Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid,a new sialic acid from horse submandibular gland

The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already know equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acidand 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.     

Monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by protease in N,N-dimethylformamide with low water content

2-O-α-d-Glucopyranosyl-l-ascorbic acid (AA-2G) laurate was synthesized from AA-2G and vinyl laurate with a protease from Bacillus subtilis in N,N-dimethylformamide (DMF) with low water content. Addition of water to DMF dramatically enhanced monoacyl AA-2G synthesis. Maximum synthetic activity was observed when 3% (v/v) water was added to the reaction medium. Under the optimal reaction conditions, 5-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid, 2-O-(6′-O-dodecanoyl-α-d-glucopyranosyl)-l-ascorbic acid, and 6-O-dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid were synthesized in yields of 5.5%, 3.2%, and 20.4%, respectively.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

Biotransformation of o- and p-aminobenzoic acids and N-acetyl p-aminobenzoic acid by cell suspension cultures of Solanum mammosum

Some new biotransformation products, p-aminobenzoic acid 7-O-β-d-glucopyranosyl ester, N-acetyl p-aminobenzoic acid 7-O-β-d-glucopyranosyl ester, o-aminobenzoic acid 7-O-β-d-(β-1,6-O-d-glucopyranosyl)glucopyranosyl ester and o-aminobenzoic acid 7-O-β-d-glucopyranosyl ester were isolated from cell suspension cultures of Solanum mammosum following administration of p-aminobenzoic acid, N-acetyl p-aminobenzoic acid or o-aminobenzoic acid respectively. N-acetyl p-aminobenzoic acid and N-formyl p-aminobenzoic acid were also identified as cell suspension metabolites of p-aminobenzoic acid.     

N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

Chemistry of glycosylamines. Isopropylidene acetals of N-acetyl-α-d-glucofuranosylamine

The reaction of N-acetyl-α-d-glucofuranosylamine with 2,2-dimethoxypropane, catalyzed by p-toluenesulfonic acid, gave 1-acetamido-2,3:5,6-di-O-isopropylidene-1-O-methyl-d-glucitol (65.6%), 1-acetamido-2,3-O-isopropylidene-1-O-methyl-d-glucitol (3.7%), and N-acetyl-5,6-O-isopropylidene-α-d-glucofuranosylamine (3.2% yield). The structures of these compounds were determined by chemical and spectroscopic methods, and their relation to the pattern of n.m.r. resonances of the isopropylidene methyl groups is discussed.     

Phosphono-sphingomyelins. I: Synthesis of ceramide (trimethyl) aminoethyl phosphonate

The synthesis of the phosphono-analogue of sphingomyelin is described. The N-acyl-D-erythro-sphingosyl-1-(N,N,N-trimethyl-2-aminoethyl) phosphonate was obtained by phosphonylation of N-acyl-3-O-benzoyl-D-erythro-sphingosine with (2-bromoethyl)phosphonic acid chloride and triethylamine, subsequent quaternisation with anhydrous trimethylamine and benzene at 55–60°C for four days, and finally, consecutive removal of the protective group by mild alkaline hydrolysis. Comparison of the CD spectra of both, natural sphingomyelin and its phosphono-analogue, confirmed that their structures and configurations were identical.     

Versatile methods for the synthesis of mixed-acid 1,2-diacylglycerols

Two methods for synthesizing mixed-acid 1,2-diacylglycerols starting from 2,3-epoxy-1-propanol (glycidol) or 3-chloro-1,2-propanediol have been described. This first method involves fatty acid addition to a protected glycidol derivative and solid-state isomerization. The second approach exploits the specificity of the trityl group for primary alcohols and the nucleophilic replacement of chlorine by a carboxylate ion in an aprotic solvent. The second method proves to be more general: with 3-chloro-1-O-trityl-1,2-propanediol as an intermediate compound apparently all types of mixed-acid, saturated and unsaturated, chiral and racemic 1,2-diacylglycerols can be prepared in good yields. In the first method tritylglycidol is a good starting compound. The use of this method, however, is restricted because only the 2-position of glycerol can be occupied with an unsaturated fatty acid. For de-blocking protected 1,2-diacylglycerols, the trityl group and other protecting groups were exchanged for the trifluoroacetyl group, which group could then be removed without any detectable acyl migration (< 1%). To this end, the 1,2-diacyl-3-trifluoroacetyl-glycerols were dissolved at room temperature in methanol containing pyridine, whereby the trifluoroacetyl group was split off, giving the 1,2-diacylglycerol.     

