Enantio-Complementary Continuous-Flow Synthesis of 2-Aminobutane Using Covalently Immobilized Transaminases

Chiral amines are a common feature of many active pharmaceutical ingredients. The synthesis of very small chiral amines is particularly challenging, even via biocatalytic routes, as the level of discrimination between similarly sized R-groups must be exceptional, yet their synthesis creates attractive building blocks that may then be used to prepare diverse compounds in further steps. Herein, the synthesis of one of the smallest chiral amines, 2- aminobutane, using transaminases, is being investigated. After screening a panel of mainly wild-type transaminases, two candidates were identified: an (S)-selective transaminase from Halomonas elongata (HEwT) and a precommercial (R)-selective transaminase from Johnson Matthey (*RTA-X43). Notably, a single strategic point mutation enhanced the enantioselectivity of HEwT from 45 to >99.5% ee. By covalently immobilizing these candidates, both enantiomers of 2-aminobutane were synthesized on a multigram scale, and the feasibility of isolation by distillation without the need for any solvents other than water was demonstrated. The atom economy of the process was calculated to be 56% and the E-factors (including waste generated during enzyme expression and immobilization) were 55 and 48 for the synthesis of (R)-2-aminobutane and (S)-2-aminobutane, respectively

GPhos Ligand Enables Production of Chiral N-Arylamines in a Telescoped Transaminase-Buchwald-Hartwig Amination Cascade in the Presence of Excess Amine Donor

The combination of biocatalysis and chemocatalysis can be more powerful than either technique alone. However, combining the two is challenging due to typically very different reaction conditions. Herein, chiral N-aryl amines, key features of many active pharmaceutical ingredients, are accessed in excellent enantioselectivity (typically>99.5 % ee) by combining transaminases with the Buchwald-Hartwig amination. By employing a bi-phasic buffer-toluene system as well as the ligand GPhos, the telescoped cascade proceeded with up to 89 % overall conversion in the presence of excess alanine. No coupling to alanine was observed

Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats

Background: Anxiety is a heterogeneous behavioral domain playing a role in a variety of neuropsychiatric diseases. While anxiety is the cardinal symptom in disorders such as panic disorder, co-morbid anxious behavior can occur in a variety of diseases. Stiff person syndrome (SPS) is a CNS disorder characterized by increased muscle tone and prominent agoraphobia and anxiety. Most patients have high-titer antibodies against glutamate decarboxylase (GAD) 65. The pathogenic role of these autoantibodies is unclear. Methodology/Principal Findings: We re-investigated a 53 year old woman with SPS and profound anxiety for GABA-A receptor binding in the amygdala with (11)C-flumazenil PET scan and studied the potential pathogenic role of purified IgG from her plasma filtrates containing high-titer antibodies against GAD 65. We passively transferred the IgG fraction intrathecally into rats and analyzed the effects using behavioral and in vivo electrophysiological methods. In cell culture, we measured the effect of patient IgG on GABA release from hippocampal neurons. Repetitive intrathecal application of purified patient IgG in rats resulted in an anxious phenotype resembling the core symptoms of the patient. Patient IgG selectively bound to rat amygdala, hippocampus, and frontal cortical areas. In cultured rat hippocampal neurons, patient IgG inhibited GABA release. In line with these experimental results, the GABA-A receptor binding potential was reduced in the patient’s amygdala/hippocampus complex. No motor abnormalities were found in recipient rats. Conclusion/Significance: The observations in rats after passive transfer lead us to propose that anxiety-like behavior can be induced in rats by passive transfer of IgG from a SPS patient positive for anti-GAD 65 antibodies. Anxiety, in this case, thus may be an antibody-mediated phenomenon with consecutive disturbance of GABAergic signaling in the amygdala region

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

Broadband Multi-wavelength Properties of M87 during the 2017 Event Horizon Telescope Campaign

Abstract: In 2017, the Event Horizon Telescope (EHT) Collaboration succeeded in capturing the first direct image of the center of the M87 galaxy. The asymmetric ring morphology and size are consistent with theoretical expectations for a weakly accreting supermassive black hole of mass ∼6.5 × 109 M ⊙. The EHTC also partnered with several international facilities in space and on the ground, to arrange an extensive, quasi-simultaneous multi-wavelength campaign. This Letter presents the results and analysis of this campaign, as well as the multi-wavelength data as a legacy data repository. We captured M87 in a historically low state, and the core flux dominates over HST-1 at high energies, making it possible to combine core flux constraints with the more spatially precise very long baseline interferometry data. We present the most complete simultaneous multi-wavelength spectrum of the active nucleus to date, and discuss the complexity and caveats of combining data from different spatial scales into one broadband spectrum. We apply two heuristic, isotropic leptonic single-zone models to provide insight into the basic source properties, but conclude that a structured jet is necessary to explain M87’s spectrum. We can exclude that the simultaneous γ-ray emission is produced via inverse Compton emission in the same region producing the EHT mm-band emission, and further conclude that the γ-rays can only be produced in the inner jets (inward of HST-1) if there are strongly particle-dominated regions. Direct synchrotron emission from accelerated protons and secondaries cannot yet be excluded

