Preparation and characterization of pyrene modified uridine derivatives as potential electron donors in RNA

Article abstract of DOI:10.1039/c8ob02246a

Charge transfer across double stranded DNA was observed for the first time about 20 years ago, and ever since it has been the subject of a large number of studies. RNA has been hardly investigated in this regard, which not least is due to the lack of suitably functionalized ribonucleotide building blocks to serve as electron sources upon incorporation into oligoribonucleotides. We have synthesized two uridine derivatives carrying pyrene or dimethylaminopyrene linked to C5 of the nucleobase. The key to successful synthesis was the adaptation of Suzuki-Miyaura conditions to the coupling of the pyrene moiety with the ribonucleoside. Final decoration of the pyrenylated nucleosides with standard 5′-O- and 2′-O-protecting groups and subsequent 3′-O-phosphitylation delivered the building blocks for incorporation into RNA. Spectroscopic analysis of the two pyrenylated uridines and of the accordingly modified oligonucleotides showed that in particular the dimethyaminopyrene functionalized nucleoside is a promising candidate as an electron source for RNA charge transport studies.

Full text of DOI:10.1039/c8ob02246a

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DOI: 10.1039/C8OB02246A
Organic & Biomolecular Chemistry
ARTICLE
Preparation and characterization of pyrene modified uridine
derivatives as potential electron donors in RNA
Received 00th January 20xx,
Accepted 00th January 20xx
Jennifer Frommer, Beatrice Karg, Klaus Weisz and Sabine Müller*
Charge transfer across double stranded DNA has been observed for the first time about 20 years ago, and ever since has
been subject to a large number of studies. RNA has been hardly investigated in this regard, which not at last is due to the
lack of suitably functionalized ribonucleotide building blocks to serve as electron sources upon incorporation into
oligoribonucleotides. We have synthesized two uridine derivatives carrying pyrene or dimethylaminopyrene linked to C5 of
the nucleobase. The key to successful synthesis was the adaptation of Suzuki-Miyaura conditions to the coupling of the
pyrene moiety with the ribonucleoside. Final decoration of the pyrenylated nucleosides with standard 5'-O- and 2'-O-
protecting groups and subsequent 3'-O-phosphitylation delivered the building blocks for incorporation into RNA.
Spectroscopic analysis of the two pyrenylated uridines and of accordingly modified oligonucleotides showed that in
particular the dimethyaminopyrene functionalized nucleoside is a promising candidate as electron source for RNA charge
transport studies.
DOI: 10.1039/x0xx00000x
www.rsc.org/
an acceptor are needed for charge transfer, both of which attached
to suitable sites of synthetic nucleic acid strands. Due to the
Introduction
The attachment of fluorophores to nucleic acids is a long-recorded progress in nucleic acid chemistry, nucleic acid based donor-bridge-
procedure usually applied for structural characterization and/or acceptor systems of desired composition can be generated and
measuring reaction kinetics by fluorescence spectroscopy used for molecular electronics studies. For reductive charge
1
, 2
techniques. Pyrene has less frequently been used in this regard. transfer, pyrene derivatives, linked to the constituents of
However, it has appealing advantages over commonly used deoxyoligonucleotides in manifold ways, have proven being very
1
2
fluorophores, like high quantum yield, long fluorescence lifetime useful electron sources. Wagenknecht and co-workers have been
3
-6
and the formation of excimers. Accordingly, over the past years focussing on pyrenes linked to the nitrogenous bases of
1
3-15
the importance of pyrene modified nucleosides has increased in the deoxynucleosides.
Notably, a sugar linked pyrene-nucleoside
field of nucleic acid research, and based on their characteristic conjugate, 2’-O-(pyren-1-yl)methylribonucleoside, has so far been
fluorescence properties, pyrene modified nucleic acids have gained the only known example of an electron donor that was applied to
7
, 8
11, 16
potential for applications as biosensor components. Beyond that, induce reductive transfer in RNA.
The photophysical properties
pyrene moieties attached to appropriate sites of oligonucleotides of pyrenes are strongly dependent on the nucleoside position, to
1
7
have attracted attention as electron donating entities in nucleic acid which the pyrene moiety is linked. Hence, choosing the optimal
charge transfer studies. Charge transport across DNA has been conjugation site is an important aspect to achieve efficient electron
9
18
known for more than 20 years. By formation of a B-form DNA helix transfer over defined distances.
the π-stacking arrangement of the nucleobases constitutes the
prerequisite for conducting charge transfer through a molecular
We have been interested in reductive charge transfer through RNA.
According to the results obtained with pyrene modified
wire, which can proceed as oxidative hole transport or reductive
1
4
1
0
deoxyoligonucleotides, we chose pyrene connected to the C5-
atom of uridine as electron donating entity. To the best of our
knowledge, such derivatives have not yet been synthesized and
used for RNA charge transfer studies. Synthesis of a C5-pyrenyl
uridine building block for incorporation into RNA requires the
covalent attachment of pyrene to the nucleobase, standard
protection of the 5'- and 2'-sugar hydroxyl groups with DMT and
TBDMS, and functionalization of the 3'-OH group as
phosphoramidite. For the initial C-C bond formation, the Suzuki-
Miyaura reaction appears to be a very good choice, since the
electron transfer. Both charge transfer processes are specified by
various mechanisms, though there is still some lack of
1
0
understanding. Very little is known about the ability of RNA to
transport charges, either in the same way as DNA, or possibly
1
1
through alternative mechanisms. An electron donating source and
a.
University of Greifswald, Institute of Biochemistry, Feilx-Hausdorff-Str. 4, 17487
Greifswald. E-mail: smueller@uni-greifswald.de
b.
Address here.
Electronic Supplementary Information (ESI) available: UV/VIS and fluorescence
measurements as well as quantum yields of pyrene and the nucleosides
in MeOH. NOESY NMR spectra for nucleoside and 12. NMR spectra for
nucleosides 15. See DOI: 10.1039/x0xx00000x
4, 10 and
1
2
4
4
-
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Scheme 1 Synthesis of PyU: (a) 0.05 equiv. [Ir(OMe)(COD)] , 1.15
2
equiv. bis(pinacolato)diborone, 0.1 equiv. dtbpy, cyclohexane, 80 Scheme 2 Synthesis of DMAPyU: (a) HNO (dropwise), glacial acetic
3
°
C, 20 h, 46%; (b) 0.8 equiv. pyrene derivative 3, 0.17 equiv. acid, 60 °C, 30 min, 99%; (b) 2.5 equiv. NaSH (1.82 M), EtOH, reflux,
Pd(PPh ) , 8 equiv. NaOH, THF/MeOH/H O (2:1:1), 70 °C, 24 h, 37%; 90 min, 95%; (c) 5 equiv. methyl iodide, 2.2 equiv. NEt , 2.2 equiv.
3
4
2
3
(
c) 1.5 equiv. DMTCl, pyridine, room temperature, 6 h, 77%; (d) 1.5 K CO , DMF, 120 °C, 4 h, 71%; (d) 0.05 equiv. [Ir(OMe)(COD)] , 1.15
2 3 2
equiv. tBDMSCl, 1.4 equiv. AgNO , 4 equiv. pyridine, THF, room equiv. Bis(pinacolato)diborone, 0.1 equiv. dtbpy, cyclohexane, 80
3
temperature,
6 h, 54%; (e) 2.5 equiv. 2-cyanoethyl-N,N- °C, 20 h, 62%; (e) 1.1 equiv. pyrene derivative 11, 0.1 equiv.
diisopropylchlorophosphoramidite,
temperature, 2 h, n/a.
