[Show abstract][Hide abstract] ABSTRACT: The use of biomass as raw material is becoming a major alternative to fossil fuels, as it is widely abundant and relatively inexpensive. Carbohydrates, such as cellulose, starch, and sucrose, are important raw materials in the chemical industry, as they are produced from biomass that is readily available in large amounts, facilitating their large-scale application. It is necessary to define the concept of green chemistry and the principles that govern it to adapt the chemistry of carbohydrates to sustainable production and processing. Green chemistry also offers the tools to build a sustainable industrial and research effort.
Chemical Reviews 06/2015; 115(14):6811–6853. DOI:10.1021/cr500719h · 46.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII-Arixtra complex. IM measurements of ATIII and ATIII-Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII-Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein-carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.
The Analyst 06/2015; DOI:10.1039/c5an00908a · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Chemical Communications 06/2015; 51(55). DOI:10.1039/c5cc02008e · 6.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A biotinylated heparosan hexasaccharide was synthesized using one-pot multi-enzyme strategy, in situ activation and transfer of N-trifluoroacetylglucosamine (GlcNTFA) to heparin backbone significantly improved the synthetic efficiency. The biotinylated hexasaccharide could serve as a flexible core to diversify its conversion into heparan sulfate isoforms with potential biological applications and therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Covering: up to May 2014Heparan 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.
[Show abstract][Hide abstract] ABSTRACT: The exquisite control of growth factor function by heparan sulfate (HS) is dictated by tremendous structural heterogeneity of sulfated modifications. How specific HS structures control growth factor-dependent progenitor expansion during organogenesis is unknown. We isolated KIT+ progenitors from fetal salivary glands during a stage of rapid progenitor expansion and profiled HS biosynthetic enzyme expression. Enzymes generating a specific type of 3-O-sulfated-HS (3-O-HS) are enriched, and fibroblast growth factor 10 (FGF10)/FGF receptor 2b (FGFR2b) signaling directly regulates their expression. Bioengineered 3-O-HS binds FGFR2b and stabilizes FGF10/FGFR2b complexes in a receptor- and growth factor-specific manner. Rapid autocrine feedback increases 3-O-HS, KIT, and progenitor expansion. Knockdown of multiple Hs3st isoforms limits fetal progenitor expansion but is rescued with bioengineered 3-O-HS, which also increases adult progenitor expansion. Altering specific 3-O-sulfated epitopes provides a mechanism to rapidly respond to FGFR2b signaling and control progenitor expansion. 3-O-HS may expand KIT+ progenitors in vitro for regenerative therapy.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate (HS) is a linear polysaccharide expressed on cell surfaces, in extracellular matrices and cellular granules in metazoan cells. Through non-covalent binding to growth factors, morphogens, chemokines and other protein families, HS is involved in all multicellular physiological activities. Its biological activities depend on the fine structures of its protein-binding domains, the determination of which remains a daunting task. Methods have advanced to the point that information-rich product ion mass spectra may be produced on purified HS saccharides. However, the interpretation of these complex product ion patterns has emerged as the bottleneck to the dissemination of these HS sequencing methods. To solve this problem, we designed HS-SEQ, the first comprehensive algorithm for HS de novo sequencing using high-resolution tandem mass spectra. We tested HS-SEQ using negative electron transfer dissociation (NETD) tandem mass spectra generated from a set of pure synthetic saccharide standards with diverse sulfation patterns. The results showed that HS-SEQ rapidly and accurately determined the correct HS structures from large candidate pools.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate and heparin are highly sulfated polysaccharides that consist of a repeating disaccharide unit of glucosamine and glucuronic or iduronic acid. The 2-O-sulfated iduronic acid (IdoA2S) residue is commonly found in heparan sulfate and heparin; however, 2-O-sulfated glucuronic acid (GlcA2S) is a less abundant monosaccharide (∼<5% of total saccharides). Here, we report the synthesis of three GlcA2S-containing hexasaccharides using a chemoenzymatic approach. For comparison purposes, additional IdoA2S-containing hexasaccharides were synthesized. NMR analyses were performed to obtain full chemical shift assignments for the GlcA2S- and IdoA2S-hexasaccharides. These data show that GlcA2S is a more structurally rigid saccharide residue than IdoA2S. The antithrombin binding affinities of a GlcA2S- hexasaccharide and an IdoA2S-hexasaccharide were determined by affinity co-electrophoresis. In contrast to IdoA2S-hexasaccharides, the GlcA2S-hexasaccharide does not bind to antithrombin, confirming that the presence of IdoA2S is critically important for the anticoagulant activity. The availability of pure synthetic GlcA2S-containing oligosaccharides will allow the investigation of the structure and activity relationships of individual sites in heparin or heparan sulfate.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3-phosphoadenosine 5-phosphate, and a heptasaccharide. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg80, Lys350, and Arg190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.
