Synthesis, characterization, thermokinetic analysis and biological application of novel allyl glucosamine based glycopolymers.

The synthesis of glycopolymers by copolymerising an allyl glucosamine (AG) monomer with co-monomers methyl methacrylate (MMA), acrylonitrile (AN) and 2-hydroxyethyl methacrylate (HEMA) was investigated via free-radical polymerisation of 2,2-azobisisobutyronitrile (AIBN) in dimethylformamide (DMF). Three new copolymers, poly(AG-co-MMA), poly(AG-co-AN) and poly(AG-co-HEMA), were obtained. The chemical structures of the glycopolymers were analysed using 1H-NMR, 13C-NMR and FTIR. The thermal properties and degradation kinetics of the three glycopolymers were examined by thermogravimetric (TG) analysis at different heating rates. The effects of different co-monomers on the copolymerisation yield, thermal properties and biological activities of the resulting glycopolymers were investigated. The activation energies of the decomposition stages were calculated using the Flynn-Wall-Ozawa (FWO) and Kissinger methods. Furthermore, the biological activity of AG monomers and glycopolymers was studied and compared to chitosan. Poly(AG-co-HEMA) had the most significant effect on MCF-7 cell viability, and all glycopolymers have a low toxic effect profile on MCF-7 cell lines.

Unexpected Diastereomer Formation and Interconversions in Cyclohexane-1,2-diacetal Derivatization of a Glucuronic Acid Thioglycoside.

Reactions of a glucuronic acid (GlcA) ß-thioglycoside with cyclohexadione show initial formation of the two anticipated all-trans decalin-type O2,O3 and O3,O4 cyclohexane-1,2-diacetals (CDAs) along with an epimer of the main O2,O3 acetal. This trans-cis isomer is then interconverted leading to higher amounts of the two all-trans products. Isomerization studies indicate slow interconversion between the all-trans CDA acetals, with only one undergoing significant interconversion with the minor 2,3-diastereomer. Crystal structures of all three isomers are included. These findings are relevant to other uses of CDA protections where occurrence of apparently disfavored isomers may be occurring, along with interconversions between CDA isomers.

Synthesis and structure of d-glucuronolactone derived carboxamides.

5-O-Protected and 1,2-acetonide-protected D-glucurono-6,3-lactone furanosides were converted into novel furano-glucuronamides through treatment with ammonia. Several O3 protections and O5-deprotection routes afford new primary gluconamide derivatives. However, attempted O3-benzylations of O5-protected intermediates led instead to silyl migration (from O5-TDBMS), competitive N-benzylation or reclosure to the lactone are observed as competing processes. This is not seen the using 5-O-PMB protection which the provides the method of choice for obtaining a fully protection-differentiated glucofuranamide. X-ray crystal structures of a fully-protected glucurono-6,3-lactone lactone and a glucuronamide derivatives are reported.

Solid state structure of sodium ß-1-thiophenyl glucuronate identifies 5-coordinate sodium with three independent glucoronates.

Glucuronic acid is a key component of the glycosaminoglycans (GAGs) Chrondroitin Sulfate (CS), Heparin/Heparan sulfate (HS) and Hyaluronic Acid (HA), as well an important metabolite derivative. In biological systems the carboxylate of uronic acids in GAGs is involved in important H-binding interactions, and the role of metal coordination, such as sodiated systems, has indications associated with a number of biological effects, and physiological GAG-related processes. In synthetic approaches to GAG fragments, thioglycoside intermediates, or derivatives from these, are commonly employed. Of the reported examples of sodium coordination in carbohydrates, 6-coordinate systems are usually observed often with water ligands involved, Herein we report an unexpected 5-coordinate sodiated GlcA crystal structure of the parent GlcA, but as a thioglycoside derivative, whose crystal coordination differs from previous examples, with no involvement of water as a ligand and containing a distorted trigonal bypramidal sodium with each GlcA having five of 6 oxygens sodium-coordinated.

Synthesis of Heparan Sulfate- and Dermatan Sulfate-Related Oligosaccharides via Iterative Chemoselective Glycosylation Exploiting Conformationally Disarmed [2.2.2] l-Iduronic Lactone Thioglycosides.

