Oligonucleotides containing new fluorescent 1-phenylethynylpyrene and 9,10-bis(phenylethynyl)anthracene uridine-2′-carbamates: Synthesis and properties

Article abstract of DOI:10.1016/j.tet.2004.03.076

The synthesis of two novel fluorescent uridine-2′-carbamate phosphoramidites is described. The reagents carrying fluorescent polyaromatic hydrocarbons 1-phenylethynylpyrene (PEPy) or 9,10-bis(phenylethynyl)anthracene (BPEA) are suitable for oligonucleotide synthesis. Prepared oligonucleotide conjugates show strong dye emissions at 401 and 485 nm, but low FRET rate when located in the oligonucleotide duplex. The dyes show considerable compensation of the usual carbamate duplex destabilization. The possible explanation of both effects is binding of PEPy and BPEA to the minor groove of the DNA duplex.

Full text of DOI:10.1016/j.tet.2004.03.076

Tetrahedron 60 (2004) 4617–4626
Oligonucleotides containing new fluorescent
1-phenylethynylpyrene and 9,10-bis(phenylethynyl)anthracene
uridine-20-carbamates: synthesis and propertiesq
*
Natalia N. Dioubankova, Andrei D. Malakhov, Zakhar O. Shenkarev and Vladimir A. Korshun
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10,
Moscow 117997, Russia
Received 19 December 2003; revised 1 March 2004; accepted 25 March 2004
Abstract—The synthesis of two novel fluorescent uridine-20-carbamate phosphoramidites is described. The reagents carrying fluorescent
polyaromatic hydrocarbons 1-phenylethynylpyrene (PEPy) or 9,10-bis(phenylethynyl)anthracene (BPEA) are suitable for oligonucleotide
synthesis. Prepared oligonucleotide conjugates show strong dye emissions at 401 and 485 nm, but low FRET rate when located in the
oligonucleotide duplex. The dyes show considerable compensation of the usual carbamate duplex destabilization. The possible explanation
of both effects is binding of PEPy and BPEA to the minor groove of the DNA duplex.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
an energy donor–acceptor pair, and were also able to form
excimers. Her0e, we report the introduction of PEPy and
BPEA in the 2 -position of uridine as well as the fluorescent
and thermal denaturation properties of modified
oligonucleotides.
Oligonucleotides conjugates with fluorescent dyes have
numerous and diverse applications in molecular biology,
bioorganic chemistry, and medicine. The sugar part of
nucleosides, particularly the 20-position, is an attractive site
for modification, for example, for potential antisense
applications.1 Examples of the introduction of hetero- and
polyaromatic residues at the 20 position of nucleosides
include dansyl,2 anthracene,3 anthraquinone,4 fluorescein,5
nalidixic acid,6 porphyrins,7 1,8-naphthalimide,8 and
pyrene.9 Aromatic ligands attached to the 20-positions of
ribonucleotides in oligonucleotides are placed in the minor
groove and can affect duplex stability. Pyrene, a fluorescent
tetracyclic aromatic hydrocarbon, is of particular interest
because of its abilities to give a response in the fluorescent
spectrum upon hybridization of pyrene-labeled oligo-
nucleotides9b,e–i,k,n and to form excimers.9n,o,q Recently,
we developed a convenient method of 20-carbamate
modification of uridine with pyrene,9n and non-nucleotide
modifying reagents for introducing new fluorescent poly-
aromatic hydrocarbons 1-phenylethynylpyrene (PEPy) and
9,10-bis(phenylethynyl)anthracene (BPEA) into oligo-
nucleotides.10 PEPy and BPEA were shown to constitute
2. Results and discussion
2.1. Syntheses of phosphoramidites
The first step in the preparation of PEPy-modified uridine-
20-carbamate phosphoramidite 6 is alkynylation of the
starting 20-O-(4-iodobenzylaminocarbonyl)-30,50-O-(tetra-
isopropyldisiloxan-1,3-diyl)uridine 19n with 1-ethynyl-
pyrene in the conditions of Sonogashira coupling
(Pd(PPh3)4/CuI in DMF in the presence of triethylamine)11
(Scheme 1). The reaction was performed under Ar to reduce
the quantity of side product 3 from Glaser acetylene
coupling. It is worthy of note, that the reacton mixture
should be washed with water many times upon workup to
remove the solvent (DMF), otherwise traces of 3 can
accompany the main product in the course of 0 column
purification on silica gel. The Markiewicz’ 30,5 -O-silyl
group from 2 was removed with triethylamine trihydro-
fluoride in THF.9n Crystalline nucleoside 4 (UP) was 50-O-
dimethoxytritylated, and then 30-O-phosphitylated using
standard methods of nucleoside chemistry.12 Pyridine (not
triethylamine) should be added to the eluent for neutraliz-
ation of silica gel in chromatographic purification of 50-O-
Dmt derivative 5, because more basic conditions can lead to
q Supplementary data associated with this article can be found in the online
version, at doi: 10.1016/j.tet.2004.03.076
Keywords: Modified oligonucleotides; Polycyclic aromatic hydrocarbons;
Fluorescence; Duplex stability.
*
Corresponding author. Tel./fax: þ7-95-3306738;
e-mail address: korshun@mail.ibch.ru
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.03.076

4618
N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
Scheme 1. Synthesis of PEPy uridine-20-carbamate phosphoramidite 6.
20!30 migration of the carbamoyl group.9n A small amount
of bis-30,50-O-Dmt nucleoside 5a was also isolated.
Phosphoramidite 6 was isolated as a solid suitable for
prolonged storage (2 years).
similar to PEPy compounds (Scheme 2). 9-Ethynyl-10-
phenylethynylanthracene10 was used for the assembly of a
dye molecule.
The structures of modified nucleosides UP and UA were
1
It was also tempting to synthesize a similar fluorescent
nucleoside which could serve as an energy acceptor for the
PEPy nucleoside UP. BPEA nucleoside UA and its 50-O-
Dmt-30-O-phosphoramidite derivative 11 were prepared
confirmed by H–13C NMR correlations. The first step in
the assignment of 13C NMR spectra was distinguishing the
proton-bound carbon atoms taking into account cross-peaks
on HMQC spectra. The signals of non-protonated carbons
Scheme 2. Synthesis of BPEA uridine-20-carbamate phosphoramidite 11.

