Ω-amino Acids Research Articles

Optimized Spatial Configuration of Heterogeneous Biocatalysts Maximizes Cell-Free Biosynthesis of ω-Hydroxy and ω-Amino Acids.

Cell-free biocatalysis is gaining momentum in producing value-added chemicals, particularly in stepwise reaction cascades. However, the stability of enzyme cascades in industrial settings is often compromised when free enzymes are involved. In this study, we have developed a stable multifunctional heterogeneous biocatalyst coimmobilizing five enzymes on microparticles to transform 1,ω-diols into 1,ω-hydroxy acids. We improved the operational efficiency and stability of the heterogeneous biocatalyst by fine-tuning the enzyme loading and spatial organization. Stability issues are overcome through postimmobilization polymer coating. The general applicability of this heterogeneous biocatalyst is demonstrated by its scale-up in both batch and packed bed reactors, allowing a product yield of >80%. The continuous process is fed with H2O2 as the oxygen source, reaching a space-time yield (STY) of 0.76 g·L-1·h-1, maintained for the first 12 h. Finally, this flow system is telescoped with a second plug-flow reactor packed with a different heterogeneous biocatalyst integrating an additional transaminase. As a result, this 6-enzyme 2-reactor system sequentially transforms 1,ω-diols into 1,ω-amino acids while in situ recycling NAD+, depleting H2O2, and generating O2.

Functional characterization of Capsicum chinense vanillin aminotransferase: Detection of vanillylamine-forming activity from vanillin

In capsaicin biosynthesis, vanillin aminotransferase (VAMT; EC 2.6.1.119) catalyzes the conversion of vanillin (V) to vanillylamine (VA). In vitro analysis of the recombinant VAMT enzyme has been reported; however, this enzyme catalyzed only the V-forming reaction and not the VA-forming reaction, which is inconsistent with the postulated pathway for capsaicin biosynthesis. In this study, we expressed, purified, and characterized functional recombinant VAMT of Capsicum chinense cv. Habanero from an Escherichia coli strain. The enzyme catalyzed reversible transamination between V and VA, and its VA-forming activity was high when γ-aminobutyric acid (GABA) was used as an amino donor. The enzyme exhibited maximum activity at pH 8.0 and 55 °C, and was stable up to 60 °C over a pH range from 4.5 to 8.0. The enzyme was stable in the presence of various chemicals and metal ions. The enzyme accepted several 4–8-carbon long primary amines and ω-amino acids with carbon chains longer than 4 as amino donors despite the narrow specificity of the amino acceptor. Based on its kinetic attributes and localization, VAMT appears to have evolved from GABA-aminotransferase to catalyze reversible transamination between V and VA, and is responsible for VA biosynthesis using GABA as an amino donor in the cytosol of capsicum fruit cells.

Adapting an acyl CoA ligase from Metallosphaera sedula for lactam formation by structure-guided protein engineering

The CoA ligase from Metallosphaera sedula (MsACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of MsACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of MsACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction.

Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes.

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize, in yields of 72-86%, 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids (Aa) of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, 12 NBD-Aa-triterpene conjugates have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these NBD-Aa-triterpene conjugates enabled their uptake and subcellular distribution to be followed in order to probe in detail their biological properties at the cellular and molecular level.

Building Flexible Escherichia coli Modules for Bifunctionalizing n-Octanol: The Byproduct of Oleic Acid Biorefinery

Artificial biorefinery of oleic acid into 1,10-decanedioic acid represents a revolutionizing route to the sustainable production of chemically difficult-to-make bifunctional chemicals. However, the carbon atom economy is extremely low (56%) due to the formation of unifunctional n-octanol. Here, we report a panel of recombinant Escherichia coli modules for diverse bifunctionalization, where the desired genetic parts are well distributed into different modules that can be flexibly combined in a plug-and-play manner. The designed ω-functionalizing modules could achieve ω-hydroxylation, consecutive ω-oxidation, or ω-amination of n-octanoic acid. By integrating these advanced modules with the reported oleic acid-cleaving modules, high-value C8 and C10 products, including ω-hydroxy acid, ω-amino acid, and α,ω-dicarboxylic acid, were produced with 100% carbon atom economy. These ω-functionalizing modules enabled the complete use of all of the carbon atoms from oleic acid (released from plant oil) for the green synthesis of structurally diverse bifunctional chemicals.

