Síntese enantiosseletiva de beta-aminoésteres quirais através de sistemas enzimáticos envolvendo transaminases

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Chemoenzymatic approaches to obtain chiral-centered selenium compounds

The synthesis of chiral-centered selenium compounds is presented. Enantioselective oxidations of these organoselenium compounds were performed using a wide range of biocatalysts, including Baeyer-Villiger monooxygenases, oxidoreductases-containing Aspergillus terreus and lipase (Cal-B) in the presence of oxidants. Finally, efficient synthesis of enantiopure organoselenium compounds using a kinetic resolution approach mediated by Cal-B was achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Kinetic resolution of alpha-bromophenylacetamides using quinine or Cinchona alkaloid salts

The kinetic resolution of racemic alpha-bromophenylacetamides 1 was achieved in the presence of benzenethiolate and Cinchona alkaloid salts as phase-transfer catalysts or benzenethiol and quinine, yielding (S)-enantioenriched alpha-sulfanylated products. The observed stereoselection was rationalized on the basis of the best fitting of 1 and the resolving agent in the ternary complexes. (C) 2012 Elsevier Ltd. All rights reserved.

New routes to enantioenriched substances through small organic molecules

In this thesis we will disclose the results obtained from the diastereoisomeric salt formation (n salt, p salt and p1,n1 salt) between non-racemic trans-chrysanthemic acid (trans-ChA) and pure enantiomers of threo-2-dimethylamino-1-phenyl-1,3-propanediol (DMPP). The occurrence of p1,n1 salt formation can have profound effects on enantiomer separation of scalemic (non-racemic) mixtures. This phenomenon when accompanied by substrate self-association impedes the complete recovery of the major enantiomer through formation of an inescapable racemate cage. A synthetic sequence for the asymmetric synthesis of bicyclo[3.2.0]heptanones and bicyclo[3.2.0]hept-3-en-6-ones through a cycloaddition strategy is reported. The fundamental step is a [2+2]-cycloaddition of an enantiopure amide derived from the reaction between a set of acids and an oxazolidinone as the chiral auxiliary. The inter- and intramolecular cycloaddition of in situ-generated keteniminium salts gives bicycles with a good enantioselection. A key intermediate of Iloprost, a chemically stable and biologically active mimic of prostacyclin PGI2 is synthesized following a ‘green approach’. An example of simple optical resolution of this racemic intermediate involving the diastereoisomeric salt formation is described.

Asymmetrische Synthese von 2-substituierten N-Galactosyl-piperidinderivaten und deren Verwendung zum Aufbau bicyclischer Heterocyclen

Ziel dieser Arbeit war es, ausgehend von auxiliargebundenen Piperidinderivaten, unterschiedliche chirale bi- und tricyclische Verbindungen darzustellen. Dazu wurde das 2,3,4,6-Tetra-O-pivaloyl--D-galactosylamin durch Kondensation mit Aldehyden in die entsprechenden Galactosylaldimine überführt, die in einer Lewissäure-katalysierten hochdiastereoselektiven Tandem-Mannich-Michael-Reaktionssequenz mit Danishefsky-Dien zu 2-substituierten Dehydropiperidinonen umgesetzt wurden. Die auf diese Weise zugänglichen chiralen Heterocyclen wurden diastereoselektiv in trans-konfigurierte 5-Bromverbindungen überführt. In einer Thiazolsynthese nach Hantzsch konnten die -Bromketone mit ambidenten Nukleophilen, wie Thiobenzamiden und unsymmetrischen Thioharnstoffderivaten, in niedrigen Ausbeuten zu bicyclischen Tetrahydro-thiazolo[5,4-c]pyridinen umgesetzt werden. Weitere bicyclische Heterocyclen mit einem Tetrahydro-thieno[2,3-c]pyridin-System konnten durch eine Gewald-Cyclisierung an 2-substituierten N-Galactosyl-piperidinonen erhalten werden. Durch Palladium-katalysierte Kreuzkupplungen an heterocyclischen Enoltriflaten, die ausgehend von den N-Galactosyl-dehydropiperidinonen synthetisiert wurden, gelang die Einführung von Aryl-, Alkinyl- und Alkenylsubstituenten in 4-Position des Piperidinringes. Zur Freisetzung der 2,4-disubstituierten Dehydropiperidinen wurde die N-glycosidische Bindung im sauren Milieu gespalten. Verbindungen mit einer exocyclischen Doppelbindung wurden einer Diels-Alder-Reaktion mit N-Phenylmaleinimid zum Aufbau von Isochinolinderivaten eingesetzt. Des Weiteren gelang die Synthese von 2-benzylsubstituierten N-Galactosyl-dehydropiperidinonen, wobei ortho-halogensubstituierte Phenylacetaldehyde eingesetzt wurden. Die in hohen Diastereomerenüberschüssen gebildeten Dehydropiperidinone wurden in die entsprechenden Enoltriflate überführt und einer Domino-Suzuki-Heck-Reaktion unterworfen. In dieser Kaskadenreaktion konnten tricyclische diastereomerenreine Benzomorphanderivate synthetisiert werden.

