Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Synthesis and Antiviral Evaluation of (1,4-Disubstituted-1,2,3-Triazol)-(E)-2-Methyl-but-2-Enyl Nucleoside Phosphonate Prodrugs

A series of hitherto unknown (1,4-disubstituted-1,2,3-triazol)-(E)-2-methyl-but-2-enyl nucleosides phosphonate prodrugs bearing 4-substituted-1,2,3-triazoles were prepared in a straight approach through an olefin acyclic cross metathesis as the key synthetic step. All novel compounds were evaluated for their antiviral activities against HBV, HIV and SARS-CoV-2. Among these molecules, only compound 15j, a hexadecyloxypropyl (HDP)/(isopropyloxycarbonyl-oxymethyl)-ester (POC) prodrug, showed activity against HBV in Huh7 cell cultures with 62% inhibition at 10 μM, without significant cytotoxicity (IC₅₀ = 66.4 μM in HepG2 cells, IC₅₀ = 43.1 μM in HepG2 cells) at 10 μM.     

Could humans recognize odor by phonon assisted tunneling?

Our sense of smell relies on sensitive, selective atomic-scale processes that occur when a scent molecule meets specific receptors in the nose. The physical mechanisms of detection are unclear: odorant shape and size are important, but experiment shows them insufficient. One novel proposal suggests receptors are actuated by inelastic electron tunneling from a donor to an acceptor mediated by the odorant, and provides critical discrimination. We test the physical viability of this mechanism using a simple but general model. With parameter values appropriate for biomolecular systems, we find the proposal consistent both with the underlying physics and with observed features of smell. This mechanism suggests a distinct paradigm for selective molecular interactions at receptors (the swipe card model): recognition and actuation involve size and shape, but also exploit other processes.     

In situ X‐ray data collection and structure phasing of protein crystals at Structural Biology Center 19‐ID

A prototype of a 96‐well plate scanner for in situ data collection has been developed at the Structural Biology Center (SBC) beamline 19‐ID, located at the Advanced Photon Source, USA. The applicability of this instrument for protein crystal diffraction screening and data collection at ambient temperature has been demonstrated. Several different protein crystals, including selenium‐labeled, were used for data collection and successful SAD phasing. Without the common procedure of crystal handling and subsequent cryo‐cooling for data collection at T = 100 K, crystals in a crystallization buffer show remarkably low mosaicity (<0.1°) until deterioration by radiation damage occurs. Data presented here show that cryo‐cooling can cause some unexpected structural changes. Based on the results of this study, the integration of the plate scanner into the 19‐ID end‐station with automated controls is being prepared. With improvement of hardware and software, in situ data collection will become available for the SBC user program including remote access.     

Facile Preparation of Mn3O4 Hausmanite Nanoplates from a New Octahedral Manganese (III) Schiff Base Complex

A novel bidentate Schiff base ligand L (L = N-(4-amino-2-chloro-phenyl)-2-hydroxybenzaldehyde) and the subsequent octahedral manganese(III) Schiff base complex MnL ₃ have been synthesized and characterized by, FT-IR spectroscopy and elemental analyses (CHN). Additionally, Schiff base ligand has been characterized by ¹H NMR spectroscopy. Thermogravimetric analysis of the ligand and its metal complexes reveals their thermal stability and decomposition pattern. Thus, the MnL ₃ complex has been investigated as a novel precursor for the facile preparation of Mn₃O₄ nanoparticles via solid-state thermal decomposition under aerobic conditions, at a temperature of ca. 450 °C The resulting Mn₃O₄ nanocrystals were characterized by FT-IR spectroscopy, X-ray powder diffraction (XRPD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The XRPD studies reveal the characteristic diffraction peaks indexed to the Mn₃O₄ hausmanite structure, while the absence of additional peaks tends to clearly indicate the high purity of the sample. In addition, the TEM/SEM investigations displayed the nanoplate shape of the rather monodisperse crystalline Mn₃O₄ nanoparticles, with an average diameter of ca. 10 nm. The statistical distribution of the nanoparticles size has to be provided with an histogram graphic.     

Discrimination between Olfactory Receptor Agonists and Non‐agonists

A joint approach combining free‐energy calculations and calcium‐imaging assays on the broadly tuned human 1G1 olfactory receptor is reported. The free energy of binding of ten odorants was computed by means of molecular‐dynamics simulations. This state function allows separating the experimentally determined eight agonists from the two non‐agonists. This study constitutes a proof‐of‐principle for the computational deorphanization of olfactory receptors.     

Gram‐Scale Enantioselective Formal Synthesis of Morphine through an ortho–para Oxidative Phenolic Coupling Strategy

A gram‐scale catalytic enantioselective formal synthesis of morphine is described. The key steps of the synthesis involve an ortho–para oxidative phenolic coupling and a highly diastereoselective “desymmetrization” of the resulting cyclohexadienone that generates three of the four morphinan ring junction stereocenters in one step. The stereochemistry is controlled from a single carbinol center installed through catalytic enantioselective hydrogenation. These transformations enabled the preparation of large quantities of key intermediates and could support a practical and scalable synthesis of morphine and related derivatives.     

Antibacterial Activity of Synthetic Curcumin Derivatives: 3,5-Bis(benzylidene)-4-Piperidone (EF24) and EF24-Dimer Linked via Diethylenetriaminepentacetic Acid (EF2DTPA)

Curcumin is well known for its antimicrobial and anti-inflammatory properties. However, since systemic absorption and bioavailability of curcumin from gastrointestinal tract is considerably poor, synthetic curcuminoids are being developed as better alternatives. Two curcumin derivatives: 3,5-bis(benzylidene)-4-piperidone (EF24) and EF24-dimer linked via diethylenetriaminepentacetic acid (EF₂DTPA), were included in this study. We investigated the antibacterial activity of EF24 and EF₂DTPA against Gram-negative (Escherichia coli) and Gram-positive (Enterococcus faecalis, Staphylococcus aureus) bacteria. We also studied the effects of EF24 and EF₂DTPA on uptake and localization of pHrodo-labeled E. coli in the acidic compartments (phagolysosomes) of dendritic cells (DCs) under in vitro conditions. Our results demonstrate that treatment with EF24 and EF₂DTPA directly suppresses the bacterial growth. However, these compounds do not affect the bacterial uptake or localization in the DCs.     

Polyesters by a Radical Pathway: Rationalization of the Cyclic Ketene Acetal Efficiency

Radical ring-opening polymerization (rROP) of cyclic ketene acetals (CKAs) combines the advantages of both ring-opening polymerization and radical polymerization thereby allowing the robust production of polyesters coupled with the mild polymerization conditions of a radical process. rROP was recently rejuvenated by the possibility to copolymerize CKAs with classic vinyl monomers leading to the insertion of cleavable functionality into a vinyl-based copolymer backbone and thus imparting (bio)degradability. Such materials are suitable for a large scope of applications, particularly within the biomedical field. The competition between the ring-opening and ring-retaining propagation routes is a major complication in the development of efficient CKA monomers, ultimately leading to the use of only four monomers that are known to completely ring-open under all experimental conditions. In this article we investigate the radical ring-opening polymerization of model CKA monomers and demonstrate by the combination of DFT calculations and kinetic modeling using PREDICI software that we are now able to predict in silico the ring-opening ability of CKA monomers.