Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Induction of 3'-O-beta-D-ribofuranosyl adenosine during compatible, but not during incompatible, interactions of Arabidopsis thaliana or Lycopersicon esculentum with Pseudomonas syringae pathovar tomato

All hitherto identified aromatic compounds accumulating in leaves of Arabidopsis thaliana (L.) Heynh. upon infection with virulent or avirulent strains of Pseudomonas syringae pathovar tomato (Pst) were indolic metabolites. We now report the strong accumulation of a novel type of natural product, 3-O--d-ribofuranosyl adenosine (3RA), exclusively during compatible interactions. In contrast to the various indolic metabolites, 3RA was undetectable in incompatible interactions of A. thaliana leaves with an avirulent Pst strain, as well as in uninfected control leaves. A similar, strong induction of 3RA was observed in compatible but, again, not in incompatible interactions of Pst with its natural host, Lycopersicon esculentum. The strength of the effect and its confinement to compatible interactions suggests that it may be applicable as a diagnostic tool.Abbreviations Pst Pseudomonas syringae pathovar tomato - 3RA 3-O--d-ribofuranosyl adenosine     

Effects of cyclic nucleotides on morphogenesis inNostoc muscorum

The filamentous cyanophyteNostoc muscorum A grew aseriately in light in a mineral salts (sugar-free) culture medium supplemented with adenosine 3:5-cyclic-monophosphate or N⁶, O²-dibutyryl adenosine 3:5-cyclic-monophosphate (1 mM). The aseriate morphology thus formed in the light on the 10th day following inoculation was similar to that formed in the dark after 20–30 days growth in cAMP-free medium containing glucose or sucrose. Inoculum previously grown in sucrose- or glucose-containing medium displayed aseriate morphology with lesser proliferation of coccoid cells as compared to inoculum grown in the absence of glucose or sucrose. cGMP, ADP, AMP and inhibitors of phosphodiesterase (theophylline and caffeine) did not have any effect on the persistence of aseriate morphology. However they stimulated cell division at the aseriate stage and delayed the release of hormogonia.Abbreviations cAMP adenosine 3:5-cyclic-monophosphate - db cAMP N⁶, O²-dibutyryl adenosine 3:5-cyclic-monophosphate - cGMP guanosine 3:5-cyclic-monophosphate - ATP adenosine 5-triphosphate - ADP adenosine5-diphosphate - AMP adenosine 5-monophosphate     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

Relationships betweenin vitro andin vivo biotransformation of drugs in humans and animals: pharmaco-toxicological consequences

Given the crucial role played by hepatocytes in the detoxification/toxification processes of drugs, these cells have been increasingly used during the last decade in various pharmaco-toxicological areas. The majority of these studies have, however, dealt with animal cells, although examples of failures in the extrapolation of the data to man are frequent. This drawback, together with the ethical considerations in performingin vivo experiments, makes the application of the human hepatocyte model critical in the preclinical evaluation of new compounds. However, before making extensive use of these promising tools for prospective pharmaceutical research, one must ensure that they can generate data that correlate well with those obtainedin vivo. This is only possible through extensive studies on drugs showing a variety of phase I and phase II metabolic pathways in hepatocytes from different species, including man, and comparison within vivo data. Providing this validation step is undertaken, the use of such systems in drug research and development may greatly enhance the rational design of safe and effective drugs, allowing savings in time, cost and test materials as well as minimizing the use of animals.Abbreviations AMT 3-amino-3-deoxythymidine - AZT 3-azido-3-deoxythymidine - GAMT 5-glucuronyl-3-amino-3-deoxythymidine - GAZT 3-azido-3-deoxy-5-O--d-glucopyranuronosylthymidine - HIV human immunodeficiency virus - MAO monoamine oxidase     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Involvement of cyclic adenosine-3′, 5′-monophosphate in chloronema differentiation in protonema cultures of Funaria hygrometrica

The role of purine and pyrimidine ribosides, nucleotides and substituted xanthines in the differentiation of chloronema filaments in suspension cultures of protonema of the moss Funaria hygrometrica Hedw. has been examined. Cyclic adenosine-3,5-monophosphate (cAMP) and mono-and dibutyryl cAMP evoked the maximum response in wild-type protonema. ADP and ATP also enhanced chloronema differentiation but were less active than cAMP; pyrimidine derivatives were completely inactive. Inhibitors of cyclic-nucleotide phosphodiesterase aminophylline, theophylline and ICI 58, 301 (3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine)-mimicked the effect of cAMP. A leaky, chloronema-repressed mutant was isolated and in this mutant cAMP was much more active than cyclic guanosine monophosphate and ADP in enhancing chloronema differentiation. These results strongly indicate that cAMP is involved in chloronema differentiation in Funaria, and a hypothesis on growth regulation in protonema cell cultures is proposed.Abbreviations cAMP, cyclic AMP cyclic adenosine-3, 5-monophosphate - cCMP, cGMP, cIMP cyclic cytosine-, guanosine-and inosine-3, 5-monophosphates, respectively - IAA indole-3-acetic acid - ICI 58,301 3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine     

Influence of the glycosidic torsion angle on 13C and 15N shifts in guanosine nucleotides: Investigations of G-tetrad models with alternating syn and anti bases

