Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.     

Biosynthesis of cholestanol in higher plants

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.     

Molecular orbital study of complexes of zinc(II) with sulphide, thiomethanolate, thiomethanol, dimethylthioether, thiophenolate, formiate, acetate, carbonate, hydrogen carbonate, iminomethane and imidazole. Relationships with structural and catalytic zinc in some metallo-enzymes.

Geometry optimization and energy calculations have been performed at the density functional B3LYP/LANL2DZ level on hydrogen sulfide (HS-), dihydrogensulfide (H2S), thiomethanolate (CH3S-), thiomethanol (CH3SH), thiophenolate (C6H5S-), methoxyde (CH3O-), methanol (CH3OH), formiate (HCOO-), acetate (CH3COO-), carbonate (CO3(2-)), hydrogen carbonate (HCO3-), iminomethane (NH=CH2), [ZnS], [ZnS2]2-, [Zn(HS)]+, [Zn(H2S)]2+, [Zn(HS)4]2-, [Zn(CH3S)]+, [Zn(CH3S)2], [Zn(CH3S)3]-, [Zn(CH3S)4]2-, [Zn(CH3SH)]2+, [Zn(CH3SCH3)]2+, [Zn(C6H5S)]+, [Zn(C6H5S)2], [Zn(C6H5S)3]-, [Zn(HS)(NH=CH2)2]+, [Zn(HS)2(NH=CH2)2], [Zn(HS)(H2O)]+, [Zn(HS)(HCOO)], [Zn(HS)2(HCOO)]-, [Zn(CH3O)]+, [Zn(CH3O)2], [Zn(CH3O)3]-, [Zn(CH3O)4]2, [Zn(CH3OH)]2+, [Zn(HCOO)]+, [Zn(CH3COO)]+, [Zn(CH3COO)2], [Zn(CH3COO)3]-, [Zn(CO3)], [Zn(HCO3)]+, and [Zn(HCO3)(Imz)]+ (Imz, 1,3-imidazole). The computed Zn-S bond distances are 2.174A for [ZnS], 2.274 for [Zn(HS)]+, 2.283 for [Zn(CH3S)]+, and 2.271 for [Zn(C6H5S)]+, showing that sulfide anion forms stronger bonds than substituted sulfides. The nature of the substituents on sulfur influences only slightly the Zn-S distance. The optimized tetra-coordinate [Zn(HS)2(NH=CH2)2] molecules has computed Zn-S and Zn-N bond distances of 2.392 and 2.154A which compare well with the experimental values at the solid state obtained via X-ray diffraction for a number of complex molecules. The computed Zn-O bond distances for chelating carboxylate derivatives like [Zn(HOCOO)]+ (1.998A), [Zn(HCOO)]+ (2.021), and [Zn(CH3COO)]+ (2.001) shows that the strength of the bond is not much influenced by the substituent on carboxylic carbon atom and that CH3- and HO- groups have very similar effects. The DFT analysis shows also that the carboxylate Ligand has a preference for the bidentate mode instead of the monodentate one, at least when the coordination number is small.     

Stereochemical aspects of the formation of double bonds in abscisic acid

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.     

Simultaneous quantification of prostaglandins,isoprostane and thromboxane in cell-cultured medium using gas chromatography-mass spectrometry

We have developed a simultaneous quantification method for prostaglandin (PG) E(2), PGD(2), PGF(2 alpha), 8-epi-PGF(2 alpha), 6-keto-PGF(1 alpha) and thromboxane (TX) B(2). Using [3,3,4,4-(2)H(4)]PGE(2), [3,3,4,4-(2)H(4)]PGD(2), [3,3,4,4-(2)H(4)]8-epi-PGF(2 alpha), [3,3,4,4-(2)H(4)]PGF(2 alpha), [3,3,4,4-(2)H(4)]6-keto-PGF(1 alpha) and [18,18,19,19-(2)H(4)]TXB(2) as internal standards (I.S.), the eicosanoids and their I.S. were simultaneously extracted by solid-phase extraction from cell-cultured medium, derivatized to methyl ester/methoxim/tert.-butyldimethylsilyl ether derivatives and analyzed using gas chromatography-mass spectrometry in the selected ion monitoring mode. The accuracy for the added eicosanoids ranged from 92 to 113%, and coefficients of variation ranged from 0.1 to 12.2%. Increased eicosanoids in RAW264.7 and U937 cells stimulated by lipopolysaccharide were suppressed by NS-398 and indometacin. This simultaneous quantification method can be applied routinely for assaying eicosanoids in vitro.     

Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp

In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.     

