BIOCATALYTIC SYNTHESIS OF CHIRAL PHARMACEUTICAL INTERMEDIATES

Questa Tesi di Dottorato \ue8 stata strutturata in due parti differenti precedute da un\u2019introduzione riguardante i principi fondamentali della biocatalisi con particolare attenzione sull\u2019applicazione di cellule intere di lieviti non convenzionali e una nuova chetoreduttasi proveniente dal lievito Pichia glucozyma. Tutte queste tematiche rappresentano il \u201cbackground\u201d per poter meglio comprendere le parti sperimentali descritte nei successivi paragrafi. Parte 1 Il potenziale biocatalitico rappresentato da cellule intere di P. glucozyma \ue8 stato studiato usando substrati di differente natura chimica interessanti sia dal punto di vista farmaceutico che sintetico. Dopo l\u2019ottimizzazione delle condizioni di reazione, effettuata sulla trasformazione dell\u2019acetofenone in (S)-1-feniletanolo, l\u2019attenzione \ue8 stata focalizzata sulla riduzione di differenti chetoni aromatici. Tutte le reazioni generalmente avvengono con elevate rese ed elevata selettivit\ue0 per il corrispondente (S)-alcol. Parte 2 Riduzioni catalizzate da una benzil-reduttasi (KRED1-Pglu) NADPH-dipendente proveniente da P. glucozyma, rappresentano un\u2019 interessante strategia sintetica per la riduzione stereoselettiva di chetoni aromatici. Dopo identificazione, la caratterizzazione e l\u2019over-espressione della proteina in E. coli, un metodo di purificazione e conservazione dell\u2019enzima \ue8 stato messo a punto. Dopo ottimizzazione dei parametri di reazione, \ue8 stato effettuato un accurato studio riguardante i substrati accettati da KRED1-Pglu. Questa proteina preferisce substrati stericamente ingombrati, spesso convertiti con elevata stereoelettivit\ue0. La differente enantioselettivit\ue0 sperimentalmente osservata, \ue8 stata spiegata mediante studi di docking molecolare. In conclusione, due differenti metodi efficienti ed eco-sostenibili per la riduzione stereoselettiva di diversi chetoni aromatici, utilizzando sia cellule intere di lieviti non convenzionali tra cui P. glucozyma CBS 5766 e l\u2019enzima isolato KRED1-Pglu sono stati presentati in questo lavoro di tesi.The Doctoral Thesis has been structured into two parts preceded by a general introduction that covers basic concepts on biocatalysis with special focus on application of whole cells of unconventional yeasts and a new ketoreducatese from Pichia glucozyma. All these thematic issues give a background to the following two parts, where the experimental results have been deeply discussed. Part 1 The biocatalytic potential of whole cells of P. glucozyma have been studied through biotransformations using substrates of different chemical nature having interesting pharmaceutical and synthetic aspects. After optimization of the reaction conditions through the transformation of acetophenone in (S)-1-phenylethanol, the attention was focused on the reduction of different aromatic ketones. All the reactions occurred with high yields and high enantiomeric excess for the corresponding (S)-alcohols. Part 2 Bioreduction catalysed by NADPH-dependent benzil-reductase (KRED1-Pglu) from Pichia glucozyma provides an attractive approach for selectively reducing a broad range of aromatic ketones. After protein identification, characterization and over-expression in E. coli, we have established a procedure for the purification and storage of KRED1-Pglu. After optimization of the reaction parameters, a thorough study of the substrate range of KRED1-Pglu was conducted. KRED1-Pglu prefers space demanding substrates which are often converted with high stereoselectivity. The observed activities and enantioselectivities were explained with a molecular modelling study for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrate. In conclusion, different efficient and sustainable methods for the stereoselective reduction of prochiral ketones using both whole cells of unconventional yeasts such as P. glucozyma CBS 5766 and the isolated enzyme KRED1-Pglu have been proposed in this PhD work

Efficient methodology to produce a duloxetine precursor using whole cells of Rhodotorula rubra

