Preparative Anion-exchange Chromatography Research Articles

Alleviative effects of sulfated polysaccharide from Ishige Okamurae against DSS-induced ulcerative colitis via inhibiting inflammation and modulating gut microbiota

A water-soluble polysaccharide from the brown alga Ishige Okamurae, designated IOP-0, was obtained by preparative anion-exchange and size-exclusion chromatography. Chemical and spectroscopic investigations revealed that IOP-0 was a sulfated fucoidan with a backbone primarily composed of 3-linked and 4-linked-L-fucose with sulfate groups at C-2/C-4 of the 3-linked-L-fucose. The protective effect of IOP-0 on ulcerative colitis was evaluated in this work. The results showed that IOP-0 could significantly alleviate the symptoms of ulcerative colitis by preventing weight loss, preserving the structure of intestinal tissues, and ameliorating the dysregulation of inflammatory cytokines (TNF-α, IL-6, and IL-10). Meanwhile, IOP-0 protected the colonic mucosal barrier by promoting the tight junction protein ZO-1 and occludin expression. In addition, IOP-0 was able to maintain intestinal homeostasis and improve intestinal function by regulating the gut microbiota and their metabolites, such as short-chain fatty acids (SCFAs). These results suggest that IOP-0 might be a potential dietary supplement for the prevention and treatment of ulcerative colitis.

Effect of plasmid DNA isoforms on preparative anion exchange chromatography.

Increased need for plasmid DNA (pDNA) with sizes above 10kbp (large pDNA) in gene therapy and vaccination brings the need for its large-scale production with high purity. Chromatographic purification of large pDNA is often challenging due to low process yields and column clogging, especially using anion-exchanging columns. The goal of our investigation was to evaluate the mass balance and pDNA isoform composition at column outlet for plasmids of different sizes in combination with weak anion exchange (AEX) monolith columns of varying channel size (2, 3 and 6µm channel size). We have proven that open circular pDNA (OC pDNA) isoform is an important driver of reduced chromatographic performance in AEX chromatography. The main reason for the behaviour is the entrapment of OC pDNA in chromatographic supports with smaller channel sizes. Entrapment of individual isoforms was characterised for porous beads and convective monolithic columns. Convective entrapment of OC pDNA isoform was confirmed on both types of stationary phases. Porous beads in addition showed a reduced recovery of supercoiled pDNA (on an 11.6kbp plasmid) caused by diffusional entrapment within the porous structure. Use of convective AEX monoliths or membranes with channel diameter >3.5µm has been shown to increase yields and prevent irreversible pressure build-up and column clogging during purification of plasmids at least up to 16kbp in size.

Antidiabetic-activity sulfated polysaccharide from Chaetomorpha linum: Characteristics of its structure and effects on oxidative stress and mitochondrial function

A water-soluble polysaccharide from the green alga Chaetomorpha linum, designated CHS2, was obtained by water extraction, preparative anion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that CHS2 was a sulfated rhamnogalactoarabinan, and its backbone was mainly constituted by 4-linked and 3,4-linked β-l-arabinopyranose with sulfate groups at C-2/C-3 of 4-linked β-l-arabinopyranose. The branching contained 4-linked, 6-linked β-d-galactopyranose and terminal rhamnose residues. Based on the inhibition of human islet amyloid polypeptide (hIAPP) aggregation and morphology change of hIAPP aggregates in in vitro tests, it was proved that CHS2 effectively inhibited the hIAPP aggregation and possessed strong antidiabetic activity. CHS2 was nearly no toxicity in NIT-1 cells and could attenuate hIAPP-induced cytotoxicity. CHS2 may significantly reduce the generation of intracellular reactive oxygen species and hIAPP aggregation-induced oxidative stress in NIT-1 cells. CHS2 was co-localized with mitochondria, and largely protected mitochondria function from hIAPP aggregation-induced damage through stabilizing mitochondrial membrane potential and enhancing the mitochondrial complex I, II or III activity and ATP level. The data demonstrated that CHS2 could have potential prospect to become an antidiabetic drug for type 2 diabetes mellitus treatment.

Polysaccharides from Basidiocarps of the Polypore Fungus Ganoderma resinaceum: Isolation and Structure.

