Synthesis of unusual alpha-amino acids and study of the effect of their incorporation into antimicrobial peptides. Total synthesis of biactive marine natural products and analogues thereof

The principle theme of this thesis was the synthesis of bioactive compounds. To this end, this work was focus on two main projects. The first one, which was carried out in the Department of Chemistry of the University of Girona under the supervision of Dr Montserrat Heras, concerned the synthesis of new unnatural amino acids bearing a pyrimidine ring within their side chain for incorporation into the antimicrobial peptide BP100 following a rational design in order to improve its biological profile. On the other hand, the second chapter of this thesis was developed in collaboration with the Laboratoire de Chimie Organique (ESPCI-ParisTech, Paris, France) under the guidance of Pr Janine Cossy and Dr Arseniyadis. This chapter was centered on the total synthesis of three marine natural products with complex structures and interesting biological activities: acremolide B, (–) bitungolide F and lyngbouilloside.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

Dietary supplements of whole linseed and vitamin E to increase levels of alpha-linolenic acid and vitamin E in bovine milk

The potential to increase the concentrations of n-3 polyunsaturated fatty acids (PUFAs) in milk fat was investigated by studying the effects of feeding a xylose-treated, whole cracked linseed supplement ( rich in alpha-linolenic acid) to dairy cows. Also the effect of increasing the dietary intake of vitamin E on the vitamin E status of milk was investigated. The effect of pasteurisation on milk fatty acid composition was also examined. Using a 3 x 2 factorial design, a total of 60 Holstein dairy cows were fed a total mixed ration based on grass silage supplemented with one of three levels of whole cracked linseed (78, 142 or 209 g . kg(-1) diet dry matter (DM); designated LL, ML or HL, respectively) in combination with one of two levels of additional dietary vitamin E intake ( 6 or 12 g vitamin E . animal(-1) . day(-1); designated LE or HE, respectively). Increasing lipid supplementation reduced (P < 0.01) diet DM intake and milk yield, and increased (P < 0.001) the overall content of oleic, vaccenic, alpha-linolenic and conjugated linoleic acids, and total PUFAs and monounsaturated fatty acids (MUFA). Myristic and palmitic acids in milk fat were reduced ( P < 0.001) through increased lipid supplementation. While α-linolenic acid concentrations were substantially increased this acid only accounted for 0.02 of total fatty acids in milk at the highest level of supplementation (630 g α-linolenic acid &BULL; animal(-1) &BULL; day(-1) for HL). Conjugated linoleic acid concentrations in milk fat were almost doubled by increasing the level of lipid supplementation (8.9, 10.4 and 16.1 g &BULL; kg(-1) fatty acids for LL, ML and HL, respectively). Although milk vitamin E contents were generally increased there was no benefit (P > 0.05) of increasing vitamin E intake from 6 to 12 g . animal(-1) . day(-1). The fatty acid composition of milk was generally not affected by pasteurisation.

Nitrogen fertilizer and seed rate effects on Hagberg failing number of hybrid wheats and their parents are associated with alpha-amylase activity, grain cavity size and dormancy

Field experiments were carried out to assess the effects of nitrogen fertilization and seed rate on the Hagberg falling number (HFN) of commercial wheat hybrids and their parents. Applying nitrogen (200 kg N ha(-1)) increased HFN in two successive years. The HFN of the hybrid Hyno Esta was lower than either of its parents (Estica and Audace), particularly when nitrogen was not applied. Treatment effects on HFN were negatively associated with a-amylase activity. Phadebas grain blotting suggested two populations of grains with different types of a-amylase activity: Estica appeared to have a high proportion of grains with low levels of late maturity endosperm a-amylase activity (LMEA); Audace had a few grains showing high levels of germination amylase; and the hybrid, Hyno Esta, combined the sources from both parents to show heterosis for a-amylase activity. Applying nitrogen reduced both apparent LMEA and germination amylase. The effects on LMEA were associated with the size and disruption of the grain cavity, which was greater in Hyno Esta and Estica and in zero-nitrogen treatments. External grain morphology failed to explain much of the variation in LMEA and cavity size, but there was a close negative correlation between cavity size and protein content. Applying nitrogen increased post-harvest dormancy of the grain. Dormancy was greatest in Estica and least in Audace. It is proposed that effects of seed rate, genotype and nitrogen fertilizer on HFN are mediated through factors affecting the size and disruption of the grain cavity and therefore LMEA, and through factors affecting dormancy and therefore germination amylase. (c) 2004 Society of Chemical Industry.

