Positioning of pyrimidine motifs around cassette exons defines their PTB-dependent splicing in Arabidopsis.

Alternative splicing (AS) is a complex process that generates transcript variants from a single pre-mRNA and is involved in numerous biological functions. Many RNA-binding proteins are known to regulate AS; however, little is known about the underlying mechanisms, especially outside the mammalian clade. Here, we show that polypyrimidine tract binding proteins (PTBs) from Arabidopsis thaliana regulate AS of cassette exons via pyrimidine (Py)-rich motifs close to the alternative splice sites. Mutational studies on three PTB-dependent cassette exon events revealed that only some of the Py motifs in this region are critical for AS. Moreover, in vitro binding of PTBs did not reflect a motif's impact on AS in vivo. Our mutational studies and bioinformatic investigation of all known PTB-regulated cassette exons from A. thaliana and human suggested that the binding position of PTBs relative to a cassette exon defines whether its inclusion or skipping is induced. Accordingly, exon skipping is associated with a higher frequency of Py stretches within the cassette exon, and in human also upstream of it, whereas exon inclusion is characterized by increased Py motif occurrence downstream of said exon. Enrichment of Py motifs downstream of PTB-activated 5' splice sites is also seen for PTB-dependent intron removal and alternative 5' splice site events from A. thaliana, suggesting this is a common step of exon definition. In conclusion, the position-dependent AS regulatory mechanism by PTB homologs has been conserved during the separate evolution of plants and mammals, while other critical features, in particular intron length, have considerably changed.

Multifactorial and Species-Specific Feedback Regulation of the RNA Surveillance Pathway Nonsense-Mediated Decay in Plants.

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that detects aberrant transcript features and triggers degradation of erroneous as well as physiological RNAs. Originally considered to be constitutive, NMD is now recognized to be tightly controlled in response to inherent signals and diverse stresses. To gain a better understanding of NMD regulation and its functional implications, we systematically examined feedback control of the central NMD components in two dicot and one monocot species. On the basis of the analysis of transcript features, turnover rates and steady-state levels, up-frameshift (UPF) 1, UPF3 and suppressor of morphological defects on genitalia (SMG) 7, but not UPF2, are under feedback control in both dicots. In the monocot investigated in this study, only SMG7 was slightly induced upon NMD inhibition. The detection of the endogenous NMD factor proteins in Arabidopsis thaliana substantiated a negative correlation between NMD activity and SMG7 amounts. Furthermore, evidence was provided that SMG7 is required for the dephosphorylation of UPF1. Our comprehensive and comparative study of NMD feedback control in plants reveals complex and species-specific attenuation of this RNA surveillance pathway, with critical implications for the numerous functions of NMD in physiology and stress responses.

Synthesis, characterisation and in vitro cytotoxicity of mixed ligand Pt(ii) oxadiazoline complexes with hexamethylenetetramine and 7-nitro-1,3,5-triazaadamantane.

trans-Platinum(ii) oxadiazoline complexes with 7-nitro-1,3,5-triazaadamantane (NO2-TAA) or hexamethylenetetramine (hmta) ligands have been synthesised from trans-[PtCl2(PhCN)2] via cycloaddition of nitrones to one of the coordinated nitriles, followed by exchange of the other nitrile by NO2-TAA or hmta. Stoichiometric control allows for the selective synthesis of mono- and dinuclear complexes where 7-NO2TAA and hmta act as mono- and bidentate ligands, respectively. Precursors and the target complexes trans-[PtCl2(hmta)(oxadiazoline)], trans-[PtCl2(NO2-TAA)(oxadiazoline)] and trans-[{PtCl2(oxadiazoline)}2(hmta)] were characterised by elemental analysis, IR and multinuclear (1H, 13C, 195Pt) NMR spectroscopy. DFT (B3LYP/6-31G*/LANL08) and AIM calculations suggest a stronger bonding of hmta with the [PtCl2(oxadiazoline)] fragment, in agreement with the experimentally observed reactivity in the ligand exchange (hmta > 7-NO2TAA). Replacement of the nitrile by hmta is predicted to be more exothermic than that with 7-NO2-TAA, although the activation barriers are similar. Protonation of the non-coordinated N atoms is anticipated to weaken the Pt-N bond and lower the activation barrier for ligand exchange. This effect might help activate these compounds in a slightly acidic environment such as some tumour tissues. Ten of the new compounds were tested for their in vitro cytotoxicity in the human cancer cell lines HeLa and A549. Some of the mononuclear complexes are more potent than cisplatin, and their activity is still high in A549 where cisplatin shows little effect. The dinuclear complexes are inactive, presumably due to their lipophilicity and reduced solubility in water.

Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells.

Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.

Synthesis of alkynyl-substituted camphor derivatives and their use in the preparation of paclitaxel-related compounds.

Compounds containing two alkyne groups in close vicinity at the rigid skeleton of camphorsulfonamide show unique reactivities when treated with electrophiles or catalytic amounts of platinum(II). The formed product structures depend not only on the reagents used but also on the substituents attached to the triple bonds. Cycloisomerisations with perfect atom economy lead to polycyclic heterocycles that resemble to some extent the AB ring system of paclitaxel. Herein, we present practical synthetic methods for the selective synthesis of precursor dialkynes bearing different substituents (alkyl, aryl) at the triple bonds, based on ketals or an imine as protecting groups. We show for isomeric dialkynes that the reaction cascade induced by Pt(II) includes ring annulation, sulphur reduction, and ring enlargement. One isomeric dialkyne additionally allows for the isolation of a pentacyclic compound lacking the ring enlargement step, which we have proposed as a potential intermediate in the catalytic cycle.

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Brg1 chromatin remodeling ATPase balances germ layer patterning by amplifying the transcriptional burst at midblastula transition.

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning.

Alternative Splicing Substantially Diversifies the Transcriptome during Early Photomorphogenesis and Correlates with the Energy Availability in Arabidopsis.

Plants use light as source of energy and information to detect diurnal rhythms and seasonal changes. Sensing changing light conditions is critical to adjust plant metabolism and to initiate developmental transitions. Here, we analyzed transcriptome-wide alterations in gene expression and alternative splicing (AS) of etiolated seedlings undergoing photomorphogenesis upon exposure to blue, red, or white light. Our analysis revealed massive transcriptome reprogramming as reflected by differential expression of ∼20% of all genes and changes in several hundred AS events. For more than 60% of all regulated AS events, light promoted the production of a presumably protein-coding variant at the expense of an mRNA with nonsense-mediated decay-triggering features. Accordingly, AS of the putative splicing factor REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1, previously identified as a red light signaling component, was shifted to the functional variant under light. Downstream analyses of candidate AS events pointed at a role of photoreceptor signaling only in monochromatic but not in white light. Furthermore, we demonstrated similar AS changes upon light exposure and exogenous sugar supply, with a critical involvement of kinase signaling. We propose that AS is an integration point of signaling pathways that sense and transmit information regarding the energy availability in plants.

Strecker degradation of amino acids promoted by a camphor-derived sulfonamide.

A camphor-derived sulfonimine with a conjugated carbonyl group, oxoimine 1 (O2SNC10H13O), reacts with amino acids (glycine, L-alanine, L-phenylalanine, L-leucine) to form a compound O2SNC10H13NC10H14NSO2 (2) which was characterized by spectroscopic means (MS and NMR) and supported by DFT calculations. The product, a single diastereoisomer, contains two oxoimine units connected by a -N= bridge, and thus has a structural analogy to the colored product Ruhemann´s purple obtained by the ninhydrin reaction with amino acids. A plausible reaction mechanism that involves zwitterions, a Strecker degradation of an intermediate imine and water-catalyzed tautomerizations was developed by means of DFT calculations on potential transition states.

Abiotic stress protection by ecologically abundant dimethylsulfoniopropionate and its natural and synthetic derivatives: insights from Bacillus subtilis.

