Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed

) Maceil, C. E.; In The Encyclopedia of Nuclear Magnetic Resonance

Evanescent-wave cavity ring-down spectroscopy has been applied to a planar fused-silica surface covered with crystal violet (CV + ) cations to characterize the silanol groups indirectly. A radiation-polarization dependence of the adsorption isotherm of CV + at the CH 3 CN/silica interface is measured and fit to a two-site Langmuir equation to determine the relative populations of two different types of isolated silanol groups. CV + binding at type I sites yields a free energy of adsorption of -29.9 ( 0.2 kJ/mol and a saturation surface density of (7.4 ( 0.5) × 10 12 cm -2 , whereas the values of -17.9 ( 0.4 kJ/mol and (3.1 ( 0.4) × 10 13 cm -2 are obtained for the type II sites. The CV + cations, each with a planar area of ∼120 Å 2 , seem to be aligned randomly while lying over the SiOtype I sites, thereby suggesting that this type of site may be surrounded by a large empty surface area (>480 Å 2 ). In contrast, the CV + cations on a type II sites are restricted with an average angle of ∼40°tilted off the surface normal, suggesting that the CV + cations on these sites are grouped closely together. The average tilt angle increases with increasing concentration of crystal violet so that CV + cations may be separated from each other to minimize the repulsion of nearby CV + and SiOH sites. Adsorption behavior of organic molecules on silica surfaces has been the major theme of interface studies for improving the efficiency of chromatographic separations. When cationic molecules are involved, the strong electrostatic interaction with the negatively charged silanol (SiOH) groups on the surface of the stationary-phase silica may cause unwanted peak broadening and tailing, mainly from a slow kinetic response of the electrostatic adsorption. [1][2][3][4][5][6] The surface charge density is one of the primary factors influencing the strength of electrostatics. Accordingly, insight into how the cationic molecules interact with the local silanol groups of the silica surface should aid in the improvement of the design of surface modifications. Silanol groups play the main role in influencing the interfacial adsorption behavior, possessing an average surface density of ∼4.9 × 10 14 cm -2 on the silica surface 7-9 or an average surface area of 20.4 Å 2 per silanol group. As compared to silica sol particles, which have higher surface areas of (0.1-5) × 10 22 Å 2 /g, 7-9 only a few studies focus on characterization of silanol groups on a planar silica surface. 10-12 Ong et al. 10 first reported that isolated and vicinal silanol groups both exist at the water/silica interface possessing different pK a values of 4.9 and 8.5, with corresponding surface populations of 19 and 81%, respectively. These results were confirmed by means of cross-polarization magic angle spinning NMR 13 and fluorescence microscopy. 14 The isolated silanol groups with pK a ) 4.9 are anticipated to be separated far from each other (>5.5 Å), permitting proton dissociation. The vicinal silanol groups are located so closely as to form hydrogen bonds directly with their neighbors (<3.3 Å), which share 46% of the surface population, or through a water-molecule bridge (3.5-5.5 Å), which covers ∼35% of the surface population. 12,[15][16][17] By using second harmonic generation (SHG) with a cationic crystal violet (CV + ) molecular probe to investigate the local density distribution of the isolated silanols (pK a ) 4.9) on the planar fusedsilica surface, Xu and co-workers 12 classified them into two types. The first type of silanol group is anticipated to be surrounded by a large empty surface area (g120 Å 2 ) with a surface density o

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L−1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein. Keywords: Pichia pastoris, Metabolic engineering, Terpene cyclase, Triterpenoid, Squalene, (+)-ambrei

Synthesis and Catalytic Properties of New Polymeric Monometallic Composites Based on Copolymers of Polypropylene Glycol Maleate Phthalate with Acrylic Acid

Metal-polymer composites based on copolymers of polypropylene glycol maleate phthalate with acrylic acid and metallic nickel and silver were synthesized for the first time. The objects obtained were characterized by infrared (IR) and Raman spectroscopies, thermogravimetry, a scanning electron microscope with energy dispersive spectroscopy, and atomic emission spectrometry. The catalytic activity of new metal-polymer composites that exhibited a rather high efficiency in the reactions of electrocatalytic hydrogenation of pyridine was studied. It is shown that nanoparticles of metals are evenly distributed in the volume of the polymer matrix; more than 80% of nanoparticles are in the range from 25 to 40 nm and have spherical and rhombic shapes. The reusability of the obtained composites is shown