Tailored-BODIPY/Amphiphilic Cyclodextrin Nanoassemblies with PDT Effectiveness.

Amphiphilic cyclodextrins (aCDs) are an intriguing class of carrier systems which, recently, have been proposed to deliver porphyrinoids and anticancer drugs or combined dose of both for dual therapeutic applications. The design of nanoassemblies based on aCD and photosensitizers (PSs) aims to preserve the photodynamic therapy (PDT) efficacy of PS, reducing the tendency of PS to self-aggregate, without affecting the quantum yield of singlet oxygen (1O2) production, and, not less importantly, minimizing dark toxicity and reducing photosensitization effects. With this idea in mind, in this paper, we focus on nanoassemblies between a non-ionic aCD (SC6OH) and halo-alkyl tailored iodinated boron-dipyrromethenes (BODIPY) dye, a class of molecules which recently have been successfully proposed as a stimulating alternative to porphyrinoids for their high photodynamic efficacy. Nanoassemblies of BODIPY/aCD (BL01I@SC6OH) were prepared in different aqueous media by evaporation of mixed organic film of aCD and BODIPY, hydration, and sonication. The nanostructures were characterized, measuring their hydrodynamic diameter and ξ-potential and also evaluating their time-stability in biological relevant media. Taking advantage of emissive properties of the not-iodinated BODIPY analogue (BL01), nanoassemblies based on aCD and BL01 were investigated as model system to get insight on entanglement of BODIPY in the amphiphile in aqueous dispersion, pointing out that BODIPY is well-entrapped in monomeric form (τ ≅ 6.5 ns) within the colloidal carriers. Also morphology and fluorescence emission properties were elucidated after casting the solution on glass. BL01@SC6OH is easily detectable in cytoplasm of HCT116 cell lines, evidencing the remarkable intracellular penetration of this nanoassembly similar to free BODIPY. On the same cell lines, the photodynamically active assembly BL01I/aCD shows toxicity upon irradiation. Despite the fact that free BL01I is more PDT active than its assembly, aCD can modulate the cell uptake of BODIPY, pointing out the potential of this system for in vivo PDT application.

Effect of zinc cations on the kinetics of supramolecular assembly and the chirality of porphyrin J-aggregates.

Dilute aqueous solutions of anionic meso-4-sulfonatophenyl-porphyrin (TPPS) extract zinc(ii) ions from glass or quartz surfaces at room temperature and efficiently form the corresponding metal complex (ZnTPPS). The partial or complete formation of ZnTPPS has been probed by UV/Vis spectroscopy and both static and time-resolved fluorescence. The source of zinc(ii) ions has been clearly identified through inductively coupled plasma optical emission spectrometry. The presence of increasing amounts of ZnTPPS slows down the rate of TPPS J-aggregate formation in acid solution. This influences the nucleation step and has a profound impact on the onset of chirality in these species. This evidence indicates the important role of this adventitious metal ion in the interpretation of various spectroscopic and kinetic data for the self-assembly of the TPPS porphyrin and provides some insights into controversial findings on their chirality. The use of this metal derivative as the starting compound for in situ formation of monomeric TPPS is suggested.

Viruses: As mediators in "Élan vital" of the "creative" evolution.

The understanding of the processes occurring in Nature has been a continuing concern throughout the history of mankind. Intellectual tools employed towards this goal were specific for each period and have been largely based on the prevailing paradigms that reigned in the past. In this work we present evidence that supports the idea of viruses as key agents mediating natural processes linked to the evolution of organisms, particularly those involved in the flux of genes in the environment. This point of view tinges our perception of Nature and prompts us to include "viral" creativity and plasticity among the tools we employ to analyze those processes far beyond actual paradigms. Experimental data to support this proposal arose during the study of the interaction of the human pathogen, herpes simplex virus (HSV) with sulfated polysaccharides during multiplication of the virus in vitro. Sulfated polysaccharides are the main chemical structures found in carrageenans (CGNs) that are natural products obtained from seaweeds, which proved to be strong inhibitors for the virus. Here we describe the interaction between virus and CGNs as a suitable scenario for the emergence of viral variants which proved to be markedly attenuated for mice. A striking feature of these variants is that they showed changes at the level of conserved regions of the genome such as the DNA polymerase and thymidine kinase genes. In view of these findings, the importance of HSV evolution towards attenuated variants by the action of polysaccharides is also discussed. Attenuation may be considered part of a natural evolutionary process enabling the virus to contribute with valuable information for the host.

