Liu, Yi. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 154-175). / Abstracts also in Chinese. / Title from PDF title page (viewed on 25, October, 2016).

Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.

The isoprenoid biosynthetic pathway is targeted in the treatment of several diseases, including hypercholesteremia and bone related disorders. Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are isoprenoid biosynthetic pathway intermediates that are utilized during post-translational modification of proteins termed farnesylation and geranylgeranylation, respectively, together known as prenylation. The Ras and Rho GTPase family members are examples of proteins that are prenylated. Prenylation is essential for proper membrane localization and function of these small GTPases. Activating mutations or over-expression of these proteins promote oncogenic events, such as increased proliferation and migration. Studies have demonstrated that farnesyl transferase inhibitors and geranylgeranyl transferase inhibitors possess anti-cancer effects in humans and animal models of cancer, respectively. An alternative way to impair protein prenylation is through the depletion of FPP and GGPP. Statins and nitrogenous bisphosphonates (NBPs) deplete FPP and GGPP leading to impaired protein prenylation by inhibiting HMG-CoA Reductase (HMGCR) and FPP synthase (FDPS), respectively. These drugs have been shown to induce apoptosis, inhibit cancer cell migration, and induce cell cycle arrest. The anti-cancer effects of statins and NBPs can be prevented by GGPP addition, suggesting that GGPP depletion may be the mechanism by which these agents interfere with cancer cell survival. We and our collaborators have developed bisphosphonate inhibitors of GGPP synthase (GGDPS), an enzyme that produces GGPP from the substrates FPP and isopentenyl pyrophosphate. The goal of this research was to identify novel GGDPS inhibitors and to assess the effects of specific inhibition of GGDPS on cancer cell survival and function. Two aromatic bisphosphonates were identified as potent inhibitors of GGDPS in enzyme and cellular assays. Apoptosis hallmarks such as PARP cleavage and DNA fragmentation demonstrated that GGDPS inhibition induces apoptosis in K562 chronic myeloid leukemia cells through GGPP depletion and FPP accumulation. Isobologram analysis and enhanced impairment of protein geranylgeranylation showed that GGDPS inhibition is synergistic with the inhibition of HMGCR. Migration assays, transwell assay and large scale digital cell analysis system microscopy, demonstrated that GGDPS inhibition interferes with MDA-MB-231 breast cancer cell migration. Increased LC3-II expression showed that FDPS and GGDPS inhibition induces autophagy in PC3 prostate and MDA-MB-231 breast cancer cells. Inhibition of autophagy enhances the toxic effects of GGDPS inhibition as measured by MTT assay. Propidium iodine staining of DNA and immunostaining of cell cycle proteins such as p27 did not show significant effects of GGDPS inhibition on cell cycle progression. Importantly, exogenous addition of GGPP prevented most of the effects observed with GGDPS inhibition, suggesting specific inhibition of GGDPS by our bisphosphonate inhibitors. The data obtained herein suggest that GGDPS can be targeted to interfere with the progression of cancer cells.