Kohlenhydrat-Auxiliare in der asymmetrischen Synthese chiraler Stickstoffheterocyclen und Alkaloide

In dieser Arbeit wurden durch Verwendung eines stereodifferenzierenden Kohlenhydrat-Auxiliars chirale Stickstoffheterocyclen und enantiomerenreine Piperidin-Alkaloide synthetisiert. Alkaloide mit einer Piperidin-Grundstruktur sind in der Natur weit verbreitet und weisen vielfältige biologische Aktivitäten auf. Zusammen mit synthetischen Derivaten sind sie daher von großem Interesse für die Wirkstoffforschung. Mit dem aus D-Arabinose zugänglichen 2,3,4-Tri-O-pivaloyl-D-arabinosylamin wurden mit hoher Stereoselektivität N-Glycosyl-dehydropiperidinone aufgebaut, die vielfältig modifizierbare Ausgangsverbindungen zur Synthese unterschiedlich substituierter Stickstoffheterocyclen darstellen. In einer Vielzahl vor allem metallorganischer Reaktionen waren regio- und stereoselektive Derivatisierungen an allen Positionen der N-glycosidisch gebundenen Dehydropiperidinone möglich. Durchgeführt wurden z. B. die Addition aktivierter Cuprate, elektrophile Substitutionen, Reduktionen, Iod-Magnesium-Austausch sowie palladium- und kupferkatalysierte Kupplungen. Die Kombination dieser Methoden führte zu mehrfach substituierten Piperidinen. In einer Ringschlussmetathese wurde zudem ein Zugang zu bicyclischen Heterocyclen geschaffen. Das Kohlenhydrat-Auxiliar steuert den stereochemischen Verlauf der Bildung der Dehydropiperidinone und der daran durchgeführten Funktionalisierungen. Die Konfigurationen der neu gebildeten Stereozentren wurden mittels Röntgenstrukturanalysen und NMR-Spektroskopie sowie durch die Überführung der Piperidin-Derivate in Alkaloide mit bekanntem Drehwert ermittelt. Die Stickstoffheterocyclen können nach Entfernen der Enamin-Doppelbindung durch milde Acidolyse vom Kohlenhydrat-Auxiliar abgespalten werden, wodurch man die enantiomerenreinen Alkaloide erhält.

Stereoselektive Synthesen von Stickstoffheterocyclen aus 4-substituierten N-Galactosyl-dehydropiperidinonen

