Sodium Borodeuteride Research Articles

Extending the diversity of Myceliophthora thermophila LPMOs: Two different xyloglucan cleavage profiles

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new MtLPMO9F was characterized for its substrate (xyloglucan) specificity, and MtLPMO9H was further delineated. Aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis, we found that both MtLPMOs released predominately C4-oxidized, and C4/C6-double oxidized xylogluco-oligosaccharides. Further characterization showed that MtLPMO9F, having a short active site segment 1 and a long active site segment 2 (−Seg1+Seg2), followed a “substitution-intolerant” xyloglucan cleavage profile, while for MtLPMO9H (+Seg1−Seg2) a “substitution-tolerant” profile was found. The here characterized xyloglucan specificity and substitution (in)tolerance of MtLPMO9F and MtLPMO9H were as predicted according to our previously published phylogenetic grouping of AA9 LPMOs based on structural active site segment configurations.

Regioselective C4 and C6 Double Oxidation of Cellulose by Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard‐to‐convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello‐oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H2 18O, it was confirmed that the C6 gem‐diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem‐diol, could not be excluded. These findings can be extended to how the reactive LPMO‐cosubstrate complex is positioned towards the substrate.

Synthesis of deuterated chlorophyll derivatives and their physical properties

The 3-vinyl group of methyl pyropheophorbide-a, one of the chlorophyll-a derivatives, was chemically transformed into partially or fully deuterated vinyl, 1-hydroxyethyl, acetyl, formyl, and hydroxymethyl groups via oxidation, oxidative cleavage, reduction, dehydration, or Grignard reaction. Using commercially available sodium borodeuteride and trideuteromethyl iodide as deuterium sources, a variety of regioselectively deuterated pyropheophorbides were obtained with either no or a slight loss of the deuteration degree compared with those of the starting deuterated materials. The deuteration affected largely the related infrared vibrational bands, slightly the chemical shifts of specific proton signals, and no change of visible absorption bands in a solution.

Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4-O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

Quantitative Comparison of Pyranose Dehydrogenase Action on Diverse Xylooligosaccharides.

Pyranose dehydrogenases (PDHs; EC 1.1.99.29; AA3_2) demonstrate ability to oxidize diverse carbohydrates. Previous studies of these enzymes have also uncovered substrate-dependent regioselectivity, along with potential to introduce more than one carbonyl into carbohydrate substrates. Enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials with higher value; accordingly, PDHs that show activity on xylooligosaccharides could offer a viable approach to extract higher value from hemicelluloses that are typically fragmented during biomass processing. In this study, AbPDH1 from Agaricus bisporus and AmPDH1 from Leucoagaricus meleagris were tested using linear xylooligosaccharides, along with xylooligosaccharides substituted with either arabinofuranosyl or 4-O-(methyl)glucopyranosyluronic acid residues with degree of polymerization of two to five. Reaction products were characterized by HPAEC-PAD to follow substrate depletion, UPLC-MS-ELSD to quantify the multiple oxidation products, and ESI-MSn to reveal oxidized positions. A versatile method based on product reduction using sodium borodeuteride, and applicable to carbohydrate oxidoreductases in general, was established to facilitate the identification and quantification of oxidized products. AbPDH1 activity toward the tested xylooligosaccharides was generally higher than that measured for AmPDH1. In both cases, activity values decreased with increasing length of the xylooligosaccharide and when using acidic rather than neutral substrates; however, AbPDH1 fully oxidized all linear xylooligosaccharides, and 60–100% of all substituted xylooligosaccharides, after 24 h under the tested reaction conditions. Oxidation of linear xylooligosaccharides mostly led to double oxidized products, whereas single oxidized products dominated in reactions containing substituted xylooligosaccharides. Notably, oxidation of specific secondary hydroxyls vs. the reducing end C-1 depended on both the enzyme and the substrate. For all substrates, however, oxidation by both AbPDH1 and AmPDH1 was clearly restricted to the reducing and non-reducing xylopyranosyl residues, where increasing the length of the xylooligosaccharide did not lead to detectable oxidation of internal xylopyranosyl substituents. This detailed analysis of AbPDH1 and AmPDH1 action on diverse xylooligosaccharides reveals an opportunity to synthesize bifunctional molecules directly from hemicellulose fragments, and to enrich for specific products through appropriate PDH selection.

Contribution of ketone/aldehyde-containing compounds to the composition and optical properties of Suwannee River fulvic acid revealed by ultrahigh resolution mass spectrometry and deuterium labeling.