Formation of a stable l-ascorbic acid α-glucoside by mammalian α-glucosidase-catalyzed transglucosylation

Enzymatic transglucosylation from maltose to l-ascorbic acid (AA) with mammalian tissue homogenates was determined by a high-performance liquid chromatography method and compared with the reaction catalyzed by α-glucosidase from Aspergillus niger. The homogenates of small intestine and kidney had a high transglucosylase activity to form a new type of glucosylated AA, which was associated with α-glucosidase activity. The new compound was demonstrated to be an equimolar conjugate of AA and glucose by the spectral and quantitative analyses. In particular, it showed a high stability in a neutral solution and no reducing activity toward cytochrome c and a dye. These properties were very different from those of AA and l-ascorbic acid α-glucoside formed with α-glucosidase form A. niger, but they were consistent with those of l-ascorbic acid 2-O-phosphate and l-ascorbic acid 2-O-sulfate. Moreover, it exhibited a reducing power associated with AA after mild acid hydrolysis or treatment with rat intestinal α-glucosidase. These results indicate that it should be assigned the 2-O-α-glucoside structure. Consequently, i should be assigned the 2-O-α-glucoside structure. Consequently, it is concluded that mammalian α-glucosidase is able to form a very stable and nonreducing form of glucosylated AA through a specific transglucosylation reaction distinct from that of microbial α-glucosidase.     

Depolymerization of heparin with diazomethane. Structure of N,O-methylated,even-numbered oligosaccharides produced by β-eliminative cleavage of the 2-amino-2-deoxyglycosylic linkage

The long-period reaction of heparin with excess diazomethane at 20° resulted in cleavage at the β-position of the uronic acid carboxyl group to give a mixture of methyl α- and β-glycosides of N,O-methylated di-, tetra-, and hexa-saccharides having a 4,5-unsaturated uronic acid, nonreducing end-group. The major disaccharides obtained were methyl O-(4-deoxy-3-O-methyl-α-l-threo-hex-4-enopyranosyluronic acid 2-sulfate)-(1→4)-2-deoxy-3-O-methyl-2-(N-methylsulfoamino)-α- and -β-d-glucopyranoside. The reaction of heparin at 4° yielded a mixture of methylated, higher-molecular-weight oligosaccharides, which retained some affinity for antithrombin III-Sepharose.     

Synthèse de l'acide 5-acétamido-3,5-didésoxy-4-O-méthyl- d-glycéro-d-galacto-2-nonulosonique (acide 4-O-méthyl-N-acétylneuraminique). Partie II

3-Acetamido-3-deoxy-4,5:6,7-di-O-isopropylidene-d-glycero-d-galacto-heptose diethyl dithioacetal was transformed into 3-acetamido-3-deoxy-4,5:6,7-di-O-isopro-pylidene-2-O-methyl-aldehydro-d-glycero-d-galacto-heptose after O-methylation followed by desulfuration. A Wittig reaction with an excess of [ethoxy(ethoxycarbonyl)-methylene]triphenylphosphorane in the presence of benzoic acid gave a mixture of ethyl 5-acetamido-3.5-dideoxy-2-O-ethyl-6,7:8,9-di-O-isopropylidene-4-O-methyl-d-glycero-d-galacto-non-2-enonate (23 %) and the d-glycero-d-talo (22 %) isomer. An ethoxymercuration-demercuration reaction, followed by acid hydrolysis, converted the former into ethyl 4-O-methyl-N-acetylneuraminate and the latter into the C-4 stereoisomer. 4-O-Methyl-N-acetylneuraminic acid was then obtained in crystalline form, and its structure ascertained by mass spectrometry and ¹H- and ¹³C-nuclear magnetic resonance.     

Acetonation of some pentoses with 2,2-dimethoxypropane-N,N-dimethylformamide-p-toluenesulfonic acid

d-Xylose, d-arabinose, and d-ribose were each treated with 2,2-dimethoxypropane in N,N-dimethylformamide containing a trace of p-toluenesulfonic acid. d-Xylose gave 3,5-O-isopropylidene-d-xylofuranose, 1,2:3,5-di-O-isopropylidene-α-d-xylofuranose, 1,2-O-isopropylidene-α-d-xylopyranose, and two acyclic di-O-isopropylidene derivatives. d-Arabinose gave the known 3,4-O-isopropylidene-β-d-arabinopyranose and 1,2:3,4-di-O-isopropylidene-β-d-arabinopyranose. d-Ribose gave 2,3-O-isopropylidene-d-ribofuranose almost exclusively.     

