Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

alpha-Tocopherol enhances tumour growth inhibition by cis-dichlorodiammine platinum (II)

Present studies indicate that alpha-tocopherol enhances the efficacy of cisplatin as demonstrated by inoculation of Dalton's lymphoma cells incubated with either cisplatin (5 or 10 µg/ml) alone or cisplatin + alpha-tocopherol (25 or 50 µg/ml) into C3H/He mice. Tumour cells (3 x 10(6) cells/mouse) incubated with cisplatin grow slowly in syngeneic mice as indicated by the late appearance of tumour. However, mice failed to develop tumour when inoculated with tumour cells incubated with cisplatin + alpha-tocopherol. When the animals were challenged with tumour cells (3 x 10(6) cells/mouse) on the 15th day after the initial inoculation, 30-50% survived more than 60 days, with 10% tumour-free survivors being observed in some groups. Antitumour activity was higher in mice receiving lymphoma cells (3 x 10(6) cells/mouse) preincubated with cisplatin + alpha-tocopherol compared to cisplatin alone. Tumour-bearing mice receiving cisplatin in combination with different concentrations of alpha-tocopherol exhibited significantly higher (P<0.001) intratumour platinum content (123-306%) but without any change in the kidney platinum content as compared to those receiving cisplatin (5 or 10 µg/ml) alone. Enhancement of cisplatin-induced tumour growth inhibition is probably due to the modulation of tumour cell membrane permeability by alpha-tocopherol. alpha-Tocopherol might increase the influx of cisplatin into tumour cells, causing the DNA repair machinery to be less efficient due to increased efficiency of adduct formation in the DNA molecule. This effect of alpha-tocopherol can render cisplatin more effective as an antitumour agent.

Towards the enantioselective synthesis of lycoricidine alkaloids /

Two efficient, regio- and stereo controlled synthetic approaches to the synthesis of racemic analogs of pancratistatin have been accomplished and they serve as the model systems for the total synthesis of optically active 7-deoxy-pancratistatin. In the Diels-Alder approach, an efficient [4+2] cycloaddition of 3,4-methylenedioxyco- nitrostyrene with Danishefsky's diene to selectively form an exo-nitro adduct has been developed as the key step in the construction of the C-ring of the target molecule. In the Michael addition approach, the key step was a conjugate addition of an organic zinc-cuprate to the 3,4-methylenedioxy-(B-nitrostyrene, followed by a diastereocontroUed closure to form the cyclohexane C-ring of the target molecule via an intramolecular nitro-aldol cyclization on a neutral alumina surface. A chair-like transition state for such a cyclization has been established and such a chelation controlled transition state can be useful in the prediction of diastereoselectivity in other related 6-exo-trig nitroaldol reactions. Cyclization of the above products fi^om both approaches by using a Bischler-Napieralski type reaction afforded two lycoricidine derivatives 38 and 50 in good yields. The initial results from the above modeling studies as well as the analysis of the synthetic strategy were directed to a chiral pool approach to the total synthesis of optically active 7-deoxy-pancratistatin. Selective monsilylation and iodination of Ltartaric acid provided a chiral precursor for the proposed key Michael transformation. The outlook for the total synthesis of 7-deoxy-pancratistatin by this approach is very promising.A concise synthesis of novel designed, optically pure, Cz-symmetrical disulfonylamide chiral ligands starting from L-tartaric acid has also been achieved. This sequence employs the metallation of indole followed by Sfj2 replacement of a dimesylate as the key step. The activity for this Cz-symmetric chiral disulfonamide ligand in the catalytic enantioselective reaction has been confirmed by nucleophilic addition to benzaldehyde in the disulfonamide-Ti (0-i-Pr)4-diethylzinc system with a 48% yield and a 33% e.e. value. Such a ligand tethered with a suitable metal complex should be also applicable towards the total synthesis of 7-deoxy-pancratistatin.

