Controlled sulfation of mixed-linkage glucan by Response Surface Methodology for the development of biologically applicable polysaccharides.

Endogenous and exogenous sulfated polysaccharides exhibit potent biological activities, including inhibiting blood coagulation and protein interactions. Controlled chemical sulfation of alternative polysaccharides holds promise to overcome limited availability and heterogeneity of naturally sulfated polysaccharides. Here, we established reaction parameters for the controlled sulfation of the abundant cereal polysaccharide, mixed-linkage ß(1,3)/ß(1,4)-glucan (MLG), using Box-Behnken Design of Experiments (BBD) and Response Surface Methodology (RSM). The optimization of the degree-of-substitution (DS) was externally validated through the production of sulfated MLGs (S-MLGs) with observed DS and Mw values deviating less than 20% and 30% from the targeted values, respectively. Simultaneous optimization of DS and Mw resulted in the same range of deviation from the targeted value. S-MLGs with DS > 1 demonstrated a modest anticoagulation effect versus heparin, and a greater P-selectin affinity than fucoidan. As such, this work provides a route to medically important polymers from an economical agricultural polysaccharide.

Potent, Selective, Allosteric Inhibition of Human Plasmin by Sulfated Non-Saccharide Glycosaminoglycan Mimetics.

Although plasmin inhibitors could be used in multiple disorders, their use has been restricted to preventing blood loss in hemostatic dysregulation because of poor efficacy and adverse effects of current agents. We reasoned that a new class of direct inhibitors that offer better efficacy, selectivity, and safety could be discovered by exploiting allosterism in plasmin, a protease homologous to other allosteric serine proteases. We report on the synthesis, biological activity, and mechanism of action of a group of small molecules, called non-saccharide glycosaminoglycan mimetics (NSGMs), as direct allosteric plasmin inhibitors. Our results show that distinct NSGMs selectively inhibit human full-length plasmin. The molecule inhibited clot lysis, alluding to its promise as an allosteric regulator of plasmin. We show that direct allosteric inhibition of plasmin could led to new antifibrinolytic agent(s) that may exhibit better efficacy, potency, selectivity, and safety in comparison to current therapy.

Fluorous-Tag Assisted Syntheses of Sulfated Keratan Sulfate Oligosaccharide Fragments.

The first block iteration strategy for iterative solution-phase synthesis of protected keratan sulfate (KS)-like fragments is reported. Obstacles in a strategy using galactose-glucosamine (Gal-GlcNAc) modules led to the discovery of a differentially protected GlcNAc-Gal module that could be used to synthesize KS-like fragments using a fluorous tag that maintained solubility in organic solvents for purification of all intermediates via fluorous solid-phase extraction.

Novel Polyanions Inhibiting Replication of Influenza Viruses.

Novel sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) and N-sulfonated chitosan (NSCH) have been synthesized, and their activity against influenza A and B viruses has been studied and compared with that of a series of carrageenans, marine polysaccharides of well-documented anti-influenza activity. NSPAHs were found to be nontoxic and very soluble in water, in contrast to gel-forming and thus generally poorly soluble carrageenans.In vitroandex vivostudies using susceptible cells (Madin-Darby canine kidney epithelial cells and fully differentiated human airway epithelial cultures) demonstrated the antiviral effectiveness of NSPAHs. The activity of NSPAHs was proportional to the molecular mass of the chain and the degree of substitution of amino groups with sulfonate groups. Mechanistic studies showed that the NSPAHs and carrageenans inhibit influenza A and B virus assembly in the cell.

Sulfated phenolic acids from Dasycladales siphonous green algae.

Sulfated aromatic acids play a central role as mediators of chemical interactions and physiological processes in marine algae and seagrass. Among others, Dasycladus vermicularis (Scopoli) Krasser 1898 uses a sulfated hydroxylated coumarin derivative as storage metabolite for a protein cross linker that can be activated upon mechanical disruption of the alga. We introduce a comprehensive monitoring technique for sulfated metabolites based on fragmentation patterns in liquid chromatography/mass spectrometry and applied it to Dasycladales. This allowed the identification of two new aromatic sulfate esters 4-(sulfooxy)phenylacetic acid and 4-(sulfooxy)benzoic acid. The two metabolites were synthesized to prove the mass spectrometry-based structure elucidation in co-injections. We show that both metabolites are transformed to the corresponding desulfated phenols by sulfatases of bacteria. In biofouling experiments with Escherichia coli and Vibrio natriegens the desulfated forms were more active than the sulfated ones. Sulfatation might thus represent a measure of detoxification that enables the algae to store inactive forms of metabolites that are activated by settling organisms and then act as defense.

