RNA as scaffold for pyrene excited complexes

Article abstract of DOI:10.1016/j.bmc.2007.04.058

Synthesis and spectral properties of 1-ethynylpyrene base modified RNA are reported. The fluorophore attached to the 2-position of adenosine is directed into the easily accessible minor groove in RNA. Through an intermolecular interaction of the pyrene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence maxima around 450 and 480 nm, respectively. This behaviour allows the kinetic investigation of RNA hybridisation and folding by fluorescence spectroscopy.

Full text of DOI:10.1016/j.bmc.2007.04.058

Bioorganic & Medicinal Chemistry 16 (2008) 19–26
RNA as scaffold for pyrene excited complexes
Christian Grunewald,a Taewoo Kwon,a Nelly Piton,a Ute Forster,b
¨
¨
Josef Wachtveitlb,* and Joachim W. Engelsa,*
aInstitute of Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University,
Max-von-Laue-Str. 7, 60348 Frankfurt, Germany
bInstitute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University,
Max-von-Laue-Str. 7, 60348 Frankfurt, Germany
Received 11 October 2006; revised 26 February 2007; accepted 27 April 2007
Available online 3 May 2007
Abstract—Synthesis and spectral properties of 1-ethynylpyrene base modified RNA are reported. The fluorophore attached to the 2-
position of adenosine is directed into the easily accessible minor groove in RNA. Through an intermolecular interaction of the pyr-
ene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence maxima around 450 and
480 nm, respectively. This behaviour allows the kinetic investigation of RNA hybridisation and folding by fluorescence
spectroscopy.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
deoxy-uridine.4 This rigid label is expected to minimally
disturb the duplex geometry and to align the fluoro-
phore in a predictable fashion due to its rigid acetylenic
linker. Pyrene and 1-ethynylpyrene are commonly intro-
duced into the base at the 5-position of pyrimidines and
at the 8-position of the purines.5 Pyrene was also intro-
duced at the 20-sugar-position in DNA, RNA and LNA6
using flexible linkers. Alternatively pyrene can also sub-
stitute the whole nucleic base or nucleotide.7
DNA as a building block for nanoscale materials1 has
attracted considerable interest due to its possible appli-
cation in molecular devices. RNA, on the other hand,
has much less been utilized.2 Both nucleic acids form
predictable three dimensional structures based on Wat-
son-Crick complementarity. These duplexes are either
of the B-type with a wide major and a narrow minor
groove mostly for DNA or with a deep and narrow ma-
jor groove and a shallow minor groove for the A-type
mostly found in double-stranded RNA. Here, the con-
cept of site specifically mono-functionalized oligonucle-
otides led us to utilize the easily accessible minor
groove site in RNA. In order to achieve this, the group
of interest has to be attached to the purine 2-position,
which is possible with A and G.3 We started to deriva-
tize adenosine in RNA with pyrene using an acetylenic
linker.
A first simple molecular model constructed with Insight-
II (Fig. 1) using an ideal, unperturbed RNA structure as
scaffold showed that two 1-ethynylpyrene residues at-
tached to the 2-position in different strands can be
placed in the minor groove of double-stranded RNA
without sterical hindrance. In addition we predicted
good stacking between two pyrenes only when fixed on
opposite strands. Otherwise, only a partial overlap is ex-
pected. The distance between the two pyrene residues in
˚
the model is about 3 A, a distance suitable for complex
formation.
1-Ethynylpyrene as a highly fluorescent chromophore
has initially been introduced into the 5-position of
Herein we report the introduction of 1-ethynylpyrene
into the 2-position of adenosine in RNA oligonucleo-
tides, their characterization and their fluorescence prop-
erties. The site specific functionalization is achieved by
Sonogashira cross-coupling on solid support in the
course of oligonucleotide synthesis,8 an innovative
method rarely used by now.9
Keywords: Fluorescence; Pyrene; Excited complex; Interstrand
stacking.
*
Corresponding authors. Tel.: +49 0 69 798 29150; fax: +49 0 69 798
29148 (J.W.E); tel.: +49 0 69 798 29351; fax: +49 0 69 798 29709
(J.W.); e-mail addresses: wveitl@theochem.uni-frankfurt.de;Joachim.