Heparan sulfate (HS) and dermatan sulfate (DS) are l-iduronic acid containing glycosaminoglycans (GAGs) which are implicated in a number of biological processes and conditions including cancer and viral infection. Chemical synthesis of HS and DS is required to generate structurally defined oligosaccharides for a biological study. Herein, we present a new synthetic approach to HS and DS oligosaccharides using chemoselective glycosylation which relies on a disarmed [2.2.2] l-ido lactone motif. The strategy provides a general approach for iterative-reducing end chain extension, using only shelf-stable thioglycoside building blocks, exploiting a conformational switch to control reactivity, and thus requires no anomeric manipulation steps between glycosylations.

Evaluation of neomycin analogues for HIV-1 RRE RNA recognition identifies enhanced activity simplified neamine analogues.

Synthetic neamine mimetics have been evaluated for binding to the HIV-1 Rev response element. Modified neamine derivatives, obtained from reductive amination of neamine, led to identification of new 6-amino modified neamine-type ligands with HIV-1 RRE binding affinity up to 20× that of neamine and up to 6× that of the more complex neomycin itself. This provides a noteworthy structure-activity increase and a useful lead to simplified, chemically accessible mimetics.

C3 and C6 Modification-Specific OYE Biotransformations of Synthetic Carvones and Sequential BVMO Chemoenzymatic Synthesis of Chiral Caprolactones.

The scope for biocatalytic modification of non-native carvone derivatives for speciality intermediates has hitherto been limited. Additionally, caprolactones are important feedstocks with diverse applications in the polymer industry and new non-native terpenone-derived biocatalytic caprolactone syntheses are thus of potential value for industrial biocatalytic materials applications. Biocatalytic reduction of synthetic analogues of R-(-)-carvone with additional substituents at C3 or C6, or both C3 and C6, using three types of OYEs (OYE2, PETNR and OYE3) shows significant impact of both regio-substitution and the substrate diastereomer. Bioreduction of (-)-carvone derivatives substituted with a Me and/or OH group at C6 is highly dependent on the diastereomer of the substrate. Derivatives bearing C6 substituents larger than methyl moieties are not substrates. Computer docking studies of PETNR with both (6S)-Me and (6R)-Me substituted (-)-carvone provides a model consistent with the outcomes of bioconversion. The products of bioreduction were efficiently biotransformed by the Baeyer-Villiger monooxygenase (BVase) CHMO_Phi1 to afford novel trisubstituted lactones with complete regioselectivity to provide a new biocatalytic entry to these chiral caprolactones. This provides both new non-native polymerization feedstock chemicals, but also with enhanced efficiency and selectivity over native (+)-dihydrocarvone Baeyer-Villigerase expansion. Optimum enzymatic reactions were scaled up to 60-100 mg, demonstrating the utility for preparative biocatalytic synthesis of both new synthetic scaffold-modified dihydrocarvones and efficient biocatalytic entry to new chiral caprolactones, which are potential single-isomer chiral polymer feedstocks.

Inversion dimers dominate the crystal packing in the structure of trimethyl citrate (trimethyl 2-hy-droxy-propane-1,2,3-tri-carboxyl-ate).

Trimethyl citrate, C9H14O7 (systematic name: trimethyl 2-hy-droxy-propane-1,2,3-tri-carboxyl-ate), 2, was prepared by the esterification of citric acid and methanol in the presence of thionyl chloride at 273 K. The bond lengths and angles in 2 compare closely with those observed in citric acid. The C-C bonds adjacent to the terminal carboxyl groups are significantly shorter than those around the central C atom. The central carboxyl-ate group and the hy-droxy group occur in the normal planar arrangement with an r.m.s. deviation of 0.0171 Šfrom the mean plane involving all six atoms in the central unit. The crystal structure is almost completely dominated by the formation of inversion dimers through an O-H⋯O hydrogen bond, together with an extensive array of weaker C-H⋯O contacts. These generate a three-dimensional network structure with mol-ecules stacked along the c-axis direction.

Chemoenzymatic Synthesis of the Intermediates in the Peppermint Monoterpenoid Biosynthetic Pathway.