Dimethylsulfoniopropionate (DMSP) is an abundant osmolyte and anti-stress compound produced primarily in marine ecosystems. After its release into the environment, microorganisms can exploit DMSP as a source of sulfur and carbon, or accumulate it as an osmoprotectant. However, import systems for this ecophysiologically important compatible solute, and its stress-protective properties for microorganisms that do not produce it are insufficiently understood. Here we address these questions using a well-characterized set of Bacillus subtilis mutants to chemically profile the influence of DMSP import on stress resistance, the osmostress-adaptive proline pool and on osmotically controlled gene expression. We included in this study the naturally occurring selenium analogue of DMSP, dimethylseleniopropionate (DMSeP), as well as a set of synthetic DMSP derivatives. We found that DMSP is not a nutrient for B. subtilis, but it serves as an excellent stress protectant against challenges conferred by sustained high salinity or lasting extremes in both low and high growth temperatures. DMSeP and synthetic DMSP derivatives retain part of these stress protective attributes, but DMSP is clearly the more effective stress protectant. We identified the promiscuous and widely distributed ABC transporter OpuC as a high-affinity uptake system not only for DMSP, but also for its natural and synthetic derivatives.

Polypyrimidine tract binding protein homologs from Arabidopsis are key regulators of alternative splicing with implications in fundamental developmental processes.

Alternative splicing (AS) generates transcript variants by variable exon/intron definition and massively expands transcriptome diversity. Changes in AS patterns have been found to be linked to manifold biological processes, yet fundamental aspects, such as the regulation of AS and its functional implications, largely remain to be addressed. In this work, widespread AS regulation by Arabidopsis thaliana Polypyrimidine tract binding protein homologs (PTBs) was revealed. In total, 452 AS events derived from 307 distinct genes were found to be responsive to the levels of the splicing factors PTB1 and PTB2, which predominantly triggered splicing of regulated introns, inclusion of cassette exons, and usage of upstream 5' splice sites. By contrast, no major AS regulatory function of the distantly related PTB3 was found. Dependent on their position within the mRNA, PTB-regulated events can both modify the untranslated regions and give rise to alternative protein products. We find that PTB-mediated AS events are connected to diverse biological processes, and the functional implications of selected instances were further elucidated. Specifically, PTB misexpression changes AS of PHYTOCHROME INTERACTING FACTOR6, coinciding with altered rates of abscisic acid-dependent seed germination. Furthermore, AS patterns as well as the expression of key flowering regulators were massively changed in a PTB1/2 level-dependent manner.

Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) and those for the choline-specific OpuB transporter (opuB) but not of that for the promiscuous OpuC transporter. GbsR acts genetically as a repressor and functions as an intracellular choline sensor. Spectroscopic analysis of the purified GbsR protein showed that it binds the inducer choline with an apparent K(D) (equilibrium dissociation constant) of approximately 165 µM. Based on the X-ray structure of a protein (Mj223) from Methanococcus jannaschii, a homology model for GbsR was derived. Inspection of this GbsR in silico model revealed a possible ligand-binding pocket for choline resembling those of known choline-binding sites present in solute receptors of microbial ABC transporters, e.g., that of the OpuBC ligand-binding protein of the OpuB ABC transporter. GbsR was not only needed to control gbsAB and opuB expression in response to choline availability but also required to genetically tune down glycine betaine production once cellular adjustment to high osmolarity has been achieved. The GbsR regulatory protein from B. subtilis thus records and integrates cellular and environmental signals for both the onset and the repression of the synthesis of the osmoprotectant glycine betaine.

Antihypertensive treatment and risk of dementia: a retrospective database study.