A prior method of mass labeling ketone-/aldehyde-containing species in natural dissolved organic matter (DOM) is further developed and applied. This application involved the treatment of Suwannee River fulvic acid (SRFA) with increasing concentrations of sodium borodeuteride (NaBD4), followed by detection of reduced species via negative mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). The extent of reduction, as determined by ESI FTICR MS, resulting from increasing concentrations of NaBD4 correlated well with changes in the absorption and emission spectra of the corresponding untreated and borodeuteride-reduced samples, providing evidence that ketone/aldehyde functional groups contribute substantially to the bulk optical properties of SRFA. Furthermore, the differences in the reactivity and abundance of ketone-/aldehyde-containing species for various regions in Van Krevelen plots were revealed, thus showing how this mass labeling method can be used to provide more detailed structural information about components within complex DOM samples than that provided by the determination and analysis of molecular formulae alone. Graphical abstract.

Synthesis of Deuterium-Labeled Steroid 3,6-Diols

A facile synthesis of a set of deuterium-labeled steroid 3,6-diols with different steroid A/B ring fusion, unsaturations, and configurations of hydroxyl groups at C-3 and C-6 is described. Reduction and deuteration, based on deuterium-exchange of the obtained the cholest-4-ene-3,6-dione from cholesterol using sodium borodeuteride and deuterium water, were used. The obtained steroid diols are intended to be used as precursors in the studies on biosynthesis of some marine polar steroids.

Cross-Course Collaboration in the Undergraduate Chemistry Curriculum: Isotopic Labeling with Sodium Borodeuteride in the Introductory Organic Chemistry Laboratory

A microscale isotopic labeling experiment is described for the introductory organic chemistry laboratory course wherein half of the students use sodium borohydride (NaBH4) and the other half use sodium borodeuteride (NaBD4) to reduce acetophenone to 1-phenylethanol and then compare spectral data. The cost is reasonable, and the experiment is well-suited to serve as a first experience with 1H NMR spectroscopy, infrared spectroscopy, and/or mass spectrometry. No special apparatus are required for the reaction. The entire experiment, including an extensive prelab lecture and acquisition of 1H NMR data, is completed in a 3 h period. The experiment can stand alone, but lends itself to cross-course collaboration between the organic and physical chemistry laboratories. The succeeding companion article describes the measurement of a kinetic isotope effect using the product from this experiment. The physical chemistry students report their experimental results back to the organic class.

Identification and comparative quantitation of glycation by stable isotope labeling and LC–MS

Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC-MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2Da using sodium borohydride or a molecular weight increase of 3Da using sodium borodeuteride. The molecular weight difference of 1Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels.

Selective Mass Labeling for Linking the Optical Properties of Chromophoric Dissolved Organic Matter to Structure and Composition via Ultrahigh Resolution Electrospray Ionization Mass Spectrometry

The mass spectra acquired by ESI FT-ICR MS of untreated, borohydride-reduced, and borodeuteride-reduced samples of Suwannee River fulvic acid (SRFA) and a C18 extract from the upper Delaware Bay were compared to one another. Treatment of these samples with sodium borodeuteride was shown to produce unique mass labels for species which contain one or two ketone/aldehyde moieties. Approximately 30% of all identified peaks in the two samples were shown to comprise ketone/aldehyde-containing species. The molecular formulas of the majority of these species had O/C and H/C molar ratios typically attributed to lignin-derived compounds and/or carboxylic rich alicyclic molecules (CRAM). However, the significant loss of UV-vis absorption following reduction supports a lignin-based origin for the optical (and photochemical) properties of these samples. The mass-labeling method described and tested herein shows great promise as a means to further characterize the structure and composition of complex natural samples, especially in terms of identifying specific subsets of chemical species that contribute significantly to the optical and photochemical properties of such samples.

The complete targeted profile of the organic acid intermediates of the citric acid cycle using a single stable isotope dilution analysis, sodium borodeuteride reduction and selected ion monitoring GC/MS

The quantitative profiling of the organic acid intermediates of the citric acid cycle (CAC) presents a challenge due to the lack of commercially available internal standards for all of the organic acid intermediates. We developed an analytical method that enables the quantitation of all the organic acids in the CAC in a single stable isotope dilution GC/MS analysis with deuterium-labeled analogs used as internal standards. The unstable α-keto acids are rapidly reduced with sodium borodeuteride to the corresponding stable α-deutero-α-hydroxy acids and these, along with their unlabeled analogs and other CAC organic acid intermediates, are converted to their tert-butyldimethylsilyl derivatives. Selected ion monitoring is employed with electron ionization. We validated this method by treating an untransformed mouse mammary epithelial cell line with well-known mitochondrial toxins affecting the electron transport chain and ATP synthase, which resulted in profound perturbations of the concentration of CAC intermediates.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-013-0521-1) contains supplementary material, which is available to authorized users.

Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4,5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen–deuterium exchange reaction of a β-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,β-enone in perdeuteromethanol was used.

