O-acetyl Substituents Research Articles

Characterization and biological evaluation of Crataegus azarolus fruit polysaccharides

The polysaccharide obtained from Crataegus azarolus fruit, by water extraction followed by ethanol precipitation and fractionation using copper complex, shows physiological activities: prebiotic and antioxidant. The total acid hydrolysis shows that the polysaccharide consists of galactose, glucose, mannose and rhamnose. The employed physico-chemical techniques of the polysaccharide sequences approach (FT-IR, MALDI-TOF-MS spectrometry, 1H and 13C NMR) indicate that it is a Galactoglucomannan, the main chain consists of major amounts of β-(1→4)-linked glucose and mannose residues with monosaccharide branches of α-(1→6) galactose and O-acetyl substituents. The properties of this new glucomannan suggest that it could find an application as a prebiotic in the food industry.

Mass Spectrometric Analysis of Acidic Exo-Oligosaccharides of Root Nodule Bacterium Rhizobium leguminosarum bv. viciae VF39

The structures of exo-oligosaccharides produced by a symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae VF39 were determined using the methods of mass spectrometry and HCD fragmentation of ions. Exo-oligosaccharides were shown to be monomers, dimers and trimers of an octasaccharide repeating unit of the high-molecular-weight acidic exopolysaccharide. The major fraction was represented by octasaccharide monomers with two glucuronic acid residues at the reducing end. Heptasaccharides corresponding to an octasaccharide unit without GlcA-c or Glc-b residues in the main chain were also found in the culture liquid. Heterogeneity of exo-oligosaccharides according to the content of O-acetyl and hydroxybutanoyl substituents was revealed.

Distribution of the O-acetyl groups and β-galactofuranose units in galactoxylomannans of the opportunistic fungus Cryptococcus neoformans.

Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that β-galactofuranose (β-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.

Looking for new antiplasmodial quinazolines: DMAP-catalyzed synthesis of 4-benzyloxy- and 4-aryloxy-2-trichloromethylquinazolines and their in vitro evaluation toward Plasmodium falciparum

A DMAP catalyzed synthesis of new 4-benzyloxy- and 4-aryloxy-2-trichloromethylquinazolines was studied, in a view to react 4-chloroquinazolines with poorly nucleophilic alcohols such as benzylic alcohols, via a simple and cheap SNAr reaction approach. A fast (1 h) general operating procedure, affording good reaction yields, was achieved under microwave irradiation. Thus, a series of 35 molecules was obtained and evaluated in vitro on the K1 multi-resistant Plasmodium falciparum strain, in parallel with a cytotoxicity assessment on the human HepG2 cell line. 5 hit-molecules were identified, presenting both promising antiplasmodial activity (1.5 μM < IC50 < 2 μM) and low cytotoxicities (25 μM < CC50 < 45 μM). Apart for 2 molecules, the global series displayed a satisfying solubility in the aqueous biological media. Structure-activity relationships showed that the molecules presenting a benzyloxy moiety were less cytotoxic than the ones bearing a phenoxy moiety at position 4 of the quinazoline ring. It also appeared that the introduction of a heteroaryl moiety afforded inactive compounds. Finally, the most active and selective molecules (Selectivity Index = 22–27) were the ones presenting either an unsubstituted benzyloxy group or a phenoxy group, this last bearing a p-bromo or an o-acetyl substituent.

O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

O-acetylation of Arabidopsis hemicellulose xyloglucan requires AXY4 or AXY4L, proteins with a TBL and DUF231 domain.