OBJECTIVE: Vascular risk factors play an important role in the pathogenesis of vascular dementia, as well as in Alzheimer disease. The effect of antihypertensive medication on risk of dementia is unclear. The aim was to investigate the association between antihypertensive prescriptions and incident dementia, using a primary care database. METHODS: The analysis was based on 575 general and internal practices in Germany (10/2003 - 09/2008) (Disease analyzer database). Antihypertensive medication (ATC codes) during 3 years before newly diagnosed dementia (ICD codes or specific medication) in 1,297 patients was compared to 1,297 controls without dementia after matching for age (mean age: 80.6 ± 8.6 y), sex (females: 62%) and date of diagnosis. Conditional logistic regression was used to calculate crude and adjusted odds ratios (95% confidence intervals). RESULTS: Betablocker prescriptions (≥ 1 per year over 3 y) showed a significant inverse association with newly diagnosed dementia (Odds ratio, OR: 0.79 95% CI 0.61 - 0.99) after adjusting for demographic covariates, health care use, and cardiovascular and neurological comorbidity. In the fully adjusted model, ACE inhibitors also tended to be inversely associated with incident dementia, but failed statistical significance (OR 0.84 95% CI 0.65 - 1.08). Calcium channel blockers were positively related to cognitive impairment only in the crude analysis. The other drug groups were not significantly related to dementia (diuretics OR: 0.89; 0.67 - 1.19; angiotensin- 1-inhibitors OR: 1.04; 0.66 - 1.64). CONCLUSIONS: This practice-based case-control study indicated a possible protective effect of some antihypertensive agents (betablockers, ACE-inhibitors) on the development of dementia. Randomized controlled trials are required to confirm this finding.

PlexinA1 interacts with PTK7 and is required for neural crest migration.

Members of the plexin protein family are known regulators of axon guidance, but recent data indicate that they have broader functions in the regulation of embryonic morphogenesis. Here we provide further evidence of this by showing that PlexinA1 is expressed in Xenopus neural crest cells and is required for their migration. PlexinA1 expression is detected in migrating cranial neural crest cells and knockdown of PlexinA1 expression using Morpholino oligonucleotides inhibits neural crest migration. PlexinA1 likely affects neural crest migration by interaction with PTK7, a regulator of planar cell polarity that is required for neural crest migration. PlexinA1 and PTK7 interact in immunoprecipitation assays and show phenotypic interaction in co-injection experiments. Considering that plexins and PTK7 have been shown to genetically interact in Drosophila axon guidance and chick cardiac morphogenesis, our data suggest that this interaction is evolutionary conserved and may be relevant for a broad range of morphogenetic events including the migration of neural crest cells in Xenopus laevis.

Polypyrimidine tract-binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control.

Alternative splicing (AS) of precursor mRNAs is a widespread phenomenon in plants; however, many questions, especially regarding its regulation and functional implications, remain to be elucidated. In vertebrates, polypyrimidine tract-binding proteins (PTBs) have been identified as key splicing factors influencing splice site selection and orchestrating coordinated splicing programmes during developmental processes. Here, we analysed three PTB homologues from Arabidopsis thaliana and provide evidence for their gene regulatory potential based on AS and a splicing-independent mechanism. Our data reveal that Arabidopsis PTB homologues are subject to extensive auto- and cross-regulation via AS-coupled nonsense-mediated decay, thereby establishing a basis for interlinking their expression. Furthermore, the multiple modes of action of Arabidopsis PTB homologues are reflected in their subcellular localization in the nucleus, cytosol and processing bodies. This work provides insight into the regulation of AS in plants and highlights the regulatory potential of the multifunctional plant PTB homologues, which might have important implications in diverse biological processes.

Design, synthesis, characterisation and chemical reactivity of mixed-ligand platinum(II) oxadiazoline complexes with potential cytotoxic properties.

