Very Important Publication DOI: 10.1002/adsc.201500916 Towards Keratan Sulfate – Chemoenzymatic Cascade Synthesis of Sulfated N-Acetyllactosamine (LacNAc) Glycan Oligomers Bastian Lange, a Anna S ˇ imonovµ, b Thomas Fischçder, a Helena Pelantovµ, b Vladimír Kr ˇen, b and Lothar Elling a, * a Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, 52074 Aachen, Germany Fax: (+ 49)-241-802-387; phone: (+ 49)-241-802-8350; e-mail: l.elling@biotec.rwth-aachen.de b Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, CZ 142 20 Prague 4, Czech Republic Received: September 29, 2015; Revised: November 30, 2015; Published online: January 22, 2016 Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500916. Abstract: We report on a novel chemoenzymatic cas- cade for the synthesis of sulfated N-acetyllactos- amine [(3Galb1,4GlcNAcb1,) n , LacNAc] oligomer structures. Starting from a linker modified GlcNAc substrate di- and trisaccharides were first synthesized by sequential use of human b4-galactosyltransferase- 1(b4GalT-1) and b3-N-acetylglucosaminyltransferase from Helicobacter pylori (b3GlcNAcT). Subsequent regioselective chemical sulfation rendered the C-6 mono-, di-, and tri-O-sulfated products in good yields. Further enzymatic elongation by b4GalT- 1 and b3GlcNAcT in a sequential mode yielded 6-O- sulfated LacNAc oligomers up to hexasaccharide length with variable degrees of sulfation. These car- bohydrate structures mimic the sulfation pattern found in keratan sulfate and are potential ligands for different classes of glycan binding proteins. Keywords: biocatalysis; carbohydrates; glycoconju- gates; keratan sulfate; sulfation Introduction Eukaryotic cells are covered with a dense layer of gly- cans, which is involved in cell–cell and cell–extracellu- lar matrix (ECM) interactions. Galectins have been shown to interact with glycoproteins/glycolipids of the cell surface and extracellular matrix (ECM). They recognize poly-N-acetyllactosamine [poly-LacNAc; (3Galb1,4GlcNAcb1,) n ] glycans of cell surface recep- tors such as integrins and ECM glycoproteins such as laminin, fibronectin, and collagen IV. [1] In this way ga- lectins cross-link the cell surface to the ECM. We re- cently demonstrated the beneficial utilization of a ga- lectin-mediated artificial ECM, based on immobilized poly-LacNAc glycoconjugates. [2] In order to extend our strategy towards 6-O-sulfated poly-LacNAc struc- tures (keratan sulfate, KS) we present here a chemo- enzymatic cascade synthesis. KS is a major glycosami- noglycan of eukaryotes predominantly found in the cornea, cartilage and brain. Maintenance of corneal matrix structure is a crucial role of KS. [3] This is espe- cially evident in the development of macular corneal dystrophy, a disease which is caused by a mutation in the sulfotransferase GlcNAc6ST-5 [4] causing increas- ing opaqueness of the cornea with ultimate loss of vision and the need of corneal transplantation. The sulfation pattern across the polysaccharide KS is not uniform. [5] It consists of di-sulfated, mono-sulfated and non-sulfated parts of disaccharide units. Sulfation patterns of other glycosaminoglycans (chondroitin sul- fate, heparin, heparan sulfate) have been demonstrat- ed to encode binding sites for growth factors. [6] Recent studies suggest also growth-factor binding for KS isolated from bovine cornea. [7] However, binding motifs are still elusive. In contrast, the ability to sup- press IL-12 production by macrophages has been demonstrated for the keratan sulfate disaccharide, (6- O-sulfo)Galb1-4(6-O-sulfo)GlcNAc. Interestingly, neither polymeric KS, nor KS tetrasaccharides, LacNAc or other glycosaminoglycan disaccharides were able to alter interleukin production indicating that also the size is relevant for biological function. [8] Strategies for the chemical synthesis of sulfated LacNAc were described for up to a tetrasaccharide structure. [9] They involve multiple protection and de- protection steps and are thus not well suited for the synthesis of oligomeric keratin-like structures. (Che- mo)enzymatic synthesis approaches for KS synthesis 584 # 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2016, 358, 584 – 596 FULL PAPERS