NMR studies of the exchange reactions of CH3CN.BX3 with excess CH3CN /|nJoseph Fogelman. -- 260 St. Catharines, Ont. : [s. n.],

Exchange reactions between molecular complexes and excess acid or base are well known and have been extensively surveyed in the literature(l). Since the exchange mechanism will, in some way involve the breaking of the labile donor-acceptor bond, it follows that a discussion of the factors relating to bonding in molecular complexes will be relevant. In general, a strong Lewis base and a strong Lewis acid form a stable adduct provided that certain stereochemical requirements are met. A strong Lewis base has the following characteristics (1),(2) (i) high electron density at the donor site. (ii) a non-bonded electron pair which has a low ionization potential (iii) electron donating substituents at the donor atom site. (iv) facile approach of the site of the Lewis base to the acceptor site as dictated by the steric hindrance of the substituents. Examples of typical Lewis bases are ethers, nitriles, ketones, alcohols, amines and phosphines. For a strong Lewis acid, the following properties are important:( i) low electron density at the acceptor site. (ii) electron withdrawing substituents. (iii) substituents which do not interfere with the close approach of the Lewis base. (iv) availability of a vacant orbital capable of accepting the lone electron pair of the donor atom. Examples of Lewis acids are the group III and IV halides such (M=B, AI, Ga, In) and MX4 - (M=Si, Ge, Sn, Pb). The relative bond strengths of molecular complexes have been investigated by:- (i) (ii) (iii) (iv) (v] (vi) dipole moment measurements (3). shifts of the carbonyl peaks in the IIIR. (4) ,(5), (6) .. NMR chemical shift data (4),(7),(8),(9). D.V. and visible spectrophotometric shifts (10),(11). equilibrium constant data (12), (13). heats of dissociation and heats of reactions (l~), (16), (17), (18), (19). Many experiments have bben carried out on boron trihalides in order to determine their relative acid strengths. Using pyridine, nitrobenzene, acetonitrile and trimethylamine as reference Lewis bases, it was found that the acid strength varied in order:RBx3 > BC1 3 >BF 3 • For the acetonitrile-boron trihalide and trimethylamine boron trihalide complexes in nitrobenzene, an-NMR study (7) showed that the shift to lower field was. greatest for the BB~3 adduct ~n~ smallest for the BF 3 which is in agreement with the acid strengths. If electronegativities of the substituents were the only important effect, and since c~ Br ,one would expect the electron density at the boron nucleus to vary as BF3adducts and the shift in the carbonyl I.R. stretch, Drago (22) et al have proposed that the angular di~tortion of the X-B-X bonds from sp2 (12 ) to sp3 (10 hybridization is proportional to the amount of charge transferred, i.e. to the nature of the base, and they have rejected the earlier concept of reorganization energy in explaining the formation of the adduct bond (19).

ESI-MS[n] of anticancer pt[iv] organoamido complexes and their interactions with DNA-model compounds /

