Tautomers of a Fluorescent G Surrogate and Their Distinct Photophysics Provide Additional Information Channels.

Thienoguanosine ((th) G) is an isomorphic nucleoside analogue acting as a faithful fluorescent substitute of G, with respectable quantum yield in oligonucleotides. Photophysical analysis of (th) G reveals the existence of two ground-state tautomers with significantly shifted absorption and emission wavelengths, and high quantum yield in buffer. Using (TD)-DFT calculations, the tautomers were identified as the H1 and H3 keto-amino tautomers. When incorporated into the loop of (-)PBS, the (-)DNA copy of the HIV-1 primer binding site, both tautomers are observed and show differential sensitivity to protein binding. The red-shifted H1 tautomer is strongly favored in matched (-)/(+)PBS duplexes, while the relative emission of the H3 tautomer can be used to detect single nucleotide polymorphisms. These tautomers and their distinct environmental sensitivity provide unprecedented information channels for analyzing G residues in oligonucleotides and their complexes.

Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer.

The HIV-1 nucleocapsid protein (NCp7) is a nucleic acid chaperone required during reverse transcription. During the first strand transfer, NCp7 is thought to destabilize cTAR, the (-)DNA copy of the TAR RNA hairpin, and subsequently direct the TAR/cTAR annealing through the zipping of their destabilized stem ends. To further characterize the destabilizing activity of NCp7, we locally probe the structure and dynamics of cTAR by steady-state and time resolved fluorescence spectroscopy. NC(11-55), a truncated NCp7 version corresponding to its zinc-finger domain, was found to bind all over the sequence and to preferentially destabilize the penultimate double-stranded segment in the lower part of the cTAR stem. This destabilization is achieved through zinc-finger-dependent binding of NC to the G(10) and G(50) residues. Sequence comparison further revealed that C•A mismatches close to the two G residues were critical for fine tuning the stability of the lower part of the cTAR stem and conferring to G(10) and G(50) the appropriate mobility and accessibility for specific recognition by NC. Our data also highlight the necessary plasticity of NCp7 to adapt to the sequence and structure variability of cTAR to chaperone its annealing with TAR through a specific pathway.

Fully regiocontrolled polyarylation of pyridine.

Starting from commercially available 2-chloro-3-hydroxypyridine, a new route leading to the first protypical pentaarylpyridine bearing five different substituents is reported. This strategy involves a set of five sequential but fully regiocontrolled Suzuki-Miyaura reactions and highlights the 2-OBn pyridine protecting group as a key intermediate. The 2-OBn group played a double role: (i) it allowed additional bromination at position 5 and (ii) it could afford the reactive OTf species for the last C-arylation step at the less hindered 2 position of the tetraarylpyridine. The photophysical properties of the novel compounds are also described. The synthesized pentaarylpyridine derivative exhibit a large Stokes shift, strong solvatochromism, and quantum yield values up to 0.47; thus, they constitute promising building blocks for the design of environment-sensitive probes.

Impact of ambient conditions on the minor nutrients content in spirulina "Arthrospira platensis" (AP) extracted from Lake Chad revealed by fluorescence spectroscopy coupled with chemometric methods.

In this study, the technique of fluorescence spectroscopy coupled with chemometric methods is used to analyse samples of Lake Chad Spirulina "Arthrospira platensis" (AP), either harvested and conditioned by using the traditional method at different seasons or industrially processed. The content of minor fluorescent nutrients is investigated. To this end, fluorescence excitation-emission matrices (EEMs) of 46 AP samples are recorded in aqueous solution. Synchronous fluorescence (SF) spectra are extracted from these EEMs and their important features are compared to those of PARAFAC methods. Synchronous fluorescence scanning allows different AP samples to be characterized in a single scan. The SF and PARAFAC methods yielded two groups of fluorescent compounds; the first group, consisting of vitamin-like molecules, shows excitation/emission (ex/em) peaks at 340/460, 390/462, 370/440 and 450/526 nm, attributed to caffeic acid, vitamin K, E and riboflavins respectively, while the second group, consisting of pigments, shows ex/em peaks at 610/654, 590/630 and 570/644 nm, attributed to phycocyanins, C-phycocyanin and allophycocyanin. Our fluorescence data showed that while both vitamins and pigments are present in AP during the rainy season, only fluorescent components of vitamin-like compounds are present during the dry season. PCA methods allowed classifying different AP samples according to their geographic origin and harvesting season. Fluorescence spectroscopy therefore appears to be a powerful technique for rapidly assessing the chemical composition of AP.

Identification of potential dipeptidyl peptidase IV inhibitors from the ConMedNP library by virtual screening, and molecular dynamics methods.

