6 mL (solvent system A)]; 'H NMR (DMSOd ~ 6 )2.4 (s, 3, CH3), 2.7 (s, br, 4, succinoyl CHz), 2.95 (s, br, 4, NSu CHZ),4.47 (d, 2H, glycyl CHz), 6.4 (s, 1,vinyl), 8.1 (s, 4, aromatic). The spectrum was unchanged after 8 days a t room temperature. Anal. Calcd for CzoH21N&S: C, 47.3; H, 4.17; S, 6.32. Found: C, 47.5; H, 4.18; S, 6.43. N4-[3-[[ (5-Carboxypentyl)amino]carbonyl]propanoyl]sulfamethoxazole (9c). A solution of +aminohexanoic acid (2.2 g, 16.8 mmol) and NaHC03 (2.35 g, 28 mmol) in 16 mL of water was treated with a solution of 7 (4.95 g, 11mmol) in 13 mL of DMF as described for the preparation of 9b: 3.65 g (70%); mp 190 "C dec. It was 87% homogeneous on HPLC [elution volume 17.1 mL (solvent system A)] and contained 1 2 % of 8. A second crop of 300 mg (mp 195-197 "C) was also obtained. Impurity 8 was still present after two recrystallizations from 30-40 parts of MeOH. The recrystallized material was used for conversion to ester 1Oc. For analysis a solution of 146 mg of the product in 1 mL of DMF was applied to a small column prepared from 4 g of silica (Fisher, grade 12,28-200 mesh). Elution was performed with EtOAc in 5-mL fractions. The product was located by TLC: 40 mg; mp 193-197 "C. It was recrystallized from DMF-Et20 without change in melting point and was homogeneous on HPLC and TLC [Rf0.09 (solvent system D)]. Anal. Calcd for C ~ O H Z ~ N ~C,O 51.5; ~ S : H, 5.62; S, 6.87. Found: C, 51.3; H, 5.73; S, 6.76. N4-[3-[ [[5-[(Succinimidooxy)carbonyl]pentyl]amino]carbonyl]propanoyl]sulfamethoxazole ( 1Oc). The reaction of 9c (508 mg, 1.1 mmol) with HONSu (127 mg, 1.1mmol) and DCC (232 mg, 1.1mmol) in 3 mL of DMF was carried out as described for the preparation of lob. The precipitate was washed twice with 0.5 mL of DMF, then twice with 10 mL of EtZO. An oil that settled after being washed four times with 5 mL of Et20 furnished 170 mg (28%) of a solid, mp 155-210 "C. The product [TLC, R f 0.24 (solvent system D)] was contaminated with 8 and was not improved by recrystallization from THF-i-PrOH. A second crop obtained from the filtrate and washings [341 mg (55%), mp 155-160 "C] on recrystallization furnished 283 mg, mp 173-178 "C. HPLC showed 1Oc [elution volume, 26.9 mL (solvent system A)], 9c,and 8

R/N NH2 molar ratio

purifcn methodb A B

in the ratio 81:9:10. Anal. Calcd for C ~ ~ H S N ~ OC, ~S: 51.2; H, 5.19; S, 5.69. Found: C, 50.7; H, 5.05; S, 6.60.

