Synthesis of two repeat units corresponding to the backbone of the pectic polysaccharide rhamnogalacturonan I

A tetrasaccharide corresponding to a sequence of the rhamnogalacturonan I backbone has been synthesized. This synthesis relies on only two protected monosaccharides and proceeds through a common disaccharide intermediate. Synthesis of this tetrasaccharide has been designed to allow for the addition of branching elements at the 4-positions of the rhamnosyl units, or further chain elongation at the 2-position.     

Structural characterization of red wine rhamnogalacturonan II

The pectic polysaccharide rhamnogalacturonan II (RG-II), which accounts for ˜ 20% of the ethanol-precipitable polysaccharides in red wine, has been isolated from wine polysaccharides by anion-exchange chromatography. Four fractions enriched with RG-II were obtained and the RG-II then purified to homogeneity by Concanavalin A affinity and size-exclusion chromatographies. The glycosyl-residue compositions of the four RG-IIs are similar; all the RG-IIs contain the monosaccharides (apiose, , , Kdo, Dha, and aceric acid) that are diagnostic of RG-II. The glycosyl-linkages of the neutral and acidic sugars, including aceric acid, were determined simultaneously by GC-EIMS analysis of the methylated alditol acetates generated from per-O-methylated and carboxyl-reduced RG-II. Two of the RG-IIs contain boron, most likely as a borate di-ester that cross-links two molecules of RG-II together to form a dimer. The dimer contains 3′- and 2,3,3′-linked apiosyl residues whereas the monomer contains only 3′-linked apiosyl residues which suggests that the borate di-ester is located on at least one of the apiosyl residues of RG-II. Although the wine RG-IIs all have similar structures they are not identical since they differ in the length and degree of methyl-esterification of the RG-II backbone and in the presence or absence of borate di-esters. Nevertheless, these studies show that the major structural features of wine and primary cell wall RG-II are conserved.     

The pectic polysaccharide rhamnogalacturonan II is present as a dimer in pectic populations of bilberries and black currants in muro and in juice

Rhamnogalacturonan II (RG II) can play an important role during processing of berries due to its enzyme resistance and its possible role as a pectic cross-linker. This article describes the presence of RG II in cell walls, in juice and in press cake of bilberries and black currants. RG II was identified and quantified via its diagnostic sugar residues. RG II, which was released from homogalacturonan, was probably present in its dimeric form in muro. Juice contained the free RG II dimer, while from press cake dimeric RG II was released by enzymatic degradation of homogalacturonan. A higher amount of RG II was present in juice than in press cake. During juice processing a cross-linker RG II might improve gel formation, which hinders the processability of berries. In addition, enzymes used during juice processing release dimeric RG II from pectin molecules and accumulate RG II in the juice.     

Synthesis of an -quinovose-containing disaccharide

The synthesis of a versatile -rhamnose monosaccharide synthon is described. This synthon is used in the synthesis of a disaccharide containing the rare sugar, 6-deoxy- -glucose, linked to the 3-C-hydroxymethyl group of methyl 2,3-O-isopropylidene 3-C-(hydroxymethyl)-β- -erythrofuranoside.     

Synthesis of fluorescence-labelled disaccharide substrates of glucosidase II

The fluorescence-labelled disaccharides Glcalpha(1-->3)GlcalphaOR and Glcalpha(1-->3)ManalphaOR, both substrates for the glycoprotein-processing enzyme glucosidase II, were synthesised via the use of a n-pentenyl-derived linker at the anomeric position. This allowed incorporation of a pyrenebutyric acid label, via a sequence of oxidative hydroboration, mesylation, azide displacement, reduction with concomitant global deprotection, and peptide coupling. Selective activation of a fully armed thioglycoside in the presence of n-pentenyl glycosides was readily achieved by the use of methyl triflate as promoter.     

