DNA with stable fluorinated da and dG substitutes: Syntheses, base pairing and 19F-NMR spectra of 7-fluoro-7-deaza-2′-deoxyadenosine and 7-fluoro-7-deaza-2′-deoxyguanosine

Article abstract of DOI:10.1039/b806145a

Fluorinated DNA containing stable fluorine substituents in the "purine" base were synthesized for the first time. For this, the phosphoramidites of 7-fluoro-7-deaza-2′-deoxyadenosine and 7-fluoro-7-deaza-2′-deoxyguanosine were prepared and oligonucleotide

Full text of DOI:10.1039/b806145a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
DNA with stable fluorinated dA and dG substitutes: syntheses, base pairing
and 19F-NMR spectra of 7-fluoro-7-deaza-2ꢀ-deoxyadenosine and
7-fluoro-7-deaza-2ꢀ-deoxyguanosine
Frank Seela*a,b and Kuiying Xua,b
Received 11th April 2008, Accepted 6th June 2008
First published as an Advance Article on the web 17th July 2008
DOI: 10.1039/b806145a
Fluorinated DNA containing stable fluorine substituents in the “purine” base were synthesized
for the first time. For this, the phosphoramidites of 7-fluoro-7-deaza-2ꢀ-deoxyadenosine and
7-fluoro-7-deaza-2ꢀ-deoxyguanosine were prepared and oligonucleotides were synthesized. The 7-fluoro
substitution leads to increased duplex stability and more selective base pairing compared to the
non-functionalized 7-deazapurine oligonucleotides. 19F NMR spectra of fluorinated nucleosides, single
stranded oligonucleotides and DNA duplex show only a single signal for one fluorine modification. The
NMR sensitive 19F spin or the positron emitting 18F isotope make these compounds applicable for
DNA detection or imaging in vitro and in vivo.
stable, 5-fluorinated uracil/thymine and cytosine nucleosides have
been incorporated in DNA or RNA either chemically by solid-
Introduction
phase synthesis or enzymatically employing polymerases.13 Single
crystal X-ray analyses showed that a fluorine atom present in the
5-position of a pyrimidine moiety is well accommodated in the
duplex structures.14,15 It was also reported that a 5-fluorouridine
residue at a single site of RNA stabilizes the duplex due to more
favorable stacking interactions.16
Fluorinated nucleosides develop extraordinary pharmacologi-
cal activities as chemotherapeutic agents for the treatment of
cancer or as antiviral drugs.1 Fluorinated oligonucleotides are
stabilized against enzymatic degradation and polyfluorinated
oligonucleotides are employed in fluorous affinity purification.2
The fluorine isotopes are used for molecular imaging or as
molecular tags. The positron emitting isotope [18F] (s = 109.8 min)
finds clinical application in in vivo imaging by positron emission
tomography (PET) using [18F] labelled molecules. By this means,
the hypermetabolic activity of proliferating cells can be followed
in tissues. On the other hand, the NMR sensitive 19F isotope
is a valuable tool to study DNA or RNA secondary structure3
by NMR spectroscopy or to investigate the binding of nucleic
acids to proteins or other cellular components. Sugar fluori-
nated nucleosides,4 in particular the 2ꢀ-deoxy-2ꢀ-fluoroarabino
nucleosides have been used as nucleoside mimics in DNA and
RNA chemistry, and molecular biology.5–8 However, a fluorine
atom in the 2ꢀ-position of the sugar moiety of nucleosides and
oligonucleotides induces conformational changes depending on
the antipodal position of incorporation (2ꢀ-ribo or 2ꢀ-arabino).8–12
This effect can be circumvented when the fluorine atom is
introduced into the nucleobase.
Compared to the easier access to fluorinated pyrimidine nucle-
osides, the introduction of fluorine atoms into purine nucleosides
encounters difficulties, as fluorine substituents in the 2- or 8-
position are labile and subject to nucleophilic displacement reac-
tions (purine numbering is used throughout the general section).
A fluorine in the 2-position of adenosine or 2ꢀ-deoxyadenosine17
is easily displaced by nucleophiles such as amines.18 2-Fluoro-
7-deaza-2ꢀ-deoxyadenosine is more stable and oligonucleotides
with this modification were prepared recently.19 Nevertheless, the
replacement of a hydrogen atom by fluorine in the 2-position
has an unfavorable effect on the dA–dT base pair stability.19
By changing the fluorination side of a purine base from the
2- to the 8-position, the situation becomes even more complex
already on the nucleoside level.20 The preparation encounters
synthetic difficulties and defluorination reactions are occurring
in basic and acidic media.21 Thus, there has been no report on the
incorporation of a purine nucleobase fluorinated in the 8-position
of the adenine or the guanine moiety, neither in DNA, RNA or in
short oligonucleotides.
Our laboratory and others have selected the 7-position of 7-
deazapurines as the target site for fluorination reactions. Recently,
the syntheses of 7-deaza-7-fluoro-2ꢀ-deoxyadenosine (1b) and 7-
deaza-7-fluoro-2ꢀ-deoxyguanosine (2b) have been reported22 and
the X-ray analysis of 1b was performed.23 Now, we are studying
the behavior of oligonucleotides incorporating the fluorinated
7-deazapurine nucleosides 1b and 2b. The non-functionalized
nucleosides 1a and 2a, as well as other halogenated derivatives
including 1c and 2c, were already introduced into the DNA
molecule (Scheme 1).19,24–26 This manuscript describes for the
From a steric point of view, the fluorine atom introduced into
a nucleobase is expected to lead only to minor perturbation of
the molecule shape. This results from the small van der Waals
˚
radius of fluorine (1.47 A) compared to other halogens and
˚
being similar to that of hydrogen (1.20 A). As 5-fluorinated
pyrimidine nucleosides are readily accessible and reasonably
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 48149 Mu¨nster, Germany. E-mail:
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. E-mail: Frank.Seela@uni-osnabrueck.de
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Scheme 1 Structure of nucleosides and the corresponding phosphoramidites.
first time the solid-phase synthesis of stable fluorinated oligonu-
cleotides with fluorine atoms in the “purine” moiety employing
the phoshoramidites 3 and 4. It reports on the thermal and
chemical stability of base pairs formed by the 7-fluorinated
nucleosides 1b or 2b. It demonstrates that the 19F NMR signal
can be used to detect fluorinated “purine” bases in oligonucleotide
single strands and duplexes by 19F-NMR spectroscopy. Thus the
modification has the potential to be employed in DNA detection or
imaging.
