Tpa Complexes Research Articles

Synthesis and characterization of azolate gold(i) phosphane complexes as thioredoxin reductase inhibiting antitumor agents

Following an increasing interest in the gold drug therapy field, nine new neutral azolate gold(I) phosphane compounds have been synthesized and tested as anticancer agents. The azolate ligands used in this study are pyrazolates and imidazolates substituted with deactivating groups such as trifluoromethyl, nitro or chloride moieties, whereas the phosphane co-ligand is the triphenylphosphane or the more hydrophilic TPA (TPA = 1,3,5-triazaphosphaadamantane). The studied gold(I) complexes are: (3,5-bis-trifluoromethyl-1H-pyrazolate-1-yl)-triphenylphosphane-gold(I) (1), (3,5-dinitro-1H-pyrazolate-1-yl)-triphenylphosphane-gold(I) (2), (4-nitro-1H-pyrazolate-1-yl)-triphenylphosphane-gold(I) (5), (4,5-dichloro-1H-imidazolate-1-yl)-triphenylphosphane-gold(I) (7), with the related TPA complexes (3), (4), (6) and (8) and (1-benzyl-4,5-di-chloro-2H-imidazolate-2-yl)-triphenylphosphane-gold(I) (9). The presence of deactivating groups on the azole rings improves the solubility of these complexes in polar media. Compounds 1-8 contain the N-Au-P environment, whilst compound 9 is the only one to contain a C-Au-P environment. Crystal structures for compounds 1 and 2 have been obtained and discussed. Interestingly, the newly synthesized gold(I) compounds were found to possess a pronounced cytotoxic activity on several human cancer cells, some of which were endowed with cis-platin or multidrug resistance. In particular, among azolate gold(I) complexes, 1 and 2 proved to be the most promising derivatives eliciting an antiproliferative effect up to 70 times higher than cis-platin. Mechanistic experiments indicated that the inhibition of thioredoxin reductase (TrxR) might be involved in the pharmacodynamic behavior of these gold species.

Efficient aerobic oxidation of hydrocarbons promoted by high-spin nonheme Fe(II) complexes without any reductant

Fe(II)–tris(2-pyridylmethyl)amine complexes, Fe(II)–tpa, having different co-existing anions, [Fe(tpa)(MeCN) 2](ClO 4) 2 ( 1), [Fe(tpa)(MeCN) 2](CF 3SO 3) 2 ( 2) and [Fe(tpa)Cl 2] ( 3), were prepared. Effective magnetic moments (evaluated by the Evans method) revealed that while 1– 3 in acetone and 3 in acetonitrile (MeCN) have a high-spin Fe(II) ion at 298 K, the Fe(II) ions of 1 and 2 are in the low-spin state in MeCN. The aerobic oxidation of 1– 3 was monitored by UV–Vis spectral changes in acetone or MeCN under air at 298 K. Only the high-spin Fe(II)–tpa complexes were oxidized with rate constants of k obs = 0.1–1.3 h −1, while 1 and 2 were stable in MeCN. The aerobic oxidation of 1 or 2 in acetone was greatly accelerated in the presence of pure, peroxide-free cyclohexene (1000 equiv.) and yielded a large amount of oxidized products; 2-cyclohexe-1-ol (A) and 2-cyclohexene-1-one (K) (A + K: 23 940% yield based on Fe; A/K = 0.3), and cyclohexene oxide (810%). Besides cyclohexene, aerobic oxidation of norbornene, cyclooctene, ethylbenzene, and cumene proceeded in the presence of 1 in acetone at 348 K without any reductant. Essential factors in the reaction are high-spin Fe(II) ion and labile coordination sites, both of which are required to generate Fe(II)-superoxo species as active species for the H-atom abstraction of hydrocarbons.

