New synthetic strategies towards indolizines and pyrroles

Indolizines and pyrroles are considered as “privileged” structures since their skeletons were found in many biologically active natural products and they possess a wide range of pharmaceutical properties. Syntheses of these small drug-like molecules are very important in medicinal chemistry. However, most existent methodologies are usually limited to specific substitution patterns or require impractical starting materials or expensive catalysts. Therefore, developing new methodologies for the synthesis of indolizines and pyrroles from commercially available or readily accessible sources is highly desirable.rnIn this PhD thesis, several methods has been described for the synthesis of indolizines and pyrroles. In the first part, indolizines carrying substituents in positions 1-3 were synthesized via a formal [3+2]-cycloaddition of pyridinium ylides and nitroalkenes. Pyridinium salts were prepared by N-alkylation of pyridines with cyanohydrin triflates which could be prepared from corresponding aldehydes via a Strecker reaction followed by O-triflylation. Nitroalkenes were simply prepared from the corresponding aldehydes and nitroalkanes in a nitroaldol condensation. Overall, this modular approach allows to construct the indolizine framework with various substitution patterns starting from a pyridine, two different aldehydes and a nitroalkane. In contrast to reported methods, the produced indolizines do not have to contain an electron-withdrawing group.rnIt has also been found that nitrile-stabilized 2-alkylpyridinium ylides cyclize to unstable 2-aminoindolizines via an intramolecular 5-exo-dig cyclization. Using an in situ acetylation of the amino group, N-protected 2-aminoindolizines could be synthesized. As a less common substitution pattern, indolizines carrying substituents in positions 5–8 were synthesized from enones and 2-(1H-pyrrol-1-yl)nitriles obtained from α-aminonitriles using a modified Paal-Knorr pyrrole synthesis. The decoration of the pyridine unit in the indolizine skeleton has been achieved by a one-pot conjugate addition/cycloaromatization sequence.rnIn the second part of the thesis, the diversity-oriented synthesis of pyrroles from 3,5-diaryl substituted 2H-pyrrole-2-carbonitriles (cyanopyrrolines) obtained in a cyclocondensation of enones with aminoacetonitrile hydrochloride is being discussed. 2,4-Di-, 2,3,5-trisubstituted pyrroles, pyrrole-2-carbonitriles and 2,2’-bipyrroles were synthesized in a one- or two-step protocol. While the microwave-assisted thermal elimination of HCN from cyanopyrrolines gave 2,4-disubstituted pyrroles, DDQ-oxidation of the same intermediates furnished pyrrole-2-carbonitriles. Furthermore, 2,3,5-trisubstituted pyrroles were obtained via a C-2-alkylation of the deprotonated cyanopyrrolines followed by the elimination of HCN. Finally, it has also been found that tetraaryl substituted 2,2’-bipyrroles could be synthesized by the oxidative dimerization of cyanopyrrolines using copper (II) acetate at 100 °C.rn

Synthese von Crinan-Alkaloid-Vorstufen : Studien zum stereoselektiven Aufbau quartärer Kohlenstoffzentren

In einer Ex-chiral-Pool-Synthese konnten wichtige optisch aktive Vorstufen für die Substanzklasse der Crinan-Alkaloide hergestellt werden. Diese Alkaloid-Klasse zeichnet sich durch ihre zahlreichen physiologischen Eigenschaften (Antitumor-, Antiviral-, Antimalaria-Aktivität etc.) aus und stellt deshalb ein interessantes Syntheseziel für eine Totalsynthese dar. Ausgehend von der chiralen Information des L-Serins konnte dabei das quartäre, arylierte Kohlenstoffzentrum gezielt durch eine Stetter-Reaktion (Umpolungs-Reaktion) aufgebaut werden. Der Aufbau des dafür benötigten trisubstituierten Olefins wurde über eine Horner-Olefinierung und Heck-Cyclisierung erreicht. Hierbei konnten Bedingungen erarbeitet werden, die eine racemisierungsfreie Synthese gewährleisten.

