Figure - uploaded by Laszlo Poppe
Content may be subject to copyright.
Scheme 2. Preparation of a-aminophosphonic acids (R)(+ +)-3 and (S)-(À)-3 with absolute configuration assignment by the X-ray method. Reaction conditions: a) Me 2 NH, H 2 O 2 ;b )T MSBr, allylTMS; c) H 2 O; d) HCl, dioxane; e) 4BrC 6 H 4 NCO,pyridine.
Source publication
![Figure3.Production curves of (E)-cinnamic acid [(E)-CA] formationf rom...](profile/Laszlo-Poppe/publication/316648342/figure/fig4/AS:522579153821696@1501603987022/Figure3Production-curves-of-E-cinnamic-acid-E-CA-formationf-rom-l-Phe-S-1c_Q320.jpg)
![Figure 4. Thermodynamic bindinge nergieso fd-Phe [(S)-1], and each...](profile/Laszlo-Poppe/publication/316648342/figure/fig5/AS:522579153571840@1501603987114/Thermodynamic-bindinge-nergieso-fd-Phe-S-1-and-each-enantiomero-ft-he_Q320.jpg)
Aromatic amino acid ammonia‐lyases and aromatic amino acid 2,3‐aminomutases contain the post‐translationally formed prosthetic 3,5‐dihydro‐4‐methylidene‐5 H ‐imidazol‐5‐one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α‐ or β‐amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Chara...
Citations
... [33] Prominent examples include (R)-phosphaleucine, [34] (R)-and (S)-phosphaalanine (as a component of the drugs alafosfalin and fotemustine), [35] (R)-phosphatyrosine (as component of the bioactive compound K26), [36] and phosphaphenylalanine, to name but a few. [37] Due to their interesting properties, today stereoselective syntheses for some phospha-analogs to proteinogenic α-aminocarboxylic acids are known, but they have never been prepared by the same strategy. Usually, completely different approaches are needed for each target compound, and often both enantiomers have to be synthesized by different sequences. ...
Aminophosphonic acids have a remarkably broad bioactivity spectrum. They can function as highly efficient transition state mimics for a variety of hydrolytic and angiotensin‐converting enzymes, which makes them interesting target structures for synthetic chemists. In particular, the phosphonic acid analogs to α‐aminocarboxylic acids (PaAAs) are potent enzyme inhibitors, but many of them are only available by chiral or enzymatic resolution; sometimes only one enantiomer is accessible, and several have never been prepared in enantiopure form at all. Today, a variety of methods to access enantiopure α‐aminophosphonic acids is known but none of the reported approaches can be generally applied for the synthesis of PaAAs. Here we show that the phosphonic acid analogs of many (proteinogenic) α‐amino acids become accessible by the catalytic, stereoselective asymmetric transfer hydrogenation (ATH) of α‐oxo‐phosphonates. The highly enantioenriched (enantiomeric excess (ee) ≥ 98 %) α‐hydroxyphosphonates obtained are important pharmaceutical building blocks in themselves and could be easily converted to α‐aminophosphonic acids in most studied cases. Even stereoselectively deuterated analogs became easily accessible from the same α‐oxo‐phosphonates using deuterated formic acid (DCO2H).
... In addition, D-phenylalanine (D-1a) was known as a competitive inhibitor of PALs, and the determined inhibitory constant (K i ) was in the same order of magnitude as the K m of L-1a, which would result in an attainable V max , but a significantly increased K m . 34,35 Therefore, we would refer to previous data reported for rac-amino acids for rough evaluation only, not for the direct comparison to our results. ...
Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of l-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward l-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55-60 °C and pH 9.0-9.5. However, four of them, LsPAL1-LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.
... Co-crystallization with 10-fold excess of (R)-APEP or (S)-APPA resulted in full occupancy of the active sites of PcPAL (Figure 3a,b). In the structures, both (R)-APEP and (S)-APPA are covalently attached to the MIO residue by their amino group, despite the reversibility of the inhibition 37 (Figure 3c: N−C distances are 1.3 and 1.4 Å for (R)-APEP and (S)-APPA, respectively). The inhibitor (R)-APEP is the phosphonic acid equivalent of the natural substrate L-Phe; hence, the binding conformation and the reaction mechanism of the AL reaction with L-Phe may be inferred from this crystal structure. ...
