Copper binding motifs with their molecular mechanisms of selective copper(I) recognition are essential molecules for acquiring copper ions, trafficking copper to specific locations and controlling the potentially damaging redox activities of copper in biochemical processes. The redox activity and multiple Cu(I) binding of an analog methanobactin peptide-2 (amb2) with the sequence acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 was investigated using ion mobility-mass spectrometry (IM-MS) and UV-Vis spectrophotometry analyses. The Cu(II) titration of amb2 showed oxidation of amb2 via the formation of intra- and intermolecular Cys-Cys disulfide bridges and the multiple Cu(I) coordination by unoxidized amb2 or the partially oxidized dimer and trimer of amb2. The principal product of these reactions was [amb2 + 3Cu(I)](+) which probably coordinates the three Cu(I) ions via two bridging thiolate groups of Cys2 and Cys6 and the δN6 of the imidazole groups of His6, as determined by geometry optimized structures at the B3LYP/LanL2DZ level of theory. The products observed by IM-MS showed direct correlation to spectral changes associated with disulfide bond formation in the UV-Vis spectrophotometric study. The results show that IM-MS analysis is a powerful technique for unambiguously determining the major ion species produced during the redox and metal binding chemistry of oligopeptides.

The Cu(II) and pH titrations of four structurally similar 2His-2Cys motif peptides were investigated by electrospray ionization-ion mobility-mass spectrometry. The results provided insight into the pH dependent redox processes that took place in solution and identified the number of inter- or intramolecular disulfide bridges, the number of Cu(I) or Cu(II) ions, the deprotonation sites, and likely Cu(I/II) coordination of the various products. Competitive Cu(II) titrations of binary peptide mixtures at pH 5 indicated which species would preferably bind Cu(I) ions over forming the intramolecular disulfide bridge. Moreover, these reactions were pH dependent and included the formation of various multimers and multiple Cu(I/II) binding. For example, for the mildly acidic solution (pH ∼ 3-6) each monomer (whether it was free or in a multimer) primarily bound up to 3 Cu(I) ions, whereas at pH ∼ 8-11 the fully oxidized monomer or multimer (where all Cys formed a disulfide bond) primarily bound up to 2 Cu(II) ions. This behavior was indicative of linear bridging of Cu(I) by Cys thiolate and His imidazole groups, whereas the coordination of Cu(II) involved His and the nitrogens of deprotonated backbone amide groups, resulting in either distorted T-shaped or square planar geometries.

Background: Fulvestrant is a new estrogen receptor antagonist available in the market globally for the treatment of hormone receptor-positive metastatic breast cancer in postmenopausal women. Methods: The chromatographic separation of fulvestrant was carried out by using ACQUITY UPLC and a BEH Shield RP18, 50 mm × 2.1 mm, i.d 1.7-μm column with a prepared mobile phase consisting of water, acetonitrile, and methanol in the ratio of 300:400:300 (v/v/v), respectively. 1.0 mL orthophosphoric acid was added to the prepared mobile phase. The wavelength for detection was made at 220.0 nm using a PDA-UV detector with a flow rate of 0.3 mL min − 1. Results:The system suitability parameters were found within the limits. The coefficient of correlation was found not less than 0.999. The percent recoveries of fulvestrant from 80, 100, to 120% levels are 100.1, 100.4, and 99.7 respectively. The LOD (0.51 μg mL − 1 ) and LOQ (1.54 μg mL − 1 ) values from the study demonstrate that the method is sensitive. The samples were subjected to forced degradation conditions of acidic and alkaline hydrolysis, oxidation, photolysis, metallic and thermal degradation in all conditions; peak was found pure (purity angle less than that of threshold). Conclusion:A rapid, simple, stability-indicating, and validated RP-UPLC method was developed with 6 min of run time for the quantification of fulvestrant in oil-based injection formulations. This is the first stability-indicating method with the capability of resolving all the fulvestrant degradation impurities in the drug products. The method was validated for system suitability, linearity, precision, accuracy, specificity, intermediate precision, ruggedness, robustness, and solution stability.