Pyrene Residues Research Articles

Oligo(2′-O-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-O-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5'-Pyrenylmethylphosphoramidite and 5'-bispyrenylmethylphosphordiamidite derivatives of oligo(2'-O-methylribonucleotides) and their analogues with thymidine attached at their 3'-termini by a 3'-3'-phosphodiester internucleotide bond (inverted thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5'-mono- and 5'-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5'-Pyrenylmethylphosphoramidite derivatives of oligo(2'-O-methylribonucleotides) and their inverted analogues were shown to serve as sensitive probes for the detection of oligonucleotide substitutions in RNA and DNA by the method of thermal denaturation of the formed duplexes detected according to changes in their fluorescence.

The Thermodynamics of 3‘-Terminal Pyrene and Guanosine for the Design of Isoenergetic 2‘-O-Methyl-RNA-LNA Chimeric Oligonucleotide Probes of RNA Structure

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.

Carboxylate anion diminishes chloride transport through a synthetic, self-assembled transmembrane pore.

Six amphiphilic heptapeptides with the structure (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-(Gly)m-O(CH2)6CH3, in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in approximately (Gly)3-Pro-(Xxx)3 approximately was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl(-) over K+ was maintained even when the carboxylate anion was present within it. Complexation of Cl(-) by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.

Inside Cover: Helical Arrangement of Interstrand Stacked Pyrenes in a DNA Framework (Angew. Chem. Int. Ed. 24/2007)

A self-organizing system composed of two oligopyrene strands that leads to the formation of an interstrand helical stack embedded in a double strand of DNA is reported by R. Häner and co-workers in their Communication on page 4464 ff. Fluorescence and CD spectroscopy show that helical organization takes place in a hybrid containing 14 consecutive achiral pyrene building blocks but not within the respective single strands nor in hybrids containing 6 or less pyrene residues.

Spectroscopic properties of pyrene-containing DNA mimics

DNA mimics containing non-nucleosidic pyrene building blocks are described. The modified oligomers form stable hybrids, although a slight reduction in hybrid stability is observed in comparison to the unmodified DNA duplex. The nature of the interaction between the pyrene residues in single and double stranded oligomers is analyzed spectroscopically. Intra- and interstrand stacking interactions of pyrenes are monitored by UV-absorbance as well as fluorescence spectroscopy. Excimer formation is observed in both single and double strands. In general, intrastrand excimers show fluorescence emission at shorter wavelengths (approx. 5–10 nm) than excimers formed by interstrand interactions. The existence of two different forms of excimers (intra- vs. interstrand) is also revealed in temperature dependent UV-absorbance spectra.

RNA as scaffold for pyrene excited complexes

Synthesis and spectral properties of 1-ethynylpyrene base modified RNA are reported. The fluorophore attached to the 2-position of adenosine is directed into the easily accessible minor groove in RNA. Through an intermolecular interaction of the pyrene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence maxima around 450 and 480 nm, respectively. This behaviour allows the kinetic investigation of RNA hybridisation and folding by fluorescence spectroscopy.

13C NMR Analysis of Biologically Produced Pyrene Residues by Mycobacterium sp. KMS in the Presence of Humic Acid

Cultures of the pyrene degrading Mycobacterium sp. KMS were incubated with [4-13C]pyrene or [4,5,9,10-14C]pyrene with and without a soil humic acid standard to characterize the chemical nature of the produced residues and evaluate the potential for bonding reactions with humic acid. Cultures were subjected to a "humic acid/ humin" separation at acidic pH, a duplicate separation followed by solvent extraction of the humic acid/humin fraction, and a high pH separation. 13C NMR analysis was conducted on the resulting solid extracts. Results indicated that the activity associated with solid extracts did not depend on pH and that approximately 10% of the added activity was not removed from the solid humic acid/humin fraction by solvent extraction. 13C NMR analysis supported the conclusion that the majority of pyrene metabolites were incorporated into cellular material. Some evidence wasfound for metabolite reaction with the added humic material, but this did not appear to be a primary fate mechanism.

Phosphoramidites and solid supports based on N-substituted 2,4-dihydroxybutyramides: universal reagents for synthesis of modified oligonucleotides

Abstract A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-α-hydroxy-γ-butyrolactone or (R)-(−)-pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues) into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.

