Selective,pH-Dependent Colorimetric and Fluorimetric Detection of Quadruplex DNA with 4-Dimethylamino(phenyl)-Substituted Berberine Derivatives

The 9- and 12-dimethylaminophenyl-substituted berberine derivatives 3 a and 3 b were readily synthesized by Suzuki-Miyaura reactions and shown to be useful fluorescent probes for the optical detection of quadruplex DNA (G4-DNA). Their association with the nucleic acids was investigated by spectrometric titrations, CD and LD spectroscopy, and with DNA-melting analysis. Both ligands bind to duplex DNA by intercalation and to G4-DNA by terminal π stacking. At neutral conditions, they bind with higher affinity (K_b=10⁵−10⁶ M⁻¹) to representative quadruplex forming oligonucleotides 22AG , c-myc , c-kit , and a2 , than to duplex calf thymus (ct) DNA (K_b=5-7×10⁴ M⁻¹). At pH 5, however, the affinity of 3 a towards G4-DNA 22AG is higher (K_b=1.2×10⁶ M⁻¹), whereas the binding constant towards ct DNA is lower (K_b=3.9×10³ M⁻¹) than under neutral conditions. Notably, the association of the ligand with DNA results in characteristic changes of the absorption and emission properties under specific conditions, which may be used for optical DNA detection. Other than the parent berberine, the ligands do not show a noticeable increase of their very low intrinsic emission intensity upon association with DNA at neutral conditions. In contrast, a fluorescence light-up effect was observed upon association to duplex (Φ_fl=0.01) and quadruplex DNA (Φ_fl=0.04) at pH 5. This fluorimetric response to G4-DNA association in combination with the distinct, red-shifted absorption under these conditions provides a simple and conclusive optical detection of G4-DNA at lower pH.     

Studies on the Interactions of 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium and Insulin with the Quadruplex-Forming Oligonucleotide Sequence a2 from the Insulin-Linked Polymorphic Region

Recently, several quadruplex-DNA-forming sequences have been identified in the insulin-linked polymorphic region (ILPR), which is a guanine-rich oligonucleotide sequence in the promoter region of insulin. The formation of this non-canonical quadruplex DNA (G4-DNA) has been shown to be involved in the biological activity of the ILPR, specifically with regard to its interplay with insulin. In this context, this contribution reports on the investigation of the association of the quadruplex-forming ILPR sequence a2 with insulin as well as with the well-known G4-DNA ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium (1), also named RHPS4, by optical and NMR spectroscopy. CD- and NMR-spectroscopic measurements confirmed the preferential formation of an antiparallel quadruplex structure of a2 with four stacked guanine quartets. Furthermore, ligand 1 has high affinity toward a2 and binds by terminal π stacking to the G1–G11–G15–G25 quartet. In addition, the spectroscopic studies pointed to an association of insulin to the deoxyribose backbone of the loops of a2.     

Spectral properties and G-quadruplex DNA binding selectivities of a series of unsymmetrical perylene diimides

A series of new unsymmetrical perylene diimides have been synthesized to investigate their binding selectivities to G-quadruplex DNA structure, a unique four-stranded DNA motif, which is significant to the regulation of telomerase activity. The structures of the perylene diimides have been characterized by IR spectrophotometer, ¹H NMR, ¹³C NMR, MS, TGA and time-resolved instruments. Spectrochemical behaviors have been investigated by visible absorption and fluorescence emission spectra. The spectral characterization of the compounds has been investigated in five common organic solvents of different polarity and in water (in 170 mM phosphate buffer at pH 6). Marked red shifts of absorbance and fluorescence emission bands of the compounds in aqueous solution are compared with the other organic solutions. The fluorescence quantum yields are determined low in more polar solvents and also calculated to be about less than about 0.05 in aqueous solution because of the aggregation effects. Photodegradation rate constants (k_p) of the synthesized compounds have been compared under xenon lamp irradiation in acetonitrile solution.Binding abilities of the synthesized perylene diimides to different form of DNA strands have been investigated by visible absorption and fluorescence spectroscopy in the phosphate buffer solutions. Also, pH-dependent aggregation and G-quadruplex DNA binding selectivity of these ligands have been compared. Among these ligands, N-(2,6-diisopropylphenyl)-N′-(4-pyridyl)-perylene-3,4,9,10-tetracarboxylic diimide (PYPER) has been found to be the most selective interactive ligand for G-quadruplex formed in the G4′-DNA structure. PYPER has shown a significant selectivity to G4′-DNA which is comprised of d(TTAGGG) repeats, known as human telomeres, in the phosphate buffer at pH 6. The absorption maximum of the PYPER/G4′-DNA complex has given bathochromic shift of 7 nm with respect to the absorption maximum of DNA-free solution of PYPER in phosphate buffer at pH 6. Fluorescence quenching experiments between PYPER and G4′-DNA show that PYPER demonstrates about a 9.3-fold selectivity for binding to G4′-DNA versus ds-DNA base pairs with the bimolecular rate constant of 0.95 × 10¹² M⁻¹ s⁻¹.     