archives@tulane.edu / Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain injury. nNOS and eNOS have been shown to uncouple in pathological conditions to produce superoxide. Oxidative stress is believed to be the underlying cause of several cardiovascular diseases including ischemic stroke. However, the role of eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective of the study was to determine the effect of eNOS and nNOS inhibition on reactive oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain microvascular endothelial cells (BMECs) and rat cortical neurons following oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific inhibition of NOS in endothelial cells reduced ROS levels in BMECs but increased ROS levels in neurons under normoxia. This suggests that a pool of uncoupled NOS exists in the BMECs whereas the dominant functional NOS in neurons produces NO. We observed increased levels of ROS following OGD/R that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced mitochondrial respiration while it improved cell survival rate in both BMECs and neurons following OGD/R. Thus, it is possible that decreased mitochondrial respiration in the immediate aftermath (4 hours) of OGD/R could be protective against reoxygenation injury. Moreover, we identified the expression of nNOS in BMECs from rat, human, and mouse. We observed that the nNOS in the BMECs constitutively produces superoxide under physiological conditions instead of NO. In contrast, nNOS in the neurons produces NO and doesn’t contribute to ROS. We also confirmed the nNOS expression and its function in freshly isolated rat brain microvessels. In addition, we developed a novel method to measure mitochondrial respiration in freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We validated the method by demonstrating impaired mitochondrial respiration in cerebral microvessels isolated from old mice compared to young mice. In summary, the present doctoral research investigated the distinct role of NOS isoforms in BMECs and Neurons leading to the identification of novel functional variant of nNOS in BMECs and brain microvessels. / 1 / RAMARAO SVNL

Antibiotic resistance represents a real threat in the modern world. The problem of resistance is brought about by the fast evolution of bacteria, accelerated by misuse and over-prescription of antibiotics and compounded by the decline in the discovery and development of new classes of antibiotics. Consequently, new targets for antibiotics are in high demand. Flavin-dependent thymidylate synthase (FDTS), which is not present in humans and is responsible for the biosynthesis of a DNA building block in several human pathogens (e.g., M. tuberculosis, B. anthracis, H. pylori), is one such novel target. FDTS catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to produce 2'-deoxythymidine-5'-monophosphate (dTMP), with N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄fol) serving as the carbon source and a nicotinamide cofactor as the electron source. No efficient inhibitors of FDTS are known, despite high-throughput screening attempts to find them. Intermediate and transition-state mimics are likely to bind the enzyme with greater affinity and hence have a better chance at inhibiting FDTS. Therefore, the understanding of the chemical mechanism of FDTS is critical to the informed design of compounds capable of disrupting its function in bacteria. We utilized various techniques, including chemical trapping of reaction intermediates, substrate isotope exchange and stopped-flow, to investigate the FDTS mechanism and determine what sets it apart from other pyrimidine methylases. We found that at least two different intermediates kinetically accumulate in the FDTS-catalyzed reaction. Both of these intermediates are trapped in acid in the form of 5-hydroxymethyl-dUMP, which has never been isolated in other uracil-methylating enzymes. Under basic conditions, however, the earlier intermediate is converted to a species with an unusual flavin-derived adduct, while the later intermediate is converted to dTMP product. Our experiments also suggest that dUMP is activated for the reaction by the reduced flavin - a substrate activation mechanism distinct from the one employed by the classical pyrimidine-methylating enzymes.