→ 5b, 7b, Scheme 2). The amino-protected intermediates 5b and
7b were converted into the 5ꢀ-O-DMT derivatives 6 and 8 under
standard conditions with DMT-Cl in pyridine. Phosphitylation
with 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite in
anhydrous CH2Cl2 (i-Pr2EtN) furnished the phosphoramidites 3
and 4 (Scheme 2).
The structures of all new compounds were established by
1H-, 13C-, 19F- and 31P-NMR spectroscopy, as well as by mass
spectrometry and elemental analyses (Table 1 and experimental
part). Assignment of the 13C-NMR resonance was made on the
1
basis of gated-decoupled H-13C-NMR spectra and by using JCF
coupling constants as described in earlier publications.22,31 Table 1
summarizes the 13C NMR chemical shifts and Table 2 the 19F
chemical shifts and the JCF coupling constants. The observed C-
7 chemical shifts of the 7-fluorinated 7-deazapurine nucleosides
1b and 2b22(d ≈ 140 ppm) are found downfield compared to
the non-functionalized nucleosides 1a and 2a (d ≈ 100 ppm).24,25
The other 7-halogen substitutions lead either to a small down-
field shift (D = 1–2 ppm upon 7-chlorination)24,25 or an up-field
Results and discussion
1. Synthesis and properties of the monomers
The synthesis of 7-fluoro-7-deaza-2ꢀ-deoxyadenosine (1b) and 7-
fluoro-7-deaza-2ꢀ-deoxyguanosine (2b) has been already reported
by our laboratory.22 The phosphoramidites 3 and 4 were prepared
from 1b and 2b in a three-step route (Scheme 2). As amino
protecting groups, the benzoyl residue (Bz) was chosen for
compound 1a27 and the isobutyryl residue (i-Bu) for 2a.28,29 These
protecting groups were also employed here for the 7-fluorinated
nucleosides using the protocol of transient protection30 (1b, 2b
shift (D ≈ −15 ppm upon bromination and D ≈ −50 ppm upon
1
iodination).24,25,32,33 The 7-fluoro atom causes JC7,F
,
2JC8,F, and
2JC5,F couplings, as was observed for the sugar modified nucleosides
Scheme 2 Reagents and conditions: (i) TMSCl, BzCl, pyridine; (ii) 4,4ꢀ-dimethoxytriphenylmethyl chloride, anhydrous pyridine; (iii) 2-cya-
noethyl-N,N-diisopropylphosphoramidochloridite, N,N-diisopropylethylamine, dichloromethane; (iv) TMSCl, isobutyric anhydride, pyridine.
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Table 1 13C NMR chemical shifts (d) of nucleosidesa
C(2)b
C(2)c
C(4)
C(6)
C(4a)
C(5)
C(5)
C(7)
C(6)
C(8)
C(7a)
C(4)
C(1ꢀ)
C(2ꢀ)
C(3ꢀ)
C(4ꢀ)
C(5ꢀ)
CO
OMe
d
d
d
d
d
d
d
d
1a
151.6
152.7
150.0
150.1
150.1
152.5
153.0e
152.5
147.4
147.4
157.5
155.8
151.0
151.2
151.2
158.5
156.9e
158.5
154.9
154.9
102.9
92.3
109.4
101.9
102.0
100.0
89.9
100.0
93.6
93.8
99.6
142.6
103.1
141.6
141.6
102.1
145.6
102.1
145.4
145.3
121.6
103.9
124.3
108.9
108.8
116.7
99.5
116.7
102.2
102.2
149.7
145.8
152.0
147.3
147.3
150.5
147.6e
150.5
143.5
143.5
83.3
82.4
82.9
82.5
82.5
82.2
82.6
82.2
82.3
82.3
71.2
70.9
70.9
70.9
70.6
70.8
70.8
70.8
70.9
70.5
87.3
87.2
87.3
87.4
85.5
86.9
87.4
86.9
87.3
85.4
62.2
61.9
61.9
61.8
64.2
61.9
61.8
61.9
61.8
64.2
—
—
165.7
166.5
166.6
—
1b22
5a24
5b
6
55.0
54.9
2a25
2b22
7a25
7b
—
34.8
34.8
180.2
180.2
8
a Measured in d6-DMSO at 298 K. b Systematic numbering. c Purine numbering. d Superimposed by DMSO. e Tentative.
c
Table 2 19F NMR chemical shifts (d)a,b and JFC coupling constants of
observed in the case of the non-halogenated derivative 7a or the
corresponding 7-iodo derivative 7c (Fig. 1c). Consequently, mild
deprotection conditions were chosen for 7b and deprotection was
complete after 24 hours’ treatment in 25% ammonia, as monitored
by HPLC, without significant decomposition (Fig. 1d). Thus,
for the synthesis of oligonucleotides containing 2b, canonical
phosphoramidites with tert-butylphenoxyacetyl (tac) protecting
groups were employed, which can be deprotected in aqueous
ammonia at room temperature.
nucleosides
Compound
d (F-7)b
1JC7,F7
2JC8,F7
2JC5,F7
3JC4,F7
3JC6,F7
d
d
d
d
d
d
d
d
d
d
d
d
1b
9
10
5
6
2b
7b
8
−167.95
−168.30
−168.00
−163.79
−163.60
−167.89
−165.93
−165.94
248.1
245.3
244.8
248.1
247.8
247.8
247.8
247.9
28.4
30.2
26.8
28.4
27.9
27.1
26.8
26.5
15.8
15.7
15.7
15.8
15.3
12.4
13.8
13.8
3.2
3.1
2.6
2.8
2. Synthesis and duplex stability of oligonucleotides containing
the fluorinated nucleosides 1b and 2b
◦
a Measurements were performed in d6-DMSO at 25 C. b Data are taken
from 19F NMR spectra with CFCl3 as reference. c Data are taken from 13
C
NMR spectra. d Smaller than 1 Hz.
