ABSTRACT

Abstract

Abstract

A set of cyclic tetranuclear complexes of the metallacalix[4]arene type with formula [{Pt(en)(L)}₄]⁴⁺ (en=ethylenediamine; 2: LH=5-chloro-2-hydroxypyrimidine (5-Cl-Hpymo); 3: LH=5-bromo-2-hydroxypyrimidine (5-Br-Hpymo); 4: LH=5-iodo-2-hydroxypyrimidine (5-I-Hpymo)) have been obtained from the reaction between cis-protected square-planar [Pt(en)(H₂O)₂]²⁺ metal entities and LH in aqueous media. Additionally, the binding properties of 2, 3, 4 and their congener [{Pt(en)(L)}₄]⁴⁺ (1: LH=2-hydroxypyrimidine (Hpymo)) with calf thymus-DNA (ct-DNA) have been studied by using different techniques including circular and linear dichroism (CD and LD, respectively) and UV-visible absorbance spectroscopies, gel electrophoresis, fluorescence competitive-binding studies and atomic force microscopy (AFM). The results are consistent with significant non-covalent interactions taking place between the polynuclear cyclic species and ct-DNA. Moreover, gel electrophoresis, linear dichroism titrations and AFM images of ct-DNA with metallacalixarenes show ct-DNA coiling at low metallacalixarene concentrations and upon subsequent increments in metallacalixarene concentration ct-DNA can be seen to uncoil with concomitant formation of long and inflexible ct-DNA structures.

Abstract

The linear dichroism of the visible wavelength transitions of retinal have been used to analyse linear dichroism spectra to determine the orientation of aromatic and peptide structural motifs of Bacteriorhodopsin incorporated into unilamellar soy bean liposomes. The results are consistent with the available X-ray data. This proves that visible light absorbing chromophores can be used to analyse linear dichroism data to give the orientation of membrane proteins in membrane mimicking environments. The work has been extended by screening a wide range of hydrophobic molecules with high extinction coefficients in transitions above 300 nm to find molecules that could be used as independent probes of liposome orientation for experiments involving proteins incorporated into liposomes. Three probes were found to have potential for future work: bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC₄), retinol and rhodamine B. All three can be used to determine the orientation of the porphyrin of cytochrome c, the aromatic residues of gramicidin and the helices of both proteins. The orientation parameter, S, for the liposomes varied from batch to batch of unilamellar liposomes prepared by extruding through a 100 nm membrane. The value and variation in S was 0.030 ± 0.010. Repeat experiments with the same batch of liposomes showed less variation. Film LD data were measured for DiBAC₄ and rhodamine B to determine the polarisations of their long wavelength transitions.

The DNA binding of a dicationic pyridylimine-based dicopper(I) metallosupramolecular cylinder is reported together with its ability to act as an artificial nuclease. The cylinder binds strongly to DNA; more strongly than the spherical dication [Ru(phen)₃]²⁺ (phen=1,10-phenanthroline), but more weakly than the corresponding tetracationic cylinders. DNA coiling effects are not observed with this dication, in contrast to the situation with the previously reported tetracationic cylinder involving a similar ligand. Linear dichroism (LD) data suggests that the dicopper cylinder binds in a different orientation from that of the tetracationic iron cylinder. Furthermore, the dicopper cylinder shows DNA-cleavage activity in the presence of peroxide. Of particular note is that the cylinder displays a marked and unusual ability to cleave both DNA strands at the same site, probably reflecting its dinuclear nature and possibly its mode of binding to the DNA.

A platinum metal complex in which terpyridine joins estradiol (via an ethynyl link) to a platinum with a labile ligand (chloride) has been designed, synthesised and its X-ray crystal structure determined. The aim of this work was to link a targeting motif (in this case estrogen) to a metal-based biomolecule recognition unit (the platinum moiety). The target molecule: 17α-[4′-ethynyl-2,2′:6′,2′-terpyridine]-17β-estradiol platinum(II) chloride (PtEEtpy) has been shown to bind to both human and bovine serum albumin (SA) and to DNA. FTICR mass spectrometry shows that the bimolecular units are in each case linked through coordination to the platinum with displacement of the chloride ligand. Circular dichroism indicates that a termolecular entity involving PtEEtpy, SA and DNA is formed. A range of electrospray mass spectrometry experiments showed that the PtEEtpy complex breaks and forms coordination bonds relatively easily. A whole cell estrogen receptor assay in an estrogen receptor positive cell (MCF-7) confirms binding of both EEtpy and PtEEtpy to the estrogen receptor in cells. The work demonstrates the concept of linking a targeting moiety (in this case estrogen) to a DNA binding agent.

A thermostatted micro volume Couette cell has been designed to enable linear dichroism (LD) data to be collected at a range of temperatures. The cell is a development of the traditional Couette flow LD cell and includes the recent development of micro-volume LD (20–40 μL) coupled with the addition of a heating element, temperature probe and controller. This new micro volume Couette LD cell opens the way not only to the LD analysis of systems where sample volume is critical, but also for the LD analysis of temperature sensitive samples. The polymerization of the microtubule protein tubulin has been followed in a range of different conditions using the thermostatted micro volume Couette LD cell. The focusing lenses on the cell, which are required for the microvolume cell, have the side benefit of significantly reducing the light-scattering artifacts caused by the large size of tubulin microtubules. It is now possible to monitor real-time polymerization and depolymerization kinetics, and any structural rearrangements of chromophores within the polymer. In the case of tubulin, the LD spectra revealed a greater change in the orientation of tryptophan residues at ∼290 nm during polymerization compared to other contributing chromophores—guanine, phenylalanine, and tyrosine. The improvements in instrumental design have also allowed LD spectra of tubulin to be collected down to ∼230 nm (previous data have only been available from the near UV region), which means that some indication of protein backbone-orientation changes are now available. It was observed during this work that apparent LD intensity maxima are in fact artifacts when the high-tension voltage is high. The onset of such artifacts has been observed at much lower voltages with light-scattering fibrous proteins (including tubulin) than with nonscattering samples. Therefore, caution must be used when interpreting LD data collected with medium to high photomultiplier tube voltages. Chirality, 2006. © 2006 Wiley-Liss, Inc.

