Multivalent Conjugates of Poly-γ - American Chemical Society

Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K., and Tenovus Research Laboratory,. General Hospital, Tremona Road, Southampton ...
0 downloads 0 Views 145KB Size
Download PDF
1148

Bioconjugate Chem. 2003, 14, 1148−1155

Multivalent Conjugates of Poly-γ-D-glutamic Acid from Bacillus licheniformis with Antibody F(ab′) and Glycopeptide Ligands Emmanuel J. F. Prodhomme,† Alison L. Tutt,‡ Martin J. Glennie,‡ and Timothy D. H. Bugg*,† Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K., and Tenovus Research Laboratory, General Hospital, Tremona Road, Southampton SO16 6YD, U.K.. Received February 22, 2002; Revised Manuscript Received August 28, 2003

Poly-γ-D-glutamic acid from Bacillus licheniformis is a water-soluble, nontoxic, nonimmunogenic exopolymer. Using synthetic linkers, the R-carboxylate side chains of PGA were conjugated to an exposed thiol side chain of an antibody F(ab′) fragment, Mc109F4. Analysis of the PGA-Mc109F4 conjugate by gel filtration HPLC revealed a mixture of multivalent conjugates. The PGA-Mc109F4 conjugate retained biological activity, but showed a lower binding affinity to target BCL3B3 cells than free Mc109F4 F(ab′)2 by flow cytometry, and a lower efficacy for BCL3B3 growth inhibition than free Mc109F4 F(ab′)2. PGA was also conjugated with the free amino group of glycopeptide antibiotic vancomycin. The PGA-vancomycin conjugate showed slightly lower antibacterial activity than free vancomycin versus susceptible Bacillus subtilis, but slightly higher activity versus intrinsically resistant Leuconostoc mesenteroides.

INTRODUCTION

Microorganisms which can survive in the human body commonly utilize nonimmunogenic cell surface exopolymers to evade the human immune system. These biopolymers are therefore attractive reagents for the formation of conjugates which could operate successfully in the human body. One such exopolymer is poly-γ-D-glutamic acid (Figure 1), a γ-linked polypeptide of D-glutamic acid of molecular mass 100-300 kDa, found in several strains of Bacillus (1), including soil bacterium Bacillus licheniformis (1, 2), and the human pathogen Bacillus anthracis (1 ,3). We have previously shown that γ-PGA from Bacillus licheniformis can be covalently modified in aqueous solution with amines bearing a UV chromophore (4), giving a modified polymer of reduced molecular weight (10-15 kDa). Early literature reports of PGA conjugates (5, 6) suggested that this biopolymer would be a useful template for functionalization with bioactive molecules. In this paper we describe methods for conjugation of PGA with an antibody F(ab′), and with a glycopeptide antibiotic. Targetting of cytotoxic drugs via comodification of the drug to a water-soluble polymer with a cell-specific antibody is an attractive approach for anticancer chemotherapy (7). Tumor-specific antibody F(ab′) fragments have been successfully conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) with cytotoxic agents adriamycin (8) or doxorubicin (9) to give cytotoxic conjugates. Methods have also been developed for the attachment of antibody F(ab′) fragments, via a reactive thiol group, to poly-R-lysine (10,11), poly[2-(dimethylamino)ethyl methacrylate] (12), and poly(ethylene oxide)-poly(propylene oxide) triblock copolymers (13). In this study, we used the F(ab′) fragment of antibody Mc109G4, a monoclonal * To whom correspondence should be addressed. Tel 02476573018. Fax 02476-524112. E-mail [email protected]. † University of Warwick. ‡ Tenovus Research Laboratory. 1 Abbreviations used: PGA, poly-γ-D-glutamic acid; FITC, fluorescein isothiocyanate; EDC, N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride.

Figure 1. Structure of poly-γ-D-glutamic acid.

rat IgG2a antibody (mAb) with activity against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma (14). The glycopeptide antibiotic vancomycin exerts its antibacterial effects via complexation of the peptidyl-D-AlaD-Ala termini of peptidoglycan on the surface of Grampositive bacterial cell walls (15). Recent studies by Williams et al. have demonstrated that members of the vancomycin group of glycopeptide antibiotics form dimers, and that the dimerization is cooperative with ligand binding (15, 16). Thus, multivalent derivatives of vancomycin which are able to bind the target ligands cooperatively might have enhanced affinity for bacterial peptidoglycan, particularly for vancomycin-resistant strains containing peptidyl-D-Ala-D-Lac termini, for which vancomycin shows low affinity (15). Synthetic dimers (17) and trimers (18, 19) of vancomycin have been reported to show enhanced binding of L-Lys-D-Ala-D-Ala and enhanced antibacterial activity against vancomycin-resistant strains. Polymers derived from a vancomycin-containing monomer have also been reported to show enhanced antibacterial activity (20, 21). We therefore also investigated the effects of conjugating vancomycin to γ-PGA. EXPERIMENTAL SECTION

General. Poly-γ-D-glutamic acid was prepared from Bacillus licheniformis ATCC 9945a by the method of Troy (2) and was stored as a powder at 4 °C. N-butoxycarbonylethane 1,2-diamine was prepared by the method of Krapcho and Kuell (22). Chemicals and biochemicals were purchased from Sigma-Aldrich. N-Butoxycarbonyl-[2,2′-(ethylenedioxy)bis-ethylamine] was prepared by a modification of the method of Krapcho and Kuell (22). A solution of di-tert-butyl dicarbonate (2.18 g, 10 mmol) in dioxane (30 mL) was added

10.1021/bc020019m CCC: $25.00 © 2003 American Chemical Society Published on Web 11/01/2003