Um die Grundlagen für die in klinischen Studien beim Einsatz des standardisierten Kiefernrindenextraktes (Pycnogenol®) gefundenen Effekte auf einer mechanistischen zellulären Ebene aufzuklären, wurde in der hier vorliegenden Arbeit der Einfluss der Komponenten des Extraktes und dessen Metabolit M1 (chemisch benannt δ-(3,4-Dihydroxyphenyl)-γ-valerolacton bzw. 5 (3,4 Dihydroxybenzyl)dihydrofuran 2(3H) on) hinsichtlich der Wirkung auf Stickstoffmonoxid(= NO)-produzierende Systeme untersucht. NO ist an einer Vielzahl von physiologischen und pathophysiologischen Prozessen in lebenden Organismen beteiligt. Im Menschen sind bislang drei NO-Synthasen bekannt: die induzierbare (iNOS), die hinsichtlich der Pathologie vor allem mit entzündlichen Vorgängen assoziiert wird, die endotheliale (eNOS), die bei Gefäß- und Herzkreislauferkrankungen eine Rolle spielt, und die neuronale (nNOS), die mit der Gedächtnisbildung, aber auch mit zytotoxischen Prozessen im Gehirn etwa bei Morbus Alzheimer oder der Parkinson-Krankheit in Verbindung gebracht wird. Der nach peroraler Einnahme des Extraktes im Darm durch metabolisierende Kolonbakterien entstehende und darauf im Plasma erscheinende Metabolit M1, dem bei allen durchgeführten Untersuchungen besonderes Augenmerk zuteil wurde, zeigte eine starke konzentrationsabhängige Inhibierung der NO-Freisetzung der iNOS aus einer durch einen Entzündungsreiz stimulierten murinen Makrophagenzellkultur (IC50= 1,28 µg/mL). Im Vergleich mit Fraktion I des Kiefernrindenextraktes, die vor allem monomere Extraktbestandteile enthält, und Hydrocortison zeigte M1 zusätzlich einen stärkeren Hemmeffekt auf die NO-Freisetzung nach dem Entzündungsreiz. Die Zytotoxizität von M1 im Testsystem war dabei als gering einzustufen. Interessanterweise wurde neben den NO-Radikalfängereigenschaften von M1 auch ein deutlich hemmender konzentrationsabhängiger Effekt auf die iNOS-Proteinexpression gefunden (IC50= 3,78 µg/mL). Da die bislang im Plasma bestimmten M1-Konzentrationen deutlich geringer als die in Zellkulturversuchen wirksamen waren, wurde eine mögliche Anreicherung von M1 in Gegenwart von Serumproteinen in humanen Endothelzellen, primären Monozyten und murinen Makrophagen untersucht. Dabei wurde eine starke Bindung von M1 an die Zellen gezeigt und Hinweise für eine potentiell erleichterte Aufnahme von M1 durch membranständige Transporter unter Einsatz eines Influx-Hemmers (Phloretin) gefunden. Zur Untersuchung der eNOS, die sehr geringe Mengen NO produziert, wurden neue methodische Ansätze entwickelt. In diesem Zusammenhang wurden zuvor unbekannte Fallstricke bei der Verwendung der Fluoreszenzsonde DAF-2 (4,5-Diaminofluorescein) zur NO-Detektion und dem Einsatz unterschiedlicher Detektionssysteme entdeckt. DAF-2 zeigte unter verschiedenen Bedingungen auch ohne extern zugegebene NO-Quelle und besonders beim Einfrieren/Auftauen unerwarteterweise eine Konversion zum korrespondierenden NO-Addukt (DAF-2T). Die eingesetzten monomeren Testsubstanzen ((+)-Catechin, (-)-Epicatechin, Resveratrol, M1) waren über die Testzeiträume deutlich instabil mit dynamischer Eigenfluoreszenz. Sowohl über kurze (≤ 45 min) als auch über längere Zeiträume (14-20 h) wurde entsprechend der Redoxaktivität der eingesetzten Polyphenole eine konzentrationsabhängige scheinbar hemmende Wirkung auf die extrazelluläre NO-Freisetzung der eNOS gezeigt. Die eNOS-Proteinexpression blieb durch die verwendeten Monomere weitestgehend unbeeinflusst. Durch eine hohe Konzentration der Fraktion I des Kiefernrindenextraktes wurde eine Steigerung der eNOS-Proteinkonzentration in Endothelzellen gefunden, wobei zytotoxische Artefakte dabei nicht auszuschließen waren. Als kompetitive endogene Inhibitoren der NOS wurden in vivo in jüngster Zeit methylierte Arginine (ADMA= asymmetrisches, SDMA= symmetrisches Dimethylarginin) entdeckt. In einer randomisierten, kontrollierten, doppelt-blinden klinischen Studie mit einem Cross-over Design am Universitätsklinikum Zürich mit 28 Patienten, die an einer koronaren