Pyrene-appended α-Cyclodextrin as a Fluorescent pH Probe Responding to a Wide Range

Pyrene-appended alpha-cyclodextrin (3) in which a trimethylenediamine linker connected the pyrene residue to the alpha-cyclodextrin moiety showed pH-dependent fluorescence intensity changes. The fluorescence intensity was almost linearly changed within the pH range of 5 - 10. The unique fluorescence response of 3 to the pH was due not only to the favorable pK(a) values (pK(a1) = 6.4 and pK(a2) = 8.8), but also to the almost equal contributions of the amino groups to the pyrene's fluorescence quenching.

Comparison of stir bar sorptive extraction and membrane-assisted solvent extraction as enrichment techniques for the determination of pesticide and benzo[a]pyrene residues in Brazilian sugarcane juice

The present work compares two modern and environmentally friendly enrichment techniques, stir bar sorptive extraction (SBSE) and membrane-assisted solvent extraction (MASE) for the determination of 18 organic contaminant residues in Brazilian sugarcane juice. Stir bar sorptive extraction and thermal desorption coupled to capillary gas chromatography–mass spectrometry using the selected ion monitoring mode [SBSE–TD–GC–MS(SIM)] and membrane-assisted solvent extraction combined with large volume injection [MASE–LVI–GC–MS(SIM)] methods were assessed taking into account the time of extraction [SBSE (3 h) and MASE (30 min)], the recoveries [SBSE (0.2–55.3%) and MASE (13.6–103.1%)], the repeatability [SBSE (0.3–19.2%) and MASE (2.6–18.4%)] and the limits of detection [SBSE (0.002–0.71 μg L −1) and MASE (0.004–0.56 μg L −1)] of the selected triazine, organochlorine and organophosphorus pesticides as well as benzo[a]pyrene in sugarcane juice. In general, faster analyses and much better analyte recovery results were achieved with MASE, whereas greater sensitivity and repeatability were obtained with SBSE. SBSE and MASE procedures were applied to the analysis of six sugarcane juice samples from the city São Carlos, in the state of São Paulo, Brazil. A comparison of the results of the pesticide and benzo[a]pyrene residues at μg L −1 level obtained by the two methods showed good agreement.

Supramolecular Probe for Bicarbonate Exhibiting Anomalous Pyrene Fluorescence in Aqueous Media

Highly selective HCO3- sensing is realized in a pH 8.6 aqueous solution on the basis of the HCO3--induced anomalous pyrene fluorescence of an association dimer that consists of pyrene-appended gamma-cyclodextrins having a triamine linker. The anomalous fluorescence that is produced by the HCO3--induced twisted conformation of the pyrene residues in the association dimer is insensitive to the presence of other anions.

Intramolecular Energy Transfer in Pyrene–Bodipy Molecular Dyads and Triads

Molecules bearing a 4,4-difluoro-8-(aryl)-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene (bodipy) core and 1-pyrenyl-1-phenyl-4-(1-ethynylpyrene), or 1-phenyl-4-[1-ethynyl-(6-ethynylpyrene)pyrene] units were constructed in a step-by-step procedure based on palladium(0)-promoted cross-coupling reactions with the required preconstructed modules. X-ray structures of single crystals reveal a twisted arrangement of the two chromophores. In one case, an almost perfect orthogonal arrangement is found. These dyes are strongly luminescent in solution and display rich electrochemistry in which all redox processes of the bodipy and pyrene fragments are clearly resolved. The absorption spectra indicate that the bodipy and pyrene chromophores are spectrally isolated, thereby inducing a large "virtual" Stokes shift. The latter is realised by efficient transfer of intramolecular excitation energy by the Förster dipole-dipole mechanism. The rate of energy transfer depends on the structure of the dual-dye system and decreases as the centre-to-centre separation increases. The energy transfer efficiency, however, exceeds 90 % in all cases. The linkage of two pyrene residues by an ethyne group leads to a decrease in the energy-transfer efficiency, with the two polycycles acting as a single chromophore. The directly linked bodipy-pyrene dual dye binds to DNA and operates as an efficient solar concentrator when dispersed in plastic.

