Highly fluorescent conjugated pyrenes in nucleic acid probes: (Phenylethynyl)pyrenecarbonyl-functionalized locked nucleic acids

Article abstract of DOI:10.1002/chem.200801077

In recent years, fluorescently labeled oligonucleotides have become a widely used tool in diagnostics, DNA sequencing, and nanotechnology. The recently developed (phenylethynyl)pyrenes are attractive dyes for nucleic acid labeling, with the advantages of long-wave emission relative to the parent pyrene, high fluorescence quantum yields, and the ability to form excimers. Herein, the synthesis of six (phenylethynyl)pyrene-functionalized locked nucleic acid (LNA) monomers M¹-M⁶ and their incorporation into DNA oligomers is described. Multilabeled duplexes display higher thermal stabilities than singly modified analogues. An increase in the number of phenylethynyl substituents attached to the pyrene results in decreased binding affinity towards complementary DNA and RNA and remarkable bathochromic shifts of absorption/emission maxima relative to the parent pyrene fluorochrome. This bathochromic shift leads to the bright fluorescence colors of the probes, which differ drastically from the blue emission of unsubstituted pyrene. The formation of intra- and interstrand excimers was observed for duplexes that have monomers M¹-M⁶ in both complementary strands and in numerous single-stranded probes. If more phenylethynyl groups are inserted, the detected excimer signals become more intense. In addition, (phenylethynyl)pyrenecarbonyl- LNA monomers M⁴, M⁵, and M⁶ proved highly useful for the detection of single mismatches in DNA/ RNA targets.

Full text of DOI:10.1002/chem.200801077

DOI: 10.1002/chem.200801077
Highly Fluorescent Conjugated Pyrenes in Nucleic Acid Probes:
(Phenylethynyl)pyrenecarbonyl-Functionalized Locked Nucleic Acids
Irina V. Astakhova,[a, b] Vladimir A. Korshun,*[a] and Jesper Wengel*[b]
Abstract: In recent years, fluorescently
labeled oligonucleotides have become
a widely used tool in diagnostics, DNA
sequencing, and nanotechnology. The
recently developed (phenylethynyl)pyr-
enes are attractive dyes for nucleic acid
labeling, with the advantages of long-
wave emission relative to the parent
pyrene, high fluorescence quantum
yields, and the ability to form excimers.
Herein, the synthesis of six (phenyl-
than singly modified analogues. An in-
crease in the number of phenylethynyl
substituents attached to the pyrene re-
sults in decreased binding affinity to-
wards complementary DNA and RNA
and remarkable bathochromic shifts of
absorption/emission maxima relative to
the parent pyrene fluorochrome. This
bathochromic shift leads to the bright
fluorescence colors of the probes,
which differ drastically from the blue
emission of unsubstituted pyrene. The
formation of intra- and interstrand ex-
cimers was observed for duplexes that
have monomers M1–M6 in both com-
plementary strands and in numerous
single-stranded probes. If more phenyl-
ethynyl groups are inserted, the detect-
ed excimer signals become more in-
tense. In addition, (phenylethynyl)pyr-
enecarbonyl–LNA monomers M4, M5,
and M6 proved highly useful for the de-
tection of single mismatches in DNA/
RNA targets.
ACHTUNGTRENNUNGethynyl)pyrene-functionalized locked
Keywords: (phenylethynyl)pyrenes·
fluorescent probes · locked nucleic
nucleic acid (LNA) monomers M1–M6
and their incorporation into DNA olig-
omers is described. Multilabeled du-
plexes display higher thermal stabilities
acids
· long-wave fluorescence ·
oligonucleotides
Introduction
long lifetime of its excited state, sensitivity towards micro-
environmental changes, and propensity to p-stacking.[1]
A
Currently, there is remarkable growth in the use of synthetic
oligonucleotide analogues in bioorganic chemistry, medicine,
and molecular biology. Thus, fluorescently labeled oligonu-
cleotides have now widely been introduced for the detection
and study of nucleic acids in complex environments. Pyrene-
based dyes are of increasing interest for these applications
because of pyreneꢀs photophysical properties, such as the
pyrene label covalently attached to the sugar part of nucleo-
sides has been used for the detection of nucleic acid hybridi-
zation.[2] Recently, 2’-amino variants of locked nucleic acid
(LNA) derivatives that contain pyrene fluorochrome have
been reported (Scheme 1). It has been shown that the bicy-
clic skeleton of 2’-amino-LNA and a short linkage act to
direct hydrophobic pyrene residues into the minor groove of
nucleic acid duplexes.[3] Oligonucleotide probes with
pyrene–LNA modifications displayed a high affinity towards
DNA/RNA complements,[4] high fluorescence quantum
yields,[3] remarkable fluorescence energy resonance transfer
(FRET) efficiency,[5] an ability to form interstrand exci-
mers,[6] and sensitivity of the pyrene fluorescence to nucleic
acid hybridization.[3,7] These properties make these probes a
promising tool for homogeneous fluorescence assays[4b–f,5]
and ꢁngstrçm-scale chemical engineering.[6,8]
[a] I. V. Astakhova, Dr. V. A. Korshun
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry
Miklukho-Maklaya 16/10
117997 Moscow (Russia)
Fax : (+7)495-330-6738
E-mail: korshun@mail.ibch.ru
[b] I. V. Astakhova, Prof. Dr. J. Wengel
Nucleic Acid Center
Department of Physics and Chemistry
University of Southern Denmark
5230 Odense M (Denmark)
Fax : (+45)6-550-4385
However, the relatively short absorption wavelengths of
pyrene are an obstacle for its use in in vivo experiments be-
cause of the cell autofluorescence observed upon excitation
at the same wavelengths.[1a,9] This limitation of pyrene can
be overcome by altering its chemical functionalization and
E-mail: jwe@ifk.sdu.dk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200801077.
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FULL PAPER
sorbance and fluorescence maxima of 1,6-bisPEPy were red-
shifted by lꢀ25 and 30 nm, respectively, compared with
monoPEPy, and lꢀ75 and 60 nm, respectively, compared
with unsubstituted pyrene. In turn, attachment of bulky ary-
lethynyl groups to positions 6 and 8 of the pyrene dimers
DP1–DP3 resulted in a ꢀ45 nm red shift of the fluorescence
maxima compared to the reference unsubstituted compound
(Scheme 2; R=H). Additionally, compounds DP1–DP3 ex-
hibited high fluorescence quantum yields (FF =0.88 for DP2
in cyclohexane) and remarkable excimer emission at lmax
ꢀ525 nm. The fluorescence quantum yields of 1,6-bisPEPy
and 1-PEPy in EtOH were observed to be FF =0.79 and
0.90, respectively, and the lifetimes of the excited state of 1-
PEPy and 1,6-bisPEPy in EtOH were 7.72 ns and 1.65 ns, re-
spectively. Thus, a short excited lifetime implies that PEPy
excimers should confirm the preassociation of two PEPy
moieties, not just their mutual spacial attainability, which
makes PEPy a specified probe of choice for structural stud-
ies of biomolecules.[10e]
Scheme 1. The chemical structures of the pyrene fluorochrome and LNA,
2’-amino-LNA, and its 1-pyrenemethyl[4b] and 1-pyrenoyl[3] derivatives;
T=thymin-1-yl.
thereby affecting the spectroscopic and photophysical prop-
erties of the fluorochrome. Recently, the 1-(phenylethynyl)-
pyrene (1-PEPy) fluorochrome was introduced as an im-
proved dye for nucleic acid labeling.[9a,10] Thus, the presence
of one or more phenylethynyl groups induced an increase in
the pyreneꢀs absorption together with a bathochromic shift
in the absorption and emission maxima (lꢀ50 and 30 nm,
respectively), a decrease in the Stokesꢀ shift and excited life-
time, and an increased emission quantum yield. More re-
cently, systematic photophysical evaluations of 1-PEPy, 1,6-
bisPEPy, and DP1–DP3 were reported (Scheme 2).[9] Upon
incorporation of the second phenylethynyl group, the ab-
The high-affinity hybridization of 2’-amino-LNAs func-
tionalized with PEPy has recently been realized, but the
spectral and photochemical properties of these conjugates
were not thoroughly investigated (Scheme 2, monomers X1–
X3 and Y1–Y3).[4e] Thus, stimulated by the attractive fea-
tures of modified LNAs[3,4,6–8] and of the PEPy fluoro-
AHCTUNGTRENNUNG
chrome,[9a,10] we have investigated mono-, bis-, and tris(phe-
nylethynyl)pyrene dyes attached to 2’-amino-LNAs at the
first or second position of the pyrene nuclei via amide link-
age. Herein, we describe the synthesis of mono-, bis-, and
trisACTHNUGRTENUNG(phenylethynyl)pyrenecarbonyl (mono-, bis-, and trisPE-
Pyc) LNA monomers (3-phenylethynyl-1-carbonyl (M1), 6-
phenylethynyl-2-carbonyl (M2), 3,6-bis(phenylethynyl)-1-car-
bonyl (M3), 6,8-bis(phenylethynyl)-2-carbonyl (M4), 3,6,8-
trisACHTNUGRTNEUNG
(phenylethynyl)-1-carbonyl (M5), 3,6,8-tris(phenylethyn-
yl)-2-carbonyl (M6)) and their incorporation into DNA olig-
omers and thermal denaturation studies, and also the spec-
tral properties of conjugates that contain PEPyc moieties
positioned either in a probe strand or in each of two com-
plementary strands (“zipper” duplexes).
Results and Discussion
Synthesis of PEPyc-modified LNA phosphoramidites: The
general approach to the preparation of PEPy carboxylic
acids and their conjugation with 2’-amino-LNA is shown in
Schemes 3 and 4, respectively. The bromination of starting
1-acetylpyrene 1 with one or two equivalents of bromine
was performed according to the published procedure,[11] with
modified temperature and time conditions. Hereby, we ob-
tained mixtures of isomeric bromo products that were used
directly in the next step, which was followed by purification
of the corresponding PEPy derivatives. The reaction of 1
with three equivalents of bromine at 1208C[12] resulted in
full conversion of the starting compound to tribromopyrene
derivative 4. In turn, mono-, di-, and tri-brominations of iso-
meric 5[13] afforded the desired products 6–8 in high yields
Scheme 2. The chemical structures of 1-PEPy, 1,6-bisPEPy,[9a] DP1–DP3,
[9b]
and 1-, 2-, and 4-PEPy–LNA monomers X1–X3 and Y1–Y3;[4e] T=
thymin-1-yl.
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V. A. Korshun, J. Wengel and I. V. Astakhova
Scheme 3. Synthesis of compounds 9–20. Reagents and conditions: i) Br2, CCl4, 608C; ii) Br2, PhNO2, 1208C; iii) phenylacetylene, [PdCl
CuI, NEt3, THF, 708C; iv) NaOH, Br2, 1,4-dioxane, water, 08C.
2ACHTUNGERTN(NUNG PPh3)2], PPh3,
lene under Sonogashira reac-
tion conditions[14] to give
mono-, bis-, and tris-PEPy de-
rivatives 9–14. The yields of
compounds 9 and 11 after sep-
aration of the isomers were
29% and 53%, respectively,
for two steps, whereas the So-
nogashira reaction of pure bro-
mopyrenes 4–8 resulted in
overall yields of 40–80%. The
structure of compound 11 was
confirmed by HMQC and
HMBC NMR spectroscopy. In
our strategy, the attachment of
PEPy fluorochromes had to be
carried out by acylation, and
therefore products 9–14 were
Scheme 4. Synthesis of compounds 22–24 from 21. Reagents and conditions: i) 15–20, HBTU, DIEA, DMF (or
DMF-1,1-dichloroethane); ii) NC(CH2)2OP(N(iPr)2)2, diisopropylammonium tetrazolide, CH2Cl2; iii) DNA
synthesizer. DMT=4,4’-dimethoxytrityl.
G
ACHTUNGTRENNUNG
subsequently converted to the
corresponding carboxylic acids
under the same conditions as for 1-acetylpyrene. In the case
of exhaustive bromination of 2-acetylpyrene, formation of
the ortho isomer with respect to the acetyl group might take
place due to a significant contribution from radicals to the
bromination mechanism at high temperature.
The obtained acetylbromopyrenes are prospective build-
ing blocks for further modification by a wide range of transi-
tion-metal-catalyzed reactions, including possible transfor-
mations of the acetyl groups. Phenylethynyl substitution was
of interest to us because it would improve the spectroscopic
and photophysical properties of the pyrene. Thus, com-
pounds 2–4 and 6–8 were cross-coupled with phenylacety-
15–20 under haloform oxidation conditions[15] to prepare for
conjugation by amide bond formation.
