Sayaka Masuko

Chemoenzymatic Synthesis of Homogeneous Ultralow Molecular Weight Heparins

An enzymatic synthesis may ultimately offer a more efficient means of producing an important class of anticoagulant drugs. Ultralow molecular weight (ULMW) heparins are sulfated glycans that are clinically used to treat thrombotic disorders. ULMW heparins range from 1500 to 3000 daltons, corresponding from 5 to 10 saccharide units. The commercial drug Arixtra (fondaparinux sodium) is a structurally homogeneous ULMW heparin pentasaccharide that is synthesized through a lengthy chemical process. Here, we report 10- and 12-step chemoenzymatic syntheses of two structurally homogeneous ULMW heparins (MW = 1778.5 and 1816.5) in 45 and 37% overall yield, respectively, starting from a simple disaccharide. These ULMW heparins display excellent in vitro anticoagulant activity and comparable pharmacokinetic properties to Arixtra, as demonstrated in a rabbit model. The chemoenzymatic approach is scalable and shows promise for a more efficient route to synthesize this important class of medicinal agent.

Orthogonal analytical approaches to detect potential contaminants in heparin

Heparin is a widely used anticoagulant and antithrombotic agent. Recently, a contaminant, oversulfated chondroitin sulfate (OSCS), was discovered within heparin preparations. The presence of OSCS within heparin likely led to clinical manifestations, most prevalently, hypotension and abdominal pain leading to the deaths of several dozens of patients. Given the biological effects of OSCS, one continuing item of concern is the ability for existing methods to identify other persulfonated polysaccharide compounds that would also have anticoagulant activity and would likely elicit a similar activation of the contact system. To complete a more extensive analysis of the ability for NMR and capillary electrophoresis (CE) to capture a broader array of potential contaminants within heparin, we completed a systematic study of NMR, both mono- and bidimensional, and CE to detect both various components of sidestream heparin and their persulfonated derivatives. We show that given the complexity of heparin samples, and the requirement to ensure their purity and safety, use of orthogonal analytical techniques is effective at detecting an array of potential contaminants that could be present.

Chemoenzymatic synthesis of uridine diphosphate-GlcNAc and uridine diphosphate-GalNAc analogs for the preparation of unnatural glycosaminoglycans.

Eight N-acetylglucosamine-1-phosphate and N-acetylgalactosamine-1-phosphate analogs have been synthesized chemically and were tested for their recognition by the GlmU uridyltransferase enzyme. Among these, only substrates that have an amide linkage to the C-2 nitrogen were transferred by GlmU to afford their corresponding uridine diphosphate(UDP)-sugar nucleotides. Resin-immobilized GlmU showed comparable activity to nonimmobilized GlmU and provides a more facile final step in the synthesis of an unnatural UDP-donor. The synthesized unnatural UDP-donors were tested for their activity as substrates for glycosyltransferases in the preparation of unnatural glycosaminoglycans in vitro. A subset of these analogs was useful as donors, increasing the synthetic repertoire for these medically important polysaccharides.

Fluorous-assisted chemoenzymatic synthesis of heparan sulfate oligosaccharides.

The chemoenzymatic synthesis of heparan sulfate tetrasaccharide (1) and hexasaccharide (2) with a fluorous tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate (FBoc) tag did not interfere with enzymatic recognition for both elongation and specific sulfation, and flash purification was performed by standard fluorous solid-phase extraction (FSPE). Based on an FBoc attached disaccharide as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate oligosaccharides were successfully synthesized employing fluorous techniques.

Structure/Function Analysis of Pasteurella multocida Heparosan Synthases: TOWARD DEFINING ENZYME SPECIFICITY AND ENGINEERING NOVEL CATALYSTS*

Background: The Pasteurella heparosan synthase isozymes are highly homologous yet have different catalytic properties. Results: Chimeric enzymes bearing combinations of desirable traits were produced, and structure/function relationships were studied. Conclusion: Distinct regions of the isozymes are important for high specific activity, sugar analog usage, and chain initiation and elongation. Significance: New catalysts with utility for production of defined heparan sulfate polysaccharides were created. The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

Chemoenzymatic synthesis of the next generation of ultralow MW heparin therapeutics.