Engels@chemie.uni-frankfurt.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.058

20
C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
Figure 1. Molecular model of a 12-membered RNA oligomer containing an AU-core with an interstrand pyrene dimer in the minor groove, red:
adenosine nucleoside, green: 1-ethynylpyrene.
2. Results and discussion
yielded 20-ACE protected 13. Protection of the 50-hydro-
xyl group with benzhydryloxy-bis(trimethylsilyl-
oxy)chlorsilane (BzH-Cl) and reaction with methyl-
2.1. Synthesis
N,N,N0,N0-tetraisopropylphosphordiamidite
afforded
Whereas 5-halo-pyrimidines and 8-halo-purines used
by other research groups are commercially available,
2-iodoadenosine has to be synthesized according to liter-
ature procedures. Compound 9 was synthesized in four
steps from guanosine in an overall yield of 45%. For
the preparation of 6, the use of acetonitrile as solvent
and DMAP/triethylamine as catalyst/co-catalyst10 was
preferred to DMF/pyridine as solvent as better yields
could be achieved and the interference of residual
DMF with POCl3 (formation of Vilsmeier-Haack-re-
agent) in the synthesis of 7 could be avoided. 20,30,50-
Tri-O-acetylguanosine 6 was treated with POCl3 to af-
ford the chloro-compound 7.11 Conversion to the 2-
iodo-6-chloro-purine derivative 8 was performed with
a variant of the Sandmeyer reaction of then in situ gen-
erated diazonium salt of 7.12 Nucleophilic aromatic sub-
stitution at the 6-position of the purine with methanolic
ammonia and subsequent treatment with NaOMe in
MeOH to deprotect the remaining acetyl groups yielded
2-iodoadenosine 9 (Scheme 1).
the 20-ACE-50-BzH-protected phosphoramidite 15 suit-
able for solid-phase synthesis in an overall yield of
49% starting with 2-iodoadenosine (Scheme 2).
Solid-phase RNA synthesis was performed on a
0.2 lmol scale using standard Dharmacon protocols.
The introduction of 1-ethynylpyrene was achieved by
Sonogashira cross-coupling on solid support: the oligo-
nucleotide solid-phase synthesis was interrupted after
the incorporation of 2-iodoadenosine without deprotec-
tion of the 50-hydroxyl group. The column was then re-
moved from the synthesizer and the reagent mixture for
Sonogashira cross-coupling of 1-ethynylpyrene with the
iodinated base was loaded to the column using syringes.9
The coupling was performed twice for 2.5 h each to en-
sure quantitative reaction (Fig. 2).
After each Sonogashira cross-coupling the solid support
was washed thoroughly with CH2Cl2, dried in vacuo
and the solid-phase synthesis was resumed. After remov-
ing the protecting groups, the oligonucleotides were
purified by anion exchange chromatography. Following
desalting and deprotection of the residual 20-protecting
group, the oligonucleotides were analysed by MALDI-
TOF mass spectroscopy.
As we have had experienced excellent results with the re-
cently established ACEꢁ chemistry used for our spin-
labelling experiments, this method was also applied to
the solid-phase synthesis of the pyrene-modified RNA.
The ACEꢁ chemistry13 developed by Dharmacon uses
an inverted protection group strategy with a fluoride-la-
bile 50-silyl-group and an acid labile 20-orthoester group
in comparison to the established TBDMS-strategy. The
synthesis of the 20-bis-acetoxyethyloxy-50-silyl protected
amidite14 15 for solid-phase RNA synthesis starts with
the protection of the exocyclic amine with N,N-dimethy-
lacetamide-dimethylacetal in DMF as the corresponding
amidine 10.15 For discrimination of 20- and 30-hydroxy
function the Markiewicz strategy16 was used. Reaction
of the N-protected 2-iodoadenosine with 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane affords 11. Reaction of
11 with tris-(acetoxyethyloxy)methylester (ACE) and
subsequent treatment with TEMED-HF in acetonitrile
We synthesized self-complementary oligonucleotides
with an AU core and 50- and 30-GC pairs as well as
non self-complementary sequences to be able to investi-
gate singly 1-ethynylpyrene-modified single and double
strands in addition to the twofold modified duplexes.