Miniature β-Hairpin Mimetic by Intramolecular Hydrogen Bond and C–H···π Interactions

Canonically, protein β-hairpin motifs are stabilized by intramolecular hydrogen bonds. Here, we attempt to develop a rational design recipe for a miniature hairpin structure stabilized by hydrogen bonding as well as C–H···π interaction and try to understand how such a stabilization effect varies with different functional groups at each terminus. Database analysis shows that the α-amino acids with an aromatic side chain will not favor that kind of C–H···π stabilized hairpin structure. However, hybrid tripeptides with an N-terminal Boc-Trp-Aib corner residue and C-terminal aromatic ω-amino acids fold into the hairpin conformation with a central β-turn/open-turn that is reinforced by a C–H···π interaction. The CCDC database analysis further confirms that this C–H···π stabilized hairpin motif is general for Boc-protected tripeptides containing Aib in the middle and aromatic functionality at the C-terminus. The different α-amino acids like Leu/Ala/Phe/Pro/Ser at the N-terminus have a minor influence on the C–H···π interaction and stabilities of the folded structures in solid-state. However, the hybrid peptides exhibit different degrees of conformational heterogeneity both in the solid and solution phase, which is common for this kind of flexible small molecule. Conformational heterogeneity in the solution phase including the C–H···π stabilized β-hairpin structures are characterized by the molecular dynamics (MD) simulations explaining their plausible origin at an atomistic level.

The Importance of 6-Aminohexanoic Acid as a Hydrophobic, Flexible Structural Element.

6-aminohexanoic acid is an ω-amino acid with a hydrophobic, flexible structure. Although the ω-amino acid in question is mainly used clinically as an antifibrinolytic drug, other applications are also interesting and important. This synthetic lysine derivative, without an α-amino group, plays a significant role in chemical synthesis of modified peptides and in the polyamide synthetic fibers (nylon) industry. It is also often used as a linker in various biologically active structures. This review concentrates on the role of 6-aminohexanoic acid in the structure of various molecules.

Electrochemistry of Quinones with Respect to their Role in Biomedical Chemistry.

Quinones are ubiquitous in nature and form one of the largest class of antitumor agents approved for clinical use. They are known to be efficient in inhibiting cancer cells growth. Under physiological conditions they can undergo non-enzymatic one-electron reduction to give the moderately toxic species of semiquinone radical-anion. Thus, electrochemical study of quinones might provide a basic knowledge on semi-quinone radicals formation in both in vivo and in vitro under different media. Several processes are outlined briefly and discussed in the present article. Previously we investigated the electrochemical and spectral properties of ω-N-quinonyl amino acids. Such quinone-bearing peptides are known to be cytotoxic and of potential clinical significance. We were able to prove that the ω-amino quinonyl compounds are very effective in producing stable semiquinone radicals. Moreover, a direct relation was found between the first reduction potentials of the quinonyl moiety and their reactivity towards the ω-amino acids. In order to increase our knowledge of such amino quinonyl compounds and enlarge the arsenal of such cytotoxic compounds, a series of N,N-diquinonyl amines (1-6) bearing an internal proton (stems from the NH moiety) were synthesized. Their electron-transfer capabilities were probed by cyclic voltammetry measurements, in dichloromethane. It was found that the acidic NH group linking the two quinonyl moieties undergoes an initial electrochemical reduction step and generates a nitride anion. This step is followed by further reductions to yield quasi-stable semiquinone radicals and polyanions, Since these acidic diquinones (1-6) serve also as a source of internal proton donors even in non-polar medium, they might cause protonation of basic radical-anions and polyanion intermediates during the various electrochemical stages. The processes are demonstrated and discussed by analyzing different mechanistic schemes. The successful generation of relatively stable semiquinone radicals is a prerequisite for the manifestation of site directed antitumor activity by these bis-quinonyl amino derivatives. Based on the values of their redox potentials some of them could be promising candidates for clinical development.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4–C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants—AVA6 and AVA10—with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

