Base-base recognition of nonionic dinucleotide analogues in an apolar environment studied by low-temperature NMR spectroscopy

Article abstract of DOI:10.1021/ja910220s

Two self-complementary dinucleotide analogues T_SiA and A _si with a nonionic diisopropylsilyl-modified backbone were synthesized, and their association in a nonaqueous aprotic environment was studied by NMR spectroscopy. Using a CDClF₂/CDF₃ solvent mixture, measurements at temperatures as low as 113 K allowed the observation and structural characterization of individual complexes in the slow exchange regime. The A_siT analogue associates to exclusively form a dinucleotide antiparallel duplex with regular Watson-Crick base pairing, but both A and T nucleosides exhibit a predominant C3'-endo sugar pucker reminiscent of an A-type conformation. In contrast to A_siT the T_SiA dinucleotide is found to exhibit significant variability and flexibility. Thus, different secondary structures with weaker hydrogen bonds for all T _SiA structures are observed at low temperatures. Although a B-like Watson-Crick antiparallel dinucleotide duplex with a preferred C2'-endo sugar pucker largely predominates at temperatures above 153 K, two additional species, namely a dinucleotide Hoogsteen duplex with a syn glycosidic torsion angle of the adenosine nucleoside and a presumably intramoleculariy folded structure, are increasingly populated upon further cooling. By adding typical DNA intercalators like anthracene or benz[c]acridine derivatives to the A _siT dinucleotide duplex in the aprotic solvent environment, no binding of the polycyclic aromatic molecules can be detected even at lower temperatures. Obviously, van der Waals and stacking interactions are insufficient to compensate for the other unfavorable contributions to the overall free energy of binding, and only in the presence of additional hydrophobic effects in an aqueous environment does binding occur.

Full text of DOI:10.1021/ja910220s

Published on Web 02/24/2010
Base-Base Recognition of Nonionic Dinucleotide Analogues
in an Apolar Environment Studied by Low-Temperature NMR
Spectroscopy
Zhou Xiao† and Klaus Weisz*
Institute of Biochemistry, Ernst-Moritz-Arndt UniVersity Greifswald, Felix-Hausdorff-Str. 4,
D-17489 Greifswald, Germany
Received December 3, 2009; E-mail: weisz@uni-greifswald.de
Abstract: Two self-complementary dinucleotide analogues TSiA and ASiT with a nonionic diisopropylsilyl-
modified backbone were synthesized, and their association in a nonaqueous aprotic environment was studied
by NMR spectroscopy. Using a CDClF2/CDF3 solvent mixture, measurements at temperatures as low as
113 K allowed the observation and structural characterization of individual complexes in the slow exchange
regime. The ASiT analogue associates to exclusively form a dinucleotide antiparallel duplex with regular
Watson-Crick base pairing, but both A and T nucleosides exhibit a predominant C3′-endo sugar pucker
reminiscent of an A-type conformation. In contrast to ASiT, the TSiA dinucleotide is found to exhibit significant
variability and flexibility. Thus, different secondary structures with weaker hydrogen bonds for all TSiA
structures are observed at low temperatures. Although a B-like Watson-Crick antiparallel dinucleotide
duplex with a preferred C2′-endo sugar pucker largely predominates at temperatures above 153 K, two
additional species, namely a dinucleotide Hoogsteen duplex with a syn glycosidic torsion angle of the
adenosine nucleoside and a presumably intramolecularly folded structure, are increasingly populated upon
further cooling. By adding typical DNA intercalators like anthracene or benz[c]acridine derivatives to the
ASiT dinucleotide duplex in the aprotic solvent environment, no binding of the polycyclic aromatic molecules
can be detected even at lower temperatures. Obviously, van der Waals and stacking interactions are
insufficient to compensate for the other unfavorable contributions to the overall free energy of binding, and
only in the presence of additional hydrophobic effects in an aqueous environment does binding occur.
Introduction
chloroform have indicated the formation of a parallel duplex
with both interstrand and intrastrand hydrogen bonds much
The reliable storage and readout of genetic information in
nucleic acids are based on their specific hydrogen bond mediated
base-base recognition. These interactions will generally result
in the formation of the well-known DNA double helix with
adenine-thymine (AT) and guanine-cytosine (GC) base pairs.
Consequently, much emphasis has been placed on the nucleo-
bases as the major determinants of DNA secondary structure,
and only a small amount of attention has been paid in the past
to the role of the sugar phosphate backbone that carries the
nucleic acid bases. However, the design of backbone-modified
nucleic acids, e.g., for use in antisense technologies for improved
cellular uptake and nuclease resistance,1 has greatly stimulated
interest in backbone-induced structural effects recently. Thus,
different from regular base pairing.3 Interestingly, the stabilizing
hydrogen bond network was found to also include backbone
hydroxyl groups in addition to base-base interactions.
Obviously, a string of phosphate negative charges along the
chain associated with significant charge repulsion will invariably
promote a more extended conformation and suppress the
collapse to a coiled structure with largely intramolecular
interactions. Yet another widely acknowledged idea assumes a
more prominent role of the backbone in the base recognition
scheme and a direct impact of the charged phosphodiester on
the molecular base-base recognition by directing interstrand
interactions toward the Watson-Crick edge of the heterocycles
to maximize the separation between the negative backbones of
the contacting strands4 (for an excellent review, see ref 4b).
Consequently, much can be learned about the inherent critical
properties of the natural DNA backbone for the storage and
release of genetic information by looking at DNA analogues
with a nonphosphate scaffold. Also, questioning the ability of
nucleic acids to still serve as a carrier of genetic information in
a nonaqueous environment directly calls for a better understand-
a regular Watson-Crick duplex was reported for a short GSO2
C
dinucleotide analogue with a nonionic dimethylenesulfone
linkage in the crystal.2 In contrast, NMR structural studies on
a dimeric dimethylenesulfone-linked USO2C RNA analogue in
† Present address: Nanyang Technological University, Singapore.
(1) Selected reviews: (a) Nielsen, P. E. Annu. ReV. Biophys. Biomol. Struct.
1995, 24, 167–183. (b) Cook, P. D. In Antisense Drug Technology:
Principles, Strategies, and Applications; Crooke, S. T., Ed.; Marcel
Dekker: New York, 2001; pp 29-56. (c) Micklefield, J. Curr. Med.
Chem. 2001, 8, 1157–1179. (d) Sanghvi, Y. S. In DNA and Aspects
of Molecular Biology; Kool, E. T., Ed.; Elsevier Science Ltd.: Oxford,
2002; Vol. 7, pp 285-311. (e) Kurreck, J. Eur. J. Biochem. 2003,
270, 1628–1644.
(2) Roughton, A. L.; Portmann, S.; Benner, S. A.; Egli, M. J. Am. Chem.
Soc. 1995, 117, 7249–7250.
(3) Steinbeck, C.; Richert, C. J. Am. Chem. Soc. 1998, 120, 11576–11580.
(4) (a) Huang, Z.; Benner, S. A. J. Org. Chem. 2002, 67, 3996–4013. (b)
Benner, S. A.; Hutter, D. Bioorg. Chem. 2002, 30, 62–80.
9
3862 J. AM. CHEM. SOC. 2010, 132, 3862–3869
10.1021/ja910220s  2010 American Chemical Society

Base-Base Recognition of Nonionic Dinucleotide Analogues
A R T I C L E S
respect to type and strength of formed hydrogen bonds.5c,d The
low-temperature NMR experiments on the association of silyl-
linked dinucleotides ASiT and TSiA not only provide detailed
structural information in terms of base-base recognition and
the formation of specific hydrogen bonds but also allow for an
assessment of the major forces promoting the binding of ligands
to DNA.
Experimental Section
NMR experiments were performed on a Bruker AVANCE 600
spectrometer. Temperatures were adjusted by a Eurotherm variable-
temperature unit to an accuracy of (1.0 °C. The temperatures used
for determining equilibrium constants for self-association were
calibrated by using a standard solution of 4% CH3OH in CD3OD
in the range 293-233 K. 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 ) 7.18 ppm).
The deuterated freonic mixture CDClF2/CDF3 was prepared as
described6 and handled on a vacuum line which was also used for
the sample preparation. Chemicals were purchased from Sigma-
Aldrich, Deisenhofen, Germany. Detailed procedures for the
preparation of ASiT and TSiA and their characterization are provided
as Supporting Information.
Figure 1. Nonionic triisopropylsilyl backbone with atom numbering of
the 2′-deoxyribose (left) and natural anionic phosphodiester backbone (right).
ing of the impact of a nonionic backbone on structure and
recognition. A nucleic acid in a more hydrophobic environment
will probably rely on a neutral backbone to ensure sufficient
solubility. In this case, will the nonpolar backbone still allow
for a well-defined recognition code between two strands with
only a few mispairings to allow for occasional mutations?
Another important issue within this context deals with the
binding of ligands under such conditions. Clearly, ligand binding
allows for gene regulation, a prerequisite for a controlled
expression of genomic sequences. Excluding water molecules
and thus hydrophobic effects from the binding event, studies
on ligand binding to double-helical DNA in a nonaqueous
environment will also yield valuable information on the various
contributions that determine the affinity toward duplex DNA
under physiological conditions.
To study the structure and recognition of nucleic acids with
a nonionic backbone, we here report on NMR structural studies
of diisopropylsilyl-linked dinucleotides as model systems for a
neutral nucleic acid. A silyl backbone seems to be an attractive
phosphodiester surrogate given its high Si-O bond energy,
achirality, and lipophilicity and the similarity of silicon in size,
bond angles, and bond lengths to phosphorus (Figure 1). In
addition, silicon is the eighth most common element in the
universe by mass and thus of particular interest when considering
constituents of nucleic acid analogues having potentially evolved
as carriers of genetic information in an extraterrestrial
environment.
With only a limited number of interactions, intermolecular
forces between short dinucleotides tend to be weak, and
consequently hydrogen-bonded complexes in solution are often
in fast exchange on the NMR chemical shift time scale at
ambient temperatures preventing the unambiguous characteriza-
tion of coexisting associates. To circumvent averaging effects
and to characterize hydrogen-bonded complexes by liquid-state
NMR in detail, NMR measurements have been performed in
the past at very low temperatures by employing a deuterated
Freon mixture CDClF2/CDF3 as solvent.5 Thus, with measure-
ments in the liquid state as low as 113 K individual hydrogen-
bonded complexes of free nucleosides in slow exchange could
be observed and unambiguously characterized in solution with
Results and Discussion
Synthesis. An internucleotide diisopropylsilyl linkage was
chosen as a neutral phosphodiester analogue based on its
favorable stability and reactivity.7 Also, additional silyl protect-
ing groups at the 5′- and 3′-OH of nucleosides have previously
been found to promote solubility in freonic solvents especially
at low temperatures.8 Synthetic methods for silyl-linked oligo-
nucleotides have been reported in the past.7,9 In a typical
procedure, a 5′-protected nucleoside is 3′-silylated with a
silylating reagent and a base. Finally, this silyl-linked nucleoside
continues to react with a second 3′-protected nucleoside to
produce a fully protected 3′,5′-silyl-linked dinucleotide. Prob-
lems with this strategy often involve low yields due to the
formation of both undesired 3′,3′-symmetrical dimers as byprod-
ucts and a significant loss of product upon deprotection and
final purification.9a To reduce the formation of 3′,3′-coupling
products and to allow direct coupling of a 3′-silyl intermediate
to an unprotected nucleoside, a hindered base procedure was
developed and successfully employed for the preparation of
various silyl-linked nucleosides.9b For our structural studies, the
use of high-purity products was of major concern. In this respect,
the best results in our own efforts to synthesize fully protected
5′- and 3′-O-di-(tert-butyldimethylsilyl) dinucleotides with a
diisopropylsilyl linkage were obtained with slightly modified
protocols for the mixed thymidine-adenosine (TSiA) or
adenosine-thymidine (ASiT) dinucleotide.
