1-(Phenylethynyl)pyrene and 9,10-bis(phenylethynyl)anthracene, useful fluorescent dyes for DNA labeling: Excimer formation and energy transfer

Article abstract of DOI:10.1002/ejoc.200300677

A series of novel modifying reagents, including phosphoramidites and solid supports, have been synthesized, and used for the introduction of 1-(phenylethynyl)pyrene (PEPy) and 9,10-bis(phenylethynyl)anthracene (BPEA) fluorescent dyes into predetermined positions of synthetic oligonucleotides. These two fluorophores have been shown to constitute an energy donor-acceptor pair, and can be used as such in fluorescent oligonucleotide probes, designed for the detection and structural studies of nucleic acids. The sensitivity of the probe to duplex formation is demonstrated. The formation of the PEPy and BPEA excimers is reported for the first time on nucleic acids. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300677

FULL PAPER
1-(Phenylethynyl)pyrene and 9,10-Bis(phenylethynyl)anthracene, Useful
Fluorescent Dyes for DNA Labeling: Excimer Formation and Energy Transfer
Andrei D. Malakhov,[a] Mikhail V. Skorobogatyi,[a] Igor A. Prokhorenko,[a]
Sergei V. Gontarev,[b] Dmitry T. Kozhich,[c] Dmitry A. Stetsenko,[a][‡] Irina A. Stepanova,[a]
Zakhar O. Shenkarev,[a] Yuri A. Berlin,[a][†] and Vladimir A. Korshun*[a]
Keywords: Bioorganic Chemistry / DNA / Fluorescence / FRET / Excimers
A series of novel modifying reagents, including phosphor-
amidites and solid supports, have been synthesized, and
used for the introduction of 1-(phenylethynyl)pyrene (PEPy)
and 9,10-bis(phenylethynyl)anthracene (BPEA) fluorescent
dyes into predetermined positions of synthetic oligonucleo-
tides. These two fluorophores have been shown to constitute
fluorescent oligonucleotide probes, designed for the detec-
tion and structural studies of nucleic acids. The sensitivity of
the probe to duplex formation is demonstrated. The forma-
tion of the PEPy and BPEA excimers is reported for the first
time on nucleic acids.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
an energy donor−acceptor pair, and can be used as such in Germany, 2004)
Introduction
lexes,[2a,2b,2e,2k] and triplexes.[2g] Excimer emission from oli-
gonucleotides containing
5-(1-pyrenylethynyl)uracil,[3]
trans-stilbene,[4] and perylene[5] has also been reported.
In the cases where two different dyes were used, a relative
proximity of the two moieties and an overlap of the wave-
lengths of one dye’s emission with those of the other dye’s
excitation may result in fluorescence resonance energy
transfer (FRET). FRET is useful in the homogeneous de-
tection of biomolecular interactions.[1a,6] Recent examples
of the application of FRET in nucleic acid chemistry in-
clude structural studies of the complexes of nucleic acids[7]
and of ribozymes,[8] detection of specific sequences based
on energy transfer,[9] and FRET on DNA with multiple[10]
and ‘‘tunable’’[11] fluorophores.
It is interesting, therefore, to broaden the range of fluor-
escent dyes suitable for nucleic acid labeling, and to develop
new methods for their site-specific introduction into oligo-
nucleotides. Herein we describe the synthesis of func-
tionalized derivatives of two fluorescent aromatic hydro-
carbons, 1-(phenylethynyl)pyrene (PEPy) and 9,10-bis-
(phenylethynyl)anthracene (BPEA), and the fluorescence
properties of single- and double-stranded labeled oligonu-
cleotides. Preliminary communications on the PEPy and
BPEA reagents have been published.[12]
Fluorescent techniques are used extensively in nucleic
acid research. Fluorescent labels give a direct, simple-to-
register signal, and the processes involving such groups can
be easily monitored in real time, repeatedly, in biomolecule-
friendly conditions, and even in vivo.[1]
Currently, methods based on the detection of changes in
the emission of interacting pairs of fluorophores (identical
or not) make up a growing field, both in terms of the devel-
opment of these methods and of their application. An ex-
ample from this field is the formation of excimers (exci-
plexes) from two spatially proximal planar fluorophores
linked to a biomolecule. Excimer fluorescence has been ob-
served in aqueous solutions of pyrene-linked nucleic acids
and their analogues.[2] Changes in the excimer/monomer
fluorescence intensity ratio allowed the detection of
complementary
complexes,
including
antiparallel
duplexes,[2c,2d,2f,2h,2i,2mϪ2o,2r,2tϪ2v,2x,2y]
parallel dup-
[a]
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences,
Miklukho-Maklaya 16/10, Moscow 117997, Russia
Fax: (internat.) ϩ 7-095-3306738
E-mail: korshun@mail.ibch.ru
Institute of Physical Organic Chemistry, National Academy of
Sciences of Belarus,
[b]
Surganova 13, Minsk 220072, Belarus
STC of Medico-Biotechnological Institute,
Bjaduli 10, Minsk 220034, Belarus
[c]
[‡]
Results and Discussion
Present address: Medical Research Council Laboratory of
Molecular Biology
9,10-Bis(phenylethynyl)anthracene (BPEA) is one of the
most efficient fluorescent compounds known.[1b,13] Several
substituted bis(phenylethynyl)anthracenes have been report-
ed,[1b,14] but to the best of our knowledge, this dye has been
Hills Road, Cambridge CB2 2QH, UK
Deceased November 23, 2001
[†]
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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DOI: 10.1002/ejoc.200300677
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1-(Phenylethynyl)pyrene and 9,10-Bis(phenylethynyl)anthracene
FULL PAPER
Scheme 1. Preparation of (iodophenyl)-diol 6
never used for the covalent labeling of biomolecules. 2,2-dimethyl-1,3-dioxane-4,6-dione (3) (Meldrum’s acid),[25]
Another parent dye, 1-(phenylethynyl)pyrene (PEPy), is a resulting in the (4-iodophenyl)acetyl derivative 4. Al-
considerably less-studied fluorophore.[3] The introduction coholysis of the cyclic acylal 4 with 2-propanol, ac-
of the phenylethynyl group into a fluorescent molecule usu- companied by decarboxylation, led to ester 5, which crys-
ally leads to a bathochromic shift in absorbance and emis- tallized as an individual keto tautomer (only two-proton
sion, as well as an increase in the quantum yield of emis- singlets at δ ϭ 3.62 and 3.84 ppm, and no lower-field one-
1
sion. This has been shown for naphthalene,[15] anthracene proton singlets, were present in the H NMR spectrum).
and tetracene,[16] pyrene,[17] perylene,[18] 2-phenylbenz- Reduction with NaBH4 in a boiling MeOH/THF mix-
oxazole,[19] bimanes,[20] EuIII chelates,[21] coumarins,[22] ture[26] gave the desired 4-(4-iodophenyl)-1,3-butanediol (6)
BODIPY,[23] and fluorescein.[24]
in 99% yield.