Presence of rhamnogalacturonan II in the juices produced by enzymatic liquefaction of Agave pulquero stem (Agave mapisaga)

Rhamnogalacturonan II (RG-II), a small complex pectic polysaccharide is released from Agave pulquero stems (Agave mapisaga), after the production period of aguamiel. RG-II was obtained by treatment with two commercial liquefying enzyme preparations, it was isolated by size-exclusion chromatography and characterized. RG-IIs contains diagnostic sugars such as apiose, 2-O-methyl-l-fucose, 2-O-methyl-d-xylose, aceric acid, Kdo and Dha. Glycosyl-linkage compositions of the Agave pulquero RG-II like structures are similar to the theoretical model described through sycamore RG-II structure. The presence of 3′-linked apiose indicates that the obtained juice from Agave pulquero plant contains the free RG-II dimer. Thus, when pectinolytic enzyme preparations are used to process Agave pulquero (A. mapisaga), RG-II is released as one of the main soluble polysaccharide fraction.     

Synthesis of a spacer-containing disaccharide fragment of Bordetella pertussis lipopolysaccharide

The disaccharide 2-(p-aminophenyl)ethyl 4-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-2,3-diacetamido-2 ,3-dideoxy-alpha-D-mannopyranoside uronate, which is assumed to be a partial structure of the Bordetella pertussis polysaccharide, was synthesized starting from D-glucose and D-glucosamine, respectively. The major synthetic transformations were conversion of D-glucosamine into the donor ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-beta-D-glucopyranoside and conversion of glucose, by a sequence involving 2,3-epoxide formation/opening, nucleophilic triflate displacement in the 3-position, and necessary protecting group manipulations, into the acceptor 2-(p-trifluoroacetamidophenyl)ethyl 6-O-benzyl-2,3-diazido-2,3-dideoxy-alpha-D-mannopyranoside. Coupling of the donor and acceptor units promoted by dimethyl(methylthio)sulfonium triflate followed by selective oxidation of the 6'-position and deprotection gave the target disaccharide.     

Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.     

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly.

Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II     

Synthesis of a C-linked hyaluronic acid disaccharide mimetic

The synthesis of a C-disaccharide that is designed as a mimetic for the repeating unit disaccharide of hyaluronic acid is described. The target compound was obtained via the SmI2-promoted coupling reaction of the sulfone, 2-acetamido-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1,2-dideoxy-1-pyridinylsulfonyl-beta-D-glucopyranose (6), and the aldehyde, p-methoxyphenyl 2,3-di-O-benzyl-4-deoxy-4-C-formyl-6-O-p-methoxybenzyl-beta-D-glucopyranoside (14).     

Indirect approach to C-3 branched 1,2-cis-glycofuranosides: synthesis of aceric acid glycoside analogues

Aceric acid (3-C-carboxy-5-deoxy-alpha-l-xylofuranose) residues are present in pectic polysaccharide rhamnogalacturonan II (RG II) in the form of synthetically challenging 1,2-cis-glycofuranosides. To access synthetic fragments of RG II incorporating aceric acid, a four-step procedure based on C-2 epimerisation of initially prepared 1,2-trans-glycofuranoside was developed. Readily available derivatives of branched-chain l-lyxofuranose bearing a 3-C-vinyl group as a masked 3-C-carboxyl group were investigated as potential precursors of aceric acid units. In the first step of the procedure, installation of a participating group at C-2 of the furanose ring ensured stereocontrol of the O-glycosylation, which was carried out with the thioglycoside of 2-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-L-lyxofuranose. After the glycosylation step, the 2-O-acetyl group was removed, the free 2-OH group was oxidised and the resulting ketone was finally reduced to form the C-3-vinyl-L-xylofuranoside. The use of L-Selectride in the key reduction reaction was essential to achieve the required stereoselectivity to generate 1,2-cis-furanoside.     

