ATP-Independent and Cell-Free Biosynthesis of β-Hydroxy Acids Using Vinyl Esters as Smart Substrates

In vitro biosynthetic pathways that condense and reduce molecules through coenzyme A (CoASH) activation demand energy and redox power in the form of ATP and NAD(P)H, respectively. These coenzymes must be orthogonally recycled by ancillary reactions that consume chemicals, electricity, or light, impacting the atom economy and/or the energy consumption of the biosystem. In this work, we have exploited vinyl esters as dual acyl and electron donor substrates to synthesize β-hydroxy acids through a non-decarboxylating Claisen condensation, reduction and hydrolysis stepwise cascade, including a NADH recycling step, catalyzed by a total of 4 enzymes. Herein, the chemical energy to activate the acyl group with CoASH and the redox power for the reduction are embedded into the vinyl esters. Upon optimization, this self-sustaining cascade reached a titer of (S)-3-hydroxy butyrate of 24 mM without requiring ATP and simultaneously recycling CoASH and NADH. This work illustrates the potential of in vitro biocatalysis to transform simple molecules into multi-functional ones.     

Self‐Assembled Submonolayer of β‐Cyclodextrins on Gold Electrode for the Selective Determination of 4‐Aminobiphenyl

The formation of an inclusion complex between 4‐aminobiphenyl (4‐AB) and β‐cyclodextrin molecules (β‐CD), allows the use of thiolated β‐CDs as chemi‐adsorbed material on a Au electrode as a self‐assembled submonolayer for the selective square wave voltammetric determination of 4‐AB. The submonolayer was characterized by reductive desorption and an association constant of 1.2×10⁴ L/mol was obtained. The optimization of variables yielded a linear dependence of ip/4‐AB concentration in the range of 10^?5 to 10^?4 mol/L. The selectivity of the method was evaluated in the presence of other aromatic amines obtaining better results with the modified electrode. This methodology was applied to the voltammetric determination of 4‐AB in wastewater samples.     

Polyphenolic profile by FIA/ESI/IT/MSn and antioxidant capacity of the ethanolic extract from the barks of Maytenus cajalbanica (Borhidi & O. Muñiz) Borhidi & O. Muñiz

Abstract

Medium and high polarity extracts from Maytenus species are known to contain polyphenolic compounds such as proanthocyanidins. The high polarity and structural complexity of these compounds make very difficult their isolation even by modern chromatographic techniques. Maytenus cajalbanica (Borhidi & O. Muñiz) Borhidi & O. Muñiz is endemic from Cuba. So far, there are reports neither of phytochemical work nor of biological evaluation of extracts from this subspecies. The goal of this work is to determine the polyphenolic profile and the antioxidant capacity of the ethanolic extract from the barks of Maytenus cajalbanica. FIA/ESI/IT/MSⁿ analysis allowed the identification of 5 flavan-3-ol monomers, 33 proanthocyanidins, 2 free flavonoids and their respective glycosides as major compounds of the ethanolic extract, which showed a strong radical scavenging capacity and a significant ferric reduction power. FIA/ESI/IT/MSⁿ technique led the rapid, effective and sensitive determination of the polyphenolic profile of Maytenus cajalbanica without previous separation.     

Influence of the Chain Length of Alkanethiols in Self‐Assembled Submonolayers on the Electrochemical Response of Aromatic Amine Mixtures

Alkanethiols, such as di‐n‐octadecyldisulfide, octanethiol, and ethanethiol, have been employed for self‐assembled monolayers on a gold electrode to study the influence of their chain length on the electrochemical response of aniline and 4‐aminobiphenyl. The responses of individual solutions and mixtures of both compounds show that the organization of the submonolayer built can improve the selectivity in the electrochemical analysis of those aromatic amines. The use of C8 10^?2 M allows the analysis of 4‐aminobiphenyl avoiding the aniline response. A better resolution for the analysis of those amine mixtures was obtained if a C18 10^?6 M modified gold electrode was used instead of an unmodified one.     

Solvent effects and alkali metal ion catalysis in phosphodiester hydrolysis

The kinetics of the alkaline hydrolysis of bis(p-nitrophenyl) phosphate (BNPP) have been studied in aqueous DMSO, dioxane, and MeCN. In all solvent mixtures the reaction rate steadily decreases to half of its value in pure water in the range of 0-70 vol % of organic cosolvent and sharply increases in mixtures with lower water content. Correlations based on different scales of solvent empirical parameters failed to describe the solvent effect in this system, but it can be satisfactorily treated in terms of a simplified stepwise solvent-exchange model. Alkali metal ions catalyze the BNPP hydrolysis but do not affect the rate of hydrolysis of neutral phosphotriester p-nitrophenyl diphenyl phosphate in DMSO-rich mixtures. The catalytic activity decreases in the order Li+ > Na+ > K+ > Rb+ > Cs+. For all cations except Na+, the reaction rate is first-order in metal ion. With Na+, both first- and second-order kinetics in metal ions are observed. Binding constants of cations to the dianionic transition state of BNPP alkaline hydrolysis are of the same order of magnitude and show a similar trend as their binding constants to p-nitrophenyl phosphate dianion employed as a transition-state model. The appearance of alkali metal ion catalysis in a medium, which solvates metal ions stronger than water, is attributed to the increased affinity of cations to dianions, which undergo a strong destabilization in the presence of an aprotic dipolar cosolvent.