Precursor-Directed Biosynthesis and Biological Testing of omega-Alicyclic- and neo-Branched Tunicamycin N-Acyl Variants.

Tunicamycins (TUNs) are Streptomyces-derived natural products, widely used to block protein N-glycosylation in eukaryotes or cell wall biosynthesis in bacteria. Modified or synthetic TUN analogues that uncouple these activities have considerable potential as novel mode-of-action antibacterial agents. Chemically modified TUNs reported previously with attenuated activity on yeast have pinpointed eukaryotic-specific chemophores in the uridyl group and the N-acyl chain length and terminal branching pattern. A small molecule screen of fatty acid biosynthetic primers identified several novel alicyclic- and neo-branched TUN N-acyl variants, with primer incorporation at the terminal omega-acyl position. TUNs with unique 5- and 6-carbon ω-cycloalkane and ω-cycloalkene acyl chains are produced under fermentation and in yields comparable with the native TUN. The purification, structural assignments, and the comparable antimicrobial properties of 15 of these compounds are reported, greatly extending the structural diversity of this class of compounds for potential medicinal and agricultural applications.

Assessing the diversity of anthocyanin composition in various tissues of purple corn (Zea mays L.).

Anthocyanins are natural pigments used in various foods, beverages, textiles, and nutraceuticals. Anthocyanins in the grain of purple corn (Zea mays L., Poaceae) have been a focus of many studies, but not much is known about anthocyanins in other maize tissues. In this study, purple corn variety Apache Red Cob was crossed to genetic stock 320 N, which is recessive for anthocyanin 3. The result was intense anthocyanin production in portions of the plant not normally pigmented. Anthocyanin extracts from anthers, cob glumes, husks, kernels, leaf sheaths, seedlings, silks, and tassels were assessed using UHPLC. A previously undescribed pigment produced in anthers was determined by NMR to be anthocyanidin 3-6″-phenylacetylglucoside. Multivariate analysis classified maize anthocyanins into 8 major compositional profiles. Results of this study show that maize produces anthocyanins abundantly in non-grain portions of the plant and that maize anthocyanin extracts have numerous applications due to the diversity in pigment profiles and hues.

Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide phosphates, and sugar nucleotides.

Nucleosides and nucleotides are a group of small molecule effectors and substrates which include sugar nucleotides, purine and pyrimidine-based nucleotide phosphates, and diverse nucleotide antibiotics. We previously reported that hydrogenation of the nucleotide antibiotic tunicamycin leads to products with reduced toxicity on eukaryotic cells. We now report the hydrogenation of diverse sugar nucleosides, nucleotide phosphates, and pyrimidine nucleotides. UDP-sugars and other uridyl and thymidinyl nucleosides are quantitatively reduced to the corresponding 5,6-dihydro-nucleosides. Cytidyl pyrimidines are reduced, but the major products are the corresponding 5,6-dihydrouridyl nucleosides resulting from a deamination of the cytosine ring.

Unique Flavanol-Anthocyanin Condensed Forms in Apache Red Purple Corn.

Anthocyanin pigments from purple corn are being explored as a potential alternative to artificial colorants and for their health-promoting properties. However, all pericarp-pigmented corn varieties examined to date primarily contain cyanidin-derived anthocyanins, which produce bluish-red or pink extracts. Here we describe the first pelargonidin-dominant pericarp-pigmented corn lines from the landrace Apache Red (AR). Anthocyanins were characterized from six AR families using high-performance liquid chromatography-mass spectrometry (HPLC-MS). From this, we identified two new flavanol-anthocyanin condensed forms in corn: catechin-(4,8)-pelargonidin 3,5-diglucoside and afzelechin-(4,8)-pelargonidin 3,5-diglucoside, which were subsequently confirmed with NMR. Additionally, several apigenin-derived C-glycosyl flavones were identified in abundance. With a diverse flavonoid profile containing an array of different anthocyanin species and flavones, Apache Red will be an important line in which to study control of the flavonoid biosynthesis pathway.

