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Only a few analytical techniques are available for the characterization of mechanochemical synthetic reaction products. We demonstrate here that DESI-MS is a powerful technique for this purpose, combining the selectivity of MS-based assays with the simplicity and in situ analysis capability of ambient ionization methods. In this work, we report that auranofin, a gold-based drug, and its precursor triethylphosphine gold(I) chloride undergo a complex array of ligand exchange/scrambling reactions with thiol-containing amino acids in the solid state. The products were readily characterized by DESI-MS analysis from the solid-phase reaction, clearly exhibiting ligand exchange and scrambling, with independent confirmation by solid state 13C-NMR. The thioglucose and triethylphosphine moieties exchanged with cysteine and its derivatives, whereas the glutathione replaced 2,3,4,6-tetra-o-acetyl-β-1-d-glucopyranose only. It was concluded that ligand exchange and scrambling reactions can be carried out in the solid state, and some of the unique products reported in this study can be conveniently prepared through mechanochemical synthesis in good yields (> 98%), as demonstrated by synthesis of (l-cysteinato-S)-triethylphosphine gold(I) from triethylphosphine gold(I) chloride and l-cysteine.

The nature of acidic and basic (alkaline) oxides can be easily illustrated via a series of three straightforward classroom demonstrations for high school and general chemistry courses. Properties of carbon dioxide, sulfur dioxide, and magnesium oxide are revealed inexpensively and safely. Additionally, the very different kinetics of hydration of SO2 (rapid) and CO2 (slow) are evident. The pH changes observed by use of universal indicator provide striking visual evidence that makes the concepts of acidic and basic oxides less abstract and more concrete than verbal or written descriptions alone. By using the MgO solution for the SO2 hydration reaction, one can mimic environmental interactions that lead to the neutralization of alkaline and acidic species. Interestingly, the SO2 and CO2 demonstrations can easily be adapted to environmental chemistry courses and especially the very relevant realm of climate change science. The difference in hydration rates explains why CO2 is a greenhouse gas, but SO2 is not ...

Interaction of auricyanide, an important metabolite of anti-arthritic gold-based drug auranofin, was studied in vitro with a pharmacologically active ligand n-acetylcysteine with a view to understand reactivity of gold in vivo. Formation of reduction product aurocyanide occurred through mono- and di-n-acetylcysteine-substituted intermediates. The product and intermediates were identified and monitored spectrophotometrically and by electrospray ionization mass spectrometry. This study suggests successive substitution with n-acetylcysteine through trans effect. At equimolar concentrations of auricyanide and n-acetylcysteine, only mono-substituted mixed-ligand complex was formed. Substitution of the data obtained to various kinetic models suggested that the reaction orders are 0.6 in terms of n-acetylcysteine, 1.5 in terms of auricyanide, and 2 overall. The intermediates detected in this work may help to synthesize more effective and less toxic gold drugs.

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mossbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

Cellular metallothionein (MT) protects against Cd(2+) exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn(7)-MT-2 with Cd(2+) was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn(4)-alpha domain revealed that only the fastest step involved the Zn(3)-beta domain. Each step of the Zn(4)-alpha domain reaction with Cd(2+) displayed hyperbolic dependence of the observed rate constant on Cd(2+) concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd(2+) to the Zn((4-n)),Cd(n)-thiolate cluster (n = 0-3) of the alpha domain and the first order rate constant for the exchange of Cd(2+) for Zn(2+) in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn((4-n)),Cd(n)-thiolate clusters (n = 0-3) was prepared by titration of the Zn(4)-alpha domain with (113)Cd(2+). The products were analyzed by one-dimensional (113)Cd(2+) NMR spectroscopy to define the distribution of (113)Cd(2+) among the four cluster binding sites. Each of these species was also reacted with Cd(2+). The properties of these reactions were similar to those extracted from the reaction of Cd(2+) with the overall domain. Thus, the kinetic results were linked to (113)Cd(2+) occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd(2+) with the Zn(4)-alpha domain in relation to the alpha domain cluster structure.