The sequences and analytic data of the synthesized oli-
gonucleotides are listed in Table 1.
2.2. Thermal stability and CD spectra
To determine the influence of 1-ethynylpyrene in the 2-
position of adenosine on the stability of the synthesized
oligonucleotides, melting curves and CD spectra were

C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
21
O
O
N
Cl
N
N
NH
N
N
N
N
NH
NH2
N
N
N
NH2
O
NH2
i
ii
HO
O
AcO
O
O
O
O
O
OH OH
OAc OAc
O
O
5
6
7
Cl
NH2
N
N
N
N
N
N
N
I
N
I
AcO
HO
iii
iv
O
O
OAc OAc
OH OH
8
9
Scheme 1. Synthesis of 2-iodoadenosine 9. Reagents and conditions: (i) Ac2O, MeCN, Et3N, N,N-dimethylaminopyridine, 30 min, rt, 91%; (ii)
POCl3, MeCN, 15 min, 110 ꢂC, 80%; (iii) iso-pentylnitrite, THF, I2, CH2I2, CuI, 45 min reflux, 89%; (iv) NH3/MeOH, 24 h, rt, 70%.
N
N
N
I
N
N
N
I
N
N
N
I
NH2
N
N
N
N
N
N
N
N
N
N
N
N
O
N
I
O
HO
HO
Si
iii
ii
i
O
O
O
O
O
Si
O
O
O
O
Si
O
O
OH OH
O
OH
OH OH
O
Si
O
O
9
10
11
12
N
N
I
N
N
N
I
N
N
I
N
N
N
N
N
N
N
N
O
Si
O
N
N
N
O
HO
Si O
Si
O
Si
O
O
O
O
O
O
vi
iv
v
O
O
O
Si
Si
O
N
O
O
OH
O
O
OH
O
O
O
O
O
O
P
O
O
O
O
O
O
O
O
O
13
14
15
Scheme 2. Synthesis of ACE-protected phosphoramidite 15 starting with 2-iodoadenosine 9. Reagents and conditions: (i) DMF-dimethylacetal,
DMF, 1 h, 50 ꢂC, 86%; (ii) tetraisopropyldisiloxandichloride (TIPS-Cl2), pyridine, 2 h, 0 ꢂC ! rt, 83%; (iii) tri-(2-acetoxyethyl)-ortho formate,
CH2Cl2, 48 h, rt, 89%; (iv) TEMED-HF, MeCN, 3 h, rt, 96%; (v) benzhydryloxy-bis-(trimethylsilyloxy)chlorsilane, CH2Cl2, diisopropylamine, 1 h,
rt, 92%; (vi) bis-(N,N-diisopropylamino)-methoxyphosphine, CH2Cl2, 5 min, 0 ꢂC, 13 h, rt, 86%.
measured. Melting temperatures (Tm) of synthesized
double-stranded oligonucleotides are given in Table 2.
Modified self-complementary oligonucleotides are sig-
nificantly more stable (+11.1, +11.0 and +8.5 ꢂC for
IB, IIB and IIIB, respectively) than the corresponding
unmodified duplexes. In case of the non self-comple-
mentary sequence the double-stranded singly modified
oligonucleotide duplexes IVB–VA and IVA–VB are
drastically less stable (ꢀ6.3 ꢂC and ꢀ12.2 ꢂC, respec-
tively) than the unmodified sequence in contrast to the
twofold modified duplex IVB–VB that is thermally more
stable (+3.2 ꢂC) than the unmodified sequence. The sta-
bilisation of inherently twofold modified RNA could be
attributed to an attractive p–p-interaction between the
aromatic fluorophores as it is also the case for the two-
fold modified non self-complementary duplex IVB–VB.
In contrast, a single pyrene residue, lacking attractive
p–p-interaction, seems to disturb the stack and therefore
lowers the melting temperature with respect to the
unmodified sequence.

