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.

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.

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.

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.

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.

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.

Heparosan-derived heparan sulfate/heparin-like compounds: one kind of potential therapeutic agents.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan and exists in all animal tissues. HS and heparin are very similar, except that heparin has higher level of sulfation and higher content of iduronic acid. Despite the fact that it is a century-old drug, heparin remains as a top choice for treating thrombotic disorders. Pharmaceutical heparin is derived from porcine intestine or bovine lung via a long supply chain. This supply chain is vulnerable to the contamination of animal pathogens. Therefore, new methods for manufacturing heparin or heparin-like substances devoid of animal tissues have been explored by many researchers, among which, modifications of heparosan, the capsular polysaccharide of Escherichia coli K5 strain, is one of the promising approaches. Heparosan has a structure similar to unmodified backbone of natural HS and heparin. It is feasible to obtain HS or heparin derivatives by modifying heparosan with chemical or enzymatic methods. These derivatives display different biological activities, such as anticoagulant, anti-inflammatory, anticancer, and antiviral activities. This review focuses on the recent studies of synthesis, activity, and structure-activity relationship of HS/heparin-like derivatives prepared from heparosan.

Synthesis of a targeted library of heparan sulfate hexa- to dodecasaccharides as inhibitors of ß-secretase: potential therapeutics for Alzheimer's disease.

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure-activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the ß-site cleaving enzyme ß-secretase (BACE1), a protease responsible for generating the toxic Aß peptides that accumulate in Alzheimer's disease (AD), with 6-O-sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa- to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure-activity relationships for optimal BACE1 inhibition, and yielded a number of potent non-anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aß peptide levels.

Toward the solid-phase synthesis of heparan sulfate oligosaccharides: evaluation of iduronic acid and idose building blocks.

Glycan arrays have been established as the premier technical platform for assessing the specificity of carbohydrate binding proteins, an important step in functional glycomics research. Access to large libraries of well-characterized oligosaccharides remains a major bottleneck of glycan array research, and this is particularly true for glycosaminoglycans (GAGs), a class of linear sulfated polysaccharides which are present on most animal cells. Solid-supported synthesis is a potentially powerful tool for the accelerated synthesis of relevant GAG libraries with variations in glycan sequence and sulfation pattern. We have evaluated a series of iduronic acid and idose donors, including a couple of novel n-pentenyl orthoester donors in the sequential assembly of heparan sulfate precursors from monosaccharide building blocks in solution and on a polystyrene resin. The systematic study of donor and acceptor performance up to the trisaccharide stage in solution and on the solid support have resulted in a general strategy for the solid-phase assembly of this important class of glycans.

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach.

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3 nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells.

BACKGROUND: Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important. DESIGN AND METHODS: Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation. RESULTS: Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice. CONCLUSIONS: This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.

Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies.

The rare N-unsubstituted glucosamine (GlcNH (3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. However, it is difficult to prepare naturally-occuring, GlcNH(3)(+)-containing oligosaccharides from HS because of their low abundance, as well as the inherent problems in both excising and identifying them. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH(3)(+) residues would greatly contribute to investigating their natural role in HS. In this study, a series of heparin/HS oligosaccharides, from dp6 up to dp16 in length that possess internal GlcNH(3)(+) residues were prepared by a combination of chemical modification and heparinase I digestion. Purification and structural analysis of the major species derived from the octa- to dodeca-saccharide size fractions indicated the introduction of between 1 and 3 internal GlcNH(3)(+) residues per oligosaccharide. In addition, a GlcNH(3)(+) residue was selectively introduced into an internal position in a tetrasaccharide species by direct chemical modification. This selectivity has potential as an alternative procedure for preparing internally-modified oligosaccharides of various lengths. The utility of such oligosaccharides was demonstrated by a comparison of the binding of three different tetrasaccharide species containing 0, 1 and 2 free amino groups to the NK1 truncated variant of hepatocyte growth factor/scatter factor.

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.

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.

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.

Modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies.

Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides. To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease.

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.

Solution structures of chemoenzymatically synthesized heparin and its precursors.

We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [(13)C,(15)N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA(1,4)GlcNS-); enzyme-catalyzed C5-epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [(13)C,(15)N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved (13)C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.