Z-Selective ruthenium metathesis catalysts: Comparison of nitrate and nitrite X-type ligands

Two new Ru-based metathesis catalysts, 3 and 4, have been synthesized for the purpose of comparing their catalytic properties to those of their cis-selective nitrate analogues, 1 and 2. Although catalysts 3 and 4 exhibited slower initiation rates than 1 and 2, they maintained high cis-selectivity in homodimerization and ring-opening metathesis polymerization reactions. Furthermore, the nitrite catalysts displayed higher cis-selectivity than 2 for ring-opening metathesis polymerizations, and 4 delivered higher yields of polymer

Cellular uptake and anticancer activity of carboxylated gallium corroles

We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC_(50) values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC_(50) values (> 3 > 2 >> 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging

Toxicity of Transition Metal Complex-based Nanophotoswitches in Retina

Purpose: Nanophotoswitches (NPSs) based on transition metal complexes offer a new tool for optical stimulation of neural activity in photoreceptor degenerated retina. We previously reported robust light-elicited neural activity in degenerate retinae exposed to ruthenium bipyridine based NPSs (Rubpy-C17) and its iridium analog (Irbpy-C17). Irbpy-C17 was developed as an alternative to Rubpy-C17 for the biosafety properties of the iridium complexes. Here we present a study of the toxicity of both NPSs in rodent retinae. Methods: Toxicity of Rubpy-C17 was tested in wildtype C57BL/6J mice and Irbpy-C17 in wildtype Long Evans rats. Animals were intravitreally injected with the test molecules (up to 50 µM) and sacrificed at different time points post injection: 3, 7, 14 and 28 days, respectively. Retinae were obtained, fixed and sliced for histological analysis immediately after animal euthanization. H&E staining was performed to examine morphological integrity of retina and TUNEL staining performed to detect apoptosis of retinal cells. For comparison, Ru(bpy)3]Cl2 injection and sham surgery were included for control. Results: H&E staining revealed no detectable sign of morphological or structural changes in the retinae after prolonged exposure to either Rubpy-C17 or Irbpy-C17 versus the control. There was no significant reduction in the thickness of different nuclear and plexiform retinal layers or the density of retinal neurons (p<0.05), nor was there evidence of significant aggregation of immune cells. TUNEL staining showed minimal occurrence of cell apoptosis in the NPS treated retinae, similar to the control (p<0.05). No longitudinal changes in either the morphology or the cell apoptosis was observed with the post injection time. Conclusions: Overall our data did not find ocular toxicity associated with either the ruthenium or the iridium based NPSs within the concentration range tested. The results obtained with both complexes are similar to that obtained with the control molecule [Ru(bpy)3]Cl2, which lacks a membrane-anchoring 17 carbon chain attached to the bipyridine group, indicating that the inclusion of the carbon chain did not enable NPSs entry into the cells, nor did it cause apoptotic response. The present study provides new evidence of biosafety of our NPSs in rodent retinae, further encouraging developing NPS-based molecular retinal prosthesis to potentially restore high-acuity prosthetic vision in the blind

Toxicity of Transition Metal Complex-based Nanophotoswitches in Retina

Purpose: Nanophotoswitches (NPSs) based on transition metal complexes offer a new tool for optical stimulation of neural activity in photoreceptor degenerated retina. We previously reported robust light-elicited neural activity in degenerate retinae exposed to ruthenium bipyridine based NPSs (Rubpy-C17) and its iridium analog (Irbpy-C17). Irbpy-C17 was developed as an alternative to Rubpy-C17 for the biosafety properties of the iridium complexes. Here we present a study of the toxicity of both NPSs in rodent retinae. Methods: Toxicity of Rubpy-C17 was tested in wildtype C57BL/6J mice and Irbpy-C17 in wildtype Long Evans rats. Animals were intravitreally injected with the test molecules (up to 50 µM) and sacrificed at different time points post injection: 3, 7, 14 and 28 days, respectively. Retinae were obtained, fixed and sliced for histological analysis immediately after animal euthanization. H&E staining was performed to examine morphological integrity of retina and TUNEL staining performed to detect apoptosis of retinal cells. For comparison, Ru(bpy)3]Cl2 injection and sham surgery were included for control. Results: H&E staining revealed no detectable sign of morphological or structural changes in the retinae after prolonged exposure to either Rubpy-C17 or Irbpy-C17 versus the control. There was no significant reduction in the thickness of different nuclear and plexiform retinal layers or the density of retinal neurons (p<0.05), nor was there evidence of significant aggregation of immune cells. TUNEL staining showed minimal occurrence of cell apoptosis in the NPS treated retinae, similar to the control (p<0.05). No longitudinal changes in either the morphology or the cell apoptosis was observed with the post injection time. Conclusions: Overall our data did not find ocular toxicity associated with either the ruthenium or the iridium based NPSs within the concentration range tested. The results obtained with both complexes are similar to that obtained with the control molecule [Ru(bpy)3]Cl2, which lacks a membrane-anchoring 17 carbon chain attached to the bipyridine group, indicating that the inclusion of the carbon chain did not enable NPSs entry into the cells, nor did it cause apoptotic response. The present study provides new evidence of biosafety of our NPSs in rodent retinae, further encouraging developing NPS-based molecular retinal prosthesis to potentially restore high-acuity prosthetic vision in the blind

