Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: Synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne click reaction

Article abstract of DOI:10.1039/b719459e

5-(Octa-1,7-diynyl)-2′-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C≡?C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(i)-catalyzed Huisgen-Sharpless-Meldal cycloaddition click reaction . Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative 3 and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b719459e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple
bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the
azide–alkyne “click” reaction†
Frank Seela*a,b and Venkata Ramana Sirivolua,b
Received 17th December 2007, Accepted 1st February 2008
First published as an Advance Article on the web 26th March 2008
DOI: 10.1039/b719459e
5-(Octa-1,7-diynyl)-2ꢀ-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed
cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7
as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C≡C bonds were
conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed
Huisgen–Sharpless–Meldal cycloaddition “click reaction”. Strongly fluorescent 1H-1,2,3-triazole
conjugates (30–33) are formed incorporating two fluorescent reporters—the pyrdC nucleoside and the
coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl
7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase
synthesis using the phosphoramidite building blocks 10 and 13; the pyrrolo-dC oligonucleotides are
formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related
derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more
stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are
even more efficient. The click reaction was also performed on oligonucleotides containing the
pyrdC-derivative 3 and the fluorescence properties of nucleosides, oligonucleotides and their coumarin
conjugates were studied.
the regioselective nucleobase anion glycosylation6 at nitrogen N1,
Introduction
as was exemplified on compound 2a. PyrC or pyrdC has been
Fluorescent reporter groups are widely used to explore the
incorporated into oligoribo- and oligodeoxyribonucleotides using
the standard protocol of solid-phase synthesis.10 Fluorescent 6-
unsubstituted 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one derivatives
and related bicyclic analogues were reported to develop stable
base pairs with dG.14,15 A tridentate base pair is formed between
compound 2c and dG which can mimic the dC–dG base pair.9
Nucleoside 2d has been used to study nucleic acid duplex and
triplex formation or to probe DNA/RNA interactions with
proteins.16–21
This manuscript reports on pyrdC-derivatives bearing side
chains at the 6-position with a terminal triple bond. This
functionality allows further derivatisation of the whole molecule
by the copper(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar
cycloaddition reaction, the so-called “click” reaction.22–24 This
protocol is orthogonal to other reactions and can be performed
efficiently in aqueous media with high functional group tolerance.
Earlier investigations by our laboratory have demonstrated that a
set of all four canonical bases, pyrimidines and purines—the latter
in the form of their 7-deaza derivatives—can be functionalized by
this route.25–27 The terminal C≡C bonds of the pyrdC-derivative
3 and its open-chain analogue 4 were conjugated with the non-
fluorescent azido–coumarin 5 generating strongly fluorescent
1,4-disubstitued 1H-1,2,3-triazole conjugates (Scheme 2). These
molecules contain two fluorescent moieties: the pyrrolo-dC base
and the coumarin dye. The “click” reaction was also performed
on the oligonucleotide level. After hybridization the duplex DNA
contains one fluorescent reporter (pyrdC) being part of the
base pair stack while the other fluorescent residue (coumarin)
structure, dynamics and interactions of nucleic acids. As canonical
bases of nucleic acids are non-emissive, numerous fluorescent
nucleobase analogues have been synthesized and incorporated
into DNA.1a Among the modified purine derivatives, 2-amino
purines,1b 2-amino- and 2-hydroxy pyrrolo[2,3-d]pyrimidines2–4
as well as 1,N6-etheno compounds5,6 are all fluorescent. Var-
ious cytosine nucleosides, such as etheno-dC7,8 and pyrrolo-C
(pyrC) or pyrrolo-dC (pyrdC) develop significant fluorescence.
PyrC (2a, 2b) or pyrdC (2c, 2d) (Scheme 1) are related to
the pyrrolo[2,3-d]pyrimidine nucleoside 1,4 which is strongly
fluorescent. PyrC/dC are prepared from the furano-U or furano-
dU,9–11 which are accessible from the 5-alkynyl compounds by
cyclization with copper iodide. The bicyclic furano pyrimidine
nucleoside analogues posses significant biological activity against
Varicella Zoster virus.12,13 Treatment of the furo[2,3-d]pyrimidine
nucleosides (e.g. 7) with ammonia generates the pyrrolo[2,3-
d]pyrimidine nucleosides (e.g. 3), a reaction which takes place dur-
ing oligonucleotide deprotection. An alternative synthetic route
makes use of the low nucleophilicity of the pyrrol nitrogen allowing
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 4814, Mu¨nster, Germany. E-mail:
Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; Web: www.seela.
net; Fax: +49 (0)251 53 406 857; Tel: +49 (0)251 53 406 500
bLaboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r
Chemie, Universita¨t Osnabru¨ck, Barbarastraße 7, 49069, Osnabru¨ck,
Germany
† Electronic supplementary information (ESI) available: Table 5. See DOI:
10.1039/b719459e
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Scheme 1 Structures of nucleosides.
Scheme 2 Copper(I)-catalyzed alkyne–azide cycloaddition.
is protruding into the major groove of the DNA duplex. The
duplex stability of modified base pairs was compared with those
incorporating dC–dG.
Results and discussion
1. Synthesis and properties of monomers
The syntheses of 5-alkynylated 2ꢀ-deoxyuridine 6 and the 5ꢀ-
O-(4,4ꢀ-dimethoxytrityl) derivatives 8 and 11 have already been
reported from our laboratory using the Sonogashira cross-
coupling reaction followed by dimethoxytritylation.25,26 Earlier, a
one-pot preparation of bicyclic furano nucleoside 7 was described;
however, as the yield was below 20%28 the 5-(octa-1,7-diynyl)-
2ꢀ-deoxyuridine intermediate 6 was isolated first, which was
then cyclized in the presence of CuI to give the 6-hexynyl-
3H-furo[2,3-d]pyrimidin-2-one nucleoside 7 in 87% yield, which
undergoes conversion in aqueous ammonia to form the 6-hexynyl-
7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleoside 3 in 90% yield
(Scheme 3). An alternative synthetic route was reported by our
laboratory for the synthesis of the pyrC-derivative 2a using
the regioselective glycosylation of the 7-deazapurine-2-one at
nitrogen-1.6 Compound 2e was prepared using the same cycliza-
tion reaction as reported above and their fluorescence properties
were investigated.6
To study the effect of 6-alkynyl and 6-alkyl substitution of
pyrdC on the DNA duplex stability, oligonucleotides containing
3 and 2f were synthesized. For that purpose the phosphoramidite
building blocks 10 and 13 were prepared and employed in solid-
phase oligonucleotide synthesis. Two routes were performed for
the preparation of the dimethoxytrityl derivative 9. The first
method starts with the dimethoxytritylation of the 6-hexynyl 3H-
furo[2,3-d]pyrimidin-2-one nucleoside 7, while the second begins
Scheme 3 Reagents and conditions: (i) Et3N/MeOH, CuI, reflux 70 ◦C;
(ii) 25% aq. NH3, overnight, r.t.
with the cyclization of the 5-octa-1,7-diynylated dimethoxytrityl
derivative 8 to form 9 (85% yield). In a similar way the 5-
oct-1-ynylated dimethoxytrityl derivative 11 was cyclized into
12 in 87% yield. The above compounds were further trans-
formed into the phosphoramidite building blocks 10 and 13
by phosphitylation reaction using chloro-(2-cyanoethoxy)-N,N-
diisopropylaminophosphine (Scheme 4). For comparison the
phosphoramidite building block of 2d was prepared according
to the reported procedure.20
The target compounds and all intermediates were characterized
by 1H, 13C NMR and 31P-NMR spectroscopy as well as by
elemental analyses. The cyclized 7 shows a signal (6.44 ppm)
consistent with the chemical shift of a vinyl proton and no signals
beyond 10 ppm, revealing the absence of an imino proton. The
presence of an NH signal at 11 ppm indicates the formation of
pyrrolo-dC derivative 3. The 13C NMR signals were assigned on
the basis of related compounds (2a, 2d, 2e), which were assigned
earlier6,11 by their gated-decoupled spectra, and are displayed in
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Scheme 4 Reagents and conditions: (i) DMTr-Cl, pyridine, 6 h, r.t.; (ii) Et3N/MeOH, CuI, reflux 70 ◦C; (iii) iPr2NP(Cl)OCH2CH2CN, iPr2EtN, CH2Cl2,
45 min, r.t.
