1H, 13C, and 31P nuclear magnetic resonance spectral assignments for tobramycin, 2″-(Adenosine-5′-phosphoryl)-tobramycin and 2″-(Adenosine-5′-thiophosphoryl)-tobramycin

Abstract

Two enzymatically modified derivatives of tobramycin have been prepared by gentamicin nucleotidyl transferase-catalyzed adenylylation of tobramycin, using ATP and ( S p)-ATPαS as adenylylation substrates. (EC 2.7.7.46). The 1H, 13C, and 31P NMR spectra have been assigned for tobramycin, 2″-(adenosine-5′-phosphoryl)-tobramycin (TbAMP) and 2″-(adenosine-5′-thiophosphoryl)-tobramycin (TbAMPS). Several one -and two-dimensional NMR techniques have been utilized, notably, 1H 1H homonuclear correlation spectroscopy at 470 or 500 MHz and 13C 1H heteronuclear correlation spectroscopy at 50.3 MHz. The 1H assignments for tobramycin are similar to those previously reported for rings I and III of kanamycin A. The 13C assignments for tobramycin were similar to those previously reported, except for reversal of the assignments for anomeric carbons in the glycosyl rings. The 1H and 13C assignments for tobramycin were used to guide the assignments of the spectra for TbAMP and TbAMPS. Nearly complete assignments were obtained for these two derivatives of tobramycin. From the measured proton coupling constants, only small conformational changes were observed upon modification of tobramycin by adenylylation. From the proton and carbon spectra of the adenylylated derivatives the 2″ position is shown to be the site of adenylation. Large downfield shifts of the 2″ proton and carbon resonances are easily observed and are more pronounced for TbAMPS than for TbAMP.