Glycosidic Cleavage Research Articles

Electron Transfer Dissociation of Milk Oligosaccharides

For structural identification of glycans, the classic collision-induced dissociation (CID) spectra are dominated by product ions that derived from glycosidic cleavages, which provide only sequence information. The peaks from cross-ring fragmentation are often absent or have very low abundances in such spectra. Electron transfer dissociation (ETD) is being applied to structural identification of carbohydrates for the first time, and results in some new and detailed information for glycan structural studies. A series of linear milk sugars was analyzed by a variety of fragmentation techniques such as MS/MS by CID and ETD, and MS(3) by sequential CID/CID, CID/ETD, and ETD/CID. In CID spectra, the detected peaks were mainly generated via glycosidic cleavages. By comparison, ETD generated various types of abundant cross-ring cleavage ions. These complementary cross-ring cleavages clarified the different linkage types and branching patterns of the representative milk sugar samples. The utilization of different MS(3) techniques made it possible to verify initial assignments and to detect the presence of multiple components in isobaric peaks. Fragment ion structures and pathways could be proposed to facilitate the interpretation of carbohydrate ETD spectra, and the main mechanisms were investigated. ETD should contribute substantially to confident structural analysis of a wide variety of oligosaccharides.

Kinetics and mechanism of 2,3,5,4′-tetrahydroxystilbene-2-O-β- d-glycoside (THSG) degradation in aqueous solutions

The hydrolytic kinetics and degradation mechanism of 2,3,5,4′-tetrahydroxystilbene-2-O-β- d-glycoside (THSG) extracted from Radix Polygoni Multiflori (a commonly used officinal Chinese herbal Heshouwu), were investigated using reversed-phase high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS). The influences of pH (1.5–9.9), temperature (25–60 °C) and irradiation on the hydrolysis of THSG were studied in aqueous solutions. The results showed that the degradation of THSG was pH-, temperature- and irradiation-dependent and all followed first-order kinetics. The effect of temperature on the rate of THSG degradation was characterized using the Arrhenius equation. Maximum stability of THSG was found at pH 1.5 ( t 0.5 = 47.57 d). THSG was unstable in alkaline and irradiation conditions. The active energy ( E a) of THSG degradation in aqueous solution at pH 6.8 (most frequently adopted extract solvent) under lucifugal and irradiation conditions was 47.7 kJ mol −1 and 25.3 kJ mol −1, respectively. Three hydrolytic products of THSG were identified by LC–MS. Cis– trans isomerism took place under irradiation, and hydrolysis took place in acid–base conditions. Moreover, further oxidation on aglycon occurred after hydrolytic cleavage of phenolic glycoside in acidic conditions. The possible hydrolytic pathways of THSG are proposed.

ChemInform Abstract: Reduction of Daunomycin in Dimethyl Sulfoxide. Long-Lived Semiquinones and Quinone Methide and Formation of an Enolate at the 14-Position via the Quinone Methide.

Anaerobic reduction of daunomycin (1, daunorubicin) in 5%/95% H 2 O/DMSO (DMSO=dimethyl sulfoxide) or in DMSO with sodium dithionite or bi(3,5,5-trimethyl-2-oxomorpholin-3-yl) (TM-3 dimer), respectively, yields 7-deoxydaunomycinone (7) and a mixture of the diastereomers of bi(7-deoxydaunomycinon-7-yl) (8). A precursor to both 7 and 8 is 7-deoxydaunomycinone quinone methide (4) formed from glycosidic cleavage of daunomycin hydroquinone (3). The hydroquinone 3 is estabished as a precursor to the quinone methide 4 from relative rates

OH-radical induced degradation of hydroxybenzoic- and hydroxycinnamic acids and formation of aromatic products—A gamma radiolysis study

The OH-radical induced degradation of hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCiA) and methoxylated derivatives, as well as of chlorogenic acid and rosmarinic acid was studied by gamma radiolysis in aerated aqueous solutions. Primary aromatic products resulting from an OH-radical attachment to the ring (hydroxylation), to the position occupied by the methoxyl group (replacement –OCH 3 by −OH) as well as to the propenoic acid side chain of the cinnamic acids (benzaldehyde formations) were analysed by HPLC–UV and LC–ESI–MS. A comparison of the extent of these processes is given for 3,4-dihydroxybenzoic acid, vanillic acid, isovanillic acid, syringic acid, cinnamic acid, 4-hydroxycinnamic acid, caffeic acid, ferulic acid, isoferulic acid, chlorogenic acid, and rosmarinic acid. For all cinnamic acids and derivatives benzaldehydes were significant oxidation products. With the release of caffeic acid from chlorogenic acid the cleavage of a phenolic glycoside could be demonstrated. Reaction mechanisms are discussed.