The general solution behaviour and" the major fragmentation pathways of the anticanceractive PtIV coordination complexes, trans, trans, cis, cis-[PtCIOH{N(pFC6F4) CH2h(pY)2] (1), trans, cis, cis-[Pt(OH)2{N(p-FC6F4)CH2h(Py)2] (2), trans, cis, cis-[Pt(OH)2{N(p-HC6F4)CH2h(Py)2] (3), trans, trans, cis, cis-[PtCIOH{N(pHC6F4) CH2h(Py)2] (4), and trans, trans, cis, cis-[PtOH(OCH3){N(p-HC6F4)CH2h(PY)2] (5) (Py = pyridine) have been deduced by positive-ion tandem-in-time ESI-MS. Overall, the acquired full-scan, positive-ion ESI-MS spectra of 2, 3, and 5 were characterized by the presence of relatively low-intensity [M+Nar and [M+Kt mass spectral peaks, whereas those of 1 and 4 were dominated by extremely intense [M+Hr peaks. Complexes 2 and 3 were also noted to form [2M+Ht and [2M+Nat dilneric cations. The source of Na + and K+ ions is believed to be the sample, the solvent systems used or the transport line carrying the sample solutions into the ES ion source. Further, the fragmentation pathway of all complexes studied was found to be almost identical with concurrent loss of py and H20 molecules, loss of a {N(p-YC6F4)CH2} (Y = F, H) group and/or concomitant release of the latter group and a py ligand being the most conunon. The photochemical degradation behaviour of 1 and 2 was also investigated using either fluorescent or ultraviolet light and some products of that degradation were positively identified. Altogether, light irradiation of solutions of both complexes resulted in cation cationisation, reductive-elimination, ligand-release, ligand-exchange and ligand-addition reactions. Finally, positive- and negative-ion ESI-MSn spectra of 5' -GMP, guanosine, inosine and products of their reactions with 1, 2,3, and 4 were also recorded. On the whole, full-scan ESI-MS spectra of the pure nucleobases revealed the presence of cationic and anionic species that are highly reflective of both their solution ionic composition and their propensity t9 form polymeric clusters. Analyses of mass spectra acquired from their reaction solutions with the aforementioned platinum complexes indicated very slow kinetics. However, all complexes investigated formed, to various degrees, Pt-nucleobase adducts with guanosine and inosine, but not with 5'-GMP. The products included species having coordination numbers of III, IV, V, and VI, among which the first-time· observed, coordinatively saturated, jive-coordinate PtlI-nucleobase complexes were of most interest. The latter complexes are presumably stabilized by 7tback- donation involving the filled d orbitals of the PtII centre and the empty pz· orbital of MeCN. All products, whose peaks appeared inlull-scan ESI-MS spectra, are believed to represent solution species rather than artifacts of gas-phase processes. Finally, negativeion ESI-MSn spectra recorded in reaction solutions of 1 and 4 with guanosine and of the latter complex with inosine revealed the negative-ion-ESI-MS first-time observed, noncovalent, nucleoside-chloride adducts, with the source of chloride anion being complexes 1 and 4 theillselves. In contrast, no such adducts were observed to form with Na25'-GMP or its protonated fonn. Few suggestions are offered for the possible cause(s) behind the absence of such adduct ions.

Nuclear magnetic resonance studies of boron trihalide complexes of 1,1-BIS (dimethylamino) ethylene and related bases

Boron tribalide complexes of 1,1-bis(dimethylamino)ethylene (DME) , t etramethylurea (TMU), tetramethylguanidine (TMG) , and pentamethylguanidine (PMG) and also mixed boron t r ihalide adducts of DME have been investigated by 1H and 19F NMR spectroscopy. Both nitrogen and the C-Q-H carbon of DME are possible donor a toms to boron trihal ides but complexation has been found to occur only at carbon of DME. The initial adduct acts as a Bronsted acid and gives up a proton to free DME in solut ion. A side reaction in the DME-BF, system gives rise to trace amounts of a complex aSSigned as (DME)2BF2+. (DME)2BF2+ is produced in much larger quantities in t he DME-BF3-BC13 and DME-BF,-BBr, systems by reaction of free DME with DME:BF2X (X = Cl, Br). Restricted r otation about the C-N bonds of TMUlBC13 and n1U:BBr3 has been observed at low temperatures. This complements previous work in this system and confirms oxygen donation of TMU to boron trihalides . Restricted rotation at low temperatures also has been observed in DMEboron trihalide systems

The mechanism of formation of the mixed boron trihalide adducts of trimenthylamine

Boron trihalide and mixed boron trihalide adducts of trimethylamine have been prepared, and characterized by proton and fluorine N.M.R. spectroscopy. The acceptor power of the boron trihalides was seen to increase in the order BF3 < BC13 < BBr3 < BI3, corroborating previous evidence. The mixed boron trihalides had intermediate Lewis acidities. Solution reactions between adducts and free boron trihalides rapidly led to the formation of mixed adducts when the free boron trihalide is a stronger Lewis acid than that in the adduct. A slower reaction is observed when the free BX3 is a weaker Lewis aoid than that complexed. The mechanism of halogen exchange leading to the mixed (CH3)3NBX3 adducts was investigated. 10B labelling experiments precluded B-N bond rupture as a possible mechanism in solution; results are discussed in terms of halogen-bridged intermediates. Pre-ionization may be important for some systems. At higher temperatures, during gas phase reactions,B-N coordinate bond rupture may be the initial step of reaction. Two mixed adduots, namely (CH3)3NBClBr2 and (CH3)3NBHOIBr were prepared and characterized by Mass Spectrometry