The role of counter-anions in the kinetics and chirality of porphyrin J-aggregates.

Kinetics of the growth of TPPS4 porphyrin J-aggregates slow down in the order H2SO4 > HCl > HBr > HNO3 > HClO4, in agreement with the Hofmeister series. The rate constants and the extent of chirality correlate with the structure-making or breaking abilities of the different anions with respect to the hydrogen bonding network of the solvent.

Experimental in vitro infection of rat osteoblasts with measles virus stimulates osteogenic differentiation.

In this work we characterized the infection of a primary culture of rat osteoblastic lineage cells (OBCs) with measles virus (MeV) and the effect of infection on cell differentiation and maturation. Infection of OBCs with MeV led to high titers of infectivity released early after infection. Also, analysis of mRNAs corresponding to osteogenic differentiation markers like alkaline phosphatase (ALP), bone sialo-protein (BSP) and bone morphogenetic proteins (BMPs) 1-4-5-7 in OBCs revealed higher values (2-75-fold of increment) for infected cells in comparison with uninfected controls. Differentiation of OBCs in osteogenic medium prior to infection influenced the level of stimulation induced by MeV. Furthermore, treatment of OBCs with Ly294002, a PI3K/AKT inhibitor, increased viral titers, whereas treatment with 10µM or 100µM ATPγS diminished MeV multiplication. In addition, increments of osteogenic differentiation markers induced by MeV infection were not modified either by treatment with Ly294002 or ATPγS. These data provide the first evidence demonstrating that MeV can infect osteoblasts in vitro leading to osteoblastic differentiation, a key feature in bone pathogenic processes like otosclerosis.

Selection of supramolecular chirality by application of rotational and magnetic forces.

Many essential biological molecules exist only in one of two possible mirror-image structures, either because they possess a chiral unit or through their structure (helices, for example, are intrinsically chiral), but so far the origin of this homochirality has not been unraveled. Here we demonstrate that the handedness of helical supramolecular aggregates formed by achiral molecules can be directed by applying rotational, gravitational and orienting forces during the self-assembly process. In this system, supramolecular chirality is determined by the relative directions of rotation and magnetically tuned effective gravity, but the magnetic orientation of the aggregates is also essential. Applying these external forces only during the nucleation step of the aggregation is sufficient to achieve chiral selection. This result shows that an almost instantaneous chiral perturbation can be transferred and amplified in growing supramolecular self-assemblies, and provides evidence that a falsely chiral influence is able to induce absolute enantioselection.

Virus driven evolution: a probable explanation for "Similia Similibus Curantur" philosophy.

Despite the advances in biomedical knowledge, there remain many challenging and significant unsolved problems among which are included viral pathogenesis and antiviral therapy, as main topics in human health. On this respect, for instance, our knowledge about human immunodeficiency virus and AIDS is still insufficient to deal with problems of immense significance, such as the possible "natural cure" for a chronic infection or the induction of protective immunity against this agent. At the same time, new viral diseases of humans and animals continue to emerge or re-emerge, due to changes in host susceptibility and/or in virus virulence as well as to re-introduction of a virus that had disappeared from a defined population. These changes, at least in part, may appear as a consequence of antiviral therapies and lead to the selection of viral mutants. Moreover, taking into account that viruses have been studied as causative agents of conspicuous diseases a broad spectrum of uncertainty is still present when unapparent persistent infections are considered. Based on Hippocrates (460-357 b.C.E) natural philosophy, "Natura Morborum Medicatrix" which represents the natural healing force, i.e.: "Nature cures diseases"; and "Similia Similibus Curantur" which means "like cure like", we propose the use of natural compounds with chemical structures similar to cellular membrane components. On this approach, sulfated polysaccharides obtained from marine algae may act as a driving force for the emergence of attenuated viruses, enabling this way a practical approach for preventive therapies for herpes simplex virus infection. At the same time, viruses would be creative tools and their contribution by adding new genetic identity to their host are set points of genesis in the growth of the tree of life.

