A novel two-step enzymatic synthesis of blastose, a β-d-fructofuranosyl-(2↔6)-d-glucopyranose sucrose analogue

- A novel two-step biosynthetic route to obtain blastose is described.
- Trehalose is regioselectively fructosylated by B. subtilis levansucrase.
- B. subtilis levansucrase may use sucrose or levan as fructosyl donor.
- Fructose is transferred to trehalose to yield O-β-d-Fruf-(2↔6)-trehalose.
- O-β-d-Fruf-(2→6)-trehalose is hydrolyzed by trehalase to yield blastose.

Blastose, a natural disaccharide found in honey, is usually found as a byproduct of fructo-oligosaccharide synthesis from sucrose with fructosyltransferases. In this study, we describe a novel two-step biosynthetic route to obtain blastose, designed from a detailed observation of B. subtilis levansucrase (SacB) acceptor structural requirements for fructosylation. The strategy consisted first in the synthesis of the trisaccharide O-β-d-Fruf-(2↔6)-O-α-d-Glcp-(1↔1)-α-d-Glcp, through a regioselective β-d-transfructosylation of trehalose (Tre) which acts as acceptor in a reaction catalyzed by SacB using sucrose or levan as fructosyl donor. In this reaction, levansucrase (LS) transfers regioselectively a fructosyl residue to either C6-OH group of the glucose residues in Tre. The resulting trisaccharide obtained in 23% molar yield based on trehalose, was purified and fully characterized by extensive NMR studies. In the second step, the trisaccharide is specifically hydrolyzed by trehalase, to obtain blastose in 43.2% molar yield based on the trisaccharide. This is the first report describing the formation of blastose through a sequential transfuctosylation-hydrolysis reaction.