A robust and sensitive ultra-low flow liquid chromatography (UFLC) method that can reproducibly, at reasonable cost, detect low concentrations of piperine from human plasma is necessary. Piperine in plasma was separated and quantified by a gradient method using ultraviolet detection at a maximal absorbance wavelength of 340 nm. An aliquot was injected onto a reversed-phase column Waters SymmetryShield, 2.1 × 100 mm, 3.5 μm, C18 column, attached to a Waters absorbosphere, 4.6 × 30 mm, C18 guard column and eluted with a mobile phase containing a mixture of acetonitrile/water/ acetic acid (25:74.9:0.1, v/v/v) on line A and acetonitrile/acetic acid (99.9:0.1, v/v) on line B. The flow rate was 0.3 mL/min. The gradient method consisted of an opening condition of 20% pump B, with a linear increase to 37% pump B over 8 min, then a linear increase to 100% pump B at 11 min, 2 min at 100% pump B, and then a return to the opening condition (20% pump B) via a linear gradient over 2 min, followed by 5 min re-equilibration at opening conditions. The total run time was 20 min for each sample. All samples were processed protected from ambient light to avoid isomerization of piperine. The plasma assay was linear with R = 0.9995, with a lower limit of detection [signal-to-noise (S/N) > 5:1] of 100 pg of piperine loaded into the analytical system with acceptable accuracy and precision. Extraction recoveries of piperine from human plasma were 88% for quality control high (QCH), 93% for quality control medium (QCM), and 90% for quality control low (QCL), and the matrix effect was <12%. Piperine was quantifiable from a 50 mg oral dose given to human volunteers. A UFLC method for the rapid assay of human plasma with sensitivity to detect as low as 5 ng/mL piperine was developed. The method sensitivity equals that of liquid chromatography/tandem mass spectrometry (LC/MSMS) methods with much less cost.
Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications, to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses of these bifunctional constructs, owing to the abundance and favorable distribution of their target, FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syntheses and/or multiple protection-deprotection schemes prior to installation into CIDs. In this work, we describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated, cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified with an array of functional groups, including primary amines and carboxylic acids, which make the resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the synthesis of a heterobifunctional HIV protease inhibitor.
S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.
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