Studies on chemoenzymatic synthesis: Lipase-catalyzed acylation in multistep organic synthesis

In this thesis, biocatalysis is defined as the science of using enzymes as catalysts in organic synthesis. Environmental aspects and the continuously expanding repertoire of available enzymes have firmly established biocatalysis as a prominent means of chemo-, regio- and stereoselective synthesis. Yet, no single methodology can solve all the challenges faced by a synthetic chemist. Therefore, the knowledge and the skills to combine different synthetic methods are relevant. Lipases are highly useful enzymes in organic synthesis. In this thesis, an effort is being made to form a coherent picture of when and how can lipases be incorporated into nonenzymatic synthesis. This is attempted both in the literature review and in the discussion of the results presented in the original publications contained in the thesis. In addition to lipases, oxynitrilases were also used in the work. The experimental part of the thesis comprises of the results reported in four peer-reviewed publications and one manuscript. Selected amines, amino acids and sugar-derived cyanohydrins or their acylated derivatives were each prepared in enantio- or diastereomerically enriched form. Where applicable, attempts were made to combine the enzymatic reactions to other synthetic steps either by the application of completely separate sequential reactions with isolated intermediates (kinetic and functional kinetic resolution of amines), simultaneously occurring reactions without intermediate isolation (dynamic kinetic resolution of amino acid esters) or sequential reactions but without isolating the intermediates (hydrocyanation of sugar aldehydes with subsequent diastereoresolution). In all cases, lipase-catalyzed acylation was the key step by which stereoselectivity was achieved. Lipase from Burkholderia cepacia was a highly selective enzyme with each substrate category, but careful selection of the acyl donor and the solvent was important as well.Tutkimuksia kemoentsymaattisista synteeseistä: Lipaasien katalysoima asylaatio monivaiheisessa orgaanisessa synteesissä Tässä väitöskirjassa biokatalyysillä tarkoitetaan entsyymien käyttöä katalyytteinä orgaanisessa synteesissä. Ympäristöystävällisyys ja jatkuvasti laajeneva saatavilla olevien entsyymien kirjo ovat vakiinnuttaneet biokatalyysin kemo-, regio- ja stereoselektiivisen synteesin keskeiseksi keinoksi. Kuitenkaan mikään yksittäinen menetelmä ei pysty ratkaisemaan kaikkia synteesikemistin kohtaamia haasteita. Näin ollen eri synteesimenetelmien yhdistämiseen tarvittavat tiedot ja taidot ovat olennaisia. Lipaasit ovat orgaaniseen synteesiin erityisen hyvin soveltuvia entsyymejä. Väitöskirjassa pyritään muodostamaan kokonaisvaltainen kuva siitä, milloin ja miten lipaaseja voidaan hyödyntää perinteisen synteesin osana. Tätä päämäärää tavoitellaan sekä kirjallisuuskatsauksessa että väitöskirjaan sisältyvissä alkuperäisjulkaisuissa. Lipaasien lisäksi työssä hyödynnettiin oksinitrilaasi-entsyymejä. Väitöstyön kokeellinen osa koostuu tuloksista, jotka on raportoitu neljässä vertaisarvioidussa tutkimusartikkelissa ja yhdessä toistaiseksi julkaisemattomassa käsikirjoituksessa. Tutkimuskohteiksi valitut amiinit, aminohapot ja sokeriperäiset syanohydriinit tai niiden asyloidut johdannaiset valmistettiin enantio- tai diastereomeerisesti rikastuneessa muodossa. Soveltuvin osin tutkittiin mahdollisuutta yhdistää entsymaattisia reaktioita synteesin muihin vaiheisiin joko suorittamalla reaktiot täysin erillisinä välituotteet eristäen (amiinien kineettinen ja funktionaalinen kineettinen resoluutio), samanaikaisina reaktioina eristämättä välituotteita (aminohappoestereiden dynaaminen kineettinen resoluutio) tai erillisinä reaktioina mutta eristämättä välituotteita (sokerialdehydien hydrosyanaatio ja tätä seuraava diastereoresoluutio). Kussakin tapauksessa lipaasin katalysoima asylaatio oli keskeinen stereoselektiivisyyden määräävä reaktio. Burkholderia cepacia -bakteerista eristetty lipaasi oli hyvin selektiivinen entsyymi kullekin lähtöaineluokalle, mutta myös asyylinluovuttajan ja liuottimen huolellinen valitseminen oli tärkeää.Siirretty Doriast

Analysis of mandelonitrile lyase and 0-glucosidase from sweet almonds by combined electrophoretic techniques