ChemInform Abstract: Synthesis of 2-Substituted Polyhydroxytetrahydropyrimidines (N-Hydroxy Cyclic Guanidino-Sugars): Transition-State Mimics of Enzymatic Glycosidic Cleavage.

The synthesis of 2'-substituted polyhydroxytetrahydropyrimidines as transition-state mimics of enzymatic glycosidic cleavage has been achieved by using guanylation and cyclization methodologies. The D-galacto type N-hydroxy cyclic guanidino-sugar 21 was synthesized in six steps from amine 7 and thiourea 14 in an overall yield of 59%. To further derivatize compound 21 to incorporate the leaving group moiety, we have synthesized 2-methylsulfanyl compounds 26-29 as key intermediates. The 2-methylsulfanyl group in 29 was displaced with amines, assisted by silver tetrafluoroborate as Lewis acid, to give protected cyclic guanidines 30-32 in moderate yields (60-67%). Removal of the protecting groups in 32 gave the D-galacto-type N-hydroxy cyclic guanidino-sugar 34. The key steps in the synthesis of the 6-deoxy-DL-galacto type N-hydroxy cyclic guanidino-sugars 49, 54, and 64-66 involve cyclization of the appropriate acetal intermediates (45, 50, and 58-60) followed by removal of the protecting groups.

Identification of the Unpaired Cysteine Status and Complete Mapping of the 17 Disulfides of Recombinant Tissue Plasminogen Activator Using LC−MS with Electron Transfer Dissociation/Collision Induced Dissociation

Recombinant tissue plasminogen (rt-PA) with 35 cysteine residues has been completely assigned by mapping the 17 disulfide linkages and the unpaired cysteine. The result is consistent with the prediction from homology except for the unassigned cysteine, which was identified at Cys83. This cysteine was found to be blocked and paired with either a glutathione or cysteine residue in an approximately 60:40 ratio, respectively. The analysis was conducted using a multifragmentation approach consisting of electron transfer dissociation (ETD) and collision induced dissociation (CID), in combination with a multienzyme digestion strategy (Lys-C, trypsin, and Glu-C). The disulfide-linked peptides, even those containing N- or O-linked glycosylation, could be assigned since the disulfide bonds were still preferably cleaved over the glycosidic cleavages under ETD fragmentation. The use of a multiple and sequential enzymatic digestion strategy was important in producing fragment sizes suitable for analysis. For the analysis of complex intertwined disulfides, the use of CID-MS(3) to target partially disulfide-dissociated peptides from the ETD fragmentation was necessary for linkage assignment. The ability to identify the exact location and status of the unpaired cysteine (free or blocked with a glutathione or cysteine) could shed light on the activation of rt-PA, upon stimulation by either oxidative or ischemic stress.

Negative electron transfer dissociation of glycosaminoglycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even- and odd-electron products. The loss of SO(3) can be minimized and an increase in cross-ring cleavages is observed if a negatively charged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.

Disposition of [1′-14C]Stavudine after Oral Administration to Humans

The disposition of stavudine, a potent and orally active nucleoside reverse transcriptase inhibitor, was investigated in six healthy human subjects. Before dosing humans with [1'-(14)C]stavudine, a tissue distribution study was performed in Long-Evans rats. Results from this study showed no accumulation of radioactivity in any of the tissues studied, indicating that the position of the (14)C-label on the molecule was appropriate for the human study. After a single 80-mg (100 microCi) oral dose of [1'-(14)C]stavudine, approximately 95% of the radioactive dose was excreted in urine with an elimination half-life of 2.35 h. Fecal excretion was limited, accounting for only 3% of the dose. Unchanged stavudine was the major drug-related component in plasma (61% of area under the plasma concentration-time curve from time zero extrapolated to infinite time of the total plasma radioactivity) and urine (67% of dose). The remaining radioactivity was associated with minor metabolites, including mono- and bis-oxidized stavudine, glucuronide conjugates of stavudine and its oxidized metabolite, and an N-acetylcysteine (NAC) conjugate of the ribose (M4) after glycosidic cleavage. Formation of metabolite M4 was shown in human liver microsomes incubated with 2',3'-didehydrodideoxyribose, the sugar base of stavudine, in the presence of NAC. In addition, after similar microsomal incubations fortified with GSH, two GSH conjugates, 3'-GS-deoxyribose and 1'-keto-2',3'-dideoxy-3'-GS-ribose, were observed. This suggests that 2',3'-didehydrodideoxyribose underwent cytochrome P450-mediated oxidation leading to an epoxide intermediate, 2',3'-ribose epoxide, followed by GSH addition. In conclusion, absorption and elimination of stavudine were rapid and complete after oral dosing, with urinary excretion of unchanged drug as the predominant route of elimination in humans.