Conjugates of Sulfamethoxazole and H u m a n Ser u m Albumin,Transferrin,and Polylysines. N-e [N4(3-Carbonylpropanoyl)sulfamethoxazole],-HSA(9f).To a solution of HSA (100 mg) and NaHC03 (23 mg) in 1mL of water was added with magnetic stirring over 10 min a solution of 7 (77 mg) in 0.45 mL of DMF (Table I, expt 1). The somewhat turbid solution was stirred for 4 h and remained near pH 7. Highly substituted or other material that had precipitated out of the reaction mixture (Table I, expts 5 and 6) dissolved upon addition of 0.2 M sodium phosphate buffer, pH 8. Low molecular weight reagents and byproducts were removed by dialysis, concentration dialysis, or gel filtration as specified in Table I in the manner described under "General Chemical Procedures". The product was obtained on lyophilization. N-e-[N4-[3-[ [ (Carbonylmethyl)amino]carbonyl]propanoy llsulfamet hoxazole] 53-HSA (11b) and N-E- [N4-[3[ [ (5-Carbonylpentyl)amino]carbonyl]propanoyl]su~famethoxazole1~1-HSA(1IC). A solution of 50 mg of HSA and 38 mg of NaHC03 in 0.5 mL of water was treated with 10b or 1Oc in 0.55 mL of DMF essentially as described for 9f. Within 30 min the reaction mixture became very thick. After 3 h the suspension was solubilized by addition of 0.2 M sodium phosphate buffer, pH 8, and clarified by centrifugation. It was then dialyzed and lyophilized (Table 11). N - c[N4-(3-Carbony1propa~yl)sulfamethoxazole],-polylysines (9e and 9 4 . A solution of PLaHBr (n = 430, MW 90 000,4623mg, 0.22 mmol of NH2) in 0.6 mL of water was adjusted to pH 8 with 10 pL of 1 N NaOH. Sodium bicarbonate (24.5 mg, 0.29 mmol) was then added with magnetic stirring, followed by a solution of 7 (109 mg, 0.24 mmol) in 0.5 mL of DMF over 15 min. A few minutes later a white solid precipitated out. After a total period of 1.5 h, the solid was collected by centrifugation, washed successively (3X) with 2 mL of 0.1 N NaHC03, water, MeOH, 2 X 2 mL of DMF, and MeOH, and then was dried. The solid was suspended in 1.5 mL of water, and succinic anhydride (56.2 mg, 0.56 mmol) was added in 3 portions with stirring along with 1N NaOH to maintain pH 8-8.5. The solid dissolved as the reaction progressed. Low molecular weight reagents and byproducts were removed from the clarified reaction mixture by dialysis, and product 9e was obtained on lyophilization. Essentially the same procedure was used to obtain 9d from PL-HBr (n = 16, MW 3300,49.5 mg, 0.26 mmol of NH2). The insoluble product was collected and washed with sodium bicarbonate solution and water and dried,

Sulfonamide Drug-Protein Conjugates

Bioconjugate Chem., Vol. 2, No. 2, 1991

129

Scheme I

1

2

HCI

1

kOOC(CHl)I 4

5

3

Scheme I1 NHCOCH,CH,COOH

b -0 NH COC H,C HICOO

HONSu

0

f 3 H 1

DCC. THF

SOzNH

6

lNH2 OQO

BNH&cH3

7

S

0

6

SO,NH 10 b m = l c m=5

9 a

HSA

n

RNH,

I

NH,CH,COOCH, NH,CH,COOH

b

I

C

I

d

I1

e

71 2-21 25f6

f g

HSA

Transferrin

I tiO;rCH,

S O I N H ~ II b C

m

n

I 5

53 51

then with DMF and MeOH and dried, then succinylated, dialyzed, and lyophilized (Table IT). RESULTS AND DISCUSSION In the present study the use of sulfanilyl fluoride (1) as reagent in place of N4-acetylsulfanilyl chloride (5) allowed sulfanilyl groups to be introduced directly into HSA, making subsequent deprotection of the conjugate unnecessary. The degree of substitution in the sulfanilyl-HSA conjugate 2 was determined by hydrolysis in acid followed by analysis by reverse-phase HPLC for N-c-sulfanilyllysine (3) and sulfanilic acid. Quantitation of 3 was based upon comparison with an authentic sample of its crystalline dihydrochloride prepared by acylation of a-BOC-L-lysine (4) with N4-acetylsulfanilylchloride (5), followed by deprotection in acid. N-c-Sulfanilyl-L- and DL-lysines had been prepared before through a less convenient acylation of the copper chelate of lysine reported without detail (6, 18). The routes to compounds 2 and 3 are given in Scheme I. Preliminary work showed that the introduction of a sulfonamide drug into HSA was not a routine procedure. Attempts to couple SMX and HSA with the carbodiimide DCC or EDC were unpromising, a5 was the use of disuccinimidyl suberate. The low reactivity was probably due

