2.1Synthesis of NAG–thiazoline
NAG–Thiazoline was prepared in 46% overall yield from peracetylated GlcNAc by treatment with Lawesson's reagent according to the procedure of Knapp et al.  with modifications. Thus, a solution of 1.03 g (2.66 mmol) of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-β-d-glucopyranose (Toronto Research Chemicals, Toronto, Ont.) in 10 ml of dry toluene was treated with 0.68 g (1.68 mmol) of Lawesson's reagent (Sigma–Aldrich, St. Loius, MO), and the reaction mixture was heated to 60 °C for 1.5 h. The reaction mixture was then cooled to room temperature, washed with H2O, dried over anhydrous Na2SO4, and concentrated. The resulting yellow syrup containing the crude thiazoline triacetate was chromatographed on silica gel with ethyl acetate/hexane (7:1) as the solvent to give 0.42 g (46%) of purified peracetate.
A portion of the thiazoline triacetate (0.42 g) was treated with 0.2 M methanolic sodium methoxide (10 ml) with stirring for 10 min. After de-ionization with Bio-Rad 50W-X4 (H+ form; Bio-Rad Laboratories) cation-exchange resin, the reaction mixture was filtered and the filtrate was concentrated to give 0.27 g NAG–thiazoline as a hygroscopic brownish solid. Purity and identity to the known NAG–thiazoline  was confirmed by TLC and NMR analysis. Rf 0.21 (2:1:1:1 ethyl acetate/isopropanol/H2O/acetic acid); 1H NMR (400 MHz, CD3OD) δ 6.37 (d, 1H, J=7.0 Hz, H-1), 5.60 (d, 0.1H, J=4.7 Hz, residual GlcNAc H-1) 4.30 (m, 1H, H-2), 4.19 (t, 1H, J=4.0 Hz, H-3), 3.78 (dd,1H, J=2.6, 12 Hz, H-61), 3.65 (dd, 1H, J=6.1, 12 Hz, H-6), 3.62 (ddd, 1H, J=1.0, 3.8, 9.1 Hz, H-4), 3.28 (m, 1H, H-5), 2.22 (d, 3H, J=1.6 Hz, Thiazoline CH3). 13C NMR (100 MHz, CD3OD) δ 170.0 (SC=N), 90.80, 80.65, 76.40, 74.23, 71.43, 63.45, 24.25.
Growth of E. coli DH5α (Clonetech) cultures in the presence and absence of NAG–thiazoline was examined by culturing one colony in Luria–Bertani (LB) broth (1% tryptone peptone, 1% NaCl, 0.5% yeast extract) at 37 °C with shaking. After 16 h, a 1/50 dilution of the culture was performed in triplicate into fresh LB broth containing NAG–thiazoline and growth was monitored turbidometrically (OD600). Identical dilutions of the starter culture were inoculated into fresh LB broth alone to serve as controls.
In other experiments to examine the effect of NAG–thiazoline on penicillin-induced autolysis, cultures of E. coli DH5α were grown in LB broth at 37 °C to a cell density of OD600 of 0.18 (early exponential phase) and divided into three aliquots. Each aliquot was then supplemented with either nothing (to serve as controls), 5 μg/ml (1.7 × MIC) ampicillin, or 5 μg/ml ampicillin and 300 μg/ml NAG–thiazoline. Subsequent growth at 37 °C was monitored turbidometrically for 5 h while samples were withdrawn periodically for microscopic examination.
2.3Morphological effect of NAG–thiazoline on E. coli
Phase contrast microscopy of unstained wet mounts of cells was conducted using Nomarski optics on a Leica microscope with differential interference contrast. Images were captured and then processed with Adobe Photoshop software.
For scanning electron microscopy, cells were harvested from 1 ml samples of cultures by centrifugation at 8000g for 10 min at 4 °C. The bacterial cell pellet was washed in phosphate buffered saline to remove any residual broth. The pellet was then suspended in 70 mM Sorensen's phosphate, pH 6.8, placed on a 0.2 μm polycarbonate membrane filter (Poetics Corp. Livermore, CA), and fixed with 2% gluteraldehyde in phosphate-buffered saline for 1 h and post-fixed with OsO4. The samples were then rinsed in several changes of buffer, dehydrated through a series of ethanol washes, critical point dried (Ladd Industries) using carbon dioxide, and sputter coated with 20 nm of gold/palladium in a Hummer VII sputter coater (Anatech Corp. Alexandria, VA). To visualize the organisms, the filters were scanned using a Hitachi S-570 SEM (Tokyo, Japan), and images were collected directly from the SEM using Quartz PCI software (Quartz Imaging Corp. Vancouver, BC).
2.4Affect of NAG–thiazoline on LPS profile of E. coli
Cultures of E. coli DH5α grown for 6 h (late exponential phase) in LB containing 0 or 600 μg/ml NAG–thiazoline (each in triplicate) were harvested by centrifugation (6000g, 10 min, 4 °C) and the cells were resuspended in 100 μl of NuPAGE® (Invitrogen Life Technologies Inc.) sample buffer for analysis of their LPS profiles . After incubation at 100 °C for 10 min, 50 μg of Proteinase K was added and incubated at 60 °C for 1h. Samples were then boiled at 100 °C for 10 min and diluted 1/10 before loading onto a NuPAGE® 4–12% gradient electrophoresis gel. Samples were subjected to electrophoresis for 80 min at 120 V and the LPS was detected by silver staining.
2.5Cell-surface hydrophobicity assay
Surface hydrophobicity of cells grown in the presence and absence of NAG–thiazoline was assessed using the bacterial adhesion to hydrocarbon (BATH) assay of Rosenberg et al. . Cells grown to late exponential phase in LB broth containing 0, 100, 300, or 600 μg/ml of NAG–thiazoline were harvested by centrifugation and washed with 3 ml of 150 mM phosphate buffer, pH 7.1 containing 30 mM urea and 0.8 mM MgSO4. Triplicate samples of the washed cells were resuspended in the same buffer to an OD400 of 1.0, transferred to glass test tubes, and treated with 0.1 ml hexadecane. Following incubation for 15 min at 37 °C with shaking, the samples were vortexed for 2 min and the phases were allowed to separate for 10 min. The OD400 of each of the aqueous phases was measured and expressed as a percentage of the difference between the adhesion to the glass and adhesion to hexadecane.