For the ASiT synthesis, a strategy with 5′- and 3′-TBDMS-
protected nucleosides was employed to avoid byproducts and
thus difficult separations. After N-benzoylation, reaction of
(5) (a) Siegel, J. S.; Anet, F. A. L. J. Org. Chem. 1988, 53, 2629–2630.
(b) Golubev, N. S.; Denisov, G. S. J. Mol. Struct. 1992, 270, 263–
276. (c) Weisz, K.; Ja¨hnchen, J.; Limbach, H.-H. J. Am. Chem. Soc.
1997, 119, 6436–6437. (d) Dunger, A.; Limbach, H.-H.; Weisz, K.
Chem.sEur. J. 1998, 4, 621–628. (e) Smirnov, S. N.; Benedict, H.;
Golubev, N. S.; Denisov, G. S.; Kreevoy, M. M.; Schowen, R. L.;
Limbach, H.-H. Can. J. Chem. 1999, 77, 943–949. (f) Schlund, S.;
Mladenovic, M.; Bas´ılio Janke, E. M.; Engels, B.; Weisz, K. J. Am.
Chem. Soc. 2005, 127, 16151–16158. (g) Hupp, T.; Sturm, C.; Bas´ılio
Janke, E. M.; Pe´rez Cabre, M.; Weisz, K.; Engels, B. J. Phys. Chem.
A 2005, 109, 1703–1712.
(6) Golubev, N. S.; Smirnov, S. N.; Gindin, V. A.; Denisov, G. S.;
Benedict, H.; Limbach, H.-H. J. Am. Chem. Soc. 1994, 116, 12055–
12056.
(7) Cormier, J. F.; Ogilvie, K. K. Nucleic Acids Res. 1988, 16, 4583–
4594.
(8) Bas´ılio Janke, E. M.; Limbach, H.-H.; Weisz, K. J. Am. Chem. Soc.
2004, 126, 2135–2141.
(9) (a) Ogilvie, K. K.; Cormier, J. F. Tetrahedron Lett. 1985, 26, 4159–
4162. (b) Saha, A. K.; Sardaro, M.; Waychunas, C.; Delecki, D.;
Kuntny, R.; Cavanaugh, P.; Yawman, A.; Upson, D. A.; Kruse, L. I.
J. Org. Chem. 1993, 58, 7827–7831.
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J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3863

A R T I C L E S
Xiao and Weisz
Scheme 1. Synthesis Scheme of ASiTa
Figure 2. 1H NMR spectra of ASiT in Freon at 243 and 123 K showing
base proton spectral regions.
its free 3′-OH, and the benzoyl group was finally removed by
treatment with ammonia.
Final HPLC-purified dinucleotides were subjected to a
detailed NMR spectroscopic characterization using COSY and
NOESY experiments. For an unambiguous confirmation of a
1
3′-5′ diisopropylsilyl linkage, a gradient-enhanced H-29Si
HMQC experiment was acquired with a delay optimized for
long-range 1H-29Si couplings of 3 Hz.10 Correlations observed
between 29Si within the internucleotide linkage and H3′ of the
5′-terminal nucleoside as well as H5′/H5′′ of the 3′-terminal
nucleoside confirm the correct structure (see the Supporting
Information).
NMR Structural Studies on AsiT. To obtain information on
low energy structures of potential associates under aprotic
solvent conditions, the ASiT dinucleotide was dissolved in a
freonic mixture and studied by NMR measurements down to
temperatures low enough to reach the slow exchange regime.
When the sample was cooled to 123 K, the thymine imino
proton signal shifted downfield and all resonances exhibited
some gradual line broadening as expected for slower molecular
reorientation rates associated with a decreased spectral resolu-
tion. However, as seen in Figure 2, with only a single set of
proton resonances even under slow exchange conditions at 123
K and with no indication for coalescence over the wide
temperature range studied, a single well-defined structure is
formed and favored at all temperatures.
a DMTr ) dimethoxytrityl, TMS ) trimethylsilyl, Bz ) benzoyl,
TBDMS ) tert-butyldimethylsilyl, iPr ) isopropyl.
The downfield-shifted T imino resonance at 15 ppm and 123
K points to its participation in a hydrogen bond to adenine. In
general, assignment to either a Watson-Crick or Hoogsteen
adenosine with tert-butyldimethylsilyl chloride yielded the 5′-
protected nucleoside with high regioselectivity. On the other
hand, 3′-silylation of thymidine was achieved following a
transient 5′-tritylation. It was found out that using imidazole in
the absence of a hindered base gives better yields and formed
byproducts are mostly 3′-3′ adenosine dimers. With this
strategy, only the benzoyl group has to be removed after
separation of the desired 5′-3′ coupling product from the
reaction mixture. The synthesis scheme of ASiT is outlined in
Scheme 1.
1
geometry can be achieved through the observation of H-1H
NOE contacts of the imino resonance to either A H2 or A H8
protons, respectively. Because of this critical information, the
adenine base protons were unambiguously assigned making use
of the 1H-13C 3J scalar couplings within the purine ring system
for coherence transfer experiments.11,12
As seen in Figure 3 (bottom), adenine C4 at 150 ppm shows
1
crosspeaks to both H2 and H8 protons in a H-13C HMBC
For the synthesis of TSiA, 5′-O-tert-butyldimethylsilyl thy-
midine was directly coupled with the nonsilylated N6-benzoyl-
2′-deoxyadenosine in the presence of the hindered base 2,6-di-
tert-butyl-4-methylpyridine and diisopropyldichlorosilane as the
silylating reagent. The latter was found to give better results
compared to bis(trifluoromethanesulfonyl)diisopropylsilane upon
addition of the amino-protected 2′-deoxyadenosine as the second
nucleoside. Following the coupling reaction, some 3′,3′-thymi-
dine dimer can be isolated as a byproduct after column
separation. The 5′-3′-dinucleotide was TBDMS-protected at
experiment establishing an H2-H8 through-bond correlation
via the C4 atom. In addition, H8 exhibits another crosspeak to
C5 at ∼120 ppm, whereas H2 is correlated through scalar
(10) Hewitt, J. M.; Lenhart, W. C.; Moore, R. N.; Saha, A. K.; Weis, A. L.
