1-, 2-, and 4-ethynylpyrenes in the structure of twisted intercalating nucleic acids: structure, thermal stability, and fluorescence relationship

A postsynthetic, on-column Sonogashira reaction was applied on DNA molecules modified by 2- or 4-iodophenylmethylglycerol in the middle of the sequence, to give the corresponding ortho- and para-twisted intercalating nucleic acids (TINA) with 1-, 2-, and 4-ethynylpyrene residues. The convenient synthesis of 2- and 4-ethynylpyrenes started from the hydrogenolysis of pyrene that has had the sulfur removed and separation of 4,5,9,10-tetrahydropyrene and 1,2,3,6,7,8-hexahydropyrene, which were later converted to the final compounds by successive Friedel-Crafts acetylation, aromatization by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and a Vilsmeier-Haack-Arnold transformation followed by a Bodendorf fragmentation. Significant alterations in thermal stability of parallel triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in homopyrimidine TINAs. Thus, for para-TINAs the bulge insertion of an intercalator led to high thermal stability of Hoogsteen-type parallel triplexes and duplexes, whereas Watson-Crick-type duplexes were destabilized. In the case of ortho-TINA, both Hoogsteen and Watson-Crick-type complexes were stabilized. Alterations in the thermal stability were highly influenced by the ethynylpyrene isomers used. This also led to TINAs with different changes in fluorescence spectra depending on the secondary structures formed. Stokes shift of approximately 100 nm was detected for pyren-2-ylethynylphenyl derivatives, whereas values for 1- and 4-ethynylpyrenylphenyl conjugates were 10 and 40 nm, respectively. In contrast with para-TINAs, insertion of two ortho-TINAs opposite each other in the duplex as a pseudo-pair resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues, which was also accompanied with higher thermal stability.     

Substance P conjugated to CdTe quantum dots triggers cytosolic calcium concentration oscillations and induces quantum dots internalization in the pancreatic carcinoma cell line AR4-2J

Highly fluorescent CdTe quantum dots (QDs) stabilized by 3-mercaptopropionic acid were prepared by an aqueous solution approach and used as a fluorescent label to link substance P (SP) in studying the interaction of SP with NK-1 receptor, which was expressed on the AR4-2J cell line. Nonspecific adsorptions of CdTe QDs on the AR4-2J cell membrane were observed, whereas the QD-SP conjugates successfully crossed the cell membrane and entered the cytosol. SP is a neurotransmitter, and neurotransmitter-induced calcium concentration oscillation is a common phenomenon in diverse cells especially of secretory type. Cytosolic calcium concentration responses were studied in the AR4-2J cell line during stimulation with SP and QD-SP conjugates. The oscillations triggered by SP and QD-SP conjugates were dose-dependent and very similar. Such QD-SP conjugates readily internalized into the cytosol as would be expected of an active NK-1 ligand. Therefore QD-SP conjugates could be used successfully to study ligand and NK-1 receptor interactions in live cells. Our research may provide a meaningful reference for congener research.     

三联吡啶-萘酰亚胺二元化合物的合成及其与DNA的相互作用

本文合成了两种三联吡啶修饰的萘酰亚胺化合物NPI1和NPI2,并利用紫外-可见吸收光谱(UV-Vis)、圆二色光谱(CD)、荧光共振能量转移(FRET)等方法研究了它们与双链CT DNA和Htelo G-四链体DNA的相互作用。实验结果表明,化合物NPI1和NPI2对G-四链体DNA具有很好的结合能力和选择性,溶液中的碱金属离子种类和萘酰亚胺基团上的取代基对NPI1和NPI2与DNA的作用有很大的影响。在含K+的缓冲液中,NPI2与G-四链体的结合常数达到1.06×108 L/mol,是与双链CT DNA结合常数的268倍。圆二色谱结果表明在不含碱金属离子的溶液中,NPI1和NPI2可诱导Htelo DNA形成反平行结构G-四链体。Autodock分子对接模拟表明NPI1和NPI2可以通过堆积作用、静电作用、氢键等作用方式与G-四链体结合,使得它们对G-四链体具有很高亲和性(Ka>107 L/mol)。     

Highly sensitive electrochemical detection of proteins using aptamer-coated gold nanoparticles and surface enzyme reactions

A novel electrochemical detection methodology is described for the femtomolar detection of proteins which utilizes both DNA aptamer-functionalized nanoparticles and a surface enzymatic reaction. Immunoglobulin E (IgE) was used as a model protein biomarker, which possesses two distinct epitopes for antibody (anti-IgE) and DNA aptamer binding. A surface sandwich assay format was utilized involving the specific adsorption of IgE onto a gold electrode surface that was pre-modified with a monolayer of aptamer-nanoparticle conjugates followed by the specific interaction of alkaline phosphatase (ALP) conjugated anti-IgE. To clearly demonstrate the signal enhancement associated with nanoparticle use, anodic current measurements of the ALP catalyzed oxidation of the enzyme substrate 4-aminophenylphosphate (APP) were also compared with electrode surfaces upon which the aptamer was directly attached. The detection of an unlabelled protein at concentrations as low as 5 fM is a significant improvement compared to conventional electrochemical-based immunoassay approaches and provides a foundation for the practical use and incorporation of nanoparticle-enhanced detection into electrochemical biosensing technologies.     

