1.A novel form of ficin from Ficus carica latex: Purification and characterization.
Baeyens-Volant D1, Matagne A2, El Mahyaoui R1, Wattiez R3, Azarkan M4. Phytochemistry. 2015 Sep;117:154-67. doi: 10.1016/j.phytochem.2015.05.019. Epub 2015 Jun 12.
A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS-PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS-PAGE, around 27 kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% β-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.
2.Sulfate Radical Anion (SO4(•-)) Mediated C(sp(3))-H Nitrogenation/Oxygenation in N-Aryl Benzylic Amines Expanded the Scope for the Synthesis of Benzamidine/Oxazine Heterocycles.
Laha JK1, Tummalapalli KS1, Nair A1, Patel N1. J Org Chem. 2015 Nov 20;80(22):11351-9. doi: 10.1021/acs.joc.5b01872. Epub 2015 Nov 9.
A transition-metal-free, K2S2O8-mediated intramolecular oxidative nitrogenation/oxygenation of C(sp(3))-H in N-aryl benzylic amines followed by oxidation at the benzylic center has been developed for the synthesis of benzamidine/benzoxazine heterocycles, providing an expedient access to quinazolin-4(3H)-ones, N-aryl-2-arylbenzimidazoles, and 4H-3,1-benzoxazin-4-ones. A considerable amount of work dealing with the mechanistic study to understand the crucial intramolecular cyclization step largely favors an iminium ion as the key intermediate.
3.Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.
Kim Y1, Hilty C. Angew Chem Int Ed Engl. 2015 Apr 13;54(16):4941-4. doi: 10.1002/anie.201411424. Epub 2015 Feb 20.
Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.
4.Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.
Nimishakavi S1, Raymond WW1, Gruenert DC2, Caughey GH3. PLoS One. 2015 Oct 20;10(10):e0141169. doi: 10.1371/journal.pone.0141169. eCollection 2015.
Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation.