Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

6-Dimethylaminopurine and cyclohexamide are mutagenic and alter reproductive performance and intrauterine development in vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemProA (R) medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Our data show that Ca2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

Highly Substituted Isoxazoles: The Baylis-Hillman reaction of substituted 4-isoxazolecarbaldehydes and attempted cyclization to isoxazole-annulated derivatives

In an attempt to understand the effect of position of the formyl group on the efficiency of Baylis-Hillman reaction within isoxazolecarboxaldehydes, the reactions of substituted 4-isoxazolecarboxaldehydes to obtain highly substituted isoxazoles are described. Attempts to obtain isoxazole-annealed derivatives from these Baylis-Hillman adducts involving SNR’-SNAr substitu-tion strategy are also described.

Facile Baylis-Hillman reaction of substituted 3-isoxazolecarbaldehydes: The impact of proximal heteroatom within a heterocycle on the acceleration of reaction

The fast and facile Baylis-Hillman reaction in substi-tuted 3-isoxazolecarbaldehydes confirms the impact of the proxi-mal heteroatom within a heterocycle towards enhanced reactivity of the formyl group for this reaction

Production of triploid Kuruma shrimp, Marsupenaeus (Penaeus) japonicus (Bate) nauplii through inhibition of polar body I, or polar body I and II extrusion using 6-dimethylaminopurine

This study investigated the chromosome ploidy level of Marsupenaeus (Penaeus) japonicus (Bate) non-viable (unhatched) embryos and nauplii after exposure to 6-dimethylaminopurine (6-DMAP), timed to stop either polar body (PB) I, or PBI and II extrusion. Embryos from eight separate families or spawnings were exposed to 150 or 200 mu M 6-DMAP from 1- to 3-min post-spawning detection (psd) for a 4- to 5-min duration (timed to stop PBI extrusion). Separate aliquots of embryos from five of the same spawnings were also exposed to 200 mu M of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). For one spawning, a third aliquot of embryos was exposed to 400 p M of 6-DMAP from 1- to 3-min psd for a 16-min duration (timed to stop both PBI and II extrusion). At 18-h psd, non-viable embryo and nauplii samples were taken separately for fluorescent activated cell sorting (FACS). FACS revealed that there were diploids and triploids among all treated non-viable embryos and nauplii. All control non-viable embryos and nauplii were diploid. Percentages of triploid induction for the 4- to 5-min and 16-min durations were not significantly different (P > 0.05). Additionally, no difference was found in the triploidy level of nonviable embryos compared to nauplii in these treatments. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 4- to 5-min duration ranged from 29.57% to 99.23% (average 55.28 +/- 5.45%) and from 5.60% to 98.85% (average 46.70 +/- 7.20%), respectively. The percentage of triploid embryos and nauplii when exposed to 6-DMAP for a 16-min duration ranged from 11.71% to 98.96% (average 52.49 +/- 11.00%) and from 47.5% to 99.24% (average 79.38 +/- 5.24%), respectively. To our knowledge, this is the first documentation of successful PBI or PBI and II inhibition in shrimp. This study conclusively shows that treatment of M. japonicus embryos with 6-DMAP at 1- to 3-min pscl for either a 4- to 5-min duration (timed to stop PBl extrusion) or 16-min duration (timed to stop both PBI and II extrusion) results in viable triploid nauplii. (c) 2006 Elsevier B.V. All rights reserved.

Novel materials for membrane separation processes

The aim of this work was to synthesise a series of hydrophilic derivatives of cis-1,2-dihydroxy-3,5-cyclohexadiene (cis-DHCD) and copolymerise them with 2-hydroxyethyl methacrylate (HEMA), to produce a completely new range of hydrogel materials. It is theorised that hydrogels incorporating such derivatives of cis-DHCD will exhibit good strength and elasticity in addition to good water binding ability. The synthesis of derivatives was attempted by both enzymatic and chemical methods. Enzyme synthesis involved the transesterification of cis-DHCD with a number of trichloro and trifluoroethyl esters using the enzyme lipase porcine pancreas to catalyse the reaction in organic solvent. Cyclohexanol was used in initial studies to assess the viability of enzyme catalysed reactions. Chemical synthesis involved the epoxidation of a number of unsaturated carboxylic acids and the subsequent reaction of these epoxy acids with cis-DHCD in DCC/DMAP catalysed esterifications. The silylation of cis-DHCD using TBDCS and BSA was also studied. The rate of aromatisation of cis-DHCD at room temperature was studied in order to assess its stability and 1H NMR studies were also undertaken to determine the conformations adopted by derivatives of cis-DHCD. The copolymerisation of diepoxybutanoate, diepoxyundecanoate, dibutenoate and silyl protected derivatives of cis-DHCD with HEMA, to produce a new group of hydrogels was investigated. The EWC and mechanical properties of these hydrogels were measured and DSC was used to determine the amount of freezing and non-freezing water in the membranes. The effect on EWC of opening the epoxide rings of the comonomers was also investigated