In this study, we screened novel dipeptidyl peptidase IV (DPP4) inhibitors from the ConMedNP library consisting of 3507 molecules. Interestingly, molecular docking, ADMET, and the anti-diabetic activity predictions suggest that three molecules, namely OTH_UD_XX06_1, GB19, and BMC_000104, have a high binding affinity toward DPP4. The molecular dynamics (MD) simulation results suggest that these hit molecules have a stable binding pose and occupy the binding pockets throughout the 200 ns simulation. The presence of intermolecular H-bonding between the ligands and DPP4 was observed throughout the simulation period. Thus, docking and MD results, predicted that the three compounds were the most potent DPP4 inhibitors that could putatively bind to the DPP4 active site via both conventional H-bonding and hydrophobic interactions. These results could aid the discovery of new drugs to treat type 2 diabetes.

Binding of Mammea A/AA (MA) to calf thymus DNA revealed by the ratiometric absorbance of MA in the UV-visible range molecular dynamic simulations and TD-DFT calculations.

The in vitro anti-proliferative activity of MA (5,7-dihydroxy-8-(3-methylbut-2-enyl)-6-(3-methyl-1-oxobutyl)-4-phenyl[1]2H-[1]benzopyran-2-one)on a variety of cancer cells was previously demonstrated. This work strives to understand the mechanisms by which MA exerts this biological activity. Thereafter, the binding of MA to calf thymus DNA was studied by monitoring the change in the UV-visible absorbance of MA. It was found that, the response of MA to binding with calf thymus DNA is characterised by an increase in the AS/AL ratio of the absorbance of the longest wavelength absorption band to the shortest one, and the appearance of a new band at about 377 nm assigned to S0→S1 transition, which is red shifted as compared to free MA. From the bands ratio, the binding constant is found to be 4.3x105 M-1, indicating strong binding. The deduced binding free energy, enthalpy and entropy are -7.7 kcal/mol, -10.89 ± 0.28 kcal/mol and -54.46 ± 4 J/K, respectively, indicating that MA binds to DNA by a non-bonding Van der Waals type interactions and hydrogen bonds. Further study with classical molecular dynamics shows that MA binds to DNA by intercalation, where it is positioned between two AT base pairs. Unlike isolated MA, TDDFT calculations on ten images extracted from the MD trajectory show that, the frontier molecular orbitals of the complex are distributed over the DNA and MA. This indicates a strong stacking interaction and then explains the hypochromism and the red shift of the S0→S1 transition. The present work demonstrates the potency of MA as antitumor compound and as absorbance-based molecular probe.Communicated by Ramaswamy H. Sarma.

Ab initio study of the solvent H-bonding effect on ESIPT reaction and electronic transitions of 3-hydroxychromone derivatives.

The electronic transitions occurring in 4-(N,N-dimethylamino)-3-hydroxyflavone (DMAF) and 2-furanyl-3-hydroxychromone (FHC) were investigated using the TDDFT method in aprotic and protic solvents. The solvent effect was incorporated into the calculations via the PCM formalism. The H-bonding between solute and protic solvent was taken into account by considering a molecular complex between these molecules. To examine the effect of the H-bond on the ESIPT reaction, the absorption and emission wavelengths as well as the energies of the different states that intervene during these electronic transitions were calculated in acetonitrile, ethanol and methanol. The calculated positions of the absorption and emission wavelengths in various solvents were in excellent agreement with the experimental spectra, validating our approach. We found that in DMAF, the hydrogen bonding with protic solvents makes the ESIPT reaction energetically unfavourable, which explains the absence of the ESIPT tautomer emission in protic solvents. In contrast, the excited tautomer state of FHC remains energetically favourable in both aprotic and protic solvents. Comparing our calculations with the previously reported time-resolved fluorescence data, the ESIPT reaction of DMAF in aprotic solvents is reversible because the emitting states are energetically close, whereas in FHC, ESIPT is irreversible because the tautomer state is below the corresponding normal state. Therefore, the ESIPT reaction in DMAF is controlled by the relative energies of the excited states (thermodynamic control), while in FHC the ESIPT is controlled probably by the energetic barrier (kinetic control).

Impact of temperature and sunlight exposition on locally brewed beers composition revealed by fluorescence spectroscopy coupled with chemometric methods.