to the weak basicity of the p-amino group of SMX: PKa’S of the protonated amine of sulfonamide drugs are near 2-2.5 (19). For construction of protein conjugates of sulfamethoxazole, a hemisuccinate route (20)was chosen, shown as Scheme 11, in which the key intermediate was N4-succinylsulfamethoxazole(6). This route served to convert the poorly reactive aromatic p-amino group of sulfamethoxazole to a fully reactive aliphatic carboxyl group in 6 that when activated by a conventional route proved to be capable of acylating protein nucleophiles effectively. Compound 6 was prepared in 92% yield by treatment of SMX with succinic anhydride in the manner used for succinylsulfathiazole (211. To react 6 with protein nucleophilesthe active ester approach was considered more promising than carbodiimidewith its risk of protein crosslinking side reactions. Compound 6 was converted to its succinimidoester (7)by a carbodiimide-catalyzedcoupling with HONSu in THF. When the reaction of HONSu and 6 was attempted in DMF, in place of 7 its cyclized derivative, 3-(4-succinimidobenzenesulfonamido)-5-methylisoxazole (8), was isolated in 70% yield. Active ester 7 was reasonably stable in the cold in the solid state. In solution in DMF, DMSO, or T H F under anhydrous conditions, however, it tended to undergo ring closure with the elimination of HONSu to give 8. Almost

Tatake et al.

130 Bioconjugate Chem., Vol. 2, No. 2, 1991

E L U T I O N

V O L U M E ,

IL

Figure 2. Characterization by HPLC of various dialyzed conjugates of HSA containing sulfamethoxazole or sulfanilyl groups, distinguishing them from HSA and confirming the absence of low molecular weight sulfonamides. Materials were filtered over a column of Protein-Pak 1-125 in 0.05 M potassium phosphate buffer, pH 6.8, at a flow rate of 0.2 or 0.3 mL/min. Absorbance was determined at 254 nm. (A) HPLC of (sulfanilyl)lz-HSA: - -, 0.5 pg of (sulfanilyl)l2-HSA; - -, 5 pg of HSA; -, mixture of 0.5 pg of (sulfanilyl)l2-HSA and 5 pg of HSA. (B) HPLC of (~UCSMX)~~--HSA: - -, 3 pg of (sucSMX)ls-HSA;- -, 10pg of HSA; -, mixture of 3 pg of (sucSMX)ls-HSA and 10 of pg of HSA. SucSMX elutes at 9.7 mL. (C) HPLC of a mixture of 0.8 pg of (sucSMX-NHzhex)sl-HSA and 19.2 pg of HSA, respectively. (D) Composite chromatogram from HPLC of polylysine and a conjugate of it containing sulfamethoxazolegroups, each chromatographed separately at a flow rate of 1.5 mL min: - -, 1.7 pg of succinylated (sucSMX),lLysm with absorbance determined at 254 nm; -, 5 pg of (Lys-HBr)bNwith absor ance determined at 210 nm.

-

l

complete conversion of compound 7 to 8 was observed in a solution of 7 in DMSO-d,j prepared for determination of 'H NMR and reexamined after 8 days. In the coupling reactions between active ester 7 and proteins and polylysines in aqueous DMF the tendency of ester 7 to cyclize and to hydrolyze to acid 6, also observed as a side reaction, were compensated by the use of an excess of the active ester. As a model reaction to introduce SMX groups covalently into HSA, ester 7 was allowed to react with methyl glycinate in aqueous DMF at pH 6.8-7. The expected product sucSMX-glycinemethyl ester (9a),obtainedinsatisfactory yield, agreed in melting point and chromatographic behavior with the product derived from a carbodiimide coupling of acid 6 and methyl glycinate. Treatment of methyl glycinate with 8 in aqueous DMF failed to furnish 9a as indicated by TLC of the isolated product, making it unlikely that 8 was an intermediate in the reaction of 7 and methyl glycinate. HSA was then treated with 7 under a variety of conditions of pH, temperature, time, and modes of addition and molar ratios of reactants. Reaction mixtures were subjected to gel filtration, concentration dialysis, or dialysis. The final conjugates were examined by gelfiltration HPLC. An increase above 0.88 in the 254:280 nm absorbancy ratio of the protein peak suggested successful conjugation. Low-voltage electrophoresis on cellulose acetate strips provided another rapid way to assess conjugation. As shown in Figure 1, (sucSMX),-HSA conjugates (n = 2, 3, 6, 9) were readily distinguishable from HSA, even when only a few residues of SMX, increasing the molecular weight by only 176, had been incorporated. The products then were isolated by lyophilization. For quantitative analysis of covalently bound SMX, conjugates were subjected to a novel mild alkaline hy-