Oligonucleotide synthesis was carried out on a solid phase with
an ABI 392-08 synthesizer at a 1 lmol scale employing the
phosphoramidites 3 and 4, as well as standard building blocks. The
coupling yields were always higher than 95%. For oligonucleotides
without modification, or those containing 1b, the synthesis of
oligonucleotides was performed by employing the DMT-on mode.
After cleavage from the solid support, the oligonucleotides were
deprotected in 25% aq. NH3 for 16 h at 60 ◦C. The synthesis
of oligonucleotides incorporating 2b used tert-butylphenoxyacetyl
(tac) protected canonical phosphoramidites employing the DMT-
off mode and mild deprotection conditions (25% aq. NH3,
room temperature, 24 h). The oligonucleotides were purified by
reversed-phase HPLC. The detailed procedure for oligonucleotide
purification is reported in the experimental part. The molecular
masses of all synthesized oligonucleotides were determined by
922 and 10.34 The protected 7-fluoro-7-deaza-2ꢀ-deoxyguanosine
derivatives show long range couplings (3JC4,F and 3JC6,F) (Table 2).
Next, the stability of compounds 5b and 7b was studied.
The half life values of both compounds were determined UV-
spectrophotometrically in 25% aqueous NH3 at 40 ◦C in a sealed
vessel. In the “dG” series, the half life is 94 min for 7b, which is
similar to that of 7a (109 min).28 The corresponding values for
the “dA” derivatives are significantly longer with 530 min for 5b,
compared to 320 min for 5a28 (Scheme 3). Both 5b and 5a were
fully deprotected at 60 ◦C in 25% aqueous NH3. However, in
the case of 7-fluoro-7-deaza-2ꢀ-deoxyguanosine, the deprotection
of 7b was accompanied by partial decomposition (see HPLC
profile of Fig. 1a compared to 1b). This decomposition was not
Scheme 3 Structure of nucleosides.
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Fig. 1 Reversed-phase HPLC (RP-18) profiles of deprotection of 7b with 25% aqueous ammonia at 60 ◦C for 16 h (a); an artificial mixture of compounds
2a–c (b); deprotection of 7c with 25% aqueous ammonia at 60 ◦C for 16 h (c) and deprotection of 7b with 25% aqueous ammonia at room temperature
(d). The mixtures were analyzed by reversed-phase HPLC at 260 nm on a RP-18 column (250 × 4 mm). Gradient: 4% MeCN, 96% buffer [buffer: 0.1M
(Et3NH)OAc (pH 7.0)–MeCN, 95 : 5]. Flow rate = 0.7 cm3 min−1
.
MALDI-TOF mass spectra (Table 6, experimental part). They
were in agreement with the calculated values.
7-iodo derivative 2c (Table 3), which also stabilizes the duplexes
under the same conditions. The step-wise stability increase by the
various halogen substituents is most clearly seen in the case of
the self-complementary dodecamers 29–32. Both the fluorinated
and the iodinated residues have a positive effect on the base pair
stability. The duplex stability increases with the increasing van der
Waals radii of the 7-halogens (Table 3).
Fig. 2 shows the space-filling molecular models of adenine, 7-
deazaadenine and the halogenated 7-deazapurines. It is apparent
that the fluorine substituent is less space demanding than the
other halogens. So, in enzymatic reactions within the limited space
in a catalytic active center of an enzyme, the fluoro compounds
are expected to be excellent substrates with favorable properties
over the other halogenated derivatives. As the non-functionalized
7-deazapurine nucleosides (1a and 2a) in the form of their
triphosphates are already excellent substrates for the polymerase
chain reaction35 or sequencing protocols36, it is expected that the
7-fluoro compounds 1b and 2b are also excellent mimics of the
canonical DNA constituents.
The base pair stabilities of halogenated 7-deazapurine nu-
cleosides with Cl, Br, I in the 7-position of 7-deaza-2ꢀ-
deoxyadenosines and 7-deaza-2ꢀ-deoxyguanosines have already
been reported.19,24–26,33 However, due to synthetic problems, noth-
ing is known about fluorinated derivatives. Therefore, the effect
of 7-fluorine substitution in 7-deazapurines has now been studied
for the first time by comparing the duplex stabilities and mismatch
discrimination of the 7-fluorinated nucleosides (1b, 2b) with the
parent compounds (1a, 2a) and the 7-iodo derivatives (1c, 2c).
The hybridization experiments were performed using the duplexes
5ꢀ-d(TAGGTCAATACT) (11) and 3ꢀ-d(ATCCAGTTATGA) (12)
as references. The modified duplexes contain single or multiple
incorporations of fluorine-modified molecules at various posi-
tions. The base pairing of the oligonucleotides with the four
canonical nucleosides opposite to the modification sites was also
investigated.