Ziel dieser Arbeit war es, an in 4-Position substituierten N-Galactosyl-dehydropiperidinonen die übrigen Positionen des Heterocycluses selektiv zu funktionalisieren und die erarbeiteten Methoden im Rahmen von Total- und Partialsynthesen biologisch aktiver Verbindungen anzuwenden. Ausgehend von N-Galactosyl-2-pyridon, welches sich in drei Stufen aus D-Galactose im Gramm-Maßstab erhalten lässt, konnten die in Position 4-substituierten Dehydropiperidinone in regio- und diastereoselektiv verlaufenden Additionen von Grignard-Reagenzien und Organocupraten synthetisiert werden. Es gelang die Einführung sowohl unverzweigter als auch sekundärer, tertiärer und cyclischer Alkylreste. Ebenfalls gute Ausbeuten und exzellente Diastereoselektivitäten wurden bei der konjugierten Addition verschieden substituierter Aryl- und Benzyl-Grignard-Reagenzien erhalten. Das Kohlenhydratauxiliar kontrolliert dabei nicht nur die faciale Selektivität, sondern es bestimmt gleichzeitig die Regioselektivität. Die absolute Konfiguration der 4-substituierten 2-Pyridone konnte durch Röntgenstrukturanalysen zweier Produkte zweifelsfrei geklärt werden. Dass die so dargestellten Heterocyclen wertvolle Synthone zur asymmetrischen Synthese mehrfach substituierter Piperidinverbindungen sind, konnte gezeigt werden durch die Ausarbeitung verschiedener Methoden zur weitergehenden Funktionalisierung an den Positionen C-2, C-3, C-5 und C-6 sowie durch die Entwicklung eines Verfahrens zur Freisetzung der stereoselektiv synthetisierten Heterocyclen. Diese systematisch untersuchten Synthesewege konnten in Partial- und Totalsynthesen von pharmakologisch relevanten Verbindungen erfolgreich beschritten werden. So gelang die Synthese des biologisch aktiven (3S)-Piperidinols, sowie die des 3-Hydroxy-4-(4-fluorphenyl)-piperidin-Derivates. Weiterhin gelang die formale Totalsynthese von (+)-Paroxetin, welches einen pharmakologisch interessanten Wirkstoff mit der Struktur eines 3,4-trans-disubstituierten Piperidins darstellt. Ein weiterer Themenschwerpunkt dieser Arbeit war die regio- und stereoselektive Synthese von Benzomorphan-Derivaten. Diese gelang durch intramolekulare Amino-Alkylierung der 4-Benzyl-substituierten Dehydropiperidinone. Durch Anwendung dieser Methodik konnte eine Reihe verschieden substituierter 7,8-Benzomorphan-Derivate synthetisiert werden, die interessante Zwischenstufen in der asymmetrischen Benzomorphansynthese darstellen. In einer exemplarischen Synthese wurde so das 7,8-Benzomorphan hergestellt.

Studio sull'applicazione di derivati dell'ammina di betti chirali in sistemi catalitici organici

In this thesis, the development of asymmetric α-alkylation of aldehydes using two new organocatalysts is described. Nowadays organocatalized asymmetric synthesis uses preferentially primary or secondary amines. In our case two new Betti bases derivatives have been used as organocatalysts. We tried to find a method based on resolution to obtain both enantiomers with ee major than 90%. At the end we tried them in an organocatalytic processes which involve indole derivatives and aldehydes as substrates. In questa tesi è descritto lo sviluppo del processo di alfa-alchilazione di aldeidi utilizzando due nuovi catalizzatori organici chirali. Al giorno d’oggi la sintesi asimmetrica organo catalitica sfrutta principalmente ammine primarie e secondarie chirali. Come organo catalizzatori, sono stati utilizzati due nuovi derivati della base di Betti. E’ stato ricercato un metodo che permettesse di risolvere entrambi gli enantiomeri e che permettesse di ottenere un eccesso enantiomerico maggiore del 90%. Infine questi catalizzatori sono stati utilizzati e ottimizzati in micro-processi che utilizzano un substrato indolico e di un’aldeide.

Auxiliar-vermittelte stereoselektive Totalsynthese von neurotoxischen Tetraponerinen

