- Efficient chemoenzymatic synthesis of O-linked sialyl oligosaccharides
-
The tumor associated Tn (GalNAcα(1-1)-Thr/Ser)- and T (Galβ(1-3)-GalNAcα(1-1)Thr/Ser)-antigens and their sialylated derivatives are present on the surface of many cancer cells. Preparative synthesis of these sialylated T- and Tn-structures has been achieved mainly from a chemical synthetic approach due to the lack of the required glycosyltransferases. We demonstrate a flexible and efficient chemoenzymatic approach for using recombinant sialyltransferases including a chicken GalNAcα2,6-sialyltransferase (chST6GalNAc I) and a porcine Galβ(1-3)GalNAcα-2,3-sialyltransferase (pST3Gal I). Using these enzymes, the common O-linked sialosides Neu5Acα(2-6)GalNAcα(1-1)Thr, Galβ(1-3)[Neu5Acα(2-6)] GalNAcα(1-1)Thr, Neu5Acα(2-3)Galβ(1-3)GalNAcα(1-1)Thr, and Neu5Acα(2-3)Galβ(1-3)[Neu5Acα(2-6)]GalNAcα(1-1)Thr were readily prepared at preparative scale. The chST6GalNAc I was found to require at least one amino acid (Thr/Ser) for optimal activity, and is thus an ideal catalyst for synthesis of synthetic glycopeptides and glycoconjugates with O-linked glycans.
- Blixt, Ola,Allin, Kirk,Pereira, Laura,Datta, Arun,Paulson, James C.
-
-
Read Online
- Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: Comparison of substrate flexibility of three microbial CMP-sialic acid synthetases
-
Three microbial CMP-sialic acid synthetases were cloned from Neisseria meningitidis, Streptococcus agalactiae, and Escherichia coli, respectively. Their activities in the production of CMP-sialic acid analogs were compared by HPLC analysis. The N. meningitidis synthetase was used in the preparative synthesis of eight CMP-sialic acid derivatives in a one-pot two-enzyme system. Three C terminal His6-tagged recombinant microbial CMP-sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP-sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP-sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP-sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100-200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.
- Yu, Hai,Yu, Hui,Karpel, Rebekah,Chen, Xi
-
p. 6427 - 6435
(2007/10/03)
-
- Synthesis of CMP-sialic acid conjugates: Substrates for the enzymatic synthesis of natural and designed sialyl oligosaccharides
-
The syntheses of several congeners of CMP-NeuAc are described. These compounds are substrates for enzymatic glycosylation.
- Chappell, Mark D.,Halcomb, Randall L.
-
p. 11109 - 11120
(2007/10/03)
-