NMR spectroscopy of Cu(II) complexes with acrylamide and sodium acrylate copolymer and ω-amino acids

Macromolecular complexes of acrylamide and sodium acrylate copolymer with microelements, including Cu(II), may form at preparation of crop protection and stimulation compositions, where the copolymer serves as an adhesive, water-retaining and film-forming agent. Preparations for crop production may also contain amino acids that protect plants under stressful conditions (cold, dry, etc.). Carboxylic groups of copolymer, carboxylic and amino groups of amino acids may be involved in mixed Cu(II) ions complexes formation. Number of methylene groups separating carboxylic and amino group of amino acids affects its ability to form a stable chelate cycle and, therefore, ligand composition of mixed Cu(II) ions complexes with acrylamide and sodium acrylate copolymer and amino acid. This work is aimed at determining the ligand composition of mixed macromolecular Cu(II) ion complexes with acrylamide and sodium acrylate copolymer and ω-amino acids (β-alanine, γ-aminobutyric acid, ε-aminocaproic acid). 13C and 1H NMR spectroscopy was used to clarify complexes composition. A complex where carboxylic groups of amino acids are ligands has been found to form in aqueous solutions of Cu(II) ions and ω-amino acid (β-alanine, γ-aminobutyric acid, ε-aminocaproic acid) at molar ratio of Cu(II) ions – amino acid equal to 1 : 6. A chelate complex where both carboxylic and amino groups of β-alanine are involved in coordination has been discovered to form in the solution containing Cu(II) ions, β-alanine, as well as acrylamide and sodium acrylate copolymer at molar ratio of Cu(II) – β-alanine – copolymer COO− equal to 1 : 6 : 30. Carboxylic groups of copolymer participate in complex formation as well. Carboxylic groups of both amino acids and the copolymer have been shown to participate in complex formation in aqueous solutions containing Cu(II) ions, either γ-aminobutyric or ε-aminokaproic acid and also acrylamide and sodium acrylate copolymer.

An Integrated Cofactor/Co-Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines.

We report a highly atom-efficient integrated cofactor/co-product recycling cascade employing cycloalkylamines as multifaceted starting materials for the synthesis of nylon building blocks. Reactions using E. coli whole cells as well as purified enzymes produced excellent conversions ranging from >80 and 95 % into desired ω-amino acids, respectively with varying substrate concentrations. The applicability of this tandem biocatalytic cascade was demonstrated to produce the corresponding lactams by employing engineered biocatalysts. For instance, ϵ-caprolactam, a valuable polymer building block was synthesized with 75 % conversion from 10 mM cyclohexylamine by employing whole-cell biocatalysts. This cascade could be an alternative for bio-based production of ω-amino acids and corresponding lactam compounds.

Synthesis of Bifunctional Molecules for the Production of Polymers Based on Unsaturated Fatty Acids as Bioderived Raw Materials