In der vorliegenden Arbeit wurde eine neue Strategie zur stereoselektiven Synthese von Tetraponerinen entwickelt. Hierzu wurde die Kohlenhydrat-Auxiliar gesteuerte Tandem-Mannich-Michael-Reaktion als Schlüsselschritt der Totalsynthese eingesetzt. Die synthetisierten Tetraponerine wurden in biologischen Prüfungen auf neurotoxische und insektizide Aktivität untersucht. Zur Darstellung von (+)-Tetraponerin-8 wurde zunächst ausgehend von trans-2-Octensäure in sieben linearen Stufen ein chiraler beta-Amino-Aldehydbaustein synthetisiert. Dieser wurde in einer Tandem-Mannich-Michael-Reaktion mit dem Auxiliar 2,3,4-Tri-O-pivaloyl-alpha-D-arabinopyranosylamin und dem Danishefsky-Dien zur Reaktion gebracht und lieferte stereoselektiv das entsprechende 2-substituierte N-Arabinosyl-dehydropiperidinon. Nach weiterer Umsetzung des Dehydropiperidinons konnte (+)-Tetraponerin-8 in einer Gesamtausbeute von 0.6 % über 17 lineare Stufen dargestellt werden. Der Ringschluss zum tricyclischen Aminal wurde alternativ auch über die Freisetzung eines cyclischen Acetals durch saure Hydrolyse durchgeführt. Diese Syntheseroute lieferte (+)-Tetraponerin-8 in einer Gesamtausbeute von 3 % über 19 lineare Stufen.rnrnUm den Einfluss eines Stereozentrums im Aldehyd auf den stereochemischen Verlauf der Tandem-Mannich-Michael-Reaktion zu untersuchen, wurde auch das spiegelbildliche Enantiomer des chiralen beta-Amino-Aldehydbausteins synthetisiert und zur Darstellung von N-Arabinosyl-dehydropiperidinon herangezogen. Hierbei ergab sich ein „mismatched“-Fall unter Bildung eines Diastereomeren-Gemisches im Verhältnis 2:1. Nach einer Cyclisierung über ein cyclisches Acetal konnte (+)-Tetraponerin-7 über 19 lineare Stufen in einer Gesamtausbeute von 0.6 % dargestellt werden. Darüber hinaus wurde auch die racemische Form von Tetraponerin-8 nach einer literaturbekannten Synthese dargestellt, um dessen biologische Wirksamkeit mit der der enantiomerenreinen Verbindung vergleichen zu können.

Bioconjugation and synthetic approach towards enantioenriched gem-difluoromethylene compounds through carbenium ions

Bioconjugation of peptides and asymmetric synthesis of gem-difluoromethylene compounds are areas of the modern organic chemistry for which mild and selective methods continue to be developed. This thesis reports new methodologies for these two areas based on the use of stabilized carbenium ions. The reaction that makes the bioconjugation of peptides possible takes place via the direct nucleophilic substitution of alcohols and is driven by the spontaneous formation of stabilized carbenium ions in water. By reacting with the thiol group of cysteine in very mild conditions and with a high selectivity, these carbenium ions allow the site-specific ligation of polypeptides containing cysteine and their covalent derivatization with functionalized probes. The ligation of the indole ring of tryptophan, an emerging target in bioconjugation, is also shown and takes place in the same conditions. The second area investigated is the challenging access to optically active gem-difluoromethylene compounds. We describe a methodology relying on the synthesis of enantioenriched 1,3-benzodithioles intermediates that are shown to be precursors of the corresponding gem-difluoromethylene analogues by oxidative desulfurization-fluorination. This synthesis takes advantage of the highly enantioselective organocatalytic α-alkylation of aldehydes with the benzodithiolylium ion and of the wide possibilities of synthetic transformations offered by the 1,3-benzodithiole group. This approach allows the asymmetric access to complex gem-difluoromethylene compounds through a late-stage fluorination step, thus avoiding the use of fluorinated building blocks.

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

Binap-gold (I) versus Binap-silver trifluoroacetate complexes as catalysts in 1,3-dipolar cycloadditions of axomethine ylides

The 1,3-dipolar cycloaddition between azomethine ylides and alkenes is efficiently catalysed by [{(Sa)-Binap-Au(tfa)}2] (Binap=2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; tfa=trifluoroacetyl). Maleimides, 1,2-bis(phenylsulfonyl)ethylene, chalcone and nitrostyrene were suitable dipolarophiles even when using sterically hindered 1,3-dipole precursors. The results obtained in these transformations improve the analogous ones obtained in the same reactions catalysed by [Binap–Ag(tfa)]. In addition, computational studies have also been carried out to demonstrate both the high enantioselectivity exhibited by the chiral gold(I) complex, and the non-linear effect observed in this transformation.