Zn(2+) and Cd(2+) ion exchange between transcription factor IIIA (TFIIIA) and apo-metallothionein (MT) were studied using a combination of methods including chromatography, ultrafiltration and UV spectroscopy. Under near stoichiometric conditions, apoMT was able to remove most if not all of the zinc ions from TFIIIA, whether or not the TFIIIA was bound to the 5S DNA internal control region (ICR), and concomitantly inhibit its DNA-binding activity as indicated by an electrophoretic mobility shift assay. The kinetics of the two processes were similar. The rate of the metal exchange reaction increased with the concentrations of both reactants. A second-order rate constant of 30+/-10 M(-1)s(-1) was calculated. Similar observations were made for the reaction between apoMT and Cd-substituted TFIIIA, which proceeded without observable intermediates according to a spectrophotometric analysis. A very slow metal ion exchange occurred between Cd-TFIIIA and Zn-MT, but not between Cd-MT and Zn-TFIIIA. Comparative studies on the reaction of TFIIIA with a small competing ligand, ethylenedinitrilo-tetraacetic acid (EDTA), were also conducted. Although EDTA reacts with free Zn-TFIIIA, under similar conditions it failed to compete for Zn(2+) bound as Zn-TFIIIA-ICR.

Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal–thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal–thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing experiments were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5′-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, respectively. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Con–MT (n=1–7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric analysis of the titration suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titration of free sulfhydryl groups (S) in Con–MT (n=1–7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Con–MT (n=1–3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.

Kinetic studies of the reactions of isolated α- and β-domains of rabbit liver MT-II with DTNB (5,5‘-dithio-2,2‘-dinitrobenzoic acid) and AuSTm (aurothiomalate, Myochrysine) were carried out in 5 mM Tris·HCl/0.1 M KCl at pH 7.4 and 25 °C. These results demonstrate that the kinetics of the DTNB reaction with the β-domain are monophasic, with observed rates similar to those of the slow step of the reaction of the holo-protein, Cd7MT, which confirms previous findings that the α-domain is the site of the kinetically fast step. DTNB concentration dependence studies resulted in the following rate law, rate = {k1s + k2s[DTNB]}[MT], that corresponds to two of the four terms in the holoprotein rate law, those of the slow step. The reaction of aurothiomalate with the β-domain is independent of the AuSTm concentration and described by a rate function with a single rate constant, rate = k1s[MT]. The α-domain reaction with AuSTm, also AuSTm-concentration independent, involves slow and fast phases with rate = {k1s + k1f...

The mechanisms by which metals induce activation of T cells and thus produce allergic and/ or autoimmune reactions are still obscure, and the same is true for the mechanisms that underly T cell cross-reactivity to different heavy metal ions. In the present study, we investigated induction by metals of T cell reactions to cryptic peptides of bovine RNase A. Murine CD4+ T cell hybridomas specific for cryptic RNase peptides presented from Au(III)-treated RNase were used as detection probes. We showed that in vitro treatment of RNase with Pd(II), Pd(IV), Ni(IV), and partially Pt(IV), but not Au(I), Ni(II), or Pt(II), induced presentation of the same cryptic peptides as those presented from Au(III)-treated RNase. That the former heavy metal ions, but not the latter, were able to alter the antigenicity of RNase was reflected by their ability to induce conformational changes of RNase, as detected by circular dichroism spectroscopy. Furthermore, upon immunization against RNase pretreated with these metals, CD4+ T cell hybridomas specific for unidentified cryptic peptides were obtained. In conclusion, "metal-specific" T cell reactions may be directed against cryptic peptides, and metal cross-reactivity in allergic individuals might be due to metal-induced presentation of overlapping, but not identical, panels of cryptic peptides.

Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Copper and zinc analyses were carried out by atomic absorption spectrophotometry. Consistent quantitative thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concentration. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titration of the reducing equivalents of the protein with ferricyanide. The stoichiometry of reaction was consistent with the oxidation of all the sulphydryl groups to disulphides and all of the bound Cu from the cuprous to the cupric oxidation state. According to these methods, total numbers of zinc plus copper ions bound to metallothionein isolated from a number of calf livers centred on about 7, 10–12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zinc metallothionein reacted almost entirely in two slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70–80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.

