Phase transitions in phospholipid dispersions studied with an intramolecular excimer forming fluorescent probe, 1,3-bis (beta-naphthyl)propane

We introduce the use of an intramolecular excimer forming, non-conjugated bichromophoric molecule: 1,3-bis (β-naphthyl)propane as a new probe for measuring thermal phase transitions in aqueous dispersions of phospholipids.Intermolecular excimer forming systems, such as pyrene, have been intensely studied as probes for the “microfluidity” of phospholipid dispersions.The probe we used has the added advantage that intramolecular excimer formation follows a pseudomonomolecular mechanism. This makes observations independent of probe concentration and allows for minute concentrations of the probe to be used, lowering the risk of perturbation of the phospholipid phase.Phase transition temperatures determined from 1,3-bis (β-naphthyl) fluorescence are in good agreement with differential scanning calorimetry and light scattering measurements.     

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.     

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.     

Activation energy and entropy for intramolecular excimer formation in a dipyrenylphosphatidylcholine probe in lamellar and hexagonal lipid phases.

Intramolecular excimer formation in pyrene-labeled phosphatidylcholine was used as a tool to determine thermodynamic characteristics of the lamellar to hexagonal phase transitions in a binary lipid system dilinoleoylphosphatidylethanolamine (DLPE)/palmitoyloleoylphosphatidylcholine (POPC). Upon an L alpha/HII phase transition, the activation energy Ea for excimer formation increased from 5.6 +/- 0.2 kcal/mol to 6.3 +/- 0.2 kcal/mol, while the activation entropy delta S decreased from -40.0 +/- 0.8 cal/K.mol to -38.4 +/- 0.8 cal/K.mol. The results are consistent with the idea of molecular splaying of the acyl chains in the hexagonal phase. It is estimated that the molecular area at the terminal carbon of the lipid acyl chains increases by a factor of 2.2 upon the L alpha HII transition in DLPE/POPC.     

Pressure effects on the apparent viscosity of artificial dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine membranes using intramolecular excimer probe

Emission spectroscopy of intramolecular excimer probes allows the determination of ‘equivalent viscosity’ of membranes. While increasing the pressure on artificial membrane suspensions, variations in viscosity — essentially related to an increase in the order parameter in the membranes — are observed. In the case of mixed phospholipids, the effect of pressure is amplified, probably due to the existence of holes on the molecular scale between the two lipidic layers.     

Fluorescence probe properties of intramolecular charge transfer diphenylbutadienes in micelles and vesicles

This paper reports absorption and fluorescence spectral studies of methyl 4-[(1E,3E)-4-phenylbuta-1,3-dienyl]benzoate (1), N,N-dimethyl-N-[4-[(1E,3E)-4-phenylbuta-1,3-dienyl]phenyl]amine (2), methyl 4-[(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl]benzoate (3) and 1-methyl-4-[(1E,3E)-4-[4-methoxyphenyl]buta-1,3-dienyl]benzoate (4) in homogeneous media of 1,4-dioxane and 1,4-dioxane-water binary mixtures, and in microheterogeneous media of cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton-X-100 micelles, and dipalmotoyl phosphatidylcholine (DPPC) vesicles. The binding site of the diene probes in micelles and vesicles has been determined and it has been found that while in micelles dienes occupy the polar interfacial regions, in vesicles the probes are located deep inside the hydrophobic bilayer. The binding of dienes to the vesicles is stronger than their binding to the micelles as indicated by the binding constant values. The fluorescence emission of the probe dienes in micelles is from a conformationally relaxed intramolecular charge transfer excited state. However, in vesicles, since the excited state conformational motions are restricted due to the rigidity of the alkyl chain, the dienes fluoresce from their planar locally excited states.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Pressure and temperature dependence of the intramolecular excimer formation rate.

We have measured the pressure dependence of the intramolecular excimer formation rate, K(p), for di-(1'-pyrenedecanoyl)-phosphatidylcholine (dipy10PC) probes in single-component lipid multilamellar vesicles (MLV) as a function of temperature. Apparent volumes of activation (V(a)) for intramolecular excimer formation are obtained from the slopes of plots of log K(p) versus P. For liquid-crystalline saturated lipid MLV (DMPC and DPPC), these plots are linear and yield a unique V(a) at each temperature, whereas for unsaturated lipids (POPC and DOPC) they are curvilinear and V(a) appears to decrease with pressure. The isothermal pressure induced phase transition is marked by an abrupt drop in the values of K(p). The pressure to temperature equivalence values, dPm/dT, estimated from the midpoint of the transitions, are 47.0, 43.5, and 52.5 bar degree C-1 for DMPC, DPPC, and POPC, respectively. In liquid-crystalline DMPC, V(a) decreases linearly as a function of temperature, with a coefficient -dVa/dT = 0.65 +/- 0.11 ml degree C-1 mol-1. Using a modified free volume model of diffusion, we show that this value corresponds to the thermal expansivity of DMPC. Both the apparent energy and entropy of activation, Ea and delta Sa, increase with pressure in DMPC, whereas both decrease in POPC and DOPC. This difference is attributed to the sensitivity of the dynamics and/or packing of the dipy10PC probes to the location of the cis-double bonds in the chains of the unsaturated host phospholipids. Finally, the atmospheric pressure values of Ea and delta Sa for the four host MLV examined are shown to be linearly related. The relevance of this finding with respect to the structure of the excimers formed by the dipy10PC probes is briefly discussed.     

