Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups

E2 enzymes catalyze the ATP-dependent polymerization of polyubiquitin chains which function as molecular signals in the regulation of numerous cellular processes. Here we present a method that uses genetically encoded unnatural amino acids to halt and re-start ubiquitin polymerization providing access to natural-linkage, precision-length ubiquitin chains that can be used for biochemical, structural, and dynamics studies.     

Oxidation of Tyrosine Photoinduced by Pterin in Aqueous Solution

Pterins, heterocyclic compounds widespread in biological systems, accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. Pterins have been previously identified as good photosensitizers under UV‐A irradiation. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the oxidation of tyrosine (Tyr) in aqueous solutions. Tyr is an important target in the study of the photodynamic effects of UV‐A radiation because it is oxidized by singlet oxygen (¹O₂) and plays a key role in polymerization and cross‐linking of proteins. Steady UV‐A irradiation of solutions containing Ptr and Tyr led to the consumption of Tyr and dissolved O₂, whereas the Ptr concentration remained unchanged. Concomitantly, hydrogen peroxide (H₂O₂) was produced. By combining different analytical techniques, we could establish that the mechanism of the photosensitized process involves an electron transfer from Tyr to the triplet excited state of Ptr. Mass spectrometry, chromatography and fluorescence were used to analyze the photoproducts. In particular, oxygenated and dimeric compounds were identified.     

Screening of non-polar heterocyclic amines in urine by microextraction in packed sorbent-fluorimetric detection and confirmation by capillary liquid chromatography

A rapid and simple procedure for the direct screening of urine samples is described. The method involves microextraction in a packed sorbent (MEPS) that is on-line coupled to a capillary liquid chromatograph with fluorimetric detection. The overall arrangement works as a screening/confirmatory system for monitoring non-polar heterocyclic aromatic amines (HAAs) in urine samples. This configuration allows the selective retention of HAAs from urine on a C₁₈ MEPS cartridge integrated in the needle of a micro-well plate autosampler. Retained HAAs were eluted with methanol/water (90:10, v/v) and directly injected into the fluorimetric detector. This screening method provides a yes/no binary response that may require confirmation. The samples for which the concentration of HAAs was close to or above the established threshold limit (30 ng mL⁻¹) were subjected to capillary liquid chromatography (CLC) for confirmation purposes. A mobile phase of acetonitrile and triethylamine (25 mM) at pH 2.5, through a gradient of composition at a flow rate of 20 μL min⁻¹, resulted in good separations between the analytes in less than 11 min. This confirmation method allowed the determination of the analytes in the 10-100 ng mL⁻¹ range for harmane and norharmane and from 20 to 200 ng mL⁻¹ for 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b] indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido-[4,3-b] indole (Trp-P-2), 2-amino-9H-pyrido-[2,3-b] indole (AαC) and 2-amino-3-methyl-9H-pyrido-[2,3-b] indole (MeAαC), with relative standard deviation (RSD) values between 2.12% and 3.73%, and limits of detection between 1.6 and 5.6 ng mL⁻¹ for all the HAAs.     

Direct C-4 arylation of crotonaldehyde with arenediazonium tetrafluoroborates

Arenediazonium tetrafluoroborates react with crotonaldehyde (2-butenal) in methanol in the presence of catalytic amounts of Pd(OAc)₂ to yield mainly 4,4-dimethoxy-1-butenylarenes. In most of the examples studied, small amounts of an isomeric byproduct were formed, presumably 3,3-dimethoxy-1-methylenepropylarenes. Because crotonaldehyde and arenediazonium salts are cheap and readily available, this reaction is a convenient access to protected 4-arylbutenals.     

Density dependent interactions and structure of charged colloidal dispersions in the weak screening regime

We determine the structure of charge-stabilized colloidal suspensions at low ionic strength over an extended range of particle volume fractions using a combination of light and small angle neutron scattering experiments. The variation of the structure factor with concentration is analyzed within a one-component model of a colloidal suspension. We show that the observed structural behavior corresponds to a nonmonotonic density dependence of the colloid effective charge and the mean interparticle interaction energy. Our findings are corroborated by similar observations from primitive model computer simulations of salt-free colloidal suspensions.