The synthesis of regiospecifically 13C-labeled compounds of a biosynthetic precursor of lipid A and its analogue with shorter acyl chains is described. d-(6-13C)Glucose was converted into a suitably protected glucosamine derivative via 1,6-anhydro-β-d-(6-13C)glucose. After coupling with glycosyl donors, the desired compounds were synthesized through a 6-step reaction sequence. The total yields were 1.7% for the biosynthetic precursor, and 6.4% for the short acyl analogue, respectively, for a total of 18 steps from d-(6-13C)glucose.

The detailed conformational analysis of a single molecule of the tetraacyl biosynthetic precursor-type lipid A and its characteristic supramolecular assembly in aqueous SDS-micelles are described. Regular molecular arrangements were observed by detailed analysis of the NMR spectra of synthetically pure specimens, including regiospecifically 13C-labeled ones. NMR analysis of a biologically inactive precursor-type analogue with four shorter acyl chains demonstrated its conformational flexibility, indicating the importance of hydrophobic interactions for maintaining the conformation of such molecules.

During the course of a conformational study of lipid A, which is a bioactive entity of lipopolysaccharide of the Gram-negative bacterial cell surface, the molecular conformation of its tetraacyl biosynthetic precursor in dimethyl sulfoxide was unambiguously determined by means of NMR using both 6-13 C-labeled and nonlabeled synthetic specimens. The conformation of the hydrophilic moiety was determined by an NMR analysis based on the spin-coupling constants and nuclear Overhauser enhancement data around the glycosidic linkage. The whole molecular shape of the glycolipid was then elaborated with the aid of molecular mechanics calculations.

In order to investigate precise structural requirements for expression of the biological activity of lipid A, new structural analogs were synthesized via improved, highly efficient preparative routes. The number and chain length of the fatty acyl groups proved to have crucial influence on the biological activity. For the study of conformation relevant to activity of lipid A by means of NMR spectrometry, lipid A analogs [13C]-labeled at the 6-position were then synthesized starting from 6-[13C]-glucose. Precise analysis of J CH and NOE data disclosed a particular molecular conformation and characteristic supramolecular assembly in an aqueous SDS micelle solution of the tetraacyl biosynthetic precursor molecule. The observed mode of assembly may reflect the actual behavior of lipid A molecules in the cell surface outer membrane of living bacteria. A biologically inactive, artificial, tetra-acyl analog having shorter acyl chains exhibits, by contrast, distinct conformations with no sign of supramolecule formation. The acyl moieties in lipid A are thus found to play an important role in regulating the overall conformation of the hydrophilic backbone.

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