Enantioselective alkylation of β-keto esters promoted by dimeric Cinchona-derived ammonium salts as recoverable organocatalysts

Dimeric anthracenyldimethyl-derived Cinchona ammonium salts are used as chiral organocatalysts in 5 mol% for the phase-transfer enantioselective alkylation reaction of 2-alkoxycarbonyl-1-indanones with activated bromides. The corresponding adducts bearing a new all-carbon quaternary center are obtained usually in high yield and with moderate and opposite enantioselectivity (up to 55%) when using ammonium salts derived from quinidine and its pseudoenantiomer quinine as organocatalysts. These catalysts can be almost quantitatively recovered by precipitation in ether and reused.

Theoretical study of chloroperoxidase catalyzed chlorination of beta-cyclopentanedione and role of water in the chlorination mechanism

Chloroperoxidase (CPO) is a potential biocatalyst for use in asymmetric synthesis. The mechanisms of CPO catalysis are therefore of interest. The halogenation reaction, one of several chemical reactions that CPO catalyzes, is not fully understood and is the subject of this dissertation. The mechanism by which CPO catalyzes halogenation is disputed. It has been postulated that halogenation of substrates occurs at the active site. Alternatively, it has been proposed that hypochlorous acid, produced at the active site via oxidation of chloride, is released prior to reaction, so that halogenation occurs in solution. The free-solution mechanism is supported by the observation that halogenation of most substrates often occurs non-stereospecifically. On the other hand, the enzyme-bound mechanism is supported by the observation that some large substrates undergo halogenation stereospecifically. The major purpose of this research is to compare chlorination of the substrate β-cyclopentanedione in the two environments. One study was of the reaction with limited hydration because such a level of hydration is typical of the active site. For this work, a purely quantum mechanical approach was used. To model the aqueous environment, the limited hydration environment approach is not appropriate. Instead, reaction precursor conformations were obtained from a solvated molecular dynamics simulation, and reaction of potentially reactive molecular encounters was modeled with a hybrid quantum mechanical/molecular mechanical approach. Extensive work developing parameters for small molecules was pre-requisite for the molecular dynamics simulation. It is observed that a limited and optimized (active-site-like) hydration environment leads to a lower energetic barrier than the fully solvated model representative of the aqueous environment at room temperature, suggesting that the stable water network near the active site is likely to facilitate the chlorination mechanism. The influence of the solvent environment on the reaction barrier is critical. It is observed that stabilization of the catalytic water by other solvent molecules lowers the barrier for keto-enol tautomerization. Placement of water molecules is more important than the number of water molecules in such studies. The fully-solvated model demonstrates that reaction proceeds when the instantaneous dynamical water environment is close to optimal for stabilizing the transition state.

Chemoenzymatic routes to enantiopure hydroxytetrahydrofurans: muscarine and its analogues

Muscarine was identified as an active principle of the poisonous mushroom Amanita muscaria over 170 years ago and has been identified as an agonist of acetylcholine. The synthesis of all stereoisomers of muscarine have been accomplished at this stage by chemical methods and the biological activity of these compounds tested. A number of synthetic routes to enantiomerically pure muscarine and its analogues have been published. In this work, we are focussed on the use of a novel biotransformation strategy to access these compounds. Asymmetric synthesis involves targeting a synthetic pathway leading to one enantiomer of a compound and biocatalysis is one strategy used in asymmetric synthesis. Chapter 1 consists of a review of the relevant literature pertaining to the synthesis and stereoselective transformations of 3-hydroxytetrahydrofuranss. A review of synthetic routes to these compounds is presented, with a particular focus on routes to the natural product muscarine and its analogues. Chapter 2 discusses the preparative routes to the 3-hydroxytetrahydrofurans via 3(2H)- furanones. Steps amongst which include Rh(II) mediate cyclisation and kinetic resolution via baker’s yeast mediated carbonyl reduction, resulting in enantioenriched 3- hydroxytetrahydrofuran derivatives. Finally, application of this methodology to the preparation of all four enantiomers of an analogue of desmethylmuscarine and the synthesis of epimuscarine is described. Chapter 3 consists of a detailed experimental section outlining the synthetic procedures employed.