Search

Five new bifunctional conjugates of pyropheophorbide a with 17-substituted testosterone, dihydrotestosterone and epitestosterone differing in the length of linker (1–5) and two new complex conjugates 6 and 7 (containing three functional units: pyropheophorbide a, 17α-substituted testosterone, and lipophylic hexadecyl chain) were synthesized. Mutual influence of steroidal and macrocyclic fragments in conjugates 1–7 was established by analysis of 1H NMR spectra and molecular models of conjugates. The uptake and internalization of conjugates 1–5 by prostate carcinoma cells were dependent on the stereochemical configuration of 17-hydroxyl group in steroidal moiety, and the length of linker. Conjugates 1–5 significantly decreased the LNCaP and PC-3 cells growth and proliferation at 96 h incubation; the anti-proliferative activity of epitestosterone derivative comprising short linker 3 was superior. Irradiation of cells labeled with conjugates with light (λ = 660 nm) significantly increased cytotoxicity. Trifunctioal conjugates 6 and 7 easily formed mixed micells with phosphatidyl choline and pluronic F68; these mixed micelles efficiently internalized by human hepatocarcinoma Hep G2 cells, herewith the internalization was dependent on the conjugate structure, rather than on the method of solubilization.

Cell analysis by optochemical sensing represents large and important niche in life and biomedical sciences. We present advanced multi-modal, multi-analyte sensing platform and dedicated materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of the different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of the ratiometric version of the pH sensor, assessment of photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration of sensor performance in Oxygen Consumption Rate (OCR) and Extracellular Acidification (ECA) measurements with relevant cell models. The main outcomes include the elaboration of the structure-function relationships for this biosensor system, development of the self-referenced tandem O2/pH and OCR/ECA sensing system which enables calibration-free operation, demonstration of the sensor operation on common plastic substrates in cell analysis, benchmarking against the existing platforms and real-life experiments with cells. This new sensing platform shows potential for wide practical use.

The features of porphyrins defining their functionality are related to their conformational flexibility. The degree of nonplanarity of metalloporphyrins depends directly on the number of substituents, their size and their location. The introduction of substituents in themesopositions of β-substituted porphyrins increases the steric interaction and leads to distortions of the porphyrin core. Increasing the distortion of the porphyrin core would augment the bathochromic (red) shift of the electronic absorption spectra. A new nonsymmetrical 2,3,7,8,12,13,17,18-octaethyl-5-[(methylimino)methyl]porphyrin complex of palladium(II), [Pd(C38H47N5)], was synthesized and characterized by NMR, mass spectrometry and X-ray analysis. The features of the electronic absorption spectrum of the synthesized complex are explained by the planarity of the porphyrin core and the π-system of the imino group orthogonal to it.

The effective synthesis of Pd(II) monofunctional azomethine derivatives of coproporphyrin isomers I and II is reported. The proposed method is based on the interaction of a “phosphorus complex”, generated in situ by Vilsmeier-Haack reaction, with various amines. The reaction with palladium complex of coproporphyrin II gave Schiff bases as a mixture of two structural isomers in 4:1 ratio. The structure of the obtained compounds was determined using 1H, 13C, 1H-1H NOESY and UV-vis spectroscopy and mass spectrometry data.

The features of porphyrins defining their functionality are related to their conformational flexibility. The degree of nonplanarity of metalloporphyrins depends directly on the number of substituents, their size and their location. The introduction of substituents in the meso positions of β-substituted porphyrins increases the steric interaction and leads to distortions of the porphyrin core. Increasing the distortion of the porphyrin core would augment the bathochromic (red) shift of the electronic absorption spectra. A new nonsymmetrical 2,3,7,8,12,13,17,18-octaethyl-5-[(methylimino)methyl]porphyrin complex of palladium(II), [Pd(C

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

Several phosphorescent platinum(II) and palladium(II) complexes of tetrapyrrole dyes are prepared in an attempt to improve the compatibility with red laser diodes. The nature of meso-substituents in benzoporphyrin macrocycle is found to affect mainly the position of the Soret band and has little influence on the Q-bands. In rigid polymeric matrices these dyes possess more favourable photochemical properties than corresponding meso-tetraphenyltetrabenzoporphyrins. Platinum(II) chlorins show good compatibility with 635 nm laser diode and 632.8 line of He–Ne laser, and have moderate brightness with emission yields ∼2%. Photophysical properties and photostability of these dyes is assessed and compared with the known NIR-emitting oxygen indicators.

In this work the synthesis, optical and luminescence spectroscopic characterization of the new ytterbium complexes with asymmetric porphyrins of hematoporphyrin group, modified by two β-diketone fragments are presented. All ligands and complexes are investigated by the methods of molecular mechanics, elemental analysis, UV-vis, IR, luminescence, mass and 1H NMR spectroscopy. Influence of some factors (such as location of β-diketones substituents, value of charges on ytterbium ion, etc.) on the value of 4f-luminescence is discussed.

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca2+ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.

Vilsmeier formylation of the oporphyrin-I tetraethyl ester Ni-complex (III) followed by reduction of the intermediary imine salt (IV) with NaBH4 gave the o-dimethylaminomethyl-coproporphyrin-I Ni-complex (V). Heating the latter in methyl iodide gave 65% of the ethane- bis-porphyrin (I), which has been characterized by NMR-spectroscopy and a molecular and crystal structure investigation. The crystal structure shows an extended structure of the bisporphyrin with a parallel arrangement of the porphyrin subunits. Crystal data: I - C90H106N8Ni2O16. Triclinic, a = 12.319(3) Å. b = 13.297(3) Å, c = 14.444(3) Å, α = 105.56(2)°, β = 100.82(2)°, γ = 112.98(2)°, V = 1981.0(7) Å3, Cu Kα radiation, λ = 1.54178 Å, space group P1̄, Z = 1, R = 0.07.

Research on cell-penetrating peptides for the intracellular delivery of porphyrin compounds has mainly focused on the use of trans-activator of transcription (TAT)-derived peptides and, to a lesser extent, on proline-rich peptides and phosphorescent metalloporphyrins. In this article, we describe a novel phosphorescent oxygen-sensitive probe for intracellular use which comprises a bactenecin 7 peptide fragment (15-24) conjugated with the uncharged monofunctional derivative of Pt(II) coproporphyrin I (PEPP0). This probe provides efficient loading of various mammalian cells, including PC12, HCT116, SH-SY5Y and HeLa, via cell-type-dependent uptake mechanisms. The conjugate displays a similar distribution in cytoplasm and mitochondria which allows local oxygen levels to be monitored. Respiratory responses of PC12 cells loaded with the conjugate, measured on a time-resolved fluorescent reader, showed significant cell deoxygenation in response to uncoupling by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone and external hypoxia. Treatment with mitochondrial inhibitors led to a decrease in cell deoxygenation. Although the biophysical properties of this conjugate are similar to those of the phosphorescent intracellular oxygen-sensitive probes described previously, it possesses a number of advantages, including ease of synthesis, high loading efficiency and reliability in physiological experiments with cells. This intracellular probe can be employed for the measurement of intracellular O(2) levels in samples containing mammalian cells using the phosphorescence quenching technique. In addition, the responses to metabolic stimuli can be assessed in a wide range of cells, as can the levels of relative cell oxygenation under external hypoxia.

Several new derivatives of the phosphorescent Pt(II)-coproporphyrin (PtCP) were evaluated with respect to the sensing of intracellular oxygen by phosphorescence quenching. Despite the more favorable molecular charge compared to PtCP, self-loading into mammalian cells was rather inefficient for all the dyes, while cell loading by facilitated transport using transfection reagents produced promising results. The PtCP-NH(2) derivative, which gave best loading efficiency and S/N ratio, was investigated in detail including the optimisation of loading conditions, studies of sub-cellular localization, cytotoxicity, oxygen sensitivity and long-term signal stability. Being spectrally similar to the macromolecular MitoXpress™ probe currently used in this application, the PtCP-NH(2) demonstrated higher loading efficiency and phosphorescent signals, suitability for several problematic cell lines and a slightly increased lifetime scale for the physiological range (0-200 μM O(2)). In physiological experiments with different cell types, mitochondrial uncouplers and inhibitors performed on a time-resolved fluorescence plate reader, this probe produced the anticipated profiles of intracellular oxygen concentration and responses to cell stimulation. Therefore, PtCP-NH(2) represents a convenient probe for the experiments and applications in which monitoring of cellular oxygen levels is required.