Synthesis of pyrene labeled RNA for fluorescence measurements

Article abstract of DOI:10.1080/15257770701534048

The fluorophores 1-ethynylpyrene and 1-(p-ethynyl-phenylethynyl)-pyrene were attached to RNA through a Sonogashira cross-coupling with 5-iodocytidine either in solution through phosphoamidite synthesis or via on-column conjugation during solid-phase oligonucleotide synthesis. Six probes with the sequence 5′-CUU UUC UUU CUU-3′ were derivatized with both fluorophores, whereby the position of the modified cytidine was varied. Fluorescence measurements showed sensitivity of the pyrene group to its environment in the single strands and corresponding duplexes. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701534048

Nucleosides, Nucleotides, and Nucleic Acids, 26:1381–1386, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701534048
SYNTHESIS OF PYRENE LABELED RNA FOR FLUORESCENCE
MEASUREMENTS
Taewoo Kwon, Nelly Piton, Christian Gru¨ newald, and Joachim W. Engels
Institut fu¨r Organische Chemie und Chemische Biologie (OCCB), Johann Wolfgang
Goethe-Universita¨t, Frankfurt am Main, Germany
2
The fluorophores 1-ethynylpyrene and 1-(p-ethynyl-phenylethynyl)-pyrene were attached to RNA
2
through a Sonogashira cross-coupling with 5-iodocytidine either in solution through phosphoamidite
synthesis or via on-column conjugation during solid-phase oligonucleotide synthesis. Six probes
with the sequence 5ꢁ-CUU UUC UUU CUU-3ꢁ were derivatized with both fluorophores, whereby the
position of the modified cytidine was varied. Fluorescence measurements showed sensitivity of the
pyrene group to its environment in the single strands and corresponding duplexes.
Keywords Pyrene; RNA; fluorescence
INTRODUCTION
Fluorescence is a powerful tool for the detection and structural inves-
tigation of biomolecules like oligonucleotides.[1] Selected examples for
the use of pyrene modified oligonucleotides include their application as
RNA recognition probes,[2a] to detect the B-Z-DNA transition in twofold
modified oligonucleotides[2b] and in the detection of single nucleotide
polymorphism.[2c] The predictable structure of DNA and RNA can also
serve as scaffold for the construction of fluorescent nano-architectures.[3]
1-Ethynylpyrene itself was first introduced to the 5-position of
2ꢁ-deoxyuridine by Pd-catalyzed cross-coupling, subsequent formation
of corresponding phosphoramidite and incorporation into DNA.[4]
SYNTHESIS
Fluorophores
1-Ethynylpyrene 4 was synthesized in three steps from pyrene accord-
ing to procedures described in literature: selective mono-bromination,[5a]
Address correspondence to Joachim W. Engels, Institut fu¨r Organische Chemie und Chemische
Biologie (OCCB), Johann Wolfgang Goethe-Universita¨t, Max-von-Laue Straße 7, D-60438, Frankfurt am
Main, Germany. E-mail: joachim.engels@chemie.uni-frankfurt.de
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SCHEME 1 Synthesis of 1-ethynylpyrene
4 and 1-(p-ethynyl-phenylethynyl)-pyrene 8, reaction
conditions: a) HBr/H2O2, MeOH/Et2O, 16 hours, rt, 96%; b) 2-Methyl-3-butin-2-ol, morpholine,
10 hours, 110◦C, 72%; c) NaH, toluene, 15 minutes, reflux, 86%; d) (PPh3)2PdCl2, CuI, TEA, toluene,
3 hours, 40◦C, 47%; e) (PPh3)2PdCl2, CuI, TEA, THF, 20 hours, rt, 97%; f) NaH, toluene, 15 minutes,
reflux, 86%.
Sonogashira coupling with 2-methyl-3-butin-2-ol[5b] and final deprotection
of the tert-butanol group. 1-(p-ethynyl-phenylethynyl)-pyrene 8 was obtained
in three steps from diiodobenzene through two successive cross-couplings
as described in Scheme 1.
Phosphoramidites
To introduce these fluorophores into RNA, we used two different meth-
ods: A labeled cytidine-phosphoramidite was prepared and incorporated
into the oligonucleotide. The multi-step reaction affords the product in
an overall yield of 11% (R = 4) and 13% (R = 8) starting from protected
cytidine. Critical step is the Pd(0) catalyzed cross-coupling between the
protected 5-iodocytidine and the corresponding fluorophore (Scheme 2).
Indeed, using the usual but bulkier tert-dimethylsilyl group as 2ꢁ-OH
protecting group instead of the cyanoethoxymethyl (CEM) groups[6]
decreases the yield drastically. The coupling yield in RNA is comparable to
the natural bases but the final deprotection of the CEM group with TBAF
is only partial, which demands a further separation of both products with
anion-exchange HPLC.
5-iodocytidine phosphoramidite 13 was easily synthesized using the
ACE chemistry recently put forward by Dharmacon[7] and incorporated
into RNA. The fluorophores were post-synthetically introduced during the
oligonucleotide solid-phase synthesis, as shown in Figure 1 with the same
procedure already used in our laboratory for nitroxides.[8] In this case Pd(0)
was preferred to Pd(II) and DCM to DMF.[9] This method presents several
advantages in comparison to the derivatization in solution and was favored:

