8-Aza-7-deazaguanine nucleosides and oligonucleotides with octadiynyl side chains: Synthesis, functionalization by the azide-alkyne 'click' reaction and nucleobase specific fluorescence quenching of coumarin dye conjugates

Article abstract of DOI:10.1039/b822041g

Oligonucleotides incorporating 7-(octa-1,7-diynyl) derivatives of 8-aza-7-deaza-2′-deoxyguanosine (2d) were prepared by solid-phase synthesis. The side chain of 2d was introduced by the Sonogashira cross coupling reaction and phosphoramidites (3a, 3b) wer

Full text of DOI:10.1039/b822041g

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
8-Aza-7-deazaguanine nucleosides and oligonucleotides with octadiynyl side
chains: synthesis, functionalization by the azide-alkyne ‘click’ reaction and
nucleobase specific fluorescence quenching of coumarin dye conjugates†
Frank Seela,*a,b Hai Xiong,a,b Peter Leonarda and Simone Budowa
Received 9th December 2008, Accepted 15th January 2009
First published as an Advance Article on the web 2nd March 2009
DOI: 10.1039/b822041g
Oligonucleotides incorporating 7-(octa-1,7-diynyl) derivatives of 8-aza-7-deaza-2¢-deoxyguanosine (2d)
were prepared by solid-phase synthesis. The side chain of 2d was introduced by the Sonogashira cross
coupling reaction and phosphoramidites (3a, 3b) were synthesized. Duplexes containing 2d are more
stabilized compared to those incorporating the non-functionalized 8-aza-7-deaza-2¢-deoxyguanosine
(2a) demonstrating that these side chains have steric freedom in duplex DNA. Nucleoside 2d as well as
2d-containing oligonucleotides were conjugated to the non-fluorescent 3-azido-7-hydroxycoumarin 15
by the Huisgen-Meldal-Sharpless ‘click’ reaction. Pyrazolo[3,4-d]pyrimidine nucleoside conjugate 16
shows a much higher fluorescence intensity than that of the corresponding pyrrolo[2,3-d]pyrimidine
derivative 17. The quenching in the dye conjugate 17 was found to be stronger on the stage of
monomeric conjugates than in single-stranded or duplex DNA. Nucleobase-dye contact complexes are
suggested which are more favourable in the monomeric state than in the DNA chain when the
nucleobase is part of the stack. The side chains with the bulky dye conjugates are well accommodated in
DNA duplexes thereby forming hybrids which are slightly more stable than canonical DNA.
d]pyrimdines. An electron transfer from the nucleobase to the
Introduction
dye moiety was suggested.14 Thus, it was anticipated that other
The Huisgen-Meldal-Sharpless ‘click’ reaction has emerged as
nucleobases having different redox potentials may not show such
a convenient and effective approach for the preparation of a
drawback. Pyrazolo[3,4-d]pyrimidine nucleosides offer properties
which make them applicable as alternative systems.15 They show
similar base pairing characteristics and can be functionalized at
the 7-position. 8-Aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine)
nucleosides have already been incorporated into DNA including
7-propynyl and 7-halogeno derivatives.16–18 It was shown that
the 8-aza-7-deaza-2¢-deoxyguanosine residues 2a–c form stable
base pairs with dC giving steric freedom to 7-substituents to
be well accommodated in the major groove of duplex DNA.18
Usually, 7-substituted nucleosides form more stable duplexes than
the non-functionalized ones.18–22 Thus, pyrazolo[3,4-d]pyrimidine
nucleosides can be considered as purine nucleoside shape mimics
which will not disturb the DNA duplex structure.
This manuscript reports on the synthesis of 8-aza-7-deaza-
7-(octa-1,7-diynyl)-2¢-deoxyguanosine (2d), its conversion into
the phosphoramidite building blocks (3a,b) and their ap-
plication in solid-phase oligonucleotide synthesis (Fig. 1).
Studies regarding base pairing and fluorescence properties
were carried out on oligonucleotides incorporating nucleo-
side 2d. The azide-alkyne ‘click’ reaction for the dye con-
jugation with 3-azido-7-hydroxycoumarin on nucleosides and
oligonucleotides with side chains having terminal triple bonds
was studied according to Scheme 1. Fluorescence enhance-
ment of the oligonucleotide coumarin dye conjugates is ex-
pected for oligonucleotides containing pyrazolo[3,4-d]pyrimidine
dye conjugates compared to pyrrolo[2,3-d]pyrimidine deriva-
tives.
diversity of new molecules with various functionalities performed
under simple reaction conditions. The reaction is orthogonal to
most of the common biomolecule substituents and chemoselective
under Cu(I) catalysis.1 Thus, the protocol is particularly attractive
to be applied in drug design,2 molecular diagnostics3,4 polymer
synthesis, material science,5–10 and other areas of chemistry and
chemical biology.11–13
Recently, we reported on the synthesis of oligonucleotides
incorporating 7-deaza-7-(octa-1,7-diynyl)-2¢-deoxyguanosine (1)
(purine numbering is used in the results and discussion section).
The side chains bearing terminal triple bonds were conjugated
with various azides including the dye 3-azido-7-hydroxycoumarin
by the Huisgen-Meldal-Sharpless ‘click’ reaction.11c,14 Spectro-
scopic studies showed that the fluorescence is strongly quenched
when the DNA nucleobase belongs to the class of pyrrolo[2,3-
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center
for Nanotechnology, Heisenbergstraße 11, 48149, Mu¨nster, Germany.
E-mail: Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; Web:
www.seela.net; Fax: +49 (0)251-53406-857; Tel: +49 (0)251-53406-500
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: UV spectrum of
nucleoside 2d, pKa-values of nucleosides, HPLC profile of an artificial
mixture of nucleosides and 13C NMR spectra of compounds 2d, 4a, 5a, 4b,
5b, 16 and 17. See DOI: 10.1039/b822041g
1374 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Fig. 1
Scheme 1 Copper(I)-catalyzed azide-alkyne cycloaddition.
of distortionless enhancement by polarization transfer (DEPT-
135) spectra. From this it was concluded that the triple bonds are
not affected by the Pd-assisted Sonogashira cross coupling (allene
formation).
Results and discussion
1. Synthesis and characterisation of nucleosides
The 7-iodinated 8-aza-7-deaza-2¢-deoxyguanosine 2b served as
starting material for the synthesis of the phosphoramidites
3a,b.15–17 Compound 2b was employed in the Sonogashira cross-
coupling reaction resulting in the formation of derivative 2d
(Scheme 2). The reaction was performed in dry DMF in the
presence of Et3N, [Pd0(PPh3)4] and CuI, with a five-fold excess
of 1,7-octadiyne.23,24 Due to the excessive diyne only the terminal
Pyrazolo[3,4-d]pyrimidine nucleosides show significantly al-
tered pKa values compared to pyrrolo[2,3-d]pyrimidine or purine
counterparts. Consequently, the pKa-value of compound 2d was
measured UV-spectrophotometrically and compared with already
existing data of 7-deazapurine and 8-aza-7-deazapurine nucleo-
sides (Table S1, see supporting information). The representative
titration profile of compound 2d is shown in Figure S1 (supporting
information). From the data of Table S1 it is obvious that lactam
protons of the pyrazolo[3,4-d]pyrimidine nucleosides are more
acidic than those of pyrrolo[2,3-d]pyrimidine nucleosides. The
7-substituents have almost no influence on the pKa-values which
is different in case of 5-substituted pyrimidine nucleosides, e.g.
2¢-deoxyuridine derivatives which are strongly affected by the
electronic properties of the side chain.14
∫
C C bond was functionalized and compound 2d was isolated in
52% yield. Next, derivative 2d was protected at the 2-amino group
with either the N,N-dimethylaminomethylidene or isobutyryl
residue affording the protected intermediates 4a or 4b in similar
yields (88% vs. 89%). Both compounds were converted to the 5¢-O-
DMT-derivatives 5a and 5b under standard reaction conditions.
Phosphitylation yielded the phosphoramidites 3a and 3b (63% and
72%, respectively).
All compounds were characterized by either elemental analyses
or mass spectra, 1H- and 13C-NMR spectra. The 13C-NMR
chemical shifts are listed in Table 1, the 1H-13C-coupling constants
were determined from 1H-13C gated decoupled spectra (see Table 6,
experimental section). These assignments are in agreement with
literature values of propynyl compounds.18 The intact structure of
the octa-1,7-diynyl side chain was confirmed by 13C-NMR spectra
showing four signals of the methylene groups (27.1, 27.0, 18.3 and
17.3 ppm) and four signals for the triple bond carbons (2d: 71.4,
73.2, 84.4, 100.1 ppm; Table 1), confirmed by inverted signals
2. Synthesis, characterization and duplex stability of
oligonucleotides
We reported on the influence of substituents of 5-substituted
pyrimidines and 7-substituted 7-deazapurines containing halogens
or alkynyl groups on the duplex stability.14,27 It was shown that nu-
cleoside analogues with bromo or iodo substituents and propynyl
chains attached to the 7-position of pyrrolo[2,3-d]pyrimidines or
pyrazolo[3,4-d]pyrimidines are well accommodated in the major
groove of DNA and increase the thermal stability of duplexes by
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1375
©

View Article Online
Scheme 2 Reagents and conditions: i) octa-1,7-diyne, [Pd0[P(Ph3)4], CuI, DMF, Et3N, rt, 12 h; (ii) N,N-dimethylformamide dimethyl acetal, MeOH, rt,
2 h; (iii) HMDS, rt, 3 h; (iv) i-Bu2O, rt, overnight; (v) MeOH, 1 h; (vi) 4,4¢-dimethoxytriphenylmethyl chloride, anhydrous pyridine, (i-Pr)2EtN, rt;
(vii) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, anhydrous CH2Cl2, (i-Pr)2EtN, rt.
