Ruthenium Complexes

Introduction

Experimental protocols

Materials
DNA synthesis, purification, and quantification
Rhodium complex synthesis
Photocleavage titrations
Binding constant determination
MALDI-TOF mass spectrometry
Fluorescence spectroscopy
Structural modeling
Cell culture
Cellular proliferation ELISA

The solvent was removed in vacuo and the product was purified by alumina column chromatography. The resulting midpoint value (ie, the logarithm of [rhodium complex] . at the inflection point of the curve) was converted to concentration units ([Rh50 %)]).

Figure 1.6. Chemical structures of [Rh(L 2 )(chrysi)] 3+ complexes surveyed in this study.

Results

Binding affinities of metal complexes at single base mismatches

In the case of Rh(DIP)2chrysi3+, the quantification of the fraction of DNA bound to the metal complex is complicated by the presence of a slower band above the base. Based on the binding constant of Rh(bpy)2chrysi3+, the binding constant of Rh(NH3)4chrysi3+.

Figure 1.8. Autoradiogram of a denaturing 20% polyacrylamide electrophoresis gel show- show-ing a representative photocleavage titration

Inhibition of cellular proliferation by enzyme-linked immunosorbent

The difference spectra (ie, the difference in intensity, I, between the spectra recorded in the presence of 100 nM and 1 µM DNA, as well as the difference in I between the spectra in the absence and presence of 1 µM DNA) are shown in the bottom row. The inhibition difference is the difference of the normalized percentages of cell proliferation for the two cell lines, HCT116O vs. HCT116N.

Figure 1.14. Emission spectra of Rh(DAP) 2 chrysi 3+ in the absence and presence of matched (top left) or mismatched (top right) DNA (excitation wavelength = 470 nm).

Discussion

Rh(phen)2chrysi3+, intermediate in size, shows binding affinities for the mismatches that are an order of magnitude lower than that of the bpy derivative but more than an order of magnitude higher than that of the DIP complex. Importantly, the DNA mismatch binding affinities of the Rh(L)2chrysi3+ family correspond well with the differential biological effects seen between the repair-proficient HCT116N and repair-deficient HCT116O cell lines.

Figure 1.21. Crystal and model structures of rhodium metalloinsertors bound to the mismatch site

Conclusions

Materials

Ruthenium complexes were prepared and enantiomers separated by previously reported methods; all complexes were used as chloride salts.22–24,39 The oligonucleotides used for measurements of steady state luminescence and excited state lifetimes were synthesized on an ABI 3400 DNA synthesizer (Applied Biosystems) and purified as previously described.40 The copper complex Cu (phen) )22+ was generated in situ by reacting the phen ligand with CuCl2 in a ratio of 3:1.41,42.

Steady state fluorescence

Time-resolved fluorescence

Results and discussion

Steady state luminescence of rac-, ∆-, and Λ-Ru(bpy) 2 dppz 2+ bound
Comparison with other DNA-binding fluorophores
Luminescence behavior of Ru with different base mismatches
Excited state lifetimes of Ru
Luminescence response of Ru toDNA in the presence of Cu(phen) 2+ 2 46

To further elucidate the luminescent features of the Ru complexes bound to DNA defects, we investigated their excited state lifetimes in the presence of the 27-mer oligonucleotides. Several observations suggest that binding of Ru in the mismatch or abasic sites occurs in a manner analogous to that of the metalloinsertor Rh(bpy)2chrysi3+ (that is, insertion from the minor groove): (i) the correlation of the luminescent enhancement with the thermodynamic instability of mismatched sites, (ii) the preferential quenching of the enhanced luminescence at defects with Cu(phen)22+. Simple fluorescent pyrimidine analogs detect the presence of DNA abasic sites. Journal of the American Chemical Society.

First observation of the key intermediate in the “light switch” mechanism of [Ru(phen)2dppz]2+.

Figure 2.3. Comparisons with common DNA-binding fluorophores. Structures of EB and TO-PRO-3 are shown along with plots of the integrated emission intensity of EB (100 nM) and TO-PRO-3 (100 nM) in the presence of 27-mer duplex DNAs (Figure 2.2; λ ex for EB

Experimental procedures

Materials
Crystallization and data collection
Structure determination and refinement
Steady state fluorescence

The data for crystal 1 were collected from a flash-cooled crystal at 100 K on an R-axis IV imaging plate using Cu Kα radiation generated by a Rigaku RU-H3RHB rotating-anode generator with dual-focus mirrors and a Ni filter is produced. High-resolution data were subsequently collected from another crystal on beamline 12-2 at the Stanford Synchrotron Radiation Laboratory (Menlo Park, CA; λ = 0.7749 ˚A, 100 K, PILATUS 6M detector). The data was processed with MOSFLM or XDS22, and SCALA from the CCP4 suite of programs.23.

