Specificity Toward Primary and Secondary Hydroxyls of Monosaccharides as Acceptor Substrates in Transphosphatidylation Catalyzed by Streptomyces sp. Phospholipase D

ABSTRACT

Glycophospholipids are a rather unexplored type of novel synthetic phospholipid that has shown interesting self‐assembly behavior in aqueous solution. The type of carbohydrate used in these conjugates has a surprisingly large effect on lipid self‐assembly and the type of vesicle structures formed when dispersed in excess aqueous solution. These lipids can be produced through enzymatic transphosphatidylation catalyzed by phospholipase D (PLD), but the tautomeric equilibria of carbohydrates lead to conjugates with varied linkage positions and anomeric forms. To clarify how carbohydrate structure affects both lipid self‐assembly and PLD selectivity, we performed detailed structural characterization of three previously unexplored glycophospholipid conjugates: phosphatidyl glucose (P‐Glu), phosphatidyl fructose (P‐Fru), and phosphatidyl xylose (P‐Xyl). Using mass spectrometry and NMR spectroscopy, we identified distinct and unexpected conjugation patterns. P‐Glu formed exclusively via the C6 primary hydroxyl, while P‐Fru yielded two products, 1‐P‐Fru and 6‐P‐Fru, demonstrating high PLD specificity for both primary positions. Surprisingly, P‐Xyl conjugated via the secondary C4 hydroxyl, indicating that PLD can preferentially react with a secondary alcohol even when minor amounts of xylofuranose containing a primary hydroxyl are present. These results provide new insight into the catalytic flexibility of PLD toward complex carbohydrate substrates and establish a structural basis for understanding the self‐assembly behavior of these novel glycophospholipids.

Practical applications : Understanding how phospholipase D selects different hydroxyl groups on complex carbohydrates enables more predictable and strategic synthesis of glycophospholipids. Such control is valuable for designing lipids with tailored self‐assembly properties for use in drug delivery, formulation science, and biomaterials. The demonstrated ability of PLD to form linkages at secondary hydroxyls expands the range of feasible carbohydrate substrates, opening new routes for enzymatic production of functional amphiphiles without relying on protecting‐group chemistry. The structural insights generated here can support the rational design of vesicle‐forming lipids with specific stability, morphology or encapsulation characteristics, facilitating their application in nanocarriers, emulsifiers, and sustainable surfactant systems.