Synthesis of 5-(2-pyrenyl)-2′-deoxyuridine as a DNA modification for electron-transfer studies: The critical role of the position of the chromophore attachment

Article abstract of DOI:10.1002/ejoc.200700818

5-(2-Pyrenyl)-2′-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 con

Full text of DOI:10.1002/ejoc.200700818

FULL PAPER
DOI: 10.1002/ejoc.200700818
Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine as a DNA Modification for
Electron-Transfer Studies: The Critical Role of the Position of the
Chromophore Attachment
Claudia Wanninger-Weiß[a] and Hans-Achim Wagenknecht*[a]
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-posi-
tion 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-tet-
to explore the optical properties of the 2PydU label in duplex
DNA with respect to the counterbase opposite the 2PydX
modification site and in comparison with 1PydU-modified
DNA. The studies with the 2PydU nucleoside and the 2PydU-
modified DNA clearly allow the conclusion to be drawn that
ramethyl-1,3,2-dioxaborolan-2-yl)pyrene (4) by using an Ir the pyrene and the uridine moieties, as the two aromatic
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/H2O mixture as the sol-
vent. The modified nucleoside 2 was characterized by ab-
sorption and fluorescence spectroscopy. The results were
compared with the strongly electronically coupled 5-(1-pyr-
enyl)-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
groups in this modified nucleoside, are only weakly electron-
ically coupled. Accordingly, the 2PydU label behaves op-
tically like a pyrene derivative, in contrast to the 1PydU la-
bel. The chromophore shows the ability for Watson–Crick
base-pairing inside the DNA, as revealed by the absorption
and fluorescence spectra. 2PydU represents an optical label
for DNA that changes its absorption properties upon DNA
hybridization and undergoes fluorescence quenching by
charge transfer.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
opment of versatile and tunable optical probes for
DNA.[11,12]
Introduction
The analytical problems typical of biomedicinal diagnos-
tics and chemical bioanalysis demand powerful and versa-
tile DNA labels for optical spectroscopy. A variety of or-
ganic dyes have been investigated and applied as artificial
DNA base substitutes for fluorescent nucleic acid analy-
sis.[1–3] Recently, the chemical detection of single nucleotide
polymorphisms without the application of any enzymes was
achieved by fluorescent DNA base substitutions based on,
for example, ethidium,[4] thiazole orange[5] or indole.[6]
Moreover, there is an increasing demand for optical DNA
hybridization labels that change not only their emission but
also their absorption properties as a result of duplex forma-
tion. One important way to create this kind of duplex-sensi-
tive optical property is to attach organic chromophores co-
valently to DNA bases. Over the last few years, we have
used this kind of modification strategy to investigate DNA-
mediated electron-transfer processes[7–10] and in the devel-
Pyrenes have been widely used for nucleic acid label-
ling.[12–18] We used 5-(1-pyrenyl)-2Ј-deoxyuridine (1PydU,
1) and other pyrene-modified nucleosides as models for un-
derstanding electron transfer in DNA.[7,8] Furthermore, in
1PydU-modified DNA duplexes, photochemically induced
electron transfer from the PydU group can be studied by
chemical and spectroscopic techniques.[9] Moreover, we re-
cently described 8-(1-pyrenyl)-2Ј-deoxyguanosine as a du-
plex-sensitive probe for absorption and fluorescence spec-
troscopy with DNA and for the detection of base mis-
matches.[6,12] Pyrene-modified nucleosides have also been
described as base-discriminating fluorescent probes.[14] The
characteristic excimer fluorescence of two and more stacked
pyrene moieties has also been applied in the diagnosis of
genetic variations.[15] Helical π arrays of pyrene-modified
nucleosides along DNA and RNA duplexes have been de-
scribed by our group[16] and others.[17] DNA has also been
used as a framework for the helical arrangement of in-
terstrand stacked pyrenes.[18]
[a] Institute for Organic Chemistry, University of Regensburg,
93040 Regensburg, Germany
A critical issue for the optical properties of pyrene is the
linkage between this chromophore and the oligonucleotide.
Fax: +49-941-943-4617
E-mail: achim.wagenknecht@chemie.uni-regensburg.de
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Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine
In our previously described pyrene-modified nucleosides, electron picture the LUMO of the uridine moiety has to
such as 1PydU (1), the two chromophores are linked coval- accept an electron from an occupied pyrene π orbital. The
ently by a single C–C bond.[8] According to the observed LUMOs of 1PydU (1) and 2PydU (2) were calculated with
unstructured fluorescence bands with solvent-dependent the semi-empirical AM1 method (Figure 1). They were ob-
maxima, strong electronic coupling exists between the aro- tained after full geometry optimization. The results show
matic parts of the molecule. These intramolecular (bonded) clearly that 1PydU (1) has a large coefficient on the carbon
exciplexes exhibit both excited-state and charge-separated- at the 1-position that connects the pyrene to the uridine
state character.[19] Herein, we present the synthesis of 5-(2- moiety. This leads to a largely delocalized orbital and, as
pyrenyl)-2Ј-deoxyuridine (2PydU, 2) as an alternative py- already pointed out in the Introduction, to exciplex states
rene-labelled uridine and the spectroscopic evaluation of its that show unstructured, solvent-dependent fluorescent
optical properties. In the modified nucleosides, 1PydU (1) bands. In contrast, the LUMO of 2PydU (2) is strictly local-
and 2PydU (2), the two aromatic groups are connected ized on the aromatic system of the pyrene with a low coeffi-
through a single C–C bond. However, in contrast to 1PydU cient on the carbon atom (at the 2-position) connecting the
(1), the uridine moiety in 2PydU (2) is connected to the 2- uridine moiety. The overlap of the uridine and pyrene do-
position of the pyrene chromophore which alters the optical nor orbital is negligible and no significant electronic coup-
properties of the pyrene label significantly. Based on this ling takes place between the aromatic moieties. Hence, in
molecular design, 2PydU (2) should show optical properties 2PydU (2) the typical optical properties of pyrene and the
typical of pyrene together with a base-pairing ability in du- base-pairing ability of the uridine group should reflect that
plex DNA due to the presence of the uridine group.
the pyrene is connected covalently but not coupled electron-
ically.