Table 1. A significant chemical shift difference is observed for the
C2 and C4 signals (purine numbering is used) in the 13C NMR
spectra of pyrrolo-dC 3 and its furano precursor 7. Moreover, C4,
which is connected to the pyrdC, appears at 159 ppm, while C4
of the furano compound 7 is in the range of 171–172 ppm. The
assignments of the C1ꢀ and C4ꢀ sugar signals uses differences in
the coupling constants taken from the gated-decoupled spectra.6
The side chain of 3 shifts C8 upfield (14) ppm when compared to
the methyl compound.6
2. Synthesis and characterization of oligonucleotides
Oligonucleotide synthesis was carried out on solid phase with
an ABI 392–08 synthesizer at a 1 lmol scale employing the
Table 1 13C-NMR chemical shifts of modified nucleosides and their derivatives measured in d6-DMSO at 298 K
C2a
C2b
C4a
C5a
C6a
C4b
C7a
C5b
C8a
C6b
C7ab
C4ab
C≡Cc/triazoled
C1ꢀ
C2ꢀ
C3ꢀ
C4ꢀ
C5ꢀ
2a6
2d6
2e6
3
154.1
153.7
153.7
153.8
157.9
158.0
159.0
158.3
153.9
149.4
153.5
158.7
159.2
159.1
159.2
171.2
171.2
172.0
171.3
159.2
—
107.4
108.9
108.6
108.7
106.3
106.2
107.0
106.6
108.8
—
137.3
134.0
134.5
134.5
136.8
136.4
137.2
137.0
134.5
161.7
164.3
100.5
96.8
96.5
96.4
99.9
99.3
99.9
100.0
96.5
98.9
90.4
127.4
137.6
141.1
142.0
153.7
153.7
154.5
154.7
142.2
142.7
143.5
—
—
83.7
84.3
84.2
84.2
—
—
71.6
71.3
71.3
71.3
91.1
86.5
86.6
86.6
87.3
86.0
86.7
87.6
86.7
84.6
85.2
74.9
41.2
41.3
41.3
41.2
41.2
42.0
41.3
68.4
69.8
69.6
69.9
69.6
69.0
69.8
69.8
69.9
70.1
70.1
84.1
87.6
87.6
87.7
88.0
87.3
88.0
88.2
87.7
87.5
87.4
59.8
60.9
61.9
61.0
60.7
62.5
63.3
60.9
61.1
60.9
61.0
7
9
12
30
31
32
33
—
—
147.0
146.9
146.8
146.8
123.0
122.8
122.8
122.8
41.3
e
e
—
—
a Purine numbering. b Systematic numbering. c Triple bond carbons for compounds 2e, 3, 7, 9. d Triazole carbons for compounds 30–33. e Superimposed
by the signal of DMSO-d6.
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Fig. 1 HPLC profile a) of the reaction products obtained after enzymatic hydrolysis of the oligomer 5ꢀ-d(AGT AT3 GAC CTA) with snake venom
phosphodiesterase and alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.5) at 37 ◦C, b) of an artificial mixture of nucleosides 3, 6 and 7. Column:
LiChrosphere 100 RP-18 (5 lm). The following solvent systems were used: MeCN (A) and 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5 (B); gradient for a:
25 min 100% B, 50 min 0–50% A in B; gradient for b: 50 min 5–60% A in B.
synthesized phosphoramidites 10, 13 as well as standard building
blocks. The coupling yields were always higher than 95%. The
synthesis of oligonucleotides was performed by employing the
DMT-on mode. After cleavage from the solid support, the
oligomers were deprotected in 25% aqueous ammonia solution
3. The duplex stability of oligonucleotides incorporating base
pairs formed by the bycylic compounds 3, 2f, 2d and the open-chain
analogue 5-(octa-1,7-diynyl)-2ꢀ-deoxycytidine (4)
The duplex stability of non-self complementary duplexes contain-
ing various 6-substituted pyrrolo-dC nucleosides was investigated
(Table 2). For that the duplex 5ꢀ-d[TAG GTC AAT ACT] (14) and
3ꢀ-d[ATC CAG TTA TGA] (15) was used as a reference and the
modified nucleosides shown in Scheme 6 were incorporated in the
place of dC.
◦
for 14–16 h at 60 C. The purification of 5ꢀ-dimethoxytrityl
oligomers was carried out on reverse-phase HPLC (see Exper-
imental section). The base composition of the oligonucleotides
was determined by tandem enzymatic hydrolysis (for details see
Experimental section). The HPLC profile of the reaction products
obtained after enzymatic digestion clearly demonstrates that the
newly incorporated nucleoside 3 migrate much slower than the
canonical DNA constituents (Fig. 1a). The molecular masses of
the oligonucleotides were determined by MALDI-TOF Biflex-
III mass spectrometry (Bruker Saxonia, Leipzig, Germany) and
Applied Biosystems Voyager DE PRO with 3-hydroxypicolinic
acid (3-HPA) as a matrix. The detected masses were identical with
the calculated values (see ESI, Table 5†). During deprotection
of oligonucleotides the incorporated furano pyrimidines were
converted into pyrrolo pyrimidines (Scheme 5). In order to confirm
this, the structure of the modified nucleoside was not altered
during oligonucleotide synthesis. The mobility of the nucleosides
on reverse-phase HPLC refers to the hydrophobic character of
the side chains of the nucleosides with compound 7 as the slowest
migrating compound when compared with the nucleosides 6 and
3 (Fig. 1b).
A single replacement of pyrrolo-dC nucleosides shows a positive
influence on the DNA duplex stability with a tendency of higher
stabilization for the open-chain compounds (Scheme 6). The
nucleoside conjugate 31 containing the 1,2,3-triazole moiety as
well as the coumarin dye residue was only slightly destabilized,
showing that the bulky side chain does not interfere with base-
pairing significantly. To study the effect of multiple incorporations
of the pyrrolo-dC nucleosides and corresponding open-chain com-
pounds, a series of oligonucleotides was prepared by incorporating
the modified bases at different positions (Table 2). From this a
significant difference in the Tm values was observed. It is apparent
that the replacement of the canonical 2ꢀ-deoxycytidine within
DNA duplex by the side chain derivatives leads to the following
conclusions. (i) The open-chain compound 4 forms a more stable
base pair with dG than the bicyclic pyrrolo-dC derivative 3. (ii)
The DNA duplexes containing a terminal triple bond in the side
chain (nucleoside 3) are more stable than that of 2f without
terminal triple bond. The higher stability of the pyrdC-derivative
3 compared to its saturated analogue 2f results from the better
solvation of the side chain in aqueous solution. It is likely that
the triple bond acts as proton acceptor while the terminal proton
is a potential H-donor. Thus, water molecules can interact with
the side chain terminus by hydrogen bonding which is supported
by the fact that the saturated compound 2f does not show such
favorable properties. A similar phenomenon was already observed
for the corresponding 2ꢀ-deoxyuridine derivatives.25
Next, the mismatch formation was studied by incorporating
compound 3 against the four canonical nucleosides. For that a
similar set of experiments were performed by using a standard
Scheme 5 Conversion of the furano moiety of compound (7) into the
pyrrole ring system (3) after treatment with aqueous NH3.