Table 3 summarizes the Tm values of duplex oligonucleotides
containing 1a–c, 2a–c with one to twelve modifications. According
to the Table, the replacement of one or two dA-residues by 2ꢀ-
deoxytubercidin (1a) has a slight destabilizing effect on the non-
self complementary duplex stability (Tm = 50 ◦C for duplex
13·12 and 11·14). For comparison, compound 1b introduced at
the same positions stabilizes the duplexes (Tm = 51 ◦C, 53 ◦C
for 15·12 and 11·16) as well as compound 1c. Also, the 7-
deaza-2ꢀ-deoxyguanosine (2a) has a destabilizing effect on the
duplex in place of dG (Tm = 50 or 49 ◦C with one or more
modifications). Like in the “2ꢀ-deoxyadenosine” series, the 7-
fluorination of the 2ꢀ-deoxyguanosine analogue (→2b) led to the
stabilization of the duplexes (Tm = 51–53 ◦C with one or more
modifications). This stabilizing effect is comparable to that of the
Apart from Watson–Crick base pairing, we wanted to investi-
gate the mismatch discrimination of the fluorinated nucleosides 1b
and 2b. Therefore, the base pairing of compounds 1b, 2b against
the four canonical nucleosides was studied. For this investigation,
the Tm values of 12-mer duplexes containing the modified
nucleosides located opposite the four canonical constituents were
prepared and Tm values were measured (Table 4 and Table 5). From
earlier investigations19 and the data shown in Table 5, it is apparent
that 7-deaza-2ꢀ-deoxyadenosine (1a) generates a strong base pair
not only with dT (Tm = 50 ◦C) but also with dG (Tm = 47 ◦C) and
dC (Tm = 44 ◦C), while a significant discrimination occurs against
dA (Tm = 33 ◦C). Although the 7-fluoro derivative 1b strengthens
◦
◦
the base pairs with dT (Tm = 53 C), dG (Tm = 51 C) and dA
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Table 3 Tm values and thermodynamic data of oligonucleotide duplexes containing 1a–c, 2a–ca
Duplexes
Tm/◦C
DGb
Tm/◦C
DGb
5ꢀ-d(TAGGTCAATACT)
3ꢀ-d(ATCCAGTTATGA)
11
12
51
−13.0
5ꢀ-d(TAGGTCAATACT)
11
21
50
50
49
49
51
52
52
53
−12.0
3ꢀ-d(ATCCA2aTTATGA)
5ꢀ-d(TAGGTC1aATACT)
1319
12
50
50
48
51
53
52
53
52
53
51
54
54
33
36
−12.7
−11.3
−11.0
−10.7
−13.0
−11.8
−12.4
−12.0
−11.7
−11.9
−13.2
−12.2
0.22
5ꢀ-d(TAGGTCAATACT)
11
−11.8
−12.0
−11.5
−10.7
−11.5
−11.6
−12.2
3ꢀ-d(ATCCAGTTATGA)
3ꢀ-d(ATCCA2aTTAT2aA)
2226
5ꢀ-d(TAGGTCAATACT)
11
5ꢀ-d(TA2a2aTCAATACT)
2326
12
3ꢀ-d(ATCC1aGTT1aTGA)
1419
3ꢀ-d(ATCCAGTTATGA)
5ꢀ-d(TAGGTC1aATACT)
3ꢀ-d(ATCC1aGTT1aTGA)
13
14
5ꢀ-d(TA2a2aTCAATACT)
3ꢀ-d(ATCCA2aTTAT2aA)
23
22
5ꢀ-d(TAGGTC1bATACT)
15
12
5ꢀ-d(TAGGTCAATACT)
11
24
3ꢀ-d(ATCCAGTTATGA)
3ꢀ-d(ATCCA2bTTATGA)
5ꢀ-d(TAGGTCAATACT)
11
16
5ꢀ-d(TAGGTCAATACT)
11
25
3ꢀ-d(ATCC1bGTT1bTGA)
3ꢀ-d(ATCCA2bTTAT2bA)
5ꢀ-d(TAGGTC1bATACT)
3ꢀ-d(ATCC1bGTT1bTGA)
15
16
5ꢀ-d(TA2b2bTCAATACT)
26
12
3ꢀ-d(ATCCAGTTATGA)
5ꢀ-d(TAGGTC1b1bTACT)
17
12
5ꢀ-d(TA2b2bTCAATACT)
3ꢀ-d(ATCCA2bTTAT2bA)
26
25
3ꢀ-d(ATCCAGTTATGA)
5ꢀ-d(T1bGGTC1b1bT1bCT)
18
12
3ꢀ-d(ATCCAGTTATGA)
5ꢀ-d(T1bGGTC1b1bT1bCT)
3ꢀ-d(ATCC1bGTT1bTGA)
18
16
5ꢀ-d(TAGGTC1cATACT)
1919
12
5ꢀ-d(TAGGTCAATACT)
11
52
51
53
48
59
−11.9
−11.6
−12.1
−1.9
3ꢀ-d(ATCCAGTTATGA)
3ꢀ-d(ATCCA2cTTAT2cA)
2728
5ꢀ-d(TAGGTCAATACT)
11
5ꢀ-d(TA2c2cTCAATACT)
2828
12
3ꢀ-d(ATCC1cGTT1cTGA)
2019
3ꢀ-d(ATCCAGTTATGA)
5ꢀ-d(TAGGTC1cATACT)
3ꢀ-d(ATCC1cGTT1cTGA)
19
20
5ꢀ-d(TA2c2cTCAATACT)
3ꢀ-d(ATCCA2cTTAT2cA)
28
27
5ꢀ-d(ATATATATATAT)c
3ꢀ-d(TATATATATATA)
2924
29
5ꢀ-d(1bT1bT1bT1bT1bT1bT)c
3ꢀ-d(T1bT1bT1bT1bT1bT1b)
31
31
5ꢀ-d(1aT1aT1aT1aT1aT1aT)c
3ꢀ-d(T1aT1aT1aT1aT1aT1a)
3024
30
0.0
5ꢀ-d(1cT1cT1cT1cT1cT1cT)
3ꢀ-d(T1cT1cT1cT1cT1cT1c)
3233
32
−11.4
a Measured at 260 nm in 1 M NaCl, 0.1 M MgCl2, 60 mM Na-cacodylate, pH 7, at 5 lM + 5 lM of single strand concentration. b DG values are given in
Kcal mol−1 and within 10% errors. c 15 lM of single strands.