Nucleosides Nucleotides 1992, 11, 1661–1666.
(11) Hilbers, C. W.; Wijmenga, S. S. In Encylcopedia of Nuclear Magnetic
Resonance; Grant, D. M., Harris, R. K., Eds.; Wiley: Chichester, U.K.,
1996; pp 3346-3359.
(12) Van Dongen, M. J. P.; Wijmenga, S. S.; Eritja, R.; Azorin, F.; Hilbers,
C. W. J. Biomol. NMR 1996, 8, 207–212.
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Base-Base Recognition of Nonionic Dinucleotide Analogues
A R T I C L E S
T H6 and A H8 with their own sugar protons clearly suggest
an anti conformation for both nucleosides.14,15 Also, in line with
a helix-type duplex structure, T H6 exhibits additional medium
to strong sequential contacts to adenosine H2′, H2′′, and H3′
protons (for all proton chemical shifts of ASiT, see the
Supporting Information).15
A semiquantitative evaluation of NOE crosspeak intensities
allows us to gain additional information on the dinucleotide
duplex conformation. There are several lines of evidence that
support the formation of an A-type duplex. These include
moderate to strong internucleotide crosspeaks between T H6
and A H3′ and A H2′, a weak sequential crosspeak between T
H6 and A H1′, and the presence of another moderately strong
intrastrand contact between A H2 and T H1′. Also, a stronger
cross peak of A H8 with H3′ compared to H2′/H2′′ and a strong
cross peak between H2′′ and H4′ in the adenine nucleotide
indicate a C3′-endo sugar pucker.15
A sugar conformational analysis was also performed on the
1
basis of measured H-1H scalar couplings at 243 K (see the
Supporting Information). With resolution-enhancing window
functions for processing, multiplet structures of deoxyribose
proton resonances are well resolved at these higher temperatures
and allow for a fairly accurate determination of coupling
constants without resorting to crosspeak simulations. Assuming
a two-state equilibrium between North (C3′-endo) and South
(C2′-endo) conformers, a coupling constant analysis with the
optimized Karplus relationship of Altona et al.16 indicates a
nearly pure C3′-endo sugar pucker for adenosine, while the T
nucleoside is mixed with ∼20-40% of a C2′-endo conforma-
tion. This again supports an A-type dinucleotide duplex for ASiT
under these conditions.
The A-typical conformation of the ASiT dimer contrasts with
B-DNA duplexes generally observed in an aqueous environment.
It is well-known that DNA adopts a conformation that signifi-
cantly depends on the presence of water molecules, and X-ray
diffraction studies have repeatedly shown that low relative
humidity favors A conformations. The observation of an A-type
duplex in the present study may thus be taken as the result of
lacking hydration. However, a partial impact by the modified
silyl backbone on the observed preference of a C3′-endo pucker
for the deoxyribonucleosides, a major determinant of an A-type
duplex structure, cannot be excluded.
The ASiT self-association was further characterized by its
equilibrium constant Ka determined by a nonlinear least-squares
fit of the thymine imino proton chemical shift measured as a
function of dinucleotide concentration in methylene chloride.
Thus, an association constant of 135 M-1 is obtained at 294 K.
A subsequent van’t Hoff analysis of Ka(T) determined in a
temperature range between 249 and 294 K yields a ∆H° ) -64
( 2.5 kJ/mol and a ∆S° ) -178 ( 9.4 J/K ·mol. Interestingly,
∆H° for the ASiT association mediated by a total of four
hydrogen bonds is approximately three times as large compared
to the association enthalpy of a free adenosine and uridine
nucleoside under the same solution conditions.17 On the other
hand, with a nearly 5-fold increase in -∆S° the entropic penalty
1
Figure 3. H2 and H8 crosspeak region of a H-13C HMBC spectrum of
ASiT with long-range coupling 3JCH optimized to 10 Hz (bottom); 2D NOE
spectrum of ASiT with an 80 ms mixing time showing crosspeaks between
imino and base protons (top). Both spectra were acquired at 133 K in Freon.
Insets show the adenine base (bottom) and an AT Watson-Crick base pair
(top) with atom numbering.
coupling with C6 at ∼157 ppm. With HMBC spectra taken at
different temperatures we also noticed that H2 and H8 ex-
changed positions with decreasing temperature. Above 243 K,
H2 is more downfield shifted but becomes more upfield shifted
compared to H8 at lower temperatures.
With A H2 and H8 assigned, an NOE crosspeak found for
the T imino to a hydrogen-bonded adenine amino proton as well
as a strong contact to A H2 established a Watson-Crick
arrangement in a symmetrical, antiparallel dimer (Figure 3, top).
The complete assignment of all resonances follows established
strategies for nucleosides and oligonucleotides.13 With only a
single species present, it is essentially based on the analysis of
a 2D NOE experiment performed at 163 K to take advantage
of well-resolved signals (see the Supporting Information).
Compared to even lower temperatures, exchange crosspeaks
between labile protons can still be observed at 163 K but do
not compromise the assignments and the structural analysis
based on the nonlabile protons. As expected for a Watson-Crick
geometry with antiparallel strands, intranucleotide contacts for
(14) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag:
New York, 1984.
(15) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley-Interscience:
New York, 1986; pp 205-214.
(13) (a) Feigon, J.; Wright, J. M.; Leupin, W.; Denny, W. A.; Kearns, D. R.
J. Am. Chem. Soc. 1982, 104, 5540–5541. (b) Scheek, R. M.; Russo,
N.; Boelens, R.; Kaptein, R.; van Boom, J. H. J. Am. Chem. Soc. 1983,
105, 2914–2916.