A chemoenzymatic approach providing access to all four intermediates in the peppermint biosynthetic pathway between limonene and menthone/isomenthone, including noncommercially available intermediates (-)- trans-isopiperitenol (2), (-)-isopiperitenone (3), and (+)- cis-isopulegone (4), is described. Oxidation of (+)-isopulegol (13) followed by enolate selenation and oxidative elimination steps provides (-)-isopiperitenone (3). A chemical reduction and separation route from (3) provides both native (-)- trans-isopiperitenol (2) and isomer (-)- cis-isopiperitenol (18), while enzymatic conjugate reduction of (-)-isopiperitenone (3) with IPR [(-)-isopiperitenone reductase)] provides (+)- cis-isopulegone (4). This undergoes facile base-mediated chemical epimerization to (+)-pulegone (5), which is subsequently shown to be a substrate for NtDBR ( Nicotiana tabacum double-bond reductase) to afford (-)-menthone (7) and (+)-isomenthone (8).

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

The realization of a synthetic biology approach to microbial (1R,2S,5R)-(-)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase.

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

Metabolism of steroidal lactones by the fungus Corynespora cassiicola CBS 161.60 results in a mechanistically unique intramolecular ring-D cyclization resulting in C-14 spiro-lactones.

The fungus Corynespora cassiicola metabolises exogenous steroids in a unique and highly specific manner. Central to this, is the ability of this organism to functionalise substrates (androgens, progestogens) at the highly stereochemically hindered 8ß-position of the steroid nucleus. A recent study has identified that 8ß-hydroxylation occurs through inverted binding in a 9α-hydroxylase. In order to discern the metabolic fate of more symmetrical molecules, we have investigated the metabolism of a range of steroidal analogues functionalised with ring-D lactones, but differing in their functional group stereochemistry at carbon-3. Remarkably, the 3α-functionalised steroidal lactones underwent a mechanistically unique two step intramolecular cyclisation resulting in the generation of a ring-D spiro-carbolactone. This rapid rearrangement initiated with hydroxylation at carbon 14 followed by transesterification, resulting in ring contraction with formation of a butyrolactone at carbon-14. Remarkably this rearrangement was found to be highly dependent on the stereochemistry at carbon-3, with the ß-analogues only undergoing 9α-hydroxylation. The implications of these findings and their mechanistic bases are discussed.

Excited-State Properties of Protochlorophyllide Analogues and Implications for Light-Driven Synthesis of Chlorophyll.

Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate for the light-driven enzyme protochlorophyllide oxidoreductase. Pchlide has excited-state properties that allow it to initiate photochemistry in the enzyme active site, which involves reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical behavior has been investigated here using a combination of time-resolved spectroscopies and density functional theory calculations of a number of Pchlide analogues with modifications to various substituent groups. A keto group on ring E is essential for excited-state charge separation in the molecule, which is the driving force for the photoreactivity of the pigment. Vibrational "fingerprints" of specific regions of the Pchlide chromophore have been assigned, allowing identification of the modes that are crucial for excited-state chemistry in the enzyme. This work provides an understanding of the structural determinants of Pchlide that are important for harnessing light energy.

Pyrazolobenzothiazine-based carbothioamides as new structural leads for the inhibition of monoamine oxidases: design, synthesis, in vitro bioevaluation and molecular docking studies.

Two new series of pyrazolobenzothiazine-based carbothioamides (3a-o and 4a-o) were synthesized using saccharin as the starting material. The synthesized derivatives were investigated for their ability to inhibit monoamine oxidases (MAO). Compound 3b was found to be a very potent MAO-A inhibitor with an IC50 value of 0.003 ± 0.0007 µM, while compound 4d was the most effective inhibitor of MAO-B having an IC50 value of 0.02 ± 0.001 µM. Molecular docking studies were performed to identify the probable binding modes in the active site of the monoamine oxidase enzymes. The synthetic and computational investigations in the current work suggested that these newly identified inhibitors may serve as a powerful starting point for the exploration and optimization of potential therapeutic agents targeting Parkinson's disease.

Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,ß-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.

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.