N2-acylation of 2’-amino-LNA monomer 21[16] with 15–20
was performed in DMF by using HBTU (O-(benzotriazol-1-
yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate)[17]
as the coupling reagent. For compounds 17–20, 1,1-dichloro-
ethane had to be added because of the low solubility of the
bis- and trisPEPy acids in DMF. The steric effect of phenyl-
ethynyl substituents on the accessibility of the carboxylic
group may explain why the highest coupling yield (90%)
was obtained for least-hindered 16, whereas the most-hin-
dered 20 gave a yield of only 42%.
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Functionalized Locked Nucleic Acids
FULL PAPER
Surprisingly, the 13C NMR spectra of modified nucleosides
22c–f did not show signals from the C1’–C5’ and C5’’ carbon
atoms, whereas the 13C NMR spectra of monoPEPyc ana-
logues 22a–b displayed unusually broad nucleoside peaks.
However, 13C NMR spectroscopy experiments for deriva-
tives 22c–f, recorded by using 8192 scans, showed broad sig-
nals in the d=100–50 ppm region that were similar to those
in the 13C NMR spectra of 22a–b (Figure S1, Supporting In-
formation) for the corresponding nucleoside carbon atoms.
The same effect has been previously reported for 3’,5’-silyl-
protected ara-uridine-2’-carbamates that contain 1-PEPy.[10e]
Thus, attachment of bulky modifications to 2’-amino-LNA
results in unusual shielding of nucleoside carbon atoms, in
the same way as the presence of large silyl groups and aryl
carbamate residues does in
each coupling step, were 85–95%. The coupling efficiencies
of commercial DNA and LNA amidites varied between 98
and 100%. All ONs were purified by reverse-phase HPLC
(RP-HPLC) and their identity confirmed by MALDI-TOF
mass spectrometry (Table S1, Supporting Information).
Hybridization of ONs containing monomers M1–M6 with
complementary DNA and RNA: Thermal denaturation tem-
peratures were determined in medium salt buffer by using
1.0 mm of the two complementary strands, and were com-
pared to the denaturation temperatures of the correspond-
ing
unmodified
duplexes
(Table 1).
Except
for
ON24,ON25:DNA/RNA and ON32,ON33:DNA/RNA, the
thermal denaturation curves of all the duplexes displayed S-
ara-uridine.
nature of this shielding still
needs to be clarified.
However,
the
Table 1. Thermal denaturation temperatures (Tm) for duplexes of modified ONs and DNA/RNA comple-
AHCTUNGTRENNUNG
ments.[a]
5’!3’ sequence
DNA duplex
RNA duplex
Tm (DTm/mod) [8C]
The nucleoside derivatives
22a–f were phosphitylated
with bis(N,N-diisopropylami-
no)-2-cyanoethoxyphosphine
in CH2Cl2 and in the presence
of diisopropylammonium tetra-
zolide to give phosphorami-
dites 23a–f.
Tm (DTm/mod)[b] [8C]
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
GTGAM1ATGC
20.0 (ꢁ8.0)
21.5 (ꢁ6.5)
42.5 (+14.5)
34.5 (+3.3)
29.0 (+0.5)
33.5 (+5.5)
34.0 (+6.0)
32.5 (+4.5)
45.0 (+8.5)
43.5 (+7.8)
25.0 (ꢁ3.0)
<10 (>ꢁ18)
41.0 (+13.0)
>70 (>+21)
42.0 (+7.0)
32.5 (+4.5)
28.0 (0.0)
23.0 (ꢁ3.0)
44.5 (+8.3)
37.0 (+4.5)
<10 (>ꢁ18)
<10 (>ꢁ18)
<10 (>ꢁ18)
n.t.
19.5 (ꢁ6.5)
24.5 (0.0)
22.5 (ꢁ2.0)
33.0 (+3.5)
32.0 (+3.8)
34.0 (+8.0)
33.5 (+9.0)
32.0 (+7.5)
43.5 (+8.8)
43.5 (+9.5)
<10 (>ꢁ16)
24.0 (ꢁ0.5)
<10 (>ꢁ14)
47.5 (+10.8)
37.0 (+6.3)
30.0 (+4.0)
29.0 (+5.5)
23.0 (ꢁ1.5)
40.5 (+7.3)
33.0 (+4.3)
<10 (>ꢁ16)
<10 (>ꢁ14)
<10 (>ꢁ14)
n.t.
GCAM1ATCAC
GCATAM1 CAC
GTGAM1AM1GC
GCAM1AM1 CAC
GTGAM2ATGC
GCAM2ATCAC
GCATAM2 CAC
ON9
GTGAM2AM2GC
GCAM2AM2 CAC
GTGAM3ATGC
Synthesis of modified oligonu-
cleotides (ONs): PEPyc-modi-
fied LNA monomers 23a–f
were used in automated ON
synthesis to prepare a series of
modified ONs. The sequences
of mixed base 9-mer ONs were
similar to those previously de-
signed for studies of pyrene-
functionalized LNA.[3,4e,6] In
addition, 15-mer ONs were de-
signed based on the reported
13-mer sequence used for the
study of 2’-N-(pyren-1-yl)car-
bonyl–2’-amino-LNA.[3]
Synthesis of modified ONs
was performed by following
standard protocols, except for
incorporation of sterically hin-
dered monomers M5 and M6,
for which a double coupling
procedure with an extended
coupling time (2ꢃ30 min) was
used. For all the modified
monomers, 1H-tetrazole was
used as the activator. The re-
sulting stepwise coupling yields
of monomers M1–M6, based on
the absorbance of the dimeth-
ON10
ON11
ON12
ON13
ON14
ON15
ON16
ON17
ON18
ON19
ON20
ON21
ON22
ON23
ON24
ON25
ON26
ON27
ON28
ON29
ON30
ON31
ON32
ON33
ON34
ON35
ON36
ON37
ON38
ON39
GCAM3ATCAC
GCATAM3 CAC
GTGAM3AM3GC
GCAM3AMCAC
GTGAM4ATGC
GCAM4ATCAC
GCATAM4 CAC
GTGAM4AM4GC
GCAM4AM4 CAC
GTGAM5ATGC
GCAM5ATCAC
GCATAM5 CAC
GTGAM5AM5GC
GCAM5AM5 CAC
CGLTTTLATLAM5 ATLCAMeCLG
CGLTTTLAM5AM5 ATLCAMeCLG
CGLTTM5ATLATL AM5CAMeCLG
GTGAM6A TGC
n.t.
n.t.
44.0 (ꢁ11.5)
32.5 (ꢁ9.0)
42.0 (ꢁ4.3)
30.5 (+2.5)
<10 (>ꢁ18)
32.0 (+4.0)
n.t.
54.5 (ꢁ7.5)
45.0 (ꢁ6.0)
34.0 (ꢁ11.5)
<10 (>ꢁ16)
<10 (>ꢁ14)
<10 (>ꢁ14)
n.t.
n.t.
>70 (>+8)
26.0
GCAM6ATCAC
GCATAM6 CAC
GTGAM6AM6GC
GCAM6AM6 CAC
CGLTTTLATLAM6 ATLCAMeCLG
GTGATATGC
n.t.
54.0 (ꢁ1.5)
28.0
GCATATCAC
28.0
55.5
50.5
50.5
24.5
62.0
57.0
57.0
CGLTTTLATLATATLCAMeCLG
CGLTTTLATATATLCAMeCLG
CGLTTTATLATL ATC AMeCLG
[a] TL =thymin-1-yl LNA monomer, MeCL =5-methylcytosin-1-yl LNA monomer, GL =guanin-9-yl LNA mono-
mer; see Scheme 4 for the structures of monomers M1–M6. n.t.=no clear melting transition detected by UV
melting experiment. [b] Tm values were measured as the maximum of the first derivatives of the melting
curves (A260 vs. temperature), and reported Tm values are the average of at least two measurements. All Tm
values were recorded in medium salt buffer. DTm/mod: the change in Tm per modification relative to the corre-
sponding reference duplex).
ACHTUNGTRENNUNGoxytrityl cation released after
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11013

V. A. Korshun, J. Wengel and I. V. Astakhova
shaped monophasic transitions similar to those of the un-
modified reference duplexes (Figure S2, Supporting Infor-
mation). In the case of ON24, ON25, ON32, and ON33, for-
mation of duplexes with DNA and RNA complements was
confirmed by fluorescence spectroscopy and circular dichro-
ism (CD).
underlines an effective communication between PEPy
groups in the double stranded constructs (Table 1; e.g., du-
plexes of ON14, ON15, ON19, and ON20 with DNA/RNA
complements, compared with the duplexes formed by
ON11–ON13 and ON16–ON18). In turn, thermal denatura-
tion curves of duplexes that contain two insertions of M5 or
M6 do not exhibit a clear melting transition, whereas CD
spectra of the single-stranded probes and the corresponding
duplexes are rather similar (Figure S9H, Supporting Infor-
mation). This suggests that interaction between two PEPyc
monomers might stabilize single-stranded helical conforma-
tion of the doubly M5 or M6-labeled ONs, which resembles
the conformation of the probes within the duplexes.
Previously, it was reported that insertion of an unsubsti-
tuted 2’-amino-LNA monomer into a 9-mer mixed-base se-
quence induces a significant increase in the thermal stability
of duplexes with both complementary DNA and RNA.[4a]
Conjugation of the pyrene to 2’-amino-LNA by a short rigid
amide linker does not decrease binding affinity of the ONs
due to a double helix geometry that allows accommodation
of the polyaromatic group in the minor groove of the du-
plexes.[3] However, monomer M1 showed a destabilizing
effect, except for an extraordinarily high stabilization in the
case of the ON3:DNA duplex (DTm/mod=+14.58C). Per-
haps the presence of a phenylethynyl group in M1 that is
close to the site of attachment of PEPy to 2’-amino-LNA
causes difficulties for accommodation of the modification in
the narrow minor groove of the resulting duplexes. Addi-
tionally, DNA:RNA hybrids, and especially LNA-modified
ones, are duplexes of the A/B transition type.[18] This implies
alterations of the double-helix parameters from the pure B-
type, such as widening of the minor groove, which correlates
with the higher Tm values of M1-modified DNA:RNA du-
plexes.
Hybridization of ONs containing monomers M1–M6 with
mismatched DNA and RNA: In addition, we evaluated the
Watson–Crick selectivity of singly modified probes ON16,
ON26, and ON34 towards DNA and RNA strands that con-
tained a single mismatched nucleotide at the central three
positions (Table 2). Probe ON16 exhibits excellent mismatch
discrimination, except against its DNA target with mis-
matched nucleotides at position 6. In this case, the thermal
stability of the mismatched duplexes is similar to or higher
than the one for fully matched duplex ON16:DNA. This
suggests that the presence of mismatched nucleotides direct-
ly opposite monomer M4 might promote an intercalation of
the PEPy residue into the duplexes. At the same time,
probe ON16 does not bind to any other mismatched DNA/
RNA targets at temperatures above 108C, which makes it a
promising tool for mismatch-sensitive nucleic acid detection.
As expected, longer sequences ON26 and ON34, which
contain five affinity-enhancing LNA monomers, bind com-
plementary DNA and RNA more strongly and are thus less
mismatch-selective than ON16 (Table 2; Tm values for ON26
and ON32 against complementary and singly mismatched
DNA/RNA, compared with duplexes of ON16). Neverthe-
less, the presence of mismatches decreases the thermal de-
naturation temperatures of the complexes compared with
the corresponding fully matched duplexes. Probe ON34 con-
taining monomer M6 showed better mismatch discrimination
relative to M5-modified analogue ON26, and both ON26
and ON34 exhibit the best Watson–Crick discrimination of
DNA targets with mismatched nucleotides at position 10.
This is in disagreement with previously reported data that
shows that mismatches opposite the LNA monomers are
more efficiently discriminated against than those opposite
unmodified nucleotides.[19] For RNA targets, probe ON26
showed the best Watson–Crick discrimination with mis-
matches that were opposite modification M5 (position 9). In
turn, single mismatches at positions 8–10 of the RNA target
were all equally well discriminated by probe ON32, as dem-
onstrated by a decrease in Tm values of more than 158C.
Insertion of two M1 monomers results in increased bind-
ing affinity of ONs towards both complementary DNA and
RNA, which suggests a stabilizing interaction between PEPy
residues in the complementary complexes (Table 1; data for
the duplexes of ON4 and ON5 with DNA/RNA comple-
ments). This interaction might arise due to the spatial pre-
association of two pyrene residues in the duplex. Addition-
ally, replacement of modification M1 with isomeric, less ster-
ically hindered M2 in a 9-mer mixed-base sequence resulted
in thermal stabilities of the same magnitude as for 2’-N-
(pyren-1-yl)carbonyl–2’-amino-LNAs (Table 1; data for
ON6–ON8:DNA/RNA).[3]
If the number of phenylethynyl substituents attached to
the pyrene core is increased, the singly labeled duplexes
formed with both complementary DNA and RNA are dra-
matically destabilized, which most likely results from the
distortion of the double helix geometry by the bulky modifi-
cations M3, M5, and M6 (Table 1; Tm values for ON11,
ON12, ON21–ON23, and ON29–ON31 towards DNA/RNA
complements). Interestingly, a M3-modified duplex with a
constitution analogous to that of ON3:DNA displayed a re-
markable stabilizing effect as well, which was probably due
to the intercalation of the modifications into the double
helix (Table 1, ON13:DNA). It is worth noting that duplexes
that contain M4 display higher thermal denaturation temper-
atures than those that contain M3, M5, and M6, which might
be explained by less steric hindrance from modification M4.
