Glycosides of Nadifloxacin-Synthesis and Antibacterial Activities against Methicillin-Resistant Staphylococcus aureus.

The increase in the number of bacteria that are resistant to multiple antibiotics poses a serious clinical problem that threatens the health of humans worldwide. Nadifloxacin (1) is a highly potent antibacterial agent with broad-spectrum activity. However, its poor aqueous solubility has limited its use to topical applications. To increase its solubility, it was glycosylated herein to form a range of trans-linked (3a-e) and cis-linked (7a,b) glycosides, each of which was prepared and purified to afford single anomers. The seven glycoside derivatives (3a-e, 7a,b) were examined for potency against eight strains of S. aureus, four of which were methicillin-resistant. Although less potent than free nadifloxacin (1), the α-L-arabinofuransoside (3a) was effective against all strains that were tested (minimum inhibitory concentrations of 1-8 µg/mL compared to 0.1-0.25 µg/mL for nadifloxacin), demonstrating the potential of this glycoside as an antibacterial agent. Estimation of Log P as well as observations made during preparation of these compounds reveal that the solubilities of the glycosides were greatly improved compared with nadifloxacin (1), raising the prospect of its use in oral applications.

Topological impact of noncanonical DNA structures on Klenow fragment of DNA polymerase.

Noncanonical DNA structures that stall DNA replication can cause errors in genomic DNA. Here, we investigated how the noncanonical structures formed by sequences in genes associated with a number of diseases impacted DNA polymerization by the Klenow fragment of DNA polymerase. Replication of a DNA sequence forming an i-motif from a telomere, hypoxia-induced transcription factor, and an insulin-linked polymorphic region was effectively inhibited. On the other hand, replication of a mixed-type G-quadruplex (G4) from a telomere was less inhibited than that of the antiparallel type or parallel type. Interestingly, the i-motif was a better inhibitor of replication than were mixed-type G4s or hairpin structures, even though all had similar thermodynamic stabilities. These results indicate that both the stability and topology of structures formed in DNA templates impact the processivity of a DNA polymerase. This suggests that i-motif formation may trigger genomic instability by stalling the replication of DNA, causing intractable diseases.

Structural Studies Reveal Enantiospecific Recognition of a DNA G-Quadruplex by a Ruthenium Polypyridyl Complex.

By using X-ray crystallography, we show that the complexes Λ/Δ-[Ru(TAP)2 (11-CN-dppz)]2+ (TAP=1,4,5,8-tetraazaphenanthrene, dppz=dipyridophenazine) bind DNA G-quadruplex in an enantiospecific manner that parallels the specificity of these complexes with duplex DNA. The Λ complex crystallises with the normally parallel stranded d(TAGGGTTA) tetraplex to give the first such antiparallel strand assembly in which syn-guanosine is adjacent to the complex at the 5' end of the quadruplex core. SRCD measurements confirm that the same conformational switch occurs in solution. The Δ enantiomer, by contrast, is present in the structure but stacked at the ends of the assembly. In addition, we report the structure of Λ-[Ru(phen)2 (11-CN-dppz)]2+ bound to d(TCGGCGCCGA), a duplex-forming sequence, and use both structural models to provide insight into the motif-specific luminescence response of the isostructural phen analogue enantiomers.

X-ray Crystal Structures Show DNA Stacking Advantage of Terminal Nitrile Substitution in Ru-dppz Complexes.

The new complexes [Ru(TAP)2 (11-CN-dppz)]2+ , [Ru(TAP)2 (11-Br-dppz)]2+ and [Ru(TAP)2 (11,12-diCN-dppz)]2+ are reported. The addition of nitrile substituents to the dppz ligand of the DNA photo-oxidising complex [Ru(TAP)2 (dppz)]2+ promote π-stacking interactions and ordered binding to DNA, as shown by X-ray crystallography. The structure of Λ-[Ru(TAP)2 (11-CN-dppz)]2+ with the DNA duplex d(TCGGCGCCGA)2 shows, for the first time with this class of complex, a closed intercalation cavity with an AT base pair at the terminus. The structure obtained is compared to that formed with the 11-Br and 11,12-dinitrile derivatives, highlighting the stabilization of syn guanine by this enantiomer when the terminal base pair is GC. In contrast the AT base pair has the normal Watson-Crick orientation, highlighting the difference in charge distribution between the two purine bases and the complementarity of the dppz-purine interaction. The asymmetry of the cavity highlights the importance of the purine-dppz-purine stacking interaction.

Delta chirality ruthenium 'light-switch' complexes can bind in the minor groove of DNA with five different binding modes.