Thiazolidine Peracetates: Carbohydrate Derivatives that Readily Assign cis-, trans-2,3-Monosaccharides by Gas Chromatography-Mass Spectrometry Analysis.

A novel group of carbohydrate derivatives is described that uniquely assign cis/ trans-2,3-aldose stereoisomers at low nanomolar concentrations. Aldopentoses, aldohexoses, or component aldoses from hydrolysis of polysaccharides or oligosaccharides react with cysteamine in pyridine to give quantitative formation of thiazolidines, which are subsequently peracetylated in a one-pot reaction. The nonpolar thiazolidines peracetate (TPA) derivatives are analyzed by gas chromatography and electron impact mass spectrometry (GC/EI-MS), each aldose giving rise to two TPA geometric isomers. The quantitative ratio of these diastereomers is dependent upon whether the parent monosaccharide is cis-2,3-(Rib, Lyx, Man, All, Gul, and Tal), or trans-2,3-aldose (Xyl, Ara, Glc, Gal, Ido, and Alt). TPAs generate observed EI-MS fragment ions characteristic of C1-C2 and C3-C4 bond cleavage of the parent sugars. This has been used to estimate the extent of metabolic labeling of microbial cell-wall carbohydrates, especially into the defining anomeric carbons and during aldolase / ketolase -catalyzed rearrangements.

Biosynthesis and Conformational Properties of the Irregular Sesquiterpenoids Isothapsadiene and ß-Isothapsenol.

A carbocation cyclization/rearrangement mechanism for the biosynthesis of isothapsadiene and ß-isothapsenol is shown to be energetically viable on the basis of density functional theory (DFT) calculations. In addition, for both isothapsadiene and ß-isothapsenol, variable-temperature NMR experiments reveal two equilibrium conformers that undergo hindered exchange. The identities of these conformers, which are related by a chair-flip, are confirmed by DFT calculations on their structures, energies, 1H and 13C chemical shifts, and interconversion pathways.

Selective catalytic hydrogenation of the N-acyl and uridyl double bonds in the tunicamycin family of protein N-glycosylation inhibitors.

Tunicamycin is a Streptomyces-derived inhibitor of eukaryotic protein N-glycosylation and bacterial cell wall biosynthesis, and is a potent and general toxin by these biological mechanisms. The antibacterial activity is dependent in part upon a π-π stacking interaction between the tunicamycin uridyl group and a specific Phe residue within MraY, a tunicamycin-binding protein in bacteria. We have previously shown that reducing the tunicamycin uridyl group to 5,6-dihydrouridyl (DHU) significantly lowers its eukaryotic toxicity, potentially by disrupting the π-stacking with the active site Phe. The present report compares the catalytic hydrogenation of tunicamycin and uridine with various precious metal catalysts, and describe optimum conditions for the selective production of N-acyl reduced tunicamycin or for tunicamycins reduced in both the N-acyl and uridyl double bonds. At room temperature, Pd-based catalysts are selective for the N-acyl reduction, whereas Rh-based catalysts favor the double reduction to provide access to fully reduced tunicamycin. The reduced DHU is highly base-sensitive, leading to amide ring opening under mild alkaline conditions.

A one-pot synthesis of 1,6,9,13-tetraoxadispiro(4.2.4.2)tetradecane by hydrodeoxygenation of xylose using a palladium catalyst.