22
C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
N
N
N
N
N
N
N
N
N
N
N
N
I
O
O
BzH
BzH
O
O
O
O
O
O
Pd(PPh3)4, CuI
ACE
ACE
CH2Cl2/Et3N, 2.5 h, RT
Figure 2. Conditions for the solid-phase Sonogashira cross-coupling between the polymer bound oligonucleotide and 1-ethynylpyrene.
Table 1. Sequences and calculated masses of synthesized modified and
unmodified oligonucleotides together with the corresponding analytic
data found by MALDI-TOF mass spectroscopy
Cotton effect with a minimum around 430 nm and a
maximum at 380 nm, which implies an ordered arrange-
ment of the pyrene groups in the chiral environment.
ON Sequence
Mass calcd Mass found
In the case of the modified non self-complementary se-
quences no CD signal can be observed in this range if
just one strand is modified (as in IVB–VA and IVA–
VB) in contrast to the twofold modified duplex IVB–
VB which also exhibits a signal similar to the self-com-
plementary duplexes (Fig. 3).
IA
IB
50-GCGCA UGCGC-30
3174.99
3399.62
3159.98
3384.23
3144.97
3369.22
3632.15
3174.02
3398.95
3157.66
3382.04
3142.63
3367.68
3631.40
3855.05
3913.16
4136.69
50-GCGCAPyUGCGC-30
50-GCGAA UUCGC-30
50-GCGAAPyUUCGC-30
IIA
IIB
IIIA 50-GCAAA UUUGC-30
IIIB 50-GCAAAPyUUUGC-30
IVA 50-CUUUUCA UUCUU-30
IVB 50-CUUUUCAPyUUCUU-30 3856.42
These findings imply that two pyrene residues are
needed for an ordered arrangement presumably as a
p–p-stack in the minor groove inhibiting rotation
around the acetylenic bond for both 1-ethynylpyrene
residues. This is in contrast to results presented for 5-
(1-ethynylpyrene)-20-deoxyuridine modified DNA where
it is stated that at least three pyrene residues are needed
for a detectable CD signal.5c In the duplexes with only
one pyrene modification an extended rotational freedom
can be assumed, which explains the absence of a CD sig-
nal due to the lack of ordered structure.
VA
VB
30-GAAAAGUA AGAA-50
30-GAAAAGUAPyAGAA-50 4137.78
3913.51
Table 2. Thermal denaturation studies of modified oligonucleotides
Duplex
Sequence
Tm (ꢂC)
DTM
IA–IA
IB–IB
50-GCGCA UGCGC-30
50-GCGCAPyUGCGC-30
68.3 0.9
—
79.4 0.6 +11.1
42.0 0.2
53.0 0.6 +11.0
26.5 0.2
35.0 0.2 +8.5
42.2 0.4
IIA–IIA
IIB–IIB
50-GCGAA UUCGC-30
—
50-GCGAAPyUUCGC-30
IIIA–IIIA 50-GCAAA UUUGC-30
—
2.3. Fluorescence spectra
IIIB–IIIB
IVA–VA
50-GCAAAPyUUUGC-30
In order to investigate the changes of pyrene fluores-
cence in the twofold modified RNA upon hybridisation,
temperature dependent fluorescence spectra were re-
corded. The self-complementary sequence IIIB (50-
GCAAAPyUUUGC-30) shows a broad unstructured
fluorescence spectrum with a maximum at 471 nm.
Upon thermal denaturation of the double strand, this
maximum is blue-shifted by 27 nm to 444 nm (Fig. 4).
These findings are comparable to the results of Wagen-
knecht and co-worker where, in contrast to the pre-
sented work, 20-deoxyuridine in DNA is modified with
1-ethynylpyrene.5c
50-CUUUUCAUUCUU-30
30-GAAAAGUAAGAA-50
—
IVB–VA
IVA–VB
50-CUUUUCAPyUUCUU-30
30-GAAAAGU AAGAA-50
50-CUUUUCAU UCUU-30
30-GAAAAGUAPyAGAA-50
35.9 0.1 ꢀ6.3
30.0 0.1 ꢀ12.2
IVB–VB
50-CUUUUCAPyUUCUU-30
30-GAAAAGUAPyAGAA-50
45.4 0.1 +3.2
CD spectra of both modified and unmodified self-com-
plementary as well as non self-complementary sequences
show maxima at 265 nm (global), 222 nm (local) and
minima at 245 nm (global), 211 nm (local) correspond-
ing to an A-form RNA. CD spectra indicate that the
overall structure of the modified oligonucleotides com-
pared to the corresponding unmodified ones is con-
served. This is even the case for the destabilised singly
modified non self-complementary duplexes IVA–VB
and IVB–VA (Fig. 3). Furthermore, the twofold modi-
fied oligonucleotides show a CD signal between 350
and 450 nm for the pyrene absorption with a negative
The shift of the emission band is biggest in the region
of the oligonucleotide melting point (35 ꢂC for IIIB).
