Maria Ngu-Schwemlein

A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide.

The reaction of ozone (approximately 5% in oxygen) with sodium azide (0.02-0.2 M in water) at pH 12 and 0-4 degrees C is shown to yield concentrated, stable peroxynitrite solutions of up to 80 mM. The product of this reaction is identified based on a broad absorption spectrum with a maximum around 302 nm and by its first-order rate of decomposition (k = 0.40 +/- 0.01 s-1 at pH 7.05 and 25 degrees C). These peroxynitrite solutions can be obtained essentially free of hydrogen peroxide (detection limit 1 microM) and only traces of azide (detection limit 0.1 mM). They are low in ionic strength and have a pH of about 12 but without buffering capacity; therefore, they can be adjusted to any pH by addition of buffer. These preparations of peroxynitrite frozen at -20 degrees C show negligible decomposition for about 3 weeks of storage and follow a first-order decomposition with a halflife of about 7 days at refrigerator temperatures (approximately 5 degrees C). These preparations give reactions that are characteristic of peroxynitrite. For example, at pH 7.0, they react with L-tyrosine to give a 7.3 mol % yield of nitrotyrosine(s), and with dimethyl sulfoxide to give a 8.2 mol % yield of formaldehyde, based on starting peroxynitrite concentration.

Thermodynamics and fluorescence studies of the interactions of cyclooctapeptides with Hg2+, Pb2+, and Cd2+.

The purpose of this work is to characterize the interactions of cyclooctapeptides (CP) containing glutamyl and/or cysteinyl residues with common heavy-metal ions in order to facilitate the design of cyclopeptides as sensors for metal ions. Isothermal titration calorimetry studies show that cyclooctapeptides containing glutamyl and/or cysteinyl residues bind these Hg(2+) and Pb(2+) over Cd(2+) and other common metal ions. Differential binding isotherms, in their interactions with Hg(2+), support a two-binding site model, whereas pertinent interactions with Pb(2+) support a 2:1 stoichiometry, suggesting a CP/Pb(2+)/CP mode of complexation. The cyclooctapeptide containing both glutamyl and cysteinyl residues shows a significant binding affinity for Hg(2+) (K(a)=7.6x10(7)M(-1)), which is both enthalpically and entropically driven. The fluorescence of these cyclooctapeptides showed pronounced fluorescence quenching responses to Hg(2+) over Pd(2+) and Cd(2+). Stern-Volmer analyses of the dependence of fluorescence intensity on Hg(2+) and Pb(2+) are reported. The observed trends are useful for the design of Hg(2+) sensors based on fluorophore-tagged cyclooctapeptides.

Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides

In order to gain more insight into the associations of mercury(II) with cysteinyl peptides, we investigated the effect of increasing cysteinyl residues on complex type formations. Three series of di-, tri-, and tetra-cysteinyl peptides, D[CGD]nCG (CP 2A, CP 3A, and CP 4A), E[CEG]nCG (CP 2B, CP 3B, and CP 4B) and E[CDG]nCG (CP 2C, CP 3C, and CP 4C), where n=1, 2, or 3, were prepared by microwave-assisted solid phase peptide synthesis. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides were characterized by electrospray orbitrap mass spectrometry utilizing complex specific mercury isotopic patterns. In equimolar mercury(II) to peptide ratio, all three series of di-, tri-, and tetra-cysteinyl peptides form predominantly the 1:1Hg(peptide) complex type, indicating that the intervening amino acid residues do not elicit preferential complex type formation. However, in non-equivalent mercury(II) to peptide ratio, the number of cysteinyl residues has a significant effect on the Hg:peptide stoichiometry in the complex formed. For example, in four times excess peptide, the 1:2Hg(peptide)2 and 1:1Hg(peptide) complexes are formed for di-cysteinyl peptides but not for the tri- and tetra-cysteinyl peptides. In contrast, the 2:1Hg2(peptide) and 1:1Hg(peptide) complexes are formed for the tri- and tetra-cysteinyl peptides. In excess mercury(II), CP 4C formed exclusively the 2:1Hg2(peptide) complex. The exact number of deprotonations observed for each complex could be derived from its signature mercury isotope pattern and monoisotopic peak mass. These multi-cysteinyl peptides present an attractive option for mercury chelation or environmental heavy metal remediation.

In Vitro Synergy Between Some Cationic Amphipathic Cyclooctapeptides and Antibiotics

The antimicrobial activities of some cationic amphipathic cyclooctapeptides in combination with select antibiotics are investigated. Accordingly, cyclooctapeptides (CPs 1–11) derived from the sequence cyclo[Leu-d-Leu-Leu-d-Leu-Lys-d-Lys-Lys-d-Lys] were tested against Escherichia coli and Staphylococcus aureus. Synergistic effects were evaluated in combination with tetracycline, chloramphenicol, oflaxacin, rifampicin, colistin, clindamycin, and vancomycin by the checkerboard titration assay. The results show that these cyclooctapeptides are fast acting bactericidals and are comparable to the related α-helical peptides in their activities. Significantly, some of these cyclooctapeptides exert selective synergistic effects in combination with the bacteriostatic tetracycline against S. aureus; a greater than 4-fold decrease in the minimum inhibition concentration was observed. Their synergism with the other evaluated antibiotics was generally partial or additive. Cationic amphipathic cyclooctapeptides in combination with some antibiotics could offer a possible solution to the increasing antimicrobial resistance predicament.