Synthèse de sulfilimines et de sulfoximines catalysée par les métaux de transition

Les sulfilimines et les sulfoximines sont des motifs structuraux dont l’intérêt synthétique est grandissant, notamment du fait de leurs applications en chimie médicinale et en agrochimie. Les travaux rapportés dans cet ouvrage décrivent le développement de nouvelles méthodes de synthèse efficaces pour la production de ces unités atypiques. Ces méthodes sont basées sur la réactivité d’une source d’azote électrophile, vis-à-vis de thioéthers et de sulfoxydes. L’utilisation d’un complexe métallique introduit en quantité catalytique a permis de favoriser le processus réactionnel. En tirant bénéfice de l’expertise de notre groupe de recherche sur le développement de réactions d’amination stéréosélectives de liaisons C-H et d’aziridination de styrènes, nous avons d’abord étudié la réactivité des N-mésyloxycarbamates comme source d’azote électrophile. Après avoir optimisé sa synthèse sur grande échelle, ce réactif chiral a été utilisé dans des réactions d’amination de thioéthers et de sulfoxydes, catalysées par un dimère de rhodium (II) chiral. Un processus diastéréosélectif efficace a été mis au point, permettant de produire des sulfilimines et des sulfoximines chirales avec d’excellents rendements et sélectivités. Au cours de l’optimisation de cette méthode de synthèse, nous avons pu constater l’effet déterminant de certains additifs sur la réactivité et la sélectivité de la réaction. Une étude mécanistique a été entreprise afin de comprendre leur mode d’action. Il a été observé qu’une base de Lewis telle que le 4-diméthylaminopyridine (DMAP) pouvait se coordiner au dimère de rhodium(II) et modifier ses propriétés structurales et redox. Les résultats que nous avons obtenus suggèrent que l’espèce catalytique active est un dimère de rhodium de valence mixte Rh(II)/Rh(III). Nous avons également découvert que l’incorporation de sels de bispyridinium avait une influence cruciale sur la diastéréosélectivité de la réaction. D’autres expériences sur la nature du groupe partant du réactif N-sulfonyloxycarbamate nous ont permis de postuler qu’une espèce nitrénoïde de rhodium était l’intermédiaire clé du processus d’amination. De plus, l’exploitation des techniques de chimie en débit continu nous a permis de développer une méthode d’amination de thioéthers et de sulfoxydes très performante, en utilisant les azotures comme source d’azote électrophile. Basée sur la décompositon photochimique d’azotures en présence d’un complexe de fer (III) simple et commercialement disponible, nous avons été en mesure de produire des sulfilimines et des sulfoximines avec d’excellents rendements. Le temps de résidence du procédé d’amination a pu être sensiblement réduit par la conception d’un nouveau type de réacteur photochimique capillaire. Ces améliorations techniques ont permis de rendre la synthèse plus productive, ce qui constitue un élément important d’un point de vue industriel.

Greener methodology for the synthesis of alpha-diazocarbonyl compounds and a novel approach to dioxinone derivatives

This thesis outlines a more environmentally benign approach to diazo transfer, and the investigation of the reactivity of -diazocarbonyl compounds when subjected to transition metal and lanthanide catalysis. Extensive studies were carried out to find the optimum conditions for a greener diazo transfer methodology, and this was also applied to a continuous process for the synthesis of -diazo--ketoesters. The first chapter includes a literature review of the synthesis and subsequent reactivity of -diazocarbonyl compounds. An overview of the applications of flow chemistry for the synthesis of hazardous intermediates is also included. The applications of lanthanide catalysts in organic synthesis is also discussed. The second chapter outlines the extensive studies undertaken to determine the optimum conditions for a greener diazo transfer methodology, including base and solvent studies. Use of water as a viable solvent for diazo transfer was successfully investigated. Diazo transfer to a range of -diazo--ketoesters was achieved using 5 mol% triethylamine or DMAP in water with high conversions. Polystyrene-supported benzenesulfonyl azide as an alternative diazo transfer reagent was also explored, as well as investigations into cheaper generation of this safer reagent. This polymer-supported benzenesulfonyl azide was used with 25 mol% of base in water to achieve successful diazo transfer to a range of -diazo--ketoesters. The third chapter describes the application of the new methodology developed in Chapter 2 to a continuous processing approach. Various excellent conditions were identified for both batch and flow reactions. A series of -diazo--ketoesters were synthesised with excellent conversions using 25 mol% triethylamine in 90:10 acetone water using flow chemistry. Successful diazo transfer was also achieved using a polymer-supported benzenesulfonyl azide in water under flow conditions. The fourth chapter discusses the reactivity of -diazo--ketoesters under transition metal and lanthanide catalysis. This chapter describes the synthesis of a range of -ketoesters via transesterification, which were used to synthesise a range of novel -diazo--ketoesters that were used in subsequent decomposition reactions. A novel route to dioxinones via rhodium(II) catalysis is reported. Attempted OH and SH insertion reactions in the presence of various lanthanide(II) catalysts are outlined, leading to some unexpected and interesting rearrangement products. The experimental details, including spectroscopic and analytical data for all compounds prepared, are reported at the end of each chapter.