A series of mixed ligand platinum(II) oxadiazoline complexes bearing 7-nitro-1,3,5-triazaadamantane (7-NO(2)TAA) as a labile and reactive nitrogen ligand has been synthesised from easily accessible starting materials. [2+3] cycloaddition of nitrones R(1)R(2)C-N(+)(Me)O(-) to only one of the nitrile ligands in trans-[PtX(2)(PhCN)(2)] (X = Cl, Br) results in the selective formation of mono-oxadiazoline complexes trans-[PtX(2)(PhCN){N=C(Ph)-O-N(Me)-CR(1)R(2)}] from which the remaining nitrile can be replaced by 7-NO(2)TAA. The resulting complexes trans-[PtX(2)(7-NO(2)TAA) {N=C(Ph)-O-N(Me)-CR(1)R(2)}] and their precursors were characterised by elemental analysis, IR and multinuclear NMR spectroscopy.The suitability of the target complexes as anticancer agents was extrapolated from their general chemical reactivity. They are stable in DMSO, but react with thiols and undergo aquation of a chloro ligand. In the absence of a competing ligand, the coordinated 7-NO(2)TAA ligand slowly hydrolyses in an aqueous medium under release of formaldehyde, and this could induce bioactivity independent of the one typically found with platinum compounds. With nitrogen heterocycles such as pyridine a slow exchange of the 7-NO(2)TAA ligand occurs. A combined DFT/AIM study confirms the reaction observed in the experiment and predicts that other nitrogen heterocycles such as DNA nucleobases should react in the same way. Moreover, the 7-NO(2)TAA should be even more labile in an aqueous medium where protonation of the remaining amines can occur. A PM6 molecular modelling study suggests that the PtCl(oxadiazoline) fragment formed after release of one chloro and the labile 7-NO(2)TAA ligand fits well into the DNA groove and is able to form d(GpG) intrastrand crosslinks similar to the ones observed with cisplatin.

Synthesis and characterisation of mixed ligand Pt(II) and Pt(IV) oxadiazoline complexes.

The nitrile ligands in trans-[PtX2(PhCN)2] (X = Cl, Br, I) undergo sequential 1,3 dipolar cycloadditions with nitrones R1R2C=N+(Me)-O(-) (R1 = H, R2 = Ph; R1 = CO2Et, R2 = CH2CO2Et) to selectively form the Delta4-1,2,4-oxadiazoline complexes trans-[PtX2(PhCN) (N=C(Ph)-O-N(Me)-CR1R2)] or trans-[PtX2(N=C(Ph)-O-N(Me)-CR1R2)2] in high yields. The reactivity of the mixed ligand complexes trans-[PtX2(PhCN)(N=C(Ph)-O-N(Me)-CR1R2)] towards oxidation and ligand substitution was studied in more detail. Oxidation with Cl2 or Br2 provides the Pt(IV) species trans-[PtX2Y2(PhCN)(N=C(Ph)-O-N(Me)-CH(Ph))] (X, Y = Cl, Br). The mixed halide complex (X = Cl, Y = Br) undergoes halide scrambling in solution to form trans-[PtX(4-n)Yn(PhCN)(N=C(Ph)-O-N(Me)-CH(Ph))] as a statistical mixture. Ligand substitution in trans-[PtCl2(PhCN)(N=C(Ph)-O-N(Me)-CR1R2)] allows for selective replacement of the coordinated nitrile by nitrogen heterocycles such as pyridine, DMAP or 1-benzyl-2-methylimidazole to produce mixed ligand Pt(II) complexes of the type trans- [PtX2(heterocycle)(N=C(Ph)-O-N(Me)-CR1R2)]. All compounds were characterised by elemental analysis, mass spectrometry, IR and 1H, 13C and 195Pt NMR spectroscopy. Single-crystal X-ray structural analysis of (R,S)-trans-[PtBr2(N=C(Ph)-O-N(Me)-CH(Ph))2] and trans-[PtCl2(C5H5N)(N=C(Ph)-O-N(Me)-CH(Ph))] confirms the molecular structure and the trans configuration of the heterocycles relative to each other.

Synthesis and characterization of platinum(II) oxadiazoline complexes and their in vitro antitumor activity in platinum-sensitive and -resistant cancer cell lines.

