Figure 1.
SOAT catalyzes the synthesis of AcGD1b.
(A) TLCs comparing the products obtained using two methods of O-acetylation for GD1b: chemical synthesis (CS) with trimethylortoacetate and enzymatic synthesis (ES) with SOAT. (B) HPLC chromatogram of the enzymatic reaction and TLC of products purified by HPLC.
Figure 2.
SOAT O-acetylates GD1b in the alpha-2,8 bound sialic acid.
Negative electrospray-MS spectra of GD1b (A) and AcGD1b (B) gangliosides species that contain a ceramide with a LCB of 18∶1 and F.A. of 18∶0. (d18∶1/18∶0). The m/z values are indicated for every peak.
Figure 3.
Chemical structures of GD1b and AcGD1b (Neurostatin).
Figure 4.
SOAT O-acetylates GD1b in the C-9 position of the alpha-2,8 bound sialic acid.
(A) Comparison of the 700 MHz 1H-NMR spectra of GD1b and AcGD1b in DMSO-d6/D2O 98∶2 at 298 and 295 K, respectively. (B) Magnified 1H-NMR spectra in the anomeric and acetyl region of GD1b and AcGD1b. (C) Comparison of the anomeric region in the 2D 1H-13C multiplicity-edited HSQC spectra of GD1b and AcGD1b. In this spectrum signals arising from CH3 and CH groups have different sign (positive) to those of CH2 groups (negative). Cross peaks are positive (several contours) for CH2 groups and negative (one contour) for CH and CH3 groups. In both cases the corresponding 1D 1H specrum is shown above the 2D spectrum.
Figure 5.
SOAT catalyzes the synthesis of AcGD1a.
TLC of the products obtained in the reactionof GD1a with SOAT (ES).
Figure 6.
SOAT O-acetylates GD1a in a alpha-2,3 bound sialic acid.
Negative electrospray-MS spectra of GD1a (A) and AcGD1a (B) gangliosides species that contain a ceramide with a LCB of 18:1 and F.A. of 18:0. (d18:1/18:0). The m/z values are indicated for every peak.
Figure 7.
SOAT O-acetylates GD1a in the outer alpha-2,3 bound sialic acid.
TLC of GD1a and AcGD1a after neuraminidase and alkaline treatment compared to standard gangliosides (GM3, GM2, GM1a and GD1a), Stds. Neuraminidase digestion of AcGD1a (lane 7) or neuraminidase digestion plus alkaline hydrolysis of AcGD1a (lane 8) gives only GM1a as product, demonstrating that AcGD1a is specifically O-acetylated in the outermost alpha-2,3 bound sialic acid and not in the inner alpha-2,3 bound sialic acid to the galactose of lactosyl-ceramide.
Table 1.
Summary of the products obtained in the reactions of different gangliosides with SOAT.