Our approach for the synthesis of the pyrene-modified
nucleosides such as 1 and 2 was to apply the palladium-
Results and Discussion
Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine (2PydU, 2): To catalyzed Suzuki–Miyaura-type cross-coupling of pyrenyl-
understand the electronic differences between 1PydU (1) boronic acids or acid esters to the corresponding haloge-
and 2PydU (2) we have studied the topology of the relevant nated nucleosides. In general, Suzuki–Miyaura-type coup-
molecular orbitals that are involved in the intramolecular lings are highly suitable for nucleoside synthesis because
electron-transfer processes in these nucleosides. The locally they work in moist or even aqueous solutions, and they
excited state of pyrene Py* is a good electron donor and tolerate the presence of unprotected hydroxy and amino
can induce electron transfer yielding the charge-separated groups which are the characteristic functional groups of nu-
state that consists of the pyrenyl radical cation Py·+ and the cleosides.[22] Hence, Suzuki–Miyaura-type couplings have
corresponding uridine radical anion dU·–. This assumption been applied for the preparation of a broad variety of aryl-
is based on the redox potential (1.5 V vs. NHE) and the ated and alkenylated nucleosides.[23,24] We have developed a
singlet energy of pyrene (E00 = 3.25 eV).[20] The driving special Suzuki–Miyaura coupling protocol to synthesize all
force of this intramolecular electron transfer is a maximum four 1-pyrenyl-modified nucleosides.[8] In order to apply
0.5–0.6 eV on the basis of the reported reduction potentials this protocol to the synthesis of 2 the bromination of pyrene
of –1.1 to –1.2 V for uracil.[21] According to this simple one- (3) in the 2-position is required as the first step towards the
Figure 1. Calculated LUMOs of 1PydU (1) and 2PydU (2).
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C. Wanninger-Weiß, H.-A. Wagenknecht
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corresponding pyrene-2-boronic acid or acid ester. Stan- drogenation.[26] This biphenyl aromatic system can be bro-
dard electrophilic substitution of pyrene (3) produces minated in the 2-position and subsequently reoxidized to
mainly derivatizations in the 1-position and subsequently the fully aromatic 2-bromopyrene.[27]
1,3-, 1,6- and 1,8-disubstituted products.[25] Pyrenes that are
In accord with a recent related report,[28] we chose the
substituted in ring position 2 are difficult to obtain. The direct approach to the synthesis of a pyrene-2-boronic acid
most convenient synthetic strategy for the introduction of ester as potential precursor for the subsequent Suzuki–
functional groups in the 2-position involves the conversion Miyaura cross-coupling (Scheme 1). This approach is based
of pyrene (3) into 4,5,9,10-tetrahydropyrene by catalytic hy- on studies by Hartwig and Miyaura who have shown that
Scheme 1. Synthesis of 2PydU (2) and the corresponding DNA building block 7 for automated oligonucleotide synthesis: Reagents and
conditions: a) NaOMe (3.0 equiv.), cyclohexane, 60 min, room temp.; b) 3 (1.0 equiv.), bis(pinacolato)diborane (1.1 equiv.), dtbpy
(0.1 equiv.), [Ir(OMe)(cod)]2 (0.05 equiv.), cyclohexane, 48 h, 80 °C (37%, a+b); c) 4 (1.2 equiv.), [Pd(dppf)2Cl2] (0.11 equiv.), THF/H2O/
MeOH = 2:1:1, NaOH (19.5 equiv.), 60 h, 65 °C (62%); d) DMTCl (1.0 equiv.), pyridine, 48 h, room temp. (78%); e) cyanoethyl N,N-
diisopropylchlorophosphoramidite (1.0 equiv.), EtN(iPr)2 (3.6 equiv.), CH2Cl2, 1 h, room temp. (95%).
Figure 2. Assigned NOESY spectrum of the aromatic region of 4.
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Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine
iridium catalysts are particularly efficient for the direct bo-
rylation of aromatic compounds.[29] We started the synthe-
sis of 2 by converting pyrene (3) into 2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrene (4) using the catalyst that
was prepared in situ from [IrCl(cod)]2 and 4,4Ј-di-tert-bu-
tyl-2,2Ј-bipyridine (dtbpy) in the presence of NaOMe. The
product 4 was obtained in 37% yield by using 5 mol-% of
the precursor for the active Ir catalyst in cyclohexane. In
order to prove the successful derivatization at the 2-position
of the pyrene chromophore spectroscopically we first as-
signed all pyrene protons by 2D NMR spectroscopy. As
expected, the corresponding spectrum shows one singlet for
the protons 1-H and 3-H which are adjacent to the modifi-
cation site. Subsequently, a NOESY spectrum of 4 was re-
corded to support this result. This spectrum shows the
characteristic NOE pattern of a 2-substituted pyrene (Fig-
ure 2). Accordingly, the protons 1-H and 3-H show a strong
NOE to each other and a weak NOE to 4-H and 10-H. On
the other side, 7-H exhibits a NOE with 6-H and 8-H.
The subsequent Suzuki–Miyaura cross-coupling of 4
with 5-iodo-2Ј-deoxyuridine (5) was performed using our
previously described protocol: 1,1Ј-bis[(diphenylphosphan-
yl)ferrocene]dichloropalladium(II) [Pd(dppf)2Cl2] (0.11
equiv.) was used as the catalyst in a THF/MeOH/H2O mix-
ture as solvent. After 60 h at 65 °C the product 2 was iso-
lated in 62% yield.
