2-Pyrenyl-DNA: Synthesis, pairing, and fluorescence properties

Article abstract of DOI:10.1021/ol302150a

Multiple 2-pyrenyl-C-nucleosides were incorporated into the center of a DNA duplex resulting in stable pyrene self-recognition and excimer formation. This helical pyrene array may find use in DNA-mediated charge transfer and in the creation of DNA-based sensors.

Full text of DOI:10.1021/ol302150a

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
2‑Pyrenyl-DNA: Synthesis, Pairing, and
Fluorescence Properties
Filip Wojciechowski, Jory Lietard, and Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
CH-3012 Bern, Switzerland
leumann@ioc.unibe.ch
Received August 2, 2012
ABSTRACT
Multiple 2-pyrenyl-C-nucleosides were incorporated into the center of a DNA duplex resulting in stable pyrene self-recognition and excimer
formation. This helical pyrene array may find use in DNA-mediated charge transfer and in the creation of DNA-based sensors.
Non-natural bases capable of forming WatsonꢀCrick
base pairs have been pivotal in understanding the struc-
ture-to-function relationship in nucleic acids and the
intricate forces stabilizing the double helix.1 Similarly, ortho-
gonal bases and aromatic base replacements that lack
hydrogen-bonding functional groups, and interact through
edge-on or face-on stacking, have played an important role
in understanding the recognition/processing by DNA polym-
erases and in attempting to expand the genetic code.2 In
this context, simple polycyclic aromatic hydrocarbons such
as naphthalene, phenanthrene, and pyrene were introduced
into DNA.3 Pyrene was chosen to replace an entire adenine/
thymine base pair, demonstrating for the first time that a
WatsonꢀCrick base pair may be replaced by an arene
without a large decrease in thermal stability.4
DNA materials and DNA-multichromophore systems.5
For example, pyrene’s ability to form fluorescent excimers
via πꢀπ interactions has led to the detection of nucleic
acids, alkali metal ions, and proteins and has been used as
reporters for esterases and lipases.6 In a nonbiological
context, pyrene is used as a photoexcitable electron donor
to study charge transfer, in the construction of aromatic
π-arrays, and in the formation of novel helical structures.7
Recently, we incorporated non-hydrogen bonding/non-
shape complementary base surrogates such as bipyridine,
biphenyl, and phenanthrene into the middle of a DNA
duplex, and we discovered that they recognize each other
€
(5) (a) Malinovskii, V. L.; Wenger, D.; Haner, R. Chem. Soc. Rev.
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(d) Malinovskii, V. L.; Nussbaumer, A. L.; Haner, R. Angew. Chem., Int.
Pyrene-functionalized oligonucleotides are of consider-
able interest in diagnostic applications, in creating functional
Ed. 2012, 51, 4905–4908.
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Wagenknecht, H. A. Angew. Chem., Int. Ed. 2005, 44, 1636.
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Chem. Soc. 2010, 132, 7466.
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T.; Malyshev, D. A.; Romesberg, F. E. Chem.;Eur. J. 2012, 18, 1231.
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r
10.1021/ol302150a
XXXX American Chemical Society