In an Arabidopsis thaliana forward genetic screen aimed at identifying mutants with altered structures of their hemicellulose xyloglucan (axy mutants) using oligosaccharide mass profiling, two nonallelic mutants (axy4-1 and axy4-2) that have a 20 to 35% reduction in xyloglucan O-acetylation were identified. Mapping of the mutation in axy4-1 identified AXY4, a type II transmembrane protein with a Trichome Birefringence-Like domain and a domain of unknown function (DUF231). Loss of AXY4 transcript results in a complete lack of O-acetyl substituents on xyloglucan in several tissues, except seeds. Seed xyloglucan is instead O-acetylated by the paralog AXY4like, as demonstrated by the analysis of the corresponding T-DNA insertional lines. Wall fractionation analysis of axy4 knockout mutants indicated that only a fraction containing xyloglucan is non-O-acetylated. Hence, AXY4/AXY4L is required for the O-acetylation of xyloglucan, and we propose that these proteins represent xyloglucan-specific O-acetyltransferases, although their donor and acceptor substrates have yet to be identified. An Arabidopsis ecotype, Ty-0, has reduced xyloglucan O-acetylation due to mutations in AXY4, demonstrating that O-acetylation of xyloglucan does not impact the plant's fitness in its natural environment. The relationship of AXY4 with another previously identified group of Arabidopsis proteins involved in general wall O-acetylation, reduced wall acetylation, is discussed.

Structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl exclusion zone

Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC–FID, HPAEC–PAD), determination of glycosylation sites (GLC–EIMS) and 1D/2D NMR ( 1H, 13C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of d-galactose, d-glucose, d-mannose and d-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure: [Display omitted]

Agrobacterium rubiT DSM 6772 Produces a Lipophilic Polysaccharide Capsule whose Degree of Acetylation is Growth Modulated

The structure of the capsular polysaccharide produced from the type strain of Agrobacterium rubi DSM 6772 is demonstrated by means of chemical and spectroscopical methodologies. It is constituted from the quite rare monosaccharide 6-deoxy-L-talose, involved in alternating alpha-(1 --> 2) and alpha-(1 --> 3) linkages. This simple backbone is further complicated from the occurrence of O-acetyl substituents located always at O-2 of the O-3 substituted 6-deoxy-talose. This decoration is not stoichiometric and it depends on the growth stadium of the bacterium, leading to an almost regular acetylation pattern only at the stationary phase, where all the potential positions are substituted.

Structural differences of xylans affect their interaction with cellulose

The affinity of xylan to cellulose is an important aspect of many industrial processes, e.g. production of cellulose, paper making and bio-ethanol production. However, little is known about the adsorption of structurally different xylans to cellulose. Therefore, the adsorption of various xylans to bacterial cellulose (BC) was studied. Also, the relationship between xylan size and adsorption was analysed. BC was used as cellulosic material, because of its high specific surface area and homogeneous structure. In general, unsubstituted linear xylan parts favoured adsorption to BC. Xylan affinity for BC was also related to xylan size. The presence of arabinosyl and O-acetyl substituents to xylan decreased the adsorption of xylan to BC considerably. Removing substituents resulted in higher amounts of adsorbed material. Most likely, increasing the number of unsubstituted xylosyl residues induced the formation of xylan–xylan interactions, which contributed to adsorption to BC. Schematic models are proposed showing the adsorption of structurally different xylans to BC.

Structure and Activity of Two Metal Ion-dependent Acetylxylan Esterases Involved in Plant Cell Wall Degradation Reveals a Close Similarity to Peptidoglycan Deacetylases

The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a alpha/beta hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 A resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a beta-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co2+. Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" esterases involved in plant cell wall degradation are shown to be closely related to the de-N-acetylases involved in chitin and peptidoglycan degradation (Blair, D. E., Schuettelkopf, A. W., MacRae, J. I., and Aalten, D. M. (2005) Proc. Natl. Acad. Sci. U. S. A., 102, 15429-15434), which form the NodB deacetylase "superfamily."

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700 ± 1500 Da. The enzyme catalyses a β-elimination reaction of the disaccharide side chain β- d-Gal p-(1 → 2)-α- d-Rha p-(1→ from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.

NMR characterization of endogenously O-acetylated oligosaccharides isolated from tomato ( Lycopersicon esculentum) xyloglucan