Furthermore, duplexes with two insertions of M3 or M4 ex-
hibit impressive increases in the thermal denaturation tem-
peratures compared with singly modified analogues, which
Spectral and photophysical properies of PEPyc-modified
oligomers and their duplexes with complementary DNA and
RNA: The UV/Vis absorption and steady-state fluorescence
emission spectra were obtained in medium salt buffer with
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Functionalized Locked Nucleic Acids
FULL PAPER
Table 2. Thermal denaturation temperatures for duplexes of ON19, ON26, and ON32 and their DNA/RNA
Table 3 lists the spectral and
photophysical properties of the
complements with single mismatches.[a]
ON:target[b]
Tm [8C]
M1–M4-functionalized
ONs
DNA target, B:
RNA target, B:
(SSP) and their duplexes with
DNA and RNA complements.
All the UV/Vis spectra for
A
C
G
T
A
C
G
U
ON16 5’-GTGAM4ATGC
<10
<10
<10
32.5 <10
<10
<10
30.0
target 3’-CACBATACG
monoACHTNUGRTNEUNG
PEPyc monomers M1 and
5’-GTGAM4ATGC
32.5
<10
40.5
33.0
<10
43.0
33.0 36.0
<10 32.5 <10
41.5 44.0
30.0 <10
<10
50.0
<10
<10
<10
M2 contain two main absorp-
tion bands near the visible
region, with lmax ꢀ391–399 and
369–381 nm. Absorbances by
M2-containing ONs are blue-
shifted by ꢀ5 nm relative to
M1-containing ONs, and both
modifications display a 1–5 nm
shift in lmax upon hybridization
to complementary DNA, but
not RNA. The absorption
maxima are slightly redshifted
and the absorption coefficients
3’-CACTBTACG
5’-GTGAM4ATGC
30.0
54.5
47.5
54.5
3’-CACTABACG
ON26 5’-CGLTTTLATLAM5 ATLCAMeCLG
target 3’-GCAAATABATAGTGC
5’-CGLTTTLATLAM5 ATLCAMeCLG
3’-GCAAATATBTAGTGC
5’-CGLTTTLATLAM5 ATLCAMeCLG
3’-GCAAATATABAGTGC
ON32 5’-CGLTTTLATLAM6 ATLCAMeCLG
target 3’-GCAAATABATAGTGC
5’-CGLTTTLATLAM6 ATLCAMeCLG
3’-GCAAATATBTAGTGC
5’-CGLTTTLATLAM6 ATLCAMeCLG
3’-GCAAATATABAGTGC
51.0
54.5
50.0
53.0
52.5
44.0
34.0
43.5
54.0
41.0
41.0
37.0
43.5
47.0
44.5
44.0 42.0
36.0 44.0
45.0 54.0
48.0
48.5
53.0
51.0
51.0
47.0
49.0
55.0 >70
47.0 48.5 >70
45.0 54.0 51.5
52.5
52.5
53.0 >70
[a] For the conditions of the thermal denaturation experiments, see Table 1; the melting temperatures of fully
matched duplexes are shown in bold. The melting temperatures of unmodified duplexes are 28.0 and 26.08C
increase
with
increasing
number of incorporated mono-
mers M1 and M2. There is a
minor difference in the excita-
tion spectra obtained for ONs
for ON16:DNA and ON16:RNA, respectively, and 55.5 and 62.08C for ON26
ACHTUNGTREN(NUNG ON32):DNA and ON26-
ACHTUNGTRENNUNG(ON32):RNA, respectively. [b] DNA targets are shown. RNA targets have the same sequences and contain U
nucleosides instead of T.
0.1–1.0 mm of the single stranded probe or the two comple-
mentary strands. For recording fluorescence spectra, excita-
tion wavelengths of l=375 (M1), 370 (M2), 415 (M3), 395
(M4), 425 (M5), and 420 nm (M6) were used. Fluorescence
emission quantum yields of PEPyc-modified ONs and du-
plexes were determined relative to reference standards. A
highly diluted solution of 9,10-diphenylanthracene (FF =
0.95)[20] in cyclohexane was the first reference standard. The
fluorescence quantum yields of 5-(pyren-1-ylethynyl)-2’-de-
oxyuridine[21] in ethanol and perylene (FF =0.93)[22] in cyclo-
hexane were measured to be FF =0.45 and 0.93, respective-
ly, relative to the first standard. The optical densities of the
solutions used for quantum yield measurements were kept
between 0.1–0.01 to avoid uncertainties. The FF values were
corrected with the refractive index of the solvents. The
quantum yields of several samples were also measured in
degassed buffer solutions, but no significant difference was
found when compared with nondegassed solutions. Thus, all
the FF values presented were determined for air-saturated
solutions.
and duplexes that contain monomers M1 and M2 compared
to their absorbance spectra (Figure S3, Supporting Informa-
tion).
The fluorescence spectra of ONs and duplexes that con-
tain single insertions of M1 and M2 lie within the l=400–
480 nm region, and none of them exhibit significant vibra-
tional features (Figure S4, Supporting Information). The
Stokesꢀ shift for monoPEPyc monomers is ꢀ25 nm. Singly
labeled ON1 and ON6 reveal a broad unstructured band at
lmax ꢀ430 nm, which slightly shifts (1–4 nm) to shorter wave-
lengths upon hybridization. If the emission spectra of single-
stranded probes are compared with those of the correspond-
ing duplexes, no perceptible changes in the shape of the
spectral curves or the fluorescence intensities are observed.
Doubly modified ON9 displays a broad, unstructured band
of excimer emission at lmax ꢀ490 nm, which does not appear
upon hybridization with DNA/RNA complements (Fig-
ure S4, Supporting Information). However, excimer forma-
tion is not taking place in the case of M1-mofidied ON4. Al-
though excimer emission is not observed, there might be an-
Table 3. Spectroscopic and photophysical properties of modified ONs and duplexes that contain monomers M1–M4.
max
abs
max
fl
l
bands I, II [nm]
DNA
l
[nm]
FF
FB[a]
DNA
SSP[b]
RNA
SSP
DNA
RNA
SSP
DNA
RNA
SSP
RNA
ON1
ON4
ON6
ON9
ON11
ON14
ON16
ON19
398, 378
399, 381
393, 372
397, 375
435, 413
433, 413
414, 395
411, 392
394, 379
397, 376
390, 369
392, 370
435
428
417, 396
410, 392
397, 379
398, 378
393, 373
391, 370
n.d.[c]
432, 419
408, 391
403, 388
423
444
423
417
443
452
425
500
422
425
420
418
443
466
427
422
424
425
419
418
n.d.
453
423
420
0.43
0.09
0.27
0.07
0.65
0.11
0.21
0.07
0.39
0.09
0.45
0.08
0.39
0.04
1.00
0.03
0.32
0.07
0.28
0.08
n.d.
0.03
0.75
0.03
18.7
5.1
14.9
4.6
32.8
6.2
8.8
25.4
5.4
26.6
4.6
27.1
2.9
33.0
2.0
18.0
5.2
22.4
4.6
n.d.
1.8
22.6
1.8
5.9
[a] FB=fluorescence brightness FB=e425 ꢃFF. [b] SSP=Single-strand probe. [c] n.d.=no duplex formed above 108C; see Table 1.[3]
Chem. Eur. J. 2008, 14, 11010 – 11026
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11015

V. A. Korshun, J. Wengel and I. V. Astakhova
other mechanism of fluorescence quenching that results in
the reduced quantum yields of ON4 and its duplexes (e.g.,
interaction with nucleobases). It is also worth noting that
the fluorescence spectrum of single-stranded ON4 is entirely
redshifted by 19 nm with regard to ON4:DNA/RNA. At the
same time, the fluorescence quantum yields of ONs and du-
plexes with single incorporations of M1 or M2 are remark-
Figure S5, Supporting Information). However, the emission
spectra of ONs and duplexes with double insertions of M4
are similar to those of ON14 and its duplexes (spectra are
not shown).
As mentioned above, probe ON16 displays remarkable
Watson–Crick selectivity for singly mismatched DNA and
RNA targets. Therefore, there are no duplexes formed
above 108C for all the targets except for DNA strands that
contain mismatches at position 6 (Table 2). We recorded the
fluorescence spectra of the duplexes, and found that the
presence of single mismatches opposite monomer M4 does
not significantly affect fluorescence intensity relative to that
of fully matched duplexes (Figure S5E, Supporting Informa-
tion). Moreover, the fluorescence spectrum of the monomer
is seen to be independent of the nature of the mismatch.
This might be explained by intercalation of the fluoro-
chrome within a double helix in the presence of a single mis-
match. Intercalation might also explain the increased ther-
mal stabilities of the mismatched duplexes compared with
the fully matched one.
ACHTUNGTRENNUNG
ably higher than typical quantum yields[23] of pyrene on
DNA (Table 3; data for ON1, ON6, and the corresponding
duplexes). The high quantum yields measured for PEPyc–
LNAs can be explained by the decreased lifetime of the ex-
cited state accompanied by the direction of the functionali-
ties attached to 2’-amino-LNA by an amide linker to the
minor groove of the nucleic acid helix, thus reducing the
quenching of fluorescence.[3,4,24]
As can be seen from data presented in Table 3, expansion
of the p-system of pyrene by two phenylethynyl groups has
a significant effect on its spectral and photophysical proper-
ties. Thus, there is a complete shift in both the absorption
maxima to the visible region on going from mono- to bisPE-
Pyc modifications. The absorption bands of ONs and du-
plexes with monomer M3 are observed at lmax ꢀ433–435 and
413–419 nm, and at ꢀ20 nm shorter wavelengths for M4-
modified analogues. Absorbance spectra by the M3 conju-
gates are faintly affected by hybridization, whereas the
maxima of the M4 ones are 5–6 nm blueshifted upon hybrid-
ization with RNA. As expected, the emission shifts to
longer wavelengths on increasing the number of attached
phenylethynyl groups, whereas the Stokesꢀ shifts are de-
creased from l=25 nm for monoPEPyc to l=8 nm for bis-
PEPyc modifications. Similarly to M1- and M2-modified ONs
and duplexes, there is a minor difference between the exci-
tation spectra obtained for M3- and M4-modified ONs and
duplexes compared with their absorbance spectra (Fig-
ure S3, Supporting Information).
Singly modified ON11 and ON11:DNA conjugates display
essential superiority in their fluorescence quantum yields
and fluorescence brightness compared with monoPEPyc
monomers. However, in the same manner as described
above, insertion of a second M3 monomer leads to quenched
structureless fluorescence and decreased fluorescence quan-
tum yields of single-stranded probes and duplexes (Table 3;
data for ON14, ON19, and their duplexes with complemen-
tary DNA and RNA). Interestingly, this correlates with the
higher thermal stabilities of the doubly modified duplexes
relative to the singly modified ones (Table 1). Additionally,
the fluorescence spectra of M3- and M4-containing conju-
gates have a shoulder (lmax ꢀ468 and 450 nm, respectively)
that is less clear for isomeric monomer M3.
As described above, single incorporations of M5 and M6
into 9-mer ONs dramatically reduces their binding affinity,
whereas upon double insertion these monomers result in in-
creased Tm values for the duplexes (Table 1). Singly labeled
ON21–23 do not bind to the DNA and RNA complements
above 108C and, therefore, can be studied by spectroscopy
only as single strands. In turn, M6-modified ON29 and
ON31 form duplexes with complementary DNA at room
temperature and, hence, are characterized by UV/Vis ab-
sorption and fluorescence spectroscopy.
The spectral and photophysical properties of the conju-
gates that contain modifications M5 and M6 are collected in
Table 4. Representative absorption spectra, steady-state
emission spectra and photographs of fluorescence of 9-mer
probes singly modified with monomers M1–M6 are shown in
Figure 1. Absorption bands of ONs that contain trisPEPyc
monomers are observed at lmax ꢀ449–461 and 421–435 nm.
Modifications M4–M6 exhibit an additional absorption band
at lmax ꢀ310–340 nm (Figure 1). The spectral broadening at
longer wavelengths exhibited by M5 and M6 indicates the in-
crements of different vibrational levels of the p-expanded
system.[9a] As in the case of M1–M4, we observed a slight dif-
ference in the excitation spectra for the trisPEPyc mono-
mers compared with their absorbance spectra (Figure S3,
Supporting Information).
