A synthetic approach to palmerolides via Negishi cross coupling. The challenge of the C15-C16 bond formation

The esterification of fragment C1-C8 (2) with fragment C16-C23 (3) to give iodo derivative 4, followed by a Pd-catalysed coupling with a C9-C15 fragment (7 or 8), may provide a common precursor of most palmerolides. Ligands and reaction conditions were exhaustively examined to perform the C15-C16 bond formation via Negishi reaction. With simple models, pre-activated Pd-Xantphos and Pd-DPEphos complexes were the most efficient catalysts at RT. Zincation of the C9-C15 fragment (8) and cross coupling with 4 required 3 equiv of t-BuLi, 10 mol % of Pd-Xantphos and 60 °C.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Carbon-carbon bond formation : from transition metal catalysis to base-promoted homolytic aromatic substitution

Cette thèse de doctorat porte sur la catalyse à partir de métaux de transition et sur la substitution homolytique aromatique favorisée par une base visant à former de nouvelles liaisons C–C, et à ainsi concevoir de nouvelles structures chimiques. Au cours des vingt dernières années, des nombreux efforts ont été réalisés afin de développer des méthodologies pour la fonctionnalisation de liens C–H, qui soient efficaces et sélectives, et ce à faible coût et en produisant le minimum de déchets. Le chapitre d'introduction donnera un aperçu de la fonctionnalisation directe de liens C–H sur des centres sp2 et sp3. Il sera également discuté dans cette partie de certains aspects de la chimie radicalaire reliés a ce sujet. Les travaux sur la fonctionnalisation d’imidazo[1,5-a]pyridines catalysée par des compleces de ruthénium seront présentés dans le chapitre 2. Malgré l'intérêt des imidazo[1,5-a]azines en chimie médicinale, ces composés n’ont reçu que peu d'attention dans le domaine de la fonctionnalisation de liens C–H. L'étendue de la réaction et l'influence des effets stériques et électroniques seront détaillés. Les cyclopropanes représentent les 10ème cycles carbonés les plus rencontrés dans les petites molécules d’intérêt pharmacologique. Ce sont aussi des intermédiaires de synthèse de choix pour la création de complexité chimique. Malgré de grands progrès dans le domaine de la fonctionnalisation de liens C(sp3)–H, l'étude des cyclopropanes comme substrats dans les transformations directes est relativement nouvelle. Le chapitre trois présentera l'arylation intramoléculaire directe de cyclopropanes. Cette réaction est réalisée en présence de palladium, en quantité catalytique, en combinaison avec des sels d’argent. Des études mécanistiques ont réfuté la formation d'un énolate de palladium et suggéreraient plutôt une étape de métallation - déprotonation concertée. En outre, les cycles de type benzoazepinone à sept chaînons ont été synthétisés par l'intermédiaire d'une séquence d'activation de cyclopropane/ouverture/cyclisation. Une arylation directe intermoléculaire des cyclopropanes a été réalisée en présence d'un auxiliaire de type picolinamide (Chapitre 4). Les deux derniers chapitres de ce mémoire de thèse décriront nos études sur la substitution homolytique aromatique favorisée par une base. Le mécanisme de la réaction de cyclisation intramoléculaire d'halogénures d'aryle, réalisée en présence de tert-butylate de potassium, a été élucidé et se produit via une voie radicalaire (Chapitre 5). La transformation, exempte de métaux de transition, ne nécessite que la présence d’une base et de pyridine comme solvant. Cette réaction radicalaire a été étendue à la cyclisation d'iodures d'alkyle non activés en présence d'un catalyseur à base de nickel et de bis(trimethylsilyl)amidure de sodium comme base (Chapitre 6). Des études de RMN DOSY ont démontré une association entre le catalyseur, la base et le matériel de départ.

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

The Pair-Bond Formation and Its Role in the Stimulation of Reproductive Function in Female Common Marmosets (Callithrix jacchus)

SILVA, H.P.A.; SOUSA, M.B.C. The pair-bond formation and its role in the stimulation of reproductive function in female common marmosets (collithrix Jacchus). International Journal of Primatology, v, 18, n.3, p.387-400, 1997.