Steroidal saponins from roots of Asparagus officinalis

Sarsasapogenin M (1) and sarsasapogenin N (2), two new oligospirostanosides with a unique aglycone moiety, (25S)-5beta-spirostan-3beta, 17alpha-diol, along with seven known compounds (25S)-5beta-spirostan-3beta-ol-3-O-beta-d-glucopyranosyl-(1,2)-[beta-d-xylopyranosyl-(1,4)]-beta-d-glucopyranoside (3), (25S)-5beta-spirostan-3beta-ol-3-O-beta-d-glucopyranosyl-(1,2)-beta-d-glucopyranoside (4), (25S)-5beta-spirostan-3beta-ol-3-O-alpha-l-rhamnopyranosyl-(1,2)-[alpha-l-rhamnopyranosyl-(1,4)]-beta-d-glucopyranoside (5), (25S)26-O-beta-d-glucopyranosyl-5beta-furost-20 (22)-ene-3beta,26-diol-3-O-beta-d-glucopyranosyl-(1,2)-beta-d-glucopyranoside (6), yamogenin (7), beta-sitosterol (8), and sitosterol-beta-d-glucoside (9) were isolated from the roots of Asparagus officinalis L. Their structures were determined by spectral analysis, including extensive 1D and 2D NMR experiments.     

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol,a useful intermediate for the preparation of deuterated isotopomers of sterols

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.     

Novel synthesis of cholesterol analogs: condensation of pregnenolone with dihydropyran or dihydrofuran.

Pregnenolone 3-(2'-tetrahydropyranyl) ether (1) was condensed with 3,4-[2H]dihydropyran to mainly give (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (20R-3), according to nuclear magnetic resonance (NMR). Cold, dilute HCl in ethanol removed the tetrahydropyranyl group at C-3 and also opened the dihydropyranyl ring at the C-20 position of 20R-3 to give (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol (20R-5). Analogous results were obtained by condensing pregnenolone 3-acetate with 3,4-[2H]dihydropyran to provide (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-acetate (20R-4). Acid-catalyzed opening of the dihydropyranyl ring at C-20 in 20R-4 yielded 20R-7, which, on acetylation followed by crystallization, provided (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol 3,26-diacetate (20R-8), identical to the diacetate made from 20R-5. Varying the reaction sequence beginning with 20(R,S)-4 gave an 84:16 ratio of 20R to 20S in a mixture of 20(R,S)-8, according to NMR analysis. Crystallization of the mixture from methanol provided pure 20R-8. Condensing 2,3-dihydrofuran and 1 for producing (20R)-[5'-(2',3'-dihydrofuranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (6) gave instead (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol 3-(2'-tetrahydropyranyl) ether (20R-9) by partial hydrolysis during workup. Treating 20R-9 briefly with dilute HCl produced (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol (20R-10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemistry and mechanism of reactions catalyzed by tryptophanase Escherichia coli.

Several beta replacement and alpha,beta elimination reactions catalyzed by tryptophanase from Escherichia coli are shown to proceed stereospecifically with retention of configuration. These conversions include synthesis of tryptophan from (2S,3R)- and (2s,3s)-[3(-3H)]serine in the presence of indole, deamination of these serines in D2O to pyruvate and ammonia, and cleavage of (2S,3R)-and (2S,3S)-[3(-3H)]tryptophan in D2O to indole, pyruvate, and ammonia. A coupled reaction with lactate dehydrogenase was used to trap the stereospecifically labeled [3-H,2H,3H]pryuvates as lactate, which was oxidized to acetate for chirality analysis of the methyl group. During deamination of tryptophan there is significant intramolecular transfer of the alpha proton of the amino acid to C-3 of indole. To determine the exposed face of the cofactor.substrate complex on the enzyme surface and to analyze its conformational orientation, sodium boro[3H]hydride was used to reduce tryptophanase-bound alaninepyridoxal phosphate Schiff's base. Degradation of the resulting pyridoxylalanine to (2S)-[2(-3H)]alanine and (4'S)-[4'(-3H)]pyridoxamine demonstrates that reduction occurs from the exposed si face at C-4' of the complex and that the ketimine double bond is trans.     

The first structural study on a cyclic tricoordinate phosphorochloridite and a pentacoordinate phosphorane based on 1,2,3,5-protected myo-inositol--a new conformation of 1,3,2-dioxaphosphorinane ring

Treatment of the phosphoramidite [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)P-NH-i-Pr)] with o-chloranil affords the first example of inositol-based pentacoordinate phosphorane [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)P-NH-i-Pr)(1,2-O(2)C(6)Cl(4))] (9) (X-ray structure) with a trigonal bipyramidal geometry at phosphorus. The six-membered 1,3,2-dioxaphosphorinane ring with the inositol residue has an unusual boat conformation in 9 which is quite different from that found in unrestrained rings investigated before, but is similar to that of its P(III) chloro precursor [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)PCl)] (X-ray structure). Also, a convenient and chromatography-free procedure for the protected myo-inositol derivative [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(OH)(2)] is reported.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Two triacylated and tetraglucosylated anthocyanins from Ipomoea asarifolia flowers