through interstrand stacking interactions which results in a
gradual increase in duplex stability.8 We refer to this type
of stacking interaction as the zipper-like recognition motif,
confirmed by an NMR structure for a DNA duplex con-
taining one biphenyl pair.9
Scheme 1. Synthesis of 2-Pyrene Deoxynucleoside
Phosphoramidite 4
2-pyrenylmagnesium bromide.11 This was then transmeta-
lated with copper iodide and reacted with Hoffer’s chloro-
sugar (3,5-ditoluoyl-1-R-chloro-2-deoxy-D-ribose)12 to give
1 as a mixture of R/β anomers. Next, acid-catalyzed
epimerization13 of the R-anomer gave the corresponding
desired β-C-nucleoside. The 10-β-configuration was veri-
fiedby1H NMR NOE experiments. Thetoluoyl protecting
groups were removed, and the 50-hydroxyl group was
protected with DMT-triflate/2,6-di-tert-butylpyridine14
followed by phosphitylation using standard conditions.
Phosphoramidite 4 (Py) along with the abasic site (φ) was
incorporated into oligodeoxynucleotides using standard
automated DNA synthesis. Oligodeoxynucleotides were
purified by RP-HPLC and characterized by ESI-MS (see
the Supporting Information).
Figure 1. (a) Chemical structures of 2-pyrenyl deoxynucleoside
(Py) and the abasic site (φ). (b) A cartoon of the zipper-like
recognition motif highlighting pyrene interstrand stacking in-
teractions; the vertical arrows represent a phosphodiester back-
bone. (c) Molecular model (HyperChem 8.0) of a duplex
containing two interstrand stacked Py residues (left: side view,
right: top view), pyrenes are shown in yellow and magenta.
Sequences containing one, two, or three consecutive Py
residues were hybridized to the complementary sequence
containing Py residues (duplexes 3ꢀ5 in Table 1) or φ
residues (duplexes 6ꢀ11 in Table 1).
When a single Py residue was paired against an abasic
site, as in duplex 6 or 9, a minor decrease in thermal
stability was obtained (ꢀ0.4 and ꢀ1.1 °C compared to
the unmodifiedsequenceorꢀ2.0 and ꢀ2.7 °C compared to
the duplex containing an A:T base pair). These results are
comparable to those obtained with a 1-pyrene deoxynu-
cleoside (ꢀ1.6 or ꢀ2.2 °C compared to an AꢀT base pair)4
demonstrating that both the 1-pyrenyl- and 2-pyrenyl-C-
nucleotides can be paired against an abasic site without a
large difference in thermal stability. Sequences containing
two or three intrastacked Py residues paired against two or
three abasic sites (duplexes 7, 8, 10, and 11) resulted in a
gradual decrease in thermal stability reaching ꢀ9.8 and
ꢀ10.3 °C. In contrast, duplexes containing interstrand
stacked Py residues (duplexes 3, 4, and 5) were all more
stable than the unmodified sequence. Duplex 5 is 9.6 °C
We now report the hybridization properties and excimer
formation of oligodeoxynucleotides containing a 2-pyrene-
C-nucleoside as a non-natural base surrogate. Pyrene was
attached at its 2-position to deoxyribose in order to max-
imize πꢀπ interactions through interstrand stacking inter-
actions and minimize conformational isomerism around the
C-glycosidic bond, as depicted in Figure 1.
Synthesis of 2-pyrene-C-nucleoside phosphoramidite
4 follows established routes in C-aryl-nucleoside
preparation (Scheme 1).10 Treatment of 2-bromopyr-
ene with magnesium metal led to the formation of
(8) (a) Brotschi, C.; Haberli, A.; Leumann, C. J. Angew. Chem., Int.
Ed. 2001, 40, 3012. (b) Brotschi, C.; Leumann, C. J. Angew. Chem., Int.
Ed. 2003, 42, 1655. (c) Grigorenko, N. A.; Leumann, C. J. Chem.;Eur.
J. 2009, 15, 639.
(9) Johar, Z.; Zahn, A.; Leumann, C. J.; Jaun, B. Chem.;Eur. J.
2008, 14, 1080.
(12) Hoffer, M. Chem. Ber. 1960, 93, 2777.
(13) Jiang, Y. L.; Stivers, J. T. Tetrahedron Lett. 2003, 21, 4051.
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2006, 8, 3915.
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72, 8811.
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Commun. 1988, 18, 2207.
B
Org. Lett., Vol. XX, No. XX, XXXX

