Application of partial-filling capillary electrophoresis using lectins and glycosidases for the characterization of oligosaccharides in a therapeutic antibody

Oligosaccharides in therapeutic recombinant antibodies play important roles in regulation of various biological functions. To monitor the glycosylation profiles of antibody pharmaceuticals in the manufacturing process, a highly sensitive and specific method is required. We extended partial-filling techniques using lectins and exoglycosidases in capillary electrophoresis for the characterization of 8-aminopylene-1,3,6-trisulfonic acid labeled N-linked oligosaccharides derived from the therapeutic antibody rituximab. In the lectin-filling method, Galb1–4GlcNAc-specific Erythrina cristagali agglutinin, a1, 6-linked Fuc-specific Aleuria aurantia lectin and Neu5Aca2–3Gal-specific Maackia amurensis lectin were used. The oligosaccharides migrated through the lectin plug during separation; the changes in separation profiles were observed according to the interaction with the lectins. The glycosidase-filling method allowed rapid digestion as suggested by the electropherograms. Partial-filling CE methods can avoid tedious hands-on procedures such as overnight incubation and optimization reaction condition with lectins and exoglycosidases. Combination of these partial-filling capillary electrophoresis methods makes the characterization of oligosaccharide profiles of therapeutic antibodies easier and faster.     

Benchmarking of density functionals for the kinetics and thermodynamics of the hydrolysis of glycosidic bonds catalyzed by glycosidases

In recent years, there has been an increased interest in understanding the enzymatic mechanism of glycosidases resorting mostly to DFT and DFT/MM calculations. However, the performance of density functionals (DFs) is well known to be system and property dependent. Trends drawn from general studies, despite important to evaluate the quality of the DFs and to pave the way for the development of new DFs, may be misleading when applied to a single specific system/property. To overcome this issue, we carried out a benchmarking study of 40 DFs applied to the geometry optimization and to the electronic barrier height (E _Barrier) and electronic energy of reaction (E _R) of prototypical glycosidase‐catalyzed reactions. Additionally, we report calculations with SCC‐DFTB and four semiempirical MO methods applied to the same problem. We have used a universal molecular model for retaining glycosidases, comprising only a 22‐atoms system that mimics the active site and substrate. High accuracy reference geometries and energies were calculated at the CCSD(T)/CBS//MP2/aug‐cc‐pVTZ level of theory. Most DFs reproduce the reference geometries extremely well, with mean unsigned errors (MUE) smaller than 0.05 Å for bond lengths and 3° for bond angles. Among the DFs, wB97X‐D, CAM‐B3LYP, B3P86, and PBE1PBE have the best performance in geometry optimizations (MUE = 0.02 Å). Conversely, semiempirical MO and SCC‐DFTB methods yielded less accurate geometries (MUE between 0.09 and 0.17 Å). The inclusion of D3 correction has a small, but still relevant, influence in the geometry predicted by some DFs. Regarding E _Barrier, 11 DFs (MPW1B95, CAM‐B3LYP, M06 ‐ 2X, PBE1PBE, wB97X ‐ D, B1B95, BMK, MN12 – SX, M05, M06, and M11) presented errors below 1 kcal.mol^?1, in relation to the reference energy. Most of these functionals belong to the family of hybrid functionals (H‐GGA, HH‐GGA, and HM‐GGA), which shows a positive influence of HF exchange in the determination of E _Barrier. The inclusion of D3 correction has not affected significantly the E _Barrier and E _R. The use of geometries at the accurate but expensive MP2/aug‐cc‐pVTZ level of theory has a small, albeit not insignificant, influence in the E _Barrier when compared with energies calculated with geometries determined with the DFs (usually a few tenths of kcal.mol^?1, with exceptions). In general, semiempirical MO methods and DFTB are associated with larger errors in the determination of E _Barrier, with unsigned errors from 6.9 to 24.7 kcal.mol^?1.     

Effect of natural polysaccharides and oligosaccharides on the permeability of cell membranes

Glycyrrhizic acid, arabinogalactan, and β-cyclodextrin are capable of forming complexes with various drug compounds significantly enhancing their solubility and bioavailability. To elucidate the mechanism of action of oligo- and polysaccharides, the exchange time of transporting formate ions through the membrane of myeloblastic leukemia cells (K562) was measured by NMR spectroscopy with the addition of paramagnetic ions. It was found that glycyrrhizic acid and arabinogalactan substantially increased the membrane permeability.     