Above 40 ꢂC and under 28 ꢂC only a slight change
in fluorescence maximum can be observed. This
behaviour is visualized in more clarity by plotting
the fluorescence maxima against temperature
(Fig. 5). The estimated melting temperature computed
from a fitted curve is 34.9 ꢂC 3.9 ꢂC, which is in
good agreement with the melting temperature ob-
tained by UV measurements.

C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
23
5
4
Compound 16 only exhibits a broad unstructured fluo-
rescence spectrum with a red-shifted maximum around
437 nm at a concentration of 10ꢀ5 M in methanol com-
pared to 1-ethynylpyrene with its three maxima between
380 and 430 nm (Fig. 6).
IVA-VA
IVB-VA
IVA-VB
IVB-VB
3
2
1
0
A similar behaviour for conjugated pyrene-modified
nucleosides was also found for 8-(1-ethynylpyrene)-
adenosine modified oligonucleotides.17
200
250
300
350
400
450
-1
-2
-3
-4
Quantum yield for 16 estimated on the basis of quinine
sulfate in sulfuric acid is 0.72. Quantum yields of the oli-
gonucleotides are generally lower in the range of 0.49
(for IVB in duplex with VA) to 0.68 for self-complemen-
tary IIIB.
wavelength [nm]
Figure 3. CD spectra of unmodified (IVA–VA), singly modified (IVB–
VA, IVA–VB) and twofold pyrene labelled (IVB–VB) double strands in
phosphate buffer, oligonucleotide concentration 5 lM.
Considering the fluorescence spectrum of 1-ethynylpy-
rene-modified nucleoside 16 and the concentration of
5 lM of oligonucleotides in thermal denaturation stud-
ies what excludes formation of an intermolecular exci-
mer of the pyrene residues, the broad fluorescence can
be assigned to the fluorescence of the singly pyrene-
modified single strand without an interstrand interaction
of the two pyrene residues. The loss of vibronic structure
leads to a broad emission band for 2-(1-ethynylpyrene)-
adenosine derivative presumably due to a strong elec-
tronic coupling in the conjugated system.
The fluorescence spectrum of the single strand also
shows no structure as it would be expected for pyrene
with its three maxima around 385, 405 and 425 nm. 1-
Ethynylpyrene itself shows the typical structured pyrene
emission spectrum and formation of an excimer band at
490 nm at concentrations above 10ꢀ4 M (Fig. 6). The
protected 2-(1-ethynylpyrene)-adenosine derivative 16
exhibits a different behaviour.
N
N
N
In the case of the non self-complementary oligonucleo-
tides it is possible to investigate the fluorescence spectra
of singly modified double strands together with the two-
fold modified strands. Both 1-ethynylpyrene-modified
single strands IVB (50-CUUUUCAPyUUCUU-30) and
VB (50-AAGAAPyUGAAAAG-30) showed broad emis-
sion spectra with maxima at 451 and 452 nm, respec-
tively. Upon hybridisation with the complementary
unmodified strand VA and IVA, respectively, the wave-
lengths of emission maxima did not change within the
experimental error. There is, however, a significant drop
of approximately 50% in fluorescence intensity. This can
most likely be attributed to quenching mechanisms, for
example by means of an energy transfer into the base
stack and a subsequent radiationless deexcitation. Like
N
N
N
O
O
Si
O
O
OH
Si
16
475
10°C
22°C
28°C
33°C
34°C
37°C
38°C
40°C
44°C
48°C
54°C
62°C
fluorescence maximum
470
465
460
455
450
445
440
350
400
450
500
550
600
10
20
30
40
50
60
70
wavelength [nm]
temperature [°C]
Figure 4. Temperature dependent emission spectra of pyrene-modified
self-complementary strand IIIB (50-GCAAAPyUUUGC-30) in phos-
phate buffer, oligonucleotide concentration 5 lM, kexc = 380 nm.