Spectroscopic and Electrochemical Characterization of
2PydU (2): The synthesized nucleoside 2 was characterized
by optical spectroscopy. All measurements were compared
with the pyrene nucleoside 1, which was synthesized accord-
ing to the literature,[8] and commercially available pyrene
(3). The steady-state optical characterization of 2PydU (2)
was performed in MeOH and MeCN as representative sol-
vents with and without hydrogen-bonding capabilities (Fig-
ure 3). We have previously shown that the dynamics of in-
tramolecular electron transfer in 1PydU (1) in a non-protic
solvent (MeCN) and in a protic one (MeOH) are signifi-
cantly different, although both solvents have similar dielec-
tric properties.[8] The UV/Vis spectra of both modified com-
pounds, 1 and 2, in both solvents exhibit shapes typical of
pyrene derivatives (Figure 3). Compared with pyrene (3),
the absorption spectra of 1PydU (1) are significantly more
redshifted than those of 2PydU (2), indicating the stronger
electronic coupling between the two aromatic parts in 1
compared with 2.
Figure 3. UV/Vis absorption spectra of 2PydU (2) in comparison
with 1PydU (1) and pyrene (3), measured in MeCN (top) and
MeOH (bottom). The samples were adjusted to an identical optical
density at the excitation wavelength of 338 nm (in MeCN: 1: 30 µ;
2: 35 µ; 3: 55 µ; in MeOH: 1: 40 µ; 2: 45 µ; 3: 100 µ).
with maxima at 399 and 421 nm). In contrast, 1PydU (1)
shows a broad fluorescence band in MeCN (425 nm) that is
redshifted in MeOH (447 nm) which is typical of a strongly
coupled bonded exciplex state, as discussed above. In
MeCN, some of the locally excited state of the pyrene group
in 1 persists (side-band at 388 nm). The relative fluores-
cence intensities of both nucleosides 1 and 2 are signifi-
cantly reduced in MeOH compared with MeCN. The mea-
sured quantum yields reflect the fluorescence observations
quantitatively (Table 1). The quantum yield of 1 is signifi-
cantly reduced from 24% in MeCN to 2.5% in MeOH. This
is also observed in the case of 2, however, not to such an
extent. As previously shown by time-resolved studies, the
electron-transfer yield in 1 was greatly enhanced in
MeOH.[8] Clearly, this effect is much weaker in 2 as a result
of the weaker electronic coupling in this nucleoside. This
result emphasizes the critical role of hydrogen bonding in
electron transfer involving DNA bases.
Based on the recorded absorbance spectra, the excitation
wavelength for the steady-state fluorescence measurements
was chosen to be 338 nm. It is important to point out that
the samples were adjusted to an identical optical density at
this excitation wavelength in order to elucidate exclusively
the influence of hydrogen bonding on the emission. The
corresponding fluorescence spectra of 1–3 show remarkable
differences (Figure 4). The emission of pyrene (3) is nearly
identical in MeCN and MeOH; no influence of the solvent
was observed. The fluorescence spectra of 2PydU (2) exhi-
bit the typical pyrene band structure, although significantly
redshifted by approximately 30 nm. The emission maxima
Figure 4. Fluorescence spectra of 2PydU (2) in comparison with
1PydU (1) and pyrene (3), measured in MeCN (black) and MeOH
(grey). The samples were adjusted to an identical optical density at
the excitation wavelength of 338 nm (in MeCN: 1: 30 µ; 2: 35 µ;
of 2 in MeCN and MeOH are nearly the same (double band 3: 55 µ; in MeOH: 1: 40 µ, 2: 45 µ; 3: 100 µ).
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Table 1. Quantum yields of 2PydU (2) in comparison with 1PydU (Figure 5) and by their melting temperatures Tm (Table 2).
(1) and pyrene (3), measured at 338 nm.
The CD spectra of the duplexes 8a–8d and 9a–9d confirm
the normal right-handed helical B-DNA conformation. The
melting temperatures of the duplexes 9a–9d are all lower
than those of the duplexes 8a–8d, indicating that the steric
demand and local perturbation of the pyrene group is
higher in 1PydU than in 2PydU. The “mismatched” du-
plexes 8b and 8c, but not 8d, exhibit increased melting tem-
peratures which indicate a partial intercalation causing
hydrophobic stabilization of the pyrene. However, the Tm
data does not clearly reveal a preferred Watson–Crick base-
pairing for 2PydU.
Compound
Φ (MeCN)
Φ (MeOH)
1
2
3
0.235
0.067
0.019
0.025
0.027
0.018
It is remarkable to note that the optical properties of
2PydU (2) reflect clearly the electronic properties that were
expected from its design. This interpretation is based
mainly on three observations: (i) only a small redshift of
the absorption compared with pyrene (3), (ii) an emission
that exhibits the structure of the locally excited state of the
pyrene chromophore, and (iii) no solvent-dependence of the
fluorescence maxima and a smaller effect of the solvent on
the fluorescence intensity. Hence, in contrast to 1PydU (1),
the two aromatic moieties in 2PydU (2) are only weakly
electronically coupled. Hence, the absorption and fluores-
cence properties of 2 are similar to those of pyrene (3).
Synthesis and Characterization of 2PydU-Modified DNA:
In order to explore the optical properties of the pyrene-
modified nucleoside 2 in DNA we synthesized the duplex
set 8a–8d bearing
a random DNA base sequence
(Scheme 2). Measurements were also performed with the
duplex set 9a–9d which contain identical sequences except
with the 1PydU modification instead of the 2PydU group.
