Detection of agar, by analysis of sugar markers, associated with Bacillus anthracis spores, after culture

Detection of small quantities of agar associated with spores of Bacillus anthracis could provide key information regarding its source or growth characteristics. Agar, widely used in growth of bacteria on solid surfaces, consists primarily of repeating polysaccharide units of 3,6-anhydro-l-galactose (AGal) and galactose (Gal) with sulfated and O-methylated galactoses present as minor constituents. Two variants of the alditol acetate procedure were evaluated for detection of potential agar markers associated with spores. The first method employed a reductive hydrolysis step, to stabilize labile anhydrogalactose, by converting to anhydrogalactitol. The second eliminated the reductive hydrolysis step simplifying the procedure. Anhydrogalactitol, derived from agar, was detected using both derivatization methods followed by gas chromatography-mass spectrometry (GC-MS) analysis. However, challenges with artifactual background (reductive hydrolysis) or marker destruction (hydrolysis) respectively lead to the use of an alternative agar marker. A minor agar component, 6-O-methyl galactose (6-O-M gal), was readily detected in agar-grown but not broth-grown bacteria. Detection was optimized by the use of gas chromatography-tandem mass spectrometry (GC-MS-MS). With appropriate choice of sugar marker and analytical procedure, detection of sugar markers for agar has considerable potential in microbial forensics.     

Enzymatic synthesis of L-6-hydroxynorleucine

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.     

NAD(P)H依赖型氧化还原酶不对称还原胺化制备手性胺的研究进展

不对称还原胺化反应是制备医药中间体手性胺结构单元的重要反应。目前已有许多不同种类的酶被应用于合成手性胺,其中NAD(P)H依赖型氧化还原酶催化的还原胺化反应最为引人注目,因为其能够一步将潜手性酮化合物完全转化为光学纯的手性胺化合物。文中以亚胺还原酶、氨基酸脱氢酶、冠瘿碱脱氢酶和还原性酮胺化酶为例,从NAD(P)H依赖型氧化还原酶的结构特征、作用机理、分子改造及催化应用等方面,综述了其在不对称还原胺化合成手性胺领域的研究进展。     

Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, was converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-6-O-benzyl-2-deoxy-beta-D-glucopyranoside (6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-alpha-L-xylo-hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected tetrasaccharide, which is functionalized for conjugation.     

Resolution of the enantiomers of aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates

Acyclic diastereoisomers, namely, 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates are readily obtained by reductive amination of aldoses with chiral α-methylbenzylamine (MBA) in the presence of sodium cyanoborohydride, and may be separated by reversed-phase 1.c. and, more effectively, by adsorption 1.c. According to this procedure, enantiomers of the common, neutral aldoses are resolved. In adsorption 1.c., l-l_* [defined as an adduct of an l-aldose and l-(-)-MBA] is eluted before d-l_* for erythrose, xylose, ribose, glucose, 4-O-methylglucose, galactose, and fucose, and the elution order is the reverse for arabinose, lyxose, mannose, rhamnose, and glyceraldehyde. This behavior is probably related to the configuration of C-2 of the aldoses.     

Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines

The peptides DRPVPY and MDWNMHAA, which were identified as mimics of the cell-surface polysaccharides of Streptococcus Group A and Shigella flexneri Y, respectively, were used in this study to develop experimental vaccines directed against these two bacteria. Both oligopeptides were synthesized employing the Fmoc solid-phase strategy and linked via the amino end to a bifunctional linker, diethylsquarate. These adducts were then conjugated to the two carrier proteins, bovine serum albumin (BSA) and tetanus toxoid (TT) to yield the peptide conjugate vaccines. The average level of incorporation of DRPVPY and MDWNMHAA on TT was 65% and 75%, respectively, whereas that of both peptide haptens on BSA was 100%. A polysaccharide conjugate against S. flexneri Y, which comprises about 10 tetrasaccharide repeating units, was also prepared based on reductive amination at the reducing end with 1,3-diaminopropane, followed by coupling of the aminated polysaccharide to diethylsquarate, and subsequent coupling of the adduct to TT. An average incorporation of 73% of polysaccharide haptens was achieved. The glycoconjugate and the oligopeptide conjugates were shown to bind effectively to the respective monoclonal antibodies directed against the cell-surface polysaccharides.     

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.     