Data for crystal2 were collected from a flash-cooled crystal at 100 K on the Rigaku diffractometer described above, processed with XDS,22 POINTLESS and SCALA.23 3.2.3 Structure determination and refinement.

Results and discussion

Cocrystallization of ∆-Ru(bpy) 2 dppz 2+ with DNA
Structure 1
Structure 2
Differences and similarities between the two structures
Comparisons to other structures
Solution luminescence

Interestingly, despite an overall similarity between the two structures, the orientation of the dppz ligand is quite different. An AT base pair (blue) is extruded from the helix by an inserted ruthenium complex. The intercalation of the dppz ligand is so deep that the end most distal to the ruthenium center projects into the major groove.

However, in this structure the cytidine is positioned in the minor groove and sandwiched between the ruthenium end cap and the first complex of the next repeating unit.

Figure 3.2. Crystal structure (1) of ∆-Ru(bpy) 2 dppz 2+ (red) bound to the oligonucleotide d(CGGAAATTACCG) 2 (gray)

Conclusions

Synthesize and characterize a ruthenium-dye conjugate that only exhibits observable acceptor emission through RET in the presence of DNA base mismatch. Synthesize and characterize a rhodium-dye conjugate that luminesces only in the presence of DNA base mismatches. TO-3 emission through RET can only be detected in the presence of a mismatch (mismatched bases shown in red), when both ruthenium and TO-3 are bound in the minor groove.

TO-3 emission via RET is only detectable in the presence of a mismatch (red) when both the ruthenium and TO-3 are bound in the minor groove.

Experimental protocols

Materials

Synthesize and characterize nonconjugated ruthenium molecules that exhibit luminescence enhancement in the presence of DNA base mismatches. Since ruthenium complexes such as Ru(bpy)2(dppz)2+ fluoresce in the presence of both well-matched and mismatched DNA, although more strongly with the latter, a strategy to selectively register the signal of ruthenium bound to. A good candidate for an RET acceptor is TO-PRO-3, a trimethycyanine dye that binds DNA from the minor groove and exhibits significantly enhanced fluorescence in the bound form.13 It has been shown to achieve RET with.

However, when a mismatch is present, ruthenium binds the mismatch from the minor groove, allowing the tethered TO-3 to also bind in the minor groove.

Synthesis of dye, metal complex, and conjugates

Since match binding of DNA occurs from the major groove and mismatch binding occurs from the minor groove, we propose to attach a RET acceptor, which releases only when it binds to DNA in the minor groove, to the route, so to observe the emission from the RET receiver. only when ruthenium is also in the minor groove, i.e., as illustrated in Figure 2, without a mismatch, ruthenium binds to DNA nonspecifically from the major groove and the TO-3 bound moiety is unable to bind DNA- in from the main groove. The shift in the emission maximum of the resulting emission profile and the changed intensity will be indicative of mismatch coupling.

Second, ruthenium fluorescence has a long lifetime, which will translate into a longer TO-3 emission lifetime via RET.

Figure 4.2. Chemical structures of Ru-dye conjugates: Ru-10-TO (1), Ru-1-TO (2), Ru- Ru-7-TO (3), Ru-Eth (4).

Steady state fluorescence

Results and discussion

Ru-TO-3 conjugates

The conjugate emits extremely weakly in acetonitrile, indicating that FRET serves to quench Ru luminescence in the absence of DNA. This is consistent with the idea that TO-3 only emits when bound to DNA in a rigid form.13 Upon excitation at 440 nm in the presence of DNA, the conjugated emission shows a characteristic profile of TO-3 fluorescence, and the resulting emission intensity is much higher than the older Ru complex at the same concentration. The linker in 2 is so short that it is not expected to allow binding of the conjugate in both grooves simultaneously, so TO-3 must also be able to bind in the major groove.

In other words, TO-3 is actually in the minor groove with ruthenium when DNA defects are present, but it still binds with ruthenium in the major groove in the absence of DNA defects.

Figure 4.4. Scheme showing the synthesis of trisheteroleptic Ru(L)(L’)dppz 2+ complexes 6 and 7

Ru-Eth conjugate

Since Ru emission is already at longer wavelength than ethidium absorption, the blue-shift of the acceptor absorption will undoubtedly reduce FRET efficiency. The Turro system therefore has better spectral overlap, as the acceptor absorption is moved closer to donor emission. Finally, binding by the terminal amines on ethidium with a rather short linker probably prevented the proper intercalation of ethidium into the DNA duplex.

