Prior to NMR analyses, purified Tf GL2, 1-P-myo-inositol (Sigma-Aldrich), l -threo-sphinganine C18 (Sigma-Aldrich) and d -erythro-sphinganine C18 (Sigma-Aldrich) were dissolved in deuterated methanol (Deutero GmbH, Kastellaun, Germany) and placed in 5-mm NMR tubes.
All 1D (1H, 13C and 31P) and 2D NMR 1H,1H COSY, TOCSY as well as 1H,13C HSQC, 1H,13C HMBC (only for Tf GL2), and 1H,31P HMQC experiments performed on Tf GL2 and 1-P-myo-inositol were recorded at 27 °C on a Bruker DRX Avance III 700 MHz spectrometer (operating frequencies of 700.75 MHz for 1H NMR, 176.2 MHz for 13C NMR and 283.7 MHz for 31P) and standard Bruker software (TopSpin ver. 3.2). COSY and TOCSY experiments were recorded using data sets (t1 by t2) of 4096 by 512 points, with 6 scans for COSY, and 16 for TOCSY. The TOCSY experiments were carried out in the phase-sensitive mode with mixing times of 120 ms. The 1H,13C correlations measured in the 1H-detected mode via HSQC-DEPT with proton decoupling in the 13C domain and HMBC spectra were acquired using data sets of 4096 by 512 points and 64 scans (HSQC) and 80 scans (HMBC) for each t1 value. HMBC spectra were adjusted to J coupling constant value of 145 Hz and long range proton carbon coupling constant of 10 Hz. Chemical shifts were reported relative to internal methanol (δH 3.34, δC 49.86) [24 ] or external phosphoric acid (δP 0.0) [25 (link)].
All 1D (1H, 13C and 31P) and 2D NMR 1H,1H COSY, TOCSY as well as 1H,13C HSQC, 1H,13C HMBC (only for Tf GL2), and 1H,31P HMQC experiments performed on Tf GL2 and 1-P-myo-inositol were recorded at 27 °C on a Bruker DRX Avance III 700 MHz spectrometer (operating frequencies of 700.75 MHz for 1H NMR, 176.2 MHz for 13C NMR and 283.7 MHz for 31P) and standard Bruker software (TopSpin ver. 3.2). COSY and TOCSY experiments were recorded using data sets (t1 by t2) of 4096 by 512 points, with 6 scans for COSY, and 16 for TOCSY. The TOCSY experiments were carried out in the phase-sensitive mode with mixing times of 120 ms. The 1H,13C correlations measured in the 1H-detected mode via HSQC-DEPT with proton decoupling in the 13C domain and HMBC spectra were acquired using data sets of 4096 by 512 points and 64 scans (HSQC) and 80 scans (HMBC) for each t1 value. HMBC spectra were adjusted to J coupling constant value of 145 Hz and long range proton carbon coupling constant of 10 Hz. Chemical shifts were reported relative to internal methanol (δH 3.34, δC 49.86) [24 ] or external phosphoric acid (δP 0.0) [25 (link)].