Recent advances in the synthesis of extensive libraries of heparan sulfate oligosaccharides for structure-activity relationship studies.

Heparan sulfate (HS) is a linear, sulfated and highly negatively-charged polysaccharide that plays important roles in many biological events. As a member of the glycosaminoglycan (GAG) family, HS is commonly found on mammalian cell surfaces and within the extracellular matrix. The structural complexities of natural HS polysaccharides have hampered the comprehension of their biological functions and structure-activity relationships (SARs). Although the sulfation patterns and backbone structures of HS can be major determinants of their biological activities, obtaining significant amounts of pure HS from natural sources for comprehensive SAR studies is challenging. Chemical and enzyme-based synthesis can aid in the production of structurally well-defined HS oligosaccharides. In this review, we discuss recent innovations enabling the syntheses of large libraries of HS and how these libraries can provide insights into the structural preferences of various HS binding proteins.

Synthetic heparan sulfate ligands for vascular endothelial growth factor to modulate angiogenesis.

We report the discovery of a potential heparan sulfate (HS) ligand to target several growth factors using 13 unique HS tetrasaccharide ligands. By employing an HS microarray and SPR, we deciphered the crucial structure-binding relationship of these glycans with the growth factors BMP2, VEGF165, HB-EGF, and FGF2. Notably, GlcNHAc(6-O-SO3-)-IdoA(2-O-SO3-) (HT-2,6S-NAc) tetrasaccharide showed strong binding with the VEGF165 growth factor. In vitro vascular endothelial cell proliferation, migration and angiogenesis was inhibited in the presence of VEGF165 and HT-2,6S-NAc or HT-6S-NAc, revealing the potential therapeutic role of these synthetic HS ligands.

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis.

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Probing Amyloid ß Interactions with Synthetic Heparan Sulfate Oligosaccharides.

Heparan sulfate (HS) can play important roles in the biology and pathology of amyloid ß (Aß), a hallmark of Alzheimer's disease. To better understand the structure-activity relationship of HS/Aß interactions, synthetic HS oligosaccharides ranging from tetrasaccharides to decasaccharides have been utilized to study Aß interactions. Surface plasmon resonance experiments showed that the highly sulfated HS tetrasaccharides bearing full 2-O, 6-O, and N-sulfations exhibited the strongest binding with Aß among the tetrasaccharides investigated. Elongating the glycan length to hexa- and deca-saccharides significantly enhanced Aß affinity compared to the corresponding HS tetrasaccharide. Solid state NMR studies of the complexes of Aß with HS hexa- and deca-saccharides showed most significant chemical shift perturbation in the C-terminus residues of Aß. The strong binding HS oligosaccharides could reduce the cellular toxicities induced by Aß. This study provides new insights into HS/Aß interactions, highlighting how synthetic structurally well-defined HS oligosaccharides can assist in biological understanding of Aß.

Continuous-Flow Accelerated Sulfation of Heparan Sulfate Intermediates.

We report for the first time a continuous-flow strategy to execute O-sulfation modification of heparan sulfate (HS) oligosaccharides. A systematic investigation of the influence of the flow parameters on the installation of the sulfate group on glucosamine monosaccharide can aid the development of a comprehensive, quick, and reliable strategy for O-sulfation of HS oligosaccharide precursors. Deprotection of the sulfated heparin intermediates led to the development of a comprehensive biologically inspired oligosaccharide library to understand the crucial structure-function relationship of HS.

Concise chemoenzymatic synthesis of heparan sulfate analogues as potent BACE-1 inhibitors.

Heparan sulfate (HS) and heparin, representative members of the glycosaminoglycans, possess distinct biological functions in terms of their specific interactions with hundreds of binding proteins. However, the structural properties of HS and heparin are complex due to their variable repeating motifs, different chain lengths and sulfation patterns. A concise chemoenzymatic approach has been developed to obtain well-defined low molecular weight (LMW) HS analogues. Pasteurella multocida heparosan synthase-2 (PmHS2) was utilized to fabricate the HS backbones with controllable chain lengths ranging from 14mer to 26mer. Moreover, regioselective and overall sulfation were conducted by chemical approach. The persulfated HS analogues exhibited more potent beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE-1) inhibitory activity than heparin and enoxaparin, and enhanced BACE-1 inhibitions were also found with the increasing molecular size of the HS analogues. This approach supplies the promising LMW HS analogues for the potential development of novel anti-Alzheimer's drugs.