Herzerkrankung litten, wurden die Plasmaspiegel methylierter Arginine vor und nach 8 wöchiger Einnahme des Kiefernrindenextraktes bestimmt. Es zeigte sich dabei trotz einer Verbesserung der flussinduzierten Gefäßerweiterungskapazität (Flow-mediated dilation) und Verringerung der 15-F2t-Isoprostan-Plasmaspiegel keine signifikante Veränderung der Plasmakonzentrationen von ADMA, SDMA und ET-1 (Endothelin-1) durch die Einnahme des Extraktes. Die nNOS kommt vor allem im Gehirn, aber auch in Muskelzellen vor. Der Einsatz des Metaboliten M1 führte zu keinen deutlichen Effekten auf die konstitutive nNOS-Expression in einem Rhabdomyosarkom(A-673)-Zellkulturmodell. Zur Beantwortung der Frage, wie wahrscheinlich es ist, dass zur möglichen Beeinflussung von (patho)-physiologischen zerebralen Prozessen Polyphenole in vivo das Gehirn erreichen, wurde erstmals ein in silico-Modell zur Vorhersage der Verteilung von ausgewählten polyphenolischen Substanzen zwischen Blut und Gehirn entwickelt. Damit wurde anschließend eine Reihenfolge mit logBB-Werten (logarithmierter Quotient aus Konzentration im Blut und im Gehirngewebe) geordnet nach einer entsprechend dem Modell wahrscheinlich höheren Verteilung ins Gehirn für die untersuchten Substanzen berechnet: Protocatechusäure < Quercetin < Cyanidin < (+) Catechin < (-)-Epicatechin < Phloretin < M1. Insgesamt schienen die untersuchten polyphenolischen Substanzen eher schwach bluthirnschrankengängig zu sein. Der Metabolit M1 zeigte den höchsten logBB-Wert und somit die höchste Wahrscheinlichkeit der untersuchten Polyphenole, die Blut-Hirnschranke in vivo zu überwinden. Im Kontext einer möglichen Anwendung bei chronisch-entzündlichen Erkrankungen wurde zusätzlich ein Extrakt aus der Frucht von Morinda citrifolia L. in einem primären Monozyten-Zellkulturmodell auf seine Eigenschaften hin die Sekretion der Matrix-Metalloprotease-9 (MMP-9) aus Immunzellen nach einem Entzündungsreiz zu beeinflussen untersucht. Dabei zeigten die Extraktverdünnungen deutliche konzentrationsabhängige Hemmeffekte um bis zu ~50 % der maximalen MMP-9 Sekretion, die mit dem Einsatz von Hydrocortison vergleichbar waren. Somit konnten in der vorliegenden Arbeit neue Beiträge zur Wirkungsweise der untersuchten Pflanzenextrakte und vor allem zum Verständnis der möglichen Effekte von Polyphenolen auf physiologisch relevante NO-Systeme sowie zur methodischen Wissenserweiterung der komplexen NO-Analytik geleistet werden. / Many clinical trials with the administration of a standardized maritime pine bark extract (Pycnogenol®) have shown benefical effects. In order to study the basic mechanistic principles of how the extract might work on a cellular level the impact of the extract components and especially of the metabolite M1 (chemically named δ-(3,4-Dihydroxyphenyl)-γ-valerolactone or 5 (3,4 Dihydroxybenzyl)dihydrofuran 2(3H) one) on nitric oxide (NO) producing systems was tested and evaluated. NO is involved in many physiological and pathophysiological processes in living organisms. There are three different isoforms of NO-synthases (NOS) known in humans so far: the inducible NOS (iNOS) is pathologically associated with inflammatory processes, the endothelial NOS (eNOS) is linked to (cardio-)vascular diseases, and the neuronal NOS (nNOS) plays a role in memory formation, but is also put in context to neuronal cytotoxicity within the course of Alzheimer’s and Parkinson’s disease. The metabolite M1 is generated by microbiota in the human colon after per os ingestion of the pine bark extract and thereafter enters the circulation. M1 showed a strong concentration dependent inhibition of NO-release by iNOS from murine macrophages after an inflammatory stimulus (IC50= 1.28 µg/mL). M1 was even more potent at inhibiting NO-release from macrophages than fraction I of the pine bark extract, which predominantly contains monomeric extract compounds, and slightly more potent than hydrocortisone. Cytotoxicity of M1 was found to be modest in the test system. In addition to the NO-radical scavenging activities of M1 there were also profound concentration dependent inhibitory effects on iNOS protein expression (IC50= 3.78 