(16) (a) De Leeuw, F. A. A. M.; Altona, C. J. Chem. Soc., Perkin Trans.
2 1982, 375–384. (b) Rinkel, L. J.; Altona, C. J. Biomol. Struct. Dyn.
1987, 4, 621–649.
(17) Xiao, Z.; Weisz, K. J. Phys. Org. Chem. 2007, 20, 771–777.
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A R T I C L E S
Xiao and Weisz
Figure 4. 1H NMR spectra of TSiA in Freon showing base proton spectral
regions as a function of temperature.
of forming a base-paired complex has significantly increased
for the ASiT duplex when compared to the free AU base pair.
Disregarding minor differences between uridine and thymidine
nucleosides, these data indicate that in addition to the formation
of four Watson-Crick hydrogen bonds stacking interactions
considerably contribute to the stability of the dinucleotide duplex
in the apolar environment. However, reorganization of the silyl
backbone upon duplex formation adds a significant entropic cost
to the dinucleotide self-association.
NMR Structural Studies on TsiA. Upon preparing a TSiA
solution in Freon we noticed a lower solubility compared to
ASiT, yet the poorer solubility did not compromise low-
temperature 1H NMR measurements. As shown in Figure 4, TSiA
resonances start to considerably broaden at temperatures below
243 K. Interestingly, two more upfield shifted imino signals
appear in addition to the major imino resonance and gradually
become more intense with decreasing temperature below a
clearly noticeable coalescence point for the imino protons at
∼183 K.
Figure 5. Portion of a 2D NOE spectrum of TSiA in Freon showing regions
with amino/H2/H8-amino/H2/H8/H1′ (top) and amino/H2/H8-imino (bot-
tom) crosspeaks. The boxed region encloses base-H1′ contacts. The spectrum
was acquired at 133 K with an 80 ms mixing time.
nucleoside. Surprisingly, however, the generally favored S-type
sugar pucker for the A and T nucleosides in TSiA stands in
striking contrast to the N-type pucker found for ASiT at 243 K.
To unambiguously assign the three imino resonances to
corresponding complex structures, a 2D NOE experiment at 133
K was performed. At this temperature, no crosspeaks due to
chemical exchange are observed and NOE contacts directly
identify protons in close spatial proximity. Regions of the 2D
NOE spectrum showing imino to amino/H2/H8 NOE contacts
are shown in Figure 5 (bottom). The imino signal for structure
1TSiA at 14.48 ppm exhibits three strong crosspeaks in the
amino/H2/H8 region at 9.47, 8.64, and 8.48 ppm. The two
downfield shifted resonances at 9.47 and 8.64 ppm can easily
be assigned to the two adenine amino protons since they are
mutually connected by a strong crosspeak as shown in the top
spectrum of Figure 5. Assignment of the remaining signal at
8.48 ppm to either H2 or H8 is again crucial for the determi-
nation of the hydrogen-bonding pattern in the complex. With
only a very weak NOE contact in the H1′ region and no
connectivities to any other sugar protons, the signal must be
attributed to the H2 proton, thus establishing a symmetric
antiparallel Watson-Crick duplex for 1TSiA. Additional assign-
ments make use of a moderately strong NOE contact between
adenine amino protons and adenine H8 at 7.91 ppm and
intraresidue NOE connectivities between H8 and its sugar
Upon closer inspection, three sets of signals can also be found
in the base proton region between 10 and 7.5 ppm and H1′
region between 7 and 6 ppm. Apparently, different structures
for TSiA are formed and coexist under slow exchange conditions
at low temperatures. In the following, the three species are
denoted 1TSiA, 2TSiA, and 3TSiA and refer to structures with their
imino signals resonating at increasingly higher field, respec-
tively. Integration of the three imino signals at 133 K results in
a relative population of approximately 9:6:5 for structures 1, 2,
and 3.
Due to significant signal broadening, a reliable sugar pucker
1
analysis of TSiA based on H-1H scalar coupling constants
required higher temperatures and was performed at 273 K.
1
Therefore, it should mostly reflect the conformation of TSiA.
Again, assuming a dynamic equilibrium between the two major
N- and S-type sugar conformations, the J coupling constants
determined for the T and A nucleosides in TSiA indicate a B-like
sugar conformation with a population of approximately 90%
and 60% C2′-endo sugar pucker, respectively. Similar to ASiT,
the pyrimidine nucleoside seems to have a stronger propensity
to adopt an S-type conformation when compared to the purine
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Base-Base Recognition of Nonionic Dinucleotide Analogues
A R T I C L E S
Å. Changing the glycosidic torsion angle to syn will considerably
shorten the H8-H1′ distance to ∼2.6 Å. On the other hand,
adenine H2 is expected to show only a weak or no NOE contact
to its own H1′ proton depending on the presence of anti and
syn conformations, respectively. Apparently, the strong NOE
contact of the resonance at 8.0 ppm in the H1′ region is only
compatible with an H8 proton and a syn glycosidic torsion angle
suggesting an antiparallel Hoogsteen arrangement for the 2TSiA
self-associate. Unfortunately, sugar protons of the 2TSiA complex
could only partially be assigned due to significant signal overlap
(see the Supporting Information for a list of chemical shift
2
assignments of TSiA). A schematic model of a Hoogsteen
2
dinucleotide duplex for TSiA is depicted in Figure 6b.
3
For the remaining least populated TSiA structure the rather
upfield shifted imino proton again exhibits NOE contacts to
adenine amino protons at 9.89 and 7.37 ppm as well as to either
A H2 or A H8 at 8.24 ppm. With no prominent NOE contact
to any sugar proton, the signal at 8.24 ppm can be attributed to
A H2. Accordingly, another resonance at 8.31 ppm exhibiting
a crosspeak to the amino proton at 9.89 ppm and also showing
a strong crosspeak to an H1′ proton must be assigned to A H8
3
of TSiA. With H8-H1′ being the strongest crosspeak of this
H8 signal in the sugar proton region, a glycosidic torsion angle
close to a syn-like conformation can be derived for this adenine
nucleoside.