Fluorescence emission by modifications M5 and M6 ob-
served within the l=440–540 nm region (with lmax
ꢀ465 nm) revealed the bathochromic shift to be around
100 nm compared to the emission by the parent pyrene.[9a]
Such a significant redshift of fluorescence results in bright
green and blue emissions by M5 and M6, respectively
(Figure 1). Insertion of three phenylethynyl groups into the
pyrene core also results in an additional emission band at
lmax ꢀ490 nm and an even shorter Stokesꢀ shift than ob-
served for bisPEPyc modifications (lꢀ5 nm).
If incorporated once in ONs and duplexes, monomer M4
displays the same emission wavelengths as monoPEPyc fluo-
rochrome M1 (lmax ꢀ420 nm). The fluorescence intensity of
ON16 at l=415 nm is found to be about 3 times and 3.6
times increased upon hybridization with RNA and DNA
complements, respectively, and the fluorescence quantum
yields of the resulting ON16:DNA and ON16:RNA duplex-
es are extraordinarily high (FF =1.00 and 0.75, respectively;
Single incorporations of monomers M5 and M6 into 9-mer
ONs leads to brightly fluorescent probes. In good agreement
11016
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Functionalized Locked Nucleic Acids
FULL PAPER
Table 4. Spectroscopic and photophysical properties of modified ONs and duplexes that contain monomers M5 and M6.
max
fl
lmabasx, bands I, II [nm]
l
[nm]
FF
FB[a]
SSP
DNA
RNA
SSP
DNA
RNA
SSP
DNA
RNA
SSP
DNA
RNA
ON21
ON24
ON26
ON27
ON28
ON29
ON32
ON34
461, 434
460, 432
456, 428
460, 434
462, 435
455, 428
456, 432
449, 425
n.d.[b]
433
454, 430
454
462, 435
454, 430
435
n.d.
432
448, 426
515
461, 434
n.d.
466
520
468
520
520
492, 461
461
n.d.
520
462
515
520
489, 460
520
n.d.
520
462
0.42
0.02
0.50
0.09
0.20
0.18
0.03
0.14
n.d.
0.04
0.62
0.13
0.20
0.37
0.06
0.59
n.d.
0.04
0.62
0.14
0.27
n.d.
0.04
0.65
16.7
1.6
21.2
10.8
20.5
9.1
n.d.
3.2
n.d.
3.2
37.6
16.6
22.6
17.7
2.8
41.3
17.8
33.9
n.d.
1.9
510
520, 66
n.d.
520
434
441, 421
1.5
12.2
443, 420
460
455
456
31.9
36.9
[3]
[a] FB=fluorescence brightness FB=e425 ꢃFF. [b] n.d.=no duplex formed above 108C; see Table 1.
formation (Figure S6, Supporting Information). Taking into
account the large destabilization effect of M5 and M6, we de-
signed 15-mer probes that contained an additional number
of affinity-enhancing LNA monomers (Table 1; data for
ON26–ON28 and ON34). Then we investigated the fluores-
cence properties of trisPEPyc monomer M5 incorporated
into the 15-mer ONs (Table 4).
Singly modified ON26 and its duplexes with complemen-
tary DNA and RNA exhibit high fluorescence quantum
yields (FF =0.50–0.65) and brightness values close to those
of the analogues duplexes with four insertions of pyrenecar-
bonyl–LNA.[3] On the contrary, doubly modified ON27
shows quenched fluorescence for both the single-stranded
probe and its duplexes with DNA/RNA, and this was ac-
companied by remarkable excimer emission at lmax ꢀ520 nm
(Figure S6, Supporting Information). Interestingly, fluores-
cence spectra of ON28 and its duplexes show excimer sig-
nals as well, even though there are five base pairs separating
the PEPyc-modified M5 nucleotides. Emission of the trisPE-
Pyc excimer reaches l=590 nm, which results in a bright
yellow fluorescence of ON28 and its duplexes. Probably in-
termolecular stacking between phenyl groups of the PEPyc
residues is a key interaction that determines the structural
preference of the excimer formation even with a larger dis-
tance separating the fluorochromes. At the same time, exci-
mer formation in solution is a diffusion-controlled collision
process.[25] Therefore, the increased molecular radii of the
PEPyc fluorochromes may significantly affect the rate pa-
rameters involved in excimer association and dissociation in
solution.
Figure 1. A) Absorption spectra of ONs modified with monomers M1–M6
recorded in medium salt buffer at 198C with ONs (1.0 mm) and normal-
ized at 260 nm, and B) steady-state emission spectra normalized at the
fluorescence maximum, obtained in medium salt buffer at 198C with ex-
citation wavelengths of l=375 (ON1), 370 (ON6), 415 (ON11), 395
(ON16), 425 (ON21), or 420 nm (ON29) and 0.1 mm of ONs and comple-
mentary strands. For both sets of spectra, c: ON1, c: ON6, c:
ON11, c: ON16, c: ON21, c: ON29. C) Photographs of the fluo-
rescence in medium salt buffer of 2.0 mm ON1 (vial 1), ON6 (vial 2),
ON11 (vial 3), ON16 (vial 4), ON21 (vial 5), and ON29 (vial 6). Photo-
Formation of duplexes with DNA and RNA by the M6-
containing probe ON34 leads to about a 6-fold increase of
the quantum yield (Table 4). Thus, long-wave emission of
trisPEPyc fluorochromes attached to 2’-amino-LNA may
become a useful tool for the detection of nucleic acid hy-
bridization.
Fluorescence of PEPy dyes in duplexes of modified ONs
with mismatched DNA and RNA: Owing to the promising
fluorescence properties of trisPEPyc monomers, we wanted
to apply their long-wave emission for detection of single
mismatched nucleotides in DNA and RNA target strands.
Thus, we investigated duplexes of probes ON26 and ON34
with singly mismatched DNA/RNA complements. The ob-
graphs were taken with a digital camera and a laboratory UV lamp (lex
=
365 nm).
with the properties of mono- and bisPEPyc analogues, inser-
tion of a second fluorochrome into 9-mer probes results in
dramatic quenching of fluorescence and remarkable excimer
Chem. Eur. J. 2008, 14, 11010 – 11026
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11017

V. A. Korshun, J. Wengel and I. V. Astakhova
served fluorescence intensities and representative fluores-
cence spectra are shown in Figure 2. As can be seen, the
fluorescence intensity of the duplex ON26:DNA is two
times greater with the presence of an A:C mismatch at posi-
tion 8 and two times smaller with a T:G mismatch at posi-
tion 9. On the contrary, the fluorescence intensity of probe
ON34 is slightly affected by the presence of mismatched nu-
cleotides in the DNA target. As regards duplexes with
RNA, ON26 is seen to be sensitive to A:A, A:C, and T:G
mismatches, whereas ON34 displays the strongest fluores-
CD spectra obtained for the duplexes of ON4 with both
DNA and RNA have an additional negative band at l
ꢀ300 nm, which is not observed for either unmodified or
singly modified analogues. This suggests that upon double
insertion of monomer M1, some preorganized duplex struc-
ture might be formed, which also correlates with increased
thermal denaturation temperatures and quenched fluores-
cence of ON4:DNA/RNA compared with singly modified
ON1:DNA/RNA (Table 1, Table 3). CD spectra of 15-mer
duplexes ON26–ON27:DNA/RNA indicate similar confor-
mation to the parent LNA-modified duplexes ON37:DNA
and ON37:RNA (Figure S9, Supporting Information). Hy-
bridization of ON26 and ON27 with complementary DNA
results in intermediate A/B-type geometry of the duplexes,
whereas ON26:RNA and ON27:RNA exhibit remarkable
conformational changes towards the A-form. It is worth
noting that the CD spectra are not significantly affected by
the introduction of an additional monomer M5 (ON27),
which suggests successful accommodation of both trisPE-
Pyc-modified monomers in the minor groove of the 15-mer
duplexes.
cence in response to
a T:G mismatch at position 8
(Figure 2). Thus, the fluorescence of M5- and M6-labeled
probes is sensitive to the nature of the neighboring nucleo-
tides and can be used to discriminate matched from mis-
matched base pairs.
CD studies with duplexes that contain modifications in one
strand: CD is a convenient method for determination of nu-
cleic acid conformations in solution. Therefore, we em-
ployed CD spectroscopy for the structural characterization
of modified duplexes. Duplexes ON1:DNA and ON6:DNA
exhibit CD spectra of normal B-type duplexes, which seems
to change towards an intermediate A/B form upon hybridi-
zation of ON1 and ON6 with the RNA complement (Fig-
ure S9, Supporting Information).
Thermal denaturation studies on zipper duplexes: Thermal
denaturation temperatures of duplexes with two strands
each modified by a PEPyc monomer M1–M6 were deter-
Figure 2. Fluorescence intensities of duplexes between complementary or singly mismatched DNA/RNA targets and A) ON26 or B) ON34. Representa-
tive steady-state fluorescence emission spectra of C) ON26 and D) ON34 and their duplexes with complementary and singly mismatched DNA. The con-
ditions were as described in Figure 1B. The green and black loops on the zipper schematic indicate M5–M6 and LNA, respectively.
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Functionalized Locked Nucleic Acids
FULL PAPER
mined as described above. The measured Tm values were
compared with those of the corresponding unmodified
dsDNA (dsDNA=double-stranded DNA) and are collected
in Table 5. In general, the positioning of two modified mon-
and ꢁ1 zippers are listed in Table 6. Fluorescence spectra of
the various multilabeled duplexes are shown in Figure 3. As
one can see, the fluorescence quantum yield of the M1-con-
taining ꢁ1 zipper is of the same magnitude as of the parent
ON1:DNA (Table 3). All duplex-
es with monomer M1 in both com-
Table 5. Thermal denaturation temperatures for modified duplexes with monomers M1–M6 in both comple-
plementary strands displayed an
unstructured monomer emission
band at lmax ꢀ420 nm, whereas
doubly modified ON4 exhibits an
excimer peak at lmax ꢀ500 nm,
which is not detected for ꢁ1 and
2+1 zippers (Figure 3A).
Previously, it was reported that
the 2+2 zipper arrangement of
the pyrene–LNA is the most pref-
erable for the excimer formation
compared with the other zip-
pers.[6] Indeed, the 2+2 zipper
duplex that contains monomer M1
displayed remarkable excimer for-
mation that correlates with the
high thermal denaturation tem-
mentary strands.[a]
Tm (DTm/mod) [8C]
5’-d(GTGAMATGC)
3’-d(GCATAMCAC)
5’-d(GTGAMATGC)
3’-d(GCAMATCAC)
5’-d(GTGAMATGC)
3’-d(GCAMAMCAC)
5’-d(GTGAMAMGC)
3’-d(GCAMAMCAC)
+1 zipper
ꢁ1 zipper
1+2 zipper
2+2 zipper
M1 <10 (>ꢁ9)
M2 39.5 (+5.8)
M3 30.5 (+1.3)
M4 31.0 (+1.5)
M5 34.3 (+3.2)
M6 36.5 (+4.3)
31.9 (+2.0)
60.0 (+16.0)
33.5 (+2.8)
34.0 (+3.0)
50.5 (+11.3)
40.0 (+6.0)
36.7 (+2.9)
62.5 (+11.5)
39.0 (+3.7)
41.5 (+4.5)
n.t.[b]
48.0 (+5.0)
85.0 (+14.3)
41.0 (+3.3)
43.0 (+3.8)
n.t.
40.0 (+4.0)
>70 (>+11)
[a] Thermal denaturation temperatures were recorded under the same conditions as described in Table 1. The
black loops represent monomers M1–M6. [b] n.t.=no clear transition.
omers in various zipper constitutions increase the thermal
stabilities compared with duplexes with modifications in one
strand only (data presented in Table 5 compared with data
in Table 1). For all monomers M1–M6, the Tm values increase
in the order +1<ꢁ1<1+2<2+2 zipper duplexes, which
suggests an effective stabilizing interstrand communication
between PEPyc functionalities in the created motifs.
In addition, we studied thermal stability of +1 and ꢁ1
zipper duplexes that contained all possible combinations of
monomers M1–M6 in both complementary strands
(Table S2). As for the above-mentioned zipper constructs,
the ꢁ1 constitution proved to be more stable than the +1
constitution, and for both of them the thermal stability in-
creases as the number of attached phenylethynyl groups in-
creases. Similarly, interstrand communication between M5-
residues in 15-mer zippers (together with incorporation of
additional LNA monomers) significantly compensate for the
destabilizing effect of the modification (data in Table 6 com-
pared with Table 1; e.g., 44.0 and 668C for ON26:DNA and
ON26:ON41, respectively).
perature of ON4:ON5. Incorporation of monomer M2 into
the ꢁ1 zipper motif resulted in the same quantum yield as
for the singly labeled ON4:DNA. However, for the +1
zipper analogue the quantum yield was two times lower. It
is also worth noting that M2-modified duplexes displayed
more effective excimer formation than the analogous M1-
modified duplexes (Figure 3B). Fluorescence quantum
yields of both M3-containing +1 and ꢁ1 zippers were of the
same magnitude as for ON11:DNA, and both these com-
plexes exhibited intensive monomer emission (Figure 3C).