µg/mL). As there is a gap between determined plasma concentrations of M1 after pine bark extract ingestion and required bioactive concentrations in cell culture test systems the binding of M1 to human endothelial cells, human monocytes and murine macrophages in the presence of serum proteins was determined in order to test for cellular M1 accumulation. There was a strong binding of M1 to these cells and a possible facilitated uptake via distinct membrane transporters was shown with using an influx-inhibitor (phloretin). In order to analyze the very small amounts of NO produced by eNOS new methodic approaches were established. Formerly unkown pitfalls and limitations with using the fluorescence dye DAF-2 (4,5-diaminofluorescein) and with applying different detection systems for NO were revealed. DAF-2 was subject to conversion to the NO adduct triazolofluorescein (DAF-2T) even without an externally added NO source under certain assay and storage conditions, especially after freezing and thawing sample solutions. Thus control of spontaneous reagent conversion is advisable. The monomers (+)-catechin, (-)-epicatechin, resveratrol, and M1 were not stable over time and displayed highly dynamic auto-fluorescence. In accordance with their radical scavenging activity the polyphenols seemingly showed a concentration dependent inhibitory effect on NO-release from endothelial cells over short (≤ 45 min) and longer incubation periods (14-20 h). After incubation with the monomers eNOS-protein expression was not markedly affected. When applying very high concentrations of fraction I of the extract an increase of cellular eNOS protein content was observed, but cytotoxic artefacts could not be ruled out though. Recently, competitive endogenous in vivo inhibitors of NOS were identified as methylated arginines (ADMA= asymmetric-, SDMA= symmetric dimethylarginine). In the course of a randomized, double-blind, placebo-controlled and cross-over designed clinical trial, which was conducted at the University of Zurich and enrolled 28 coronary heart disease patients, plasma concentrations of methylated arginines before and after the 8-week intake of pine bark extract were determined. Despite of improvements in flow-mediated dilation parameters and decrease of 15-F2t-Isoprostane plasma concentrations there was no significant alteration of plasma concentrations of ADMA, SDMA and ET-1 (endothelin-1) after the pine bark extrakt intake. The nNOS is found in neuronal cells but is also expressed in other cell types like muscle cells. In a rhabdomyosarcoma (A-673) cell culture model the metabolite M1 had no meaningful effect on nNOS expression. In order to contribute answering the question whether polyphenols are likely to cross the blood brain barrier an in silico model for predicting polyphenol distribution between blood and brain tissue (logBB) was developed. logBB values for selected polyphenols were calculated and resulted in the following rank order with increasing probability of an uptake into the brain: protocatechuic acid< quercetin < cyanidin < (+) catechin < (-)-epicatechin < phloretin < M1. The metabolite M1 showed the highest logBB value of the tested polyphenols and thus had the highest predicted feasibility for crossing the blood brain barrier as described with the newly established computer based model. The extract of the fruits from Morinda citrifolia L. is traditionally utilized for the treatment of inflammatory diseases. Applying a primary human monocyte cell culture model the extract was tested for its ability to inhibit the secretion of matrix-metalloprotease-9 after an inflammatory stimulus. Dilutions of the extract in cell culture media showed profound concentration dependent inhibitory effects with up to a ~50 % decrease of metalloprotease secretion that were comparable to hydrocortisone. Thus, new and substantial contributions were made in this work allowing a better understanding of the mode of action of the tested plant extracts, especially of the effects of polyphenols on physiological relevant nitric oxide systems, and gaining a deeper methodical knowledge in the challenging field of NO-analysis.