˚
Fig. 2 Structure of nucleobases (left) and the corresponding space-filling models (right). Van der Waals radii (A) are: H = 1.2; F = 1.47; Cl = 1.75; Br =
1.85; I = 1.98. Electronegativities are: H = 2.2; F = 3.98; Cl = 3.16; Br = 2.96; I = 2.66.
(Tm = 39 ◦C) when compared to the parent compound 1a, the
pairing discrimination against dC is significantly better in the case
of the fluorinated nucleoside 1b than 1a. The mispairing of 1b with
dG is not unexpected as stable dA–dG base pairs (Fig. 3, motif I)
have already been detected in many other cases including dA–dG
or c7Ad–dG pairs (Fig. 3, motif II).37–40 It was already reported that
the stability of this mispair is sequence dependent. Also, tandem
base pairs are better stabilized than single substitutions. Table 4
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Table 4 Tm values (◦C) of duplexes 5ꢀ-d(TAGGTXAATACT)-3ꢀ·3ꢀ-
d(ATCCAYTTATGA) with mismatches opposite to dG and 2a,ba,b
In the case of 1a, it was found that a decreasing pH value19
resulted in higher Tm values, which is in line with protonated
base pair formation (Fig. 4, motifs IV and V). As the pH value
of protonation of compound 1a is higher (pKa = 5.08) compared
to dA (pKa = 3.5), it is obvious that at neutral pH, compound
1a forms stronger mispairs with dC than dA. Table 5 shows the
base discrimination of oligonucleotides with 1a–c against dC. The
DTm values are calculated on the basis of the corresponding dA
(c7Ad)–dT base-pair Tm values to eliminate the effect of the second
modification. Compared to the 7-non functionalized compound
X·Y
Duplex
Tm/◦C
DTm/◦C
DG/Kcal mol−1
C·G
G·G
A·G
T·G
11·12
33·12
34·12
35·12
11·21
33·21
34·21
35·21
11·24
33·24
34·24
35·24
51
30
35
36
50
30
36
38
51
33
31
38
−13.0
−6.4
−7.3
−7.8
−12.0
−5.9
−7.5
−8.3
−10.7
−7.1
−6.5
−7.8
−21
−16
−15
C·2a
G·2a
A·2a
T·2a
C·2b
G·2b
A·2b
T·2b
−20
−14
−12
1a (DTm = −6 ◦C), the 7-fluorinated compound 1b (DTm
=
−10 ◦C) shows a better mismatch discrimination being similar
to dA (DTm = −14 ◦C). Therefore, at pH 7.0, a higher proportion
of protonated species is formed in the case of compound 1a (pKa
5.1) compared to 1b (pKa 4.4).18 Thus, the 7-fluorinated 7-deaza-
2ꢀ-deoxyadenosine (1b) behaves more as a dA analogue than the
non-fluorinated 1a. The proposed base pair motifs are shown in
Fig. 4.
−18
−20
−13
a Measured at 260 nm in 1 M NaCl, 0.1 M MgCl2, 60 mM Na-cacodylate
buffer, pH 7.0, with 5 lM single-strand concentration. b The data in
base mismatch
parentheses are DTm (Tm
− Tmbase match).
Table 5 Tm values (◦C) of duplexes 5ꢀ-d(TAGGXCAATACT)·3ꢀ-
d(ATCCYGTTYTGA) with mismatches opposite to A and 1a–ca,b
X·Y
Duplex
Tm/◦C
DTm/◦C
DG/Kcal mol−1
T·A
11·12
36·12
37·12
38·12
11·14
36·14
37·14
38·14
11·16
36·16
37·16
38·16
11·20
36·20
37·20
38·20
51
40
47
37
50
33
47
44
53
39
51
43
54
41
53
43
−13.0
−8.7
A·A
G·A
C·A
−11
−4
−10.1
−7.9
−14
T·1a
A·1a
G·1a
C·1a
T·1b
A·1b
G·1b
C·1b
T·1c
A·1c
G·1c
C·1c
−11.3
−6.9
−16
−3
−10.6
−9.7
−6
Fig. 4 Base pare motifs for dA and 1b with dC.
−13.0
−8.4
−14
−2
19F NMR spectroscopy is widely used for the structure elucida-
tion of monomeric nucleosides as well as for oligonucleotides.3,10,42
As the fluorine nucleus is surrounded on average by 9 electrons,
rather than by a single electron in the case of hydrogen, the range
of fluorine chemical shifts and the sensitivity to local environment
changes are used as sensitive reporters. At present, fluorinated
purine nucleosides cannot be used for this purpose due to stability
problems. The 19F spectra of the fluorinated nucleosides 2b and the
oligonucleotide 24 incorporating 2b can be used, and spectra were
measured in aqueous buffer solution. The data for the monomers
measured in DMSO-d6 are already summarized in Table 2. All
7-fluorinated 7-deazapurine nucleosides show only one 19F NMR
signal at approximately −165 ppm when measured in d6-DMSO
(see Table 2). This signal is not split when the proton-coupled
mode is used. As we wanted to confirm that the measurement
was correct, the corresponding spectrum of the bis-fluorinated
compound 9 (Scheme 3, see above) was undertaken. The latter
shows only one singlet for the 7-fluorine atom at −168.30 ppm
(Table 2) and a multiplet at −199.68 ppm for the 2ꢀ-fluoro
substituent with a 2JF,H2 ꢀ coupling and two 3JF,H couplings.22 Thus,
it was confirmed that a possible coupling of the 7-fluorine atom
with any proton is smaller than 1 Hz, which is the detection limit
of our NMR machine.
−11.0
−9.8
−10
−13.2
−8.8
−13
−1
−12.1
−10.0
−11
a Measured at 260 nm in 1 M NaCl, 0.1 M MgCl2, 60 mM Na-cacodylate
buffer, pH 7.0, with 5 lM single-strand concentration. b The data in
base mismatch
parentheses are DTm (Tm
− Tmbase match).