... The clear-cut electron densities in the structure complexed with the inhibitors confirm the absolute configuration determination of the (S)-APPA inhibitor (Figure 3b) and reveal a surprising enantiomer preference switch generated by the methylidene group in (R)-APEP at the β-position ( Figure 3b). 37 Inferring D-Phe binding from this structure, we propose that the positioning of the β-carbon atom compared to Tyr A (Y110, Figure 3c) is the most important structural feature determining enantioselectivity. ...
... 58 Our previous study showed that the methylidene group at the β-position in APPA altered the enantiopreference of binding of this phosphonic acid analogue of Phe in PALs. 37 Our recent set of experiments introducing the binding pathway-blocking residues from the (S)-selective PaPAM to the (R)-selective TcPAM indicated the potential of structure-based enzyme access tunnel engineering to alter the enantioselectivity of MIO-enzymes ( Figure 6). The strict enantiomer preference of the (R)-selective TcPAM altered significantly in both the ligand access tunnel mutants of TcPAM, whereas the double mutant became an (S)-selective β-AL. ...
The enzyme family harboring the post-translationally formed 5-methylene-3,5-dihydro-4H-imidazol-4-one (MIO) catalytic residue comprises both aromatic amino acid ammonia-lyases (ALs) and 2,3-aminomutases (AMs). The structural origin of the different functions and the role of the inner loop region in substrate binding are not fully understood. Here, we provide the three-dimensional structures for Petroselinum crispum phenylalanine AL (PcPAL) with fully resolved inner loops in a catalytically competent conformation. Using molecular modeling, we demonstrate that in both ALs and AMs of eukaryotic origin, just a small opening of the inner loop is sufficient for ligand egress. Furthermore, we show that ligand-binding tunnels are analogous to eukaryotic MIO-enzymes and that the critical initial part of these tunnels is present across the whole enzyme family. Engineering of these binding tunnels converts an (R)-AM to a highly selective (S)-β-AL thus establishing a nonclassified enzyme function.
... Within this class of drugs, hybrids molecules introducing two potentially pharmacophores, including allylic amine moieties and α-aminophosphonic acid functional groups, such as allylic α-aminophosphonic acid derivatives (IV and V), have attracted scarce attention since only a few examples have been reported in the literature. For instance, (1-amino-2propenyl)phosphonic acid (IV) inhibit alanine racemase and D-alanine:D-alanine ligase [30,31], while α-aminophosphonic acid analogue (V) of the natural phenylalanine bearing a methylidene at the βposition acts as an inhibitor of phenylalanine ammonia-lyases (PAL) [32] (Figure 1). ...
This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C–N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N–C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.
... The rabbit skeletal myosin subfragment-S1 was a kind gift of Máté Gyimesi, Eötvös University, Budapest, Hungary. These proteins were expressed and purified as described previously [23][24][25]. The proteins were dialyzed against a buffer pH 7.5 comprising 20 mM HEPES, 100 mM NaCl and 1 mM TCEP. ...
EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments.
... Email address: Jeannie.Horak@uni-tuebingen.de (J. Horak) [17][18][19], the need for fast and robust chiral separation techniques persist. ...
Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC). In addition, throughout this study a u- ¹³ C ¹⁵ N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u- ¹² C ¹⁴ N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.
... Generally, analogues of phenylglycine exhibit inhibitory activity towards buckwheat enzyme higher than towards potato PAL. They are also equipotent with or even more active than aminobenzylphosphonic acid 2, a formal analogue of phenylglycine, against parsley (Petroselinum crispum) enzyme [21,23]. Additionally, the most active compounds: 5, 6, 17, 18 and 20 were significantly less active than cyclic derivative 29 (Scheme 1B) towards buckwheat enzyme [22]. ...
... The process of movement of substrates and inhibitors along entrance channel has not been, however, analyzed in he literature yet. This speculation is to some extend supported by recent finding that some inhibitors of phenylalanine ammonia lyase act as slow-binding ones [23]. ...
A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato L-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site.
The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-¹³C¹⁵N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated.
It was found that a 0.4 M borate buffer with a pH of 8.0 +/− 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-¹³C¹⁵N]-Met as well as its oxidation products, AQC-DL-[U-¹³C¹⁵N]-Met-O and AQC-DL-[U-¹³C¹⁵N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC–ESI–QTOF–MS analysis in positive ionization mode.