Synthesis, reactivity and applications of 3,5-dimethyl-4-nitroisoxazole derivatives

3,5-dimethyl-4-nitroisoxazole derivatives are useful synthetic intermediates as the isoxazole nucleus chemically behaves as an ester, but establish better-defined interactions with chiral catalysts and lability of its N-O aromatic bond can unveil other groups such as 1,3-dicarbonyl compounds or carboxylic acids. In the present work, these features are employed in a 3,5-dimethyl-4-nitroisoxazole based synthesis of the γ-amino acid pregabalin, a medication for the treatment of epilepsy and neuropatic pain, in which this moiety is fundamental for the enantioselective formation of a chiral center by interaction with doubly-quaternized cinchona phase-transfer catalysts, whose ability of asymmetric induction will be investigated. Influence of this group in cinchona-derivatives catalysed stereoselective addition and Darzens reaction of a mono-chlorinated 3,5-dimethyl-4-nitroisoxazole and benzaldehyde will also be investigated.

Synthesis of novel ribose-based thioimidate n-oxides

This work is based on the study of new synthetic paths to obtain thioimidate N-oxides (TINOs) from D-ribose and to study their reactivity with the purpose to obtain ketonitrones. TINOs, aren’t well known molecules, but these enantiomerically pure backbones could be valuable intermediates in the synthesis of novel ketonitrones which are key intermediates in the synthesis of iminosugars. TINOs were discovered from the study of glucoraphanin, a particular glucosinolate, that unexpectedly cyclized into a TINO after desulfatation, by a spontaneous intramolecular Michael addition. The first part of this work was to synthetize the TINO 3 from D-ribose 1. The key step was the desilylative cyclisation of a suitably functionalized thiohydroximate 2. Based on precedent work developed in the laboratory, we could obtain the thiohydroximate from D-ribose. We then focused our studies on the cyclisation step trying to find the suitable substituents that could give the TINO in good yield by desilylative cyclisation. The second part of the project is to obtain ketonitrones 4 by palladiumcatalyzed coupling reaction.

Synthesis of (-)-dactylolide and 13-desmethylene-(-)-dactylolide and their effects on tubulin

An efficient new synthesis has been elaborated for non-natural (-)-dactylolide ((-)-2) and its 13-desmethylene analogue 4, employing a HWE-based macrocyclization approach with beta-keto-phosphonate/aldehyde 19 and the respective 13-desmethylene derivative as the key intermediates. Both (-)-2 and 4 as well as the corresponding C20 alcohols inhibit human cancer cell proliferation with IC(50) values in the sub-micromolar range and induce the polymerization of tubulin in vitro.

Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.

Synthesis of the Sugar Building Block of Bicyclo-RNA

We present the novel synthesis of two sugar units that are central intermediates for the formation of members of the bicyclo-DNA and -RNA family. The synthesis starts from commercially available 1,2: 5,6-di-O-isopropylidene-alpha-D-glucofuranose. The key step involves the elaboration of a carbocyclic ring in a furanoside by rhodium(I)-catalyzed hydroacylation. Via this pathway, one of the sugar units is available in 8 steps and in an overall yield of 27%, while its deoxy derivative is obtained in 11 steps, which is 5 steps fewer than in our previous synthesis of this compound.

Investigation of the Potential Energy Surface for the First Step in the Alkaline Hydrolysis of Methyl Acetate

The potential energy surface for the first step of the alkaline hydrolysis of methyl acetate was explored by a variety of methods. The conformational search routine within SPARTAN was used to determine the lowest energy am1 and pm3 structures for the anionic tetrahedral intermediate. Ab initio single point and geometry optimization calculations were performed to determine the lowest energy conformer, and the linear synchronous transition (lst) method was used to provide an initial structure for transition state optimization. Transition states were obtained at the am1, pm3, 3-21G, and 3-21 + G levels of theory. These transition states were compared with the anionic tetrahedral intermediates to examine the assumption that the intermediate is a good model for the transition state. In addition, the Cramer/Truhlar sm3 solvation model was used at the semiempirical level to compare gas phase and aqueous alkaline hydrolysis of methyl acetate.