In an effort to expand the number of biobased chemicals available from sugars, xylose has been converted to 1,6,9,13-tetraoxadispiro(4.2.4.2)tetradecane in a one-pot reaction using palladium supported on silica-alumina as the catalyst. The title compound is produced in 35-40% yield under 7 MPa H2 pressure at 733 K using 3-10 wt%Pd on silica-alumina catalyst. It is isolated using a combination of liquid-liquid extractions and flash chromatography. This dimer can be converted to its monomer, 2-hydroxy-(2-hydroxymethyl)tetrahydrofuran, which ring opens under acid conditions to 1,5-dihydroxy-2-pentanone. This diol can then be esterified with vinylacetate in phosphate buffer to produce 1,5-bis(acetyloxy)-2-pentanone which is an inhibitor of mammalian 11ß-hydroxysteroid dehydrogenase 1. (1)H and (13)C nmr spectra of each of these species are reported. The single crystal X-ray structure of the title compound is also reported. These data were collected in a temperature range of 100 K-273 K and show a solid state phase change from triclinic to monoclinic between 175 K and 220 K without a conformational change.

Quinovosamycins: new tunicamycin-type antibiotics in which the α, ß-1″,11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.

Octadecyl ferulate behavior in 1,2-Dioleoylphosphocholine liposomes.

Octadecyl ferulate was prepared using solid acid catalyst, monitored using Supercritical Fluid Chromatography and purified to a 42% yield. Differential scanning calorimetry measurements determined octadecyl ferulate to have melting/solidification phase transitions at 67 and 39°C, respectively. AFM imaging shows that 5-mol% present in a lipid bilayer induced domains to form. Phase behavior measurements confirmed that octadecyl ferulate increased transition temperature of phospholipids. Fluorescence measurements demonstrated that octadecyl ferulate stabilized liposomes against leakage, maintained antioxidant capacity within liposomes, and oriented such that the feruloyl moiety remained in the hydrophilic region of the bilayer. Molecular modeling calculation indicated that antioxidant activity was mostly influenced by interactions within the bilayer.

Frost Grape Polysaccharide (FGP), an Emulsion-Forming Arabinogalactan Gum from the Stems of Native North American Grape Species Vitis riparia Michx.

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified ßGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at >15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a ß1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

Nickel-Catalyzed Proton-Deuterium Exchange (HDX) Procedures for Glycosidic Linkage Analysis of Complex Carbohydrates.

The structural analysis of complex carbohydrates typically requires the assignment of three parameters: monosaccharide composition, the position of glycosidic linkages between monosaccharides, and the position and nature of noncarbohydrate substituents. The glycosidic linkage positions are often determined by permethylation analysis, but this can be complicated by high viscosity or poor solubility, resulting in under-methylation. This is a drawback because an under-methylated position may be misinterpreted as the erroneous site of a linkage or substituent. Here, we describe an alternative approach to linkage analysis that makes use of a nonreversible deuterium exchange of C-H protons on the carbohydrate backbone. The exchange reaction is conducted in deuterated water catalyzed by Raney nickel, and results in the selective exchange of C-H protons adjacent to free hydroxyl groups. Hence, the position of the residual C-H protons is indicative of the position of glycosidic linkages or other substituents and can be readily assigned by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) or, following suitable derivatization, by gas chromatography-mass spectroscopy (GC/MS) analysis. Moreover, because the only changes to the parent sugar are proton/deuterium exchanges, the composition and linkage analysis can be determined in a single step.

Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations.

Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan was hydrolyzed by the action of two commercial enzyme cocktails, in the presence and absence of an additional α-arabinofuranosidase enzyme. The two most abundant enzymatic digestion products from each commercial enzyme treatment were separated and characterized by LC-MS(n), linkage analysis, and NMR. The most abundant oligosaccharide from each commercial cocktail was susceptible to hydrolysis when supplemented with a GH62 α-arabinofuranosidase enzyme; further characterization confirmed the presence of (1→3)-α-arabinose linkages. These results demonstrate the lack of the required selectivity for arabinose-containing substrates in the commercial enzyme preparations tested. One product from each condition remained intact and was found to contain (1→2)-ß-xylose-(1→3)-α-arabinose side chains; this linkage acts as a source of oligosaccharide recalcitrance.

Determination of the stereochemistry of the aggregation pheromone of harlequin bug, Murgantia histrionica.