Structural and magnetic characterization of a novel series of dinuclear Cu(II) complexes bridging by 2,5-pyrazine dicarboxylate

A new series of dinuclear 2,5-pyrazine dicarboxylato-bridged copper(II) complexes were synthesized and characterized by spectroscopic techniques. The complexes have the general structural formula [Cu 2(L) 2(μ-pyzdc)](ClO 4) 2· nH 2O where L = TPA, n = 2 ( 1); L = pmedien, n = 2 (2); L = aepn, n = 3 ( 3); L = dpt, n = 2 ( 4); L = Medpt, n = 0 ( 5); L = dien, n = 0 ( 6) and L = MeDPA, n = 2 ( 7) with TPA = tris(2-pyridylmethyl)amine, pmdien = N,N,N′,N′′,N′′-pentamethyldiethylenetriamine, aepn = N-(2-aminoethyl)-1,3-diaminopropane, dpt = dipropylene-triamine, Medpt = 3,3′-diamino-N-methyldipropylamine, dien = diethylenetriamine, MeDPA = N,N-di(2-pyridylmethyl)methylamine. In these complexes, the bridging nature of the 2,5-pyrazine dicarboxylato ligand (pyzdc) was confirmed by single-crystal X-ray crystallography. The structure of the TPA complex 1 consists of μ-pyzdc bridging two Cu(II) centers in a bis(monodentate) bonding fashion through a single oxygen atom supplied by each carboxylate group of the bridged pyzdc in a distorted trigonal bipyramidal geometry achieved by the four nitrogen atoms from the TPA ligand. In the complexes 2– 5 derived from tridentate amines, the bridged pyzdc acts as a bis(bidentate) ligand in a distorted square pyramidal geometry achieved by one nitrogen and one carboxylate-oxygen of pyzdc, and by the three N-atoms of the amine coligands. The intradimer Cu ⋯ Cu distances in the complexes 2– 5 are in the range 6.97–7.45 Ǻ and in it is 10.96 Ǻ in 1. The corresponding intermolecular distances are even shorter (5.34–7.99 Ǻ). The susceptibility measurements at variable temperatures over the 5–300 K range reveal weak antiferromagnetic coupling with J values ranging from −0.61 to −4.78 cm −1.

Bis(thiocyanato-κN)[tris(2-pyridylmethyl)amine-κ4N,N′,N′′,N′′′]nickel(II) methanol hemisolvate

The title complex, [Ni(NCS)2(C18H18N4)]·0.5CH3OH, consists of two crystallographically distinct complexes and a methanol solvent mol­ecule. The NiII complexes are pseudo-octa­hedral six-coordinate, with the tris­(2-pyridylmeth­yl)amine (TPA) ligand providing four N atoms and two N-bound thio­cyanates providing the final two N atoms. The distances and angles are typical for NiII–TPA complexes. The compound has unit-cell parameters that are surprisingly similar to the previously reported hydrate.

Human Neuroserpin: Structure and Time-Dependent Inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA) . Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 Å resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS–tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin–cognate protease complexes.

Alkane hydroxylation catalyzed by a series of mononuclear nonheme iron complexes containing 4-nitropyridine ligands

We have prepared a series of mononuclear nonheme iron(II) complexes supported by TPA (TPA = tris(2-pyridylmethyl)amine) and its derivatives that have one to three nitro-groups at 4-position of the pyridine ligand, and evaluated the effect of the nitro-groups on the catalytic oxidation activity of these complexes. The introduction of nitro-group to the TPA ligand proved to decrease the product yield of cyclohexane oxidation with hydrogen peroxide as well as the product ratio of alcohol to ketone. During the catalytic reaction, a significant amount of Fe(II) species exists in cases of nitro-substituted complexes, while the parent Fe–TPA complex was rapidly oxidized to Fe(III) state. These results suggest that the introduction of nitro-groups prevents from the generation of cis-oxo-hydroxo-iron(V) species, but induces homolysis of the Fe O bond of Fe(III)OOH species to initiate radical oxidation of cyclochexane.

Dinuclear fumarato- and terephthalato-bridged copper(II) complexes: Structural characterization and magnetic properties

