Nuclear Magnetic Resonance Spectroscopy Titration Research Articles

Preparation and chromatographic evaluation of 9-oxa-10-phosphophenanthrene 10-oxide bonded phenyl stationary phase and investigation of its retention mechanism through nuclear magnetic resonance spectroscopy

BackgroundIn reversed-phase liquid chromatography, the C18 alkyl bonded phase, as the primary stationary phase, is widely used in pharmaceutical and food analysis. The phenyl bonded phase often serves as a complementary choice to the C18 phase to enhance the separation performance of specific categories of compounds. However, both C18 and the currently available phenyl bonded phase chromatography columns show room for further optimization in improving the separation efficiency of specific compound classes, such as dihydroflavonoids. Additionally, the potential role and impact of introducing phosphorus groups into chromatographic stationary phases have not been fully explored, indicating a promising direction for research. ResultsIn the present work, we prepared a novel phenyl stationary phase by bonding 9-oxa-10-phosphaphenanthrene 10-oxide onto silica gel. The obtained material was characterized by scanning electron microscopy, fourier transforms infrared spectroscopy, and elemental analysis. The results show that 9-oxa-10-phosphaphenanthrene 10-oxide was successfully bonded on the silica surface with a load of 3.90 %. Further chromatographic characterization in high-performance liquid chromatography exhibited high column efficiency (40,792 plates m-1 for the determination of biphenyl) and good stability (RSD of 0.28 %∼5.38 %). Moreover, we made a detailed study of the column separation mechanism by nuclear magnetic resonance spectroscopy titration experiment. Comparing to commercial phenyl column, the proposed stationary phase showed shorter retention time and higher throughput. In addition, the stationary phase has a strong ability to separate multiple types of compounds, which provides a new strategy for the separation of complex samples, such as active ingredients in traditional Chinese medicine. SignificanceWe have developed a novel phenyl column and conducted a comprehensive examination of its chromatographic performance, demonstrating excellent separation capabilities and high efficiency for both nonpolar and moderately polar aromatic compounds. Additionally, we explored the impact of phosphorus-containing groups on the separation performance of chromatographic stationary phases.

Defining the epitope of a blood–brain barrier crossing single domain antibody specific for the type 1 insulin-like growth factor receptor

Ligand-activated signaling through the type 1 insulin-like growth factor receptor (IGF1R) is implicated in many physiological processes ranging from normal human growth to cancer proliferation and metastasis. IGF1R has also emerged as a target for receptor-mediated transcytosis, a transport phenomenon that can be exploited to shuttle biotherapeutics across the blood–brain barrier (BBB). We employed differential hydrogen–deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) to characterize the interactions of the IGF1R ectodomain with a recently discovered BBB-crossing single-domain antibody (sdAb), VHH-IR5, in comparison with IGF-1 binding. HDX-MS confirmed that IGF-1 induced global conformational shifts in the L1/FnIII-1/-2 domains and α-CT helix of IGF1R. In contrast, the VHH-IR5 sdAb-mediated changes in conformational dynamics were limited to the α-CT helix and its immediate vicinity (L1 domain). High-resolution NMR spectroscopy titration data and linear peptide scanning demonstrated that VHH-IR5 has high-affinity binding interactions with a peptide sequence around the C-terminal region of the α-CT helix. Taken together, these results define a core linear epitope for VHH-IR5 within the α-CT helix, overlapping the IGF-1 binding site, and suggest a potential role for the α-CT helix in sdAb-mediated transcytosis.

Equilibrium, structural and antibacterial characterization of moxifloxacin-β-cyclodextrin complex

Moxifloxacin (MOX), a novel fourth-generation fluoroquinolone antibacterial was characterized in terms of β-cyclodextrin (β-CD) complexation in order to improve its antibacterial activity. The inclusion complexation has been examined with a wide variety of state-of-the-art analytical techniques, such as nuclear magnetic resonance spectroscopy (NMR), affinity capillary electrophoresis (ACE), mass spectrometry (MS), infrared spectroscopy (IR) and differential scanning calorimetry (DSC). The stoichiometry of the complex was investigated by two different techniques. NMR Job plot method indicated 1:1 stoichiometry in liquid state, however MS study revealed that a complex with 2:1 (MOX:β-CD) stoichiometry can also be formed in gas phase. The stability constants were determined by 1H NMR titration and ACE at different pH values, where MOX exists predominantly in monocationic, neutral and monoanionic form, respectively, indicating that the neutral macrospecies forms the most stable complex with the host (logK = 2.51 ± 0.03). Geometric aspects of the inclusion complex were assessed by 2D ROESY NMR and proved that the tricyclic moiety of guest can enter the host cavity. To understand the interaction of different protonation forms of MOX with β-CD at atomic level molecular modeling studies were also performed. Solid state complexation in 1:1 M ratio was carried out by lyophilization and investigated by DSC and IR, which also confirmed the inclusion complex formation in solid state. The antibacterial activity of the complex was tested against Gram-negative and Gram-positive bacteria by determination of minimum inhibitory concentrations, which revealed that supramolecular interactions do not affect significantly the antibacterial activity of the drug.

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.