Heparin, a sulfated glycosaminoglycan, is a widely used injectable anticoagulant. This polysaccharide is a natural product extracted from porcine intestinal tissue. A specific pentasaccharide sequence is responsible for heparins high affinity towards anti-thrombin III, which undergoes a conformational change and, as a result, inhibits the blood coagulation Factor Xa, a critical serine protease at the convergence on the intrinsic and extrinsic activation pathway of the coagulation cascade. Due to its structural complexity and heterogeneity, the synthesis of the anti-thrombin III-binding sequence of heparin has been limited to a few approaches. The heparin contamination crisis in 2007 has motivated the development of alternative methods for the efficient preparation of safe heparin products. In this article, we discuss the current methods and recent advances in heparin and low MW heparin syntheses and the recent successful chemoenzymatic preparation of ultralow MW heparins.

Signal Amplification by Glyco‐qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology

Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery.

Glycosaminoglycans of the Porcine Central Nervous System

Glycosaminoglycans (GAGs) are known to participate in central nervous system processes such as development, cell migration, and neurite outgrowth. In this paper, we report an initial glycomics study of GAGs from the porcine central nervous system. GAGs of the porcine central nervous system, brain and spinal cord were isolated and purified by defatting, proteolysis, anion-exchange chromatography, and methanol precipitation. The isolated GAG content in brain was 5 times higher than in spinal cord (0.35 mg/g of dry sample, compared to 0.07 mg/g of dry sample). In both tissues, chondroitin sulfate (CS) and heparan sulfate (HS) were the major and the minor GAG, respectively. The average molecular masses of CS from brain and spinal cord were 35.5 and 47.1 kDa, respectively, and those for HS from brain and spinal cord were 56.9 and 34 kDa, respectively. The disaccharide analysis showed that the compositions of CS from brain and spinal cords are similar, with uronic acid (1→3) 4-O-sulfo-N-acetylgalactosamine residue corresponding to the major disaccharide unit (CS type A) along with five minor disaccharide units. The major disaccharides of both brain and spinal cord HS were uronic acid (1→4) N-acetylglucosamine and uronic acid (1→4) 6-O-sulfo-N-sulfoglucosamine, but their composition of minor disaccharides differed. Analysis by (1)H and two-dimensional NMR spectroscopy confirmed these disaccharide analyses and provided the glucuronic/iduronic acid ratio. Finally, both purified CS and HS were biotinylated and immobilized on BIAcore SA biochips. Interactions between these GAGs and fibroblast growth factors (FGF1 and FGF2) and sonic hedgehog (Shh) were investigated by surface plasmon resonance.

Fibroblast Growth Factor-based Signaling through Synthetic Heparan Sulfate Blocks Copolymers Studied Using High Cell Density Three-dimensional Cell Printing

Background: Fibroblast growth factor-receptor-heparan sulfate (FGF-HS-FGFR) signals cell proliferation. Results: HS synthesized with sulfated domains at its non-reducing ends actively promotes cellular proliferation in a three-dimensional cell microarray. Conclusion: A symmetric 2:2:2 FGF-HS-FGFR complex is preferred over an asymmetric 2:1:2 model by these data. Significance: This paper suggests a preference for symmetry in the signal transduction complex having two FGF-FGFR on the non-reducing end of two HS chains. Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ∼40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

Antimicrobial mechanism of resveratrol-trans-dihydrodimer produced from peroxidase-catalyzed oxidation of resveratrol.

Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol‐trans‐dihydrodimer and pallidol) produced were tested for antimicrobial activity. The resveratrol‐trans‐dihydrodimer displayed antimicrobial activity against the Gram‐positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 μM, respectively) and against Gram‐negative Escherichia coli (MIC = 123 μM, upon addition of the efflux pump inhibitor Phe‐Arg‐β‐naphthylamide). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometric analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol‐trans‐dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide, and a platform for the development of polyphenolic antimicrobials. Biotechnol. Bioeng. 2015;112: 2417–2428.