A series of platinum(II) complexes bearing Delta (4)-1,2,4-oxadiazoline ligands have been synthesized and characterized. Their in vitro antitumor activity has been assessed in platinum-sensitive and -resistant human ovarian cancer cell lines (PEO1, PEOCisR, PEOCarboR, and SK-OV3), as well as in colon cancer (SW948) and testicular cancer cell lines (N-TERA). All compounds tested showed potent cytotoxicity in the platinum-sensitive cell lines and retained activity in the cisplatin- and carboplatin-resistant lines, with IC 50 values similar to the parental drug sensitive counterpart. We propose, therefore, that platinum(II) oxadiazoline complexes may possess a novel mechanism of action, which render them active in tumor cells, with resistance to currently used platinum anticancer agents.

ESR and NMR spectroscopy studies on protein oxidation and formation of dityrosine in emulsions containing oxidised methyl linoleate.

Oxidised lipids are reported to interact with proteins causing undesirable changes in nutritional and functional properties including a loss of amino acids, cross-linking and damage to proteins and DNA. ESR spectroscopy with spin trapping was used to study the type of radical species generated in methyl linoleate and the transfer of the radical to protein beta-lactoglobulin. Antioxidants vitamins C and E reduced lipid oxidation and subsequent transfer of the radical to the protein as shown by the shape and size of the radical adduct. Changes to protein molecular structure due to oxidation were investigated by multidimensional NMR spectroscopy and liquid chromatography. NMR spectra indicated that as a result of oxidation and protein denaturation, there was an increase in structural flexibility and some initially protected backbone amide groups were exposed as they become sharper and easily identifiable. Dityrosine was detected in all samples tested which is indicative of oxidative damage to proteins. Monitoring tyrosyl radicals and formation of dityrosine is of practical value in order to enhance the acceptability, nutritional and safety aspects of proteins.

DFT-calculation of the structural isomers of (1-methyl-4-phenyl-1-azabuta-1,3-diene)tetracarbonyliron(0) and (4-phenyl-1-oxabuta-1,3-diene)tetracarbonyliron(0).

DFT calculations (B3LYP/LANL2DZ/6-31 G*) were used to investigate the ways in which 1-methyl-4-phenyl-1-azabuta-1,3-diene and 4-phenyl-1-oxabuta-1,3-diene bind to a Fe(CO)(4) moiety. As possible coordination modes, eta(2)-coordination across the C=C or C=N/C=O bond, sigma-coordination to the lone pair of the heteroatom, or eta(3)-coordination through the C=C-C or the N=C-C/O=C-C moiety were considered. The latter forms involve coupling of the non-coordinated atom of the heterodiene with one of the carbonyl ligands to an acyl species. The calculated geometric parameters of all structures compare well with X-ray crystallographic data of similar complexes. The species in which the ligand is transoid and sigma-coordinated is lowest in energy, for both compounds studied. However, the eta(2)-alkene bound 1-oxabuta-1,3-diene complex is practically equal in energy to the sigma-transoid form and thus competes. This agrees with experimental observations that the heterodiene is sigma-bonded in Fe(CO)(4)(1-methyl-4-phenyl-1-azabuta-1,3-diene) but eta(2)-coordinated in Fe(CO)(4)(4-phenyl-1-oxabuta-1,3-diene). The solvent dependence was estimated from single point PCM calculations, for CH(2)Cl(2) as solvent. For the 1-azabuta-1,3-diene complexes, the relative energies of eta(2)-olefin and eta(3)-allyl forms are inverted, with the eta(3)-allyl form being more stable in polar solvents. The 1-oxabuta-1,3-diene complexes in their eta(2)-olefin and sigma-O forms change order of relative energy, and conversion to the sigma-O form is expected in a polar medium for these complexes. Calculated IR vibrational stretching frequencies of the carbonyl ligands and the C[double bond, length as m-dash]N/C[double bond, length as m-dash]O bond were compared with experimental data, to produce the best fits for the sigma-transoid form of Fe(CO)(4)(1-methyl-4-phenyl-1-azabuta-1,3-diene) and eta(2)-olefin bonded Fe(CO)(4)(4-phenyl-1-oxabuta-1,3-diene). These results are again consistent with the experiment and show that the DFT method applied in this work can be used as an aid for structural validation.