Designing heparan sulfate-based biocompatible polymers and their application for intracellular stimuli-sensitive drug delivery.

Heparan sulfate (HS) is a kind of natural polysaccharides with good biocompatibility. And as drug carriers, it has some advantages compared to heparin. However, the preparation of HS is cumbersome and difficult, which limits its application in drug delivery. Here, we use modern separation technique combined with chromatography to establish a new preparation method of HS. The molecular weight and degree of dispersion of HS were (1.03 × 104 ±â€¯107) kDa and 1.106, respectively. HS also showed low anticoagulation activity in comparison with heparin. Subsequently, novel redox-sensitive heparan sulfate-cystamine-vitamin E succinate (HS-SS-VES, HSV) micelles were designed to increase tumor selectivity and improve the therapeutic effect of doxorubicin (DOX). DOX-loaded HSV micelles (DOX/HSV) with spherical morphology had average particle size of 90-120 nm and good redox-triggered release behavior. The cell viabilities of blank micelles were >90% in both human breast cancer (MCF7) cells and African green monkey SV40-transformed kidney fibroblast (COS7) cells. However, the cytotoxicity of DOX/HSV in MCF7 cells was higher than that of COS7 cells. Flow cytometry analyses and confocal laser scanning microscopy observation indicated that DOX/HSV micelles were internalized by endocytosis, and then the drug was released quickly and entered the nuclei of tumor cells. The results demonstrate that high-purity HS can be prepared and has the potential to be further used for drug delivery in antitumor applications.

Design and synthesis of structurally defined heparan sulfate (HS)-FK506 conjugates as an exogenous approach to investigate biological functions of nucleus HS.

Although heparan sulfate (HS) is widely implicated in numerous physiological and pathological processes, the biological function of nucleus HS remains underexplored, largely due to its complex structure and high hydrophilic property. To supplement these efforts, ideal vehicles are drawing attention as they combine attractive features including lipid solubility for penetrating cell membrane, high affinity binding to its target receptor, metabolic stability, and no cellular actions resulting in toxicity. Herein, we develop a convenient and promising strategy to prepare HS-FK506 conjugates for membrane transport and entry into nucleus, where click chemistry takes easily place between the exocyclic allyl group of a clinic drug FK506 and thiol as a handle incorporated into HS analogues. HS derivatives for constructing the conjugates were synthesized using a cutting-edge chemoenzymatic method. Meantime, [35S] labeled 3'-phosphoadenosine 5'-phosphosulfate (PAP35S) and [14C] glucuronic acid (Glc A) were adopted to label HS-FK506 conjugates, respectively, to evaluate their efficiency of nucleus entry, as a result, 14C Glc A was sensitive, effective and reliable whereas PAP35S gave rise to a mixture of labeled compounds, hampering the understanding of structure-function relationship of nucleus HS. Compared with the corresponding HS, the amount of HS-FK506 conjugates to translocate into nucleus from radioactive assay experiments sharply increased, e.g. tridecasaccharide-FK506 1d increased by approximate 10 folds, offering a simple and robust platform for enabling hydrophilic compounds including carbohydrates to translocate into nucleus and shedding light on their biological functions.

Improved de novo sequencing of heparin/heparan sulfate oligosaccharides by propionylation of sites of sulfation.

The structure of heparin and heparan sulfate (Hep/HS) oligosaccharides, as determined by the length and the pattern of sulfation, acetylation, and uronic acid epimerization, dictates their biological function through modulating interactions with protein targets. But fine structural determination is a very challenging task due to the lability of the sulfate modifications and difficulties in separating isomeric HS chains. Previously, we reported a strategy for chemical derivatization involving permethylation, desulfation, and trideuteroperacetylation, combined with standard reverse phase LC-MS/MS that enables the structural sequencing for heparin/HS oligosaccharides of sizes up to dodecasaccharide by positionally replacing all sulfates with more stable trideuteroacetyl groups, allowing for robust MS/MS sequencing. However, isomeric oligosaccharides that contain both N-sulfation and N-acetylation become isotopomers after labeling, differing only in the sites of deuteration. This prevents chromatographic separation of these different mixed domain sequences post-derivatization, and makes sequencing by MS/MS difficult due to co-fragmentation of the isotopomers leading to chimeric product ion spectra. In order to improve chromatographic separation of mixed domain oligosaccharides, we have introduced a propionylation step in place of trideuteroacetylation for labeling of sites of sulfation. HS standard disaccharides have been used to evaluate the efficiency of this improved chemical derivatization. The results show that we can quantitatively replace sulfation with propionyl groups with the same high efficiency as the previously reported trideuteroacetylation. After derivatization, we demonstrate the ability to chromatographically separate two mixed domain tetrasaccharide isomers differing solely by the order of N-sulfation and N-acetylation, allowing for full sequencing of each by MS/MS. These results represent a marked improvement in the ability of our previously reported derivatization strategy to analyze complex mixtures of Hep/HS oligosaccharides without a decrease in sensitivity.