Multivalent Conjugates of Poly-γ-D-glutamic Acid

to 2,2′-(ethylenedioxy)bis-ethylamine (7.3 mL, 5 equiv) in dioxane (30 mL) over 2.5 h. The reaction mixture was stirred at room temperature for 22 h. Solvent was then removed under reduced pressure. Water (50 mL) was added to the residue, and the insoluble bis-substituted product removed by filtration. The filtrate was extracted with dichloromethane (3 × 50 mL), the extracts were combined and dried (MgSO4), and the solvent was evaporated at reduced pressure to give the product as a clear oil (2.2 g, 63%). Rf 0.10 (9:1 CH2Cl2/EtOH); δH (300 MHz, CDCl3) 5.65 (1H, br s, NH), 3.55 (4H, s, OCH2CH2O),3.45(4H,m,NHCH2CH2O),3.25(2H,m,BocNHCH2CH2O), 2.80 (2H, t, J ) 7 Hz, H2NCH2CH2O), 1.40 (9H, s, tBu) ppm; δC (75 MHz, CDCl3) 156.0, 78.7, 73.3, 70.1, 66.9, 41.6, 40.2, 28.3 ppm. Preparation of N-Carboxyalkylmaleimides 1a,b. A solution of maleic anhydride (2.94 g, 30 mmol) in acetic acid (100 mL) was added to a solution of 6-aminocaproic acid (3.94 g, 30 mmol) in acetic acid (100 mL). The reaction was stirred for 3 h at room temperature, yielding a white precipitate, which was filtered, yielding N-maleamyl-6-aminocaproic acid (4.6 g, 67%). Rf 0.25 (9:1 CH2Cl2/EtOH); mp 159-161 °C; δH (300 MHz, d6-DMSO) 9.15 (1H, br, COOH), 6.40 (1H, d, J ) 11 Hz, CHdC), 6.25 (1H, d, J ) 11 Hz, CHdC), 3.15 (2H, q, J ) 6 Hz, NCH2), 2.20 (2H, t, J ) 7 Hz, CH2COOH), 1.45 (4H, m), 1.30 (2H, m) ppm; δC (75 MHz, d6-DMSO) 174.5, 165.5, 165.4, 133.1, 131.8, 40.4, 33.6, 28.2, 26.0, 24.2 ppm; m/z (ES+) 230.1 (MH+, 60%). N-Maleamyl-6-aminocaproic acid (0.92 g, 4.0 mmol) was dissolved in dry toluene (100 mL), to which was added triethylamine (1.1 mL, 8.0 mmol). The reaction was refluxed for 1 h in a Dean-Stark apparatus, with removal of water. The toluene solution was decanted and evaporated under reduced pressure to give a solid, which was dissolved in water (20 mL), acidified (HCl) to pH 2.0. The product was extracted with ethyl acetate (2 × 20 mL), washed with sat. sodium chloride solution (5 mL), dried (MgSO4), and evaporated at reduced pressure to give N-carboxypentylmaleimide 1b as a white solid (0.42 g, 50%). Rf 0.72 (9:1 CH2Cl2/EtOH); mp 79-80 °C; UV/ vis λmax 319 nm; δH (300 MHz, d6-acetone) 10.1 (1H, br, COOH), 6.70 (2H, s, CHdC), 3.30 (2H, t, J ) 6 Hz, NCH2), 2.15 (2H, t, J ) 7 Hz, CH2COOH), 1.45 (4H, qui, J ) 7 Hz), 1.20 (2H, qui, J ) 7 Hz) ppm; δC (75 MHz, d6-acetone) 206.0, 174.3, 134.9, 37.7, 33.7, 28.7, 26.6, 24.9 ppm. The same procedure was used to prepare N-carboxymethylmaleimide 1a from glycine, in 46% overall yield. Rf 0.35 (9:1 CH2Cl2/EtOH); mp 113-114 °C; UV/vis λmax 318 nm; δH (300 MHz, d6-acetone) 6.35 (2H, s, CHdC), 4.15 (2H, s, CH2) ppm; δC (75 MHz, d6-acetone) 206.0, 168.8, 135.4, 38.6 ppm. Preparation of N-Boc-ethylenediaminylcarboxyalkylmaleimides 2a,b. Isobutyl chloroformate (129 µl, 1.0 mmol) was added to a solution of N-carboxypentylmaleimide (0.21 g, 1.0 mmol) and triethylamine (0.2 mL) in THF (10 mL) at 0 °C, and the reaction was stirred for 15 min at room temperature. A solution of N-butoxycarbonylethylenediamine (0.16 g, 1.0 mmol) and triethylamine (0.2 mL) in THF (10 mL) was then added, and the reaction was stirred for 16 h at room temperature. The solution was filtered and solvent evaporated under reduced pressure. The resulting oil was triturated with diethyl ether to give N-butoxycarbonyl-N-6-(N-maleimyl)aminohexanoylethylenediamine 2b as a pale yellow gum (0.165 g, 45%). Rf 0.70 (9:1 CH2Cl2/EtOH); δH (300 MHz, CDCl3) 6.60 (2H, s, CHdC), 6.15 (1H, br s, NH), 4.90 (1H, br s, NH), 3.45 (2H, t, J ) 6 Hz, NCH2), 3.2-3.3

