Structural analysis of underivatized neutral human milk oligosaccharides in the negative ion mode by nano-electrospray MS(n) (part 2: application to isomeric mixtures)

A complex mixture of isomeric neutral oligosaccharides from pooled human milk was analyzed by nano-electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer (QIT-MS) in the negative ion mode. Since deprotonated molecules of neutral oligosaccharides follow distinct fragmentation rules, which have been elucidated by using model compounds (see [1]), spectra obtained from consecutive CID experiments allowed the differentiation of isomers out of this highly complex mixture. With this method new human milk oligosaccharides of previously unknown isomeric structures have been identified, e.g., the occurence of three isomeric fucosylated lacto-N-hexaoses could be determined precisely, which have not been described before: (1) Fuc (alpha1-->2) Gal (beta1-->3) GlcNac (beta1-->3) Gal (beta1-->4) GlcNac (beta1-->3) Gal (beta1-->4) Glc, (2) Gal (beta1-->4) GlcNAc [(alpha1-->3) Fuc] (beta1-->3) Gal (beta1-->4) GlcNac (beta1-->3) Gal (beta1-->4) Glc, (3) Gal (beta1-->4) GlcNAc (beta1-->3) Gal (beta1-->4) GlcNac [(alpha1-->3) Fuc] (beta1-->3) Gal (beta1-->4) Glc.     

Differentiation of type 1 and type 2 chain linkages of native glycosphingolipids by positive-ion fast-atom bombardment mass spectrometry with collision-induced dissociation and linked scanning.

Two underivatized glycosphingolipids, Le(b) and Le(y), isomeric in carbohydrate structure (Fuc alpha 1-->2Gal beta 1--> 3[Fuc alpha 1-->4]GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer and Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3Gal beta 1--> 4Glc beta 1-->1Cer, respectively), were analyzed by positive-ion fast-atom bombardment (FAB) mass spectrometry with high energy collision-induced dissociation (CID) and linked scanning. The two isomers were distinguishable by the abundance of product ions derived from the non-reducing terminal tetrasaccharide fragment via sequential beta-eliminations of vicinally linked saccharide residues. Following earlier studies from other laboratories, which have dealt primarily with positive-ion FAB-CID mass spectrometry of simple model oligosaccharides, these results exemplify the practical application of two-sector methodology to underivatized complex glycoconjugates commonly encountered in the biomedical field.     

Facile method for the preparation of lyso-GM1 and lyso-GM2

This paper reports a facile method for the preparation of lyso-GM1 [Gal beta1-->3GalNAc beta1--> 4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-sphingosine] and lyso-GM2 [GalNAc beta1-->4(Neu5Ac alpha2-->3)Gal beta1-->4Glc beta1-->sphingosine], respectively, from GM1 [Galbeta1-->3GalNAc beta1-->4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-Cer] and GM2[GalNAc beta1-->4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-Cer], using sphingolipid ceramide deacylase and high performance anion-exchange chromatography (HPAEC). The enzymatically released lyso-GM1 and/or lyso-GM2 was effectively separated from its parent ganglioside by HPAEC using a Mono Q HR 5/5 column with an Amersham Biosciences fast protein liquid chromatography system. The yield was almost quantitative and the separation completed in approximately 3 h. This method is more convenient and effective than the conventional method using alkaline hydrolysis and silicic acid chromatography to generate and purify lyso-gangliosides.     

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.     

Triterpene glycosides from the seeds of Astragalus sinicus L.

From the seeds of Astragalus sinicus L. (Leguminosae), seven triterpene glycosides were isolated and identified as soyasaponin I-III methyl esters (1-3) which were treated with CH2N2 during the separation procedure, soyasaponin IV (4), soyasapogenol B 3-O-beta-D-glucuronopyranoside (5), 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-xylopyranosyl(1-->2)-beta-D- glucuronopyranosyl 3 beta, 22 beta, 24-trihydroxy-11-oxoolean-12-ene (6), whose sapogenol (8) was obtained by enzymatic hydrolysis using glycyrrhizinic acid hydrolase, unambiguously characterized and designated as complogenin, and 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D - glucuronopyranosyl complogenin (7).     

Triterpenoid saponins from the roots of Sophora koreensis

From the roots of Sophora koreensis (Fabaceae), three new oleanene-type triterpene glycosides, echinosophorosides A(1) (1) and B (2), and acetyl-subproside II (5), were isolated as their methyl esters, together with the four known ones sophoraflavoside I, kudzusaponin SA(3), subproside II, and azukisaponin V. The structures of the new saponins were elucidated to be 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl kudzusapogenol A 22-O-alpha-L-arabinopyranoside (1), 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucuronopyranosyl abrisapogenol C 22-O-alpha-L-arabinopyranoside (2), and 3-O-alpha-L-rhamnopyranosyl(1-->2)-alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl kudzusapogenol A 22-O-acetate (5), respectively. It is noteworthy that two arabinopyranosyl moieties in the same molecule, echinosophoroside B (2), have different conformations. The conformation of the arabinopyranosyl moiety existing in the trisaccharide moiety was determined to be (1)C(4), whereas that of the arabinopyranosyl unit at C-22 was identified as (4)C(1).     

Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports

Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.     

A highly stereoselective construction of beta-glycosyl linkages by reductive cleavage of cyclic sugar ortho esters

The preparation of beta-glycosides by the reductive cleavage of spiro sugar ortho esters is described in this report. This procedure is based on a concept completely different from those of other methods for glycosylation. Twelve sugar ortho esters that commonly possess perhydrospiro[2H-pyran-2,2'-pyrano[3,2-d][1,3]dioxin] ring systems in their molecules were reduced by LiAlH(4)/AlCl(3) or NaCNBH(3)/AlCl(3). Among these ortho esters, those (9a-12a) prepared from the D-sugar lactones (1-4) and 2, 3-di-O-benzyl-alpha-D-glucopyranoside (7) or those (19a, 20a) prepared from the L-sugar lactones (5, 6) and 2, 3-di-O-benzyl-alpha-D-galactopyranoside (8) were selectively converted into beta-(1 --> 4)-glycosides (9b-12b or 19b, 20b) in excellent yields by the treatment of LiAlH(4)/AlCl(3). In contrast, the ortho esters (13a-16a or 17a, 18a) that were prepared from combinations of the D-sugar lactones and 8 or those of the L-sugar lactones and 7 were efficiently reduced with NaBH(3)CN/AlCl(3) to afford beta-(1 --> 6)-glycosides (13b-16b or 17b, 18b) selectively. It was remarkable that the resulting disaccharides were obtained with extremely high beta-selectivity even in the cases with mannosyl or rhamnosyl glycosides. Moreover, these products would be useful units for the construction of branched saccharides, because the newly formed hydroxy groups could be again glycosylated without further deprotection procedures. The high regio- and stereoselectivity was totally explained by considering the structures and the conformations of these ortho ester molecules and the stereoelectoronic effects of their spiro ring systems. In addition, the preparation of the sugar ortho esters with glucosamine derivatives and the reactivity of these ortho esters are described in this report. N-Phthaloyl glucosamine derivatives (21, 22) were efficiently reacted with the benzyl-protected gluconolactone (1) in the presence of TMSOMe and TMSOTf to afford ortho esters (23a-c). After the conversion of the phthalimido functionality to the dibenzyl amino group, glucosylideneglucosamine (25) was reduced with LiAlH(4)/AlCl(3) to afford beta-(1 --> 4)-glycoside (26) selectively.     

Two new glycosides from the soft coral Sinularia firma

Two new glycosides, named Firmacosides A and B, together with known fatty esters, batyl alcohol, Delta(5,20) sterol and sphingosine derivatives have been isolated from the soft coral Sinularia firma TIX-DUR. On the basis of spectroscopic analysis ((1)H-NMR, (13)C-NMR, (1)H-(1)H COSY, HMQC, HMBC and FAB-MS), Firmacoside A was established as hexadecanyl-1-O-alpha-D-arabinopyranosyloxy (1-->4)-alpha-D-arabinopyranosyloxy (1-->4)-alpha-D-arabinopyranoside (1), and firmacoside B was elucidated as docosanyl-1-O-alpha-D-arabinopyranosyloxy (1-->4)-3-O-acetyl-alpha-arabinopyranosyloxy (1-->4)-alpha-D-arabinopyranoside (2).     

Synthesis, Antigenicity Against Human Sera and Structure-Activity Relationships of Carbohydrate Moieties from <em>Toxocara</em> larvae and Their Analogues

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galβ1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meβ1-3GalNAcα1-OR (A), Fucα1-2Gal4Meβ1-3GalNAcα1-OR (B), Fuc2Meα1-2Galβ1-3GalNAcα1-OR (C), Fucα1-2Galβ1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meβ1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1?3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.     

Iron-catalyzed esterification of allylic sp3 C–H bonds with carboxylic acids: Facile access to allylic esters

The first general and efficient iron-catalyzed esterification of allylic sp³ C–H bonds with carboxylic acids using ionic iron(III) complexes (1–4) as a catalyst and DTBP (DTBP = di-tert-butyl peroxide) as an oxidant is achieved. A variety of allylic esters were synthesized in good to excellent yields using the ionic iron(III) complex 2 as a catalyst in a 5 mol% loading. This reaction is characterized by its high efficiency, broad substrate scope with excellent steric hindrance tolerance and good functional group compatibility.     