The duplexes 9a–9d were prepared according to the litera-
ture.[9] In both duplex sets, 8a–8d and 9a–9d, the
counterbase Y opposite the pyrene modification site X was
varied in order to explore the Watson–Crick base-pairing
properties of 1PydU and 2PydU inside duplex DNA.
Figure 5. CD spectra of DNA 8a–8d (top) and 9a–9d (bottom)
[2.5 µ duplex in 10 m Na-Pi-buffer (pH 7.0), 250 m NaCl,
20 °C].
Table 2. Melting temperatures (Tm) of DNA duplex sets 8a–8d and
9a–9d [2.5 µ duplex, 10 m Na-Pi-buffer (pH 7.0), 250 m NaCl,
260 nm, 10–90 °C, heating rate: 0.7 °C/min].
Counterbase
Y
DNA
Tm [°C]
DNA
Tm [°C]
A
C
T
8a
8b
8c
8d
55.2
58.2
56.0
54.3
9a
9b
9c
9d
52.5
55.8
54.0
53.1
G
Scheme 2. Sequences of DNA duplex sets 8a–8d and 9a–9d.
The pyrene-modified uridine 2 was converted into the
The UV/Vis absorption spectra of both duplex sets, 8a–
DMT-protected compound 6 and then to the completely 8d and 9a–9d, show clearly the presence of the pyrene chro-
protected nucleoside 7 carrying the phosphoramidite group mophore in the range between 300 and 400 nm (Figure 6).
in the 3Ј-position (Scheme 1). Standard procedures were ap- However, the shapes of the pyrene absorption bands are
plied to these two synthetic steps. By using the DNA build- significantly different for the two chromophore-modified
ing block 7, the 2PydU-modified oligonucleotide 8 was pre- nucleosides. The single-stranded oligonucleotide 8 has an
pared by automated solid-phase synthesis using the Expe- absorption maximum at 344 nm that is maintained after hy-
dite 8909 DNA synthesizer. Nearly quantitative coupling of bridization with the mismatched counterstrands yielding
the monomer 7 was achieved with the standard coupling the DNA duplexes 8b–8d. It is important to point out that
time of 1.6 min. The HPLC-purified oligonucleotide was only in DNA 8a, bearing an adenine as the correct Watson–
identified by ESI mass spectrometry and quantified by its Crick counterbase opposite the 2PydU modification, is the
UV/Vis absorption spectrum by using ε260 = 18600 –1 cm–1 absorption maximum blueshifted to 338 nm. Together with
for the 2PydU modification. By using the 2PydU-modified the melting temperatures discussed above, this blueshift can
oligonucleotide 8 we prepared the corresponding DNA du- be interpreted as the result of the destacking of the pyrene
plexes 8a–8d by slow cooling in the presence of 1.2 equiv. chromophore out of the DNA helix as a result of the base-
of the unmodified complementary strands. These DNA du- pairing of the uridine group of 2PydU with the adenine
plexes were subsequently characterized by CD spectroscopy counterbase. This result is not observed with the duplex set
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Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine
9a–9d. The single-stranded oligonucleotide 9 exhibits ab- the charge-separated state. As a result of direct π-orbital
sorption maxima at 335 and 350 nm, similar to the absorp- overlap between the radical cation Py·+ and the radical
tion of 1PydU (1) in MeOH. After hybridization with the anion dU·– the transient absorption exhibits broad spectral
counterstrands, the four duplexes 9a–9d show a similar ab- signals that range from around 450 to around 730 nm which
sorption with a broad maximum at around 355 nm. The are difficult to assign and to correlate with the dynamics of
absorption differences between the duplexes 8a and 8b–8d electron transfer. Based on the steady-state studies of the
on the one hand and the similarity of the duplexes 9a–9d nucleoside 2PydU (2) and the representative synthesized
on the other are remarkable. In fact, the 2PydU moiety be- 2PydU-modified DNA 8a we can conclude a weak elec-
haves like a pyrene covalently attached but not electroni- tronic coupling between the two aromatic groups in the new
cally coupled to the DNA base uridine. According to the label. Hence, we expect that the dynamics of electron trans-
absorption spectra the chromophore shows an ability for fer can be assigned to the transient absorption signals of
Watson–Crick base-pairing in DNA. In contrast, the the 2PydU label in DNA more clearly than was the case
strongly coupled pyrene chromophore in the 1PydU group with the 1PydU-modified DNA.
does not exhibit this property.
Figure 7. Fluorescence spectra of DNA 8a–8d (top) and DNA 9a–
9d (bottom) [2.5 µ duplex, 10 m Na-Pi-buffer (pH 7.0), 250 m
NaCl, 20 °C].
Figure 6. UV/Vis absorption spectra of DNA 8a–8d (top) and
DNA 9a–9d (bottom) [2.5 µ duplex in 10 m Na-Pi-buffer
(pH 7.0), 250 m NaCl, 20 °C].