MAGIC and H.E.S.S. detect VHE gamma rays from the blazar OT081 for the first time: a deep multiwavelength study

https://pos.sissa.it/395/815/pdfPublished versio

Biocatalytic synthesis of chiral amine building blocks

Many commercially important molecules, such as agrochemicals and active pharmaceutical ingredients (APIs), contain chiral amines. However, the synthesis of chiral amines by chemical means is often challenging and in particular in the case of aliphatic amines only low enantiomeric excesses (ees) are achieved. By using enzymes, high pressure hydrogen, high temperatures, precious metals, and organic solvents can often be avoided. In the production of chiral amines, lipases are the most important class commercially, catalysing the enantioselective acylation of chiral amines, resulting in a kinetic resolution. However, while their enantio-selectivity is usually excellent when substituents on the α-carbon are sufficiently different in size, it tends to be poor if substituents are similar (e.g. 2-aminobutane). Additionally, while it is possible to recover both enantiomers and racemizeand recycle the unwanted enantiomer, a synthesis of just the desired enantiomer from a prochiral precursor, such as a ketone, has advantages. Here, amine transaminases are the most developed class of enzymes, with several examples of enzyme engineering and scale-up in the literature. While most research with transaminases is focussed on bulky-bulky ketones, in this work the use of transaminases for the synthesis small chiral amines is being explored. Here, a significant limitation of wild-type transaminases proved to be an advantage: the small pocket that typically does not accept substituents significantly larger than a methyl-group allows for excellent enantioselectivity (> 99.5% ee) even for very small chiral amines, such as 2-aminobutane, for which a multi-gram scale synthesis in continuous flow is described.However, attempts at engineering a transaminase for the synthesis of 2,2-dimethylhexan-3-amine were less successful, with only traces of activity being observed. With the cyclic pro-chiral ketone, the enantiomeric outcome of the reaction depended on the reaction conditions (ionic strength and concentration of organic molecules), with ees ranging from 70% (S) to 19% (R). The discovery of the tetrameric quaternary structure of two (R)-selective transaminases (RTA) (from Aspergillus terreus and Thermomyces stellatus) is also described. Using this information, a rational mutation stabilizing the tetramer was introduced, which resulted in an overall more stable catalyst that could be used at higher substrate concentrations compared to the wild-type. Finally, a sequential cascade involving transaminases followed by a Buchwald-Hartwig amination (BHA) is described, which allows access to chiral N-arylamines without the need for purification of the intermediate. Employing a biphasic water-toluene system and using a 3rd-generation Buchwald precatalyst, the BHA showed excellent formation of the desired amine also in the presence of excess amine donor (in particular with alanine), allowing for the quick generation of diverse libraries of compounds which may be of use during drug discovery

Biocatalytic synthesis of chiral amine building blocks

Many commercially important molecules, such as agrochemicals and active pharmaceutical ingredients (APIs), contain chiral amines. However, the synthesis of chiral amines by chemical means is often challenging and in particular in the case of aliphatic amines only low enantiomeric excesses (ees) are achieved. By using enzymes, high pressure hydrogen, high temperatures, precious metals, and organic solvents can often be avoided. In the production of chiral amines, lipases are the most important class commercially, catalysing the enantioselective acylation of chiral amines, resulting in a kinetic resolution. However, while their enantio-selectivity is usually excellent when substituents on the α-carbon are sufficiently different in size, it tends to be poor if substituents are similar (e.g. 2-aminobutane). Additionally, while it is possible to recover both enantiomers and racemizeand recycle the unwanted enantiomer, a synthesis of just the desired enantiomer from a prochiral precursor, such as a ketone, has advantages. Here, amine transaminases are the most developed class of enzymes, with several examples of enzyme engineering and scale-up in the literature. While most research with transaminases is focussed on bulky-bulky ketones, in this work the use of transaminases for the synthesis small chiral amines is being explored. Here, a significant limitation of wild-type transaminases proved to be an advantage: the small pocket that typically does not accept substituents significantly larger than a methyl-group allows for excellent enantioselectivity (> 99.5% ee) even for very small chiral amines, such as 2-aminobutane, for which a multi-gram scale synthesis in continuous flow is described.However, attempts at engineering a transaminase for the synthesis of 2,2-dimethylhexan-3-amine were less successful, with only traces of activity being observed. With the cyclic pro-chiral ketone, the enantiomeric outcome of the reaction depended on the reaction conditions (ionic strength and concentration of organic molecules), with ees ranging from 70% (S) to 19% (R). The discovery of the tetrameric quaternary structure of two (R)-selective transaminases (RTA) (from Aspergillus terreus and Thermomyces stellatus) is also described. Using this information, a rational mutation stabilizing the tetramer was introduced, which resulted in an overall more stable catalyst that could be used at higher substrate concentrations compared to the wild-type. Finally, a sequential cascade involving transaminases followed by a Buchwald-Hartwig amination (BHA) is described, which allows access to chiral N-arylamines without the need for purification of the intermediate. Employing a biphasic water-toluene system and using a 3rd-generation Buchwald precatalyst, the BHA showed excellent formation of the desired amine also in the presence of excess amine donor (in particular with alanine), allowing for the quick generation of diverse libraries of compounds which may be of use during drug discovery

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40°C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50°C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and D-alanine as well as aromatic and aliphatic ketones and aldehydes

Release of Soybean Isoflavones by Using a β‐Glucosidase from Alicyclobacillus herbarius

β‐Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel β‐glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5–11) and temperatures (4–55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7‐fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C