Photophysical and Duplex‐DNA‐Binding Properties of Distamycin Dimers Based on 4,4′‐ and 2,2′‐Dialkoxyazobenzenes as the Core

Distamycin‐based tetrapeptide ( 1 ) was covalently tethered to both ends of the central dihydroxyazobenzene moiety at either the 2,2′ or 4,4′ positions. This afforded two isomeric, distamycin–azobenzene–distamycin systems, 2 (para) and 3 (ortho), both of them being photoisomerizable. Illumination of these conjugates in solution at approximately 360 nm induced photoisomerization and the time course of the process was followed by UV/Vis and ¹H NMR spectroscopy. The kinetics of the thermal reversion at various temperatures of cis to trans isomers of the conjugates obtained after photoillumination were also examined. This afforded the respective thermal‐activation parameters. Both the molecular architecture and the location of the substituent around the core azobenzene determined the rate and activation‐energy barrier for the cis‐to‐trans back‐isomerization of these conjugates in solution. Duplex–DNA binding of the conjugates and the changes in DNA‐binding efficiency upon photoisomerization was also examined by CD spectroscopy, thermal denaturation studies, and a Hoechst displacement assay. The conjugate 2 showed higher DNA‐binding affinity and a greater change in the DNA‐binding efficiency upon photoisomerization compared with its 2,2′‐disubstituted counterpart. The experimental findings were substantiated by using molecular‐docking studies involving each conjugate with a model duplex d[(GC(AT)₁₀CG)]₂ DNA molecule.     

吖啶-多胺类缀合物的合成、 抗肿瘤活性及与DNA键合作用

设计合成了系列吖啶-多胺类衍生物(ACP1~ACP6), 并通过四甲基偶氮唑蓝(MTT)染色法研究了化合物对K562(人白血病细胞)、 A549(人肺癌细胞)和Hela(人宫颈癌细胞)细胞株的体外抗肿瘤活性. 结果显示, 化合物对肿瘤细胞具有一定的抑制作用, 尤其是化合物ACP2的抗肿瘤活性优于阳性对照顺铂. 通过UV-Vis光谱、 荧光光谱、 圆二色谱和热变性实验研究了合成化合物与小牛胸腺DNA(Ct-DNA)的键合作用. 结果表明, 三乙烯四胺修饰的化合物ACP2具有较好的抗肿瘤活性, 与DNA分子具有较强的结合能力.     

In vitro pH-dependent drug release from N4-(4-carboxybutyryl)-1-beta-D-arabinofuranosylcytosine and its conjugate with poly-L-lysine or decylenediamine-dextran T70

N4-(4-Carboxybutyryl)-1-beta-D-arabinofuranosylcytosine (glu-ara-C), the conjugate of glu-ara-C and poly-L-lysine (PLL), (PLL-glu-ara-C), and the conjugate of glu-ara-C and decylenediamine-dextran T70 (T70-C10), (T70-C10-glu-ara-C), were prepared. Drug regeneration from glu-ara-C and the conjugates was investigated in buffered solutions of pH 4,5,7,7.4 and 8. The character of the drug release from the conjugates was different from that from glu-ara-C. Namely, the release of 1-beta-D-arabinofuranosylcytosine (ara-C) from glu-ara-C was accelerated under both weakly acidic and weakly basic conditions, while it was accelerated only under weakly basic conditions from the conjugates. Overall, the drug release profiles from the conjugates showed similar patterns. However, under neutral and weakly basic conditions, ara-C was regenerated more rapidly from PLL-glu-ara-C than from T70-C10-glu-ara-C. During the incubation of glu-ara-C and the conjugates under weakly acidic conditions, 1-beta-D-arabinofuranosyluracil (ara-U) was detected and its amount increased with time to similar extents.     

DNA dissociation and degradation at gold nanoparticle surfaces

The instability of DNA-functionalized gold nanoparticles (Au-DNA conjugates) upon exposure to high temperatures is characterized using fluorescence spectroscopy, gel electrophoresis and ion-exchange chromatography. Above 70 °C, aqueous Au-DNA conjugates decompose within hours due to both desorption of thiol-terminated DNA from the gold nanoparticle surface and chemical degradation of DNA in the presence of colloidal gold. Although the chemical mechanism for DNA degradation was not identified in this study, the gold surface participates directly in the cleavage reaction. These results have important implications for the use of Au-DNA conjugates in biotechnological and clinical applications that require high temperatures, such as polymerase chain reaction (PCR).     