Conclusions
The fluorescence spectra of the duplexes were recorded at
an excitation wavelength of 340 nm for the 2PydU-modified
The critical issue for the optical properties of the 1PydU
DNA 8a–8d and 360 nm for the 1PydU-modified DNA 9a– label that we have studied extensively over the last few years
9d (Figure 7). In both duplex sets, the emission increases was the strong electronic coupling between the pyrene and
significantly compared with the single-stranded oligonucleo- the uridine as the two aromatic groups in this modified nu-
tides 8 and 9. The emission of the 2PydU chromophore cleoside. Hence, the 2PydU label was designed, synthesized
allows the right counterbase (A in 8a) to be discriminated and studied as an alternative pyrene-modified nucleoside
against the wrong ones (8b–8d); the emission of the former and optical label for DNA. In both uridines, 1PydU and
is greater than the latter by a factor of at least 2. This is 2PydU, the pyrene is attached covalently through a single
not the case for the 1PydU-modified duplexes 9a–9d. This C–C bond. However, due to the fact that the uridine moiety
result is similar to that already pointed out above for the in 2PydU is connected to the 2-position of the pyrene chro-
absorption differences. However, one has to take into ac- mophore and not to the 1-position as in 1PydU the elec-
count the fact that the quantum yields of all the duplexes tronic coupling is significantly weaker. The absorption and
lie in the range between 1.0 and 1.5%. In comparison with fluorescence spectroscopy studies described above clearly
the quantum yield for the isolated nucleoside 2 of 7% in allow this conclusion to be drawn. Accordingly, the proper-
MeCN this result indicates a significant quenching of the ties of 2PydU exhibit characteristics typical of a pyrene de-
2PydU fluorescence in DNA. Based on our previous experi- rivative. 2PydU behaves optically and electrochemically
ments with 1PydU-modified duplexes,[9] we assume that this more like a pyrene label for DNA than does 1PydU. Strong
fluorescence quenching can be assigned to a photoinduced fluorescence quenching of the 2PydU label is observed in
electron transfer inside the DNA, preferably to the adjacent duplex DNA presumably due to a charge transfer into the
thymines. This result makes the 2PydU label a highly prom- DNA.
ising one-electron donor for spectroscopic studies of charge
In general, the observed differences between 1PydU and
transfer in DNA. The critical issue of the 1PydU label that 2PydU underscore the fact that the position of attachment
we have applied until now to elucidate the dynamics of elec- of large aromatic chromophores is critical and determines
tron transfer in DNA was the strong electronic coupling in the optical properties. Pyrene as an artificial DNA base sur-
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rogate has previously been used as a fluoroside[30] and in the
investigation of DNA–protein interactions.[31] The 2PydU
modification represents a pyrene label that retains the
unique and characteristic optical properties of the pyrene
chromophore and connects it, but does not couple it elec-
tronically, to the DNA base. The chromophore shows the
ability for Watson–Crick base-pairing inside DNA, as re-
vealed by the absorption and fluorescence spectra.
0.330 mmol, 1.0 equiv.) and 1,1Ј-bis[(diphenylphosphanyl)ferro-
cene]dichloropalladium(II) (29.4 mg, 0.036 mmol, 0.11 equiv.) were
dissolved in degassed THF/H2O (2:1, 40 mL) under nitrogen. After
addition of MeOH (10 mL) and NaOH (257 mg, 6.44 mmol,
19.5 equiv.) the mixture was stirred at 65 °C for 60 h, neutralized
with 2  HCl and extracted with EtOAc (150 mL). The organic
phase was dried with Na2SO4 and the solvent was removed under
vacuum. The crude product was purified by flash chromatography
(CH2Cl2/acetone = 4:1, eluent: EtOAc/MeOH = 10:3) to give 89 mg
(62%) of a pale brown solid. TLC (EtOAc/MeOH/H2O = 10:1:0.5):
1
Rf = 0.56. H NMR (300 MHz, [D6]DMSO): δ = 11.67 (br. s, 1 H,
Experimental Section
NH), 8.53 [s, 1 H, 6-H (dU)], 8.48 [s, 2 H, 1-H, 3-H (pyrene)], 8.29
3
Materials and Methods: 1H, 13C, 31P and 2D NMR spectra were
recorded at 300 K with a Bruker Avance 300 or 400 MHz spec-
trometer. NMR signals were assigned on the basis of 2D NMR
measurements (HSQC, HMBC, NOESY). ESI, EI and HR-EI
mass spectra were recorded in the analytical facility of the institute.
The HR-ESI mass spectra (ESI-FTICR) were recorded by Coring
System Diagnosticx GmbH. Analytical chromatography was per-
formed on Merck silica gel 60 F254 plates. Flash chromatography
was performed on Merck silica gel (40–63 µm). C18-RP analytical
and semi-preparative HPLC columns (300 Å) were purchased from
Supelco. Solvents were dried according to standard procedures. All
reactions were carried out under dry nitrogen. Commercial chemi-
cals were purchased by Fluka, Sigma–Aldrich and Alpha Aesar
and were used without further purification. All spectroscopic mea-
surements were performed in quartz glass cuvettes (1 cm) using Na-
Pi-buffer (10 m). Absorption spectra and the melting tempera-
tures (2.5 µ duplex, 250 m NaCl, 260 nm, 10–90 °C, interval
0.7 °C) were recorded with a Varian Cary 100 spectrometer. The
B-DNA conformation of all the duplexes was confirmed by CD
spectroscopy (2.5 µ duplex, 200–350 nm) performed with a Jasco
J-715 spectropolarimeter. The fluorescence spectra (2.5 µ duplex)
were recorded with a Fluoromax-3 fluorimeter (Jobin–Yvon) and
corrected for Raman emission from the buffer solution. All emis-
sion spectra were recorded with a band-pass of 2 nm for both exci-
tation and emission and are intensity-corrected. The fluorescence
quantum yields were determined according to literature methods
using quinine sulfate as the standard.[32]
[d, JHH = 7.7 Hz, 2 H, 6-H, 8-H (pyrene)], 8.20 [s, 4 H, 4-H, 5-H,
3
3
9-H, 10-H (pyrene)], 8.06 [dd, JHH = 7.1, JHH = 7.9 Hz, 1 H, 7-
3
H (pyrene)], 6.30 (t, JHH = 6.6 Hz, 1 H, 1Ј-H), 5.30 (m, 1 H, 3Ј-
OH), 5.24 (m, 1 H, 5Ј-OH), 4.36 (m, 1 H, 4Ј-H), 3.86 (m, 1 H, 3Ј-
H), 3.66 (m, 2 H, 5Ј-H), 2.25 (m, 2 H, 2Ј-H) ppm. 13C NMR
(75.4 MHz, [D6]DMSO): δ = 162.3, 150.0, 139.2 [C-5 (dU)], 131.2,
130.6, 130.4, 127.5 [C-4, C-5, C-9, C-10 (pyrene)], 126.2 [C-7 (pyr-
ene)], 125.1 [C-6, C-8 (pyrene)], 124.5 [C-1, C-3 (pyrene)], 123.6,
122.8, 113.5, 87.5 (C-3Ј), 84.7 (C-1Ј), 70.0 (C-4Ј), 60.8 (C-5Ј), 40.2
(C-2Ј) ppm. ESI-MS: m/z (%) = 427.2 (100) [M – H+]–, 463.2 [M
+ Cl]–, 487.2 [M + CH3COO]–. HR-MS (ESI-FTICR): calcd. for
C25H20N2O5 428.1372; found 427.1301.