Suitably functionalized phenylethynyl derivatives of py-
We envisaged that the iodine atom could easily be dis-
rene and anthracene could be promising tools for the devel- placed by ethynyl derivatives of polyaromatic hydrocarbons,
opment of fluorescent probes for structural studies of nu- such as 9-ethynyl-10-(phenylethynyl)anthracene (12) (pre-
cleic acids. These compounds exhibit fluorescence within a pared as shown in Scheme 2) or 1-ethynylpyrene (13) (com-
range of 400Ϫ550 nm, have high quantum yields, and are mercially available), under Sonogashira reaction con-
chemically stable and capable of various non-covalent inter- ditions.[27] The resulting pseudonucleosides, after routine di-
actions. In addition, their spectral characteristics suggest methoxytritylation and phosphitylation (Scheme 3), may
that they can potentially be used as a donorϪacceptor pair then serve as monomers in automated oligonucleotide syn-
in non-radiative energy transfer. We therefore embarked on thesis by the phosphoramidite method.
the synthesis of functionalized derivatives of PEPy and
1-Ethynylpyrene (13) was coupled with 4-(4-iodophenyl)-
BPEA. Such derivatives could be used to prepare specifi- 1,3-butanediol (6) in the presence of tetrakis(triphenylphos-
cally modified oligonucleotides, or to study the spectral phane)palladium, copper() iodide, and triethylamine in
properties of these dyes’ conjugates. We tried to mimic the DMF, affording 4-[4-(1-pyrenylethynyl)phenyl]-1,3-butane-
3Ј- and 5Ј-hydroxy groups of natural nucleosides by intro- diol (14) in high yield. For all of the diol 6 to react, a mod-
ducing a 1,3-butanediol group into these fluorophores erate excess of 13 was required, as the alkyne was also con-
(compounds 14 and 17). 4-(4-Iodophenyl)-1,3-butanediol sumed in a side-reaction: the Glaser oxidative dimerization,
(6) was a key compound in the synthesis (Scheme 1).
leading to 1,4-bis(1-pyrenyl)buta-1,3-diyne (15). Although
(4-Iodophenyl)acetyl chloride (2), obtained from the acid the reaction was carried out under argon, the formation
1 by treatment with oxalyl chloride, was used to acylate of 5Ϫ10% of this diacetylene, which is sparingly soluble in
Scheme 2. Preparation of alkyne derivative 12
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V. A. Korshun et al.
FULL PAPER
Scheme 3. Preparation of PEPy and BPEA phosphoramidites and solid supports; reagents: i: [Pd(PPh3)4]/CuI/Et3N/DMF; ii: DmtCl/Py;
iii: (iPr2N)2POCH2CH2CN/diisopropylammonium tetrazolide/MeCN; iv: LCAA-CPG-NHCOCH2CH2CO2H/N,NЈ-diisopropylcarbodi-
imide/DMAP/DMF
common solvents, could not be avoided. Using standard two steps. Compound 11 was also obtained by an alterna-
methods of nucleoside chemistry, pseudonucleoside 14 was tive method, starting from 9-bromo-10-(3-hydroxy-3-meth-
converted into phosphoramidite 19a and support 20a. ylbutyn-1-yl)anthracene (10). Precursor 10 reacted with
These materials were then used in automated DNA syn- phenylacetylene under Sonogashira conditions to give, after
thesis to incorporate the pseudonucleoside into a number chromatographic purification, a 55% yield of 11. Deprotec-
of predetermined positions in oligonucleotide chains. We tion of the ethynyl residue and formation of 9-ethynyl-10-
describe here how these labeled oligonucleotides can be (phenylethynyl)anthracene (12) were achieved by heating
used as fluorescent probes, with the label moiety being po- with powdered KOH in benzene in the presence of dibenzo-
tentially sensitive to changes in its microenvironment; for 18-crown-6. After flash chromatography, compound 12 was
example, the interaction of such a labeled probe with an used in the next Sonogashira reaction with aryl iodide 6 to
oligonucleotide with the complementary nucleic acid give the BPEA-substituted 1,3-butanediol 17 in 67% yield.
sequence.
We were tempted to synthesize a fluorescent pseudonu- support 20b, similarly to the PEPy derivatives (Scheme 3).
cleoside which could serve as an energy acceptor for the The excitation and emission spectra of pseudonucleosides
substituted pyrene 14. We chose to prepare BPEA deriva- 14 and 17 are shown in Figure 1. The compounds exhibit
The diol 17 was converted into phosphoramidite 19b and
max
tives, compounds that emit in the visible region and exhibit strong fluorescence in MeOH, with
λ
ϭ 389 and
em
high quantum yields.[1b] The starting material, 9,10-di- 410 nm for the pyrene derivative 14, and 471 and 501 nm
bromoanthracene (7), was allowed to react with phenyl- for the anthracene derivative 17. It is noteworthy that the
acetylene in the presence of bis(triphenylphosphane)palla- emission band of 14 overlaps with the excitation band of
dium dichloride, copper() iodide, and triethylamine, in a 1,4- 17, and in fact, these dyes constitute
a potential
dioxane/DMF mixture under argon (Scheme 2), resulting in donorϪacceptor pair in non-radiative energy transfer.
a mixture of 9-bromo-10-(phenylethynyl)anthracene (8), un- Therefore, this pair may be useful in the construction of
changed 7, and the parent BPEA (9). Whereas 9 was easily probes for FRET-based determination of intra- and inter-
removed by flash chromatography, dibromide 7 proved to molecular distances on biomolecules.[1a]
be difficult to separate completely. Therefore, the mixture
Starting from pyrene-diol 14, we prepared dimethoxytri-
of mono- and dibromides 7 and 8 was used directly in the tyl derivative 18a, phosphoramidite 19a, and solid support
next coupling with 2-methyl-3-butyn-2-ol, from which the 20a (Scheme 3), using conventional methods of nucleoside
disubstituted anthracene 11 was obtained in 38% yield over chemistry.[28] Similar BPEA reagents 19b and 20b were ob-
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1-(Phenylethynyl)pyrene and 9,10-Bis(phenylethynyl)anthracene
FULL PAPER
reduced their gel mobility. Within a set of equally long oli-
gonucleotides (i.e. 21, 23, 24, and 25; or 21, 29, 30, and
31), mobility decreased with an increase in the number of
modified units. The contributions of the PEPy and BPEA
moieties were almost equal, and roughly corresponded to
one nucleotide increase in length.
