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1. Introduction

Chiral nitrogen-containing molecules and their derivatives have gained importance in fine chemical, pharmaceutical, agrochemical industries and also in electronics and optics, since N-containing structures frequently occur in many biologically active small molecules and peptidomimetics. Amongst them, amino alcohols are extensively used as chiral auxiliaries of many biologically active compounds and natural products, whereas protected and non-protected amino acids play an important role in artificial peptide design. Therefore, synthesis of enantiopure nitrogen-containing compounds is an attractive and prominent research field providing direct applicability on both laboratory and industrial scales.

Additionally, enormous effort has also been devoted to the development of N-containing ligands to suit organometallic and coordination chemistry. Once integrated into metal complexes, amino moieties should provide important structural benefits. Transition metal complexes with nitrogen-containing ligands proved to be stable, easily separable and recyclable catalysts for asymmetric synthesis, homogeneous and heterogeneous catalysis.

Numerous chemical and biochemical processes have been developed as providing practical route to enantiomerically pure amino-containing compounds. Biocatalysis often offers several advantages over chemical synthesis, e.g. high stereo-, regio- and chemoselectivity and mild reaction conditions (ambient temperature, atmospheric pressure), allowing the transformation of sensitive substrates. Moreover, it is a green, sustainable alternative for some transformations impossible to perform via traditional organic synthesis.

In chemo-enzymatic processes biocatalysts are often used either as whole microorganisms or isolated enzymes. When whole cell systems are used, there is no need for enzyme isolation or purification, thus the enzyme stability can be enhanced. Regarding the cofactor dependent enzymes, cofactor regeneration is typically provided by the cell’s own metabolic machinery. Although cellular systems provide all these benefits, they usually have several drawbacks: lower selectivities could be achieved (due to the competition from other similar enzymes present in the cell) and the control and maximization of the synthetic processes could be difficult. In contrast, in vitro approaches are less complex and, therefore, reaction conditions are easier to improve and optimize by enzyme or substrate concentration, co-solvents, pH or temperature.

Isolated wild-type enzymes are often highly selective catalysts of a certain transformation, but are very sensitive and generally not sufficiently tolerant to process conditions or not sufficient active to ensure a high productivity. Protein engineering is the most important method to overcome the limitation of natural enzymes as biocatalysts. Directed evolution technologies and new developments in genomics offer many possibilities for the manufacture of tailor-made biocatalysts. Today we are able to tailor to engineer the enzyme structure, in order to fit the process specifications. Beside protein engineering techniques, enzyme immobilization is a frequently used approach toward enzyme stabilization and reuse, thereby lowering the biocatalysts cost contribution to the process.  Normally, immobilization involves attaching the enzyme to an inert and usually insoluble support. The result is a recoverable, stable and specific biocatalyst.

Amongst all enzymes, hydrolases are frequently used for the resolution processes where one of the enantiomers in a racemic mixture is selectively modified, to afford a separable derivative. They catalyse many organic reactions including esterification, transesterification, acylation and hydrolysis. Popularity of hydrolases in general and lipases in particular relies on their selectivity, mild reaction conditions, ability to utilize wide range of substrates and high stability towards extreme temperature and pH. Lipases are the most important biocatalysts frequently employed to perform new reactions both in non-aqueous, aqueous media and in the synthesis of various biologically active and pharmaceutically important molecules.

Ammonia-lyases catalyse the reversible addition of ammonia and amines to double bonds of the corresponding α,β-unsaturated carboxylic acids, which are often cheap substrates. In the past decade, a wide range of biocatalytic and therapeutic applications of ammonia-lyases have been emerged. Ammonia-lyase catalysed reactions can be used for the stereoselective production of valuable synthetic amino acids.

This thesis is divided into two separate parts.

The first part describes two chemo-enzymatic approaches for the total synthesis of bufuralol enantiomers, a widely used β-adrenoceptor antagonist. One procedure involves the baker’s yeast whole cells-catalysed enantiotopselective transformation of α-hydroxy and α-acetoxymethyl ketones, with opposite stereopreference, into the corresponding diol enantiomers. In the second approach, stereoselectivity relies on the lipase-mediated enantioselective O-acylation of the corresponding bromo-alcoholic intermediates.