Structural properties of DNO investigated with pyrene excimer formation.

Four diamino acid-Nalpha-substituted oligopeptide (DNO) oligomers substituted with pyrenyl as photophysical probes were synthesized. The excimer formation and ground-state association of the pyrenyl groups were investigated by means of absorption and steady-state fluorescence spectroscopy together with time-resolved fluorescence techniques. The photophysical parameters obtained from the different derivatives reflect the secondary structural properties of the DNO backbones.     

Oligonucleotides with strongly fluorescent groups pi-conjugated to a nucleobase: syntheses,melting temperatures,and conformation

Phosphoramidite 1 was prepared, incorporated into oligonucleotides, and these were studied via thermal denaturation and circular dichroism.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Relationship between intramolecular and intermolecular excimer formation rates.

In the intramolecular excimeric membrane probe, dipyrenylphosphatidylcholine (dipyn PC), pyrene moieties are linked to the terminal carbons of the two acyl chains, each of which contains n carbons. We show here how the probe intramolecular excimer production rate, K, may be determined from the excimer/monomer intensity ratio, rl, by making use of the fluorescence titrations of the related monopyrenyl probe, pyn PC, analyzed according to the milling crowd model. rl and the rate K of dipy10 PC in four model membrane systems were measured over a wide temperature range and both parameters are shown to be sensitive functions of the lateral fluidity of the host matrix. A model for relating the intramolecular and intermolecular excimer formation rates is proposed according to which both processes are limited by the reorientational rate of the pyrene moiety. Above the fluid-gel transition temperature, Tc, the diffusion rate (f) of the monopyrenyl probe (pyn PC) is accordingly related to K by: pE approximately K/(K + 1/2f + tau -1M), where pE is the probability of excimer formation between nearest neighbor pyn PC probes, and tau M is the monomer lifetime. Values of pE derived in this way are found to be consistent with pE values derived from the milling crowd analysis of fluorescence yield titration experiments. K for dipy10 PC in DMPC multibilayers ranges from 0.21 x 10(7) s-1 at 10 degrees C in the gel phase, to 5.7 x 10(7) s-1 at 60 degrees C in the fluid phase, whereas the lateral diffusion coefficient, D, for py10 PC in the same bilayers ranged from 8 to 34 microns2 s-1, when calculated with D = fL2/4, L being the average lipid-lipid spacing of the host membrane. Above Tc and at the same reduced temperature, (T - Tc)/Tc, both f for py10 PC, and K for dipy10 PC were found to have relative magnitudes in the order: DPPC greater than DMPC greater than POPC greater than DOPC. This and the similarity of the activation energies for f and K suggest that the rotation of the the pyrene moiety is the rate-limiting step for both the lateral mobility of py10 PC and intramolecular excimer formation in dipy10 PC.     

Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4,5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen–deuterium exchange reaction of a β-ketophosphonate intermediate catalyzed by ND₄Cl in D₂O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,β-enone in perdeuteromethanol was used.     

Characterization of the aldehyde reactive probe reaction with AP-sites in DNA: influence of AP-lyase on adduct stability

Alkoxyamines react with the open-chain aldehyde form of AP-sites in DNA to produce open-chain aldehyde oximes. Here we characterize the effect of AP-site cleavage by yeast AP-endonuclease 1 (APN1) or T4 pyrimidine dimer DNA glycosylase/AP-lyase (T4 Pdg) on the efficiency and stability of the alkoxyamine aldehyde reactive probe (ARP) condensation reaction with AP-sites. The results indicate that (1) reaction of ARP with the open-chain aldehyde equilibrium form of the AP-site was less efficient than with the 3'-alpha,beta-unsaturated aldehyde produced by T4 Pdg; (2) the dRP moiety was least reactive with ARP; (3) both the AP-site and 3'-alpha,beta-unsaturated aldehyde were stable with regard to reaction with ARP over a 30-min incubation period at 37 degrees C; and (4) ARP adducted to the open-chain aldehyde form of the AP-site could be replaced by methoxyamine, but the 3'-alpha,beta-unsaturated aldehyde ARP oxime was stable against methoxyamine attack.     