Table 1 13C-NMR chemical shifts of 8-aza-7-deaza-2¢-deoxyguanosine derivativesa
C(2)b
C(6) c
C(4)b
C(5)b
C(6)b
C(4)c
C(7)b
C(3)c
C(7a)c
C(3a)c
C C
CH2
CH3
—
C(1¢)
83.2
83.3
84.3
83.6
83.8
C(2¢)
38.5
38.5
38.5
38.7
38.7
C(3¢)
71.0
71.1
71.4
70.8
70.3
C(4¢)
87.5
87.6
83.0
87.7
85.5
C(5¢)
62.5
62.5
64.3
62.2
64.0
∫
2d
4a
5a
4b
5b
155.2
154.6
154.6
150.4
150.3
155.5d
158.7d
158.7d
152.8d
152.7d
93.4
93.6
93.1
94.4
94.1
157.2d
159.5d
159.5d
155.1d
155.1d
130.5
130.6
130.3
130.7
130.6
100.1, 84.4,
73.2, 71.4
102.5, 84.4,
73.7, 71.4
102.5, 85.2,
73.4, 70.6
103.0, 84.2,
72.5, 71.3
103.2, 84.2,
72.6, 71.3
27.1, 27.0 18.3,
17.3
27.2, 27.1, 18.3,
17.4
27.1, 27.0, 18.3,
17.3
27.1, 26.9, 18.2,
17.2
34.9
34.8
19.5
18.8
19.5
18.7
27.0, 26.9, 18.2,
17.2
a Measured in [D6]DMSO at 298 K. b Purine numbering. c Systematic numbering. d Tentative.
stabilizing the ‘dG–dC’ base pair.19–22,25 While 7-alkynyl chains
with one triple bond and three to six carbon atoms led to
stabilization of duplex DNA, longer chains are destabilizing
due to the increased hydrophobic character.19–22,25 Recently, we
demonstrated that long chain linkers with two triple bonds like
octadiynyl residues or propargyl ether moieties show a positive
influence on the duplex stability similar to that of a propynyl
residue.14,27 The advantage of such linkers is the presence of the
additional terminal triple bond which enhances the hydrophilic
character of the linker. Water molecules can be bound to the side
chain. We anticipate hydrogen bonding between water and triple
bonds.
was performed in a 1 mmol scale employing phosphoramidite
chemistry. The coupling yields were always higher than 95%.
Deprotection of the oligomers was performed in 25% aqueous
NH3 at 60 ◦C for 18–24 h. The oligonucleotides were purified
before and after detritylation by reversed-phase HPLC. The base
composition of the oligonucleotides was determined by tandem
enzymatic hydrolysis with snake-venom phosphodiesterase fol-
lowed◦by alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.5)
at 37 C (for details see experimental section). The homogeneity
of the oligonucleotides was confirmed by MALDI-TOF mass
spectrometry. The detected masses were in good agreement with
the calculated values (Table 7).
To evaluate the potential of the base modification on the
duplex stability and fluorescence properties, oligonucleotides were
prepared by solid-phase synthesis using the phosphoramidites
3a and 3b as well as standard building blocks. Their synthesis
Single and multiple incorporations of 2d, replacing dG-residues
within various positions of the reference duplex 5¢-d(TAG GTC
AAT ACT) (6) and 3¢-d(ATC CAG TTA TGA) (7) were per-
formed. As shown in Table 2, the replacements of dG by nucleoside
1376 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Table 2 Tm-values and thermodynamic data of oligonucleotide duplexes
containing 1, 2c and 2da
3. Functionalization of nucleosides and oligonucleotides with
3-azido-7-hydroxycoumarin (15) by the Huisgen-Meldal-Sharpless
[2 + 3] cycloaddition
DG◦ c/kcal
310
Duplexes
Tm/◦C DTm b/◦C mol-1
The dye functionalization of nucleoside 2d as well as oligonu-
cleotides 5¢-d(AGT ATT 2dAC CTA) (12) and 5¢-d(A2dT ATT
GAC CTA) (13) by the azide-alkyne ‘click reaction’ was per-
formed. The octa-1,7-diynyl nucleoside 2d with a terminal triple
bond was functionalized with the non-fluorescent coumarin azide
15 in an ethanol–water mixture to form the ‘click’ adduct 16 in
90% yield when sodium ascorbate and CuSO4 as a catalyst were
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA TGA) (7)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CA2c TTA TGA) (8)
5¢-d(TA2c 2cTC AAT ACT) (9)
3¢-d(ATC CAG TTA TGA) (7)
5¢-d(TA2c 2cTC AAT ACT) (9)
3¢-d(ATC CA2c TTA TGA) (8)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T1A) (10)
5¢-d(TA1 1TC AAT ACT) (11)
3¢-d(ATC CAG TTA TGA) (7)
5¢-d(TA1 1TC AAT ACT) (11)
3¢-d(ATC CAG TTA T1A) (10)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CA2d TTA TGA) (12)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T2dA) (13)
5¢-d(TA2d 2dTC AAT ACT) (14)
3¢-d(ATC CAG TTA TGA) (7)
5¢-d(TA2d 2dTC AAT ACT) (14)
3¢-d(ATC CAG TTA T2dA) (13)
5¢-d(TA2d 2dTC AAT ACT) (14)
3¢-d(ATC CA2d TTA TGA) (12)
50
—
—
+3
+2
-11.8
-10.9
-12.5
(47)
5318
(49)
5618
(52)
5818
(56)
5214
+3
-14.5
-14.0
-11.8
-12.3
-12.7
+2.5
+2.7
+3
used (Scheme 3). For details see the experimental section. The
+2
13
∫
C-NMR spectra show the absence of the terminal C C carbon
5314
5514
+1.5
+1.7
atom signals, and the new appearing double bond signals of
the 1,2,3-triazole moiety identified at d = 122.9 ppm and d =
146.8 ppm (Table 5). Due to steric hindrance, a bis-function-
alization of the second triple bond is not observed.23,24,28 The struc-
ture of the conjugate 16 was assigned on the basis of 13C-NMR
spectra and JC,H coupling constants (Table 5 and Table 6, see
experimental section). The 13C-NMR chemical shifts of the side
chain for the dye conjugate 16 are almost identical to those of the
7-deaza-2¢-deoxyguanosine coumarin conjugate 17 (Scheme 3). In
both cases the JC,H coupling constants for triazole-C5, H-C5 are
199 Hz. Moreover, the resonance of the triazole ring carbons is
clearly evidenced by 1H-NMR, gated decoupled 1H-13C as well as
DEPT-135 NMR spectroscopy.
The 7-octadiynyl side chain of compound 2d has a strong in-
fluence on the UV spectrum of 8-aza-7-deaza-2¢-deoxyguanosine.
UV data are shown in Table 3 and spectra are given in Fig. 2. The
short wavelength maximum of the alkynyl nucleoside is shifted
hypsochromically from 254 to 243 nm compared to the non-
functionalized compound 2a or the iodo derivative 2b. The UV
spectra of the ‘click’ products 16 and 17 are a combination of the
UV spectra of either compound 1 or 2d, the coumarin azide 15
and the 1,2,3-triazole moiety (Fig. 2a). Thus, the conjugate can
be easily identified by the long wavelength absorption maximum
of coumarin moiety at 346 nm in methanol. This is also valid for
oligonucleotides incorporating the dye conjugate 16 (356 nm in
water).
52
(49)
53
+2
+2
+4
-12.9
-11.7
-14.2
54
58
+2
-12.5
-14.6
+2.7
60
(54)
+3.3
+2.3
-15.0
-13.6
a Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-
cacodylate (pH 7.0) with 5 mM + 5 mM single-strand concentration.
Data in parentheses were measured in 100 mM NaCl, 10 mM MgCl2
and 10 mM Na-cacodylate (pH 7.0) at the same concentration. b Refers
to the contribution of the modified residues divided by the number of
replacements. c DG◦ values are given with 15% error.
310
2d result in a Tm increase of 2–3 ◦C per modification. These values
are close to the Tm values found for oligonucleotide duplexes
containing 8-aza-7-deaza-7-propynyl-dG (2c) or 7-deaza-7-octa-
1,7-diynyl-dG (1) at identical positions with the tendency of duplex
stabilization. Thus, the space demanding octadiynyl side chain
has a positive influence on the DNA duplex stability as already
observed for propynyl residues (2c = 2d > 1).
Scheme 3 Functionalization of nucleoside 2d with 3-azido-7-hydroxycoumarin (15).
The Royal Society of Chemistry 2009 Org. Biomol. Chem., 2009, 7, 1374–1387 | 1377
This journal is
©

View Article Online
Table 3 UV maxima and molar absorptivity (mol. abs.) of compound 2d
not affected during oligonucleotide synthesis and conjugation. As
expected, the lipophilicity of a compound increases by increasing
the length of the side chain from the propynyl derivative 2c to the
octadiynyl compound 2d as displayed by the reversed-phase HPLC
profile (Figure S2, see supporting information). The introduction
of the hydrophilic coumarin residue makes the conjugate less
lipophilic than the starting material 2d; thus the conjugate 16
is migrating faster than 2d. The information obtained from the
artificial nucleoside mixture was beneficial for the peak assignment
of the enzymatic digest (Fig. 3).
and corresponding derivativesa
Wavelength
Cpd. (lmax)/nm
Wavelength
Mol. Abs. (e) Cpd. (lmax)/nm
Mol. Abs. (e)
17
17
220
240 (sh)
274
26900
19000
7200
240
273
294
20200
9000
8100
298
7300
346
258
342
243
13300
10800
12300
25200
13200
2a26
2c18
2d
254
242
243
14200
27300
28000
2b16
15
16
346
a Measured in methanol.