The second part of the thesis deals with the preparation of α- and β-amino acids through biocatalytic processes. First, a microfluidic, continuous-flow system filled with phenylalanine ammonia-lyase from Petroselinum crispum immobilized on magnetic nanoparticles (PcPAL-MNP) was developed and tested for the efficient kinetic resolution of five unnatural α-amino acids. Reliability and reproducibility assessments of the measurements were also performed in order to validate the designed system. Finally, a lipase-catalysed stereoselective hydrolytic procedure was developed which enables the efficient kinetic resolution of poorly soluble and highly unstable β-amino acid ethyl esters containing a phenylfuran skeleton. These compounds were resolved, for the first time, in their hydrochloric salt forms, which adumbrating possible applicability of this method to the manipulation of unstable amino acids and amino esters.

2. PART I. Synthesis of bufuralol

2.1. Importance of vicinal amino alcohols as β-blockers

Enantiomerically pure 1,2-amino alcohols, also named as vicinal amino alcohols, are important and versatile building blocks of biologically important compounds, natural products and chiral auxiliaries for asymmetric synthesis . A large number of asymmetric synthetic methods have been developed for the preparation of vicinal amino alcohols, relying on the functional group of amino acids manipulation , introduction of the amino alcohol moiety on a pre-existing carbon skeleton , or the coupling of two molecules . Besides of the organocatalytic processes, the importance of biocatalysis as an efficient, environmentally friendly and sustainable alternative has been growing rapidly. Lipases  and transaminases  have successfully been used for the asymmetric synthesis of 2-amino-1-arylethanol derivatives.

The β-blocking activity of 1,2-aminoalcohols was first documented in the early 1960’s, when Black and Stephenson demonstrated that pronethalol antagonizes the myocardial beta adrenergic receptors, decreasing heart rate and increasing exercise tolerance in people with angina.  (Scheme 1) In 1964 Pritchard described the antihypertensive effect of pronethalol , however, it never came into clinical use due to its side-effects in mice and man.  Propranolol was the first β-blocker approved as antihypertensive agent. Moreover, it became an accepted treatment for arrhythmia and hypertrophic cardiomyopathy. Due to their multiple pharmacodynamics and pharmacokinetic actions including β1-selectivity, partial agonist activity, α-adrenergic activity, direct vasodilator activity, the introduction of β-adrenoceptor blocking drugs in medicine has been a huge impact on the treatment of many cardiovascular diseases (systemic hypertension, angina pectoris, arrhythmias, etc.).

Scheme 1. Beta blockers with 1,2-aminoalcochol moiety

2.1.1. Bufuralol

Bufuralol is a chiral benzofuran-based 1,2-aminoalcohol, first described by Fothergill and developed by Roche.  It is a potent, non-selective β-adrenoceptor antagonist closely resembles propranolol which has in addition β-adrenoceptor agonistic properties.  It is successfully used for the treatment of hypertension  and presents inhibitory effect toward testosterone 6β-hydroxylase in bacterial membranes.

The metabolism of bufuralol is very complex; it is mediated by cytochrome P-450 system in human liver microsomes  and undergoes a series of transformations to alcohol and ketone metabolites also possessing β-receptor blocking activities.  Therefore it is also important and widely used for polymorphism studies of these enzyme complexes.

Although the β-blocking potency of the (S)-bufuralol is significantly higher than that of the (R)-enantiomer, the (R)-bufuralol is a generally used as marker of hepatic CYP2D6 activity.  Thus, there is a high demand for the stereoselective synthesis of both isomer of bufuralol in enantiopure form.