Synthesis of an apiose-containing disaccharide fragment of rhamnogalacturonan-II and some analogues

Beta-rhamnosylation of methyl 2-C-hydroxymethyl-2,3-O-isopropylidene-beta-D-erythrofuranoside and methyl 2,3-O-isopropylidene-beta-D-ribofuranoside was achieved using 4-O-acetyl-2,3-O-carbonyl-alpha-L-rhamnopyranosyl bromide and Ag2O as a promoter. Deprotected disaccharides beta-L-Rhap-(1-->3')-beta-D-Apif-OMe and beta-L-Rhap-(1-->3')-beta-D-Ribf-OMe were compared to their alpha-rhamnosyl isomers which were prepared using conventional Helferich glycosylation.     

Isolation and structural characterization of a novel oligosaccharide from the rhamnogalacturonan of Gossypium hirsutum L

An alkali extract of cell walls of Gossypium hirsutum L. was sequentially digested by endo-polygalacturonase (EC 3.2.1.15), arabinofuranosidase AN1571.2 (EC 3.2.1.55), endo-arabinase (EC 3.2.1.99), and rhamnogalacturonan hydrolase AN9314.2 (EC 3.2.1.15). The rhamnogalacturonan hydrolase-generated oligosaccharides were separated by ultrafiltration, size-exclusion, and anion exchange chromatography. Fractions from the anion exchange chromatography were pooled, lyophilized, and screened by MALDI-TOFMS. A new oligosaccharide (RGS29), which contained a rhamnogalacturonan dimer backbone with two galactose and two arabinose residues in the side chains, was found. Its structure was identified by 1D and 2D NMR spectra as follows: [FORMULA: SEE TEXT].     

Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.     

l-Galactose replaces l-fucose in the pectic polysaccharide rhamnogalacturonan II synthesized by the l-fucose-deficient mur1 Arabidopsis mutant

Arabidopsis thaliana mur1 is a dwarf mutant with altered cell-wall properties, in which l-fucose is partially replaced by l-galactose in the xyloglucan and glycoproteins. We found that the mur1 mutation also affects the primary structure of the pectic polysaccharide rhamnogalacturonan II (RG-II). In mur1 RG-II a non-reducing terminal 2-O-methyl l-galactosyl residue and a 3,4-linked l-galactosyl residue replace the non-reducing terminal 2-O-methyl l-fucosyl residue and the 3,4-linked l-fucosyl residue, respectively, that are present in wild-type RG-II. Furthermore, we found that a terminal non-reducing l-galactosyl residue, rather than the previously reported d-galactosyl residue, is present on the 2-O-methyl xylose-containing side chain of RG-II in both wild type and mur1 plants. Approximately 95% of the RG-II from wild type and mur1 plants is solubilized as a high-molecular-weight (>100 kDa) complex, by treating walls with aqueous potassium phosphate. The molecular mass of RG-II in this complex was reduced to 5–10 kDa by treatment with endopolygalacturonase, providing additional evidence that RG-II is covalently linked to homogalacturonan. The results of this study provide additional information on the structure of RG-II and the role of this pectic polysaccharide in the plant cell wall.Abbreviations AIR Alcohol-insoluble residue - d-Gal d-Galactosyl - EPG Endopolygalacturonase - ESI–MS Electrospray ionization mass spectrometry - GC–MS Gas chromatography–mass spectrometry - ¹H-NMR Proton nuclear magnetic resonance spectroscopy - l-Fuc l-Fucosyl - l-Gal l-Galactosyl - 2-O-MeFuc 2-O-Methyl l-fucosyl - 2-O-MeGal 2-O-Methyl l-galactosyl - 2-O-MeXyl 2-O-Methyl d-xylosyl - MWCO Molecular weight cut-off - RG-II Rhamnogalacturonan II - ppm Parts per million - RI Refractive index - SEC Size-exclusion chromatography - TFA Trifluoroacetic acid - WT Wild type     