Preparation of a complete stereoisomeric library of 1,10-bisaboladien-3-ols and selected 10,11-epoxy-1-bisabolen-3-ols was pivotal for the identification of the aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys. Herein, we describe syntheses of the remaining 10,11-epoxy-1-bisabolen-3-ols, and provide additional evidence on the assignment of relative and absolute configurations of these compounds by single-crystal X-ray crystallography of an intermediate, (3S,6R,7R,10S)-1-bisabolen-3,10,11-triol. To demonstrate the utility of this stereoisomeric library, we revisited the aggregation pheromone of the harlequin bug, Murgantia histrionica, and showed that the male-produced pheromone consists of two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol. Employment of eight cis-10,11-epoxy-1-bisabolen-3-ol stereoisomeric standards, two enantioselective GC columns, and NMR spectroscopy enabled the identification of these compounds as (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol and (3S,6S,7R,10R)-10,11-epoxy-1-bisabolen-3-ol, which are produced by M. histrionica males in 1.4:1 ratio.

Structural characterization of (1→2)-ß-xylose-(1→3)-α-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with α-arabinofuranosidase and ß-endo-xylanase.

Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation and to reduce inhibitory effects xylooligomers may have on cellulolytic glycoside hydrolase enzymes. To identify structural features of switchgrass xylan that are not distinguishable by mass spectrometry alone, a α-arabinofuranosidase enzyme was used to remove the arabinose side chains from alkali-extracted switchgrass xylan from three cultivars with simultaneous hydrolysis by ß-endo-xylanase to enrich for oligosaccharide products with extended branching. The two most abundant enzymatic digestion products were separated and characterized by LC-MS(n), linkage analysis, and NMR. These two oligosaccharides were present in all three switchgrass cultivars and found to contain (1→2)-ß-xylose-(1→3)-α-arabinose side chains, a linkage not previously reported in switchgrass.

Discovery of the aggregation pheromone of the brown marmorated stink bug (Halyomorpha halys) through the creation of stereoisomeric libraries of 1-bisabolen-3-ols.

We describe a novel and straightforward route to all stereoisomers of 1,10-bisaboladien-3-ol and 10,11-epoxy-1-bisabolen-3-ol via the rhodium-catalyzed asymmetric addition of trimethylaluminum to diastereomeric mixtures of cyclohex-2-enones 1 and 2. The detailed stereoisomeric structures of many natural sesquiterpenes with the bisabolane skeleton were previously unknown because of the absence of stereoselective syntheses of individual stereoisomers. Several of the bisabolenols are pheromones of economically important pentatomid bug species. Single-crystal X-ray crystallography of underivatized triol 13 provided unequivocal proof of the relative and absolute configurations. Two of the epoxides, (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (4), were identified as the main components of a male-produced aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys, using GC analyses on enantioselective columns. Both compounds attracted female, male, and nymphal H. halys in field trials. Moreover, mixtures of stereoisomers containing epoxides 3 and 4 were also attractive to H. halys, signifying that the presence of additional stereoisomers did not hinder attraction of H. halys and relatively inexpensive mixtures can be used in monitoring, as well as control strategies. H. halys is a polyphagous invasive species in the U.S. and Europe that causes severe injury to fruit, vegetables, and field crops and is also a serious nuisance pest.

Glucansucrase acceptor reactions with d-mannose.

The main acceptor product of glucansucrases with d-mannose has not previously been identified. We used glucansucrases that form water-insoluble α-d-glucans to produce increased yields of acceptor products from d-mannose, and identified the major product as 6-O-α-d-glucopyranosyl-d-mannose. Glucansucrases that synthesize insoluble α-d-glucans produced higher yields of the disaccharide compared to typical dextransucrases.

Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases.