-

drolysis. This cleaved the succinanilic linkage in the protein and polylysine conjugatessmoothly to release SMX that was then readily determined by reverse-phase HPLC. The alkaline hydrolysis procedure applied to SMX and 6 afforded a quantitative recovery of SMX. It was more satisfactory than the usual acid hydrolysis that led to sulfanilic acid which was not as accurately determined, because of its highly polar nature, by reverse-phase HPLC under our conditions. The degrees of substitution of HSA by 6 under the various coupling conditions are given in Table 1. A reaction pH of 6.8 seemed superior to pH 9.5 (expts 1and 2). Drug substitution into HSA increasedas the mole ratio of active ester 7:HSA amino groups increased from two to eight (expts 1,4,5). Conjugates were obtained containing 2,6, 9, 13, and 21 mol of SMX/mol of HSA. The maximal introduction of SMX into HSA observed represents acylation of 22-35 ?!& of the available amino groups. This was attained by reaction of HSA at pH 6.8 in aqueous DMF for 4 h at room temperature with an 8-fold molar excess of 7. These conditions were adopted for the synthesis of analogues. Treatment of transferrin with ester 7 in a similar way yielded the corresponding conjugates of transferrin (succinylSMX),-TR (9g), some of which were more highly substituted than those of HSA. Two runs on a 50-mg scale led to about 46% substitution; two on a 100-mgscale led to 29 % substitution. Treatment of polylysine (n= 16 and 430) with ester 7 at a lower mole ratio yielded conjugates 9d and 9e of polylysine covalently bound to sucSMX with 65 and 17% substitution, respectively. Both polylysine conjugates had limited solubility and were solubilized by succinylation. Conjugates (sucSMX),-HSA (9f)have spacer chains of four carbons between SMX and the protein lysine +amino groups. Two additional activated esters, sucSMX-Gly-