Leptin-based glycopeptide induces weight loss and simultaneously restores fertility in animal models.

AIM: To design, manufacture and test a second generation leptin receptor (ObR) agonist glycopeptide derivative. The major drawback to current experimental therapies involving leptin protein is the appearance of treatment resistance. Our novel peptidomimetic was tested for efficacy and lack of resistance induction in rodent models of obesity and appetite reduction. METHODS: The glycopeptide containing two additional non-proteinogenic amino acids was synthesized by standard solid-phase methods. Normal mice were fed with peanuts until their blood laboratory data and liver histology showed typical signs of obesity but not diabetes. The mice were treated with the peptidomimetic at 0.02, 0.1 or 0.5 mg/kg/day intraperitoneally side-by-side with 0.1 mg/kg/day leptin for 11 days. After termination of the assay, the blood cholesterol and glucose amounts were measured, the liver fat content was visualized and quantified and the remaining mice returned to normal diet and were allowed to mate. In parallel experiments normal rats were treated intranasally with the glycopeptide at 0.1 mg/kg/day for 10 days. RESULTS: The 12-residue glycosylated leptin-based peptidomimetic E1/6-amino-hexanoic acid (Aca) was designed to target a principal leptin/ObR-binding interface. E1/Aca induced leptin effects in ObR-positive cell lines at picomolar concentrations and readily crossed the blood-brain barrier (BBB) following intraperitoneal administration. The peptide initiated typical leptin-dependent signal transduction pathways both in the presence and absence of leptin protein. The peptide also reduced weight gain in mice fed with high-fat peanut diet in a dose-dependent manner. Obese mice receiving peptide E1/Aca at a 0.5 mg/kg/day dose lost weight, corresponding to a net 6.5% total body weight loss, while similar mice treated with leptin protein did not. Upon cessation of the weight loss treatment, several obesity-related pathologies (i.e. abnormal metabolic profile and liver histology as well as infertility) normalized in peptide-, but not leptin-treated, mice. Peptide E1/Aca added intranasally to growing normal rats decelerated normal weight gain corresponding to a net 6.8% net total body weight loss with statistical significance. CONCLUSIONS: No resistance induction to peptide E1/Aca or toxicity in either obese or healthy rodents was observed, indicating the potential for widespread utility of the peptidomimetic in the treatment of leptin-deficiency disorders. We provide additional proof for the hypothesis that difficulties in current leptin therapies reside at the BBB penetration stage, and we document that by either glycosylation or intranasal peptide administration we can overcome this limitation.

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro.

In this paper we demonstrate that infection of cell cultures with the arenavirus Junín (JUNV), agent of the argentine haemorrhagic fever, leads to the activation of PI3K/Akt signalling pathway. Phosphorylation of Akt occurs early during JUNV infection of Vero cells and is blocked by the PI3K inhibitor, Ly294002. Infection of cells with UV-irradiated JUNV redeemed the pattern of stimulation observed for infectious virus indicating that an early stage of multiplication cycle would be enough to trigger activation. Treatment of cells with chlorpromazine abrogated phosphorylation of Akt upon JUNV infection suggesting virus internalization as responsible for activation. Inhibition of Akt phosphorylation by Ly294002 impaired viral protein synthesis and expression leading to a reduced infectious virus yield without blocking the onset of persistent stage of infection. This impairment is linked to a reduced amount of virus bound to cells probably due to a blockage on the recycling of transferrin cell-receptor, employed by the virus to adsorb to the cell surface. Early Akt activation was also observed in BHK-21 and A549 JUNV infected cells suggesting an important role of PI3K/Akt signalling in JUNV multiplication in vitro.

Focus on the aggregation processes of Photosystem II complexes.

In this work the effect of temperature and n-dodecyl-beta-d-maltoside (DM) on PSII complexes organization was investigated. An aggregation process of PSII monomers and dimers was documented at different temperatures and low DM concentration by steady-state fluorescence, absorption, circular dichroism, Rayleigh and dynamic light-scattering experiments. Measures of oxygen evolution enabled us to estimate the change in photoactivity of PSII during the aggregation. This process was found to be extensively reversed by increasing DM concentration as proved by means of steady-state fluorescence and dynamic light-scattering experiments.