Table 1. Tm Data (°C), Obtained from UV-Melting Curves at
260 nm, for Duplexes 1ꢀ12a
Table 2. Thermodynamic Parameters of Duplex Formationa
50ꢀd(GATGAC(Py)nGCTAG)
50ꢀd(GATGAC(X)nGCTAG)
30ꢀd(CTACTG(Py)nCGATC)
30ꢀd(CTACTG(Y)nCGATC)
K
ΔH
ΔS
ΔG298
b
c
no. [kcal molꢀ1
]
ΔΔHb [cal Kꢀ1 molꢀ1
]
ΔΔSc [kcal molꢀ1
]
duplex
n
X
Y
Tm
ΔTm
ΔTm
1
2
3
0
1
1
ꢀ
ꢀ
48.0
49.6
51.6
ꢀ
ꢀ
0
1
2
3
ꢀ72.6
ꢀ69.7
ꢀ73.6
ꢀ78.3
ꢀ
ꢀ199.2
ꢀ187.4
ꢀ201.2
ꢀ209.5
ꢀ
ꢀ13.3
ꢀ13.8
ꢀ13.7
ꢀ15.9
T
A
þ1.6
þ3.6
ꢀ
þ2.9
ꢀ1.0
ꢀ5.7
þ11.8
ꢀ2.0
ꢀ10.3
Py
Py
þ4.0/
þ4.7
þ8.1/
þ4.0
þ19.4/
þ19.9
ꢀ
4
5
2
3
Py
Py
Py
Py
49.6
57.6
þ1.6
þ9.6
a Enthalpies, entropies, and Gibb’s free energies were obtained from
van’t Hoff plots (concentrationꢀvariation method). b ΔΔH = ΔHn ꢀ
ΔHn=0 and are in kcal molꢀ1
.
c ΔΔS = ΔSn ꢀ ΔSn=0 and are in cal
K
ꢀ1 molꢀ1
.
6
1
2
3
1
2
3
1
Py
Py
Py
φ
φ
47.6
41.5
38.2
46.9
45.6
37.7
29.7
ꢀ0.4
ꢀ6.5
ꢀ9.8
ꢀ1.1
ꢀ2.4
ꢀ10.3
ꢀ18.3
7
φ
ꢀ
8
φ
ꢀ
Next, the sequences were investigated for their ability to
9
Py
Py
Py
φ
ꢀ
undergo excimer formation (Figure 2). Duplex 3 exhibited
weak monomer emission at 378 and 398 nm and a broad
structureless emission centered at 490 nm that is typical for
pyrene excimer formation. Increasing the number of Py
base pairs lead to a concomitant increase in excimer
intensity in all duplexes (Figure 2a,b) and single strands
(see Supporting Information). Surprisingly, duplex 5 (530
au) had an approximate 4- or 5-fold increase in excimer
intensity when compared to duplex 8 (138 au) or 11 (107
au). It likely occurs because of a cooperative interaction of
the Py residues and not simply from the sum of the excimer
intensities originating from intramolecular excimer forma-
tion in the single strands. Similar behavior has been
observedwithpyrene substituted atthe 20-sugarposition.7b
Next, we measured the change in excimer intensity with
increasing temperature and, as expected, found a gradual
decrease in excimer emission. A shift from excimer emis-
sion to monomer emission was observed, in duplex 3,
between 50 and 60 °C which is consistent with the obtained
melting temperature (Tm = 51.6 °C). Duplex 5 also under-
went a gradual decrease in excimer emission. However,
even at 80 °C, which is well above the melting temperature,
there was no monomer emission present and only weak
excimer emission from intramolecular stacked pyrenes in
the single strands was observed.
CD measurements were performed on both the Py
containing duplexes and single strands in order to gain
further insight into the structural preference (Figure 3).
Duplex 3 exhibited a typical B-type conformation with
almost equal positive (275 nm) and negative (250 nm)
ellipticities zeroing at 263 nm (Figure 3a). At wavelengths
above 300 nm, the CD spectra are biphasic due to exciton
coupling between the pyrenes.16 Interestingly, a negative-
positive (-) cotton effect for the pyrene band was observed
inthecaseof duplex3 whichwas inverted(() induplexes7,
10 or the single strands. This change in sign can most likely
be attributed to the change in distance and dihedral angle
between the pyrenes.17 The exciton coupling intensities
10
11
12
φ
ꢀ
φ
ꢀ
φ
ꢀ
a Conditions: 2.5 μM strand concentration in 10 mM NaH2PO4, 150
mM NaCl, pH 7.0. Estimated error in Tm = ( 0.5 °C. b ΔTm is the
diffrence in Tm relative to the duplex 1 lacking X and Y (n = 0). c ΔTm is
the diffrence in Tm between duplexes 3, 4, or 5 containing interstrand
stacked pyrenes and the corresponding duplex containing intrastrand
stacked pyrenes facing an abasic site φ (duplexes 6ꢀ8 and 9ꢀ11). The
values are obtained by subtracting the Tm of duplex 3 from duplex 6 or 9,
subtracting the Tm of duplex 4 from duplex 7 or 10, and subtracting the
Tm of duplex 5 from duplex 8 or 11.
more stable than the unmodified sequence or 19.4 °C/
19.9 °C more stable than duplex 8 or 11. These results
confirm that interstrand stacking interactions play a domi-
nant role in stabilizing a DNA duplex containing non-
shape complementary/non-hydrogen bonding base pairs.
Furthermore, these results differ from interstrand stacked
pyrenes in DNA using non-nucleosidic linkers, in which
stable hybrids were formed but with a minor gradual
decrease in thermal stability compared to the unmodified
sequence.15
To gain further insight into the hybridization behavior
we measured the thermodynamic parameters of duplex
formation, Table 2. Formation of duplex 3 (n = 1) was
driven by a favorable entropic change (ΔΔS = þ11.8 cal
K
ꢀ1 molꢀ1) and an unfavorable change in enthalpy (ΔΔH =
þ2.9 kcal molꢀ1) when compared to the unmodified
sequence. Formation of duplex4 (n = 2) was accompanied
with an unfavorable change in entropy (ΔΔS = ꢀ2.0 cal
Kꢀ1 molꢀ1) that was compensated for by a favorable
change in enthalpy (ΔΔH = ꢀ1.0 kcal molꢀ1). A similar
trend was observed for duplex 5. Subtraction of the
enthalpies (ΔHn=3 ꢀ ΔHn=1) compensates for the inter-
action of Py with the neighboring natural bases and
revealed that duplex 5 is stabilized by ꢀ8.6 kcal molꢀ1
.
This suggests that overall a duplex containing more than
one Py base pair is enthalpically driven.
€
(15) (a) Langenegger, S. M.; Haner, R. Chem. Commum. 2004, 2792.
(16) Berova, N.; Nakanishi, K. Circular Dichroism: Principles and
Applications; Berova, N., Nakanishi, K., Woody, R. W., Eds.; Wiley-VCH:
New York, 2000; p 337.
€
(b) Malinovskii, V. L.; Samain, F.; Haner, R. Angew. Chem., Int. Ed.
2007, 46, 4464.
Org. Lett., Vol. XX, No. XX, XXXX
C