4. Fluorescence properties of nucleoside and oligonucleotide
3-azido-7-hydroxycoumarin conjugates and DNA duplex stability
Next, the cycloaddition ‘click’ reaction was performed on
the oligonucleotide level using 5 A260 units of the purified
oligonucleotides 5¢-d(AGT ATT 2dAC CTA) (12) and 5¢-d(A2d
T ATT GAC CTA) (13) each containing one 8-aza-7-deaza-
7-(octa-1,7-diynyl)-2¢-deoxyguanosine residue. Both oligonu-
cleotides were separately functionalized with the non-fluorescent
coumarin azide 15.29 The reaction was performed in aqueous
solution (H2O–t-BuOH–DMSO) in the presence of a com-
plex of CuSO4–TBTA (tris(benzyltriazoylmethyl)amine) (1 : 1),
TCEP (tris(carboxyethyl)phosphine) and NaHCO3 (Scheme 4).
NaHCO3 was essential for the completion of the reaction within
12 h yielding the strongly fluorescent oligonucleotides 5¢-d(AGT
ATT 16AC CTA) (18) and 5¢-d(A16 T ATT GAC CTA) (19). For
details see the experimental section. The oligonucleotides were
purified by reversed-phase HPLC and characterized by MALDI-
TOF mass spectra (Table 7), UV-spectroscopy (Fig. 2b) as well as
by the HPLC elution profile (Scheme 4). The UV/VIS spectrum
of oligonucleotide 19 shows two maxima which can be attributed
to the absorption of the canonical nucleobases at 260 nm and the
coumarin dye at 356 nm.
Pyrrolo[2,3-d]pyrimidine nucleoside dye conjugates show signifi-
cantly lower fluorescence than those of pyrimidine nucleosides;
a phenomenon which was studied with 7-hydroxycoumarin as
reporter group.14 Now the fluorescence properties of compound
16 were investigated and compared with those of 17 at pH 8.5.
Under alkaline conditions (pH 8.5), the 7-hydroxycoumarin dye
exists predominantly in the anionic form.14,30 Both compounds
show similar excitation maxima (398 nm for dye 16 vs. 393 nm for
dye 17) with emission maxima at 478 nm and 477 nm, respectively,
both measured in 0.1 M Tris-HCl-buffer at pH 8.5 (Fig. 4a and
b). For solubility reasons the coumarin 1,2,3-triazolyl conjugates
16 and 17 were dissolved in 0.5 ml of DMSO and then diluted
with 99.5 ml 0.1 M Tris-HCl buffer, pH 8.5. In all experiments
the concentration of the nucleoside dye conjugates was identical
(9.4 ¥ 10-3 mol l-1).
From Fig. 4b, it is apparent that the 8-aza-7-deaza-2¢-
deoxyguanosine dye conjugate 16 develops a much stronger
fluorescence than the conjugate 17 (Fig. 4a). This is clearly
evidenced by their direct comparison in Fig. 4c showing that
the fluorescence maximum of 16 is about 10-times higher than
that of 17. Next, the Cu(I)-catalyzed azide-alkyne cycloaddition
of 1 and 2d with non-fluorescent 3-azido-7-hydroxycoumarin (15)
affording the nucleoside dye conjugates 17 and 16 was followed by
fluorescence measurements (Fig. 4d). It is evident that during the
formation of the pyrrolo[2,3-d]pyrimidine dye conjugate 17 only a
small fluorescence increase is observed, whereas the formation of
Composition analyses of the oligonucleotides 13 and 19
were performed using snake venom phosphodiesterase (Crotallus
adamanteus, EC 3.1.15.1) and alkaline phosphatase (E. coli, EC
3.1.3.1).14 The composition is shown in the HPLC profile of the
digest demonstrating that all monomeric components including
the modified nucleoside 2d as well as the ‘click’ product 16 are
Fig. 2 (a) UV/VIS spectra of nucleoside 2d, coumarin azide 15 and the coumarin conjugate 16 measured in methanol. (b) UV/VIS spectrum of the
oligonucleotide coumarin conjugate 5¢-d(A16 T ATT GAC CTA) (19) measured in distilled water.
1378 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Scheme 4 Huisgen-Meldal-Sharpless [2 + 3] cycloaddition of oligonucleotide 12 incorporating nucleoside 2d with 3-azido-7-hydroxycoumarin (15) and
HPLC profile of oligonucleotide 18 on a RP-18 (250 ¥ 4 mm) column at 260 nm. Gradient: 0–20 min 0–20% A in B, 20–30 min 20–30 A in B with a flow
rate of 0.7 ml min-1. Solvent systems: MeCN (A) and 0.1 M (Et3NH)OAc (pH 7.0)–MeCN, 95 : 5 (B).
Fig. 3 HPLC profiles of the enzymatic hydrolysis products of (a) the oligonucleotide 13 and (b) the oligonucleotide 19 obtained after digestion with
snake-venom phosphodiesterase and alkaline phosphatase in 0.1 M Tris-HCl buffer (pH 8.5) at 37 ◦C. Gradient: 0–25 min 100% B, 25–60 min 0–50% A
in B; flow rate 0.7 ml min-1. The following solvent systems were used: MeCN (A) and 0.1 M (Et3NH)OAc (pH 7.0)–MeCN, 95 : 5 (B).
the pyrazolo[3,4-d]pyrimidine dye conjugate 16 leads to a much
stronger increase of fluorescence.
fluorescence intensities of single-stranded and duplex DNA. This
observation is contrary to that of the related oligonucleotide
pyrrolo[2,3-d]pyrimidine nucleoside coumarin conjugate 17. Here,
a strong fluorescence decrease (85%) for the coumarin dye was
found upon duplex formation.14
The single-stranded oligonucleotide 5¢-d(A16 T ATT GAC
CTA) (19) containing the 8-aza-7-deaza-2¢-deoxyguanosine dye
conjugate 16 was then subjected to enzymatic phosphodiester
hydrolysis by snake venom phosphodiesterase (0.1 M Tris-HCl
buffer, pH 8.5). The fluorescence was measured at 479 nm
with a fixed excitation wavelength (396 nm). Within 20 min,
a fluorescence increase for oligonucleotide 19 by a factor of
3.4 was observed (Fig. 6a). Dephosphorylation with alkaline
phosphatase (incubation at 37 ◦C for 9 h) did not lead to
Next, the fluorescence properties were studied on the oligonu-
cleotides 18 and 19 containing the 1,2,3-triazole coumarin deriva-
tive 16 as monomeric building block. The fluorescence spectra of
both single-stranded oligonucleotides 18 and 19 show a strong
fluorescence with emission maxima at 479 nm and excitation
maxima at 401 nm (Fig. 5a and c). Upon hybridization with
the non-modified complementary strands the duplexes 5¢-d(TAG
GTC AAT ACT) (6) · 3¢-d(ATC CAG TTA T16A) (19) and 5¢-
d(TAG GTC AAT ACT) (6) · 3¢-d(ATC CA16 TTA TGA) (18) are
formed where 16 is located either near the 5¢-terminus (Fig. 5b) or
in a central position (Fig. 5d). No significant fluorescence change
is observed during duplex formation resulting in almost identical
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1379
©

View Article Online
Fig. 4 The excitation and emission spectra of (a) the octa-1,7-diynylated pyrrolo nucleoside coumarin conjugate 17 and (b) the octa-1,7-diynylated
pyrazolo nucleoside coumarin conjugate 16. (c) The direct comparison of the emission spectra of the coumarin dye conjugates 16 and 17. All spectra
were measured in a mixture of DMSO (0.5 ml) and 99.5 ml of 0.1 M Tris-HCl buffer at pH 8.5 with a concentration of 9.4 ¥ 10-3 mol l-1. (d) Time
dependent fluorescence increase during the Cu(I)-catalyzed azide-alkyne cycloaddition of 1 and 2d with 3-azido-7-hydroxycoumarin (15). The reaction
was performed in THF : t-BuOH : H2O (3 : 1 : 1) at 12.5 mM nucleoside concentration with 30 mM dye concentration and 100 mM Na-ascorbate (pH 8.5).
The reaction was started with aq. CuSO4 (25 mM final concentration).
a further fluorescence change. When these experiments were
performed with the oligonucleotide 5¢-d(A17 T ATT GAC CTA)
(20) incorporating the 7-deaza-2¢-deoxyguanosine dye conjugate
17, a completely different observation was made. The hydrolysis of
oligonucleotide 20 incorporating the 7-deaza-2¢-deoxyguanosine
dye conjugate 17 resulted in a strong fluorescence quenching
(Fig. 6b).
The fluorescence phenomena observed on the nucleoside
coumarin dye conjugates 16 and 17 as well as on the oligonu-
cleotides 19 and 20 incorporating 16 and 17 need justification.