The binding of single-stranded DNAs and a neutral DNA analogue (peptide nucleic acid, PNA) to single-walled carbon nanotubes in solution phase has been probed by absorbance spectroscopy and linear dichroism. The nanotubes are solubilised by aqueous sodium dodecyl sulfate, in which the nucleic acids also dissolve. The linear dichroism (LD) of the nanotubes, when subtracted from that due to the nanotubes/nucleic acid samples, gives the LD of the bound nucleic acid. The binding of the single-stranded DNA to the single-walled nanotubes is quite different from that previously observed for double-stranded DNA. It is likely that the nucleic acid bases lie flat on the nanotube surface with the backbone wrapping round the nanotube at an oblique angle in the region of 45°. The net effect is like beads on a string. The base orientation with the single-stranded PNA is inverted with respect to that of the single-stranded DNA, as shown by their oppositely signed LD signals.

[Fe(1,10-phenanthroline)₃]²⁺ is known to racemise in solution and DNA is known to shift the equilibrium point of the Δ/Λ-[Fe(1,10-phenanthroline)₃]²⁺ mixture from 50:50 to a position favouring a slight excess of the Δ enantiomer. In this paper it is shown that DNA catalyses the racemisation reaction, presumably by providing vibrational energy to species that bind in a less favoured mode for that enantiomer. The racemisation of [Co(1,10-phenanthroline)₃]³⁺, however, is not enhanced by DNA unless [Co(1,10-phenanthroline)₃]²⁺ is also added to the solution. In this case, the DNA acts as a template to facilitate the Co^II Co^III electron transfer. Racemisation occurs while the [Co(1,10-phenanthroline)₃]ⁿ⁺ is in the labile Co^II form.

The polyamines spermine and spermidine cause a range of structural effects on DNA. [Co(NH₃)₆]³⁺ is known to mimic many of these effects and we have shown previously that (+)-[Co(en)₃]³⁺ and (−)-[Co(en)₃]³⁺ (en = ethylenediamine) do so as well. In this paper the effects of mono- and disubstitution of the Co^III ammines by aqua, halo and nitro groups is explored. The overall effectiveness of the amines in conferring thermal stability on the ct-DNA proceeds in the order: spermine > (+)-[Co(en)₃]³⁺ ≈ (−)-[Co(en)₃]³⁺ ≈ [Co(NH₃)₆]³⁺ ≈ spermidine > [Pt(NH₃)₄]²⁺ > [Co(NH₃)₅H₂O]³⁺ ≈ [Co(NH₃)₅Cl]²⁺ ≈ [Co(NH₃)₅Br]²⁺ > cis-[Co(en)₂NH₃Cl]²⁺ > [Co(NH₃)₅(NO₂)]²⁺ ≈ trans-[Co(NH₃)₄(H₂O)₂]³⁺; whereas the BZ transition-induction ordering is: spermine ≈ [Co(NH₃)₆]³⁺ > (+)-[Co(en)₃]³⁺ ≈ [Co(NH₃)₅(H₂O)]³⁺ > (−)-[Co(en)₃]³⁺ > [Co(NH₃)₅Br]²⁺ > [Co(NH₃)₅Cl]²⁺, with the other amines failing to cause the transition under the 11 mM salt concentration and temperature ramp conditions used; the DNA bending ranking is: spermine > [Co(NH₃)₆]³⁺ > (+)-[Co(en)₃]³⁺ > (−)-[Co(en)₃]³⁺ > [Co(NH₃)₅(H₂O)]³⁺ > trans-[Co(NH₃)₄(H₂O)₂]³⁺ > spermidine, with the other 2+ amines (the aquo complexes will be 2+ at neutral pH), especially the bromo and nitro compounds, having comparatively little effect. While the presentation of NNN triangular faces is found to be important in the BZ transition, the fact that the halo ligands are probably substituted by the DNA phosphates plays a role, especially in the DNA bending, where deep penetration of the major groove is concluded to be required. A favourable interaction between the backbone and the aquo ligands may enhance the BZ transition.

Many polycationic species bind to DNA and induce structural changes. The work reported here is the first phase of a program whose long-term aim is to create a class of simple and inexpensive sequence-selective compounds that will enable enhanced DNA structure control for a wide range of applications. Three classes of molecule have been included in this work: the polyamine spermine (charge: 4⁺) and spermidine (charge: 3⁺) (which are known to induce a wide range of DNA conformational changes but whose binding modes are still not well understood); cobalt (III) ammine transition metal complexes as potential polyamine mimics and [Fe(H₂O)₆]³⁺; and the first member of a new class of di-metallo tris-chelated cylinders of helical structure (charge 4⁺). Temperature-dependent absorption, circular dichroism, linear dichroism, gel electrophoresis, and molecular modeling data are presented. The cobalt ammines prove to be effective polyamine mimics, although their binding appears to be restricted to backbone and major groove. All the ligands stabilize the DNA, but the 4⁺ di-iron tris-chelate does so comparatively weakly and seems to have a preference for single-stranded DNA. All the molecules studied bend the DNA, with the di-iron tris-chelate having a particularly dramatic effect even at very low drug load. Chirality 12:221–236, 2000. © 2000 Wiley-Liss, Inc.