Csaba Paizs

Optically active 1-(benzofuran-2-yl)ethanols and ethane-1,2-diols by enantiotopic selective bioreductions

Abstract Enantiotopic selective reduction of 1-(benzofuran-2-yl)ethanones 1a – d , 1-(benzofuran-2-yl)-2-hydroxyethanones 4a – c and 2-acetoxy-1-(benzofuran-2-yl)ethanones 3a – c was performed by bakers yeast for preparation of optically active (benzofuran-2-yl)carbinols [( S )- 5a – d , ( S )- 6a – c and ( R )- 6a – c , enantiomeric excess from 55 to 93% ee].

Candida antarctica lipase A in the dynamic resolution of novel furylbenzotiazol-based cyanohydrin acetates

A series of novel (R)-furylbenzotiazol-based cyanohydrin acetates were prepared in over 90% isolated yields from the corresponding furancarbaldehydes. The one-pot method combines a basic resin to produce hydrogen cyanide from acetone cyanohydrin, an equilibrium between the formation and decomposition of furylbenzotiazol-based cyanohydrins and the unique enantioselectivity of Candida antarctica lipase A, allowing the acylation of (R)-cyanohydrins in the presence of vinyl acetate in anhydrous acetonitrile.

Preparation of novel phenylfuran-based cyanohydrin esters: lipase-catalysed kinetic and dynamic resolution

A series of novel (R)-5-phenylfuran-2-yl cyanomethyl butanoates were prepared by Pseudomonas cepacia lipase- catalysed dynamic kinetic resolution in toluene. The method exploits a basic resin both for the racemization and formation of phenylfuran-based cyanohydrins and for the decomposition of acetone cyanohydrin in one-pot with enzymatic enantioselective acylation using vinyl butanoate. The lipase-catalysed methanolysis of racemic 5-phenylfuran-2-yl cyanomethyl butanoates in toluene with E 100 was shown to be usable when the corresponding (S)-butanoates are needed. Candida antarctica lipase A provided racemic cyanohydrin butanoates with quantitative chemical yields under mild conditions.

Kinetic resolution of 1-(benzofuran-2-yl)ethanols by lipase-catalyzed enantiomer selective reactions

Abstract Kinetic resolution of racemic 1-(benzofuran-2-yl)ethanols rac - 1a – d was performed by lipase-catalyzed enantiomer selective acylation ( E ≫100) yielding (1 R )-1-acetoxy-1-(benzofuran-2-yl)ethanes ( R )- 2a – d and (1 S )-1-(benzofuran-2-yl)ethanols ( S )- 1a – d in highly enantiopure form. The degree of enantiomer selectivity for enzymatic alcoholysis/hydrolysis processes starting from racemic 1-acetoxy-1-(benzofuran-2-yl)ethane rac - 2 was also tested under various conditions including supercritical CO 2 medium. Racemization-free lipase-catalyzed ethanolysis of the (1 R )-1-acetoxy-1-(benzofuran-2-yl)ethanes ( R )- 2a – d yielded almost quantitatively the enantiopure (1 R )-1-(benzofuran-2-yl)ethanols ( R )- 1a – d .

Immobilization of Phenylalanine Ammonia‐Lyase on Single‐Walled Carbon Nanotubes for Stereoselective Biotransformations in Batch and Continuous‐Flow Modes

Carboxylated single‐walled carbon nanotubes (SwCNTCOOH) were used as a support for the covalent immobilization of phenylalanine ammonia‐lyase (PAL) from parsley by two different methods. The nanostructured biocatalysts (SwCNTCOOH‐PALI and SwCNTCOOH‐PALII) with low diffusional limitation were tested in the batch‐mode kinetic resolution of racemic 2‐amino‐3‐(thiophen‐2‐yl)propanoic acid (1) to yield a mixture of (R)‐1 and (E)‐3‐(thiophen‐2‐yl)acrylic acid (2) and in ammonia addition to 2 to yield enantiopure (S)‐1. SwCNTCOOH‐PALII was a stable biocatalyst (>90 % of the original activity remained after six cycles with 1 and after three cycles in 6 M NH3 with 2). The study of ammonia addition to 2 in a continuous‐flow microreactor filled with SwCNTCOOH‐PALII (2 M NH3, pH 10.0, 15 bar) between 30–80 °C indicated no significant loss of activity over 72 h up to 60 °C. SwCNTCOOH‐PALII in the continuous‐flow system at 30 °C was more productive (specific reaction rate, rflow=2.39 μmol min−1 g−1) than in the batch reaction (rbatch=1.34 μmol min−1 g−1).

Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production.

Carboxylated single-walled carbon nanotubes (SWCNTCOOH) were used as support for covalent immobilization of Candida antarctica lipase B (CaL-B) using linkers with different lengths. The obtained nanostructured biocatalysts with low diffusional limitation were tested in batch mode in the ethanolysis of the sunflower oil. SWCNTCOOH-CaL-B proved to be a highly efficient and stable biocatalyst in acetonitrile (83.4% conversion after 4h at 35°C, retaining >90% of original activity after 10 cycles).

Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.

Preparation of unnatural amino acids with ammonia-lyases and 2,3-aminomutases

Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.

Bisepoxide Cross‐Linked Enzyme Aggregates—New Immobilized Biocatalysts for Selective Biotransformations

Glycerol diglycidyl ether (GDE) is a convenient and inexpensive bisepoxide cross‐linker as demonstrated by the preparation of cross‐linked enzyme aggregates (CLEAs) from two enzyme classes. The GDE CLEAs of lipase from Pseudomonas fluorescens (AK), lipase from Burkholderia cepacia (PS), and lipase B from Candida antarctica (CaL B) as well as of phenylalanine ammonia‐lyase (PAL) from Petroselinum crispum demonstrated improved properties as compared with their glutaraldehyde (GA) cross‐linked counterparts. Ultrasonication studies indicated that the GDE CLEAs of lipase PS and PAL were mechanically more stable than the GA CLEAs. In the kinetic resolution of rac‐1‐phenylethanol, the catalytic activity of the GDE–lipase CLEAs (U=69.6, 134.8, and 127.4 U g−1 for AK, CaL B, and PS prepared at 22 °C, respectively) surpassed that of the corresponding GA–lipase CLEAs (U=24.4, 131.0, and 119.2 U g−1 for AK, CaL B, and PS prepared at 22 °C, respectively). The GDE co‐CLEAs from PAL and bovine serum albumin (BSA) could be recycled at least three times if used for the stereoselective ammonia addition in 6 M ammonia to (E)‐3‐(thiophen‐2‐yl)acrylic acid, whereas the recycling of the conventional GA–PAL CLEAs from this medium failed.

Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l‐PG and l‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.