Four-way-branched DNA-porphyrin conjugates for construction of four double-helix-DNA assembled structures

[reaction: see text] DNA-porphyrin conjugates having four DNA strands were designed and synthesized. Four double helices were assembled using two DNA-porphyrin conjugates and their complementary strands, and the formation of the four double-helix assembled structures with the two DNA-porphyrin units was examined by gel electrophoresis and spectroscopic analysis. The interaction between two porphyrin chromophores in the complex was investigated by measurement of fluorescence lifetimes, and the singlet energy transfer between the two different phorphyrin units (Zn-porphyrin and H2-porphyrin) was observed. These results indicate that multiple and different porphyrin chromophores can be integrated into the DNA structures by programming the sequences of the DNA strands.     

Photocontrolled DNA binding of a receptor-targeted organometallic ruthenium(II) complex

A photoactivated ruthenium(II) arene complex has been conjugated to two receptor-binding peptides, a dicarba analogue of octreotide and the Arg-Gly-Asp (RGD) tripeptide. These peptides can act as "tumor-targeting devices" since their receptors are overexpressed on the membranes of tumor cells. Both ruthenium-peptide conjugates are stable in aqueous solution in the dark, but upon irradiation with visible light, the pyridyl-derivatized peptides were selectively photodissociated from the ruthenium complex, as inferred by UV-vis and NMR spectroscopy. Importantly, the reactive aqua species generated from the conjugates, [(η(6)-p-cym)Ru(bpm)(H(2)O)](2+), reacted with the model DNA nucleobase 9-ethylguanine as well as with guanines of two DNA sequences, (5')dCATGGCT and (5')dAGCCATG. Interestingly, when irradiation was performed in the presence of the oligonucleotides, a new ruthenium adduct involving both guanines was formed as a consequence of the photodriven loss of p-cymene from the two monofunctional adducts. The release of the arene ligand and the formation of a ruthenated product with a multidentate binding mode might have important implications for the biological activity of such photoactivated ruthenium(II) arene complexes. Finally, photoreactions with the peptide-oligonucleotide hybrid, Phac-His-Gly-Met-linker-p(5')dCATGGCT, also led to arene release and to guanine adducts, including a GG chelate. The lack of interaction with the peptide fragment confirms the preference of such organometallic ruthenium(II) complexes for guanine over other potential biological ligands, such as histidine or methionine amino acids.     

Spectroscopic binding studies of novel fluorescent distamycin derivatives

Novel distamycin-porphyrin conjugates were synthesized and their interaction with calf thymus DNA was studied. Minor groove binding of the distamycin part of the molecule was confirmed. The porphyrin part of the conjugates exhibited intercalation and the non-specific electrostatic interaction with the phosphate groups of DNA.     

Preparation of an IMI dye (imidazole functional group) containing a 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole fluorophore for labeling of phosphomonoesters

We are studying dye-imidazole conjugates ("IMI dyes") as reagents for labeling phosphomonoesters such as nucleotides. Previously we have employed a BODIPY dye in our IMI reagents, and analyzed the labeled products by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) involving an argon ion laser. (The BODIPY fluorophore is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Here we broaden the technology by preparing a DBD-IMI dye [DBD = 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole], and using a helium-cadmium laser. While DBD-IMI (IMI3) is about 50x more stable photolytically than a BODIPY-IMI dye (IMI2, a conjugate of a BODIPY dye with histamine, was tested), the detection limit for IMI2 (5.10(-11) M; S/N = 5, CE-LIF with an argon ion laser) is tenfold better than that for IMI3 (5.10(-10) M, S/N = 5, helium-cadmium laser). IMI3 conjugates of the four major DNA nucleotides were prepared and detected by CE-LIF.     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

A Novel Molecule: 1-(2,6 Dichlorobenzyl)-4-(2-(2-4-hydroxybenzylidene)hydrazinyl)pyridinium Chloride and its Interaction with DNA

Herein, a novel pyridine derivative, 1-(2,6 dichlorobenzyl)-4-(2-(2-4-hydroxybenzylidene)hydrazinyl)-pyridinium chloride (DHPC), was synthesized as a candidate drug molecule. Interaction of DHPC with DNA was used to explore its effect on DNA via Differential Pulse Voltammetry, Cyclic Voltammetry, and Electrochemical Impedance Spectroscopy. We demonstrated that oxidation signal of guanine bases of DNA decreased significantly while that of DHPC increased after its interaction with one another. Our candidate drug molecule exhibits LOD and LOQ, e.g., 1.5 μg/mL and 4.9 μg/mL, respectively. Toxicity effect value for DHPC (S%) was calculated as %31, demonstrating the candidate drug molecule's toxic effect on DNA.     

