N2-functionalized blue luminescent guanosines by 2,2'-dipyridylamino and 2-(2'-pyridyl)benzimidazolyl chelate groups and their interactions with Zn(II) ions

The syntheses of new blue luminescent N(2)-modified guanosine derivatives with chromophores p-4,4'-biphenyl-NPh2 (1a), p-4,4'-biphenyl-N(2-py)2 (1b), and p-4,4'-biphenyl-2-(2'-pyridyl)benzimidazolyl (1c), respectively, have been achieved. These new N(2)-guanosines are moderate blue emitters with lambda(max) = 395 nm (1a), 370 nm (1b), and 403 nm (1c) and Phi = 0.13, 0.07, and 0.10 in tetrahydrofuran, respectively. Spectroscopic studies and density-functional theory calculations established that the guanine moiety and the new chromophore in all three molecules are involved in the luminescent process. We have also established that guanosines 1a-1c can interact with metal ions such as Zn(II). The interactions of Zn(II) ions with the three guanosines were examined via absorption, fluorescence, circular dichroism (CD), and NMR spectroscopic analyses. We have found that these guanosines display a distinct fluorescent response toward Zn(II) ions which can be attributed to the presence of the chelate chromophore N(2-py)2 in 1b and 2-py-benzimidazolyl in 1c. For 1a and 1b, the addition of Zn(II) ions causes straight fluorescent quenching while for 1c the addition of Zn(II) ions causes quenching initially, which is followed by a distinct spectral red shift and the intensity enhancement of the new emission peak. NMR and CD studies demonstrated that the Zn(II) ions bind preferentially to the guanine moiety in 1a and 1b but to the 2-(2'-py)benzimidazolyl chelate site in 1c. Moreover, the anion-dependent CD response of 1a-1c toward Zn(II) salts points to the possible involvement of intramolecular hydrogen bonding between the acetate bound to the Zn(II) ion and the hydroxyl groups of the guanosine.     

Enzymatically modified peptide surfaces: towards general electrochemical sensor platform for protein kinase catalyzed phosphorylations

We hereby present an electrochemical approach for monitoring the three protein kinases sarcoma-related kinase (Src), extracellular signal-regulated kinase 1 (Erk1), and cyclin A-dependent kinase 2 (CDK2/cyclin A). The electrochemical sensor is based on the ability of kinases to transfer a redox-labeled phosphoryl group to surface-bound peptides that are highly specific substrates for the particular protein kinase (EGIYDVP, EPLTPSG, and HHASPRK, respectively). The detection method relies on the use of 5'-γ-ferrocenoyl-ATP (Fc-ATP) as a co-substrate for peptide phosphorylation. The peptides themselves are attached to a Au substrate, which acts as the working electrode. In this process a Fc-phosphoryl group is transferred to the peptide and the presence of the redox active Fc group is detected electrochemically. All peptide films were fully characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Particular attention was given to the electron transfer rates, k(ET), in peptide films after Fc-phosphorylation which were found to be on the order of seconds. The slow ET kinetics is presumably a result of the negative charge on the phosphoryl group. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) experiments based on the peptide modified Au surfaces reveal significant ferrocene and phosphate group content introduced using the kinase-catalyzed phosphorylation reaction.     

Interactions of drugs and an oligonucleotide with charged membranes analyzed by immobilized liposome chromatography

We studied the effect of charged lipids or detergent on the retention of drugs and an oligonucleotide by immobilized liposome chromatography to characterize solute-membrane interactions. This is a novel approach in analysis of oligonucleotide-liposome interactions. The charged lipids (phosphatidylserine or distearoyltrimethylammoniumpropane) or detergent (sodium dodecylsulfate) interacted electrostatically in a concentration-dependent matter with the solutes. The oligonucleotide ions presumably bound to the liposomes by multipoint interactions, which was saturable. Sodium dodecylsulfate seemed to affect the drug-membrane interactions more strongly than phosphatidylserine did, probably due to different positioning in the bilayer.     

Electrochemical investigations into Tau protein phosphorylations

Hyperphosphorylation of Tau, a protein that stabilizes microtubules, leads to the breakdown of the microtubular structure and ultimately to the formation of neurofibrillar tangles within neurons. Here, we report monitoring of Tau phosphorylations electrochemically, using Tau protein films chemically linked to gold surfaces and 5'-γ-ferrocenyl (Fc) adenosine triphosphate (Fc-ATP) as a co-substrate. Fc-phosphorylation reactions of Tau are explored using the three protein kinases, glycogen synthase kinase (GSK-3β), sarcoma (Src)-related kinase, and protein kinase A (PKA), which catalyze Fc-phosphorylation of different residues and regions within Tau. The kinetic parameters of the biochemical process (K(M) and V(max)) were determined.