Preparation of dehydrobenzylisoquinolines by immobilized (S)-tetrahydroprotoberberine oxidase from plant cell cultures

(S)-Tetrahydroprotoberberine oxidase (STOX) has been isolated in enriched (7.4-fold) form from a high yielding cell suspension of Berberis wilsoniae var. subcaulialata in a three step procedure and was immobilized by several different methods. The properties of immobilized STOX were compared with immobilized Berberis cells and with the soluble enzyme. Although pH and temperature optima were shifted by immobilization, K_m-values with (S)-norreticuline remained unaffected. The stability of immobilized STOX was 50 times better than the free enzyme. A cyclic process is described using the stereospecific enzymatic oxidation of (S)norreticuline to 1,2-dehydronorreticuline followed by sodium borohydride reduction for the transformation of racemic norreticuline to the (R)-enantiomer.     

Stereoselectivity of sodium borohydride reduction of saturated steroidal ketones utilizing conditions of Luche reduction

A series of keto steroids were reduced with sodium borohydride in the presence of cerium(III) chloride or samarium(III) iodide (Luche reduction). The ratios of axial and equatorial alcohols were determined by HPLC and the results were compared with those obtained by a standard sodium borohydride reduction. The best results were obtained with the 2-keto derivative 1, 7-keto derivatives 5 and 6, and 12-keto derivative 8 where the cerium(III) ion addition resulted in the inversion of the axial/equatorial ratios. The Luche reduction of the 20-keto derivative 11 improved the proportion of the (20S)-alcohol in a mixture of (20S)/(20R) alcohols up to 35% from 5% in a standard sodium borohydride reduction.     

Reactions of the bis(p-tolylsulfonylhydrazone) of periodate-oxidized methyl 4,6-O-benzylidene-α-D-glucopyranoside with borohydride and cyanoborohydride: novel syntheses of unsaturated and deoxy sugars

Periodate-oxidized methyl 4,6-O-benzylidene-α-D-glucopyranoside (1) reacted with p-toluenesulfonylhydrazine to give the substituted bis(hydrazone) 2, which was converted into an N-substituted epimino derivative (3) by treatment with sodium borohydride in ethanol. Compound 3 was further converted into the glyc-2-enoside 4 by heating it with sodium borohydride in 1,4-dioxane. Sodium cyanoborohydride in ethanol reduced 2 to an epimeric mixture of 2-deoxy-D-arabino (5) and D-ribo (6)-hexoside derivatives. In the presence of an acidic resin in the same solvent, however, compound 2 underwent hydrogenation to the bis(hydrazino) derivative (7). The mechanisms of these reactions are discussed.     

Role of carbohydrates in insect resistance in Phaseolus vulgaris

The bruchid beetles Acanthoscelides obtectus (Say) and Zabrotes subfasciatus Boheman, are major storage pests of the economically important legume Phaseolus vulgaris (haricot bean). In the present study, resistance of a wild line of P. vulgaris G12953, towards these two insects is confirmed. Mature seeds from both resistant and susceptible lines were fractionated and tested in feeding trials in an attempt to elucidate the basis of resistance. The heteropolysaccharide fraction isolated from the resistant line and a susceptible line were each incorporated into artificial beans over a concentration range up to 10% dry wt. At a concentration of 4%, the approximate physiological concentration within the seed, the heteropolysaccharide fraction from the resistant line was very toxic resulting in 80–85% larval mortality of A. obtectus with an LC₅₀ of 2.5%. Furthermore, surviving larvae showed a marked increase in their development period. The corresponding fraction from a susceptible line, on the other hand, even at twice the physiological concentration, had a negligible effect upon larval development both in terms of adult emergence and development period. The effects of albumin proteins and globulin proteins on larval development of A. obtectus were also tested by incorporation into artificial beans at a range of concentrations up to 10%. Whilst those from the susceptible line had little effect upon development both the albumins and globulins from the resistant line were found to cause a slight decrease in adult emergence and a small increase in the median development period. These effects, however, are not sufficient to account for the observed seed resistance of G12953. Mature seeds from the resistant line and nine susceptible lines were also screened for the following secondary plant compounds: proteinase inhibitors, α-amylase inhibitors, lectins (phytohaemagglutinins), saponins, tannins and alkaloids. Although some of these antimetabolites were found to be present none, except for α-amylase inhibitors, were thought to play any significant role in conferring seed resistance. It is concluded that this example of insect resistance in seeds of P. vulgaris, line G12953, is due, at least in part, to the presence of a heteropolysaccharide which has an unusually high arabinose and fucose content. In the absence of other antimetabolic compounds the levels of this heteropolysaccharide present in G12953 are sufficiently high to account for the observed resistance. However, it is possible regarding seed resistance towards Z. subfasciatus, that α-amylase inhibitors may be having a synergistic effect.     

Mapping the non-darkening trait from ‘Wit-rood boontje’ in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>)

Key message

A QTL for non-darkening seed coat from ‘Wit-rood boontje’ was mapped in pinto bean population on chromosome Pv10, comprising 40 candidate genes.

