Electrochemical detection of nucleic acids using pentaferrocenyl phosphoramidate α-oligonucleotides

Article abstract of DOI:10.1039/c0nj00902d

We report the synthesis of α-oligonucleotides exhibiting five phosphoramidate linkages bearing a ferrocenyl (Fc) moiety. Three different linkers: ethyl, propyl and 4-methyl-1-ethyl-1,2,3-triazol between the ferrocene (Fc) residue and the phosphoramidate f

Full text of DOI:10.1039/c0nj00902d

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PAPER
Electrochemical detection of nucleic acids using pentaferrocenyl
phosphoramidate a-oligonucleotidesw
a
b
¸
ois Morvan,*b Jean-Jacques Vasseurb
´
Gregory Chatelain, Albert Meyer, Franc
and Carole Chaix*a
Received (in Montpellier, France) 16th November 2010, Accepted 31st January 2011
DOI: 10.1039/c0nj00902d
We report the synthesis of a-oligonucleotides exhibiting five phosphoramidate linkages bearing a
ferrocenyl (Fc) moiety. Three different linkers: ethyl, propyl and 4-methyl-1-ethyl-1,2,3-triazol
between the ferrocene (Fc) residue and the phosphoramidate function have been studied.
We demonstrated that these Fc5-a-DNA conjugates exhibit similar or even better stability than
the wild type b-DNA when hybridized to their complementary b-DNA. Their electrochemical
behavior studied in solution by cyclic voltammetry showed that this original structure leads
to a strong difference in the Fc environment when the probe is alone or hybridized with a
complementary strand. These preliminary electrochemical analyses are very encouraging and
augur a good sensitivity of these probes for DNA detection after grafting on an electrode
microsystem.
nucleobases19–21 or at the ribose 20-O-position.11,22 Alternatively,
Introduction
more complicated sandwich systems have been reported, where
hybridization of the probe and target is visualized using a third
partner bearing several Fc moieties to increase the intensity of
the electrochemical response.23,24 Gold nanoparticles have been
functionalized by numerous ferrocene caps in order to amplify
voltammetric detection of a sandwich DNA complex.25,26 In all
these approaches, the ferrocene oxidation current is amplified by
the multiple labels of the third partner. The improved gain of the
current response upon target hybridization leads to increased
general sensitivity of the assays. These approaches, however,
typically require time consuming, multistep processing, such as
washing or amplification steps, in order to reduce false positive
or negative signal arising from the non-specific adsorption of the
signalling entity.
Nucleic acids (DNA and RNA) play a key role in living
organisms and are currently the most targeted biomolecules
in the field of medical diagnostics,1 genetic screening,2 drug
design,3 forensics,4 food and agriculture analysis or environ-
mental control.5,6 With the aim of developing fast and selective
DNA-based diagnostic systems, much research is currently
devoted to the elaboration of microarrays which allow real-time,
labelless and multi-sequence detection.7 Among the different
methodologies, electrochemistry receives considerable attention
due to its intrinsic properties: easy integration, sensitivity,
rapidity and low cost.8
One strategy for elaborating an electrochemical biodevice is
to label the probe which hybridizes the target with a redox
compound like ferrocene (Fc).9,10 Several works have reported
the synthesis of Fc-oligonucleotide conjugates for electrochemical
detection.1,11–17 A Fc moiety has been introduced on either
the 30- or 50-end,12,13,18 or in the sequence using modified
In our laboratory, we focused on developing multiferrocenyl
hairpin probes which offer significant improvement of detec-
tion limit.27–29 We have synthesized a ferrocene bispropanol
phosphoramidite synthon that can be incorporated several
times in an oligonucleotide sequence, directly during DNA
solid-phase synthesis. By this strategy, a multilabel tag can be
bound to a DNA probe. We succeeded in introducing up to
8 Fc, either between two nucleotides in the sequence or
at the oligonucleotide extremities. Unlike fluorescence, whose
quenching processes do not permit the accumulation of nu-
merous dyes in order to increase signal intensity, we observed
that the incorporation of numerous ferrocenes in close proximity
within the oligonucleotide sequence enhances their response
by cyclic voltammetry (CV) in an exponential manner.