Ortho Effect in the Bergman Cyclization: Electronic and Steric Effects in Hydrogen Abstraction by 1-Substituted Naphthalene 5,8-Diradicals

We present a detailed theoretical study of geometries, electronic structure, and energies of transition states and intermediates completing the full Bergman cycloaromatization pathway of ortho-substituted enediynes with a focus on polar and steric contributions to the kinetics and thermodynamics of hydrogen abstraction. This study provides a rare unambiguous example of remote substitution that affects reactivity of a neutral reactive intermediate through an σ framework.

A Computationally Efficient Procedure for Modeling the First Step in the Alkaline Hydrolysis of Esters

A computationally efficient procedure for modeling the alkaline hydrolysis of esters is proposed based on calculations performed on methyl acetate and methyl benzoate systems. Extensive geometry and energy comparisons were performed on the simple ester methyl acetate. The effectiveness of performing high level single point ab initio energy calculations on the geometries obtained from semiempirical and ab initio methods was determined. The AM1 and PM3 semiempirical methods are evaluated for their ability to model the transition states and intermediates for ester hydrolysis. The Cramer/Truhlar SM3 solvation method was used to determine activation energies. The most computationally efficient way to model the transition states of large esters is to use the PM3 method. The PM3 transition structure can then be used as a template for the design of haptens capable of inducing catalytic antibodies.

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

A Computational Analysis of the Collision-Induced Dissociation Mechanisms of the Dipeptide Glycine-Serine Under Mass Spectrometry

Collision-induced dissociation (CID) of peptides using tandem mass spectrometry (MS) has been used to determine the identity of peptides and other large biological molecules. Mass spectrometry (MS) is a useful tool for determining the identity of molecules based on their interaction with electromagnetic fields. If coupled with another method like infrared (IR) vibrational spectroscopy, MS can provide structural information, but in its own right, MS can only provide the mass-to-charge (m/z) ratio of the fragments produced, which may not be enough information to determine the mechanism of the collision-induced dissociation (CID) of the molecule. In this case, theoretical calculations provide a useful companion for MS data and yield clues about the energetics of the dissociation. In this study, negative ion electrospray tandem MS was used to study the CID of the deprotonated dipeptide glycine-serine (Gly-Ser). Though negative ion MS is not as popular a choice as positive ion MS, studies by Bowie et al. show that it yields unique clues about molecular structure which complement positive ion spectroscopy, such as characteristic fragmentations like the loss of formaldehyde from the serine residue.2 The increase in the collision energy in the mass spectrometer alters the flexibility of the dipeptide backbone, enabling isomerizations (reactions not resulting in a fragment loss) and dissociations to take place. The mechanism of the CID of Gly-Ser was studied using two computational methods, B3LYP/6-311+G* and M06-2X/6-311++G**. The main pathway for molecular dissociation was analyzed in 5 conformers in an attempt to verify the initial mechanism proposed by Dr. James Swan after examination of the MS data. The results suggest that the loss of formaldehyde from serine, which Bowie et al. indicates is a characteristic of the presence of serine in a protein residue, is an endothermic reaction that is made possible by the conversion of the translational energy of the ion into internal energy as the ion collides with the inert collision gas. It has also been determined that the M06-2X functional¿s improved description of medium and long-range correlation makes it more effective than the B3LYP functional at finding elusive transition states. M06-2X also more accurately predicts the energy of those transition states than does B3LYP. A second CID mechanism, which passes through intermediates with the same m/z ratio as the main pathway for molecular dissociation, but different structures, including a diketopiperazine intermediate, was also studied. This pathway for molecular dissociation was analyzed with 3 conformers and the M06-2X functional, due to its previously determined effectiveness. The results suggest that the latter pathway, which meets the same intermediate masses as the first mechanism, is lower in overall energy and therefore a more likely pathway of dissociation than the first mechanism.

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.