Antiviral activity of a carrageenan from Gigartina skottsbergii against intraperitoneal murine herpes simplex virus infection.

The partially cyclized mu/nu-carrageenan 1C3, isolated from the red seaweed Gigartina skottsbergii, was previously shown to be a potent inhibitor of the in vitro replication of Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here the protective effect of 1C3 in a murine model of intraperitoneal ( i. p.) HSV-1 infection was evaluated. OF1 mice were i. p. infected with 5 x 10 (5) PFU of HSV-1 KOS strain, and the effects of different treatments with 1C3 were studied. When 30 mg/kg of body weight of 1C3 was administered by the i. p. route immediately after HSV-1 infection, 87.5 % survival of the animals was achieved (p < 0.005), associated with a delay in the mean day of death in 1C3-treated non-surviving mice. Animal survival was not improved when multiple doses of 1C3 were also given in the period 1 - 48 h post-infection, and no protection was afforded when treatment was started after 24 h of infection. When virus and compound were injected by different routes, i. p. and intravenous ( i. v.), respectively, a still significant protection was achieved (40 % survival, p < 0.05). No toxicity of 1C3 for the animals was recorded. The pharmacokinetic properties were analyzed after injection of 1C3 into the tail vein by monitoring of [ (3)H]-1C3 in plasma and organs and by a bioassay of the anti-HSV-1 activity remaining in serum after non-radioactive 1C3 inoculation. A very rapid disappearance of the compound from the blood was observed since only 5.9 - 0.9 % of the radioactivity of the initially administered [ (3)H]-1C3 appeared in the plasma between 5-300 minutes after administration. A transient peak of radioactivity was detected in the kidney 15 minutes after inoculation. The bioassay confirms the presence of the compound circulating in a biologically active form up to 1 hour after injection.

Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice.

In the present study, the protective effect of 1T1, a lambda-carrageenan extracted from the red seaweed Gigartina skottsbergii was evaluated in a murine model of herpes simplex virus type 2 (HSV-2) genital infection. Six to eight-week-old female BALB/c mice were intravaginally inoculated with a lethal dose of HSV-2 (MS strain) and pre- or post-infection treated with different doses of a 10mg/ml solution of 1T1. A single topical administration of 1T1 shortly before infection of BALB/c mice with HSV-2 protected 9 out of 10 mice from HSV-2-induced lesions and mortality, compared with only 10% survival in control mice. In addition, 1T1 produced a total blockade in virus shedding in the vaginal secretions. When 1T1 pre-treatment was reinforced with a second dose 2h after infection, total protection was observed even when the prophylactic administration had taken place at 60min before infection. The irreversible virucidal action of 1T1 against herpes virus seems to be responsible of its protective effect against virus replication and mortality following vaginal HSV-2 infection.

Resistance to superinfection of Vero cells persistently infected with Junin virus.

Two non-virogenic Vero cell lines persistently infected with the arenavirus Junin (JUNV), named V3 and V7, were characterized with respect to their resistance to superinfection with homologous and antigenically related viruses. Both lines were refractory to JUNV multiplication and partially resistant to other arenaviruses. JUNV was able to adsorb and penetrate persistently infected cells and, although V3 and V7 were able to support synthesis of antigenomic sense viral RNA, protein production of superinfecting virus was totally blocked. This resistance was not mediated by defective interfering particles but rather by a "cell-associated factor" that could be cell to cell transmitted. Prolonged thermal treatment of V3 and V7 abrogated expression of the viral nucleoprotein (N) and turned persistently infected cells permissive to JUNV multiplication. Thermal treated cells cultured at 37 degrees C resumed the expression of N in association to the recovery of resistance. Results strongly suggest a correlation between the presence of the viral nucleoprotein and superinfection exclusion.

Herpes simplex virus type 1 variants arising after selection with an antiviral carrageenan: lack of correlation between drug susceptibility and syn phenotype.