Next, insertion of the third monomer, M3, induced a weak
excimer peak, whereas quadruply modified ON14:ON15
showed dramatical quenching of the fluorescence together
with ꢀ1:1 excimer-to-monomer fluorescence intensity ratio
(Figure 3C). Double incorporation of M4-monomers into +/
ꢁ1 zippers resulted in reduced fluorescence quantum yields
for both ON16:ON17 and ON16:ON18 compared with an
extraordinarily high quantum yield for ON16:DNA (FF =
1.0). The latter was accompanied by a remarkable excimer
Table 6. Spectroscopic and photophysical properties of duplexes that contain monomers M1–M6 in +1 zipper
and ꢁ1 zipper constitutions.[a]
Spectral properties of zipper
duplexes: The UV/Vis absorp-
tion and steady-state fluores-
cence emission spectra of the
various interstrand zipper ar-
rangements of monomers M1–
M6 were obtained by using the
same conditions as described
for the ONs and duplexes that
contained modifications in one
strand. The spectroscopic and
photophysical data evaluated
for monomers M1–M6 in +1
max
abs
max
fl
max
fl
l
bands I, II [nm]
l
[nm] FF
FB[b]
lmabasx bands I, II [nm]
l
[nm]
FF
FB[b]
M1 n.d.[c]
n.d.
425
444
520, 424
468
n.d. n.d.
0.27 22.1
0.45 58.3
0.37 24.5
0.23 39.1
0.16 16.1
395, 378
393, 372
433
421, 402
438
424
427
443
518
468
0.32
0.41
0.51
0.35
0.23
0.18
30.5
38.5
60.0
25.0
39.2
18.0
M2 391, 369
M3 434
M4 415, 396
M5 457, 435
M6 455, 430
490, 461
450, 432
491, 460
[a] Thermal denaturation temperatures were recorded under the same conditions as described in Table 1. The
black loops represent monomers M1–M6. [b] FB=fluorescence brightness FB=e425 ꢃFF. [c] n.d.=no duplex
formed above 108C.[3]
Chem. Eur. J. 2008, 14, 11010 – 11026
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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11019

V. A. Korshun, J. Wengel and I. V. Astakhova
Figure 3. Steady-state fluorescence emission spectra of multilabeled duplexes that contain monomers M1–M6 in various zipper constitutions. For condi-
tions, see Figure 1B.
signal at lmax ꢀ512 nm, which is much more intense for the
ꢁ1 zipper constitution than for the +1 constitution (Fig-
ure 3D).
yields, especially in the case of monomer M6. As for M1–M4-
modified analogues, the ꢁ1 zippers with trisPEPyc fluoro-
chromes exhibit more intensive excimer signals than their
+1 analogues (Figure 3D,E). Note that increasing the
number of phenylethynyl substitutes attached to the pyrene
core leads to more effective interstrand excimer formation
Furthermore, hybridization of M5- and M6-modified
probes with complementary DNA strands that contained
trisPEPyc monomers dramatically decreased the quantum
11020
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ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 11010 – 11026

Functionalized Locked Nucleic Acids
FULL PAPER
(Figure 3; ON16:ON18, ON32:ON33, compared with
ON1:ON5, ON4:ON5).
15-Mer duplexes containing monomer M5 in both comple-
mentary strands showed increasing thermal stabilities and
more effective excimer formation in the order
ON26:ON40<ON26:ON41<ON27:ON40<ON27:ON41
(Table 5; Figure S7, Supporting Information). Long-wave ex-
cimer emission of M5-modified duplexes exceeds 600 nm,
which leads to vivid yellow fluorescence, whereas less red-
shifted bis- and monoPEPyc excimers result in yellow-green
and blue-green solutions, respectively.
Next, we studied PEPyc interstrand excimer formation for
combined modifications M1–M6 in DNA duplexes of +1 and
ꢁ1 zipper arrangements. The steady-state fluorescence emis-
sion spectra of the duplexes were recorded by using the
same conditions as described above, except for the use of
excitation wavelengths of l=350 (combinations of mono-/
trisPEPyc monomers), 390 (combinations of mono-/bisPE-
Pyc monomers), or 420 nm (combinations of bis-/trisPEPyc
monomers); the representative fluorescence spectra of com-
binarorial zipper duplexes are shown in Figure S8 (Support-
ing Information). Excimer fluorescence was detected as a
broad structureless band at lmax ꢀ500–530 nm, whereas mo-
nomer emission by combined duplexes was observed at lmax
ꢀ440–460 nm. We used the excimer-to-monomer fluores-
cence intensity ratio, Iex/Im, as a criterion of the excimer for-
mation. The resulting Iex/Im values are presented in Figure 4.
As can be seen, the intensity of excimer fluorescence is
always higher for combinations of monomers with increased
numbers of phenylethynyl groups. The configuration of the
ꢁ1 zipper is more preferable for excimer formation than
that of the +1 zipper, which is in good agreement with the
increased thermal denaturation values of ꢁ1 zippers relative
to +1 zippers (Table S2, Supporting Information). Weak ex-
cimer signals were detected for two monoPEPyc monomers
M1 and M2 in dsDNA, and higher signals for various combi-
nations of M4, M5, and M6. This might be caused by inter-
strand stacking of phenyl groups that could promote the
spatial preorganization of two pyrene residues, and also by
the increased molecular radii of bis- and trisPEPyc modifi-
cations.
Figure 4. Fluorescent properties of A) +1 and B) ꢁ1 zipper duplexes
with different combinations of monomers M1–M6 in their complementary
strands. The spectra were recorded in medium salt buffer at 198C with
0.1 mm complementary strands and at the excitation wavelengths men-
tioned in the text.
due to significant improvement in the spectral and photo-
physical characteristics of pyrene. As the number of phenyl-
ethynyl substitutuents increases, the absorption/emission
maxima are drastically redshifted. This results in the change
in the fluorescence colors from purple for monoPEPyc to
vivid green and blue for trisPEPyc fluorochromes. At the
same time, fluorescence quantum yields and the ability to
form excimers were enchanced. Duplexes with a single in-
corporation of M1–M6 display lower thermal stabilities and
higher fluorescence quantum yields than their doubly modi-
fied analogues. On this basis, we propose an effective elec-
tronic interaction between PEPy residues in the multila-
beled constructs, which was confirmed by the detection of
excimer emission. The fluorescence of a single-stranded
probe that contains a single insertion of monomer M4 is
quenched, whereas upon hybridization with complementary
DNA and RNA, the fluorescence quantum yields increase
up to FF =1.00 and 0.75, and this accompanied by excellent
Conclusion
Development of highly emissive fluorochromes for nucleic
acid labeling is of considerable interest due to the possibility
of their use in both in vitro and in vivo fluorescence assays.
Herein, the synthesis of six long-wave emission 2’-(phenyl-
ACHTUNGTRENNUNG
ethynyl)pyrenecarbonyl–2’-amino-LNA monomers, M1–M6,
their incorporation into ONs, their thermal denaturation
studies, and their spectral and photophysical properties are
described. For the preparation of monomers M1–M6, meth-
ods for the selective electrophilic mono, di, and tri substitu-
tions of 1- and 2-acetylpyrene were developed by employing
transition-metal-catalyzed reactions.
Probes containing phenylethynyl-substituted pyrene
monoACHTUNGTRENNUNG
mers M1–M6 were demonstrated to be very promising
Chem. Eur. J. 2008, 14, 11010 – 11026
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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11021

V. A. Korshun, J. Wengel and I. V. Astakhova
tained from commercial suppliers and used without further purification,
and all the modified ONs were purified by using DMT-ON RP-HPLC
with the Waters Prep LC 4000 equipped with Xterra MS C18-column
(10 mm, 300ꢃ7.8 mm). Elution was performed with an initial isocratic
hold of A-buffer (95% NH4HCO3 (0.1m), 5% CH3CN) for 5 min, fol-
lowed by a linear gradient to 55% B-buffer (25% NH4HCO3 (0.1m),
75% CH3CN) over 75 min at a flow rate of 1.0 mLminꢁ1. RP-purification
was followed by detritylation (80% aq. AcOH, 20 min), precipitation
(abs. EtOH, ꢁ188C, 12 h), and washing three times with abs. EtOH. The
identity of ONs was verified by MALDI-TOF mass spectrometry
(Table S1, Supporting Information). UV/Vis absorption spectra and ther-
Watson–Crick mismatch discrimination. Selectivity against
singly mismatched DNA/RNA targets is likewise considera-
ble for longer 15-mer ONs that contain M5 and M6, which in
turn display significant changes in fluorescence intensity in
the presence of mismatched nucleotides compared with fully
matched duplexes. Duplexes with monomers M1–M6 in both
complementary strands revealed remarkable thermal stabili-
ties accompanied by high fluorescence quantum yields and
effective excimer formation.
mal denaturation experiments were performed on
a Perkin–Elmer
The long-wave emission of these novel probes allows exci-
tation at longer wavelengths without irradiation of intrinsic
cell fluorochromes. Taking this advantage into account to-
gether with an increased ability to form excimers, sensitivity
to the neighboring base pairs, and high fluorescence quan-
tum yields of singly labeled probes, we propose the possibili-
ty of applying PEPyc fluorochromes attached to 2’-amino-
LNA for both in vitro and in vivo nucleic acid fluorescence
assays. Applications of such probes for the in vivo detection
of single-nucleotide polymorphisms, fluorescence in situ hy-
bridization including the detection of specific RNAs in
living cells, and analysis of gene expression will be especially
interesting.
Lambda 35 UV/Vis spectrometer equipped with a Peltier Temperature
Programmer 6 in a medium salt buffer (NaCl (100 mm), Na-phosphate
(10 mm), EDTA (0.1 mm), pH 7.0). The concentrations of ONs were cal-
culated by using the following extinction coefficients (OD260/mmol): G
10.5, A 13.9, T/U 7.9, C 6.6, M1 33.5, M2 34.2, M3 31.7, M4 30.2, M5 35.1,
M6 34.9. ONs (1.0 mm per strand) were thoroughly mixed, denaturated by
heating, and subsequently cooled to the starting temperature of experi-
ment. Thermal denaturation temperatures (Tm, 8C) were determined to
be the maximum of the first derivative of the thermal denaturation curve
(A260 vs. temperature). Reported Tm values are an average of two meas-
urements within ꢂ1.08C. Fluorescence spectra were obtained in
a
medium salt buffer by using a Perkin–Elmer LS 55 luminescence spec-
trometer equipped with a Peltier temperature controller. For recording
fluorescence spectra, 0.1 mm concentrations of the single-stranded probe
or corresponding duplex were used. For weakly fluorescent samples the
concentration was increased to 0.5 mm. The fluorescence quantum yields
were measured by the relative method using 9,10-diphenylantracene
(FF =0.95)[20] in cyclohexane as the first standard and 5-(pyrene-1-yle-
thynyl)-2’-deoxyuridine[21] in abs. EtOH and perylene (FF =0.93)[22] in cy-
clohexane as the second standard. For fluorescence quantum yield deter-
minations, 0.5mm solutions were used. CD spectra were recorded by using
a JASCO J-815 CD spectrometer equipped with a CDF 4265/15 tempera-
ture controller.
Experimental Section
General: Reagents were obtained from commercial suppliers (Sigma–Al-
drich–Fluka) and were used as received; 2-acetylpyrene,[26] 5-(pyren-1-
ylethynyl)-2’-deoxyuridine,[21] and diisopropylammonium tetrazolide[27]
were synthesized as described. Perylene, 5-(pyren-1-ylethynyl)-2’-deoxy-
uridine and 9,10-diphenylantracene were used as standards for emission
quantum yield measurements after recrystallization. HPLC-grade toluene
and acetone were distilled and stored over activated 4 ꢁ molecular
sieves. CH2Cl2 was always freshly distilled over CaH2 before being used.
Other solvents were used as received. Photochemical studies were per-
formed with spectroscopy-grade cyclohexane and absolute (abs.) ethanol.
NMR spectra were recorded at 303 K by using Varian Gemini 2000
300 MHz and Bruker DRX 500 MHz instruments. Chemical shifts are re-
ported in ppm relative to solvent peaks (CDCl3: 7.26 ppm for 1H and
77.0 ppm for 13C; [D6]DMSO: 2.50 ppm for 1H and 39.5 ppm for 13C;
General procedure for the monobromination of acetylpyrenes: A solu-
tion of bromine (769 mL, 15 mmol) in CCl4 (25 mL) was added dropwise
over 30 min to a stirred solution of the corresponding acetylpyrene (1 or
5; 2.44 g, 10 mmol) in CCl4 (50 mL) at RT. After the addition was com-
plete, the mixture was heated to 608C, kept at this temperature for 1 h,
and then allowed to cool to RT to yield a precipitate that was filtered,
washed with ethanol (2ꢃ30 mL), and dried in vacuo.