NOS1, das für die neuronale Stickstoffmonoxidsynthase (NOS-I) kodierende Gen, konnte bislang durch eine stetig wachsende Zahl an Untersuchungen mit verschiedenen Pathomechanismen bedeutsamer neurologischer und psychiatrischer Erkrankungen in Verbindung gebracht werden. Der Dinukleotid-Polymorphismus im Promotergen von NOS1, für welchen in der Population verschieden lange Allelen existieren, war bislang bezüglich der durch ihn vermittelten Wirkungen kaum untersucht. Um die Relevanz und Funktionalität des Promoterpolymorphismus NOS1 Ex1f-VNTR zu erforschen, wurde in der vorliegenden Arbeit ein Reportergen-Assay durchgeführt, der den Einfluss verschieden langer Allele auf transkriptionaler Ebene verdeutlichen sollte. Hierfür wurden NOS1 Exon 1f-Promoterregionen mit unterschiedlich langen VNTRs in einen Luciferase-Genvektor kloniert und der Einfluss der verschiedenen Allellängen auf die Aktivität des Reportergens ermittelt. Hierbei zeigte sich der Einfluss der Allele dergestalt, dass das Vorhandensein kurzer Allele des NOS1 Ex1f-VNTR in verminderter Aktivität des Reportergens resultierte. Durchgeführte Stimulationsversuche mit Östrogen und Forskolin ergaben hingegen keine signifikante Änderung der Transkriptionsaktivität. Im DNA-Microarray konnten mit kurzen Allelen des NOS1 Ex1f-VNTR assozierte Alterationen im Transkriptom des humanen Brodmann-Areals 46 nachgewiesen werden, was Wechselwirkungen zwischen dem Vorhandensein kurzer Allele des NOS1 Ex1f-VNTR und der Gentranskription psychiatrisch relevanter Gene demonstriert. Die Ergebnisse der vorliegenden Arbeit zeigen, dass der NOS1 Ex1f-VNTR Einfluss auf transkriptionaler Ebene ausübt und mit psychiatrischen Krankheiten assoziiert ist, was ihn weiterhin zu einem wichtigen Forschungsobjekt macht und in die Gruppe klinisch bedeutsamer Polymorphismen einreiht. / NOS1, the coding gene for the neuronal nitric oxide synthase (NOS-I), has been linked to various pathomechanisms of neurologic and psychiatric disorders by a steadily growing body of work. The dinucleotide polymorphism contained in the NOS1 promotergene, existing in a variety of allelic lengths, has so far been rarely investigated in terms of influence. To determine transcriptional functionality and relevance of different lengths of the promoter polymorphism NOS1 Ex1f-VNTR, a reporter gene assay has been carried out in the herein presented study. By cloning NOS1 Ex1f-VNTR promoter regions containing different lenghts oft the VNTR in a Luciferase-gene vector, the influence of different promoter alleles on reporter gene activity has been demonstrated. A reduced reporter gene activity has been shown in the presence of short alleles of the NOS1 Ex1f-VNTR. Accomplished trials of stimulating activity via estrogene and forskoline treatment resulted in no further changes of repoter gene activity. In DNA-Microarray studies an association of short NOS1 Ex1f-VNTR alleles and transcriptome alterations in human Brodmann area 46 has been detected, indicating a correlation of short NOS1 Ex1f-VNTR alleles and transcription of genes involved in psychiatric disorders. These results demonstrating the functionality of the NOS1 Ex1f-VNTR confirm its clinical relevance and the importance of its further investigation.

共著者あり。共著者名:Hizawa Nobuyuki, Yamaguchi Etsuro, Jinushi Eisei, Nishimura Masaharu. / Hokkaido University (北海道大学) / 博士 / 医学

[N,N??-bis-(mercaptoethyl)-1,5-diazacyclooctanato]NiII, Ni-1, is known to undergo metallation reactions with numerous metals. [N,N??-bis-(mercaptoethyl)-1,5-diazacycloheptanato]NiII, (bme-dach)Ni or Ni-1??, differs from Ni-1 by one less carbon in its diazacycle backbone ring producing subtle differences in N2S2Ni geometry. Metallation of Ni-1?? with PdCl2, Pd(NO3)2, and NiBr2 produced three structural forms: Ni2Pd basket, Ni4Pd2 C4-paddlewheel, and Ni3 slant chair. In attempts to provide a rationale for the heterogeneity in the active site of Acetyl coA Synthase, metal ion capture studies of Ni-1 in methanol found a qualitative ranking of metal ion preference: Zn2+ < Ni2+ < Cu+. Formation constants for metal ion capture of Ni-1?? in water were determined for