Interestingly, the 3T imino proton exhibits another strong NOE
contact to a signal at 3.54 ppm as well as a very broad crosspeak
to a resonance centered at ∼8.96 ppm and only observable upon
further lowering the 2D threshold level (not shown). Additional
medium to weak NOE contacts connect the resonance at 3.54
3
3
ppm to A H2 at 8.24 ppm, to the two A amino protons at
9.89 and 7.37 ppm, and to the very broad signal at 8.96 ppm
(see the Supporting Information). Also, whereas this signal
appears as a sharp singlet at higher temperatures it resolves into
a doublet below 200 K before broadening again at very low
temperatures. Based on its chemical shift and its resolved
coupling at lower temperatures, the signal may be attributed to
methyl protons of tightly bound residual methanol within the
sample. Consequently, the broad signal observed at 8.96 ppm
at 133 K must arise from the methanol OH proton. Given these
Figure 6. Schematic model for (a) 1TSiA antiparallel Watson-Crick duplex,
2
3
(b) TSiA antiparallel Hoogsteen duplex, and (c) putative TSiA-folded
dinucleotide structure; R ) isopropyl, R′ ) tert-butyldimethylsilyl.
protons. A weaker NOE contact observed between A H8 and
A H3′ when compared to A H8 and A H1′ again supports data
from the J coupling analysis in having a predominant C2′-endo
sugar conformation.15 The B-type Watson-Crick dinucleotide
3
assignments, the observed NOE contacts for TSiA can be
rationalized by methanol-mediated base pairing with OH of
methanol bridging the adenine N1 acceptor and the thymine
imino donor (see Figure 6c). Although possible, an alternative
methanol-bound symmetric dimer is hardly conceivable. Rather,
folding with methanol-mediated intramolecular AT base pairing
seems plausible for this additional TSiA structure. Indeed,
molecular models suggest that such a bridging function of
methanol enables the formation of an otherwise overly strained
intramolecular base pair.
1
duplex structure of TSiA is schematically shown in Figure 6a
1
(for a full list of chemical shift assignments of TSiA, see the
Supporting Information).
Likewise, the T imino proton of structure TSiA located at
2
13.51 ppm shows crosspeaks to adenine amino protons at 9.31
and 7.96 ppm mutually connected by a strong NOE contact.
Another strong contact to a resonance at ∼8.0 ppm obviously
overlaps with one of the imino-amino crosspeaks. Depending
on a Watson-Crick or Hoogsteen hydrogen bond geometry,
this crosspeak may be attributed to either H2 or H8, respectively.
Unfortunately, due to the lower concentration of TSiA in the
Freon sample and signal broadening at low temperatures, a
1H-13C HMBC experiment as has been successfully applied
for ASiT failed to unambiguously distinguish H2 and H8 in this
case. Therefore, alternative efforts were directed toward the H1′
region. The signal at 8.0 ppm connected by a crosspeak to the
2T imino exhibits an unusually strong NOE contact to an H1′
anomeric proton without exhibiting noticeable contacts to other
sugar protons (Figure 5 top). For an anti glycosidic torsion angle,
the distance between adenine H8 and H1′ is expected to be ∼3.8
The presumed presence of residual methanol and non-
nucleosidic protons raises the question of impurities within the
sample and their influence on the complex formation of TSiA
at low temperatures. With TSiA well characterized and found
1
to be pure by both mass spectrometry and H NMR, those
impurities must come from the freonic solvent. However, upon
codissolving TSiA and ASiT in Freon (vide infra), no change in
the respective complex formation of either TSiA or ASiT is
observed, indicating that traces of methanol or other impurities
cannot be attributed to the different association behavior of TSiA
3
and ASiT. Consequently, the TSiA species may depend on
residual CH3OH but seems to nevertheless be based on an
intrinsic tendency of TSiA to intramolecularly fold into compact
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3867

A R T I C L E S
Xiao and Weisz
structures as often observed for nonionic oligonucleotides in
aqueous solution.
ASiT and TSiA spectra (not shown). Obviously, the favored
formation of antiparallel structures as typically found in natural
systems persists with the backbone-modified nucleic acids in
aprotic solvents, indicating that neither solvation effects nor
steric or electrostatic effects of the backbone have a major
impact on this widely observed preference. Yet, parallel-stranded
duplexes are known to also form under specific conditions and
can be stabilized at neutral pH by reverse Watson-Crick A-T
base pairs.23 Alternatively, parallel-stranded structures can also
be formed through Hoogsteen A-T base pairing.24 However,
reported duplexes with A-T sequences only form under
conditions where a parallel strand orientation is enforced or
extensive mismatches in the antiparallel orientation limit
competition with a regular antiparallel structure.
Hydrogen Bond Interactions. Comparison of proton chemical
shifts in the slow exchange regime at 133 K can be used as a
sensitive measure for the relative strength of hydrogen bonds.
A more deshielded proton in the hydrogen bridge originates
from a displacement of the proton toward the acceptor atom
and is associated with a lengthening of the covalent A-H bond
with an attenuation of shielding effects by the sigma electron
pair and a shortening of the H · · ·B hydrogen bond in the
A-H· · ·B hydrogen bridge.5e,25 With an imino proton chemical
shift of 15 ppm in the Watson-Crick duplex, ASiT exhibits the
most downfield shifted proton indicating the strongest hydrogen
bond interaction among all dinucleotide duplexes. In contrast,
the T imino signal of the Watson-Crick duplex formed by the
TSiA dinucleotide is shifted upfield by 0.5 ppm compared to
the imino proton in ASiT. Interestingly, these data again suggest
the presence of a more flexible TSiA structure held together by
weaker hydrogen bonds when compared to ASiT. The TSiA imino
resonance of the additional Hoogsteen duplex resonating at 13.5
ppm is further upfield shifted indicating a stronger Watson-Crick
NH· · ·N1 hydrogen bond when compared to the corresponding
Hoogsteen NH · · ·N7 hydrogen bond. Such a relative strength
of AT Watson-Crick and Hoogsteen hydrogen bonds is also
suggested by previous scalar coupling analyses for Hoogsteen-
Watson-Crick T-A-T triplets in an intramolecular DNA
triplex26 and has also been found as a result of corresponding
low temperature NMR studies in Freon for a mixture of free
uridine and adenosine nucleosides that have shown the formation
of minor amounts of Hoogsteen base pairs in addition to
predominant Watson-Crick base pairs.25 Finally, the most
shielded imino proton with a chemical shift of 12.8 ppm
assigned to the third structure of TSiA supports its tentative
assignment to an intramolecularly folded structure with methanol-
mediated AT base pairing.