N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
Table 1. Properties of modified and unmodified oligodeoxyribonucleotides
4619
#
Sequence, 50!30
MALDI MS
Calculated mass [MþH]þ
ON01
ON02
ON03
ON04
ON05
ON06
ON07
ON08
ON09
ON10
ON11
ON12
ON13
ON14
ON15
ON16
CTCCCAGGCTCAAAT
ATTTGAGCCTGGGAG
4490.4
4643.8
4930.4
4927.8
5013.4
4853.8
4855.2
5214.2
4935.8
6185.1
5005.9
5002.7
5007.5
6229.7
6228.7
6228.2
4496.0
4643.0
4930.9
4930.9
5013.4
4854.9
4854.9
5214.0
4934.9
4186.1
5005.9
5005.9
5005.9
6229.2
6229.2
6229.2
CTCCCAGGCUACAAAT
CUACCCAGGCTCAAAT
CTCCCAGGCTCAAAUApa
CTCCCAGGCUPCAAAT
CUPCCCAGGCTCAAAT
CUPCCCAGGCUPCAAAT
CTCCCAGGCTCAAAUPpa
CTCCCAGGCTCAAAUPCTGGpa
AUPTTGAGCCTGGGAG
ATUPTGAGCCTGGGAG
ATTUPGAGCCTGGGAG
CCAGAUPTTGAGCCTGGGAG
CCAGATUPTGAGCCTGGGAG
CCAGATTUPGAGCCTGGGAG
a
30-phosphate.
were assigned using 1H–13C interactions through two, three
and four bonds visualized as cross-peaks on HMBC
spectra.†
The presence of a fluorescent label in the oligonucleotides
can be confirmed by UV spectra. Modified oligonucleotides,
in addition to the inherent oligonucleotide absorption
maximum around 260 nm, show characteristic maxima of
the fluorophore, in the region of 350–400 nm (for PEPy) or
420–500 nm (for BPEA) (Fig. 4).
2.2. Oligonucleotide synthesis
Fluorescent uridine-20-carbamate phosphoramidites 6 and
11 were used in solid-phase oligodeoxyribonucleotide
synthesis. The coupling time for the modifying reagents
was increased to 10 min; the coupling yield was .95%. The
sequence chosen was a 15-mer complementary to the 22–36
sequence (ON02) of the trans-activation responsive region
of the human immunodeficiency virus type 1 (HIV-1) TAR
RNA (ON01).13 The sequences are given in Table 1.
Fluorescent uridine-20-carbamates were placed instead of
internal thymidine (ON03, ON006, ON14-ON16), the
thymidine located closely to the 5 -end (ON04, ON07), or
at the both sites (ON08). Complementary unmodified
(ON02) and modified (ON11-ON16) oligodeoxyribo-
nucleotides were also prepared. The integrity and purity of
modified oligonucleotides was analyzed by MALDI mass-
spectrometry (Fig. 1, Table 1) and HPLC (Fig. 2). An
insertion of such bulky moieties into oligonucleotides
reduces their mobility on polyacrylamide gel (data not
shown) and increases the retention time of conjugates on
reverse-phase column (Fig. 2); BPEA containing oligo-
nucleotide ON03 is considerably more hydrophobic than
PEPy labelled oligomer ON06.
The absorption maxima of the fluorophore are shifted upon
changing of the solvent to less polar one. An even greater
bathochromic shift was observed for a dye with drastically
changed microenvironment. The ‘oligonucleotide-bound’
2.3. Absorbance properties of PEPy and BPEA
nucleosides
1-Phenylethynylpyrene14 and 9,10-bisphenylethynylanthra-
cene15 have characteristic absorbance spectra. Figure 3
presents the comparison of UV spectra of fluorescent
nucleosides UP and UA in polar solvent (water–ethanol)
and Markiewicz-protected nucleosides 2 and 7 in non-polar
solvent (DCM). The spectra of both chromophores in DCM
have negligible long-wavelength shifts (ca. 2.5–3 nm) in
comparison with their spectra in water–ethanol.
Figure 1. Examples of MALDI-TOF mass spectra: BPEA-modified
oligomer ON04 (A) and PEPy-modified oligomer ON06 (B).
†
See Supplementary DataApplication 1.

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N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
Figure 2. Examples of HPLC profiles of crude oligonucleotides:
unmodified 15-mer ON01 (1), PEPy-modified oligomer ON06 (2) and
BPEA-modified oligomer ON03 (3). For HPLC conditions see Section 4.
PEPy has a 15 nm shift as compared to the PEPy nucleoside
(plot 2 in Fig. 5). This may indicate significant hydrophobic
environment of the dye and the decrease in the number of
degrees of freedom for the rotational movement, for
example, location in the minor groove.
Figure 4. UV spectra of (A) PEPy-modified oligonucleotides ON06 (1),
ON07 (2), ON07 (3) in water, normalized by pyrene absorbance, and (B)
unmodified ON01 and BPEA-modified oligonucleotide ON03, normalized
by oligonucleotide absorbance at 260 nm; 1260 for BPEA is approx.
22,000.16
Figure 3. UV spectra of PEPy derivatives (A), normalized at dye
absorbance around 360 nm and BPEA derivatives (B), normalized at dye
absorbance around 460 nm. Conditions: solution UP (A, 1) or UA (B, 1) in
water/ethanol 1:1 (v/v), solution of 2 (A, 2) or 7 (B, 2) in DCM.
Figure 5. Comparison of the UV spectrum of PEPy-modified nucleoside
UP in water/ethanol 1:1 (v/v) (1) and the difference between UV spectra of
oligonucleotides ON08 and ON06 (2) in water, normalized by PEPy
absorbance.