The oligonucleotides were then analysed by MALDI-
TOF mass spectrometry and reverse-phase HPLC. Not sur-
prisingly, the highly hydrophobic modifications consider-
ably increased the retention time values of the conjugates
(Table 1).
We showed that the modified pseudonucleosides also af-
fected the thermal stability of oligonucleotide duplexes. A
PEPy or BPEA residue dangling in the terminal position of
a duplex produced a stabilizing effect, as estimated from the
melting curves for duplexes 22·26 and 22·32 (Table 2). Simi-
lar results were obtained upon studying pyrene-containing
complementary heptanucleotides,[31] where the stabilizing
effect was ascribed to stacking of an aromatic polycyclic
system on the adjacent nucleobases.
Figure 1. Normalized excitation spectra of diols 14 (1, λem
ϭ
440 nm) and 17 (3, λem ϭ 520 nm), and fluorescence spectra of 14
(2, λex ϭ 320 nm) and 17 (4, λex ϭ 400 nm) in MeOH (10Ϫ6 )
tained from the anthracene pseudonucleoside 17. These re-
agents were used for introduction of single or multiple
PEPy or BPEA residues into predetermined positions of
oligonucleotides.
We synthesized a series of modified oligonucleotides
21Ϫ34 corresponding to a fragment of the gene for the hy-
The lower degree of stabilization in our case may be due
to the shorter linker arm, and the fact that only one ter-
minus is stabilized, which may be less favourable for stack-
pothetical eukaryotic transcription factor fet5ϩ of Schizo- Table 2. Duplex stability of modified oligonucleotides
saccharomyces pombe[29] (Table 1). Between one and three
Duplex Sequence (Y Ϫ PEPy, Z Ϫ BPEA)[a] Tm [°C] ∆Tm [°C]
substitutions of the PEPy or BPEA pseudonucleosides were
made for dA or dT units. An identical coupling cycle was
21·22
22·26
22·23
22·32
22·29
(5Ј) ACGAGGAAAGCGTAA
(3Ј) TGCTCCTTTCGCATT
57.4
61.9
56.0
59.0
50.6
used for all of the phosphoramidites, which were added as
0.1  solutions in MeCN. Under these conditions, the ef-
ficiency of the coupling of a modified phosphoramidite to
the next standard nucleoside phosphoramidite, as deter-
mined by the acid-catalyzed release of Dmtϩ cation,[30] was
ca. 99% for 19a and 95% for the somewhat bulkier 19b.
After standard ammonia-mediated deprotection, all the
synthetic oligonucleotides 21Ϫ34 were isolated by 20% de-
naturing PAGE. A control treatment of compounds 14 and
17 with ammonia did not reveal (TLC) any transformation
products. The modifications introduced into the oligomers
(5Ј) ACGAGGAAAGCGTAAY
(3Ј) TGCTCCTTTCGCATT
ϩ4.5
Ϫ1.4
ϩ1.6
Ϫ6.8
(5Ј) ACGAGGYAAGCGTAA
(3Ј) TGCTCCTTTCGCATT
(5Ј) ACGAGGAAAGCGTAAZ
(3Ј) TGCTCCTTTCGCATT
(5Ј) ACGAGGZAAGCGTAA
(3Ј) TGCTCCTTTCGCATT
[a]
For conditions, see Exp. Sect.
Table 1. Oligonucleotides synthesized for fluorescence measurements
Compound
Sequence (Y Ϫ PEPy, Z Ϫ BPEA)
Rt[a]
MALDI-TOF mass spectra,
[M ϩ H]ϩ, found/calcd.
21
22
23
24
25
26
27
28
29
30
31
32
33
34
(5Ј) ACGAGGAAAGCGTAA
(3Ј) TGCTCCTTTCGCATT
(5Ј) ACGAGGYAAGCGTAA
(5Ј) ACGAGGYYAGCGTAA
(5Ј) ACGAGGYYYGCGTAA
(5Ј) ACGAGGAAAGCGTAAY
(3Ј) TGCTCCYTTCGCATT
(3Ј) TGCTCCYYTCGCATT
(5Ј) ACGAGGZAAGCGTAA
(5Ј) ACGAGGZZAGCGTAA
(5Ј) ACGAGGZZZGCGTAA
(5Ј) ACGAGGAAAGCGTAAZ
(3Ј) TGCTCCZTTCGCATT
(3Ј) TGCTCCZZTCGCATT
10.2
11.8
4658.8/4660.1
4483.5/4485.9
4797.3/4799.3
4936.0/4938.5
5078.2/5077.8
5112.1/5112.5
4633.1/4634.2
4780.0/4782.4
4876.7/4875.4
5089.4/5090.7
5304.9/5306.1
5189.9/5188.6
4709.8/4710.3
4933.1/4934.6
19.9
32.8, 34.3[b]
44.1, 44.5[b]
25.8
19.7
29.9
26.9
32.5, 33.1, 33.6[b]
37.1, 37.7, 38.3[b]
39.5
23.5
30.9
[a]
[b]
For HPLC conditions, see Exp. Sect. Peaks of diastereomers.
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V. A. Korshun et al.
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ing. At the same time, the dye residues in the middle of the leads to the complete disappearance of the residual mono-
strand somewhat destabilize the duplexes, with this effect meric fluorescence of a single-stranded doubly-modified
being much more pronounced in the case of BPEA oligomer (curve 4; cf. curve 3).
(Table 2). The PEPy pseudonucleoside can effectively re-
place deoxyadenosine (duplex 22·23). However, the melting
temperatures remained sufficiently high for any spectral
studies to be performed.
The introduction of both fluorescent labels into the oli-
gonucleotides was confirmed by the appearance, in addition
to the inherent oligonucleotide absorption maximum
around 260 nm, of maxima due to the dyes, in the region
of 350Ϫ400 nm for PEPy, and 420Ϫ500 nm for BPEA.
Next, we studied the luminescent characteristics of the
fluorescently labeled oligonucleotides and duplexes. The
fluorescence spectra were registered in aqueous phosphate
buffer (pH ϭ 7.0) at an excitation wavelength of 370 nm for
PEPy- and 430 nm for BPEA-containing oligonucleotides.