Synthèse diastéréosélective et énantiosélective de dérivés cyclopropaniques 1,2,3-substitués à l’aide de carbénoïdes gem-dizinciques

Les dérivés cyclopropaniques 1,2,3-substitutés sont des composés intéressants dans de nombreux domaines de la chimie. Au cours de cet ouvrage, nous nous sommes intéressés à la synthèse, tout d’abord diastéréosélective puis énantiosélective de ces composés. Nous nous sommes en particulier intéressés à l’utilisation de la zinciocyclopropanation pour l’obtention de ces dérivés cyclopropaniques 1,2,3-substitutés. Cette méthode consiste en l’utilisation d’un carbénoïde gem-dizincique pour effectuer une réaction de type Simmons-Smith. Cette stratégie a l’avantage d’être diastéréosélective favorisant la formation du zinciocyclopropane dont l’atome de zinc est dans une configuration cis avec le groupement directeur qu’est l’oxygène allylique basique. Lors de cette réaction, l’existence d’une réaction compétitive avec un réactif monozincique diminuait l’utilité de la zinciocyclopropanation. L’issue de la réaction s’est avérée dépendre fortement de la nature du carbénoïde utilisé, de la température réactionnelle et de la présence de ZnI2 dans le milieu. L’étude par GCMS de nombreuses conditions pour la formation des différents carbénoïdes a permis d’identifier les paramètres clés conduisant à la zinciocyclopropanation. Ces découvertes ont notamment permis d’étendre la réaction de zinciocyclopropanation aux alcools allyliques portant un seul groupement directeur (ie. non dérivé du 1,4-buténediol). Dans ces conditions, la réaction s’est avérée extrêmement diastéréosélective favorisant la formation du zinciocyclopropane dont l’atome de zinc est dans une configuration cis avec le groupement directeur. Afin de fonctionnaliser les zinciocyclopropanes ainsi obtenus, plusieurs réactions de fonctionnalisation in situ ont été développées. Chacune de ces méthodes a montré une conservation totale de la diastéréosélectivité obtenue lors de la réaction de zinciocyclopropanation. La versatilité de la zinciocyclopropanation a donc été démontrée. Avec une méthode diastéréosélective efficace pour la formation de zinciocyclopropanes à partir d’alcools allyliques ne portant qu’un seul groupement directeur, il est devenu possible d’envisager le développement énantiosélectif de la réaction. L’utilisation d’un dioxaborolane énantiopur a permis la zinciocyclopropanation avec de très bons excès énantiomères de divers alcools allyliques chiraux. La présence sur la même molécule d’un lien C–Zn nucléophile est d’un atome de bore électrophile a conduit à un échange bore-zinc in situ, formant un cyclopropylborinate énantioenrichi. La formation de ce composé bicyclique a permis d’obtenir une diastéréosélectivité parfaite. De nombreux alcools allyliques ont pu ainsi être convertis en cyclopropylborinates. Une réaction de Suzuki subséquente a permis la formation de dérivés cyclopropaniques 1,2,3-trisubstitués avec de très bons excès énantiomères et une excellente diastéréosélectivité. Les cyclopropylborinates obtenus à l’issue de la zinciocyclopropanation énantiosélective se sont avérés être des unités très versatiles puisque de nombreuses méthodes ont pu être développés pour leur fonctionnalisation.

Synthesis, properties and characterization of N-Alkyl substituted b-Diketiminato copper(I) Complexes