N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
4621
Figure 6. Fluorescence spectra of PEPy (A), and BPEA derivatives (B),
normalized at fluorescence maximum. Conditions: solution UP (A, 1) or
UA (B, 1) in water/ethanol 1:1 (v/v), solution 2 (A, 2) or 7 (B, 2) in DCM,
hybridization buffer (Section 4).
Figure 7. Fluorescence spectra of single stranded (A) PEPy-modified
oligonucleotides ON07 (mono-labeled, 1) and ON08 (double-labeled, 2),
(B) BPEA-modified oligonucleotide ON04.
2.4. Fluorescent measurements
fluorescence resonance energy transfer (Fig. 8A, plot 3).
The effect slightly depends on the distance between
fluorophores within duplexes (Fig. 8B).
The changes in fluorescent spectra of the dyes upon
replacement of the solvent are similar to those for
absorption. There are negligible long-wave shifts of the
fluorescent bands in DCM compared to those in ethanol–
water 1:1 solution for both PEPy (about 1.5 nm) and BPEA
(about 4 nm) derivatives (Fig. 6).
2.5. Thermal denaturation studies
The negative effect of 20-carbamate function on the thermal
stability of DNA–DNA and DNA–RNA duplexes is well
known.1,9n Because of the aromatic nature of dyes (PEPy,
BPEA) the ‘carbamate’ destabilization is reduced and the
melting temperatures remained sufficiently high for spectral
studies to be performed (Table 2). This is caused
presumably by fluorophore interaction with grooves of
DNA duplexes. In cases of near-terminal positions of
chromophores duplexes are more stabilized (ON04£ON11
and ON07£ON11). On the other hand, a close proximity of
two fluorophores placed in different strands leads to an
increase in the melting temperature (cf. ON03£ON13 and
ON03£ON11 or ON07£ON13 and ON07£ON11). More-
over, the combination of BPEA and PEPy results in better
stabilization than two PEPy residues (compare
ON03£ON13 and ON07£ON13 or ON03£ON12 and
ON07£ON12 or ON03£ON11 and ON07£ON11).
We studied the luminescent characteristics of the labelled
oligonucleotides and their duplexes. The fluorescence
spectra were registered in aqueous phosphate buffer (pH
7.0) at the excitation wavelength of 375 nm for PEPy-
containing oligonucleotides and 440 nm for BPEA-contain-
ing oligonucleotides. As Figure 7 shows, the introduction of
a dye residue results in a characteristic monomeric
fluorescence with maxima at 401, 423 nm (PEPy, A), and
at 485, 516 nm (BPEA, B).
A study of the spectral properties of duplex ON03£ON11
revealed an irradiative energy transfer. It turned out that
BPEA is an energy acceptor for PEPy: hybridization of the
above sequence, containing these residues, results in a

4622
N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
Table 2. Thermal denaturation studies of modified oligonucleotides
#
Sequence
Tm (8C)
DTm (8C)
ON01
ON02
50-CTCCCAGGCTCAAAT
30-GAGGGTCCGAGTTTA
57.6
—
ON03
ON11
50-CTCCCAGGCUACAAAT
30-GAGGGTCCGAGTTUPA
57.2
57.5
59.2
56.0
56.1
57.5
62.1
58.0
60.7
62.0
60.8
59.5
55.0
54.7
55.5
63.3
58.7
58.8
62.2
59.0
58.7
20.4
20.1
þ1.6
21.6
21.5
20.1
þ4.5
þ0.4
þ3.1
þ4.4
þ3.2
þ1.9
22.6
22.9
22.1
þ5.7
þ1.1
þ1.2
þ4.6
þ1.4
þ1.1
ON03
ON12
50-CTCCCAGGCUACAAAT
30-GAGGGTCCGAGTUPTA
ON03
ON13
50-CTCCCAGGCUACAAAT
30-GAGGGTCCGAGUPTTA
ON07
ON11
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGTTUPA
ON07
ON12
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGTUPTA
ON07
ON13
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGUPTTA
ON04
ON11
50-CUACCCAGGCTCAAAT
30-GAGGGTCCGAGTTUPA
ON04
ON12
50-CUACCCAGGCTCAAAT
30-GAGGGTCCGAGTUPTA
ON04
ON13
50-CUACCCAGGCTCAAAT
30-GAGGGTCCGAGUPTTA
ON07
ON11
50-CUPCCCAGGCTCAAAT
30-GAGGGTCCGAGTTUPA
ON07
ON12
50-CUPCCCAGGCTCAAAT
30-GAGGGTCCGAGTUPTA
ON07
ON13
50-CUPCCCAGGCTCAAAT
30-GAGGGTCCGAGUPTTA
ON07
ON14
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGTTUPAGACC
ON07
ON15
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGTUPTAGACC
ON07
ON16
50-CTCCCAGGCUPCAAAT
30-GAGGGTCCGAGUPTTAGACC
ON10
ON11
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGTTUPA
ON10
ON12
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGTUPTA
ON10
ON13
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGUPTTA
ON10
ON14
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGTTUPAGACC
ON10
ON15
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGTUPTAGACC
ON10
ON16
50-CTCCCAGGCTCAAAUPCTGG
30-GAGGGTCCGAGUPTTAGACC
3. Summary
The results presented indicate that fluorescent hydrocarbons
1-phenylethynylpyrene (PEPy) and 9,10-bis(phenyl-
ethynyl)anthracene (PEPy), when attached through a
carbamate spacer to the 20-position of uridine and directed
to the minor groove of DNA duplex, show weak FRET
signal in comparison with PEPy–BPEA pair from non-
nucleotide reagents. PEPy and BPEA covalently bound to
duplexes give increase in Tm thus suggesting the
Figure 8. Fluorescence spectra of (A) single stranded BPEA-modified
oligonucleotide ON03 lex 440 nm (1), PEPy-modified oligonucleotide
ON11 lex 375 nm (2), and their duplex lex 375 nm (3), lex 440 nm (4);
(B) duplexes ON03£ON11 (1), ON03£ON12 (2), ON03£ON13 (3), lex
375 nm; (C) duplexes ON04£ON11 (1), ON04£ON12 (2), ON04£ON13
(3), lex 375 nm. Conditions: hybridization buffer, concentration of each
oligo 1027 M.