It has to be noted that, being less stabilized by stacking
interactions, TA steps in nucleic acids are generally found to
exhibit more flexibility and are more deformable compared to
more rigid AT steps.18 Obviously, the formation of additional
TSiA structures at low temperatures again points to the higher
structural variability of this dinucleotide even under nonaqueous
apolar solution conditions. Thus, a second symmetric duplex
structure with antiparallel strands and AT Hoogsteen pairing is
formed through a syn glycosidic torsion angle of the adenosine
nucleoside. The Hoogsteen dinucleotide duplex is increasingly
populated at very low temperatures and is not significantly less
populated compared to the regular Watson-Crick duplex at 113
K. Isolated Hoogsteen base pairs have frequently been observed
in aqueous solution, such as in the closing loop of DNA hairpins,
in circular oligonucleotides, in chemically modified nucleic acid
structures, or upon protein binding and intercalation by drugs
like echinomycin.19 Most noticeably, a fully Hoogsteen base-
paired antiparallel duplex DNA with adenosines in the syn
conformation has been found for a d(ATATAT) sequence by
single-crystal X-ray crystallography demonstrating its ability to
constitute an alternative secondary structure to the classical
B-DNA double helix.20 It might be argued that AT sequences
are strongly polymorphic and special packing effects may be
responsible for the Hoogsteen structures seen in the crystal.
However, an antiparallel Hoogsteen duplex in polyd(AT)
sequences was found to be stable in an aqueous solution during
molecular dynamics simulations with an energy similar to that
of the B-type duplex.21 Also, a dynamic conversion of the fully
Watson-Crick to a partly Hoogsteen base-paired structure was
reported as a result of a single-point substitution of the O4′
oxygen by CH2 at an adenosine sugar residue in a self-
complementary DNA double helix.22 Overall these data suggest
that an antiparallel Hoogsteen duplex exhibiting a smaller strand
separation is an accessible structure for an AT-alternating
sequence and might compete with normal B-DNA for natural
nucleic acids and even more so for nonionic nucleic acid
analogues with no electrostatic repulsions between the two
backbones.
For the homoassociation of the self-complementary TSiA and
ASiT dinucleotides, duplexes formed through two AT base pairs
are only possible with an antiparallel strand orientation. To also
test the potential formation of parallel-stranded structures with
the backbone-modified analogues in an aprotic environment, a
1:1 mixture of ASiT and TSiA were dissolved in the freonic
solvent and subjected to additional low temperature NMR
experiments. In case of heteroassociation, a parallel duplex
stabilized by two AT base pairs is expected to form. However,
even when the temperature is decreased to 123 K, no additional
signals of a heteroassociate are observed for the mixture and
spectra are always a mere superposition of the two individual
Interaction of Polycyclic Mutagens with AsiT in Aprotic
Solvents. Since ASiT has been shown to form a single well-
defined Watson-Crick duplex structure in a freonic solvent, it
was employed as a potential target for DNA binding ligands.
Thus, the polycyclic aromatic hydrocarbon 9-(methylaminom-
(18) (a) Packer, M. J.; Dauncey, M. P.; Hunter, C. A. J. Mol. Biol. 2000,
295, 71–83. (b) Mack, D. R.; Chiu, T. K.; Dickerson, R. E. J. Mol.
Biol. 2001, 312, 1037–1049.
(23) (a) van de Sande, J. H.; Ramsing, N. B.; Germann, M. W.; Elhorst,
W.; Kalisch, B. W.; v. Kitzing, E.; Pon, R. T.; Clegg, R. C.; Jovin,
T. M. Science 1988, 241, 551–557. (b) Otto, C.; Rippe, K.; Thomas,
K.; Ramsing, N. B.; Jovin, T. M. Biochemistry 1991, 30, 3062–3069.
(c) Parvathy, V. R.; Bhaumik, S. R.; Chary, K. V. R.; Govil, G.; Liu,
K.; Howard, F. B.; Miles, H. T. Nucleic Acids Res. 2002, 30, 1500–
1511.
(19) Mendel, D.; Dervan, P. B. Proc. Natl. Acad. Sci. U.S.A. 1987, 84,
910–914.
(20) (a) Abrescia, N. G. A.; Thompson, A.; Huynh-Dinh, T.; Subirana,
J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2806–2811. (b) Abrescia,
N. G. A.; Gonza´lez, C.; Gouyette, C.; Subirana, J. A. Biochemistry
2004, 43, 4092–4100.
(24) Raghunathan, G.; Miles, H. T.; Sasisekharan, V. Biopolymers 1994,
34, 1573–1581.
(21) Cubero, E.; Abrescia, N. G. A.; Subirana, J. A.; Luque, F. J.; Orozco,
M. J. Am. Chem. Soc. 2003, 125, 14603–14612.
(25) Dunger, A.; Limbach, H.-H.; Weisz, K. J. Am. Chem. Soc. 2000, 122,
10109–10114.
(22) Isaksson, J.; Zamaratski, E.; Maltseva, T. V.; Agback, P.; Kumar, A.;
Chattopadhyaya, J. J. Biomol. Struct. Dyn. 2001, 18, 783–806.
(26) Dingley, A. J.; Masse, J. E.; Peterson, R. D.; Barfield, M.; Feigon, J.;
Grzesiek, S. J. Am. Chem. Soc. 1999, 121, 6019–6027.