1-Acetyl-3-bromopyrene (2): The monobromination of 1 resulted in a
mixture of isomeric 3-, 6-, and 8-bromopyrenes (yellow solid; 3.05 g,
95%), which was used directly in the next step without further purifica-
tion. IR (KBr; signals for intense bands are given): n˜ =3436, 1672, 1587,
1503, 1349, 1235, 846, 713, 674, 632 cmꢁ1; ESI-MS (70 eV): m/z (%):
calcd for C18H10BrO+: 321, 322, 323, 324 [M+ꢁH]; found: 321 (100%),
323 (98%), 322 (60%), 324 (22%).
1
85% aq. H3PO4 (external standard): 0.00 ppm for 31P). H NMR spectros-
copy coupling constants are reported in Hz and refer to apparent multi-
plicities. ESI mass spectra were obtained by using a Finnigan SSQ 710
mass spectrometer. High-resolution mass spectra were recorded in posi-
tive-ion mode by using a IonSpec Fourier Transform ICR mass spectrom-
eter (MALDI) or PESCIEX QSTAR pulsar mass spectrometer (ESI). IR
spectra were recorded by using a Perkin–Elmer 1720 infrared fourier
transform spectrometer. Melting points were determined by using a Boe-
tius heating table and are uncorrected. Analytical thin-layer chromatog-
raphy was performed on Kieselgel 60 F254 precoated aluminium plates
(Merck). Silica-gel column chromatography was performed by using
Merck Kieselgel 60 0.040–0.063 mm. ON synthesis was carried out by
using a PerSpective Biosystems Expedite 8909 instrument on a 200 nmol
scale with the manufacturerꢀs standard protocols. In the case of LNA
phosphoramidites (monomers TL, GL, MeCL), the coupling-step time was
extended to 15 min. Additionally, in the case of monomers M3–M6, a
double-coupling procedure was applied. Step-wise coupling yields of 85–
95% for monomers M1–M6 were obtained based on the absorbance of
the dimethoxytrityl cation released after each coupling. The coupling effi-
ciencies of standard DNA and LNA amidites varied between 98 and
100%. Cleavage from solid support and removal of nucleobase protect-
ing groups was performed by using standard conditions (32% aqueous
ammonia for 12 h at 558C). Unmodified DNA/RNA strands were ob-
2-Acetyl-6-bromopyrene (6): The title compound was obtained as
a
yellow solid (2.88 g, 90%). Rf =0.46 (40% petroleum ether/toluene);
m.p. 155–1568C (recrystallized from PhNO2, washed with 96% aq.
EtOH); IR (KBr; signals for intense bands are given): n˜ =3434, 1695,
1590, 1454, 1293, 1169, 1014, 842, 820, 704, 658, 600 cmꢁ1; ESI-MS
(70 eV): m/z (%): calcd for C18H10BrO+: 321, 322, 323, 324 [M+ꢁH];
found: 321 (100), 323 (100), 322 (67), 324 (33).
General procedure for the dibromination of acetylpyrenes: A solution of
bromine (1.28 mL, 25 mmol) in CCl4 (50 mL) was added dropwise over
30 min to a vigorously stirred solution of the corresponding acetylpyrene
(2.44 g, 10 mmol) in CCl4 (50 mL) at RT. The reaction mixture was
heated to 608C, kept at this temperature for 6 h, and then cooled to RT
to yield a precipitate that was filtered, washed with ethanol (2ꢃ30 mL)
and dried in vacuo.
1-Acetyl-3,6-dibromopyrene (3): The dibromination of 1 resulted in a
mixture of isomeric 3,6-, 3,8- and 6,8-dibromoproducts (3.29 g, red solid),
which was used directly in the next step without further purification. IR
(KBr; signals for intense bands are given): n˜ =3435, 1682, 1581, 1514,
1365, 1242, 844, 755, 701, 654, 611 cmꢁ1; ESI-MS (70 eV): m/z (%): calcd
11022
www.chemeurj.org
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 11010 – 11026

Functionalized Locked Nucleic Acids
FULL PAPER
for C17H7Br2O+: 387, 385, 389, 388 [M+ꢁCH3]; found: 387 (100%), 389
1-Acetyl-3,6-bis(phenylethynyl)pyrene (11): Prepared from
3 (1.21 g,
3 mmol), phenylacetylene (988 mL, 9 mmol), [PdCl2A(PPh3)2] (421 mg,
CTHUNGTRENNUNG
(48%), 385 (43%), 388 (26%).
0.6 mmol), PPh3 (315 mg, 1.2 mmol), and CuI (114 mg, 0.6 mmol). The
product was purified by column chromatography on silica gel by using a
gradient elution of 20!80% CHCl3/petroleum ether. The combined frac-
tions containing the product were evaporated and purified by column
chromatography on silica gel by using a gradient elution of 40!50% tol-
uene/petroleum ether to give the product as an orange solid (858 mg,
53% for two steps). Rf =0.63 (5% EtOAc/toluene); m.p. 195–1998C
(CHCl3); 1H NMR (300 MHz, CDCl3): d=9.02 (d, J=9.6 Hz, 1H), 8.69
(d, J=9.6 Hz, 1H), 8.55 (d, J=9.0 Hz, 1H), 8.50 (s, 1H), 8.17–8.09 (m,
3H), 7.68–7.65 (m, 4H), 7.40–7.37 (m, 6H), 2.87 ppm (s, 3H); 13C NMR
(300 MHz, CDCl3): d=201.6, 134.6, 134.3, 133.9, 132.2, 132.1, 132.0,
131.6, 131.5, 131.3, 131.2, 130.7, 130.6, 130.5, 129.5, 129.1, 129.0, 128.9,
128.8, 128.6, 126.7, 126.2, 126.0, 124.9, 124.2, 123.6, 123.4, 120.0, 117.9,
96.7, 96.2, 88.6, 88.2, 30.8 ppm; MALDI-HRMS: m/z calcd for C34H20O+:
444.1509 [M+]; found: 444.1528.
2-Acetyl-6,8-dibromopyrene (7): The title compound was obtained as a
yellow solid (3.61 g, 90%). Rf =0.56 (40% petroleum ether/toluene);
m.p. 2188C (10% EtOH (96% aq.)/CCl4); IR (KBr; signals for intense
bands are given) n˜ =3435, 1698, 1589, 1458, 1291, 1172, 1024, 821, 702,
661 cmꢁ1; ESI-MS (70 eV): m/z (%): calcd for C18H10Br2O+: 402, 400,
404, 403 [M+]; found: 400 (100%), 403 (68%), 402 (57%), 404 (37%).
General procedure for the tribromination of acetylpyrenes: Bromine
(2.05 mL, 40 mmol) was added dropwise under vigorous stirring to a solu-
tion of corresponding acetylpyrene (2.44 g, 10 mmol) in nitrobenzene
(20 mL) at 1208C over a period of 15 min. The mixture was kept at
1208C for 4 h, then allowed to cool to RT yielding a precipitate, which
was filtered, washed with ethanol (2ꢃ30 mL) and dried in vacuo.
1-Acetyl-3,6,8-tribromopyrene (4): The title compound was obtained as a
yellow solid (4.43 g, 92%). Rf =0.60 (50% petroleum ether/toluene);
m.p. 188–1908C (recrystallized from PhNO2, washed with 96% aq.
EtOH); IR (KBr; signals for intense bands are given): n˜ =3435, 1695,
1596, 1524, 1348, 1227, 1021, 976, 876, 814, 706, 659, 621 cmꢁ1; ESI-MS
(70 eV): m/z (%): 478 (60), 480 (100), 482 (44), 484 (40) [M+].
2-Acetyl-6,8-bis(phenylethynyl)pyrene (12): Prepared from
7 (1.21 g,
3 mmol), phenylacetylene (988 mL, 9 mmol), [PdCl2A(PPh3)2] (421 mg,
CTHUNGTRENNUNG
0.6 mmol), PPh3 (315 mg, 1.2 mmol), and CuI (114 mg, 0.6 mmol). The
product was purified by column chromatography on silica gel using a gra-
dient elution of 30!50% CHCl3/petroleum ether. The combined frac-
tions containing the product were evaporated and purified by column
chromatography on silica gel using a gradient elution of 70!90% tolu-
ene/petroleum ether to give the product as a yellow solid (587 mg, 44%).
Rf =0.39 (5% EtOAc/toluene); m.p. 2218C (50% CHCl3/96% aq.
2-Acetyl-1,6,8-tribromopyrene (8): The title compound was obtained as a
yellow solid (3.79 g, 79%). Rf =0.52 (50% petroleum ether/toluene);
m.p. 215–2168C (recrystallized from o-dichlorobenzene, washed with
96% aq. EtOH); IR (KBr; signals for intense bands are given) n˜ =3436,
1722, 1589, 1455, 1283, 1055, 994, 878, 812, 760, 673, 608 cmꢁ1; ESI-MS
(70 eV): m/z (%): 477 (60%), 479 (97%), 481 (100%), 483 (46%) [M+
ꢁH].
1
EtOH); H NMR (300 MHz, CDCl3): d=8.55 (s, 2H), 8.38 (d, J=9.0 Hz,
2H), 8.24 (s, 1H), 7.99 (d, J=9.0 Hz, 2H), 7.72–7.64 (m, 4H), 7.52–7.38
(m, 6H), 2.79 ppm (s, 3H); 13C NMR (300 MHz, CDCl3): d=198.6, 134.8,
134.2 (2C), 132.4, 132.3 (4C), 131.3 (2C), 129.4 (2C), 129.0 (2C), 128.9
(4C), 126.4 (2C), 126.3, 125.6 (2C), 124.2, 123.5 (2C), 118.6 (2C), 96.1
(2C), 87.8 (2C), 27.2 ppm; MALDI-HRMS: m/z calcd for C34H20O+:
444.1509 [M+]; found: 444.1505.
General procedure for the Sonogashira coupling of bromopyrenes: Phe-
nylacetylene, [PdCl2ACHTUNGTRENNUNG(PPh3)2], PPh3, and CuI were successively added to a
degassed solution of the corresponding starting compound in NEt3
(20 mL) and THF (20 mL). The reaction mixture was stirred under argon
at 708C for 16 (9, 10), 24 (11, 12), or 48 h (13, 14). After conversion of
the starting material was complete (as monitored by TLC), the mixture
was poured into CHCl3 (150 mL). The resulting solution was washed with
1-Acetyl-3,6,8-tris(phenylethynyl)pyrene (13): Prepared from 4 (1.44 g,
3 mmol), phenylacetylene (1.49 mL, 13.5 mmol), [PdCl2ACHTNUGTRENUNG(PPh3)2] (632 mg,
0.9 mmol), PPh3 (472 mg, 1.8 mmol), and CuI (171 mg, 0.9 mmol). The
product was purified by column chromatography on silica gel using a gra-
dient elution of 50!80% toluene/petroleum ether to give the product as
a red solid (1.37 g, 84%). Rf =0.63 (5% EtOAc/toluene); m.p. 2348C
(30% CHCl3/ethanol); 1H NMR (300 MHz, CDCl3): d=8.88 (d, J=
9.3 Hz, 1H), 8.47–8.34 (m, 4H), 8.18 (s, 1H), 7.67–7.60 (m, 6H), 7.35–
7.34 (m, 9H), 2.80 ppm (s, 3H); 13C NMR (300 MHz, CDCl3): d=201.0,
133.9, 133.5, 132.1, 132.0, 131.8 (5C), 131.6 (2C), 131.3, 131.0, 130.7,
129.1, 128.6 (3C), 128.5 (5C), 127.8 (2C), 126.1, 126.0, 124.0, 123.4, 123.1,
122.7, 119.4, 119.2, 117.8, 96.4, 96.3, 96.2, 87.8, 87.6, 87.5, 29.7 ppm;
MALDI-HRMS: m/z calcd for C42H24O+: 544.1822 [M+]; found:
544.1806.
3% Na2ACHTUNGTRENNUNG(edta) (4ꢃ100 mL) and water (2ꢃ100 mL), dried over Na2SO4,
and evaporated to dryness. The crude products were purified by column
chromatography on silica gel as indicated in the individual cases.