Single-molecule fluorescence detection of a tricyclic nucleoside analogue

Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C-linked 8-(diethylamino)benzo[b][1,8]naphthyridin-2(1H)-one nucleoside, which we name ABN, exhibits ε₄₄₂ = 20 000 M⁻¹ cm⁻¹ and Φ_em,540 = 0.39 in water, increasing to Φ_em = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities.

Fluorescent nucleoside analogue ABN is readily detected at the single-molecule level and retains a quantum yield >50% in duplex DNA oligonucleotides.     

Polycyclic Azoniahetarenes: Assessing the Binding Parameters of Complexes between Unsubstituted Ligands and G‐Quadruplex DNA

Polycyclic azoniahetarenes were employed to determine the effect of the structure of unsubstituted polyaromatic ligands on their quadruplex‐DNA binding properties. The interactions of three isomeric diazoniadibenzo[b,k]chrysenes ( 4 a – c ), diazoniapentaphene ( 5 ), diazoniaanthra[1,2‐a]anthracene ( 6 ), and tetraazoniapentapheno[6,7‐h]pentaphene ( 3 ) with quadruplex DNA were examined by DNA melting studies (FRET melting) and fluorimetric titrations. In general, penta‐ and hexacyclic azoniahetarenes bind to quadruplex DNA (K_b≈10⁶ M ^?1) even in the absence of additional functional side chains. The binding modes of 4 a – c and 3 were studied in more detail by ligand displacement experiments, isothermal titration calorimetry, and CD and NMR spectroscopy. All experimental data indicate that terminal π stacking of the diazoniachrysenes to the quadruplex is the major binding mode; however, because of different electron distributions of the π systems of each isomer, these ligands align differently in the binding site to achieve ideal binding interactions. It is proposed that tetraazonia ligand 3 binds to the quadruplex by terminal stacking with a small portion of its π system, whereas a significant part of the bulky ligand most likely points outside the quadruplex structure, and is thus partially placed in the grooves. Notably, 3 and the known tetracationic porphyrin TMPyP4 exhibit almost the same binding properties towards quadruplex DNA, with 3 being more selective for quadruplex than for duplex DNA. Overall, studies on azonia‐type hetarenes enable understanding of some parameters that govern the quadruplex‐binding properties of parent ligand systems. Since unsubstituted ligands were employed in this study, complementary and cooperative effects of additional substituents, which may interfere with the ligand properties, were eliminated.     

Arene Ru(II) Complexes Acted as Potential KRAS G-Quadruplex DNA Stabilizer Induced DNA Damage Mediated Apoptosis to Inhibit Breast Cancer Progress

A series of arene Ru(II) complexes, [(η⁶-MeC₆H₅)Ru(L)Cl]Cl, (L=o-ClPIP, 1; m-ClPIP, 2 and p-ClPIP, 3) (o-ClPIP=2-(2-chlorophenyl)imidazo[4,5-f][1,10]phenanthroline; m-ClPIP=2-(3-chlorophenyl)imidazo[4,5-f][1,10]phenanthroline; p-ClPIP=2-(4-chlorophenyl)imidazo[4,5-f][1,10]phenanthroline) was synthesized and investigated as a potential apoptosis inducer in chemotherapy. Spectroscopy and molecular docking simulations show that 1 exhibits moderated binding affinity to KRAS G-quadruplex DNA by groove mode. Further, in vitro studies reveal that 1 displays inhibitory activity against MCF-7 growth with IC₅₀ = 3.7 ± 0.2 μM. Flow cytometric analysis, comet assay, and immunofluorescence confirm that 1 can induce the apoptosis of MCF-7 cells and G0/G1 phase arrest through DNA damage. In summary, the prepared arene Ru(II) complexes can be developed as a promising candidate for targeting G-quadruplex structure to induce the apoptosis of breast cancer cells via binding and stabilizing KRAS G-quadruplex conformation on oncogene promoter.     