Eight oligosaccharide subunits, generated by endoglucanase treatment of the plant polysaccharide xyloglucan isolated from the culture filtrate of suspension-cultured tomato ( Lycopersicon esculentum) cells, were structurally characterized by NMR spectroscopy. These oligosaccharides, which contain up to three endogenous O-acetyl substituents, consist of a cellotetraose core with α- d-Xyl p residues at O-6 of the two β- d-Glc p residues at the non-reducing end of the core. Some of the α- d-Xyl p residues themselves bear either an α- l-Ara p or a β- d-Gal p residue at O-2. O-Acetyl substituents are located at O-6 of the unbranched (internal) β- d-Glc p residue, O-6 of the terminal β- d-Gal p residue, and/or at O-5 of the terminal α- l-Ara p residue. Structural assignments were facilitated by long-range scalar coupling interactions observed in the high-resolution gCOSY spectra of the oligosaccharides. The presence of five-bond scalar coupling constants in the gCOSY spectra provides a direct method of assigning O-acetylation sites, which may prove generally useful in the analysis of O-acylated glycans. Spectral assignment of these endogenously O-acetylated oligosaccharides makes it possible to deduce correlations between their structural features and the chemical shifts of diagnostic resonances in their NMR spectra.

Structural characterisation of hemicellulosic polysaccharides from Benincasa hispida using specific enzyme hydrolysis, ion exchange chromatography and MALDI-TOF mass spectroscopy

Benincasa hispida fruit is an important source of water-soluble polysaccharides. Hemicellulosic polysaccharides extracted with 1- and 4-M KOH represented 120 mg/g of alcohol insoluble residues. The intrinsic viscosity of the extracted polymers (named B1OH and B4OH fractions) was 284 and 262 ml/g, respectively. The extracted polymers, which were further characterised by size exclusion chromatography, give one broad peak. The apparent molecular weight of the isolated polysaccharide was estimated to be around 130±40 kDa. Sugar composition analysis of both fractions demonstrates the presence of xyloglucan (XG) and xylan. Structural characterisation of hemicellulosic polysaccharides using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectroscopy showed that B. hispida fruit XG is of XXXG type and contained XXXG, XXLG, XXFG and XLFG (named according to Fry et al. 1993) in the ratio of 31:28:41 as the major oligomeric subunits. This fucogalactoxyloglucan carries O-acetyl substituents exclusively on galactose-containing oligosaccharides subunits. Finally, enzyme hydrolysis together with HPAEC and MALDI-TOF-mass spectrometric analysis of the generated fragments shows that the xylans, also present in both fractions, exhibit a classical structure with a backbone of β-(1→4)-linked xylopyranosyl residues substituted with three 4- O-methyl glucuronic acid per 97 xylopyranosyl unit.

Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood

A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)- 1H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC–NMR was performed in order to study the structural features of the acetylated oligomers ‘on-line’ avoiding O-acetyl migration. Finally, the precise location of the 2- O- or 3- O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.

Use and validation of NMR assays for the identity and O-acetyl content of capsular polysaccharides from Neisseria meningitidis used in vaccine manufacture

We describe a validated NMR (nuclear magnetic resonance) spectroscopic assay for the identity of the capsular polysaccharides (CPSs) from Neisseria meningitidis Groups A, C, W135 and Y used in vaccine manufacture, and to determine the proportion of residues carrying an O-acetyl substituent. Proof of structural identity and quantitation of the O-acetyl content are key control parameters for these vaccines. The meningococcal CPSs have variable levels of O-acetylation, present at multiple sites in the repeat unit, leading to complex NMR spectra. Base-catalysed de- O-acetylation of the Groups A, C, W135 and Y CPSs yields simplified and reproducible spectra suitable for comparison with reference data. The degree of O-acetylation of the original CPS can be determined by integration of the acetate anion resonance and a suitable resonance from the saccharide moiety. The assay was validated using 46 independent samples from five manufacturers, and is shown to be robust and reproducible.

Characterization of the phosphocholine-substituted oligosaccharide in lipopolysaccharides of type b Haemophilus influenzae.