4 equiv. TEA, DCM, room Pd(PPh ) , 10 equiv. NaOH, THF/MeOH/H O (2:1:1), 70 °C, 20 h,
3
4
2
43%; (f) 1.3 equiv. DMTCl, pyridine, room temperature, 5 h, 72%;
g) 1.4 equiv. tBDMSCl, 1.3 equiv. AgNO , 4 equiv. pyridine, THF,
(
3
linkage is formed directly between the aromatic nucleobase and the room temperature, 6 h, 55%; (h) 1.2 equiv. 2-cyanoethyl-N,N-
1
9
diisopropylchlorophosphoramidite,
temperature, 1 h, 85%.
4 equiv. TEA, DCM, room
polycyclic aromatic pyrene. The typical reagents of the Suzuki-
Miyaura reaction are an aromatic halide and an aromatic
organoboron derivative. Here we describe the synthesis of C5-
pyrene modified uridine derivatives by reaction of 5-iodouridine
with the pyrene moiety as the boronic acid compound in form of a
to obtain 1-dimethylaminopyrene (DMAPy) 10. For the generation
of the boronic acid ester on C7, the Iridium catalyst in combination
with dtbpy was used. The modified pyrene 11 was obtained with a
yield of 41 % over four reactions steps. Both boronic acid esters 2
and 11 were used individually with the halogenated nucleoside 3
for synthesis of the two pyrenylated nucleosides, requiring five
reaction steps for the pyrene functionalized derivative PyU 4
1
3, 14, 19
pinacol ester. This strategy is widespread in the literature,
although it has so far been used only in the deoxy series.
Furthermore, we report on the spectroscopic characterization of
the pyrenylated derivatives, as monomers and upon incorporation
into RNA.
(
Scheme 1) and eight reaction steps for the dimethylaminopyrene
functionalized derivative DMAPyU 12 (Scheme 2). The most
challenging part was defining appropriate conditions for the Suzuki-
Miyaura reaction of 2 and 11 with 5-iodouridine 3 to yield the
target compounds PyU 4 and DMAPyU 12. A number of protocols
have been described in the literature, employing different types of
Results and discussion
Synthesis of pyrene modified uridine building blocks
For our studies we decided to use unsubstituted pyrene (Py) and 1-
dimethylamino pyrene (DMAPy) as electron donors. Therefore, as a
first step, both pyrene moieties had to be converted into the
boronic acid derivatives 2 and 11 (Scheme 1 and 2). 5-Iodouridine 3
was chosen as the halide component and synthesized from uridine,
following a known protocol, with satisfying yield of 75% over three
19
Pd catalysts together with various ligands and bases. We mainly
followed the protocol developed by Wagenknecht and co-workers
14
for the attachment of pyrene derivatives to C5 of deoxyuridine.
However, we varied conditions for adaptation and optimization
regarding the conversion of ribonucleosides (Table 1). All
approaches delivered the expected products 4 and 12, although
with differing yields (Table 1). Clearly, the solvent was found to
2
0
steps. The boronic acid ester was introduced at C2 of pyrene to
generate the pyrene derivative 2 suitable for a Suzuki-Miyaura
reaction. Formation of the B-C bond was mediated by an Iridium
catalyst, whose active form was generated using 4,4’-di-tert-butyl-
2
affect the reaction yield, with THF/MeOH/H O apparently providing
the most suitable environment for the reaction. The Pd catalyst
apparently has less influence, giving comparable yields independent
of the nature of the ligands. Nevertheless, in combination with the
2
1
2
,2’-bipyridine (dtbpy) as an additional ligand (for details see ). The
synthesis of 1-dimethylaminopyrene 10 was accomplished by
2
preferred solvent mixture (THF/MeOH/H O), NaOH as base, and a
regioselective nitration of pyrene to give 1-nitropyrene 9, followed
reaction temperature of 70 °C, triphenylphosphine ligands at Pd
appeared to be most suitable (Entry 4). The nature of the ligand
turned out to significantly affect purification of 4 and 12.
2
2
by reduction to 1-aminopyrene 10. Subsequently, the aromatic
amino group was methylated with methyl iodide according to a
23
protocol developed in the Wagenknecht laboratory,
2
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Table 1 Variation of Suzuki-Miyaura conditions for synthesis of different pattern caused by the loss of symmetry. Three scalar
pyrene modified uridine derivatives 4 and 12.
couplings correlating H2" with H3'', H4'' with H5'', and H9'' with
H10'' are observed (Figure 1A). No contacts are detected for H6’’
and H8’’, demonstrating that dimethylaminopyrene is linked to
uridine via C7'' as expected. Additional NOESY spectra of both
compounds 4 and 14 corroborate these results by corresponding
NOE contacts between the adjacent protons (see Supporting
Information Figure S1 and S2).
Spectroscopic characterization of uridine derivatives PyU and
DMAPyU
Optical properties were characterized by UV/Vis and fluorescence
spectroscopy. The synthesized nucleoside derivatives 4 and 12 were
compared to the corresponding free pyrene moieties using the
same concentration in solvents expected to differently affect
1
4, 24
intramolecular electron transfer processes.
acetonitrile (MeCN) as a polar aprotic solvent and aqueous sodium
phosphate buffer (Na-P ), pH 7, as a polar protic solvent (Figure 2).
as catalyst, purification was considerably In addition, spectra were recorded in methanol (see Supporting
hampered, because the ferrocene-type ligand could not be fully Information Figure S3) to allow direct comparison with the deoxy
We chose
i
2 2
With Pd(dppf) Cl
1
4, 24
removed from the product, thus significantly decreasing the final series previously measured in methanol and MeCN.
yield after purification (Entry 3 and 6). On the contrary, no selected with respect to future RNA charge transfer studies to be
problems emerged when reaction was carried out with Pd(PPh as carried out in this buffer system.
i
Na-P was
3 4
)
catalyst. Hence, Suzuki-Miyaura reactions for conjugation of both
pyrene derivatives 2 and 11 to uridine were conducted under the
conditions given above, providing slightly different but in both cases
moderate yields (Entries 2 and 4).
Generally, UV/Vis spectra of the pyrenylated nucleosides largely
14
match those of their deoxynucleoside counterparts. However,
observed traces of beginning aggregation of pyrene and DMAPy 10
in sodium phosphate buffer lead to significantly lower absorption
Formation of the desired linkage between the pyrene derivatives values and hamper interpretation of corresponding spectra. DMAPy
1
and the pyrimidine base was confirmed through H NMR DQF-COSY 10 and its nucleoside 12 do not exhibit the typical absorption bands
2
5
measurements (Figure 1). For PyU 4, the presence of H6’’/8’’-H7’’ of unmodified pyrene. In acetonitrile, absorption maxima of both
scalar couplings and the absence of scalar couplings for H1’’/H3’’ pyrenylated nucleosides 12 and 4 show a bathochromic shift when
protons indicate that pyrene is indeed linked to uridine via its C2'' compared to the spectra of the corresponding free pyrene moieties.
atom (Figure 1B). The aromatic region of DMAPyU 14 shows a
Figure 1 DQF-COSY spectra showing the aromatic region of the pyrene modified nucleosides 4 and 14. A) As expected, three scalar
couplings are observed for nucleoside 14 confirming the C7”-C5-linkage of dimethylaminopyrene and the nucleobase. B) Scalar
couplings observed between H6”/8” and H7” serve as evidence for the C2”-C5-linkage between pyrene and the pyrimidine base of
nucleoside 4.
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Figure 2 UV/Vis absorption spectra of pyrene, DMAPy 10, and of Figure 3 Fluorescence spectra of free pyrene, DMAPy 10, and of
pyrene modified nucleosides PyU 4 and DMAPyU 12. All spectra pyrene modified nucleosides PyU 4 and DMAPyU 12. All spectra
were recorded at 20 °C at a concentration of 25 µM in Na-P
and MeCN (bottom).
i
(top) were recorded at 20 °C in Na-P (top) and MeCN (bottom). The
i
concentration of all compounds was adjusted to reach the same
optical density at the individual excitation wavelength. In Na-Pi:
This implies that the pyrimidine base and the linked pyrene are pyrene (334 nm, 5 µM); 10 (350 nm, 6 µM); 12 (355 nm; 1.5 µM); 4
2
5, 26
(
1
337 nm, 1.5 µM). In MeCN: pyrene (334 nm, 1 µM); 10 (355 nm,
.5 µM); 12 (363 nm; 1.5 µM); 4 (337 nm, 1.5 µM).
electronically coupled,
which is encouraging, because electronic
coupling is a fundamental requirement for an intramolecular charge
2
5
transfer process in an excited-state complex or exciplex. Notably,
the red shift of DMAPyU 12 exceeds that of PyU 4. Generally,
absorptions of 10 and 12 were bathochromically shifted in
comparison to pyrene and 4 in all solvents including methanol (see
Supporting Information). The formation of an exciplex after
excitation to achieve a charge separated state only occurs, if the
donor and acceptor part are featured with strong electronic
coupling. If this is not the case, the intramolecular charge separated
state could be achieved also by another mechanism named TICT
yields were estimated according to standard procedures with
4
quinine sulfate used as a reference. Determined quantum yields
for all compounds are summarized in Table 2. Most notably, the
DMAPyU 12 fluorescence is almost completely quenched
irrespective of the type of solvent. Likewise, the quantum yield of
PyU 4 is reduced, albeit to a smaller extent as seen for DMAPyU.
This indicates that, after excitation, the pyrene moiety of the
nucleoside forms an exciplex with the pyrimidine base in its ground
state, such that intramolecular charge transfer can occur. As a
result, a charge separated exciplex forms, exerting a quenching
(
Twisted Intramolecular Charge Transfer), which relies on a twist
2
7
24
between the donor and acceptor part of the excited molecule.
effect. This process is favoured in protic solvents, because
The local excitation of the donor part leads to a twist of the single
bond, which connects the two aromatic systems and leads either to
hydrogen bond interactions stabilize the charge-separated
1
4, 24
state.
Both pyrenylated nucleosides 4 and 12 fulfil general
2
7
a full charge transfer or none. Requirement for this mechanism is
that electronic coupling between the two aromatic compounds is
rather weak. This applies to pyrene moieties linked to nucleosides
via the C2-position, as was determined by calculation of the LUMOs
requirements for a charge transfer. In general, the bathochromic
shift of the UV-maxima is rather small, indicating rather weak
electronic coupling. Hence, the charge-separated state most likely is
reached through a TICT mechanism, which apparently is more
pronounced in 12, as indicated by the strong fluorescence
quenching independent of the solvent.
1
4
in 2PydU. For fluorescence measurements and determination of
fluorescence quantum yields, appropriate excitation wavelengths
were derived from the UV/Vis spectra, and all compounds were
adjusted to the same optical density (Figure 3 and Table 2). To Table 2 Fluorescence quantum yields for pyrene, DMAPy 10,
DMAPyU 12, and PyU 4 in Na-P
determined using quinine sulfate in 0.1 N H
i
and MeCN. Quantum yields were
ensure comparability of spectra recorded in different solvents, all
fluorescence measurements were carried out with identical
2
SO as a reference.
4
instrument settings. Fluorescence spectra of pyrene and 4 in Na-P
and MeCN exhibit the typical double band shape, whereas 10 and
2 display an unresolved broad fluorescence band similar to their
i
1
UV/Vis absorptions. Again, the modified nucleosides 4 and 12 were
red-shifted in both solvents when compared to the corresponding
free pyrene moieties. Because fluorescence quantum yields of the
compounds may deliver valuable information on their ability to
undergo reductive charge transfer, quantum
4
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Table 3 Conditions of phosphitylation of 7 and 14.
ARTICLE
Synthesis and spectroscopic characterization of DMAPyU-modified
RNA
The DMAPyU phosphoramidite 16 was coupled under standard
conditions in solid phase RNA synthesis to generate the modified
RNA1 (Table 4). The modified oligonucleotide was hybridized to a
non-modified oligoribonucleotide of complementary sequence, and
the melting temperature of the resulting duplex was measured in
comparison to the unmodified duplex (Table 4). The decrease in
melting temperature observed for the modified duplex can be
attributed to the DMAPy modification and indicates a destabilizing
effect of the pyrene moiety. Intercalation of the pyrene moiety has
been suggested in the literature for DNA duplexes, but not for RNA
30
duplexes, which implies that indeed charge transfer through RNA
11
occurs by a distinct mechanism as compared to DNA. Comparison
of the UV-Vis spectra of RNA1 hybridized to the unmodified
complementary strand (RNA1_ds in Figure 4), and of the monomer
The preferential formation of a TICT-state in 12 can be attributed to
the electron donating effect of the dimethylamino group,
enhancing the electron density of the pyrene moiety.
1
2 shows a small bathochromic shift of the absorption maximum
from 355 nm to 358 nm and the appearance of a second maximum
at 347 nm. This shift is somewhat less pronounced for the single
Synthesis of pyrenylated phosphoramidite building blocks
To convert the pyrenylated nucleosides to building blocks for solid stranded RNA (ssRNA1 in Figure 4). A red shift was also observed
phase RNA synthesis, the 5’- and 2’-hydroxyl groups of 4 and 12 for the monomer nucleoside 12 when compared to free DMAPy 10
were protected with DMT and TBDMS, respectively. Both reaction (Figure 2) and taken as evidence for electron coupling between the
steps were achieved with satisfying yields resulting in the target pyrene moiety and the nucleobase (see above).
compounds 6 and 14. Subsequent conversion into the respective
phosphoramidites was performed according to protocols for
2
8, 29
phosphitylation of modified or natural nucleosides
with
variation of reaction conditions including solvent, reaction time,
amount of phosphitylation reagent, and eluent composition during
chromatographic purification. The initial phosphitylation of 6 was
carried out with a 2.5-fold excess of the phosphitylation reagent 2-
cyanoethyl-N,N-diisopropylchlorophosphoramidite over two hours
(
Table 3). For chromatographic purification, dichloromethane/
acetone/ trimethylamine (90:9:1) was used as a rather polar eluent
to quickly elute the product and avoid long contacts of the
nucleoside phosphoramidite with the silica gel matrix.