The ligand substitution reaction of EDTA with Cd7-metallothionein (Cd7-MT) has been reinvestigated. NMR titration of the 111Cd-protein with EDTA showed that the ligand interacts preferentially and cooperatively with Cd2+ ions in the beta-domain cluster. NMR and ultrafiltration kinetic analysis of this reaction using 5.6 mM Cd2+ as 111Cd7-MT and 56 mM EDTA indicated that cadmium-EDTA formed less rapidly than 111Cd peak intensity declined. Spectrophotometric and gel filtration studies of the reaction with 20 microM Cd2+ as Cd7-MT with various concentrations of EDTA revealed biphasic kinetics with much larger rate constants than observed in the NMR experiments. The fraction of total ligand substitution occurring in each kinetic step varied with EDTA concentration. The EDTA concentration dependence of both kinetic steps was consistent with the initial formation of protein.EDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd4-alpha- and Cd3-beta-domains with EDTA. The Cd4 domain reacted with EDTA with biphasic kinetics, in which one Cd2+ was removed rapidly with first-order kinetics, which were zero-order in EDTA. The other three reacted with kinetics like those for the slower step of the holoprotein. Cd3-beta reacted with EDTA like the faster rate process associated with the Cd7-protein. The observed rate constants for the reaction of Cd7-metallothionein with EDTA and the fraction of reaction in the faster rate process were sensitive to protein concentration. These results are consistent with the hypothesis that the monomer-dimer equilibrium of the protein controls its kinetic reactivity with EDTA.

Metallopharmaceuticals have a long history in the development of chemotherapy. The more recent success of cisplatin and six related Pt-based antitumor drugs, and longer histories of chrysotherapy (gold treatments) for arthritis, bismuth antiulcer agents, and silver-, antimony- and arsenic-based antimicrobial agents demonstrate that the periodic table represents a potential wealth of medicinal agents to be explored and developed in the future. This article reviews the use of twelve elements (Ag, As, Au, Bi, Ga, Li, Pt, Ru, Sb, Sn, Ti, V) for a wide variety of diseases and disorders. The current state of research on particular applications varies widely – from promising treatments that have not yet reached the clinic to those that are well established empirically despite uncertain mechanisms of action. The array of antitumor agents licensed or in clinical trials includes compounds of As, Ga, Ru, and Ti, in addition to platinum. There are also exciting efforts to apply known treatments or biological properties to new diseases by taking advantage of extensive databases, for example, developing antitumor agents from organotin complexes that have long been used as fungicides and antifouling agents, and antimicrobial agents from gold complexes. The ability to modulate the properties of metal complexes by choice of the oxidation state (AuI vs AuIII; PtII vs PtIV; VIII, VIV & VV, etc.) and design of the medical carrier ligands (e.g. 1,2-diaminocyclohexane vs two ammine ligands for Pt antitumor agents) allows targeting of particular tissues or cells and balancing of lipophilicity, solubility, and reactivity to balance therapeutic activity against toxicity. Many, if not most, metallopharmaceuticals are prodrugs that undergo redox changes and/or ligand exchange reactions in vivo to generate the active species. Hence, research on metallodrug metabolism and pharmacology is as important as the initial medicinal screening of the agents. Keywords: metallopharmaceuticals; metallodrugs; chrysotherapy; cisplatin; silver antimicrobials; platinum antitumor agents; bismuth antiulcer agents

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Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mössbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

Systematic spectroscopic titrations of bovine serum albumin (62% mercaptalbumin, AlbSH, content) with Et 3 -PAuCl demonstrate evidence for the formation of a cysteine-34μ-thiolato species, AlbS(AuPEt 3 ) 2 +, characterized by a 31 P NMR chemical shift of 35.6 ppm vs trimethyl phosphate (TMP). When iodoacetate-modified albumin (AlbSCH 2 COO-) is similarly titrated, this species does not form, verifying its association with Cys-34.

Lobster metallothionein (MT), which has two three-metal clusters instead of the four-metal and three-metal clusters found in mammalian MTs, was used to determine the origin of the biphasic reactions of MT with DTNB. Cd-rich MTs (CdsCu-MT-2 and Cd 6 -MT-2) were isolated from lobster hepatopancreas after Cd treatment preceded by dexamethasone injection or an applied stress. Each lobster MT-2 preparation reacts biphasically with DTNB [5,5'-dithiobis(2-nitrobenzoic acid)], as do mammalian MTs, and the reaction proceeds about 1 order of magnitude easter than that of mammalian MTs

The ligand-scrambling reaction of R 3 PAuCN (to form (R 3 P) 2 Au + and Au(CN) 2 −) has been studied for R=Ph, Me, Et, i-Pr, and Cy (Cy=cyclo-C 6 H 11 ). The reactions are conveniently studied by 31 P and 13 C NMR spectroscopy. The phenyl complex undergoes rapid ligand exchange and must be cooled to approximately 240 K to observe sharp resonances for the individual equilibrium components. All of the aliphatic complexes are in the slow exchange limit allowing observation by NMR spectroscopy at room temperature