Humic Acid Toxicity in Biologically Treated Soil Contaminated with Polycyclic Aromatic Hydrocarbons and Pentachlorophenol

Contaminated soil from a land treatment unit at the Libby Groundwater Superfund Site in Libby, MT, was amended with 14C pyrene and incubated for 396 days to promote biodegradation and the formation of soil-associated bound residues. Humic and fulvic acids were extracted from the treated soil microcosms and analyzed for the presence of pyrene residues. Biologic activity promoted 14C association with the fulvic acid fraction, but humic acid-associated 14C did not increase with biologic activity. The Aboatox flash toxicity assay was used to assess the toxicity of humic and fulvic acid fractions. The fulvic acid gave no toxic response, but the humic acid showed significant toxicity. The observed toxicity was likely associated with pentachlorophenol, a known contaminant of the soil that was removed by solvent extraction of the humic acid and that correlated well with toxicity reduction.

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

dUTP and dCTP derivatives containing a 4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy group were incorporated into the 3′-end of the DNA primer within complexes with the DNA-matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA-binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28–47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross-links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA polymerase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found.

Conformational biosensor for diagnosis of prion diseases

A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-beta-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.

Novel nucleic acid architectures involving locked nucleic acid (LNA) and pyrene residues: Results from an Indo-Danish collaboration

Abstract We report herein our results for locked nucleic acid (LNA)-type oligonucleotides containing pyrene residues. Pyrene has a large hydrophobic and planar surface area and is therefore a potential intercalating unit; furthermore, it is interesting as a fluorescent tag when covalently bound to DNA. Synthesis and hybridization of conformationally locked universal base surrogates are described together with efficient interstrand communication as shown by the formation of pyrene excimer bands for duplexes containing 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomers positioned in a zipper-like manner within a DNA duplex.

Multiple-pyrene residues arrayed along DNA backbone exhibit significant excimer fluorescence

Oligodeoxyribonucleotides (ODNs) carrying multi-pyrene clusters were chemically prepared by introducing a novel nucleoside–pyrene conjugate into ODNs. The multi-pyrene residues arrayed on DNA strands induced significant excimer fluorescence and the intensity was exponentially increased as the number of pyrene residues in clusters increased. The excimer fluorescence of the arrays was stable and was not quenched by duplex formation.

Detection of point mutations using pyrene-labeled DNA probes

Pyrene-labeled oligodeoxyribonucleotide probes were shown to be suitable for the detection of point mutations. Reagents based on homochiral 2,4-dihydroxybutyramides were used to introduce pyrene residues at the 3"- and 5"- ends of oligonucleotide pairs. The oligonucleotide pair forms a tandem complex with a complementary target, giving rise to an excimer signal (λmax 470—490 nm) in the fluorescence spectra when the pyrene residues come into close proximity. The maximum ratio of the intensity of the excimer signal to the monomer signal (λmax 380—400 nm) is attained when (S)-N-(1-pyrenylmethyl)-3,3-dimethyl-2,4-dihydroxybutyramide is used to introduce the pyrene residue. The excimer fluorescence completely disappears with an increase in the distance between the pyrene residues (upon the introduction of an additional nucleotide in the target) or in the presence of a mismatch near the contact site of the probes.

The Role of Hydrophobic Interactions in Amyloidogenesis: Example of Prion-Related Polypeptides

Conversion of the non-infectious, cellular forom of the prion protein (PrPC) to the infectious form (PrPSc) is thought to be driven by an α-helical to β-sheet conformational transition. To reveal the sequence determinants which encourage the transition to (β-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and sleady stale fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant β-strand formation, i.e., before the appearance of any ordered secondary structure delectable by circular dichroism. At moderate concentrations of trifluo-roethanol and/or acetonitrile, the conformational ensemble shifts towards β-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid (β-sheet structures. Encouragement of β-folds requires the deslabilizalion of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms

Helix formation of oligodeoxyribonucleotides containing base-aromatic ring conjugates.

Novel nucleoside analogues with base moieties containing pyrene groups have been synthesized and incorporated into oligodeoxyribonucleotides (ODNs). ODNs containing tandem pyrene residues at the 5'-end gave fluorescence spectra characteristic of eximers that were diminished by duplex formation of the ODNs with complementary strands.