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Table 2 Tm values of oligonucleotide duplexes containing the pyrrolo-
dC nucleosides 3, 2f, 2d and the open-chain 2ꢀ-deoxycytidine derivative 4
located opposite dGa
Table 3 Tm values and thermodynamic data of oligonucleotide duplexes
containing the nucleoside 3 located opposite canonical nucleosidesa
Duplex
Tm/◦C
34
DTm
—
DG◦310/kcal mol−1
Duplex
Tm/◦C
50
DTm
—
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(ATC CAG CTA TGA) (25)
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(ATC CAG 3TA TGA) (26)
5ꢀ-d(TAG GTC TAT ACT) (27)
3ꢀ-d(ATC CAG CTA TGA) (25)
5ꢀ-d(TAG GTC TAT ACT) (27)
3ꢀ-d(ATC CAG 3TA TGA) (26)
5ꢀ-d(TAG GTC GAT ACT) (28)
3ꢀ-d(ATC CAG CTA TGA) (25)
5ꢀ-d(TAG GTC GAT ACT) (28)
3ꢀ-d(ATC CAG 3TA TGA) (26)
5ꢀ-d(TAG GTC CAT ACT) (29)
3ꢀ-d(ATC CAG CTA TGA) (25)
5ꢀ-d(TAG GTC CAT ACT) (29)
3ꢀ-d(ATC CAG 3TA TGA) (26)
−7.3
−7.7
−7.6
−8.0
−13.0
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GT3 AAT ACT) (16)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(AT3 3AG TTA TGA) (22)
5ꢀ-d(TAG GT3 AAT ACT) (16)
3ꢀ-d(AT3 3AG TTA TGA) (22)
5ꢀ-d(TAG GT31 AAT ACT) (21)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GT4 AAT ACT) (19)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(AT4 4AG TTA TGA) (23)
5ꢀ-d(TAG GT4 AAT ACT) (19)
3ꢀ-d(AT4 4AG TTA TGA) (23)
5ꢀ-d(TAG GT2f AAT ACT) (17)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GTC AAT ACT) (14)
3ꢀ-d(AT2f 2fAG TTA TGA) (24)
5ꢀ-d(TAG GT2f AAT ACT) (17)
3ꢀ-d(AT2f 2fAG TTA TGA) (24)
5ꢀ-d(TAG GT2d AAT ACT) (18)
3ꢀ-d(ATC CAG TTA TGA) (15)
5ꢀ-d(TAG GTX AAT ACT) (20)
3ꢀ-d(ATC CAG TTA TGA) (15)
37
+3.0
—
52
+2
−3
−1
−2
+3
+5
+7
+1
−6
−5
+1
+3
36
47
39
+3.0
—
49
56
48
54
−2.0 −12.1
53
32
—
−6.9
−7.3
55
35
+3.0
57
a Measured at 260 nm in 1 M Nacl, 100 mM MgCl2 and 60 mM Na-
cacodylate (pH 7.0) with 5 lM single-strand concentration.
51
44
45
concluded that pyrdC (3) shows a higher base discrimination
than dC. Nevertheless, duplexes incorporating pyrdC (3) generally
showed higher Tm-values against canonical nucleosides than those
containing dC. This might be caused by the increased stacking
interactions due to the larger surface area of the pyrdC base
compared to dC as well as the stabilizing effect of the side chain.
51
53
a Measured at 260 nm in 1 M NaCl, 100 mM MgCl2 and 60 mM Na-
cacodylate (pH 7.0) with 5 lM single-strand concentration. X = 5-
Propynyl-2ꢀ-deoxycytidine.
4. Photophysical properties of pyrdC-derivatives and their
coumarin conjugates formed by the copper(I)-catalyzed
azide–alkyne cycloaddition (“click reaction”); one dye vs two dyes
fluorescence
oligonucleotide containing pyrrolo-dC nucleoside 3 3ꢀ-d(ATC
CAG 3TA TGA) 26. The complementary strands are modified
in such a way that the modified base located opposite to dA,
dC, dT or dG. All mismatches led to a decrease of the duplex
stability compared to the 3–dG base pair. From Table 3, it is
The bicyclic furano nucleoside 7 and its corresponding pyrdC
analogue 3 exhibit a strong purple luminescence on TLC plates
under the UV lamp. Fluorescence spectra of compound 7 shows
Scheme 6
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Fig. 2 The emission and excitation spectra of nucleosides 7 and 3 measured at room temperature in bi-distilled water.
an excitation maximum at 327 nm with an emission at 410 nm,
where as for the nucleoside 3 longer excitation and emission
wavelengths were observed (excitation, 340 nm; emission, 466 nm).
A representative emission curves for 7 and 3 are shown in Fig. 2.
Also, the Stokes shift is increased from 7 (Dk = 83 nm) to 3
(Dk = 126 nm). The fluorescence quantum yield (U) of 3 in
bi-distilled water was determined to be 0.05, which is almost
similar to that observed for 7 (0.06). The introduction of a side
chain at the 6-position of 3 does not influence the quantum yield
significantly when compared to the unsubstituted 2a, but shifts the
excitation as well as the emission maxima to longer wavelength
(Table 4).29
molecules and the extremely stable 1,2,3-triazole product can have
only small interactions with biological structures. The stacking of
1,2,3-triazoles in the major groove of DNA led to slightly enhanced
duplex stability.39b The 1,2,3-triazole coumarin formed from the
terminal alkynes and 3-azido coumarins is found to be highly
fluorescent.40 Among many fluorophores, coumarin derivatives
have been extensively studied because of their photophysical
properties. Coumarins were used as potential fluorescent probes
to investigate the structural dynamics of DNA,41 for the specific
labeling of nucleic acids42 and mainly as nucleobase-specific
quenchers.43 The derivatives of 7-hydroxycoumarin are generally
good fluorophores that are used as dyes for the blue spectra region.
The presence of electron-donating groups at the 7-position of 3-
azido coumarins show a strong enhancement in the fluorescence.40
Moreover, it is well documented that the fluorescence proper-
ties of coumarin derivatives are pH dependent. Different ab-
sorption and emission bands can be observed at various pH
values.
Although pyrrolo-dC (3) and its alkynyl derivatives show
significant fluorescence, their extinction coefficients and quantum
yields are low when compared to fluorescent dyes like coumarins.
Therefore, we intended to conjugate a fluorescent dye to the pyrdC
derivative bearing an alkynyl side chain. This would allow to detect
possible quenching effects of stacked (pyrdC 3) and non-stacked
(coumarin) fluorescence reporters within ss-oligonucleotides or
duplex DNA. The modified version of Huisgen 1,3-dipolar
cycloaddition22 so called “click” chemistry is used for the dye
conjugation. Recently, the Sharpless23 and the Meldal groups24
demonstrated this chemistry for the functionalization of alkyny-
lated compounds with different azides. The reaction proceeds via
copper(I)-catalyzed 1,3-dipolar cycloaddition of a terminal alkyne
to an organic azide resulting in the exclusive formation of 1,4-
disubstituted-1,2,3-triazole. The advantage of this reaction is its
insensitivity to oxygen, water and tolerance to a variety of func-
tional groups, which has emerged as the most versatile reaction
and found many applications in nucleic acids,31–33 carbohydrates,34
drug discovery, polymers and surface chemistry.35–37
The nucleosides 7 and 3 containing terminal C≡C bonds were
selectively conjugated with the azide residue of the coumarin dye
5 in the presence of copper sulfate and sodium ascorbate to form
the strongly fluorescent 1,4-disubstituted 1,2,3-triazole products
30 and 31 in 82 and 85% yields. In a similar way the open-
chain nucleoside–coumarin conjugates 32 (81%) and 33 (61%)
were prepared by the copper(I)-catalyzed cycloaddition reaction
between 5-(octa-1,7-diynyl)-2ꢀ-deoxyuridine (6)25 or 5-(octa-1,7-
diynyl)-2ꢀ-deoxycytidine (4)27 with 5 (Scheme 7).