The dinuclear dicarboxylato-bridged copper(II) complexes [Cu 2(TPA) 2(μ-tp)](ClO 4) 2 · H 2O ( 1), [Cu 2(TPA) 2(μ-fum)](ClO 4) 2 · 2H 2O ( 2) and [Cu 2(pmedien) 2(μ-fum)(H 2O) 2](ClO 4) 2 ( 3) (tp = terephthalate dianion, fum = fumarate dianion, TPA = tris(2-pyridylmethyl)amine and pmedien = N, N, N′, N″, N″-pentamethyldiethylenetriamine) were synthesized and structurally characterized by X-ray crystallography. The structures of the TPA complexes 1 and 2 consist of μ-tp or μ-fum bridging two Cu(II) centers in a bis(monodentate) bonding fashion. The coordination geometry around the Cu(II) ions in these compounds has a distorted trigonal bipyamidal geometry, TBP with four nitrogen atoms from the TPA ligand and a coordinated oxygen atom supplied by the carboxylate group of the bridged dicarboxylato ligand. Complex 3 has a distorted square pyramidal geometry achieved by the three N-atoms of the pmedien, one fum-carboxylate-oxygen and by an oxygen atom from a coordinated water molecule. The intradimer Cu…Cu distances in these complexes are 11.078(3), 8.663(4) and 9.520(3) Å for 1, 2 and 3, respectively. The electronic spectra of the complexes in aqueous solutions are in complete agreement with the assigned X-ray geometry around the Cu(II) centers. Also, analysis of the infrared spectral data for the ν(COO −) stretching frequencies of the tp-carboxalato groups reveals the existence of the bis(mondentate) coordination mode for the bridged dicarboxylato ligands in compounds 1 and 2. The susceptibility measurements at variable temperature over the 2–300 K range are reported. For 1– 3, it has been observed slight antiferromagnetic coupling with J values of −0.8, −3.0 and −2.9 cm −1, respectively.

Generation of a Nonheme Oxoiron(IV) Intermediate and Its Reactivities in Oxidation Reactions

High-valent iron(IV)-oxo species have been invoked as the key reactive intermediates in the catalytic cycles of nonheme iron enzymes involved in dioxygen metabolism. Direct experimental evidence for such intermediates has been obtained in the chemistry of methane monooxygenase and taurine-2-oxoglutarate dioxygenase. To gain insight into this interesting chemistry, we have made much effort in the design and synthesis of functional models for such systems. Recently, it has been reported that synthetic mononuclear oxoiron(IV) complexes were generated using tetradentate N4 ligands such as macrocyclic TMC and tripodal TPA ligands and pentadentate N5 ligands such as N4Py and Bn-tpen. While the TMC oxoiron(IV) intermediate can effect oxygen-atom transfer to PPh3 and the TPA complex is able to epoxidize cyclooctene, the N4Py and Bn-tpen complexes are capable of activating C-H bonds of alkanes. As the TMC ligand provides relatively weak reactivity to oxoiron(IV) species, we have tried to modify cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane) with other substituents on nitrogens of the ligand, in order to understand the effect of ligand environment by cyclam derivatives on the spectroscopic properties and reactivities of oxoiron(IV) species. We now report the synthesis and characterization of an Fe(IV)-oxo complex bearing a tetradentate macrocyclic TBC (TBC = 1,4,8,11-tetrabenzyl-1,4, 8,11-tetraazacyclotetradecane) which has benzyl groups instead of methyl on TMC. This new Fe=O species exhibits an ability to oxidize a variety of organic substrates such as triphenylphosphine, thioanisole, N,N-dimethylaniline, and triphenylmethane. The tetradentate TBC ligand (1) was prepared from cyclam by alkylation with benzyl bromide (Scheme 1). After purification by column chromatography, the TBC ligand was obtained in 60% yield. Reaction of 1 with Fe(OTf)2 afforded white powder of [Fe(TBC)(CH3CN)2](OTf)2 (2). Complex 2 collected by recrystallization was subjected to comprehensive spectroscopic analysis. We then tried to test the generation of oxoiron(IV) species

Characterization of a small molecule PAI-1 inhibitor, ZK4044

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC 50 values of 644±255 and 100±90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, α 1-antitrypsin and α 2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was ∼2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.

Nonheme FeIVO complexes that can oxidize the C-H bonds of cyclohexane at room temperature.

Nonheme oxoiron(IV) complexes of two pentadentate ligands, N4Py (N,N-bis(2-pyridylmethyl)-bis(2-pyridyl)methylamine) and Bn-tpen (N-benzyl-N,N',N'-tris(2-pyridylmethyl)-1,2-diaminoethane), have been generated and found to have spectroscopic properties similar to the closely related tetradentate TPA (tris(2-pyridylmethyl)amine) complex reported earlier. However, unlike the TPA complex, the pentadentate complexes have a considerable lifetime at room temperature. This greater thermal stability has allowed the hydroxylation of alkanes with C-H bonds as strong as 99.3 kcal/mol to be observed at room temperature. Furthermore, a large deuterium KIE value is found in the oxidation of ethylbenzene. These observations lend strong credence to postulated mechanisms of mononuclear nonheme iron enzymes that invoke the intermediacy of oxoiron(IV) species.

