Synthesis of methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides and carbamoyl-group migration between them

Methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides, (2 and 3), were synthesized from methyl α-D-mannopyranoside via ammonolysis of a cyclic carbonate or a p-nitrophenoxycarbonate, as shown in Charts 1 and 2. Carbamoyl-group migration between the C-2 and C-3 hydroxyl groups, in methyl α-D-mannopyranoside under alkaline conditions, was also studied.     

Détermination des sites d'oxydation des dérivés du α-D-glucofuranose utilisés comme donneurs

The sites of oxidation, by catalytic transfer of H, of derivatives of 1,2-O-isopropylidene-α-D-glucofuranose suggest a regiospecific reaction. Compounds having vicinal hydroxyl groups at C-5 and C-6, or at C-3 and C-5, are oxidized at OH-5, whereas compounds having two hydroxyl groups at C-3 and C-6 or three hydroxyl groups give first aldehydes and then lactones.     

Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-l-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX₃CX₂C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.     

Structure–Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1–2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.     

Synthesis and antiplasmodial activity of lycorine derivatives

Twenty seven lycorine derivatives were prepared and evaluated for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum. The best antiplasmodial activities were achieved with lycorine derivatives that present free hydroxyl groups at C-1 and C-2 or esterified as acetates or isobutyrates. The double bond C-2–C-3 is also important for the activity. Concerning to the antiplasmodial activity of the secolycorines, the higher values were obtained with the replacement of the methylenedioxy moiety by hydroxyl or acetate groups and with methyl substituent attached to the nitrogen atom.     

Mode of cleavage of the c-c bonds in methyl pento- and hexo-pyranosides with periodate

The course of oxidation reactions of three methyl pentopyranosides and five methyl hexopyranosides with periodate was studied by simultaneous determination of the conjugated aldehydes in the products. It was found that the C-3-C-4 bonds in all of these glycosides were cleaved rapidly, but the velocity of subsequent cleavage of the C-2-C-3 bonds depended on the configuration of the substituent groups on the pyranose ring. Equatorial C-1 methoxyl and C-2 hydroxyl groups favored cleavage of the C-2-C-3 bonds more than did the corresponding axial groups, whereas the equatorial C-5 hydroxymethyl group suppressed C-2-C-3-bond-cleavage. The 4-isomeric glycosides were oxidized at the same rate, without regard to the configuration at C-4.     

P.m.r. spectroscopy of dimethyl ethers of D-galactopyranose and its derivatives

P.m.r. parameters (determined at 100 MHz for solutions in deuterium oxide) are presented for di-O-methyl derivatives of D-galactopyranose (ten), methyl D-galactopyranoside (ten), and galactitol (five). The effects, on the methoxyl and anomeric-proton chemical-shifts, of anomeric change, methylation of neighboring hydroxyl groups, and change in configuration of adjacent carbon atoms bearing hydroxyl or methoxyl groups (other than at C-1) are discussed.     

Synthesis of methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides and carbamoyl-group migration between them

Methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides, (2 and 3), were synthesized from methyl α-D-mannopyranoside via ammonolysis of a cyclic carbonate or a p-nitrophenoxycarbonate, as shown in Charts 1 and 2. Carbamoyl-group migration between the C-2 and C-3 hydroxyl groups, in methyl α-D-mannopyranoside under alkaline conditions, was also studied.     

A sulfated glucuronofucan containing both fucofuranose and fucopyranose residues from the brown alga Chordaria flagelliformis

A fucoidan fraction composed of l-fucose, sulfate, and d-glucuronic acid in a molar proportion of about 1:1:0.25 and small amount of acetyl groups was isolated from the brown alga Chordaria flagelliformis. Several modified polysaccharides were prepared from the native fucoidan using solvolytic desulfation, carboxyl reduction, and partial acid hydrolysis. Polysaccharide structures were elucidated by methylation analysis and 1D and 2D NMR spectroscopy. The fucoidan was shown to contain a backbone of 3-linked α-l-fucopyranose residues, about one-third of which are glycosylated at C-2 by α-d-glucopyranosyluronic acid residues. About half of the latter residues are glycosylated at C-4 by single α-l-fucofuranose residues or by disaccharides α-l-Fucf-(1→2)-α-l-Fucf-(1→. Fucofuranose residues are mono- and disulfated at different positions, whereas some additional sulfate groups occupy C-2 and C-4 of the backbone, the latter position being also partially acetylated.     