1
The “click” products were identified by comparing their H
NMR spectra with those of the starting compounds. The terminal
side chain proton of the triple bond of 3 (2.75 ppm) and 7
(2.67 ppm) disappeared and newly formed triazole vinyl proton
signals of 30 and 31 are now present in the range of 8–9 ppm. More-
over, the 13C spectra shows the absence of terminal C≡C carbon
atom signals; the newly associated triazole carbons were found at
dC4 = 147 ppm, dC5 = 123 ppm (Table 1). The large difference in
triazole carbons for compounds 30–33 (D dC4–dC5 = 24–25 ppm)
indicates the chemoselective formation of the 1,4-disubstituted
triazole, which is in agreement with previous reports.44,45 Moreover,
the resonance of the triazole ring carbons is clearly evidenced
by 1H,13C distortionless enhancement by polarization transfer
(DEPT) 135 NMR spectroscopy, not displaying a signal for
quaternary carbon (dC4) and inverted signals of triazole carbon
(dC5) at around 123 ppm.
Moreover, the click reaction is used in biological systems38,39a as
the azide and alkyne components are unreactive towards biological
Table 4 Photophysical data of the base modified nucleosidesa
Compound
Excitation/nm
Emission/nm
Quantum yield (U)
2a
3
7
336
340
327
450
466
410
0.06
0.05
0.06
a Measured in H2O; quantum yields were determined using quinine sulfate
in 0.1 N H2SO4 as standard with U = 0.53.30
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Scheme 7 Reagents and conditions: (i) CuSO4, Na-ascorbate, THF–H2O–t-BuOH (3:1:1), r.t.
The mobility of the pyrrolo-dC 31 and the open-chain click
nucleoside 33 in reverse-phase HPLC was determined by injecting
an artificial mixture of the starting materials and products onto the
column; the starting 3-azido-7-hydroxycoumarin (5) moves much
slower when compared to 3, 31 (Fig. 3a) and 4, 33 (Fig. 3b)
conjugates the coumarin excitation wavelength around 347 nm
is used in the fluorescence experiments which were performed in
water.
Next, the fluorescence properties of nucleoside–coumarin 1,2,3-
triazolyl conjugates 30 and 31 were studied and compared to
the starting nucleosides. The fluorescence emission maxima for
all coumarin dye conjugates are almost identical. Differences
are observed in the excitation spectra which resulted from the
different UV absorptions of the open-chain and bicyclic analogs.
The pyrdC–coumarin conjugate 31 has an excitation maximum
at 350 nm with an emission at 476 (Stokes shift of 126 nm)
(Fig. 5), while the furano–coumarin conjugate 30 has an excitation
maximum at 338 nm with a strong emission at 474 nm with a
Stokes shift of 136 nm. The Stokes shifts of pyrrolo-dC nucleosides
3 and 31 are almost identical. However, in the case of furano
nucleosides 7 and its conjugate 30 there is a large shift in the
emission wavelength (from 410 nm to 474 nm) and also a large
Stokes shift difference is observed. The fluorescence quantum
yields of 30 and 31 in bi-distilled water were determined to be
0.15 and 0.18, which are significantly higher than that of the
starting nucleosides 7 (0.06) and 3 (0.05) (Table 4). The solvent
pH strongly influences the fluorescence of the dye conjugates.
At neutral or lower pH values the possible generation of highly
fluorescent phenolate anions was minimized.
Fig. 3 HPLC profile a) of the artificial mixture of compounds 3, 5 and
31, b) of the artificial mixture of compounds 4, 5 and 33 on a RP-18 (200 ×
10 mm) column. The following solvent systems were used: MeCN (A) and
0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5 (B). Gradient 0–50 min 0–50%
A in B.
Next, the corresponding open-chain derivatives of the mono
cyclic nucleoside–dye conjugates 32 and 33 were investigated
(Fig. 5). From the fluorescence spectra of nucleoside–coumarin
conjugates, an excitation maxima observed at around 398 nm cor-
responds to that of coumarin dye. The two excitation wavelengths
(345 and 392 nm) of coumarins is due to the presence of neutral
as well as phenolate anionic species in the solution,46 while at the
alkaline pH value predominantly the phenolate anionic structure
is observed with the excitation at 392 nm. The fluorescence
quantum yield of 2ꢀ-deoxyuridine–dye conjugate 32 (0.32) is higher
than that of 2ꢀ-deoxycytidine conjugate 33 (0.22). Moreover, the
At first, the UV-Vis absorption spectra of terminal alkynyl
nucleosides (3, 4, 6, 7) and their dye conjugates (30–33) were
measured (Fig. 4). It is apparent that in the case of open-chain
nucleoside–coumarin derivatives 32 and 33 (Fig. 4c,d) a clear
differentiation in the absorption bands of the octadiynyl base and
coumarin dye can be made. For the furano conjugate (30, Fig. 4a),
there is only a slight difference in the absorption maxima of furano
base when compared to the dye, where as in the case of pyrrolo-dC
dye conjugate (31, Fig. 4b) the UV absorptions of the pyrdC base
and the coumarin dye overlap. So for all the nucleoside–coumarin
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Fig. 4 UV-Vis spectra of a) furano compound 7 and its dye conjugate 30, b) pyrdC 3 and its dye conjugate 31, c) 5-octadiynyl-2ꢀ-deoxyuridine (6) and
its dye conjugate 32, d) 5-octadiynyl-2ꢀ-deoxycytidine (4) and its dye conjugate 33 measured in methanol.
Fig. 5 The emission and excitation spectra of the bicyclic furano nucleoside–coumarin conjugate 30, the pyrrolo-dC–coumarin conjugate 31, the
open-chain 2ꢀ-deoxyuridine–coumarin conjugate 32 and the 2ꢀ-deoxycytidine–coumarin conjugate 33 measured in water (pH 7.0, upper row) and 0.1 M
Tris-HCl buffer (pH 8.5, lower row).
open-chain coumarin dye conjugates have higher quantum yields
than the corresponding cyclic compounds 30 (0.15) and 31 (0.18).
The quantum yield of fluorescence for all the nucleosides was
determined relative to quinine sulfate in 1.0 N H2SO4.
Most of the fluorophores have molar extinction coefficients
at their wavelength of maximum absorption ranging between
5000 and 200 000 cm−1 M−1.47 Fluorescence intensity per dye
molecule is proportional to the product of extinction coefficient
(e) and quantum yield (U). For comparison, the molar extinction
coefficients of the nucleoside conjugates 30–33 were measured in
methanol. The following values are found: 30 (342 nm 23 000),
31 (346 nm 17 000), 32 (346 nm 21 300), 33 (345 nm 11 000).
From these data it is apparent that the extinction coefficient of the
pyrrolo-dC coumarin conjugate 31 is higher when compared to
that of the open-chain derivative 33, however a higher quantum
yield was observed for the latter.
5. Influence of one dye and two dye pyrrolo-dC derivatives on the
fluorescence properties of ss- and ds-DNA
In the following the fluorescence properties of pyrrolo-dC nu-
cleoside 3 and its coumarin conjugate 31 were studied at the
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Scheme 8 Reagents and conditions: (i) CuSO4–TBTA (1:1), TCEP, H2O–t-BuOH–DMSO–THF, 16 h, r.t.