Currently, investigations of polymer-building blocks made from biorenewable feedstocks such as, for example, fatty acids, are of high interest for the chemical industry. An alternative synthesis of nitrile-substituted aliphatic carboxylic acids as precursors for ω-amino acids, which are useful to produce polymers, was investigated starting from biorenewable fatty acids. By hydroformylation of unsaturated fatty acids or unsaturated acids being accessible from unsaturated fatty acids by cross-metathesis reactions, aldehydes are formed. In this work, the hydroformylation of such unsaturated acids led to the formation of the corresponding aldehydes, which were afterwards converted with hydroxylamine to aldoximes. Subsequent dehydration by an aldoxime dehydratase as a biocatalyst or by CuII acetate led to the desired nitriles. Within this work, C7-, C9- and C11-carboxylic acids with a terminal nitrile functionality as well as a branched nitrile-functionalized stearate derivative were synthesized by means of this approach. As these nitriles serve as precursors for amino acids being suitable for polymerization, this work represents an alternative synthetic access to polyamide precursors, which starts directly from unsaturated fatty acids as biorenewable resources and avoids harsh reaction conditions as well as and by-product formation.

Implementation of HPLC Analysis for γ-Aminobutyric Acid (GABA) in Fermented Food Matrices

In this study, a high-performance liquid chromatography (HPLC) method was adapted to measure the γ-aminobutyric acid (GABA) and glutamate (Glu) content in different fermented foods. The best solvent to extract GABA and Glu from the food matrices was 75% EtOH and water, combined with 4% SSA as posttreatment. The extracted amino acids were derivatized with o-phthalaldehyde (OPA) for 2 min at room temperature and analyzed by HPLC with fluorescence detector (λex = 340 nm and λem = 455 nm). Animal-based fermented products had a higher GABA content, compared with plant-based and soy-based fermented products. Among 17 fermented samples, fish sauce showed the highest amount of GABA (16.29 ± 0.53 mg/kg DW). Results obtained by the HPLC method were compared with those obtained by a spectrophotometric method, which is based on the reaction of ω-amino acids with phenol. All measured GABA contents, obtained by HPLC method, were much lower than those obtained by the spectrophotometric method, which was due to the nonspecific reaction of the coloring reagent with other amino acids than GABA. It can be concluded that GABA content in food products can only be measured by HPLC.

Plant Coumarins: XVII. Synthesis and Transformations of 7-Hydroxy-2-oxo-2H-chromene-6-carboxamides

Umbelliferone-6-carbonyl chloride reacted with amines and α-, β-, and ω-amino acid methyl esters to afford the corresponding N-substituted 7-hydroxy-2-oxo-2H-chromene-6-carboxamides. The reaction of umbelliferone-6-carbonyl chloride with glycine gave 2-(7-hydroxy-2-oxo-2H-chromene-6-carboxamido)acetic acid which was converted into acid chloride, and the latter reacted with benzylamines and α-amino acid methyl esters to produce compounds containing a dipeptide fragment.

Lupane-type conjugates with aminoacids, 1,3,4- oxadiazole and 1,2,5-oxadiazole-2-oxide derivatives: Synthesis, anti-inflammatory activity and in silico evaluation of target affinity

With the purpose to improve anti-inflammatory activity, the impact of introduction of 1,2,5- and 1,3,4-oxadiazole fragments to betulonic acid core as well as hybrids tethered with short ω-amino acids has been studied. The anti-inflammatory activity of synthesized compounds was tested in vivo using models of inflammation induced by concanavalin A and histamine. The majority of new compounds demonstrated higher anti-inflammatory activity compared with starting betulonic acid. To confirm the molecular targets of new derivatives in NRf2 and NFκB pathways the docking at Kelch and BTB active sites of Keap1 as well as IKK was done. The novelty of the present work is the development of new class of low toxic anti-inflammatory substances consisting of amino acid-linked betulonic acid - oxadiazole conjugates. These compounds can be considered as prospective chemopreventive agents.

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

The biodegradation of the nylon-6 precursor caprolactam by a strain of Pseudomonasjessenii proceeds via ATP-dependent hydrolytic ring opening to 6-aminohexanoate. This non-natural ω-amino acid is converted to 6-oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5'-phosphate (PLP) enzymes. To understand the structural basis of 6-aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacteriumviolaceum (CvAT) and Vibriofluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat /KM ) with the caprolactam degradation intermediates 6-aminohexanoate and 6-oxohexanoic acid, in accordance with its proposed invivo function. Five distinct three-dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6-aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate-binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6-aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6-aminohexanoate degradation. DATABASE: The atomic coordinates and structure factors P.jessenii 6-aminohexanoate aminotransferase have been deposited in the PDB as entries 6G4B (E∙succinate complex), 6G4C (E∙phosphate complex), 6G4D (E∙PLP complex), 6G4E (E∙PLP-6-aminohexanoate intermediate), and 6G4F (E∙PMP complex).