Several asymmetric procedures have been already described using both bio- and organo-catalytic strategies (Scheme 2). (S)-bufuralol has been synthesized via (−)-B-chlorodiisopinocampheylborane ((−)-DIP-Cl) mediated stereoselective reduction of 2-bromo-1-(7-ethylbenzofuran-2-yl) ethanone  (Scheme 2, i.) or via asymmetric transfer hydrogenation of 1-(7-ethylbenzofuran-2-yl)-2-mesyloxyethanol catalysed by Cp*RhCl[(S,S)-TsDPEN] as the key step  (Scheme 2, iii.). More recently, the enantiotope selective cyanation of 7-ethylbenzofuran-2-carbaldehyde using a homochiral metal-organic catalyst has been performed affording enantiomerically enriched (S)-bufuralol (yield 95%, ee 98%, Scheme 2, iv.).  The transfer hydrogenation approach has been successfully extended to the synthesis of (R)-bufuralol (Scheme 2, ii.).  Turner have shown that lipases from Candida and Pseudomonas species can mediate the esterification, acetylation or hydrolysis of ethylbenzofuran chloroethanol, which can be further transformed into (R)-bufuralol.  A few years later, the combination of PSC Amano II lipase and a ruthenium complex as racemization agent has successfully led to the dynamic kinetic resolution of aforementioned chloro alcohol through enantioselective O-acylation, contributing, after all, to the synthesis of (R)-bufuralol in high ee and good overall yield (Scheme 2, vi.).

Scheme 2. Several asymmetric synthetic strategies for the preparation of bufuralol enantiomers

2.2. Aim of the study

The aim of the study includes the enantioselective synthesis of both (R)- and (S)-enantiomers of bufuralol by way of chemo-enzymatic procedures including lipase- or baker’s yeast catalysed reaction as the key stereoselective step. The first approach is based on the baker’s yeast-mediated enantioselective transformation of 2-(7-ethylbenzofuran-2-yl)-2-oxoethyl acetate 5 and 1-(7-ethylbenzofuran-2-yl)-2-hydroxyethanone 6, whereas in the second procedure, stereoselectivity was achieved introduced by lipase-catalysed acylation of rac-2-bromo-1-(7-ethylbenzofuran-2-yl)ethanol rac-9. The obtained enantiomerically enriched intermediates were further transformed into the desired enantiomers of bufuralol.

2.3. Synthesis of (R)- and (S)-bufuralol via baker’s yeast-mediated biotransformation

The use of baker’s yeast in biocatalytic processes is widespread due to its low cost, mild reaction conditions and availability of the whole-cell system. Nevertheless, oxido-reductases and hydrolases present in Saccharomyces cerevisiae cells offer high stereoselectivity and broad substrate acceptability.

Our group has previously demonstrated that Saccharomyces cerevisiae cells transform α-hydroxy and α-acetoxymethyl ketones with opposite stereopreference into the corresponding diol enantiomers.  The bioreduction of the α-acetoxymethyl ketones by oxidoreductases from baker’s yeast is usually followed by a subsequent enzymatic hydrolysis of the formed hydroxy-monoacetate into 1,2-ethanediol by the hydrolases also present in baker’s yeast.

Moreover it has been revealed that the enantioselective bioreduction of 1-(benzofuran-2-yl)-2-hydroxyethanones and 2-acetoxy-1-(benzofuran-2-yl)ethanones afforded the opposite enantiomers of the diols with high enantiomeric purity . Ketones bearing the relatively small and hydrophilic hydroxymethyl group were reduced from the same face, whereas acetoxymethyl ketones were converted from the opposite enantiotopic face. In this latter case, the desired 1,2-diol is obtained from a subsequent hydrolysis of the reduction product acetoxymethyl-alcohol.

The synthesis starts with the preparation of racemic 2-(7-ethylbenzofuran-2-yl)-2-oxoethyl acetate 5 and 1-(7-ethylbenzofuran-2-yl)-2-hydroxyethanone 6 used as substrates for the enzymatic step (Scheme 3). 2-Ethylphenol 1 was ortho-formylated with paraformaldehyde in the presence of magnesium chloride and triethylamine. The obtained 3-ethyl-salicylaldehyde 2 was transformed into 7-ethyl-benzofuran-2yl-ethanone 3 which was further α-brominated using pyridinium tribromide. The bromo ketone 4 was further transformed with sodium acetate under anhydrous conditions into the corresponding α-acetoxymethyl-ketone 5. Subsequent lipase-assisted ethanolysis of compound 5 afforded the desired hydroxymethyl-ketone 6.