Summary The effect of the glycosidic torsion angle on ¹³C and ¹⁵N shifts of the sugar and base moieties of guanosine nucleotides was investigated by comparing the sites in two model G-tetrad oligodeoxynucleotides that contain guanosine residues alternately with syn and anti bases. The sugar puckering has been shown to be C2-endo for both cases. It was observed that, for the instances with syn bases, the C1 through C4 carbons showed shifts that may be distinguished from those normally found in B-DNA-like structures. C1, C3 and C4 moved to lower field, while C2 moved to higher field. Effects of the change in glycosidic torsion angle were also seen in the shifts of base carbons and nitrogens in the five-membered ring portion of the base. Characterization of the shift variation associated with this conformational change may be useful in developing the use of ¹³C shifts as a tool in conformational analysis of oligonucleotides.Part of the work reported here derives from the Ph.D. Thesis of Karen L. Greene, Emory University, Atlanta, GA, 1991.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3) encoding the gene for the 3/5-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3 cDNA sequence shows about 67–85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3/5 (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

Mechanistic possibilities in prebiotic thiophosphate chemistry

Summary Two types of reactivities of thiophosphates have been demonstrated: one being nucleophilic displacement by the P-S moiety of nucleoside phosphorothioates and the other, phosphorylation via P-S cleavage as the driving force. We have designed a system where both displacement on carbon and P-S cleavage are possible. Adenosine derivatives have been synthesized with 5-deoxy-5-chloro and 5-O-tosyl substitutions as leaving groups utilizing the 3-O-phosphorothioate as the biphilic center. The main products of cyclization were 5-O-tosyl and 5-chloroadenosine 2:3-cyclic phosphate. Formation of 3:5-S-phosphorothioate was slow even using an excellent leaving group. This is possibly due to hydrogen bonding between the 2-OH and the neighboring P-O.^– KOH hydrolysis of the cyclic phosphorothioate yielded 2(3) phosphorothioates in a 1:1 ratio. The 2 and 3 isomers were separated and used to study the relative rates of cyclization. The cyclization via P-S cleavage of 2(3)-O-phosphorothioates showed that the 2 isomer was more reactive. This is the first report of superior reactivity of the 3-OH of a ribonucleoside.     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Reconstitution of cytoplasmic streaming inCharaceae

Summary The active sites of actin of oneCharaceae species were found to interact with the endoplasmic factor from a different species. Protoplasm was suqueezed out of cells ofChara australis with vacuoles that had been perfused beforehand with a medium containing EGTA and Mg · ATP. Centrifugation of this protoplasmic mixture divided it into the supernatant composed of endoplasmic granules and the precipitate composed of chloroplasts and nuclei. When the endoplasmic granular aggregates were introduced into a tonoplast-freeNitella axilliformis cell treated with NEM to inactivate the endoplasmic factor, they became attached to theNitella gel and streamed longitudinally with the polarity. Treatment of the endoplasmic granules with the strong Mg²⁺chelator CyDTA (1,2-cyclohexane diamineN, N-tetraacetic acid) irreversibly inhibited reconstitution of the cytoplasmic streaming.Abbreviations APW artificial pond water - ATP adenosine-5-triphosphoric acid - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid - HMM heavy meromyosin - NEM N-ethylmaleimide - PEP phosphoenolypyruvate - PIPES piperazine-N,N-bis-(2-ethane-sulfonic acid) - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride     

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line

The human glioma D-54MG cell line grownin vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3-isoLM1, an alteration that was dose dependent, with the highest dose (1×10⁸) resulting in a phenotype that was most like that of the inoculum. After one passagein vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grownin vitro than in those grownin vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors. Abbreviations: The gangliosides have been designated according to Svennerholm (Eur J Biochem (1977)79: 11–21) GM3, II³NeuAc-LacCer; GM2, II³NeuAc-GgOse₃Cer; GM1, II³NeuAc-GgOse₄Cer; GD3, II³(NeuAc)₂-LacCer; GD2, II³(NeuAc)₂-GgOse₃Cer; GD1a, IV³NeuAc, II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc, II³(NeuAc)₂-GgOse₄Cer; 3-LM1, IV³NeuAc-nLcOse₄Cer; 3-isoLM1, IV³NeuAc-LcOse₄Cer; 3,6-isoLD1, IV³NeuAc, III⁶NeuAc-LcOse₄Cer; 38-LM1, IV³(NeuAc)₂-nLcOse₄Cer. MAb(s), monoclonal antibody (ies); the designation LM1 is used when both 3-isoLM1 and 3-LM1 and LD1, when both 36-isoLD1 and 38-LD1 are included.     

ζ-Carotene<Emphasis Type="Italic"> cis</Emphasis> isomers as products and substrates in the plant poly-<Emphasis Type="Italic">cis</Emphasis> carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and -carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9-tricis--carotene with 15,9-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by -carotene desaturase, the 15-cis double bond of 9,15,9-tricis--carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9-dicis--carotene is utilized by -carotene desaturase via 7,9,9-tricis-neurosporene to 7,9,7,9-tetracis-lycopene. Other -carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis--carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to -carotene. The 5-cis-lycopene and the 9-cis-and 13-cis--carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.