Isolation of inulin‐type oligosaccharides from Chinese traditional medicine: Morinda officinalis How and their characterization using ESI‐MS/MS

Inulin-type oligosaccharides with different DP were prepared by size-exclusion chromatography and purity of each oligosaccharide was determined by HPLC equipped with cyclodextrin-bond column. The purities of obtained inulin-type oligosaccharides with different DP were more than 98% by one-step process. The DP and molecular weight were obtained through ESI-MS in negative mode. The characterization of the inulin-type oligosaccharides with different DP was studied by MS/MS spectra obtained by collision-induced dissociation of molecular ions ([M-H](-)). When the DP was lower, the fragment ions were formed through cross-ring cleavages of two bonds within the sugar ring and glycosidic cleavages. However, with the increase of DP, the ions resulting from glycosidic cleavages between two sugar residues were predominant.

Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose and d-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.

Complete amino acid sequence of three reptile lysozymes

To study the structure and function of reptile lysozymes, we have reported their purification, and in this study we have established the amino acid sequence of three egg white lysozymes in soft-shelled turtle eggs (SSTL A and SSTL B from Trionyx sinensis, ASTL from Amyda cartilaginea) by using the rapid peptide mapping method. The established amino acid sequence of SSTL A, SSTL B, and ASTL showed substitutions of 43, 42, and 44 residues respectively when compared with the HEWL (hen egg white lysozyme) sequence. In these reptile lysozymes, SSTL A had one substitution compared with SSTL B (Gly126Asp) and had an N-terminal extra Gly and 11 substitutions compared with ASTL. SSTL B had an N-terminal extra Gly and 10 residues different from ASTL. The sequence of SSTL B was identical to soft-shelled turtle lysozyme from STL ( Trionyx sinensis japonicus). The Ile residue at position 93 of ASTL is the first report in all C-type lysozymes. Furthermore, amino acid substitutions (Phe34His, Arg45Tyr, Thr47Arg, and Arg114Tyr) were also found at subsites E and F when compared with HEWL. The time course using N-acetylglucosamine pentamer as a substrate exhibited a reduction of the rate constant of glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution for amino acids mentioned above for substrate binding at subsites E and F. Interestingly, the variable binding free energy values occurred on ASTL, may be contributed from substitutions at outside of subsites E and F.

The role of potassium carbonate on cellulose pyrolysis

Cellulose pyrolysis with/without potassium carbonate (K 2CO 3) under relatively high heating rate was carried out to elucidate the role of alkali metals on wood pyrolysis. Gaseous species produced by wood pyrolysis were determined by TCD and FID gas chromatography. Tar was analyzed to quantify polycyclic aromatic hydrocarbons (PAHs) by GC/MS. Changes to the chemical bonds in the solid residue (char) were analyzed by 13C NMR and FTIR spectroscopies. With the addition of potassium carbonate, an increase in gaseous yield and a decrease in tar yield and a significant decrease in the initiation temperature of pyrolysis were observed. The saccharide structure in cellulose was preserved in the structure of char obtained from pyrolysis at 500 °C when potassium was not present. However, with the presence of potassium carbonate, the structure was dramatically changed. The simultaneous occurrence between the CO 2 release and the glycosidic linkage cleavage detected by NMR spectrum leads to a comprehensive understanding of the role of potassium in cellulose pyrolysis. A model which explains the mechanism of how the alkali addition enhances the biomass pyrolysis was developed based on the observed experimental results.

Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans

Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.