N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
4623
polyaromatics are interacting with DNA double helix. The
possible explanation of the results is that PEPy and BPEA
hydrocarbons are bound to the minor groove of DNA and
have unfavourable spatial orientation for resonance energy
transfer. Dye–DNA interaction in the minor groove can also
increase DNA strands affinity and thus reduce the negative
effect of 20-carbamate modification.
gradient 0!50% EtOAc in CHCl3 (v/v). Yield 2.38 g
(94.1%), yellow foam. Rf 0.31 (CHCl3/EtOAc, 1:1).
MALDI-TOF MS (matrix 2,4,6-trihydroxyacetophenone
(2,4,6-THAP)) 846.87, 868.28. Calcd 844.34 [MþH]þ,
1
866.33 [MþNa]þ. H NMR ([D6]DMSO): d 11.41 (s, 1H,
00
00
00
H-3), 8.61 (d, 1H, J9 ,10 ¼9.1 Hz, H-10 ), 8.41–8.21 (m,
7H, H-200 –600,800,900), 8.12 (apparent t, 1H, J6 ,7 ¼J7 ,8 ¼7.7
00 00
00 00
Hz, H-700), 8.05 (br t, J¼6.1 Hz, OCONH), 7.70 (m, 3H,
H-6, Hd), 7.39 (d, 2H, Jd,e¼8.0 Hz, He), 5.71 (d, 1H,
0
0
0
J1 ,2 ¼1.5 Hz, H-1 ), 5.62 (d, 1H, J5,6¼8.1 Hz, H-5), 5.41
4. Experimental
4.1. General
0
0
0
0
0
(dd, 1H, J1 ,2 ¼1.5 Hz, J2 ,3 ¼5.7 Hz, 0 H-2 ), 4.51 (dd,
0
0
0
0
J2 ,3 ¼5.7 Hz, J3 ,4 ¼8.1 Hz, 1H, H-3 ), 4.27 (m, 2H,
JNHCH¼6.1 Hz, 2J¼15.8 Hz, NCH2), 4.11–3.88 (m, 3H,
J5 a,5 b¼12.9 Hz, J4,5 a¼4.0 Hz, H-40, H-50), 1.07–0.97 (m,
28H, Pri). Anal. Calcd for C47H53N3O8Si2: C, 66.88; H,
6.33; N, 4.98. Found: C, 66.96; H, 6.21; N, 5.01.
0
0
0
4-Iodobenzylamine, 1-ethynylpyrene were from Lancaster
Synthesis; triethylamine trihydrofluoride, triethylamine,
were from Aldrich; N,N-carbonyldiimidazole (CDI) was
from Sigma, 4,40-dimethoxytritylchloride (DmtCl) was
from Avocado; bis(N,N-diisopropylamino)-2-cyanoethoxy-
phosphine was from Fluka. 20-O-(4-Iodobenzylaminocar-
bonyl)-30,50-O-(tetraisopropyldisiloxan-1,3-diyl)uridine,9n
9-ethynyl-10-phenylethynyl-anthracene,10 and diisopropyl-
ammonium tetrazolide12 were prepared as described. DCM
was always used freshly distilled over CaH2. Anhydrous
pyridine was from Aldrich; THF was freshly distilled over
4.1.2. 20-O-[4-(9-Phenylethynylanthracen-10-ylethynyl)-
phenylmethylaminocarbonyl]-30,50-O-(tetraisop0ropyl-
disiloxan-1,3-diyl)uridine (7). To a solution of 2 -O-(4-
iodobenzylaminocarbonyl)-30,50-O-(tetraisopropyldisiloxan-
1,3-diyl)uridine (0.863 g, 1.157 mmol) in DMF (25 mL),
9-ethynyl-10-(phenylethynyl)anthracene10
(350 mg,
1.157 mmol), CuI (44 mg, 0.231 mmol), Pd(PPh3)4
(134 mg, 0.157 mmol) and Et3N (0.403 mL, 2.89 mmol)
were added with stirring under argon for 24 h at room
temperature. The workup was as above; the compound was
isolated by chromatography on silica gel in step gradient of
0!35% EtOAc in CHCl3 (v/v). Yield 0.98 g (92.1%),
orange foam. Rf 0.31 (CHCl3/EtOAc, 1:1). MALDI-TOF
MS (matrix 2,4,6-THAP) 921.77, 944.23, 960.48. Calcd
˚
powdered LiAlH4 and stored over 4 A molecular sieves
under nitrogen. Other solvents were used as received.
1
500 MHz H, 125.7 MHz 13C and 202.4 MHz 31P NMR
spectra were measured on a Bruker AC-500 spectrometer.
1
Chemical shifts (d) for H, 13C and 31P are referenced to
internal solvent resonances and reported relative to SiMe4
1
921.21 [MþH]þ, 943.20 [MþNa]þ, 959.30 [MþK]þ. H
1
(1H and 13C) and 85% aq. H3PO4 (31P). H NMR coupling
NMR ([D6]DMSO): d 11.41 (s, 1H, H-3), 8.67 (m, 4H,
H-100,400,500,800), 8.07 (br t, 1H, J¼6.1 Hz, OCONH), 7.89 (m,
2H, Hc), 7.78 (m, 6H, Hj, H-200,300,600,700), 7.70 (d, 1H,
J5,6¼8.1 Hz, H-6), 7.53 (m, 3H, Ha,b), 7.41 (d, 2H,
Jj,k¼8.1 Hz, Hk), 5.71 (br s, 1H, H-10), 5.60 (m, 1H, H-5),
constants are reported in Hz and refer to apparent
multiplicities. MALDI-TOF mass spectra were measured
on
a
Voyager-DE BioSpectrometry Workstation
(PerSeptive Biosystems) in positive ion mode. Elemental
analysis was performed on CHNS-analyser/EA1112
‘ThermoFinnigan’. TLC and column chromatographies
were carried out with Macherey-Nagel silica gel
(ALUGRAMw SIL G/UV254 and Kieselgel 60 0.040–
0.063 mm). Thermal denaturation experiments with oligo-
nucleotide duplexes were performed on a Perkin–Elmer
Lambda 40 UV/VIS Spectrometer with PTP 6 (Peltier
Temperature Programmer). Fluorescence spectra were
obtained using a Perkin–Elmer LS 50B Luminescence
Spectrometer. Thermal denaturation studies for DNA
duplexes were done in a buffer containing 100 mM NaCl,
10 mM Na-phosphate, 0.1 mM EDTA, pH 7.0.