In this study, we focused on the isolation and structural characterization of polysaccharides from a basidiocarp of polypore fungus Ganoderma resinaceum. Polysaccharide fractions were obtained by successive extractions with cold water at room temperature (20 °C), hot water under reflux (100 °C), and a solution of 1 mol L−1 sodium hydroxide. The purity of all fractions was controlled mainly by Fourier transform infrared (FTIR) spectroscopy, and their composition and structure were characterized by organic elemental analysis; neutral sugar and methylation analyses by gas chromatography equipped with flame ionization detector (GC/FID) and mass spectrometry detector (GC/MS), respectively; and by correlation nuclear magnetic resonance (NMR) spectroscopy. The aqueous extracts contained two main polysaccharides identified as a branched O-2-β-d-mannosyl-(1→6)-α-d-galactan and a highly branched (1→3)(1→4)(1→6)-β-d-glucan. Mannogalactan predominated in the cold water extract, and β-d-glucan was the main product of the hot water extract. The hot water soluble fraction was further separated by preparative anion exchange chromatography into three sub-fractions; two of them were identified as branched β-d-glucans with a structure similar to the corresponding polysaccharide of the original fraction. The alkaline extract contained a linear (1→3)-α-d-glucan and a weakly branched (1→3)-β-d-glucan having terminal β-d-glucosyl residues attached to O-6 of the backbone. The insoluble part after all extractions was identified as a polysaccharide complex containing chitin and β-d-glucans.

A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity

A homogeneous polysaccharide was obtained from Monostroma angicava Kjellm by water extraction, preparative anion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that the polysaccharide was a glucuronic acid-containing rhamnan-type sulfated polysaccharide. The backbone mainly consisted of →3)-α-l-Rhap-(1→ and →2)-α-l-Rhap-(1→ residues, partially sulfated at C-2 of →3)-α-l-Rhap-(1→ and C-3/C-4 of →2)-α-l-Rhap-(1→. The branching contained unsulfated or monosulfated 3-linked, 2-linked, 4-linked α-l-rhamnose and terminal β-d-glucuronic acid residues. The polysaccharide had strong antidiabetic activity assessed by glucose consumption, total cholesterol and triglyceride levels using human hepatocellular carcinoma (HepG2) and insulin-resistant HepG2 cells. The polysaccharide exhibited high anticoagulant property by activated partial thromboplastin time and thrombin time assays, and possessed high fibrin(ogen)olytic activity evaluated by plasminogen activator inhibitior-1, fibrin(ogen) degradation products and D-dimer levels using rats plasma. The investigation demonstrated that the polysaccharide from Monostroma angicava Kjellm was a novel sulfated rhamnan and could be a potential antidiabetic and anticoagulant polysaccharide.

Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius.

A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

Enzymatically derived aldouronic acids from Cryptomeria japonica arabinoglucuronoxylan

Abstract An arabinoglucuronoxylan was extracted from the holocellulose of sugi ( Cryptomeria japonica ) wood with 10% KOH and subjected to hydrolysis by partially purified xylanase fraction from a commercial cellulase preparation “Meicelase”. Neutral sugars liberated were analyzed by size exclusion chromatography showing the presence of xylooligosaccharides up to xylohexaose. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were identified by monosaccharide analysis, comparison of their volume distribution coefficients ( Dv s) with those of the authentic samples in anion exchange chromatography and 1 H and 13 C NMR spectroscopy, resulting in the characterization of eight aldouronic acids including acids consisting of two 4- O -Me-α-D-GlcA p residues and 3–5 D-Xyl residues. 1. Fr. 1-S1: (aldohexaouronic acid, MeGlcA 3 Xyl 5 ), O -β-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )-(1 → 2)]- O -β-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 2. Fr. 1-S2: (aldopentaouronic acid, MeGlcA 3 Xyl 4 ), O -β-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )-(1 → 2)]- O -β-D-Xyl p -(1 → 4)- O -β-Xyl p -(1 → 4)-D-Xyl 3. Fr. 2-S1: (aldotetraouronic acid, MeGlcA 3 Xyl 3 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 4. Fr. 3-S1: (aldotetraouronic acid, GlcA 3 Xyl 3 ), O -(α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-Xyl p -(1 → 4)-D-Xyl, 5. Fr. 4-S1: (aldotriouronic acid, GlcA 2 Xyl 2 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)-D-Xyl 6. Fr. 4-S2: (MeGlc 4 MeGlcA 3 Xyl 5 ), O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)-[ O -(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 7. Fr. 6-S1: (MeGlcA 4 MeGlcA 3 Xyl 4 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -[(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -β-D-Xyl p -(1 → 4)-D-Xyl 8. Fr. 7-S1: (MeGlcA 3 MeGlc 2 Xyl 3 ), O -(4- O -Me-α-D-GlcA p )-(1 → 2)- O -β-D-Xyl p -(1 → 4)- O -[(4- O -Me-α-D-GlcA p )]-(1 → 2)- O -β-D-Xyl p -(1 → 4)-D-Xyl Fr. 4-S2 was a new acidic oligosaccharide. The distribution pattern of these vicinal uronic acid units along the D-xylan chain was discussed.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Enzymatically derived aldouronic acids from Eucalyptus globulus glucuronoxylan

