Figure 2.

(A) The structure of S. aureus LTA as determined from NMR spectroscopic analysis. Color coding identifies the methine group of the sn-glycerol repeating unit (green), three resonance signals of d-N-acetylglucosamine (GN, yellow), d-alanine (d-Ala, blue), gentiobiose-sn-glycerol (gray), and fatty acids (orange). (B) ¹H NMR spectrum (600 MHz, 300 K) of LTA obtained after butanol extraction. The resonance signal intensities allow for the quantification of the substituents and the average glycerophosphate chain length (45 < n < 50). Microheterogeneity leads to signal broadening as visible in the expansion of the anomeric proton of d-N-acetylglucosamine. (C) Hydrolysis of the d-alanine esters was performed at pH 8.5 in the NMR tube overnight. No cleavage of the fatty acids was observed under these conditions. The anomeric proton of N-acetylglucosamine resolved to a doublet with a vicinal coupling constant of 3.6 Hz as expected for an α-glycosidic bond. The gentiobiose resonances are highlighted in gray. (D) S. aureus LTA obtained after standard phenol extraction was characterized by a reduced chain length and significant hydrolysis of d-alanine esters. The alkyl chains of the fatty acids served as reference intensities for comparison of the three ¹H NMR spectra, B–D.