Figure 2. (a and b) Fluorescence spectra of duplexes 3, 4, 5 and 7,
8, 10, 11. (c and d) Temperature dependent fluorescence spectra
of duplexes 3 and 5. Conditions: λex, 350 nm; detector, 575 V;
excitation slit, 5 nm; emission slit, 5 nm; same buffer and con-
centration as those in Table 1.
decrease with increasing temperature, and no exciton cou-
pling occurred above the melting temperature (Figure 3b).
The single strands in Figure 3c,e also maintained exciton
coupling between the pyrenes with a positive Cotton effect
(() and approximately equal intensity at 330 and 350 nm.
Increasing the number of Py base pairs (as observed for
duplex 4 and 5) was accompanied by an increase in
intensity for the positive peaks found at 345/330/277 nm
and the negative peak at 237 nm. Overall, comparison of
the unmodified duplex 1 with duplex 5 in the 200ꢀ300 nm
region indicates that the accommodation of three Py base
pairs is accompanied by a slight change in the DNA
conformation.
In summary, the present results demonstrate that 2-pyr-
enyl containing DNA forms a duplex that is more stable
than the unmodified duplex. The thermal stability, excimer
formation, and CD spectroscopy support interstrand
stacking as the stability determining factor. We are currently
investigating if a similar behavior will be observed with
DNA containing 1-pyrenyl or 4-pyrenyl base replacements.
Figure 3. (aꢀf) CD spectra of the duplexes and single strands.
Conditions: 5.0 μM strand concentration, same buffer as that in
Table 1.
Since pyrene and substituted pyrenes can be used as photo-
excitable electron donors, the present helical pyrene array
may find use in studying DNA-mediated charge transfer.
Such research is of importance in the design of aromatic
π-arrays and in the construction of novel DNA-based sensors.
Acknowledgment. We acknowledge financial support
from the Swiss National Science Foundation (Grant No.
200020-130373) and the EU for a Marie Curie Fellowship
to F.W.
Supporting Information Available. Synthetic proce-
dures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
(17) Lewis, F. D.; Zhang, L.; Liu, X.; Zuo, X.; Tiede, D. M.; Long,
H.; Schatz, G. C. J. Am. Chem. Soc. 2005, 127, 14445.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX