Phi Ligand Research Articles

Binding Kinetics of DNA Intercalation by Small Rhodium Complexes

We characterize the kinetics and DNA binding properties of the small rhodium based molecules, [Rh(bpy)2(chrysi)]3+ (chrysi) and [Rh(bpy)2(phi)]3+ (phi). Both motifs bind to DNA. Phi contains an aromatic ring system known to intercalate between adjacent bases in a fairly sequence-neutral manner. Intercalation is weaker for chrysi due to the presence of an additional aromatic ring. Instead, chrysi has been shown to bind to DNA mismatches preferentially, ejecting and replacing the bases along the helical stack. We used optical tweezers on single DNA hairpin and long DNA molecules. Hairpin unfolding experiments on mismatch-containing sequences show that chrysi binds preferentially to sequences containing mismatches, stabilizing the hairpin. Long double stranded DNA was stretched at a constant external force in the presence of the ligand and a change in DNA extension was observed to be driven directly by ligand intercalation. Under the influence of increasing force, intercalation of the phi ligand into matched DNA is observed as expected. Surprisingly this intercalation into matched DNA was also observed for the chrysi ligand. Further analysis revealed the dissociation constants (Kd) of the chrysi and phi complexes to be on the order of 1 μM, though phi showed a significantly stronger intercalating affinity than chrysi.

Synthesis and spectroelectrochemistry of Ir(bpy)(phen)(phi)(3+), a tris(heteroleptic) metallointercalator.

A tris(heteroleptic) phenanthrenequinone diimine (phi) complex of Ir(III), Ir(bpy)(phen)(phi)(3+), was synthesized through the stepwise introduction of three different bidentate ligands, and the Lambda- and Delta-enantiomers were resolved and characterized by CD spectroscopy. Like other phi complexes, this tris(heteroleptic) iridium complex binds avidly to DNA by intercalation. Electrochemical studies show that Ir(bpy)(phen)(phi)(3+) undergoes a reversible one-electron reduction at E(0) = -0.025 V in 0.1 M TBAH/DMF (versus Ag/AgCl), and spectroelectrochemical studies indicate that this reduction is centered on the phi ligand. The EPR spectrum of electrochemically generated Ir(bpy)(phen)(phi)(2+) is consistent with a phi-based radical. The electrochemistry of Ir(bpy)(phen)(phi)(3+) was also probed at a DNA-modified electrode, where a DNA binding affinity of K = 1.1 x 10(6) M(-1) was measured. In contrast to Ir(bpy)(phen)(phi)(3+) free in solution, the complex bound to DNA undergoes a concerted two-electron reduction, to form a diradical species. On the basis of UV-visible and EPR spectroscopies, it is found that disproportionation of electrochemically generated Ir(bpy)(phen)(phi)(2+) occurs upon DNA binding. These results underscore the rich redox chemistry associated with metallointercalators bound to DNA.

Structure of a photoactive rhodium complex intercalated into DNA.

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.

Functional mimics of copper enzymes. Synthesis and stereochemical properties of the copper(II) complexes of a trinucleating ligand derived from l-histidine

