Article pubs.acs.org/JPCB
Low-Temperature NMR Studies on the Geometry of Base Pairs Involving 5‑Substituted Uracil Derivatives Eline M. Basílio Janke† and Klaus Weisz* Institute of Biochemistry, Ernst-Moritz-Arndt University Greifswald, Felix-Hausdorff-Straße 4, D-17487 Greifswald, Germany ABSTRACT: Uridine (U) imino proton chemical shifts, measured in the slow hydrogen-bond exchange regime at low temperatures in a freonic solution, show that electron-withdrawing 5-bromo and 5fluoro substituents on the uracil base strengthen NHN hydrogen bonds in XU·A base pairs formed by the free nucleosides. Whereas the halogens do not alter the preferential formation of Watson−Crick geometries, self-associates of the halouracils point to a more favorable engagement of the 2-carbonyl as proton acceptor in the cyclic hydrogen bonds, suggesting increased formation of reverse geometries and wobble base pairs with guanine when compared to the thymine base. Employing 15N-labeled 5-bromouridine, no noticeable population of minor enol tautomers is found in the freonic mixture at 113 K.
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INTRODUCTION The mutual recognition of natural nucleobases through the formation of specific hydrogen bonds constitutes one of the most fundamental processes in biology by allowing for a reliable storage and readout of genetic information. While base pair formation through Watson−Crick hydrogen bonds largely determines the structure of genomic DNA, association between nucleobases is not restricted to this specific base pairing pattern and other geometries, e.g. with reverse or Hoogsteen hydrogen bonds, are frequently observed depending on the nucleic acid system and its environment.1−5 Introducing a 5-fluorosubstituted uridine (FU) opposite adenosine in a DNA duplex was shown to still yield a canonical Watson−Crick FU·A base pair but at the same time exhibits significant differences in its dynamic behavior.6 Being of particular interest, 5-fluoro- and 5bromouridine (BrU) are mutagenic nucleoside analogues and transition mutations induced by the halouracil bases are thought to be a result of FU·G and BrU·G mispairs occurring during DNA replication. Such an increased mispair formation has been related to either the presence of rare enol tautomers based on previous studies on uracil keto−enol equilibria7,8 or to the partial ionization of the more acidic halogenated base. Thus, NMR studies on oligonucleotide duplexes pointed to a pH dependent equilibrium between ionized and wobble structures for centrally located FU·G or BrU·G mismatches.9,10 On the other hand, crystal structures on a Z-DNA fragment suggested the formation of BrU·G wobble base pairs with no indication of an enol tautomeric form or the presence of an ionized base.11 Evaluating the geometry and strength of favored hydrogen bonds within a particular base pair is associated with specific limitations depending on the system and the methodology employed. Thus, the pairing of free nucleobases has previously been studied in the gas phase with the exclusion of any potential solvent effects.12 On the other hand, base pair © 2013 American Chemical Society
formation within oligonucleotide duplexes under more physiological conditions is restricted by the mutual preorientation of bases as enforced by the sugar−phosphate backbone. Finally, studies employing free nucleosides in solution may suffer from fast-exchange processes as often observed between the weakly hydrogen-bonded associates. Consequently, data obtained from NMR measurements at ambient temperatures represent averages over all coexisting hydrogen-bonded species and even employing 13C, 17O, or 15N nuclei as more specific probes do not allow a simple and unambiguous evaluation of type and relative population of the different associates present in solution.13−17 In order to slow the hydrogen-bond exchange and to observe individual hydrogen-bonded species for their more detailed characterization, nucleosides have to be dissolved in a low-melting freonic solvent mixture that allows measurements in the slowexchange regime at temperatures as low as 103 K.18−21 As an additional benefit, the temperature-dependent dielectric constant of the Freon solvent of up to 40 at very low temperatures22 may closely mimic the relatively apolar microenvironment of the solvent-protected base pairs within a double-helical nucleic acid. In the present study we have employed low-temperature NMR meaurements in freonic solvent mixtures to determine the strength and geometry of base pairs formed by 5substituted uridine derivatives. Analysis of the NMR data provides information on complex geometries without restrictions from the sugar−phosphate backbone, reflecting intrinsic properties of the particular nucleobase and providing Received: January 11, 2013 Revised: March 1, 2013 Published: March 26, 2013 4853
dx.doi.org/10.1021/jp400348x | J. Phys. Chem. B 2013, 117, 4853−4859
The Journal of Physical Chemistry B
Article
additional insight into the origin of the mutagenic nature of halouracil bases when incorporated into genomic DNA.
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EXPERIMENTAL SECTION Materials. The deuterated freonic mixture CDClF2/CDF3 was prepared as described23 and handled on a vacuum line which was also used for the sample preparation. Deuteration of adenosine at its 8-position,24 3-15N labeling of 5-bromouridine25 and the protection of sugar hydroxyl groups of the nucleosides by either O-acetyl or O-tert-butyldimethylsilyl (TBDMS) groups was performed according to standard procedures. Methods. NMR experiments were performed on Bruker AMX 500 and Bruker AVANCE 600 spectrometers. Temperatures were adjusted by a Eurotherm variable temperature unit to an accuracy of ±1.0 °C. Temperatures in the range 293 to 233 K were calibrated by using a standard solution of 4% CH3OH in CD3OD. Low-temperature spectra were recorded with a specially designed low-temperature dual probehead. 1H NMR chemical shifts in the freonic mixture were referenced relative to CHClF2 (δH = 7.13 ppm). Phase-sensitive twodimensional nuclear Overhauser effect (2D NOE) spectra at temperatures