Solid-state and solution properties of cationic lanthanide complexes of a new neutral heptadentate N4O3 tripodal ligand.

The synthesis of the potentially heptadentate ligand tris[6-((2-N,N-diethylcarbamoyl)pyridyl)methyl]amine, tpaam, containing three pyridinecarboxamide arms connected to a central nitrogen is described. Lanthanide complexes of this ligand are prepared and characterized. The crystallographic structure of the complexes of three lanthanide ions (La, Nd, Lu) is determined. The lanthanide(III) complexes of tpaam crystallize as monomeric species (in the presence of chloride or iodide counterions) in which the ligand tpaam acts as a N4O3 donor. The crystal structures presented here show that the Ln[bond]O and Ln[bond]N(pyridyl) distances in the complexes of tpaam are similar to those found for the tpaa complexes (H(3)tpaa = alpha,alpha',alpha' '-nitrilotri(6-methyl-2-pyridinecarboxylic acid) despite the difference in charge. A lengthening of the Ln[bond]N(apical) distance is observed in the tpaam complexes compared to the tpa (tris[(2-pyridyl)methyl]amine) complexes which is more marked for larger lanthanides than for smaller ones. The solution structures of the tpaam complexes were analyzed across the 4f series and compared to the solution structures of the lanthanide complexes of the tetradentate ligand tpa. Proton NMR studies are in agreement with the presence of C(3)(v) symmetric solution species for both ligands. For the larger lanthanides, the cation moves away from the apical nitrogen compared to the position occupied in tpa complexes, whereas for the smaller lanthanides, the metal ion is located in a similar position for the two ligands. Quite surprisingly, the formation constant of the Eu(tpaam)Cl(3) complex in D(2)O at 298 K (log beta(110) = 2.34(4)) is very similar to the one reported for Eu(tpa)Cl(3) (log beta(110) = 2.49(4) at 298 K in D(2)O) indicating that the addition of three amide groups to the ligand tpa does not lead to any increase in stability of the lanthanide complexes of tpaam compared to those of tpa.

Electronic structure and reactivity of high-spin iron--alkyl- and--pterinperoxo complexes.

The spectroscopic properties and electronic structure of the four-coordinate high-spin [FeIII(L3)(OOtBu)]+ complex (1; L3 = hydrotris(3-tert-butyl-5-isopropyl-1-pyrazolyl)borate; tBu = tert-butyl) are investigated and compared to the six-coordinated high-spin [Fe(6-Me3TPA)(OHx)(OOtBu)]x+ system (TPA = tris(2-pyridylmethyl)amine, x = 1 or 2) studied earlier [Lehnert, N.; Ho, R. Y. N.; Que, L., Jr.; Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 12802-12816]. Complex 1 is characterized by Raman features at 889 and 830 cm-1 which are assigned to the O-O stretch (mixed with the symmetric C-C stretch) and a band at 625 cm-1 that corresponds to nu(Fe-O). The UV-vis spectrum shows a charge-transfer (CT) transition at 510 nm from the alkylperoxo pi v* (v = vertical to C-O-O plane) to a d orbital of Fe(III). A second CT is identified from MCD at 370 nm that is assigned to a transition from pi h* (h = horizontal to C-O-O plane) to an Fe(III) d orbital. For the TPA complex the pi v* CT is at 560 nm while the pi h* CT is to higher energy than 250 nm. These spectroscopic differences between four- and six-coordinate Fe(III)-OOR complexes are interpreted on the basis of their different ligand fields. In addition, the electronic structure of Fe-OOPtn complexes with the biologically relevant pterinperoxo ligand are investigated. Substitution of the tert-butyl group in 1 by pterin leads to the corresponding Fe(III)-OOPtn species (2), which shows a stronger electron donation from the peroxide to Fe(III) than 1. This is related to the lower ionization potential of pterin. Reduction of 2 by one electron leads to the Fe(II)-OOPtn complex (3), which is relevant as a model for potential intermediates in pterin-dependent hydroxylases. However, in the four-coordinate ligand field of 3, the additional electron is located in a nonbonding d orbital of iron. Hence, the pterinperoxo ligand is not activated for heterolytic cleavage of the O-O bond in this system. This is also evident from the calculated reaction energies that are endothermic by at least 20 kcal/mol.