The ligand piperazine-1,4-bis[4-( N-(1-acetoxy-3-(1-methyl-1 H-imidazol-4-yl))-2-propyl)- N-(1-methyl-1 H-imidazol-2-ylmethyl)aminobutyl] (PHI) was synthesized by a multistep procedure starting from N τ-methyl- l-histidine, piperazine-1,4-bis[4-(4-oxo-4-butanoic) acid] and 1-methyl-1 H-imidazole-2-carbaldehyde. This ligand has two potential tridentate, aminobis(imidazole) (A sites), and one bidentate, piperazine (B site), binding sites for metal ions and was employed for the synthesis of the binuclear [Cu 2PHI] 4+ and the trinuclear [Cu 3PHI] 6+ complexes, the latter of which features a coordination environment mimicking that present in the trinuclear clusters of the blue copper oxidases. For comparison purposes, the mononucleating ligand l- N α-(1-methyl-1 H-imidazol-2-ylmethyl)- N τ-methylhistidine methyl ester (IH) and its complex [CuIH] 2+ have been also prepared. These copper(II) model complexes are the first reported which are directly derived from chiral l-histidine residues. A detailed analysis of the UV–vis, CD and EPR spectra of the complexes has established that the Cu(II) centers bound to PHI A sites are square-pyramidal in solution, with the amino and one imidazole donor in the equatorial plane and the additional imidazole group bound axially. This arrangement implies the adoption of an unusual conformation of λ chirality by the l-histidine residue and is determined by the attempts to minimize steric interference between the substituents at the tertiary amine donor group and the histidine residue bearing the C-α substituent acetoxymethylene group of the bound PHI ligand. For the less sterically crowded secondary amine group of the bound IH ligand, the histidine C-α substituent can occupy a pseudoaxial position, so that in the complex [CuIH] 2+ the `normal' arrangement with three equatorial nitrogen donors and δ chirality in the l-histidine chelate ring occurs.

Functionalized Rhodium Intercalators for DNA Recognition.

A series of rhodium complexes containing the phenanthrenequinone diimine (phi) ligand have been prepared which bind DNA by intercalation and, upon photoactivation, promote DNA strand breaks. In this series, the ancillary, nonintercalating bipyridyl or phenanthroline ligands have been functionalized to yield complexes containing guanidinium, amido, or amino groups arranged with defined stereochemistry for site-specific interaction with the DNA bases. Lambda-1-[Rh(MGP)(2)phi](5+) (MGP = 4-(guanidylmethyl)-1,10-phenanthroline) site-specifically targets the 6-base pair sequence 5'-CATATG-3' with a binding affinity of 1 (+/-0.5) x 10(8) M(-)(1) while Delta-1-[Rh(MGP)(2)phi](5+) displays an affinity of 5 (+/-2) x 10(7) M(-)(1) for 5'-CATCTG-3'. Even though these two isomers target sites which differ by only a single base, binding is highly enantioselective. The specificity is derived chiefly from interactions of the pendant guanidinium groups with the DNA bases. For the racemates of 1-[Rh(GEB)(2)phi](5+) (GEB = (4-(2-guanidylethyl)-4'-methyl-2,2'-bipyridine) and 1-[Rh(GPB)(2)phi](5+) (GPB = (4-(2-guanidylpropyl)-4'-methyl-2,2'-bipyridine), photocleavage patterns also show the strongest site of photocleavage as 5'-CATCTG-3', the target site for Delta-1-[Rh(MGP)(2)phi](5+). Moreover, consistent with the dominance of the guanidinium groups in establishing specificity, significantly enhanced photocleavage is evident for the 1-positional isomer of these complexes, where the guanidinium moieties are directed toward the DNA (above and below the phi ligand) compared to the 2-isomer, in which the guanidinium groups are directed away from the DNA. In contrast to Lambda-1-[Rh(MGP)(2)phi](5+), Lambda-1-[Rh(GEB)(2)phi](5+) shows little cleavage at 5'-CATATG-3'; this sensitivity to linker length likely depends on the mode of recognition of 5'-CATATG-3' involving sequence-dependent unwinding of the DNA site. Analogous site-specificity or isomer-specificity is not evident with the complexes which contain pendant amido or amino functionalities. Instead these complexes appear to resemble the parent, unfunctionalized [Rh(phen)(2)phi](3+) with respect to recognition. Pendant guanidinium functionalities appear to be particularly advantageous in the construction of small molecules which bind DNA with site-specificity.

Differential DNA recognition by the enantiomers of 1-Rh(MGP)2 phi: a combination of shape selection and direct readout.