13C- and 1H-N.M.R. spectroscopy of permethylated α- and β-D-galactopyranoses

The complete assignment of the ¹³C- and ¹H-n.m.r. spectra of the permethylated α- and β-D-galactopyranoses was performed with the aid of specific trideuteriomethylation, heteronuclear spin-decoupling, and spectrum simulation. The n.m.r. data are discussed and compared with those of the permethylated glucopyranoses. Identification of partially methylated galactoses, e.g., as obtained in the methylation analysis of carbohydrates, can be carried out by conversion of the free hydroxyl functions into ²H- or ¹³C-labelled methoxyl groups, and comparison of the n.m.r. spectra of the resulting permethyl ethers with those of reference compounds.     

The carrageenans of gigartina skottsbergii s. et g. : Part III. methylation analysis of the fraction precipitated with 0.3-0.4m potassium chloride

The fraction of carrageenan from the red seaweed Gigartina skottsbergii that is precipitated with 0.3-0.4m potassium chloride has been studied by methylation analysis. The results agree qualitatively with the structure previously suggested, except that 3-linked D-galactose 4-sulfate residues are present rather than the corresponding 2-sulfate. For every ten D-galactose residues linked at C-3, there are, on the average, six residues of 3,6-anhydro-D-galactose linked at C-4 and ten sulfate groups (five as 3,6-anhydro-D-galactose 2-sulfate and five as D-galactose 4-sulfate residues).     

Studies of the selective silylation of methyl α- and β-d-aldohexopyranosides: stability of the partially protected derivatives in polar solvents

Treatment of methyl α- (1) and β-d-glucopyranoside, methyl α- (3) and β-d-galactopyranosides, and methyl α-d-mannopyranoside (5) with 2, 3, or 4 mol. equiv. of tert-butyldimethylsilyl (TBDMS) chloride under two conditions afforded mixtures of TBDMS ethers which were identified. The following compounds were isolated in synthetically useful yields, the 2,6-di-TBDMS either of 1 (70%), the 2,6-di- and 2,3,6-tri-TBDMS ethers of 3 (84% and 57%, respectively), and the 2,6-di-and 3,6-di-TBDMS ethers of 5 (50% and 80%, respectively). In dipolar solvents, no migration of the TBDMS groups was detected between partially silylated hydroxyl groups, but the addition of a base (triethylamine or imidazole) caused migration to vicinal cis positions.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Cyclic glycolipids from glandular trichome exudates of Cerastium glomeratum

Fourteen cyclic glycolipids, named glomerasides A–N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui–Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6′-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).     

Selective benzylation of some D-galactopyranosides. Unusual relative reactivity of the hydroxyl group at C-4

Partial benzylation of methyl 2,3-di-O-benzyl-α-D-galactopyranoside gave methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside as the major product, whereas the isomeric 2,6-di-O-benzyl ether gave a mixture of products in which the ratio of methyl 2,4,6- to methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside was ≈4:1. The proportion of unreacted starting-material was low in both cases, whereas after a similar reaction of methyl 2,6-di-O-benzyl-β-D-galactopyranoside more than 50% of the dibenzyl ether was recovered unchanged. In this case also, considerably higher reactivity was exhibited by the hydroxyl group at C-4 than that at C-3. Acid hydrolysis of the methyl glycosides of the tribenzyl ethers afforded crystalline 2,4,6-tri-O-benzyl-α-D-galactose and syrupy 2,3,6-tri-O-benzyl-D-galactose. Structures of intermediates were established by acetylation, examination of their n.m.r. spectra, and conversion into the known 3-O and 4-O-methyl-D-galactose.     

Studies on the koenigs-knorr reaction : Part IV: The effect of participating groups on the stereochemistry of disaccharide formation

