Small molecule antagonists of cell-surface heparan sulfate and heparin–protein interactions

Surfen, bis-2-methyl-4-amino-quinolyl-6-carbamide, was previously reported as a small molecule antagonist of heparan sulfate (HS), a key cell-surface glycosaminoglycan found on all mammalian cells. To generate structure–activity relationships, a series of rationally designed surfen analogs was synthesized, where its dimeric structure, exocyclic amines, and urea linker region were modified to probe the role of each moiety in recognizing HS. An in vitro assay monitoring inhibition of fibroblast growth factor 2 binding to wild-type CHO cells was utilized to quantify interactions with cell surface HS. The dimeric molecular structure of surfen and its aminoquinoline ring systems was essential for its interaction with HS, and certain dimeric analogs displayed higher inhibitory potency than surfen and were also shown to block downstream FGF signaling in mouse embryonic fibroblast cells. These molecules were also able to antagonize other HS–protein interactions including the binding of soluble RAGE to HS. Importantly, selected molecules were shown to neutralize heparin and other heparinoids, including the synthetic pentasaccharide fondaparinux, in a factor Xa chromogenic assay and in vivo in mice. These results suggest that small molecule antagonists of heparan sulfate and heparin can be of therapeutic potential for the treatment of disorders involving glycosaminoglycan–protein interactions.     

Synthesis of Disaccharides Containing 6-Deoxy-a-L-talose as Potential Heparan Sulfate Mimetics

A 6-deoxy-a-L-talopyranoside acceptor was readily prepared from methyl a-L-rhamnopyranoside and glycosylated with thiogalactoside donors using NIS/TfOH as the promoter to give good yields of the desired a-linked disaccharide (69-90%). Glycosylation with a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor was not completely stereoselective (a:b = 6:1), but the desired a-linked disaccharide could be isolated in good overall yield (60%) following conversion into its corresponding tribenzoate derivative. The disaccharides were designed to mimic the heparan sulfate (HS) disaccharide GlcN(2S,6S)-IdoA(2S). However, the intermediates readily derived from these disaccharides were not stable to the sulfonation/deacylation conditions required for their conversion into the target HS mimetics.     

Toward the assembly of heparin and heparan sulfate oligosaccharide libraries: efficient synthesis of uronic acid and disaccharide building blocks

The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine-glucuronic acid (GlcN-GlcA) or iduronic acid (GlcN-IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation.     

Rational designing of glyco-nanovehicles to target cellular heterogeneity

The aberrant expression of endocytic epidermal growth factor receptors (EGFRs) in cancer cells has emerged as a key target for therapeutic intervention. Here, we describe for the first time a state-of-the-art design for a heparan sulfate (HS) oligosaccharide-based nanovehicle to target EGFR-overexpressed cancer cells in cellular heterogeneity. An ELISA plate IC₅₀ inhibition assay and surface plasma resonance (SPR) binding assay of structurally well-defined HS oligosaccharides showed that 6-O-sulfation (6-O-S) and 6-O-phosphorylation (6-O-P) of HS tetrasaccharides significantly enhanced EGFR cognate growth factor binding. The conjugation of these HS ligands to multivalent fluorescent gold nanoparticles (AuNPs) enabled the specific and efficient targeting of EGFR-overexpressed cancer cells. In addition, this heparinoid-nanovehicle exhibited selective homing to NPs in cancer cells in three-dimensional (3D) coculture spheroids, thus providing a novel target for cancer therapy and diagnostics in the tumor microenvironment (TME).

Heparan sulfate oligosaccharide based nanovehicle greatly enhance the selective targeting of cancer cells in tumor microenvironment.     

Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the “sulfation code” and understanding the roles of specific GAG structures in physiology and disease.     

Differentiation of 3-O-sulfated heparin disaccharide isomers: Identification of structural aspects of the heparin CCL2 binding motif

The presence of 3-O-sulfated glucosamine residues in heparin or heparan sulfate plays a role in binding to antithrombin III and HSV infection. In this study, tandem mass spectrometry was used to differentiate between two heparin disaccharide isomers containing variable sulfate at C6 in a common disaccharide and C3 in a more rare one. The dissociation patterns shown by MS² and MS³ were clearly distinguishable between the isomers, allowing their differentiation and quantitation. Using this technique, we show that an octasaccharide with 11 sulfate groups with high affinity for inflammatory chemokine CCL2 does not contain 3-O-sulfated disaccharides.     