Static and dynamic fluorescence quenching of coumarins by
nucleobase derivatives has already been reported.31–33 Nucleobase-
specific quenching by fluorescence resonance transfer can be ruled
out by the lack of appropriate spectral properties.31 Therefore, it
becomes likely, that photoinduced electron transfer and proton
coupled electron transfer have to be considered. The redox
properties of nucleobases, nucleosides and coumarin derivatives
were already reported by Seidel et al.31 A correlation of dye
quenching and the oxidation potential of nucleobases was made.
Intermolecular quenching experiments with different dyes and
DNA constituents (conjugates) were performed. The data give
evidence for the formation of non-fluorescent ground state
complexes between the dye and the nucleobases. Only upon
contact formation, efficient fluorescence quenching occurs via
electron transfer. Considering these findings and adopting this
to the pyrrolo[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine
coumarin dye conjugates, it is evident that the stronger electron-
donating 7-deaza-2¢-deoxyguanosine conjugate (17) is a better
quencher for the coumarin moiety than the dye conjugate of 8-aza-
7-deaza-2¢-deoxyguanosine (16). When the conjugate 17 serves
as constituent of oligonucleotides the 7-deaza-2¢-deoxyguanosine
moiety participates in base stacking. Almost no contact formation
between the base and the coumarin moiety is possible thereby pre-
venting fluorescence quenching. The fluorescence decreases upon
phosphodiester hydrolysis (monomeric conjugate formation) due
to formation of a contact complex between the nucleobase and
the dye; either in an intramolecular or intermolecular way. Nev-
ertheless, the opposite behavior—increase of the fluorescence—
of the 8-aza-7-deaza-2¢-deoxyguanosine coumarin conjugate 16 is
observed when it is released from the oligonucleotide 19. Here,
the oxidation potential of the pyrazolo[3,4-d]pyrimidine moiety is
too high to transfer an electron to the coumarin dye (negligible
1380 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Fig. 5 (a) The excitation and emission spectra of the single-stranded oligonucleotide 19 (2 mM single-strand concentration); (b) comparison of the
fluorescence emission spectra of the single-stranded oligonucleotide 19 and the duplex 19 · 6 (2 mM + 2 mM single-strand concentration) when excited
at 401 nm; (c) the excitation and emission spectra of the single-stranded oligonucleotide 18 (2 mM single-strand concentration); (d) comparison of the
fluorescence emission spectra of the single-stranded oligonucleotide 18 and the duplex 18 · 6 (2 mM + 2 mM single-strand concentration) when excited at
401 nm. All spectra were measured in 1 M NaCl, 100 mM MgCl2, 60 mM Na-cacodylate buffer at pH = 8.5.
Fig. 6 Fluorescence spectra obtained after the enzymatic digestion of (a) 1.8 mM of the single-stranded oligonucleotide 5¢-d(A16 T ATT GAC CTA)
(19), (b) 2.3 mM of the single-stranded oligonucleotide 5¢-d(AGT ATT 16AC CTA) (18) and (c) 1.7 mM of the single-stranded oligonucleotide 5¢-d(A17
T ATT GAC CTA) (20) with snake venom phosphodiesterase and alkaline phosphatase in 1 ml of 0.1 M Tris-HCl buffer (pH 8.5) at 37 ◦C.14
quenching). However, a hyperchromic effect is existing when the
dye conjugate is integrated into the oligonucleotide chain. This
can cause a hyperchromic change after enzymatic digestion.
To evaluate the influence of dye modification on the duplex
stability, the Tm values of the reference oligonucleotide duplex 5¢-
d(TAG GTC AAT ACT) (6) and 3¢-d(ATC CAG TTA TGA) (7)
modified by the octadiynylated side chain derivative 2d or the dye
conjugate 16 were measured. The replacement of one dG residue
at the periphery of the duplex 6 · 7 by nucleoside 2d (6 · 13) resulted
◦
in a Tm increase of 3 C (Table 4). Replacement of a dG-residue
within the central position of the duplex (6 · 12) led to a Tm increase
of 2 ◦C. However, the effect of the nucleoside coumarin conjugates
16 or 17 on the duplex stability is not clear. The introduction of
16 in duplex 6 · 19 has almost no effect on the duplex stability
(DTm = +3 ◦C) compared to the corresponding octadiynylated
duplex (6 · 13) while for duplex 6 · 18 containing 16 and for duplex
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1381
©

View Article Online
Table 4 Tm-Values of oligonucleotide duplexes containing 8-aza-7-
deaza-2¢-deoxyguanosine derivatives
favourable behaviour is supported by earlier data obtained on
2¢-deoxypseudourine dye conjugates bearing negative charges.34
In contrast, positively charged dyes showed a tendency to interact
with the negatively charged phosphodiester backbone supported
by significant differences in the melting behaviour.
Next, molecular dynamics simulations using Amber MM+ force
field (Hyperchem 7.0/8.0; Hypercube Inc., Gainesville, FL, USA,
2001) were performed on the 12-mer duplexes 6 · 12 and 6 · 18.
The energy minimized molecular structures are built as B-type
DNA and are shown in Fig. 7. Fig. 7a displays a duplex (6 · 12)
in which a central dG-residue is replaced by the octa-1,7-diynyl
derivative of 8-aza-7-deaza-2¢-deoxyguanosine (2d). According to
Fig. 7a, the alkynyl group linked to the pyrazolo[3,4-d]pyrimidine
base is linear and in plane with the heterocyclic base as it was
observed earlier by the X-ray structure of the related molecule
7-deaza-7-propynyl-2¢-deoxyguanosine (1c).35 Moreover, the
duplex structure is not disturbed by the modification.
In Fig. 7b and Fig. 7c, the energy minimized model of the
oligonucleotide duplex containing the coumarin conjugate 16 is
shown by two different views. Also in this case the bulky residue
is well accommodated in the major groove of DNA. The triazole
moiety does not develop stacking interaction with the base pairs
of the duplex and seems to have steric freedom not disturbing the
B-DNA duplex.
a
b
c
b
Tm
/
DTm
/
Tm
/
DTm
/
Duplex
◦C
◦C
◦C
◦C
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA TGA) (7)
5¢-d(TA2a2aTC AAT ACT) (21)
3¢-d(ATC CA2a TTA T2aA) (22)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CA2d TTA TGA) (12)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T2dA) (13)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CA16 TTA TGA) (18)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T16A) (19)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T1A) (21)
5¢-d(TAG GTC AAT ACT) (6)
3¢-d(ATC CAG TTA T17A) (20)
50
—
0
47
—
48
52
49
53
—
—
—
—
+1
+5
+2
+6
—
—
50*21
52*
53
+2
+3
-3
+3
+2
-3
47
53
5214
47
a Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-
cacodylate (pH 7.0) with 2 mM + 2 mM single-strand concentration.
b Refers to the contribution of the modified residues divided by the number
of replacements. c Measured at 260 nm in 1 M NaCl, 100 mM MgCl2,
and 60 mM Na-cacodylate (pH 8.5) with 2 mM + 2 mM single-strand
concentration. Tm values marked with an asterisk were measured at a
5 mM + 5 mM single-strand concentration.
6 · 20 containing 17 a strong destabilization was observed (DTm
=
Conclusion and outlook
◦
-3 C). A destabilizing interaction of the coumarin moiety with
the duplex helix has to be considered.
8-Aza-7-deaza-7-octa-1,7-diynyl-2¢-deoxyguanosine (2d) was syn-
thesized by the Sonogashira cross-coupling reaction from 8-aza-
7-deaza-7-iodo-2¢-deoxyguanosine (2b) and excess octadiyne. Nu-
cleoside 2d was converted into phosphoramidite building blocks
(3a, b) and oligonucleotides were prepared. The replacement
of dG residues by the 7-octadiynyl nucleoside 2d increases the
dG-dC base pair stability in a similar way as 8-aza-7-deaza-7-
propynyl-2¢-deoxyguanosine (2c) and 7-deaza-7-octa-1,7-diynyl-
2¢-deoxyguanosine (1). The octa-1,7-diynylated pyrazolo[3,4-
d]pyrimidine derivative 2d bearing a partially stiff linker arm
and a terminal triple bond was further functionalized by the
For comparison, the duplex stabilities of 6 · 19 and 6 · 18
were determined at pH 8.5; a pH value at which coumarin
forms an anionic species. Under these conditions a much stronger
stabilization of 6 · 19 compared to the reference duplex 6 · 7
was observed (DTm = +6 ◦C). Moreover, duplex 6 · 18 which
was destabilized at pH 7 (DTm = -6 ◦C) shows a Tm increase
(DTm = +2 ◦C) under alkaline conditions (pH 8.5). As the
coumarin dye is negatively charged at pH = 8.5, ionic interaction
with the phosphodiester backbone can be excluded and the
coumarin dye is protruding into the major groove of B-DNA. This
Fig. 7 Molecular models (a) of duplex 5¢-d(TAG GTC AATACT) (6) · 3¢-d(ATC CA2d TTA TGA) (12), (b) and (c) duplex 5¢-d(TAG GTC AAT ACT)
(6) · 3¢-d(ATC CA16 TTA TGA) (18). The models were constructed using Hyperchem 7.0/8.0 and energy minimized using the AMBER calculations.
1382 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Table 5 13C-NMR chemical shifts of the nucleoside coumarin conjugates
16 and 17
azide-alkyne Huisgen-Meldal-Sharpless cycloaddition (‘click’ re-
action) on the stages of nucleosides and oligonucleotides.