The efflux of gold from red blood cells (RBCs) exposed to 10–100 μM auranofin [the second generation chrysotherapy agent, triethylphosphine-( 2,3,4,6- tetra -O- acetyl -1-β- d -glucopyranosato-S- ) gold(I)] was studied. RBCs in whole blood were allowed to accumulate gold, and then were placed in fresh plasma or buffered saline solution. In plasma, the kinetics of efflux were first order in gold with an apparent rate constant of 0.81 ± 0.18hr −1 . Serum albumin, in plasma or added to a buffered solution, shifted the equilibrium between intra- and extracellular gold in favor of the latter (compared to saline solution). [ 14 C]Glutathione, generated by in situ labeling, also effluxed and associated with the albumin and gold, providing the first direct evidence that the albumin-gold-glutathione complex (AlbSAuSG) may be a circulating metabolite of auranofin formed after both of the original ligands of auranofin are displaced.

A method has been developed to exploit the metallochromic dyes pyridylazoresorcinol and Zincon as monitors of the rate of zinc and cadmium displacement from Zn−, Zn, Cd−, Zn,Cd,Cu− and Cd−thioneins. The antiarthritic drug gold sodium thiomalate is the displacing agent

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.

Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES−. We report here that the permeant, pseudohalide anion [Au(CN)2]− can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100nM [Au(CN)2]− produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]− and activity was not restored by washing [Au(CN)2]− from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]−-blocked channels to excess [CN]−. The results are consistent with the hypothesis that [Au(CN)2]− can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of −1, blocks the anion conduction pathway.

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.

Biomimetic pathways for the oxidation of [Au(CN) 2 ] − , a gold metabolite, and further cyanation of the gold(III) products to form Au(CN) 4 − were investigated using 13 C NMR and UV–Visible spectroscopic methods. Hypochlorite ion, an oxidant released during the oxidative burst of immune cells, was employed. The reaction generates mixed dicyanoaurate(III) complexes, trans -[Au(CN) 2 X 2 ] − , where X − represents equilibrating hydroxide and chloride ligands, and establishes the chemical feasibility of dicyanoaurate oxidation by OCl − to gold(III) species. This oxidation reaction suggests a new procedure for synthesis of H[Au(CN) 2 Cl 2 ]. Reaction of trans- [Au(CN) 2 X 2 ] − (X − =Cl − and Br − ) or [AuCl 4 ] − with HCN in aqueous solution at pH 7.4 leads directly to [Au(CN) 4 ] − without detection of the anticipated [Au(CN) x X 4− x ] − intermediates, which is attributed to the cis - and trans -accelerating effects of the cyanides. The reduction of [Au(CN) 4 ] − by glutathione and other thiols is a complex, pH-dependent process that proceeds through two intermediates and ultimately generates [Au(CN) 2 ] − . These studies provide further insight into the possible mechanisms of an immunogenically generated gold(I)/gold(III) redox cycle in vivo.

The ligand substitution reactions of cadmium-carbonic anhydrase with EDTA and pyridine-2,6-dicarboxylic acid were compared with one in which rabbit apometallothionein was the competing metal-binding agent. This last reaction occurred more rapidly than the other two at a much smaller ratio of competing ligand to Cd-carbonic anhydrase. It was characterized as a second-order reaction, first-order in Cd-carbonic anhydrase and in apometallothionein, having a rate constant of 5.8 +/- 0.1 M-1 s-1 at 25 degrees C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 degrees C, Zn7-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 +/- 0.02 M-1 s-1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49-61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd2+ into the peptide. At pH 7.4 and 25 degrees C, Cd2+ has higher affinity for apometallothionein than for the apo-peptide.

Mammalian and crustacean metallothioneins are complex proteins which form two independent domains with 3 or 4 metal ions (Cd+2 or Zn+2) in the native domains. Synthesis of the individual domains and even smaller fragments allows investigation of specific structure-function questions that can not be addressed easily with site-directed mutagenesis of the entire two-domain structure. Residues 49–61 of mammalian MTs, which include the only sequential four cysteines bound to the same metal ion (CdI), are a putative nucleation site. We have synthesized rabbit liver MT-II49–61 (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH) and examined its binding to Cd+2 and other metal ions. Physiochemical studies (113Cd NMR, UV-vis, CD, ES-MS, LSIMS and chemical reactions) of Cd-Pep49–61 demonstrate that it exhibits properties consistent with a role in Cd-induced MT folding. To examine the possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters, the stable lob-ster βC and βN domains (each having 9 cysteines and binding 3 M+2 ions) and a modified domain βC→N (in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain) have been synthesized and characterized. The synthetic native domains (Cd3-βC and Cd3-βN) show metal binding properties and reactivity similar to the holo-protein, but the modified Cd3-βC→N domain is unusually reactive and in the presence of Chelex aggregates to a Cd5(βC→N)2 dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids.