Molecular docking analysis and spectroscopic investigations of zinc(II), nickel(II) N-phthaloyl-β-alanine complexes for DNA binding: Evaluation of antibacterial and antitumor activities

Herein, computational molecular docking, UV/visible and fluorescence spectroscopic techniques have been used to explore the DNA binding interactions of N-phthaloyl-β-alanine (NPA) ligand and its Zn(II) and Ni(II) complexes (NPAZn, NPANi). The compounds were further tested for anti-bacterial and anti-tumor activities. Docking analysis depicted that ligand NPA interacted with DNA via intercalation, while its metal complexes showed mixed mode of interactions. Spectroscopic experiments for DNA binding studies were run under physiological conditions of pH (stomach; 4.7, blood; 7.4) and temperature (37 °C). Based on changes in spectral responses, binding parameters for all the compounds were obtained which showed comparatively greater binding constant values (K_b: UV; 1.16 × 10⁵ M⁻¹, Flu; 1.35 × 10⁵ M⁻¹) and more negative free energy changes (ΔG: UV; −30.00 kJ mol⁻¹, Flu; −30.44 kJ mol⁻¹) for NPAZn at pH 4.7. The overall, binding results were also found more significant at stomach pH. Dynamic “K_D” and bimolecular “K_B” constants were evaluated, and the values affirmed the participation of static process for each compound–DNA binding. The greater binding site size values (n > 1) of metal complexes NPAZn and NPANi indicated other sites availability of intercalative compounds. DNA viscosity variation by increasing compound’s concentration further verified the compound–DNA interaction. Antibacterial and tumor inhibitory activities were observed significant for both metal complexes, while ligand has shown no activity. The greater binding affinity of metal complexes, as evaluated both computationally and spectroscopically, further validated the lower IC50 values of complexes as compared to ligand.     

Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel–Chromomycin Complex Resulting in Large-Scale DNA Deformation

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. Ni^II(Chro)₂ (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)_n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the Ni^II chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by Ni^II(Chro)₂. The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.     

Interactions between a dsDNA Oligonucleotide and Imidazolium Chloride Ionic Liquids: Effect of Alkyl Chain Length,Part I

Ionic liquids (ILs) have become nearly ubiquitous solvents and their interactions with biomolecules has been a focus of study. Here, we used the fluorescence emission of DAPI, a groove binding fluorophore, coupled with molecular dynamics (MD) simulations to report on interactions between imidazolium chloride ([Im_n,1]⁺) ionic liquids and a synthetic DNA oligonucleotide composed entirely of T/A bases (7(TA)) to elucidate the effects ILs on a model DNA duplex. Spectral shifts on the order of 500–1000 cm⁻¹, spectral broadening (~1000 cm⁻¹), and excitation and emission intensity ratio changes combine to give evidence of an increased DAPI environment heterogeneity on added IL. Fluorescence lifetimes for DAPI/IL solutions yielded two time constants 0.15 ns (~80% to 60% contribution) and 2.36–2.71 ns for IL up to 250 mM. With DNA, three time constants were required that varied with added IL (0.33–0.15 ns (1–58% contribution), ~1.7–1.0 ns (~5% contribution), and 3.8–3.6 ns (94–39% contribution)). MD radial distribution functions revealed that π-π stacking interactions between the imidazolium ring were dominant at lower IL concentration and that electrostatic and hydrophobic interactions become more prominent as IL concentration increased. Alkyl chain alignment with DNA and IL-IL interactions also varied with IL. Collectively, our data showed that, at low IL concentration, IL was primarily bound to the DNA minor groove and with increased IL concentration the phosphate regions and major groove binding sites were also important contributors to the complete set of IL-DNA duplex interactions.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

Silver(I) Complexes Based on Oxadiazole-Functionalized α-Aminophosphonate: Synthesis,Structural Study,and Biological Activities

Two silver(I) complexes, bis{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN³:κN⁴-amino) (4-trifluoromethylphenyl)methyl]phosphonate-(tetrafluoroborato-κF)}-di-silver(I) and tetrakis-{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN³-amino)(4-trifluoromethylphenyl)methyl]phosphonate} silver(I) tetrafluoroborate, were prepared starting from the diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-amino)(4-trifluoromethylphenyl)methyl]phosphonate (1) ligand and AgBF₄ salt in Ag/ligand ratios of 1/1 and 1/4, respectively. The structure, stoichiometry, and geometry of the silver complexes were fully characterized by elemental analyses, infrared, single-crystal X-ray diffraction studies, multinuclear NMR, and mass spectroscopies. The binuclear complex ([Ag₂(1)₂(BF₄)₂]; 2) crystallizes in the monoclinic asymmetric space group P2₁/c and contains two silver atoms adopting a {AgN₂F} planar trigonal geometry, which are simultaneously bridged by two oxadiazole rings of two ligands, while the mononuclear complex ([Ag(1)₄]BF₄; 3) crystallizes in the non-usual cubic space group Fd-3c in which the silver atom binds to four distinct electronically enriched nitrogen atoms of the oxadiazole ring, in a slightly distorted {AgN₄} tetrahedral geometry. The α-aminophosphonate and the monomeric silver complex were evaluated in vitro against MCF-7 and PANC-1 cell lines. The silver complex is promising as a drug candidate for breast cancer and the pancreatic duct with half-maximal inhibitory concentration (IC₅₀) values of 8.3 ± 1.0 and 14.4 ± 0.6 μM, respectively. Additionally, the interactions of the ligand and the mononuclear complex with Vascular Endothelial Growth Factor Receptor-2 and DNA were evaluated by molecular docking methods.     

Two Distinct Thermal Stabilities of DNA and Enzymatic Activities of DNase I in a Multistep Assembly with Carbazole Ligands: Different Binding Characteristics for Duplex and Quadruplex DNA

A partially hydrophobic carbazole ligand ((Im⁺)₂Cz: 2,2′‐(9‐ethyl‐9 H‐carbazole‐3,6‐diyl)bis(ethyne‐2,1‐diyl)bis(1,3‐dimethyl‐1 H‐imidazol‐3‐ium)) adopts two different binding states (binding states I and II) in its interactions with calf‐thymus (ct‐) DNA. Two distinct binding states were identified by biphasic UV/Vis and circular dichroism (CD) spectral changes during the titration of DNA into the carbazole ligand. At low concentrations of ct‐DNA, (Im⁺)₂Cz binds to nearly every part of ct‐DNA (binding state I). By contrast, an increased concentration of ct‐DNA results in a switch in the DNA‐binding state, so that the ligands are bound per five DNA base pairs. Similarly, a monocationic carbazole ligand (Im⁺Cz: 2‐((6‐bromo‐9‐ethyl‐9 H‐carbazol‐3‐yl)ethynyl)‐1,3‐dimethyl‐1 H‐imidazol‐3‐ium) also shows biphasic UV/Vis spectral changes during the titration of ct‐DNA into Im⁺Cz, which suggests two different binding states of the Im⁺Cz ligand with ct‐DNA. The stepwise equilibrium of the ligand–DNA‐complex formation is capable of switching the thermal stability of ct‐DNA, as well as the enzymatic activity of deoxyribonuclease (DNase I). In binding state I, the (Im⁺)₂Cz ligands interact with nearly every base pair in ct‐DNA and stabilize the double‐helix structure, which results in a larger increase in the melting temperature of the ct‐DNA than that observed with binding state II. On the other hand, the (Im⁺)₂Cz ligand significantly reduces the enzymatic activity of DNase I in binding state I, although the enzymatic activity is recovered once the binding state of the ligand–DNA complex is changed to binding state II. The (Im⁺)₂Cz ligand was also employed as a binder for G‐quadruplex DNA. In contrast to the stepwise complex formation between (Im⁺)₂Cz and ct‐DNA, (Im⁺)₂Cz shows a monotonous UV/Vis spectral response during the titration of G‐quadruplex DNA into (Im⁺)₂Cz, which suggests a single binding state for (Im⁺)₂Cz with G‐quadruplex DNA.     