5.40 (m, 1H, H-20), 4.52 (dd, 1H, J2 ,3 ¼5.8 Hz, J3 ,4
¼
0
0
0
0
8.1 Hz, H-30), 4.27 (m, 2H, JNHCH¼6.1 Hz, J¼15.8 Hz,
2
0
0
0
NCH2), 4.11–3.88 (m, 3H, J5 a,5 b¼12.9 Hz, J4,5 a¼3.7 Hz,
H-40, H-50), 1.04–0.98 (m, 28H, Pri). Anal. Calcd for
C53H57N3O8Si2: C, 69.18; H, 6.24; N, 4.57. Found: C,
69.04; H, 6.30; N, 4.62.
4.1.3. 20-O-[4-(Pyren-1-ylethynyl)phenylmethylamino-
carbonyl]uridine (4). To a solution of 200-O-[4-(pyren-1-
ylethynyl)phenylmethylaminocarbonyl]-3 ,50-O-(tetraiso-
propyldisiloxan-1,3-diyl)uridine (1.688 g, 2 mmol) in THF
(5 mL) in a teflon flask (Nalgene, screw-top) was added
triethylamine trihydrofluoride (0.814 mL, 5 mmol) and the
mixture was left overnight at room temperature (the
completion of deprotection was checked by TLC (15%
MeOH in CHCl3, v/v), then diluted with hexane (25 mL).
The upper layer was discarded, and the residue (oil) was
washed with 1:1 (v/v) toluene–hexane mixture (3£25 mL)
by decantation, triturated in absolute ethanol (5 mL), and
the crystalline product was filtered off, washed with ethanol
(5 mL), diethyl ether (10 mL), and dried in vacuo. Yield
1.193 g (99.1%), yellow crystals. Rf 0.4 (CHCl3/MeOH,
17:3 (v/v)), mp 144–145.5 8C. MALDI-TOF MS (matrix
2,4,6-THAP) 602.78, 625.80. Calcd 602.19 [MþH]þ,
4.1.1. 20-O-[4-(Pyren-1-ylethynyl)phenylmethylamino-
carbonyl]-30,50-O-(tetraisopropyldisiloxan-1,3-diyl)uri-
dine (2). To a solution of 20-O-(4-iodobenzyl-
aminocarbonyl)-30,50-O-(tetraisopropyldisiloxan-1,3-diyl)-
uridine9n (2.24 g, 3.0 mmol) in DMF (25 mL), 1-ethynyl-
pyrene (792 mg, 3.5 mmol), CuI (130 mg, 0.68 mmol),
Pd(PPh3)4 (390 mg, 0.34 mmol) and Et3N (1.0 mL,
7.1 mmol) were added with stirring under argon. After
24 h at room temperature, TLC showed that the reaction is
complete. The reaction mixture was diluted with DCM
(300 mL) and washed with 0.3 M EDTA – (NH4)2
(2£200 mL), water (10£200 mL) and 5% citric acid
(200 mL), the organic layer was dried and evaporated, and
the residue was chromatographed on silica gel in step
1
624.17 [MþNa]þ. H NMR ([D6]DMSO): d 11.38 (s, 1H,
00
00
H-3, exchangeable with D2O), 8.62 (d, 1H, J9 ,10 ¼9.1 Hz,

4624
N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
H-1000), 8.39–8.21 (m, 7H, H-200 –060 00,800,900), 8.13 (apparent t,
gel column in step gradient of 0.5!1!1.5!2% MeOH in
CHCl3/EtOAc 2:1þ0.5% pyridine (v/v/v/v). Fractions
containing product were combined, evaporated, and the
residue was dried in vacuo to afford 5 (1.3 g, 86.2%) as a
yellow foam. Rf 0.31 (CHCl3/EtOAc 1:1þ1% Et3N (v/v/v)).
MALDI-TOF MS (matrix 2,4,6-THAP) 905.81, 928.04,
944.55. Calcd 904.32 [MþH]þ, 926.31 [MþNa]þ, 942.28
[MþK]þ. 1H NMR ([D6]DMSO): d 11.41 (s, 1H, H-3)
00 00
00 00
1H, J6 ,7 ¼J7 ,8 ¼7.6 Hz, H-7 ), 7.98–7.93 (m, 2H,
OCONH, H-6), 7.73 (d, 2H, Je,d¼8.0 Hz, Hd), 7.37 (d,
0
0
0
2H, Je,d¼8.0 Hz, He), 6.05 (d, 1H, J1 ,2 ¼5.7 Hz, H-1 ), 5.69
(d, 1H, J5,6¼8.1 Hz, H-5), 5.56 (d, 1H, J3 ,OH¼5.1 Hz, 30-
0
OH), 5.20 (t, 1H, J¼5.7 Hz, 50-OH) (both exchangeable
0
00 00
0
0
with D2O), 5.13 (apparent t, 1H, J1 ,2 ¼J2 ,3 ¼5.7 Hz, H-2 ),
4.25 (m 3H, H-30, NCH2), 3.94 (m, 1H, H-40), 3.64 (0m, 2H,
H-500). 13C NMR ([D6]DMSO), d 43.6 (Cg), 60.8 (C5 ), 68.9
(C3 ), 74.8 (C20), 85.4 (C10), 85.7 (C40), 87.9 (Ca), 95.2
(Cb), 102.1 (C5), 117.0 (0C0 100), 120.7 (Cc), 123.3 (C10c00),
123.6 (C10b00), 010 24.7 (C3 ),00124.8 (C1000), 125.9 (2C, C600,
C800), 126.6 (C4 ), 127.1 (C7 ), 127.2 (2C, Ce), 128.3 (C500),
00
00
(exchangeable with D2O), 8.62 (d, 1H, J9 ,10 ¼9.0 Hz,
H-1000), 8.41–8.22 (m, 7H, H-200 –600,800,900), 8.14 (apparent t,
00
OCONH), 7.73 (m, 3H, H-6,d), 7.43–7.22 (m, 9H, ArH
00 00
00 00
1H, J6 ,7 ¼J7 ,8 ¼7.5 Hz, H-7 ), 8.00 (br t, 1H, J¼6.1 Hz,
(Dmt)), 6.89 (d, 4H, Jd,e¼8.6 Hz, He), 5.97 (d, 1H,
128.7 (C9 ), 129.5 (C200), 130.4, 130.7, 130.8, 130.9 (C3a00,
C5a00, C8a , C10a00), 131.4 (2C, Cd), 140.6 (2C, C6, Cf),
J1 ,2 ¼4.9 Hz, H-1 ), 5.61 (d, 1H, J3 ,OH¼5.6 Hz, OH)
00
00
0
0
0
0
(exchangeable with D2O), 5.41 (1H, J5,6¼8.1 Hz, H-5),
0
0
0
0 0
150.5 (C2), 155.4 (Ch), 162.9 (C4). Anal. Calcd for
C35H27N3O7: C, 69.88; H, 4.52; N, 6.98. Found: C, 69.91;
H, 4.49; N, 6.92.