Bioconjugate Chem., Vol. 14, No. 6, 2003 1149

(4H, m, NHCH2CH2NH), 2.10 (2H, t, J ) 7 Hz, CH2CO), 1.55 (4H, qui, J ) 7 Hz), 1.35 (9H, s, tBu), 1.25 (2H, qui, J ) 7 Hz) ppm; δC (75 MHz, CDCl3) 174.0, 171.0, 159.7, 134.2, 80.2, 40.9, 40.4, 37.8, 36.6, 28.5, 28.4, 26.5, 25.2 ppm; m/z (ES+) 354.3 (MH+, 100%). The same procedure was used to prepare N-butoxycarbonyl-N-6-(N-maleimyl)aminoethanoylethylenediamine 2a from N-carboxymethylmaleimide (1a) and N-butoxycarbonylethylenediamine, in 51% yield. Mp 133-135 °C; δH (300 MHz, CDCl3) 7.0 (1H, br s, NH), 6.70 (2H, s, CHdC), 5.05 (1H, br s, NH), 4.10 (2H, s, NCH2), 3.2-3.3 (4H, m, NHCH2CH2NH), 1.35 (9H, s, tBu) ppm; δC (75 MHz, CDCl3) 170.0, 167.0, 158.0, 134.2, 80.0, 41.7, 40.9, 40.4, 28.5 ppm; m/z (ES+) 298.2 (MH+, 100%). Preparation of Boc-protected Maleimides 3a,b. Isobutyl chloroformate (129 µL, 1.0 mmol) was added to a solution of N-carboxypentylmaleimide (0.21 g, 1.0 mmol) and triethylamine (0.2 mL) in THF (10 mL) at 0 °C, and the reaction was stirred for 15 min at room temperature. A solution of N-butoxycarbonyl-[2,2′-(ethylenedioxy)bis-ethylamine] (0.248 g, 1.0 mmol) and triethylamine (0.2 mL) in THF (10 mL) was then added, and the reaction was stirred for 16 h at room temperature. The solution was filtered and solvent evaporated under reduced pressure. The resulting oil was triturated with diethyl ether to give maleimide 3b as a pale yellow gum (0.176 g, 40%). Rf 0.75 (9:1 CH2Cl2/EtOH); δH (300 MHz, CDCl3) 6.60 (2H, s, CHdC), 6.15 (1H, br s, NH), 5.65 (1H, br s, NH), 3.55 (4H, s, OCH2CH2O), 3.50 (2H, t, J ) 6 Hz), 3.40 (4H, m), 3.25 (2H, m), 2.85 (2H, t, J ) 7 Hz, BocNHCH2-), 2.1, (2H, t, J ) 7 Hz, CH2CO), 1.55 (4H, m), 1.40 (9H, s, tBu), 1.30 (2H, m) ppm; δC (75 MHz, CDCl3) 173.8, 171.2, 157.0, 134.4, 78.7, 73.1, 70.3, 67.2, 41.9, 39.9, 37.7, 36.4, 28.4, 28.2, 26.3, 24.9 ppm; m/z (ES+) 442.3 (MH+, 45%). The same method was used to prepare N-Boc maleimide 3a from maleimide acid 2a, in 48% yield. Rf 0.64 (9:1 CH2Cl2/EtOH); δH (300 MHz, CDCl3) 6.70 (2H, s, CHd C), 6.40 (1H, br s, NH), 4.95 (1H, br s, NH), 4.15 (2H, s, NCH2CO), 3.60 (4H, s, OCH2CH2O), 3.50 (4H, m), 3.25 (4H, m), 1.40 (9H, s, tBu) ppm; δC (75 MHz, CDCl3) 170.3, 167.8, 158.0, 134.7, 79.8, 73.8, 70.3, 70.1, 41.9, 41.4, 39.9, 28.5 ppm; m/z (ES+) 387.2 (MH+, 40%). Preparation of Maleimide Linkers 4a,b and 5a,b. Boc-protected maleimide 2b (90 mg, 0.25 mmol) was stirred in a 1:1 mixture of trifluoroacetic acid and dichloromethane (10 mL) at room temperature for 30 min. Solvent was removed under reduced pressure, and the residue was coevaporated with toluene to remove excess trifluoroacetic acid, to give the deprotected maleimide 4b (trifluoroacetate salt) as a brown oil (61 mg, 96%). δH (300 MHz, CDCl3) 7.90 (2H, br s, NH), 6.60 (2H, s, CHdC), 6.35 (1H, br s, NH), 3.50 (2H, t, J ) 6 Hz, NCH2), 3.25 (2H, q, J ) 5 Hz, NHCH2), 3.05 (2H, m, NHCH2), 2.15 (2H, t, J ) 7 Hz, CH2CO), 1.60 (4H, qui, J ) 7 Hz), 1.25 (2H, qui, J ) 7 Hz) ppm; δC (75 MHz, CDCl3) 174.4, 172.1, 133.7, 40.7, 39.6, 37.5, 36.3, 28.2, 26.0, 24.7 ppm; m/z (ES+) 254.1 (MH+, 100%). The same procedure was used to prepare maleimide 4a from Boc-protected maleimide 2a, in 94% yield. δH (300 MHz, CDCl3) 8.05 (2H, br s, NH), 7.05 (1H, br s, NH), 6.70 (2H, s, CHdC), 4.10 (2H, s, NCH2), 3.20 (2H, t, J ) 5 Hz, NHCH2), 3.05 (2H, m, NHCH2) ppm; δC (75 MHz, CDCl3) 170.0, 167.0, 134.2, 41.7, 40.9, 40.4 ppm; m/z (ES+) 198.3 (MH+, 100%). The same procedure was used to prepare maleimide 5a from Boc-protected maleimide 3a, in 95% yield. δH (300 MHz, CDCl3) 7.80 (2H, br s, NH), 6.75 (2H, s, CHd C), 6.20 (1H, br s, NH), 4.10 (2H, s, NCH2CO), 3.65 (4H,