Elevated soluble urokinase plasminogen activator receptor in plasma from patients with idiopathic myelofibrosis or polycythaemia vera.

Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) system and the family of metalloproteases play a crucial role in the matrix degradation and tissue remodelling processes characteristic of malignant disorders. The receptor for urokinase plasminogen activator (uPAR) serves to localise and intensify the action of uPA and is expressed on the surface of malignant as well as tumour stromal cells including fibroblasts. A soluble form of uPAR (suPAR) cleaved from its glycosyl-phosphatidylinositol anchor is detected in plasma from healthy individuals and increased levels of suPAR have been found in advanced malignancy, suggesting that suPAR may be a marker of extensive tissue remodelling. In an attempt to clarify whether suPAR might be a marker for bone marrow tissue remodelling we measured plasma suPAR levels in a patient cohort comprising 17 with myelofibrosis (MF), 17 with polycythaemia vera (PV), 15 with essential thrombocythaemia (ET), one with a transitional myeloproliferative disorder evolving from PV and 30 controls. Compared with controls suPAR levels were significantly higher in the patients (P < 0.0001) (median 3.35 vs. 2.32 microg L(-1)). Moreover, in subgroup analyses including patients with MF, PV, and ET, respectively, suPAR levels differed significantly with the highest levels found in patients with MF and PV (MF vs. PV vs. ET; P = 0.0003). When comparing suPAR levels of the individual patient subgroups with controls, only suPAR levels of PV and MF patients were significantly increased (P < 0.0001). Sixty-five percent of patients with PV and MF (22/34) had suPAR plasma values that were above the mean +2 standard deviations (SD) of controls. The concentration of suPAR was significantly correlated to plasma lactate dehydrogenase, thrombomodulin, and complex of tPA:PAI-1 in the patients. There was no difference between patients and controls when comparing plasma uPA levels. Increased plasma suPAR levels in patients with chronic myeloproliferative disorders may reflect increased uPAR production in the bone marrow, leading to enhanced bone marrow remodelling.

Preparation, spectroscopic properties, and dynamic behavior of a μ-oxo dirhenium(III) complex [Re2(μ-O)Cl2(tpa)2](PF6)2 (tpa=tris(2-pyridylmethyl)amine)

An oxo-bridged dirhenium(III,III) complex of tris(2-pyridylmethyl)amine (tpa) and its one-electron oxidized (III,IV) species, [Re2(μ-O)Cl2(tpa)2]3+,4+ have been prepared. They are new members to a series of rhenium tpa complexes in various oxidation states. X-ray structural determination of the Re2(III,IV) complex revealed practically linear ReORe bridge (178(1)°) with short ReO distances of 1.85(2) Å indicative of some multiple bonded character. The 1H NMR spectrum disclosed the relatively slow rotation around the ReORe axis in CH3CN solution in the timescale of 1H NMR. The complex undergoes two consecutive reversible one electron oxidations Re2(III,III)/(III,IV) and Re2(III,IV)/(IV,IV) at E1/2=0.23 and 0.90 V vs Ag/AgCl, respectively. Strong visible absorption bands are observed for the Re2(III,III) species at 448 (ε=31 160) and 563 nm (19 550) which are tentatively assigned to MLCT transitions. A unique oxidation product, Re2(μ-O)(O)2Cl2(bpaO2)2 (bpaO2H=1,3-bis(2-pyridyl)-2-aza-propanedione) has also been isolated and its crystal structure was determined. The complex is dirhenium(V) species with linear OReOReO moiety. Ligand tpa has been oxidized to ketone with simultaneous dissociation of one of the 2-pyridylmethyl arms.

Stimulation of Plasminogen Activator and Inhibitor in the Lymphatic Endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at Mr of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of ∼110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.