A glucuronoxylan was extracted from the holocellulose of Eucalyputus globulus wood with 10% KOH and subjected to hydrolysis by a commercial cellulase preparation “Meicelase”. Neutral xylooligosaccharides liberated were analyzed by size exclusion chromatography. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were studied by monosaccharide analysis, comparison of volume distribution coefficients ( Dvs) in anion exchange chromatography with those of the authentic samples, and 1H and 13C NMR spectroscopy, resulting in the characterization of seven aldouronic acids including a novel one containing galactose residue. O-β- d-Xyl p-(1 → 4)-[ O-(4- O-Me-α- d-GlcA p)-(1 → 2)]- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- d-Xyl O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1→3)- O-α- l-Rha p-(1 → 2)- O-α- l-GalA p-(1 → 4)- d-Xyl O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 3)- O-α- l-Rha p-(1 → 2)- d-GalA O-β- d-Xyl p-(1 → 3)- O-α- l-Rha p-(1 → 2)- O-α- d-GalA p-(1 → 4)- d-Xyl O-β- d-Gal p-(1 → 2)- O-(4- O-Me-α- d-GlcA p)-(1 → 2)- O-β- d-Xyl p-(1 → 4)- O-β- d-Xyl p-(1 → 4)- d-Xyl. The oligosaccharides liberated provide information on multiplicity of xylanases secreted by Trichoderma viride. The presence of the last aldouronic acid shows a structural feature of E. globulus xylan.

Structures of aldouronic acids liberated from kenaf xylan by endoxylanases from Streptomyces sp.

Streptomyces sp. SK519 was cultivated in the medium containing 0.5% birch wood xylan to produce extracellular xylanase. The xylanase was isolated from the culture supernatant by ammonium sulfate precipitation and partially purified by absorbing on the insoluble xylan. Alkali-extracted kenaf glucuronoylan of which main chain primarily consisting of (1-4)-linked β- D-xylopyranose (Xyl p), some of which carry α-(1-2)-linked 4- O-methylglucopyranosyl acid (MeGlcA p) and glucopyranosyluronic acid (GlcA p) as side chain was digested with the xylanase preparation. At the final stage of digestion, main neutral oligosaccharides were xylotriose (Xyl 3) and xylobiose (Xyl 2) followed by xylotetraose (Xyl 4). Xylose was only formed in a trace amount. As acidic oligosaccharides liberated, following seven aldopentao- and aldotetraouronic acids were isolated in a pure state, respectively, by preparative anion-exchange chromatography. Their structures were determined by analysis of constitutional sugar residues and 1H and 13C NMR spectroscopy. Xyl-β-1,4-[MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl; [MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl-β-1,4-Xyl; Xyl-β-1,4-[GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl [GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl-β-1,4-Xyl [MeGlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-D-Xyl [GlcA-α-1,2-]Xyl-β-1,4-Xyl-β-1,4-Xyl Xyl-β-1,4-Xyl-β-1,3-Rha-α-1,2-GalA-α-1,4-Xyl

