G-Quadruplex (GQ) Complex Formation

To extend our study we investigated the formation of lipophilic 2ʹ-deoxyguanosine

quadruplexes of thrombin binding aptamer (5ʹ-dGGTTGGTGTGGTTGG) using

porphyrin 27 and CTAB. G-quadruplexes (GQ) are formed in G rich sequences in the presence of buffers containing potassium ions.7 A standard buffer for the formation of GQ is 10 mM sodium phosphate and 100 mM KCl, pH 7.0. Unfortunately, the addition of 27 to a solution of the above buffer containing no DNA resulted in the precipitation of the porphyrin. Further investigation showed that the porphyrin was still semi insoluble in salt concentrations as low as 0.62 mM sodium phosphate and 6.25 mM KCl.

It was observed that the GQ-porphyrin supramolecular complex could be formed by pre-forming the GQ in 10 mM sodium phosphate, 100 mM KCl then precipitating the oligonucleotide in GQ form from LiClO4 and acetone. It was found that when the DNA

precipitate was redissolved in water the GQ structure was still preserved, as shown by a characteristic CD signal with minima at 260 nm and maxima at 292 nm.135 _{On the}

addition of porphyrin 27 to the GQ solution a precipitate was formed which was isolated, dried and dissolved in CHCl3. UV-Vis spectroscopy of the resulting solution

showed a peak at 260 nm that was not present in porphyrin 27 therefore confirming the existence of both oligonucleotide and porphyrin in the chloroform solution (Figure 4.6). It was not possible to determining if the GQ structure still remained after complex formation as the CD spectrum of the porphyrin-DNA complex could not be measured due to the strong HT voltage rising from the porphyrin in the UV region. Dilution of the solution to give a reliable HT voltage showed no CD induced Cotton effects in the porphyrin region. This solution was also too dilute to observe any GQ CD signal. By repeating the experiment using CTAB, a precipitate was produced that could be dissolved in EtOH. This solution had an identical CD spectrum to the unmodified GQ which suggests that the GQ structure in the porphyrin modified GQ still remains.

0 0.5 1 1.5 2 2.5 240 340 440 540 640 740 λ _{/ nm} A b s

Figure 4.6 UV-Vis spectra of GQ-porphyrin complex (thicker line) and unreacted porphyrin 27 (thinner

4.5 Conclusion

Lipophilic porphyrin complexes could be synthesised using short sequences of single stranded, duplex and GQ oligonucleotides. Longer DNA sequences resulted in the formation of an insoluble complex. Loading studies on single stranded and duplex DNA showed slight overloading of porphyrins on DNA. Characterisation of the porphyrin- DNA supramolecular structure by CD spectroscopy was not possible due to the overpowering porphyrin signal.

Because the non-covalent complexes were either insoluble or unable to be fully characterised, focus switched to the covalent attachment of porphyrins to DNA as discussed in Chapters 5 and 6.

Chapter 5 Covalent Attachment of Porphyrins

to DNA

5.1 Introduction

Porphyrin-DNA supramolecular assemblies are important for the development of functional π-systems with tunable optical properties. As discussed previously, we have

chosen to focus on two coupling methods, Sonogashira and CuI _{catalysed azide alkyne}

Huisgen 1,3-dipolar cycloaddition reactions, also known as CuAAC reactions, as a means of the pre- or post-synthetic site-specific modification of oligonucleotides (Figure 5.1).

Contrary to the common functionalisation of the porphyrin through the meso positions38,

41-44, 46, 55 _{which results in a system orthogonal to the porphyrin core, a}

β-pyrrolic

modified porphyrin was used which provided a planar system between the porphyrin core and the adjacent benzene ring. To achieve site-specific modification β-pyrrolic

substituted porphyrins containing halogens (Br, I), alkynes or azides, as described in Chapter 2.4, were used in conjunction with pre- and post-synthetic Sonogashira and CuAAC reactions. N3 Br/I Nucleoside Nucleoside Br/I Nucleoside N Nucleoside Nucleoside N N Nucleoside DNA synthesis DNA synthesis Oligonucleotide Oligonucleotide I/Br Oligonucleotide N3 I/Br N Oligonucleotide Oligonucleotide N N Oligonucleotide Post-synthetic Pre-synthetic

+

+

+

+

+

+

A pre-synthetic approach involves the attachment of a porphyrin to nucleosides which are then converted to the appropriate porphyrin phosphoramidites or H-phosphonates. These are then incorporated into the DNA structure during DNA synthesis. Due to their chemical nature, stability of porphyrin phosphoramidites as DNA building blocks is usually limited46, 48 _{and is dependent on the structure of the molecule. H-Phosphonate}

porphyrin analogues have also been employed in DNA synthesis,46, 55 _{however coupling}

yields are usually not satisfactory for their multiple incorporations. A pre-synthetic approach can allow for the incorporation of many functionalised nucleotides in a single DNA strand using automated DNA synthesis, only if high yielding reactions occur.

In this regard, a post-synthetic modification of DNA is a more versatile approach compared to the time-consuming preparation of phosphoramidites.40 Post-synthetic modification, where a special functional group of the porphyrin reacts specifically with a pre-synthesised oligonucleotide carrying a complementary functional group, is particularly important for screening different substituents in nucleic acid structures.

5.2 Chapter Summary

This chapter investigates the pre-synthetic modification of oligonucleotides using both Sonogashira and CuAAC chemistry. Due to the problems accounted with pre-synthetic modification, focus switched to the development of a method for internal post-synthetic modification using alkyne containing ONs via CuAAC chemistry. We have screened porphyrin substituents using this method with ONs incorporating 2ʹ-deoxy-5-

ethynyluridine, 2′-O-propargyl uridine or 4-ethynylphenylmethylglycerol moieties. The synthesis of ONs possessing the internal porphyrin modifications allowed us to undertake UV-Vis and CD thermal stability studies on the resulting duplexes and triplexes.