1. Introduction
1.7. Modified Nucleotides
Due to their widespread applications in medicine, biology, chemistry, biochemistry and material science,[90] the chemistry of modified nucleosides, nucleotides or oligonucleotides continues to be a rapidly developing field. Modified nucleotides have been established for the investigation of many biochemical processes and therefore enhanced our current understanding.[90] Nucleotide analogues can not only be used to improve our understanding of many cellular processes, but can even be employed for medical applications, as the modified nucleotides can compete against their natural counterparts. The potential of different derivatives to be used for the treatment of various diseases has already been proven by their application as antiviral and anticancer drugs.[91] Thus, efficient methods for the synthesis of modified nucleotides are required, as they are of general interest and widespread importance.
1.7.1. 5´-Triphosphate Synthesis
Due to the great importance of phosphorylated biological molecules, several different phosphorylation methods were developed. The method established by Eckstein et al.[92] takes advantage of the multifunctionality of salicyl phosphorochloridite (see Figure 9a). Thereby, they developed a facile synthesis route for 5´-O-triphosphates, which could be employed for the generation of 5´-O-(1-thiotriphosphates) as well. They used 3´-OH protected nucleosides, which were reacted with salicylphosphorodichloridite to generate intermediate I as diastereomeric mixture. Treatment of I with pyrophosphate resulted in the formation of a cyclic phosphorous (III) species (II), which could be oxidised with iodine/water leading to the desired 5´-O-triphosphate in up to 72 % yield.[92] The nucleoside cyclic triphosphate resulting from oxidation of compound II cannot be detected due to immediate hydrolysis in the aqueous conditions used during oxidation. Alternatively, intermediate II can be reacted with sulphur to yield the corresponding 5´-O-(1-thiotriphosphate).[92] Despite the advantage to have one method which can result in the generation of 5´-O-triphosphates and 5´-O-(1-thiotriphosphates), this method requires the protection of the 3´-OH group thereby complicating its application. Since the 5´-OH group of nucleosides is more reactive than its 3´-OH group, selective protection of the 3´-OH group is rather challenging. To achieve selective 3´-OH protection, nucleosides need to undergo several selective protection and deprotection procedures.
1. Introduction 28
Thus, Huang et al.[93] established a protection-free variant of the described method by generation of a mild and selective phosphitylating reagent that differentiates the different functionalities present in the nucleoside (see Figure 9b). Therefore, they took advantage of the multifunctionality and high reactivity of salicyl phosphorochloridite.[92, 94] By treatment of the salicyl derivative with pyrophosphate, they managed to generate a weak phosphitylating reagent IV. The selective phosphitylation reagent IV can be generated in situ and can selectively react with the 5´-hydroxyl group of nucleosides without the need for protection groups on the sugar or nucleobases.[93]
Figure 9: Methods for 5´-O-triphosphorylation. a) Synthesis according Eckstein, b) according Huang, c) according Yoshikawa and Kovács.
Another protection-free strategy for synthesis of 5´-O-triphosphates employs highly reactive phosphorous oxychloride (POCl3) as phosphitylation reagent (see Figure 9 c). Trimethylphosphate is used as solvent to reduce the reactivity of POCl3, therefore limiting possible side reactions. During this method, dichlorophosphate V is primarily generated, which can be seen as equivalent to an activated monophosphate species. Subsequent treatment with pyrophosphate yields formation of the desired 5´-O-triphosphate in a protection-free one pot synthesis.[93] Yoshikawa et al. reported in 1969 the reaction of unprotected nucleosides with POCl3 in trialkyl phosphate solvents mainly leading to 5´-phosphorodichloridate V.[95] In situ hydrolysis results in the formation of nucleoside 5´-monophosphates.[96] The direct transformation of 5´-phosphorodichloridates to nucleoside 5´-triphosphates is obtained through treatment with an excess of tri-n-butylammonium pyrophosphate in DMF under anhydrous conditions followed by basic or neutral hydrolysis.[92] The occurrence of a highly reactive trimetaphosphate intermediate VI formed by intramolecular condensation could be proven by 31P NMR analysis.