Since intercalation of ethidium was not observed, the absorption of ethidium would not be different from that of free ethidium in solution, which is just too far away from Ru emission.

Figure 4.14. UV-vis spectra of ethidium bromide (blue) and ethidium-COOH 11.

Conclusions

Interference of the trimethine cyanine dye Cyan 2 with double-stranded DNA: study by spectral luminescence methods. In this chapter, we review unliganded ruthenium complexes and explore various modifications of the DPPZ ligand in an attempt to improve the mismatch selectivity of the resultant complexes while retaining their luminescence properties. However, they are able to take advantage of the thermodynamic destabilization of mismatches and bind to mismatched sites through metal insertion.1 In contrast, the DPPZ ligand of ruthenium complexes is long and narrow, which facilitates intercalation at both sites well-matched as well as in mismatched ones. .

In this chapter, we report several chemical modifications to the DPPZ ligand to increase its steric bulk and discuss the luminescence properties of the resultant complexes in the presence of well-matched and defective DNA.

Experimental protocols

Materials
Synthesis of metal complexes
Steady state fluorescence
Fluorescence lifetimes

The hexafluorophosphate salt of the product was obtained by adding NH4PF6 to an aqueous solution of the chloride salt and collecting the red precipitate. The chloride salt is obtained by passing an acetonitrile/water (3:2 v/v) solution of the hexafluorophosphate salt through a sephadex QAE anion exchange column. The chloride salt was obtained by passing an acetonitrile/water (3:2 v/v) solution of the hexafluorophosphate salt through a sephadex QAE anion exchange column.

For luminescence measurements at 77 K, complexes were dissolved in 10 M LiCl to form a clear glass.6 The sample cuvette was immersed in a dewar sample container filled with liquid nitrogen.

Figure 5.1. Synthesis of derivatives of the DPPZ ligand.

Results and discussion

Ru complexes bearing DPPZ derivatives

Steady-state luminescence of Ru complexes with DNA. As shown in Figure 5.3, all Ru complexes (8 – 14) show “light switch” properties with matched (M), mismatched (MM) and abasic (AB) DNA. This supports the observation from steady-state measurements that the luminescence intensity of 8 is present in the following order: AB>MM>M (Figure 5.3).8 In the case of 9, the longest lifetime corresponding to the perpendicular mode (AB >MM > M) have been observed, which may explain the ordering of the steady-state luminescence measurements of 9, as shown in Figure 5.3. Overall, excited state lifetime measurements show that complexes 8, 9, and 12 exhibit longer lifetimes or a higher fraction of the perpendicular mode in the presence of DNA containing a lesion on the well-matched DNA, showing more protection of the responsible phenazine nitrogen. for increased luminescence upon binding to DNA defects.

Based on excited-state lifetime measurements, Ru complexes 8 , 9 , and 12 exhibit longer lifetimes or a higher fraction of the mode perpendicular to MM and AB, corresponding to their increased luminosity.

Figure 5.3. Plots of integrated emission intensity of 8 – 14 with M, MM and AB (5 mM Tris, pH 7.5, 50 mM NaCl, λ ex = 440 nm)

Chrysi analogues of ruthenium

We measured the emission of Ru(bpy)2dppz2+ in the presence of the nucleic acid dye SYTO Red series. Emission spectra of Ru(bpy)2dppz2+ with matched (M) and mismatched (MM) RNA hairpin (sequence shown in Figure 6.1) in the absence and presence of FRET-acceptor SYTO 61 (λex = 440 nm). Schematic illustration of a mismatch-marked (MM) mRNA consisting of three hairpins, each with four CA mismatches (red).

In the absence of Dox, no luminescence is detected by firefly luciferase at any time, confirming the tightness of the tet-inducible expression system.

Figure 5.4. Synthesis of Ru(bpy) 2 chrysi 2+ and Ru(bpy) 2 (Me 2 chrysi) 2+ .

Conclusions

Experimental protocols

Materials

Ru(bpy)2dppz]Cl2 were laboratory stocks synthesized according to previously reported procedures. 4,5RNA was purchased from Dharmacon. SYTO Red nucleic acid stain and cell culture supplies were purchased from Invitrogen unless otherwise noted.

Steady state luminescence

We first measured erac-Ru(bpy)2dppz2+ luminescence in the presence of mismatched and well-matched RNA hairpins (Figure 6.1). In the presence of a DNA base mismatch, the ruthenium complex can also bind to a mismatched site from the minor groove through metal insertion, leading to a small increase in luminescence compared to DNA well matched. Representative emission spectra in the absence and presence of a SYTO stain (SYTO 61) are shown in Figure 6.2.

This small but significant difference in the staining pattern and intensity suggests that the more concentrated ruthenium fluorescence came from the MM tag.