Synthesis of S-Glycosyl Xanthates by Phase Transfer Catalyzed Substitution of Glycosyl Halides

Abstract

Peracetylated glycosyl- and glycobiosyl bromides and chlorides 1-4 including acetochloroneuraminic acid 5 were stereoselectively transformed into their corresponding S-glycosyl xanthates 6-10 in high yield (91-98%) under phase transfer catalyzed conditions. The reactions occurred at room temperature using tetrabutylammonium hydrogen sulfate as the catalyst. The substitutions gave complete inversion of configuration and thus proceeded by an S_N2 type mechanism. Changing the organic solvent from methylene chloride to ethyl acetate had no detrimental effect on the outcome of the reactions but avoided an undesirable side reaction between the xanthate anion and methylene chloride.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).     

Biosynthesis of anthecotuloide,an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route

Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.     

Development of an HPLC Method for Determination of Related Impurities in Prilocaine Substance

Development of a reversed phase high performance liquid chromatographic method for determination of six related impurities in prilocaine substance is reported. The test of related impurities in European Pharmacopoeia (Ph. Eur.) cannot meet the demands with the chromatographic parameters given, therefore different types of chromatographic systems and eight columns have been evaluated in the present study. A new method with a Hypercarb column was developed and validated. This method fulfils the demands in the Ph. Eur., and the validation shows that the method is selective, reproducible, linear, accurate and robust with sufficient limits of detection (0.001–0.004% of 2.5?mg prilocaine mL^?1) and quantification (0.002–0.009% of 2.5?mg prilocaine mL^?1).     

Control of lysine reactivity in four-helix bundle proteins by site-selective pKa depression: expanding the versatility of proteins by postsynthetic functionalization

Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.     

Asymmetric base-mediated epoxide isomerisation

The base-mediated rearrangement of epoxides into allylic alcohols is a well-known synthetic transformation. The first enantioselective version of the reaction using a chiral base was reported in 1980. Since then, the reaction has received a lot of attention mostly due to the great usefulness of chiral allylic alcohols in organic synthesis. Major breakthroughs in the area were the first report on using a sub-stoichiometric amount of chiral base, and the development of chiral bases for a true catalytic reaction protocol. The present review covers the time from when the first asymmetric epoxide isomerisation reaction was reported (1980) up to now, focusing on the period 1997-2001.     

Molecular mass distribution analysis of ethyl(hydroxyethyl)cellulose by size-exclusion chromatography with dual light-scattering and refractometric detection

Dual low-angle light scattering and refractometric detection coupled to size-exclusion chromatography provided proof for the presence of a low amount of stable aggregates/particles in ethyl(hydroxyethyl)cellulose. Unlike the correct size-exclusion chromatographic behavior of the parent polysaccharide itself, the aggregates exhibit variable size-dependent weak retention as a function of flow-rate and of ionic strength of the aqueous mobile phase. Therefore, determination of the molecular mass of non-aggregated polymer is possible in aqueous mobile phase containing 0.1 M NaCl under conditions at which aggregates are completely adsorbed on the column packing irrespective of the flow-rate used. Flow-rate and ionic strength-dependent variations of aggregate behavior as well as model size-exclusion experiments with latex particles indicate that they partly carry a minute charge and have a compact structure. Their weak retention under the separation conditions used suggests a difference in their surface chemistry when compared with the dissolved polymer coils which exhibit a correct size-exclusion behavior.