Different types of yeasts were employed as biocatalysts in the reduction of \u3b2-ketonitriles. The red microorganism, Rhodotorula rubra, was selected as the best performing catalyst in the reduction of different substituted ketonitriles giving total stereoselectivity in most cases (90-99% ee). In particular, its use as fresh and lyophilised cells was expanded to a semi-preparative scale for the production of the duloxetine precursor 1a. R. rubra was screened in the reduction of alkylation products in comparison with Pichia henricii for assignment of configuration of products 2a and 11a after derivatisation with S-MPA

Efficient Enzymatic Preparation of Flavor Esters in Water

A straightforward biocatalytic method for the enzymatic preparation of different flavor esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalyzed by an acyltransferase from Mycobacterium smegmatis (MsAcT) and proceeded with excellent yields (80-97%) and short reaction times (30-120 min), even when high substrate concentrations (up to 0.5 M) were used. This enzymatic strategy represents an efficient alternative to the application of lipases in organic solvents and a significant improvement compared with already known methods in terms of reduced use of organic solvents, paving the way to sustainable and efficient preparation of natural flavoring agents

Flow-based enzymatic synthesis of melatonin and other high value tryptamine derivatives: a five-minute intensified process

To increase the uptake of biocatalytic processes by industry, it is essential to demonstrate the reliability of enzyme-based methodologies directly applied to the production of high value products. Here, a unique, efficient, and sustainable enzymatic platform for the multi-gram synthesis of melatonin, projected to generate around 1.5 billion U.S. dollars worldwide by 2021, and its analogues was developed. The system exploits the covalent immobilization of MsAcT (transferase from Mycobacterium smegmatis) onto agarose beads increasing the robustness and longevity of the immobilized biocatalyst. The fully-automated process deriving from the integration between biocatalysis and flow chemistry is designed to maximize the overall yields (58-92%) and reduce reaction times (5 min), overcoming the limitation often associated with bioprocesses and bridging the gap between lab scale and industrial production

Sweet-and-salty biocatalysis : fructooligosaccharides production using Cladosporium cladosporioides in seawater

Production of fructooligosaccharides (FOS) from sucrose was obtained using a bioprocess entirely performed in seawater. The halophilic fungus Cladosporium cladosporioides MUT 5506 was grown in a seawater-based medium and mycelium displayed an optimal activity in seawater at 50\u201360 \ub0C, being stable up to 60 \ub0C. Under optimized conditions in seawater (50 \ub0C, sucrose 600 g/L, lyophilized mycelium 40 g/L), C. cladosporioides gave a maximum FOS yield of 344 g/L after 72 h with a preponderance of 1F-FOS derivatives (1-kestose 184 g/L, 1-nystose 98 g/L and 1-fructofuranosylnystose 22 g/L), and the noteworthy presence of the non-conventional disaccharide blastose (30 g/L after 144 h). Lyophilized mycelium exhibited good stability in seawater (76% of the initial activity was retained after 15 cycles of reutilization). This proof-of-concept application reports for the first time the production of FOS in a non-conventional medium such as seawater

The biocatalytic approach to the preparation of Pramipexole

(S)-Pramipexole is the most prescribed dopamine agonist endowed in the anti-Parkinson therapy, while the (R)-isomer (dexpramipexole) is currently in clinical development for the treatment of amyotrophic lateral sclerosis (ALS). The recently published asymmetric reduction of the prochiral ketone is the key step for the preparation of enantiomerically pure pramipexole and biocatalytic approach represents the smartest strategy as alternate method to the traditional fractional crystallization. The stereoselective potential of different yeasts was investigated in order to afford optically pure secondary alcohols. Saccharomyces cerevisiae ability to reduce the suitable ketone to the corresponding (R)-alcohol, the precursor of (S)-pramipexole, was proved with an e.e. > 98%. Twenty marine yeast strains were screened for ketoreductase activity with the aim of reducing the ketone with a stereochemical outcome opposite than S.cerevisiae, affording (S)-alcohol. In particular, Rhodotorula mucillaginosa strains gave (S)-alcohol with an e.e. ranging from 38% to 64%. Recombinant enzymes were also employed and interesting results were achieved by the application of a novel benzil reductase from Pichia glucozyma. Thanks to co-factor recycling system, the isolated enzyme was demonstrated to be able to produce (S)-alcohol with e.e. 84% . Moreover, an optimized system that employs biocatalytic potential of most promising yeast strains is under development in order to set up a continuous reaction based on Flow Chemistry technique

