Continuous Electrochemical Synthesis of Iso-Coumarin Derivatives from o-(1-Alkynyl) Benzoates under Metal- and Oxidant-Free

A non-oxidant and metal-free strategy for synthesizing iso-coumarin by using a continuous electrochemical microreactor to initiate an oxidative cyclization reaction of o-(1-alkynyl) benzoate and radicals. This efficient and clean continuous electrosynthesis method not only avoids the complicated gas protection operation and production of by-products in the batch processes, but also help to overcome the difficulty that batch metal catalysis and electrocatalysis are difficult to scale up, and has the potential for pilot-scale experiment.     

Second-Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

Mononuclear iron-containing enzymes are highly versatile oxidants that often react stereospecifically and/or regioselectively with substrates. Combined experimental and computational studies on heme monooxygenases, nonheme iron dioxygenases and halogenases have revealed the intricate details of the second-coordination sphere, which determine this specificity and selectivity. These second-coordination sphere effects originate from the positioning of the substrate and oxidant, which involve the binding of the co-factors and substrate into the active site of the protein. In addition, some enzymes affect the selectivity and reactivity through charge-stabilization from nearby bound cations/anions, an induced electric field or through the positioning of salt bridges and hydrogen-bonding interactions to first-coordination sphere iron ligands and/or the substrate. Examples of all of these second-coordination sphere effects in iron-containing enzymes and how these influence structure and reactivity are given.     

A thioester substrate binds to the enzyme Arthrobacter thioesterase in two ionization states: evidence from Raman difference spectroscopy

4‐Hydroxybenzoyl‐CoA (4‐HB‐CoA) thioesterase from Arthrobacter is the final enzyme catalyzing the hydrolysis of 4‐HB‐CoA to produce coenzyme A and 4‐hydroxybenzoic acid in the bacterial 4‐chlorobenzoate dehalogenation pathway. Using a mutation E73A that blocks catalysis, stable complexes of the enzyme and its substrate can be analyzed by Raman difference spectroscopy. Here we have used Raman difference spectroscopy, in the non‐resonance regime, to characterize 4‐HB‐CoA bound in the active site of the E73A thioesterase. In addition, we have characterized complexes of the wild‐type enzyme complexed with the unreactive substrate analog 4‐hydroxyphenacyl‐CoA (4‐HP‐CoA). Both sets of complexes show evidence for two forms of the ligand in the active site: one population has the 4‐hydroxy group protonated, 4‐OH; while the second has the group as the hydroxide, 4‐O⁻. For bound 4‐HP‐CoA, X‐ray data show that glutamate 78 is close to the 4‐OH in the complex and it is likely that this is the proton acceptor for the 4‐OH proton. Although the pK_a of the 4‐OH group on the free substrate in aqueous solution is 8.6, the relative populations of ionized and neutral 4‐HB‐CoA bound to E73A remain invariant between pH 7.3 and 9.8. The invariance with pH suggests that the 4‐OH and the ‐COO⁻ of E78 constitute a tightly coupled pair where their separate pK_a ‘s lose their individual qualities. Narrow band profiles are seen in the CO double bond and C‐S regions, suggesting that the hydrolyzable thioester group is rigidly bound in the active site in a syn gauche conformation. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized u reaseactually has thesame catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.     

Chemoenzymatic Synthesis of Enantiopure Amino Alcohols from Simple Methyl Ketones

This study highlights the straight-forward synthesis of substituted 1,2-amino alcohols from simple and readily available aromatic methyl ketones. Starting from acetophenone derivatives, the straight-forward synthesis strategy involved an initial bromination of the alpha-positioned methyl group in the first step, followed by a simple hydrolysis to the hydroxyketone (2-hydroxyacetophenone). The hydroxylated intermediate was subsequently converted from Silicibacter pomeroyi to the final 1,2-amino alcohol by using the transaminase. The transaminase-catalyzed reaction proceeded with yields up to 62 % and always excellent enantiomeric excess of >99 %. Interestingly, the keto-enol-tautomerism of the hydroxyl ketone yields an unexpected amino alcohol isomer.     

Phytochemical Profiling,In Vitro Biological Activities,and In Silico Molecular Docking Studies of Dracaena reflexa

Dracaena reflexa, a traditionally significant medicinal plant, has not been extensively explored before for its phytochemical and biological potential. The present study was conducted to evaluate the bioactive phytochemicals and in vitro biological activities of D. reflexa, and perform in silico molecular docking validation of D. reflexa. The bioactive phytochemicals were assessed by preliminary phytochemical testing, total bioactive contents, and GC-MS analysis. For biological evaluation, the antioxidant (DPPH, ABTS, CUPRAC, and ABTS), antibacterial, thrombolytic, and enzyme inhibition (tyrosinase and cholinesterase enzymes) potential were determined. The highest level of total phenolic contents (92.72 ± 0.79 mg GAE/g extract) was found in the n-butanol fraction while the maximum total flavonoid content (110 ± 0.83 mg QE/g extract) was observed in methanolic extract. The results showed that n-butanol fraction exhibited very significant tyrosinase inhibition activity (73.46 ± 0.80) and acetylcholinesterase inhibition activity (64.06 ± 2.65%) as compared to other fractions and comparable to the standard compounds (kojic acid and galantamine). The methanolic extract was considered to have moderate butyrylcholinesterase inhibition activity (50.97 ± 063) as compared to the standard compound galantamine (53.671 ± 0.97%). The GC-MS analysis of the n-hexane fraction resulted in the tentative identification of 120 bioactive phytochemicals. Furthermore, the major compounds as identified by GC-MS were analyzed using in silico molecular docking studies to determine the binding affinity between the ligands and the enzymes (tyrosinase, acetylcholinesterase, and butyrylcholinesterase enzymes). The results of this study suggest that Dracaena reflexa has unquestionable pharmaceutical importance and it should be further explored for the isolation of secondary metabolites that can be employed for the treatment of different diseases.     

Theoretical study of the mechanism of peptide ring formation in green fluorescent protein

Density functional calculations using hybrid functionals (B3LYP) have been performed to study the mechanism of peptide ring formation in green fluorescent protein (GFP). Several different chemical models were used ranging from a minimal model of the ring formation to a model including the full side chains of the groups involved in forming the peptide ring. The surrounding protein was described using a dielectric cavity model. The previously most accepted mechanism was found to lead to an endothermic cyclization of about 10 kcal/mol, independent of chemical model used. The formation of the required dihydro‐imidazolone intermediate was found to be even more endothermic with 16–18 kcal/mol. In contrast, another mechanism where the dehydration of residue 66 precedes cyclization was found to be exothermic by 1.9 kcal/mol and to go over an endothermic intermediate of only 6.7 kcal/mol. Correcting these results using the more accurate G2‐M scheme leads to an intermediate with an energy of only +3.7 kcal/mol and an overall exothermicity of 4.7 kcal/mol. Possible transition states involving proton transfer steps were also investigated. Comparisons are made to the similar and more well‐known deamination reaction of Asn‐Gly sequences in peptides, for which good agreement is obtained with experiments. The results are discussed with respect to available experiments. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 81: 169–186, 2001     

Structural Basis and Enzymatic Mechanism of the Biosynthesis of C9‐ from C10‐Monoterpenoid Indole Alkaloids

Cutting carbons : The three‐dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C₉‐ from C₁₀‐monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate‐specific serine esterase. It harbors the catalytic triad S87‐D216‐H244, and is a new member of the α/β‐fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.