Considering these results, one important improvement
will be to integrate several Fc in the probe’s own structure
a
´
Laboratoire des Sciences Analytiques, UMR 5180 CNRS-Universite
´
Lyon 1, Universite de Lyon, 43, Bd du 11 Nov. 1918,
69622 Villeurbanne Cedex, France.
E-mail: carole.chaix-bauvais@univ-lyon1.fr;
Fax: +33 (0)472448479; Tel: +33 (0)472448306
b
´
Institut des Biomolecules Max Mousseron (IBMM),
´
´
UMR 5247 CNRS-Universite Montpellier 1 - Universite Montpellier 2,
Place Euge`ne Bataillon, CC1704, 34095 Montpellier Cedex 5,
France. E-mail: morvan@univ-montp2.fr
w Electronic supplementary information (ESI) available: 1—HPLC
and MALDI-ToF MS analyses of 4a, 4b, 8; 2—thermodynamic
parameter determination; 3—electrochemical characterization of the
system in standard macroelectrode device; 4—electrochemical measure-
ments of hybridization reaction. See DOI: 10.1039/c0nj00902d
c
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New J. Chem., 2011, 35, 893–901 893

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to dramatically change the environment of Fc moieties when
the probe is hybridized, leading to a marked difference in the
electrochemical response between the single- and double-
stranded states. In this strategy, the ferrocenes will be carried
by the probe. It will provide direct and specific detection of the
DNA target in solution.
Herein, we report the synthesis of a-oligonucleotides exhibiting
five phosphoramidate linkages bearing Fc moieties and their
electrochemical behavior studied by cyclic voltammetry (CV).
We assume that a-DNA bearing a block of 5 Fc residues
at one end through phosphoramidate linkages will lead to a
strong difference in the Fc environment when the probe is
alone or hybridized with a complementary strand, giving our
system a high level of sensitivity.
Ideally, the Fc moiety should be introduced on the inter-
nucleosidic linkage where the difference of environment and
liberty of conformational freedom between the single- and
double-stranded states appear to be the most important.
However this has never been reported because phosphate
modified oligonucleotides are known to hybridize less efficiently
due to the chirality effect.30 Furthermore, this destabilizing
effect increases as the size of the group on the phosphate
increases. However, we have shown that this negative chirality
effect is largely eliminated when phosphate modified oligo-
nucleotides are constituted with a-anomeric deoxyribose.31
These P-modified a-DNAs exhibit similar or even better
stability32–37 than wild type b-DNA and, like phosphodiester
a-DNA, they form duplexes with complementary b-DNA or
RNA with parallel orientation.38–40 Among the P-modifications,
the synthesis of phosphoramidate linkages is straightforward,
starting from H-phosphonate diester linkages by amidative
oxidation, and allows the introduction of a substituent.41
We have reported that amidative oxidation of H-phosphonate
diester linkages is achieved with a good yield. This method
provides an efficient strategy for incorporating numerous
Fc on the internucleosidic linkages of an oligonucleotide.
Results and discussion
To evaluate the influence of the distance between the phosphor-
amidate function and the Fc moiety on the electrochemical
response, we prepared a-DNAs (12-mers) with three different
linkers: ethyl (Et), propyl (Pr) and 4-methyl-1-ethyl-1,2,3-
triazol (Tz) (Scheme 1).
Synthesis of a-DNAs
We applied a convenient strategy to introduce a Fc moiety
into a-DNAs using amidative oxidation of H-phosphonate
diester.41 Starting from solid supported a-thymidine, six
a-phosphoramidite nucleosides (T and C)42 were introduced
using phosphoramidite chemistry and then five a-H-phosphonate
nucleosides (T and C)35 using H-phosphonate chemistry,
affording solid supported a-DNA 1 with five H-phosphonate
diesters and six phosphotriester linkages (Scheme 1).