Unravelling the Structural and Molecular Basis Responsible for the Anti-Biofilm Activity of Zosteric Acid.

The natural compound zosteric acid, or p-(sulfoxy)cinnamic acid (ZA), is proposed as an alternative biocide-free agent suitable for preventive or integrative anti-biofilm approaches. Despite its potential, the lack of information concerning the structural and molecular mechanism of action involved in its anti-biofilm activity has limited efforts to generate more potent anti-biofilm strategies. In this study a 43-member library of small molecules based on ZA scaffold diversity was designed and screened against Escherichia coli to understand the structural requirements necessary for biofilm inhibition at sub-lethal concentrations. Considerations concerning the relationship between structure and anti-biofilm activity revealed that i) the para-sulfoxy ester group is not needed to exploit the anti-biofilm activity of the molecule, it is the cinnamic acid scaffold that is responsible for anti-biofilm performance; ii) the anti-biofilm activity of ZA derivatives depends on the presence of a carboxylate anion and, consequently, on its hydrogen-donating ability; iii) the conjugated aromatic system is instrumental to the anti-biofilm activities of ZA and its analogues. Using a protein pull-down approach, combined with mass spectrometry, the herein-defined active structure of ZA was matrix-immobilized, and was proved to interact with the E. coli NADH:quinone reductase, WrbA, suggesting a possible role of this protein in the biofilm formation process.

Sulfation mediates activity of zosteric acid against biofilm formation.

Zosteric acid (ZA), a metabolite from the marine sea grass Zostera marina, has attracted much attention due to its attributed antifouling (AF) activity. However, recent results on dynamic transformations of aromatic sulfates in marine phototrophic organisms suggest potential enzymatic desulfation of metabolites like ZA. The activity of ZA was thus re-investigated using biofilm assays and simultaneous analytical monitoring by liquid chromatography/mass spectrometry (LC/MS). Comparison of ZA and its non-sulfated form para-coumaric acid (CA) revealed that the active substance was in all cases the non-sulfated CA while ZA was virtually inactive. CA exhibited a strong biofilm inhibiting activity against Escherichia coli and Vibrio natriegens. The LC/MS data revealed that the apparent biofilm inhibiting effects of ZA on V. natriegens can be entirely attributed to CA released from ZA by sulfatase activity. In the light of various potential applications, the (a)biotic transformation of ZA to CA has thus to be considered in future AF formulations.

Identification of 2-substituted ethenesulfonic acid ester derivatives as novel, potent and selective inhibitors of protein tyrosine phosphatase 1B.

Sixteen 2-substituted ethenesulfonic acid ester derivatives were designed, synthesized and evaluated for the inhibitory activity against tyrosine phosphatase 1B (PTP1B) and T-Cell protein tyrosine phosphatase (TCPTP). The structural activity relationship (SAR) of these compounds demonstrated that the hydrophilic head, aromatic center and the hydrophobic tail affected the inhibitory activities against PTP1B and the selectivity over TCPTP. Most of the compounds exhibited excellent inhibitory activity against PTP1B with IC50 value of 1.0 µM - 31.2 µM. SAR analysis revealed that the hydrophilic head was indispensable in the maintain of inhibitory activity against PTP1B, the aromatic center significantly altered the selectivity of PTP1B over TCPTP, and the hydrophobic tail significantly altered the inhibitory activity against PTP1B.

Iron-catalyzed synthesis of thioesters from thiols and aldehydes in water.

The preparation of thioesters through the iron-catalyzed coupling reaction of thiols with aldehydes is described. The reactions were carried out by using tert-butyl hydroperoxide (TBHP) as an oxidant and water as a solvent in most cases. This system is compatible with a variety of functional groups.