Synthetic heparin and heparan sulfate: probes in defining biological functions.

Heparin and heparan sulfate are glycosaminoglycans that modulate numerous biological processes. The desire to capture the structural diversity responsible for their functions provides notable issues during synthesis, including site-selective sulfonation, stereoselective glycosylation and the sheer number of probable targets at hand. With current advances in synthetic approaches, carbohydrate chemists generate these complex targets by chemical and enzymatic methods. Fondaparinux and a number of polysaccharides have been synthesized to probe anticoagulation and other biological functions. Moreover, a trove of structural information could be obtained by many analytical methods, which provide hints to the potential protein-binding sequences within the sugar chain. Further structure-activity relationship studies help unveil the secrets of the heparin/heparan sulfate code, providing potential candidates for drug development.

Large scale synthesis and regioselective protection schemes of ethyl 2-azido-2-deoxy-1-thio-α-d-cellobioside for preparation of heparin thiodisaccharide building blocks.

Crystalline acetylated ethyl 2-azido-2-deoxy-1-thio-α-d-cellobioside has been prepared on a multigram scale from cellobiose in an overall yield of 23% with no chromatography required and converted after deacetylation into the 4',6'-O-benzylidene and 4',6'-O-benzylidene-6-O-TBDMS protected derivatives. Applying a number of regioselective benzylation methods on these gave access to a variety of regioselectively protected derivatives, both mono-ols (2'- and 3-OH), diols (2',6-, 2',3-, and 3,6-di-OH), and triols (2',3,6- and 2',3',3-tri-OH). A number of these derivatives were further processed by benzoylation followed by removal or opening of the benzylidene acetal and selective oxidation of the exposed primary alcohol to give heparin building block intermediates comprising a range of possible sulfation patterns.

Single-Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide.

The first single-molecule fluorescence detection of a structurally-defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface-tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single-molecule level. The diverse and dynamic nature of HS-protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof-of-principle demonstration of the feasibility of single-molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments.

Divergent Synthesis of Heparan Sulfate Oligosaccharides.

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis.

Design and synthesis of active heparan sulfate-based probes.

A chemoenzymatic approach for synthesizing heparan sulfate oligosaccharides with a reactive diazoacetyl saccharide residue is reported. The resultant oligosaccharides were demonstrated to serve as specific inhibitors for heparan sulfate sulfotransferases, offering a new set of tools to probe the structural selectivity for heparan sulfate-binding proteins.

Single-entity heparan sulfate glycomimetic clusters for therapeutic applications.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan with a variety of critical functions in cell signaling and regulation. HS oligosaccharides can mimic or interfere with HS functions in biological systems; however, their exploitation has been hindered by the complexity of their synthesis. Polyvalent displays of small specific HS structures on dendritic cores offer more accessible constructs with potential advantages as therapeutics, but the synthesis of single-entity HS polyvalent compounds has not previously been described. Herein we report the synthesis of a novel targeted library of single-entity glycomimetic clusters capped with varied HS saccharides. They have the ability to mimic longer natural HS saccharides in their inhibition of the Alzheimer's disease (AD) protease BACE-1. We have identified several single-entity HS clusters with IC50 values in the low-nanomolar range. These HS clusters are drug leads for AD and offer a novel framework for the manipulation of heparan sulfate-protein interactions in general.

Preparation of heparan sulfate-like polysaccharide and application in stem cell chondrogenic differentiation.

