Amine Acceptor Research Articles

Amine Synthesis Using the Amine Donor N-Phenyl Putrescine and the Johnson Matthey Transaminase Biocatalyst Library

Chiral amines are important building blocks in the pharmaceutical, agrochemical and chemical industries. There is a drive to augment traditional transition metal catalysts with ‘green’ alternatives such as biocatalysts. Transaminase (TA) biocatalysts can be used in combination with ‘smart’ sacrificial amine donors to synthesise a variety of aliphatic and aromatic amines from the corresponding aldehydes and ketones. Despite their enormous potential, the unfavourable reaction equilibrium often limits the widespread application of TAs for industrial synthesis. Recently we disclosed a new biomimetic amine donor N-phenyl putrescine (NPP), which was inspired by the biosynthesis of the dipyrroloquinoline alkaloids. NPP was demonstrated to have good activity with a library of commercial and wild-type TAs (total 25 TAs). This work focused on exploring the use of NPP with the Johnson Matthey TA kit (17 biocatalysts; eight S-selective and nine R-selective) and three different amine acceptors (vanillin, benzaldehyde and acetophenone). NPP worked well with all 17 TAs and gave the corresponding amine products vanillylamine, benzylamine and methylbenzylamine (MBA) in up to 85% high-performance liquid chromatography (HPLC) yield. From the screen, STA-14 was identified as a good biocatalyst for further analysis and used in a comparative screen of NPP versus the commonly used donor iPrNH2. It was found that NPP was the best amine donor and used to prepare S-methylbenzylamine in >99.5% enantiomeric excess (e.e.). This work, combined with our previous study, highlights the potential of NPP in the biocatalytic synthesis of amines.

Highly efficient synthesis of chiral lactams by using a ω-transaminase from Bacillus megaterium and its mutant enzymes

In this study, a series of chiral lactams were successfully prepared by one-pot two-step reactions. It included a transamination reaction catalyzed by a ω-transaminase from Bacillus megaterium (ω-TABm), and subsequent spontaneous cyclisation. The different amine donors were firstly screened with ethyl levulinate as an amine acceptor, and both the excellent conversion and enantiomeric excess (e.e.p) of >99% could be achieved with 4-bromophenylethylamine as an amine donor, however, the long reaction time of 24 h was unfavorable for its application. After the amino acid residue Thr120 situated in the small pocket was mutated to Ser, the T120S variant could exhibit 10.6-fold higher activity comparing to that of wild type, and achieve the reaction with both conversion and e.e.p of >99% within 3 h. The T120S variant could also be applied to prepare the other chiral γ- and δ-lactams with excellent conversion and e.e.p. Furthermore, the inclusion bodies of ω-TABm and its variants could be aggregated as “native-like structures” to continuously prepare the chiral lactam for 18 h. The aim of the study is to provide a simply and efficiently biocatalytic route for preparing chiral lactams.

A Multiplex Assay to Assess the Transaminase Activity toward Chemically Diverse Amine Donors.

The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of 5-(hydroxymethyl)furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.

Development of a glutaric acid production system equipped with stepwise feeding of monosodium glutamate by whole-cell bioconversion

In the bioproduction of glutaric acid, an emerging bioplastic monomer, α-ketoglutaric acid (α-KG) is required as an amine acceptor for 4-aminobutyrate aminotransferase (GabT)-driven conversion of 5-aminovalerate (5-AVA) to glutarate semialdehyde. Herein, instead of using expensive α-KG, an indirect α-KG supply system was developed using a relatively cheap alternative, monosodium glutamate (MSG), for l-glutamate oxidase (Gox)-based whole-cell conversion. Using 200 mM 5-AVA and 30 mM MSG initially with Gox, 67.1 mM of glutaric acid was produced. By applying the stepwise feeding strategy of MSG, the glutaric acid production capability was increased to 159.1 mM glutaric acid with a conversion yield of 79.6%. In addition, a buffer-free one-pot reaction from l-lysine was also applied in a 5 L bioreactor to evaluate its industrial applicability, resulting in a conversion yield of 54.2%. The system developed herein might have great potential for the large-scale, economically feasible production of glutaric acid by whole-cell conversion.

