Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Theoretical study of C-arylations with aryl halides to determine the reaction mechanism, the effect of substituents and heteroatoms.

Carbon arylations are very important in the pharmaceutical industry. New synthesis routes are often studied with the objective of trying to insert new bonds and substituents into an organic framework. Ullman reactions have been very useful in this context. In light of this, a wB97XD/6-311g set of Ullman-like reactions among substituted amide arylations with iodoaniline were theoretically studied in order to understand their intrinsic reactivity and their reaction mechanisms. The studied systems included unsubstituted (C), sulphur (S), synthesized by the authors in a previous experimental work. In this study, amino (NH) and butyloxycarbonyl (NBoc) amides were added. IRC calculations on catalyzed species showed that the catalyst lowers the reaction barrier, and changes the reactivity in order to lower the nitrogen charge. The reaction mechanism proceeds by binding the CuI catalyst and N,N-dimethylethylenediamine (DMEN) to the N lactam, in a barrierless reaction, thereby activating the nitrogen to bond with the aryl iodine through a nucleophilic substitution, and thus recovering the catalyst.

Surfing the Blood Coagulation Cascade: Insight into the Vital Factor Xa.

Factor Xa (FXa) plays a key role in haemostasis, it is a central part of the blood coagulation cascade which catalyzes the production of thrombin and leads to clot formation and wound closure. Therefore, FXa is an attractive target for the development of new anticoagulant agents. In this review, we will first describe the molecular features of this fundamental protein in order to understand its mechanism of action, an essential background for the design of novel inhibitors by means of synthetic organic chemistry or using peptides obtained from recombinant methodologies. Then, we will review the current state of the synthesis of novel direct FXa inhibitors along with their mechanisms of action. Finally, approved reversal agents that aid in maintaining blood haemostasis by using these commercial drugs will also be discussed.

Novel FXa Inhibitor Identification through Integration of Ligand- and Structure-Based Approaches.

Factor Xa (FXa), a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4H-1,4-benzothiazin-2-yl)-2,4-dioxo-N-phenylbutanamide) displayed an IC50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors.