Bohdan Skalski

Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells

Limitations of available indicators [such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl- are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl- and/or I- sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(β-d-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400-470 and 490-560 nm, molar extinction 11,100 M-1 ⋅ cm-1(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I- by a collisional mechanism (Stern-Volmer constant 60 M-1) and is not affected by other halides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I- (50% quenching at 38 mM I-). LZQ was used to measure forskolin-stimulated I-/Cl-and I-/[Formula: see text]exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.Limitations of available indicators [such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl(-) are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl(-) and/or I(-) sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(beta-D-ribofuranosylamino)-pyrido[2, 1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400-470 and 490-560 nm, molar extinction 11,100 M(-1). cm(-1) (424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I(-) by a collisional mechanism (Stern-Volmer constant 60 M(-1)) and is not affected by other halides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I(-) (50% quenching at 38 mM I(-)). LZQ was used to measure forskolin-stimulated I(-)/Cl(-) and I(-)/NO(-)(3) exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.

Salt- and solvent-dependent conformational transitions of ribo-CGCGCG duplex

Abstract The conformational transition of r(CpG)3 was investigated under different chemical conditions. It was found that NaCl at a high concentration induced a partial transformation of the duplex into another conformation of RNA, whereas MgCl2 and LiCl at concentrations of 2.3 and 5.4 M, respectively, caused the complete transition from A-RNA to the new conformation. 31P-NMR spectra measured in 5.0 M LiCl confirmed the conclusion that A-RNA was transformed into another conformation which at 70°C was apparently melted to single-stranded RNA. An increase in MgCl2 concentration to 0.5 M caused an apparent increase in the stability of the duplex. It was established that apolar alcohols did not influence the duplex conformation but, at 78% ( v v ), they caused the aggregation of the duplex. Trifluoroethanol and urea at 78% and 10 M, respectively, caused the melting of the duplex due to the breakage of the hydrogen bonds within the duplex. It was suggested that r(CpG)3 formed a right-handed helix which under extreme conditions was transformed into another conformation and it was presumed that it might be a left-handed Z-RNA.

Intra- and Intermolecular Electronic Relaxation of the Second Excited Singlet and the Lowest Excited Triplet States of 1,3-Dimethyl-4-thiouracil in Solution¶

Intramolecular processes of deactivation of 1,3‐dimethyl‐4‐thiouracil (DMTU) from the second excited singlet (S2) (π, π*) and the lowest excited triplet (T1) (π, π*) states have been studied using perfluoro‐1,3‐dimethylcyclohexane (PFDMCH) as a solvent. The spectral and photophysical (PP) properties of DMTU in CCl4, hexane and water have also been described. For the first time, the fluorescence from S2 state DMTU has been observed. The picosecond lifetime of DMTU in the S2 state (τ  S 2) in PFDMCH has been proposed to be determined by a very fast intramolecular reversible process of hydrogen abstraction from the ortho methyl group by the thiocarbonyl group. The shortening of τ  S 2 in CCl4 is interpreted to be caused by the intermolecular interactions between DMTU (S2) and the solvent. Results of the phosphorescence decay as a function of DMTU concentration were analyzed using the Stern–Volmer formalism, which enabled determination of the intrinsic lifetime of the T1 state (τ0  T 1) and rate constants of self‐quenching (ksq). The lifetimes, τ0  T 1, of DMTU in PFDMCH and CCl4 are much longer than the values hitherto obtained in more reactive solvents. The PP properties of DMTU both in the S2 and T1 states have been shown to be determined by the thiocarbonyl group.

Photophysical and photochemical properties of 4-(1,2,4-triazol-1-yl)-pyrimidin-2(1H)-ones

Abstract The solvent and pH dependence of the absorption and fluorescence spectra, fluorescence quantum yields, fluorescence lifetimes, low temperature luminescence spectra and quantum yields and photochemical reactivity of a series of 4-(1,2,4-triazol-1-yl)-pyrimidin-2(1H)-ones have been examined and the data obtained were compared with the results reported previously for uracil and thymine nucleosides. Stacking interaction occurs between triazolyl-pyrimidinones and common nucleic acid bases and was detected by UV absorption and fluorescence spectroscopy. The compounds studied undergo [2+2] photocycloaddition. This was determined on the basis of photochemical studies of synthesized dinucleotide analogues.

Photophysical studies of luminarosine: a new, highly fluorescent ribonucleoside with pteridine-like betaine as the aglycone

The solvent and pH dependence of the absorption and fluorescence spectra, fluorescence quantum yields and lifetimes, and fluorescence quenching behaviour of 4-(2,3,5-tri-O-acetyl-β-D-ribofuranosylamino)pyrido[2,1-h]pteridin-11-ium-6-olate, its α-anomer, the parent ribonucleoside 4-(β-D-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (luminarosine) and the aglycone 4-aminopyrido[2,1-h]-pteridin-11-ium-5-olate (luminarine) are reported. Spectroscopic properties of these compounds in solvents of different polarities are characterized by evidence of the occurrence of a highly dipolar, charge-transfer excited state within the heterocyclic betaine system. The pH dependence of the absorption spectra of luminarosine and luminarine infer the occurrence of ground state prototropic equilibria in solution involving protonation of the negatively charged oxygen atom followed by tautomerism to a lactam. The excited state prototropic equilibria, as shown by fluorescence properties, seem to be more complex and suggest the possible occurrence of proton-transfer and/or phototautomerization reactions.

Photophysical and photochemical properties of C-linked ribosides of pyridin-2-one

Abstract Absorption and emission properties as well as photochemical behavior of C-ribosides bearing pyridone as the aglycone, namely 5-(β- d -ribofuranosyl)-pyridin-2-one ( 1 ) and 3-(β- d -ribofuranosyl)-pyridin-2-one ( 2 ) and pyridone-uridine bichromophoric compound ( 6 ) have been investigated. Photocycloisomerization of the pyridone ring leading to diastereomeric Dewar type photoproducts is the major photochemical pathway both in C-nucleosides 1 , 2 and the bichromophoric compound 6 . In the case of the photocycloisomerization of 1 diastereomeric excess of 26% was determined by HPLC.

Photocycloaddition of 5-bromouracil to uracil in a dinucleotide model compound

Abstract The photochemical reactivity of 5-bromouracil towards uracil in a dinucleotide model compound 1 has been examined. The formation of a cyclobutane adduct 2 as a major product (>90%), without debromination of the 5-bromouracil moiety was observed upon irradiation of 1 with near UV light ( λ >300 nm) under both anaerobic and aerobic conditions. Upon heating, 2 undergoes cyclobutane ring cleavage with concomitant hydrolysis to form 5-hydroxyuracil analogue 3 as a major product.

POSTSYNTHETIC TRANSFORMATIONS OF OLIGODEOXYNUCLEOTIDES ORIGINATED AT 6-METHYLTHIO-PURINE SITE

Abstract New route to oligodeoxynucleotides labeled with fluorescent luminarine was explored. Regioselective oxidation of 6-methylthio-purines to 6-methylsulphoxides reactive toward pyridine was achieved. Upon UV irradiation of 6-pyridinium-purines oligonucleotide cleavage instead of phototransformation to luminarine was observed.

The further investigation of physical and photochemical properties of quasimetacyclophane derived from thymine.

The thymine derived quasimetacyclophane exist in two conformers a and b. The absorption spectra of a and b were evaluated and the conformational equilibrium in different solvents /H2O : EtOH/ were examined. The rate constant k−1 for reaction b→a was established as well as Ea.

Ground and excited state prototropic behavior of 1-(purin-6-yl)-3-methylimidazolium chloride

Abstract The ground and excited state acid-base properties of the compound 1-(purin-6-yl)-3-methylimidazolium chloride, 2 , have been studied in aqueous solutions by means of UV absorption and fluorescence (steady state and time resolved) measurements. The ground state exhibits cation-zwitterion and cation-divalent cation equilibria with p K a = 7.25±0.05 and −1.3±0.1, respectively. Within the pH range 5.3–3, the cationic form of 2 undergoes deprotonation in the excited singlet state leading to a highly fluorescent zwitterionic species. However, the equilibrium is not established within the excited singlet state lifetime and it is estimated that only a fraction (≈12%) of the excited molecules undergoes deprotonation. Using the Forster cycle, an excited singlet state p K a * = 1.5 is estimated from the steady state absorption and fluorescence emission spectral data. In the range 1 3 , and 1-(3-methylpurin-6-yl)-3methylimidazolium chloride, 4 .