ω-Transaminase-Mediated Asymmetric Synthesis of (S)-1-(4-Trifluoromethylphenyl)Ethylamine

The pivotal role played by ω-transaminases (ω-TAs) in the synthesis of chiral amines used as building blocks for drugs and pharmaceuticals is widely recognized. However, chiral bulky amines are challenging to produce. Herein, a ω-TA (TR8) from a marine bacterium was used to synthesize a fluorine chiral amine from a bulky ketone. An analysis of the reaction conditions for process development showed that isopropylamine concentrations above 75 mM had an inhibitory effect on the enzyme. Five different organic solvents were investigated as co-solvents for the ketone (the amine acceptor), among which 25–30% (v/v) dimethyl sulfoxide (DMSO) produced the highest enzyme activity. The reaction reached equilibrium after 18 h at 30% of conversion. An in situ product removal (ISPR) approach using an aqueous organic two-phase system was tested to mitigate product inhibition. However, the enzyme activity initially decreased because the ketone substrate preferentially partitioned into the organic phase, n-hexadecane. Consequently, DMSO was added to the system to increase substrate mass transfer without affecting the ability of the organic phase to prevent inhibition of the enzyme activity by the product. Thus, the enzyme reaction was maintained, and the product amount was increased for a 62 h reaction time. The investigated ω-TA can be used in the bioconversion of bulky ketones to chiral amines for future bioprocess applications.

Palladium-Catalyzed O - and N -Glycosylation with Glycosyl Chlorides

Despite the significant progress in carbohydrate chemistry, there remains a pressing need for efficient and practical glycosylation methods using simple glycosyl donors and with high atom economy. ...

Screening and Comparative Characterization of Microorganisms from Iranian Soil Samples Showing ω-Transaminase Activity toward a Plethora of Substrates

In this study, soil microorganisms from Iran were screened for ω-transaminase (ω-TA) activity based on growth on minimal media containing (rac)-α-methylbenzylamine (rac-α-MBA) as a sole nitrogen source. Then, for the selection of strains with high enzyme activity, a colorimetric o-xylylendiamine assay was conducted. The most promising strains were identified by 16S rDNA sequencing. Five microorganisms showing high ω-TA activity were subjected to determine optimal conditions for ω-TA activity, including pH, temperature, co-solvent, and the specificity of the ω-TA toward different amine donors and acceptors. Among the five screened microorganisms, Bacillus halotolerans turned out to be the most promising strain: Its cell-free extract showed a highly versatile amino donor spectrum toward aliphatic, aromatic chiral amines and a broad range of pH activity. Transaminase activity also exhibited excellent solvent tolerance, with maximum turnover in the presence of 30% (v/v) DMSO.

Asymmetric synthesis of l-phosphinothricin using thermostable alpha-transaminase mined from Citrobacter koseri

α-Transaminase (α-TA) responsible for catalyzing the reversible transfer of amino groups between amine donors and amine acceptors, is applicable to enzymatic route for asymmetric synthesis of herbicide l-phosphinothricin (l-PPT). In the search for α-TAs with better catalysis performance, three α-TAs were discovered by genome mining approach using a known sequence encoding Escherichia coli tyrosine TA (TyrB) as probe. Through detailed comparison of their expression amount, activities and characteristics, Citrobacter koseri TA (CkTA) exhibited better activity and thermostability, which retain 65.9% of initial activity after incubation at 57 °C for 4 h. The Km and kcat/Km values of CkTA were 36.75 mM and 34.29 mM−1 min−1, respectively. In addition, recombinant CkTA cells were immobilized onto Celite 545 using tris(hydroxymethyl)phosphine as crosslinker. During five repetitive asymmetric synthesis of l-PPT from 20 g/L prostereogenic ketone using l-Glu as amine donor, all the yields of l-PPT reached up to 91.2% (＞99% ee). These characteristics made CkTA a valuable addition to the currently scarce α-TA library for stereospecific synthesis of l-PPT.

Biochemical and structural investigation of taurine:2-oxoglutarate aminotransferase from Bifidobacterium kashiwanohense.

Taurine aminotransferases catalyze the first step in taurine catabolism in many taurine-degrading bacteria and play an important role in bacterial taurine metabolism in the mammalian gut. Here, we report the biochemical and structural characterization of a new taurine:2-oxoglutarate aminotransferase from the human gut bacterium Bifidobacterium kashiwanohense (BkToa). Biochemical assays revealed high specificity of BkToa for 2-oxoglutarate as the amine acceptor. The crystal structure of BkToa in complex with pyridoxal 5'-phosphate (PLP) and glutamate was determined at 2.7 Å resolution. The enzyme forms a homodimer, with each monomer exhibiting a typical type I PLP-enzyme fold and conserved PLP-coordinating residues interacting with the PLP molecule. Two glutamate molecules are bound in sites near the predicted active site and they may occupy a path for substrate entry and product release. Molecular docking reveals a role for active site residues Trp21 and Arg156, conserved in Toa enzymes studied to date, in interacting with the sulfonate group of taurine. Bioinformatics analysis shows that the close homologs of BkToa are also present in other anaerobic gut bacteria.

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPIp ), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPIp exhibits a rigid, thermo-resistant double-psi-beta-barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln-6 as the only amine acceptor site on SPIp accessible for MTG. Substitution of Lys-7 demonstrated that small and hydrophobic residues in close proximity to Gln-6 favor MTG-mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPIp for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG-substrates used in biomedical applications.