Figure 5. Temperature dependency of the fluorescence maxima, same
conditions as in Figure 4.

24
C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
located on different strands. In agreement with this
interpretation, the temperature dependent fluorescence
measurements showed no shift for the fluorescence maxi-
ma wavelength in case of the singly modified duplexes
IVA–VB or IVB–VA but a hypsochromic shift from
481 to 451 nm of the twofold modified duplex IVB–VB
upon thermal denaturation.
1-ethynylpyrene c(0.1 mM)
1-ethynylpyrene c(1 mM)
16
Comparison of the absorption spectra shows small dif-
ferences in the region of the S0 ! S1 transition. For
the thermally denatured case, a small blue shift can be
seen and the two absorption maxima are more pro-
nounced. Further measurements to clarify the signifi-
cance of these differences in the absorption spectrum
are currently being carried out.
350
400
450
500
550
wavelength [nm]
Figure 6. Fluorescence spectra of 1-ethynylpyrene (blue) at concen-
trations of 10ꢀ3 M and 10ꢀ4 M in MeCN and 16 (green) at a
concentration of 10ꢀ5 M in MeOH, kexc = 365 nm.
3. Conclusion
In summary, we described a convenient way to intro-
duce fluorophores like 1-ethynylpyrene into RNA oligo-
nucleotides by site specific functionalization on solid
support during oligonucleotide synthesis using Sono-
gashira cross-coupling. Upon bonding 1-ethynylpyrene
to adenosine in the 2-position the structured pyrene
fluorescence spectrum changed to a broad unstructured
band. This is attributed to an electronic coupling be-
tween pyrene and the nucleic base18 in the extended con-
jugated system. The nature of this electronic coupling
has still to be investigated in detail.
self-complementary sequences, but in contrast to the
singly modified double strands, the twofold modified
duplex IVB–VB emits with a maximum at 481 nm corre-
sponding to a bathochromic shift of 30 nm. This wave-
length shift can be followed by the naked eye (Fig. 7).
These findings indicate that the bathochromic shift of
27 nm for self-complementary strands and 30 nm in case
of twofold labelled non self-complementary duplexes is
not just an effect of hybridisation with the complemen-
tary strand but gives rise to the assumption of an inter-
action of the two pyrene residues located on the different
strands. Twofold ethynylpyrene-modified uridine in
DNA has already been investigated by femtosecond
time resolved spectroscopy and an electronic interaction
of the two chromophores was reported for adjacent
chromophores as well as across an intermediate base
pair, leading to a wavelength shift half as large as the
one we observed for our system.18 In contrast to this
work, where chromophores are located on the same
strand, in our system energy transfer through the base
stack is not expected since the two pyrene residues are
The increase in Tm values and the CD spectra presented
together with the fluorescence spectra of singly and two-
fold 1-ethynylpyrene-modified oligonucleotides allows
us to assume that the two pyrene residues can actually
interact with each other and show a broad red-shifted
fluorescence on the RNA as a scaffold. The fluorescence
wavelength shift is quite pronounced and can be fol-
lowed by the naked eye. By this fluorescence shift, one
can distinguish between single-stranded and double-
stranded RNA during thermal denaturation. This
0.8
0.7
IVB-VB at 25ºC
IVB-VB at 70ºC
VB
0.6
0.5
IVA-VB
0.4
IVB-VB
0.3
0.2
0.1
0
200
250
300
350
400
450
wavelength [nm]
350
400
450
500
wavelength [nm]
550
600
650
ss-VB
IVA-VB IVB-VB
Figure 7. (Left) Fluorescence spectra of single-stranded VB, double-stranded singly pyrene-modified IVA–VB and double-stranded twofold modified
IVB–VB, phosphate buffer, oligonucleotide concentration 5 lM, kexc = 385 nm; inset: UV spectrum of duplex IVB–VB, same conditions. (Right)
Photographs of cuvettes containing the corresponding solutions of oligonucleotides, excitation with a standard UV lamp at kexc = 365 nm.