Le ligand nacnacxylH (xyl = C6Me2H3) et les ligands dikétimines N-alkyle substitués (nacnacCH(Me)PhH, nacnacBnH and nacnaciPrH) ont été préparés avec de bons rendements à l’exception du nacnaciPrH (23%) en utilisant un protocole en une étape et à l’aide d’un montage Dean-Stark. La réaction du S,S-nacnacCH(Me)PhH et du nacnacBnH avec le nBuLi dans le THF conduit au S,S-nacnacCH(Me)PhLi(THF) et au nacnacBnLi(THF). Les tentatives de bromation de ces composés par le N-bromosuccinimide conduisent plutôt aux ligands S,S-succnacnacCH(Me)PhH et succnacnacBnH (succ = succinimido) substitués par un groupement succinimido sur le carbone  La chloration par le N-chlorosuccinimide conduit au produit désiré, mais avec des impuretés. La réaction de ces ligands avec le CuOtBu (ou bien MesCu, où Mes = C6Me3H2, et une quantité catalytique de CuOtBu) en présence de bases de Lewis donne les (nacnacxylCu)2(-toluène), nacnacxylCuCNC6H3(Me)2, nacnacCH(Me)PhCuL (L = PPh3, PMe3, CNC6H3(Me)2, DMAP, lutidine, Py, MeCN), nacnacBnCuL (L = PPh3, CNC6H3(Me)2, styrène, trans-stilbene, phenylvinylether, acrylonitrile, diphenylacetylène), nacnaciPrCuL (L = PPh3, CNC6H3(Me)2, MeCN) et le succnacnacCH(Me)PhCuL (PPh3, CNC6H3(Me)2, pyridine). Tous ces complexes sont jaunes et sensibles à l’air et à l’humidité. En l’absence de fortes bases de Lewis, on n’observe pas de réaction entre les précurseurs de cuivre et les ligands N-alkyle substitués. Les études RMN des complexes dans le C6D6 ne présentent pas de complexe de toluène mais un mélange à l’équilibre du (nacnacxylCu)2(-C6D6) et nacnacxylCu(C6D6) dans une proportion de 2 pour 1. Alors que l’addition de plus de cinquante équivalents soit de THF, soit de toluène n’induit aucun changement des spectres RMN, l’addition de 2 équivalents de MeCN conduit instantanément au complexe nacnacxylCu(MeCN). De plus, le (nacnacxylylCu)2(-C6D6) ne se coordone ni ne réagit avec le N2O, même après avoir été chauffé à 60°C pendant treize jours. En présence de DPA (diphenylacétylène), la réaction du nacnacBnH avec le CuOtBu conduit au dimère ponté (nacnacBnCu)2(µ-DPA). L’addition d’un excès de DPA (10-12 équivalents) transforme le dimère ponté en complexe lié en position terminale nacnacBnCuDPA. Les nacnacRH (R = CH(Me)Ph et i-Pr) ne forment pas de complexe ni avec les oléfines ni avec le DPA. Une réactivité similaire a été observée avec les complexes de nacnacCH(Me)PhCu(NCMe) et nacnaci-PrCu(NCMe). Tandis que le complexe lié en position terminale par MeCN a été isolé et caractérisé, l’équilibre en solution nous laisse suspecter la formation d’un complexe d’acétonitrile ponté. Des études de réactivité comparatives ont été menées sur quelques complexes de cuivre. La Morpholine ne réagit pas avec le nacnacBnCu(acrylonitrile) contrairement à l’acrylonitrile libre. L’expérience de l’échange d’oléfine montre que l’acrylonitrile (une oléfine électro-attractrice) se lie plus fortement que les autres oléfines, mettant ainsi en évidence l’importance de la rétrodonation  face à la donation La rétrodonation est cependant faible comparée aux autres complexes de styrène structurellement caractérisés. Les complexes nacnacCH(Me)PhCuL (L = PPh3 et MeCN) ont été employés dans la cyclopropanation catalytique du styrène et dans l’addition conjuguée du ZnEt2 sur la 2-cyclohexénone, mais les résultats indiquent que le ligand dikétimine est éliminé avant son entrée dans le cycle catalytique. Par conséquent, il n’y a pas d’induction chirale. Les complexes tétra coordinées de cuivre avec les nacnacRCu(phen) (R = Bn, CH(Me)Ph et Phen = 1,10-phenanthroline, 2-Mes-1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline (dmp) et 2,9-diphenyl-1,10-phenanthroline (dpp)) ont été synthétisés. Ces complexes sont d’une intense couleur bleue et des interactions d’empilement entre l’un des cycles phényle des ligands nacnac et la phénanthroline ont été observées dans les structures à l’état solide. Les mesures en absorption UV-visible ont été effectuées dans le toluène et les bandes MLCT sont déplacées vers le rouge par rapport à celles des complexes de cuivre et bisphénanthroline. Tous ces composés émettent à l’état solide mais les complexes 1,10-phenanthroline et 2-Mes-1,10-phenanthroline n’émettent pas en solution. Pour renforcer les interactions d’empilement , les nouveaux ligands nacnacRH (R = CH2C6H2(OMe)3, CH2C6F5) et leurs complexes de cuivre respectifs ont été préparés avec du dmp et dpp. Afin de permettre la comparaison, le nacnaciBuCu(dmp) a été synthétisé. Alors que les complexes dmp montrent une augmentation des interactions intramoléculaires - avec les substituants phényle du ligand dikétimine et de la phénanthroline, les complexes dpp ne révèlent pas de telles interactions. Les complexes perfluorés montrent, en absorption et en émission, un déplacement significatif vers le bleu, alors que les complexes substitués par un groupements isobutyle présentent des transitions déplacées vers le rouge. Alors que les intensités de luminescence et les durées de vie sont faibles, les déplacements réduits de Stokes et les pics étroits de luminescence comparables indiquent une réduction des distorsions de l’état excité.

