Improved curve fitting procedures to determine equilibrium binding constants

For ligand-biomacromolecule titration experiments it has been traditional practice to extract parameters such as the equilibrium binding constant K and the number of bases per ligand binding site n with relatively labour intensive methods, usually based on single wavelength data, such as the difference method by Rodger and Nordén coupled together with a Scatchard plot. Presented in this paper are both the theory and a least squares fitting method to derive parameters such as K and n more directly from all spectral non-linear experimental data. Both the case of non competitive binding of a metal complex ligand to DNA and the case of displacement by a metal complex ligand of an ethidium marker attached to the DNA are considered. This work may be applied directly to reduce experimental data produced by a spectropolarimeter (for circular or linear dichroism) or a spectrophotometer (for fluorescence or UV-Vis spectroscopy).     

Intramolecular Fluorocyclizations of Unsaturated Carboxylic Acids with a Stable Hypervalent Fluoroiodane Reagent

A new class of fluorinated lactones was prepared by the intramolecular fluorocyclizations of unsaturated carboxylic acids by using the stable fluoroiodane reagent in combination with AgBF₄. This unique reaction incorporates a cyclization, an aryl migration, and a fluorination all in one step. The fluoroiodane reagent, prepared easily from fluoride, can also be used without a metal catalyst to give moderate yields within just 1 hour, thus demonstrating that it is a suitable reagent for developing new ¹⁸F‐labelled radiotracers for PET imaging.     

Pyrroles as antioxidants: solvent effects and the nature of the attacking radical on antioxidant activities and mechanisms of pyrroles, dipyrrinones, and bile pigments

The absolute rate constants, k(inh), and stoichiometric factors, n, of pyrroles, 2-methyl-3-ethylcarboxy-4,5-di-p-methoxyphenylpyrrole, 6, 2,3,4,5-tetraphenylpyrrole, 7, and 2,3,4,5-tetra-p-methoxyphenylpyrrole, 8, compared to the phenolic antioxidant, di-tert-butylhydroxyanisole, DBHA, during inhibited oxidation of cumene initiated by AIBN at 30 degrees C gave the relative antioxidant activities (k(inh)) DBHA > 8 > 7 > 6 and n = 2, whereas in styrene, 8 > DBHA. These results are explained by hydrogen atom transfer, HAT, from the N-H of pyrroles to ROO(*) radicals. The k(inh) values in styrene of dimethyl esters of the bile pigments of bilirubin ester (BRDE), of biliverdin ester (BVDE), and of a model compound (dipyrrinone, 1) gave k(inh) in the order pentamethylhydroxychroman (PMHC) > BRDE > 1 > BVDE. These antioxidant activities for BVDE and the model compound, 1, and PMHC dropped dramatically in the presence of methanol due to hydrogen bonding at the pyrrolic N-H group. In contrast the k(inh) of BRDE increased in methanol. We now show that pyrrolic compounds may react by HAT, proton-coupled electron transfer, PCET, or single electron transfer, SET, depending on their structure, the nature of the solvent, and the attacking radical. Compounds BVDE and 1 react by the HAT or PCET pathway (HAT/PCET) in styrene/chlorobenzene with ROO(*) and with the DPPH(*) radical in chlorobenzene according to N-H/N-D kH/kD of 1.6, whereas the DKIE with BRDE was only 1.2 with ROO(*). The antioxidant properties of polypyrroles of the BVDE class and model compounds (e.g., 1) are controlled by intramolecular H bonding which stabilizes an intermediate pyrrolic radical in HAT/PCET. According to kinetic polar solvent effects on the monopyrrole, 8, and BRDE, which gave increased rates in methanol, some pyrrolic structures are also susceptible to SET reactions. This conclusion is supported by some calculated ionization potentials. The antioxidant mechanism for BRDE with peroxyl radicals is described by the PCET reaction. Experiments using the 2,6-di-tert-butyl-4-(4'-methoxyphenyl)phenoxyl radical (DBMP(*)) showed this to be a better radical to monitor HAT activities in stopped-flow kinetics compared to the use of the more popular DPPH(*) radical.     

Lewis acid stabilized methylidene and oxoscandium complexes

The methylidene scandium complex (PNP)Sc(mu3-CH2)(mu2-CH3)2[Al(CH3)2]2 (PNP = N[2-P(CHMe2)2-4-methylphenyl]2-) can be prepared from the reaction of (PNP)Sc(CH3)2 and 2 equiv of Al(CH3)3. The Lewis acid stabilized methylidenes candium complex has been crystallographically characterized, and its bonding scheme analyzed by DFT. In addition, we report preliminary reactivity studies of the Sc-CH2 ligand with substrates such as H2NAr and OCPh2. While the former results in an Br?nsted acid-base reaction, the latter reagent produces the olefin H2C CPh2 along with the novel oxoscandium complex (PNP)Sc(mu3-O)(mu2-CH3)2[Al(CH3)2]2, quantitatively.     

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.     

Regioselective reactivity of an asymmetric tetravalent di[dihydroxotin(IV)] bis-porphyrin host driven by hydrogen-bond templation

Local molecular environment effects on the rates of ligand exchange at an asymmetric di[dihydroxotin(IV)] bis-porphyrin 5 are examined. The host 5 possesses four non-equivalent tin(IV)-ligand binding sites that are distinguished by their position relative to a shallow cavity, by the steric environment at each binding site and by electronic-structure differences between the constituent porphyrin and quinoxalinoporphyrin macrocycles. These design features of the asymmetric host are confirmed by X-ray crystal structure analysis. Binding experiments with monodentate carboxylic acids and bidentate dicarboxylic acids show significant differences in the rate of ligand exchange at each of the four tin(IV) binding sites. For monodentate carboxylic acids, binding preferentially occurs at the exterior porphyrin site. Further addition of carboxylic acid results in sequential binding at the quinoxalinoporphyrin sites and lastly at the interior site on the porphyrin, with high regioselectivity. These selective binding outcomes are immediately apparent by NMR spectroscopy. A series of 2D NMR spectroscopy experiments allowed identification of the preferred binding sites at the host. This positively identifies that steric hindrance and electron-withdrawing functionality on the porphyrin macrocycle impede ligand exchange. However, these effects are overcome by dicarboxylic acid guests, which form ditopic hydrogen-bond interactions between the intracavity hydroxo ligands in the initial stage of ligand exchange, leading to regioselective binding between the tin(IV) sites within the cavity. It is envisaged that the factors identified herein that define regioselective ligand exchange at host 5 will find wider application in supramolecular systems incorporating tin(IV) porphyrins.     

Tandem mass spectrometry of deprotonated iodothyronines

In order to assist with the development of more selective and sensitive methods for thyroid hormone analysis the [M-H]- anions of the iodothyronines T4, T3, rT3, (3,5)-T2 and the non-iodinated thyronine (T0) have been generated by negative ion electrospray mass spectrometry. Tandem mass spectra of these ions were recorded on a triple-quadrupole mass spectrometer and show a strong analogy with the fragmentation pathways of the parent compound, tyrosine. All iodothyronines also show significant abundances of the iodide anion in their tandem mass spectra, which represents an attractive target for multiple reaction monitoring (MRM) analysis, given that iodothyronines are the only iodine bearing endogenous molecules. Characteristic fragments are observed at m/z 359.7 and 604.5 for rT3 but are absent in the spectrum of T3, thus differentiating the two positional isomers. The striking difference in the fragmentation patterns of these regioisomeric species is attributed to the increased acidity of the phenol moiety in rT3 compared with T3.     

Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.