Sterol content analysis suggests altered eburicol 14 alpha-demethylase (CYP51) activity in isolates of Mycosphaerella graminicola adapted to azole fungicides

The recent decline in the effectiveness of some azole fungicides in controlling the wheat pathogen Mycosphaerella graminicola has been associated with mutations in the CYP51 gene encoding the azole target, the eburicol 14 alpha-demethylase (CYP51), an essential enzyme of the ergosterol biosynthesis pathway. In this study, analysis of the sterol content of M. graminicola isolates carrying different variants of the CYP51 gene has revealed quantitative differences in sterol intermediates, particularly the CYP51 substrate eburicol. Together with CYP51 gene expression studies, these data suggest that mutations in the CYP51 gene impact on the activity of the CYP51 protein.

Changes in oestrogen receptor-alpha and -beta during progression to acquired resistance to tamoxifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breast cancer cells

Cell culture models of antioestrogen resistance often involve applying selective pressures of oestrogen deprivation simultaneously with addition of tamoxifen or fulvestrant (Faslodex, ICI 182,780) which makes it difficult to distinguish events in development of antioestrogen resistance from those in loss of response to oestrogen or other components. We describe here time courses of loss of antioestrogen response using either oestrogen-maintained or oestrogen-deprived MCF7 cells in which the only alteration to the culture medium was addition of 10(-6) M tamoxifen or 10(-7) M fulvestrant. In both oestrogen-maintained and oestrogen-deprived models, loss of growth response to tamoxifen was not associated with loss of response to fulvestrant. However, loss of growth response to fulvestrant was associated in both models with concomitant loss of growth response to tamoxifen. Measurement of oestrogen receptor alpha (ER alpha) and oestrogen receptor beta (ER beta) mRNA by real-time RT-PCR together with ER alpha and ER beta protein by Western immunoblotting revealed substantial changes to ER alpha levels but very little alteration to ER beta levels following development of antioestrogen resistance. In oestrogen-maintained cells, tamoxifen resistance was associated with raised levels of ERa mRNA/protein. However by contrast, in oestrogen-deprived MCF7 cells, where oestrogen deprivation alone had already resulted in increased levels of ERa mRNA/protein, long-term tamoxifen exposure now reduced ER alpha levels. Whilst long-term exposure to fulvestrant reduced ERa. mRNA/protein levels in the oestrogen-maintained cells to a level barely detectable by Western immunoblotting and non-functional in inducing gene expression (ERE-LUC reporter or pS2), in oestrogen-deprived cells the reduction was much less substantial and these cells retained an oestrogen-induction of both the ERE-LUC reporter gene and the endogenous pS2 gene which could still be inhibited by antioestrogen. This demonstrates that whilst ER alpha can be abrogated by fulvestrant and increased by tamoxifen in some circumstances, this does not always hold true and mechanisms other than alteration to ER must be involved in the development of antioestrogen resistant growth. (c) 2006 Elsevier Ltd. All rights reserved.

The photodimerisation of the alpha- and beta-forms of trans-cinnamic acid: a study of single crystals by vibrational microspectroscopy

Infrared and Raman microspectroscopy have been used to follow the photodimerisation reactions of single crystals, the alpha- and beta-forms of trans-cinnamic acid. This approach allows the starting materials and products -alpha-truxillic acid that has C-i symmetry and beta-truxinic acid, which has C-s symmetry-to be identified. It also allows the topotactic nature of the reaction to be confirmed. Attempts to produce the poorly-defined unreactive gamma-form of trans-cinnamic acid resulted only in a mixture of the alpha- and beta-forms. The findings suggest a wide role for these spectroscopic methods in monitoring solid-state organic reactions. (C) 2002 Elsevier Science B.V. All rights reserved.

From alpha-cedrene to crinipellin B and onward: 25 years of the alkene-arene meta-photocycloaddition reaction in natural product synthesis

Wender and Howbert's remarkable synthesis of alpha-cedrene in 1981 brought the attention of the synthetic community to the alkene - arene meta-photocycloaddition reaction. Here we review the natural product syntheses that have been achieved, over the last 25 years, utilising this strategic level reaction.

21-Deoxycortisone (17 alpha-hydroxy4-pregnene-3,11,20-trione)

The title compound, C21H28O4, a synthetic glucocorticoid, crystallizes with a single molecule in the asymmetric unit. Ring A is almost in a half-chair conformation, rings B and C are almost in chair conformations, and ring D is between a twist and a 13 beta-envelope conformation. The A/B ring junction is quasi-trans, whereas the B/C and C/D ring junctions both approach trans characteristics. The molecule as a whole is slightly convex towards the beta side, with an angle of 9.60 (2)degrees between the C10-C19 and C13-C18 vectors. Molecular-packing and hydrogen-bonding (both intra- and inter-molecular) interactions play a major role in the structural association of the compound.

Cycloadditions of chiral carbonyl ylides with imine dipolarophiles as a route to enantiomerically pure alpha-amino-beta-hydroxy acids

The preparation of enantiomerically pure threo-beta-amino-alpha-hydroxy acids via 1,3-dipolar cycloadditions of imine dipolarophiles with the chiral isomunchnone derived from (5R)-5-phenylmorpholin-3-one 1 is described. The cycloadducts were obtained with excellent diastereofacial- and exo-selectivity. Subsequent hydrolysis and chemoselective exocyclic amide cleavage afforded the threo-beta-amino-alpha-hydroxy acids with recovery of the initial chiral auxiliary. (C) 2009 Published by Elsevier Ltd.

alpha-Aminoisobutyric acid modified protected analogues of beta-amyloid residue 17-20: a change from sheet to helix

The strong intermolecular interactions mediated by short hydrophobic sequences (e.g., 17-20, -L-Leu-L-Val-L-Phe-L-Phe-) in the middle of A beta are known to play a crucial role in the neuropathology of Alzheimer's disease. FTIR, TEM and Congo red binding studies indicated that a series of L-Ala substituted terminally protected peptides related to the sequence 17-20 of the beta-amyloid peptide, adopted D-sheet conformations. However, the Aib-modified analogues disrupt the D-sheet structure and switch over to a 310 helix with increasing number of Aib residues. X-ray crystallography shed some light on the change from sheet to helix at atomic resolution. (c) 2006 Elsevier Ltd. All rights reserved.

Organic acid formation in the gas-phase ozonolysis of alpha-pinene

The mechanism of formation of pinonic and norpinonic acids from alpha-pinene ozonolysis has been investigated by studying the products of the ozonolysis of an enone derived from alpha-pinene using gas chromatography coupled to mass spectrometry.

Gas-phase rate coefficients for the reactions of NO3, OH and O-3 with alpha,beta-unsaturated esters and ketones: structure-activity relations (SARs)