C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
25
behaviour could be used for the time resolved investiga-
tion of RNA hybridisation and folding. The exact nat-
ure of the pyrene–pyrene-interaction remains yet to be
examined. Femtosecond fluorescence measurements are
currently performed to investigate this point.
(m, 3H, H40, H50), 3.33 (s, 3H, CH3), 3.27 (s, 3H,
CH3), MALDI-TOF m/z (%) = 790.73 (100) [M+H]+.
4.3. Oligonucleotide synthesis and purification
The oligonucleotides were synthesized on a 0.2 lmol
scale on a rebuilt ABI 392 synthesizer (Applied Biosys-
tems) with phosphoramidites purchased from Dharma-
con. Every RNA synthesis was stopped after
incorporation of the 2-iodoadenosine phosphoramidite
without deprotecting the 50-hydroxyl group. The col-
umn was removed from the synthesizer and maintained
under argon atmosphere. In the meantime CuI (6.0 mg)
was dissolved in dried and deoxygenated CH2Cl2/Et3N
(17.5:7.5 mL). 150 lL of this solution was added under
argon to a mixture of (PPh3)4Pd (3.5 mg, 15 equiv)
and 1-ethynylpyrene (2.1 mg, 45 equiv) with syringe
(equivalents related to the 0.2 lmol scale). The orange
solution was given onto the column and moved in it
back and forth using two syringes. After a reaction time
of 2.5 h the column was washed with 10 mL of dried
CH2Cl2, dried for 10 min in vacuo and flushed with ar-
gon. The Sonogashira cross-coupling was performed a
second time as described above. After the second cou-
pling the column was reinstalled on the synthesizer to
end the oligonucleotide synthesis. Afterwards the phos-
phate protecting groups were cleaved on solid support
by treatment with 2 mL of a 0,4 M solution of diso-
dium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate-trihy-
drate (S2Na2) in DMF/H2O = 98:2 within 60 min.
Deprotection of the exocyclic protection groups and
cleavage of acetate of the 20-ACE group as well as cleav-
age of the oligonucleotides from solid support are ef-
fected by treatment with a 40% aqueous solution of
methylamine for 10 min at 55 ꢂC. The crude oligonu-
cleotide was purified by anion exchange chromatogra-
phy (A: demineralised water, B: 1 M LiCl, gradient: 0–
80% B within 40 min) on JASCO HPLC-system 900
with a semi-preparative Dionex DNAPacꢁ PA-100 col-
umn (9 · 250 mm). After desalting with PD-10 Sepha-
dex columns from Amersham Biosciences, the final
deprotection of the 20-ACE protecting group was per-
formed under sterile conditions with a TEMED/acetic
acid buffer adjusted to pH 3.8 for 30 min at 60 ꢂC.
The obtained oligonucleotides were characterized by
MALDI-TOF mass spectrometry (VOYAGER DE-
PRO mass spectrometer from Applied Biosystems).
4. Experimental
The synthesis of compounds 7–15 will be reported else-
where.19 The reactions were monitored by thin-layer
chromatography (TLC) analysis on silica gel aluminium
plates (silica gel 60 F254, 0.2 mm, Merck). Column chro-
matography was performed on silica gel (40–63 lm,
Merck). Technical solvents were used after distillation
for chromatography; absolute solvents, dried over
molecular sieve, were purchased from FLUKA. 1H
and 13C NMR spectra were recorded with a Bruker
AMX250 at 250 MHz. Electron Spray Ionisation (ESI)
masses were collected on a VG Platform II (Fisons
Instruments).
