James W. Webb

Structural and spectral studies of copper(II) and nickel(II) complexes of pyruvaldehyde mixed bis{N(4)-substituted thiosemicarbazones}

Abstract Pyruvaldehyde mixed bis(thiosemicarbazones) have been prepared in which the two thiosemicarbazone moieties have different N(4)-substituents. The mixed bis(thiosemicarbazones) and their copper(II) and nickel(II) complexes have been characterized with IR, electronic, mass, 1H NMR (Ni) and EPR (Cu) spectra. Representative crystal structures have been solved of nickel(II) complexes with either a pyruvaldehyde mixed bis(thiosemicarbazone) or a bis(thiosemicarbazone) with identical N(4)-substituents acting as a tetradentate ligand. [Ni(Pu4M4DE)] has an N(4)-methylthiosemicarbazone substituent on the keto “arm” and N(4)-diethylthiosemicarbazone substituent on the aldehyde arm. [Ni(Pu4M)] contains two N(4)-methylthiosemicarbazone moieties. Both bis(thiosemicarbazones) form square-planar N2S2 complexes with nickel(II) and copper(II).

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstroms macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.

Antagonism of Pseudomonas cepacia against phytopathogenic fungi

Two strains ofPseudomonas cepacia, RJ3 and ATCC 52796, have been identified as potential antagonists of fungal plant pathogens. We have compared the antagnonistic activity of these two strains against various fungal pathogens. Although both strains displayed high levels of antagonism, ATCC 52796 was slightly more antagonistic than RJ3. The antagonist from RJ3 has been identified as the antifungal compound pyrrolnitrin after purification by HPLC and characterization by UV, IR, NMR, and mass spectroscopy. Both strains also antagonized the fungi by production of volatile compound(s), which have not yet been identified. Both strains are similar with respect to in vitro antagonism, mechanism of antagonism, and sensitivity to antibiotics.

Glutathionato-S-Gold(III) complexes formed as intermediates in the reduction of auricyanide by glutathione ☆

The reduction of auricyanide ([Au(CN)(4)](-), a potential gold(III) metabolite of antiarthritic gold(I) compounds), by glutathione (G(-)SH, an anionic biological reductant) proceeds through two intermediates (I(230) and I(290)) which have previously been identified by their UV-vis spectra, but not isolated. Negative-ion electrospray ionization-mass spectroscopy (ESI-MS) has unambiguously identified them as [Au(CN)(3)(SG)](2-) and [Au(CN)(2)(SG)(2)](3-), respectively, and allowed their formation and decay to be monitored. The spectra also confirm that the products are aurocyanide ([Au(CN)(2)](-), a known metabolite of chrysotherapy agents) and oxidized glutathione (GSSG(2-)). The reactions are dependent on the presence or absence of buffering agents and the pH of the reaction media. The reaction can be driven to the first intermediate by using an excess of auricyanide or by running the reaction at low pH which prevents further reaction. At neutral pH and/or with excess of glutathione present, the reaction proceeds to the second intermediate, which is then reduced to aurocyanide. The monoanions, [Au(CN)(3)(SGH)](-) at m/z=581.2 and [Au(CN)(2)(SGH)(2)](-) at m/z=861.5 generate more intense signals than their respective dianions, [Au(CN)(3)(SG)](2-) at m/2=290.2 and [Au(CN)(2)(SG)(SGH)](2-)m/2=430.9, respectively, whereas the trianion [Au(CN)(2)(SG)(2)](3-) (m/3=281.2) was not observed. These studies demonstrate the value of ESI-MS methods for characterizing reactions of metallopharmaceuticals under biomimetic conditions and suggest that they will be useful for other systems which give strong ESI-MS signals.

Comparison of isolation methods of urinary organic acids by high-performance liquid chromatography.

Four methods for extracting organic acids from human urine prior to analysis by high-performance liquid chromatography (HPLC) were compared. The methods were manual solvent extraction with ethyl acetate and diethyl ether, continuous solvent extraction, anion exchange with pyridinium acetate as the eluting solvent and anion exchange with hydrochloric acid as the eluting solvent. All four methods produced samples that could be analyzed by reversed-phase HPLC, but the continuous solvent extraction and anion exchange with pyridinium acetate methods gave the best reproducibilities (approximately 6% relative standard deviations). Pretreatment of the urine with barium hydroxide and hydroxylamine hydrochloride prior to anion exchange did not markedly alter the HPLC profiles.

High Resolution Separation of Urinary Organic Acids by High Performance Liquid Chromatography

Abstract Reverse phase, anion exchange, and two-dimensional HPLC techniques were studied in order to increase resolution of organic urinary acids for eventual quantitative measurements. Reverse phase HPLC with a phosphate buffer/acetonitrile gradient yielded a separation of over 85 components in forty minutes and a peak area reproducibility of better than 5%. Connecting two reverse phase columns together resulted in the separation of 110 components. Anion exchange chromatography was determined to be of little use in resolving urinary acids in a resonable time except as the first stage in two-dimensional chromatography where fractions from the anion exchange column were injected into a reverse phase column. Over 139 components were separated by this two-dimensional method.