Unfortunately, side products resulting from hydrolysis of excess
phosphitylation reagent could not be sufficiently separated under
3
1
those conditions, as detected by
P
NMR. Therefore,
phosphitylation of 14 was conducted under changed reaction and
purification conditions (Table 3). In addition to a reduced amount of
Figure 4 UV/Vis absorption spectra of pyrene modified RNA1 (ds
phosphitylation reagent, a shorter reaction time and a less polar and ss) and of DMAPyU 12 and. All spectra were recorded at 22 °C
at a concentration of 2.5 µM in Na-P for the RNAs and 1.5 µM for
DMAPyU 12. The apsorption of all measurements between 330 and
eluent system for column chromatography was used. The desired
nucleoside phosphoramidite 16 was obtained without further
impurities in 85% yield.
i
4
00 nm are displayed in the extract.
Table 4 Synthesized oligonucleotide sequences and analytical data.
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1
4
comparable results to pyrenylated deoxyribonucleosides, which
have already been used as electron donors in DNA charge transfer
1
5, 32, 33
studies.
Thus, upon their incorporation into RNA the
pyrenylated ribonucleotides are promising candidates to serve as
electron donors for charge transfer through RNA, a field hardly
investigated so far. Based on the superior spectroscopic properties,
DMAPyU 12 appears to be advantageous over PyU 4, and thus
should be the preferred electron donating unit in future studies.
The successful incorporation of 12 in RNA allowed spectroscopic
characterization of a pyrene-modified single stranded (RNA1_ss)
and double stranded RNA (RNA1_ds). DMAPy conjugated to the
nucleobase of 12 seems to be oriented towards the groove, as
concluded from higher fluorescence of RNA1_ds in comparison to
RNA1_ss, and decreased melting temperature for RNA1-ds as
compared to the non-modified duplex (RNA0_ds). This observation
is notable, because the location of the electron donor can be
expected to significantly affect charge transfer efficiency. The here
Figure 5 Fluorescence spectra of pyrene modified RNA1 (ds and ss)
and of DMAPyU 12. All spectra were recorded at 22 °C in Na-Pi with
a concentration of 2.5 µM for RNAs (excitation at 350 nm) and 1.5 reported UV/Vis and fluorescence data suggests that upon
µM for DMAPyU 12 (excitation at 355 nm).
excitation of the pyrene moiety, a charge separated state occurs
within the RNA duplex, and thus is encouraging for further
investigation into charge transfer through RNA.
Therefore, it may be concluded that in the duplex electronic
coupling is slightly stronger than in the single strand. Comparison of
the fluorescence spectra of the double stranded RNA1 (RNA1_ds in
Figure 5) with the monomer DMAPyU 12 shows a pronounced
hypsochromic shift and a marginally smaller quantum yield (Figure
5
). This implies that the charge separated state is available in the Experimental Section
dsRNA. The injection of the electron into the duplex-structure
would be favoured through the U-rich sequence, considering that
among the four natural nucleotides uridine is most easily
Triethylamine (TEA) was stored over calcium hydride and distilled
before use. All other reagents, chemicals and solvents were
obtained as the highest commercially available grade and used
without further purification. All reactions were carried out in dry
solvents under argon atmosphere, for the Suzuki-Miyaura reactions
the solvents were additionally degassed in an ultrasonic bath. Silica
gel for column chromatography (0.063–0.2 mm) was obtained from
Merck. TLC chromatography was performed on pre-coated
1
6, 31
reduced.
Fluorescence of the single stranded DMAPy modified
RNA (RNA1_ss) is weaker than that of the duplex (RNA1_ds). This
has been reported for pyrene-modified RNA, although with the
pyrene moiety attached to the sugar, and was interpreted as
3
0
evidence for a non-intercalating behaviour of the pyrene moiety.
Together with the decreased melting temperature for the DMAPy
modified duplex in comparison to the corresponding non-modified
duplex (Table 4), this suggests that the pyrene moiety of DMAPy in
RNA1_ds does not interact with the nucleobases of the duplex by
intercalation, but rather is extended into the groove. Comparison
of the fluorescence spectra of RNA1_ss and RNA1_ds again shows a
small but clearly detectable bathochromic shift, which further
supports the interpretation made above, that electronic coupling of
the pyrene moiety with the nucleobase is slightly stronger in the
duplex than in the single strand.
1
13
aluminium silica gel 60 F254 plates (Merck). NMR spectra ( H, C,
DQF-COSY, NOESY, HMBC, HSQC) were acquired on a Bruker Avance
6
00 MHz spectrometer, assignment of the NMR signals was carried
out by 2D NMR measurements (DQF-COSY, NOESY, HMBC, HSQC).
Mass spectra were recorded on a Bruker microflex MALDI-TOF MS.
UV/Vis absorption measurements were recorded with a JASCO V-
6
6
50. Fluorescence measurements were performed with a JASCO FP-
500 spectrofluorometer. UV melting curves were determined
using the Cary 100 spectrophotometer equipped with
temperature control unit (Varian), applying a heating/cooling rate
of 0.2 °C/min in a temperature range from 10 °C to 90°C. Na-P
a
i
buffer was prepared to contain 20 mM sodium phosphate and 100
mM NaCl at pH 7.
Conclusions
Synthetic protocols were developed for two pyrene modified
uridine derivatives and for their conversion into fully protected
phosphoramidite building blocks. Careful tuning of reaction
conditions, in particular for the Suzuki-Miyaura reaction, was the
key to achieve efficient syntheses and to obtain the two nucleoside
derivatives 7 and 15 in satisfying yields. Analysis of the fluorescence
properties of the unprotected ribonucleosides 4 and 12 showed
5
-(Pyren-2-yl)-uridine (4)
3 4
-Iodouridine 3 (0.278 g, 0.75 mmol), Pd(PPh ) (0.121 g, 0.105
5
mmol), NaOH (0.24 g, 6 mmol) and pyrene derivative 2 (0.197, 0.6
mmol) were filled into a two-necked flask and purged three times
with argon. The compounds were dissolved under argon
atmosphere in degassed THF/MeOH/H O (80 ml, 2:1:1, v/v/v), and
2
6
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the resulting mixture was heated at 70 °C (reflux) over night. The 112.89 (CH, DMT) 112.86 (CH, DMT) 88.69 (CH, C1‘) 85.59 (q, DMT)
reaction was stopped by addition of NH Cl (0.32 g, 6 mmol) after 83.13 (CH, C4‘) 73.33 (CH, C2‘) 69.93 (CH, C3‘) 63.57 (CH , C5‘) 54.69
cooling to room temperature. Afterwards 100 ml ethyl acetate and (OCH 54.58 (OCH ); MALDI-TOF m/z 747.37 ([M
00 ml H O was added, and the two phases were separated in a C H N O H calc. 747.27).
