raffinose and melezitose

The hemolytic activity and solubilizing capacity of two families of non-reducing trisaccharide fatty acid monoesters have been studied to assess their usefulness as surfactants for pharmaceutical applications. The carbohydrate-based surfactants investigated included homologous series of raffinose and melezitose monoesters bearing C10 to C18 acyl chains prepared by lipase-catalyzed synthesis in organic media. The hemolytic activity was determined in vitro using a static method based on the addition of the surfactants to an erythrocyte suspension and subsequent spectrophotometric determination of the released hemoglobin. The effect of the carbohydrate head group, the acyl chain length and the regioisomeric purity was investigated. In all cases, the carbohydrate monoester surfactants decreased their hemolytic activity (with respect to their critical micelle concentration) when increasing the length of the acyl chain. A very similar behaviour was observed either the carbohydrate head-group (raffinose and melezitose) or regardless of the regioisomeric purity. Interestingly, decanoyl (C10) and lauroyl (C12) monoesters were just marginally hemolytic at their critical micelle concentrations while the longer palmitoyl (C16) and (C18) stearoyl monoesters become hemolytic at concentrations much higher than their respective cmc. The palmitoyl and stearoyl monoesters also displayed higher solubilization capacity than the shorter acyl chain monoesters in a solubilization assay of a hydrophobic dye as a model drug mimic. These results suggest that raffinose and melezitose monoesters with long-chain fatty acids (C16 to C18) are promising surfactants for pharmaceutical applications and could be an alternative to the use of current commercial nonionic polyoxyethylene-based surfactants in parenteral formulations.

Topics: Carbohydrates; Chemistry, Pharmaceutical; Coloring Agents; Fatty Acids; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Lipase; Micelles; Raffinose; Surface-Active Agents; Trisaccharides

Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control.

Topics: Bacterial Proteins; Bifidobacterium; Gene Expression Regulation, Bacterial; Multigene Family; Operator Regions, Genetic; Promoter Regions, Genetic; Raffinose; Repressor Proteins; Transcription, Genetic; Trisaccharides

A new instrument that combines ion mobility spectrometry (IMS) separations with tandem mass spectrometry (MS(n)) is described. Ion fragmentation is achieved with vacuum ultraviolet photodissociation (VUV PD) and/or collision-induced dissociation (CID). The instrument is comprised of an approximately 1 m long drift tube connected to a linear trap that has been interfaced to a pulsed F(2) laser (157 nm). Ion gates positioned in the front and the back of the primary drift region allow for mobility selection of specific ions prior to their storage in the ion trap, mass analysis, and fragmentation. The ion characterization advantages of the new instrument are demonstrated with the analysis of the isomeric trisaccharides, melezitose and raffinose. Mobility separation of precursor ions provides a means of separating the isomers and subsequent VUV PD generates unique fragments allowing them to be distinguished.

Topics: Ions; Isomerism; Models, Molecular; Raffinose; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Trisaccharides

Overtone mobility spectrometry (OMS) is examined as a means of determining the collision cross sections for multiply charged ubiquitin and substance P ions, as well as for singly charged rafinose and melezitose ions. Overall, values of collision cross section measured by OMS for stable ion conformations are found to be in agreement with values determined by conventional ion mobility spectrometry (IMS) measurements to within ∼1% relative uncertainty. The OMS spectra for ubiquitin ions appear to favor different conformations at higher overtones. We propose that the changes in the distributions as a function of the overtone region in which they are measured arise from the elimination of ions that undergo structural transitions in the drift regions. Kinetics simulations suggest that structural transitions occurring on the order of a few ms and resulting in an ∼4% change in ion collision cross sections are detected by OMS measurements. The unique method of distinguishing ion mobilities with OMS reveals these structural transitions which are not readily apparent from traditional IMS measurements.