4.1. 20,30,50-Tri-O-acetylguanosine (6)
Guanosine (5 g, 17.7 mmol) and N,N-dimethylamino-
pyridine (162 mg, 1.3 mmol, 7 mol%) were dissolved in
220 mL acetonitrile. After addition of TEA (9.7 mL,
69.9 mmol, 1.1 equiv) and acetic acid anhydride (6 mL,
63.5 mmol, 3.6 equiv) the mixture was stirred at room
temperature for 30 min. The reaction was quenched by
addition of 3 mL MeOH and the solvent evaporated in
vacuo. The oily residue was recrystallized from 2-propa-
nol to afford 2 as a white powder (6.6 g, 91%). Rf = 0.60
(CH2Cl2/MeOH = 9:1). 1H NMR (250 MHz, DMSO-d6)
d [ppm] 10.73 (s, 1H, NH), 7.92 (s, 1H, H8), 6.53 (s, 2H,
NH2), 5.97 (d, 1H, H10), 5.78 (t, 1H, H20), 5.48 (dd, 1H,
H30), 4.39–4.21 (m, 3H, H40, H50), 2.10 (s, 3H, OAc),
2.03 (s, 3H, OAc), 2.02 (s, 3H, OAc). 13C NMR
(63 MHz, DMSO-d6) d [ppm] 170.05, 169.41, 169.24,
156.58, 153.58, 151.08, 135.50, 116.80, 84.37, 79.51,
72.01, 70.28, 63.04, 20.50, 20.35, 20.16; ESI-MS(+) m/z
(%) = 410.0 (100) [M+H]+, 258.7 (50) [tri-O-acetylri-
bose+H]+, 152.5 (60) [guanine+H]+.
4.2. 2-(1-Ethynylpyrenyl)-30,50-O-(tetraisopropyldisilox-
ano)-N6-(N0,N0-dimethylamino-methyleno)-adenosine
(16)
Protected 2-iodoadenosine 11 (90 mg, 0.13 mmol) and 1-
ethynylpyrene (40 mg, 0.18 mmol, 1.36 equiv), CuI
(5 mg, 26 lmol, 15 mol%) and (PPh3)4Pd (20 mg,
17 lmol, 10 mol%) were dissolved in a degassed solvent
mixture of 5 mL DMF and 300 lL TEA. After 24 h of
stirring at room temperature, the reaction mixture was
diluted with 30 mL CH2Cl2 and extracted twice with
10 mL 0.3 M EDTA (aqueous solution) and the aqueous
phase extracted with CH2Cl2. After evaporation of the
solvent and column chromatography (CH2Cl2/
MeOH = 90:10; Rf = 0.58) the product was obtained as
a yellow solid (80 mg, 78%) 1H NMR (250 MHz,
DMSO-d6) d [ppm] 9.08 (s, 1H, CH), 8.80 (m, 2H,
ArH, H8), 8.35–8.04 (m, 8H, ArH), 6.17 (d, 1H, H10),
5.04 (m, 1H, H20), 4.74 (m, 1H, H30), 4.22-4.07
4.4. Spectroscopic measurements
All spectroscopic measurements were performed in a
phosphate buffer containing 10 mM NaH2PO4, 10 mM
Na2HPO4 and 140 mM NaCl adjusted to pH 7.0. Oligo-
nucleotide concentration was 5 lM. Melting curves were
recorded on a Cary UV–vis spectrophotometer from
Varian by detecting the optical density at 260 nm. The
solution was heated to 80 ꢂC for 1 min and then cooled
to 10 ꢂC within 10 min to form the duplex. The melting
curves were recorded using a heating rate of 0.5 ꢂC/min.
CD spectra were recorded on a JASCO J-710 spectropo-
larimeter at a temperature of 10 ꢂC between 200 and
450 nm. The fluorescence spectra were recorded on a

26
C. Gru¨newald et al. / Bioorg. Med. Chem. 16 (2008) 19–26
7. (a) Kool, E. T.; Younjin, C. Chembiochem 2006, 7, 669–
672; (b) Ha¨ner, R.; Langenegger, S. M. Chem. Commun.
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Hitachi F-4500 fluorescence spectrophotometer with
excitation at 385 or 365 nm.
Quantum yields were estimated on the basis of quinine
sulfate quantum yield of 0.55 in 0.5 M sulfuric acid.
For organic solvents a correction based on diffractive
indices was done, for aqueous solutions no such correc-
tion was carried out.
8. Grinstaff, M. W.; Khan, S. I. J. Am. Chem. Soc. 1999, 121,
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