Synthesis of oligonucleotide derivatives containing a bis-pyrene residue in the main chain.

The oligonucleotide having the bis-pyrene residue in the main chain was synthesized. The preparation of the bis-pyrene was started from the conversion of 2,2-bis-(bromomethyl)-1,3-propanediol into the protected bis-amino derivative. The reaction of the bis-amino derivative with 1-pyrenebutyric acid using DCC/HOBT afforded the desired bis-pyrene. This compound was then converted to the protected phosphormidite. The oligonucleotides possessing the bis-pyrene were synthesized by using the amidite. The oligonucleotides having the bis-pyrene residue can bind to DNA sequence in an aqueous solution to give the duplex with comparable thermal stability as that of the unmodified DNA/DNA duplex. The significantly enhanced pyrene-excimer fluorescence was observed upon hybridization of the bis-pyrene modified oligonucleotides with DNA.     

Frequency-resolved intramolecular excimer fluorescence study of lipid bilayer and nonbilayer phases

Frequency-domain fluorescence intensity decays of the intramolecular excimer forming (DipyPE) in a fully hydrated dioleoyl-phosphatidylethanolamine (DOPE) suspension have been measured at the monomer (395 nm) and excimer (475 nm) emissions and at different temperatures (0-30°C). A classical Birks (two-state) and a new three-state kinetics models were used to analyze the frequency-domain data. The three-state model allowed us to resolve various intramolecular dynamics parameters of DipyPE in the host DOPE suspension. Those parameters are the excimer association (K_dm) and dissociation (K_md) rate constants, effective concentration (C), and lateral diffusion rate (f) of the pyrene moieties in the DipyPE. In contrast, only CK_dm and K_md were determined based on the two-state model. We observed that K_dm declined while C increased abruptly at ∼12°C, the known thermotropic lamellar liquid crystalline-to-inverted hexagonal (L_α-H_II) phase transition temperature of DOPE. No abrupt changes in K_md and f were observed at all temperatures. We concluded that the rotation of the lipid acyl chains is hindered and the free volume available for the lipid terminal methyl ends is reduced as the lipid membrane enters the highly curved H_II phase from the planar L_α phase.     

BON-BONs: cyclic molecules with a boron-oxygen-nitrogen backbone. Computational studies of their thermodynamic properties

Although they were first reported in 1963, molecules with a boron-oxygen-nitrogen dimeric backbone do not seem to have been investigated seriously in terms of thermodynamic properties. Here we report on the calculated structures and properties, including thermodynamics, of several so-called “BON-BON” molecules. With the popularity of nitrogen-containing substituents on new high-energy materials, nitro-substituted BON-BONs were a focus of our investigation. A total of 42 BON-BON molecules were evaluated, and thermochemical analysis shows a decrease in the specific enthalpy of combustion or decomposition with increasing NO₂ content, consistent with other systems.     

Oligonucleotides with pyrene fluorophore at the sugar fragment: synthesis and properties in binding to complementary polynucleotide

Oligonucleotides with 1-pyrenylmethyl substituent at the designated sugar residue were synthesized by using 5'-dimethoxytrityl 2'-(1-pyrenylmethyl)uridine 3'-phosphorobisdiethylamidite 1. It was shown that the pyrene-oligonucleotides have enhanced affinity in binding to the complementary polynucleotide sequence. The fluorescence yield and life time of the pyrene-oligonucleotide was drastically enhanced when bound by the complementary polynucleotide.     

Cannabinols and the rosette forming properties of lymphocytes in vitro.

Preincubation of lymphocytes, for 60 minutes at 37°C with tetrahydrocannabinol (THC), obtained from controls produced reduction in early but not total T cell rosettes compared to aliquots treated with vehicle alone. Similar reductions in early rosettes were seen after preincubation with cannabinol, cannabidiol, and olivetol. The marijuana smokers' lymphocytes were less affected by preincubation with THC than those from controls. Presumably their lymphocytes may already have been affected by prior exposure to cannabinols. These data show that THC and other cannabinols can affect the rosetting properties of T cells $\text{in vitro}$ and may provide a model for the study of THC effects on these immunocompetent cells.     

Bioactive sulfoximines: syntheses and properties of Vioxx analogs

The syntheses and biological profiles of sulfoximine-based Vioxx® analogs 2 are described. Interesting data have been obtained for 2a, which shows a selective COX-2 inhibition (albeit not as strong as Vioxx® itself) exhibiting reduced hERG activity compare to the parent sulfone Vioxx® (1).