Effective Sugar Nucleotide Regeneration for the Large-Scale Enzymatic Synthesis of Globo H and SSEA4

Abstract

We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

“…In the preliminary enzymatic activity screening assay, the reaction of MtGalK was performed in a solution (100-200 mL) containing 8 mM Gal, 10 mM ATP, 5 mM MgCl 2 , 50 mM HEPES (pH 7.5), and 1 mg mL À1 purified MtGalK. The reaction was incubated at 37 8C for 0, 1, 2, 3, 4,5,6,7,8,9,10,15,20,25,30,40,50, 60, 90 and 120 min and was quenched by the addition of 200 mM EDTA to a final EDTA concentration of 20 mM. In a further optimal reaction condition analysis (temperature, metal ion cofactor and pH), the reaction was incubated for 5 min then quenched by the addition of EDTA.…”

“…To solve this problem, the first enzyme-based strategy for the synthesis of Globo H was developed in 2008, [29] and the gram-scale syntheses of Globo H, SSEA-3, and SSEA-4 by more effective sugar nucleotide regeneration methods were achieved in 2013. [30] Compared to the existing chemical synthetic methods for a-1,4-galactosidic bond construction, single step enzymatic routes bypass the required multistep synthetic manipulations. However, the aforementioned effective enzymatic syntheses require the preparation and screening of an array of enzymes for sugar nucleotide regeneration, which could be time-consuming and might not be easy to handle by a general organic synthesis laboratory.…”

Efficient enzymatic production of rebaudioside A from stevioside

“…To reduce the cost of supplying of UDP-glucose in such reactions, in situ regeneration of UDP-glucose is an advantageous option. 26,27) Here, recombinant E. coli containing pEUGT-SUS was induced and crude extracts (2 mU/mL enzymes) from the E. coli cells expressing UGT76G1 and AtSUS1 were added to the reaction mixtures, where the concentrations of stevioside was 0.6 mM, and the initial stevioside:UDP-glucose:sucrose mole ratios were 1:0.2:1 and 1:0.2:0, respectively (Fig. 5).…”

Design of Disaccharide Modules for a Programmable One‐Pot Synthesis of Building Blocks with LacNAc Repeating Units for Asymmetric N‐Glycans

“…The characteristics of the poly‐LacNHAc derivatives including molecular weight and structure were studied by HRMS, 1D and 2D NMR. Additionally, we carried out enzymatic glycosylations to further elongate the poly‐LacNAc moiety according to strategies reported by our group . Compound 33 was treated with β‐1, 4‐galactosyltransferase and uridine diphosphate galactose (UDP‐galactose) at 37 °C to install the galactose residue onto the terminus of compound 34 , then α‐2,3‐sialyltransferase was utilized to install the Neu5Ac residue onto the galactosyl terminal to obtain sialic acid‐containing compound 35 in a total yield of 71 %.…”