Activity of iridium pyridine-based nanophotoswitches in retina

Purpose : Nanophotoswitches (NPSs) offer a new tool for optical stimulation of neuronal activity, in vitro and potentially in vivo. Our group previously reported a ruthenium bipyridine-based NPS (Rubpy-C17) that, after injection into the eyes of photoreceptor degenerated blind rats, elicited electrical activity in the contralateral superior colliculus upon light exposure. Compared with the ruthenium complexes, the family of iridium complexes has been more widely used in clinics, owing to its biosafety profile. We have thus synthesized and tested Irbpy-C17, an analog of Rubpy-C17 with the ruthenium core replaced by iridium. Methods : Rubpy molecules can be visualized by their luminescence upon visible wavelength illumination. To examine membrane incorporation, fluorescence imaging of HEK cells was obtained after incubation with Irbpy-C17. Activity of Irbpy-C17 was studied by MEA recording from wholemount retina after intravitreal injection. The test molecules were administered into the vitreous of blind RCS rats at the concentration of 200 µM. Animals were kept in dark after injection until the surgical dissection of retina. Acutely isolated retina was mounted on the MEA with the ganglion cell layer facing down to capture the spiking activity in response to light stimuli. Results : Irbpy-C17 exhibited good membrane incorporation similar to that of Rubpy-C17. Interestingly, despite that Irbpy-C17 elicited minimal light response initially, subsequent application of synaptic blocker cocktail that pharmacologically isolated RGCs substantially enhanced the light activation of RGCs (1.8 ± 0.3 fold increase in spike frequency). In comparison with the 3-hour incubation between injection and dissection, prolonged 24-hour incubation led to a more pronounced 2.5 ± 0.5 fold increase in spike frequency. Conclusions : Our data suggest that Irbpy-C17 may act on multiple components of the retinal neural circuitry that could suppress its direct action on RGCs via synaptic transmission. These molecules intravitreally administered remain stable and active in the ocular environment up to at least one day post injection. These data will prompt us to further study the iridium complexes in parallel with the ruthenium counterparts, particularly for the underlying mechanism of their differential behavior. The NPSs obviates the need for gene manipulation or toxic UV illumination, highlighting its potential in generating high-acuity prosthetic vision in the blind

Unfolded States of Heme Proteins

Cytochromes adopt discrete structures when the heme cofactor is present. Since the heme provides crucial contacts in the native protein, this hydrophobic prosthetic group plays an important role in the folding mechanism and introduces interesting features into the folding landscape. Since the iron-porphyrin is sensitive to protein structure and environment, it also acts an optical probe and will be used in photophysical and photochemical studies of three alpha-helical proteins in the electron transport family of cytochromes: both horse heart and Saccharomyces cerevisiae iso-1 cytochrome c (cyt c), Rhodopseudomonas palustris cytochrome c' (cyt c'), and the engineered cytochrome cb₅₆₂(cyt cb₅₆₂) from E. coli cytochrome b₅₆₂. The work in this thesis expands upon previous research in the Gray Group that has shown heterogenous unfolded populations in these three alpha-helical proteins. Triplet state decay kinetics of Zn-substituted cytochrome c (Zn-cyt c) reveal the level of solvent exposure of the heme through an isotope effect and bimolecular quenching of lifetimes. Differences of the GuHCl, urea, and thermally-induced unfolded states between Zn- and Fe-cyt c, cyt c' and cb₅₆₂ were uncovered, revealing a distinct hydrophobic effect. The inability of elevated temperature to disrupt hydrophobic pockets was studied further for DNS(C102)-cyt c through FET kinetics that reveal a significant compact population at high temperature. Finally, initial experiments on macromolecular crowding of cyt c revealed only a slight effect on the equilibrium unfolded states.</p

Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochrome c

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes:  the decay in the folded protein (τ_H_2_O ∼ 10 ms) is only modestly affected by isotopically substituted buffers (k_H_2_O/k_D_2_O = 1.2), whereas a reduced triplet lifetime (∼1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1−0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH_3)_6^(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 × 10^8 M^(-1) s^(-1) in GdmCl, 6.3 × 10^8 M^(-1) s^(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 × 10^7 M^(-1) s^(-1) in GdmCl, 8.6 × 10^6 M^(-1) s^(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions

DNA interactions with cytotoxic platinum-corrole conjugates

One of the great challenges in cancer treatment is the selective targeting of cancer cells over normal cells. The specific nuclear penetration of functionalizable corroles introduces the possibility of targeted, efficient delivery of covalently-tethered chemotherapy drugs. Indeed, previous expts. demonstrate a sulfonated corrole can act as a carrier mol. for chemotherapeutic agents, specifically the DNA-intercalating anthracycline drug doxorubicin, resulting in enhanced drug cytotoxicity. Anticancer drugs due to their notorious lack of specificity for cancerous cells over normal cells and their need to be localized in the nucleus to be effective. Exploiting the selective uptake of the sulfonated corrole into the nucleus of brain metastatic prostate carcinoma by synthesizing a platinum-corrole conjugate could result in a highly specific and effective treatment for this type of metastases. Synthethic routes to bioactive platinum-corrole conjugates and their interactions with DNA will be presented