Évaluation de la cytogénotoxicité humaine induite par l’exposition à de faibles doses de benzo-a-pyrène, à l’aide de biomarqueurs précoces

Le benzo-a-pyrène (BaP) est un hydrocarbure aromatique polycyclique (HAP) cancérogène pour l’homme, qui contamine toutes les sphères de notre environnement. Son métabolite, le BaP-7,8-diol-9,10-époxyde (BPDE) est considéré comme son cancérogène ultime. Le BPDE se lie à l’ADN, formant des adduits qui doivent être réparés et qui seraient responsables des dommages à l’ADN et de la cancérogenèse induite par le BaP. Les adduits BPDE-ADN et les dommages à l’ADN (bris simple-brin [BSB] à l’ADN, aberrations chromosomiques [AC], échanges entre chromatides-sœurs [ÉCS] et micronoyaux [MN]) ont été mesurés dans les lymphocytes humains exposés à de faibles concentrations de BaP, provenant de jeunes volontaires non-fumeurs et en santé. Suite à l’exposition au BaP, le niveau d’adduits BPDE-ADN et la fréquence des AC et des MN augmentent significativement, puis diminuent aux concentrations les plus élevées de BaP testées, suggérant une induction du métabolisme de phase II du BaP. Lors de la mesure des ÉCS, nous obtenons une courbe dose-réponse linéaire, indiquant la production d’un autre type de lésions devant être réparées par le système de réparation par recombinaison homologue. Ces lésions pourraient être des bris à l’ADN ou des bases oxydées (8-OH-dG), ce qui est suggéré par l’analyse des corrélations existant entre nos biomarqueurs. Par ailleurs, la comparaison de la courbe dose-réponse des hommes et des femmes montre que des différences existent entre les sexes. Ainsi, les ÉCS, les AC et les MN sont significativement augmentés chez les hommes à la plus faible concentration de BaP, alors que chez les femmes cette augmentation, quoique présente, est non significative. Des différences interindividuelles sont également observées et sont plus importantes pour les adduits BPDE-ADN, les MN et les AC, alors que pour les ÉCS elles sont minimes. Les analyses statistiques effectuées ont permis d’établir que quatre facteurs (niveau d’exposition au BaP, adduits BPDE-ADN, fréquence des AC et nombre de MN par cellule micronucléée) expliquent jusqu’à 59 % de la variabilité observée dans le test des ÉCS, alors qu’aucun facteur significatif n’a pu être identifié dans le test des AC et des MN. L’analyse du mécanisme de formation de nos biomarqueurs précoces permet de suggérer que les bris à l’ADN et les bases oxydées devraient être classées comme biomarqueurs de dose biologique efficace, au sein des biomarqueurs d’exposition, dans le continuum exposition-maladie du BaP, étant donné qu’ils causent la formation des biomarqueurs de génotoxicité (ÉCS, AC et MN). Par ailleurs, le test des AC et des MN ont permis de confirmer l’action clastogénique du BaP en plus de mettre en évidence des effets aneugènes affectant surtout la ségrégation des chromosomes lors de la division cellulaire. Ces effets aneugènes, reliés à l’étape de progression dans la cancérogenèse, pourraient être particulièrement importants puisque l’exposition au BaP et aux HAP est chronique et dure plusieurs années, voire des décennies. La compréhension des mécanismes régissant la formation des biomarqueurs étudiés dans cette étude, ainsi que des relations existant entre eux, peut être appliquée à de nombreux contaminants connus et émergents de notre environnement et contribuer à en évaluer le mode d’action.

Palladium Catalyzed Carbon-Carbon/Carbonheteroatom Bond Formation Reactions Utilizing Pentafulvene Derived Bicyclic Hydrazines

The reactions involving fulvenes and its derivatives have received a great deal of attention over the years in synthetic organic chemistry. Functionalizations of fulvenes provide versatile and powerful approaches to various polycyclic systems and natural products. They serve as versatile intermediates in the construction of various ring systems through inter- as well as intramolecular cycloadditions. Compared to the rich literature on the cycloaddition reactions of pentafulvenes, much less attention has been paid to the synthetic utilization of their cycloadducts. Tactical manipulations on the chosen adduct offer the prospects for designing a variety of useful molecular skeletons. Addition of heterodienophiles to fulvenes offers an efficient strategy towards the synthesis of azabicyclic olefins. However, there have been no serious attempts to study the synthetic utility of these substrates. In this context and with the intention of utilizing pentafulvenes towards synthetically important molecules, author decided to explore the reactivity of pentafulvene derived azabicyclic olefins. Our attention was focused on the synthetic potential associated with the ring opening of fulvene derived bicyclic hydrazines under palladium catalysis. It was envisioned that the desymmetrization of these adducts using various soft nucleophiles will provide a novel access to synthetically and biologically important alkylidene cyclopentenes. The investigations along this line form the focal theme of this thesis entitled “PALLADIUM CATALYZED CARBONCARBON/ CARBON-HETEROATOM BOND FORMATION REACTIONS UTILIZING PENTAFULVENE DERIVED BICYCLIC HYDRAZINES

Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

Molecular mechanism involved in the mutagenicity induced by Aflatoxin B1

The aflatoxin B1 (AFB1) is a mycotoxin that has been identified as the most potent hepatocarcinogen. The metabolite resulting from detoxification process of AFB1 in liver, has the ability to react with the genomic DNA, generating AFB1-DNA adducts; during DNA replication process, this adduct induced the G:C→T:A transversion. Polymorphism in genes encoding for enzymes involved in the activation and detoxification of AFB1 and DNA repair enzymes has been associated with the risk of hepatocellular carcinoma (HCC) development. Additionally, in populations of high exposure to aflatoxin and high prevalence of hepatitis B virus (HBV) infection, has been demonstrated a synergism between these two risk factors for the development of HCC.

Genotipificación de HLA-B en pacientes colombianos afectados por el síndrome Stevens-Johnson y la Necrólisis Epidérmica Tóxica

Las reacciones alérgicas a medicamentos cutáneas severas (RAM) como el Síndrome Stevens Johnson (SJS) y la Necrólisis Epidérmica Tóxica (NET),caracterizadas por exantema, erosión de la piel y las membranas mucosas, flictenas, desprendimiento de la piel secundario a la muerte de queratinocitos y compromiso ocular. Son infrecuentes en la población pero con elevada morbi-mortalidad, se presentan luego de la administración de diferentes fármacos. En Asia se ha asociado el alelo HLA-B*15:02 como marcador genético para SJS. En Colombia no hay datos de la incidencia de estas RAM, ni de la relación con medicamentos específicos o potenciales y tampoco estudios de aproximación genómica de genes de susceptibilidad.

Liquid ultraviolet matrix-assisted laser desorption/ionization - mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet -- matrix-assisted laser desorption/ionisation -- mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. The low-femtomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and low-mass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.