Triplet Emitting C^N^C Cyclometalated Dibenzo[c,h]Acridine Pt(II) Complexes

In a series of Pt(II) complexes [Pt(dba)(L)] containing the very rigid, dianionic, bis-cyclometalating, tridentate C^N^C²⁻ heterocyclic ligand dba^2– (H₂dba = dibenzo[c,h]acridine), the coligand (ancillary ligand) L = dmso, PPh₃, CNtBu and Me₂Imd (N,N’-dimethylimidazolydene) was varied in order to improve its luminescence properties. Beginning with the previously reported dmso complex, we synthesized the PPh₃, CNtBu and Me₂Imd derivatives and characterized them by elemental analysis, ¹H (and ³¹P) NMR spectroscopy and MS. Cyclic voltammetry showed partially reversible reduction waves ranging between −1.89 and −2.10 V and increasing along the series Me₂Imd < dmso ≈ PPh₃ < CNtBu. With irreversible oxidation waves ranging between 0.55 (L = Me₂Imd) and 1.00 V (dmso), the electrochemical gaps range between 2.65 and 2.91 eV while increasing along the series Me₂Imd < CNtBu < PPh₃ < dmso. All four complexes show in part vibrationally structured long-wavelength absorption bands peaking at around 530 nm. TD-DFT calculated spectra agree quite well with the experimental spectra, with only a slight redshift. The photoluminescence spectra of all four compounds are very similar. In fluid solution at 298 K, they show broad, only partially structured bands, with maxima at around 590 nm, while in frozen glassy matrices at 77 K, slightly blue-shifted (~580 nm) bands with clear vibronic progressions were found. The photoluminescence quantum yields Φ_L ranged between 0.04 and 0.24, at 298 K, and between 0.80 and 0.90 at 77 K. The lifetimes τ at 298 K ranged between 60 and 14040 ns in Ar-purged solutions and increased from 17 to 43 µs at 77 K. The TD-DFT calculated emission spectra are in excellent agreement with the experimental findings. In terms of high Φ_L and long τ, the dmso and PPh₃ complexes outperform the CNtBu and Me₂Imd derivatives. This is remarkable in view of the higher ligand strength of Me₂Imd, compared with all other coligands, as concluded from the electrochemical data.     

Spectroscopic Studies of Quinobenzothiazine Derivative in Terms of the In Vitro Interaction with Selected Human Plasma Proteins. Part 1

Plasma proteins play a fundamental role in living organisms. They participate in the transport of endogenous and exogenous substances, especially drugs. 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium salts, have been synthesized as potential anticancer substances used for cancer treatment. Most anticancer substances generate a toxic effect on the human body. In order to check the toxicity and therapeutic dosage of these chemicals, the study of ligand binding to plasma proteins is very relevant. The present work presents the first comparative analysis of the binding of one of the 5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazinium derivatives (Salt1) with human serum albumin (HSA), α-1-acid glycoprotein (AGP) and human gamma globulin (HGG), assessed using fluorescence, UV-Vis and CD spectroscopy. In order to mimic in vivo ligand–protein binding, control normal serum (CNS) was used. Based on the obtained data, the Salt1 binding sites in the tertiary structure of all plasma proteins and control normal serum were identified. Both the association constants (K_a) and the number of binding site classes (n) were calculated using the Klotz method. The strongest complex formed was Salt1–AGP_complex (K_a = 7.35·10⁴ and 7.86·10⁴ mol·L⁻¹ at excitation wavelengths λ_ex of 275 and 295 nm, respectively). Lower values were obtained for Salt1–HSA_complex (K_a = 2.45·10⁴ and 2.71·10⁴ mol·L⁻¹) and Salt1–HGG_complex (K_a = 1.41·10⁴ and 1.33·10⁴ mol·L⁻¹) at excitation wavelengths λ_ex of 275 and 295 nm, respectively, which is a positive phenomenon and contributes to the prolonged action of the drug. Salt1 probably binds to the HSA molecule in Sudlow sites I and II; for the remaining plasma proteins studied, only one binding site was observed. Moreover, using circular dichroism (CD), fluorescence and UV-Vis spectroscopy, no effect on the secondary and tertiary structures of proteins in the absence or presence of Salt1 has been demonstrated. Despite the fact that the conducted studies are basic, from the scientific point of view they are novel and encourage further in vitro and in vivo investigations. As a next part of the study (Part 2), the second new synthetized quinobenzothiazine derivative (Salt2) will be analyzed and published.     

Antioxidant and Starch-Hydrolyzing Enzymes Inhibitory Properties of Striga-Resistant Yellow-Orange Maize Hybrids