The conjugation of the terminal triple bond with the non-
fluorescent 3-azido-7-hydroxycoumarin 15 resulted in the for-
mation of highly fluorescent dye conjugates. The fluorescence
intensity of the 8-aza-7-deaza-2¢-deoxyguanosine conjugate 16 was
found to be about ten times higher than of the corresponding
7-deaza-2¢-deoxyguanosine conjugate 17. The quenching induced
by the pyrrolo[2,3-d]pyrimidine base on the coumarin dye vanishes
in the case of the pyrazolo[3,4-d]pyrimidine conjugate. According
to our data14 and the observation by others,31,36,37 an electron
transfer from the pyrrolo[2,3-d]pyrimidine nucleobase to the dye
is suggested which is not occuring in the case of pyrazolo[3,4-
d]pyrimidines. Fig. 8 displays possible intramolecular contact
complexes within one nucleoside dye conjugate (a, b) and
intermolecular contact complexes between two nucleoside dye
conjugates.
Cpd. C(2) bC(6)c C(4) bC(7a)c C(5) bC(3a)c C(6) bC(4)c C(7) bC(3)c
17
16
154.6
156.4
150.1d
155.3d
99.5
93.3
157.9d
156.9d
89.7
130.7
Cpd. C(1¢)
C(2¢)
C(3¢)
C(4¢)
C(5¢)
17
16
82.2
83.1
38.7
38.7
70.9
70.9
87.1
87.5
61.9
62.4
∫
Cpd. C C
Triazole
Coumarin
17
16
101.0, 74.7 147.0, 122.9 162.3, 119.5, 136.1, 110.4, 130.8,
114.2, 156.4, 102.1, 154.6
100.0, 73.2 146.8, 122.9 162.9, 119.2, 136.2, 110.1, 130.4,
114.4, 155.4, 102.2, 154.6
a Measured in [D6]DMSO at 298 K. b Purine numbering. c Systematic
numbering. d Tentative.
Experimental
General
All chemicals were purchased from Acros, Fluka, or Sigma-
Aldrich (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
Solvents were of laboratory grade. Thin layer Chromatography
(TLC): aluminium sheets, silica gel 60 F254 (0.2 mm; Merck,
Darmstadt, Germany). Flash column chromatography (FC): silica
gel 60 (VWR, Germany) at 0.4 bar; sample collection with
an Ultra Rac II fractions collector (LKB Instruments, Swe-
den). UV spectra: U-3200 spectrometer (Hitachi, Tokyo, Japan);
Reversed-phase HPLC was carried out on a 250 ¥ 4 mm PR-
18 column (Merck) with a Merck-Hitachi HPLC pump (model
L-7100) connected with a variable wavelength monitor (model
L-7400). NMR spectra: Avance-DPX-300 spectrometer (Bruker,
1
Rheinstetten, Germany), at 300 MHz for H and 13C; d in ppm
relative to Me4Si as internal standard or external 85% H3PO4 for
31P. The J values are given in Hz. MALDI-TOF mass spectra were
recorded with Applied Biosystems Voyager DE PRO spectrometer
with 3-hydroxypicolinic acid (3-HPA) as a matrix. Elemental
analyses were performed by Mikroanalytisches Laboratorium
Beller (Go¨ttingen, Germany). The melting temperature curves
were measured with a Cary-100 Bio UV-VIS spectrophotome-
ter (Varian, Australia) equipped with a Cary thermoelectrical
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 (version 3.0)
program using the curve fitting of the melting profiles according
to a two-state model.40
Fig. 8 Intramolecular and intermolecular contact complexes within
nucleoside dye conjugates.
The fluorescence measurements were performed in bidistilled
water at 20 ◦C. Fluorescence spectra were recorded in the
wavelength range between 320 to 600 nm using the fluorescence
spectrophotometer F-250 (Hitachi, Tokyo, Japan).
8-Aza-7-deaza-2¢-deoxyguanosine
and
7-deaza-2¢-deoxy-
guanosine are the most ideal shape mimics of 2¢-deoxyguanosine
allowing functionalization at the 7-position not disturbing the
DNA helix structure. Their coumarin conjugates (16 and 17) show
almost identical base pairing properties but strong differences in
their fluorescence intensity. The differences of nucleobase specific
quenching has the potential to be applied in DNA and RNA
detection or sequencing.38,39
Synthesis, purification, and characterization of the
oligonucleotides 6–14
The syntheses of oligonucleotides were performed on
DNA synthesizer, model 392–08 (Applied Biosystems, Weiter-
stadt, Gemany) at 1 mmol scale using the phosphoramidites
a
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1383
©

View Article Online
Table 6 Coupling constants J(C,H) [Hz] of 8-aza-7-deaza-2¢-
deoxyguanosine and 7-deaza-2¢-deoxyguanosine derivativesa,b
Table 7 Molecular mass [M - H]- of selected oligonucleotides determined
by MALDI-TOF mass spectrometry
J/Hz
Oligonucleotides
[M - H]- (calc.) [M - H]- (found)
Coupling
2d
17
16
5.6
5¢-d(AGT ATT 2dAC CTA) (12)
5¢-d(TA2d 2dTC AAT ACT) (14)
5¢-d(A2dT ATT GAC CTA) (13)
5¢-d(AGT ATT 16AC CTA) (18)
5¢-d(A16T ATT GAC CTA) (19)
3748.6
3853.7
3745.7
3948.8
3948.8
3746.9
3849.7
3746.3
3951.1
3950.0
3950.3a
C1¢¢∫
3J(C1¢¢, H-C3¢¢)
3.6
—
145
c
C3¢¢
1J(C3¢¢, H-C3¢¢)
130.0
5.4
69.6
57.7
129.0
5.0
132.0
2J(C3¢¢, H-C4¢¢)
1J(C4¢¢, H-C4¢¢)
—
—
127.7
—
—
128.5
5¢-d(A17T ATT GAC CTA)14 (20) 3950.2a
C4¢¢
C5¢¢
C6¢¢
1J(C5¢¢, H-C5¢¢)
1J(C6¢¢, H-C6¢¢)
c
a Determined as [M + H]+ in the linear positive mode.
2J(C6¢¢, H-C5¢¢)
C7¢¢∫
∫C8¢¢
2J(C7¢¢, H-C8¢¢)
4.2
—
—
—
—
1J(C8¢¢, H-C8¢¢)
248.0
4.2
—
—
—
—
—
—
160
149
149
140
3J(C8¢¢, H-C6¢¢)
fluorescence spectra were measured at different time intervals with
an emission at 478 nm.
Triazole-C5
Coumarin-C4
Coumarin-C5
Coumarin-C6
Coumarin-C7
Coumarin-C8
C1¢
1J(triazole-C5, H-C5)
1J(coumarin-C4, H-C4)
1J(coumarin-C5, H-C5)
1J(coumarin-C6, H-C6)
1J(coumarin-C7, H-C7)
1J(coumarin-C8, H-C8)
1J(C1¢, H-C1¢)
198.9
168.5
168.7
162.9
120.0
164.3
163
156
148
140
199.0
168.4
164.9
158.6
114.3
163.7
163
151
147
142
6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihydro-3-
(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one (2d)
A solution of 2b (1.0 g, 2.55 mmol) in dry DMF (5 cm3) was treated
with CuI (50 mg, 0.25 mmol), [Pd(PPh3)4] (150 mg, 0.125 mmol),
dry Et3N (0.5 cm3, 0.25 mmol) and 5 equiv. of octa-1,7-diyne
(12.75 mmol). The reaction mixture which slowly became black
was stirred under N2 for 12 h (TLC monitoring). Then, the mixture
C3¢
1J(C3¢, H-C3¢)
C4¢
1J(C4¢, H-C4¢)
C5¢
1J(C5¢, H-C5¢)
a Measured in [d6]DMSO at 298 K. b Purine numbering. c Tentative.
was diluted with MeOH–CH2Cl2 (1: 1, 25 cm3), and Dowex HCO3
-
form (100 200 mesh; 1.5 g) was added. After stirring for 15 min,
the evolution of gas ceased. Stirring was continued for another
30 min, the resin was filtered off and washed with MeOH–CH2Cl2
(1 : 1, 250 cm3). The combined filtrate was evaporated and the
oily residue was adsorbed on silica gel and loaded on the top
of a column. FC (silica gel, column 15 ¥ 3 cm, eluted with
CH2Cl2–MeOH 95 : 5 → 90 : 10 → 85 : 15) afforded one main zone.
Evaporation of the solvent gave 2d as colourless solid (500 mg,
52%) (Found: C, 57.98; H, 5.82; N, 18.57%. C18H21N5O4 requires
C, 58.21; H, 5.70; N, 18.86%); TLC (silica gel, CH2Cl2–MeOH,
9 : 1): Rf 0.4; lmax (MeOH)/nm 243 (e/dm3 mol-1 cm-1 28 000);
dH(250 MHz; [d6]DMSO; Me4Si) 1.64 (4 H, t, J 1.5, 2 ¥ CH2),
2.13 (1 H, m, 2¢-Ha), 2.19 (2 H, m, C∫C-CH2), 2.50 (2 H, t, J 6.5,
3a and 3b following the synthesis protocol for 3¢-O-(2-
cyanoethyl)phosphoramidites. After cleavage from the solid sup-
port, the oligonucleotides were deprotected in 25% aqueous
ammonia solution for 12–16 h at 60 ◦C; The purification of
the ‘trityl-on’ oligonucleotides was carried out on reversed-phase
HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system
(A = MeCN, B = 0.1 M (Et3NH)OAc(pH 7.0)–MeCN, 95 : 5):
3 min 15% A in B, 12 min 15–50% A in B, and 5 min 50–
10% A in B; flow rate 1.0 cm3 min-1. The purified ‘trityl-on’
oligonucleotides were treated with 2.5% CHCl2COOH–CH2Cl2
for 5 min at 0 ◦C to remove the 4,4¢-dimethoxytrityl residues.