1150 Bioconjugate Chem., Vol. 14, No. 6, 2003

s, OCH2CH2O), 3.55 (4H, m), 3.35 (2H, m), 3.15 (2H, m, NCH2) ppm; δC (75 MHz, CDCl3) 170.8, 168.3, 134.2, 71.5, 70.8, 70.2, 41.3, 41.1, 38.9 ppm; m/z (ES+) 287.5 (MH+, 60%). The same procedure was used to prepare maleimide 5b from Boc-protected maleimide 3b, in 93% yield. δH (300 MHz, CDCl3) 8.30 (2H, br s, NH), 6.60 (2H, s, CHd C), 6.10 (1H, br s, NH), 3.60 (4H, s, -OCH2CH2O-), 3.45 (2H, t, J ) 6 Hz), 3.35 (2H, m), 3.25 (2H, m), 3.15 (2H, m), 2.65 (2H, t, J ) 7 Hz, NHCH2), 2.10 (2H, t, J ) 7 Hz, CH2CO), 1.50 (4H, m), 1.20 (2H, m) ppm; δC (75 MHz, CDCl3) 173.6, 170.7, 134.1, 73.9, 70.2, 67.7, 41.7, 39.1, 37.1, 36.5, 28.9, 27.1, 25.3 ppm; m/z (ES+) 342.5 (MH+, 50%). Preparation of N-Succinylethane 1,2-Diamine (6). Succinic anhydride (0.86 g, 1.1 equiv) was added to a solution of N-Boc-ethylene 1,2-diamine (1.25 g, 7.85 mmol) in toluene (250 mL). The reaction was heated at 60 °C for 4 h, then cooled. The product, which crystallized upon cooling, was filtered and dried to give N-Boc N-succinylethylene 1,2-diamine as white crystals (1.826 g, 90%). Mp 124-126 °C; δH (300 MHz, d6-DMSO) 12.05 (1H, br s, COOH), 7.85 (1H, br s, NH), 6.75 (1H, br s, NH), 3.05 (2H, t, J ) 7 Hz, NCH2), 2.95 (2H, t, J ) 7 Hz, NCH2), 2.40 (2H, t, J ) 7 Hz, COCH2), 2.30 (2H, t, J ) 7 Hz, COCH2), 1.40 (9H, tBu) ppm; δC (75 MHz, CD3OD) 176.2, 174.8, 158.4, 80.0, 40.7, 40.5, 31.4, 30.2, 28.7 ppm; m/z (ES+) 261.4 (MH+, 100%). N-Boc-N-succinylethylene 1,2-diamine (0.2 g, 0.77 mmol) was added to a 1:1 mixture (10 mL) of trifluoroacetic acid and dichloromethane at room temperature for 30 min. Solvent was removed under reduced pressure, and the residue was coevaporated with toluene to remove excess trifluoroacetic acid, to give N-succinylethylene 1,2-diamine 6 (trifluoroacetate salt) as a brown oil (0.117 g, 95%). δH (300 MHz, d6-DMSO) 11.70 (1H, br s, COOH), 8.20 (1H, br s, NH), 7.85 (2H, br s, NH), 6.75 (1H, br s, NH), 3.25 (2H, t, J ) 7 Hz, NCH2), 2.85 (2H, t, J ) 7 Hz, NCH2), 2.40 (2H, t, J ) 7 Hz, COCH2), 2.30 (2H, t, J ) 7 Hz, COCH2) ppm; δC (75 MHz, d6-DMSO) 175.0, 172.2, 39.4, 38.7, 31.2, 29.7 ppm; m/z (ES+) 161.0 (MH+, 10%). Toxicity of Poly-γ-D-glutamic Acid. PGA was incubated with the EHRB human B cell line at 20 and 100 µg/mL concentration for 24 h, then stained with propidium iodide to detect apoptotic cells, as described by Nicoletti et al. (23). Uptake of 3H-thymidine into EHRB cells was also assayed, at 100 and 10 µg/mL concentrations of PGA, as previously described (24). Animal toxicity was assessed by injection of PGA (1 mg) intra-peritoneally into mice. No signs of toxicity (morbidity, coat appearance, diarrhea) were observed. Modification of Poly-γ-D-glutamic Acid with Maleimide Linkers. Poly-γ-D-glutamic acid (35 mg, 0.27 mmol available sites) was dissolved in water (10 mL), to which was added the appropriate amine (0.27 mmol), triethylamine (38 µl, 0.3 mmol), N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (53 mg, 0.27 mmol), and hydroxybenzotriazole (37 mg, 0.27 mmol). The reaction mixture was stirred for 24 h at room temperature, then dialyzed against water (1 l), and freeze-dried, to give the PGA-maleimides as pale brown solids. Maleimide-modified polymers were characterized by UV and NMR spectroscopy. Observed λmax values: PGA-4a, 306 nm; PGA-4b, 317 nm; PGA-5a, 304 nm; PGA-5b, 299 nm. Conjugation of Poly-γ-D-glutamic Acid with Antibody Mc109F4 F(ab′). Buffers: 1 M NTE8 buffer stock contains 25 mM Tris, 12.5 mM EDTA, and 1 M NaCl, adjusted to pH 8.0, which was diluted 5-fold for use. AE

Prodhomme et al.

buffer contains 50 mM sodium acetate and 0.6 mM EDTA, adjusted to pH 5.2 with acetic acid. PBS buffer contains 10 mM sodium phosphate (pH 7.4) and 150 mM NaCl. F(ab′)2 fragments were obtained by proteolytic digestion of antibody Mc109F4, as previously described (25). F(ab′)2 fragments (5-12 mg/mL, 1.0 mL) in 0.2 M NTE8 buffer were reduced by addition of 100 µL of 200 mM β-mercaptoethanol, followed by incubation at 30 °C for 30 min, then cooled to 4 °C. Reduced F(ab′)SH was separated from reducing agent by passage through a Sephadex G25 column (80 × 2.4 cm) in 50 mM AE buffer. Fractions from the G25 column were analyzed by HPLC gel filtration (Phenomenex BioSep-SEC-S3000 column, 1.0 mL/min flow rate, monitored at 280 nm, retention times F(ab′)2 8.6 min, F(ab′)SH 9.4 min, β-mercaptoethanol 12.5 min). Fractions containing F(ab′)SH and free of β-mercaptoethanol were pooled and immediately treated with a solution of maleimide-activated PGA (160 mg) in water (1 mL). The molar ratio of reagents was 5:1 PGAmaleimide (moles of glutamic acid):antibody F(ab′). The mixture was left to react at 4 °C for up to 14 days and monitored by HPLC gel filtration (retention time of conjugate 7.3 min). The conjugate was purified by passage through two AcA44 gel filtration columns (120 × 2.0 cm) in series, in 0.2 M NTE8 buffer. Two main protein peaks were observed: the first being the PGA-F(ab′) conjugate, and the second being unreacted F(ab′)2 and F(ab′)SH. The PGA-F(ab′) conjugate was pooled, concentrated using an Amicon device, and dialyzed into PBS. HPLC gel filtration was carried out using a Phenomenex BioSep-SEC-S3000 column, eluting with 50 mM sodium acetate buffer pH 5.2 containing 0.5 mM EDTA at a flow rate of 1.0 mL/min. Molecular weight calibration was carried out using authentic samples of poly-R-Dglutamic acid (Sigma): 12000-14000 Da (9.1 min); 16000-32000 (8.2 min); 45000-65000 (6.6 min). Analysis of Interaction of PGA-5b-Mc109F4 Conjugate with BCL3B3 Cells. BCL1 3B3 cells were analyzed by indirect immunofluorescent staining using a FACS vantage (Becton Dickinson, Mountain View, CA), as described previously (14). BCL3B3 cells were incubated for 30 min with the conjugate or control antibody, then washed and incubated with goat anti-rat IgG-FITC (Stratech Scientific Ltd, Luton UK), then washed and analyzed by flow cytometry. Protein concentration of 25 µg/mL was used in each assay. Concentration of F(ab′) in PGA-5b-Mc109F4 estimated by measurement of A280 and comparison with standard sample of Mc109F4. A growth assay to assess direct effects of mAb or derivatives on BCL13B3 target tumor cells in vitro was carried out as described previously (24). The cells (5 × 104) were grown in various concentrations (1-25 µg/mL) of antibodies for 42 h, then pulsed with 3H-thymidine and harvested 6 h later, and growth was assessed by 3H incorporation. Preparation of PGA-Vancomycin Conjugates. Poly-γ-D-glutamic acid (30 mg, 0.23 mmol available sites) was dissolved in water (5 mL), to which was added the N-succinylethane 1,2-diamine (6, 30 mg, 0.23 mmol), triethylamine (32 µl, 0.25 mmol), N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride (45 mg, 0.23 mmol), and hydroxybenzotriazole (32 mg, 0.23 mmol). The reaction mixture was stirred for 15 min at room temperature. Vancomycin hydrochloride (35 mg, 0.23 mmol, Sigma) and triethylamine (32 µl, 0.25 mmol) were then added, and the reaction mixture was stirred overnight at room temperature. The mixture was then