UCHP Method for Oligosaccharide Combinatorial Library Synthesis

A new methodology for oligosaccharide combinatorial library synthesis using a special hydroxy protecting group, the uni-chemo hydroxy protection (UCHP) group, was developed. The UCHP group was composed of oligomeric amino acid derivatives. The amino terminals of UCHP groups were protected by either Boc or Fmoc groups. By using these two types of UCHP, five kinds of trigalactoses [Galβ1-3Galβ1-3Gal, Galβ1-3(Galβ1-4)Gal, Galβ1-4Galβ1-3Gal, Galβ1-3Galβ1-4Gal, and Galβ1-4Galβ1-4Gal] were successfully synthesized on a solid support as a model of oligosaccharide combinatorial library. Each step of all reactions was also successfully monitored using a combination of two colorimetric tests, chloranil and methyl red-DIC.     

Cholestane glycosides from Solanum abutiloides. III

Four new cholestane glycosides, named abutilosides D (1), E (2), F (3) and G (4), were isolated from the fresh roots of Solanum abutiloides. By chemical and spectroscopic evidence, their structures were elucidated as 26-O-beta-D-glucopyranosyl 3beta,16alpha,26-trihydroxy-5alpha-cholestan-22-one 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-rhamnopyranosyl-(1-->4)-beta-D- glucopyranoside and its desxylosyl compound (1 and 3, respectively), 26-O-beta-D-glucopyranosyl 3beta,16alpha,26-trihydroxycholest-5-en-22-one 3-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-rhamnopyranosyl-(1-->4)-beta-D- glucopyranoside and its desxylosyl compound (2 and 4, respectively). These compounds were regarded as precursors of a dormantinone type compound on a hypothetical pathway of steroid biogenesis.     

Studies related to carba-pyranoses: a radical decarboxylation approach to monocarba-disaccharides

(1--> 1), (1--> 3) and (1--> 4) acetal-linked monocarba-disaccharides have been synthesised from a series of glucosylated gamma- and delta-lactonic acids prepared from common intermediate, obtained from the Diels-Alder reaction of maleic anhydride and (E)-1-(2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyloxy)-3-(trimethylsiloxy)buta-1,3-diene 1. Thiohydroxamic ester 14, prepared from gamma-lactonic acid 9, gave, upon treatment with tert-butyl thiol and light, the lactone 15. Subsequent lithium aluminium hydride reduction and acetylation gave the (1--> 3) acetal-linked monocarbadisaccharides 1,6-di-O-acetyl-3-O-(2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyl)-2,4-dideoxy-5a-carba-beta-L-threo-hexopyranose 16. In a similar manner, protected monocarba-disaccharides 13, 19, 30, and 35 possessing L-ido, L-xylo, D-arabino and L-ido configurations of the carba-pyranose ring have been prepared. Treatment of thiohydroxamic esters 14 and 17 with either tert-butyl thiol or trityl thiol, dimethyl sulfide, oxygen and light gave alcohols 20 and 22. Subsequent lithium aluminium hydride reduction and aceytlation gave the monocarbadisaccharides 1,4,6-tri-O-acetyl-3-O-[2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyl]-2-deoxy-5a-carba-beta-L-arabino-hexopyranose 21 and 1,2,4,6-tetra-O-acetyl-3-O-(2',3',4',6'-tetra-O-acetyl-beta-D-glucopyranosyl)-5a-carba-beta-L-glucopyranose 23 respectively.     

A general method for the synthesis of enantiopure aliphatic chain alcohols with established absolute configurations. Part 2, via catalytic reduction of acetylene alcohol MαNP esters

A general method for synthesizing enantiopure (100% ee) aliphatic alcohols with established absolute configurations has been developed and applied to alcohols CH₃(CH₂)_n–CH(OH)–(CH₂)_mCH₃, the enantiomeric discrimination of which is the most difficult, if m = n + 1 and n is large. Racemic saturated alcohols with short chains could be directly enantioresolved as (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid) esters by HPLC on silica gel, and their absolute configurations were simultaneously determined by ¹H NMR diamagnetic anisotropy. However, the application of this powerful MαNP ester method to alcohols with long chains was difficult, because of smaller values of the separation factor α. In such cases, the use of the corresponding acetylene alcohol MαNP esters was crucial. Acetylene alcohol MαNP esters were largely separated by HPLC on silica gel, and their absolute configurations were unambiguously determined by ¹H NMR as reported in the Part 1 paper. The MαNP esters obtained with established absolute configurations were catalytically hydrogenated to yield saturated alcohol MαNP esters. It was evidenced that no racemization occurred at the stereogenic center of the alcohol moiety during catalytic hydrogenation, by the coinjection of MαNP esters in HPLC. From the MαNP esters obtained, enantiopure (100% ee) aliphatic chain alcohols with established absolute configurations were recovered. Although the [α]_D values of these alcohols were too small for the identification of the enantiomers, it was clarified that the analytical HPLC of MαNP esters is useful for identification in most cases.     