Sulfonamide Drug-Protein Conjugates

NSu (lob) and sucSMX-NHzHex-NSu (lOc), synthesized and allowed to react with HSA, furnished conjugates (sucSMX-Gly)53-HSA (1l b ) and (sucSMX-NHzHex)51-HSA (llc) with spacer chains of 7 and 11 units, respectively. The route to 10b involved the condensation of 7 with free glycine in aqueous DMF to give sucSMX-Gly (9b), followed by treatment of the latter with HONSu and DCC in the usual way. A similar route from 7 and e-NH2 hexanoic acid yielded sucSMX-NHzHex (9c) and from it, sucSMX-NH2Hex-NSu (1Oc). In the synthesis of 9b and 9c from ester 7 the readily available glycine and c-aminohexanoic acid were used in 30-50% excess. All coupling and esterification reactions in the series leading to the activated ligands used for conjugation generally proceeded in good yield and afforded homogeneous compounds as judged by elemental analysis, TLC, and HPLC. The exceptions were in the e-aminohexanoic acid series. Compound 9c and 1Oc prepared from it were both contaminated with about 10% of 8 that was not readily removed; 1Oc also contained 9 % 9c. These products were used without further purification since the impurities were unreactive in the subsequent coupling with HSA and were readily removed during dialysis of the resulting conjugate. Conjugates l l b and l l c , synthesized by treatment of HSA with 10b and 1Oc under conditions optimal for (sucSMX)p,-HSA, were ca. 87 % substituted. Thus virtually all the amino groups of HSA are accessible to the mild active ester reagents. The higher acylation yields of l l b and 1IC,as compared to the 22-35 % in (sucSMX),-HSA, were probably determined by the greater stability of the active esters 10b and 1Oc as compared to 7 and the better solubility of their conjugation reaction mixtures. Table I1 summarizes the yields and degrees of substitution attained under various conditions in the synthesis of conjugates of sulfamethoxazole and HSA, transferrin, and two polylysines linked via a succinyl, succinylglycyl, or succinyl-6-aminohexanoyl spacer and of conjugates of sulfanilic acid linked directly to HSA or lysine. When the dialyzed conjugate (SA)lzHSAwas examined by HPLC gel filtration to confirm the absence of unbound ligand before estimating bound ligand, it became evident that the conjugate was distinguishable from HSA in the same system and that it was essentially free of unsubstituted HSA as well as of unbound ligand. The efficiency of an 1-125 gel-filtration column is evaluated in practice partly by its ability to resolve a mixture of OVA and HSA that differ in molecular weight by 60%. In our hands these albumins separated partially with a = 1.67. It was therefore unexpected to find that HSA and its sulfanilyl conjugate 2, differing in molecular weight by only 2.676, could be distinguished chromatographically on this sizeexclusion column. Figure 2A shows the elution patterns on a Protein Pak 1-125 column of (SA)lz-HSA, HSA, and a synthetic mixture of the two substances. The conjugate eluted ca. 1 min earlier than HSA, and a synthetic 1:l mixture clearly partially resolved, with a = 1.26. Chromatographic observations were similar with HSA and (sucSMX)ls-HSA (9f), differing in molecular weight by 6.5% (Figure 2B), and with HSA and (sucSMX-c-NHzHex)51HSA (llc), which differ in molecular weight by 33% (Figure 2C). HSA and 9f separated partially with a = 1.29, HSA and l l c with a = 1.69. TR and (sucSMX)25TR likewisewere distinguishable, with a = 2.0 (not shown). In the same gel-filtration system polylysine and its highly succinylated (sucSMX),~-LYS~~O conjugate (9e) separated very effectively, with a = 46 (Figure 2D). It is unlikely that such separations are based on size

Bioconjugate Chem., Vol. 2, No. 2, 1991

131

exclusion alone. Other factors that may have contributed to the observed separations include the anionic charges introduced by the SMX residues and in 9e by the additional succinyl residues. Derivatization of PL, while increasing molecular weight by more than 2-fold, has converted the polyvalent cation into a polyvalent anion. Clear evidence for the anionic nature of the sulfonamide group is the electrophoretic migration a t pH 5.6 of conjugates (sucSMX),-HSA (n = 2, 3, 6, 9) toward the anode with mobilities dependent on epitope density (Figure l ) , behavior that corresponds to the relative charge density provided by the sulfonamide anion of the SMX residues whose pK, is near 5.6 (19). Hydrophobicity of the sulfanilyl and spacer-linked SMX residues may also have contributed to the HPLC separations. Newer sizeexclusion matrices designed to minimize ionic and hydrophobic interactions may lack the chromatographic advantage for examining protein- and polylysine-drug conjugates encountered with the SEC support used in this work. ACKNOWLEDGMENT