5Ј-O-[Bis(4-methoxyphenyl)phenylmethyl]-5-(2-pyrenyl)-2Ј-deoxyur-
idine (6): Compound 2 (60 mg, 0.140 mmol, 1.0 equiv.) was dis-
solved in dry pyridine (5 mL) under nitrogen. After addition of
4,4Ј-dimethoxytriphenylmethyl chloride (47 mg, 0.140 mmol,
1.0 equiv.) the solution was stirred at room temp. for 48 h. The
reaction was quenched with MeOH (2 mL) and stirred for 1 h at.
room temp. The solvent was removed under vacuum. The crude
product was purified by flash chromatography (CH2Cl2/acetone =
4:1) to give 80 mg (78%) of a brown solid. TLC (EtOAc/MeOH/
1
H2O = 10:1:0.5): Rf = 0.68. H NMR (600 MHz, [D6]DMSO): δ =
3
11.67 (br. s, 1 H, NH), 8.25 [d, JHH = 7.7 Hz, 2 H, 6-H, 8-H
3
(pyrene)], 8.19 [s, 2 H, 1-H, 3-H (pyrene)], 8.08 [d, JHH = 9.3 Hz,
3
3
2 H, 4-H, 10-H (pyrene)], 8.04 [dd, JHH = 7.9, JHH = 7.7 Hz, 1
3
H, 7-H (pyrene)], 7.98 [s, 1 H, 6-H (dU)], 7.80 [d, JHH = 9.1 Hz,
2 H, 5-H, 9-H (pyrene)], 7.29–7.07 [m, 9 H, arom. (DMT)], 6.53
3
2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyrene (4):[28] Bis-
(1,5-cyclooctadiene)diiridium(I) dichloride (132 mg, 0.197 mmol,
0.05 equiv.) was suspended in dry cyclohexane (10 mL) under nitro-
gen. NaOMe (33 mg, 0.591 mmol, 3 equiv.) was added and the sus-
pension was stirred at room temp. for 60 min. Pyrene (3) (797 mg,
3.94 mmol, 1.0 equiv.), bis(pinacolato)diborane (1.10 g, 4.33 mmol,
1.1 equiv.) and dtbpy (106 mg, 0.394 mmol, 0.1 equiv.) were dis-
solved in dry cyclohexane. The prepared catalyst solution was
added through a cannula and the solution turned dark immediately.
After stirring at 80 °C for 48 h the solvent was removed under vac-
uum. The crude product was purified by flash chromatography
(SiO2, hexane/CH2Cl2 = 1:1) to give 486 mg (37%) of a pale yellow
solid. TLC (hexane/CH2Cl2 = 1:1): Rf = 0.60. 1H NMR (300 MHz,
[m, 4 H, arom. (DMT)], 6.30 (t, JHH = 6.6 Hz, 1 H, 1Ј-H), 5.33
(m, 1 H, 3Ј-OH), 4.28 (m, 1 H, 4Ј-H), 3.95 (m, 1 H, 3Ј-H), 3.44 (s,
3 H, OCH3), 3.39 (s, 3 H, OCH3), 3.17 (m, 2 H, 5Ј-H), 2.41 (m, 1
H, 2Ј-H), 2.27 (m, 1 H, 2Ј-H) ppm. 13C NMR (150 MHz, [D6]-
DMSO): δ = 162.3, 157.8, 157.7, 150.0, 144.5, 138.2 [C-6 (dU)],
135.3, 135.2, 130.7, 130.5, 130.2, 129.5, 149.4, 127.6 [arom.
(DMT)], 127.5 [arom. (DMT)], 127.2 [C-5, C-9 (pyrene)], 126.4
(DMT), 126.1 [C-7 (pyrene)], 125.0 [C-6, C-8 (pyrene)], 124.9 [C-
1, C-3 (pyrene)], 123.5, 122.9, 114.4, 112.9 [arom. (DMT)], 85.9,
85.6 (C-3Ј), 85.0 (C-1Ј), 70.6 (C-4Ј), 63.7 (C-5Ј), 54.6 (OCH3), 45.7,
40.0 (C-2Ј), 30.6 ppm. ESI-MS: m/z (%) = 729.4 (100) [M –
H+]–, 765.4 [M + Cl]–, 789.4 [M + CH3COO]–.
5Ј-O-[Bis(4-methoxyphenyl)phenylmethyl]-5-(2-pyrenyl)-2Ј-deoxyur-
idin-3Ј-yl 2-Cyanoethyl N,N-Diisopropylphosphoramidite (7): Com-
pound 6 (80 mg, 0.109 mmol, 1.0 equiv.) was dissolved in dry
CH2Cl2 (3.3 mL) under nitrogen. Dry EtN(iPr)2 (55 µL,
0.394 mmol, 3.6 equiv.) and 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite (24 µL, 0.109 mmol) were added. The solution
was stirred at room temp. for 1 h. The reaction was quenched with
dry EtOH (100 µL) and quickly washed with freshly prepared aq.