The enantiomers of the symmetric metallointercalator complex 1-Rh(MGP)2phi5+ [MGP = 4-(guanidylmethyl)-1,10-phenanthroline; phi = phenanthrenequinone diimine] bound to DNA decamer duplexes containing their respective 6 bp recognition sequences have been investigated using 1H NMR. Shape selection due to the chirality of the metal center and hydrogen-bonding contacts of ancillary guanidinium groups to 3'-G N7 atoms define the recognition by complexes which bind by intercalation to duplex DNA. The titration of Lambda-Rh into the self-complementary decamer containing the recognition sequence (5'-GACATATGTC-3', L1) resulted in one symmetric bound conformation observed in the 1H NMR spectrum, indicating that the DNA duplex retains its symmetry in the presence of the metal complex. Upfield chemical shifts of duplex imino protons and the disruption of the NOE base-sugar contacts defined the central T5-A6 intercalation site. The downfield shift of the G8 imino proton supports the conclusion that the pendant guanidinium arms make simultaneous H-bonding contacts to the N7 atoms of 3'-G8 bases on either side of the site. A variable-temperature study of a partially titrated sample (2:3 Lambda-Rh/L1) showed the exchange rate (kobs) at 298 K to be 68 s-1 and the activation barrier to exchange (DeltaG of association) to be 2.7 kcal/mol, a value comparable to the stacking energy of one base step. The results presented coupled with biochemical data are therefore consistent with binding models in which Lambda-1-Rh(MGP)2phi5+ (Lambda-Rh) traps the recognition site 5'-CATATG-3' in an unwound state, permitting intercalation centrally and hydrogen bonding to guanines at the first and sixth base pair positions. The data suggest a different model of binding and recognition by Delta-Rh. The titration of Delta-Rh into a DNA decamer containing the 6 bp recognition site (D1, 5'-CGCATCTGAC-3'; D2, 5'-GTCAGATGCG-3') resulted in two, distinct conformers, in slow exchange on the NMR time scale. The rate of exchange between the two conformers (kobs) at 298 K is 37 s-1, most likely due to partial dissociation between binding modes. The slower rate relative to Lambda-Rh association reflects the relative rigidity of the D1 and/or D2 sequence in comparison to L1. NOE cross-peaks between the intercalating phi ligand and protons of T5-C6, as well as the upfield shifts observed for imino protons at this step, serve to define the central T5-C6 step as the single site of intercalation. The downfield shift of the 3'-G imino protons indicates the complex makes hydrogen bond contacts with these bases. The complex, which is too small to span a 6 bp B-form DNA sequence, nonetheless makes major groove contacts with 3'-G bases to either side of the site. Notably, both 3'-guanine bases are necessary to impart site specificity and slow dissociation kinetics with the 5'-CATCTG-3' site, as evidenced by the extremely exchange-broadened two-dimensional NOESY spectra of Delta-Rh bound to modified duplexes containing N7-deazaguanine at either G8 or G18; the loss of one major groove contact completely abolishes specificity for 5'-CATCTG-3'. DNA chemical shifts upon binding and intermolecular NOE contacts therefore support a model in which Delta-Rh intercalates in one of two canted binding conformations. Within this model, each intercalation mode allows one guanidinium-guanine hydrogen bond at a time, while bringing the other arm close to the phosphate backbone.

Solution Structure of a Metallointercalator Bound Site Specifically to DNA

The solution structure of the complex of the metallointercalator Δ-α-[Rh[(R,R)-Me2trien]phi]3+ ((R,R)-Me2trien = 2R,9R-diamino-4,7-diazadecane; phi = 9,10-phenanthrenequinone diimine) bound to d(GAGTGCACTC)2 at 4 °C has been determined using 1H NMR spectroscopy coupled with restrained molecular dynamics calculations. Δ-α-[Rh[(R,R)-Me2trien]phi]3+ binds specifically to its designed site, 5‘-TGCA−3‘, with slow exchange kinetics. Using interproton distance restraints (including 70 intermolecular restraints) derived from NOESY spectra in both D2O and H2O and torsional restraints derived from ECOSY data, 20 disparate starting structures converged to a set of final structures with an average RMSD (between structures) of 1.0 A. As shown previously, binding occurs by deep intercalation of the phi ligand at the central 5‘-GC-3‘ step of the decamer, positioning the ancillary Me2trien ligand in the major groove. C2 symmetry is maintained with no kinking or bending of the DNA double helix. The binding site is deforme...