Abstract

The seed coat colour darkens with age in some market classes of dry beans (Phaseolus vulgaris), including pinto bean. Beans with darkened seed coats are discounted in the market place, since they are believed to be associated with lower nutritional quality, increased cooking time, and decreased palatability. The objective of this research was to map a non-darkening gene from a cranberry-like bean ‘Wit-rood boontje’ using a recombinant inbred line population, derived from a cross between ’Wit-rood boontje’ and a slow-darkening pinto bean (1533-15). The population was characterized for seed phenotype and genotyped with an Illumina BeadChip. A genetic linkage map was constructed with 1327 informative SNP markers plus an STS marker (OL4S₅₀₀) and an SSR marker (Pvsd-0028), previously associated with the J gene and Sd gene, respectively, as well as non-darkening and slow-darkening phenotypes. The linkage map spanned 1253.2 cM over 11 chromosomes. A major QTL for the non-darkening trait was flanked by SNP 715646341 and SNP 715646348 on chromosome Pv10. The region, which spanned 13.2 cM, explained 48% of the phenotypic variation for seed coat darkening. Forty candidate genes were identified in the QTL interval. This information can be used to develop a gene-based marker to facilitate breeding non-darkening pinto beans and may lead to a better understanding of the molecular mechanism for the postharvest darkening phenomenon in pinto bean.

     

Determination of the number-average molecular weight of polysaccharides by end-group reduction with borohydride-t

The reduction with sodium borohydride-t of a series of maltose oligosaccharides yields products whose residual radioactivity (after deduction of a blank) has a linear dependence on degree of polymerisation. The procedure has been extended to measure the average degree of polymerisation (or alternatively M_n) of hydrolysed amyloses. For this purpose, it is necessary to use a washing procedure to remove a radioactive impurity in the sodium borohydride. The method should be applicable to measurement of number-average molecular weights of any polysaccharide which has a reducing end-group.     

Determination of the degree of methyl esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose

A procedure for the determination of the degree of methyl esterification of pectin in virtually any sample is described. Samples were dissolved or suspended in 1 M imidazole buffer, pH 7.0, cooled on ice, and reduced with sodium borohydride. Quantitative reduction of samples was accomplished after 1 h using at least 20 mg sodium borohydride/mg sample. The degree of methyl esterification was determined by either the increase in galactose content as determined by GLC of the sugar or by the change in galacturonic acid content by colorimetric uronic acid analyses. Sample requirements were at least as low as 100 micrograms per determination by GLC or 2 to 3 mg per determination by colorimetric uronic acid analysis compared to 5 mg or more per determination for other published procedures. The degrees of methyl esterification determined by the methods described have compared very favorably with those determined by established methods.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

A novel method for the methylation of heterocyclic amino groups. Conversion of guanosine into its 2-N-methyl- and 2-N,2-N-dimethyl derivatives

The heterocyclic amino-compounds 11a, 13a, 13b, and 17 reacted with formaldehyde and p-thiocresol (14) in alcoholic solution to give the corresponding N-methylphenylthiomethyl derivatives (16, 15a, 15b, and 18a, respectively) in satisfactory to good yields. The reactions were catalyzed by acetic acid. 2-N-Methylguanosine (6a) was obtained in good yield by treatment of 15b with sodium borohydride followed by acidic hydrolysis, or alternatively by Raney nickel desulfurization of 15a followed by ammonolysis of the product. Sodium borohydride reduction of 18a gave 21 in good yield. 2-N,2-N-Dimethylguanosine (6b) was obtained from 19a in three steps.     

Cross-inoculation specificity ofPsophocarpus tetragonolobus (L.) DC

Summary Only legumes of the cowpea cross-inoculation group, including the winged bean (Psophocarpus tetragonolobus) were found to form nodules in a temperate zone soil with no previous history of legume cropping. Isolates from root nodules from uninoculated winged beans grown in the field only nodulated legumes in the cowpea cross-inoculation group.Rhizobium japonicum formed ineffective nodules with the winged bean. Contribution No.5852, Scientific Article No.A2802 of the Maryland Agricultural Experiment Station, Department of Botany.     

Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant, Pongamia pinnata L.

A new full length cDNA clone encoding stearoyl-ACP desaturase (SAD) was isolated from seeds of Pongamia pinnata, an oil yielding legume plant. The cDNA clone (PpSAD) contained a single open reading frame of 1182-bp coding for 393 amino acids with a predicted molecular mass of 45.04 kDa, and shares similarity with SAD from other plants. Characteristics of the deduced protein were predicted and analyzed using molecular homology modeling; its three dimensional structure strongly resembled the crystal structure of Ricinus communis (RcSAD). Southern blot analysis indicated that ‘sad’ is a multiple copy gene and was a member of a small gene family. Expression analysis using quantitative real-time PCR revealed that the gene showed marked distinct expression during different stages of seed developments. The results of the expression analysis in this study, combined with existing research, suggest that ‘sad’ gene may be involved in the regulation of plant seed growth and development.     

Inheritance of seed condensed tannins and their relationship with seed-coat color and pattern genes in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol–HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.     

