The behavior of 9-aminoacridine as an indicator of transmembrane pH difference in liposomes of natural bacterial phospholipids

The behavior of 9-aminoacridine as an indicator of pH differences artificially set across a membrane has been reexamined in liposomes prepared from bacterial phospholipids extracted from chromatophores ofRhodopseudomonas capsulata grown photoheterotrophically. The dye behaves as an ideal indicator for pH differences lower than about three units; at higher pH's the expected linear dependence of Q/(100-Q) vs. pH is no longer strictly observed. Similarly a linear dependence upon the volume of the liposomes added has been verified. The amine ceases to respond to pH changes when the pH of the external medium exceeds the value of 10, corresponding to the pK_a of 9-aminoacridine. The apparent volume of the inner phase of liposomes, as calculated from fluorescence quenching, but not the slope of dependence of fluorescence on pH, appears to be affected by several factors, including the ionic composition, the osmolarity of the external medium, and the microscopic structure of the liposomes. Millimolar concentrations of earth-alkaline cations diminish the apparent internal volume of liposomes, in agreement with the complexing effect of these ions on phospholipid bilayers. The osmotic response of the apparent inner volume has also been verified; this parameter decreases linearly with the reciprocal of the external osmolarity, as expected from the van't Hoff relation; an osmolarity exceeding 0.3 M is, however, necessary in order to observe this effect.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Fluorescence decay studies of the DNA-3,6-diaminoacridine complexes

The interaction of several 3,6-diaminoacridines with DNAs of various base composition has been studied by steady-state and transient fluorescence measurements. The acridine dyes employed are of the following two classes: class I - proflavine, acriflavine and 10-benzyl proflavine; class II - acridine yellow, 10-methyl acridine yellow and benzoflavine. It is found that the fluorescence decay kinetics follows a single-exponential decay law for free dye and the poly[d(A-T)]-dye complex, while that of the dye bound to DNA obeys a two-exponential decay law. The long lifetime (tau 1) for each complex is almost the same as the lifetime for the poly[d(A-T)]-dye complex, and the amplitude alpha 1 decreases with increasing GC content of DNA. The fluorescence quantum yields (phi F) of dye upon binding to DNA decrease with increasing GC content; the phi F values for class I are nearly zero when bound to poly(dG) X poly(dC), but those for class II are not zero. This is in harmony with the finding that GMP almost completely quenches the fluorescence for class I, whereas a weak fluorescence arises from the GMP-dye complex for class II. The fluorescence spectra of the DNA-dye complexes gradually shift toward longer wavelengths with increasing GC content. In this connection, the fluorescence decay parameters show a dependence on the emission wavelength; alpha 1 decreases with an increase in the emission wavelength. In view of these results, it is proposed that the decay behavior of the DNA-dye complexes has its origin in the heterogeneity of the emitting sites; the long lifetime tau 1 results from the dye bound to AT-AT sites, while the short lifetime tau 2 is attributable to the dye bound in the vicinity of GC pairs. Since GC pairs almost completely quench the fluorescence for class I, partly intercalated or externally bound dye molecules may play an important role in the component tau 2.     

The 'delta pH'-probe 9-aminoacridine: response time, binding behaviour and dimerization at the membrane

The fluorescence quenching of 9-aminoacridine (9-AA) after imposition of a transmembrane pH gradient (inside acidic) in liposomes has been investigated for a number of different lipid systems. The initial fluorescence decrease after a rapid pH jump, induced in the extravesicular medium by a stopped-flow mixing technique, was ascribed to a response of 9-AA to the imposed pH gradient and not to changes in the vesicular system itself. Time constants for this fluorescence quenching are in the range of several hundred milliseconds at 25 degrees C. Fluorescence recovery which should be correlated to the dissipation of the pH gradient occurs in the 100 s time range and is 10-30-times faster than the delta pH decay monitored with the entrapped hydrophilic pH-indicator dye pyranine. The quenching was severely hindered below the lipid phase transition of dipalmitoylphosphatidylglycerol. No delta pH-induced quenching was obtained in lipid vesicles containing only zwitterionic, net uncharged phosphatidylcholine headgroups. For the occurrence of quenching, the presence of negatively charged headgroups, i.e. phosphatidylglycerol or phosphatidylserine, was necessary. The extent of quenching, at a specific pH difference applied, had a cooperative dependency (Hill coefficient approximately 2) on the number of negative headgroups in the membrane and on the concentration of unquenched (unbound) 9-AA molecules. The concentration of quenched 9-AA molecules was furthermore proportional to the number of dimer-excimer complexes of 9-AA which are formed during the quenching process.     