Pyrene Labeled RNA
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SCHEME 2 Synthesis of modified phosphoramidite 12, reaction conditions: a) abs. pyridine, DMTrCl,
rt, 5 hours, 52%; b) 1,2-dichloroethane, cyanoethoxymethylchloride (CEMCl), Bu2SnCl2, EtN(Pr)2,
90 minutes rt, 30 minutes 70◦C, 49%; c) 4 or 8, abs. DMF, 10 mo1% Pd(PPh3)4, 20 mol% Cul, Et3N, rt,
24 hours, 90% (4), 85% (8); d) abs. DCM, DIPEA, 0◦C → rt, 3 hours, 47% (4), 58% (8).
the amount of fluorophore necessary is smaller and the yields are quantita-
tive. Furthermore, the synthesis of only one phosphoramidite is necessary to
introduce different and various fluorophores what represents a large gain of
time.
Six modified RNAs were synthesized by the method on-column with
high purity (Figure 1b). Melting curve measurements showed a noticeable
destabilization up to 6◦C of the labeled RNAs in comparison to the un-
modified duplexes (Table 1) due to the orientation of the bulky pyrene
group into the narrow major groove. Yet, due to a dangling end effect[10]
a slight stabilization of 3.7◦C and 2.0◦C was observed for RNA 3 and RNA
FIGURE 1 a) Sonogashira cross-coupling during the solid-phase synthesis (ACE chemistry); b) MALDI
of RNA 5, calc. mass: 3933.5 g/mol.

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T. Kwon et al.
TABLE 1 Synthesized RNAs with the corresponding masses and Tm-values. Cꢁ: cytidine labeled with
1-ethynylpyrene, C∗: cytidine labeled with 1-(p-ethynyl-phenylethynyl)-pyrene
RNA
1
Sequence
Calc. mass Found mass (MALDI) [m/z]
T
m[◦C]
ꢀTm
5ꢁ-CUU UUC UUU CꢁUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-CUU UUCꢁ UUU CUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-CꢁUU UUC UUU CUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-CUU UUC UUU C∗UU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-CUU UUC∗ UUU CUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-C∗UU UUC UUU CUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
5ꢁ-CUU UUC UUU CUU-3ꢁ
3ꢁ-GAA AAG AAA GAA-5ꢁ
3834.4
3834.4
3834.4
3933.5
3933.5
3933.5
3833.0
3835.4
3837.1
3941.0
3932.6
3933.5
38.3 0.2 −3.4
36.1 0.1 −5.3
45.1 0.2 +3.7
35.4 0.2 −6.0
38.2 0.2 −3.2
43.4 0.2 +2.0
2
3
4
5
6
7
3609.1
3936.6
3607.2
3936.0
41.4 0.2
/
6, respectively, where the fluorophore is situated at the end of the strand
and can interact through π-π stacking with the neighboring nucleobase of
the complementary strand. The CD spectrum of unmodified RNA7 shows
maxima at 270 nm (global) and 225 nm (local) and minima at 245 nm
(local) and 210 nm (global) typical for A-form RNA oligonucleotides.[11]
The pyrene labeled RNAs exhibit extrema at the same wavelengths with
only slight decrease in intensity.
FLUORESCENCE SPECTRA
Emission spectra of the single stranded (ss) and double stranded (ds)
RNAs were measured in phosphate buffer (140 mM NaCl, 10 mM phos-
phate, 2.5 µM oligonucleotide concentration) to study the influence of the
pyrene residues 4 and 8 depending on the position in the strand (Figure 2).
Emission spectra are broadened and red-shifted compared to pyrene
monomer Fluorescence (Bande at 400 nm is an artefact and results from
scatered excitation). This can be explained with an electronic coupling
through the triple bond in the conjugated π-system.[12]
Upon hybridisation the fluorescence intensity largely increases espe-
cially in the case of 1-ethynylpyrene. Indeed, in the ssRNA the emission
is partially quenched through electron transfer between pyrene and the
neighboring pyrimidines[13] what is not valid anymore after formation of
the duplex. Due to the longer phenyl linker this quenching effect is not
so pronounced for 1-(p-ethynyl-phenylethynyl)-pyrene, in particular when
the fluorophore is situated in the middle of the strand (Figure 2e). In the
case of dangling ends (RNA3 and RNA6) the fluorescence intensity does not
change or even slightly decreases. Indeed the positive effect of hybridisation

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1385
FIGURE 2 Fluorescence spectra of a) RNA1, b) RNA2, c) RNA3, d) RNA4, e) RNA5 and f) RNA6;
ssRNA (solid), dsRNA (dashed); λ
= 395 nm.
exc.
is compensated by the electron transfer due to the stacking interaction with
the neighboring nucleobase.
OUTLOOK
The pyrene residues in the presented oligonucleotides are placed in the
less accessible major groove of RNA explaining the thermal destabilization
of modified oligonucleotides. Using the adenosine 2-position it could be
possible to address the more accessible minor groove in the case of RNA.
Interactions in twofold pyrene modified oligonucleotides could be used to
investigate the kinetics of RNA hybridization and folding.
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