Fig. 3 Base pair motifs of dA–dG, c7Ad–dG and dA–c7Gd.
shows the mispairing of dA–2a,b (Fig. 3, motif III). Obviously a
dA (or 1a–c)–dG (or 2a,b) pair between two dA–dT pairs (dA–dG,
Tm = 35 ◦C, Table 4) is less stable than that between two dG–dC
base pairs (dA–dG, Tm = 47 ◦C, Table 5).
In aqueous solutions (0.1 M NaCl, 10 mM MgCl2, and 10 mM
Na-cacodylate buffer), the chemical shift of 7-fluorine of 2b
is shifted downfield to −165.96 ppm compared to −166.67 in
d6-DMSO (Fig. 5). Next, the oligonucleotide 24 incorporating
one 2b residue was measured also showing only one signal at
The base pairing of protonated dA with dC41 or of the proto-
nated 7-deaza-2ꢀ-deoxyanosine with dC19 was already reported.
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for 19F, or 85% H3PO4 for 31P. The J-values are given in Hz.
Reversed-phase HPLC was carried out on a 4 × 250 mm RP-18
(10 lm) LiChroscorb column (VWR International) with a Merck-
Hitachi HPLC pump (Model 655 A-12) connected with a variable
wavelength monitor (model 655-A), a controller (model L-500)
and an integrator (model D-2000). Elemental analyses were per-
formed by the Mikroanalytisches Laboratorium Beller, Go¨ttingen,
Germany. MALDI-TOF mass spectra were recorded on a Biflex-
III spectrometer (Bruker, Leipzig, Germany) in the reflector mode
and on an Applied Biosystems Voyager DE PRO spectrometer
in the linear mode. Half-life values (s) and the melting curves
were measured with a Cary-100 Bio UV/Vis spectrophotometer
(Varian, Australia) and all values were measured more than twice
and they were repeatable.
Fig. 5 19F NMR spectra (CFCl3 as external standard and measured at
298 K) of compound 2b (a); oligonucleotide 24 (b) and duplex 11·24 (c).
Samples were dissolved in 0.4 mM in 1 M NaCl, 100 mM MgCl2 and
60 mM Na-cacodylate buffer, pH 7.0.
Synthesis and characterization of oligonucleotides
The oligonucleotide synthesis was performed on an ABI 392–
08 synthesizer, model 392–08 (Applied Biosystems, Weiterstadt,
Germany) at a 1 lmol scale (trityl on mode) employing phospho-
ramidites and following the synthesis protocol for 3ꢀ-cyanoethyl
phosphoramidites (User’s Manual of the DNA Synthesizer, Ap-
plied Biosystems, Weiterstadt, Germany). The average coupling
yield was always higher than 95%. After cleavage from the solid-
support, the oligonucleotides were deprotected in 25% aq. NH3
for 16 h at 60 ◦C or 24 h at r.t. for oligonucleotides containing 2b.
The DMT-containing oligonucleotides were purified by reversed-
phase HPLC (RP-18) with the following solvent gradient system
[A: 0.1 M (Et3NH)OAc (pH 7.0)–MeCN 95 : 5; B: MeCN;
gradient I: 0–3 min 10–15% B in A, 3–15 min 15–50% B in A,
15–20 min 50–10% B in A, flow rate 1.0 cm3 min−1]. Then, the
mixture was evaporated to dryness, and the residue was treated
with 2.5% Cl2CHCOOH–CH2Cl2 for 3 min at 0 ◦C to remove
the 4,4ꢀ-dimethoxytrityl residues. Oligonucleotides containing 2b
were synthesized in the DMT-off mode to avoid acidic conditions
after deprotection of the amino group. The detritylated oligomers
were purified by reversed-phase HPLC with the gradient II: 0–
25 min 0–20% B in A, 25–30 min 20% B in A, 30–35 min 20–0%
B in A, flow rate 1.0 cm3 min−1. The oligomers were desalted on
a short column (RP-18, silica gel) using H2O for elution of the
salt, while the oligomers were eluted with MeOH–H2O (3 : 2). The
oligonucleotides were lyophilized on a Speed Vac evaporator to
yield colourless solids which were stored frozen at −24 ◦C.
−165.86 ppm with an almost identical chemical shift to that of
the monomeric 2b measured in the same solvent (Fig. 5). This
indicates that in the single-stranded chain, the microenvironment
and the solvation of the 7-fluoro substituent are rather similar to
that of the monomeric nucleoside. The situation changes when
compound 2b is part of a base pair stack as in the duplex 11·24.
Now, the fluorine signal is shifted upfield to −166.21 ppm.
3. Conclusion
7-Fluoro substituents were introduced in 7-deaza-2ꢀ-
deoxyadenosine (→1b) and 7-deaza-2ꢀ-deoxyguanosine (→2b)
and corresponding phosphoramidites (3, 4) were prepared
as well as oligonucleotides containing this modification. The
7-fluoro substituent introduced in the 7-position of 7-deaza-2ꢀ-
deoxydenosine (1b) stabilizes the base pair with dT and improves
mismatch discrimination. A stabilization of the base pair of
2b with dC is observed. While fluorine in the 2- or 8-position
of purine nucleosides is labile, the 7-fluorinated 7-deazapurine
nucleosides are stable enough to be detected by 19F NMR
spectroscopy in single stranded oligonucleotides or duplex
DNA. Thus, the fluorinated 7-deazapurines are applicable to
DNA detection and imaging in vitro as well as in living systems.
The molecular masses of the oligonucleotides were determined
in positive mode by MALDI-TOF Biflex-III mass spectrometry
(Bruker Saxonia, Leipzig, Germany) for compounds containing
1b or in negative mode by an Applied Biosystems Voyager DE
PRO spectrometer for compounds containing 2a or 2b with 3-
hydroxypicolinic acid (3-HPA) as a matrix. The detected masses
were identical with the calculated values (Table 6).
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) was performed on a TLC aluminium sheet covered with
silica gel 60 F254 (0.2 mm, VWR International, Germany).