1-Acetyl-3-(phenylethynyl)pyrene (9): Prepared from 2 (1.29 g, 4 mmol),
phenylacetylene (658 mL, 6 mmol), [PdCl2ACHTNUTRGNE(UNG PPh3)2] (280 mg, 0.4 mmol),
PPh3 (210 mg, 0.8 mmol), and CuI (76 mg, 0.4 mmol). The product was
purified by column chromatography on silica gel by eluting with 50%
CHCl3/petroleum ether. Combined fractions containing the product were
evaporated and purified by column chromatography on silica gel by using
a gradient elution of 50!70% petroleum ether/toluene to give the prod-
uct as a yellow solid (426 mg, 29% for two steps). Rf =0.55 (5% EtOAc/
toluene); m.p. 1608C (CHCl3); 1H NMR (500 MHz, CDCl3): d=8.98 (d,
J=7.6 Hz, 1H), 8.57 (d, J=7.6 Hz, 1H), 8.52 (s, 1H), 8.25–8.18 (m, 2H),
8.17–8.15 (m, 2H), 8.02 (app. t, J=6.2 Hz, 1H), 7.74 (d, J=6.4 Hz, 2H),
7.49–7.41 (m, 3H), 2.90 ppm (s, 3H); 13C NMR (500 MHz, CDCl3): d=
201.4, 134.2, 131.7 (2C), 131.5, 130.9, 130.8, 130.6, 130.3, 130.2 (2C),
129.3, 128.7, 128.5, 126.9, 126.6, 126.5, 125.2, 124.9, 124.8, 123.9, 123.2,
117.0, 95.5, 87.9, 30.4 ppm; MALDI-HRMS: m/z calcd for C26H16O+:
344.1196 [M+]; found 344.1193.
2-Acetyl-1,6,8-tris(phenylethynyl)pyrene (14): Prepared from 8 (1.44 g,
3 mmol), phenylacetylene (1.49 mL, 13.5 mmol), [PdCl2ACHTNUGTRENUNG(PPh3)2] (632 mg,
0.9 mmol), PPh3 (472 mg, 1.8 mmol), and CuI (171 mg, 0.9 mmol). The
product was purified by column chromatography on silica gel using a gra-
dient elution of 40!50% CHCl3/petroleum ether. The combined frac-
tions containing the product were evaporated and purified by repeated
column chromatography on silica gel using a gradient elution of 80!
100% toluene/petroleum ether to give the product as a brown solid
(1.25 g, 77%). Rf =0.50 (5% EtOAc/toluene); m.p. 171–1738C (CHCl3);
1H NMR (300 MHz, CDCl3): d=8.51 (d, J=9.0 Hz, 1H), 8.36 (d, J=
9.0 Hz, 1H), 8.32 (d, J=9.3 Hz, 1H), 8.25 (s, 1H), 8.16 (s, 1H), 7.85 (d,
J=9.3 Hz, 1H), 7.65–7.61 (m, 6H), 7.38–7.34 (m, 9H), 2.92 ppm (s, 3H);
13C NMR (300 MHz, CDCl3): d=201.2, 139.0, 133.8, 132.4, 131.8, 131.7–
131.5 (8C), 131.4, 130.2, 128.9, 128.6, 128.5–128.4 (6C), 126.9, 126.6,
126.5, 125.4, 125.3, 124.6, 123.2, 123.1, 123.0, 118.7, 118.6, 116.1, 101.7,
96.1, 96.0, 87.5, 87.4, 87.3, 30.5 ppm; MALDI-HRMS: m/z calcd for
C42H24O+: 544.1822 [M+]; found: 545.1907).
2-Acetyl-6-(phenylethynyl)pyrene (10): Prepared from 6 (1.29 g, 4 mmol),
phenylacetylene (658 mL, 6 mmol), [PdCl2ACHTNUTRGNE(UNG PPh3)2] (280 mg, 0.4 mmol),
PPh3 (210 mg, 0.8 mmol), and CuI (76 mg, 0.4 mmol). The product was
purified by column chromatography on silica gel by using a gradient elu-
tion of 50!80% toluene/petroleum ether to give the product as a yellow
solid (728 mg, 53%). Rf =0.39 (5% EtOAc/toluene); m.p. 140–1428C
(CHCl3); 1H NMR (300 MHz, CDCl3): d=8.46 (s, 2H), 8.40 (d, J=
9.0 Hz, 1H), 8.04 (d, J=8.1 Hz, 1H), 7.93–7.80 (m, 4H), 7.65 (d, 2H),
7.39–7.36 (m, 3H), 2.77 ppm (s, 3H); 13C NMR (300 MHz, CDCl3): d=
198.3, 134.1, 132.2, 131.7 (2C), 131.5, 130.8, 130.6, 130.4, 128.5 (2C), 128.4
(2C), 128.3, 127.8, 126.2, 126.1, 124.8 (2C), 124.7 (2C), 124.1, 123.8, 95.5,
88.1, 27.0 ppm; MALDI-HRMS: m/z calcd for C26H16O+: 344.1196 [M+];
found: 344.1190.
General procedure for haloform oxidation: Sodium hydroxide (870 mg,
22 mmol) was dissolved in water (12 mL). The solution was cooled to
08C and bromine (308 mL, 6 mmol) was added. After stirring for 10 min
at RT, the resulting yellow solution was diluted with 1,4-dioxane (12 mL),
Chem. Eur. J. 2008, 14, 11010 – 11026
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11023

V. A. Korshun, J. Wengel and I. V. Astakhova
and the corresponding ketone (1 mmol) was added in five portions over
a period of 10 min. The solution was stirred at 08C for 3 h and then left
overnight at RT. Saturated Na2SO3 (2 mL) was added, and the mixture
stirred at RT for 1 h. After acidification with 1m aq. citric acid (to pH 3–
4), the reaction mixture was transferred into a separating funnel and ex-
tracted three times with CHCl3. The combined organic phases were dried
over Na2SO4 and evaporated. Recrystallization from the solvents men-
tioned below in each case gave analytical-quality samples of the com-
pounds, whereas the main portions of the products were used directly in
the next step without further purification.
127.6, 125.7, 124.7, 124.3, 123.2, 117.6, 114.3 (4C), 109.2, 96.5, 88.8, 88.5,
86.7, 86.6, 69.9, 66.0, 60.2, 56.0 (2C), 52.3, 13.3 ppm; MALDI-HRMS:
m/z calcd for C57H45N3O8Na+: 922.3090 [M++Na]; found: 922.3137.
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-(6-phenyl-
ACHUTNGRENUNeG thynylpyren-2-carbonyl)-3-(thymin-1-yl)-2-oxa-5-azabicycloACHUTNGTREN[NUGN 2.2.1]heptane
(22b): Prepared from 16 (135 mg) to give the title compound as a yellow
foam (rotameric mixture ꢀ9:1 by 1H NMR; 284 mg, 90%). Rf =0.44
(1:50% NEt3/acetone/toluene); 1H NMR (300 MHz, [D6]DMSO; the sig-
nals are given for the major rotamer): d=11.34 (brs, 1H), 8.74–8.10 (m,
8H), 7.73–7.15 (m, 15H), 6.90–6.84 (m, 4H), 6.01 (s, 1H), 5.93 (d, J=
3.6 Hz, 1H), 4.46 (s, 1H), 4.14–4.12 (m, 1H), 3.88 (s, 6H), 3.71–3.40 (m,
4H, partial overlap with H2O), 1.39 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO; the signals are given for the major rotamer): d=169.0, 161.2,
158.1 (2C), 150.2, 144.5, 135.2, 134.9, 134.1, 133.5, 132.7, 132.4, 132.1,
131.5 (2C), 130.9, 130.4, 130.1, 130.0, 129.7 (2C), 129.1, 128.9, 128.8 (2C),
128.4, 127.9 (2C), 127.6 (2C), 126.8 (2C), 125.3 (2C), 124.7, 124.4, 123.8,
122.8, 120.4, 120.0, 113.2 (4C), 108.6, 97.7, 89.0, 87.9, 86.7, 85.8, 69.0, 67.5,
66.8, 65.9, 54.9 (2C), 12.2 ppm; MALDI-HRMS: m/z calcd for
C57H45N3O8Na+: 922.3135 [M++Na]; found: 922.309).
3-Phenylethynylpyrene-1-carboxylic acid (15): By using the general proce-
dure, 9 (344 mg, 1 mmol) was converted into the title product (yellow
solid; 342 mg, 95%). Rf =0.60 (5% MeOH/CH2Cl2); m.p. >2508C (1%
DMF/MeNO2); MALDI-HRMS: m/z calcd for C25H14O2+: 346.0989 [M+];
found: 346.0991.
6-Phenylethynylpyrene-2-carboxylic acid (16): Prepared from 10 (344 mg,
1 mmol) to give the title compound as a yellow solid (331 mg, 92%). Rf =
0.48 (5% MeOH/CH2Cl2); m.p. >2508C (1% DMF/MeNO2); MALDI-
HRMS: m/z calcd for C25H14O2+: 346.0989 [M+]; found: 346.0988.
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-[3,6-bis-
AHCTUNGTRENNUNG
3,6-Bis(phenylethynyl)pyrene-1-carboxylic acid (17): Prepared from 11
(444 mg, 1 mmol) to give the title compound as an orange solid (420 mg,
AHCTUNGTRENNUNG
94%). Rf =0.60 (5% MeOH/CH2Cl2); m.p. >2508C (1% DMF/MeNO2);
pound as an orange foam (rotameric mixture ꢀ1:0.2:0.9 by 1H NMR;
262 mg, 75%). Rf =0.49 (1:60% NEt3/acetone/toluene); 1H NMR
(300 MHz, [D6]DMSO; the signals are given for the major rotamer): d=
11.45 (brs, 1H), 8.78–8.68 (m, 1H), 8.48–8.34 (m, 3H), 7.82–7.80 (m,
1H), 7.67–7.49 (m, 17H), 7.39–7.16 (m, 5H), 6.96–6.92 (m, 4H), 6.30 (d,
J=3.9 Hz, 1H), 5.98 (s, 1H), 5.31 (s, 1H), 4.23 (d, J=4.5 Hz, 1H), 3.87
(s, 6H), 3.71–3.60 (m, 2H), 3.41–3.20 (m, 2H, partial overlap with H2O),
1.43 ppm (s, 3H); 13C NMR (75 MHz, [D6]DMSO; the signals are given
for the major rotamer): d=169.1, 164.8, 159.2 (2C), 150.9, 145.6, 136.3
(2C), 136.0, 135.9 (2C), 135.8, 134.3, 133.0 (2C), 132.9 (2C), 132.7 (2C),
132.6 (2C), 132.5 (2C), 132.3 (2C), 131.8, 130.8 (2C), 130.6 (2C), 129.8
(2C), 129.7 (2C), 129.5 (2C), 129.1 (2C), 128.6 (3C), 124.2 (2C), 123.2
(2C), 119.1, 114.3 (4C), 109.2, 97.1, 97.0, 88.9, 88.4, 87.4, 87.0, 86.7, 70.0,
66.0, 60.2, 56.0 (2C), 46.6, 13.2 ppm; ESI-HRMS: m/z calcd for
C65H49N3O8Na+: 1022.3412 [M++Na]; found: 1022.3365.
+
MALDI-HRMS: m/z calcd for C33H18O2
:
446.1301 [M+]; found:
446.1303.
6,8-Bis(phenylethynyl)pyrene-2-carboxylic acid (18): Prepared from 12
(444 mg, 1 mmol) to give the title compound as a yellow solid (402 mg,
90%). Rf =0.41 (5% MeOH/CH2Cl2); m.p. >2508C (1% DMF/MeNO2);
+
MALDI-HRMS: m/z calcd for C33H18O2
:
446.1301 [M+]; found:
446.1305.
3,6,8-Tris(phenylethynyl)pyrene-1-carboxylic acid (19): By using the gen-
eral procedure, 13 (545 mg, 1 mmol) was converted into the title com-
pound (red solid; 544 mg, 99%). Rf =0.50 (5% MeOH/CH2Cl2); m.p.
+
>2508C (1% DMF/MeNO2); MALDI-HRMS: m/z calcd for C41H22O2
546.1614 [M+]; found: 546.1615.
:
3,6,8-Tris(phenylethynyl)pyrene-2-carboxylic acid (20): Prepared from 14
(545 mg, 1 mmol) to give the title compound as a red solid (502 mg,
92%). Rf =0.40 (5% MeOH/CH2Cl2); m.p. 163–1668C (1% DMF/
MeNO2); MALDI-HRMS: m/z calcd for C41H22O2+: 546.1614 [M+];
found: 546.1610.