The polysaccharides of red wine: total fractionation and characterization

Ethanol-precipitated red wine polysaccharides were fractionated by a combination of anion-exchange, size-exclusion and affinity chromatography steps. This comprehensive fractionation allowed us to prepare a collection of wine polysaccharides in sufficient amount to permit the determination of their intrinsic properties. Glycosyl-residue composition of each polysaccharide fraction was determined by GC–EI–MS of the per-O-trimethylsilylated methyl glycoside derivatives (TMS), a method that has been recently developed and adapted to suit simultaneous determination of neutral and acidic glycosyl-residue compositions of polysaccharides present in plant-derived products. The results showed that mannoproteins released by yeast during fermentation, and grape derived arabinogalactan-proteins, rhamnogalacturonans I and II are the main wine polysaccharides and accounted for 35, 42, 4 and 19%, respectively, of the total polysaccharides. Structural characterization revealed that rhamnogalacturonan I fractions were linked with xyloglucan-like polysaccharides. This finding represents compelling evidence of the existence of cross-linking between pectin and hemicellulose domains in plant primary cell walls.     

Synthesis of mono- and disaccharide analogs of moenomycin and lipid II for inhibition of transglycosylase activity of penicillin-binding protein 1b

Three types of mono- and disaccharides 3a,b, 4a–c, 5, and some chaetomellic acid A analogs 6 and 42–44 were synthesized as potential inhibitors of the transglycosylase activity of penicillin-binding protein 1b (PBP1b), a key bacterial enzyme responsible for the formation of the polysaccharide backbone of peptidoglycan as well as for cross-linking of its peptide portions. The target compounds combine structural features of both the active portion of moenomycin and the natural PBP1b substrate, lipid II. The desired skeletons were obtained in a convergent fashion involving attachment of the lipid-alkylated glyceric acid moieties 11a,b to the corresponding carbohydrate-containing phosphonic acids 23, 24a, and 24b. Compounds 3a,b were prepared to verify the distance requirements between the sugar and the noncleavable C-phosphonate moieties. Compounds 4a–c were synthesized to examine the importance of the first sugar unit of moenomycin, a known inhibitor of transglycosylase catalysis by PBP1b, with respect to antibiotic activity. These were prepared by condensation of 11a,b with 28a and 28c, which were made by glycosylation of 3-bromopropanol with oxazolines 25a,b, and Arbuzov reaction with triethyl or trimethyl phosphite, followed by dealkylation with bromotrimethylsilane. Compound 5 was generated to verify the possibility of using a dicarboxylate group to mimic the diphosphate of lipid II. It was synthesized by coupling of alcohol 31 with -trichloroacetimidate 34. Chaetomellic acid A analogs were prepared by a Michael addition to dimethyl acetylenedicarboxylate. With the exception of 3b, all of the target compounds were found to inhibit PBP1b, albeit with modest potency.     

Functional characterisation of a putative rhamnogalacturonan II specific xylosyltransferase

An Arabidopsis thaliana gene, At1g56550, was expressed in Pichia pastoris and the recombinant protein was shown to catalyse transfer of d-xylose from UDP-alpha-d-xylose onto methyl alpha-l-fucoside. The product formed was shown by 1D and 2D (1)H NMR spectroscopy to be Me alpha-d-Xyl-(1,3)-alpha-l-Fuc, which is identical to the proposed target structure in the A-chain of rhamnogalacturonan II. Chemically synthesized methyl l-fucosides derivatized by methyl groups on either the 2-, 3- or 4 position were tested as acceptor substrates but only methyl 4-O-methyl-alpha-l-fucopyranoside acted as an acceptor, although to a lesser extent than methyl alpha-l-fucoside. At1g56550 is suggested to encode a rhamnogalacturonan II specific xylosyltransferase.     

Synthesis and cytotoxic evaluation of novel pyrimidine deoxyapiothionucleosides

The synthesis of novel pyrimidine deoxyapiothionucleosides of d- and l-series was realized following application of a versatile and high-yielding scheme, which utilized inexpensive l- and d-arabinose as starting materials, respectively, and which makes use of a regio- and stereo-selective Pummerer rearrangement reaction for the coupling of the nucleobase with the thiosugar moiety. Some of the targeted compounds have shown selective cytotoxic effects (with IC₅₀ <10 μM) against specific cancer cell lines. All of the tested compounds had no cytotoxic effect on the normal cell line tested.