Metabolic profiles of rats of different ages

Urine samples from 6- to 31-month-old male Fischer F344 rats were analyzed using a high performance liquid chromatograph and a unique computer-based data analysis and quality control system in order to discover substances that could be used as markers in the aging process. Metabolic profiles of the organic acids from these urines yielded 42 peaks whose areas could be measured reliably. Of the 42 peaks, 10 were found by analysis of variance to vary significantly (p less than 0.05) with age. Rats from the four ages could also be distinguished using a multivariate statistic (discriminant analysis).

Proton-linked bi- and tri-metallic gold cyanide complexes observed by ESI-MS spectrometry

Electrospray ionization spectra of potential cyanide-containing gold-drug metabolites revealed additional, weak, unanticipated peaks at approximately twice the mass of the gold(I) and gold(III) cyanide complexes. The exact masses correspond to proton-linked bimetallic complexes, [H[Au(CN)(m)](2)](-), (m=2,4). Further investigation revealed a total of 12 examples, including trimetallic complexes, [H(2)[Au(CN)(m)](3)](-); mixed species with two complexes, [H[Au(CN)(2)][Au(CN)(4)]](-); and thiolato species, [H[(RS)Au(CN)(3)](2)](-). trans-[AuX(2)(CN)(2)Cl(2)](-) and trans-[AuX(2)(CN)(2)Br(2)](-) generated (35)Cl/(37)Cl and (79)Br/(81)Br isotopic patterns for the protonated bi- and tri-metallic analogues which were in good agreement with the presence of four or six halide ligands, respectively. Concentration-dependent studies demonstrated that the signals are independent of the solution concentrations of mono-metallic precursors, suggesting formation in the gas phase during or following droplet desolvation.

Preliminary water quality assessment of Spunky Bottoms restored wetland

The approximately 1200-acre “Spunky Bottoms” wetland in Southern Illinois has been undergoing restoration to conditions prior to levying of the Illinois River and draining of adjacent floodplain for intensive agriculture (circa 1900). As part of a long-term water quality impact assessment of this restoration project, baseline water quality monitoring was conducted soon after restoration began. During this baseline/preliminary assessment, water samples were taken every 2–4 weeks from 10 sampling wells and seven surface water sites throughout the wetlands area for a period of 18 months. Measured parameters include nutrients (nitrate (NO3 −) and phosphate (PO4 3−), cations and anions (SO4 2 −, Cl−, Na+, K+, Mg2 +, Ca2 +) commonly found in surface and well water, trace metals (Al, Cd, Cu, Fe, Mn, Ni, Pb, Se, Zn), total dissolved solids (TDS), pH, and trace organics (triazine herbicides and their metabolites). In general, highest concentrations of ions were found in the southwest and northeast perimeter of the wetland area for both surface and ground water samples. Primarily low concentrations of heavy metals and organic compounds were found throughout the wetland sampling area. Distribution of NO3 −-N suggests that this restored wetland, even at its infant age, may still contribute to biogeochemical (particularly N) element cycling. Continued monitoring and further research is necessary to determine long-term specific contribution of restored wetland to biogeochemical cycles.In the present work changes in the adsorption of the pesticide chlorpyrifos-methyl (CLP-m) on soil colloids induced by application of surfactants were determined using a batch equilibrium method. The surfactants used were sodium dodecyl sulphate (SDS), Tween 20, and dihexadecyldimethylammonium bromide (DHAB). The adsorption isotherms of CLP-m in aqueous medium and in surfactant solutions at concentration equal to the critical micelle concentration (CMC) fitted the Freunlich adsorption equation generally with R(2) values greater than 0.96. While the addition of SDS and DHAB decreased the pesticide adsorption, the addition of Tween 20 increased the pesticide adsorption. The increases or decreases in the adsorption in the experiment revealed that the behavior of CLP-m in soil water-systems mainly depends on the type of surfactant. Moreover water solubility of CLP-m changes by the three surfactants below and above their CMC were studied. While the solubility of CLP-m was enhanced by SDS both below and above the CMC, the solubility of the pesticide was enhanced by DHAB only above the CMC. Tween 20 did not influence the solubility of CLP-m.

Synthesis and spectral characterization of 1-phenylglyoxal bis{N(4)ethylthiosemicarbazone} and its metal complexes

Nickel(II), copper(II), zinc(II), and cadmium(II) complexes of 1-phenylglyoxal bis{N(4)-ethylthiosemicarbazone} have been synthesized and characterized spectroscopically. The bis(thiosemicarbazone) and its diamagnetic complexes have been characterized by their melting points, i.r., electronic, mass and 1H n.m.r. spectra. The four-coordinate copper(II) complex was also studied by e.s.r. spectroscopy. Upon coordination, the loss of a proton from each thiosemicarbazone moiety occurs, and the bis(thiosemicarbazone) coordinates to nickel(II) and copper(II) as a dianionic, tetradentate N2S2 ligand; but for zinc(II) and cadmium(II), mass spectral results suggest the formation of binuclear complexes.