Kinetics and coverage dependent reaction mechanisms of the copper atomic layer deposition from copper dimethylamino-2-propoxide and diethylzinc

Atomic layer deposition (ALD) has been recognized as a promising method to deposit conformal and uniform thin film of copper for future electronic devices. However, many aspects of the reaction mechanism and the surface chemistry of copper ALD remain unclear. In this paper, we employ plane wave density functional theory (DFT) to study the transmetalation ALD reaction of copper dimethylamino-2-propoxide [Cu(dmap)2] and diethylzinc [Et2Zn] that was realized experimentally by Lee et al. [ Angew. Chem., Int. Ed. 2009, 48, 4536−4539]. We find that the Cu(dmap)2 molecule adsorbs and dissociates through the scission of one or two Cu–O bonds into surface-bound dmap and Cu(dmap) fragments during the copper pulse. As Et2Zn adsorbs on the surface covered with Cu(dmap) and dmap fragments, butane formation and desorption was found to be facilitated by the surrounding ligands, which leads to one reaction mechanism, while the migration of ethyl groups to the surface leads to another reaction mechanism. During both reaction mechanisms, ligand diffusion and reordering are generally endothermic processes, which may result in residual ligands blocking the surface sites at the end of the Et2Zn pulse, and in residual Zn being reduced and incorporated as an impurity. We also find that the nearby ligands play a cooperative role in lowering the activation energy for formation and desorption of byproducts, which explains the advantage of using organometallic precursors and reducing agents in Cu ALD. The ALD growth rate estimated for the mechanism is consistent with the experimental value of 0.2 Å/cycle. The proposed reaction mechanisms provide insight into ALD processes for copper and other transition metals.

Síntese de novos N-acilaminoácidos graxos: estudo das propriedades gelificantes

Neste trabalho é descrita a síntese de novos N-acilaminoácidos e Nacilaminoésteres graxos derivados de ácidos graxos saturados e insaturados como, por exemplo, esteárico e ricinoléico, respectivamente. Após, foi avaliada a capacidade destes compostos para a gelificação de hidrocarbonetos e as propriedades dos géis formados foram estudadas pelas técnicas de Espectroscopia de Infravermelho (IV) e Calorimetria de Varredura Diferencial (DSC). A síntese dos N-acilaminoésteres graxos foi realizada na presença de DCC, DMAP e dos respectivos aminoésteres e ácidos graxos. Os N-acilaminoésteres graxos 11-15a-d foram isolados em rendimentos que variaram de 50-84%, após 12h de reação a temperatura ambiente. Os N-acilaminoácidos graxos 16-20a-d foram obtidos a partir da hidrólise básica de 11-15a-d realizada a temperatura ambiente, em rendimentos que variaram de 34-80%. A seguir foi realizado o estudo de gelificação com todos os compostos sintetizados para verificar a influência das diferentes cadeias graxas e dos grupos laterais dos aminoácidos e aminoésteres na gelificação de tolueno, hexano e gasolina. As análises de DSC e IV mostraram que a estabilidade dos géis dependeu do tipo e do tamanho da cadeia lipofílica e também da natureza dos grupos laterais ligados ao átomo de nitrogênio. Dentre os compostos testados os compostos 17a-b de cadeia satura (C16:0 e C18:0) e 19a (C16:0), derivados da alanina e da fenilalanina, respectivamente, formaram géis em hexano, gasolina e tolueno. Pela primeira vez foram citadas as capacidades de gelificação de hidrocarbonetos pelos Nacilaminoésteres graxos 15a-b de cadeia saturada, derivados da serina, sendo os géis derivados destes compostos os mais estáveis termicamente dentre todos os obtidos.

