Structural studies of a phosphocholine substituted beta-(1,3);(1,6) macrocyclic glucan from Bradyrhizobium japonicum USDA 110

Abstract

In our previous in vivo 31P study of intact nitrogen-fixing nodules (Rolin, D.B., Boswell, R.T., Sloger, C., Tu, S.I. and Pfeffer, P.E., 1989 Plant Physiol. 89, 1238-1246), we observed an unknown phosphodiester. The compound was also observed in the spectra of isolated bacteroids as well as extracts of the colonizing Bradyrhizobium japonicum USDA 110. In order to characterize the phosphodiester in the present study, we took advantage of the relatively hydrophobic nature of the material and purified it by elution from a C-18 silica reverse-phase chromatography column followed by final separation on an aminopropyl silica HPLC column. Structural characterization of this compound with a molecular weight of 2271 (FAB mass spectrometry), using 13C-1H and 31P-1H heteronuclear 2D COSY and double quantum 2D phase sensitive homonuclear 1H COSY NMR spectra, demonstrated that the molecule contained beta-(1,3); beta-(1,6); beta-(1,3,6) and beta-linked non-reducing terminal glucose units in the ratio of 5:6:1:1, respectively, as well as one C-6 substituted phosphocholine (PC) moiety associated with one group of (1,3) beta-glucose residues. Carbohydrate degradation analysis indicated that this material was a macrocyclic glucan, (absence of a reducing end group) with two separated units containing three consecutively linked beta-(1,3) glucose residues and 6 beta-(1,6) glucose residues. The sequences of beta-(1,3)-linked glucose units contained a single non-reducing, terminal, unsubstituted glucose linked at the C-6 position and a PC group attached primarily to an unsubstituted C-6 position of a beta-(1,3)-linked glucose.