Characterization of the interaction of plasminogen activator inhibitor type 1 with vitronectin by surface plasmon resonance

In plasma all plasminogen activator inhibitor type 1 (PAI-1) is found in complex with vitronectin (VN). We have investigated the interaction of human PAI-1 with monomeric native human VN by size exclusion chromatography with VN in solution and using the surface plasmon resonance technique (SPR) with immobilised VN. With these techniques PAI-1 is found to bind in a tight one-to-one complex. VN in solution competed with amine coupled VN in binding free active PAI-1 with aKD of 20 nM. A KDof 0.1 nM at 25°C was instead found for PAI-1 binding to immobilised VN. The extremely rapid binding of PAI-1 to immobilised VN had an association rate constant, κonof 20μ M–1s–1. By the immobilisation of VN the affinity for PAI-1 thus increased manifold. The stable substrate mutant PAI-1-Ala335Glu had similar binding characteristics for immobilised VN as active PAI-1. Upon injection of human α-thrombin onto PAI-1 captured on VN the ternary complex was formed and PAI-1 rapidly dissociated from immobilised VN, indicating that VN was not converted into the denatured form by amine coupling. Monoclonal antibodies against PAI-1, belonging to five different classes did all bind to PAI-1 captured on immobilised VN. There is thus no direct overlap of either of these five antibody binding epitopes on PAI-1 with the binding site for VN. PAI-1 was released from immobilised VN by addition of tissue plasminogen activator (tPA) (E.C. 3.4.21.68), with a dissociation rate depending on the tPA concentration. The preformed complex of tPA and PAI-1 did not bind to immobilised VN. An intermediate ternary complex with tPA and active PAI-1 captured on VN is not detected with SPR. On the contrary, a transient ternary complex was observed with tPA and PAI-1-Ala335Glu captured on VN. In conclusion, these results reject that VN is bound to any of the five antibody binding epitopes, and support the proposal that the binding surface for VN on PAI-1 is located near the epitope of tPA in the final complex, including parts of α-helices C, E and β-strand 1A.

Solvatochromism in ligand-field absorption bands and solvent-dependent deuteron nuclear magnetic resonance spectra of [CrF 3(tri(2-pyridyl)amine)]: implication of trigonal splitting for the first spin-allowed d–d band and evaluation of π bonding peculiarities

The correlation of the remarkable solvatochromism and solvent-dependent deuteron NMR spectra of fac-[CrF 3(tpa)] (tpa=tri(2-pyridyl)amine) with the acceptor numbers ( A Ns) for solvents demonstrated the sensitivity of the Crligand bond length or the angular overlap model (AOM) parameters to the embedding solvent, and also suggested a significant trigonal splitting in the first 4T 2← 4A 2 ligand-field band. The inverse correlation of the 2H NMR shifts for 4- and 3-deuterons of the pyridyl in the tpa complex with the solvent electron A N gives direct evidence for the π interaction with the pyridyl groups in the tpa ligand that varies from strong acceptor to weak donor behavior depending upon the solvent.

Transformed lepidopteran insect cells: New sources of recombinant human tissue plasminogen activator

Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 μg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/μg. In serum-containing medium, this line expressed 160 μg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/μg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 426–433, 1999.

Transformed lepidopteran insect cells: New sources of recombinant human tissue plasminogen activator

Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 microg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/microg. In serum-containing medium, this line expressed 160 microg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/microg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.

Syntheses and characterization of a series of carbonylruthenium(I) and (II) complexes containing pyridyl ligands

The reaction of dodecacarbonyltriruthenium (Ru 3(CO) 12) with tris(2-pyridylmethyl)ammonium perchlorate (tpa·3HClO 4) in toluene, in the presence of acetic acid, produced a new ruthenium(II) complex of tpa [Ru(CH 3CO 2)(CO)(tpa)]ClO 4·C 6H 5CH 3 ( 1). A similar reaction of Ru 3(CO) 12 with 2-pyridylcarboxylic acid (pyCO 2H) in toluene yielded a ruthenium(II) complex Ru(pyCO 2) 2(CO) 2 ( 2). A related ruthenium(III) compound, [Ru(pyCO 2) 3]·H 2O ( 3), was obtained from the reaction of RuCl 3· nH 2O and sodium 2-pyridinecarboxylate in a mixture of water and ethanol (1:1 by volume). The reaction of Ru 3(CO) 12, benzoic acid and pyridine (py) in toluene resulted in the formation of a dinuclear ruthenium(I) complex [Ru 2(C 6H 5CO 2) 2(CO) 4(py) 2]·0.5C 6H 5CH 3 ( 4). Compounds 1– 4 have been characterized by IR, NMR and X-ray crystallography.