Multivalent Conjugates of Poly-γ-D-glutamic Acid

Bioconjugate Chem., Vol. 14, No. 6, 2003 1151

Scheme 1. Synthesis of Maleimide Linkers 4, 5a

a a, AcOH; b, NEt , toluene, ∆, 46% (n ) 1) and 34% (n ) 5); c, BocNHCH CH NH , isobutyl chloroformate, NEt , THF, 51% (n 3 2 2 2 3 ) 1) and 45% (n ) 5); d, BocNH(CH2CH2O)2CH2CH2NH2, isobutyl chloroformate, NEt3, THF, 48% (n ) 1), 40% (n ) 5); e, CF3COOH, CH2Cl2, 90-96%; e, PGA, EDC, HOBT, NEt3, H2O.

dialyzed against water (1 l) and freeze-dried, to give the PGA-6-vancomycin conjugate (30 mg) as a fluffy off white solid. The PGA-vancomycin conjugate was prepared using the same method, omitting linker 6, and using 0.1 equiv of EDC, HOBT, vancomycin, and triethylamine. PGA-vancomycin conjugates were analyzed by BioSep S3000 HPLC gel filtration, as described above. Assay of Antibacterial Activity of PGA-Vancomycin Conjugates. Overnight cultures of Bacillus subtilis W23 and Lactobacillus casei subsp. casei (ATCC393) were grown in Luria broth at 37 °C and then spread onto the surface of an LB plate, and excess liquid was removed. Filter disks (Whatman no.1, 20 mm) were placed onto the plate, and an aliquot (100 µl) of a 1 mg/ mL solution of the antibiotic or conjugate was added to the disk. The plate was then grown overnight at 37 °C. A zone of growth inhibition was observed around each filter disk, whose radius from the outside of the disk was measured. RESULTS

Toxicity of Native PGA. Poly-γ-D-glutamic acid (PGA) was prepared from Bacillus licheniformis ATCC 9945a using the procedure of Troy (2), yielding approximately 120 mg of pure polymer from a 100 mL culture, as a freeze-dried powder. The toxicity of PGA was tested in cell culture, against a sensitive EHRB cell line. Using the method of Nicoletti et al. to measure % apoptotic cells (23), no toxicity was observed at 20 µg/ mL PGA (11.2%) compared to a control (11%), although slight toxicity was observed at 100 µg/mL (18.1%). Using a 3H-thymidine uptake assay (24) on EHRB cells, 100 and 10 µg/mL PGA showed 78% and 88% counts compared to untreated cells. Injection of a 1 mg dose of PGA into mice resulted in no observable toxicity. Synthesis and Attachment of Maleimide Linkers to PGA. Proteolytic digestion of IgG immunoglobulins, followed by reduction of the F(ab′)2 dimer product (100 kDa), generates an antibody F(ab′) fragment (50 kDa) bearing a unique reactive thiol group (25). To conjugate an antibody F(ab′) to PGA, a synthetic linker is required

which can be attached to the R-carboxyl group of PGA and subsequently selectively with the thiol group. Preliminary experiments using bromoacetyl- and iodoacetylcontaining linkers revealed that thiol conjugation with these linkers required high pH conditions and proceeded in low yield; therefore, the synthesis of linkers containing reactive maleimide groups was undertaken. The maleimide functional group, known to react selectively with thiol nucleophiles (26), was attached via a variable length chain to a primary amine group, which could be attached to PGA. The synthesis of four maleimide-containing linkers is illustrated in Scheme 1. Maleic anhydride was reacted with glycine to give the corresponding maleamic acid, which was converted to maleimide 1a by treatment with triethylamine in refluxing toluene (27). In a similar fashion, 6-aminohexanoic acid was converted to maleimide lb. Each maleimide acid was coupled to mono-Bocethylenediamine, to give maleimides 2a,b, and also to a longer, more polar linker containing an ethylene glycol spacer, yielding maleimides 3a,b. Deprotection under acidic conditions gave the deprotected maleimide linkers 4a,b and 5a,b containing 9, 13, 15, and 19 atom spacers, respectively. Each linker was attached to PGA using water-soluble carbodiimide EDC, and the modified polymers were dialyzed and lyophilised. The attachment of maleimide linkers to PGA was monitored by UV and NMR spectroscopy. Maleimides 4 and 5 showed characteristic UV/ visible absorption peaks at λmax 300-315 nm, due to the maleimide chromophore. PGA-maleimide conjugates showed peaks at 300-315 nm by UV/visible spectroscopy, demonstrating that immobilization of the maleimide had taken place (see Figure 2). NMR signals for the maleimide group could also be observed at δH 6.75 ppm and δC 135 ppm in the 1H and 13C NMR spectra of the modified polymer (see Figure 3). After storage for >24 h, the UV absorption of the modified polymers gradually decreased, suggesting that intramolecular reaction was taking place between the maleimide groups and free carboxylate side chains of the polymer. Therefore, samples of activated

1152 Bioconjugate Chem., Vol. 14, No. 6, 2003

Prodhomme et al.