Column flash chromatography (FC): silica gel 60 (VWR In-
ternational, Darmstadt, Germany) at 0.4 bar; UV-spectra were
recorded on a U-3200 spectrophotometer (Hitachi, Japan), kmax
in nm, e in dm3 mol−1 cm−1. NMR spectra: Avance DPX 250
or DPX 300 spectrometers (Bruker Germany); d values in ppm
relative to Me4Si as internal standard, CFCl3 as external standard
N4-(Benzoyl)-7-(2-deoxy-b-D-erythro-pentofuranosyl)-5-fluoro-
7H-pyrrolo[2,3-d]pyrimidin-4-amine (5b)
To a stirred suspension of 4-amino-7-(2-deoxy-b-D-erythro-
pentofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine
(1b,22
220 mg, 0.82 mmol) in 4.1 cm3 of dry pyridine was added
trimethyl chlorosilane (TMSCl, 0.53 cm3, 4.1 mmol) at r.t. After
30 min, benzoyl chloride (0.49 cm3, 4.1 mmol) was added. Stirring
3558 | Org. Biomol. Chem., 2008, 6, 3552–3560
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(0.26 cm3, 1.48 mmol) and 2-cyanoethyldiisopropylphosphora-
midochloride (167 ll, 0.75 mmol) while stirring at r.t. The stirring
was continued for 30 min and the reaction mixture diluted with
CH2Cl2 (15 cm3). The solution was washed with an aqueous
solution of 5% NaHCO3 (2 × 20 cm3). The organic layer was
separated and dried over Na2SO4, filtrated and evaporated to an
oil. The residue was applied to FC (silica gel, column 2.5 × 8 cm),
elution with CH2Cl2–acetone–Et3N 10 : 1 : 0.2) gave a colorless
foam (338 mg, 77%); TLC (silica gel, CH2Cl2–acetone 10 : 1): Rf
0.5; dP(101 MHz; CDCl3; H3PO4) 149.9 and 150.0.
Table 6 Molecular masses determined from MALDI-TOF mass spectra
Mass (calcd)
Mass (found)
5ꢀ-d(TAGGTC1bATACT)-3ꢀ (15)
5ꢀ-d(AGT1bTTG1bCCTA)-3ꢀ (16)
5ꢀ-d(TAGGTC1b1bTACT)-3ꢀ (17)
5ꢀ-d(T1bGGTC1b1bT1bCT)-3ꢀ (18)
5ꢀ-d(1bT1bT1bT1bT1bT1bT)-3ꢀ (31)
5ꢀ-d(AGTATT2aACCTA)-3ꢀ (21)
5ꢀ-d(AGTATT2bACCTA)-3ꢀ (24)
5ꢀ-d(A2bTATT2bACCTA)-3ꢀ (25)
5ꢀ-d(TA2b2bTCAATACT)-3ꢀ (26)
3659.7
3676.6
3676.6
3711.6
3742.6
3641.6
3659.7
3676.6
3676.6
3659.0
3677.4
3677.3
3711.3
3743.0
2642.0
3660.5
3677.0
3677.1
7-(2-Deoxy-b-D-erythro-pentofuranosyl)-3,7-dihydro-2-
(isobutyrylamino)-5-fluoro-4H-pyrrolo[2,3-d]pyrimidin-4-one (7b)
was continued for 2 h and the mixture was cooled in an ice bath.
Water (0.82 cm3) was added and after 5 min, aqueous ammonia
(1.64 cm3, 25%) was added and the mixture was kept stirring at r.t.
for 30 min. The reaction mixture was then evaporated to dryness,
dissolved in methanol, adsorbed on silica gel (1.5 g) and applied
to flash chromatography (FC) on the top of a silica gel column
(10 × 2 cm). Elution with CH2Cl2–CH3OH (30 : 1–20 : 1) yielded
5b as colourless solid (284 mg, 93%) (Found: C, 58.10; H, 4.70; N,
14.86%. C18H17FN4O4 requires C, 58.06; H, 4.60; N, 15.05%); TLC
(silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.31; kmax(MeOH)/nm
312 (e/dm3 mol−1 cm−1 6100), 276 (6400) and 227 (25300); dH
(250 MHz; [d6]DMSO; Me4Si) 2.20–2.30 (1 H, m, 2ꢀ-Ha), 2.44–2.54
(1 H, m, 2ꢀ-Hb), 3.54 (2 H, m, 5ꢀ-H), 3.84 (1 H, m, 4ꢀ-H), 4.36 (1 H,
m, 3ꢀ-H), 4.98 (1 H, m, 5ꢀ-OH), 5.32 (1 H, s, 3ꢀ-OH), 6.73 (1 H, t, J
6.3, 1ꢀ-H), 7.48–7.68 (3 H, m, arom. H), 7.75 (1 H, d, J 1.9, 6-H),
8.66 (1 H, s, 2-H), 8.05 (2 H, m, arom. H) and 11.27 (1 H, br s, NH).
Compound 2b22 (180 mg, 0.63 mmol) was dried by repeated co-
evaporation with pyridine (3 × 5 cm3). The residue was suspended
in pyridine (3.7 cm3) and Me3SiCl (0.53 cm3, 4.2 mmol) was added
at r.t. while stirring. After 30 min, the solution was treated with
isobutyric anhydride (0.53 cm3, 3.2 mmol) and the mixture was
kept stirring at r.t. for 3 h. The mixture was cooled in an ice
bath, H2O (0.6 cm3) was added, followed by the addition of 25%
aqueous ammonia (1.2 cm3) after 5 min. The solution was stirred
for another 15 min and evaporated. The residue was applied to FC
(silica gel, column 2.5 × 8 cm, eluted with CH2Cl2–CH3OH 30 :
1→15 : 1) to yield 7b as a colourless solid (186 mg, 83%) (Found: C,
50.74; H, 5.30%. C15H19FN4O5 requires C, 50.84; H 5.40%); TLC
(silica gel, CH2Cl2–CH3OH, 10 : 1): Rf 0.19; kmax(MeOH)/nm 294
(e/dm3 mol−1 cm−1 12 600); dH(250.13 MHz; [d6]DMSO; Me4Si)
1.08 (6 H, m, 2 × CH3), 2.13 (1 H, m, 2ꢀ-H), 2.35 (1 H, m, 2ꢀ-H),
2.73 (1 H, m, Me2CH), 3.50 (2 H, m, 5ꢀ-H), 3.77 (1 H, m, 4ꢀ-H),
4.30 (1 H, m, 3ꢀ-H), 4.94 (1 H, br s, 5ꢀ-OH), 5.26 (1 H, br s, 3ꢀ-OH),
6.43 (1 H, t, J 6.6, 1ꢀ-H), 7.19 (1 H, s, 6-H), 11.59 (1 H, br s, NH)
and 12.00 (1 H, br s, NH); m/z (ESI-TOF) 377.1248 (M + Na+.