4
2
+
3
)
3
+ H] ,
+
1
2
46 38
2
8
separation funnel. The aqueous phase was extracted three times
with ethyl acetate, and the combined organic phase was dried with
Na SO , followed by evaporation to dryness. The residue was 5-(Pyren-2-yl)-2’-O-(tert-butyldimethylsilyl)-5’-O-(4,4’-
2
4
loaded on silica gel (1 g) and purified by column chromatography dimethoxytrityl)-uridine (6)
CH Cl /MeOH 98:2 to 9:1) yielding 4 as a white solid (0.64 g, 0.13 Nucleoside derivative 5 (0.2 g, 0.25 mmol) was dissolved in dry THF
mmol, 37%); R
): δ 11.71 (1H, br s, NH) 8.65 (1H, s, H6) 8.49 (2H, s, H1“/H3“) 8.29 pyridine (82 µl, fresh distilled). The reaction mixture was stirred at
2H, d, J 7.5 Hz, H6“/H8“) 8.19 (4H, s, H4“/H5“/H9“/H10“) 8.07 (1H, room temperature for 20 min in the dark, followed by addition of
dd, J 7.5 Hz, H7“) 5.93 (1H, d, J 4.4 Hz, H1‘) 5.53 (1H, d, J 5.4 Hz, tert-butyldimethylsilyl chloride (0.06 g, 0.38 mmol). The resulting
’OH) 5.38 (1H, t, J 4.8 Hz, 5’OH) 5.14 (1H, d, J 5.6 Hz, 3’OH) 4.25 suspension was stirred at room temperature for 5.5 h and was
1H, dd, J 4.9 Hz and 9.8 Hz, H2‘) 4.14 (1H, dd, J 4.9 Hz and 10.4 Hz, stopped by addition of ethyl acetate (20 ml). The precipitate was
H3‘) 3.93-3.96 (1H, m, H4‘) 3.73-3.78 (1H, m, H5‘(a)) 3.62-3.67 (1H, removed by filtration, and the remaining solution was washed twice
(
2
2
1
f
2 2
3
0.40 (CH Cl /MeOH 9:1); H-NMR (600 MHz, DMSO- (2.3 ml), followed by addition of AgNO (0.06 g, 0.35 mmol) and dry
d
6
(
2
(
1
3
m, H5‘(b)) C-NMR (150 MHz, DMSO-d ): δ 162.45 (q, C4) 150.22 with saturated aq. NaHCO . The combined aq. phase was extracted
6
3
(q, C2) 139.30 (CH, C6) 131.13 (q, C2“) 130.59 (q, pyrene) 130.45 (q, with ethyl acetate and the combined organic phase was dried with
pyrene) 127.52 (CH, C4“/C5“/C9“/C10“) 126.26 (CH, C7“) 125.16 Na SO . The solvent was removed under reduced pressure.
2
4
(CH, C6“/C8“) 124.47 (CH, C1“/C3“) 123.62 (q, pyrene) 122.85 (q, Purification of the crude product by column chromatography
pyrene) 113.41 (q, C5) 88.67 (CH, C1‘) 84.60 (CH, C4‘) 74.13 (CH, (hexane/ethyl acetate 2.5:1) delivered 6 as a white solid (0.12 g,
1
C2‘) 69.35 (CH, C3‘) 60.15 (CH
2
, C5‘); MALDI-TOF m/z 445.27 ([M + 0.14 mmol, 54%); R 0.53 (CH Cl /MeOH 97:3); H-NMR (600 MHz,
f
2
2
+
+
H] , C H N O H calc. 445.13).
6
DMSO-d ): δ 11.83 (1H, br s, NH) 8.25 (2H, d J 7.7 Hz, H6”/H8”) 8.13
2
5
20
2
6
(
2H, s, H1”/H3”) 8.02-8.07 (3H, m, H5”/H9”/H7“) 8.01 (1H, s, H6)
7
7
.74 (2H, d, J 9.0 Hz, H4”/H10”) 7.28 (2H, d, J 7.8 Hz, DMT) 7.04-
.11 (6H, m, DMT) 6.93 (1H, t, J 7.6 Hz, DMT) 6.53 (2H, d, J 8.8 Hz,
5-(Pyren-2-yl)-5’-O-(4,4’-dimethoxytrityl)-uridine (5)
Nucleoside derivative 4 (0.146 g, 0.33 mmol) was dissolved in dry DMT) 6.50 (2H, d, J 8.8 Hz, DMT) 6.02 (1H, d, J 5.4 Hz, H1’) 5.16 (1H,
pyridine (3 ml, freshly distilled), followed by addition of DMTCl d, J 5.9 Hz, 3’OH) 4.44 (1H, dd, J 5.4 Hz, H2’) 4.07-4.11 (1H, m, H4’)
(
0.15 g, 0.45 mmol) under argon atmosphere. The mixture was 4.01-4.05(1H, m, H3‘) 3.42 (3H, s, OCH
stirred at room temperature for 6 h, and the reaction was stopped (1H, m, H5‘(a)) 3.19-3.23 (1H, m, H5‘(b)) 0.90 (9H, s, SiC(CH )
3 3
3
) 3.35 (3H, s, OCH
3
) 3.27-3.31
) 0.12
C-NMR (150 MHz, DMSO-d ): δ
reduced pressure, and the residue was coevaporated three times 162.28 (q, C4) 157.85 (q, DMT) 157.76 (q, DMT) 150.25 (q, C2)
with toluene before resolving in CH Cl
. The solution was washed 144.39 (q, DMT) 137.63 (CH, C6) 135.06 (q, DMT) 135.02 (q, DMT)
with saturated aq. NaHCO
twice and the combined aq. phase was 130.55 (q, pyrene) 130.39 (q, C2“) 130.27 (q, pyrene) 129.51 (CH,
extracted with CH Cl
. The combined organic phase was dried with DMT) 129.35 (CH, DMT) 127.63 (CH, DMT) 127.47 (CH, DMT) 127.23
1
3
by addition of MeOH (5 ml). The solvent was removed under (3H, s, SiCH
3
) 0.11 (3H, s, SiCH
3
)
6
2
2
3
2
2
Na
CH
2
SO
Cl
4
and evaporated to dryness. After column chromatography 127.19 (CH, C4“/C5“/C9“/C10“) 126.49 (CH, DMT) 126.17 (CH, C7“)
(
2
2
/MeOH 100:0 to 97:3) compound 5 was obtained as a white 125.03 (CH, C6“/C8“) 124.83 (CH, C1“/C3“) 123.51 (q, pyrene)
1
solid (0.2 g, 0.26 mmol, 78%); R
600 MHz, DMSO-d
H6”/H8”) 8.20 (2H, s, H1”/H3”) 8.08 (2H, d, J 9.0 Hz, H5“/H9”) 8.04 69.84 (CH, C3‘) 63.26 (CH
1H, dd, J 7.6 Hz, H7“) 8.00 (1H, s, H6) 7.77 (2H, d, J 9.0 Hz, (CH, Si(CH ) 17.99 (q, SiC(CH
H4”/H10”) 7.31 (2H, d, J 8.1 Hz, DMT) 7.08-7.15 (6H, m, DMT) 6.95 MALDI-TOF m/z 861.42 ([M + H] , C52
1H, t, J 7.3 Hz, DMT) 6.59 (2H, d, J 8.7 Hz, DMT) 6.53 (2H, d, J 8.7
Hz, DMT) 5.97 (1H, d, J 5.3 Hz, H1’) 5.58 (1H, d, J 5.6 Hz, 2’OH) 5.19
f
0.50 (CH
2
Cl
2
/MeOH 95:5); H-NMR 122.99 (q, pyrene) 115.10 (q, C5) 112.89 (CH, DMT) 112.83 (CH,
(
6
): δ 11.80 (1H, br s, NH) 8.26 (2H, d J 7.6 Hz, DMT) 87.91 (CH, C1‘) 85.68 (q, DMT) 83.56 (CH, C4‘) 75.58 (CH, C2‘)
, C5‘) 54.66 (OCH ) 54.57 (OCH ) 25.65
) -4.72 (CH, SiCH ) -5.10 (CH, SiCH );
2
3
3
(
3
)
3
3
)
3
+
3
+
3
52 2 8
H N O SiH calc. 861.35).