Figure 3. 300 MHz 1H NMR spectra of (A) γ-PGA; (B) linker 4b; (C) PGA modified by linker 4b. The position of the maleimide CdCH is indicated by arrows in B and C. Figure 2. UV/visible spectra of (A) γ-PGA; (B) linker 4a; (C) PGA modified by linker 4a. The absorption of the maleimide chromophore can be seen at λmax 300 nm.

polymer for conjugation reactions were prepared immediately prior to use. Conjugation with Antibody Mc109F4 F(ab′). Antibody Mc109F4 IgG was subjected to proteolytic cleavage by treatment with bromelain, to give the F(ab′)2 dimer (100 kDa). Treatment with β-mercaptoethanol then gave the F(ab′)-SH monomer (50 kDa), which was separated from excess reducing agent by G25 gel filtration chromatography. Preparation of F(ab′)-SH could be conveniently monitored by HPLC gel filtration, using a Phenomenex BioSep-S3000 column, which could resolve F(ab′)2 and F(ab′) with retention times 8.6 and 9.4 min, respectively (see Figure 4B,C). Freshly reduced F(ab′)-SH was mixed with the maleimide-activated polymer, in a 1:1 ratio of reduced F(ab′)-SH to maleimide linker, in 50 mM acetate buffer pH 5.2, and left to react at 4 °C. Reaction of the maleimide-activated polymer with reduced F(ab′)-SH was monitored by HPLC gel filtration. Analysis of native PGA gave a single peak at 5.5 min (Figure 4A), whereas the maleimide-activated polymers gave peaks at 9.0-9.2 min, implying that fragmentation of the native polymer had taken place during modification, as observed previously (4). Molecular weight calibration was attempted using samples of poly-R-Dglutamic acid, which gave the following retention times: 44-64 kDa, 6.6 min; 17-31 kDa, 8.2 min; 11-13 kDa, 9.1 min. Using these data, we can tentatively assign a molecular weight for the activated PGA of 11-13 kDa, which matches previous estimates (4), and a Mr for native PGA of 110 ( 40 kDa. The reason for the fragmentation of PGA upon modification is not known: it may be due to EDC-mediated cleavage (4) or the existence of a depolymerase enzyme (28).

Figure 4. HPLC gel filtration (Bio-Sep S3000) analysis of (A) native PGA, (B) Fab2 (50 kDa), (C) Fab-SH (25 kDa), (D) FabPGA conjugate. Retention times of poly-R-D-glutamic acid standards: 44-64 kDa, 6.6 min; 17-31 kDa, 8.2 min; 11-13 kDa, 9.1 min.

Analysis of the PGA-Mc109F4 conjugate by HPLC gel filtration gave a large, broad peak at 5.8-8.7 min, followed by small peak at 9.1 min (see Figure 4D). The peak at 9.1 min probably corresponds to residual activated polymer. A slight shoulder is visible at 8.2 min, which (from the difference in retention times of the F(ab′) and F(ab′)2 peaks) probably corresponds to the 1:1 PGAF(ab′) conjugate, and the remainder of the peak to higher PGA(F(ab′))n conjugates. Maximum absorbance is observed at retention time 7.3 min, suggesting that the most abundant conjugates are PGA(F(ab′))2 and PGA(F(ab′))3.

Multivalent Conjugates of Poly-γ-D-glutamic Acid

Bioconjugate Chem., Vol. 14, No. 6, 2003 1153

Figure 6. Growth inhibition of BCL3B3 cells of Mc109F4 IgG (open squares), Mc109F4 F(ab)2 (open circles), and PGA-5bMc109F4 conjugate (filled circles) assessed by 3H-thymidine incorporation (in cpm), at 0.1-6 µg/mL protein concentration. Procedure described in Experimental Section. Result of control assay (addition of medium only) is indicated.

Figure 5. Binding of PGA-5b-Mc109F4 conjugate to BCL3B3 cells assessed by flow cytometry. Procedure described in Experimental Section. Samples of control IgG and F(ab)2 antibodies were included, which have no interaction with BCL3B3 cells. Each protein sample was assayed at 25 µg/mL concentration. Strength of binding interaction assessed by mean channel fluorescence (x-axis).

Conjugation of activated PGA to F(ab′)-SH was found to take place slowly, requiring several days at 4 °C to reach completion, with most successful conjugation using PGA-5a and PGA-5b. The reactivity of samples of activated PGA was variable, low reactivity being observed for stored samples, perhaps due to intramolecular reaction of carboxylate side chains of PGA with maleimide groups, and ‘burying’ of maleimide groups in the hydrophobic core of PGA. However, by carrying out the functionalization of PGA with the PEG-maleimide linkers rapidly, coupling of antibody F(ab′)-SH was successfully achieved. The PGA-5a-F(ab′) and PGA-5b-F(ab′) conjugates were purified by AcA44 gel filtration and concentrated prior to storage. Biological Activity of PGA-Mc109F4 Conjugate. The PGA-5b-Mc109F4 conjugate was tested for binding to target BCL3B3 cells (14) using flow cytometry, as shown in Figure 5. BCL3B3 cells were incubated for 30 min with the conjugate or control antibody, washed and incubated with anti-antibody conjugated to fluorescent dye FITC, and then washed and analyzed by flow cytometry. Antibody Mc109F4 IgG and F(ab′)2 showed a mean channel fluorescence of 600 and 400 units, respectively, at 25 µg/mL concentration, respectively, whereas the PGA-Mc109F4 conjugate recorded only 70 units at 25 µg/mL concentration. A control antibody F(ab′)2 gave a reading of only 4 units at 25 µg/mL concentration. Thus,

the PGA-Mc109F4 conjugate does bind to BCL3B3 cells, but less strongly than the parent antibody F(ab′), indicating that conjugation to the polymer has reduced its biological activity. Since antibody Mc109F4 is known to inhibit the growth of BCL3B3 (14), the effect of the PGA-5b-Mc109F4 conjugate upon BCL3B3 growth was also investigated, as shown in Figure 6. The cells (5 × 104) were grown in various concentrations of antibodies for 42 h, pulsed with 3 H-thymidine, and harvested 6 h later, and growth was assessed by 3H incorporation (24). Approximately 20% growth inhibition was observed at 1 µg/mL PGAMc109F4; however, only 25% inhibition was observed at 6.25 µg/mL conjugate (and 30% inhibition at 25 µg/mL conjugate), whereas 5 µg/mL Mc109F4 F(ab′)2 caused 50% growth inhibition. In conclusion, the conjugated antibody fragment does retain its biological activity, but has reduced efficacy compared with the free antibody. Conjugation of γ-PGA with Vancomycin. The glycopeptide antibiotic vancomycin is a very interesting ligand for immobilization to PGA, since the dimerization of the antibiotic is believed to promote its antibacterial effects (15). The presence of a free primary amine, on the vancosamine sugar of the glycopeptide, provided a convenient point of conjugation to PGA, as shown in Scheme 2. Two conjugates of PGA with vancomycin were prepared. Conjugation of PGA directly to vancomycin was carried out, using the water-soluble carbodiimide EDC. A second conjugate was also prepared, using an eightatom spacer 6, prepared from Boc-ethylene 1,2-diamine and succinic anydride, as shown in Scheme 2. Analysis of the PGA-vancomycin and PGA-6-vancomycin conjugates by BioSep S3000 gel filtration gave new single peaks at retention times 16.7 and 16.4 min, respectively (γ-PGA 5.5 min, vancomycin 11.3 min), indicating that conjugates had been formed. The high retention times are indicative of an interaction between

1154 Bioconjugate Chem., Vol. 14, No. 6, 2003

Prodhomme et al.

