Åke Pilotti

Mass spectra of partially methylated alditol acetates : Part II. Deuterium Labelling Experiments

Abstract The fragmentation pattern of some partially methylated alditol acetates on electron impact has been studied by using the technique of deuterium labelling. Detailed fragmentation mechanisms are postulated.

Syntheses of a branched heptasaccharide having phytoalexin-elicitor activity

Abstract Syntheses are described of a d -glucose heptasaccharide 1 , corresponding to a glucan structure recognised by the soybean when infected by the fungus Phytophthora megasperma f. sp. glycinea and which stimulates the formation of phytoalexins. The synthetic strategy is based upon 1,2- trans -glycoside formation assisted by participating benzoate groups in the 2-position, with silver triflate as promoter and glycosyl bromides as donors for making the smaller fragments, and with methyl triflate as promoter with thioglycosides as donors for making the larger ones. Regioselective reductive openings of 4,6-benzylidene acetals play a key role in obtaining free 6-OH groups with benzyl protection at O-4.

Cytochrome P-450K of rat kidney cortex microsomes: Further studies on its interaction with fatty acids

Abstract The cytochrome P-450K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D2O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation. C10-C16 fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450K

Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Some 250 compounds, representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects encountered for positive standards. When employing the present cell toxicity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the compounds studied, are not taken into account. The most active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation obsered between functional groups and toxicity permits, within the range of functionalities studied, prediction of the toxicity for a compound of known structure.

Characterization of soluble glutathione transferase activity in resting mononuclear leukocytes from human blood

Glutathione transferase activity in the soluble fraction of resting human mononuclear leukocytes was measured and characterized using [3H] trans-stilbene oxide as a substrate. Because of the low activity of this enzyme in these cells, a published assay procedure developed for rodent liver was slightly modified to improve its sensitivity: the substrate was highly radiolabeled (2 Ci/mmole) and carefully purified, and the incubation time was extended to 30-60 min. The activity measured was linear with cell density up to at least 6 million cells. Soluble glutathione transferase activity measured in this manner has a pH optimum around 7.4 and an optimal temperature of 40 degrees. This activity could be measured in lymphocytes, monocytes, granulocytes, erythrocytes and platelets, but not in plasma. From these measurements it could be calculated that lymphocytes account for somewhat more than half of the total activity in the mononuclear leukocyte fraction and that monocytes account for the rest. The intraindividual variation in soluble glutathione transferase activity towards trans-stilbene oxide in the mononuclear leukocyte fraction from different subjects was only about 10%, whereas the interindividual variation in this same activity was 15-fold. An explanation for this relatively large interindividual variation is now being sought.

Efficient large scale microwave assisted Mannich reactions using substituted acetophenones

A series of substituted acetophenones, paraformaldehyde, and symmetrical dialkylamines were used in microwave enhanced Mannich reactions. Appropriate reaction conditions in terms of choice of solvent, reaction temperature, and reaction time were studied to allow a fast and reproducible production of Mannich bases. Both small (2 mmol) and large scale reactions (40 mmol) were performed successfully, providing a series of substituted Mannich bases in moderate to high yields and high purity.

Induction of durg-metabolizing systems and related enzymes with metabolites and structural analogues of stilbene

trans-Stilbene oxide has been found to be a new type of inducer of drug-metabolizing systems. In order to identify the true inducer and to determine the structural requirements for induction, rats were treated with metabolites and structural analogues of stilbene. Subsequently, hepatic levels of cytochrome P-450, microsomal epoxide hydrolase, and cytoplasmic glutathione S-transferase were assayed. All three enzymes were induced by cis- and trans-stilbene and cis- and trans-stilbene oxide. In addition, epoxide hydrolase and glutathione S-transferase activities were induced by benzoin and benzil. In contrast, the diols and benzoic acid had little, if any, effect. The main conclusions drawn from these findings are that: (1) trans-stilbene oxide itself seems to be the inducer of drug-metabolizing enzymes; and (2) benzil is more selective as an inducer of epoxide hydrolase than is trans-stilbene oxide. Attempts to induce epoxide hydrolase with other structural analogues of stilbene led to the following conclusions: (1) two phenyl rings are required for induction; (2) the induction is not as great if the rings are substituted or one of the ring carbon atoms is replaced by a nitrogen; (3) a carbon bridge between the phenyl groups generally results in a greater induction, especially if the bridge contains an epoxy group or one or two keto groups.

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.

Syntheses of a Heptasaccharide β-Linked to an 8-Methoxy-Carbonyl-Oct-1-Yl Linking Arm and of a Decasaccharide with Structures Corresponding to the Phytoelicitor Active Glucan of Phytophthora Megasperma F. Sp. Glycinea

Abstract Syntheses are described of the 8-methoxycarbonyloct-1-yl β-D-glycoside of 32,34-di-β-D-glucopyranosylgentiopentaose and also of 32,34,36-tri-β-D-glucopyranosylgentioheptaose, both of which were required for phytochemical studies of the defense mechanism of the soybean plant to infection by the mould Phytophthora megasperma f.sp. glycinea. Block synthesis strategies were used, relying on promotion by methyl triflate and the use of thioglycosides as glycosyl donors in the condensation of the oligosaccharide fragments. Highly stereoselective β-D-glycosylation was ensured by the presence of benzoyl groups in the 2-positions of the glycosyl donors.

Structural studies of the O-specific side-chains of the cell-wall lipopolysaccharide from Salmonella senftenberg

Abstract The structure of the O-specific side-chains of the cell-wall lipopolysaccharide (LPS) from Salmonella senftenberg , serogroup E 4 , has been investigated. From the methylation analysis of the LPS and of a partially hydrolysed product, the linkages could be identified and the order of the sugar residues assigned. The terminal, non-reducing sugar residue in the side-chains was also identified, thereby defining the “biological” repeating units in the side-chains. The anomeric nature of the different sugar residues was determined by polarimetric studies and by graded hydrolysis, followed by isolation and characterisation of a tetrasaccharide.