Most of the health benefits derived from cereals are attributed to their bioactive compounds. This study evaluated the levels of the bioactive compounds, and the antioxidant and starch-hydrolyzing enzymes inhibitory properties of six pipeline Striga-resistant yellow-orange maize hybrids (coded AS1828-1, 4, 6, 8, 9, 11) in vitro. The maize hybrids were grown at the International Institute of Tropical Agriculture (IITA), Nigeria. The bioactive compounds (total phenolics, tannins, flavonoids, and phytate) levels, antioxidant (DPPH^• and ABTS^•+ scavenging capacity and reducing power) and starch-hydrolyzing enzymes (α-amylase and α-glucosidase) inhibitory activities of the maize hybrids were determined by spectrophotometry. At the same time, carotenoids were quantified using a reverse-phase HPLC system. The ranges of the bioactive compounds were: 11.25–14.14 mg GAE/g (total phenolics), 3.62–4.67 mg QE/g (total flavonoids), 3.63–6.29 mg/g (tannins), 3.66–4.31% (phytate), 8.92–12.11 µg/g (total xanthophylls), 2.42–2.89 µg/g (total β-carotene), and 3.17–3.77 µg/g (total provitamin A carotenoids). Extracts of the maize hybrids scavenged DPPH^• (SC₅₀: 9.07–26.35 mg/mL) and ABTS^•+ (2.65–7.68 TEAC mmol/g), reduced Fe³⁺ to Fe²⁺ (0.25 ± 0.64–0.43 ± 0.01 mg GAE/g), and inhibited α-amylase and α-glucosidase, with IC₅₀ ranges of 26.28–52.55 mg/mL and 47.72–63.98 mg/mL, respectively. Among the six clones of the maize hybrids, AS1828-9 had the highest (p < 0.05) levels of tannins and phytate and the strongest antioxidant and starch-hydrolyzing enzymes inhibitory activities. Significant correlations were observed between total phenolics and the following: ABTS^•+ (p < 0.01, r = 0.757), DPPH^• SC₅₀ (p < 0.01, r = −0.867), reducing power (p < 0.05, r = 0.633), α-amylase IC₅₀ (p < 0.01, r = −0.836) and α-glucosidase IC₅₀ (p < 0.05, r = −0.582). Hence, the Striga-resistant yellow-orange maize hybrids (especially AS1828-9) may be beneficial for alleviating oxidative stress and postprandial hyperglycemia.     

Quercetin-3-O-glucuronide in the Ethanol Extract of Lotus Leaf (Nelumbo nucifera) Enhances Sleep Quantity and Quality in a Rodent Model via a GABAergic Mechanism

Current pharmacological treatments for insomnia carry several and long-term side effects. Therefore, natural products without side effects are warranted. In this study, the sleep-promoting activity of the lotus leaf (Nelumbo nucifera) extract was assessed using ICR mice and Sprague Dawley rats. A pentobarbital-induced sleep test and electroencephalogram analysis were conducted to measure sleep latency time, duration, and sleep architecture. The action mechanism of the extract was evaluated through ligand binding experiments. A high dose (300 mg/kg) of the ethanolic lotus leaf extract significantly increased sleep duration compared to the normal group (p < 0.01). Administration of low (150 mg/kg) and high doses (300 mg/kg) of the extract significantly increased sleep quality, especially the relative power of theta waves (p < 0.05), compared to the normal group. Furthermore, caffeine and lotus leaf extract administration significantly recovered caffeine-induced sleep disruption (p < 0.001), and the sleep quality was similar to that of the normal group. Additionally, ligand binding assay using [³H]-flumazenil revealed that quercetin-3-O-glucuronide contained in the lotus leaf extract (77.27 μg/mg of extract) enhanced sleep by binding to GABA_A receptors. Collectively, these results indicated that the lotus leaf extract, particularly quercetin-3-O-glucuronide, exhibits sleep quantity- and quality-enhancing activity via the GABAergic pathway.     

FeII4L4 tetrahedron binds and aggregates DNA G-quadruplexes

Since the discovery of the G-quadruplex (G4) structure in telomeres in 1980s, studies have established the role it plays in various biological processes. Here we report binding between DNA G4 and a self-assembled tetrahedral metal-organic cage 1 and consequent formation of aggregates, whereby the cage protects the DNA G4 from cleavage by S1 nuclease. We monitor DNA–cage interaction using fluorescence spectroscopy, firstly by quenching of a fluorescent label appended to the 5′ end of G4. Secondly, we detect the decrease in fluorescence of the G4-selective dyes thioflavin-T and Zn-PPIX bound to various DNA G4 sequences following the addition of cage 1. Our results demonstrate that 1 interacts with a wide range of G4s. Moreover, gel electrophoresis, circular dichroism and dynamic light scattering measurements establish the binding of 1 to G4 and indicate the formation of aggregate structures. Finally, we find that DNA G4 contained in an aggregate of cage 1 is protected from cleavage by S1 nuclease.

We find Fe^II₄L₄ binds to G-quadruplex and forms aggregates. G-quadruplex in the aggregates is protected from digestion by S₁ nuclease.