Haemophilus influenzae expresses heterogeneous populations of short-chain lipopolysaccharide (LPS) which exhibit extensive antigenic diversity among multiple oligosaccharide epitopes. These LPS oligosaccharide epitopes can carry phosphocholine (PCho) substituents, the expression of which is subject to high frequency phase variation mediated by genes in the lic1 genetic locus. The location and site of attachment of PCho substituents were determined by structural analysis of LPS from two type b H. influenzae strains, Eagan and RM7004. The lic2 locus is involved in phase variation of oligosaccharide expression. LPS obtained from the parent strains, from mutants generated by insertion of antibiotic resistance cassettes in the lic2 genetic locus, and from phase-variants showing high levels of PCho expression was characterized by electrospray ionization-mass spectrometry (ESI-MS) and 1H NMR spectroscopy of derived O-deacylated samples. ESI-MS of O-deacylated LPS from wild-type strains revealed mixtures of related glycoform structures differing in the number of hexose residues. Analysis of LPS from PCho-expressing phase-variants revealed similar mixtures of glycoforms, each containing a single PCho substituent. O-Deacylated LPS preparations from the lic2 mutants were much less complex than their respective parent strains, consisting only of Hex3 and/or Hex2 glycoforms, were examined in detail by high-field NMR techniques. It was found that the LPS samples contain the phosphoethanolamine (PEtn) substituted inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1--> 3)-L-alpha-D-He pp-(1-->5)-alpha-Kdo in which the major glycoforms carry a beta-D-Glcp or beta-D-Glcp-(1-->4)-beta-D-Glcp at the O-4 position of the 3-substituted heptose (HepI) and a beta-D-Galp at the O-2 position of the terminal heptose (HepIII). LPS from the lic2 mutants of both type b strains were found to carry PCho groups at the O-6 position of the terminal beta-D-Galp residue attached to HepIII. In the parent strains, the central heptose (HepII) of the LPS inner-core element is also substituted by hexose containing oligosaccharides. The expression of the galabiose epitope in LPS of H. influenzae type b strains has previously been linked to genes comprising the lic2 locus. The present study provides definitive evidence for the role of lic2 genes in initiating chain extension from HepII. From the analysis of core oligosaccharide samples, LPS from the lic2 mutant strain of RM7004 was also found to carry O-acetyl substituents. Mono-, di-, and tri-O-acetylated LPS oligosaccharides were identified. The major O-acetylated glycoforms were found to be substituted at the O-3 position of HepIII. A di-O-acetylated species was characterized which was also substituted at the O-6 postion of the terminal beta-D-Glc in the Hex3 glycoform. This is the first report pointing to the occurrence of O-acetyl groups in the inner-core region of H. influenzae LPS. We have previously shown that in H. influenzae strain Rd, a capsule-deficient type d strain, PCho groups are expressed in a different molecular environment, being attached at the O-6 position of a beta-D-Glcp, which is in turn attached to HepI.

Phenolic antioxidants-effect of ortho-substituents

The effect of o-substituents, other than alkyls, of phenols was studied. In this case, an α-hydrogen of an o-substituent was found to be abstracted by a phenoxy radical, resulting in the regeneration of a new phenol to enhance the activities, especially n value, of a phenol. The substituent effect proposed so far was not observed on ortho position. New effects, however, are found; it can probably change the distribution of the electron of a phenoxy radical and an electron-withdrawing group on the α-carbon of the o-substituent can enhance the activity of a phenol, or an o-substituent such as acetyl deactivates a phenol by hydrogen bonding. In conclusion, an o-acetyl substituent lowered the activity of phenol ( n=0.7) compared with that of BHT, but an o-monochloroacetyl substituent having electron-withdrawing nature enhanced the activity ( n=3.6) considerably.

Re-evaluation of the serotypes of Serratia marcescens and separation into two schemes based on lipopolysaccharide (O) and capsular polysaccharide (K) antigens.

Chemical and serological analysis has revealed that many of the 29 O serotype reference strains of Serratia marcescens contain both neutral and acidic polysaccharides which correspond to LPS O antigens and capsular K antigens, respectively. New O and K antigen typing schemes have therefore been devised, based on the known chemical structures of the surface polysaccharides of the organism. These schemes were designed to allow the specific detection of these antigens on unknown strains using ELISAs. O antigens were detected using whole cells cultured in broth then autoclaved to remove capsular material, while K antigens were detected using formolized whole cells which had been cultured on glycerol agar to enhance capsule production. After testing with the 29 reference strains as well as 423 distinct clinical strains, it was apparent that different aspects of chemical structure were associated with different degrees of serological reactivity and the typing schemes were modified further to accommodate this. In general, the O antigen repeating unit structures were chemically simple with di- or trisaccharide backbones. Serological specificity was often provided solely by the presence or absence of an O-acetyl substituent, or a change in the linkage between two sugar residues. Five of the O serotypes in the new scheme were represented by 12 of the 29 reference strains, while three reference strains lacked O antigens altogether, resulting in the elimination of 10 of the original O types. In contrast, the K antigen repeating unit structures were more complex and chemically diverse, having at least four sugar residues. Three K types were each seen in two reference strains while 12 of the 29 reference strains were acapsular. Thus, the resulting schemes contain 19 O types and 14 K types and allow the definitive serotype identification of S. marcescens.

