Combined chemoenzymatic synthisis of modified neo-glycosaminoglycans - ways to new glyco-drugs and -materials
Final Report Abstract
Joint preliminary work proposes that the combination of glycosyltransferases with galactose oxidase and sulfotransferases should be an ideal multi-enzyme system to obtain novel modified neo-glycosaminoglycans. LacdiNAc, GalNAc(beta1-4)GlcNAc(beta1-R, containing disaccharide structures should be synthesized by the combination of a UDP-GalNAc specific mutant of human beta1,4galactosyltransferase-1 (beta4GalT-1(Y284L). Further modification reactions including the use galactose oxidase/NaClO2 for oxidation and sulfotransferases for sulfation of terminal GalNAc moieties, respectively, should lead to novel glycoconjugates. Presentation of LacdiNAc glyco-ligands as repeating linear glycosamino-structures or on multivalent glyco-clusters should enhance their bioactivity. The multilinear presentation of N-acetyl-D-hexosamines should be realized by synthesis of poly-LacdiNAc combining the mutant beta4GalT-1(Y284L) with beta3GlcNAcT from H. pylori. We first elaborated optimized reaction conditions for the enzymatic synthesis of poly-LacNAc oligomers employing human beta4GalT-1 and bacterial beta3GlNAcT. Combination of both glycosyltransferases in a one-pot synthesis yielded poly-LacNAc oligomers with up to six LacNAc units. The transfer of reaction conditions to the synthesis of poly-LacdiNAc turned out to be difficult. The main obstacle was the poor acceptance of beta3GlNAcT from Helicobacter pylori for LacdiNAc. However, we succeeded in synthesizing the corresponding trisaccharide with very low yield. The structure was confirmed by mass spectrometry and NMR spectroscopy. Finally, the reaction conditions with a high excess of UDP-GlcNAc over the acceptor substrate and high enzyme activities turned out to be unfavorable for further enzymatic elongation reactions. The synthesis of multivalent LacdiNAc glyco-clusters was difficult to achieve because of multi-parameter optimization of such biocatalytic reactions. For the current project we decided to focus on a novel strategy for the synthesis of bivalent LacdiNAc-linker structures as novel LacdiNAc-based glyco-structures. Four bivalent GlcNAc-linker structures were chemically synthesized and identified as novel acceptor substrates for beta4GalT-1(Y284L). The mutant galactosyltransferase worked as a perfect synthetic tool accomplishing even quantitative glycosylation at both acceptor sites with absolute regioselectivity for the C-4 position. The enzymatic oxidation reaction of bivalent LacdiNAc compounds turned out to be a challenging task and is subject of ongoing collaborative work between our groups. In a complementary work, the oxidation reaction of LacdiNAc and terminal beta-Gal moieties of poly-LacNAc oligomers by galactose oxidase was optimized. These conditions could be transferred to bivalent LacdiNAc and LacdiNAc-terminated poly-LacNAc structures. In order to accomplish enzymatic sulfation of the synthesized LacdiNAc glycoconjugates two sulfotransferase CHST1 and CHST6 were cloned. The expression of active sulfotransferases in E. coli turned out to be not possible giving exclusively insoluble protein (inclusion bodies) in E. coli. Complementary ongoing work investigates the expression and secretion of these sulfotransferases by Pichia pastoris. In addition, recombinant arylsulfotransferase (AST-IV) was successively produced for the in situ regeneration of the chemically labile co-substrate PAPS (3'-phosphoadenosine-5'-phosphosulfate).
Publications
- Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - way to new glycodrugs and materials. Glycobiology, Vol. 19. 2009, Issue 5, pp. 509–517.
Bojarová, P., Křenek, K., Wetjen, K., Adamiak, K., Pelantová, H., Bezouška, K., Elling, L., Křen, V.
(See online at https://doi.org/10.1093/glycob/cwp010) - (2011). Combinatorial One-Pot Synthesis of Poly-N-acetyllactosamine Oligosaccharides with Leloir-Glycosyltransferases.
Advanced Synthesis & Catalysis, Vol. 353. 2011, Issue 13 (Special Issue: Biocatalysis), pp. 2492–2500.
Rech, C., Rosencrantz, R. R., Křenek, K., Pelantová, H., Bojarová, P., Römer, C. E., Hanisch, F.-G., Křen, V., Elling, L.
(See online at https://dx.doi.org/10.1002/adsc.201100375) - Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors. Carbohydrate Research, Vol. 346. 2011, Issue 12, pp. 1599–1609.
Drozdová, A., Bojarová, P., Křenek, K., Weignerová, L., Henßen, B., Elling, L., Christensen, H., Jensen, H., Pelantová, H., Kuzma, M., Bezouska, K., Krupová, M., Adámek, D., Slámová, K., Křen, V.
(See online at https://doi.org/10.1016/j.carres.2011.04.043) - Chemo-enzymatic modification of poly-N-acetyllactosamine (LacNAc) oligomers and N,N-diacetyllactosamine (LacDiNAc) based on galactose oxidase treatment. Beilstein journal of organic chemistry, Vol. 8. 2012. pp. 712-725.
Kupper, C. E., Rosencrantz, R. R., Henßen, B., Pelantová, H., Thönes, S., Drozdová, A., Křen, V., Elling, L.
(See online at https://doi.org/10.3762/bjoc.8.80) - Corrigendum to “Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors” [Carbohydr. Res. 346 (2011) 1599–1609]. Carbohydrate Research, Vol. 373. 2013, page 108.
Drozdová, A., Bojarová, P., Křenek, K., Weignerová, L., Henßen, B., Elling, L., Christensen, H., Jensen, H. H., Pelantová, H., Kuzma, M., Bezouška, K., Krupová, M., Adámek, D., Slámová, K., Křen, V.
(See online at https://doi.org/10.1016/j.carres.2013.04.020)