The first convergent total synthesis of penarolide sulfate A2, a novel α-glucosidase inhibitor.

Penarolide sulfate A2, a 31-membered macrolide encompassing a proline residue and three sulfate groups, was firstly synthesized in 16 linear steps with 4.8% overall yield. Three consecutive stereogenic centers in penarolide sulfate A2 were efficiently derived from natural chiral template l-arabinose. The crucial assembly reactions included Brown asymmetric allylation, olefin cross-metathesis, alkyne-epoxide coupling, and macrolactamization. The anti-yeast α-glucosidase activities of penarolide sulfate A2 and its fully desulfated derivative were examined showing IC50 values of 4.87 and 10.74 µg mL(-1), respectively.

Studies on esterification and sulfonation of riboflavin via semi-empirical methods.

The article presents extended computer investigations of various sulfate derivatives of riboflavin. A number of physicochemical parameters such as total energy, binding energy and formation heat were calculated via semi-empirical methods AM1 and PM3 for the different derivatives of riboflavin. Their analysis made it possible to determine the sequence of formation of sulfate derivatives--esterification is the easiest at hydroxyl groups at the farthest positions from the ring. This methodology may be used to study biologically active compounds.

Chemical synthesis and characterization of epicatechin glucuronides and sulfates: bioanalytical standards for epicatechin metabolite identification.

The monoglucuronides and sulfates of epicatechin, 3'-O-methylepicatechin, and 4'-O-methylepicatechin, respectively, were synthesized as authentic bioanalytical standards. Reversed-phase HPLC methods capable of baseline separation of the glucuronides and sulfates have been developed. Both the epicatechin glucuronides and sulfates were stable in the solid state when stored under ambient conditions and in aqueous solution when stored refrigerated. These results should prove invaluable to the research community as analytical standards as well as in future studies of the biological and pharmacological effects of epicatechin in humans.

Preparation, characterization and in vitro anticoagulant activity of highly sulfated chitosan.

This work is the first to report sulfation of chitosans with different molecular weight (Mw, 51.5-112.4 kDa) via their trimethylsilylated derivatives as the precursors. Different from original chitosan, silylated chitosan was easily sulfated with sulfur trioxide-pyridine complex under homogeneous conditions in DMSO, and highly sulfated products with degree of sulfation (DS) in the range of 1.65-2.46 and Mw in the range of 18.1-54.5 kDa were obtained at 20-40°C. Degradation of chitosan was inevitable in the reaction process. However, the extent of degradation reduced with increasing of degree of trimethylsilylation (DT). The results of UV-vis and fluorescence emission spectra indicated that the chitosan degradation involved elimination reaction. Clotting assays showed that highly sulfated chitosans significantly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT), but not prothrombin time (PT). A high DS (>2.1) value was essential for the anticoagulant activity.

Synthesis and biological evaluation of nonaprenylsulfates.

A naturally occurring nonaprenylsulfate (1) and its synthetic analogue (2) were synthesized from substituted phenolic precursors in three steps with an overall yield of 40-45%. Both compounds exhibited potent anti-inflammatory activity against 5-lipoxygenase, and potent brine shrimp lethality. They also showed moderate anti-oxidant activity in the super oxide radical scavenging model. Nonaprenylsulfate (1) showed moderate inhibition of paw edema in Freund's Complete Adjuvant (FCA) induced model of arthritis, thus confirming its anti-inflammatory activity.

Radioiodination of aryl-alkyl cyclic sulfates.

Among the currently available positron emitters suitable for Positron Emission Tomography (PET), (124)I has the longest physical half-life (4.2 days). The long half-life and well-investigated behavior of iodine in vivo makes (124)I very attractive for pharmacological studies. In this communication, we describe a simple yet effective method for the synthesis of novel (124)I labeled compounds intended for PET imaging of arylsulfatase activity in vivo. Arylsulfatases have important biological functions, and genetic deficiencies of such functions require pharmacological replacement, the efficacy of which must be properly and non-invasively evaluated. These enzymes, even though their natural substrates are mostly of aliphatic nature, hydrolyze phenolic sulfates to phenol and sulfuric acid. The availability of [(124)I]iodinated substrates is expected to provide a PET-based method for measuring their activity in vivo. The currently available methods of synthesis of iodinated arylsulfates usually require either introducing of a protected sulfate ester early in the synthesis or introduction of sulfate group at the end of synthesis in a separate step. The described method gives the desired product in one step from an aryl-alkyl cyclic sulfate. When treated with iodide, the source cyclic sulfate opens with substitution of iodide at the alkyl center and gives the desired arylsulfate monoester.