Fluorescence excitation-emission matrix (EEM) and synchronous scanning fluorescence (SF), coupled with parallel factor (PARAFAC) analysis, principal component analysis (PCA) and Linear discriminant analysis (LDA) methods were used to differentiate 49 lager beer samples and monitor the effects of temperature and sunlight exposition on their composition. EEMs were decomposed into independent fluorescent components. The beer samples were characterized by the presence of excitation/emission (exc/em) peaks at 290/350, 315/345, 340/410, 375/455, 360/420, 400/460, and 437/525 nm, which were ascribed, according to the known beer fluorescent components, respectively to aromatic amino acids, vitamin B6 (pyridoxal), vitamin B6 (pyridoxic acids), vitamin B3, iso-α-acids, vitamin B1, and vitamin B2. The variation of the relative concentration of iso-α-acids in the different beer brands presented the same trend with that of their relative IBU, thus revealing the potency of our method in the assessment of beer bitterness. The impact of temperature and sunlight was assessed by separately monitoring the modifications of the EEMs after 5 h exposition to 40°C temperature and sunlight respectively. Noticeably a variation of the peaks intensity of the iso-α-acids, carbonyl and polyphenols compounds were observed, accompanied by a decrease of the alcohol content, thus indicating beer aging. This method can be useful for the identification and monitoring of beer state during the technological production cycle and storage. PRACTICAL APPLICATION: The present work demonstrates the potency of the fluorescence technique used together with chemometric methods to give valuable information on beer bitterness. Development of rapid quantitative methods for beer bitterness assessment is of great importance for brewing industries.

Electronic transitions and ESIPT kinetics of the thienyl-3-hydroxychromone nucleobase surrogate in DNA duplexes: a DFT/MD-TDDFT study.

The fluorescent nucleobase surrogate M (2-thienyl-3-hydroxychromone fluorophore) when imbedded in DNA opposite an abasic site exhibits a two colour response highly sensitive to environment changes and base composition. Its two colour emission originates from an excited state intramolecular proton transfer (ESIPT), which converts the excited normal N* form into its T* tautomer. To get deeper insight on the spectroscopic properties of M in DNA duplexes, quantum chemical calculations were performed on M stacked with different base pairs in model trimers extracted from MD simulations. The photophysics of M in duplexes appeared to be governed by stacking interactions as well as charge and hole transfer. Indeed, stacking of M in DNA screens M from H-bonding with water molecules, which favours ESIPT and thus, the emission of the T* form. With A and T flanking bases, the electronic densities in the frontier MOs were localized on M, in line with its effective absorption and emission. In addition, reduction of the free rotation between the thienyl and chromone groups together with the shielding of the dye from water molecules largely explain its enhanced quantum yield in comparison to the free M in solution. By contrast, the localisation of the electron density on the flanking G residues in the ground state and the energetically favorable hole transfer from M to G in the excited state explains the reduced quantum yield of M sandwiched between CG pairs. Finally, the much higher brightness of M as compared to 2-aminopurine when flanked by A and T residues could be related to the much stronger oscillator strength of its S0 → S1 transition and the ineffective charge transfer from M to A or T residues.

Time-resolved fluorescent properties of 8-vinyl-deoxyadenosine and 2-amino-deoxyribosylpurine exhibit different sensitivity to their opposite base in duplexes.

8-Vinyl-deoxyadenosine (8VA) has been recently introduced as a fluorescent analogue of adenosine that is less perturbing and less quenched than the well-established 2-amino-deoxyribosylpurine (2AP) probe when inserted in oligonucleotides. To further validate 8VA as a fluorescent substitute of A, we compared the ability of 8VA and 2AP in sequences of the type d(CGT TTT XNX TTT TGC) (with N=8VA or 2AP and X=T and C) to discriminate the nature of the opposite base (Y) in duplexes. For both probes, systematic variations in the amplitudes of the short- and long-lived lifetimes of the fluorescence intensity decays as well as in the amplitude of the fast rotational correlation time of the fluorescence anisotropy decays were observed as a function of the nature of Y. From these parameters, we inferred a stability order 8VA-T > 8VA-G > 8VA-A > 8VA-C, similar to the stability order with the native A base, but different from the stability order with 2AP. Using a combination of molecular mechanics and ab initio calculations, we found that the time-resolved parameters of 8VA, but not the 2AP ones, correlate well with the geometry and the strength of the A-Y base-pairing interaction. This may be rationalized by the smaller structural and electronic perturbations induced by the vinyl group in position 8 as compared to the amino group at position 2. As a consequence, substitution of A by 8VA in a base pair was found to only minimally modify the structure and interaction energy of the base pair. Thus, 8VA can be used as a fluorescent substitute of the natural A, to straightforwardly discriminate the nature of the opposite base. This may find interesting applications notably in the elucidation of the mechanisms and dynamics of the DNA mismatch repair system.

Hypolipidemic effect and activation of Lecithin Cholesterol Acyl Transferase (LCAT) by aqueous extract of Spirulina platensis during toxicological investigation.