Decreased endo-lysosomal acidification capacity in methylene diphosphonate-resistant mutants of Dictyostelium discoideum

Abstract The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideum amoebae with acridine orange, a fluorescent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 10⁵ and 2.5 × 10⁶ cells ml⁻¹ and independent of acridine orange concentration in the 1.5 to 7.5 μM range. The dye response was sensitive to agents that perturb endo-lysosomal acidification such as NaN₃, nigericin or imidazole. Several mutant cell lines whose growth was resistant to methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lysosomal acidification was altered in these mutants.     

Measurements of transmembrane pH differences of low extents in bacterial chromatophores

In chromatophores from photosynthetic bacteria the interaction of the fluorescent monoamine, 9-amino, 6-chloro, 2-methoxyacridine (ACMA), with the membrane is evaluated and described by an S-shaped adsorption isotherm. This phenomenon is hysteretic, as indicated by the difference between the adsorption and desorption branches of the binding isotherm. Maximal saturation of adsorption is reached at one ACMA per one to four lipid molecules, indicating that the probe binds in its neutral form. Adsorption of the probe on the membrane causes a large quenching of its fluorescence, which is explaind as being due to hypochromic effects following stacking and aggregation in a medium of low dielectric constant. A further quenching of fluorescence is brought about by imposing artificially induced transmembrane pH's. This latter phenomenon titrates in at increasing pH values and approaches saturation when pH is 2. The dependence of pH on the observed quenching of fluorescence is predicted by considering a model based on the equilibrium distribution of the amine between two phases at different pH's, in which adsorption of the probe on the membrane is used to evaluate its free concentration in the inner and outer compartments of the chromatophore vesicle. It is proposed that the equation thus obtained should be used to measure pH from the quenching of ACMA fluorescence.Abbreviations pH transmembrane pH difference between the inner and outer compartments - Q quenching of fluorescence - BChl Bacteriochlorophyll - ACMA 9-amino-6-chloro-2-methoxyacridine - 9AA 9-aminoacridine - Tricine N-tris-(hydroxymethyl)methylglycine - MES 2-morpholinoethanesulfonic acid - FCCP carbonylcyanide-p-trifluoro-methoxy-phenylhydrazone - CCCP carbonylcyanide-m-chloro-phenylhydrazone     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

Na+/H+ exchange is present in basolateral membranes from rabbit small intestine

Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger.     

Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA

Interactions of several acridine dyes with DNA from different species were studied by measuring fluorescence lifetimes in the 2–30-nsec range, using the single-photon counting technique, and by measuring fluorescence quantum yields in the steady state. The results confirm the existence of two principal site classes, one in which the dye fluorescence is quenched by interaction with guanine and another in which fluorescence results from the hydrophobic environment of the A·T base pairs. The emitting sites are found, in some cases, to exhibit fluorescent decay curves which can be resolved into two exponential components corresponding to a short and to a long lifetime. The deviation from one exponential component is particularly clear with rivanol, 9-aminoacridine, and quinacrine, with which one component is two or three times longer than the other. The relative proportion of these two components depends only slightly on the DNA base composition and does not depend on the nature of the acridine derivatives. We postulate that this lifetime heterogeneity corresponds to the two discrete steps in the complex formation elucidated by kinetic studies: the first step corresponds to a semi-intercalated, or “external,” dye with a short fluorescence lifetime and the second step corresponds to a totally intercalated dye with a long lifetime. In this model, we assumed that a transient opening of the site near a semi-intercalated dye induces solvent diffusion which in turn is responsible for its short-lived fluorescence.     

Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect

The antitumor agent amsacrine, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), when bound to double-stranded DNA, particularly poly(deoxyadenylicthymidylic acid), reduced the fluorescence of bound ethidium without physically displacing it from DNA. Fluorescence lifetime measurements showed that the reduction of fluorescence was not due to reduction of the lifetime of the excited state of ethidium. Rather, a proportion of the DNA-bound ethidium changed to a state where the fluorescence was highly quenched. Several other 9-anilinoacridine derivatives, and also 9-hydroxyellipticine, caused quenching of ethidium-DNA fluorescence, whereas 9-aminoacridine, proflavin, and ellipticine had no effect. Resonance energy transfer (F?rster transfer) is not responsible for the effect since there is no spectral overlap between the absorption spectrum of any of the agents and the fluorescence emission spectrum of ethidium. It is suggested that quenching may occur as a result of reversible formation of electron-transfer complexes between the intercalating drug and the excited state of ethidium.     