Fig. 6 UV-Vis spectra of oligonucleotide coumarin conjugates. a) 5ꢀ-d(TAG GT31 AAT ACT), b) 5ꢀ-d(AGT ATT GA33 CTA) measured in bi-distilled
water.
oligonucleotide level. We already demonstrated the application of
Huisgen–Sharpless–Meldal “click” cycloaddition for the alkynyl
modified oligonucleotides on the solid-support as well as in the
solution.25,27 Here, we applied this methodology to the 12-mer
oligonucleotide 5ꢀ-d(TAG GT3 AAT ACT)-3ꢀ (16) in which the
central dC was replaced by 3 and performed the “click” reaction
in solution. 4 OD (A260 units) of the purified oligonucleotide 16 and
non-fluorescent 3-azido-7-hydroxycoumarin (5) dissolved in a t-
BuOH–H2O–DMSO–THF mixture were treated in the presence of
1:1 complex of CuSO4–TBTA (tris(benzyltriazoylmethyl)amine)
and TCEP (a water-soluble reducing agent). This resulted in
the formation of the strongly fluorescent ss-oligonucleotide 21
(Scheme 8). After completion of the reaction within 16 h, the
reaction mixture was centrifuged and the supernatant solution was
taken, concentrated and further purified by reverse-phase HPLC
(trityl-off modus; see Experimental section). The oligonucleotide
dye conjugate 21 was characterized by MALDI-TOF (see ESI,
Table 5†). Compared to oligonucleotide 16 the formation of the
conjugate 21 was accompanied by a strong fluorescence increase.
Moreover, the UV-Vis spectrum of 21 (Fig. 6a) shows a clear differ-
entiation between the absorption of the nucleobases (260 nm) and
the coumarin dye (around 350 nm), a similar differentiation was
made on the oligonucleotide containing dC–coumarin nucleoside
3327 (Fig. 6b). Therefore, the UV-Vis measurement is a useful tool
to control the performance of the “click” reaction.
Next, the fluorescence of the pyrrolo-dC nucleoside 3 as well
as its coumarin conjugate 31 was studied on DNA duplexes. All
fluorescence measurements of oligonucleotides were performed
at a constant pH value (8.5) to ensure anion formation of the
dye. As illustrated in Fig. 7a, the single-stranded oligonucleotide
containing 3 displayed a strong fluorescence quenching at 462 nm
upon duplex formation. Relative to the single strand 16, the
fluorescence of duplex 15·16 decreases by approximately 50%. We
reasoned that the modified base 3 experiences less fluorescence
quenching in the solvent-exposed unstructured single strand,
while the base-stacked hydrophobic environment of duplex caused
fluorescence quenching (Fig. 7). A similar result was observed for
pyrC (2b) and pyrdC (2d) nucleosides when they are incorporated
into DNA and RNA duplexes.29
In contrast to this, the pyrrolo-dC coumarin conjugate 31 still
retains the fluorescence with a slight increase in the emission
intensity after forming a duplex (Fig. 7b, Scheme 9). It is evident
from previous reports that the higher energy blue fluorescence
of 7-hydroxycoumarin derivatives at neutral and alkaline pH is
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Fig. 7 Fluorescence emission a) of single-stranded 16 (2.5 lM) and duplex DNA 15·16 (2.5 lM + 2.5 lM), b) of click functionalized single-stranded
DNA 21 (1.5 lM) and duplex DNA 15·21 (1.5 lM + 1.5 lM) measured in 0.1 M Tris-HCl buffer (pH 8.5).
Scheme 9 a) DNA duplex containing a pyrrolo-dC nucleoside 3, b) DNA duplex containing a pyrrolo-dC–coumarin conjugate 31.
due to the formation of the phenolate anion. The pKa value
of 7-hydroxycoumarin was found to be in the range of 8,46
while the presence of electron-donating groups at the 3-position
influences this value. In order to confirm this, the pKa value
of 7-hydroxycoumarin of nucleoside 31 was determined UV-
spectrophotometrically48 from pH 1.5 to 11.0 at 220–350 nm and
found to be in the range of 6.5. From the previous reports the
pKa of pyrC 2b appears to be <4, slightly lower than cytosine’s
pKa of 4.2.29 From these results we can conclude that apart from
the restricted freedom of the major groove coumarin dye in DNA
duplex, other factors such as generation of negatively charged phe-
nolate anion of coumarin hydroxyl group also play an important
role in the fluorescence increase. It is assumed that the coumarin
tether protrudes stretched-out from the major groove of the double
helix and does not fold back owing to the charge repulsion between
the negatively charged dye and the phosphodiester backbone. Such
interactions are more predominant in duplex DNA rather than the
single strand (Fig. 7b).
hydrolysis of oligonucleotides 16 and 21 was performed using
snake venom phosphodiesterase followed by alkaline phosphate
and their fluorescence emission was recorded at different time
intervals (Fig. 8). In the case of oligonucleotide containing pyrdC
(3) a strong enhancement in the fluorescence emission at 466 nm
was observed after complete enzymatic digestion, when excited
at 347 nm, whereas the oligonucleotide with pyrdC–coumarin
conjugate experiences a strong fluorescence quenching at an
emission wavelength of 478 nm, when excited at 395 nm.
Based on the above results, it is apparent that the pyrrolo-dC
base of 31 strongly quenches the fluorescence of the coumarin
dye attached to it, unlike its open-chain derivative 33. A
similar quenching was observed for the related 7-deazapurine
nucleosides.27 The quenching is small when the pyrdC moiety
of the conjugate is part of the oligonucleotide stack, while the
generation of the free monomeric coumarin dye conjugate 31
formed after enzymatic hydrolysis leads to the quenching of the dye
fluorescence. Apparently, in this situation, the dye and modified
base (the 7-deazapurine moiety) can come in close proximity either
by back-folding or by dimer formation. The excited coumarin
To determine the fluorescence characteristics of pyrdC (3) and
its coumarin conjugate 31 in single-stranded DNA, the enzymatic
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Fig. 8 Enzymatic digestion a) of 1.5 lM single-stranded oligonucleotide 16 b) of 1.5 lM single-stranded oligonucleotide 21 with snake venom
phosphodiesterase followed by alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.5) at 37 ◦C.
Fig. 9 The emission and excitation spectra of the pyrrolo-dC nucleoside 3 and the pyrrolo-dC coumarin conjugate 31 measured in 0.1 M Tris-HCl buffer
(pH 8.5).
serves as an electron acceptor and the nucleobase as a ground-
state donor.43
is not observed when the nucleobase unit of 31 is part of an
oligonucleotide. However, the local environment of the ionisable
dye might have an influence on the dye deprotonation when it is
near to the DNA backbone or liberated into the solution after
enzymatic digestion. Among the two fluorescent dye moieties
the coumarin shows much stronger fluorescence than the pyrdC
moiety (Fig. 9). The two-dye reporter system has the potential to
be used in cellular uptake studies employing copper-mediated or
copper-free click chemistry.49
Conclusions and outlook
When the pyrdC (3) derivative bearing an alkynyl side chain is
incorporated into DNA, it forms as stable base pair with dG
as the non-functionalized pyrdC and shows a good mismatch
discrimination against the canonical nucleosides (dG > dT >
dA > dC). The presence of the terminal acetylene group in 3
enhances the stability of DNA duplexes opposite to canonical
nucleosides, when compared with 2ꢀ-deoxycytidine. The open-
chain dC derivative 4 is found to be more duplex-stabilizing
than that of pyrdC. The terminal alkynyl group of compound 3
allows the functionalization with almost any azido reporter when
applying the Huisgen–Sharpless–Meldal “click” chemistry. Here,
the non-fluorescent coumarin azide 5 was studied to generate the
highly fluorescent coumarin conjugate 31, the reaction studied
on the nucleoside and the oligonucleotide level thereby generating
two fluorescent dye reporters. In ss-oligonucleotides one (pyrdC) is
part of the base stack while the other (coumarin) is free, protruding
into the solvent. Enzymatic phosphodiester hydrolysis of an
oligonucleotide containing pyrdC (3) or the conjugate 31 resulted
in a strong fluorescence increase of the pyrdC fluorescence while
the coumarin dye became quenched during monomer formation.