Synthetic Site-Selectively Mono-6-O-Sulfated Heparan Sulfate Dodecasaccharide Shows Anti-Angiogenic Properties In Vitro and Sensitizes Tumors to Cisplatin In Vivo.

Heparan sulphate (HS), a ubiquitously expressed glycosaminoglycan (GAG), regulates multiple cellular functions by mediating interactions between numerous growth factors and their cell surface cognate receptors. However, the structural specificity of HS in these interactions remains largely undefined. Here, we used completely synthetic, structurally defined, alternating N-sulfated glucosamine (NS) and 2-O-sulfated iduronate (IS) residues to generate dodecasaccharides ([NSIS]6) that contained no, one or six glucosamine 6-O-sulfates (6S). The aim was to address how 6S contributes to the potential of defined HS dodecasaccharides to inhibit the angiogenic growth factors FGF2 and VEGF165, in vitro and in vivo. We show that the addition of a single 6S at the non-reducing end of [NSIS]6, i.e. [NSIS6S]-[NSIS]5, significantly augments the inhibition of FGF2-dependent endothelial cell proliferation, migration and sprouting in vitro when compared to the non-6S variant. In contrast, the fully 6-O-sulfated dodecasaccharide, [NSIS6S]6, is not a potent inhibitor of FGF2. Addition of a single 6S did not significantly improve inhibitory properties of [NSIS]6 when tested against VEGF165-dependent endothelial cell functions.In vivo, [NSIS6S]-[NSIS]5 blocked FGF2-dependent blood vessel formation without affecting tumor growth. Reduction of non-FGF2-dependent ovarian tumor growth occurred when [NSIS6S]-[NSIS]5 was combined with cisplatin. The degree of inhibition by [NSIS6S]-[NSIS]5 in combination with cisplatin in vivo equated with that induced by bevacizumab and sunitinib when administered with cisplatin. Evaluation of post-treatment vasculature revealed that [NSIS6S]-[NSIS]5 treatment had the greatest impact on tumor blood vessel size and lumen formation. Our data for the first time demonstrate that synthetic, structurally defined oligosaccharides have potential to be developed as active anti-angiogenic agents that sensitize tumors to chemotherapeutic agents.

Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,ß-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.

An Updated Synthesis of the Diazo-Transfer Reagent Imidazole-1-sulfonyl Azide Hydrogen Sulfate.

Imidazole-1-sulfonyl azide and salts thereof are valuable reagents for diazo-transfer reactions, most particularly conversion of primary amines to azides. The parent reagent and its HCl salt present stability and detonation risks, but the hydrogen sulfate salt is significantly more stable. An updated procedure for the large-scale synthesis of this salt avoids isolation or concentration of the parent compound or HCl salt and will facilitate the use of hydrogen sulfate salt as the reagent of choice for diazo transfer.

The influence of few-layer graphene on the gas permeability of the high-free-volume polymer PIM-1.

Gas permeability data are presented for mixed matrix membranes (MMMs) of few-layer graphene in the polymer of intrinsic microporosity PIM-1, and the results compared with previously reported data for two other nanofillers in PIM-1: multiwalled carbon nanotubes functionalized with poly(ethylene glycol) (f-MWCNTs) and fused silica. For few-layer graphene, a significant enhancement in permeability is observed at very low graphene content (0.05 vol.%), which may be attributed to the effect of the nanofiller on the packing of the polymer chains. At higher graphene content permeability decreases, as expected for the addition of an impermeable filler. Other nanofillers, reported in the literature, also give rise to enhancements in permeability, but at substantially higher loadings, the highest measured permeabilities being at 1 vol.% for f-MWCNTs and 24 vol.% for fused silica. These results are consistent with the hypothesis that packing of the polymer chains is influenced by the curvature of the nanofiller surface at the nanoscale, with an increasingly pronounced effect on moving from a more-or-less spherical nanoparticle morphology (fused silica) to a cylindrical morphology (f-MWCNT) to a planar morphology (graphene). While the permeability of a high-free-volume polymer such as PIM-1 decreases over time through physical ageing, for the PIM-1/graphene MMMs a significant permeability enhancement was retained after eight months storage.