[Ru(phen)2(dppz)]2+ has been studied since the 1990s due to its 'light-switch' properties. It can be used as a luminescent DNA probe, with emission switched on through DNA binding. The luminescence observed is dependent on the solvent accessibility of the pyrazine nitrogen atoms, and therefore is sensitive to changes in both binding site of the cation and chromophore orientation. The compound is also chiral, and there are distinct differences between the enantiomers in terms of the emission behaviour when bound to a variety of DNA sequences. Whilst a number of binary DNA-complex X-ray crystal structures are available, most include the Λ enantiomer and there is very little structural information about binding of the Δ enantiomer. Here, we present the first X-ray crystal structure of a Δ enantiomer bound to well-matched DNA, in the absence of the other, Λ enantiomer. We show how the binding site observed here can be related to a more general pattern of motifs in the crystallographic literature and propose that the Δ enantiomer can bind with five different binding modes, offering a new hypothesis for the interpretation of solution data.

Guanine Can Direct Binding Specificity of Ru-dipyridophenazine (dppz) Complexes to DNA through Steric Effects.

X-ray crystal structures of three Λ-[Ru(L)2 dppz]2+ complexes (dppz=dipyridophenazine; L=1,10-phenanthroline (phen), 2,2'-bipyridine (bpy)) bound to d((5BrC)GGC/GCCG) showed the compounds intercalated at a 5'-CG-3' step. The compounds bind through canted intercalation, with the binding angle determined by the guanine NH2 group, in contrast to symmetrical intercalation previously observed at 5'-TA-3' sites. This result suggests that canted intercalation is preferred at 5'-CG-3' sites even though the site itself is symmetrical, and we hypothesise that symmetrical intercalation in a 5'-CG-3' step could give rise to a longer luminescence lifetime than canted intercalation.

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.

Long-Lived Excited-State Dynamics of i-Motif Structures Probed by Time-Resolved Infrared Spectroscopy.

UV-generated excited states of cytosine (C) nucleobases are precursors to mutagenic photoproduct formation. The i-motif formed from C-rich sequences is known to exhibit high yields of long-lived excited states following UV absorption. Here the excited states of several i-motif structures have been characterized following 267 nm laser excitation using time-resolved infrared spectroscopy (TRIR). All structures possess a long-lived excited state of ∼300 ps and notably in some cases decays greater than 1 ns are observed. These unusually long-lived lifetimes are attributed to the interdigitated DNA structure which prevents direct base stacking overlap.

Controlled dehydration of a ruthenium complex-DNA crystal induces reversible DNA kinking.

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognized that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fiber diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyrido[3,2-a:2',3'-c]phenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven data sets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation.

X-ray crystal structure of rac-[Ru(phen)2dppz]2+ with d(ATGCAT)2 shows enantiomer orientations and water ordering.

We report an atomic resolution X-ray crystal structure containing both enantiomers of rac-[Ru(phen)2dppz](2+) with the d(ATGCAT)2 DNA duplex (phen = phenanthroline; dppz = dipyridophenazine). The first example of any enantiomeric pair crystallized with a DNA duplex shows different orientations of the Λ and Δ binding sites, separated by a clearly defined structured water monolayer. Job plots show that the same species is present in solution. Each enantiomer is bound at a TG/CA step and shows intercalation from the minor groove. One water molecule is directly located on one phenazine N atom in the Δ-enantiomer only.

Anticancer evaluation of new organometallic ruthenium(ii) flavone complexes.

Targeting multiple malignancy features such as angiogenesis, proliferation and metastasis with one molecule is an effective strategy in developing potent anticancer agents. Ruthenium metal complexation to bioactive scaffolds is reported to enhance their biological activities. Herein, we evaluate the impact of Ru chelation on the pharmacological activities of two bioactive flavones (1 and 2) as anticancer candidates. The novel Ru complexes (1Ru and 2Ru) caused a loss of their parent molecules' antiangiogenic activities in an endothelial cell tube formation assay. 1Ru enhanced the antiproliferative and antimigratory activities of its 4-oxoflavone 1 on MCF-7 breast cancer cells (IC50 = 66.15 ± 5 µM and 50% migration inhibition, p < 0.01 at 1 µM). 2Ru diminished 4-thioflavone's (2) cytotoxic activity on MCF-7 and MDA-MB-231 yet significantly enhanced 2's migration inhibition (p < 0.05) particularly on the MDA-MB-231 cell line. The test derivatives also showed non-intercalative interaction with VEGF and c-myc i-motif DNA sequences.

The influence of loops on the binding of the [Ru(phen)2dppz]2+ light-switch compound to i-motif DNA structures revealed by time-resolved spectroscopy.