As Figure 2 shows, the introduction of a single PEPy resi-
due (oligomer 23) results in a characteristic monomeric flu-
orescence spectrum, with maxima at 401 and 423 nm (curve
Figure 3. Fluorescence spectra of modified oligonucleotides 23 (1)
and 24 (3), and duplexes 23·27 (2) and 24·28 (4); λex ϭ 370 nm
1). When a second, adjacent, PEPy residue appears (i.e. in
On the other hand, a dramatic change in the emission
spectrum of the bis(modified) oligonucleotide 24 occurred
upon formation of a duplex with unmodified oligonucleo-
tide 22: the excimer fluorescence that was seen in a spec-
trum of 24 alone (Figure 4, curve 1) almost completely dis-
appeared, giving way to intense monomer fluorescence
(Figure 4, curve 2). This change may be caused by the fact
that duplex formation impedes the interaction of the two
PEPy residues, and, as a consequence, diminishes the prob-
ability of the formation of an excited dimer. Hence, oligonu-
cleotides bearing two adjacent fluorophore residues may be
promising tools in constructing sensitive and specific probes
for nucleic acids. It should be noted that in the case of the
similar oligonucleotide 25, which contains three neighbor-
ing dye residues, duplex formation with 22 is not sufficient
to decouple all the interactions of the dye residues: excimer
fluorescence of 25 remains virtually unchanged, with no
monomeric fluorescence detectable (Figure 4, curve 3).
24), the monomeric fluorescence decreases approximately
sixfold, and an intense excimer fluorescence with a maxi-
mum at 507 nm (curve 2) can be seen. Moving to the case
of three adjacent PEPy residues (i.e. in 25) results in a com-
plete disappearance of the monomeric fluorescence, a slight
hypsochromic shift of the excimer maximum to 505 nm
(curve 3), and some decrease in its intensity, apparently be-
cause of self-quenching.
Figure 2. Fluorescence spectra of modified oligonucleotides 23 (1),
24 (2), and 25 (3); λex ϭ 370 nm
Since a single-stranded oligonucleotide 24 containing two
adjacent PEPy residues showed an intense excimer fluor-
escence, we then checked whether an excimer would be
formed in the case of a duplex in which two dye residues,
one in each of the complementary strands, were positioned
opposite one another (duplex 23·27). It can be seen from
Figure 3 that formation of a duplex (curve 2) does not sub-
stantially affect the intrinsic luminescent properties of the
PEPy residues (curve 1), nor does any major change occur
in the case of duplex 24·28, which has two adjacent PEPy
Figure 4. Fluorescence spectra of modified oligonucleotide 24 (1)
and duplexes 22·24 (2) and 22·25 (3); λex ϭ 370 nm
In contrast to the PEPy derivatives, the fluorescence spec-
residues in each strand (curve 3). Duplex formation only tra of the BPEA conjugates 27Ϫ29 are essentially indepen-
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1-(Phenylethynyl)pyrene and 9,10-Bis(phenylethynyl)anthracene
FULL PAPER
dent of the number of dye residues in the molecule (they all spectrum of BPEA; the ratio of fluorescence intensity at
display maxima at 487 and 517 nm),[12b] and are not affec- 490 nm versus 540 nm gradually decreases from 5 (curve 3)
ted by hybridization with complementary oligonucleotides. to 2.2 (curve 2) to 1.5 (curve 4). The appearance of emission
At the same time, modification of both strands of a du- in the region of 530Ϫ580 nm may reflect the contribution
plex (duplex 29·33), leads to a strong quenching of fluor- of the PEPyϪBPEA exciplex to the overall fluorescence.
escence and the appearance of a new band in the region of
530Ϫ630 nm (Figure 5, curve 1), probably due to excimer
formation. Excimer emission has previously been detected
for some fluorophores whose excited state has a nanose-
cond lifetime (which is characteristic of BPEA
derivatives),[14c,32] namely, trans-stilbene[4b,4c] and perylene[5]
residues that were attached to nucleic acids and were spati-
ally close due to duplex formation. In duplex 30·34, where
two pairs of adjacent fluorophores are in the opposite
strands, the excimer band is absent (Figure 5, curve 2).
Apparently, the formation of an excimer, which is possible
in the case of duplex 29·33, is precluded in duplex 30·34,
possibly because of increased steric hindrance.
Figure 6. Fluorescence spectra of a mixture of non-complementary
modified oligonucleotides 23 and 29 (1), and duplexes 23·33 (2),
26·33 (3), and 24·34 (4); λex ϭ 370 nm
Figure 5. Fluorescence spectra of modified duplexes 29·33 (1) and
30·34 (2); λex ϭ 430 nm
Figure 7. Excitation spectra of modified oligonucleotides 23 (1),
λem 440 nm, 29 (2), and duplexes 23·33 (3), 29·33 (4), λem ϭ 520 nm
A study of the spectral properties of duplexes 23·33,
26·33, and 24·34 (Figures 6 and 7) revealed an non-radiative
energy transfer. Apparently, BPEA is an efficient energy ac-
ceptor for PEPy. Hybridization of the sequences containing
these dyes results in FRET (Figure 6, curves 2Ϫ4); exci-
tation in the wavelength range in which PEPy absorbs leads
to distinct BPEA fluorescence. Such a donorϪacceptor pair
could allow the efficient detection of duplex formation, and
could therefore become a useful tool in structural studies.
It should be noted that when the donor and acceptor are
placed opposite each other in complementary strands (du-
plex 23·33), the energy transfer between two residues is less
effective (curve 2) than when the donor is at the end of one
oligonucleotide (duplex 26·33), separated from acceptor by
In the case of duplex 24·34 (Figure 6, curve 4), which
`
contains two donors vis-a-vis two acceptors in the com-
plementary strands, excitation with wavelength correspond-
ing to the PEPy absorption maximum leads only to fluor-
escence corresponding to BPEA. This illustrates an es-
pecially effective energy transfer between two fluorophores.
The occurrence of non-radiative energy transfer was also
confirmed by recording the excitation spectra (Figure 7).
Curves 1 and 2 display spectral characteristics of the PEPy
oligonucleotide 23 and the BPEA oligonucleotide 29,
respectively. At the same time, the excitation spectrum of
duplex 23·33, whose fluorescence occurs outside the emis-
sion region of PEPy (λem ϭ 520 nm), clearly contains two
peaks corresponding to the excitation maxima of the parent
hydrocarbon (curve 3).
˚
ca. 25 A (curve 3). This may be due to a higher mobility of
the donor moiety in the terminal position, which in turn
increases the κ2 factor value (i.e. the possibility of attaining
the most favorable orientation of dipole moments of the
donor and acceptor within the lifetime of the donor’s ex-
cited state). It is noteworthy that this effect leads to signifi-
Conclusion
We have described the chemical synthesis of reagents suit-
cant changes in the characteristic structure of the emission able for the introduction of 1-(phenylethynyl)pyrene (PEPy)
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V. A. Korshun et al.