H-phosphonate diester linkages were oxidized by CCl4
either in the presence of ferrocenylethylamine (2a) or
Scheme 1 General synthetic scheme for the preparation of Fc5-a-DNAs of sequence 50T*T*C*T*C*CTTCTTT30, with five phosphoramidate
linkages at the 50 end, 4a: ferrocenylethylphosphoramidate, 4b: ferrocenylpropylphosphoramidate, 8: 1-(ferrocenylethyl)-1,2,3-triazol-4-
methylphosphoramidate.
c
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Table 1 Principal features of the Fc5-a-DNAs (4a, 4b, 8), PNHEtFc = ferrocenylethylphosphoramidate. PNHPrFc = ferrocenylpropyl-
phosphoramidate. PNHTzFc = 1-(ferrocenethyl)-1,2,3-triazol-4-methylphosphoramidate
Fc5-a-DNAs
Internucleotide linkages
Exp. massa/g molꢀ1
Theo. massa/g molꢀ1
Quantityb/nmol
4a
4b
8
PNHEtFc
PNHPrFc
PNHTzFc
4577.7
4647.6
4983.0
4578.0
4648.0
4983.8
28
84
57
a
MALDI-TOF MS analysis in negative mode [M ꢀ H]ꢀ of the Fc5-a-DNAs after HPLC purification (experimental and theoretical mass
b
values). Quantity of Fc5-a-DNAs obtained after purification from a 500 nmol scale synthesis.
ferrocenyl-propylamine (2b) which, after ammonia treatment,
provided the Fc5-a-DNA with Et (4a) and Pr (4b) linkages,
respectively, or in the presence of propargylamine giving
a-DNA (5) with five propargyl groups. This latter was con-
jugated by 1,3-dipolar cycloaddition (‘‘click’’ chemistry) catalyzed
by Cu(I)43,44 assisted by microwaves45 using ferrocenylethyl-
azide (6) affording 7. A final ammonia treatment yielded
the expected Fc5-a-DNA conjugate with Tz linkages (8).
Each conjugate was purified by HPLC and characterized by
MALDI-TOF mass spectrometry (Table 1).
Table 2 Thermodynamic parameters of Fc5-a-DNAs 4a, 4b and 8
with complementary sequence 10 in comparison with PO a-DNA 9
and PO b-DNA 11
Duplexa
Tmb/1C
DTm
DTm/Fc
DG298/kcal molꢀ1
b
9/10
46.6 ꢁ 0.4
48.4 ꢁ 0.4
47.1 ꢁ 0.2
45.0 ꢁ 0.5
43.1 ꢁ 0.2
—
—
ꢀ13.9 ꢁ 0.2
ꢀ12.6 ꢁ 0.3
ꢀ12.7 ꢁ 0.2
ꢀ12.8 ꢁ 0.6
ꢀ13.2 ꢁ 0.1
4a/10
4b/10
8/10
+1.8
+0.5
ꢀ1.6
ꢀ3.5
+0.4
+0.1
ꢀ0.3
—
11/10
a
Sequences—9: 50a-TTCTCCTTCTTT30, 10: 50b-AAGAGGAAGA-
b
AA30, 11: 50b-TTTCTTCCTCTT30. Experiments were carried out at
Amidative oxidation with 2b was more efficient than that
with 2a (84 and 28 nmol of full length Fc5-a-DNA, respectively,
after HPLC purification), presumably because the greater
distance between the amine group and the bulky ferrocene
led to less steric hindrance disfavouring the reaction. The
last approach in two steps through the ‘‘click’’ reaction was
investigated as an alternative way to incorporate a very high
number of Fc in the oligonucleotide sequence. The first step of
amidative oxidation with smaller propargylamine revealed to
be quantitative.45 But the second reaction of 1,3-cycloaddition
with (2-azidoethyl)ferrocene 6 assisted by microwaves was
slightly less efficient, leading to non-fully-modified oligo-
nucleotides in the crude synthesis that must be eliminated.