The detritylated oligomers were purified again by reversed-phase
HPLC (gradient: 0–25 min 0–20% A in B; flow rate 1.0 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.
∫
C∫C-CH2), 2.68 (1 H, m, 2¢-Hb), 2.80 (1 H, t, J 2.4 Hz, C CH),
3.50 (2 H, m, 5¢-H), 3.78 (1 H, t, J 2.9, 4¢-H), 4.38 (1 H, m, 3¢-H),
4.76 (1 H, t, J 5.4, 5¢-OH), 5.25 (1 H, m, 3¢-OH), 6.30 (1H, t, J
6.0, 1¢-H), 6.75 (2 H, s, NH2) and 10.70 (1 H, s, NH).
The enzymatic hydrolysis of oligonucleotide 13 and 19 was per-
formed as described before using 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) at 37 ◦C, which
was analyzed by reversed-phase HPLC (RP-18, 260 nm) showing
the peaks of the modified and unmodified nucleosides.14
The synthesis of the oligonucleotide dye conjugates 18 and 19
was performed in aqueous solution with the non-fluorescent
3-azido-7-hydroxycoumarin 15 by the azide-alkyne ‘click’ reac-
tion. The molecular masses of the oligonucleotides 6–14 and 18,
19 were determined by MALDI-TOF mass spectrometry in the
linear negative mode (see Table 7).
1-(2-Deoxy-b-D-erythro-pentofuranosyl)-6-
{[(dimethylamino)methylidene]amino}-1,5-dihydro-3-
(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one (4a)
To a solution of 2d (200 mg, 0.54 mmol) in MeOH (10 cm3)
was added N,N-dimethylformamide dimethyl acetal (1.5 cm3).
The reaction mixture was stirred for 2 h at rt, the solvent was
evaporated and the oily residue was adsorbed on silica gel (50 g)
and applied to FC (silica gel, column 10 ¥ 4 cm, eluted with
CH2Cl2–MeOH 95 : 5 → 90 : 10). Evaporation of the main zone
afforded compound 4a as colourless solid (202 mg, 88%) (Found:
C, 59.12; H, 6.01; N, 19.60%. C21H26N6O4 requires C, 59.14; H,
6.14; N, 19.71%); TLC (silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.6;
lmax (MeOH)/nm 260 (e/dm3 mol-1 cm-1 29 300), 300 (26 100);
dH(250 MHz; [d6]DMSO; Me4Si) 1.64 (4 H, m, 2 ¥ CH2), 2.22
(1 H, m, 2¢-Ha), 2.49 (4 H, m, 2 ¥ C∫-CH2), 2.65 (1 H, m, 2¢-Hb),
For the fluorescence quenching experiments, the enzymatic
hydrolysis was performed using 1.7 mM single-stranded oligonu-
cleotides dissolved in 1 cm3 of 0.1 M Tris-HCl buffer (pH 8.5). To
this solution, 0.005–0.010 cm3 of snake venom phosphodiesterase
and 0.005–0.010 cm3 alkaline phosphatase were added and the
∫
2.77 (1 H, t, J 2.3, C CH), 3.05 (3 H, s, NCH3), 3.19 (3 H, s,
1384 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is The Royal Society of Chemistry 2009
©

View Article Online
NCH3), 3.43 (2 H, m, 5¢-H), 3.78 (1 H, m, 4¢-H), 4.39 (1 H,
m, 3¢-H), 4.74 (1 H, t, J 5.6, 5¢-OH), 5.25 (1 H, d, J 4.2,
3¢-OH), 6.44 (1 H, t, J 6.4, 1¢H), 8.67 (1 H, s, N CH) and 11.31
1-(2-Deoxy-b-D-erythro-pentofuranosyl)-6-[(2-
methylpropanoyl)amino]-1,5-dihydro-3-(octa-1,7-diynyl)-4H-
pyrazolo[3,4-d]pyrimidin-4-one (4b)
=
(1 H, s, NH).
Compound 2d (100 mg, 0.21 mmol) was dried by coevapora-
tion with pyridine, dissolved in DMF (1 cm3) and 1,1,1,3,3,3-
hexamethyldisilazane (0.5 cm3) was added to the solution. The
reaction mixture was stirred for 3 h at rt, then pyridine (1 cm3)
and isobutyric anhydride (1 cm3, 6.03 mmol) were added. The
solution was stirred overnight at rt, methanol (2 cm3) was added
under cooling (ice-bath) and stirring was continued for 1 h at rt.
The solvent was evaporated, and the remaining oily residue was
applied to FC (silica gel, column 8 ¥ 3 cm, eluted with CH2Cl2–
MeOH 100 : 0 → 95 : 5). Evaporation of the main zone afforded
4b as colorless foam (106 mg, 89%) (Found: C, 59.87; H, 6.27;
N, 15.67%. C22H27N5O5 requires C, 59.85; H, 6.16; N, 15.86%);
TLC (silica gel, CH2Cl2–MeOH 9 : 1): Rf 0.45; lmax (MeOH)/nm
252 (e/dm3 mol-1 cm-1 20 700); dH(250 MHz; [d6]DMSO; Me4Si)
1.10–1.12 (6 H, s, (CH3)2CH), 1.62 (4 H, m, 2 ¥ CH2), 2.21 (5 H,
1-(2-Deoxy-5-O-(4,4¢-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl)-6-{[(dimethylamino)methylidene]amino}-1,5-
dihydro-3-(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-
one (5a)
Compound 4a (140 mg, 0.33 mmol) was dried by repeated co-
evaporation with dry pyridine (3 ¥ 5 cm3). The residue was
dissolved in dry pyridine and stirred with 4,4¢-dimethoxytrityl
chloride (200 mg, 0.64 mmol) in the presence of (i-Pr)2EtN
(0.1 cm3, 0.58 mmol) at rt for 12 h. The solution was poured
into 5% NaHCO3 soln. and extracted with CH2Cl2 (3 ¥ 30 cm3).
The combined extracts were dried (Na2SO4) and the solvent
was evaporated. The residual foam was subjected to FC (silica
gel, column 10 ¥ 4 cm, eluted with CH2Cl2–acetone 95 : 5 →
90 : 10). Evaporation of the main zone afforded compound 5a
as colourless foam (184 mg, 77%) (Found: C, 69.02; H, 6.10; N,
11.42%. C42H44N6O6 requires C, 69.21; H, 6.08; N, 11.53%); TLC
(silica gel, CH2Cl2–acetone 9 : 1): Rf 0.6; lmax (MeOH)/nm 260
(e/dm3 mol-1 cm-1 25 500), 300 (22 000); dH(250 MHz; [d6]DMSO;
Me4Si) 1.62 (4 H, m, 2 ¥ CH2), 2.20 (1 H, m, 2¢-Ha), 2.50 (4 H,
∫
m, 2¢-Ha, 2 ¥ C∫C-CH2), 2.70 (1 H, m, 2¢-Hb), 2.77 (2 H, s, C CH,
CH(CH3)2), 3.44 (2 H, m, 5¢-H), 3.78 (1 H, m, 4¢-H), 4.38 (1 H, m,
3¢-H), 4.74 (1 H, m, 5¢-OH), 5.29 (1 H, m, 3¢-OH), 6.36 (1 H, t, J
6.0, 1¢-H) and 11.85 (2 H, s, 2 ¥ NH).
1-(2-Deoxy-5-O-(4,4¢-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl)-6-[(2-methylpropanoyl)amino]-1,5-dihydro-3-
(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one (5b)
∫
∫
m, 2 ¥ C CH2), 2.68 (1 H, m, 2¢-Hb), 2.78 (1 H, m, C CH), 3.06
(5 H, m, 5¢-H, NCH3), 3.19 (3 H, s, NCH3), 3.71 (3 H, s, OCH3),
3.72 (3 H, s, OCH3), 3.87 (1 H, m, 4¢-H), 4.46 (1 H, m, 3¢-H), 5.25
(1 H, d, J 4.5, 3¢-OH), 6.47 (1 H, m, 1¢-H), 6.80–7.35 (13 H, m,
Compound 4b (195 mg, 0.44 mmol) was dried by repeated co-
evaporation with pyridine and then dissolved in dry pyridine
(3 cm3). 4,4¢-dimethoxytrityl chloride (196 mg, 0.58 mmol) and
N,N-diisopropylethylamine (0.12 cm3, 0.65 mmol) were added
and the reaction mixture was stirred for 2 h at room temperature.
The solution was poured into 5% aqueous NaHCO3 (30 cm3) and
extracted with CH2Cl2, dried (Na2SO4) and evaporated to dryness.
Co-evaporation with toluene (3 ¥ 10 cm3) afforded a foamy residue
which was applied to FC (silica gel, column 10 ¥ 4 cm, eluted with
CH2Cl2–acetone 90 : 10). From the main zone 5b was obtained
as colourless foam (180 mg, 55%) (Found: C, 69.53; H, 6.01; N,
9.33%. C43H45N5O7 requires C, 69.43; H, 6.10; N, 9.42%); TLC
(silica gel, CH2Cl2–acetone 9 : 1): Rf 0.22; lmax (MeOH)/nm 235
(e/dm3 mol-1 cm-1 34 200); dH(250 MHz; [d6]DMSO; Me4Si) 1.10–
1.14 (6 H, s, (CH3)2CH), 1.63 (4 H, m, 2 ¥ CH2), 2.20 (4 H, m,
=
arom.H), 8.71 (1 H, s, N CH) and 11.36 (1 H, s, NH).