Two decades later, Kovács et al.[97] reported that hydrogen chloride, formed during hydrolysis of POCl3, resulted in several side reactions if modified nucleosides containing unsaturated side chains were transformed. As the very reactive nature of unsaturated side chains is known in acidic conditions, they performed the 5´-O-triphosphorylation reaction in presence of a base. During their studies, proton sponge (1,8-bis(dimethylamino)naphthalene) proved to be the best suited base as it accelerated the reaction significantly. Due to its steric effects, proton sponge is known as very strong base with weak nucleophilic character.[97] Thus, they could show that even modified nucleosides with highly reactive unsaturated side chains could be converted to the corresponding 5´-O-triphosphates in the presence of proton sponge.[97] Besides the drawback by usage of the very reactive POCl3 this method holds the disadvantage that different phosphorylated derivatives are obtained which are challenging to separate during purification. Nevertheless, this method holds a great potential as various modified nucleosides can be converted and different 5´-O-phosphorylated (mono-, di- and triphosphate) species can be obtained.
2. Aim of This Work 30
2. Aim of This Work
As several modifications in nucleic acids can be related to human diseases, new methods are strongly needed for their detection. Nowadays, several detection methods are known and rely mainly on selective chemical modifications[5a, 31] or the ability of different enzymes to distinguish between modified nucleotides and their unmodified counterparts.[28, 30] However, those technologies hold several disadvantages. In particular detection systems, which require chemical modifications prior to sequencing proved to be time-consuming, tedious and error-prone.[34-36] Especially for the application in personalised medicine convenient methods are required that allow multiplexing.
The aim of this thesis was to establish new approaches for the detection of the epigenetic markers 5mC and 5hmC without the need to perform modification reactions prior to sequencing. No DNA polymerase was known to be able to directly discriminate C against 5mC or 5hmC without the use of mismatched primers as direct sequencing of those epigenetic markers is rather challenging due to their unaltered Watson-Crick face.[116] DNA polymerases are known to discriminate against incorporation of non-canonical nucleotides very efficiently.[76] Nevertheless, different DNA polymerases are known to accept modified nucleotides with reduced efficiencies compared to their natural counterparts.[70] The aim of this thesis was to take advantage of the discrimination machinery of DNA polymerases as well as the ability of those DNA polymerases to incorporate modified nucleotides. By the introduction of various modifications at different sites of the nucleobase moiety of dGTP, dNTP derivatives which enable the DNA polymerase to discriminate between C and the epigenetic markers 5mC and 5hmC should be found. By enhancing the size and the steric hindrance of the introduced modification, a dNTP analogue should be found that is still accepted by the DNA polymerase but leads to a steric rearrangement of the primer/template complex in the active site of the enzyme in a way that the DNA polymerase will sense the presence or absence of the small methyl-group in 5mC. Following, a novel assay should be established that allows sensing of nucleic acid modifications without the need to perform modification reactions prior to sequencing. For this purpose, a tool box of variously modified nucleotides should be synthesised. Subsequently, the latter should be tested in combination with different DNA polymerases to find a combination of enzyme and nucleotide analogue, which leads to diverging incorporation efficiencies opposite C and the epigenetic markers 5mC and 5hmC in DNA polymerase catalysed reactions.
As modified nucleotides are not only known to be present in DNA, but can be found in RNA as well,[2]
the generated tool box should be applied in combination with the KlenTaq variant RT-KTq2. This variant is known to exhibit reverse transcriptase activity,[125] which could be used to sense RNA modifications by incorporation of modified nucleotides.
Once identified, the most promising combinations of DNA polymerase and modified nucleotide should be further studied by the measurement of steady-state kinetics and exploited for the establishment of new detection approaches.
A second approach aimed at finding and characterising DNA polymerase variants with increased discrimination in incorporating dGMP opposite 5mC compared to C. If variants possessing this ability