After HPLC purification, 57 nmol of 8 were obtained from a
500 nmol scale solid-phase synthesis. This yield could be
considered relatively low, however this strategy allows multi-
modification of the oligonucleotide probes unlike the methods
for the 30/50-end labeling. The base or the 20-OH modifications
could be used for multi-labeled probes but these methods are
limited to one or two ferrocene moieties due to the thermo-
dynamic destabilization that they induce.46
1 mM concentration for each strand in 25 mM sodium phosphate
buffer, 250 mM NaCl, pH 6.4. Data are the mean values of minimum
five experiments. DTm are calculated from the Tm control of 9/10
duplex with the a-ODN 9.
This consideration was confirmed by the DG1298 values which
were very similar for all the duplexes studied (Table 2). Note
that b-DNA with ferrocenylethylphosphoramidate linkages
gave a DTm/Fc value of ꢀ4.4 1C (data not shown). These
results confirm the need to synthesize Fc-DNAs with a rather
than b anomeric nucleotides and the interest of the phosphor-
amidate method.
Electrochemical characterization
The electrochemical characterization of each Fc5-a-DNA was
achieved by cyclic voltammetry (CV) on a gold electrode
microarray.27 This electrochemical biochip consisted of a
plastic-based support with eight working electrodes specially
designed to be a low cost, low density biodevice. Measurements
were made in a 10 mL droplet of probe solution in a phosphate
buffer. The electrochemical response of each Fc5-a-DNA
(4a, b and 8) studied alone revealed similar behaviour. The
peak-to-peak separation (DEp = Epa ꢀ Epc) was relatively low
(between 0 and 40 mV) and an equivalent intensity was
observed (Fig. 1A). Current intensity at the oxidation peak
of Fc (Ipa) was recorded at different scan rates (n). For each
Fc5-a-DNA, a linear relationship with n was found from
50 mV sꢀ1 to 300 mV sꢀ1, indicating that in this range of scan
rates, Fc5-a-DNAs were adsorbed on the microarray.47 The
confinement of the redox species on the electrode surface with
a slow exchange rate mainly controlled the electrochemical
behaviour of the system (Fig. 1B). This adsorption of the
probes onto the gold surface provided the system’s very stable
electrochemical response after a 60 min stabilisation phase in a
phosphate buffer. It is worth noting that the same studies
achieved with hairpin probes modified with four or six bis-
propanol ferrocenes resulted in a DEp up to 200 mV and a
linear relationship between Ipa and Ov, indicating that no
Thermodynamic characterization
To evaluate the hybridization properties of the Fc5-a-DNAs,
the base pairing of 4a, 4b, 8 and the fully phosphodiester (PO)
a-DNA 9 [50-a-d(TTCTCCTTCTTT)] with the complementary
DNA sequence 10 [50-b-d(AAGAGGAAGAAA)] with parallel
orientation39 was studied by UV melting experiments.29
Tm and DG1298 of these duplexes were compared to those of
the natural duplex formed between 10 and complementary
b-DNA 11 [50-b-d(TTTCTTCCTCTT)] with antiparallel
orientation (Table 2).
The stability of a-DNA/b-DNA duplexes was higher than
that of the natural b/b duplex (11/10). The duplex formed with
4a was the most stable, with a Tm increase per Fc of +0.4 1C
compared to the a/b-duplex (9/10). The duplexes formed with
longer linkers were less stabilized with only +0.1 1C for 4b and
slightly destabilized for 8 (ꢀ0.3 1C). However, the Tm variations
can be considered as minor (ꢀ0.3 1C o DTm/Fc o +0.4 1C).
c
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Fig. 1 (A) CV of 10 mM Fc5-a-DNA 4a, b and 8 solutions in sodium phosphate buffer (25 mM)/NaClO4 (250 mM)/pH 6.4. Scan rate: 100 mV sꢀ1
.
(B) Anodic current variation (Ipa) as a function of the scan rate carried out using an external reference electrode of Ag/AgCl, a gold working
electrode and a counter electrode of Pt. Experiments were achieved 80 min after the deposition of the Fc5-a-DNA 4a, b and 8 solutions on the
electrodes of the biochip.
adsorption phenomena of the Fc-b-ODNs occurred on the micro-
array surface.29 These differences tend to prove that ferrocene
phosphoramidate moieties on a single-strand a-DNA are not
embedded in the DNA structure but adsorbed on the gold surface
allowing better electron transfer.
non-complementary sequence 12 did not induce any signal
variation. The resulting DEp and Ipa variations of CV response
obtained upon target addition for 4a, 4b and 8 are gathered in
Table 3.