Scheme 2. Preparation of PGA-Vancomycin Conjugatea

vancomycin and the terminal D-alanine is absent. Synthetic vancomycin dimers (17) and vancomycin-based polymers (20) have been reported to show higher activity against vancomycin-resistant strains. Therefore, the antibacterial activity of the PGA-vancomycin conjugates was also assayed against a vancomycin-resistant strain of Lactobacillus casei, using the filter disk assay. Both conjugates showed a 5 mm zone of inhibition, whereas free vancomycin showed a 3 mm zone of inhibition. It appears, therefore, that the PGA-vancomycin conjugates possess slightly higher antibacterial activity against vancomycin-resistant strains, although not markedly higher activity. DISCUSSION

a a. Succinic anhydride, toluene, 90%; b. CF COOH, CH Cl , 3 2 2 95%; c. γ-PGA, EDC, HOBT, Et3N, H2O; d., vancomycin, EDC, HOBT, Et3N, H2O.

Table 1. Antibacterial Activities of PGA-Vancomycin Conjugates zone of inhibition vs vancomycin (1 mg/mL) PGA-vancomycin conjugate (1 mg/mL) PGA-6-vancomycin conjugate (1 mg/mL)

Bacillus subtilis Lactobacillus casei (Vm-sensitive), mm (Vm-resistant), mm 14 8

3 5

9

5

the glycoside portion of vancomycin and the column, as observed for other glycoside derivatives (data not shown); therefore, it is not possible to estimate an Mr for the conjugates. Antibacterial Activity of PGA-Vancomycin Conjugates. The antibacterial activities of the two PGAvancomycin conjugates were assessed by fliter disk assays on agar plates and compared directly with free vancomycin. Measurement of the zone of growth inhibition around the filter disk containing the antibiotic provides a measure of antibacterial potency. The results are summarized in Table 1. Against a vancomycin-susceptible strain of Bacillus subtilis, the PGA-vancomcyin and PGA-6-vancomycin conjugates were found to show antibacterial activity (8 mm and 9 mm zones, respectively) at 1 mg/mL concentration. At the same concentration, free vancomycin showed a 14 mm zome of inhibition. Thus, it appears that the immobilized vancomycin retains its biological activity, but with somewhat reduced biological efficacy. High-level vancomycin resistance, both plasmid-mediated resistance in Enterococcus strains (29) and intrinsic resistance in lactic acid bacteria (30), is known to be associated with the presence of peptidyl-D-Ala-D-Lac termini in the cell wall peptidoglycan, for which vancomycin has low affinity, since one hydrogen bond between

We have previously shown that small molecular weight amines can be immobilized onto PGA in aqueous solution using a water-soluble carbodiimide, EDC (4). In this paper we describe the preparation of linkers for the attachment of protein ligands to PGA via thiol side chains. The most successful linker was found to be maleimide linker 5b, containing an ethylene glycol spacer capable of hydrogen bonding with the aqueous solvent. Using this linker, we were able to conjugate the Mc109F4 F(ab′), to give a mixture of PGA(F(ab′))n conjugates. The conjugated antibody F(ab′) was found to retain its biological recognition properties, but showed reduced binding efficacy and cell growth inhibition, compared with the free antibody F(ab′). We have also immobilized the glycopeptide antibiotic vancomycin to PGA and have shown that the immobilized ligand also retains its biological activity, but with slightly reduced efficacy. In this case, however, there is some indication of enhanced biological activity against vancomycin-resistant strains, against which the free ligand shows low affinity. This work establishes the feasibility of attaching bioactive molecules and macromolecules such as cell-specific antibody F(ab′) fragments to nontoxic, nonimmunogenic biopolymers such as PGA. The observation that the immobilized ligands retain their biological activity implies that antibody-polymer conjugates could in principle be used to target cytotoxic drugs in the human body (7), although the reduced biological efficacy of the immobilized F(ab′) implies that there are inherent practical difficulties in this approach. Biopolymers such as PGA provide interesting alternatives to man-made polymers for antibody F(ab′) immobilization (8-13), which may prove to have certain advantages for biocompatability. ACKNOWLEDGMENT

We would like to thank EPSRC for the award of a Project Studentship (GR/L60968) to E.J.P. LITERATURE CITED (1) Thorne, C. B., Gomez, C. G., Noyes, H. E., and Housewright, R. D. (1954). Production of glutamyl polypeptide by Bacillus subtilis. J. Bacteriol. 68, 307-315. (2) Troy, F. A. (1973). Chemistry and biosynthesis of poly(γ-Dglutamyl) capsule in Bacillus licheniformis. J. Biol. Chem. 248, 305-315. (3) Uchida, I., Makino, S., Sasakawa, C., Yoshikawa, M., Sugimoto, C., and Terakado, N. (1993). Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis. Mol. Microbiol. 9, 487-496. (4) King, E. C., Watkins, W. J., Blacker, A. J., and Bugg, T. D. H. (1998). Covalent Modification in Aqueous Solution of Polyγ-D-Glutamic Acid from Bacillus licheniformis. J. Polym. Sci. Part A: Polym. Chem. 36, 1995-1999.