A Versatile Synthetic Approach to Rhodium(III) Diimine Metallointercalators: Condensation of o-Quinones with Coordinated cis-Ammines

A new route to the preparation of rhodium(III) diimine complexes which bind DNA by intercalation was developed by condensation of coordinated ammine ligands with o-quinones. Starting from cis-[Rh(L∧L)2(NH3)2]3+ (L∧L: 2,2‘-bipyridine or 1,10-phenanthroline) and the appropriate quinones the ligands 9,10-phenanthrenequinone diimine (phi) and 5,6-chrysenequinone diimine (chrysi) were introduced in high yields. The reactions are completed within hours at ambient temperature in MeCN/water mixtures, 0.1 M in NaOH. Experiments with enantiomerically pure Δ-[Rh(phen)2(NH3)2]3+ and 9,10-phenanthrenequinone showed that the configuration at the metal center is retained during the course of the reaction. Condensation of rhodium(III) tetraammine starting material with 9,10-phenanthrenequinone allows the selective introduction of one or two phi ligands, depending on the reaction conditions. The ability to incorporate specifically only one phi ligand makes this a promising approach for the synthesis of tris(heteroleptic)...

Structural examination of enantioselective intercalation: 1H NMR of Rh(en)2phi3+ isomers bound to d(GTGCAC)2.

The enantioselective recognition of d(GTGCAC)2 by delta- and lambda-Rh(en)2phi3+ (en = ethylenediamine; phi = 9,10-phenanthrenequinone diimine) has been examined in a series of one-dimensional (1D) and two-dimensional (2D) 500 MHz 1H NMR experiments both to extend our understanding of the basis for the enantioselective DNA binding and to gain structural information concerning intercalation by the octahedral metal complexes. delta-Rh(en)2phi3+ forms a symmetric 1:1 complex with d(GTGCAC)2, and the metal complex is in slow exchange with the oligodeoxynucleotide bound form at 295 K. The strong upfield shifts of the phi ligand's aromatic protons (0.6-1.3 ppm) are consistent with full intercalation of the phi ligand into the DNA base stack. 2D-NOESY experiments reveal a loss in internucleotide connectivity between G3 and C4 bases, while new NOE cross peaks are observed between the phi ligand and the G3 deoxyribose sugar. In contrast to binding by delta-Rh(en)2phi3+, the 1:1 lambda-Rh(en)2phi(3+)-d(GTGCAC)2 complex shows much broader resonances, and both metal complex and DNA protons appear to be in the intermediate exchange regime. The loss of C2 symmetry in the 1:1 complex is consistent with binding by lambda-Rh(en)2phi3+ at the T2G3 step. Although the enantiomeric metal complexes display different sequence selectivities and exchange characteristics, lambda- and delta-Rh(en)2phi3+ interact with the oligonucleotide duplex in a fundamentally similar manner, through the full intercalation of the phi ligand. Upfield movements in chemical shifts of phi protons are nearly identical for the two enantiomers, and both lambda- and delta-Rh(en)2phi3+ stabilize the duplex to melting by 5-10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Electric dichroism evidence for intercalation of optically active [Ru(bpy)2(phi)]2+(phi = 9,10-phenanthrenequinone diimine, bpy = 2,2′-bipyridyl) by DNA

Electric dichroism measurements on a solution containing DNA and enantiomeric [Ru(bpy)2(phi)]2+(phi = 9,10-phenanthrenequinone diimine, bpy = 2,2′-bipyridyl) at pH 7.0 have revealed that the phi ligand of the bound metal complex orients perpendicularly to the helical axis of DNA, supporting a structure where the metal complex is bound to DNA with the phi ligand intercalated between the base pairs.