Gas-phase rate coefficients for the atmospherically important reactions of NO3, OH and O-3 are predicted for 55 alpha,beta-unsaturated esters and ketones. The rate coefficients were calculated using a correlation described previously [Pfrang, C., King, M.D., C. E. Canosa-Mas, C.E., Wayne, R.P., 2006. Atmospheric Environment 40, 1170-1179]. These rate coefficients were used to extend structure-activity relations for predicting the rate coefficients for the reactions of NO3, OH or O-3 with alkenes to include alpha,beta-unsaturated esters and ketones. Conjugation of an alkene with an alpha,beta-keto or alpha,beta-ester group will reduce the value of a rate coefficient by a factor of similar to 110, similar to 2.5 and similar to 12 for reaction with NO3, OH or O-3, respectively. The actual identity of the alkyl group, R, in -C(O)R or -C(O)OR has only a small influence. An assessment of the reliability of the SAR is given that demonstrates that it is useful for reactions involving NO3 and OH, but less valuable for those of O-3 or peroxy nitrate esters. (c) 2006 Elsevier Ltd. All rights reserved.

Noncompetitive plasma biokinetics of deuterium-labeled natural and synthetic alpha-tocopherol in healthy men with an apoE4 genotype

Previous studies comparing the biokinetics of deuterated natural (RRR) and synthetic (all-rac) α-tocopherol (vitamin E) used a simultaneous ingestion or competitive uptake approach and reported relative bioavailability ratios close to 2:1, higher than the accepted biopotency ratio of 1.36:1. The aim of the current study was to compare the biokinetics of deuterated natural and synthetic vitamin E using a noncompetitive uptake model both before and after vitamin E supplementation in a distinct population. Healthy men (n = 10) carrying the apolipoprotein (apo)E4 genotype completed a randomized crossover study, comprised of two 4-wk treatments with 400 mg/d (RRR-α-tocopheryl and all-rac-α-tocopheryl acetate) with a 12-wk washout period between treatments. Before and after each treatment period, the subjects consumed a capsule containing 150 mg deuterated α-tocopheryl acetate in either the PRR or all-rac form depending on their treatment regimen. Blood was obtained up to 48 h after ingestion, and tocopherols analyzed by LC/MS. After deuterated all-rac administration, plasma deuterated tocopherol maximum concentrations and area under the concentration vs. time curves (AUC) were lower than those following RRR administration. The RRR:all-rac ratios determined from the deuterated biokinetic profiles (maximum concentration; C-max) and AUCs were 1.35:1 &PLUSMN; 0.17 and 1.33:1 &PLUSMN; 0.18, respectively. The 4-wk supplementation with either PRR or all-rac significantly increased plasma a-tocopherol concentrations (P < 0.001), but decreased the plasma response to newly absorbed deuterated RRR or all-rac α-tocopherol. Using a noncompetitive uptake approach, the relative bioavailability of natural to synthetic vitamin E in apoE4 males was close to the currently accepted biopotency ratio of 1.36:1.

Roseisalinus antarcticus gen. nov., sp nov., a novel aerobic bacteriochlorophyll a-producing alpha-proteobacterium isolated from hypersaline Ekho Lake, Antarctica

A Gram-negative, aerobic to microaerophilic rod was isolated from 10 m depths of the hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica). The strain was oxidase- and catalase-positive, metabolized a variety of carboxylic acids and sugars and produced lipase. Cells had an absolute requirement for artificial sea water, which could not be replaced by NaCl. A large in vivo absorption band at 870 nm indicated production of bacteriochlorophyll a. The predominant fatty acids of this organism were 16:0 and 18:1omega7c, with 3-OH 10:0, 16:1omega7c and 18:0 in lower amounts. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylcholine. Ubiquinone 10 was produced. The DNA G + C content was 67 mol%. 16S rRNA gene sequence comparisons indicated that the isolate represents a member of the Roseobacter clade within the alpha-Proteobacteria. The organism showed no particular relationship to any members of this clade but clustered on the periphery of the genera Jannaschia, Octadecabacter and 'Marinosulfonomonas' and the species Ruegeria gelatinovorans. Distinct morphological, physiological and genotypic differences to these previously described taxa supported the description of a new genus and a novel species, for which the name Roseisalinus antarcticus gen. nov., sp. nov. is proposed. The type strain is EL-88(T) (= DSM 11466(T) = CECT 7023(T)).