BACKGROUND: Spirulina platensis produced in Nomayos (Cameroon) is used as a dietary supplement. S. platensis is known as a neutraceutical with many beneficial effects on humans like lipid-lowering action. This study aims to investigate the mechanism of hypolipidemic action of aqueous extract of Spirulina platensis (S. platensis) through the toxicological studies. METHODS: In this study, we included two month old Wistar rats, weighing between 180 and 200 g. Aqueous S. platensis was extracted and prepared using standard methods. The rats received a supplementation of S. platensis at 5000 mg/Kg of body weight as single dose in acute toxicity whereas different doses (250, 500, 1000 mg / kg body weight) were administered in subacute toxicity compared to control. Acute and subacute toxicities were determined according to the guidelines 420 (14 days) and 407 (28 days) of the Organization for Economic Cooperation and Development (OECD) respectively. Biochemical parameters such as urea, creatinine, total and direct bilirubin, lipid profile and transaminases; and histopathological analysis of the liver and kidneys were used to evaluate the toxicity of S. platensis on these Wistar rats. Plasmatic hydroxymethyl glutaryl coenzyme A reductase (HMG CoA reductase) and lecithine cholesterol acyl transferase (LCAT) were performed to explain the lipid-lowering action of S. platensis. Histopathological analysis of the liver and kidneys was performed. RESULTS: Our results show a decrease in total cholesterol for male rats (from 84 to 74 mg/dl) when the dose of S. platensis increased; this reduction of the total cholesterol level in male rats was significant at 500 mg/kg. There was also a significant inhibition of HMG CoA reductase in a dose dependent manner between 25 and 84.5 fold compared to the control in both male and female groups. At the dose of 250 mg/kg bw, the level of LCAT was higher compared with other groups and control, but the difference was not statistically significant. A slight inflammation in the liver and the mesangial hyperplasia of the renal glomeruli was revealed by the histopathological investigation in subacute toxicity. CONCLUSION: Spirulina platensis from Cameroon appears to have little toxic effects and may demonstrate hypolipidemic activity through the activation of LCAT.

Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex.

Recently, a 3-hydroxychromone based nucleoside 3HCnt has been developed as a highly environment-sensitive nucleoside surrogate to investigate protein-DNA interactions. When it is incorporated in DNA, the probe is up to 50-fold brighter than 2-aminopurine, the reference fluorescent nucleoside. Although the insertion of 3HCnt in DNA was previously shown to not alter the overall DNA structure, the possibility of the probe inducing local effects cannot be ruled out. Hence, a systematic structural and dynamic study is required to unveil the 3HCnt's limitations and to properly interpret the data obtained with this universal probe. Here, we investigated by NMR a 12-mer duplex, in which a central adenine was replaced by 3HCnt. The chemical shifts variations and nOe contacts revealed that the 3HCnt is well inserted in the DNA double helix with extensive stacking interactions with the neighbor base pairs. These observations are in excellent agreement with the steady-state and time-resolved fluorescence properties indicating that the 3HCnt fluorophore is protected from the solvent and does not exhibit rotational motion. The 3HCnt insertion in DNA is accompanied by the extrusion of the opposite nucleobase from the double helix. Molecular dynamics simulations using NMR-restraints demonstrated that 3HCnt fluorophore exhibits only translational dynamics. Taken together, our data showed an excellent intercalation of 3HCnt in the DNA double helix, which is accompanied by localized perturbations. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing.

Excited-state properties and transitions of fluorescent 8-vinyl adenosine in DNA.

8-vinyl-adenosine (8VA) is a new fluorescent nucleoside analogue with improved spectroscopic properties as compared to 2-aminopurine (2AP). To further understand its photophysics, we analyzed by the time-dependent density functional theory and the configuration interaction single method, the electronic properties, and transitions of 8VA in its free form and stacked with one or two flanking bases. For free 8VA, the predicted excited-state energy gaps, absorbance peak position, and oscillator strength were found to be in excellent agreement with the experimentally determined ones. Moreover, its high fluorescence quantum yield was found to be associated with the dipole-allowed S1 --> S0 transition. Stacking of 8VA with C, T, or A in dimers or trimers resulted in fluorescence quenching through mechanisms that depend on the nature and relative orientation of the flanking base(s). When 8VA is stacked with T, quenching mainly results from nonradiative relaxation to low-lying dark excited state(s) that do not exist in free 8VA. When 8VA is stacked with A, quenching results mainly from mixing of the molecular orbitals in the ground state. Both types of quenching are thought to accompany the stacking of 8VA with C. In addition, the C-8VA-C trimer was found to exhibit a low-lying S1 emissive state that may generate an increased fluorescence quantum yield and lifetime. The predicted photophysical properties of the trimers are highly consistent with the spectroscopic data of a series of 15-mer oligonucleotides differing only by the nature of the residues flanking the central 8VA.