Partial benzylation of methyl 2-O-benzyl-α-L-fucopyranoside afforded a mixture of methyl 2,3-, and 2,4-di-O-benzyl-α-L-fucopyranoside which were separated by means of their monoacetates. Partial benzylation of methyl α-L-fucopyranoside gave the 2,4-, and 3,4-dibenzyl ethers in the ratio of 3:2, and no 2,3-isomer could be detected in the reaction mixture. The structures of the three dibenzyl ethers were established: (a) by analysis of the n.m.r. spectra of their acetates, and (b) by methylation, removal of benzyl groups by hydrogenolysis, and characterization of the methyl ethers of the methyl glycosides. Acid hydrolysis of these compounds gave the monomethyl ethers of L-fucose, two of which were identical with known compounds, whereas the third, 4-O-methyl-L-fucose, was a new compound. Selective p-nitrobenzoylation of 2,3-, 2,4-, and 3,4-di-O-benzyl-L-fucose, followed by acetylation and treatment with hydrogen bromide in dichloromethane, gave the three possible mono-O-acetyl-di-O-benzyl-α-L-fucopyranosyl bromides, which were condensed with benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-α-D-glucopyranoside. The disaccharide derived from the 2-O-acetyl substituted bromide was enriched in β-L-fucopyranoside, whereas the other two bromides gave mainly the α-L-linked anomer. The α-directing influence of the 3- and 4-O-acetyl substituents is not less than the β-directing influence of the 2-O-acetyl group in similar bromides; participation of acyl groups and electronic-steric influences are discussed as possible explanations for the steric course of the reaction.     

Analysis of bromine-oxidised dextran by 13C-n.m.r. spectroscopy

Dextran T 10, elaborated by Leuconostoc mesenteroides NRRL B-512, was oxidised with aqueous bromine at pH 7.0. The resulting oxodextran and its methoximated derivative were analysed by ¹³C-n.m.r. spectroscopy. The total amount of keto groups and their positions were established. Assignments of the ¹³C signals were made by referring to spectra of the corresponding methyl glucosiduloses and an oxodextran having most of the carbonyl groups at position 3 of the glycopyranosyl residues. In accordance with the mechanism for bromine oxidation of mono- and di-saccharides, the glucopyranosyl residues of dextran were oxidised mainly at C-2 and C-4. Over-oxidation resulted in a small proportion of acidic, ring-cleavage products.     

Preparation and separation of methyl ethers of D-arabinose

All possible methyl ethers of methyl β-d-arabinofuranoside and methyl β-d-arabinopyranoside were prepared by partial methylation using Kuhn's method (Ag₂O and MeI in N,N-dimethylformamide) and Haworth's method (Me₂SO₄ and 40% NaOH in water). The ratios of the methyl ethers obtained from the furanoside were found to be considerably more dependent on the method of methylation than those from the pyranoside. All of the methyl ethers of d-arabinofuranose and d-arabinopyranose are separable by thin-layer chromatography on silica gel and paper electrophoresis in borate buffer.     

Stéréosélectivité de la réduction de la 3′, 5′-di-O-benzoyl-2′-bromo-2′-désoxyuridine par l'hydrure de tributylétain

Samples of sodium alginate from Laminaria digitata were oxidised in aqueous solution to different extents by the addition of calculated amounts of sodium metaperiodate. An empirical measure of the molecular stiffness of the products, both before and after reduction with sodium borohydride, was obtained by studying the sensitivity of their intrinsic viscosities to changes in the ionic strength of the solution. The results showed that, whereas the stiffness decreased very sharply with increasing degree of oxidation, it changed very little upon subsequent reduction of the products. From this, and the known structures of the inter-residue hemiacetals in the unreduced products, it was inferred that the increase in flexibility brought about by oxidation is specifically associated with rotation about the three bonds adjoining C-4, C-5, the original ring-oxygen atom, and C-1 of the oxidised hexuronic-acid residues. This rotation was considerably less-hindered than the rotation around the glycosidic linkage in both the intact alginate and in the partially oxidised and reduced products. The conversion of only a small percentage of the stiff pyranoid rings into these centres of high, local flexibility had a marked effect on the overall flexibility of the chains, showing that the possible presence of such centres should be taken into account in any discussion of the solution properties of heteropolysaccharides or of polysaccharides that may have suffered slight chemical modification.     

Partial methylation of methyl glycosides: a source of samples for analysis of methylation of carbohydrate-containing compounds.

Methyl ether mixtures were obtained by partial methylation of 15 methyl glycopyranosides using Purdie's procedure. Identification of ethers as acetates by gas-liquid chromatography-mass spectroscopy (glc-ms) techniques allowed us to assume them to be sample sources for methylation analysis.T-values were tabulated using a scale ranging from permethyl ether- (0) to peracetate methyl glycosides (100) for subsequent identification with the aid of glc in NPGS and QF-1 liquid phases.A scheme has been devised for isolating individual methyl ethers by preparative glc.