1-{[2-Deoxy-5-O-(4,4¢-dimethoxytriphenylmethyl)-3-O-[(2-
cyanoethoxy)(N,N-diisopropylamino)phosphine)]-b-D-erythro-
pentofuranosyl]}-6-[(dimethylamino)methylidene]amino-1,5-
dihydro-3-(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-
one (3a)
A soln. of 5a (156 mg, 0.21 mmol) in dry CH2Cl2 (10 cm3)
was stirred with (i-Pr)2NEt (0.07 cm3, 0.42 mmol) at rt. Then,
2-cyanoethyl diisopropylphosphoramidochloridite (0.07 cm3,
0.31 mmol) was added and the reaction mixture was stirred for
30 min. The solution was poured into 5% NaHCO3 soln. (30 cm3)
and extracted with CH2Cl2 (3 ¥ 30 cm3). The combined organic
phases were dried (Na2SO4) and the solvent was evaporated. The
residual foam was applied to FC (silica gel, column 8 ¥ 3 cm,
eluted with CH2Cl2–acetone 100 : 0 → 85 : 15). Evaporation of the
main zone afforded 3a as colourless foam (125 mg, 63%) (Found:
C, 65.81; H, 6.49; N, 11.95%. C51H61N8O7P requires C, 65.93; H,
6.62; N, 12.06%); TLC (silica gel, CH2Cl2–acetone 85 : 15): Rf 0.6;
dH(250 MHz; CDCl3; Me4Si) 1.16 (12 H, m, 4 ¥ CH3), 1.64 (6 H,
m, CH2CN), 2.15 (1 H, m, 2¢-Ha), 2.19 (2H, m, C∫C-CH2), 2.50
∫
2 ¥ C C-CH2), 2.29 (1 H, m, 2¢-Ha), 2.76–2.81 (2 H, m, 2¢-Hb,
∫
CH(CH3)2), 2.97 (1 H, m, C CH), 3.05 (2 H, m, 5¢-H), 3.71 (6 H, s,
2 ¥ OCH3), 3.90 (1 H, m, 4¢-H), 4.48 (1 H, m, 3¢-H), 5.31–5.33 (1 H,
d, J 4.5, 3¢-OH), 6.36 (1 H, t, J 4.3, 1¢-H), 6.79 (6 H, m, 2 ¥ OCH3),
6.80–7.32 (13 H, 3 m, arom.H) and 11.92 (2 H, s, 2 ¥ NH).
1-{[2-Deoxy-5-O-(4,4¢-dimethoxytriphenylmethyl)-3-O-[(2-
cyanoethoxy)(N,N-diisopropylamino)phosphino]-b-D-erythro-
pentofuranosyl]}-6-[(2-methylpropanoyl)amino]-1,5-dihydro-3-
(octa-1,7-diynyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one (3b)
∫
(3 H, m, 2¢-Hb, C∫C-CH2), 2.63 (1 H, m, C CH), 3.09 (3 H, m,
NCH3), 3.19 (3 H, s, NCH3), 3.25 (2 H, m, CH(CH3)2), 3.76–3.77
(10 H, m, 2 ¥ CH3, OCH2, 5¢-H), 4.20 (1H, m, 4¢-H), 4.72 (1 H, s,
3¢-H), 6.59 (1 H, t, J 6.3, 1¢-H), 6.75–7.55 (13 H, 3 m, arom.H),
8.39 (1 H, s, N CH) and 8.74 ppm (1H, s, NH); dP(101 MHz;
CDCl3; H3PO4) 149.4 and 149.6.
To a solution of 5b (120 mg, 0.14 mmol) in dry CH2Cl2 (6 cm3), (i-
Pr)2NEt (0.06 cm3, 0.36 mmol) and 2-cyanoethyl diisopropylphos-
phoramidochloridite (0.06 cm3, 0.23 mmol) were added. After 3 h,
the solution was washed with saturated NaHCO3 (20 cm3), and
extracted with CH2Cl2 (2 ¥ 20 cm3), the combined org. layer was
=
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1385
©

View Article Online
dried over Na2SO4 and evaporated. FC (silica gel, column 8 ¥ 3 cm,
eluted with CH2Cl2–acetone 98 : 2) afforded a colorless foam of 3b
(110 mg, 72%) (Found: C, 66.99; H, 6.65; N, 10.64%. C52H62N7O7P
requires C, 67.30; H, 6.73; N, 10.56%); TLC (silica gel, CH2Cl2–
acetone 9 : 1): Rf 0.87; dH(250 MHz; CDCl3; Me4Si) 1.10 (6 H,
m, (CH3)2CH), 1.16 (12 H, m, 4 ¥ CH3), 1.74 (6 H, m, 2 ¥ CH2,
H-5-triazole), 8.56 (1 H, s, H-4-coumarin), 10.68 (1 H, s, NH); m/z
(ESI-TOF) 597.18 (M + Na+. C27H26N8O7Na requires 597.19).
Copper(I)-catalyzed [3 + 2] cycloaddition of oligonucleotides 12 or
13 with 3-azido-7-hydroxycoumarin 15
To the single-stranded oligonucleotide (5 A260 units), a 1 : 1 CuSO4–
TBTA ligand complex (0.05 cm3 of a 20 mM stock solution in
t-BuOH–H2O 1 : 9), tris(carboxyethyl)phosphine (TCEP; 0.05 cm3
of a 20 mM stock solution in water), 3-azido-7-hydroxycoumarin
(15; 0.05 cm3 of a 20 mM stock solution in dioxane–H2O 1 : 1),
sodium bicarbonate (0.05 cm3 of a 20 mM aq. solution) and
0.035 cm3 DMSO were added, and the reaction proceeded at room
temperature for 12 h. The reaction mixture was concentrated in a
speed vac and dissolved in 1 cm3 bidistilled water and centrifuged
for 20 min at 12 000 rpm. The supernatant solution was collected
and further purified by reversed-phase HPLC in the trityl-off
modus to give the ‘click’ product (oligonucleotide 18 or 19). The
molecular masses of 18 and 19 were determined by MALDI-TOF
mass spectrometry (Table 7).
∫
CH2CN), 2.18 (1 H, m, 2¢-Ha), 2.20 (2 H, m, C C-CH2), 2.52
∫
∫
(4 H, m, 2¢-Hb, C CH2, CH(CH3)2), 2.67 (1 H, m, C CH), 3.63
(2 H, m, 5¢-H), 3.77–3.79 (8 H, m, 2 ¥ CH3, OCH2), 4.23 (1 H,
m, 4¢-H), 4.75 (1 H, m, 3¢-H), 6.41 (1 H, t, J 6.3, 1¢-H), 6.74–7.45
=
(13 H, 3 m, arom. H), 8.39 (1 H, s, N CH) and 11.70 (1 H, s, NH);
dP(101 MHz; CDCl3; H3PO4) 149.0.
6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihydro-3-
(7-hydroxycoumarin-1¢,2¢,3¢-triazol-4¢-yl)pentylidyne-4H-
pyrrolo[2,3-d]pyrimidin-4-one (17)
To a solution of 1 (368 mg, 1 mmol) and 15 (291 mg, 1.43 mmol)
in water and ethyl alcohol (v/v = 1 : 1, 30 cm3), a freshly prepared
1 M solution of sodium ascorbate (0.2 cm3, 0.20 mmol) in water
and copper(II) sulfate pentahydrate 7.5% in water (0.17 cm3,
0.05 mmol) were added. The mixture was stirred vigorously
overnight in the dark at rt for 15 h. The solvent was evaporated,
and the remaining residue was loaded on a silica gel column
FC (silica gel, column 10 ¥ 4 cm, eluted with CH2Cl2–MeOH
80 : 20). From the main zone 17 was obtained as a yellow powder
(300 mg, 53%); TLC (silica gel, CH2Cl2–MeOH 9 : 1): Rf 0.30;
lmax (MeOH)/nm 243 (e/dm3 mol-1 cm-1 17 200), 347 (13 300);
dH(250 MHz; [d6]DMSO; Me4Si) 1.60 (2 H, t, J 7.2, CH2), 1.81
(2 H, t, J 7.4, CH2), 2.08 (1 H, m, 2¢-Ha), 2.30 (1 H, m, 2¢-Hb), 2.41
Acknowledgements
We thank Dr. V. R. Sirivolu for supplying us with compound
17 and Mr. P. Ding for Fig. 4d. We also thank Mr. D. Jiang for
measuring the NMR spectra, Dr. T. Koch from Roche Diagnostics
GmbH, Penzberg, Germany, for the measurement of the MALDI
spectra and Mr. N. Q. Tran for the oligonucleotide syntheses. We
appreciate critical reading by Mr. S. Pujari. Financial support
by the Roche Diagnostics GmbH and ChemBiotech, Muenster,
Germany, is gratefully acknowledged.
∫
(2 H, m, CH2), 2.76 (2 H, t, J 7.3 Hz, C C-CH2), 3.48 (1 H, m, 5¢-
H), 3.74 (1 H, m, 4¢-H), 4.27 (1 H, m, 3¢-H), 4.89 (1 H, m, 5¢-OH),
5.19 (1 H, m, 3¢-OH), 6.23 (1 H, t, J 6.0, 1¢-H), 6.30 (2 H, s, NH2),
6.85 (1 H, s, H-5-coumarin), 6.91 (1 H, m, H-6-coumarin), 7.70–
7.74 (1 H, d, J 8.6, H-8-coumarin), 8.33 (1 H, s, H-5-triazole), 8.55
(1 H, s, H-4-coumarin) and 10.39 (1 H, s, HN); m/z (ESI-TOF)
596.18 (M + Na+. C28H27N7O7Na requires 596.20).
