Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

This study provides proof of concept for cell-based heparin without heparin-induced thrombocytopenia side effects.

Among dissociation methods, negative electron transfer dissociation (NETD) has been proven the most useful for glycosaminoglycan (GAG) sequencing because it produces informative fragmentation, a low degree of sulfate losses, high sensitivity, and translatability to multiple instrument types. The challenge, however, is to distinguish positional sulfation. In particular, NETD has been reported to fail to differentiate 4-O- versus 6-O-sulfation in chondroitin sulfate decasaccharide. This raised the concern of whether NETD is able to differentiate the rare 3-O-sulfation from predominant 6-O-sulfation in heparan sulfate (HS) oligosaccharides. Here, we report that NETD generates highly informative spectra that differentiate sites of O-sulfation on glucosamine residues, enabling structural characterizations of synthetic HS isomers containing 3-O-sulfation. Further, lyase-resistant 3-O-sulfated tetrasaccharides from natural sources were successfully sequenced. Notably, for all of the oligosaccharides in this study, the successful sequencing is based on NETD tandem mass spectra of commonly observed deprotonated precursor ions without derivatization or metal cation adduction, simplifying the experimental workflow and data interpretation. These results demonstrate the potential of NETD as a sensitive analytical tool for detailed, high-throughput structural analysis of highly sulfated GAGs. Graphical Abstract.

21Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which 22 are involved in diverse biological processes. The structural complexity brings 23 difficulties in separation, making the study of structure-function relationships 24challenging. Here we present a separation method for Hp/HS oligosaccharide 25 fractionation with cross-compatible solvent and conditions, combining size exclusion 26 chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and 27 hydrophilic interaction chromatography (HILIC) as three orthogonal separation 28 methods that do not require desalting or extensive sample handling. With this 29 method, the final eluent is suitable for structure-function relationship studies, 30including tandem mass spectrometry and microarray printing. Our data indicate that 31 high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, 32the fractions co-eluted in IPRP could be further separated by HILIC, with both 33 separation dimensions capable of resolving some isomeric oligosaccharides. We 34 demonstrate this method using both unpurified reaction products from isomeric 35 synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, 36 identifying isomers resolved by this multi-dimensional separation method. We 37 demonstrate both structural analysis by MS, as well as functional analysis by 38 microarray printing and screening using a prototypical Hp/HS binding protein: basic-39 fibroblast growth factor (FGF2). Collectively, this method provides a strategy for 40 efficient Hp/HS structure-function characterization . 41 42 43 44 45 46 47 48 Heparin and heparan sulfate (Hp/HS) are glycosaminoglycans (GAGs), highly 49 anionic unbranched polysaccharides found on the surface of essentially all 50 mammalian cells. Hp/HS consists of a repeating disaccharide structure either -D-51 glucuronic or -L-iduronic acid (GlcA and IdoA, respectively) and -D-N-acetyl-D-52 glucosamine (GlcNAc), all connected by 1→4 linkages 1 . As a part of biosynthesis, 53 monosaccharides can be differentially N-and O-sulfated after polymerization. The 54GlcA can be epimerized to IdoA and can be 2-O-sulfated, while the GlcNAc can be 55 deacetylated (usually followed by N-sulfation) and/or O-sulfated at the 6-and/or 3-56 position. Since these modifications are incomplete and untemplated, there is 57 enormous structural heterogeneity in Hp/HS chains, including many isomeric 58 structures with widely varying and dynamic compositions 2 . Different cell types will 59 often display different HS structures, and these structures can change as part of the 60 cell's physiological response 3 . This structural diversity mediates a wide range of 61 protein-GAG interactions of varying specificity and affinity. Hp/HS has been involved 62 in a wide and growing array of physiological and pathophysiological processes, 63usually mediated through interactions between proteins and a subset of Hp/HS 64 structures 4-6 . While the importance of oligosaccharide structure has been establishe...

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.