Natural carrageenans of diverse structural types isolated from the red seaweed Gigartina skottsbergii were recently identified as potent and selective inhibitors of herpes simplex virus types 1 and 2 (HSV-1 and -2). The mu/nu-carrageenan 1C3 was tested in vitro for its ability to select resistant variants. After serial passages of HSV-1 strain F in Vero cells in the presence of increasing concentrations of 1C3, viruses emerged that were approximately 2- to 10-fold more resistant to 1C3 inhibition than parental virus; these viruses formed large plaques with an altered syncytial phenotype (1C3-syn). Plaque-purified syncytial variants isolated from passages 13 and 14 have shown variable levels of resistance to 1C3, as well as to the other antiviral carrageenans isolated from G. skottsbergii and to other sulfated polysaccharides with known antiviral activity, such as heparin and dextran sulfate 8000, but all the clones were susceptible to acyclovir. The syn phenotype was not related to polysaccharide resistance. All the 1C3-syn variants formed large syncytia in Vero and CV-1 cells but did not induce fusion in other cell types. The growth efficiency in Vero cells, as well as the virulence for mice by intracerebral or intraperitoneal inoculation of 1C3-syn variants, showed no significant alterations in comparison with the parental virus. The syncytial properties were not affected by cyclosporine or melittin, suggesting that an alteration on glycoprotein gB could be responsible for the syn phenotype induced by 1C3.

Synthesis and expression of viral antigens in Vero cells persistently infected with Junin virus.

Two Vero cell lines persistently infected with XJCl3 and Cl67 strains of Junin virus and named V3 and V7, respectively, have been characterized with respect to the presence and expression of the nucleoprotein (N) and the glycoprotein precursor (GPC) viral genes. After the acute phase of infection, where a marked CPE and high titers of virus were obtained, JV persistently infected cells became morphologically undistinguishable from Vero cells and virus production dropped to undetectable levels. V3 and V7 were resistant to the superinfection with antigenically related viruses. This fact could not be attributed to the presence of defective interfering particles or non-infectious virus in the supernatant. Expression of N was consistently detected in both cultures and accumulation of two degradation products of N was evident during the late passages. Although no G1 (main surface glycoprotein) expression was observed, a marked fusogenic capacity was detected in both cultures indicating at least, the synthesis of a GPC derived fusogenic glycoprotein. Cell lysates from V3 and V7 subjected to RT-PCR, using specific primers for N gene, or to a nested RT-PCR using specific primers for GPC (G1 region) confirmed the presence of both viral genes. No viral DNA sequences could be detected in JV persistently infected cells.

Separation of scattering and absorption contributions in UV/visible spectra of resonant systems.

Resonance light scattering (RLS) is a phenomenon due to an enhancement of the scattered light in close proximity to an absorption band. The effect is easily detectable in the case of strongly absorbing chromophores, which are able to interact, thus leading to large aggregates (Pasternack, R. F.; Collings, P. J. Science 1995, 269, 935). The measurement of absorption spectra from solutions containing such resonant systems can lead to misleading results. In this paper, a simple method is described to obtain absorption spectra of aggregated species with a fairly good correction of the scattering component. The RLS spectrum, obtained using a common spectrofluorimeter, is correlated to the extinction spectrum of the same sample, allowing for an estimation of the scattering contribution to the total extinction spectrum. The method has been successfully applied both on real samples containing aggregated chromophores, such as porphyrins, chlorophyll a and gold colloids, and by simulating extinction spectra.

Fluxional behavior of the dinitrogen ligand 2,9-dimethyl-1,10-phenanthroline in cationic methyl platinum(II) complexes.