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drum’s acid (3) (6.29 g, 43.7 mmol) and pyridine (6.8 mL,
87.3 mmol) in dry CH2Cl2 (35 mL), cooled in an ice bath. The bath
was removed, and stirring was continued for 1 h. The reaction mix-
ture was diluted with CHCl3 (100 mL), washed with 1  HCl
(100 mL) and H2O (100 mL), dried with Na2SO4, and the solvents
were evaporated. The resulting crude isopropylidene 2-[2-(4-iodo-
phenyl)ethylidene]malonate (4) was mixed with 2-propanol
(100 mL) and refluxed for 4 h. After concentration, the residue was
purified by chromatography on silica gel using 1% (v/v) EtOAc in
benzene to afford ester 5 (13.76 g, 84%) as a colorless oil, which
crystallized on storage, m.p. 54 °C (benzene/hexane), Rf ϭ 0.16
(benzene). MALDI TOF MS: obsd. 346.8 [M ϩ H]ϩ, calcd. for
C13H15IO3 346.2. 1H NMR ([D6]DMSO): δ ϭ 1.18 (d, J ϭ 6.2 Hz,
6 H, CH3), 3.62 (s, 2 H, COCH2CO), 3.84 (s, 2 H, ArCH2), 4.91
[sept, J ϭ 6.2 Hz, 1 H, (CH3)2CH], 6.99 (d, J ϭ 8.2 Hz, 2 H, ArH),
7.67 (d, J ϭ 8.2 Hz, 2 H, ArH) ppm. 13C NMR ([D6]DMSO): δ ϭ
21.62 (2 C), 48.22, 48.93, 68.20, 92.83, 132.36 (2 C), 134.04, 137.16
(2 C), 166.71, 201.00 ppm.
and 9,10-bis(phenylethynyl)anthracene (BPEA) residues
into any predetermined position of synthetic oligonucleo-
tides. The properties of the conjugates thus obtained might
be useful in the construction of new fluorescent probes for
the detection of nucleic acids or for structural studies. Du-
plex formation can be monitored either by observing
changes in the monomer/excimer emission ratio, or by ob-
serving FRET between PEPy and BPEA.
Experimental Section
General Remarks: (4-Iodophenyl)acetic acid[33] (now available from
Lancaster and Aldrich), Meldrum’s acid[34] (also available from all
major companies), 1-ethynylpyrene[3] (the compound available
from Lancaster should be purified by chromatography using 15%
CHCl3 in hexanes), tetrakis(triphenylphosphane)palladium(0)[35]
(the quality of commercially available batches varies), diisopro-
pylammonium tetrazolide,[28] and (2-cyanoethoxy)bis(diisopropyl-
amino)phosphane[36] (good quality compound is available from
Fluka) were prepared as described. Other chemicals and solvents
4-(4-Iodophenyl)-1,3-butanediol (6): Sodium borohydride (1.22 g;
30 mmol) was added to a solution of isopropyl 4-(4-iodophenyl)-3-
oxobutanoate (5) (2.08 g; 6.0 mmol) in THF (25 mL). The mixture
was refluxed for 30 min and then, after dropwise addition of
MeOH (30 mL), for a further 30 min. After cooling, the reaction
mixture was diluted with an equal volume of CHCl3, washed with
1  HCl (2 ϫ 50 mL) and H2O (2 ϫ 50 mL), dried with Na2SO4,
and the solvents were evaporated to dryness. The residue was puri-
fied by chromatography on a silica gel column eluted with 1% (v/
v) MeOH in CHCl3 to give diol 6 as a colorless crystalline solid,
yield 1.74 g (99%), Rf ϭ 0.33 (5% MeOH in CHCl3), m.p. 84 °C
(benzene/hexane). MALDI-TOF MS: obsd. 292.9 [M ϩ H]ϩ;
calcd. for C10H13IO2 292.1. 1H NMR ([D6]DMSO): δ ϭ 1.48Ϫ1.54
(m, 2 H, CH2CH2O), 2.60 (m, 2 H, ArCH2), 3.49 (m, 2 H, CH2O),
3.74 (m, 1 H, CHO), 4.35 (t, J ϭ 5.1 Hz, 1 H, CH2OH), 4.49 (d,
J ϭ 5.5 Hz, 2 H, CHOH), 7.02 (d, J ϭ 8.2 Hz, 2 H, ArH), 7.61 (d,
J ϭ 8.2 Hz, 2 H, ArH), ppm.
˚
were obtained from Fluka and Aldrich. LCAA-CPG 500 A pore
size was obtained from Pierce. DMF was distilled under reduced
pressure. MeCN, pyridine, and CH2Cl2 were distilled from CaH2.
Other solvents were used as received. TLC was performed on the
Kieselgel 60 F254 pre-coated aluminium plates (Merck), spots were
visualized under UV light (254 and 366 nm) and, in the case of
dimethoxytritylated compounds, by exposing the plate to TFA va-
pours. Column chromatography was carried out using Kieselgel 60
(0.040Ϫ0.063 mm, Merck). 500 MHz 1H and 125.7 MHz 13C
NMR spectra were recorded with a Bruker DRX-500 spectrometer
and referenced to [D6]DMSO (δ ϭ2.50 ppm for 1H and 39.70 ppm
1
for 13C) and CDCl3 (δ ϭ7.25 ppm for H). 161.9 MHz 31P spectra
were recorded with a Varian XR-400 spectrometer with chemical
shifts (δ) referenced to 85% aq. H3PO4 as external standard. 1H
NMR coupling constants are reported in Hz and refer to the ap-
parent multiplicities. Full 1H and 13C signal assignment was ob-
tained by a combination of standard homonuclear and heteronu-
9-(3-Hydroxy-3-methylbutyn-1-yl)-10-(phenylethynyl)anthracene
(11). A: Phenylacetylene (1.02 g, 10 mmol), [Pd(PPh3)2Cl2] (140 mg,
0.2 mmol), CuI (95 mg, 0.5 mmol), and Et3N (2.09 mL, 15 mmol)
were added to a solution of 9,10-dibromoanthracene (7) (3.36 g,
10 mmol) in DMF (50 mL) and 1,4-dioxane (20 mL) under argon.