Hybridization and Melting Behavior of Peptide Nucleic Acid (PNA) Oligonucleotide Chimeras Conjugated to Gold Nanoparticles

Peptide nucleic acids (PNA) and PNA–DNA chimeras carrying thiol groups were used for surface functionalization of Au nanoparticles. Conjugation of PNA to citrate‐stabilized Au nanoparticles destabilized the nanoparticles causing them to precipitate. Addition of a tail of glutamic acid to the PNA prevented destabilization of the nanoparticles but resulted in loss of interaction with complementary sequences. Importantly, PNA–DNA chimeras gave stable conjugates with Au nanoparticles. The hybridization and melting properties of complexes formed from chimera–nanoparticle conjugates and oligonucleotide–nanoparticle conjugates are described for the first time. Similar to oligonucleotide–nanoparticle conjugates, conjugates with PNA–DNA chimeras gave sharper and more‐defined melting profiles than those obtained with unmodified oligonucleotides. In addition, mismatch discrimination was found to be more efficient than with unmodified oligonucleotides.     

Synthesis and DNA damaging ability of enediyne-polyamine conjugates

Polyamine-enediyne conjugates were synthesized and exhibited potent DNA damaging ability under physiological conditions. The extent of their activity was shown to depend upon the polyamine length that regulates the DNA binding affinity of the conjugates, and enhanced DNA damaging activities were observed under slightly acidic conditions.     

Highly selective mitochondria-targeting amphiphilic silicon(IV) phthalocyanines with axially ligated rhodamine B for photodynamic therapy

Two axially ligated rhodamine-Si(IV)-phthalocyanine (Rh-SiPc) conjugates, bearing one and two rhodamine B, were synthesized and their linear and two-photon photophysical, subcellular localization and photocytotoxic properties were studied. These Rh-SiPc conjugates exhibited an almost exclusive mitochondrial localizing property in human nasopharyngeal carcinoma (HK-1) cells and human cervical carcinoma (HeLa) cells. Strong photocytotoxic but low dark cytotoxic properties were also observed for the two Rh-SiPc conjugates toward the HK-1 cells. Using nuclei staining method and flow cytometric DNA content analysis, apoptotic cell death was induced by these conjugates upon photoactivation. This observation is consistent with their mitochondrial localization property. The observed properties of these conjugates qualify them as promising PDT agents.     

Molecular modeling of the binding mode of chiral metal complexes △- and (A)-[Co(phen)2dppz]3+ with B-DNA

Molecular modeling methods have been applied to the structural characterization of the interaction between chiral metal complexes [Co(phen)2dppz]3+ (where phen = 1, 10-phenanthroline, dppz = dipyrido[3,2-a: 2′, 3′-c]phenazine) and the oligonucleotide (B-DNA fragment). The natures of two kinds of the binding modes, which are currently intense controversy, have been explored. Barton proposed that there is enantio-selective DNA binding by the octahedral complexes and intercalative access by these complexes from the major groove; but Norden suggested that both enantiomers bind extremely strongly to DNA from the minor groove without any noticeable enantio-selectivity. Our results support and extend structural models based upon Norden's studies, and conflict with Barton's model.     

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

Antitumor activity of 4-amino and 8-methyl-4-(3diethylamino propylamino)pyrimido[4',5':4,5]thieno (2,3-b) quinolines

The interaction of 4-aminopyrimido [4',5':4,5] thieno (2,3-b) quinoline and 8-methyl-4-(3-diethylaminopropylamino) pyrimido [4',5':4,5] thieno (2,3-b) quinoline with DNA was studied by UV-Vis and fluorescence spectrophotometry as well as by hydrodynamic methods. On binding to DNA, the absorption spectra underwent bathochromic and hypochromic shifts and the fluorescence was quenched. These compounds are able to bind to DNA with an affinity of about 10(6) M(-1) for calf thymus DNA at ionic strength 0.01 M and their intercalating characteristic (lengthening of the DNA) depends upon the length of the chain. Binding to the GC-rich DNA of Micrococcus lysodeikticus was stronger than the binding to calf thymus DNA at ionic strength 0.01 M. The cytotoxicities of these compounds on leukemia HL-60, melanoma B16F10 and neuro 2a cells are quite similar and inhibition (IC50) is in the range of 0.992-3.968 microM. The anticancer efficacy against B16 melanoma, has provided evidence of major antitumor activity for 8-methyl-4-(3diethylaminopropylamino) pyrimido [4',5':4,5] thieno(2,3-b)quinoline. Single or multiple intraperitonial (i.p) doses of drug proved high level activity against the subcutaneous (s.c) grafted B16 melanoma, significantly increasing survival (p<0.001) and inhibiting tumor growth (T/C of 4%). This study offers a new intercalation functional group to DNA-targeted drug design.