Topics: Ions; Mass Spectrometry; Molecular Conformation; Raffinose; Substance P; Trisaccharides; Ubiquitin

A novel ion mobility spectrometry instrument incorporating a cyclotron geometry drift tube is presented. The drift tube consists of eight regions, four curved drift tubes and four ion funnels. Packets of ions are propagated around the drift tube by changing the drift field at a frequency that is resonant with the ion's drift time through each region. The approach trims each packet of ions as it leaves and enters each new region. An electrostatic gate allows ions to be kept in the drift tube for numerous cycles, increasing the ability to resolve specified ions. We demonstrate the approach by isolating the [M + 2H](2+) or [M + 3H](3+) charge state of substance P as well as individual trisaccharide isomers from a mixture of melezitose and raffinose. Resolving powers in excess of 300 are obtainable with this approach.

Topics: Cyclotrons; Isomerism; Motion; Raffinose; Spectrum Analysis; Time Factors; Trisaccharides

A new method that allows a linear drift tube to be operated as a continuous ion mobility filter is described. Unlike conventional ion mobility instruments that use an electrostatic gate to introduce a packet of ions into a drift region, the present approach uses multiple segmented drift regions with modulated drift fields to produce conditions that allow only ions with appropriate mobilities to pass through the instrument. In this way, the instrument acts as a mobility filter for continuous ion sources. By changing the frequency of the applied drift fields it is possible to tune this instrument to transmit ions having different mobilities. A scan over a wide range of drift field frequencies for a single ion species shows a peak corresponding to the expected resonance time of the ions in one drift region segment and a series of peaks at higher frequencies that are overtones of the resonant frequency. The measured resolving power increases for higher overtones, making it possible to resolve structures that were unresolved in the region of the fundamental frequency. We demonstrate the approach by examining oligosaccharide isomers, raffinose and melezitose as well as a mixture of peptides obtained from enzymatic digestion of myoglobin.

Topics: Animals; Cytochromes c; Equipment Design; Hemoglobins; Horses; Humans; Ions; Mass Spectrometry; Myoglobin; Peptide Fragments; Raffinose; Trisaccharides; Trypsin

The reactivity of melezitose and raffinose under Mitsunobu conditions was studied within the scope of the use of trisaccharides for the synthesis of fatty acid esters. Melezitose led to esters with preferential substitution at primary positions following the order of reactivity 6''>6>6'. Raffinose proved to be very reluctant toward ester formation in these conditions, leading mainly to the new 3'',6''-anhydroraffinose.

Topics: Esters; Fatty Acids; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Chemical; Molecular Structure; Palmitic Acid; Raffinose; Trisaccharides

A behavioural study on the ant Lasius niger was performed by observing its feeding responses to 85 compounds presented in a two-choice situation (tested compound versus water control or sucrose solution). Among these compounds, only 21 were phagostimulating: six monosaccharides (D-glucose, 6-deoxy-D-glucose, L-galactose, L-fucose, D-fructose, L-sorbose), four derivatives of D-glucose (methyl alpha-D-glucoside, D-gluconolactone and 6-chloro- and 6-fluoro-deoxy-D-glucose), five disaccharides (sucrose, maltose, palatinose, turanose and isomaltose), one polyol glycoside (maltitol), three trisaccharides (melezitose, raffinose and maltotriose) and two polyols (sorbitol and L-iditol). None of the 16 non-carbohydrate non-polyol compounds tested, although perceived as sweet in humans, was found to be active in ants. The molar order of effectiveness of the major naturally occuring compounds (melezitose > sucrose = raffinose > D-glucose > D-fructose = maltose = sorbitol) is basically different from the molar order of their sweetness potency in humans (sucrose > D-fructose > melezitose > maltose > D-glucose = raffinose = sorbitol). On a molar basis melezitose is in L. niger about twice as effective as sucrose or raffinose, while D-glucose and D-fructose are three and four times less effective, respectively, than sucrose or raffinose. From a structure-activity relationship study it was inferred that the active monosaccharides and polyols should interact with the ant receptor through only one type of receptor, through the same binding pocket and the same binding residues, via a six-point interaction. The high effectiveness of melezitose in L. niger mirrors the feeding habits of these ants, which attend homopterans and are heavy feeders on their honeydew, which is very rich in this carbohydrate.

Topics: Animals; Ants; Deoxyglucose; Disaccharides; Fructose; Fucose; Galactose; Glucose; Humans; Isomaltose; Maltose; Models, Chemical; Raffinose; Sorbitol; Sorbose; Structure-Activity Relationship; Sugar Alcohols; Taste; Trisaccharides