Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein‐activity‐based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α‐globin probe, which was used for the enrichment and proteomic identification of α‐globin‐modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α‐globin, an excess of which aggravates β‐thalassemia symptoms. This development opens new opportunities for activity‐based‐probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.     

Validated spectrofluorometric methods for determination of amlodipine besylate in tablets

Two simple and sensitive spectrofluorometric methods have been developed and validated for determination of amlodipine besylate (AML) in tablets. The first method was based on the condensation reaction of AML with ninhydrin and phenylacetaldehyde in buffered medium (pH 7.0) resulting in formation of a green fluorescent product, which exhibits excitation and emission maxima at 375 and 480 nm, respectively. The second method was based on the reaction of AML with 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) in a buffered medium (pH 8.6) resulting in formation of a highly fluorescent product, which was measured fluorometrically at 535 nm (lambda(ex), 480 nm). The factors affecting the reactions were studied and optimized. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9949-0.9997) were found between the fluorescence intensity and the concentrations of AML in the concentration range of 0.35-1.8 and 0.55-3.0 microg ml(-1) for ninhydrin and NBD-Cl methods, respectively. The limits of assays detection were 0.09 and 0.16 microg ml(-1) for the first and second method, respectively. The precisions of the methods were satisfactory; the relative standard deviations were ranged from 1.69 to 1.98%. The proposed methods were successfully applied to the analysis of AML in pure and pharmaceutical dosage forms with good accuracy; the recovery percentages ranged from 100.4-100.8+/-1.70-2.32%. The results were compared favorably with those of the reported method.     

Synthesis of N‐pivot lariat ethers

This review covers the main strategies for the synthesis of N‐pivot lariat ethers as well as their specific syntheses.     

Coupled transport of Ag(I) ions through triethanolamine–cyclohexanone-based supported liquid membranes

Facilitated transport of silver(I) ions in acidic medium, across a supported liquid membrane (SLM) by using triethanolamine (TEA) as carrier, dissolved in cyclohexanone, has been investigated. The parameters studied are HNO₃ concentration variation in the feed, pH of the feed solution, carrier concentration in the membrane phase, silver(I) ions concentration in the feed phase and KCN concentration in the stripping phase. Increase in H⁺ concentration by increasing HNO₃ concentration from 0.5 to 1 M results into an increase in silver ions flux but a decrease in flux has been found beyond 1 M HNO₃ concentration in the feed, providing a maximum flux of 3.21 × 10⁻⁷ mol/m² s at 1 M HNO₃. Increase in TEA concentration inside the membrane enhances flux with its maximum value at 2.25 M TEA. Further increase in the concentration of TEA leads to a decreased rate of transport due to the increase in viscosity of membrane liquid. The optimum conditions for Ag(I) ions transport are 1 M HNO₃ (feed), 2.25 M TEA (membrane) and 1.5 M KCN in the stripping phase. It has been observed that Ag(I) flux across the membrane tends to increase with increase in Ag(I) ions concentration in the feed phase. Applying the studied conditions to silver plating waste solutions, Ag ions have been removed up to 99% in a time interval of 5 h.     

Sugar-assisted glycopeptide ligation with complex oligosaccharides: scope and limitations

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.     

Efficient Palladium‐Assisted One‐Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis

The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd^II complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one‐pot fashion by native chemical ligation where the Acm moiety was used to protect the N‐terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin‐like protein UBL‐5, which contains two native Cys residues, was accomplished through the one‐pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.     

Amination of Malononitrile Dimer to Amidines: Synthesis of 6‐aminopyrimidines

Reactions of amidines with malononitrile dimer in DMF and catalyzed with piperidine are reported. The reaction occurred via amination process followed by cyclization to give racemic 6‐aminopyrimidine compounds. The reaction mechanism was discussed. The structure of products was elucidated by mass spectrometry, IR and NMR spectra together with elemental analyses.     

New Pyrimidine‐2‐thiones from Reactions of Amidrazonethiols with 2‐Amino‐1,1,2‐ethenetricarbonitrile and Investigation of Their Antitumor Activity

Reactions of thiosemicarbazones with 2‐amino‐1,1,2‐ethenetricarbonitrile were reported. The reaction occurred in the amidrazonyl site, and new pyrimidine‐2‐thiones were obtained. The reaction mechanism was discussed. The structure of products was elucidated by MS, IR, and NMR spectra together with elemental analyses. The antitumor activity was evaluated against one tumor cell line. Using a standard MTT assay, the effect of the samples on the growth of HepG2 cells was investigated. Half of these compounds showed no cytotoxic effect on HepG2 cells, while the others result in decreasing in the HepG2 cell viability.