Rapid and sensitive analysis of disaccharide composition in heparin and heparan sulfate by reversed-phase ion-pair chromatography on a 2 μm porous silica gel column

A rapid and sensitive method was developed for the analysis of disaccharide composition in heparin (HP) and heparan sulfate (HS) by reversed-phase ion-pair chromatography on a 2 μm porous silica gel column. HP and HS were digested with heparin lyase I, II and III in combination, and the produced unsaturated disaccharides were separated within 15 min. Calibration graphs were linear in the range 1 ng–1 μg with the fluorometoric post-column detection using 2-cyanoacetamide.     

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase,a Key Enzyme in Cancer and Viral Infections

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-β-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS−CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end β-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.     

Discovery of rare sulfated N-unsubstituted glucosamine based heparan sulfate analogs selectively activating chemokines

Achieving selective inhibition of chemokines with structurally well-defined heparan sulfate (HS) oligosaccharides can provide important insights into cancer cell migration and metastasis. However, HS is highly heterogeneous in chemical composition, which limits its therapeutic use. Here, we report the rational design and synthesis of N-unsubstituted (NU) and N-acetylated (NA) heparan sulfate tetrasaccharides that selectively inhibit structurally homologous chemokines. HS analogs were produced by divergent synthesis, where fully protected HS tetrasaccharide precursor was subjected to selective deprotection and regioselectively O-sulfated, and O-phosphorylated to obtain 13 novel HS tetrasaccharides. HS microarray and SPR analysis with a wide range of chemokines revealed the structural significance of sulfation patterns and NU domain in chemokine activities for the first time. Particularly, HT-3,6S-NH revealed selective recognition by CCL2 chemokine. Further systematic interrogation of the role of HT-3,6S-NH in cancer demonstrated an effective blockade of CCL2 and its receptor CCR2 interactions, thereby impairing cancer cell proliferation, migration and invasion, a step towards designing novel drug molecules.     

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.     

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II)

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.     

Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study

Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS–FGF–FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1–HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1–HS are proposed.

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Graphical Abstract ?

     

Preactivation‐Based,One‐Pot Combinatorial Synthesis of Heparin‐like Hexasaccharides for the Analysis of Heparin–Protein Interactions

Heparin (HP) and heparan sulfate (HS) play important roles in many biological events. Increasing evidence has shown that the biological functions of HP and HS can be critically dependent upon their precise structures, including the position of the iduronic acids and sulfation patterns. However, unraveling the HP code has been extremely challenging due to the enormous structural variations. To overcome this hurdle, we investigated the possibility of assembling a library of HP/HS oligosaccharides using a preactivation‐based, one‐pot glycosylation method. A major challenge in HP/HS oligosaccharide synthesis is stereoselectivity in the formation of the cis‐1,4‐linkages between glucosamine and the uronic acid. Through screening, suitable protective groups were identified on the matching glycosyl donor and acceptor, leading to stereospecific formation of both the cis‐1,4‐ and trans‐1,4‐linkages present in HP. The protective group chemistry designed was also very flexible. From two advanced thioglycosyl disaccharide intermediates, all of the required disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified building‐block preparation. Furthermore, the reactivity‐independent nature of the preactivation‐based, one‐pot approach enabled us to mix the building blocks. This allowed rapid assembly of twelve HP/HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. The speed and the high yields achieved in glycoassembly without the need to use a large excess of building blocks highlighted the advantages of our approach, which can be of general use to facilitate the study of HP/HS biology. As a proof of principle, this panel of hexasaccharides was used to probe the effect of backbone sequence on binding with the fibroblast growth factor‐2 (FGF‐2). A trisaccharide sequence of 2‐O‐sulfated iduronic acid flanked by N‐sulfated glucosamines was identified to be the minimum binding motif and N‐sulfation was found to be critical. This provides useful information for further development of more potent compounds towards FGF‐2 binding, which can have potential applications in wound healing and anticancer therapy.     

Rational Design and Expedient Synthesis of Heparan Sulfate Mimetics from Natural Aminoglycosides for Structure and Activity Relationship Studies

Heparan sulfate (HS) contains variably repeating disaccharide units organized into high- and low-sulfated domains. This rich structural diversity enables HS to interact with many proteins and regulate key signaling pathways. Efforts to understand structure-function relationships and harness the therapeutic potential of HS are hindered by the inability to synthesize an extensive library of well-defined HS structures. We herein report a rational and expedient approach to access a library of 27 oligosaccharides from natural aminoglycosides as HS mimetics in 7–12 steps. This strategy significantly reduces the number of steps as compared to the traditional synthesis of HS oligosaccharides from monosaccharide building blocks. Combined with computational insight, we identify a new class of four trisaccharide compounds derived from the aminoglycoside tobramycin that mimic natural HS and have a strong binding to heparanase but a low affinity for off-target platelet factor-4 protein.     

Synthesis of tailor-made glycoconjugate mimetics of heparan sulfate that bind IFN-gamma in the nanomolar range

We have recently described the preparation of three building blocks for the combinatorial synthesis of heparan sulfate (HS) fragments. Herein we show that one of these building blocks (disaccharide 4) allows the preparation, in high yields and with total alpha stereoselectivity, of tetra-, hexa- and octasaccharides from the heparin (HP) regular region, by using 2+2, 2+4 and 4+4 glycosylation strategies, respectively. These oligosaccharides were processed into sulfated derivatives bearing an allyl moiety in the anomeric position. The UV-promoted conjugation of these compounds with alpha,omega-bis(thio)poly(ethylene glycol) spacers of three different lengths allowed us to prepare nine benzylated glycoconjugates. After final deprotection, the glycoconjugates 1 a-c, 2 a-c and 3 a-c were obtained and their ability to inhibit the interaction between IFN-gamma and HP was tested by using surface plasmon resonance detection. Compound 3 b, containing two HP octasaccharides linked by a 50-A linker was able to inhibit the IFN-gamma/HP interaction with an IC(50) value of approximately 35 nM. In addition, the nine glycoconjugates were perfect tools in the study to ascertain the topology of the IFN-gamma binding site on HS. Compounds 1 a-c, 2 a-c and 3 a-c, by mimicking the alternating sulfated and nonsulfated regions found in HS, thus comprise the first example of a library of synthetic HS mimetics giving access to the "second level of molecular diversity" found in HS.     

Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry

Heparan sulfate, a cell surface bound glycosaminoglycan polysaccharide, has been implicated in numerous biological functions. Heparan sulfate molecules are highly complex and diverse, yet deceivingly look simple and similar, rendering structure--function correlation tedious. Current chromatographic and mass spectrometric techniques have limitations for analyzing glycosaminoglycan samples that are in low abundance and that are large in size, due to their highly acidic nature arising from a large number of sulfate and of carboxylate groups. A new methodology was developed using capillary ion-paired reverse-phase C18 HPLC directly coupled to ESI-TOF-MS to address the above issues. On the basis of HS disaccharide analysis, dibutylamine was found to be the best suited for HS analysis among many ion-pairing agents investigated. Next, analysis of oligosaccharides derived from heparosan, the precursor for heparan sulfate, was undertaken to demonstrate its greater applicability in a more complex structural analysis. The established chromatographic conditions enabled the characterization of heparosan oligosaccharides of sizes up to tetracontasaccharide with high resolution in a single run and were amenable to negative ion electrospray MS in which sodium adduction and fragmentation were avoided. To date, these are the largest nonsulfated HS precursor oligosaccharides to be characterized by LC/MS. Finally, the current methodology was applied to the characterization of the biologically important ATIII binding pentasaccharide and its precursors, which differ from each other by sulfation pattern and/or degree of sulfation. All of these pentasaccharides were well-resolved and characterized by the LC/MS system with (34)SO(4) as a mass spectral probe. This newly developed methodology facilitates the purification and rapid characterization of biologically significant HS oligosaccharides, and will thus expedite their synthesis. These findings should undoubtedly pave the way in deciphering multiple functional arrangements, ascribed to many biological activities, which are predictably embedded in a single large chaotic, yet well-organized HS polysaccharide chain. Development of newer techniques for HS oligosaccharide analysis is greatly needed in the postgenome era as attention shifts to the functional implications of proteins and carbohydrates in general and HS in particular.     