Our hypothesis to explain the major impact of hybridization
with the complementary strand on the electrochemical signal
observed by CV is that after system stabilization, single-stranded
To determine the impact of hybridization on the electro-
chemical signal, electrochemical studies were then carried out
in the presence of complementary 10 or non-complementary
12 sequences (see the oligonucleotide sequences in the ESIw)
(Fig. 2, Fig. S3, ESIw). All experiments were carried out with a
10 mM concentration of Fc5-a-DNAs. For all Fc5-a-DNAs,
the addition of 5.5 eq. of target 10 significantly modified the
ferrocenes’ electrochemical signal by increasing DEp and
decreasing Ipa. Hybridization thus induced a tremendous
change in the Fc environment, as shown by CV (Fig. 2A,
Fig. S3, ESIw). In contrast, the addition of 5.5 eq. of the
Table 3 DEp and Ipa obtained by CV of 4a, 4b and 8 solutions
deposited on the electrodes of the biochip, before addition of the
complementary target 10 (Single) and after hybridization (Duplex)
DEp/mV
Ipa/nA
Fc5-a-DNA
Single
Duplex
4a
5
89
4b
36
82
8
27
72
4a
9
4
4b
12
7
8
11
6
Fig. 2 (A) CV of Fc5-a-DNA 4a alone (solid line), with non-complementary 12 (J) and complementary 10 (+) sequences. (B) Evolution of the
NEp and the NIpa of 4a by CV after the addition of 10 or 12 in the media (10 mM concentration of Fc5-a-DNA 4a in 25 mM sodium phosphate
buffer, 250 mM NaClO4, pH 6.4 and 5.5 eq. of 10 or 12). Scan rate: 100 mV sꢀ1
.
c
896 New J. Chem., 2011, 35, 893–901
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recorded upon hybridization and the possibility of quantitative
detection. The three Fc5-a-DNAs bearing different linkers
between the Fc moiety and the phosphorous atom exhibited
similar electrochemical behaviour. Work is in progress to
immobilize these probes on the electrode with the aim of strongly
improving the system’s sensitivity and further developing DNA
biochips.
Scheme 2 Schematic view of the electrochemical signal variation
induced by binding with the complementary target.
Experimental section
Fc5-a-DNAs are adsorbed on the gold surface through their
hydrophobic Fc moieties. Thus, due to the close proximity of
the redox molecules to the surface, the charge transfer to the
electrode is very efficient. In contrast, in the duplex state, Fc
moieties of Fc5-a-DNAs are positioned in the double-stranded
DNA which significantly changes their spatial arrangement
and ionic environment (Scheme 2). Duplex formation induces
conformational changes of the probe which impede efficient
electron transfer to the electrodes during ferrocene oxidation.
This specific variation, observed for the three probes, was
used to follow the kinetics of the hybridization reaction using
parameters DEp and Ipa. Parameters had to be normalized
according to a previous method29 in order to compare the
responses obtained on different working electrodes and bio-
devices. NEp = (DEp ꢀ DEp )/DEp and NIpa = (Ipa ꢀ Ipa )/Ipa
Syntheses
General methods. Thin layer chromatography (TLC) was
carried out on aluminium sheets coated with silica-gel 60 F
(Merck 5554). TLC plates were revealed by UV light and
1
charring after spraying with 5% H2SO4 in EtOH. H NMR
spectra were recorded on a Bruker 200 MHz spectrometer.
The chemical shifts were expressed on the d scale in parts per
million (ppm). The following abbreviations were used to
explain the observed multiplicities: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; q, quadruplet; m, multiplet; br,
broad. NMR solvents were purchased from Sigma-Aldrich
(Saint Quentin-Fallavier, France). Mass spectra were measured
with an electrospray-simple quadripole API 165 from Applied
Biosystems (Courtaboeuf, France). 50-O-(4,40-Dimethoxy-
trityl)-a-20-deoxythymidine 13a and 50-O-(4,40-Dimethoxytrityl)-
4-N-benzoyl-a-20-deoxycytidine 13b were purchased from R.I.