5.23 (apparent t, 1H, J1 2 ¼J2 3 ¼5.2 Hz, H-2 ), 4.37 (m, 1H,
H-30), 4.29 (m, 2H, NHCH2), 4.01 (m, 1H, H-40), 3.74 (s,
6H, CH3), 3.32–3.22 (m, 2H, H-50). Anal. Calcd for
C56H45N3O9: C, 74.40; H, 5.02; N, 4.65. Found: C, 74.62;
H, 4.93; 0N, 4.37. A byproduct, 50,30-O-bis-(4,40-dimethoxy-
trityl)-2 -O-[4-(pyren-1-ylethynyl)phenylmethylamino-
carbonyl]uridine, was also isolated as a foam (30 mg, 1.5%).
1H NMR ([D6]DMSO): d 11.46 (s, 1H, H-3) (0e0 xchangeable
4.1.4. 20-O-[4-(9-Phenylethynylanthracen-10-ylethynyl)-
p0henylmethylaminocarbonyl]uridine (9). Prepared from
2 -O-[4-(9-phenylethynyl0anthracen-10-ylethynyl)phenyl-
methylaminocarbonyl]-3 ,50-O-(tetraisopropyldisiloxan-
1,3-diyl)uridine (0.98 g, 1.1 mmol) and triethylamine tri-
hydrofluoride (0.5 mL, 3 mmol) in similar manner to
compound 4. Yield 0.707 g (98%), orange crystals. Rf 0.4
(CHCl3/MeOH, 17:3 (v/v)), mp 165–166 8C. MALDI-TOF
MS (matrix 2,4,6-THAP) 679.17, 701.61, 717.64. Calcd
00
00
with D2O), 8.63 (d, 1H, J9 ,10 ¼9.0 Hz, H-10 ), 8.41–8.21
(m, 8H, H-200 –600,800,900, OCONH), 8.14 (apparent t, 1H,
00
00 00
00 00
J6 ,7 ¼J7 ,8 ¼7.5 Hz, H-7 ), 7.72 (d, 2H, Ja,b¼7.9 Hz, H-b),
7.52 (d, 1H, J5,6¼8.0 Hz, H-6), 7.41–7.11 (m, 18H, ArH
(Dmt)), 6.86–6.74 (m, 8H, ArH (Dmt)), 6.16 (d, 1H,
0
1
678.71 [MþH]þ, 700.69 [MþNa]þ, 716.80 [MþK]þ. H
J1 ,2 ¼6.0 Hz, H-1 ), 5.39 (1H, J5,6¼8.0 Hz, H-5), 5.10
0
0
0
0
0
0 0
NMR ([D6]DMSO): d 11.38 (s, 1H, H-3, exchangeable with
(apparent t, 1H, J1 2 ¼J2 3 ¼6.0 Hz, H-2 ), 4.34–4.26 (m,
4H, H-30, H-40, NHCH2), 3.73 (s, 6H), 3.69 (s, 6H) (CH3),
3.30 (m, 2H,‡ H-50).
00 00
00 00
00 00
00 00
D2O), 8.69 (m, 4H, J1 ,2 ¼J3 ,4 ¼J5 ,6 ¼J7 ,8 ¼6.5 Hz,
H-100,400,500,800), 7.99 (br t, 1H, OCONH), 7.93 (d, 1H,
J5,6¼8.1 Hz, H-6), 7.91 (m, 2H, Hc), 7.85 (d, 2H,
Jj,k¼8.1 Hz, Hj),00 70.08000 (00m, 4H, J1 ,2 ¼J3 ,4 ¼J5 ,6
¼
4.1.6. 50-O-(4,40-Dimethoxytrityl)-20-O-[4-(9-phenyl-
ethynylanthracen-10-ylethynyl)phenylmethylamino-
carbonyl]uridine (10). Prepared from 20-O-[4-(9-phenyl-
ethynylanthracen-10-ylethynyl)phenylmethylaminocar-
bonyl]uridine (0.68 g, 1.00 mmol) in similar manner to
compound 5. Yield 0.839 g (85.6%), orange foam. Rf 0.4
(CHCl3–EtOAc 1:1þ0.5% pyridine (v/v/v)). 1H NMR
([D6]DMSO): d 11.42 (s, 1H, H-3, exchangeable with
00 00
00 00
00 00
00 00
J7 ,8 ¼6.5 Hz, H-2 ,3 ,6 ,7 ), 7.54 (m, 3H, Ha, Hb), 7.40
0
0
0
(d, 2H, Jj,k¼8.1 Hz, Hk), 6.05 (d, 1H, J1 ,2 ¼5.7 Hz, H-1 ),
0
5.69 (d, 1H, J5,6¼8.1 Hz, H-5), 5.61 (d, 1H, J3 ,OH¼5.1 Hz,
30-OH), 5.19 (m, 1H, 50-OH) (both exchangeable with D2O),
0
0
0
0
0
5.12 (apparent t, 1H, J1 ,2 ¼J2 ,3 ¼5.7 Hz, H-2 ), 4.26 (m,
3H, H-30, NCH2), 3.93 (m, 1H, H-40), 3.63 (m, 2H, H-500).