Lipase mediated enzymatic kinetic resolution of phenylethyl halohydrins acetates: A case of study and rationalization

Racemic phenylethyl halohydrins acetates containing several groups attached to the aromatic ring were resolved via hydrolysis reaction in the presence of lipase B from Candida antarctica (Novozym\uae 435). In all cases, the kinetic resolution was highly selective (E > 200) leading to the corresponding (S)-\u3b2-halohydrin with ee > 99 %. However, the time required for an ideal 50 % conversion ranged from 15 min for 2,4-dichlorophenyl chlorohydrin acetate to 216 h for 2-chlorophenyl bromohydrin acetate. Six chlorohydrins and five bromohydrins were evaluated, the latter being less reactive. For the \u3b2-brominated substrates, steric hindrance on the aromatic ring played a crucial role, which was not observed for the \u3b2-chlorinated derivatives. To shed light on the different reaction rates, docking studies were carried out with all the substrates using MD simulations. The computational data obtained for the \u3b2-brominated substrates, based on the parameters analysed such as NAC (near attack conformation), distance between Ser-O and carbonyl-C and oxyanion site stabilization were in agreement with the experimental results. On the other hand, the data obtained for \u3b2-chlorinated substrates suggested that physical aspects such as high hydrophobicity or induced change in the conformation of the enzymatic active site are more relevant aspects when compared to steric hindrance effects

Preparation of Sterically Demanding 2,2-Disubstituted-2-Hydroxy Acids by Enzymatic Hydrolysis

Preparation of optically-pure derivatives of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid of general structure 2 was accomplished by enzymatic hydrolysis of the correspondent esters. A screening with commercial hydrolases using the methyl ester of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid (1a) showed that crude pig liver esterase (PLE) was the only preparation with catalytic activity. Low enantioselectivity was observed with substrates 1a\u2013d, whereas PLE-catalysed hydrolysis of 1e proceeded with good enantioselectivity (E = 28), after optimization. Enhancement of the enantioselectivity was obtained by chemical re-esterification of enantiomerically enriched 2e, followed by sequential enzymatic hydrolysis with PLE. The preparation of optically-pure (S)-2e was validated on multi-milligram scale

Enzymatic reduction of acetophenone derivatives with a benzil reductase from Pichia glucozyma (KRED1-Pglu): electronic and steric effects on activity and enantioselectivity

A recombinant ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for the enantioselective reduction of various mono-substituted acetophenones. Reaction rates of meta- and para-derivatives were consistent with the electronic effects described by \u3c3-Hammett coefficients; on the other hand, enantioselectivity was determined by an opposite orientation of the substrate in the binding pocket. Reduction of ortho-derivatives occurred only with substrates bearing substituents with low steric impact (i.e., F and CN). Reactivity was controlled by stereoelectronic features (C[double bond, length as m-dash]O length and charge, shape of LUMO frontier molecular orbitals), which can be theoretically calculated

Marine microorganisms as source of stereoselective esterases and ketoreductases : kinetic resolution of a prostaglandin intermediate

A screening among bacterial strains isolated from water-brine interface of the deep hypersaline anoxic basins (DHABs) of the Eastern Mediterranean was carried out for the biocatalytical resolution of racemic propyl ester of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (R,S)-1, a key intermediate for the synthesis of d-cloprostenol. Bacillus horneckiae 15A gave highly stereoselective reduction of (R,S)-1, whereas Halomonas aquamarina 9B enantioselectively hydrolysed (R,S)-1; in both cases, enantiomerically pure unreacted (R)-1 could be easily recovered and purified at molar conversion below 57\u201358 %, showing the potential of DHAB extremophile microbiome and marine-derived enzymes in stereoselective biocatalysis