Structural features of acetylated galactomannans from green Coffea arabica beans

Polysaccharides were extracted from green Coffea arabica beans with water (90 °C, 1 h). Galactomannans were isolated from the water extract using preparative anion-exchange chromatography. Almost all of the galactomannans eluted in two neutral populations, while almost all of the arabinogalactans bound to the column, indicating that these arabinogalactans contain charged groups. Analysis of the molecular weight distribution of the two neutral populations showed that they differ in their molecular weight. Further characterization of these neutral populations by NMR and by MALDI-TOF MS after enzymatic degradation with an endo-mannanase, showed the presence of acetyl groups linked to the galactomannans, a feature not previously described for this type of polysaccharides from coffee beans. It was found that the high molecular weight (ca. 2000 kDa) neutral fraction was highly substituted both with galactose residues and acetyl groups, while the low molecular weight (ca. 20 kDa) population was much less substituted. Based on these results it can be concluded that at least two distinctly different populations of galactomannans are present in green coffee beans. It was also shown that the degradation of the galactomannans from green coffee beans with an endo-mannanase from A. niger is hindered by the presence of acetyl groups.

Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4.

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.

Purification and properties of phenylalanyl aminopeptidase synthesised by Pseudomonas sp.

Intracellular aminopeptidase synthesized by a soil strain of Pseudomonas sp. was purified 323-fold using the following procedure: saturation with ammonium sulfate, separation by preparative electrophoresis, anion-exchange chromatography and gel filtration chromatography. Molecular weight of the enzyme determined according to the latter method was 57 kDa. Aminopeptidase showed a high substrate specificity and affinity to Phe-beta-naphtylamide (Phe-beta-NA) as a substrate. A considerable inhibition of the enzymatic activity by iodoacetamide and p-chloromercuribenzoate (p-CMB) led to the conclusion that it was a cysteine aminopeptidase. Hydrosulphide compounds markedly stabilised the enzyme. Ethylenediaminetetra-acetic acid (EDTA), a metalloenzyme inhibitor, caused a double increase in the phenylalanyl aminopeptidase activity.( )Mg(2+) ions activated the enzyme to a negligible extent, whereas Co(2+), Cu(2+), Cd(2+) and Pb(2+) ions contributed to its inhibition. The highest enzymatic activity was observed at 37 degrees C and pH 7.0.

Offline Coupling of Low-Pressure Anion-Exchange Chromatography with MALDI-MS to Determine the Elution Order of Human Milk Oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution “fingerprint” of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.

Influence of column design on process-scale ion-exchange chromatography

The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using Whatman Express-Ion Exchanger Q. A 16 l Side-Pack column and a 24 l IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 l feedstock containing 5–7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5 M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.

Autohydrolysis of Phytic Acid

The autohydrolysis of phytic acid at 120°C resulted in the formation of most of the phosphate esters of myo-inositol in varying amounts depending upon the reaction time. Eighteen of the 39 chromatographically distinct myo-inositol mono-, bis-, tris-, tetrakis-, pentakis-, and hexakisphosphates have been characterized using two different HPLC systems. These myo-inositol phosphates were partially purified by preparative anion-exchange chromatography under acidic and alkaline elution conditions. The combination of these two methods provides a two-tiered chromatographic approach to the rapid and sensitive identification of inositol phosphates in complex mixtures. Identification of the products was confirmed by 1D and 2D 1H NMR analysis. The analytical procedure was applied to the autohydrolysis of the mixture of inositol phosphates from corn steep water.

Isolation and characterization by NMR spectroscopy of three monosubstituted 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin)

The substitution profile of 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) (SBE-β-CD) prepared in our laboratories has been previously described. However, in those studies, no attempt was made to characterize the positional or regional isomers of this material. SBE-β-CD derivatives with degrees of substitution of two or higher represent a large number of possible isomers dependent on this positional and regional substitution. The monosubstituted SBE derivative, however, cannot have regional isomers and, therefore, has only three possible substitution products related to the 2-, 3-, and 6-hydroxyl groups of a glucose unit. In this study the isomers were fractionated by preparative anion-exchange chromatography with the progress of the elution being followed by a capillary electrophoretic (CE) method that resolved these isomers. The eluent containing the isomers was processed, and the pure materials were characterized by nuclear magnetic resonance spectroscopy ( 1H NMR, DEPT, HETCOR, HOHAHA). Through this analysis the assignment of the positional isomers was made. Three isomeric monosubstituted 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) were separated by anion-exchange chromatography and characterized by 2D NMR spectroscopy.

