László Poppe

Phenylalanine Ammonia‐Lyase: The Use of Its Broad Substrate Specificity for Mechanistic Investigations and Biocatalysis—Synthesis of L‐Arylalanines

Several fluoro- and chlorophenylalanines were found to be good substrates of phenylalanine ammonia-lyase (PAL/EC 4.3.1.5) from parsley. The enantiomerically pure L-amino acids were obtained in good yields by reaction of the corresponding cinnamic acids with 5M ammonia solution (buffered to pH 10) in the presence of PAL. The kinetic constants for nine different fluoro- and chlorophenylalanines do not provide a rigorous proof for but are consistent with the previously proposed mechanism comprising an electrophilic attack of the methylidene-imidazolone cofactor of PAL at the aromatic nucleus as a first chemical step. In the resulting Friedel-Crafts-type sigma complex the beta-protons are activated for abstraction and consequently the pro-S is abstracted by an enzymic base. Results from semi-empirical calculations combined with a proposed partial active site model showed a correlation between the experimental kinetic constants and the change in polarization of the pro-S Cbeta-H bond and heat of formation of the sigma complexes, thus making the electrophilic attack at the neutral aromatic ring plausible. Furthermore, while 5-pyrimidinylalanine was found to be a moderately good substrate of PAL, 2-pyrimidinylalanine was an inhibitor.

Methylidene-imidazolone: a novel electrophile for substrate activation.

The recent three-dimensional structure of histidine ammonia-lyase revealed that the enzyme contains a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) ring, which forms autocatalytically from an Ala-Ser143-Gly triad. This novel prosthetic group, which is also present in phenylalanine ammonia-lyase, activates substrates by electrophilic interaction. Modern analytical methods, theoretical calculations and molecular biology tools have given further insight into the mode of action of MIO.

The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases

Besides the post‐translationally cyclizing catalytic Ala‐Ser‐Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5), histidine ammonia‐lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X‐ray structures of PAL revealed that the Tyr110‐loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL (∼500 amino acids) and plant and fungal PALs (∼710 amino acids), a core PAL/HAL domain (∼480 amino acids) with ≥ 30% sequence identity along the different species is common. In plant and fungal PAL a ∼100‐residue long C‐terminal multi‐helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X‐ray structures, a Tyr83‐loop‐in arrangement has been found. Based on the HAL structures, a Tyr110‐loop‐in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop‐in/loop‐out structures were compared. To study the role of the C‐terminal multi‐helix domain, Tyr‐loop‐in/loop‐out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C‐terminal domain were also built. Molecular dynamics studies indicated that the Tyr‐loop‐in conformation was more rigid without the C‐terminal multi‐helix domain. On this basis it is hypothesized that a role of this C‐terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.

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].

Baker's yeast mediated stereoselective biotransformation of 1-acetoxy-3-aryloxypropan-2-ones

Abstract A series of 1-acetoxy-3-aryloxypropan-2-ones 1a–m were synthesized and subjected to biotransformation by bakers yeast yielding optically active monoacetates 5 or ent - 5 and/or diols 4 of moderate to excellent enantiomeric purity. The dependence of the reduction/hydrolysis ratio and stereoselectivity on the size and substitution pattern of the aromatic moiety in the substrate is also discussed.

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 .

Lipase-catalyzed enantiomer selective hydrolysis of 1,2-diol diacetates

Abstract Enantiomer selective hydrolysis of racemic 1,2-diol diacetates (rac-2a-h) was investigated by using the inexpensive commercial porcine pancreatic lipase. The hydrolysis proceeds with variable regioselectivity but with moderate to good enantioselectivity yielding a mixture of isomeric monoacetates (3a-h and 4a-h) and unchanged diacetate enantiomers (2a-h). Evidence was found that both monoacetates (3a-h and 4a-h) are formed with the same sense of enantiomer selectivity.

Convenient synthetic route to (+)-faranal and (+)-13-norfaranal : The trail pheromone of pharaoh's ant and its congener 1

Abstract (+)-Faranal 1a , the trail pheromone of Pharaohs ant, and its congener, (+)-13-norfaranal 1b were synthetized from chiral building block 4 employing diastereoselective carbon-carbon bond formation. The application of crude pig liver esterase enzyme for the preparation of 4 is also discussed.

Mechanism of the Tyrosine Ammonia Lyase Reaction—Tandem Nucleophilic and Electrophilic Enhancement by a Proton Transfer

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel-Crafts (FC) route for ammonia elimination from L-tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L-tyrosine-binding state (0.0 kcal  mol(-1)) to the product-binding state ((E)-coumarate+H(2)N-MIO; -24.0 kcal  mol(-1); MIO = 3,5-dihydro-5-methylidene-4H-imidazol-4-one) involves an intermediate (IS, -19.9 kcal  mol(-1)), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4 kcal  mol(-1)) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300-OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2 kcal  mol(-1)) indicates a concerted C-N bond breaking of the N-MIO intermediate and deprotonation of the pro-S β position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO-containing ammonia lyases and 2,3-aminomutases.