Substrate-like water soluble lipase inhibitors from Filipendula kamtschatica

Filipendula kamtschatica is a plant utilized as a traditional medicine by Ainu people in Japan, but its chemical constituents are not much studied. Pancreatic lipase inhibitors are a promising tool for the treatment of obesity. We searched for natural lipase inhibitors from F. kamtschatica and two new compounds were isolated along with the known flavonoid glycoside. The structure elucidation of new compounds revealed these two to be 2-O-caffeoyl-4-O-galloyl-l-threonic acid and 3-O-caffeoyl-4-O-galloyl-l-threonic acid, which can be recognized as a pancreatic lipase’s substrate-like structure. The isolated compounds all showed an inhibitory activity against porcine pancreatic lipase and one of the isomer, 3-O-caffeoyl-4-O-galloyl-l-threonic acid, possessed the most potent activity with IC₅₀ value showing an order lower value compared to others. The substrate-like structure of the new compounds seemed to be important for their activity.     

Chemical synthesis of 1-O-alkyl and 1-O-acyl dihydroxyacetone-3-phosphate

A method for the chemical synthesis of 1-O-hexadecyl dihydroxyacetone-3-phosphate is described. The synthesis was started with the preparation of O-hexadecyl glycolic acid by condensing 1-iodohexadecane with ethyl glycolate in the presence of silver oxide, followed by saponification and free acid liberation with HC1. O-Hexadecyl glycolic acid was converted to the acid chloride (with oxalyl chloride) which was condensed with diazomethane in diethyl ether to form hexadecyloxy diazoacetone. The diazoketone was decomposed by H₃PO₄ in dioxane to give the desired product, 1-O-hexadecyl dihydroxyacetone-3-phosphate. The product was purified by chromatography on silicic acid column followed by an acid wash. The final yield was 50% starting from O-hexadecyl glycolic acid. Analytical, spectral (IR, NMR) and chromatographic properties of 1-O-hexadecyl dihydroxyacetone-3-phosphate are described. The method described here may be used to prepare different acyl and alkyl derivatives of dihydroxyacetone phosphate in good yield as illustrated by describing the procedure for the synthesis of 1-O-palmitoyl dihydroxyacetone-3-phosphate, 1-O-hexadecyl dihydroxyacetone-3-[³²P] phosphate and the dimethyl ketal of 1-O-palmitoyl [2-¹⁴C]dihydroxyacetone phosphate.     

Synthesis of some glycodipeptides containing hydroxyamino acids, and their stabilities to acids and bases

The following new compounds were prepared and characterized: N-benzyl-oxycarbonyl-O-(tetra-O-acetyl-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (1) and L-threonine methyl ester (2), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-serine amide (3), N-benzyloxycarbonyl-O-(β-D-glucopyranosyl)-N-glycyl-L-threonine methyl ester (4) and L-threonine amide (5), N-benzyloxycarbonyl-O-(tri-O-acetyl-2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine methyl ester (6), and N-benzyloxycarbonyl-O-(2-deoxy-2-trifluoroacetamido-β-D-glucopyranosyl)-N-glycyl-L-serine amide (7). Although various modifications of the Koenigs-Knorr synthesis were used, the best, over-all yields of the deacetylated dipeptide derivatives were only 5–10%. Although the products are alkali-labile, deacetylation was accomplished with methanolic ammonia. Of the deacetylated products, the threonine derivatives (4 and 5) were more rapidly hydrolyzed by acids than phenyl β-D-glucopyranoside, which in turn was more rapidly cleaved than the serine derivatives (3 and 7). The stabilities of 3, 4, 5, and 7 to sodium hydroxide and sodium borohydride were similar, and essentially complete β-elimination of the glycosyl residue occurred for the amide derivatives (3, 5, and 7). For the ester derivative 4, pH 9 was optimal; above this pH, ester hydrolysis was more rapid than β-elimination, and the resulting carboxyl derivatives did not undergo β-elimination. Under optimal conditions with sodium borohydride, the β-elimination reaction was complete, but the corresponding alanine and α-aminobutyric acid residues were not formed; presumably reductions to the amino alcohols occurred. A mechanism for the β-elimination is proposed.     