13C NMR ([D6]DMSO), d 43.6 (Cm), 60.8 (C50), 68.8 (C3 ),
74.8 (C20), 85.4 (2C, C10, Cg), 85.7 (2C, C40, Cf), 102.1
00 00
00 00
00 00
00 00
D2O), 8.69 (m, 4H, J1 ,2 ¼J3 ,4 ¼J5 ,6 ¼J7 ,8 ¼6.4 Hz,
(C5), 102.7 (Ce), 102.8 (Ch), 117.3, 117.6 (2C, C900, C10 ),
H-100,400,500,800), 8.03 (br t, J¼6.1 Hz, 1H, OCONH), 7.91
00
00
120.4 (Ci), 122.1 (Cd), 126.7 (4C, C1 , C4 , C500, C8 ),
(d, 2H, J¼6.4 Hz), 7.85 (d, 2H, J¼8.0 Hz) (Hc,0j0), 7.80 (m,
00
00
00
00
127.3 (2C, Ck), 127.7 (4C, C200, C300, C6 , C070 ), 128.8 (2C,
Cb), 129.3 (Ca), 131.2 (4C, 4a00, C8a00, C9a , 10a00), 131.6
(4C, Cj, Cc), 140.6 (C6), 141.0 (Cl), 150.5 (C2), 155.4 (Cn),
162.9 (C4). Anal. Calcd for C41H31N3O7: C, 72.66; H, 4.61;
N, 6.20. Found: C, 72.61; H, 4.55; N, 6.24.
J1 ,2 ¼J3 ,4 ¼J5 ,6 ¼J7 ,8 ¼6.4 Hz, H-2 ,3 ,6 ,7 ), 7.73 (d,
00 00 00
00 00
00 00
00 00
00 00
1H, J5,6¼8.0 Hz, H-6), 7.57–7.26 (m, 14H, Ha,b,k, ArH
(Dmt)), 6.90 (d, J¼8.5 Hz, 4H, ArH (Dmt)), 5.98 (d, 1H,
J1 ,2 ¼4.5 Hz, H-1 ), 5.63 (d, 1H, J3 ,OH¼5.0 Hz, 30-OH),
0
0
0
0
0
0
5.41 (d, 1H, J5,6¼8.0 Hz, H-5), 5.11 (apparent t, 1H, J1 ,2
¼
0
0
0
0
J2 ,3 ¼4.5 Hz, H-2 ), 4.40–4.29 (m, 3H, H-3 , NCH2), 4.02
(m, 1H, H-40), 3.74 (s, 6H, CH3), 3.31 (m, 2H,‡ H-50). Anal.
Calcd for C62H49N3O9: C, 75.98; H, 5.04; N, 4.29. Found:
C, 76.21; H, 4.93; N, 4.36. Bis-Dmt-derivative, 50,30-O-bis-
(4,40-dimethoxytrityl)-20-O-[4-(9-phenylethynylanthracen-
10-ylethynyl)phenylmethylaminocarbonyl]uridine, was
isolated as a minor byproduct (28 mg, 2.6%). 1H NMR
([D6]DMSO): d 11.45 (s, 0.7H), 11.42 (m, 0.2H), 11.39 (m,
4.1.5. 50-O-(4,40-Dimethoxytrityl)-20-O-[4-(pyren-1-
ylethynyl)phenylmethylaminocarbonyl]uridine (5). 20-
O-[4-(Pyren-1-ylethynyl)phenylmethylaminocarbonyl]uri-
dine (1.004 g, 1.67 mmol) was coevaporated with toluene
(3£20 mL), pyridine (3£20 mL), dissolved in dry pyridine
(15 mL), cooled in an ice bath, and DmtCl (623 mg,
1.84 mmol) was added in one portion. After completion of
the reaction, the excess of DmtCl was quenched with MeOH
(1 mL), and after 10 min the mixture was diluted with
CHCl3 (100 mL), washed with water (100 mL), 5%
NaHCO3 (100 mL), and water (100 mL), then dried
(Na2SO4), evaporated, coevaporated with toluene
(3£25 mL) and the residue was chromatographed on silica
00 00
00 00
00 00
00 00
0.1H) (H-3), 8.69 (m, 4H, J1 ,2 ¼J3 ,4 ¼J5 ,6 ¼J7 ,8
¼
6.5 Hz, H-100,400,500,800), 8.03 (br t, 1H, J¼6.1 Hz,
OCONH), 7.91–7.60 (m, 32H, H-200,300,600,700, H-6,
Ha,b,c,j,k, ArH (Dmt)), 6.86–6.74 (m, 8H, ArH (Dmt)),
‡
Calculated value; the signal of water is also present in the region.

N. N. Dioubankova et al. / Tetrahedron 60 (2004) 4617–4626
4625
0
0
0
0
6.16 (d, 0.7H, J1 ,2 ¼6.5 Hz), 6.13 (d, 0.2H, J1 ,2 ¼6.5 Hz),
available
20-deoxynucleoside
phosphoramidites
0
0
0
6.09 (d, 0.1H, J1 ,2 ¼6.5 Hz) (H-1 ), 5.39 (m0 , 1H, H-5), 5.11
(Cruachem). Oligonucleotides were synthesized starting
on a 1 mmol scale. Oligonucleotides were isolated using
electrophoresis in 20% denaturing (7 M urea) PAGE in
Tris–borate buffer, pH 8.3. The mass of each oligonucleo-
tide was checked by MALDI-TOF mass spectrometry on a
Perseptive Biosystems Voyager DE mass spectrometer in
positive ion mode using a 1:1 v/v mixture of 2,6-
dihydroxyacetophenone (40 mg/mL in MeOH) and aq.
diammonium hydrogen citrate (80 mg/mL) as a matrix
premixed just before loading the samples onto a plate.