沙棘果碱提水溶多糖JS1的结构研究

沙棘（Hippophae rhamnoides L.）为胡颓子科（Elaeagnaceae）酸刺属的灌木或小乔木,它含有丰富的活性物质,具有明显的医用功效;同时已被大量用于水土保持和改善生态环境等方面,对其多糖结构的研究国内外尚未见报道。本文对沙棘果碱提水溶多糖JS1的结构进行了研究。     

Structural analysis of the N-linked oligosaccharides from human urinary trypsin inhibitor.

The N-linked oligosaccharides from human urinary trypsin inhibitor were purified and their structures were investigated by compositional analysis, the two-dimensional sugar map method and 500 MHz 1H-NMR. The results revealed that they were composed of disialosyl, monosialosyl and asialosyl oligosaccharides, which have the common biantennary core structure; Gal1-4GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)M an1-4GlcNAc1-4GlcNAc.     

Medicinal foodstuffs. XXII. Structures of oleanane-type triterpene oligoglycosides, pisumsaponins I and II, and kaurane-type diterpene oligoglycosides, pisumosides A and B, from green peas, the immature seeds of Pisum sativum L

Two new oleanane-type triterpene oligoglycosides, pisumsaponins I and II, and two new kaurane-type diterpene oligoglycosides, pisumosides A and B, were isolated from the immature seeds (green peas) of Pisum sativum L. together with soyasaponin I, bersimoside I, dehydrosoyasaponin I, and their 6'-methyl esters. The structures of pisumsaponins and pisumosides were determined on the basis of chemical and physicochemical evidence as 22-O-malonylsoyasapogenol B 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucopyranosiduronic acid (22-O-malonylsoyasaponin I), sandosapogenol 3-O-alpha-L-rhamnopyranosyl(1-->2)-beta-D-galactopyranosyl(1-->2)-beta-D-glucopyranosiduronic acid, 17-O-beta-D-glucopyranosyl-6beta,7beta,13gamma,17-tetrahydroxy-19-kauranoic acid 19-O-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranoside, and 6beta,7beta,13beta,17-tetrahydroxy-19-kauranoic acid 19-O-beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranoside, respectively.     

Effects of caffeine on stereoselectivities of high cell density biotransformations of cyclic beta-keto esters with Saccharomyces cerevisiae

Caffeine affects the stereoselectivity of microbial high cell density reductions with commercial grade Saccharomyces cerevisiae (Baker's yeast). Cyclic beta-keto esters ethyl 2-oxocyclopentanoate (1) and ethyl 2-oxocyclohexanoate (3) were shown to be reduced with increased diastereoselectivity (1: 90.1 --> 92.1% de, 3: 75.0 --> 90.0% de) after addition of caffeine. Effects on enantioselectivity were less pronounced (1: 97.3 --> 98.5% ee, 3: 90.1 --> 92.1% ee). The observations are ascribed to the action of caffeine on cellular calcium homeostasis. These effects are accompanied by caffeine-induced cell-death, which preferably takes effect on pre-stressed cells which were found to decrease diastereoselectivity.     

A general method for the synthesis of enantiopure aliphatic chain alcohols with established absolute configurations. Part 1. Application of the MαNP acid method to acetylene alcohols

A method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid 1 (MαNP acid) has been applied to acetylene alcohols 4–14 to determine their absolute configurations by ¹H NMR anisotropy and/or X-ray crystallography. Diastereomeric MαNP esters prepared from racemic acetylene alcohols and (S)-(+)-MαNP acid 1 were easily separable by HPLC on silica gel. From the ¹H NMR anisotropy Δδ data of separated diastereomeric MαNP esters {Δδ = δ (R,X) − δ(S,X) = δ(2nd fr.) − δ(1st fr.)}, the absolute configurations of the first eluted esters were determined. This MαNP acid method has been successfully applied to various acetylene alcohols 4–12 and 14. In the case of MαNP esters 21b, 24a, and 26a, their absolute configurations were unambiguously determined by X-ray crystallography, which confirmed the absolute configuration assignments performed by ¹H NMR anisotropy. These acetylene alcohol MαNP esters can serve as key intermediates for the synthesis of enantiopure aliphatic chain alcohols with established absolute configurations as described in Part 2 of this series.