NaHCO3 solution. The organic phase was dried with Na2SO4 and
the solvent was removed under vacuum to yield 96 mg (95%) of a
3
[D6]DMSO): δ = 8.59 (s, 2 H, 1-H, 3-H), 8.31 (d, JHH = 7.7 Hz,
3
2 H, 6-H, 8-H), 8.27 (d, JHH = 9.3 Hz, 2 H, 4-H, 10-H), 8.20 (d,
3JHH = 8.8 Hz, 2 H, 5-H, 9-H), 8.11 (dd, 3JHH = 7.1, 3JHH = 7.9 Hz,
1 H, 7-H), 1.40 (s, 12 H, CH3) ppm. 13C NMR (75.4 MHz, [D6]-
DMSO): δ = 131.0, 130.9 (C-1, C-3), 129.9, 127.6 (C-4, C-10), 127.3
(C-5, C-9), 126.7 (C-7), 125.4, 125.0 (C-6, C-8), 123.6, 84.0 (CO),
24.8 (CH3) ppm. EI-MS: m/z (%) = 328.1(100) [M]+. HR-MS (EI):
calcd. for C22H21BO2 [M]+: 328.1635; found 328.1629
[M]+.
5-(2-Pyrenyl)-2Ј-deoxyuridine (2): Pyrene derivative 4 (130 mg, brown, viscous liquid. TLC (CH2Cl2/acetone = 6:1): Rf = 0.56. 31P
0.396 mmol, 1.2 equiv.), 5-iodo-2Ј-deoxyuridine (5) (117 mg, NMR (121.5 MHz, [D6]DMSO): δ = 148.66, 148.31 ppm.
70
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 64–71

Synthesis of 5-(2-Pyrenyl)-2Ј-deoxyuridine
[12] L. Valis, E. Mayer-Enthart, H.-A. Wagenknecht, Bioorg. Med.
Chem. Lett. 2006, 16, 3184–3187.
Preparation and Characterization of the Oligonucleotides: The oli-
gonucleotides were prepared on a Expedite 8909 DNA synthesizer
from Applied Biosystems by standard phosphoramidite protocols
using chemicals and CPG (1 µmol) from Applied Biosystems and
Proligo. Quantitative coupling of the building block 7 was achieved
by using the standard coupling time of 1.6 min. After preparation,
the trityl of the oligonucleotide was cleaved from the resin and was
deprotected by treatment with conc. NH4OH at 60 °C for 10 h.
The oligonucleotide was dried and purified by HPLC on a semi-
preparative RP-C18 column (300 Å, Supelco) by using the follow-
ing conditions: (i) unmodified oligonucleotides A = NH4OAc
buffer (50 m), pH 6.5; B = MeCN; gradient: 0–15% B over
45 min; (ii) 2PydU-modified oligonucleotides A = NH4OAc buffer
(50 m), pH 6.5; B = MeCN; gradient: 0–30% B over 55 min. The
oligonucleotides were lyophilized and quantified by their ab-
sorbance at 260 nm by using ε260 = 18600 –1 cm–1 (MeOH) for 2
and ε260 = 17000 –1 cm–1 (MeOH) for 1 using a Varian Cary 100
spectrometer. Duplexes were formed by heating to 90 °C (10 min)
followed by slow cooling.
[13] For important recent examples, see: a) T. L. Netzel, Tetrahe-
dron 2007, 63, 3491–3514; b) Y. J. Seo, H. Rhee, T. Joo, B. H.
Kim, J. Am. Chem. Soc. 2007, 129, 5244–5247; c) I. Géc i , V. V.
Filichev, E. P. Pedersen, Chem. Eur. J. 2007, 13, 6379–6386; d)
M. V. Skorobogatyi, A. D. Malakhov, A. A. Pchelintseva,
A. A. Turban, S. L. Bondarev, V. A. Korshun, ChemBioChem
2006, 7, 810–816; e) M. Nakamura, Y. Fukunaga, K. Sasa, Y.
Ohtoshi, K. Kanaori, H. Hayashi, H. Nakano, K. Yamana,
Nucleic Acids Res. 2005, 33, 5887–5895.
[14] A. Okamoto, K. Tainaka, K. Kanatani, I. Saito, Mol. BioSyst.
2006, 2, 122–127.
[15] a) T. S. Kumar, J. Wengel, P. J. Hrdlicka, ChemBioChem 2007,
8, 1122–1125; b) H. Kashida, H. Asanuma, M. Komiyama,
Chem. Commun. 2006, 2768–2770; c) K. Yamana, Y. Fukun-
aga, Y. Ohtami, S. Sato, M. Nakamura, W. J. Kim, T. Akaike,
A. Maruyama, Chem. Commun. 2005, 2509–2511.
[16] a) E. Mayer-Enthart, H.-A. Wagenknecht, Angew. Chem. Int.
Ed. 2006, 45, 3372–3375; b) E. Mayer-Enthart, C. Wagner, J.
Barbaric, H.-A. Wagenknecht, Tetrahedron 2007, 63, 3434–
3439.
[17] a) M. Nakamura, Y. Ohtoshi, K. Yamana, Chem. Commun.
2005, 5163–5165; b) M. Nakamura, Y. Shimomura, Y. Ohtoshi,
K. Sasa, H. Hayashi, H. Nakano, K. Yamana, Org. Biomol.
Chem. 2007, 5, 1945–1951.
[18] a) V. L. Malinovskii, R. Häner, Eur. J. Org. Chem. 2006, 3550–
3553; b) V. L. Malinovskii, F. Samain, R. Häner, Angew. Chem.
Int. Ed. 2007, 46, 4464–4467.