0
0
0
0
(apparent t, 1H, J1 ,2 ¼J2 ,3 ¼4.5 Hz, H-2 ), 4.36–4.24 (m,
4H, H-30, H-40, NCH2), 3.72 (s, 6H), 3.69 (s, 6H) (CH3),
3.30 (m, 2H,‡ H-50).
4.1.7. 30-O-(N,N-Diisopropylamino-2-cyanoethoxy-
phosphinyl)-50-O-(4,40-dimethoxytrityl)-20-O-[4-(pyren-
1-ylethynyl)phenylmethylaminocarbonyl]uridine
(6).
50-O-(4,40-Dimethoxytrityl)-20-O-[4-(pyren-1-ylethynyl)-
phenylmethylaminocarbonyl]uridine (1.00 g, 1.10 mmol)
was coevaporated with dry DCM (2£20 mL), dissolved in
dry DCM, diisopropylammonium tetrazolide (285 mg,
1.66 mmol) and bis(N,N-diisopropylamino)-2-cyano-
ethoxyphosphine (0.527 mL, 1.66 mmol) were added, and
the mixture was stirred under nitrogen for 2 h. After
conversion of the starting compound was complete
(monitoring by TLC) the mixture was diluted with CHCl3,
washed with 5% NaHCO3 (100 mL), 20% NaCl (100 mL),
dried over Na2SO4, evaporated to dryness, and the residue
was chromatographed on silica gel column in stepwise
gradient 5!10!20!25% acetone in CHCl3þ1% Et3N
(v/v/v). Fractions containing product were combined,
evaporated, and the residue was dried in vacuo to afford 6
(1.3 g, 92%) as a yellow foam. Rf 0.17, 0.23 (CHCl3/EtOAc
4.2. Supplementary Data
Original 1H, 13C, 1H–13C HMQC and HMBC NMR spectra
of nucleosides 4 (UP) and 9 (UA).
Acknowledgements
This research was partially supported by RFBR grants
(00-03-32701, 02-03-32376 and 03-03-32196). V.A.K. was
also supported by Ribozyme Pharmaceutical (1999–2001),
A.D.M. was supported by Astra-Zeneca (2002–2003);
N.N.D. thanks the MRC for the support during her visit to
Cambridge in 2002. The authors are grateful to Donna
Williams (MRC Laboratory of Molecular Biology) for
oligonucleotide synthesis and to the elemental analysis
facility of Moscow Institute of Fine Chemical Technology
for elemental analysis data. We thank Professor Vladimir
A. Efimov, Igor A. Prokhorenko (Shemyakin-Ovchinnikov
Institute), Dr. Dmitry A. Stetsenko and Dr. Michael J. Gait
(MRC LMB) for their encouraging interest and generous
support.
1:1þ1%Et3N (v/v/v)). 1H NMR (5% CDCl3 in [D3]MeCN):
00
00
00
d 8.66 (d, 1H, J9 ,10 ¼8.8 Hz, H-10 ), 8.33–8.08 (m, 8H,
H-200 –900), 7.74–7.24 (m, 15H, H-6, ArH (Dmt, He,d),
OCONH), 6.87 (m, 4H0, ArH (Dmt)), 6.08 (m, 1H, H-10),
5.49–5.37 (m, 2H, H-2 ,5), 4.64 (m, 1H, H-30), 4.42–4.33
(m, 2H, NHCH2), 4.01–4.29 (m, 1H, H-40), 3.78 (s, 6H,
CH3), 3.75–3.37 (m, 6H,‡ POCH2, PNCH, H-50), 2.76 (t,
0.9H, J¼5.9 Hz), 2.51 (t, 1.1H, J¼5.9 Hz) (CH2CN,
diastereomers), 1.26–1.05 (m, 12H, CHCH3). 31P NMR
(5% CDCl3 in [D3]MeCN): d 151.13, 150.99 (dia-
stereomers, ,10:8).
4.1.8. 30-O-(N,N-Diisopropylamino-2-cyanoethoxy-
phosphinyl)-50-O-(4,40-dimethoxytrityl)-20-O-[4-(9-
phenylethynylanthracen-10-ylethynyl)phenylmethyl-
aminocarbonyl]uridine (11). 50-O-(4,40-Dimethoxytrityl)-
20-O-[4-(9-phenylethynylanthracen-10-ylethynyl)phenyl-
methylaminocarbonyl]uridine (0.8 g, 0.82 mmol) was
phosphitylated as above with diisopropylammonium tetra-
zolide (21 mg, 1.23 mmol) and bis(N,N-diisopropylamino)-
2-cyanoethoxyphosphine (0.39 mL, 1.23 mmol). The com-
pound was chromatographed on silica gel column in
stepwise gradient 5!10!20!25% acetone in
CHCl3þ1% Et3N (v/v/v). Yield 1.069 g (90.5%). Rf 0.17,
0.23 (CHCl3/EtOAc 1:1þ1%Et3N (v/v/v)). 1H NMR
([D3]MeCN): d 8.70 (m, 4H, H-100,400,500,800), 7.84 (m, 2H),
7.78 (m, 2H) (Hc,j), 7.74 (m, H-200,300,600,700), 7.67 (m, 1H,
H-6), 7.53–7.24 (m, 14H, Ha,b,k, ArH (Dmt)), 6.88 (d,
J¼8.5 Hz, 4H, ArH (Dmt)), 6.08–6.06 (m,01H, H-10), 5.44–
5.36 (m, 2H, H-5), 4.69–4.59 (m, 1H, H-2 ), 4.42–4.01 (m,
4H, H-30, NCH2, H-40), 3.84–3.37 (m, 12H,‡ OCH3,
POCH2, PNCH, H-50), 2.77 (t, 2H, J¼5.8 Hz, CH2CN),
1.28–1.01 (m, 12H, CHCH3). 31P NMR ([D3]MeCN): d
151.00, 150.89 (diastereomers, ,2:1).
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