Two triacylated and tetraglucosylated anthocyanins derived from cyanidin were isolated from the flowers of Ipomoea asarifolia and their structures elucidated using chemical, GC, MS and NMR methods (1H and 13C, TOCSY-1D, DQF-COSY, DIFFNOE and HMBC). These complex pigments were found to consist of cyanidin 3-O-[2-O-(6-O-E-caffeoyl-beta-D-glucopyranosyl)]-[6-O-[4-O-(6-O-E-3,5-dihydroxycinnamoyl-beta-D-glucopyranosyl)-E-caffeoyl]-beta-D-glucopyranosyl]-5-O-beta-D-glucopyranoside and cyanidin 3-O-[2-O-(6-O-E-p-coumaroyl-beta-D-glucopyranosyl)]-[6-O-[4-O-(6-O-E-p-coumaroyl-beta-D-glucopyranosyl)-E-caffeoyl]-beta-D-glucopyranosyl]-5-O-beta-D-glucopyranoside.     

Carotenoid biosynthesis in a Flavobacterium sp.: stereochemistry of hydrogen elimination in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin (Short Communication)

[2-(14)C,(2R)-2-(3)H(1)]- and [2-(14)C,(2S)-2-(3)H(1)]-Mevalonates were rapidly incorporated into phytoene, lycopene, rubixanthin and zeaxanthin in a Flavobacterium system obtained by disruption of the bacterial cells by shaking with glass beads. Four hydrogen atoms arising from the 2-pro-S-hydrogen atoms of mevalonate were lost in the desaturation of phytoene to lycopene, rubixanthin and zeaxanthin. The desaturation of phytoene involves trans-elimination of hydrogen in the introduction of the double bonds at C-7, C-11, C-7' and C-11'.     

Three sesquiterpene hydrocarbons from the roots of Panax ginseng C.A. Meyer (Araliaceae)

The volatile constituents of the roots of Panax ginseng C.A. Meyer have been investigated after hydrodistillation and analysed by means of different analytical methods. Besides several compounds already known three sesquiterpene hydrocarbons have been isolated from the essential oil. Structure elucidation of the bicyclic panaxene as well as of the tricyclic panaginsene and ginsinsene was performed by MS and NMR. They have been identified as (1R*,2S*,5S*)-2-ethenyl-1(1-methylethenyl)-2,6,6-trimethylbicyclo[3.2.0]heptane (panaxene), (1S*,8S*,11R*)-4,7,7,11-tetramethyltricyclo[6.3.0.0(1,5)]undec-4-ene (panaginsene) und (1R*,6R*,7R*)-3,7,10,10-tetramethyltricyclo[4.3.2.0(2,6)]undec-2-ene (ginsinsene).     

Biosynthetic studies on the tropane alkaloid hyoscyamine in Datura stramonium; hyoscyamine is stable to in vivo oxidation and is not derived from littorine via a vicinal interchange process

The conversion of littorine to hyoscyamine has been investigated by feeding deuterium labelled (RS)-[2-(2)H]-, [3, 3-(2)H(2)]-, [2, 3, 3-(2)H(3)]- phenyllactic acids to transformed root cultures of Datura stramonium. Isolation and GC-MS analyses of the isotope incorporation into the resultant hyoscyamine does not support the involvement of a vicinal interchange process operating during the isomerisation of littorine to hyoscyamine. Additionally a metabolism study with [1'-13C, 3', 3'-(2)H(2)]-hyoscyamine has established that the alkaloid is metabolically stable at C-3' with no evidence for a reversible in vivo oxidation process to the corresponding aldehyde. The data do not support an S-adenosy-L-methionine (SAM 5)/co-enzyme-B(12) mediated process for the isomerisation of littorine to hyoscyamine.     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Steroidal saponins from the fruits of Asparagus racemosus

Three steroidal saponins, racemosides A (1), B (2) and C (3), were isolated from the methanolic extract of the fruits of Asparagus racemosus, and characterized as (25S)-5beta-spirostan-3beta-ol-3-O-{beta-D- glucopyranosyl (1-->6)-[alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->4)]-beta-D-glucopyranoside}, (25S)-5beta-spirostan-3beta-ol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranoside and (25S)-5beta-spirostan-3beta-ol-3-O-{alpha-L-rhamnopyranosyl-(1-->6)-[alpha-L-rhamnopyranosyl (1-->4)]-beta-D-glucopyranoside}, respectively, by spectrometric analysis and some chemical strategies.