Almonds are a rich source of mandelonitrile lyase (oxynitrilase) and f3-glucosidase. The isolation of these two enzymes from sweet almonds requires fractional ammonium sulfate precipitation followed by ion-exchange chromatography on diethylaminoethyl-(DEAE) and carboxymethylcellulose (CMC) columns. In the present investigation different electrophoretic techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing in immobilized pH gradients (IEF-IPG), and capillary electrophoresis were used to characterize these two enzymes. For the first time, B-glucosidase and oxynitrilase were separated in an immobilized pH gradient of one pH unit. Capillary zone electrophoresis (CZE) was an excellent tool for analysis of the purity of enzyme preparations, achieving complete separation of various protein constituents in only 15 min. CZE showed a resolving capacity for the separation of enzyme forms comparable to that of isoelectric focusing in an immobilized pH gradient

(R)- and (S)-cyanohydrins : their enzymatic synthesis and their reactions

The known enzymes (R)- and (S)-oxynitrilase catalyze the enantioselective addition of hydrocyanic acid to ldehydes to give (R)- and (S)-cyanohydrins. The optical yields can distinctly be improved by the application of organic solvents (i.e. ethyl acetate or diisopropyl ether) instead of a waterjethanol mixture which was used previously in these reactions. For the enzyme (S)-oxynitrilase Sorghum bicolor evolved to be the best source. The optically active cyanohydrins can be transformed without any racemization by acid catalyzed hydrolysis into a-hydroxy acids and by hydrogenation with lithiumjaluminum hydride into 1,2-amino alcohols. Via addition of Grignard reagents to the O-protected cyanohydrins and follow-up hydrogenation, 1,2-amino alcohols are gained with very high diastereoselectivity. By O-sulfonylation of the (R)- and (S)-cyanohydrins optically active α-sulfonyloxy nitriles are obtained. These nitriles react with various nucleophiles by complete inversion of configuration to form various α-substituted carboxylic acid derivatives, α-azido nitriles, α-amino nitriles, α-amino acids, etc

Synthesis and reactions of optically active cyanohydrins

Cyanohydrins have always held a place of importance both as technical products and as reagents in organic chemistry. It is surprising, therefore, that optically active Cyanohydrins have been extensively investigated and employed for syntheses relatively recently. This can be explained by the fact that only in the past few years have enzymatic methods made chiral Cyanohydrins readily available in high optical purity. Chiral Cyanohydrins are widespread in nature in the form of the respective glycosides and serve roughly 3000 plants and many insects as antifeedants. For the preparative organic chemist, this class of compounds offers an enormous synthetic potential for making other chiral compounds accessible. In a few instances, the pharmacological principle of a drug also incorporates a chiral cyanohydrin as constitutive structural element. In the development of novel, physiologically active compounds all possible stereo-isomers must be synthesized and investigated with respect to their activity range and the pathway of their metabolic transformations and/or degradation. The development of simple synthetic procedures for such compounds, which also entail a high degree of stereoselectivity, therefore has prime importance. To this end chiral Cyanohydrins may serve as stereochemically pure starting materials. In the present review, the following topics will be addressed: enantioselective addition of hydrogen cyanide (HCN), catalysed by the enzymes (R)-and (S)-oxynitrilase, to aldehydes and Ketones yielding (R) and (S) cyanohydrins, respectivity; enantioselective addition of HCN to aldehydes catalyzed by cyclic dipeptides; enantioselective esterification of racemic ocyanohydrins and enantioselective hydrolysis of cyanohydrin esters caytalyzed by lipases and esterases, reprectively; transformation of the nitrile groups of chiral cyanohydrins to provide optically active -hydroxycarboxylic acids, aldehydes, and ketones, as well as 2-amino alcohols; sulfonylation of the OH group of chiral cyanohydrins to furnish optically active -sulfonyloxynitriles which undergo SN2 displacement of the activated OH group yielding α-azido-,α -amino, and α-fluoronitriles with inverted configuration

Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs)

Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes

Enzyme-catalyzed synthesis of (S)-cyanohydrins and subsequent hydrolysis to (S)-α-hydroxy-carboxylic acids

(S)-Cyanohydrins 2 are obtained with high enantioselectivity from aromatic aldehydes and HCN in the presence of (S)-oxynitrilase (E.C.4.1.2.11). Acid-catalyzed hydrolysis of the cyanohydrins 2 affords the corresponding (S)-α-hydroxy carboxylic acids 3 without racemization

Comparison of lipase-catalyzed synthesis of cyclopentadecanolide under organic and biphasic systems