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3868 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Base-Base Recognition of Nonionic Dinucleotide Analogues
A R T I C L E S
positive entropy upon ligand binding to DNA under physiologi-
cal conditions and cast doubt on whether such molecules may
be used as effective modulators of genetic information in a
nonaqueous environment.
Conclusions
Figure 7. Structures of 7,9-dimethylbenz[c]acridine (left) and 9-(methy-
The study of nonionic nucleic acid analogues in a non-natural
aprotic environment allows for a more detailed understanding
of the influence of backbone structure, base sequence, and
solvent on the hydrogen bond mediated association to form
different secondary structures. It also gives some insight into
the ability of nucleic acids to serve as carriers of genetic
information in various environments. But most importantly, by
significantly changing parameters affecting intra- and intermo-
lecular interactions, the reported low-temperature NMR studies
provide valuable information on the predominant factors that
govern the formation of nucleic acid structures and ligand-DNA
interactions and that otherwise are difficult to extract due to
their often inextricable influence under physiological conditions.
In particular, the present studies highlight the impact of water
playing a direct structural role, modulating electrostatic interac-
tions, or being associated with hydrophobic effects. Apparently,
a sequence-specific DNA strand association following simple
rules is still supported under our nonaqueous experimental
conditions at least for short sequences. However, the regular
Watson-Crick recognition scheme seems to be less robust upon
changing the sequence as already apparent for the TSiA
dinucleotide forming additional complexes with a different
hydrogen bond pattern. Available thermodynamic data for a
number of specific drug-DNA association reactions indicate
that hydrophobic transfer processes from aqueous solution into
the interior of DNA provides the predominant driving force for
drug binding.30 Consequently, the lack of water is suggested to
be a critical determinant of any intercalation process between
DNA base pairs essentially preventing the reversible formation
of stable DNA-intercalator complexes if not promoted by
exceptionally strong dipole-dipole and dispersion forces.
Likewise, the DNA affinity of groove binding agents interacting
with only a limited number of specific hydrogen bonds or salt
bridges is expected to be low upon the removal of hydrophobic
effects in nonaqueous environments.
laminomethyl)anthracene (right).
ethyl)anthracene27 and the heterocyclic 7,9-dimethylbenz[c]acri-
dine28 known to exert mutagenic effects on DNA by interca-
lating between nucleic acid base pairs under physiological
conditions were selected for binding studies with the backbone-
modified dinucleotide (Figure 7). By employing apolar solvent
conditions, hydrophobic effects associated with the release of
water upon complex formation are eliminated, and any potential
interaction between an intercalating ligand and the nucleic acid
analogue is expected to mostly rely on enthalpic contributions
from aromatic stacking interactions, where dipole-dipole and
dispersion energies play a major role.
The two ligands were mixed with ASiT in a 1:1 molar ratio
and dissolved in CD2Cl2 or Freon. Low temperatures are
expected to additionally promote ligand binding in the case of
negative binding enthalpies. However, due to the limited
solubility of the anthracene derivative, only the benz[c]acridine
could be measured at temperatures below 200 K in the freonic
solvent. Upon decreasing the temperature, no extra set of
dinucleotide resonances due to the formation of additional ligand
complexes in a slow exchange are observed and there are also
no obvious shifts or line broadening effects of DNA resonances
in the presence of either compound as anticipated in case of
intercalation, terminal stacking, or other nonspecific exterior
binding (see the Supporting Information). The absence of any
ligand-duplex interaction is further confirmed by additional
1H-29Si HMBC experiments showing no noticeable changes
in the 29Si chemical shifts or 1H-29Si coupling constants.
Because ligand intercalation should be accompanied by signifi-
cant distortions of the silyl backbone, intercalation can be safely
excluded.
Apparently, interactions between the nonionic ligand and
DNA are strongly disfavored under our aprotic solvent condi-
tions. Recently, stacking interactions in a simple binary
proflavine-TA base pair model were studied with van der Waals
density functional calculations.29 For the uncharged acridine
derivative, a considerable dispersion energy of <-10 kcal/mol
was found to promote stacking interactions and intercalation.
Because dispersion energies are hardly affected by the transfer
from an aqueous to an apolar environment, the lack of additional
electrostatic contributions as occurring with protonated (charged)
molecules and polyanionic DNA but mostly the lack of
hydrophobic contributions associated with a favorable binding
entropy must be responsible for the absence of duplex binding
by the present ligands under our solution conditions. These
observations highlight the crucial role of water release with its
Acknowledgment. We thank the Deutsche Forschungsgemein-
schaft, Bonn-Bad Godesberg, for financial support (Grant No. WE
1933/10-1).
Supporting Information Available: 1H-29Si HMBC spectrum
of ASiT; tables with (i) 1H NMR chemical shifts of ASiT at 163
K, (ii) 1H-1H scalar coupling constants of ASiT at 243 K, (iii)
1
1H NMR chemical shifts of TSiA at 133 K, and (iv) H-1H
scalar coupling constants of TSiA at 273 K; NOESY spectrum
of ASiT in Freon at 163 K; imino proton chemical shift of ASiT
as a function of concentration at 294 K; portion of a NOESY
1
spectrum of TSiA in Freon at 133 K; H NMR spectra of ASiT
without and with 7,9-dimethylbenz[c]acridine at 253 K; syn-
thesis and characterization of ASiT and TSiA. This material is
available free of charge via the Internet at http://pubs.acs.org.
(27) Becker, H.-C.; Norde´n, B. J. Am. Chem. Soc. 1999, 121, 11947–11952.
(28) Chan, E. W.; Ball, J. K. Biochim. Biophys. Acta 1971, 238, 31–45.
(29) Li, S.; Cooper, V. R.; Thonhauser, T.; Lundqvist, B. I.; Langreth, D. C.
J. Phys. Chem. B 2009, 113, 11166–11172.
(30) Chaires, J. B. Biopolymers 1998, 44, 201–215.
JA910220S
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