Isotopomer analysis of 3‐hydroxybutyrate by liquid chromatography tandem mass spectrometry to determine ketogenic flux

Hepatic ketogenesis maintains critical systemic fuel supply during fasting, mainly from acetyl‐CoA derived from fatty acid oxidation. Ketogenesis is coupled to beta oxidation and TCA cycle and is often dysfunctional in patho‐physiological conditions including insulin resistance and diabetes. Further, quantifying ketogenesis is challenging due to the unstable nature of acetoacetate (ACAC) and the substantial sample volume (3‐4 mL plasma) required for detecting ketones by NMR spectroscopy. This method determines both ACAC and 3‐hydroxybutyrate (3‐BHB) enrichments in ~25 µL blood or plasma by isotopomer analysis of 3‐BHB using LC‐MS/MS. Following a 90 min intravenous infusion of [3,4‐13C2]ACAC (M+3) and [1,2,3,4‐13C4]3‐BHB (M+4), the blood or plasma ACAC was reduced to 3‐BHB (M+1) with sodium borodeuteride. Steady state enrichments of the ketone tracers, 3‐BHB derived from ACAC (M+2) and ACAC derived from 3‐BHB (M+5) were used to quantify ketogenesis using a two pool model. Ketogenesis measured by LC‐MS/MS in 24 h fasted Long Evans rats yielded similar values to those measured by NMR spectroscopy. Ketogenesis in insulin resistant obese mice was lower than the controls suggesting a dysfunction in channeling fatty acid carbon through central metabolic pathways in the liver. In summary, this method provides a sensitive and reliable index of ketogenesis by monitoring only 3‐BHB in micro liters of blood or plasma.

Rapid and efficient synthesis of stable isotope labeled [13C4, D4]‐5‐(hydroxymethyl)thiazole: versatile building block for biologically interesting compounds

Abstract5‐(Hydroxymethyl)thiazole is a versatile building block for many biologically active compounds. A rapid and efficient four‐step synthesis of its stable isotope labeled counterpart with four 13C and four deuterium atoms in 32% total yield is reported. Condensation of [13C2]‐chloro acetic acid with [13C]‐thiourea gave [13C3]‐2,4‐thiazolidinedione. Reaction of [13C3]‐2,4‐thiazolidinedione with phosphorus oxybromide and [13C, D]‐DMF (Me2N13CDO) produced [13C4, D]‐2,4‐dibromo‐thiazole‐5‐carboxaldehyde. The resultant aldehyde was then reduced by sodium borodeuteride to [13C4, D2]‐(2,4‐dibromo‐thiazol‐5‐yl)‐methanol. Catalytic deuteration of [13C4, D2]‐(2,4‐dibromo‐thiazol‐5‐yl)‐methanol by palladium black with deuterium gas at 1 atm pressure and room temperature produced completely de‐brominated [13C4, D4]‐5‐(hydroxymethyl)thiazole. De‐bromination of the 2,4‐dibromothiazole by the catalysis of palladium black provides a simple and convenient synthetic method for the stable isotope labeled and potentially radioactive isotope labeled thiazole compounds. Copyright © 2009 John Wiley & Sons, Ltd.

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method—precursors for the synthesis of labeled conjugates

Isotopic labeling of the C-6 of a model glycosphingolipid (2 S, 3 R, 4 E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-β- d-galactopyranoside, GalCAda, is described. Oxidation of (2 S, 3 R, 4 E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri- O-benzoyl-β- d-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled d-GalCAda, with a cumulative yield of 35%.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Oxidation of lactose with bromine

Oxidation of lactose by bromine in an aqueous buffered solution was conducted as a model experiment to examine the glycosidic linkage cleavage occurring during the oxidation of oligosaccharides and polysaccharides. The resulting oxidation products, after reduction with sodium borodeuteride, were characterized by GLC–MS analyses of the per- O-methyl or per- O-Me 3Si derivatives. Most of the products were carboxylic acids, of which lactobionic acid was major. Minor products, identified after partial fractionation on a BioGel P-2 column, comprised oxalic acid; glyceric acid; threonic and erythronic acids; tartaric acid; lyxonic, arabinonic, and xylonic acids; galactonic and gluconic acids; galactosylerythronic acid; galactosylarabinonic acid; galactosylarabinaric acid; galacturonosylarabinonic acid; and galactosylglucaric acid. No keto acids were identified. Galactose was detected as 1-deuteriogalactitol, the presence of which, together with the C 6 aldonic acids, supported a galactosidic bond cleavage. Galactosylarabinonic acid was the major constituent (7.5%) among minors, and others constituted 0.2–3.7% of the principal lactobionic acid. These products together comprised 29% of the lactobionic acid, more than half (17%) of which were accounted for by the galactosidic linkage cleavage, supporting the significant decrease in molecular weight seen earlier in the bromine-oxidized polysaccharides by glycosidic cleavage.

Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer

Recent studies have shown that an endogenous lipoperoxidation product, 9-hydroxystearic acid (9-HSA), acts in colon carcinoma cells (HT29) as a growth inhibitor by inducing p21(WAF1) in an immediate-early, p53-independent manner and that p21(WAF1) is required for 9-HSA-mediated growth arrest in HT29 cells. It is conceivable, therefore, to hypothesize that the cytostatic effect induced by this agent is at least partially associated with a molecular mechanism that involves histone deacetylase 1 (HDAC1) inhibition, as demonstrated for sodium butyrate and other specific inhibitors, such as trichostatin A and hydroxamic acids. Here, we show that, after administration, 9-HSA causes an accumulation of hyperacetylated histones and strongly inhibits the activity of HDAC1. The interaction of 9-HSA with the catalytic site of the enzyme has been highlighted by computational modeling of the human HDAC1, using its homolog from the hyperthermophilic Aquifex aeolicus as a template. Consistent with the experimental data, we find that 9-HSA can bind to the active site of the protein, showing that the inhibition of the enzyme can be explained at the molecular level by the ligand-protein interaction.

A robust and selective method for the quantification of glycosylphosphatidylinositols in biological samples.

We have developed an assay for the quantification of glycosylphosphatidylinositol (GPI)-anchored glycoconjugates. The method is based on nitrous acid deamination and sodium borodeuteride reduction of the glucosamine residue, common to all GPI structures, to yield [1-2H]-2,5-anhydromannitol. Following acid methanolysis and trimethylsilyl derivatization, detection is by selected ion monitoring gas chromatography-mass spectrometry. The unnatural inositol isomer scyllo-inositol is used as an internal standard and the [1-2H]-2,5-anhydromannitol trimethylsilyl derivative is detected by following a characteristic electron-impact fragment ion at m/z 273. This method is more selective for GPIs than assays based on measuring myo-inositol content, which are often confounded by contaminating inositol-phospholipids. We show that the method can be applied to measure total GPI content in crude total lipid extracts and even in whole trypanosome ghosts. The method was applied to whole cell lysates of wild-type, GPI-deficient, and glycosaminoglycan-deficient CHO cells. The data revealed that proteoglycans did not interfere with total glucosamine estimates but that there are background levels of non-GPI and nonproteoglycan glucosamine-containing material in CHO cells.

Glutathione transferase zeta-catalyzed bioactivation of dichloroacetic acid: reaction of glyoxylate with amino acid nucleophiles.

Dichloroacetic acid (DCA) is a drinking water contaminant, a therapeutic agent, and a rodent carcinogen. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloalkanoates and the cis-trans isomerization of maleylacetoacetate. GSTZ1-1 catalyzes the bioactivation of fluorine-lacking dihaloacetates to S-(alpha-halocarboxymethyl)glutathione, a reactive intermediate that covalently modifies and inactivates the enzyme or is hydrolyzed to glyoxylate. The purpose of this study was to examine the GSTZ1-1-catalyzed bioactivation of DCA, including the reaction of DCA-derived glyoxylate with amino acid nucleophiles and the characterization of the structures and kinetics of adduct formation by LC/MS. The binding of [1-(14)C]DCA-derived label to bovine serum albumin required both GSTZ1-1 and GSH, whereas the binding to dialyzed rat liver cytosolic protein was increased in the presence of GSH. Studies with model peptides (antiflammin-2 and IL-8 inhibitor) indicated that glyoxylate, rather than S-(alpha-chlorocarboxymethyl)glutathione, was the reactive species that modified amino acid nucleophiles. Both addition (+74 Da) and addition-elimination (+56 Da) adducts of glyoxylic acid were observed. Addition adducts (+74 Da) could not be characterized completely by mass spectrometry, whereas addition-elimination adducts (+56 Da) were characterized as 2-carboxy-4-imidazolidinones. 2-Carboxy-4-imidazolidinones were formed by the rapid equilibrium reaction of glyoxylate with the N-terminal amino group of antiflammin-2 to give an intermediate carbinolamine (K(eq) = 0.63 mM(-1)), which slowly eliminated water to give an intermediate imine (k(2) = 0.067 hour(-1)), which rapidly cyclized to give the 2-carboxy-4-imidazolidinone. Glucose 6-phosphate dehydrogenase was inactivated partially by glyoxylate when reactants were reduced with sodium borodeuteride, which may indicate that glyoxylate reacts with selective lysine epsilon-amino groups. The results of the present study demonstrate that GSTZ1-1 catalyzes the bioactivation of DCA to the reactive metabolite glyoxylate. The reaction of glyoxylate with cellular macromolecules may be associated with the multiorgan toxicity of DCA.