[Show abstract][Hide abstract] ABSTRACT: Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights. The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average molecular weight (MW): unfractionated heparin (UFH, MWavg 14,000), low molecular weight heparin (LMWH, MWavg 3,500-6,500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.
[Show abstract][Hide abstract] ABSTRACT: Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
Nature Chemical Biology 02/2014; 10(4). DOI:10.1038/nchembio.1459 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 3D 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.
[Show abstract][Hide abstract] ABSTRACT: O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [(35)S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
[Show abstract][Hide abstract] ABSTRACT: Heparin, the first biopolymeric drug, possesses a wide range of structural heterogeneity owing to its biosynthesis. Its diverse fine structure is further complicated by an animal tissue-based recovery, leading to considerable structural differences within commercial heparin active pharmaceutical ingredients (APIs). Serious concerns about control of livestock, the primary source of heparin, have been raised since 1990s following a series of incidents involving Bovine spongiform encephalopathy, viral infections and prion contamination. Lack of quality control during initial recovery stages led to adulteration of pharmaceutical heparin with oversulfated chondroitin sulfate (OSCS), resulting in an international crisis in 2008 associated with 100 deaths in US.
The inherent problems with the animal tissue-based heparin production have motivated us to develop a commercially feasible chemoenzymatic heparin preparation process. This is based on bacterial fermentation of E. coli K5 to generate a capsular polysaccharide heparin precursor, that is then chemically N-deacetylated and N-sulfonated. A series of six recombinant enzymes, derived from heparin biosynthetic pathway and expressed in E. coli, are then used to epimerize uronic acid residues and sulfonate the C2, C3 and C6 positions. These modifications are carried out in tandem with sensitive analytical techniques and bioassays to result in a series of controlled structural changes. Bioengineered heparin consists of 86% trisulfated disaccharide resembling the highly sulfated structure of heparin. The formation of 9.4% lyase resistant tetrasaccharides upon digestion with heparin lyase 2 is consistent with the presence of antithrombin binding pentasaccharide regions in the range of USP heparins (3.9-6.7%). 1D-1H NMR and HMQC techniques were employed to elucidate presence of 3-O-sulfo and I2S peak in anomeric regions. Peaks corresponding to critical features in the IdoA and GlcN residues, including 2-O-sulfo, N-sulfo, N-acetyl, 3-O-sulfo, and 6-O-sulfo, have been fully assigned by 1H-NMR and HMQC spectra. Investigation of in-vitro anticoagulant activity confirms the biological activity of chemoenzymatically derived heparin with an anti-factor Xa activity of 205.8 U/mg and an anti-factor IIa activity of 225.2 U/mg (anti-factor Xa:anti-factor IIa ratio of 0.91) complying with USP requirements. These analyses showcase generic equivalence of bioengineered heparin to animal derived heparin API. A more robust process based on immobilized enzymes, enhancing enzymatic stability and increased reusability, is under development and will be employed to generate bioengineered heparin for in-vivo studies.