Separation and identification of enzymatic sucrose hydrolysis products by high-performance anion-exchange chromatography with pulsed amperometric detection.

An accurate carbohydrate analysis method, namely high-performance anion-exchange chromatography with pulsed amperometric detection was successfully applied to the study of sucrose hydrolysis under enzymatic (baker's yeast invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of D-glucose, D-fructose and five intermediate fructans using a CarboPac PA-100 (Dionex) analytical anion-exchange column. Highly reproducible results were obtained. The unknown fructans were collected from a semi-preparative CarboPac PA-100 (Dionex) column, neutralized and then desalted on a column containing mixed bed resin AG 501-X8 (D) before identification of the chemical structure. This procedure permitted us to obtain about 20 microg of pure product which is not enough for NMR analysis. Detailed GC-MS analytical data of the methylated compounds indicated that these oligosaccharides were beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (1-kestose), beta-D-Fru-(2 --> 6)-alpha-D-glucopyranoside (6-beta fructofuranosylglucose), beta-D-Fru-(2 --> 1)-beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose) coeluating with a disaccharide.

Modification and inhibition of vancomycin group antibiotics by formaldehyde and acetaldehyde.

It is shown that several vancomycin group antibiotics (vancomycin, eremomycin, and avoparcin) undergo spontaneous chemical modifications when kept at room temperature at neutral pH in aqueous solutions containing traces of formaldehyde or acetaldehyde. This chemical modification predominantly results in a mass increase of 12 Da in the reaction with formaldehyde and 26 Da in the case of acetaldehyde. By using tandem mass spectrometry the modification can unambiguously be identified as originating from the formation of a ring-closed 4-imidazolidinone moiety at the N-terminus of the glycopeptide antibiotics, that is, near the receptor binding pocket of the glycopeptide antibiotics. Bioaffinity mass spectrometry shows that this ring-closure results in a dramatically decreased affinity for the peptidoglycan-mimicking D-alanyl-D-alanine receptor. Additionally, in vitro inhibition measurements on two different strains of bacteria have revealed that the modified antibiotics display reduced antibacterial activity. The ring-closure is also shown to have a dissociative effect on the dimerization of the vancomycin-analogue eremomycin. The spontaneous reaction of vancomycin with formaldehyde or acetaldehyde may have implications not only for the clinical use of this class of antibiotics, but also for the effectiveness of these antibiotics when they are used in chiral separation chromatography or capillary electrophoresis.

Application of high performance anion exchange chromatography to study invertase-catalysed hydrolysis of sucrose and formation of intermediate fructan products.

Baker's yeast invertase was found to catalyse transfructosylation reactions in aqueous and anhydrous organic media with sucrose as a substrate, leading to the formation of five intermediate fructans in addition to the release of D-glucose (D-Glc)and D-fructose (D-Fru). All the reaction products were separated and quantitatively estimated using high performance anion exchange-pulsed amperometric detection equipment. The unknown products were subsequently identified by linkage analysis as beta-D-Fru-(2 --> 1)-beta-D-Fru-(2 --> 1)- alpha-D-glucopyranoside (1-kestose), beta-D-Fru- (2 --> 6)-alpha-D-glucopyranoside (6-beta-fructofuranosylglucose), beta-D-Fru-(2 -->1) -beta-D-fructofuranoside (inulobiose), beta-D-Fru-(2 --> 6)-beta-D-Fru-(2 --> 1)-alpha-D-glucopyranoside (6-kestose) and beta-D-Fru-(2 --> 6)-alpha-D-Glc-(1 --> 2)-beta-D-fructofuranoside (neokestose); and this last was eluted together with a disaccharide. The time-course of sucrose hydrolysis via fructan production in 2 ml of a 50 mM sodium acetate buffer (pH 4.5) containing 0.2 M sucrose and 25 U of invertase was different from that in 2 ml of anhydrous toluene with 1.46 M sucrose and 1,000 U of invertase as a suspended powder. Under the latter experimental conditions, invertase was found to exhibit cyclic behaviour, where sucrose was degraded and subsequently synthesised. This observation has not yet been reported, as far as we know.

Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry.

The conformational stability of vancomycin group antibiotics (i.e., vancomycin and avoparcin) in aqueous solution has been studied. These complex glycopeptide antibiotics contain many chiral centers allowing the potential formation of stereoisomers. Using capillary electrophoresis these stereoisomers could be separated and detected by UV and/or mass spectrometry. Fresh aqueous samples of both vancomycin and avoparcin already contained a plethora of stereoisomers. Thermal degradation of the antibiotics was studied as well. For vancomycin thermal degradation led primarily to the formation of CDP-I and aglycons. In the case of avoparcin thermal degradation led mainly to the interconversion between stereoisomers. These antibiotic stereoisomers may exhibit different antibacterial efficacy. Solution-phase association constants of fresh and heated samples of these antibiotics and their bacterial cell wall mimicking receptors were determined by bioaffinity mass spectrometry and revealed that the heated samples exhibited, in general, a lower affinity. Minimum inhibitory concentrations (Micrococcus flavus) were determined and confirmed the decrease in antibacterial efficacy upon heating.