Synthesis of glycosaminoglycan oligosaccharides. Part 5: Synthesis of a putative heparan sulfate tetrasaccharide antigen involved in prion diseases

The synthesis of the putative prion-associated heparan sulfate tetrasaccharide containing two d-glucuronic acid units is reported. Key to the synthesis were the differentiation of the N-acetylated and N-unsubstituted glucosamine units, the high-yielding glucosylation at O-4 of an N-acetyl-d-glucosamine derivative and the α-selective glycosylation of the 4′-OH group of a β-d-GlcpA-(1→4)-d-GlcpNAc disaccharide building block with a disaccharide thioglycoside donor.     

Preferential uptake of L- versus D-amino acid cell-penetrating peptides in a cell type-dependent manner

The use of protease-resistant D-peptides is a prominent strategy for overcoming proteolytic sensitivity in the use of cell-penetrating peptides (CPPs) as delivery vectors. So far, no major differences have been reported for the uptake of L- and D-peptides. Here we report that cationic L-CPPs are taken up more efficiently than their D-counterparts in MC57 fibrosarcoma and HeLa cells but not in Jurkat T leukemia cells. Reduced uptake of D-peptides co-occurred with persistent binding to heparan sulfates (HS) at the plasma membrane. In?vitro binding studies of L- and D-peptides with HS indicated similar binding affinities. Our results identify two key events in the uptake of CPPs: binding to HS chains and the initiation of internalization. Only the second event depends on the chirality of the CPP. This knowledge may be exploited for a stereochemistry-dependent preferential targeting of cells.     

3-O-Sulfation of Heparan Sulfate Enhances Tau Interaction and Cellular Uptake

Prion-like transcellular spreading of tau in Alzheimer's Disease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However, the structural determinants for tau–HS interaction are not well understood. Microarray and SPR assays of structurally defined HS oligosaccharides show that a rare 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1^−/− (HS 3-O-sulfotransferase-1 knockout) cells, reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In a cell culture, the addition of a 3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake. NMR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2 (R2) and proline-rich region 2 (PRR2) of tau. Tau is only the seventh protein currently known to recognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau–HS binding and likely the transcellular spread of tau, providing a novel target for disease-modifying treatment of AD and other tauopathies.     

Chemistry and biology of glycosaminoglycans in blood coagulation

Glycosaminoglycans (GAGs) are widely distributed in animal tissues where they are usually associated with proteins. Six types are commonly recognized: heparin (Hep), heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (Ch-S), keratan sulfate (KS) and hyaluronic acid (Hyal). They are structurally related with a carbohydrate backbone consisting of alternating hexuronic acid (L-iduronic acid and/or D-glucuronic acid) or galactose units and hexosamine (D-glucosamine or D-galactosamine) residues. All GAGs, except Hyal, show sulfate groups along their chains. Certain sulfate glycoaminoglycans have the ability to interfere with blood coagulation, as demonstrated by the extensive clinical use of Hep as an anticoagulant agent. HS and DS show a good anticoagulant activity, although weaker than that of Hep. In contrast, Ch-S has a low ability to inhibit plasma serine proteases, and KS and Hyal are devoid of any effect on coagulation cascade. The interaction between blood coagulation serine proteases and GAGs can be found to have two principle mechanisms: the specific “lock and key” binding and the nonspecific cooperative electrostatic association. This different ability of GAGs to interact with coagulation cascade proteins depends on the molecular weight, the ratio of iduronic/glucoronic acid and the sulfation degree. Many attempts have been made to improve or induce anticoagulant activity of natural GAGs-by chemical modification. Increasing sulfation degree of DS and Ch-S is followed by their biological activity increasing. Hyal, which is devoid of any anticoagulant effect, acquires a good ability to inactivate plasma serine proteases, i.e. thrombin and Factor Xa, when it is sulfated. This ability increases by increasing the number of sulfate groups per disaccharide unit, although the mechanism of action is different from that of Hep, but seems to be independent of its molecular weight.