Scheme 3. Chemo-enzymatic synthesis of bufuralol enantiomers using baker’s yeast-mediated biotransformations

The aforementioned enantiotop selectivity of the Saccharomyces cerevisiae cells proved valid also for bufuralol intermediates (Figure 1). The analytical-scale enzymatic reactions were performed under fermenting and non-fermenting conditions. The influence of various additives upon the enzyme selectivity was also tested in order to enhance the enantiopurity of the products (Table 1). Stereochemical progress of the biotransformations was monitored by a chiral analytical HPLC method elaborated for the racemic diol rac-7 (obtained by reduction of 6 with NaBH4 in methanol).

Figure 1. HPLC chromatograms of the products obtained from baker’s yeast mediated biotransformation: A) Reference chromatogram of racemic 1,2-diol; B) (R)-enantiomer obtained starting from acetoxymethyl-ketone 5; C) (S)-enantiomer obtained starting from hydroxymethyl-ketone 6 NU SE PREA POTRIVESC TIMPII DE RETENTIE, TREBUIE SA CAUTI ALTII, NORBI ARE IN DIZERTATIE CEVA MAI BUN de exemplu

As can be observed from Table 1, different additives have noticeable influence on the enantiomeric excess of the products. While all additives (except for ethyl bromoacetate) used in the biotransformation of acetoxymethyl-ketone 5 enhance the selectivity, in the case of hydroxymethyl-ethanone 6 the effect is reversed. Only the use of L-cysteine under fermenting conditions results in higher ee in comparison with the reaction conducted without additive (Table 1, Entry 4). The best result for the transformation of acetoxymethyl-ketone 5 was obtained in fermenting system using n-hexane as additive (Table 1, Entry 3)

Table 1. Fermenting and non-fermenting cellular biotransformations of 5 and 6

Entry Additives

(0.5% w/w) eea(R)-7a ee(S)-7b

Fermenting Non-fermenting Fermenting Non-fermenting

1 Without additive 92 90 86 87

2 Allyl alcohol 96 96 80 80

3 n-Hexane 98 95 50 78

4 L-Cysteine 96 95 96 76

5 Ethyl bromoacetate − − − −

6 MgCl2 96 92 62 50

7 DMSO 95 94 76 77

afrom prochiral acetoxymethyl-ketone 5

bfrom prochiral hydroxymethyl-ketone 6

The isolated (R)- and (S)-1-(7-ethylbenzofuran-2-yl)ethane-1,2-diols ((R)- and (S)-7) were transformed into the corresponding enantiomer of bufuralol by regioselective tosylation with para-toluenesulfonyl chloride in the presence of dibutyltin(IV) oxide  and a subsequent replace of the tosyl group with tert-butylamine (Scheme 3). Enantiopurity of the end-product isomers was slightly dropped (ee 96% for (R)-8 and 93% for (S)-8) compared to those of (R)- and (S)-heteroaryl-1,2-diols (ee  98% for (R)-7 and 96% for (S)-7), beside 40% overall yield starting from (R)- and (S)-7.

2.4. Lipase-catalysed kinetic resolution of 2-bromo-1-(7-ethylbenzofuran-2-yl)-ethanol for the synthesis of (R)- and (S)-bufuralol

The second approach for the synthesis of (R)- and (S)-bufuralol involves the lipase-catalysed stereoselective O-acylation of racemic 2-bromo-1-(7-ethylbenzofuran-2-yl)ethanol rac-9 (Scheme 4) obtained through reduction of bromo-ketone 4 with sodium borohydride.

Scheme 4. Synthesis of (R)- and (S)- bufuralol from the lipase catalysed resolution products of the racemic bromoethanol rac-9

Commercially available immobilized lipases, such as lipases A and B from Candida antarctica (CaL-A on Celite and CaL-B imobilized on hydrophobic acrylic resin commercialized as Novozyme 435), lipases from Pseudomonas species (LAK and LPS), Candida rugosa (CRL) were tested for the analytical-scale O-acylation of rac-9 using vinyl acetate and vinyl laurate as acyl donors in various organic solvents. Lipase A from Candida antarctica showed excellent reactivity but poor selectivity, whereas L-AK, CRL and LPS displayed reduced activity but good selectivity in all tested solvents.