The reaction mixture was heated at 80 °C for 24 h, then cooled to
room temperature. The mixture was diluted with CH2Cl2 (150 mL)
and washed with H2O (100 mL), 0.3  diammonium EDTA
(100 mL), and H2O (5 ϫ 100 mL), was dried with Na2SO4, and
then the solvents were evaporated. Chromatography on a short sil-
ica gel column, eluting with benzene, gave 10-bromo-9-(phenylethy-
nyl)anthracene (8) containing a considerable amount of the starting
material, 9,10-dibromoanthracene (7) (4.9 g). This mixture was dis-
solved in DMF (60 mL), and 2-methyl-3-butyn-2-ol (1.5 g,
17.8 mmol), [Pd(PPh3)2Cl2] (140 mg, 0.2 mmol), CuI (57 mg,
0.3 mmol), and Et3N (2.02 g, 20 mmol) were added. The reaction
mixture was stirred under argon at 80 °C for 48 h and then treated
as above. After chromatography eluting with benzene, the yield of
11 was 1.40 g (38%), Rf ϭ 0.66 (benzene/MeOH, 9:1, v/v), Rf ϭ
0.45 (benzene/EtOAc, 17:3, v/v), m.p. 136Ϫ139 °C (benzene/hex-
1
1
clear techniques (1H double resonance, H NOESY, H-13C gradi-
ent-selected HMQC and HMBC). Melting points were determined
using a Boetius heating table and are uncorrected. MALDI-TOF
mass spectra were recorded with a VISION 2000 spectrometer
(Thermo Bioanalysis Corp). MALDI-TOF mass spectra of oligo-
nucleotides and amidites 19a,b were recorded with a Voyager-DE
BioSpectrometry Workstation (PerSeptive Biosystems) in positive
ion mode, using a mixture (1:1, v/v) of 2,6-dihydroxyacetophenone
(40 mg/mL in MeOH) and aqueous diammonium hydrogen citrate
(80 mg/mL), mixed just before loading the samples onto a plate, as
the matrix. Thermal denaturation experiments with oligonucleotide
duplexes were performed with a Gilford 2400-2 spectrometer with
a thermostatted cell (200 µL) in a buffer containing 100 m NaCl,
10 m sodium phosphate, 0.1 m EDTA, pH ϭ 7.0. Double-
stranded DNA concentrations were 5·10Ϫ6 , heating rate 1
°C·minϪ1. Fluorescence spectra were obtained using a Hitachi F-
4000 spectrofluorometer at 20 °C.
Isopropyl 4-(4-Iodophenyl)-3-oxobutanoate (5): Oxalyl chloride
ane). MALDI-TOF MS: obsd. 360.2 [M ϩ H]ϩ, calcd. for C27H20O
1
(8.1 mL, 94.6 mmol) was added to a suspension of (4-iodophenyl)- 360.5. H NMR ([D6]DMSO): δ ϭ 1.70 (s, 6 H, CH3), 5.82 (br.s.,
acetic acid (12.4 g, 47.3 mmol) in benzene (20 mL). The acid dis-
solved within a few hours to give a greenish solution, which was ArH), 8.55 (m, 2 H, ArH), 8.66 (m, 2 H, ArH) ppm. 13C NMR
kept at room temperature for 24 h, after which time the solvents ([D6]DMSO): δ ϭ 31.84 (2 C), 64.47, 76.89, 85.86, 102.53, 110.05,
were evaporated in vacuo. The resulting (4-iodophenyl)acetyl chlo- 117.10, 118.24, 122.39, 126.84 (2 C), 126.95 (2 C), 127.61 (2 C),
ride was added dropwise within 30 min to a stirred solution of Mel- 127.84 (2 C), 129.09 (2 C), 129.49, 131.39 (2 C), 131.44 (2 C),
1 H, OH), 7.54 (m, 3 H, ArH), 7.77 (m, 4 H, ArH), 7.88 (m, 2 H,
1304
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1298Ϫ1307

1-(Phenylethynyl)pyrene and 9,10-Bis(phenylethynyl)anthracene
FULL PAPER
131.79 (2 C) ppm. B: 9-Bromo-10-(3-hydroxy-3-methylbutyn-1-yl)-
anthracene (10) (203 mg, 0.6 mmol), prepared by a modified meth-
1 H, CHO), 4.35 (t, J ϭ 5.0 Hz, 1 H, CH2OH), 4.57 (d, J ϭ 5.5 Hz,
2 H, CHOH), 7.39 (d, 2 H, J ϭ 8.2 Hz, C6H4), 7.54 (m, 3 H, Ph),
od[14a] [13C NMR ([D6]DMSO): δ ϭ 31.78 (2 C), 64.44, 76.46, 7.80 (m, 6 H, anthracene, C6H4), 7.90 (m, 2 H, Ph), 8.68 (m, 4 H,
109.40, 117.96, 122.77, 126.96 (2 C), 127.64 (2 C), 127.70 (2 C), anthracene) ppm.
128.46 (2 C), 129.64 (2 C), 132.32 (2 C) ppm] was treated in 20 mL
of DMF with phenylacetylene (73 mg, 0.72 mmol), [Pd(PPh3)4]
O1-(4,4Ј-Dimethoxytrityl)-4-[4-(1-pyrenylethynyl)phenyl]-1,3-
butanediol (18a): 4-[4-(1-Pyrenylethynyl)phenyl]-1,3-butanediol (14)
(69 mg, 0.06 mmol), CuI (29 mg, 0.12 mmol), and Et3N (167 µL,
(290 mg, 0.743 mmol) was co-evaporated with dry pyridine (2 ϫ
1.2 mmol) at 60 °C for 7 h to give 118 mg (55%) of 10, identical
20 mL), dissolved in dry pyridine (30 mL), and the solvents evapo-
by melting point and UV spectrum to the compound obtained by
rated to two thirds of their volume. 4,4Ј-Dimethoxytrityl chloride
method A.
(266 mg, 0.785 mmol) was added with vigorous stirring, and the
9-Ethynyl-10-(phenylethynyl)anthracene (12): Finely powdered
KOH (300 mg, 5.36 mmol) and dibenzo-18-crown-6 (200 mg, reaction was complete according to TLC. The mixture was diluted
0.56 mmol) were added under argon to a solution of 9-(3-hydroxy- with CH2Cl2 (100 mL) and washed with a saturated Na2CO3 solu-
3-methylbutyn-1-yl)-10-(phenylethynyl)anthracene (11) (800 mg, tion (2 ϫ 50 mL). The organic layer was dried with Na2SO4 and
reaction mixture was stirred at room temperature. After 24 h, the
2.2 mmol) in benzene (60 mL). The mixture was heated at 60 °C
for 3 h, then cooled and diluted with benzene (150 mL), then
washed with H2O (100 mL), 5% citric acid (100 mL), and H2O
(100 mL). The organic layer was dried (Na2SO4) and the solvents
were evaporated. The residue was purified by chromatography on
silica gel eluted with benzene to give alkyne 12 (490 mg, 73%), Rf ϭ
0.68 (hexane/benzene, 1:4, v/v), m.p. Ͼ 300 °C (benzene/hexane).