(
(
(
1H, d, J 5.7 Hz, 3’OH) 4.36 (1H, dd, J 5.4 Hz and 10.8 Hz, H2’) 4.09 1-N,N-dimethylaminopyrene (10)
1H, dd, J 5.2 Hz and 10.4 Hz, H3’) 4.02-4.06 (1H, m, H4‘) 3.45 (3H, s, Triethylamine (4.4 ml, 32 mmol) was added to 1-aminopyrene 9
) 3.37 (3H, s, OCH ) 3.24-3.28 (1H, m, H5‘(a)) 3.16-3.21 (1H, m, (3.15 g, 14.5 mmol) solved in 30 ml DMF under argon atmosphere.
OCH
3
3
1
3
H5‘(b)) C-NMR (150 MHz, DMSO-d ): δ 162.38 (q, C4) 157.86 (q, To this stirring solution methyl iodide (2.3 ml, 36 mmol) was added
6
DMT) 157.76 (q, DMT) 150.30 (q, C2) 144.45 (q, DMT) 138.33 (CH, dropwise. The reaction mixture was heated at 120 °C for 2 h.
C6) 135.30 (q, DMT) 135.26 (q, DMT) 130.56 (q, pyrene) 130.51 (q, Reaction was followed by TLC. After 2 h, TLC analysis showed
C2“) 130.27 (q, pyrene) 129.54 (CH, DMT) 129.38 (CH, DMT) 127.67 remaining 1-methylaminopyrene, therefore, a second portion of
(
CH, DMT) 127.59 (CH, DMT) 127.24 (CH, C4“/C5“/C9“/C10“) 126.49 methyl iodide (2.3 ml, 36 mmol) was added together with K CO3
2
(CH, DMT) 126.16 (CH, C7“) 125.02 (CH, C6“/C8“) 124.89 (CH, (4.4 g, 32 mmol). After another 2 h the completed reaction was
C1“/C3“) 123.54 (q, pyrene) 122.95 (q, pyrene) 114.65 (q, C5) allowed to cool down to room temperature, and the reaction
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mixture was evaporated to dryness under reduced pressure. The temperature. Ethyl acetate (50 ml) and H O (50 ml) were added and
2
2 2
residue was dissolved in CH Cl , and washed twice with saturated the resulting two phases were separated in a separation funnel. The
aq. NaHCO3. The combined aq. phase was extracted with ethyl aqueous phase was extracted three times with ethyl acetate and
acetate. The combined organic phase was dried with Na SO and the combined organic phase was dried with Na SO , followed by
2
4
2
4
evaporated to dryness. The residue was loaded on silica gel (5 g) evaporation to dryness. The residue was loaded on silica gel (0.5 g)
and purified by column chromatography (hexane/ethyl acetate 99:1 and purified by column chromatography (CH Cl /MeOH 98:2 to 9:1)
2
2
to 9:1) yielding 10 as a yellow solid (2.52 g, 10.3 mmol, 71%); R 0.51 yielding 12 as a yellow solid (0.64 g, 0.13 mmol, 43%); R 0.36
f
f
1
1
(
hexane/ethyl acetate 9:1); H-NMR (600 MHz, DMSO-d
6
): δ 8.37 (CH
2
Cl
2
/MeOH 9:1); H-NMR (600 MHz, DMSO-d
6
): δ 11.69 (1H, br s,
(1H, d, J 9.2 Hz, H10) 8.20 (2H, d, J 7.6 Hz, H6/H8) 8.18 (1H, d, J 8.1 NH) 8.61 (1H, s, H6) 8.40 (1H, s, H8”) 8.38 (1H, s, H6”) 8.36 (1H, d, J
Hz, H3) 8.14 (1H, d, J 9.2 Hz, H9) 8.07 (1H, d, J 8.9 Hz, H4) 8.01 (1H, 9.3 Hz, H10”) 8.20 (1H, d, J 8.2 Hz, H3”) 8.14 (1H, d, J 9.3 Hz, H9“)
d, J 7.6 Hz, H7) 7.98 (1H, d, J 8.9 Hz, H5) 7.81 (1H, d, J 8.1 Hz, H2) 8.06 (1H, d, J 8.9 Hz, H4“) 7.98 (1H, d, J 8.9 Hz, H5“) 7.79 (1H, d, J
1
3
2.98 (6H, s, N(CH
3 2 6
) ) C-NMR (150 MHz, DMSO-d ): δ 148.82 (q, C1) 8.2 Hz, H2“) 5.92 (1H, d, J 4.4 Hz, H1’) 5.52 (1H, d, J 5.4 Hz, 2’OH)
131.15 (q, pyrene) 130.77 (q, pyrene) 127.36 (CH, C4) 126.24 (CH, 5.37 (1H, t, J 4.8 Hz, 5’OH) 5.14 (1H, d, J 5.5 Hz, 3’OH) 4.25 (1H, dd, J
C7) 126.15 (q, pyrene) 126.13 (CH, C9) 125.58 (CH, C3) 125.21 (CH, 4.9 Hz and 9.9 Hz, H2’) 4.13 (1H, dd, J 5.2 Hz and 10.3 Hz, H3’) 3.92-
C5) 124.44 124.31 (CH, CH, C6/C8 ) 123.38 (q, pyrene) 123.26 (CH, 3.96 (1H, m, H4‘) 3.73-3.79 (1H, m, H5‘(a)) 3.62-3.66 (1H, m, H5‘(b))
1
3
C10) 116.76 (CH, C2) 45.23 (CH, N(CH
3
)
2
); MALDI-TOF m/z 246.26 2.99 (6H, s, N(CH
150.19 (q, C2) 148.90 (q, C1“) 139.15 (CH, C6) 131.12 (q, C7“)
30.97 (q, pyrene) 130.63 (q, pyrene) 127.51 (CH, C4“) 126.31 (CH,
3 2 6
) ) C-NMR (150 MHz, DMSO-d ): δ 162.41 (q, C4)
+
+
(
[M + H] , C H NH calc. 246.12).