Nonperturbing fluorescent labeling of polysaccharides

A fluorescent labeling procedure, which does not perturb macromolecular conformations, was employed to bind a rhodamine derivative to the reducing end of several water-soluble polysaccharides by reductive amination in the presence of sodium cyanoborohydride. Fluorescence correlation spectroscopy, atomic force microscopy, and size exclusion chromatography were used to demonstrate that the conformations of the polysaccharides schizophyllan, polygalacturonic acid (PGUA), succinoglycan, and several dextrans were maintained following the labeling procedure.     

Sulfated polysaccharides from the red seaweed Georgiella confluens.

The water-soluble polysaccharides from Georgiella confluens, collected in Antarctica, were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration. The initially extracted polysaccharide and the major fraction isolated, soluble in 0.5 M NaCl, were studied. These are sulfated xylogalactans naturally rich in methylated sugar residues, comprising of 3,6-anhydro-2-O-methyl-L-galactose, 2-O-methyl-L-galactose and 6-O-methyl-D-galactose. Structural analysis was carried out by methylation, ethylation, desulfation-ethylation, desulfation-methylation, Smith degradation, 13C NMR spectroscopy and determination of the absolute configuration of monosaccharides by gas chromatography of diastereomeric derivatives produced by reductive amination. The results indicated the presence of an agaran backbone with an unusual substitution pattern: sulfation mainly at the 3-position of the alpha-L-galactose units and the presence of xylose side chains at the 4-position of the beta-D-galactose residues.     

Absorption, fluorescence, and cd spectroscopic study of chiral recognition by a binaphthyl-derived chromogenic calixcrown host

The (R)- and (S)-enantiomers of a binaphthyl-appended calix[4]crown-6 ether with two 2,4-dinitrophenylazo chromophore units ((R)-1 and (S)-1) as chiral hosts were tested in their reactions with the enantiomers of alpha-methylbenzylamine ((R)-MBA, (S)-MBA)) and phenylglycinol ((R)-PGL, (S)-PGL) as chiral guests. The visible absorption spectra indicate a two-step process: the first is a nonenantioselective proton transfer from the host to the guest, which is followed by the enantioselective real complexation. In the visible range of the CD spectra a positive/negative band belongs to the absorption of pure (R)-1/(S)-1, and a negative/positive exciton couplet to the absorption of (R)-1-(S)-MBA/(S)-1-(R)-MBA complexes. The latter phenomenon suggests that the complexation of amines is accompanied by a chiral arrangement of the two chromophore units in the hosts. The UV fluorescence of (R)-1/(S)-1 arising from the binaphthyl moiety is quenched by K+ ions, but not by the amine guests, showing that the interaction between the binaphthyl group and the complexed amines is weak.     

125I-glycoconjugate labels for identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation

Residualizing radioactive labels are designed to remain entrapped within cells following degradation of a carrier protein, and have been used for identification of the tissue and cellular sites of plasma protein catabolism. In this study we describe a convenient synthesis and purification of a series of 125I-labeled glycoconjugates, and an evaluation of their efficiency of retention in liver following degradation of a model carrier protein, asialofetuin. Glycoconjugates were prepared in 65-90% yield by reductive amination of reducing sugars with aromatic amines using NaBH3CN. The products were purified in a single ion-exchange chromatographic step, and then labeled with 125I. The derivatives prepared were mono-and disubstituted lactitol-,cellobiitol-and glucitol-[125I]tyramine and lactitol-[125I]tyrosine. 125I-Glycoconjugates were coupled to asialofetuin using either cyanuric chloride or, for lactose-containing labels, by treatment with galactose oxidase followed by reductive amination with NaBH3CN. Attachment of labels by either procedure did not affect the normal rapid clearance of asialofetuin from the rat circulation nor its uptake and degradation in liver lysosomes. Leakage of 125I-labeled degradation products from cells was measured by following the kinetics of loss of whole-body radioactivity. We observed that degradation products from larger, disubstituted glycoconjugates were retained more efficiently than those from smaller and monosubstituted derivatives, and that glycoconjugates coupled to protein via reductive amination were retained in the body more efficiently than those coupled by cyanuric chloride. Overall, dilactitol-[125I]tyramine coupled to protein by reductive amination was entrapped most efficiently in liver.     

Analysis of enantiomers of chiral phenethylamine drugs by capillary gas chromatography/mass spectrometry/flame-ionization detection and pre-column chiral derivatization

Several important chiral phenethylamine agents such as mexiletine, fenfluramine, amphetamine, methamphetamine and N-n-propylamphetamine show stereoselective disposition in humans and large differences in therapeutic relevance and toxicity. To analyze the enantiomers of chiral amine drugs, stereoselective methods were developed to separate those enantiomers on an achiral capillary gas chromatography by pre-column chiral derivatization with S-(-)-N-(fluoroacyl)-prolyl chloride. The stereoselectivity and sensitivity can be improved by chiral derivatization. The methods established offer enantioselective, simple, flexible and economic approaches for the analysis of chiral amine drug enantiomers in biological fluids. The methods have been used to determine S-(+)-methamphetamine in human forensic samples and to analyze enantiomers of amphetamine and fenfluramine in rat liver microsomes.     

Structural features of the sulphated polysaccharide from a green seaweed,Caulerpa taxifolia

A sulphated heteropolysaccharide was isolated from a green seaweed, Caulerpa taxifolia, by extraction with acid and purified via its copper complex. Methylation analysis of both the sulphated and desulphated polysaccharides revealed the presence of 1,4-linked xylose, 1,6-linked galactose, 1,4,6-linked mannose and non-reducing galactose end group units which are all devoid of sulphate groups. In addition 1,4-linked galactose units sulphated at C-3 are also present. Quantitative periodate oxidation showed a consumption of 1.30 and 1.60 moles of oxidant per anhydrosugar unit in the sulphated and desulphated polysaccharides respectively. The oxo-polysaccharides after reduction and hydrolysis revealed the presence of glycerol, erythritol and unoxidized galactose in the mol ratio 11.6:5.1:4.9 and 11.2:5.0:1.0 respectively, besides threitol (3.9 mol) in the desulphated polysaccharide.     

A study of pectic polysaccharides in musts from various mature grapes grown in the Pech Rouge experimental vineyards

Soluble pectic polysaccharides were isolated from musts of seven mature grape cultivars by a 4-step procedure: pressing of the berries, denaturation of soluble proteins from must by emulsification with chloroform, elimination of diffusable molecules by extensive dialysis and finally discoloration onto Polyamide CC6. The polysaccharides were mainly constituted of arabinose, galactose and galacturonic acid, and their concentration in musts varied from 133 to 593 mg/l. The arabinose/galactose molar ratio was stable (0.91-1.04) for all cultivars, but one, Cinsaut (0.68). Methylation analyses showed that the polysaccharides from musts are a complex mixture of type II arabinogalactans, arabinans and rhamnogalacturonans. Similarities were observed in the relative distributions of galactose and arabinose structural features except for the Cinsaut and Grenache cultivars in which higher proportions of 3- and 3,6-linked galactose were found.     

A novel high-performance anion-exchange chromatographic method for the analysis of carrageenans and agars containing 3,6-anhydrogalactose

A novel method has been developed to determine the sugar composition of 3,6-anhydrogalactose-containing polysaccharides, such as carrageenan and agar. The method is based on reductive hydrolysis with a methylmorpholine-borane complex in the presence of acid and subsequent high-performance anion-exchange chromatography analysis of the alditols without any derivatization. The method was validated by 13C NMR analysis of six carrageenans and three agars and by a previously used method based on derivatization to alditol acetates and gas-liquid chromatography analysis. The new method was found to be superior to the gas-liquid chromatography method as the analysis time was less than half. Also it was found to be more accurate and reproducible and no derivatization was required. The analysis of the six different carrageenan samples revealed that homogeneous mu- and nu-carrageenan, theoretically without 3,6-anhydrogalactose residues, cannot be isolated from red seaweeds. Consequently, the question arose if mu- and nu-carrageenans at all are present in seaweeds and if the current hypotheses regarding biosynthesis of carrageenans in the seaweeds are correct. The data demonstrated that carrageenans are highly complex natural polysaccharides, which are more irregular than assumed hitherto. The new analytical technique will permit elucidation of the detailed structure of seaweed polysaccharides and determination of their structure-property relationships.     