Rapid screening of the aglycone specificity of glycosidases: applications to enzymatic synthesis of oligosaccharides.

BACKGROUND: Retaining glycosidases can catalyse glycosidic bond formation through transglycosylation from a donor sugar to an acceptor bound in the aglycone site. The aglycone specificity of a glycosidase is not easily determined, thereby complicating the choice of the most appropriate glycosidase for use as a catalyst for transglycosylation. We have developed a strategy to rapidly screen the aglycone specificity of a glycosidase and thereby determine which enzymes are best suited to catalyse specific transglycosylation reactions. RESULTS: The reactivation, or turnover, of a glycosidase trapped as a fluoroglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapidly screen six glycosidases with 44 potential acceptor sugars. Validation of the screening strategy was demonstrated by the identification of products formed from a transglycosylation reaction with positively screened acceptors for four of the enzymes studied. CONCLUSIONS: The aglycone specificity of a glycosidase can be rapidly evaluated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening.     

Temperature dependence of thermal diffusion for aqueous solutions of monosaccharides, oligosaccharides, and polysaccharides

We studied the thermal diffusion behavior for binary aqueous solutions of glucose, maltotriose, maltohexaose, pullulan, and dextran by means of thermal diffusion forced Rayleigh scattering (TDFRS). The investigated saccharides with molar masses between 0.180 and 440 kg mol(-1) were studied in the temperature range between 15 and 55 °C. The thermal diffusion coefficient D(T) and the Soret coefficient S(T) of all solutions increase with increasing temperature. For maltohexaose and the polymers the thermal diffusion coefficient changes sign from negative to positive with increasing temperature, whereas glucose and maltotriose show only positive values in the entire investigated temperature range. While we were able to find a master curve to describe the temperature dependence of D(T), we were not able to find a similar expression for S(T). This comprehensive study allows for the first time the determination of the interaction parameters for the polymer and the solvent within the theoretical framework suggested by Würger [Phys. Rev. Lett., 2009, 102, 078302].     

Identification of the composition of oligosaccharides and polysaccharides by pyrlysis/capillary gas chromatography

Pyrolysis/capillary gas chromatography is used for the characterization of the monomer composition of various oligosaccharides and polysaccharides including glucose-containing disaccharides,glucans, a galactomannan and an arabinogalactan. The chromatograms showed many common pyrolysis products, but also unique anhydrosugar products (e.g., 1,6-anhydroglucopyranose, 1,4-anhydroarabinopyranose, 2,6-anhydrofructofuranose) derived from each type of saccharide unit present in the samples. Reasonable values of the monomer composition of the polysaccharide can also be obtained from th pyrograms. The method is rapid and direct, requiring no sample preparation.     

Studies of the forced hydrolysis degradation of copper complexes with different oligosaccharides

Bioactive copper complexes with oligosaccharides, pullulan or dextran, are the objective of the present study, because of their possible biomedical applications. The alkaline and acid hydrolysis of the Cu(II) complexes with reduced low-molar pullulan or dextran were carried out by conductometric method. The influence of ligand constitutions on the stability of the complexes was examined on the basis of ligand property. The complexes degradation during alkaline and acid hydrolysis were carried out in sodium hydroxide and hydrochloric acid solutions of 0.1, 0.5, and 1.0 mol dm⁻³, at different temperature (25, 40, and 60°C, respectively). According to the obtained results by the conductivity investigation during forced degradation studies, it could be concluded that the Cu(II) complexes show the small pharmaceutical stability to both hydrolysis.     

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides

Application of Aloe barbadensis poly/oligosaccharides to UV-irradiated skin prevents photosuppression of delayed-type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant-derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 micrograms tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xyloglucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)-10 observed in UV-irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL-10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL-10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV-activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.     

Substrates for the histochemical localization of some glycosidases

Substrates for improved histochemical localizations of some glycosidases were required. The 2-acetamido-2-deoxy-β- -glucoside, the β- -glucoside, and the β- -galactoside of 5-bromoindoxyl were synthesised through 1-acetyl-5-bromoindoxyl and the corresponding acetohalogeno-sugars. Attempts to prepare the β- -glucuronide under a variety of conditions were unsuccessful, whereas acetobromo-methyl-glucuronate condensed with naphthoic-2-hydroxy-3-(2′:5′-dimethoxy-4′-chloro-anilide) and a potential histochemical substrate obtained: the preparation of the corresponding derivatives of β- -galactose and 2-acetamido-2-deoxy-β- -glucose are also described.     

