Keywords: Electron transfer / Fluorescence / Iridium / Oligonucleotides / Palladium / Pyrene 5-(2-Pyrenyl)-2Ј-deoxyuridine (2PydU, 2) has been prepared as a new thymidine analogue in which the 2-position of the pyrene chromophore is connected covalently to the 5-position of uridine through a single C-C bond. The synthesis of 2 starts with the conversion of pyrene (3) into 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrene (4) by using an Ir catalyst that was prepared in situ from [IrCl(cod)] 2 and 4,4Ј-di-tert-butyl-2,2Ј-bipyridine (dtbpy) in the presence of NaOMe. The subsequent Suzuki-Miyaura cross-coupling of 4 with 5-iodo-2Ј-deoxyuridine (5) was performed by using 1,1Ј-bis[(diphenylphosphanyl)ferrocene]dichloropalladium-(II) as the catalyst in a THF/MeOH/H 2 O mixture as the solvent. The modified nucleoside 2 was characterized by absorption and fluorescence spectroscopy. The results were compared with the strongly electronically coupled 5-(1-pyrenyl)-2Ј-deoxyuridine (1PydU, 1). Finally, the nucleoside 2 was converted into the corresponding phosphoramidite 7 as a DNA building block. The DNA set 8a-8d was synthesized

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The chromophores pyrene and bordipyrromethenylbenzene directly linked to the 5-position of uridine are tolerated and recognized as thymine derivatives by DNA polymerases in primer extension experiments.If fluorophores are attached to DNA bases for oligonucleotide labeling, 1 an alkyl chain linker is inserted between the chromophore and DNA base to allow the replication by DNA polymerases. However, the direct covalent attachment of chromophores to DNA bases yields unique optical properties, such as solvatochromism and exciplex-type emission 2 that are suitable for DNA probing. A critical issue about this direct linkage is the question if the canonical base recognition complementarity persists in DNA polymerase-catalyzed primer extension experiments. 3 For instance, fluorophore-labeled nucleosides and fluorosides can be applied as substrates for the DNA polymerase. 4 Over the past years, we attached synthetically pyrene 5-7 or ethynylpyrene 8 , for example, to DNA bases for electron transfer studies and as fluorescent probes for DNA. To gain more insight into the counterbase selectivity, we performed primer extension experiments with a representative set of modified oligonucleotides (Scheme 1). The templates con- 6 as single modifications. The length of the radioactively labeled primer was chosen such that the modified nucleotide in the template strand codes for the first nucleotide during primer extension. Single-base incorporations were performed with each of the four dNTPs exclusively to get information about the insertion selectivity opposite to the modified nucleotide. In addition, experiments employing all four dNTPs simultaneously were performed to study the elongation bypassing the modification site.First we investigated the Klenow fragment (exo-) of E. coli DNA polymerase I (KF-) in its propensity to insert a nucleotide opposite the modified DNA nucleobase. Gel electrophoretic analysis of the radiometric primer extension reactions revealed that the canonical bases are predominantly incorporated, that means A opposite to 1PydU, 2PydU and BodU, and C opposite to PydG (Fig. 1). Only minor amounts of misincorporation of G opposite to 2PydU and less opposite to 1PydU were observed. When all four dNTPs are present in the primer extension experiment, KF-is able to bypass all three types of uridine modifications (1PydU, 2PydU, and BodU) but not the modified guanosine (PydG). This is a remarkable result since the steric hindrance by the chromophores, especially by the bordipyrromethenylphenyl substituent, was expected to be significant.Subsequently, human DNA polymerase b (Pol b), a member of the DNA polymerase X family involved in DNA repair, and DNA polymerase Dpo4, a representative of the Y-family, were examined (Fig. 1). In the single nucleotide insertion experiments both enzymes placed the canonical nucleotides opposite the modification sites, but Pol b was unable to incorporate any nucleotide opposite PydG. In contrast to KF-, a significant amount of misincorporation was not observed. However, both enzymes...

The synthetic incorporation of indole as an artificial DNA base into oligonucleotides by two different structural approaches is described. For both types of modification, the indole moiety is attached through the C-3 position to the oligonucleotides. As a mimic of natural nucleosides, the indole nucleoside of β-2Ј-deoxyribofuranoside (In) was synthesized. The corresponding In-modified duplexes were compared with duplexes that contained the indole group connected through (S)-3-amino-1,2-propanediol as an acyclic linker between the phosphodiester bridges of the oligonucleotides. This linker was tethered to the C-3 position of the indole heterocycle either directly (InЈЈ) or by a carbamate function (InЈ). The melting temperatures of the corresponding indole-modi-

Charge transfer processes through the double helix of DNA cover a broad range of mechanistic models ranging from superexchange to hopping mechanisms. Over the last decade, these processes were studied by our group in a photoinduced fashion since (i) the starting time for the charge transfer is clearly defined by the absorption of the photon and (ii) photoexcitation delivers the necessary driving force to the DNA system. It is a prerequisite to modify oligonucleotides synthetically with suitable organic fluorophores that serve as photoinducable charge donors. In the first part of this perspective article we summarize our recent advances in the area of DNA-mediated reductive electron transfer processes over short ranges using synthetic DNA-donor-acceptor systems. The second part of this article focuses on ethidium as the photoinducable charge donor. Ethidium-modified DNA can be used to compare oxidative hole transfer with reductive electron transfer since the type of charge transfer can be controlled by choosing the right charge acceptor. Recent results showed that an efficient charge transfer through DNA using covalently bound ethidium is strongly influenced mainly by DNA dynamics but also by several other parameters that affect the electronic coupling between charge donor and acceptor.