Methyl 15-hydroxy-pentadecanate, which is made from Malana oleifera chum oil, is an ideal material to synthesize cyclopentadecanolide, an important macrocycle musk, with wide applications in the fields of perfume, cosmetic, food and medicine, etc. One kind of screened lipase from Candida sp.GXU08 strain was used to catalyze the synthesis of cyclopentadecanolide from methyl 15-hydroxy-pentadecanate. Traditionally, the catalytic reaction went on under organic solvent system, while an ultrasonic technology was innovatively used in water/organic solvent biphasic system to systhesize cyclopentadecanolide and received good catalytic effect. Both organic and biphasic systems had the same optimal conditions as: substrate concentration= 8 mmol/l, T= 40°C, ω = 180 r/min, pH= 6.0 to 6.5, ultrasonic time of 30 min, output power of 200 W and using lipase solution/lipase power and reaction system twice greatly increased the production of cyclopentadecanolide. It showed that, the maximum production of cyclopentadecanolide was 47.77×10-3mg/U in biphasic system under the optimal conditions, which was 3.285 times of that in the organic system. It was also found that the production of cyclopentadecanolide in biphasic system was always higher than that in the organic system, which saved the process of freeze drying and improved the production of cyclopentadecanolide. It was verified that methyl 15-hydroxy-pentadecanate was directly cyclized into cyclopentadecanolide in the organic system while in the biphasic system, methyl 15-hydroxy-pentadecanate was hydrolysed into 15-hydroxypentadecanoic acid firstly and then cyclized into cyclopentadecanolide.Key words: Lipase, cyclopentadecanolide, Malana oleifera chum, biphasic system, ultrasonic

Native and immobilized (R)-oxynitrilase: characterization and synthesis of optically active (R)-cyanohydrins

In den letzten Jahren wurde eine Reihe von Verfahren zur Herstellung optisch aktiver Cyanhydrine entwickelt, da über diese andere wichtige Klassen optisch aktiver Verbindungen leicht zu erhalten sind, von denen eine große Zahl pharmazeutisch und biologisch aktiv ist. In dieser Arbeit erfolgte die Herstellung von (R)-Cyanhydrinen durch enzymatische Umsetzung von Benzaldehyd mit Kaliumcyanid in einem wässrigen System (pH = 3,75, T = 20°C). Die Enantiomerenausbeuten der (R)-Cyanhydrine sind größer 99,5°%. Dem Einsatz isolierter (R)-Oxynitrilase in technischen Verfahren stehen im Allgemeinen ihr hoher Preis, ihre geringe Langzeitstabilität sowie die Schwierigkeiten bei Aufarbeitung und Wiederverwendung im Wege. Ziel dieser Arbeit war eine Immobilisierungsmethode für das Enzym zu entwickelt, welches diese Nachteile behebt. Die Immobilisierung der (R)-Oxynitrilase erfolgte in zwei Schritten. Im ersten Schritt wurde das Enzyms mit Glutardialdehyd und Chitosan coquervernetzt. Das molekulargewichtsvergrößerte Enzym zeigte eine Restaktivität von größer 70 %. Im zweiten Schritt wurde das coquervernetzte Enzym in Polyvinylalkoholgel eingeschlossen und anschließend zu LentiKats® verarbeitet. Die LentiKats® sind unter Stickstoffatmosphäre langzeitstabil. Ab einer Enzymbeladung von ca. 40 U/g(Linsen) tritt eine Diffusionslimitierung auf. Zusammenfassend lässt sich sagen, dass eine Immobilisierungsmethode für die (R)-Oxynitrilase entwickelt wurde, die optimale Eigenschaften für die enzymatische Cyanhydrinsynthese aufweist.The (R)-oxynitrilase (E.C.4.1.2.10), which catalyses the reversible condensation of hydrogen cyanide with aldehydes and ketones, is a useful and promising enzyme for biotransformation. The resulting optically active cyanohydrins play an important role, since these molecules find a wide range of pharmaceutical and agrochemical applications. The use of this enzyme in industrial application has been limited because the cost of their isolation and purification is still high. The aim of this work was to immobilise (R)-oxynitrilase by crosslinking with glutardialdehye under high retention of its activity, to entrap these complexes into lens-shaped hydrogels (LentiKats®) based on polyvinylalcohol mixture and to check the efficiency and long-term stability of these systems. In conclusion, we reported a new type of entrapped oxynitrilase catalyst which not only characterized by a long term-stability, high activity, good recycling rates, but also gives high ee of >99% ee. This new type of entrapped biocatalyst is further characterized by the following properties: (i) recyclable without loss of enzymatic activity; (ii) no catalyst leaching; (iii) macroscopic well-defined size in mm-range, thus easy filtration steps are possible; (iv) suitable for technical reactors due to high elasticity; (v) very low price of the encapsulation material polyvinylalcohol (PVAL); (vi) non-toxicity of the encapsulation material; (vii) easy encapsulation technique; and (viii) a high synthetic efficiency