1,3,4-Oxadiazole and Heteroaromatic-Fused 1,2,4-Triazole Synthesis using Diverted Umpolung Amide Synthesis.

Umpolung Amide Synthesis (UmAS) has emerged as a superior alternative to conventional amide synthesis methods based on carbonyl electrophiles in a range of situations, particularly when epimerization-prone couplings are prescribed. In an unanticipated development during our most recent studies, it was discovered that diacyl hydrazide products from UmAS were not formed as intermediates when using an acyl hydrazide as the amine acceptor. This resulted in a new preparation of 1,3,4-oxadiazoles from α-bromonitroalkane donors. We hypothesized that a key tetrahedral intermediate in UmAS was diverted toward a more direct pathway to the heterocycle product rather than through formation of the diacyl hydrazide, a typical oxadiazole progenitor. In studies reported here, diversion to 1,2,4-triazole products is described, a behavior hypothesized to also result from an analogous tetrahedral intermediate, but one formed from heteroaromatic hydrazine acceptors.

The study of metal binding properties and electronic transitions of dithienopyrole derivatives

Fluorescence chromophore (L) based on dithienopyrole derivatives (DPT) have been compared the binding site for detecting metal ions. There are consisting of methacryloyl amine (L1) and cyanoacrylic acid (L2) acceptor, respectively. Corresponding molecular geometries, orbital energies, electron contributions and absorption properties of the fluorescence sensor were investigated by using density functional theory (DFT) with B3LYP method of calculation. The DFT based studies with various solvation and absorption spectra were performed using time-dependent density functional theory (TD-DFT) with the polarized continuum model. Comparing with L2, the experimental L1 shows a red shift both absorption and emission spectra at 350 and 485 nm, respectively. The solvent environment exhibited slight shift in experimental and calculated absorption. An effect of various metal ions on absorption behavior was studied. Results indicate that the L1 and L2 exhibit excellent sensitivity for Pb2+ ion. The binding complex M-L1 and M-L2 (M: Cd, Cu, Hg, Pb, Zn) exhibited Pb<Cu<Zn<Mn<Cd<Hg.

Non-enzymatic pyridine ring formation in the biosynthesis of the rubrolone tropolone alkaloids.

The pyridine ring is a potent pharmacophore in alkaloid natural products. Nonetheless, its biosynthetic pathways are poorly understood. Rubrolones A and B are tropolone alkaloid natural products possessing a unique tetra-substituted pyridine moiety. Here, we report the gene cluster and propose a biosynthetic pathway for rubrolones, identifying a key intermediate that accumulates upon inactivation of sugar biosynthetic genes. Critically, this intermediate was converted to the aglycones of rubrolones by non-enzymatic condensation and cyclization with either ammonia or anthranilic acid to generate the respective pyridine rings. We propose that this non-enzymatic reaction occurs via hydrolysis of the key intermediate, which possesses a 1,5-dione moiety as an amine acceptor capable of cyclization. This study suggests that 1,5-dione moieties may represent a general strategy for pyridine ring biosynthesis, and more broadly highlights the utility of non-enzymatic diversification for exploring and expanding natural product chemical space.

Structural and Functional Investigation of FdhC from Acinetobacter nosocomialis: A Sugar N-Acyltransferase Belonging to the GNAT Superfamily.

Enzymes belonging to the GNAT superfamily are widely distributed in nature where they play key roles in the transfer of acyl groups from acyl-CoAs to primary amine acceptors. The amine acceptors run the gamut from histones to aminoglycoside antibiotics to small molecules such as serotonin. Whereas those family members that function on histones have been extensively studied, the GNAT enzymes that employ nucleotide-linked sugars as their substrates have not been well characterized. Indeed, though the structures of two of these "amino sugar" GNAT enzymes have been determined within the past 10 years, details concerning their active site architectures have been limited because of a lack of bound nucleotide-linked sugar substrates. Here we describe a combined structural and biochemical analysis of FdhC from Acinetobacter nosocomialis O2. On the basis of bioinformatics, it was postulated that FdhC catalyzes the transfer of a 3-hydroxybutanoyl group from 3-hydroxylbutanoyl-CoA to dTDP-3-amino-3,6-dideoxy-d-galactose, to yield an unusual sugar that is ultimately incorporated into the surface polysaccharides of the bacterium. We present data confirming this activity. In addition, the structures of two ternary complexes of FdhC, in the presence of CoA and either 3-hydroxybutanoylamino-3,6-dideoxy-d-galactose or 3-hydroxybutanoylamino-3,6-dideoxy-d-glucose, were solved by X-ray crystallographic analyses to high resolution. Kinetic parameters were determined, and activity assays demonstrated that FdhC can also utilize acetyl-CoA, 3-methylcrotonyl-CoA, or hexanoyl-CoA as acyl donors, albeit at reduced rates. Site-directed mutagenesis experiments were conducted to probe the catalytic mechanism of FdhC. Taken together, the data presented herein provide significantly new molecular insight into those GNAT superfamily members that function on nucleotide-linked amino sugars.

Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion.

BackgroundSaccharomyces cerevisiae can be engineered to perform a multitude of different chemical reactions that are not programmed in its original genetic code. It has a large potential to function as whole-cell biocatalyst for one-pot multistep synthesis of various organic molecules, and it may thus serve as a powerful alternative or complement to traditional organic synthetic routes for new chemical entities (NCEs). However, although the selectivity in many cases is high, the catalytic activity is often low which results in low space-time-yields. In the case for NADH-dependent heterologous reductive reactions, a possible constraint is the availability of cytosolic NADH, which may be limited due to competition with native oxidative enzymes that act to maintain redox homeostasis. In this study, the effect of increasing the availability of cytosolic NADH on the catalytic activity of engineered yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated.ResultsA series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b) reduction of the produced acetophenone to (S)-1-phenylethanol. For the gpd1Δgpd2Δ strain, cell metabolism was effectively used for the supply of both amine acceptors and the co-factor pyridoxal-5′-phosphate (PLP) for the ω-transaminase, as well as for regenerating NADH for the reduction. In contrast, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over-expressing SADH.ConclusionsOverall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i.e. without addition of external co-factors or prosthetic groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0430-x) contains supplementary material, which is available to authorized users.

Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β‐Fluoroamines by Formal Tandem Hydrodefluorination/Deamination

AbstractTransaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β‐fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β‐fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.

Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination.

Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.

Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin.

Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln(42) and Gln(193) serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln(42) predominantly is linked to Lys(4) and that Gln(193) participates in a cross-link with Lys(154), Lys(157), or Lys(231). The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys(154)-Gln(193) isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.

Multi-step biocatalytic strategies for chiral amino alcohol synthesis

Chiral amino alcohols are structural motifs present in sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Their chemical synthesis presents several challenges in establishing at least two chiral centres. Here a de novo metabolic pathway using a transketolase enzyme coupled with a transaminase enzyme has been assembled. To synthesise this motif one of the strategies to obtain high conversions from the transaminase/transketolase cascade is the use of hydroxypyruvate (HPA) as a two-carbon donor for the transketolase reaction; although commercially available it is relatively expensive limiting application of the pathway on an industrial scale. Alternately, HPA can be synthesised but this introduces a further synthetic step. In this study two different biocatalytic strategies were developed for the synthesis of (2S,3R)-2-amino-1,3,4-butanetriol (ABT) without adding HPA into the reaction. Firstly, a sequential cascade of three enzymatic steps (two transaminases and one transketolase) for the synthesis of ABT from serine, pyruvate and glycolaldehyde as substrates. Secondly, a two-step recycling cascade where serine is used as donor to aminate erythrulose (catalysed by a transketolase) for the simultaneous synthesis of ABT and HPA. In order to test the novel pathways, three new transaminases are described, two ω-transaminases able to accept a broad range of amine acceptors with serine as amine donor; and an α-transaminase, which showed high affinity towards serine (KM: 18mM) using pyruvate as amine acceptor. After implementation of the above enzymes in the biocatalytic pathways proposed in this paper, the two-step recycling pathway was found to be the most promising for its integration with E. coli metabolism. It was more efficient (10-fold higher conversion), more sustainable and cost-effective (use of low cost natural substrates and only two enzymes), and the reaction could be performed in a one-pot system.

Amine donor and acceptor influence on the thermodynamics of ω-transaminase reactions

In recent years biocatalytic transamination using ω-transaminase has become established as one of the most interesting routes to synthesize chiral amines with a high enantiomeric purity, especially in the pharmaceutical sector where the demand for such compounds is high. Nevertheless, one limitation for successful implementation and scale-up is that the thermodynamics of such conversions are frequently found unfavourable. Herein we report experimental measurements of apparent equilibrium constants for several industrially relevant transamination reactions in a systematic manner to better understand the effect of amine acceptor and donor choice. For example, we have found that ortho-substitution of acetophenone like molecules, had a significant impact on the thermodynamic equilibrium. Likewise, the effect of cyclic amine acceptors was evaluated and compared to similar non-cyclic structures. It was found that an aliphatic six membered ring was favourable and a conjugated bicyclic five membered ring structure, unfavourable. Finally, we evaluated and compared the use of five different donor molecules, and calculated their ΔGapp values. This is particularly important in the further implementation of such reactions because it may be used to help select suitable donor/acceptor combinations. The results presented here give guidance, with respect to thermodynamics, in order to further extend the application of biocatalytic transamination.