Highly efficient chemoenzymatic synthesis and facile purification of α-Gal pentasaccharyl ceramide Galα3nLc4βCer

Abstract

A highly efficient chemoenzymatic method for synthesizing glycosphingolipids using α-Gal pentasaccharyl ceramide as an example is reported here. Enzymatic extension of the chemically synthesized lactosyl sphingosine using efficient sequential one-pot multienzyme (OPME) reactions allowed glycosylation to be carried out in aqueous solutions. Facile C18 cartridge-based quick (<30 minutes) purification protocols were established using minimal amounts of green solvents (CH₃CN and H₂O). Simple acylation in the last step led to the formation of the target glycosyl ceramide in 4 steps with an overall yield of 57%.

Scheme 1

An efficient chemoenzymatic strategy for synthesizing complex glycosphingolipids by enzymatic extension of lactosyl sphingosine (LacβSph, 3) using sequential one-pot multienzyme (OPME) reactions with C18-cartridge purification after each glycosylation reaction followed by a simple acylation process. The structures of target α-Gal pentasaccharyl sphingosine Galα3nLc₄βSph (2) and α-Gal pentasaccharyl ceramide Galα3nLc₄βCer (1) are also shown.

Scheme 2

Synthesis of sphingosine acceptor 12. Reagents and conditions: (a) TfN₃, CuSO₄, Et₃N, MeOH, CH₂Cl₂, H₂O, r.t., 6h, > 90%; (b) TBDPSCl, Et₃N, DMAP, CH₂Cl₂, r.t., 8h, 98%; (c) SO₂Cl₂, Et₃N, CH₂Cl₂, 0°C, 0.5–1 h; (d) RuCl₃.3H₂O, NaIO₄, CCl₄:CH₃CN:H₂O (1:1:1), r.t., 2 h; 89% in two steps (e) (i) Bu₄NI, DBU, Toluene, reflux, 4 h; (ii) H₂SO₄/H₂O/THF, r.t., 45 min; 85% in two steps (f) BzCl, DMAP, Et₃N, CH₂Cl₂, 0°C to r.t., 12 h, 95%; (g) HF Pyridine, THF, 0°C to r.t., 12 h, 97%.

Scheme 3

Synthesis of lactosyl sphingosine (LacβSph, 5). Reagents and conditions: (a) BF₃^·OEt₂, CH₂Cl₂, -18 °C, 3 h, 90%; (b) NaOMe, MeOH, r.t., 14 h; (c) 1,3-propanedithiol, Et₃N, pyridine-water (1:1 v/v), 50 °C, 36 h, 94% in two steps.

Scheme 4

High-yield synthesis of α-Gal pentasaccharyl ceramide Galα3nLc₄βCer (1) by enzymatic extension of lactosyl sphingosine (LacβSph, 5) using sequential one-pot multienzyme (OPME) reactions with C18-cartridge purification for the formation of α-Gal pentasaccharyl sphingosine Galα3nLc₄βSph (2) followed by a simple acylation reaction.