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-[6,8-bis-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
pound as an orange foam (rotameric mixture ꢀ1:3.3 by 1H NMR;
227 mg, 65%). Rf =0.43 (1:50% NEt3/acetone/toluene); 1H NMR
(300 MHz, [D6]DMSO; the signals are given for the major rotamer): d=
11.41 (brs, 1H), 8.84–8.55 (m, 3H), 8.35 (s, 1H), 7.87–7.83 (m, 2H), 7.69–
7.55 (m, 19H), 7.38 (d, J=8.7 Hz, 2H), 6.99–6.96 (m, 4H), 6.14 (s, 1H),
6.05–6.03 (m, 1H), 4.56 (s, 1H), 4.28 (brs, 1H), 3.85 (d, J=11.7 Hz, 1H),
3.80 (s, 6H), 3.62–3.51 (m, 3H), 1.50 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO; the signals are given for the major rotamer): d=169.8, 164.6,
159.2 (2C), 151.2, 145.6, 136.3, 135.9, 134.3, 134.2 (2C), 134.0, 132.9 (4C),
132.8 (4C), 132.6 (2C), 132.5 (2C), 132.3 (2C), 130.0 (2C), 129.9 (2C),
129.7 (4C), 129.6 (4C), 128.9, 128.6, 127.8, 127.2, 126.2, 123.0, 118.2, 114.3
(4C), 109.6 (2C), 96.8 (2C), 88.0, 87.2, 86.7, 80.1 (2C), 70.1, 67.1, 56.0
(2C), 52.9, 13.3 ppm. ESI-HRMS: m/z calcd for C65H49N3O8Na+:
1022.3412 [M++Na]; found: 1022.3402.
General procedure for the preparation of compounds 22a–f: Diisopropy-
lethylamine (132 mL, 0.76 mmol) was added as one portion to a stirred
solution of the corresponding acid (0.39 mmol) and HBTU (137 mg,
0.36 mmol) in DMF (3 mL) (15, 16) or DMF (2 mL) and 1,1-dichloro-
ethane (1 mL) (17–20). The mixture was stirred for 10 min at RT and
then added dropwise to a stirred solution of 21[16] (200 mg, 0.35 mmol) in
DMF (3 mL). After stirring for 1.5 h, TLC monitoring showed that the
reaction was complete. The reaction mixture was diluted with CH2Cl2
(100 mL) and washed with water (2ꢃ150 mL), 5% NaHCO3 (2ꢃ
150 mL), and water (3ꢃ150 mL). The organic layer was dried over
Na2SO4 and evaporated. The residue was purified by column chromatog-
raphy on silica gel using a gradient elution of 1:5!30% Et3N/acetone/
toluene.
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-(3-phenyl-
ACHTUNGTRENNUNGethynylpyren-1-carbonyl)-3-(thymin-1-yl)-2-oxa-5-azabicycloACHUTNGTREN[NUGN 2.2.1]heptane
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-[3,6,8-tris-
(22a): Prepared from 15 (135 mg) to give the title compound as an
orange foam (rotameric mixture ꢀ1:0.5 by 1H NMR; 214 mg, 68%). Rf
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
1
0.33 (1:50% NEt3/acetone/toluene); H NMR (300 MHz, [D6]DMSO; the
pound as an orange foam (304 mg, 62%). Rf =0.25 (1:50% NEt3/acetone/
toluene); 1H NMR (500 MHz, [D6]DMSO; the signals are given for the
major rotamer): d=11.50 (brs, 1H, exchange with D2O), 8.90–8.74 (m,
4H), 8.56 (s, 1H), 8.38–8.36 (m, 1H), 7.86–7.82 (m, 5H), 7.54–7.47 (m,
11H), 7.38–7.14 (m, 9H), 6.96–6.94 (m, 4H), 6.26 (d, J=3.5 Hz, 1H),
5.94 (s, 1H), 5.18 (s, 1H), 4.20 (d, J=2.5 Hz, 1H), 3.77 (s, 6H), 3.59 (d,
J=10.0 Hz, 1H), 3.49 (d, J=11.0 Hz, 1H), 3.27–3.22 (m, 2H, partial
overlap with H2O), 1.41 ppm (s, 3H); 13C NMR (125 MHz, [D6]DMSO;
the signals are given for the major rotamer): d=168.7, 164.8, 159.2 (2C),
150.9, 145.6, 136.3 (2C), 136.0, 135.9 (2C), 135.8, 135.3, 134.8, 134.1 (2C),
133.5 (2C), 132.7 (2C), 132.6 (4C), 132.5, 132.4, 132.3, 131.9 (2C), 130.8
signals are given for the major rotamer): d=1.60 (brs, 1H), 8.77 (d, J=
9.0 Hz, 1H), 8.56–8.50 (m, 3H), 8.46–8.39 (m, 2H), 8.30–8.25 (m, 2H),
7.92–7.88 (m, 2H), 7.65–7.56 (m, 5H), 7.48–7.25 (m, 8H), 7.05–7.02 (m,
4H), 6.36 (d, J=3.6 Hz, 1H), 6.02 (s, 1H), 5.26 (s, 1H), 4.29 (d, J=
4.2 Hz, 1H), 3.81 (s, 6H), 3.70 (d, J=11.0 Hz, 1H), 3.60 (d, J=11.0 Hz,
1H), 3.45–3.26 (m, 2H, partial overlap with H2O), 1.51 ppm (s, 3H);
13C NMR (75 MHz, [D6]DMSO; the signals are given for the major rota-
mer): d=169.2, 164.8, 159.2 (2C), 150.9, 145.6, 136.3, 136.0, 135.9, 135.8,
132.6, 132.5, 132.4, 132.2, 131.5 (2C), 131.4, 130.8 (2C), 130.7 (2C), 130.5,
129.9 (2C), 129.8 (2C), 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.0, 127.8,
11024
www.chemeurj.org
ꢂ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 11010 – 11026

Functionalized Locked Nucleic Acids
FULL PAPER
(2C), 130.7 (2C), 130.5, 130.3, 129.8 (4C), 129.7, 128.9, 128.7, 128.6, 128.5,
127.6 (2C), 124.4, 124.3, 124.1, 123.0 (2C), 122.9, 119.0, 114.3 (4C), 109.6,
109.2, 97.7, 97.6, 97.5, 97.3, 88.8, 88.0, 86.8, 69.9, 66.0, 60.2, 55.9 (2C),
52.4, 13.5 ppm; ESI-HRMS: m/z calcd for C73H53N3O8Na+: 1122.3724
[M++Na]; found: 1122.3719.
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4’-
dimethoxytrityloxymethyl)-7-hydroxy-5-[3,6,8-tris(phenylethynyl)pyren-2-
carbonyl]-3-(thymin-1-yl)-2-oxa-5-azabicycloACTHNUTRGENUG[N 2.2.1]heptane (23 f): Orange
foam (231 mg, 89%). Rf =0.53, 0.48 (1:50% NEt3/acetone/toluene);
31P NMR (120 MHz, [D6]DMSO): d=149.02, 147.81 ppm (1:0.4); ESI-
HRMS: m/z calcd for C82H70N5O9PNa+: 1322.4803 [M++Na]; found:
1322.4813.
(1R,3R,4R,7S)-1-(4,4’-Dimethoxytrityloxymethyl)-7-hydroxy-5-[3,6,8-tris-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
pound as an orange foam (rotameric mixture ꢀ1:0.9:0.5 by 1H NMR;
162 mg, 42%). Rf 0.30 (1:60% NEt3/acetone/toluene); 1H NMR
(300 MHz, [D6]DMSO; the signals are given for the major rotamer): d=
11.06 (brs, 1H), 8.71–8.52 (m, 2H), 7.97–7.92 (m, 3H), 7.84–7.77 (m,
2H), 7.67–7.29 (m, 24H), 7.06–7.03 (m, 4H), 5.95 (s, 1H), 5.91 (d, J=
3.9 Hz, 1H), 4.25–4.23 (m, 1H), 4. 11 (s, 1H) 3.88 (s, 6H), 3.84–3.80 (m,
2H), 3.69–3.59 (m, 2H), 1.49 ppm (s, 3H); 13C NMR (75 MHz,
[D6]DMSO; the signals are given for the major rotamer): d=167.8, 164.5,
159.2 (2C), 150.9, 145.6, 136.3 (2C), 136.0, 135.9, 135.8, 134.0, 132.7 (4C),
132.5 (2C), 132.4, 132.3 (2C), 131.9 (2C), 130.7 (4C), 129.9 (4C), 129.7,
129.6, 129.5, 129.0 (2C), 128.9 (2C), 128.6 (2C), 127.8, 127.6, 127.5, 124.4,
124.3, 124.1, 123.0, 122.9, 119.4, 119.3, 119.2, 119.1, 119.0, 114.3 (4C),
109.7, 109.5, 97.7, 97.6, 97.5, 97.4, 88.4, 88.1, 86.8, 68.3, 66.0, 60.2, 56.0
(2C), 52.0, 11.7 ppm; ESI-HRMS: m/z calcd for C73H53N3O8Na+:
1122.3724 [M++Na]; found: 1122.3710.
Acknowledgements
We greatly appreciate funding from the Danish National Research Foun-
dation and the Sixth Framework Programme Marie Curie Host Fellow-
ships for Early Stage Research Training under contract number MEST-
CT-2004-504018. I.V.A. and V.A.K. acknowledge support from the Russi-
an Foundation for Basic Research (RFBR), grant number 06-03-32426.
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Springer, Singapore, 2006; b) F. M. Winnik, Chem. Rev. 1993, 93,
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H. Tsuchida, H. Zako, T. Yamaoka, A. Murakami, Nucleic Acids
Res. 1999, 27, 2387–2392; b) K. Yamana, H. Zako, K. Asazuma, R.
Iwase, H. Nakano, A. Murakami, Angew. Chem. 2001, 113, 1138–
1140; Angew. Chem. Int. Ed. 2001, 40, 1104–1106; c) V. A. Korshun,
D. A. Stetsenko, M. J. Gait, J. Chem. Soc. Perkin Trans. 1 2002,
1092–1104; d) N. N. Dioubankova, A. D. Malakhov, D. A. Stetsen-
ko, M. J. Gait, P. E. Volynsky, R. G. Efremov, V. A. Korshun, Chem-
BioChem 2003, 4, 841–847; e) K. Kawai, H. Yoshida, T. Takada, S.
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Nakamura, Y. Fukunaga, K. Sasa, Y. Ohtoshi, K. Kanaori, H. Haya-
shi, H. Nakano, K. Yamana, Nucleic Acids Res. 2005, 33, 5887–
5895; g) D. S. Novopashina, D. A. Stetsenko, O. S. Totskaya, M. N.
Repkova, A. G. Venyaminova, Nucleosides Nucleotides Nucleic
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V. V. Filichev, S. Van Calenbergh, E. B. Pedersen, ChemBioChem
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6904–6905.
General procedure for the preparation of compounds 23a–f: The rele-
vant LNA nucleoside derivative (0.2 mmol) was evaporated with dry
CH2Cl2 (2ꢃ30 mL), dissolved in anhydrous CH2Cl2 (20 mL), then diiso-
propylammonium tetrazolide (52 mg, 0.3 mmol) and bis(N,N-diisopropyl-
ACHTUNGTRENNUNGamino)-2-cyanoethoxyphosphine (96mL, 0.3 mmol) were added under
argon and the resulting mixture was stirred for 12 h. The mixture was
then diluted with CH2Cl2 (100 mL) and washed with saturated aqueous
solutions of NaHCO3 (2ꢃ100 mL) and brine (100 mL). The organic layer
was dried over Na2SO4 and evaporated, and the crude material was puri-
fied by chromatography on silica gel eluted with 1:30% NEt3/acetone/tol-
uene.
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4’-
dimethoxytrityloxymethyl)-7-hydroxy-5-(3-phenylethynylpyren-1-carbon-
yl)-3-(thymin-1-yl)-2-oxa-5-azabicycloACHTNUGTRNEUNG[2.2.1]heptane (23a): Yellow foam
(202 mg, 92%). Rf =0.52, 0.48 (1:50% NEt3/acetone/toluene); 31P NMR
(120 MHz, [D6]DMSO): d=149.20, 149.17 ppm (1:1); MALDI-HRMS:
m/z calcd for C66H62N5O9PNa+: 1122.4177 [M++Na]; found: 1122.4170.
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4’-
dimethoxytrityloxymethyl)-7-hydroxy-5-(6-phenylethynylpyren-2-carbon-
yl)-3-(thymin-1-yl)-2-oxa-5-azabicycloACHTNUGTRNEUNG[2.2.1]heptane (23b): Yellow foam
[3] P. J. Hrdlichka, B. R. Babu, M. D. Sørensen, N. Harrit, J. Wengel, J.
Am. Chem. Soc. 2005, 127, 13293–13299.
(188 mg, 85%). Rf =0.46, 0.42 (1:50% NEt3/acetone/toluene); 31P NMR
(120 MHz, [D6]DMSO): d=150.89, 149.12 ppm (1:1.3); MALDI-HRMS:
m/z calcd for C66H62N5O9PNa+: 1122.4177 [M++Na]; found: 1122.4194.
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Received: June 3, 2008
Published online: October 31, 2008
11026
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Chem. Eur. J. 2008, 14, 11010 – 11026