This can be explained by electron transfer from the pyrrolo[2,3-
d]pyrimidine system to the coumarin dye, a phenomenon which
Experimental
General
All chemicals were purchased from Acros, Aldrich, Sigma, or
Fluka (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
Solvents were of laboratory grade. Thin layer chromatography
(TLC): aluminium sheets, silica gel 60 F254, 0.2 mm layer (VWR
International, Darmstadt, Germany). Column flash chromatogra-
phy (FC): silica gel 60 (VWR International, Darmstadt, Germany)
at 0.4 bar; Sample collection with an UltroRac II fractions collec-
tor (LKB Instruments, Sweden). UV spectra: U-3200 spectrometer
(Hitachi, Tokyo, Japan); kmax (e) in nm. NMR Spectra: Avance-
250 or Avance-300 spectrometers (Bruker, Karlsruhe, Germany),
1
at 250.13 or 300.15 MHz for H and 13C; d in ppm relative to
Me4Si as internal standard, or external 85% H3PO4 for 31P. The
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J values are in Hz. Elemental analyses were performed by
Mikroanalytisches Laboratorium Beller (Go¨ttingen, Germany).
Electron spray ionization (ESI) MS for the nucleosides: Bruker-
Daltonics-MicroTOF spectrometer with loop injection (Bremen,
Germany).
The enzymatic hydrolysis of the oligonucleotides was performed
with snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus
adamanteus) and alkaline phosphatase (EC 3.1.3.1, Escherichia
coli from Roche Diagnostics GmbH, Germany) in 0.1 M Tris-
HCl buffer (pH 8.5), which was carried out on reverse-phase
HPLC (RP-18, at 260 nm) by gradient: 0.1 M (Et3NH)OAc
(pH 7.0)/MeCN (95:5) with a flow rate of 0.7 ml min−1 showing the
peaks of the modified and unmodified nucleosides. Quantification
of the constituents was made on the basis of the peak areas, which
were divided by the extinction coefficients of the nucleosides [(e260):
dT 8800, dC 7300, dA 15 400, dG 11 700, 3 4100 (MeOH)].
The molecular masses of the oligonucleotides were determined
by MALDI-TOF Biflex-III mass spectrometry (Bruker Saxonia,
Leipzig, Germany) and Applied Biosystems Voyager DE PRO
with 3-hydroxypicolinic acid (3-HPA) as a matrix. The detected
masses were identical to the calculated values (ESI, Table 5†).
Fluorescence measurements. All measurements were done in
bi-distilled water at 20 ◦C. Absorption spectra were measured
with a Cary 100 Bio UV-visible spectrophotometer. In order to
avoid inner filter effects the sample was not allowed to exceed
0.1 at the excitation wavelength using standard quartz cuvettes
with a path length of 1 cm. Fluorescence spectra were recorded
in the wavelength range between 320 and 600 nm using the
Fluorescence Spectrophotometer F-2500 (Hitachi, Tokyo, Japan).
For all calculations the water background was subtracted from the
sample. The fluorescence quantum yields were determined using
quinine sulfate in 0.1 N H2SO4 (fluorescence quantum yield 0.53)30
as a standard with the following relation:
3-(2-Deoxy-b-D-erythro-pentofuranosyl)-3,7-dihydro-6-hexynyl-
2H-pyrrolo[2,3-d]pyrimidin-2-one (3). A solution of compound
7 (180 mg, 0.54 mmol)28 in 25% aqueous ammonium hydroxide
(30 ml) was stirred at r.t. overnight. The solvent was evaporated
and the residue was applied to flash chromatography (FC) (silica
gel, column 8 × 2 cm, CH2Cl2–MeOH, 9:1). The main zone
afforded 3 (162 mg, 90%) as a yellowish solid. (Found: C, 61.80;
H, 6.27; N, 12.58%. C17H21N3O4 requires C, 61.62; H, 6.39; N,
12.68%); TLC (silica gel, CH2Cl2–MeOH, 9:1): Rf 0.34; kmax
(MeOH)/nm 229 (e/dm3 mol−1 cm−1 27 500), 262 (4 200), 344
(4 100); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.43–1.71 (4 H, m,
2 × CH2), 2.01 (1 H, m, 2ꢀ-Ha), 2.15–2.31 (3 H, m, 2ꢀ-Hb, CH2),
2.54 (2 H, m, CH2), 2.75 (1 H, s, C≡CH), 3.63 (2 H, m, 5ꢀ-H), 3.86
(1 H, m, 4ꢀ-H), 4.23 (1 H, m, 3ꢀ-H), 5.10 (1 H, t, J 4.71, 5ꢀ-OH),
5.25 (1 H, d, J 3.46, 3ꢀ-OH), 5.91 (1 H, s, 5-H), 6.25 (1 H, t, J
6.15, 1ꢀ-H), 8.50 (1 H, s, 4-H), 11.1 (1 H, s, NH).
Uf.sample = Uf.standard × (Fsample/Fstandard) × (A standard/Asample),
where Uf.sample is the unknown fluorescence quantum yield of the
fluorophore, F is the integrated fluorescence intensity; A is the
absorbance in 1 cm cuvettes and does not exceed 0.1 at and above
the excitation wavelength.
Synthesis, purification and characterization of oligonucleotides
The oligonucleotide synthesis was performed on a DNA syn-
thesizer, model ABI 392–08 (Applied Biosystems, Weiterstadt,
Germany) at 1 lmol scale using the phosphoramidites following
the synthesis protocol for 3ꢀ-cyanoethyl phosphoramidites (user’s
manual for the 392 DNA sythesizer, Applied Biosystems, Weiter-
stadt, Germany). The coupling efficiency was always higher than
95%. After cleavage from the solid support the oligonucleotides
were deprotected with 25% aqueous NH3 for 14–16 h at 60 ◦C. The
purification of the 5ꢀ-O-(dimethoxytrityl)oligomers was carried
out on reverse-phase HPLC (Merck-Hitachi-HPLC: 250 × 4 mm
RP-18 column with the following gradient system [A: 0.1 M
(Et3NH)OAc (pH 7.0)/MeCN 95:5 and B: MeCN]; gradient:
3 min 20% B in A, 12 min 20–50% B in A and 25 min 20%
B in A with a flow rate of 1 ml min−1. The purified ‘trityl-on’
oligonucleotides were treated with 2.5% dichloroacetic acid in
3-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-6-(hexynyl)-furo[2,3-d]pyrimidin-3H-2-one
(9).
Method A: Compound 7 (470 mg, 1.41 mmol) was dried
by repeated co-evaporation with anhydrous pyridine (2 ×
10 ml) before dissolving in anhydrous pyridine (10 ml). 4,4ꢀ-
Dimethoxytriphenylmethyl chloride (610 mg, 1.80 mmol) was
added in three portions to the remaining solution at r.t. under
stirring for 6 h. The reaction was quenched by the addition
of MeOH (2 ml) and the mixture was evaporated to dryness.
The reaction mixture was dissolved in CH2Cl2 (2 × 50 ml) and
extracted with 5% aqueous sodium bicarbonate (100 ml) followed
by water (80 ml), dried over Na2SO4 and then evaporated.
Purification by FC (silica gel, column 15 × 3 cm) eluting with
CH2Cl2–MeOH, 95:5 to give a colorless foam of 9 (0.73 g 81%).