C15H19FN4O5Na requires 377.1237).
N4-(Benzoyl)-7-[2-deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-
D-erythro-pentofuranosyl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (6)
Compound 5b (564 mg, 1.51 mmol) was dried by repeated co-
evaporation with anhydrous pyridine (3 × 3 cm3) and dissolved
in anhydrous pyridine (3 cm3). This solution was treated with
4,4ꢀ-dimethoxytriphenylmethyl chloride ((MeO)2TrCl; 623 mg,
1.84 mmol) at r.t. while stirring (3 h). The mixture was coevapo-
rated with toluene to remove pyridine and the oily residue was
applied to FC (silica gel, column 2.5 × 10 cm). Elution with
CH2Cl2–CH3OH (100 : 1) furnished 6 as slight yellow foam
(744 mg, 73%) (Found: C, 69.29; H, 5.36; N, 8.15%. C39H35FN4O6
requires C, 69.42; H, 5.23; N 8.30%); TLC (silica gel, CH2Cl2–
CH3OH, 20 : 1): Rf 0.19; kmax(MeOH)/nm 312 (e/dm3 mol−1 cm−1
6200), 275 (9900) and 229 (41 700); dH(250 MHz; [d6]DMSO;
Me4Si) 2.33 (1 H, m, 2ꢀ-Ha), 2.60 (1 H, m, 2ꢀ-Hb), 3.13–3.21 (2
H, m, 5ꢀ-H), 3.73 (6 H s, 2 × OCH3), 3.97 (1 H, m, 4ꢀ-H), 4.38 (1
H, m, 3ꢀ-H), 5.39 (1 H, d, J 4.4, 3ꢀ-OH), 6.75 (1 H, t, J 6.5, 1ꢀ-H),
6.84–6.87 (4 H, m, arom. H), 7.22–7.28 (7 H, m, arom. H), 7.38 (2
H, m, arom. H), 7.54–7.66 (4 H, m, arom. H and 6-H), 8.05 (2 H,
m, arom. H), 8.67 (1 H, s, 2-H) and 11.30 (1 H, br s, NH).
7-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5-fluoro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (8)
Compound 7b (150 mg, 0.42 mmol) was dried by repeated co-
evaporation with anhydrous pyridine and dissolved in anhy-
drous pyridine (2.3 cm3). The solution was treated with 4,4ꢀ-
dimethoxytriphenylmethyl chloride (300 mg, 0.9 mmol) at r.t.
while stirring (3 h). The reaction mixture was evaporated and the
residue was applied to FC (silica gel, column 2.5 × 10 cm, eluted
with CH2Cl2–H3OH 50 : 1) to yield 8 as a slightly yellowish foam
(225 mg, 81%) (Found: C, 65.86; H, 5.60; N, 8.54%. C36H37FN4O7
requires C, 65.84; H, 5.68; N, 8.53%); TLC (silica gel, CH2Cl2–
CH3OH, 20 : 1): Rf 0.12; kmax(MeOH)/nm 276 (e/dm3 mol−1 cm−1
15 200), 283 (15 200) and 297 (14 200); dH (250 MHz; [d6]DMSO;
Me4Si) 1.12 (6 H, m, 2 × CH3), 2.22 (1 H, m, 2ꢀ-Ha), 2.46 (1 H, m,
2ꢀ-Hb), 2.75 (1 H, m, Me2CH), 3.13 (2 H, m, 5ꢀ-H), 3.73 (6 H, s, 2 ×
OCH3), 3.90 (1 H, m, 4ꢀ-H), 4.30 (1 H, m, 3ꢀ-H), 5.34 (1 H, d, J 2.4,
3ꢀ-OH), 6.43 (1 H, t, J 6.3 Hz, 1ꢀ-H), 6.83 (4 H, m, arom. H), 7.03
(1 H, s, 6-H), 7.21–7.38 (9 H, m, arom. H) and 11.59 (2 H, br s,
2 × NH); m/z (ESI-TOF) 679.2560 (M + Na+. C36H37FN4O7Na
requires 679.2544).
N4-(Benzoyl)-7-[2-deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-
D-erythro-pentofuranosyl]-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-
amine 3ꢀ-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (3)
To a solution of compound 6 (337 mg, 0.5 mmol) in anhy-
drous CH2Cl2 (4.5 cm3) were added N,N-diisopropylethylamine
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7-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytriphenylmethyl)-b-D-erythro-
pentofuranosyl]-3,7-dihydro-2-(isobutyrylamino)-5-fluoro-4H-
pyrrolo[2,3-d]pyrimidin-4-one 3ꢀ-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (4)
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Acknowledgements
We thank Dr Peter Leonard and Dr Simone Budow for helpful
discussions during the preparation of the manuscript. We thank
also Ms Padmaja Chittipu, Mr Dawei Jiang for the measurements
of the NMR spectra and Dr T. Koch, Roche Diagnostics GmbH,
for the MALDI-TOF mass spectra. We gratefully acknowledge
financial support by Idenix Pharmaceuticals, Cambridge, US and
Montpellier, France and ChemBiotech, Mu¨nster, Germany.
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