Phase-Transfer Catalyzed Microfluidic Glycosylation: A Small Change in Concentration Results in a Dramatic Increase in Stereoselectivity

Phase-transfer catalysis (PTC) is widely used in glycochemistry for the preparation of aryl glycosides by the glycosylation reaction. While investigating the possibility of synthesis of 4-(3-chloropropoxy)phenyl sialoside (Neu5Ac-OCPP) from N-acetylsialyl chloride with O-acetyl groups (1), we have recently discovered a strong dependence of the PTC glycosylation outcome on the mixing mode: under batch conditions, only α-anomer of Neu5Ac-OCPP was obtained, albeit in low yield (13%), while under microfluidic conditions the yield of Neu5Ac-OCPP increased to 36%, although stereoselectivity decreased (α/β ≤ 6.2). Here, we report that the outcome of this reaction, performed under microfluidic conditions using a Comet X-01 micromixer (at 2 μL/min flow rate), non-linearly depends on the concentration of N-acetylsialyl chloride 1 (5–200 mmol/L). The target Neu5Ac-OCPP was obtained in a noticeably higher yield (up to 66%) accompanied by enhanced stereoselectivity (α/β = 17:1–32:1) in the high concentration range (C > 50 mmol/L), whereas the yield (10–36%) and especially, stereoselectivity (α/β = 0.9:1–6.2:1) were lower in the low concentration range (C ≤ 50 mmol/L). This dramatic stepwise increase in stereoselectivity above critical concentration (50 mmol/L) is apparently related to the changes in the presentation of molecules on the surface of supramers of glycosyl donor, which exist in different concentration ranges

Synthesis of Ganglioside Lactams Corresponding to Gm1-, Gm2-,Gm3-, and GM4-Ganglioside Lactones

Ganglioside lactams are potentially useful analogs of ganglioside lactones, which are highly immunogenic derivatives of gangliosides. The lactam corresponding to the GM3-lactone saccharide has been synthesized by sialylation of a suitably protected lactose derivative carrying an azido group in the 2'-position, followed by reduction and ring closure to form GM3-lactam. Glycosylation in the 4-position of the central saccharide unit gave the Gm2- and Gm1-lactam saccharides. By a similar route, a 2-azido-Gal derivative was sialylated and treated as above to give the GM4-lactam saccharide. Deprotection gave the GM2-4-lactam saccharides in water soluble form, whereas attempted deprotection of the Gm1-lactam caused its degradation. The GM3-lactam saccharide was coupled to ceramide, to afford the ganglioside lactam analog, and via a spacer to bovine serum albumin (BSA). The BSA conjugate was used as immunogen to raise monoclonal antibodies that cross-reacted with GM3-lactone. The antibodies were used in a histological staining of murine melanoma cells, clearly showing the presence of GM3-lactone on the cell surface. Keeping the GM2-4-lactam saccharides in D2O at 37 °C for 1 month caused marginal (0—11%) hydrolysis of the lactam ring