Mass Spectrometric Distinction of In-Source and In-Solution Pyroglutamate and Succinimide in Proteins: A Case Study on rhG-CSF

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.      

IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

A variety of IR‐active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon‐deuterium bonds (C?D), cyano groups (CN), and azides (N₃); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N‐terminal Src homology 3 domain of the murine adapter protein Crk‐II (nSH3) with C?D bonds site‐selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two‐state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N₃ probes at five of the same positions. In agreement with previous studies, the position‐dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface‐exposed positions. Thus, unlike C?D labels, which are both sensitive to their environment and non‐perturbative, CN and N₃ probes should be used with caution.     

Redox‐Mediated Synthesis of a Fe3O4–MnO2 Nanocomposite for Dye Adsorption and Pseudocapacitance

A logically chosen redox reaction of submerged Fe⁰ in an aqueous KMnO₄ solution has been reported. The template‐free reaction conditions produced gram amounts of a hierarchical flowerlike Fe₃O₄–MnO₂ nanocomposite. More precisely, freshly prepared Fe⁰ nanoparticles were prepared from air‐free hot water under submerged conditions using a door magnet. The black Fe⁰ particles were oxidized in water quantitatively by KMnO₄ in the solution phase and the nanocomposite was prepared. The material has been used as a dye adsorbent and the representative cationic dye uptake, recovery, and recycling of the dye becomes easy owing to the ferromagnetic properties and surface negative charge of the material. The nanocomposite also showed a higher specific capacitance (327 F g^?1 at 10 mV s^?1) than the reported values of pure MnO₂ and Fe₃O₄. The material exhibited a high energy density as well as a high power density, and remained stable even after a large number of charge–discharge cycles.     

Visible‐Light‐Catalyzed Direct Benzylic C(sp3)–H Amination Reaction by Cross‐Dehydrogenative Coupling

A conceptually new and synthetically valuable cross‐dehydrogenative benzylic C(sp³)–H amination reaction is reported by visible‐light photoredox catalysis. This protocol employs DCA (9,10‐dicyanoanthracene) as a visible‐light‐absorbing photoredox catalyst and an amide as the nitrogen source without the need of either a transition metal or an external oxidant.     

Antiadhesive action of a marine microbial surfactant

The antiadhesive action of a lipopeptide biosurfactant from a marine bacterium was investigated. The effect of cultivation conditions on the adhesion property of few bacterial strains was studied. It was observed that the static cultures showed greater adhesion due to scarcity of oxygen. The biosurfactant upon surface conditioning was found to be effective in removal of the microbial adhesion at a concentration as low as 0.1 g L⁻¹. The percentages of inhibition of adhesion against different test bacterial strains ranged from 15 to 89% using 0.1–10 g L⁻¹ of purified biosurfactant. These percentages of adhesion inhibition were found to be significantly higher than the previously reported values. The antiadhesive efficacy of the biosurfactant was also evident from confocal laser scanning microscopy studies.     

Quenching of nucleotide-derived radicals by bis benzimidazole derivative Hoechst-33258 in aqueous solution

The pulse radiolysis technique has been employed to investigate the reaction of DNA-minor-groove ligand bisbenzimidazole Hoechst 33258 with pyrimidine and purine nucleotide-derived radicals. Formation of an N-centred Hoechst-33258 radical is observed. Bimolecular rate constants and the yields of Hoechst-33258 radical have been evaluated. While the rate constant for the reaction of pyrimidine-derived radicals with Hoechst-33258 remained the same (1–2) × 10⁹ dm³ mol⁻¹ s⁻¹, the yields of the Hoechst-33258 radical varied from 25% (5′-cytidine monophosphate) to 75% (5′-guanosine monophosphate) under anoxic conditions. The rate constant values for the reaction of purine-derived radicals with Hoechst-33258, under oxic and anoxic conditions, remained the same whereas with pyrimidine-derived radicals, the rate constant value under oxic conditions was about two orders of magnitude lower than under anoxic conditions. The difference in the yields of Hoechst-33258 radical with various nucleotide-derived radicals suggest the formation of different types of radicals and that the reaction mainly occurs by electron transfer from Hoechst-33258 to the nucleotide radicals.