Comparative analysis of sulfated galactans from red algae by reductive hydrolysis and mild methanolysis coupled to two different HPLC techniques

The sugar determination of the sulfated galactans, agars and carrageenans of various red algae was performed using two different techniques of depolymerisation with subsequent HPLC analysis: 1) reductive hydrolysis/ HPAEC-PAD; 2) mild methanolysis/ RPLC-DR. Both techniques were optimized to release quantitatively the composite sugars (galactose, 6-O-methyl-galactose, the labile 3,6-anhydrogalactose and 2-O-methyl-3,6-anhydrogalactose residues) and precise relative response factors of authentic 3,6-anhydrogalactose were determined. The methanolysate neutralisation step, performed subsequently to methanolysis depolymerisation, was demonstrated as a key step for the quantitative recovery of the anhydrogalactose residues. The yield of the main sugars released by the two techniques were in good agreement for the commercial agarose and iota and kappa carrageenans studied. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preparation of a high-affinity photolabeling reagent for the Gal/GalNAc lectin of mammalian liver: demonstration of galactose-combining sites on each subunit of rabbit hepatic lectin

On the basis of the knowledge that the D-galactose/N-acetyl-D-galactosamine-specific lectin of rabbit liver can tolerate a large group on the C-6 hydroxyl group of a galactoside [Lee, R. T. (1982) Biochemistry 21, 1045-1050], we prepared a high-affinity photolabeling reagent for this lectin from a triantennary glycopeptide fraction of asialofetuin. The C-6 hydroxyl group of a D-galactopyranoside was converted, under mild conditions, into a primary amino group. The procedure involves conversion of the hydroxyl group to an oxo group with galactose oxidase, followed by reductive amination using benzylamine and sodium cyanoborohydride. Catalytic hydrogenolysis of the benzylamino derivative yielded the desired 6-amino-6-deoxy-D-galactoside. A 4-azidobenzoyl group was attached to the newly produced amino group to yield a photoactivatable affinity-labeling reagent. The reagent labeled the Triton-solubilized, purified hepatic lectins of rabbit and rat in a photo- and affinity-dependent manner. All the polypeptide subunits of the lectins were labeled, indicating that each subunit contains at least one D-galactose-combining site. In the case of the rabbit hepatic lectin, the minor subunit (46 kDa) was labeled more efficiently than the major one (40 kDa).     

Amine transaminases in chiral amines synthesis: recent advances and challenges

Transaminases, which catalyze the stereoselective transfer of an amino group between an amino donor and a prochiral ketone substrate, are interesting biocatalytic tools for the generation of optically pure chiral amines. In particular, amine transaminases (ATAs) are of industrial interest because they are capable of performing reductive amination reactions using a broad range of amine donors and acceptors. The most remarkable example of ATAs industrial application is in the production process of the anti-hyperglycaemic drug sitagliptin (Januvia^®/Janumet^®), which generated around 6 billion U.S. dollars of revenue to Merck in 2016. In this review, an update about the availability of microbial ATAs, discovered by both screening and database-mining approaches, or obtained by protein engineering of wild-type enzymes, will be provided. Current challenges in ATAs application and possible solutions will be also discussed. In particular, innovative biocatalytic process strategies aimed at the improvement of ATAs performances in chiral amines synthesis, e.g., using in situ product removal process strategies or flow reactors, will be presented. The progress in the industrial exploitation of these enzymes will be highlighted by selected examples of large-scale ATA-catalyzed processes.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture

A novel surface modification method of biodegradable polymers was investigated for inducing the attachment of specific cells onto the polymer surface via ligand-receptor interactions. Galactose, a targeting ligand specific to asialoglycoprotein receptors present on cell membrane of hepatocytes, was introduced on the surface of poly(D,L-lactic-co-glycolic acid) (PLGA) films. A terminal end group of carboxylic acid in PLGA was activated by dicyclohexylcarbodiimide and N-hydroxysuccinimide for the direct conjugation of lactose by reductive amination reaction. Di-block copolymers of PLGA-b-poly(ethylene glycol) (PEG) having a free terminal amine group were also synthesized and used for the conjugation of galactose for the introduction of a PEG spacer between PLGA and galactose. The presence of galactose moieties on the blend film surface was characterized by measuring water contact angle and X-ray photon spectroscopy, and the amount of galactose was indirectly determined by a specific lectin-binding assay. With increasing the galactose concentration on the blend film surface, the initial attachment as well as the cell viability of hepatocyates concomitantly increased. The introduction of PEG spacer reduced the cell attachment and viability. Albumin secretion rate from hepatocytes was enhanced for galactose modified surfaces, whereas it was reduced for the surfaces not having galactose moieties.