Measurement of chloroplast internal protons with 9-aminoacridine. Probe binding, dark proton gradient, and salt effects

A defined ratio, gamma, of the total proton uptake to the concentration change of free internal H+ is observed for illuminated envelope-free chloroplasts (Haraux, F. and de Kouchkovsky, Y. (1979) Biochim. Biophys. Acta, 546, 455-471). Proton uptake is measured by the external pH shift, free internal H+ by 9-aminoacridine fluorescence quenching. Extension of this work leads to the following conclusions, which, in the case of 9-aminoacridine behaviour, should apply to any kind of diffusible protonizable delta pH probe: 1. The gamma constancy is preserved when the internal volume (Vi) is modulated by chlorophyll and osmolarity changes: thus, 9-aminoacridine behaves as expected from the delta pH distribution of an amine of high pK; previous doubts on this point are attributed to the lack of control of the external proton uptake. 2. With variable 9-aminoacridine concentration, however, some variation of gamma confirms the existence of slight light-induced probe-membrane interactions. 3. According to the diffuse layer theory, salts decrease the negative potential at the 'plane of closest approach' of the thylakoids, thereby releasing the excess 9-aminoacridine in this diffuse layer, which increases its fluorescence. Although of equal valency, NH4+ is more potent than K+, suggesting competition between amines for specific anionic binding sites. 4. Two categories of membrane modifications are induced by salts: in addition to the above-mentioned electrical effect, mono- and divalent cations at high concentration increase the chloroplast proton binding capacity. La3+ is only able to release the excess dye in the diffuse layer and leaves gamma unchanged. Therefore the probe-membrane interactions should have limited importance for steady-state delta pH measurement. 5. A Donnan-type dark pH difference, which could seriously bias these delta pH estimates, is found experimentally to be less than 2 (no significant gamma change when Vi varies) and even theoretically less than 1 (on the basis of the concentration of the non-diffusible internal protonizable groups). Similarly, the predictable errors of Vi and its possible light-induced variations must have a small effect on delta pH under present experimental conditions.     

The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria

The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.     

Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site.

New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3''-phosphate, the 5''-phosphate, the fourth internucleotidic phosphate or to both the 3''- and 5''-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3''-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.     

Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.     

Lifetime and quenching of tryptophan fluorescence in whiting parvalbumin

The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites.     

Light-induced proton gradients and internal volumes in chromatophores of Rhodopseudomonas sphaeroides

To test the predictions of the chemiosmotic hypothesis, it is essential to have sensitive and accurate measures of the aqueous volume and pH within membrane compartments. One unique feature of the present investigation is the application of electron spin resonance probes to determine internal aqueous volume and pH changes in bacterial chromatophores under virtually identical conditions. Volumes of the chromatophores ranged from 6 to 16 microliter/mg bacteriochlorophyll among different preparations, and were sensitive to the osmolarity of the suspending buffer. pH gradients reached two units in illuminated chromatophores as determined with ESR methods, and increased when KCl and valinomycin were added to the assay. Measurements with the fluorescent dye 9-amino-acridine yielded similar pH gradients, provided that an operational vesicle volume, which corrected for the binding of the dye to the membrane, was used in the calculation. The sensitivity of the ESR method allowed the measurement of pH gradients resulting from only a few light flashes. A plot of pH gradients versus number of flashes was linear up to about 30 flashes, and intercepted the origin. This result is consistent with proton release into the bulk aqueous phase after only a single light flash. This ability to measure small pH gradients offers new opportunities for the study of energy-transducing mechanisms.     

Novel emissive bio-inspired non-proteinogenic coumarin-alanine amino acid: fluorescent probe for polyfunctional systems

Two new bio-inspired non-proteinogenic compounds L1 and L2, containing coumarin and/or acridine chromophores and bearing as spacer an alanine amino acid were successfully synthesized and fully characterized by elemental analysis, (1)H and (13)C NMR, infrared spectroscopy (KBr discs), melting point, ESI-TOF (electrospray ionization-time of flight-mass), UV-vis absorption and emission spectroscopy, fluorescence quantum yields and lifetime measurements. A relative fluorescence quantum yield of 0.02 was determined for both compounds. In L2 the presence of an intramolecular energy transfer from the coumarin to the acridine unit was observed. L1 and L2 are quite sensitive to the basicity of the environment. At alkaline values both compounds show a strong quenching in the fluorescence emission, attributed to the photoinduced electron transfer (PET). However, both deprotonated forms recover the emission with the addition of Zn(2+), Cd(2+) and Al(3+) metal ions. As multifunctional emissive probes, the titration of L1 and L2 with lanthanides (III), Eu(3+) and Tb(3+) was also explored as new visible bio-probes in the absence and in the presence of liposomes. In a liposomal environment a lower energy transfer was observed.     

Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.