The ionic methylplatinum(II) complexes [Pt(Me)(L)(dmphen)]X (dmphen = 2,9-dimethyl-1,10-phenanthroline, L = Me(2)SO, X = PF(6)(-) 1a, BF(4)(-) 1b, CF(3)SO(3)(-) 1c, ClO(4)(-) 1d, B(C(6)H(5))(4)(-) 1e, [B(3,5-(CF(3))(2)C(6)H(3))(4)](-) 1f; L = n-Bu(2)SO, X = CF(3)SO(3)(-) 1g; L = PPh(3), X = PF(6)(-) 2a, BF(4)(-) 2b, CF(3)SO(3)(-) 2c, ClO(4)(-) 2d, B(C(6)H(5))(4)(-) 2e, [B(3,5-(CF(3))(2)C(6)H(3))(4)](-) 2f; X = CF(3)SO(3)(-), L = CyNH(2) 3a, i-PrNH(2) 3b, 2,6-Me(2)py 3c, EtNH(2) 3d, AsPh(3) 3e, dimethylthiourea (Me(2)th) 3f and the uncharged [Pt(Me)(X)(dmphen)] (X = SCN(-) 4a, SeCN(-) 4b) complexes have been synthesized and fully characterized. In chloroform, as well as in acetone or methanol, complexes 1a-1g, 2a-2h (X = Cl(-) g, NO(2)(-) h, formed "in situ"), and 3e show dynamic behavior due to the oscillation of the symmetric chelating ligand dmphen between nonequivalent bidentate modes. All the other compounds feature a static structure in solution. The crystal structure of 2a shows a tetrahedral distortion of the square planar coordination geometry, a loss of planarity of the dmphen ligand, and, most notably, a rotation of the dmphen moiety, around the N1-N2 vector, to form a dihedral angle of 42.64(8) degrees with the mean coordination plane. The hexafluorophosphate ion lies on the side of the phenanthroline ligand. The interionic structures of 2a, 2b, and 2f were investigated in CDCl(3) at low temperature by (1)H-NOESY and (19)F[(1)H]-HOESY NMR spectroscopies. Whereas PF(6)(-) (2a) and BF(4)(-) (2b) show strong contacts with the cation [Pt(Me)(PPh(3))(dmphen)](+), being located preferentially on the side of the phenanthroline ligand, the [B(3,5-(CF(3))(2)C(6)H(3))(4)](-) (2f) ion does not form a tight ion pair. The dynamic process was studied by variable-temperature NMR spectroscopy for 1a-1f and 2a-2h in CDCl(3). The activation energies DeltaG(298) for the sulfoxide complexes 1a-1f are lower than those of the corresponding phosphine complexes 2a-2f by approximately 10 kJ mol(-)(1). The nature of the counteranion exerts a tangible influence on the fluxionality of dmphen in both series of complexes 1 and 2. The sequence of energies observed for 2a-2h encompasses an overall difference of about 16 kJ mol(-)(1), increasing in the order Cl(-) approximately NO(2)(-) << CF(3)SO(3)(-) < ClO(4)(-) < B(C(6)H(5))(4)(-) < BF(4)(-) approximately PF(6)(-) < B(3,5-(CF(3))(2)C(6)H(3))(4)(-). Acetone and methanol have an accelerating effect on the flipping. Concentration-dependent measurements, carried out in CDCl(3) for 2a with n-Bu(4)NPF(6) and the ligands dmphen, n-Bu(2)SO, sec-Bu(2)SO, and sec-Bu(2)S showed that the rate of the fluxional motion is unaffected by added n-Bu(4)NPF(6), whereas in the other cases this increases linearly with increasing ligand concentration, according to a pattern of behavior typical of substitution reactions. Dissociative and associative mechanisms can be envisaged for the observed process of flipping. Dissociation can be prevalent within the ion pair formed by a "noncoordinating" anion with the metallic cationic complex in chloroform. Among the possible associative mechanisms, promoted by polar solvents or by relatively strong nucleophiles, a consecutive displacement mechanism is preferred to intramolecular rearrangements of five-coordinate intermediates.

Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs.

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.

Role of cyclometalation in controlling the rates of ligand substitution at platinum(II) complexes.