The first detailed kinetic analysis and mechanistic interpretation of the reactions between serum albumin and the second-generation gold drug Auranofin [Et(3)PAuSATg = (triethylphosphine)(2,3,4,6-tetra-O-acetyl-1-beta-D-glucopyranosato-S-) gold(I)] and its triisopropylphosphine analogue, iPr(3)PAuSATg, in vitro are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer methods. Under the Penefsky chromatography conditions with 0.4-0.6 mM albumin and a wide range of Et(3)PAuSATg concentrations, the reaction is biphasic. The fast phase is apparently first order in albumin with a rate constant [k(1) = 3.4 +/- 0.3 x 10(-)(2) s(-)(1)] that decreases slightly in magnitude and becomes intermediate in order at low gold concentrations, [Et(3)PAuSATg] < [AlbSH]; it accounts for approximately 95% of the Au(I) that binds. A minor, slower step [k(2) = 2.3 +/- 0.3 x 10(-)(3) s(-)(1)), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For iPr(3)PAuSATg, only the first step was observed, k(1) = (1.4 +/- 0.1) x 10(-)(2) s(-)(1), and is first order in albumin and independent of the iPr(3)PAuSATg concentration. (31)P-NMR saturation transfer experiments utilizing iPr(3)PAuSATg, under equilibrium conditions, yielded second-order rate constants for both the forward (1.2 x 10(2) M(-)(1) s(-)(1)) and the reverse (3.9 x 10(1) M(-)(1) s(-)(1)) directions. A multistep mechanism involving a conformationally altered albumin species was developed. Albumin domain IA opens with concomitant Cys-34 rearrangement, allowing facile gold binding and exchange, and then closes. In conjunction with the steady-state approximation, this mechanism accounts for the different reaction orders observed under the two set of conditions. The rate-determining conformational change of albumin governs the reaction as monitored by the Penefsky columns. Rapid second order exchange of R(3)PAuSATg at the exposed Cys-34 residue is observed under the NMR conditions. The mechanism predicts that under physiological conditions where [Et(3)PAuSATg] is 10-25 mM, the reaction will be second order and rapid with a rate constant of 8 +/- 2 x 10(2) M(-)(1) s(-)(1). The Penefsky spun columns revealed a previously unreported and novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species, which was confirmed by Hummel-Dreyer gel chromatographic techniques under equilibrium conditions. This albumin-auranofin complex (AlbSSR-Et(3)PAuSATg) is weakly bound and readily dissociates during conventional gel exclusion chromatography.

A method to isolate the labile 1:1 and 3:1 dicyanoaurate(I) adducts of serum albumin was developed. The 197Au Mossbauer spectra of these complexes establish that intact Au(CN)2- ions bind to the protein and remain two-coordinate. This mode of binding demonstrates that dicyanoaurate(I), a known metabolite of antiarthritic gold drugs, can be transported in vivo without ligand exchange reactions at cysteine 34.

Organization, format, topics, and three representative seasons of UW-Milwaukee's successful science bag outreach program. [The second author's last name is misspelled as Greenier in the table of contents and the printed annual index.]

A demonstration based on the serendipitous discovery that magnets shaped like discs or washers will replicate, in two-dimensions, the repulsion among electron pairs that underlie VSEPR theory.

New complexes of the type R 3 PAuL or (R 3 PAu) 2 (μ-L) where R=ethyl or phenyl and L=6-thioguanine, 2, 6- dithioxanthine, 2, 4-dithiouracil and/or dithioerythritol have been prepared. These complexes have been identified by using elemental analysis, 1 H, 13 C and 31 P NMR spectroscopy. The structures have been proposed based on these spectroscopic studies. Sulfur appears to be the binding site in disubstituted complexes of 2, 4-dithiouracil and 1, 4-dithioerythritol, while the phosphine gold( I ) moieties appear to be S and N bonded in 2, 6-dithioxanthine and 6-thioguanine. The potential use of these complexes as antitumor drugs is discussed.