MALDI-TOF MS: obsd. 303.3 [M ϩ H]ϩ, calcd. for C24H14 302.4.
the solvents were evaporated. The residue was purified by chroma-
tography on a silica gel column (3.5 ϫ 15 cm) eluted with 0Ϫ5%
of EtOAc in benzene containing 0.1% Et3N. Yield of compound
18a as a yellow amorphous solid was 471 mg (91%), Rf ϭ 0.32
(benzene/EtOAc, 95:5, v/v). MALDI-TOF MS: obsd. 694.0 [M ϩ
1
H]ϩ, calcd. for C34H26O2 692.8. H NMR ([D6]DMSO): δ ϭ 1.66
(m, 2 H, CH2CH2O), 2.70 (m, 2 H, ArCH2), 3.09 (m, 2 H, CH2O),
3.73 (s, 6 H, OCH3), 3.86 (m, 1 H, CHO), 4.58 (d, J ϭ 5.5 Hz, 1
1H NMR (CDCl3): δ ϭ 4.07 (s, 1 H, ϵCH), 7.44 (m, 3 H, ArH), H, CHOH), 6.88 [d, J ϭ 8.6 Hz, 4 H, ArH (Dmt)], 7.22 [d, J ϭ
7.62 (m, 4 H, ArH), 7.77 (m, 2 H, ArH), 8.62 (m, 2 H, ArH), 8.68
(m, 2 H, ArH) ppm.
8.6 Hz, 4 H, ArH (Dmt)], 7.28Ϫ7.39 [m, 7 H, ArH (Ph, C6H4)],
7.68 [d, J ϭ 8.0 Hz, 2 H, ArH (C6H4)], 8.10Ϫ8.42 [m, 8 H, ArH
(pyrene)], 8.63 [d, J ϭ 9.2 Hz, 1 H, (pyrene 10-H)] ppm. 13C NMR
([D6]DMSO): δ ϭ 37.02, 43.92, 55.16 (2 C), 60.55, 68.66, 85.41,
87.93, 95.70, 113.27 (4 C), 117.13, 119.99, 123.60, 123.85, 125.01,
125.11, 126.11 (2 C), 126.69, 126.92, 127.40, 127.83 (2 C), 127.90
(2 C), 128.48, 128.97, 129.74 (4 C), 130.06 (2 C), 130.68, 130.96,
131.03, 131.18, 131.40 (2 C), 136.25 (2 C), 140.91, 145.41, 158.12
(2 C) ppm.
4-[4-(1-Pyrenylethynyl)phenyl]-1,3-butanediol (14): 1-Ethynylpyrene
(13) (250 mg, 1.1 mmol), Et3N (280 µL, 2 mmol), CuI (38 mg,
0.2 mmol), and [Pd(PPh3)4] (116 mg, 0.1 mmol) were added to a
stirred solution of 4-(4-iodophenyl)-1,3-butanediol (6) (292 mg,
1 mmol) in DMF (20 mL) under argon. After 24 h at room tem-
perature, TLC showed that the reaction had gone to completion.
The precipitate of butadiyne 15 was filtered off, and the reaction
mixture was diluted with CH2Cl2 (100 mL) and washed with 0.3 
O1-(4,4Ј-Dimethoxytrityl)-4-{4-[10-(phenylethynyl)anthracen-9-
diammonium EDTA (2 ϫ 50 mL) and H2O (5 ϫ 50 mL). The or- ylethynyl]phenyl}-1,3-butanediol (18b) was prepared from the bis-
ganic layer was dried (Na2SO4) and the solvents were evaporated.
The residue was purified by chromatography on a silica gel column
eluted with EtOAc/benzene (3:2, v/v) to give diol 13 (422 mg, 98%),
m.p. 120Ϫ122 °C (benzene), Rf ϭ 0.53 (THF), 0.22 (EtOAc).
(phenylethynyl)anthracene diol 17 (500 mg, 1.07 mmol) and 4,4Ј-
dimethoxytrityl chloride (360 mg, 1.07 mmol) in pyridine. The title
compound was purified by chromatography on silica gel in 0Ϫ10%
MeOH in benzene. The yield was 750 mg (91%) of an amorphous
MALDI-TOF MS: obsd. 391.9 [M ϩ H]ϩ, calcd. for C28H22O2 orange solid, Rf ϭ 0.41 (benzene/EtOAc, 95:5, v/v). MALDI-TOF
390.5. 1H NMR ([D6]DMSO): δ ϭ 1.45Ϫ1.62 (m, 2 H, CH2CH2O),
MS: obsd. 770.1 [M ϩ H]ϩ, calcd. for C55H44O4 768.9. H NMR
1
2.74 (m, 2 H, ArCH2), 3.54 (m, 2 H, CH2O), 3.85 (m, 1 H, CHO), (CDCl3): δ ϭ 1.75Ϫ1.85 (m, 2 H, CH2CH2O), 2.20Ϫ2.30 (m, 1 H,
4.35 (t, J ϭ 5.0 Hz, 1 H, CH2OH), 4.57 (d, J ϭ 5.5 Hz, 2 H, CHHO), 2.80Ϫ3.00 (m, 2 H, ArCH2), 3.40Ϫ3.45 (m, 1 H, CHHO),
CHOH), 7.35 (d, J ϭ 8.1 Hz, 2 H, C6H4), 7.68 (d, J ϭ 8.1 Hz, 2
4.04Ϫ4.10 (m, 1 H, CHO), 6.86 (d, 4 H, ArH), 7.20Ϫ7.40 (m, 9 H,
H, C6H4), 8.10Ϫ8.42 [m, 8 H, ArH (pyrene)], 8.61 (d, J ϭ 8.8 Hz, ArH), 7.45Ϫ7.54 (m, 5 H, ArH), 7.60Ϫ7.80 (m, 8 H, ArH),
1 H, pyrene 10-H) ppm. 13C NMR ([D6]DMSO): δ ϭ ca. 40, 43.95,
58.26, 68.62, 87.86, 95.71, 117.14, 119.92, 123.59, 123.85, 125.01,
125.09, 126.09 (2 C), 126.91, 127.39, 128.47, 128.96, 129.68, 130.12
(2 C), 130.68, 131.01 (2 C), 131.17, 131.35 (2 C) ppm.
8.60Ϫ8.80 (m, 4 H, ArH) ppm.