DNA photocleavage by phenanthrenequinone diimine complexes of rhodium(III): shape-selective recognition and reaction

Rh(phen)_2phi^(3+) and Rh(phi)_2bpy^(3+) (phi = 9,10-phenanthrenequinone diimine, phen = 1, 10-phenanthroline, bpy = 2,2’-bipyridyl) bind double helical DNA avidly (K ≥ 10^7 M^(-1)) by intercalation and with photoactivation promote strand cleavage. Rh(phen)_2phi^(3+) and Rh(phi)_2bpy^(3+) unwind double helical DNA by 21° and 18°, respectively, per bound complex. The quantum yields for nucleic acid base release at 313 nm are 0.0012 for Rh(phen)_2phi^(3+) and 0.0003 for Rh(phi)_2bpy^(3+). While both complexes have similar photochemical properties, overall binding modes and affinities, their cleavage patterns, observed on ^(32)P-end-labeled DNA restriction fragments and oligonucleotide substrates, indicate substantially different recognition characteristics. Rh(phen)_2phi^(3+) binds DNA with some sequence selectivity, preferring 5’-pyrimidine-pyrimidine-purine-3’ sites and cleaving with 5’-asymmetry, while Rh(phi)_2bpy^(3+) binds in a predominantly sequence-neutral fashion. These differences in recognition characteristics may be understood based upon the different shapes of the complexes. Owing to steric interactions of the ancillary phenanthroline ligands, Rh(phen)_2phi^(3+) appears to bind preferentially to sites which are more open in the major groove; since no similar steric constraints arise with an ancillary phi ligand, Rh(phi)_2bpy^(3+) binds all sites with similar affinities. The shapes of these complexes also govern their chemistry of strand scission. Chemical modification studies and HPLC analyses of the DNA termini and monomeric products formed in the Rh(phi)^(3+) induced DNA cleavage reactions have been conducted to characterize the products formed upon photoreaction of the rhodium complexes with 5’-CTGGCATGCCAG-3’. For Rh(phen)_2phi^(3+), the primary products are oligomers containing 3’- and 5’-phosphate termini and nucleic acid bases (in stoichiometric proportion). For Rh(phi)_2bpy^(3+), these same products account for approximately 70% of the reaction, but in addition base propenoic acids and a terminus assigned as a 3’-phosphoglycaldehyde are obtained in a correlated amount (30% of reaction). The formation of base propenoic acids and 3’-phosphoglycaldehydes are found furthermore to depend upon oxygen concentration, while other products are oxygen-independent. The products obtained are consistent with photoreaction of Rh(phi)^(3+), intercalated in the major groove of DNA, via abstraction of a C3’-H atom of the deoxyribose. Subsequent addition of dioxygen to the C3’-H radical or solvation would lead to the degradation products obtained. The partitioning between the oxygen dependent and independent pathways of DNA strand scission is found to correlate best with how the shape of the complex limits access of dioxygen to the C-3’ position. While Rh(phi)_2bpy^(3+) was found to promote the oxygen-dependent pathway to an extent of approximately 30%, Rh(phen)_2phi^(3+), with ancillary phenanthrolines that overhang and shelter the C3’-position, appears to disfavor this pathway of DNA degradation. These studies underscore the importance of shape-selection in governing not only recognition but also reaction of molecules on the helix. Such an intimate relationship between recognition and reaction of molecules bound selectively to DNA requires consideration in understanding the reactions of DNA-binding proteins and small molecules.

Synthesis and characterization of physical, electronic, and photochemical aspects of 9,10-phenanthrenequinonediimine complexes of ruthenium(II) and rhodium(III)

A series of ruthenium(II) and rhodium(III) complexes containing the novel 9,10-phenanthrenequinone diimine (phi) ligand have been prepared and characterized with respect to structure, spectroscopy, and/or photochemical reactivity. Synthetic approaches to mixed-ligand complexes containing the phi ligand include direct addition of the protected N,N'-bis(trimethylsilyl)-phenanthrenequinone diimine ligand to the metal center of chelation first of diaminopohenanthrene followed by oxidation to the coordinated phi ligand