154235-70-8

Upstream product

• 146-91-8 guanosine-5'-diphosphate 
• 59-56-3 mannose-1-phosphate 
• 991-43-5 guanosine 5'-diphospho-D-mannose 

Downstream Products

• 64396-27-6 Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc 

Relevant academic research and scientific papers

Multi-enzyme Cascades for the In Vitro Synthesis of Guanosine Diphosphate L-Fucose

Recombinant Leloir glycosyltransferases can be exploited to synthesize a wide range of HMOs using in vitro biocatalytic reactions. However, high costs and unavailability of bulk amounts of most nucleotide sugars, such as guanosine diphosphate L-fucose (GDP-Fuc), are major obstacles for the efficient large-scale synthesis. Here, we report two novel multi-enzyme cascades for the synthesis of GDP-Fuc from readily available and low cost precursors. The first cascade was developed to produce GDP-Fuc from guanosine (Guo), fucose (Fuc), polyphosphate (PolyPn) and catalytic amounts of adenine triphosphate (ATP). GDP-Fuc was produced with a final concentration of 7 mM (4.1 g/L) and a reaction yield of 68 % from Guo and Fuc within 48 h with a biocatalyst load of 0.34 genzyme/gproduct. A second cascade, consisting of ten enzymes and eleven reactions was developed to carry out the synthesis from mannose (Man), Guo, PolyPn, L-glutamine (L-Glu) and catalytic amounts of ATP, and nicotinamide adenine dinucleotide phosphate (NADPH). Utilizing this cascade, GDP-Fuc was produced with a final concentration of 7.6 mM (4.5 g/L) and a reaction yield of 72 % in a reaction time of 48 h with a biocatalyst load of 0.97 genzyme/gproduct. Finally, a method for chromatographic purification of GDP-Fuc was established achieving product purities of 90.5 %.

Chemical-enzymatic synthesis and conformational analysis of sialyl lewis x and derivatives

Sialyl Lewis x and derivatives have been synthesized using 0-1,4-galactosyltransferase and recombinant α-2,3-sialyltransferase and α-l,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-N-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition. The enzymatic procedures have also been used in the synthesis of 2′-deoxy-LacNAc, 2′-amino-2′-deoxy-LacNAc, 2-azido-Lac, Lewis x, the Lewis x analog with GlcNAc replaced with 5-thioglucose, [Gal-l-13C]-LacNAc, [Gal-1-13C]-sialyl Lewis x, and the corresponding terminal glycal. The synthesized 13C-labeled sialyl Lewis x and intermediates (including Lewis x and sialyl LacNAc) were used for conformational study using NMR techniques combined with calculations based on GESA and MM2 programs. GESA calculation of sialyl Lewis x gave four minimum-energy conformers, and the two (A and B) consistent with NMR results were further refined with MM2 calculation. The one (A') with lower energy was picked as the preferred conformer which had all internuclear distances and glycosidic torsional angles consistent with the NMR analysis. The glycosidic torsional angle ψ of Gal-GlcNAc, for example, was determined to be 18° on the basis of the coupling between Gal-1-13C and GlcNAc, while the predicted value was 15°. The tetrasaccharide appears to form a well-defined hydrophilic surface along NeuAc-Gal- Fuc, and a hydrophobic face underneath NeuAc-Gal-GlcNAc. Comparing the conformation of sialyl Lewis x to sialyl Lewis a indicates that the recognition domain of sialyl Lewis x mainly comes from the sialic acid - galactose - fucose residues.