Secondary Structural Preferences of Some Antibacterial Cyclooctapeptides in the Presence of Calcium(II)

The purpose of this study is to understand the interactions of some antibacterial cationic amphipathic cyclooctapeptides with calcium(II) and their secondary structural preferences. The thermodynamic parameters associated with calcium(II) interactions, between the antibacterial active cyclooctapeptides (COP 1–6) and those that did not exhibit significant activities (COP 7–9), were studied by isothermal titration calorimetry. Calcium(II) binding in the absence and presence of micellar dodecylphosphocholine (DPC), a membrane mimicking detergent, was conducted by circular dichroism (CD). Both groups of cyclopeptides showed weak binding affinities for calcium(II) (Kb ca. 10−3 M−1). However, CD data showed that the antimicrobial peptides COP 1–6 adopted a twisted beta-sheet structure (positive CD absorption band at ca. 203 nm) in the presence of calcium(II) in micellar DPC. In contrast, COP 7–9, which lacked antibacterial activity, adopted a different conformational structure (negative CD absorption band at ca. 203 nm). These results indicate that these cyclopeptides could adopt secondary structural preferences in the presence of calcium(II) amidst a hydrophobic environment to elicit their antibacterial activity. These findings could be useful in facilitating the design of cyclopeptide derivatives that can adopt this beta-sheet-like secondary structure and, thereby, provide a useful molecular template for crafting antibacterial compounds.

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

1,2-Dihydro-9H-carbazole-4(3H)-thione.

The crystal structure of the title compound, C12H11NS, features parallel chains of alternating N—H⋯S hydrogen-bonded mirror-image conformers along [10]. The molecular conformation is that of an envelope, with all of the framework atoms except one close to a mean plane (rms deviation 0.054 Å); one C atom of the cyclohexenethione ring forms the envelope flap, which makes a dihedral angle of 48.6 (1)° with the rest of the molecule. There is a π–π* interaction between pairs of enantiomers in adjacent chains; the distance between parallel planes is 3.466 (1) Å.

Fluorogenic Cyclooctapeptides and Metal Ion Sensing

Introduction Optical sensors for the detection of heavy metal ions have wide applications in environmental chemistry, and are directly relevant for the development of a more accurate and rapid identification of harmful metal ions in the environment. Although the design of chemical sensors for such metal ions has been reported [1,2] we are especially interested in developing cyclopeptide-based motifs for selective metal ion binding and sensing. Recent studies have shown that a model acidic cyclopeptide, c[D-Glu-Glu-D-Glu-Glu-D-Leu-Leu-D-Leu-Trp], (CP1), exhibited selective binding and specific fluorescent responses towards some divalent transition metal ions among the surveyed alkali, alkaline earth, transition and heavy metal ions [3]. The present study was undertaken to develop a better understanding of the underlying bimolecular interactions between cyclooctapeptides with acidic groups, (CP1) and c[Glu-D-Leu-Glu-D-Leu-Glu-D-Leu-D-Glu-D-Trp] (CP2), or sulfur containing residues, c[D-Leu-Leu-D-Phe-Met-D-His-Met-D-His-Lys(Dansyl)], (CP3), with Hg, Pb and Cd. We report here the thermodynamic parameters for the interactions between these cyclopeptides with these metal ions as determined by Isothermal Titration Calorimetry (ITC). In order to evaluate the signal transduced upon peptide-metal ion interactions, the fluorescence emission spectral changes following titration of the cyclopeptide with these metal ions was compared. Standard Stern-Volmer formalism and the dependence of fluorescence intensity on quencher concentrations are presented.

Conformational Studies of Cyclotetrapeptides [Xaa-D-Ala]2 by NMR, CD and Molecular Modeling

Small cyclopeptides are attractive molecular scaffoldings with reasonable rigidity and can be used as molecular templates for studies involving molecular recognition. Indeed, macrocyclic peptides have been investigated as probes to study molecular recognition by biomolecules, as well as ligands for metal ions. Nature also utilizes the peptide backbone to design ionophores, toxins and antibiotics, such as tentoxin, AM toxin, Gramicidin S and the depsipeptide, Valinomycin. Various different amino acid residues have been incorporated into small cyclic peptides in order to investigate their structural role, including the induction of specific peptide turns and the adoption of thermodynamically favored conformation(s) [1,2].

CD Evidence of the Conformational Transitions in rMOG[1–125] in the Presence of Membrane Mimicking Detergents

Myelin oligodendrocyte glycoprotein (MOG), an integral glycoprotein associated with the myelin in brain nerve fibers, has recently been implicated as a prime autoantigen leading to autoimmune demyelination in animal models (experimental autoimmune encephalomyelitis, EAE). Immunization of animal models, including mice and marmosets, with rMOG led to the manifestation of a disease similar to multiple sclerosis (MS) — demyelination mediated by autoantibodies directed against the extracellular domain of MOG (MOG[1-125]) [1]. The extracellular location of MOG has been identified as a member of the immunoglobulin superfamily by Gardinier et al. [2]. The amino acid sequence of rat MOG[1-125] (139 residues) is as follows; MRGS-FRVIGPGHPIRALVGDEAELPCRISPGKNATGMEVGWYRSPFSRVVHL YRNGKDQDAEQAPEYRGRTELLKESIGEGKVALRIQNVRFSDEGGYTCFFRDH SYQEEAAVELKVEDPFYWINPG-RSQSHHHHHH.