Ultrafast time resolved infrared (TRIR) is used to report on the binding site of the "light-switch" complex [Ru(phen)2(dppz)]2+1 to i-motif structures in solution. Detailed information is provided due to perturbation of the local base vibrations by a 'Stark-like' effect which is used to establish the contribution of thymine base loop interactions to the binding site of 1 in this increasingly relevant DNA structure.

Stabilization of Long-Looped i-Motif DNA by Polypyridyl Ruthenium Complexes.

A spectroscopic study of the interactions of Λ- and Δ-[Ru(phen)2(dppz)]2+ with i-motif DNA containing thymine loops of various lengths. In the presence of i-motifs, the luminescence of the Λ enantiomer was enhanced much more than the Δ. Despite this, the effect of each enantiomer on i-motif thermal stability was comparable. The sequences most affected by [Ru(phen)2(dppz)]2+ were those with long thymine loops; this suggests that long-looped i-motifs are attractive targets for potential transition metal complex drugs and should be explored further in drug design.

Incorporation of a S-glycosidic linkage into a glyconucleoside changes the conformational preference of both furanose sugars.

A glyconucleoside containing a thioglycoside linkage, namely 1-(3-S-beta-D-ribofuranosyl-2,3-dideoxy-3-thio-beta-D-ribofuranosyl)-thymine, has been prepared through condensation of a suitably protected derivative of 3'-thiothymidine with an activated ribose sugar. NMR has been used to study the conformation of the S-disaccharide and the unmodified O-disaccharide. A full pseudorotational analysis showed that for the S-disaccharide, the ribose and deoxy ribose sugars have a preference for the south and north pucker, respectively; which is the reverse of what is seen for the O-disaccharide.

Amino-functionalized DNA: the properties of C5-amino-alkyl substituted 2'-deoxyuridines and their application in DNA triplex formation.

The incorporation of C5-amino-modified 2'-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2'-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl linker. Conformational parameters of the nucleosides and their pK(a) values were deduced using 1H NMR. All of them display the expected anti-conformation of the nucleoside with 2'-endo sugar puckers for the deoxyribose ring. A preference for the cisoid conformation for the Z-alkenyl analogue is found, while the E-alkenyl analogue exists exclusively as its transoid conformation. The pK(a) values range from 10.0 for the analogue with an aliphatic propyl linker to 8.5 for the propargylamino analogue. The analogues have been used for the synthesis of triple-helix forming oligonucleotides (TFOs) in which they replace thymidine in the natural sequence. Oligonucleotides containing the propargylamino analogue display the highest stability especially at low pH, while those containing analogues with propyl and especially Z-alkenyl linkers are destabilized to a great extent. TFOs containing the analogue with the E-alkenyl linker have stability similar to the unmodified structures. The chemical synthesis of TFOs containing the analogue, 5-(3-hydroxyprop-1-ynyl)-2'-deoxyuridine that possesses a neutral but polar side chain show a remarkable stability, which is higher than that of all TFOs containing the alkylamino or alkenylamino analogues and only slightly lower than that of TFOs containing the propargylamino analogue. Both the hydroxyl and propargylamino substitutions impart enhanced triple-helix stability relative to the analogous sequences containing C5-propynyl-2'-deoxyuridine. Furthermore, a similar dependence of stability on pH is found between TFOs containing the hydroxypropynyl modifications and those containing the propargylamino side chains. This suggests that the major factor responsible for stabilizing such triple helices is due to the presence of the alkyne with an attached electronegative group.

7,8-Dihydropyrido[2,3-d]pyrimidin-2-one; a bicyclic cytosine analogue capable of enhanced stabilisation of DNA duplexes.

Incorporation of a bicyclic cytosine analogue, 3-beta-D-(2'-deoxyribofuranosyl)-7,8-dihydropyrido[2,3-d]pyrimidine, into synthetic DNA duplexes results in a greatly enhanced thermal stability (3-4 degrees C per modification) compared to the corresponding unmodified duplex.

The importance of loop length on the stability of i-motif structures.

Using UV and srCD spectroscopy it is found that loop length within the i-motif structure is important for both thermal and pH stability, but in contrast to previous statements, it is the shorter loops that exhibit the highest stability.

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy.

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl-DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium.

I-motif formation in gene promoters: unusually stable formation in sequences complementary to known G-quadruplexes.

I-motif formation has been confirmed in a number of gene promoter sequences known to form G-quadruplex structures. I-motif formation can occur close to physiological temperature and pH for h-tert and PDGF-A. The i-motif structure formed by a HIF-1α promoter sequence shows unexpected stability near neutral pH.