Asymmetric formal synthesis of schulzeines A and C.

The asymmetric formal synthesis of schulzeines A and C is described. Key features of the synthesis include the efficient and stereoselective construction of the benzoquinolizidine skeleton via the aza-Claisen rearrangement-induced ring expansion of the 1-vinyl-N-glycyl-isoquinoline, which was prepared by the highly enantioselective asymmetric allylation of the 8-benzyloxy-substituted dihydroisoquinoline and by the acid-catalyzed transannulation of the resulting 10-membered lactam.

Modification in the side chain of solomonsterol A: discovery of cholestan disulfate as a potent pregnane-X-receptor agonist.

Seven synthetic analogues of the PXR (pregnane-X-receptor) potent natural agonist solomonsterol A were prepared by total synthesis. Their activity toward PXR was assessed by transactivation and RT-PCR assays. The study discloses cholestan disulfate (8) as a new, simplified agonist of PXR. By in vitro studies on hepatic cells we have demonstrated that this compound is a potent PXR agonist and functional characterization in human macrophages and hepatic stellate cells provided evidence that cholestan disulfate (8) has the ability to modulate the immune response triggered by bacterial endotoxin as well as to counter-activate hepatic stellate cell activation induced by thrombin. Because inhibition of immune-driven circuits might have relevance in the treatment of inflammation and liver fibrosis, the present data support the development of cholestan disulfate (8) in preclinical models of inflammatory diseases.

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.

Synthesis of surface sulfated BiWO with enhanced photocatalytic performance.

Sulfated BiWO (SBiWO) was synthesized by an impregnation method to enhance the visible-light-driven photoactivities of BiWO (BiWO). The characterization results verified that sulfate anion mainly anchored on the catalyst surface greatly extended the visible-light-responsive range without destroying the crystal lattice. Moreover, the SBiWO-based photoactivities were evaluated with the removal of Malachite Green (MG) under UV-Vis irradiation emitted from two microwave-powered electrodeless discharge lamps (MPEDL2) and under visible light (lamda > 420 nm). The results demonstrated that the kinetic constant was increased 2.25 times, varying from 0.1478 (BiWO) to 0.3328 min(-1) (SBiWO-1). Similar results were also obtained for the visible light-driven reaction. Furthermore, radical scavengers such as t-butanol restricted the visible-light induced degradation of MG over BiWO and SBiWO-1. This indicated that the sulfating process increased the generation of reactive oxygen species, which was further verified by molecular probe with salicylic acid. Thus, more blue-shifting at lam = 618 nm was observed over SBiWO. On the basis of the above results, the photocatalytic mechanism over the sulfated catalyst was also discussed.

Synthesis, separation, and characterization of amphiphilic sulfated oligosaccharides enabled by reversed-phase ion pairing LC and LC-MS methods.

Synthesis of amphiphilic oligosaccharides is problematic because traditional methods for separating and purifying oligosaccharides, including sulfated oligosaccharides, are generally not applicable to working with amphiphilic sugars. We report here RPIP-LC and LC-MS methods that enable the synthesis, separation, and characterization of amphiphilic N-arylacyl O-sulfonated aminoglycosides, which are being pursued as small-molecule glycosaminoglycan mimics. The methods described in this work for separating and characterizing these amphiphilic saccharides are further applied to a number of uses: monitoring the progression of sulfonation reactions with analytical RP-HPLC, characterizing sulfate content for individual molecules with ESI-MS, determining the degree of sulfation for products having mixed degrees of sulfation with HPLC and LC-MS, and purifying products with benchtop C18 column chromatography. We believe that the methods described here will be broadly applicable to enabling the synthesis, separation, and characterization of amphiphilic, sulfated, and phosphorylated oligosaccharides and other types of molecules substituted to varying degrees with both anionic and hydrophobic groups.