Acknowledgments
Financial support by the Deutsche Forschungsgemeinschaft
(DFG) (Wa 1386-7), the Fonds der Chemischen Industrie, and the
University of Regensburg is gratefully acknowledged.
[19] a) T. R. Grabowski, K. Rotkiewicz, W. Rettig, Chem. Rev.
2003, 103, 3899–4031; b) Y. Wang, O. Haze, J. P. Dinnocenzo,
S. Farid, R. S. Farid, I. R. Gould, J. Org. Chem. 2007, 72,
6970–69812.
[1] For reviews, see: a) C. Wojczewski, K. Stolze, J. W. Engels, Syn-
lett 1999, 1667–1678; b) S. Tyagi, S. A. E. Marras, F. R.
Kramer, Nat. Biotechnol. 2000, 18, 1191–1196; c) M. K.
Johansson, R. M. Cook, Chem. Eur. J. 2003, 9, 3466–3471; d)
W. Tan, K. Wang, T. J. Drake, Curr. Opin. Chem. Biol. 2004, 8,
547–553; e) R. T. Ranasinghe, T. Brown, Chem. Commun. 2005,
5487–5502; f) A. A. Martí, S. Jockusch, N. Stevens, J. Ju, N. J.
Turro, Acc. Chem. Res. 2007, 40, 402–409.
[20] T. Kubota, K. Kano, T. Konse, Bull. Chem. Soc. Jpn. 1987, 60,
3865–3877.
[21] S. Steenken, J. P. Telo, H. M. Novais, L. P. Candeias, J. Am.
Chem. Soc. 1992, 114, 4701–4709.
[22] N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457–2483.
[23] L. A. Agrofoglio, I. Gillaizeau, Y. Saito, Chem. Rev. 2003, 103,
1875–1916.
[2] Tetrahedron 2007, 63, whole issue 17.
[3] For a review, see: J. N. Wilson, E. T. Kool, Org. Biomol. Chem.
2006, 4, 4265–4274.
ˇ
[24] For recent examples, see: a) P. Capek, H. Cahová, R. Pohl, M.
[4] L. Valis, N. Amann, H.-A. Wagenknecht, Org. Biomol. Chem.
2005, 3, 36–38.
[5] O. Köhler, D. V. Jarikote, O. Seitz, ChemBioChem 2005, 6, 69–
77.
Hocek, C. Gloeckner, A. Marx, Chem. Eur. J. 2007, 13, 6196–
6203; b) E. C. Western, K. H. Shaughnessy, J. Org. Chem. 2005,
70, 6378–6388.
[25] M. Minabe, S. Takeshige, Y. Soeda, T. Kimura, M. Tsubota,
Bull. Chem. Soc. Jpn. 1994, 67, 172–179.
[26] P. P. Fu, H. M. Lee, R. H. Harvey, J. Org. Chem. 1980, 45,
2797–2803.
[27] a) H. Lee, R. G. Harvey, J. Org. Chem. 1986, 51, 2847–2848;
b) A. Musa, B. Sridharan, J. Lee, D. L. Mattern, J. Org. Chem.
1996, 61, 5481–5484.
[28] D. N. Coventry, A. S. Batsanov, A. E. Goeta, J. A. K. Howard,
T. B. Marder, R. N. Perutz, Chem. Commun. 2005, 2172–2174.
[6] C. Wanninger-Weiß, L. Valis, H.-A. Wagenknecht, Bioorg.
Med. Chem. 2007, in press: doi:10.1016/j.bmc.2007.04.064.
[7] a) N. Amann, E. Pandurski, T. Fiebig, H.-A. Wagenknecht,
Angew. Chem. Int. Ed. 2002, 41, 2978–2980; b) R. Huber, T.
Fiebig, H.-A. Wagenknecht, Chem. Commun. 2003, 1878–1879;
c) M. Raytchev, N. Amann, E. Mayer, H.-A. Wagenknecht, T.
Fiebig, ChemPhysChem 2004, 5, 706–712.
[8] a) E. Mayer, L. Valis, R. Huber, N. Amann, H.-A. Wagen-
knecht, Synthesis 2003, 2335–2340; b) A. Trifonov, I. Buchva- [29] a) T. Ishiyama, J. Takagi, J. F. Hartwig, N. Miyaura, Angew.
rov, H.-A. Wagenknecht, T. Fiebig, Chem. Phys. Lett. 2005,
409, 277–280.
Chem. Int. Ed. 2002, 41, 3056–3058; b) T. Ishiyama, Y. Nobuta,
J. F. Hartwig, N. Miyaura, Chem. Commun. 2003, 2924–2925.
[9] a) N. Amann, E. Pandurski, T. Fiebig, H.-A. Wagenknecht, [30] J. N. Wilson, J. Gao, E. T. Kool, Tetrahedron 2007, 63, 3427–
Chem. Eur. J. 2002, 8, 4877–4883; b) P. Kaden, E. Mayer, A.
Trifonov, T. Fiebig, H.-A. Wagenknecht, Angew. Chem. Int. Ed.
2005, 44, 1636–1639.
3433.
[31] C. Beuck, I. Singh, A. Bhattacharya, W. Hecker, V. S. Parmar,
O. Seitz, E. Weinhold, Angew. Chem. Int. Ed. 2003, 42, 3958–
3960.
[10] C. Wagner, H.-A. Wagenknecht, Chem. Eur. J. 2005, 22, 1871–
1876.
[11] C. Wagner, M. Rist, E. Mayer-Enthart, H.-A. Wagenknecht,
Org. Biomol. Chem. 2005, 3, 2062–2063.
[32] J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991–1024.
Received: September 4, 2007
Published Online: November 7, 2007
Eur. J. Org. Chem. 2008, 64–71
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
71