Síntese e atividade antimicobacteriana de derivados graxos da isoniazida

Neste trabalho é descrita a síntese de hidrazidas graxas derivadas da isoniazida e de ácidos graxos saturados, insaturados, poli-insaturados e hidroxilados, os quais posteriormente tiveram sua atividade antimicobacteriana in vitro avaliada frente às cepas do Micobacterium tuberculosis H37Rv (ATCC 27294), M. tuberculosis resistentes à isoniazida (INHr, ATCC 35822) e M. tuberculosis (INHr, 1896HF), e M. tuberculosis resistente à rifampicina (RIFr, ATCC 35338). A síntese dos compostos 3a-g, derivados dos ácidos graxos C16:0, 18:0, cis- 18:1, trans-18:1, 18:1(OH), 18:2, e 18:3, respectivamente, foi realizada na presença de (COCl)2, DMAP e isoniazida, e os rendimentos variaram entre 60–90%. A maioria dos compostos testados demonstrou atividade mais potente que a isoniazida contra todas as cepas de M. tuberculosis estudadas, com valores de CIM entre 0,0019–50 µg.mL-1 . No estudo de relação estrutura vs. atividade, para a cepa resistente a isoniazida, o aumento da cadeia graxa e do número de insaturações provocou uma perda na potência dos derivados testados. Para as demais cepas testadas, os valores de CIM parecem ser dependentes da cepa em estudo, não sendo evidenciada uma relação estrutura vs. atividade sistemática com relação ao arranjo estrutural da cadeia graxa. Entre os compostos testados, o derivado do ácido palmítico 3a parece representar um protótipo promissor para o desenvolvimento de fármacos antituberculose, tendo apresentado valores de CIM entre 0,003–0,125 µg.mL-1 .

Synthèse de sulfilimines et de sulfoximines catalysée par les métaux de transition

Les sulfilimines et les sulfoximines sont des motifs structuraux dont l’intérêt synthétique est grandissant, notamment du fait de leurs applications en chimie médicinale et en agrochimie. Les travaux rapportés dans cet ouvrage décrivent le développement de nouvelles méthodes de synthèse efficaces pour la production de ces unités atypiques. Ces méthodes sont basées sur la réactivité d’une source d’azote électrophile, vis-à-vis de thioéthers et de sulfoxydes. L’utilisation d’un complexe métallique introduit en quantité catalytique a permis de favoriser le processus réactionnel. En tirant bénéfice de l’expertise de notre groupe de recherche sur le développement de réactions d’amination stéréosélectives de liaisons C-H et d’aziridination de styrènes, nous avons d’abord étudié la réactivité des N-mésyloxycarbamates comme source d’azote électrophile. Après avoir optimisé sa synthèse sur grande échelle, ce réactif chiral a été utilisé dans des réactions d’amination de thioéthers et de sulfoxydes, catalysées par un dimère de rhodium (II) chiral. Un processus diastéréosélectif efficace a été mis au point, permettant de produire des sulfilimines et des sulfoximines chirales avec d’excellents rendements et sélectivités. Au cours de l’optimisation de cette méthode de synthèse, nous avons pu constater l’effet déterminant de certains additifs sur la réactivité et la sélectivité de la réaction. Une étude mécanistique a été entreprise afin de comprendre leur mode d’action. Il a été observé qu’une base de Lewis telle que le 4-diméthylaminopyridine (DMAP) pouvait se coordiner au dimère de rhodium(II) et modifier ses propriétés structurales et redox. Les résultats que nous avons obtenus suggèrent que l’espèce catalytique active est un dimère de rhodium de valence mixte Rh(II)/Rh(III). Nous avons également découvert que l’incorporation de sels de bispyridinium avait une influence cruciale sur la diastéréosélectivité de la réaction. D’autres expériences sur la nature du groupe partant du réactif N-sulfonyloxycarbamate nous ont permis de postuler qu’une espèce nitrénoïde de rhodium était l’intermédiaire clé du processus d’amination. De plus, l’exploitation des techniques de chimie en débit continu nous a permis de développer une méthode d’amination de thioéthers et de sulfoxydes très performante, en utilisant les azotures comme source d’azote électrophile. Basée sur la décompositon photochimique d’azotures en présence d’un complexe de fer (III) simple et commercialement disponible, nous avons été en mesure de produire des sulfilimines et des sulfoximines avec d’excellents rendements. Le temps de résidence du procédé d’amination a pu être sensiblement réduit par la conception d’un nouveau type de réacteur photochimique capillaire. Ces améliorations techniques ont permis de rendre la synthèse plus productive, ce qui constitue un élément important d’un point de vue industriel.