Phosphonosphingoglycolipid,a novel sphingolipid from the viscera of Turbo cornutus

A novel lipid which contained long-chain base, fatty acid, galactose and N-methylaminoethylphosphonic acid in an equimolar was isolated from the viscera of Turbo cornutus.The methods used for the structural elucidation of this lipid were partial acid hydrolysis, alkaline hydrolysis, periodate oxidation and Smith degradation. The structure of breakdown products were mainly identified by combined gas chromatography and mass spectrometry.The structure of the novel lipid was determined to be 1-O-[6′-O-(N-methylaminoethylphosphonyl) galactopyranosyl] ceramide.Mass spectra of galactose-N-methylaminoethylphosphonate and glycerol-N-methylaminoethylphosphonate are given.     

N-Substituted acetamide glycosides from the stems of Ephedra sinica

Five new N-mono-/bis-substituted acetamide glycosides, N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (1), N-methyl-N-{4-O-[3-O-(4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (2), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (3), N-methyl-N-{4-O-[3-O-(6-O-benzoyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (4), and N-methyl-N-{4-O-[3-O-(6-O-trans-cinnamoyl-4-O-α-l-rhamnopyranosyl-β-d-glucopyranosyl)-α-l-rhamnopyranosyl]-phenethyl}-acetamide (5), along with one known acetamide derivative, N-methyl-N-(4-hydroxyphenethyl)-acetamide, the shared aglycone of 2–5, were isolated from the ethanol extract of the stems of Ephedra sinica. The structures of these new compounds were elucidated on the basis of extensive spectroscopic examination, mainly including multiple 1D and 2D NMR and HRESIMS examinations, and qualitative chemical tests. All N,N-bissubstituted acetamide glycosides were found to show the obvious rotamerism, as in the case of the isolated known N-methyl-N-(4-hydroxyphenethyl)-acetamide, under the experimental NMR conditions, with the ratios of integrated intensities between anti- and syn-rotamers always being found to be about 4 to 3.     

The use of boric and benzeneboronic acids in the partial acetonation of monosaccharides

The preparation of mono-O-isopropylidene derivatives and mono-O-isopropylidene benzeneboronates of monosaccharides in one step is described, together with their p.m.r. and mass-spectral characteristics. In particular, the use of boric acid in the synthesis of the new acetal 1,2-O-isopropylidene-β-L-arabinopyranose (8) is described, together with improved procedures for the preparation of 2,3-O-isopropylidene-D-mannofuranose (5) and 3,4-O-isopropylidene-L-arabinopyranose (10). The use of boric acid in the partial hydrolysis of 1,2:3,4-di-O-isopropylidene-β-L-arabinopyranose to give the 1,2-acetal is reported.     

Enhanced stereoselectivity of α-mannosylation under thermodynamic control using trichloroacetimidates

O-Specific polysaccharides of Vibrio cholerae O1, serotypes Inaba and Ogawa, consist of α-(1→2)-linked N-(3-deoxy-l-glycero-tetronyl)perosamine (4-amino-4,6-dideoxy-d-mannose). The blockwise synthesis of larger fragments of such O-PSs involves oligosaccharide glycosyl donors that contain a nonparticipating 2-O-glycosyl group at the position vicinal to the anomeric center where the new glycosidic linkage is formed. Such glycosyl donors may bear at C-4 either a latent acylamino (e.g., azido) or the 3-deoxy-l-glycero-tetronamido group. While monosaccharide glycosyl donors, even those bearing a nonparticipating group at O-2 (e.g., methyl), and the 4-N-(3-deoxy-l-glycero-tetronyl) side chain form α-linked oligosaccharides with excellent stereoselectivity, α-mannosylation with analogous oligosaccharide donors in this series is adversely affected by the presence of the side chain. Consequently, the unwanted β-product is formed in a considerable amount. Conducting the reaction at elevated temperature under thermodynamic control substantially enhances formation of the α-linked oligosaccharide. This effect is much more pronounced when glycosyl trichloroacetimidates, rather than thioglycosides or glycosyl chlorides, are used as glycosyl donors.     

Regioselective monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by a polymer catalyst in N,N-dimethylformamide

6-O-Dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (6-sDode-AA-2G) was synthesized from 2-O-α-d-glucopyranosyl-l-ascorbic acid and lauric anhydride with a polymer catalyst, poly(4-vinylpyridine), in N,N-dimethylformamide without the introduction of protecting groups. The optimum reaction conditions enabled 6-sDode-AA-2G to be synthesized in a yield of 49.7%. The yield and the regioselectivity in this method were far superior to those in our previous method by using an enzyme. The polymer catalyst could be recycled more than five times without any significant activity loss.