Combination of Glycosyltransferases and a Glycosynthase in Sequential and One‐Pot Reactions for the Synthesis of Type 1 and Type 2 N‐Acetyllactosamine Oligomers

Abstract

In three words,h ow would you describe your research? Retrosynthetic biocatalysis (of) glycoconjugates. What aspects of this project do you find most exciting? The presented results were only possible by the joint expertise of two research groups and their dedicatedd octoral students within the DFG InternationalR esearch TrainingG roup "SeleCa-Selectivity in Chemo-and Biocatalysis" at RWTH Aachen University and Osaka University.T he Elling group is interested in the synthesis of glycan ligands and glycan inhibitors of galec-tins, al ectin class involved in tumor biology.W ew ere wondering about the possibility to synthesize oligomers with type 1 N-acetyllactosamine (LacNAc) repeats(yellow/blue sugar combination in the cover picture),w hich is known as an unusual glycan structure in somec ancer types. For this goal the expertise of the Pietruszkag roup in the use of glycosynthases was highly valuable. The combination of two glycosyltransferases (blue and red protein structures) and one galactosynthase (yellow protein structure) renders al ibrary of novel poly-LacNAc structures (the yellow/blue and red/blue sugars) in different combinations.T he most exciting aspect is that poly-LacNAc oligomers-not yet found in nature-presenting alternating type 2/type 1L acNAc structures can now be studied for galectin binding and inhibition. What was the inspiration for this cover design? Biocatalysis and especially multi-enzyme systems are av iable alternative fort he synthesis of complex glycan structures. The propera daptation of reaction conditions makes "juggling" with as et of enzymesp ossible. We present novel glycoconju-gates, not yet realized by chemical carbohydrate synthesis. Dedication and Acknowledgements Our paper is dedicated to our colleague Prof. Dr.W olf-Dieter Fessner on the occasion of his 60 th birthday.W ea cknowledge financial supporto fo ur work, as well as the realization of this cover picture, by the DFG within the IRTG 1628 "SeleCa". The front cover artwork for Issue 19/2015 is provided by the groups of Prof. Lothar Elling and Prof. Jçrg Pietruszka (RWTH Aachen University and Heinrich-Heine-Universität Düsseldorf,r espectively). The image shows the combination ("juggling") of two glycosyltransferases(blue and red protein structures) and agalactosynthase (yellow proteinstructure) for the synthesis of ag lycan library. See the Full Paper itself at http://dx.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands

“…However, compound 3 was formed as main product (after 72 h) when β3GlcNAcT was in five-fold excess ( Figure 1 c). In comparison to the one-pot synthesis using an engineered glycosynthase and β3GlcNAcT [ 16 ] the combination of both glycosyltransferases in one-pot synthesis is limited towards the synthesis of the disaccharide and trisaccharide products.…”

“…We conclude that there is a rising need for the synthesis of glycoconjugates containing LacNAc type 1 repeats to gain novel insights into the interaction of carbohydrates and carbohydrate binding proteins, known as lectins. Recently, we reported on the synthesis of LacNAc type 1 oligomers consisting of up to four repetitive LacNAc units by combining a glycosyltransferase and an engineered glycosynthase [ 16 ]. However, the synthesis required the chemically synthesized α-galactopyranosyl fluoride (αGalF) as specific donor substrate, which is prone to hydrolysis.…”

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

“…The synthesis of lactulose and galactosyl-oligosaccharides from lactose and d -fructose was demonstrated by Grubiak et al [ 32 ]. We further studied the regiospecificity of the recombinant β-glycosidase from Pyrococcus with previously synthesized Galβ(1,3/4/6)GlcNAc-linker- t Boc regioisomers [ 33 , 34 , 35 ]. The recombinant enzyme shows higher preference for the hydrolysis of β(1,4)- than for β(1,6)-linked galactosides.…”

Synthesis of Glycosides by Glycosynthases

“…Yang et al broadened the same synthesis to a FRET-probe of lyso -G M3 in order to visualize enzymatic processing of the glycolipid [ 37 ]. Another combination of glycosyltransferases and a glycosynthase was performed in sequential and also one-pot reactions by Henze et al [ 38 ]. The report demonstrates the synthesis of N -acetyllactosamine type 1 (-3-Gal-β1,3-GlcNAc-1-) and type 2 (-3-Gal-β1,4-GlcNAc-1-) oligomers by combining the glycosynthase His 6 BgaC D233G ( B. circulans ) either in a one-pot or sequentially with the glycosyltransferases β3GlcNAcT ( H. pylori ) and the βGalT-1 (human origin) [ 39 ].…”