Table 2. CaL-B catalysed enantioselective acylation of rac-9 with vinyl acetate and vinyl dodecanoate after 16 h

Entry Solvent eep (%) ees (%) c (%) E

1 MTBEa 99 49 33 » 200

2 MTBEb 99 41 29 >200

3 DIPEa >99 >99 50 » 200

4 n-Hexanea >99 69 41 >200

5 n-Hexaneb >99 93 48 » 200

6 Acetonitrilea >99 42 30 >200

7 Toluenea >99 71 41 » 200

8 Tolueneb >99 99 50 » 200

9 n-Octanea >99 99 50 » 200

a vinyl acetate;  b vinyl dodecanoate

CaL-B proved to be the most suitable biocatalyst. The enantioselectivity was high with both vinyl esters in all tested solvents (Table 2). Excellent enantiomeric excesses and high reaction rates were obtained in diisopropyl ether (DIPE) and n-octane using vinyl acetate as acyl donor (Table 2, Entries 3 and 9) and in toluene using vinyl laurate, respectively (Table 2, Entry 8). Further the scale-up of the optimal analytical - scale reactions were performed, maintaining the same substrate-biocatalyst ratio. The reactions’ progress were checked with TLC and HPLC and stopped at an approximately 50% conversion preserving the high enantiomeric excesses of the resolutions products obtained during analytical-scale optimization. Negligible decrease of the ee (~97%) was detected when vinyl acetate was used. Both enantiomerically pure compounds were isolated in excellent yields and used for further transformations.

Figure 2. Illustrative HPLC chromatograms for the lipase-catalysed kinetic resolution of rac-9: A) Reference chromatogram of racemic bromo laurate rac-10b and bromo alcohol rac-9; B) O-acylation of bromo alcohol with vinyl laurate in toluene idem

In order to prevent the undesired secondary reactions and also the racemization of the enantiomers, two particular chemical route were developed for the efficient synthesis of (R)- and (S)- bufuralol.

The reaction of tert-butylamine with optically pure bromo alcohol (R)-9 was investigated in various conditions (data not given). Due to the instability of (R)-9 under basic conditions, in each case significant loss in enantiopurity of bufuralol was observed, associated with poor reaction yields. The mild protection of the hydroxy group with trimethylsilyl-N,N-dimethylcarbamate (DMCTMS, Scheme 2) allowed the substitution of bromine with tert-butyl amine on (R)-12, preventing racemization. The deprotection of (R)-12 was possible only with HF obtaining the desired (S)-bufuralol (S)-8 with slightly reduced enantiopurity (ee 90%) and 35% overall yield.

For the synthesis of (R)-bufuralol ((R)-8) the optically pure acylated bromohydrins (S)-10a,b were transformed into the epoxide (R)-11 in the presence of LiOH, which was further transformed in neat tert-butylamine into (R)-8 with good yields (53% overall yield) and excellent enantiomeric excess (ee 98%).

In order to improve the enantiopurity of the (S)-bufuralol, this latter strategy was applied for the transformation of (R)-9 into (S)-8. Since CaL-A proved to be a highly active but unselective biocatalyst, the acylation of (R)-9 with vinyl dodecanoate in DIPE in presence of CaL-A was performed and the quantitatively obtained ester (R)-10b (ee 99%) was further transformed into the target (S)-8 (ee 98%).

2.5. Conclusions

We developed two chemo-enzymatic approaches which involve different biocatalytic steps, i.e. baker’s yeast-catalysed biotransformation or lipase-catalysed enantioselective O-acylation, for the synthesis of (S) and (R)-2-(tert-butylamino)-1-(7-ethylbenzofuran-2-yl)ethanol (S) and (R)-bufuralol. The desired compounds were obtained with high enantipurity (ee 98%) and good overall yield (53%).

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