18 15
1
C9“) 126.21 (q, pyrene) 125.66 (CH, C3“) 125.41 (CH, C5“) 125.04 (q,
pyrene) 123.89 (CH, C8“) 123.66 (CH, C6“) 123.52 (q, pyrene)
123.43 (CH, C10“) 123.30 (q, pyrene) 116.78 (CH, C2“) 113.45 (q,
1
-N,N-dimethylamino-7-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-
2-yl)-pyrene (11)
[
Ir(OMe)(COD)]
2
(0.111 g, 0.168 mmol) was filled into a two-necked C5) 88.68 (CH, C1‘) 84.61 (CH, C4‘) 74.12 (CH, C2‘) 69.40 (CH, C3‘)
, C5‘) 45.25 (CH, N(CH ); MALDI-TOF m/z 488.31 ([M +
flask and purged three times with argon. Cyclohexane (12 ml, dried 60.23 (CH
)
3 2
2
+
+
over sodium) was added and the suspension was freed from oxygen H] , C27
25 3 6
H N O H calc. 488.18).
by purging with argon for 30 min. In a second flask, dtbpy (0.09 g,
0
.336 mmol), Bis(pinacolato)diboron (0.98 g, 3.9 mmol) and 10
0.82 g, 3.36 mmol) was weighed in and purged three times with
argon, too. Cyclohexane (32 ml, dried over sodium) was added. The
5
-(1-N,N-dimethylaminopyrene-7-yl)-5’-O-(4,4’-dimethoxytrityl)-
(
uridine (13)
resulting solution was combined with the Iridium suspension, The pyrene modified nucleoside 12 (0.18 g, 0.38 mmol) was
whereby the resulting mixture turned dark. After stirring over night dissolved in dry pyridine (3.3 ml, fresh distilled), followed by
at 80 °C the solvent was removed under reduced pressure and the addition of DMTCl (0.17 g, 0.5 mmol) to the resulting solution under
residue subjected to column chromatography (hexane/diethyl argon atmosphere. The mixture was stirred at room temperature
ether/ethyl acetate 98:2:0 to 92:2:6). The product 11 was obtained for 5 h, and the reaction was stopped by addition of MeOH (5 ml).
as a yellow solid (0.77 g, 2.07 mmol, 62%); R
f
0.51 (hexane/diethyl The solvent was removed under reduced pressure, and the residue
ether/ethyl acetate 92:2:6); H-NMR (600 MHz, CD Cl): δ 8.57 (1H, was co-evaporated three times with toluene before resolving in
s, H8) 8.54 (1H, H6) 8.43 (1H, d, J 9.2 Hz, H10) 8.09 (2H, d, J 8.7 Hz, CH Cl . The solution was washed twice with saturated aq. NaHCO3,
H3/H9) 7.94 (2H, s, H4/H5) 7.74 (1H, d, J 8.2 Hz, H2) 3.05 (6H, s, and the combined aq. phase was extracted with CH Cl . The
and evaporated to
49.05 (q, C1) 131.09 (q, pyrene) 130.97 (CH, C8) 130.91 (CH, C6) dryness. Subsequent column chromatography (CH Cl /MeOH 100:0
30.79 (q, pyrene) 127.67 (q, pyrene) 127.41 (CH, C9) 127.16 (q, to 97:3) delivered 13 as a yellow solid (0.22 g, 0.28 mmol, 72%); R
1
3
2
2
2
2
1
3
N(CH
3
)
2
) 1.45 (12H, s, BO(C(CH
3
)
2
)
2
3 2 4
C-NMR (150 MHz, CD Cl): δ combined organic phase was dried over Na SO
1
1
2
2
f
1
2 2 6
pyrene) 126.91 (CH, C4) 126.11 (q, pyrene) 126.09 (CH, C5) 125.35 0.44 (CH Cl /MeOH 95:5); H-NMR (600 MHz, DMSO-d ): δ 11.79
(
CH, C3) 125.14 (q, pyrene) 123.54 (CH, C10) 117.09 (CH, C2) 84.30 (1H, br s, NH) 8.24 (1H, d, J 9.3 Hz, H10”) 8.17 (1H, d, J 8.3 Hz, H3”)
q, BO(C(CH ) ) ) 45.23 (CH, N(CH ) ) 25.21 (CH, BO(C(CH ) ) ); 8.12 (1H, s, H8”) 8.09 (1H, s, H6”) 7.98 (1H, s, H6) 7.95 (1H, d, J 9.0
(
3
2 2
3 2
3 2 2
+
+
MALDI-TOF m/z 372.45 ([M + H] , C24
H
26BNO
2
H calc. 372.21).
Hz, H4“) 7.78 (1H, d, J 8.3 Hz, H2“) 7.72 (1H, d, J 9.3 Hz, H9“) 7.60
1H, d, J 9.0 Hz, H5“) 7.32 (2H, d, J 8.2 Hz, DMT) 7.10-7.15 (6H, m,
(
DMT) 6.98 (1H, t, J 7.5 Hz, DMT) 6.59 (2H, d, J 8.9 Hz, DMT) 6.54
(2H, d, J 8.9 Hz, DMT) 5.97 (1H, d, J 5.4 Hz, H1’) 5.57 (1H, d, J 5.7 Hz,
5-(1-N,N-dimethylaminopyrene-7-yl)-uridine (12)
2
’OH) 5.19 (1H, d, J 5.6 Hz, 3’OH) 4.36 (1H, dd, J 5.5 Hz and 10.9 Hz,
5
-Iodouridine 3 (0.111 g, 0.3 mmol), Pd(PPh (0.035 g, 0.03 mmol),
3 4
)
H2’) 4.08 (1H, dd, J 5.2 Hz and 10.3 Hz, H3’) 4.02-4.05 (1H, m, H4‘)
NaOH (0.12 g, 3 mmol) and pyrene derivative 11 (0.123, 0.33 mmol)
were filled into a two-necked flask and purged three times with
argon. The compounds were dissolved under argon atmosphere in
3
3
.46 (3H, s, OCH
3 3
) 3.38 (3H, s, OCH ) 3.23-3.27 (1H, m, H5‘(a)) 3.16-
1
3
.20 (1H, m, H5‘(b)) 2.98 (6H, s, N(CH ) ) C-NMR (150 MHz, DMSO-
3 2
d6): δ 162.37 (q, C4) 157.87 (q, DMT) 157.77 (q, DMT) 150.31 (q, C2)
degassed THF/MeOH/H O (40 ml, 2:1:1, v/v/v), and the resulting
2
1
1
48.80 (q, C1“) 144.47 (q, DMT) 138.26 (CH, C6) 135.32 (q, DMT)
35.29 (q, DMT) 130.82 (q, pyrene) 130.52 (q, pyrene) 130.45 (q,
mixture was heated at 70 °C (reflux) over night. The reaction was
4
stopped by addition of NH Cl (0.16 g, 3 mmol) after cooling to room
8
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C7“) 129.55 (CH, DMT) 129.40 (CH, DMT) 127.70 (CH, DMT) 127.60 5-(1-N,N-dimethylaminopyrene-7-yl)-2’-O-(tert-
CH, DMT) 127.27 (CH, C4“) 126.52 (CH, DMT) 126.21 (q, pyrene) butyldimethylsilyl)-5’-O-(4,4’-dimethoxytrityl)-uridine-3’-O-
26.05 (CH, C9“) 125.54 (CH, C3“) 125.15 (CH, C5“) 124.96 (q, (cyanoethyl-N,N-diisopropylphosphoramidite) (15)
(
1
pyrene) 124.29 (CH, C8“) 124.09 (CH, C6“) 123.62 (q, pyrene)
23.29 (q, pyrene) 123.15 (CH, C10“) 116.71 (CH, C2“) 114.69 (q,
C5) 112.91 (CH, DMT) 112.88 (CH, DMT) 88.64 (CH, C1‘) 85.60 (q,
DMT) 83.14 (CH, C4‘) 73.27 (CH, C2‘) 69.94 (CH, C3‘) 63.60 (CH , C5‘)
The fully protected nucleoside 14 was dissolved in dry DCM (2.8
ml), followed by dropwise addition of TEA (116 µl, fresh distilled)
and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (56 µl).
