Nucleobase Solvation and Tautomer Stability
A R T I C L E S
of an isolated base pair in various solvents; however, they cannot
address tautomerization within the DNA duplex environment.
While it is clear that the nucleobases within DNA generally
adopt the keto-amino tautomers, environmental factors within
the duplex, such as solvent and metal ion accessibility and local
polarity, may facilitate tautomerization or may even stabilize
the rare enol-imino tautomers.1,19,20 Thus, in addition to being
selectively recognized by proteins, other nucleic acid polymers,
or small molecules,21-25 the different environments of the major
and minor grooves may contribute differently to base pair
dynamics and solvation. The environments of the two grooves
differ not only as a result of their structure but also due to
differences in solvation and chemical functionalities. For
example, while the majority of the nucleobase heteroatoms along
the floor of both grooves are solvated, water molecules bound
in the minor groove are more ordered than the those in the major
groove.26 In addition, while the walls of each groove are formed
from the sugar-phosphate backbone as it spirals around the
outer surface of the duplex, the ribose O4′ oxygen atoms are
only accessible from the minor groove. These O4′ atoms are
the only components of the minor groove, other than the
nucleobase atoms, that are consistently found to interact with
proteins and small molecules, suggesting that they may be an
important component of the minor groove environment.27-33
To study how the different environments within DNA affect
tautomerization, we have developed 2-(2′-hydroxyphenyl)-
Figure 1. HBO ground-state equilibrium and photoinduced tautomerization.
When R1 ) DNA and R2 ) H, the enol is positioned in the duplex minor
groove, and when R1 ) H and R2 ) DNA, the enol is positioned in the
major groove.
transfer, ESIPT),37-43 which is a mimic of the dominant
tautomer in DNA. Because ESIPT results in a characteristic and
experimentally observable red-shift of HBO fluorescence, and
because the syn- and anti-enols have unique absorption wave-
lengths and excited-state lifetimes, the conformation and sol-
vation state of HBO may be determined spectroscopically. Thus,
by examining the photodynamics of HBO appropriately incor-
porated into DNA, the contributions of the major and minor
groove environments to tautomer stability, solvation, and
dynamics may be characterized.
Using this model base pair, we previously characterized the
major groove environment of a DNA duplex.34,36 This was
accomplished by synthesizing a C-nucleoside with HBO at-
tached via a C5 aryl-glycosidic linkage (Figure 1), converting
it to the corresponding phosphoramidite, and incorporating it
into the oligonucleotide 5′-CGTTTC(HBO)TTCTC. The single-
stranded DNA (ssDNA) was annealed to a complementary
oligonucleotide containing an abasic site at the position opposite
HBO. The circular dichroism (CD) and UV-vis spectra were
both consistent with a well-packed duplex, and the duplex was
virtually as stable to thermal denaturation as an analogous duplex
containing a dA/dT base pair at the corresponding position (Tm
) 38 and 39 °C, respectively).36 The accommodation of the
model base pair within a native-like duplex was also supported
by molecular dynamics simulations, which indicated that HBO
packs within the duplex with the phototautomerizable groups
positioned in the major groove.36
benzoxazole (HBO) as a model base pair (Figure 1).34-36
A
nucleoside-bearing HBO may be synthesized such that when
incorporated into DNA, the enol moiety of the model base pair
is positioned in either the major or minor groove. In the ground
state, HBO exists exclusively as the enol-imino tautomer, with
the enol group oriented either syn or anti with respect to the
imino nitrogen.36 In aprotic solvents, the syn-enol dominates
due to the stability of the internal H-bond, while in protic
solvents, solvation disrupts the internal H-bond and HBO adopts
a mixture of solvated syn- and anti-enols.35 The internally
H-bonded syn-enol, a mimic of the rare enol-imino tautomer
in natural DNA, is efficiently photoinduced to tautomerize to
the keto-amino tautomer (excited-state intramolecular proton
(19) Egli, M.; Gessner, R. V. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 180-184.
(20) Lippert, B. J. Chem. Soc., Dalton Trans. 1997, 21, 3971-3976.
(21) Dickerson, R. E.; Kopka, M. L.; Pjura, P. E. In DNA-Ligand Interactions;
Guschlbauer, W., Sawnger, W., Eds.; Plenum Publishing Corporation: New
York, 1987; pp 45-62.
(22) Gago, F.; Reynolds, C. A.; Graham, W. Mol. Pharm. 1989, 35, 232-241.
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1995, 24, 463-493.
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69.
Various spectroscopic techniques were used to characterize
the conformation, solvation, and tautomeric dynamics within
the major groove. In ssDNA, the model base pair populates both
the syn- and anti-enol conformations34,36,44 but is completely
driven to the internally H-bonded syn-enol upon duplex forma-
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