Properties of biopolymer compositions based on products of collagen hydrolysis and polysaccharides

Physicochemical properties of biopolymer compositions based on products of collagen hydrolysis and polysaccharides were studied. The effect of the component ratio on their compatibility was examined.     

Synthetic 6-O-methylglucose-containing polysaccharides (sMGPs): design and synthesis

With the hope of mimicking the chemical and biological properties of natural 6-O-methyl-D-glucose-containing polysaccharides (MGPs), synthetic 6-O-methyl-D-glucose-containing polysaccharides (sMGPs) were designed and synthesized from alpha-, beta-, and gamma-cyclodextrins (CDs). The synthetic route proved to be flexible and general, to furnish a series of sMGPs ranging from 6-mer to 20-mer. A practical and scalable method was discovered selectively to cleave the CD derivatives and furnish the linear precursors to the glycosyl donors and acceptors. The Mukaiyama glycosidation was adopted to couple the glycosyl donors with the glycosyl acceptors. Unlike in the 3-O-methyl-D-mannose-containing polysaccharide (sMMP) series, the amount of the Mukaiyama acid required in the sMGP series increased with an increase of substrate size; that is, for large oligomers, more than one equivalent of the acid was required.     

Synthetic 3-O-methylmannose-containing polysaccharides (sMMPs): design and synthesis

With the hope of mimicking the chemical and biological properties of natural 3-O-methylmannose-containing polysaccharides (MMPs), synthetic 3-O-methylmannose-containing polysaccharides (sMMPs) were designed and synthesized in a convergent manner. With little modification of the Mukaiyama glycosidation, high alpha-selectivity (>50:1 approximately >20:1) and yields (79 approximately 74%) were achieved for the key glycosidation steps. The exceptionally high alpha-selectivity observed was shown to be consequent to the selective anomerization of beta- to alpha-anomer under the glycosidation conditions. This glycosidation is well suited for a highly convergent oligosaccharide synthesis, particularly because of excellent chemical yields even when using approximately equal-sized donors and acceptors in an approximately 1:1 molar ratio. An iterative reaction sequence allowed the growing oligosaccharide to double in size after each cycle and led to an efficient synthesis of sMMP 8-, 12-, and 16-mers 18-20.     

Design and synthesis of activity probes for glycosidases

[structure: see text] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.     

Synthetic studies of the HIV-1 protease inhibitive didemnaketals: precise and stereocontrolled synthesis of the key mother spiroketal

The precise and stereocontrolled synthesis of the C₉-C₂₃ portion, the key mother spiroketal of the HIV-1 protease inhibitive didemnaketals from the ascidian Didemnum sp., has been carried out through multisteps starting from (R)-pulegone as the chiral template. This approach involved the distereoselective construction of eight chiral centers by intramolecular chiral inducement and the coupling of two fragments by Julia reaction.     

J‐modulated 1D directed COSY for precise measurement of proton–proton residual dipolar coupling constants of oligosaccharides

A simple selective 1D experiment for the accurate and precise determination of residual proton–proton dipolar coupling constants of oligosaccharides is proposed. The technique is based on the concept of J‐modulated spectroscopy and provides coupling constants by fitting the peak intensities from a series of spectra to known transfer functions with a precision of ±0.02 Hz for splittings >0.3 Hz. This level of precision is possible owing to limiting the number of sites for polarization transfer. Selective pulses and pulsed field gradients are used for selective excitation and coherence selection, yielding clean, artefact‐free spectra. The technique is illustrated using trisaccharide samples in Pf1 phage. Copyright © 2002 John Wiley & Sons, Ltd.     

The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis

Xylan backbones in native plant cell walls are extensively acety-lated. Previously, no direct investigations as to their role in cellulolytic enzyme resistance have been done, though indirect results point to their importance. An in vitro deesterification of aspen wood and wheat straw has been completed using hydroxylamine solutions. Yields of 90% acetyl ester removal for both materials have been accomplished, with little disruption of other fractions (i.e., lignin). Apparently, as the xylan becomes increasingly deacetylated, it becomes 5–7 times more digestible. This renders the cellulose fraction more accessible, and 2–3 times more digestible. This effect levels off near an acetyl removal of 75%, where other resistances become limiting.