After stirring for at room temperature for 1 h, progress of reaction
was controlled by TLC, showing that no starting material was left.
Reaction was stopped by addition of 20 ml DCM (stored over
1
2
54.69 (OCH
3 3 3 2
) 54.59 (OCH ) 45.22 (CH, N(CH ) ); MALDI-TOF m/z
+
+
790.95 ([M + H] , C48
43 3 8
H N O H calc. 790.31).
NaHCO
NaHCO
3
) and the resulting solution was washed with saturated
. The organic phase was dried with Na SO , and evaporated
3
2
4
5
-(1-N,N-dimethylaminopyrene-7-yl)-2’-O-(tert-
to dryness. The residue was subjected to column chromatography
DCM/acetone/TEA 96:3:1) yielding the nucleoside
Nucleoside derivative 13 (0.21 g, 0.26 mmol) was dissolved in dry phosphoramidite 15 as a yellow solid (0.19 g, 0.17 mmol, 85%); R
butyldimethylsilyl)-5’-O-(4,4’-dimethoxytrityl)-uridine (14)
(
f
3
1
THF (2.4 ml), followed by addition of AgNO
dry pyridine (86 µl, fresh distilled). The reaction mixture was stirred 151.24 (s), 149.14 (s).
min at room temperature in the dark, before tert-
3
3
(0.06 g, 0.35 mmol) and 0.3 (DCM/acetone/TEA 96:3:1); P-NMR (240 MHz, CDCl ): δ
20
butyldimethylsilyl chloride (0.06 g, 0.38 mmol) was added. The
resulting suspension was stirred at room temperature for 5.5 h, RNA preparation
reaction was stopped by addition of ethyl acetate (20 ml). The
RNAs were synthesized on a Gene Assembler special DNA/RNA
precipitate was removed by filtration, the filtrate was washed with
saturated aq. NaHCO twice. The combined aq. phase was extracted
Synthesizer following the standard protocol for oligoribonucleotide
chain assembly. Standard PAC-phosphoramidites as well as CPG
supports were obtained from ChemGenes or Link Technologies. For
removal of base and phosphate protecting groups and cleavage
from the support, the synthesized RNAs were incubated with
aqueous ammonia (32%)/ethanolic methylamine (8 M) (1:1, v/v) at
65 °C for 40 min., followed by incubation with TEA·3HF for 1.5 h at
3
with ethyl acetate and the combined organic phase was dried with
Na SO . The solvent was removed under reduced pressure and the
2
4
residue purified by column chromatography (hexane/ethyl acetate
.5:1) The product 14 was obtained as a yellow solid (0.13 g, 0.15
2
1
mmol, 55%); R 0.49 (hexane/ethyl acetate 1:2); H-NMR (600 MHz,
f
DMSO-d
6
): δ 11.82 (1H, br s, NH) 8.23 (1H, d, J 9.2 Hz, H10”) 8.16
5
5 °C for removal of the 2'-O-protecting groups. Purification of the
synthesized RNA strands was performed by denaturing PAGE
12.5%, urea and acrylamide (acrylamide/bis-acrylamide 19:1) were
(1H, d, J 8.3 Hz, H3”) 8.05 (1H, s, H8”) 8.04 (1H, s, H6”) 7.99 (1H, s,
H6) 7.93 (1H, d, J 9.0 Hz, H4“) 7.78 (1H, d, J 8.3 Hz, H2“) 7.69 (1H, d,
J 9.2 Hz, H9“) 7.55 (1H, d, J 9.0 Hz, H5“) 7.29 (2H, d, J 8.3 Hz, DMT)
(
purchased from Roth). Product containing bands were cut out,
eluted from the gel with 0.3M NaOAc, pH 6.5, and desalted via
precipitation with ethanol. dsRNA for fluorescence and UV/Vis-
measurements and for UV melting curves was prepared in Na-Pi
buffer by mixing equimolar amounts of complementary strands to a
final concentration of 2.5 µM. Hybridisation was promoted by
heating the mixture at 90 °C for 2 min followed by slow cooling at
room temperature for gradual annealing.
7
.07-7.12 (6H, m, DMT) 6.96 (1H, t, J 7.4 Hz, DMT) 6.55 (2H, d, J 8.9
Hz, DMT) 6.50 (2H, d, J 8.9 Hz, DMT) 6.02 (1H, d, J 5.5 Hz, H1’) 5.16
1H, d, J 5.9 Hz, 3’OH) 4.44 (1H, dd, J 5.3 Hz, H2’) 4.07-4.10 (1H, m,
H4’) 4.03 (1H, dd, J 5.3 Hz and 10.3 Hz, H3‘) 3.43 (3H, s, OCH ) 3.36
3H, s, OCH ) 3.26-3.30 (1H, m, H5‘(a)) 3.18-3.23 (1H, m, H5‘(b))
.97 (6H, s, N(CH
NMR (150 MHz, DMSO-d
q, DMT) 150.25 (q, C2) 148.82 (q, C1“) 144.43 (q, DMT) 137.55 (CH,
(
3
(
3
1
3
2
3
)
2
) 0.89 (9H, s, SiC(CH
3
)
3
) 0.11 (6H, s, SiCH
3
)
C-
6
): δ 162.28 (q, C4) 157.86 (q, DMT) 157.79
(
C6) 135.10 (q, DMT) 135.05 (q, DMT) 130.84 (q, pyrene) 130.47 (q,
pyrene) 130.40 (q, C7“) 129.55 (CH, DMT) 129.38 (CH, DMT) 127.68
Notes and references
(
CH, DMT) 127.49 (CH, DMT) 127.27 (CH, C4“) 126.55 (CH, DMT)
26.21 (q, pyrene) 126.01 (CH, C9“) 125.57 (CH, C3“) 125.11 (CH,
C5“) 124.93 (q, pyrene) 124.26 (CH, C8“) 124.06 (CH, C6“) 123.68 (q,
pyrene) 123.29 (q, pyrene) 123.17 (CH, C10“) 116.72 (CH, C2“) 2.
1
.
S. Sindbert, S. Kalinin, H. Nguyen, A. Kienzler, L. Clima, W.
Bannwarth, B. Appel, S. Muller and C. A. Seidel, J. Am. Chem.
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D. Strohbach, N. Novak and S. Muller, Angew. Chem. Int. Ed.,
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15.16 (q, C5) 112.85 (CH, DMT) 87.83 (CH, C1‘) 85.73 (q, DMT)
3.61 (CH, C4‘) 75.54 (CH, C2‘) 69.89 (CH, C3‘) 63.33 (CH , C5‘) 54.66
) 54.58 (OCH ) 45.22 (CH, N(CH ) 25.65 (CH, SiC(CH ) 17.99
q, SiC(CH ) ) -4.72 (CH, SiCH ) -5.10 (CH, SiCH ); MALDI-TOF m/z
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(OCH
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3 3
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(
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+
+
57 3 8
904.61 ([M + H] , C54H N O SiH calc. 904.39).
7
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