Isolation and tandem mass fragmentations of an anti-inflammatory compound from Aralia elata

One-step isolation of a saponin from Aralia elata was undertaken using high-speed countercurrent chromatography coupled with evaporative light scattering detection. A triterpenoid saponin, elatoside F, was purified with 96.8% purity using a two-phase-system comprising chloroform-methanol-water-isopropanol. The yield was 35.0 mg from 348.2 mg of the enriched saponin fraction. In vitro anti-inflammatory study demonstrated that elatoside F inhibited lipopolysaccharide-induced nitric oxide production, as well as nuclear factor kappaB activation, in a dose-dependent manner. Two types of mass ionization technique were compared on elatoside F to investigate characteristic fragmentation patterns. MALDI-TOF tandem mass spectrometric fragmentation patterns of sodiated ions provided structural information on glycosidic cleavages and on extensive cross-ring cleavages. Electrospray ionization multiple-stage tandem mass fragmentation of both sodiated and lithiated ions could provide information on glycosidic cleavages. All observed tandem mass fragmentation spectra provided valuable elatoside F structural information when unknown samples from crude extracts are under screening by mass spectrometry.

Thermal degradation of sucralose and its potential in generating chloropropanols in the presence of glycerol

Sucralose, a polychlorinated synthetic high-intensity sweetener is being increasingly used in relatively large amounts in baked products under high temperature environments. This necessitates understanding its thermal decomposition and studying the consequences of hydrogen chloride release from sucralose and its ability to chlorinate various food related ingredients such as glycerol to generate chloropropanols a potentially toxic class of compounds. Studies conducted on the thermal degradation of sucralose under dry heating conditions have indicated that the glycosidic cleavage occurs through the formation of a galactopyranosyl cation contrary to sucrose where fructofuranosyl cation dictates the major breakdown products. Consequently the major product detected was levoglucosenone and its precursor. Subsequent degradation of the two monosaccharide derivatives causes dehydrochlorination and dehydration reactions to produce furan-related products. In addition, pyrolysis of sucralose in the presence of glycerol generated significant amounts of 3-monochloropropanediol and 1,2- and 1,3-dichloropropanols based on the relative intensities of their chromatographic peaks which amounted to 15% of the total chromatographic peak area.

Novel Pseudopterosin Analog Elicits New Modality for Use in Wound Repair

The pseudopterosins are known to be anti‐inflammatory agents (Look, et. al. 1986, PNAS, 83, 6238‐6240), eliciting a reduction in neutrophil infiltration in PMA induced mouse ears and more recently, mediators in acceleration of wound healing. A new pseudopterosin semi‐synthetic analog has been developed which resists glycoside cleavage in the cell via a C‐glycoside linkage. This new compound has been used for the study of cell proliferation effects in HUVEC‐C cell lines. Binding studies indicate that this compound binds to adenosine A2a and A3 receptors with an IC50/hill coefficient of 20 μM/1.2 and 10 μM/2.3 respectively in overexpressed HEK‐293 cells (Zhong, et. al. 2008, J.Org.Chem., 73, 7011‐7016). Although an understanding of the direct molecular mechanism of action has been unknown to this point we will report a tentative theory to explain pseudopterosin effects on wound repair. Modulation of inflammation and cell division/angiogenesis can be important factors in accelerating the wound healing response. Data suggests that Pseudopterosin‐A c‐glycoside elicits a 35% increase in cell proliferation with an ED50 of 3.7 μM over 48 hours in treated HUVEC‐C cell lines. We believe that this effect is mediated by modulation of an adenosine receptor which may act to stimulate the expression and release of growth factors, key to cell division (Ethier et al. 1993, Amer. J. of Phys., 265, H131‐H138). An increase in growth factor expression and release can be potentially important in treatment of chronic wounds since high bacterial and protease loads in wound site act to increase the breakdown of endogenous growth factors. This data suggests that pseudopterosin mediated wound repair may follow two routes; a reduction in inflammatory response, and an increase in cell proliferation. We gratefully acknowledge the U.S. Army Medical Research Program (Grant No. W81XWH‐06‐1‐0089) for its support of this research.

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

Systematic sulfation: Sulfated glycoconjugates are degraded either by desulfation followed by glycoside cleavage, or by glycoside cleavage followed by desulfation. To study these processes, here we report the synthesis of four regioisomerically sulfated p-nitrophenyl glucosaminides from the common precursor p-nitrophenyl N-acetyl-beta-D-glucosaminide. These substrates allowed the rapid analysis of the substrate preferences of a set of four sulfatases and 24 hexosaminidases.Sulfated carbohydrates are components of many glycoconjugates, and are degraded by two major processes: cleavage of the sulfate ester by a sulfatase, or en bloc removal of a sulfated monosaccharide by a glycoside hydrolase. However, these processes have proved difficult to study owing to a lack of homogeneous, defined substrates. We describe here the synthesis of a series of p-nitrophenyl beta-D-glucosaminides bearing sulfate esters at the 2-, 3-, 4- or 6-positions, by divergent routes starting with p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The sulfated p-nitrophenyl beta-D-glucosaminides were used to study the substrate specificity of four sulfatases (from Helix pomatia, Patella vulgata, abalone, and Pseudomonas aeruginosa), and revealed significant differences in the preference of each of these enzymes for desulfation at different positions around the sugar ring. The 3-, 4- and 6-sulfated p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucosaminides were screened against a panel of 24 fungal beta-N-acetylhexosaminidases to assess their substrate specificity. While the 4- and 6-sulfates were substrates for many of the fungal enzymes investigated, only a single beta-N-acetylhexosaminidase, that from Penicillium chrysogenum, could hydrolyze the 3-sulfated p-nitrophenyl glycoside. Together these results demonstrate the utility of sulfated p-nitrophenyl beta-D-glucosaminides for the study of both sulfatases and glycoside hydrolases.