The rates of chloride for triphenylphosphine substitution have been measured in dichloromethane for a series of cyclometalated [Pt(N-N-C)Cl] complexes containing a number of terdentate N-N-C anionic ligands, derived from deprotonated alkyl-, phenyl-, and benzyl-6-substituted 2,2'-bipyridines. These rates have been compared with those of the corresponding [Pt(N-N)(C)Cl] (N-N = 2,2'-bipyridine; C = CH3 or C6H5) complexes having the same set of donor atoms but less constrained arrangements of the ligands. The reactions of the cyclometalated compounds occur as a single-stage conversion from the substrate to the ionic pair [Pt(N-N-C)(PPh3)]Cl products. There is no evidence by 1H and 31P(1H) NMR spectroscopy for the formation of other Pt(II) species or of concurrent ring-opening processes. In contrast, in the monoalkyl- or monoaryl-2,2'-bipyridine complexes, chloride substitution is followed by subsequent slower processes which involve the detachment of one arm of the chelated 2,2'-bipyridine, fast cis to trans isomerization of the cis-[Pt(PPh3)2(eta 1-bipy)(R)]+ transient intermediate, and, eventually, the release of free bipy, yielding trans-[Pt(PPh3)2(R)Cl] (R = Me or Ph). All reactions are first-order with respect to complex and phosphine concentration, obeying the simple rate law kobsd = k2[PPh3]. The values of the second-order rate constant k2 do not seem particularly sensitive to the nature of the bonded organic moiety (alkyl or aryl), to its structure (cyclometalated or not), to the size of the ring, or to the number of alkyl substituents on it. The effects are those foreseen on the basis of an associative mode of activation. The only exception to this pattern of behavior is constituted by the complex [Pt(bipy phi-H)Cl] (bipy phi = 6-phenyl-2,2'-bipyridine), which features a significant rate enhancement with respect to the analogue [Pt(bipy)(Ph)Cl] complex. The results of this work, together with those of a previous paper, suggest that there is not a specific role of cyclometalation in controlling the reactivity, unless an in-plane aryl ring becomes part of the pi-acceptor system of the chelated 2,2-bipyridine, behaving as a cyclometalated analogue of the nitrogen terdentate 2,2':6',2"-terpyridine.

To what extent can cyclometalation promote associative or dissociative ligand substitution at platinum(II) complexes? A combined kinetic and theoretical approach.

The ligand exchange rate constants for the reactions [Pt(bph)(SR2)2] + 2*SR2 --> [Pt(bph)(*SR2)2] + 2SR2 (bph = 2,2'-biphenyl dianion; R = Me and Et) and cis-[PtPh2(SMe2)2] + 2*SMe2 --> cis-[PtPh2(*SMe2)2] + 2SMe2 have been determined in CDCl3 as a function of ligand concentration and temperature, by 1H NMR isotopic labeling and magnetization transfer experiments. The rates of exchange show no dependence on ligand concentration and the kinetics are characterized by largely positive entropies of activation. The kinetics of displacement of the thioethers from [Pt(bph)(SR2)2] with the dinitrogen ligands 2,2'-bipyridine and 1,10-phenanthroline (N-N) to yield [Pt(bph)(N-N)], carried out in the presence of sufficient excess of thioether and N-N to ensure pseudo-first-order conditions, follow a nonlinear rate law k(obsd) = a[N-N]/(b[SR2] + [N-N]). The general pattern of behavior indicates that the rate-determining step for substitution is the dissociation of a thioether ligand and the formation of a three-coordinated [Pt(bph)(SR2)] intermediate. The value of the parameter a, which measures the rate of ligand dissociation, is constant and independent of the nature of N-N, and it is in reasonable agreement with the value of the rate of ligand exchange at the same temperature. Theoretical ab initio calculations were performed for both [Pt(bph)(SMe2)2] and cis-[PtPh2(SMe2)2], and for their three-coordinated derivatives upon the loss of one SMe2 ligand. The latter optimize in a T-shaped structure. Calculations were performed in the HF approximation (LANL2DZ basis set) and refined by introducing the correlation terms (Becke3LYP model). The activation enthalpies from the optimized vacuum-phase geometries are 52.3 and 72.2 kJ moll compared to the experimental values in CDCl3 solution, 80 +/- 1 and 93 +/- 1 kJ mol(-1) for [Pt(bph)(SMe2)2] and cis-[PtPh2(SMe2)2], respectively. The electrostatic potential maps of both parent compounds show a remarkable concentration of negative charge over the platinum atom which exerts a repulsion force on an axially incoming nucleophile. On the other hand, the strength of the organic carbanions trans to the leaving group and the stabilization of the T-shaped intermediate in the singlet ground state may also rationalize the preference for the dissociative mechanism. All of the kinetic and theoretical data support the latter hypothesis and indicate, in particular, that dissociation from the complex containing the planar 2,2'-biphenyl dianion is easier than from its analogue with single aryl ligands. Electron back-donation from filled d orbitals of the metal to empty pi* of the in-plane cyclometalated rings is weak or absent and is not operative in promoting an associative mode of activation.