Effect of hydrogen bond formation on the elastic molecular scattering: a case study with methanol

Rayleigh optical activities of small hydrogen-bonded methanol clusters containing two to five molecules are reported. For the methanol trimer, tetramer, and pentamer both cyclic and linear structures are considered. After the geometry optimizations, the dipole moments and the dipole polarizabilities (mean, interaction, and anisotropic components) are calculated using HF, MP2 and DFT (B3LYP, B3P86 and BH&HLYP) with aug-cc-pVDZ extended basis set. The polarizabilities are used to analyse the depolarization ratios and the Rayleigh scattering activities. The variations in the activity and in the depolarization for Rayleigh scattered radiation with the increase in the cluster size for both cyclic and linear structures are analysed.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30–Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA–DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of β-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli

The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (Km) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent Km for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones.

Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.

Immunological synapse: a mathematical model of the bond formation process:mathematical modelling of the immature synapse

The cell:cell bond between an immune cell and an antigen presenting cell is a necessary event in the activation of the adaptive immune response. At the juncture between the cells, cell surface molecules on the opposing cells form non-covalent bonds and a distinct patterning is observed that is termed the immunological synapse. An important binding molecule in the synapse is the T-cell receptor (TCR), that is responsible for antigen recognition through its binding with a major-histocompatibility complex with bound peptide (pMHC). This bond leads to intracellular signalling events that culminate in the activation of the T-cell, and ultimately leads to the expression of the immune eector function. The temporal analysis of the TCR bonds during the formation of the immunological synapse presents a problem to biologists, due to the spatio-temporal scales (nanometers and picoseconds) that compare with experimental uncertainty limits. In this study, a linear stochastic model, derived from a nonlinear model of the synapse, is used to analyse the temporal dynamics of the bond attachments for the TCR. Mathematical analysis and numerical methods are employed to analyse the qualitative dynamics of the nonequilibrium membrane dynamics, with the specic aim of calculating the average persistence time for the TCR:pMHC bond. A single-threshold method, that has been previously used to successfully calculate the TCR:pMHC contact path sizes in the synapse, is applied to produce results for the average contact times of the TCR:pMHC bonds. This method is extended through the development of a two-threshold method, that produces results suggesting the average time persistence for the TCR:pMHC bond is in the order of 2-4 seconds, values that agree with experimental evidence for TCR signalling. The study reveals two distinct scaling regimes in the time persistent survival probability density prole of these bonds, one dominated by thermal uctuations and the other associated with the TCR signalling. Analysis of the thermal fluctuation regime reveals a minimal contribution to the average time persistence calculation, that has an important biological implication when comparing the probabilistic models to experimental evidence. In cases where only a few statistics can be gathered from experimental conditions, the results are unlikely to match the probabilistic predictions. The results also identify a rescaling relationship between the thermal noise and the bond length, suggesting a recalibration of the experimental conditions, to adhere to this scaling relationship, will enable biologists to identify the start of the signalling regime for previously unobserved receptor:ligand bonds. Also, the regime associated with TCR signalling exhibits a universal decay rate for the persistence probability, that is independent of the bond length.

The Pair-Bond Formation and Its Role in the Stimulation of Reproductive Function in Female Common Marmosets (Callithrix jacchus)

SILVA, H.P.A.; SOUSA, M.B.C. The pair-bond formation and its role in the stimulation of reproductive function in female common marmosets (collithrix Jacchus). International Journal of Primatology, v, 18, n.3, p.387-400, 1997.

The Pair-Bond Formation and Its Role in the Stimulation of Reproductive Function in Female Common Marmosets (Callithrix jacchus)

SILVA, H.P.A.; SOUSA, M.B.C. The pair-bond formation and its role in the stimulation of reproductive function in female common marmosets (collithrix Jacchus). International Journal of Primatology, v, 18, n.3, p.387-400, 1997.