New glutathione peroxidase mimetics—Insights into antioxidant and cytotoxic activity

A series of N-alkyl benzisoselenazol-3(2H)-ones has been obtained and transformed to corresponding diselenides by the reduction with sodium borohydride. Additionally, efficient methodology for the oxidative Se–N bond formation by potassium iodate has been presented, new conversion of diselenide to benzisoselenazolone was observed. The GPx-like activity of all synthetized derivatives has been evaluated by NMR. N-Allyl diselenide was up to five times better antioxidant than ebselen. Anticancer capacity towards MCF7 and DU145 cancer cells has been also tested. The highest antiproliferative activity was obtained for N-cyclohexyl benzisoselenazolone.     

The nitrous acid deamination of glycosides and acetates of 2-amino-2-deoxy-D-glucose

A procedure is described for the nitrous acid deamination of 2-amino-2-deoxy-D-glucose hydrochloride (1), and reduction of the product with buffered borohydride, to afford crystalline 2,5-anhydro-D-mannitol (3) in 71% yield. Similar treatment of the methyl α-pyranoside (4) of 1 gives 59% of crystalline 3, and the same product is obtained in 44% yield from 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-α(or β)-D-glucopyranose hydrochloride (5 or 6) by the deamination-reduction sequence with subsequent deacetylation. These results provide a model, for a nonhydrolytic, depolymerization technique for structural characterization of glycosaminoglycans.     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

3-Hydroxypropylglucosinolate,a new glucosinolate in seeds of Erysimum hieracifolium and Malcolmia maritima

Glucosinolates from seed meals of Erysimum hieracifolium and Malcolmia maritima were treated with thioglucosidase (EC 3.2.3.1), and the resultant aglucon products investigated. A major product from E. hieracifolium was 3-hydroxypropyl isothiocyanate from the novel precursor 3-hydroxypropylglucosinolate. Other aglucon products were 3-methylsulfonylpropyl, 3-methylsulfinylpropyl, 3-methylthiopropyl, and allyl isothiocyanates. The aglucon products from M. maritima seed meal included 3-hydroxypropyl isothiocyanate, in addition to the previously known 3-methylsulfonylpropyl and 3-benzoyloxypropyl isothiocyanantes.     

Stereoselective Synthesis of 2,3-O-Isopropylidene-DL-Ribofuranose and Methyl β-DI-Ribopyranoside from furfuryl alcohol

Methyl 2,3-dideoxy-DL-pent-2-enopyranosid-4-ulose (2) and 1-O-benzoyl-2,3- dideoxy-DL-pent-2-enopyranos-4-ulose (3), obtained from furfuryl alcohol, gave methyl β-DL-erythro-pentopyranosid-4-ulose (6) and 1-O-benzoyl-β-DL-erythro-pentopyranos-4-ulose (7), respectively, on cis-hydroxylation with silver chlorate- osmium tetroxide. Reduction of the isopropylidene derivatives (8 and 9) of 6 and 7 with lithium aluminium hydride and sodium borohydride, respectively, afforded DL-ribose derivatives.     

The tritium labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate--polyacrylamide slab gels.

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [³H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.     

Synthesis of 4-O-β-D-mannopyranosyl-L-rhamnopyranose

The title disaccharide (16) has been synthesized in 50% overall yield by way of condensation of 4,6-di-O-acetyl-2,3-O-carbonyl-α-D-mannopyranosyl bromide 5 with methyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (1) in chloroform solution, in the presence of silver oxide. The disaccharide was characterized as the crystalline isopropyl alcoholate of methyl 4-O-β-D-mannopyranosyl-α-L-rhamnopyranoside (11) and as 1,2,3-tri-O acetyl-4-O- (2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)-α-L-rhamnopyranose (15). Methyl β-D-mannopyranoside isopropyl alcoholate 7 was readily obtained in 85% yield via the reaction of bromide 5 with methanol.Reduction of 2,3-di-O-methyl-L-rhamnose with sodium borohydride, followed by acetylation, may result in the formation of an appreciable proportion of a boric ester, namely 1,5-di-O-acetyl-4-deoxy-2,3-di-O-methyl-L-rhamnitol-4-yl dimethyl borate, depending on the procedure used.     

The Purification, Properties, and Localization of an Abundant Legume Seed Lectin Cross-Reactive Material from Spartium junceum

The seeds of Spartium junceum contained a large quantity of lectin-like protein that did not appear to be either a hemagglutinin or active lectin. The cross-reactive material (CRM), like most legume seed lectins, was a tetrameric glycoprotein of about 130,000 M_r. The singlesized subunits of about 33,000 M_r were not covalently associated. The amino acid composition was typical of legume lectins and was rich in hydroxy-amino acids and poor in sulfur-containing amino acids. The Spartium CRM contained about 3.5% covalently associated carbohydrate, most likely of the high-mannose type, since the CRM was precipitated by concanavalin A. The CRM was localized by electron-microscopic immunocytochemistry and found to be exclusively in protein-filled vacuoles (protein bodies). Because this protein was so similar immunologically, structurally, and in its physiology, to classic legume seed lectins, it is most likely a lectin homolog. Similar seed lectin CRMs appear to be both common and widespread in the Leguminosae.