Pb2+, Ni2+, Zn2+, Cu+, and Ag+. A quantitative estimate places copper some 15 orders of magnitude above nickel or zinc in binding affinity. Sulfur dioxide uptake by Ni-1?? is characterized by significant color change, improved adduct solubility, and reversible binding of two equivalents of SO2. These combined properties establish Ni-1?? as a suitable model for gas uptake at nickel thiolate sites and as a possibly useful chemical sensor for this poisonous gas. Comparisons of molecular structures, ν(SO) stretching frequencies, and thermal gravimetric analyses are made to reported adducts including the diazacyclooctane derivative, Ni-1·2SO2. Visual SO2 detection limits of Ni-1 and Ni-1?? are established at 25 ppm and 100 ppm, respectively. Structural studies of products resulting from reaction at the nucleophilic S-sites of (bme-dach)Ni and [(bme-dach)Zn]2 included acetyl chloride and sodium iodoacetate as electrophiles are shown. The acetyl group is a natural electrophile important to the citric acid cycle. Acetylation of (bme-dach)Ni produces a five coordinate, paramagnetic species. Iodoacetate is a cysteine modification agent known to inhibit enzymatic activity. The reaction of (bme-dach)Ni and sodium iodoacetate yields a blue, six coordinate nickel complex in a N2S2O2 donor environment. The bismercaptodiazacycloheptane ligand binds lead(II) forming an unprecedented structural form of N2S2M dimers, in which Pb2+ is largely bound to sulfur in a highly distorted trigonal geometry. Its unusual structure is described in comparison to other derivatives of the bme-daco ligand. The synthesis and structural characterization of square pyramidal (bme-dach)GaCl are also given and compared to the analogous (bme-daco)GaCl.

Electronic and steric parameters for square planar NiN2S2 complexes as bidentate, S-donor ligands have been established. According to the (CO) stretching frequencies and associated computed Cotton-Kraihanzel force constants of (NiN2S2)W(CO)4 adducts, a ranking of donor abilities and a comparison with classical bidentate ligands are as follows: Ni(ema)= > { [NiN2S2]0 } > bipy phen > Ph2PCH2CH2PPh2 > Ph2PCH2PPh2. In addition, we have demonstrated that the NiN2S2 ligands are hemilabile as evidenced from CO addition to (NiN2S2)W(CO)4, which is in equilibrium with the resulting (NiN2S2)W(CO)5 species (Keq = 2.8 M-1, G = -1.4 kJ/mole at 50C). Complete NiN2S2 ligand displacement by CO-cleavage of the remaining W-S bond to form W(CO)6 was not observed, indicating that the remaining W-S bond is considerably strengthened upon ring-opening. Several new cluster compounds based on the NiN2S2 ligands bound to CuI, RhI, PdII and W0 are reported. Structural analysis of (NiN2S2)MLn complexes show a unique structural feature defined by the dihedral angle formed by the intersection of NiN2S2/WS2C2 planes; placing the NiN2S2 ligand in closer proximity to one side of the reactive metal center. This unique orientational feature of the NiN2S2 ligands in the series of bimetallic compounds contrasts with classical diphosphine or diimine ligands. The "hinge angle" ranges in value from 136 as in the (Ni-1*)W(CO)4 to 101 in the (Ni-1)Pd(CH3)(Cl) complexes. The rigidity of the SR hinge of the nickeldithiolate ligands suggests that they might be suitable for stereochemical and regioselective substrate addition to catalytically active metals such as RhI and PdII.. The structural as well as functional similarities of the acetyl CoA synthase enzyme (ACS) and a palladium-metal based industrial type catalyst led to the preparation of a [(Ni-1)Pd(CH3)]+ bimetallic complex. This complex facilitates CO and ethylene copolymerization to produce polyketone similar to conventional (diphosphine)Pd(X)2 catalysts. However, the diphosphine ligands produce more efficient catalysts as the electron-rich character of the NiN2S2 ligand favors the resting state of the catalyst, [(Ni-1)Pd(C(O)CH3)(CO)]+, over the reactive form (Ni-1)Pd(C(O)CH3)(2-C2H4)]+. An exploratory investigation with the Ni-Pd heterobimetallic showed that this complex also facilitated the C-S coupling reaction to form a thioester similar to the ACS enzyme.