This work was aided by the Burroughs Wellcome Co., the University of Connecticut Research Foundation (Grant 229), and the National Institutes of Health (Grant AI25341), for which we express thanks. Amino acid analysis was performed by Mr. George Korza of the Amino Acid Laboratory. LITERATURE CITED (1) Mandell, G. L., and Sande, M. A. (1990) Sulfonamides, Trimethoprim-Sulfamethoxazole, Quinolones, and Agents for Urinary Tract Infections. In Goodman and Gilman’s The Pharmacological Basis of Therapeutics (A. G. Gilman, T. W. Rall, A. S. Nies, and P. Taylor, Eds.) Chapter 45, Pergamon Press, New York. (2) Sogn, D. D. (1984) Penicillin Allergy. J. Allergy Clin. Immunol. 74, 589-593. (3) Ressler, C., and Mendelson, L. M. (1987) Skin test for dignosis of penicillin allergy-current status. Ann. Allergy 59, 161-171. (4) Wedum, A. G. (1942) Immunological Specificity of Sulfonamide Azoproteins. J. Infect. Dis. 70, 173-179. ( 5 ) Tomarelli, R. M., Charney, J., and Harding, M. L. (1949) The Use of Azoalbumin as a Substrate in the Colorimetric Determination of Peptic and Tryptic Activity. J . Lab. Clin. Med. 34, 428-433. (6) Drewes, S. E., von Klemperer, M., and Sutton, D. A. (1971) Separation of isomeric lysine derivatives by ion-exchange chromatography. J . Chromatogr. 56, 171-175. (7) Berlinguet, L., and Gautier, J. (1969) Can.J.Chem. 47,36413646. (8) Fasman,G. D. (1976)Proteins. InHandbookof Biochemistry and Molecular Biology, 3rd ed., Vol. 3, p 498, CRC Press, Cleveland, OH. (9) MacGillivray, R. T. A., Mendez, E., Sinha, S. K., Sutton, M. R., Lineback-Zins, J., and Brew, K. (1982) The complete amino acid sequence of human transferrin. Proc. Natl. Acad. Sci. U.S.A. 79, 2504-2508. (10) Voller, A., and Bidwell, D. (1986) Enzyme-Linked Immunosorbent Assay. I n Manual o f Clinical Laboratory Immunology, 3rd ed. (N. R. Rose, H. Friedman, and J. L. Fahey, Eds.) American Society of Microbiology, Washington, DC. (11) Ressler, C., Knapp, M. M., Tatake, J. G., Ballow, M., and Rapacz, P. (1987) Human IgM antibody to sulfamethoxazole in an adverse reaction t o trimethoprim-sulfamethoxazole.J . Allergy Clin. Immunol. 79, 198. (12) Ressler, C., Greaney, M. D., Tatake, J. G., Brettman, L. R., andBallow, M. (1990)Immunological Cross-Reactivity in Vitro Between Sulfamethoxazole and Various Other Sulfur and Related Drugs. J. Allergy Clin. Immunol. 85, 156.

132 Bioconjugate Chem., Vol. 2, No. 2, 1991

(13) Stewart, J. M., and Young, J. D., Eds. (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co, Rockford, IL, pp 6970. (14) Ressler, C., and Knapp, M. M. (1985) Liquid Chromatog raphy of Oxidized Sulfanilamide Derivatives. J. Liq. Chromatogr. 8, 1445-1453. (15) Bratton, A. C., Marshall, E. K., Jr., Babbitt, D., and Hendrickson, A. R. (1939) A New Coupling Component for Sulfanilamide Determination. J. Biol. Chem. 537-550. (16) Morris, G. J. 0. R., and Morris P. (1976) Membrane separation methods. In Separation Methods in Biochemistry, 2nd ed., John Wiley, New York, p 950. (17) Taussky, H. H., and Shorr, E. (1953) A Microcolorimetric Method for the Determination of Inorganic Phosphorus. J. Biol. Chem. 202,675-685. (18) Shchukina, M. N., and Yuan-Chen-e (1955) Synthesis of sulfanilamide derivatives in the lysine series. Chem. Abstr. 49, 6163e.

Tatake et al. (19) Newton, D. W., and Kluza, R. B. (1989) pK. Values of Drug Substances and pH Values of Tissue Fluids. In Principles of Medicinal Chemistry (W. A. Foye, Ed.) Appendix, Lea and Febiger, Philadelphia, PA. (20) Erlanger, B. F. (1980) The Preparation of Antigenic and Hapten-Carrier Conjugates: A Survey. Methods Enzymol. 70, 85-104. (21) Moore, M. L., and Miller, C. S. (1942) Dicarboxylic Acid Derivatives of Sulfonamides. J. Am. Chem. SOC.64, 15721576.

Registry No. 1,98-62-4;3,133071-56-4;4,13734-28-6; 5,12160-8; 6, 133071-57-5;7, 133071-58-6;8,133071-59-7;9a, 13307160-0; lob, 133071-61-1;~ O C 133071-64-4; , 12,723-46-6;HONSU, 6066-82-6;PL-HBr,25988-63-0;PL.HBr(SRU), 26588-20-5;NH2Hex, 60-32-2; succinic anhydride, 108-30-5; methyl glycinate hydrochloride, 5680-79-5; glycine, 56-40-6.