Synthesis of New Purine Derivatives Containing α- and ω-Amino Acid Fragments

New conjugates of purine and 2-aminopurine with several α- and ω-amino acids have been synthesized following two approaches based on the condensation and nucleophilic substitution reactions. The enantiomeric purity of the isolated compounds has been confirmed by reversed-phase HPLC using a chiral stationary phase to demonstrate the absence of racemization during the synthesis. The conjugates are inactive against Mycobacterium tuberculosis H37Rv.

Molecular Umbrellas Modulate the Selective Toxicity of Polyene Macrolide Antifungals.

Antifungal polyene macrolide antibiotics Amphotericin B (AmB) and Nystatin (NYS) were conjugated through the ω-amino acid linkers with diwalled "molecular umbrellas" composed of spermidine-linked deoxycholic or cholic acids. The presence of "umbrella" substituents modulated biological properties of the antibiotics, especially their selective toxicity. Some of the AmB-umbrella conjugates demonstrated antifungal in vitro activity comparable to that of the mother antibiotic but diminished mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro activity of NYS-umbrella conjugates was strongly reduced and all these conjugates demonstrated poorer than NYS selective toxicity. No correlation between the aggregation state and hemolytic activity of the novel conjugates was found.

Biosynthesis of Long-Chain N-Acyl Amide by a Truncated Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthase in Fungi.

Truncated iterative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) megasynthases in which only the C domain is present are widespread in fungi, yet nearly all members have unknown functions. Bioinformatics analysis showed that the C domains of such PKS-C enzymes are noncanonical due to substitution at the second histidine in the active site HHxxxDG motif. Here, we used genome mining strategy to characterize a cryptic PKS-C hybrid from Talaromyces wortmanii and discovered the products are reduced long-chain polyketides amidated with a specific ω-amino acid 5-aminopentanoic acid (5PA). The wortmanamides resemble long-chain N-acyl-amide signaling lipids that target diverse receptors including GPCRs. The noncanonical C domain of this PKS-C hybrid was also demonstrated to be a bona fide condensation domain that specifically selects 5PA and catalyzes amidation to release polyketide chain.

Design, synthesis and tumor cell growth inhibitory activity of 3-nitro-2H-cheromene derivatives as histone deacetylaes inhibitors

As a continuous research for the discovery of coumarin-based targeted anticancer agents, we designed and synthesized a series of novel histone deacetylases (HDAC) inhibitors using the 8-ethoxy-3-nitro-2H-chromene as the surface binding or cap group, linear dicarboxylic acid or ω-amino acid moiety with different length as the linking motif, ortho-aminoanilides, amides or α-aminoamides as the zinc binding group and the internal cavity motifs. Most of these 3-nitro-2H-chromene derivatives exhibited good growth inhibitory activity against K562, A549, MCF-7, PC3 and Hela cells and were more potent than the reference drug SAHA and MS-275. At the concentration of 10µM, the ortho-aminoanilide series and the d-Phe derived α-aminoamide derivatives 16a and 16b displayed more potent activity toward HADC1 over HADC2, and only moderate to weak activity over HADC6. In contrast, the amide ZBG analogues, 12a and 12b, 14 and 15, were only moderate HDAC6 inhibitors, but more selective over HDAC1 and HDAC2. The ortho-aminoanilides 9b, 9c, 10b, 10c, 11b, and the α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC50 values in the nanomolar ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more potent than MS-275 as HADC1 inhibitors and more selective over HADC2.