Method B: To a solution of Compound 8 (200 mg, 0.31 mmol)
in 15 ml (MeOH–Et3N, 7:3) was added CuI (12 mg, 0.063 mmol)
and refluxed for 5 h. The solvent was evaporated in vacuo and the
crude product was purified by FC (silica gel, column 8 × 2 cm,
CH2Cl2–acetone 7:3) afforded 170 mg (85%) of 9. (Found: C,
72.03; H, 6.20, N, 4.30%. C38H38N2O7 requires C, 71.91; H, 6.03;
N, 4.41%); TLC (silica gel, CH2Cl2–MeOH, 9:1): Rf 0.74; kmax
(MeOH)/nm 233 (e/dm3 mol−1 cm−1 32 000), 275 (3 600), 333
(6 900); dH (250.13 MHz; [d6]DMSO; Me4Si) 1.45–150 (2 H, m,
CH2), 1.62–1.65 (2 H, m, CH2), 2.08–2.22 (3 H, m, 2ꢀ-Ha, CH2),
2.39–2.63 (3 H, m, 2ꢀ-Hb, CH2), 2.76 (1 H, s, C≡CH), 3.25–3.29
(2 H, m, 5ꢀ-H), 3.73 (6 H, s, 2 × OCH3), 4.01 (1 H, m, 4ꢀ-H)), 4.38
◦
CH2Cl2 for 5 min at 0 C to remove the 4,4ꢀ-dimethoxytrityl
residues. The detritylated oligomers were purified again by reverse-
phase HPLC (gradient: 0–20 min 0–20% B in A, flow rate
1 ml min−1). The purification of click functionalized oligonu-
cleotide 21 was performed in the trityl-off modus by reverse-phase
HPLC (gradient: 0–25 mins 0–30% B in A, flow rate 0.8 ml min−1).
The oligomers were desalted on a short column (RP-18, silica gel)
and lyophilized on a Speed-Vac evaporator to yield colorless solids
which were frozen at −24 ◦C.
Melting curves were measured with a Cary-1/3 UV/VIS spec-
trophotometer (Varian, Australia) equipped with a Cary thermo-
electrical controller. The temperature was measured continuously
in the reference cell with a Pt-100 resistor, and the thermodynamic
data of duplex formation were calculated by the Meltwin 3.0
program. Cary 100 Bio UV-Vis spectrophotometer was used for
the UV-melting curves of oligonucleotides (Table 2) with a heating
rate of 1 ◦C.
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(1 H, m, 3ꢀ-H), 5.42 (1 H, d, J 4.60, 3ꢀ-OH), 5.65 (1 H, s, 5-H), 6.14
(1 H, t, J 5.31, 1ꢀ-H), 6.87–7.38 (m, H–arom), 8.53 (1 H, s, 4-H).
(1 H-arom, d, J 8.60), 8.31 (1 H-arom, s, C CH), 8.55 (1 H-arom,
s), 8.67 (1 H, s, 4-H), 10.8 (1 H, s, OH). (Found: ESI-HR-MS:
558.16 ([M + Na])+, C26H25N5O8; requires 535.17).
=
3-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-6-(hexyl)-furo[2,3-d]pyrimidin-3H-2-one
(12).
Preparation of the conjugate 31 from nucleoside 3 and 3-azido-7-
hydroxycoumarin (5)
As described above with compound 11 (400 mg, 0.62 mmol),
MeOH–Et3N, 7:3 (30 ml) and CuI (24 mg, 0.126 mmol).
Purification by FC (silica gel, column 15 × 3 cm, CH2Cl2–acetone
7:3) gave a colorless foam of 12 (348 mg, 87%). (Found: C,
71.35; H, 6.61; N, 4.36%. C38H42N2O7 requires C 71.45; H, 6.63;
N, 4.39%); TLC (silica gel, CH2Cl2–MeOH, 9:1): Rf 0.67; kmax
(MeOH)/nm 233 (e/dm3 mol−1 cm−1 27 000), 275 (3 000), 334
(5 400); dH (250.13 MHz; [d6]DMSO; Me4Si) 0.86 (3 H, CH3),
1.15–1.54 (8 H, m, CH2), 2.11 (2 H, m, 2ꢀ-H), 2.44 (2 H, m, CH2),
3.11 (2 H, m, 5ꢀ-H), 3.73 (6 H, s, 2 × OCH3), 4.0 (1 H, m, 4ꢀ-H),
4.39 (1 H, m, 3ꢀ-H), 5.42 (1 H, d, J 3.56, 3ꢀ-OH), 5.63 (1 H, s,
5-H), 6.14 (1 H, t, J 5.31, 1ꢀ-H), 6.87–7.36 (m, H–arom), 8.55
(1H, s, 4-H).
As described for 30 a solution of 3 (80 mg, 0.24 mmol), 3-azido-7-
hydroxycoumarin (5; 57 mg, 0.28 mmol) in THF–H2O–t-BuOH,
3:1:1 (4 ml) was stirred at r.t. for 30 h with 1 M aqueous sodium
ascorbate (210 ll, 0.21 mmol) and copper(II) sulfate pentahydrate
(173 ll, 0.052 mmol of a 7.5% stock solution in water). The solvent
was evaporated and applied to FC (silica gel, column 10 × 3 cm,
CH2Cl2–MeOH 85:15) to afford compound 31 (110 mg, 85%) as a
yellowish solid. TLC (silica gel, CH2Cl2–MeOH, 88:12): Rf 0.52.
kmax (MeOH)/nm 220 (e/dm3 mol−1 cm−1 25 000), 346 (13 000);
dH (300.15 MHz; [d6]DMSO; Me4Si) 1.70 (4 H, m, 2 × CH2), 2.03
(1 H, m, 2ꢀ-Ha), 2.30 (1 H, m, 2ꢀ-Hb), 2.58–2.74 (4 H, m, 2 ×
CH2), 3.62 (2 H, m, 5ꢀ-H), 3.87 (1 H, m, 4ꢀ-H), 4.22 (1 H, m, 3ꢀ-
H), 5.10 (1 H, 5ꢀ-OH), 5.25 (1 H, 3ꢀ-OH), 6.0 (1 H, s, 5-H), 6.24
(1 H, t, J 6.2, 1ꢀ-H), 6.84 (1 H-arom, d), 6.88–6.92 (1 H-arom, dd, J
3-[2-Deoxy-5-O-(4,4ꢀ -dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-6-(hexynyl)-furo[2,3-d]pyrimidin-3H-2-one-3ꢀ-(2-
cyanoethyl-N,N-diisopropylphosphoramidite (10). A stirred solu-
tion of 9 (170 mg, 0.27 mmol) in anhydrous CH2Cl2 (5 ml) was pre-
flushed with argon and treated with (i-Pr)2EtN (93 ll, 0.54 mmol)
followed by (2-cyanoethyl)diisopropylphosphoramidochloridite
(118 ll, 0.54 mmol). After stirring for 45 min at r.t. the solution
was diluted with CH2Cl2 (30 ml) and extracted with 5% aqueous
NaHCO3 solution (20 ml). The organic layer was dried (Na2SO4),
filtered and evaporated. FC (silica gel, column 10 × 3 cm, CH2Cl2–
acetone, 9:1) gave colorless foam of 10 (140 mg, 63%). TLC (silica
gel, CH2Cl2–acetone, 95:5): Rf 0.60. 31P NMR (CDCl3): 150.12,
149.74.
=
8.41), 7.74 (1 H-arom, d, J 8.70), 8.31 (1 H-arom, s, C CH), 8.49
(1 H, s, 4-H), 8.55 (1 H-arom, s), 10.9 (1 H, s, OH), 11.1 (1 H, s,
NH). (Found: ESI-HR-MS: 557.18 ([M + Na])+, C26H26N6O7;
requires 534.19).
Preparation of the conjugate 32 from nucleoside 6 and 3-azido-7-
hydroxycoumarin (5)
As described for 30 with 6 (100 mg, 0.30 mmol), 3-azido-7-
hydroxycoumarin (5; 74 mg, 0.36 mmol) in THF–H2O–t-BuOH,
3:1:1 (4 ml) was stirred at r.t for 10 h with 1 M aqueous sodium
ascorbate (300 ll, 0.30 mmol), copper(II) sulfate pentahydrate
(260 ll, 0.078 mmol of a 7.5% stock solution in water). The solvent
was evaporated and applied to FC (silica gel, column 10 × 3 cm,
CH2Cl2–MeOH 90:10) to afford compound 32 (130 mg, 81%) as
a yellowish solid. TLC (silica gel, CH2Cl2–MeOH, 9:1): Rf 0.30.
kmax (MeOH)/nm 297 (e/dm3 mol−1 cm−1 13 000), 346 (17 000); dH
(300.15 MHz; [d6]DMSO; Me4Si) 1.60–1.80 (4 H, m, 2 × CH2),
2.11 (2 H, m, 2ꢀ-H), 2.42 (2 H, m, CH2), 2.77 (2 H, m, CH2), 3.48
(2 H, m, 5ꢀ-H), 3.78 (1 H, m, 4ꢀ-H), 4.22 (1 H, m, 3ꢀ-H), 5.10 (1
H, 5ꢀ-OH), 5.23 (1 H, 3ꢀ-OH), 6.10 (1 H, t, J 6.45, 1ꢀ-H), 6.84 (1
H-arom, d), 6.88–6.92 (1 H-arom, dd, J 8.58), 7.74 (1 H-arom,
3-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-6-(hexyl)-furo[2,3-d]pyrimidin-3H-2-one
3ꢀ-(2-
cyanoethyl-N,N-diisopropylphosphor-amidite (13). As described
for 10, with 12 (200 mg, 0.31 mmol), (i-Pr)2EtN (100 ll, 0.58
mmol) and 2-cyanoethyl-diisopropylphosphoramidochloridite
(94 ll, 0.43 mmol). FC (silica gel, column 10 × 3 cm, CH2Cl2–
acetone 95:5) afforded 13 as a colorless foam (160 mg, 61%).
TLC (silica gel, CH2Cl2–acetone 9:1): Rf 0.70. 31P NMR (CDCl3):
150.88, 150.07.
Preparation of the conjugate 30 from nucleoside 7 and 3-azido-7-
hydroxycoumarin (5)
=
d, J 8.70), 8. 12 (1 H, s, 4-H), 8.31 (1 H-arom, s, C CH), 8.55
(1 H-arom, s), 10.9 (1 H, s, OH), 11.5 (1 H, s, NH). (Found:
To a solution of compound 7 (120 mg, 0.36 mmol) and 3-azido-7-
hydroxycoumarin (5; 86.5 mg, 0.43 mmol) in THF–H2O–t-BuOH,
3:1:1, (4 ml), was added sodium ascorbate (312 ll, 0.31 mmol) of a
freshly prepared 1 M solution in water, followed by the addition of
copper(II) sulfate pentahydrate 7.5% in water (260 ll, 0.078 mmol).
The emulsion was stirred for 24 h at r.t. evaporated, and applied to
FC (silica gel, column 10 × 3 cm, CH2Cl2–MeOH, 88:12). From
the main zone compound 30 (158 mg, 82%) was isolated as a
yellowish solid. TLC (silica gel, CH2Cl2–MeOH, 9:1): Rf 0.41. kmax
(MeOH)/nm 342 (e/dm3 mol−1 cm−1 22 000); dH (250.13 MHz;
[d6]DMSO; Me4Si) 1.70 (4 H, m, 2 × CH2), 2.06 (1 H, m, 2ꢀ-Ha),
2.39 (1 H, m, 2ꢀ-Hb), 2.74 (4 H, m, 2 × CH2), 3.63 (2 H, m, 5ꢀ-
H), 3.89 (1 H, m, 4ꢀ-H), 4.22 (1 H, m, 3ꢀ-H), 5.12 (1 H, 5ꢀ-OH),
5.28 (1 H, 3ꢀ-OH), 6.16 (1 H, t, J 6.12, 1ꢀ-H), 6.45 (1 H, s, 5-H),
6.84 (1 H-arom, d, J 2.05), 6.87–6.92 (1 H-arom, dd, J 8.50), 7.74
ESI-HR-MS: 558.16 ([M + Na])+, C26H25N5O8; requires 535.17).
Preparation of the conjugate 33 from nucleoside 4 and 3-azido-7-
hydroxycoumarin (5)
Compound 33 was synthesised from 4 (60 mg, 0.18 mmol)
according to the above method. Compound 33 (59 mg, 61%)
was isolated from the main zone as a yellowish solid. TLC
(silica gel, CH2Cl2–MeOH, 9:1): Rf 0.15. kmax (MeOH)/nm 345
(e/dm3 mol−1 cm−1 9 500); dH (300.15 MHz; [d6]DMSO; Me4Si)
1.61–1.77 (4 H, m, 2 × CH2), 1.98–2.10 (2 H, m, 2ꢀ-H), 2.46 (2 H,
m, CH2), 2.75 (2 H, m, CH2), 3.56 (2 H, m, 5ꢀ-H), 3.78 (1 H, m, 4ꢀ-
H), 4.20 (1 H, m, 3ꢀ-H), 5.06 (1 H, 5ꢀ-OH), 5.20 (1 H, 3ꢀ-OH), 6.10
(1 H, t, J 6.18, 1ꢀ-H), 6.84 (1 H-arom, d), 6.74 (1 H, s, NHa), 6.85
(1 H-arom, d), 6.89–6.92 (1 H-arom, dd, J 8.46), 7.71 (1 H, s, NHb),
1686 | Org. Biomol. Chem., 2008, 6, 1674–1687
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=
7.74 (1 H-arom, d), 8.08 (1 H, C(6)), 8.31 (1 H-arom, s, C CH),
8.55 (1 H-arom, s), 10.9 (1 H, s, OH). (Found: ESI-HR-MS: 557.18
([M + Na])+, C26H26N6O7; requires 534.19).
14 H. Inoue, A. Imura and E. Ohtsuka, Nippon Kagaku Kaishi., 1987, 7,
1214–1220.
15 T. Shibata, N. J. Buurma, J. A. Brazier, P. Thompson, I. Haq and D. M.
Williams, Chem. Commun., 2006, 3516–3518.
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Brown, Chem. Commun., 2005, 2555–2557.
Copper(I)-catalyzed [3 + 2] cycloaddition of the oligonucleotide
16 with coumarin azide 5. To the single-stranded oligonucleotide
16 (4 A260 units, 31 nmol), a 1:1 complex of CuSO4–TBTA
ligand (35 ll of a 20 mM stock solution in t-BuOH–H2O 1:9),
tris(carboxyethyl)phosphine (TCEP; 35 ll of a 20 mM stock
solution in H2O), 3-azido-7-hydroxycoumarin (5; 50 ll of a 20 mM
stock solution in THF) and 35 ll DMSO was added and the
reaction mixture stirred at r.t. for 16 h. The reaction mixture was
concentrated in a speed vac and dissolved in 1 ml bi-distilled water
and centrifuged for 20 min at 12 000 rpm. The supernatant solution
was collected and further purified by reverse-phase HPLC in the
trityl-off modus (for details see above) to give 21. MALDI-TOF:
(ESI, Table 5†).
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Acknowledgements
We thank Mrs S. Budow and P. Chittepu for measuring the NMR
spectra, Dr K. I. Shaikh (Muenster) and Dr T. Koch from Roche
Diagnostics GmbH, Penzberg, Germany for the MALDI-TOF
spectra, Mrs E. Michalek and Mr Tran for the oligonucleotide
syntheses and Mr P. Leonard for reading the manuscript. Financial
support by ChemBiotech, Mu¨nster, and the BMBF is gratefully
acknowledged.
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