Heparan sulfate is a component of the extracellular matrix (ECM) that modulates individual development and cell growth through its interaction with growth factors. Structurally, heparan sulfate consists of repeating linear sulfated poly-anionic disaccharide structures. The K5 polysaccharide has the same structure as heparosan, and is the capsular polysaccharide of Escherichia coli K5 strain which serves as a precursor in heparin and heparan sulfate biosynthesis. Here, we prepared sulfated K5 polysaccharides that are structurally similar to heparan sulfate and investigated their biocompatibility and bioactivity in stem cell chondrogenic differentiation. Briefly, sulfation groups were added to -NH- and/or -OH of a precursor heparosan and the modified heparosan was qualitatively analyzed by FT-IR, (1)H NMR, and (13)C NMR techniques. Cell viability was not significantly affected by the sulfated K5 capsular polysaccharide. Relative mRNA expression of the chondrogenic differentiation marker COL2A1 was significantly upregulated in cells treated with the N,O-sulfated K5 polysaccharide confirming that the sulfated K5 capsular polysaccharide is able to stimulate chondrogenic differentiation. The main sulfation pattern for chondrogenic activity is N,6-O sulfation and the activity was not proportional to the sulfation level. This type of mimic was prepared in nearly a gram scale, supporting further structural study and 3 dimension stem cell culture. Together, the results of this study show that sulfated K5 capsular polysaccharides are able to stimulate chondrogenic differentiation without affecting cell viability.

Enzymatic synthesis of heparan sulfate and heparin.

Heparan sulfate (HS) polysaccharide chains have been shown to orchestrate distinct biological functions in several systems. Study of HS structure-function relations is, however, hampered due to the lack of availability of HS in sufficient quantities as well as the molecular heterogeneity of naturally occurring HS. Enzymatic synthesis of HS is an attractive alternative to the use of naturally occurring HS, as it reduces molecular heterogeneity, or a long and daunting chemical synthesis of HS. Heparosan, produced by E. coli K5 bacteria, has a structure similar to the unmodified HS backbone structure and can be used as a precursor in the enzymatic synthesis of HS-like polysaccharides. Here, we describe an enzymatic approach to synthesize several specifically sulfated HS polysaccharides for biological studies using the heparosan backbone and a combination of recombinant biosynthetic enzymes such as C5-epimerase and sulfotransferases.

Production of size-defined heparosan, heparan sulfate, and heparin oligosaccharides by enzymatic depolymerization.

Glycosaminoglycans (GAG) are most commonly isolated as large polymers from various animal origins, the functional units of which are oligosaccharides, which bind their target proteins to induce conformational changes, compete with other ligands, or facilitate the formation of signaling complexes. One example, the extensively studied heparin pentasaccharide sequence-which binds antithrombin-III, inducing a conformational change that increases its serpin protease activity by 1,000-fold-is unique in that no other specific GAG-protein structure-function relations have been described to the same degree. Thus, production of heparan sulfate (HS) oligosaccharides is critical for obtaining specific structural information regarding the binding interactions of GAG and their ligands (typically proteins). Purely synthetic methods of oligosaccharide synthesis are possible, but the cost, time requirement, and difficulty of their preparation prohibit library synthesis in significant amounts. Herein, the use of bacterial heparin lyases for the production of HS oligosaccharides via enzymatic depolymerization of HS polymers is discussed. The separation and purification of these oligosaccharides by liquid chromatography are also described.

Preparation of isotope-enriched heparan sulfate precursors for structural biology studies.

Heparan sulfate (HS) plays numerous important roles in biological systems through their interactions with a wide array of proteins. Structural biology studies of heparan sulfate are often challenging due to the heterogeneity and complexity of the HS molecules. Radioisotope metabolic labeling of HS in cellular systems has enabled the elucidation of HS structures as well as the interactions between HS and proteins. However, radiolabeled structures are not amenable for advanced structural glycobiology studies using sophisticated instruments such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The utilization of stable isotope-enriched HS precursors is an appealing approach to overcome these challenges. The application of stable isotope-enriched HS precursors has facilitated the HS structural analysis by NMR spectroscopy and mass spectrometry. Herein we describe a simple method to prepare isotopically enriched HS precursors.

Chemoenzymatic synthesis of heparan sulfate and heparin.

Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in their polysaccharide chain lengths and sulfation patterns. The access to structurally defined heparan sulfate and heparin is critical to probe the contribution of specific sulfated saccharide structures to the biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method uses recombinant heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method provides an opportunity to develop improved heparin-based therapeutics.