O3-[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]-O1-(4,4Ј-
dimethoxytrityl)-4-[4-(1-pyrenylethynyl)phenyl]-1,3-butanediol (19a):
O1-(4,4Ј-Dimethoxytrityl)-4-[4-(1-pyrenylethynyl)phenyl]-1,3-bu-
4-{4-[10-(Phenylethynyl)anthracen-9-ylethynyl]phenyl}-1,3- tanediol (18a) (346 mg, 0.5 mmol) was co-evaporated with dry
butanediol (17): This compound was prepared in a similar manner,
from 4-(4-iodophenyl)-1,3-butanediol (6) (292 mg, 1.0 mmol),
[Pd(PPh3)4] (23 mg, 0.02 mmol), CuI (10 mg, 0.05 mmol), Et3N
(1.4 mL, 10 mmol), and alkyne 12 (a concentrated benzene solution
after the deprotection of 1.3 mmol of compound 11) in DMF
MeCN (2 ϫ 25 mL) and dissolved in dry MeCN (150 mL). Diiso-
propylammonium tetrazolide (47 mg) and (2-cyanoethoxy)bis(di-
isopropylamino)phosphane (166 mg, 175 µL, 0.55 mmol) were ad-
ded. The reaction mixture was concentrated to half its volume
(bath temperature 30Ϫ35 °C) and stirred for a further 5 h, after
(20 mL), except that the reaction was carried out at 25 °C for 12 h. which time the solvents were evaporated. The residue was dissolved
Side product 16 was filtered off, and the subsequent workup was
similar to that for compound 14. After chromatography with a
gradient from 0 to 50% Et2O in benzene, the yield of diol 17 was
410 mg (67%), Rf ϭ 0.46 (benzene/MeOH, 4:1, v/v), m.p. 195Ϫ197
in EtOAc (120 mL) and washed successively with brine (2 ϫ
100 mL), 5% NaHCO3 (100 mL), and H2O (2 ϫ 100 mL); the or-
ganic layer was dried with Na2SO4 and the solvents were evapo-
rated. The residue was purified by chromatography on a silica gel
°C (benzene). MALDI-TOF MS: obsd. 466.1 [M ϩ H]ϩ, calcd. for column, eluting with CH2Cl2 containing 1% (v/v) Et3N, to yield
1
C34H26O2 466.6. H NMR ([D6]DMSO): δ ϭ 1.45Ϫ1.62 (m, 2 H,
amidite 19a (295 mg, 68%) as a yellow amorphous solid, Rf ϭ 0.78
CH2CH2O), 2.76 (m, 2 H, ArCH2), 3.54 (m, 2 H, CH2O), 3.86 (m, (CH2Cl2/hexane/Et3N, 9:9:2, v/v/v). MALDI-TOF MS: obsd. 894.4
Eur. J. Org. Chem. 2004, 1298Ϫ1307
www.eurjoc.org  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1305

V. A. Korshun et al.
FULL PAPER
[M ϩ H]ϩ, calcd. for C58H57N2O5P 893.1. 31P NMR (CD3CN):
δ ϭ 150.349 and 151.362 ppm (1:1 mixture of diastereomers).
achieved by RP-HPLC on a SOTA C18 column 4.5 ϫ 250 mm,
linear gradient from 5 to 50% MeCN in 0.1  NH4OAc for 60 min,
or linear gradient from 5 to 45% MeCN in 0.1  NH4OAc for
40 min. Analytical HPLC was performed under the same con-
ditions. Duplexes were prepared from the equimolar amounts of
corresponding oligonucleotides in 10 m potassium phosphate
buffer (pH ϭ 7.0) supplemented with 100 m NaCl and 0.1 m
EDTA by heating for 5 min at 95 °C and cooling down to 20 °C
within 1 h. Fluorescence spectra were recorded in this buffer at the
resulting concentrations of oligonucleotides and duplexes, which
was 1·10Ϫ7 .
O3-[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]-O1-(4,4Ј-
dimethoxytrytyl)-4-{4-[10-(phenylethynyl)anthracen-9-ylethynyl]-
phenyl}-1,3-butanediol (19b): This compound was prepared from
dimethoxytritylated diol 18b (700 mg, 0.91 mmol), diisopropylam-
monium tetrazolide (156 mg, 0.91 mmol), and (2-cyanoethoxy)bis-
(diisopropylamino)phosphane (318 µL, 1 mmol) as described
above. After chromatography on silica gel, eluting with 0Ϫ5%
EtOAc in benzene containing 1% Et3N, the yield of phosphoramid-
ite 19b was 700 mg (66%) of an amorphous orange solid, Rf ϭ 0.51
(benzene/EtOAc, 93:7, v/v). MALDI-TOF MS: obsd. 970.1 [M ϩ
H]ϩ, calcd. for C64H61N2O5P 969.2. 31P NMR (CD3CN): δ ϭ
150.31 and 151.34 ppm (1:1 mixture of diastereomers).
Acknowledgments
This project was supported by the Russian Foundation for Basic
Solid Supports 20a,b: These were prepared by a modification of a
Research,
grants
98Ϫ03Ϫ33017,
00Ϫ03Ϫ32701,
and
published method.[37] LCAA CPG-500 A (300 mg) was suspended
˚
03Ϫ03Ϫ32196. The authors are grateful to Jul. G. Molotkovsky
(ShemyakinϪOvchinnikov Institute of Bioorganic Chemistry) for
generously sharing his laboratory’s fluorometric facilities, Yu. P.
Koz’min (same Institute) for mass spectroscopic data, and E. I.
Lazhko (Institute of New Antibiotics, Moscow) for 31P NMR spec-
tra. 1H NMR spectra were kindly provided by the
ShemyakinϪOvchinnikov Institute NMR Spectrometry Facility
(registry No. 96Ϫ03Ϫ08).
in a 3% (v/v) TFA solution in CH2Cl2 (10 mL), stirred at room
temperature for 4 h, filtered, washed successively with 10-mL por-
tions of an MeCN/Et3N (9:1, v/v) mixture, CH2Cl2, and Et2O, and
dried in vacuo over P4O10. A solution of succinic anhydride
(300 mg, 3 mmol) and DMAP (80 mg, 0.66 mmol) in dry pyridine
(10 mL) was added to this support under argon, and the mixture
was left at room temperature for 24 h with occasional swirling.
After filtration, successive washes with 10-mL portions of pyridine,
CH2Cl2, and Et2O, and drying over P4O10, the succinylated LCAA
CPG was suspended in DMF/pyridine (4 mL, 1:1, v/v); monotrityl-
ated pseudonucleoside 18a (173 mg, 0.25 mmol) (or 192 mg of 18b),
1,3-diisopropylcarbodiimide (280 µL, 1.8 mmol), and DMAP
(20 mg) were added; and the suspension was left at room tempera-
ture for 48 h. To block the remaining carboxylic groups, a solution
of pentafluorophenol (100 mg) in pyridine (1 mL) was then added,
and the mixture was kept at room temperature for a further 12 h.
The support was filtered, resuspended in a 5% (v/v) solution of
pyrrolidine in pyridine (3 mL), and allowed to react for 10 min.
The support was filtered again, washed successively with 10-mL
portions of CHCl3, MeOH, MeCN, and Et2O, and dried in vacuo.
Loadings of the pseudonucleoside were determined by treating a
portion (5Ϫ7 mg) of the support with 1 mL of 3% (w/v) CCl3CO2H
in 1,2-dichloroethane and measuring the absorbance of the re-
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S.
sulting
dimethoxytrityl
cation
at
504 nm
(ε
ϭ
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