References
1 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
2 H. C. Kolb and K. B. Sharpless, Drug Discovery Today, 2003, 8, 1128–
1137.
3 N. J. Agard, J. A. Prescher and C. R. Bertozzi, J. Am. Chem. Soc., 2004,
126, 15046–15047.
6-Amino-1-(2-deoxy-b-D-erythro-pentofuranosyl)-1,5-dihydro-3-
(7-hydroxycoumarin-1¢,2¢,3¢-triazol-4¢-yl)pentylidyne-4H-
pyrazolo[3,4-d]pyrimidin-4-one (16)
4 J. M. Langenhan and J. S. Thorson, Curr. Org. Synth, 2005, 2, 59–81.
5 E. Delamarche, B. Michel, C. Gerber, D. Anselmetti, H.-J. Gu¨ntherodt,
H. Wolf and H. Ringsdorf, Langmuir, 1994, 10, 2869–2871.
6 M. J. Joralemon, R. K. O’Reilly, C. J. Hawker and K. L. Wooley, J. Am.
Chem. Soc., 2005, 127, 16892–16899.
To a solution of 2d (100 mg, 0.27 mmol) and 15 (55 mg, 0.27 mmol)
in water and ethyl alcohol (v/v = 1 : 1, 8 cm3), a freshly prepared
1 M solution of sodium ascorbate (0.054 cm3, 0.054 mmol) in
water and copper(II) sulfate pentahydrate 7.5% in water (0.045 cm3,
0.0135 mmol) were added. The mixture was stirred vigorously
in the dark at rt for 15 h. The solvent was evaporated, and the
residue was applied to FC (silica gel, column 10 ¥ 4 cm, eluted
with CH2Cl2–MeOH 80 : 20). Evaporation of the main zone gave
a yellow powder of 16 (140 mg, 90%). TLC (silica gel, CH2Cl2–
MeOH 8 : 2): Rf 0.53; lmax (MeOH)/nm 243 (e/dm3 mol-1 cm-1 46
900), 347 (25 500); dH(250 MHz; [d6]DMSO; Me4Si) 1.62 (2 H, t,
J 8.4, CH2), 1.82 (2 H, t, J 8.9, CH2), 2.14 (1 H, m, 2¢-Ha), 2.50
7 M. R. Whittaker, C. N. Urbani and M. J. Monteiro, J. Am. Chem. Soc.,
2006, 128, 11360–11361.
8 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 36, 1249–
1262.
9 D. D. D´ıaz, S. Punna, P. Holzer, A. K. McPherson, K. B. Sharpless,
V. V. Fokin and M. G. Finn, J. Polym. Sci. Part A: Polym. Chem., 2004,
42, 4392–4403.
10 M. Malkoch, K. Schleicher, E. Drockenmuller, C. J. Hawker, T. P.
Russell, P. Wu and V. V. Fokin, Macromolecules (Washington, DC,
U. S.), 2005, 38, 3663–3678.
11 (a) P. M. E. Gramlich, C. T. Wirges, A. Manetto and T. Carell, Angew.
Chem., Int. Ed., 2008, 47, 8350–8358, and references therein; (b) P. M. E.
Gramlich, S. Warncke, J. Gierlich and T. Carell, Angew. Chem., Int. Ed.,
2008, 47, 3442–3444; (c) J. Gierlich, G. A. Burley, P. M. E. Gramlich,
D. M. Hammond and T. Carell, Org. Lett., 2006, 8, 3639–3642.
12 (a) A. J. Dirks, S. S. van Berkel, N. S. Hatzakis, J. A. Opsteen, F. L.
van Delft, J. J. L. M. Cornelissen, A. E. Rowan, J. C. M. van Hest,
F. P. J. T. Rutjes and R. J. M. Nolte, Chem. Commun., 2005, 4172–4174;
(b) S. S. Gupta, J. Kuzelka, P. Singh, W. G. Lewis, M. Manchester and
M. G. Finn, Bioconjugate Chem., 2005, 16, 1572–1579; (c) A. Moulin,
L. Demange, J. Ryan, D. Mousseaux, P. Sanchez, G. Berge´, D. Gagne,
∫
(2 H, s, CH2), 2.64 (1 H, m, 2¢-Hb), 2.77 (2 H, t, J 8.4, C C-CH2),
3.35 (2 H, m, 5¢-H), 3.75 (1 H, m, 4¢-H), 4.26 (1 H, m, 3¢-H), 4.72
(1 H, m, 5¢-OH), 5.22 (1 H, m, 3¢-OH), 6.26 (1 H, t, J 7.9, 1¢-H),
6.74 (2 H, s, NH2), 6.84 (1 H, s, H-5-coumarin), 6.91 (1 H, m,
H-6-coumarin), 7.71 (1 H, d, J 10.2, H-8-coumarin), 8.34 (1 H, s,
1386 | Org. Biomol. Chem., 2009, 7, 1374–1387
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
D. Perrissoud, V. Locatelli, A. Torsello, J.-C. Galleyrand, J.-A. Fehrentz
and J. Martinez, J. Med. Chem., 2008, 51, 689–693.
13 (a) P. Kocˇalka, A. H. El-Sagheer and T. Brown, ChemBioChem, 2008, 9,
1280–1285; (b) R. Kumar, A. El-Sagheer, J. Tumpane, P. Lincoln, L. M.
Wilhelmsson and T. Brown, J. Am. Chem. Soc., 2007, 129, 6859–6864;
(c) A. El-Sagheer, R. Kumar, S. Findlow, J. M. Werner, A. N. Lane and
T. Brown, ChemBioChem, 2008, 9, 50–52.
14 F. Seela, V. R. Sirivolu and P. Chittepu, Bioconjugate Chem., 2008, 19,
211–224.
15 F. Seela, Y. He, J. He, G. Becher, R. Kro¨schel, M. Zulauf and P.
Leonard, in Methods in Molecular Biology, ed. P. Herdewijn, Humana
Press, Totowa, NJ, 2004, vol. 288, pp. 165–86.
29 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q.
Wang, Org. Lett., 2004, 6, 4603–4606.
30 F. Seela and V. R. Sirivolu, Org. Biomol. Chem, 2008, 6, 1674–1687.
31 C. A. M. Seidel, A. Schulz and M. H. M. Sauer, J. Phys. Chem., 1996,
100, 5541–5553.
32 G. Wenska and S. Paszyc, Can. J. Chem., 1988, 66, 513–516.
33 S. A. E. Marras, F. R. Kramer and S. Tyagi, Nucleic Acids Res., 2002,
30, e122.
34 N. Ramzaeva, H. Rosemeyer, P. Leonard, K. Mu¨hlegger, F. Bergmann,
H. von der Eltz and F. Seela, Helv. Chim. Acta, 2000, 83, 1108–1126.
35 F. Seela, K. Shaikh and H. Eickmeier, Acta Crystallogr. Sect. C: Cryst.
Struct. Commun., 2004, 60, o489–o491.
36 M. Torimura, S. Kurata, K. Yamada, T. Yokomaku, Y. Kamagata, T.
Kanagawa and R. Kurane, Anal. Sci., 2001, 17, 155–160.
37 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley,
C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev. (Washington,
DC, U. S.), 1997, 97, 1515–1566.
16 F. Seela and G. Becher, Synthesis, 1998, 207–214.
17 F. Seela and G. Becher, Chem. Commun., 1998, 2017–2018.
18 J. He and F. Seela, Nucleic Acids Res., 2002, 30, 5485–5496.
19 G. Becher, J. He and F. Seela, Helv. Chim. Acta, 2001, 84, 1048–1065.
20 J. He and F. Seela, Tetrahedron, 2002, 58, 4535–4542.
21 F. Seela and G. Becher, Helv. Chim. Acta, 1999, 82, 1640–1655.
22 F. Seela and G. Becher, Nucleic Acids Res., 2001, 29, 2069–2078.
23 F. Seela and M. Zulauf, Synthesis, 1996, 726–730.
24 F. Seela and V. R. Sirivolu, Helv. Chim. Acta, 2007, 90, 535–552.
25 F. Seela and K. I. Shaikh, Tetrahedron, 2005, 61, 2675–2681.
26 F. Seela and H. Steker, Helv. Chim. Acta, 1986, 69, 1602–1613.
27 F. Seela and M. Zulauf, Helv. Chim. Acta, 1999, 82, 1878–1898.
28 F. Seela and V. R. Sirivolu, Chem. Biodiversity, 2006, 3, 509–514.
38 J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs, C. W. Robertson,
R. J. Zagursky, A. J. Cocuzza, M. A. Jensen and K. Baumeister, Science
(Washington, DC, U. S.), 1987, 238, 336–341.
39 C. Seidel, K. Rittinger, J. Cote´s, R. S. Goody, M. Ko¨llner, J. Wolfrum
and K. O. Greulich, in Biomolecular Spectroscopy II, ed. R. R. Birge
and L. A. Nafie, Proc. SPIE, 1991, vol. 1432, pp. 105–116.
40 M. Petersheim and D. H. Turner, Biochemistry, 1983, 22, 256–
263.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1374–1387 | 1387
©