Fractionation and characterization of 4-sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin)

4-Sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) (SBE-β-CD) are being developed as parenterally safe solubilizing and stabilizing agents. SBE-β-CDs are a mixture of positional and regional isomers containing from one to as many as twelve sulfobutyl ether (SBE) groups per cyclodextrin. Capillary electrophoretic (CE) analysis of these mixtures resolves these isomers based on the molar degree of SBE substitution (ds), and the electropherogram shows an almost symmetrical distribution of SBE incorporation centered around the band which represents the apparent average degree of substitution for the mixture. The objectives of this study were to isolate the different substitution bands for their characterization and to evaluate their mass contribution to the mixture. Mixtures of SBE-β-CDs containing from mono- up to deca-SBE substitutions were fractionated by preparative anion-exchange chromatography with salt concentration gradient elution. The bands for each ds were well resolved as characterized by CE analysis with indirect UV detection. The isolated materials were desalted and lyophilized to obtain white solids, which were then characterized by nuclear magnetic resonance ( 1H NMR) spectroscopy, capillary electrophoresis (CE), and fast-atom-bombardment mass spectrometry (FABMS). The CE molar response factor of each ds was then determined, and the actual percent mass composition of a SBE-β-CD mixture was calculated. 4-Sulfobutyl ether derivatives of cyclomaltoheptaose (β-cyclodextrin) of ds 1–10 were prepared, separated by anion-exchange chromatography, analyzed by capillary electrophoresis, and characterized by 1H NMR spectroscopy and FAB mass spectrometry.

An Improved Synthesis of L-Ascorbate 2-Polyphosphate

Abstract L-Ascorbate (0) reacted rapidly with sodium trimetaphosphate (STMP) at 20–25°C and constant pH 9.5–10.0 in the presence of calcium ion. Barium and strontium ions also catalyzed the phosphorylation reaction, but not magnesium ion. The optimum reaction mixture was initially 1.4 M L-ascorbate, 1.8 M STMP, 1.7 M calcium hydroxide, and 0.4 M calcium chloride. Over a 20-min reaction period, pH was maintained by adding approximately 0.1 g calcium hydroxide in a 20% aqueous slurry. The initial and final Ca/P ratios were -0.36 and 0.42, respectively. The reaction products were 98.8% 2-phosphorylated L-ascorbate, 1.2% unreacted 0, and a trace of 4,5-ene, probably formed through 5,6-phosphorylated L-ascorbate 2-polyphosphate. High-performance liquid chromatography with UV detection and preparative anion-exchange chromatography showed the presence of eight 2-phosphorylated derivatives of 0 and two of the 4,5-ene skeleton. Rapid 2-phosphorylation prevented accumulation of the 4, 5-elimination reaction, and th...

Isolation and characterization of rhamnogalacturonan oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase

Digests of modified hairy regions of apple pectin (MHR) obtained after degradation by rhamnogalacturonase (RGase) were analyzed for oligomer composition using high-performance anion-exchange chromatography and pulsed amperometric detection. A series of oligomers which appear to be characteristic of RGase degradation could be recognized. These oligomers were isolated on a preparative scale by size-exclusion chromatography and preparative anion-exchange chromatography and analyzed for sugar composition. 1H NMR spectroscopy showed that the oligomers consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose residues linked to C-4 of the rhamnose moiety. The HPLC elution pattern showed that higher oligomers were also formed during incubation with RGase. These have the same basic structure but may contain other sugar units in addition to those given above. The oligomer composition of RGase digests of MHR isolated from apple, pear, leek, onion, carrot, and potato was very similar. Using anion-exchange chromatography to monitor the degradation of MHR at increasing incubation times, it was found that all the oligomers were present from the initial stages of the enzyme reaction and that the ratio between the different oligomers remained constant with time. Implications of these results for the structure of MHR and the mechanism of RGase action are discussed.