Identification of the common antigenic determinant shared by Streptococcus pneumoniae serotypes 35A and 20 capsular polysaccharides--structural analysis of the Streptococcus pneumoniae serotype 35A capsular polysaccharide.

The specific polysaccharide antigen of Streptococcus pneumoniae serotype 35A was shown, by a combination of one- and two-dimensional NMR methods and chemical analyses, to be a high-molecular-mass polymer composed of D-galactose, D-glucose, mannitol, and phosphate (3:1:1:1). The pentasaccharide repeating unit is polymerized through phosphate diester linkages to give the structure, [formula in text] O-Acetyl substituents are present at positions 5 and 6 of the 3)-beta-D-Galf residue and at position 2 of the 6)-beta-D-Galf residue. The capsular polysaccharides of S. pneumoniae serotypes 20 and 35A both contain the disaccharide unit -->3)-beta-D-Galf-(1-->3)-beta-D-Glcp-(1--> which is the probable structural determinant responsible for the serological cross reactivity of the two polysaccharides.

The structures of arabinoxyloglucans produced by solanaceous plants

Several structural features, most notably the presence of α-l-Ara f-(1 → 2)- α-d-Xyl p chains, distinguish the arabinoxyloglucans (AXGs) produced by solanaceous plants from the xyloglucans produced by other dicotyledonous plants. However, previous studies did not establish the exact order of attachment of the various side chains along the backbone of these AXGs. Therefore, oligosaccharide subunits of the AXGs secreted by suspension-cultured tobacco and tomato cells were generated by treatment of the isolated AXGs with a fungal endo- β-(1 → 4)-d-glucanase (EG). The oligosaccharides were reduced with sodium borohydride to the corresponding oligoglycosyl alditol derivatives and purified by a combination of gel-permeation chromatography, reversed-phase HPLC, and HPAE chromatography. The isolated oligoglycosyl alditols were chemically characterized by NMR spectroscopy, matrix-assisted laser-desorption/ionization time of-flight mass spectrometry (MALDITOFMS), fast-atom bombardment mass spectrometry (FABMS), FABMS/MS, and glycosyl-linkage analysis. The results confirmed that the AXGs from these species are composed of a (1 → 4)-linked β- d-Glc p backbone substituted at O-6 with various side chains. Both tobacco and tomato AXG contain α-d-Xyl p and α-l-Ara f-(1 → 2)- α-d-Xyl p side chains. However, oligosaccharide fragments of tomato AXG were also shown to contain β-d-Gal p-(1 → 2)- α-d-Xyl p and β-Ara f-(1 → 3)- α-l-Ara f-(1 → 2)- α-d-Xyl p side chains that are not present in the tobacco AXG. This is the first report of β-Ara f residues in a xyloglucan. The primary structures of 20 oligosaccharides generated by EG-treatment of tobacco AXG were determined. The generation of such a large number of oligosaccharides is due in part to the presence of O-acetyl substituents at O-6 of many of the backbone β-d-Glc p residues of tobacco AXG. The presence of either an O-acetyl or a glycosidic substituent at O-6 of β-d-Glc p residue in the AXG backbone protects the glycosidic bond of this residue from cleavage by the EG. Removal of the O-acetyl substituents prior to EG-treatment of the AXG results in oligosaccharide fragments that are smaller than those produced by EG-treatment of the O-acetylated AXG. Therefore, analysis of the complex mixture of oligosaccharides obtained by EG-treatment of native tobacco AXGs provides information regarding the distribution of AXG side chains that would be lost if the AXG is de- O-acetylated prior to EG-treatment. Furthermore, the large library of oligosaccharide fragments generated by this approach revealed additional correlations between the structural features of AXGs and diagnostic chemical shift effects in their 1H NMR spectra.