Multivalent Conjugates of Poly-γ-D-glutamic Acid (5) Alkan, S. S., Nitecki, D. E., and Goodman, J. W. (1971). Antigen recognition and the immune response: the capacity of L-tyrosine-azobenzenearsonate to serve as a carrier for a macromolecular hapten. J. Immunol. 107, 353-358. (6) Blanchard, T. G., Thorne, C. B., and MacDonald, A. B. (1988). The use of polyglutamic acid as a delivery system for anti-idiotypic vaccines. FASEB J. 2, A1257. (7) Putnam, D., and Kopecek, J. (1995). Polymer conjugates with anticancer activity. Adv. Polym. Sci. 122, 55-123. (8) Kunath, K., Kopeckova, P., Minko, T., and Kopecek, J. (2000). HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line. Eur. J. Pharm. Biopharm. 49, 11-15. (9) Jelinkova, M., Strohalm, J., Plocova, D., Subr, V., St′astny, M., Ulbrich, K., and Rihova B. (1998). Targeting of human and mouse T-lymphoctes by monoclonal antibody-HPMA copolymer-doxorubicin conjugates directed against different T-cell surface antigens. J. Controlled Release 52, 253-270. (10) Trubetskoy, V. S., Narula, J., Khaw, B. A., and Torchilin, V. P. (1993). Chemically optimized antimysoin F(ab′) conjugates with chelating polymers - importance of the nature of the protein-polymer single-site covalent bond for biodistribution and infarction localization. Bioconjugate Chem. 4, 251-255. (11) Slinkin, M. A., Klibanov, A. L., and Torchilin, V. P. (1991). Terminal-modified polylysine-based chelating polymers highly efficient coupling to antibody with minimal loss in immunoreactivity. Bioconjugate Chem. 2, 342-348. (12) van Dijk-Wolthuis, W. N. E., van de Wetering, P., Hinrichs, W. L. J., Hofmeyer, L. J. F., Liskamp, R. M. J., Crommelin, D. J. A., and Hennink, W. E. (1999). A versatile method for the conjugation of proteins and peptides to poly[2-(dimethylamino)ethyl methacrylate]. Bioconjugate Chem. 10, 687-692. (13) Li, J. T., Carlsson, J., Lin, J. N., and Caldwell, K. D. (1996). Chemical modification of surface active poly(ethylene oxide)poly(propylene oxide) triblock copolymers. Bioconjugate Chem. 7, 592-599. (14) George, A. J. T., McBride, H. M., Glennie, M. J., Smith, L. J., and Stevenson, F. K. (1991). Monoclonal Antibodies Raised Against the Idiotype of the Murine B Cell Lymphoma, BCL1 Act Primarily with Heavy Chain Determinants. Hybridoma 10, 219-227. (15) Williams, D. H. (1996). The glycopeptide story: how to kill the deadly “superbugs”. Nat. Prod. Rep. 13, 469-477. (16) Beauregard, D. A., Maguire, A. J., Williams, D. H., and Reynolds, P. E. (1997). Semiquantitation of cooperativity in binding of vancomycin group antibiotics to vancomycinsusceptible and -resistant organisms. Antimicrob. Agents Chemother. 41, 2418-2423. (17) Sundram, N., Griffin, J. H., and Nicas, T. I. (1996). Novel vancomycin dimers with activity against vancomycin-resistant enterococci. J. Am. Chem. Soc. 119, 10286. (18) Rao, J., Lahiri, J., Isaacs, L., Weis, R. M., and Whitesides, G. M. (1998). A trivalent system from vancomycin: D-Ala-

Bioconjugate Chem., Vol. 14, No. 6, 2003 1155 D-Ala with higher affinity than avidin: biotin. Science 280, 708-711. (19) Rao, J., Lahiri, J., Weis, R. M., and Whitesides, G. M. (2000). Design, synthesis and characterisation of a high affinity trivalent system derived from vancomycin and L-LysD-Ala-D-Ala. J. Am. Chem. Soc. 122, 2698-2710. (20) Arimoto, H., Nishimura, K., Kinumi, T., Hayakawa, I., and Uemura, D. (1999). Multivalent polymer of vancomycin: enhanced antibacterial activity against VRE. J. Chem. Soc. Chem. Commun. 1361-1362. (21) Arimoto, H., Oishi, T., Nishijima, M., and Kinumi, T. (2001). Affinity of a vancomycin polymer with bacterial surface models. Tetrahedron Lett. 42, 3347-3350. (22) Krapcho, A. P., and Kuell, C. S. (1990). Monoprotected diamines N-tert-butoxycarbonyl-R, ω-alkanediamines from R, ω-alkanediamines. Synth. Commun. 20, 2559-2564. (23) Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271-279. (24) Tutt, A. L., French, R. R., Illidge, T. M., Honeychurch, J., McBride, H. M., Penfold, C. A., Fearon, D. T., Parkhouse, R. M. E., Klaus G. G. B., and Glennie, M. J. (1998). Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors. J. Immunol. 161, 3176-3185. (25) Glennie, M. J., Tutt, A. L., and Greenman, J. (1993). Preparation of multispecific F(ab′)2 and F(ab′)3 antibody derivatives. In Tumour Immunobiology, a practical approach (Gallagher, G, Rees, R. C., and Reynolds C. W., Eds.) pp 22544, Oxford University Press, Oxford. (26) Wong, S. S. (1993). Chemistry of protein conjugation and cross-linking, CRC Press, New York. (27) Reddy, P. Y., Kondo, S., Toru, T., and Ueno, Y. (1997). Lewis acid and hexamethyldisilizane-promoted efficient synthesis of N-alkyl and N-arylimide derivatives. J. Org. Chem. 62, 2652-2654. (28) King, E. C., Blacker, A. J., and Bugg, T. D. H. (2000). Enzymatic Breakdown of Poly-γ-D-glutamic Acid in Bacillus licheniformis: Identification of a Polyglutamyl γ-Hydrolase Enzyme. Biomacromolecules 1, 75-83. (29) Bugg, T. D. H., Wright, G. D., Dutka-Malen, S., Arthur, M., Courvalin, P., and Walsh, C. T. (1991). Molecular Basis for Vancomycin Resistance in Enterococcus faecium BM4147: Biosynthesis of a Depsipeptide Peptidoglycan Precursor by Vancomycin Resistance Proteins VanH and VanA, Biochemistry 30, 10408-10415. (30) Handwerger, S., Pucci, M. J., Volk, K. J., Liu, J., and Lee, M. S. (1994). Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate. J. Bacteriol. 176, 260-264.

BC020019M