Identification of new trace triterpenoid saponins from the roots of Panax notoginseng by high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Triterpenoid saponins are the major bioactive constituents of Panax notoginseng. In the study reported here, the fragmentation behavior of triterpenoid saponins from P. notoginseng was investigated by electrospray ionization tandem mass spectrometry (ESI-MS(n))and high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/ESI-MS(n)). Analyses revealed that product ions from glycosidic and cross-ring cleavages can give a wealth of structural information regarding the nature of the aglycone, sugar types, the sequence and linkage information of sugar units. It is noted that different glycosylation positions remarkably influenced the fragmentation behaviors, which could assist in the differentiation of saponin analogues. To rationalize this characteristic, the collision energy required for various glycosidic cleavages was investigated. According to the summarized fragmentation rules, identification of triterpenoid saponins from the roots of P. notoginseng could be fulfilled, even when reference standards were unavailable. Furthermore, minor and trace constituents were enriched and detected by eliminating the major constituents in one of the saponin fractions. As a result, a total of 151 saponins, including 56 new trace ones, were identified or tentatively characterized from saponin fractions based on their retention times, HPLC/HRMS, HPLC/ESI-MS(n) fragmentation behaviors and comparison with literature data.

Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1

Family 18 chitinases hydrolyze chitin through a substrate-assisted catalytic mechanism and are to a variable extent able to catalyze transglycosylation reactions. Previously Aspergillus fumigatus AfChiB1 was found to be able to catalyze transglycosylation reactions. Structural analysis reveals that AfChiB1 consists of an eight-stranded beta/alpha-barrel. Like other members of the family 18 hydrolases, AfChiB1 has conserved substrate binding site and catalytic acid, while a suitable nucleophile is missing. In this study, Trp137, Asp246, and Met243, which are close to the glycosidic cleavage site, were mutated to glutamate individually. As a result, the W137E remained its hydrolytic activity and was completely devoid of transglycosyl activity, while the D246E reduced its chitinolytic activity and increased its transglycosyl activity. And the M243E showed a remarkable reduction of chitinolytic activity and complete loss of transglycosyl activity. These results suggested that the transglycosyl reaction catalyzed by the AfChiB1 is due to lacking of nucleophile.

Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2‐mers to 16‐mers. Comparison with hyaluronic acid oligomers

This study reports for the first time the complete liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses performed in negative ion mode of saturated unsulfated chondroitin oligosaccharides up to 16-mers and comparison with hyaluronic acid (HA) oligomers differing only in the nature of the hexosamine residue. MS/MS of the chondroitin disaccharide on the singly charged precursor at m/z 396.1 afforded a glycosidic cleavage C1 product ion at m/z 192.9. In the tetrasaccharide, C2 (m/z 396.0) and C3 (m/z 572.0) product anions were generated by glycosidic cleavage. A C5 [M-2H]2- product ion at m/z 475.1 was generated by the glycosidic cleavage of the hexasaccharide, and a C7 ion (m/z 664.6, charge state of -2) was produced from the octasaccharide. The same fragmentation pattern of deprotonated oligomers was observed for the largest oligosaccharides, from 10- to 16-mers. There has been no previous report of MS/MS spectra for unsulfated chondroitin oligomers of these sizes. Unsulfated saturated chondroitin oligosaccharides with x-mer units and larger than a tetrasaccharide dissociate to almost exclusively form CX-1-type ions. Saturated HA oligomers also afforded the same fragmentation pattern as deprotonated oligomers by ESI-MS and MS/MS analyses. Thus, under the experimental conditions used in the current study, we were unable to distinguish between unsulfated chondroitin and HA.