We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein sequencing indicates Cx1 is similar to but not identical to ß-xylosidases of GH52, including Q09LZ0, that have retaining mechanisms. Q09LZ0 ß-xylosidase had been characterized biochemically in kinetic reactions that contained Tris. We overproduced Q09LZ0 and demonstrated that Tris is a competitive inhibitor of the ß-xylosidase. Also, the previous work used grossly incorrect extinction coefficients for product 4-nitrophenol. We redetermined kinetic parameters using reactions that omitted Tris and using correct extinction coefficients for 4-nitrophenol. Cx1 and Q09LZ0 ß-xylosidases were thus shown to possess similar kinetic properties when acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose. kcat pH profiles of Cx1 and Q09LZ0 acting on 4-nitrophenyl-ß-d-xylopyranoside and xylobiose have patterns containing two rate increases with increasing acidity, not reported before for glycoside hydrolases. The dexylosylation step of 4-nitrophenyl-ß-d-xylopyranoside hydrolysis mediated by Q09LZ0 is not rate determining for kcat(4NPX).

Synthesis and spectral characterization of methyl 9(10)-dialkylphosphonostearates.

Dimethyl, diethyl, and di-n-butyl phosphites were reacted with methyl or ethyl oleates using thermally initiated radical reactions. Reactions were conducted with or without the presence of a dilauroyl peroxide initiator. The reactions gave mixture of isomers with the phosphorus attached at the 9 or 10 carbon of the stearates. High yields (94-97%) and high purity products (98-99% by GC) were obtained in the presence of the initiator, while without initiator, the reaction was very slow resulting in very low conversions (<50% after 6 days). The phosphonostearate products were positively identified and thoroughly characterized using GC with EI-MS, FTIR, and (1)H-, (13)C-, and (31)P NMR spectra. GC achieved only partial resolution of the positional isomers. Principal component analysis was applied to successfully separate the MS-EI spectra of fractions from the 9- and 10-isomers. A mechanism to explain the observed MS fragmentation pattern and the relative abundances is proposed. 2D-NMR data analysis was applied to assign values of (13)C- and (1)H NMR shifts as well as P-C and P-H splitting constants. The molecular volume and the refractive indices of the phosphonostearates were determined experimentally and were found to be in agreement with the computationally predicted values using the PM3 semi-empirical method and the group-contribution method of Bondi.

Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380.

Aureobasidium pullulans is a common, ubiquitous fungus, which is used industrially to produce the polysaccharide pullulan. We have previously shown that A. pullulans produces various heavier-than-water oils, first named here as liamocins, that accumulate in fermentations. Here we report the structural characterization of four liamocins, A1, A2, B1, and B2, produced by A. pullulans strain NRRL 50380 using a combination of MALDI-TOF/MS, quadrupole-TOF/MS, isotopic labeling, NMR, GC/MS, and classical carbohydrate analysis. The data showed that the liamocins are composed of a single mannitol headgroup partially O-acylated with three (for liamocin A1 and A2) or four (for liamocin B1 and B2) 3,5-dihydroxydecanoic ester groups. Liamocins A1 and B1 are non-acetylated, whereas A2 and B2 each contain a single 3'-O-acetyl group. Each of these compounds is characterized by pseudomolecular [M+Na](+) ions in the MALDI-TOF/MS spectra at m/z 763.22, 949.35, 805.22, and 991.37, respectively. The 186Da mass difference between A-type and B-type liamocins corresponds to one O-linked 3,5-dihydroxydecanoate group. HMBC NMR showed that one 3,5-dihydroxydecanoate carbonyl group is ester linked to a primary hydroxyl on the mannitol. Other long range (13)C-(1)H couplings across 1,5-ester bridges showed that the 3,5-dihydroxydecanoate groups form 1-5-linked polyester chains, similar in structure to the antibiotic substance exophilin A. Moreover, the MS analysis identified several non-conjugated poly-3,5-dihydroxydecanoate esters as minor components that are tentatively assigned as exophilins A1, A2, B1, and B2. The liamocins, and three of the exophilins, are new, previously unreported structures.