Chemicals (Orange, CA, USA). N,N-Dimethylaminomethyl-
ferrocene 15 was purchased from Alpha Aesar (Schiltigheim,
France) and all reagents and solvents used for synthesis were
purchased from Sigma-Aldrich (Saint-Quentin Fallavier,
France). b-Oligonucleotides 10, 11 and 12 were purchased
from Eurogentec (Angers, France).
0
0
0
0
are defined as the normalized values of DEp and Ipa, respectively,
and DEp and Ipa are the DEp and Ipa measurements just before
target addition.
0
0
The hybridization kinetics appeared to be rapid (Fig. 2B,
Fig. S4, ESIw). For both parameters, maximum of variation
occurred within 20 minutes after the addition of 10 (5.5 eq.)
and reached a plateau. Conversely, the signal variation observed
after the addition of 12 was totally different.
It is worth noting that the addition of the non-complementary
target slightly decreased NEp and increased NIpa of 4a, 4b and 8
responses, probably due to an electrostatic effect of the negatively
charged target on the probes after introduction in the medium.
This phenomenon was previously reported with ferrocenyl-stem–
loop oligonucleotides, in the same conditions.29 One can con-
clude that the signal variation is thus significant and specific to
the target.
Nucleoside H-phosphonates (14a, b). 1 mL of diphenylphosphite
(5 mmol, 7 eq.) was added dropwise to a solution of 13a, b
(0.7 mmol, 1 eq.) in 5 mL of anhydrous pyridine under argon.
The reaction mixture was stirred at rt for 35 min under argon
then cooled at 0 1C before adding dropwise 5 mL of a solution
of water/triethylamine 1 : 1 v/v. The reaction mixture was
stirred for an additional 1 h at rt then poured into saturated
aqueous NaHCO3 (50 mL). The aqueous layer was extracted
with CH2Cl2 (2 ꢂ 50 mL). The organic layers were combined,
dried (Na2SO4), filtered and evaporated under reduced pressure.
The residue was purified by flash silica gel column chromatography
(CH2Cl2/MeOH/triethylamine 93 : 2 : 5 then CH2Cl2/MeOH/
triethylamine 87 : 8 : 5) to afford the desired H-phosphonate
nucleotides 14a, b.
Linearity of the NEp response was observed between 0.25
and 2.1 eq. of the DNA target per Fc5-a-DNAs, for 4a, 4b
and 8 (Fig. S5, ESIw).
Conclusion
In conclusion, the synthesis of three Fc5-a-DNAs, bearing in
their own structure, five Fc moieties on the phosphoramidate
internucleosidic linkages was successfully achieved on a solid
support using direct amidative oxidation or amidative oxida-
tion combined with microwave-assisted ‘‘click’’ chemistry.
Thermodynamic experiments demonstrated that Fc moieties
introduced into a-DNAs have little influence on the thermo-
dynamic stability of the duplexes formed between the resulting
Fc5-a-DNAs and their complementary b-DNA target. The
electrochemical study performed by CV demonstrated the high
potential of these Fc5-a-DNAs for use as probes for the
electrochemical detection of nucleic acids in view of their
affinity, the high variation of the electrochemical parameters
50-O-(4,40-Dimethoxytrityl)-a-20-deoxythymidin-30-yl hydrogen
phosphonate triethylammonium salt (14a). Compound 14a
(593 mg, 100%) was obtained as a white powder according to
the general procedure starting from 13a (400 mg, 0.73 mmol)
and diphenylphosphite (1 mL, 5 mmol).
RMN 1H (200 MHz, CDCl3): d (ppm) = 1.3 (t, 9H,
0
(CH3CH2)3N), 1.92 (s, 3H, H7), 2.35 (m, 1H, H2 ), 2.87
00
(m, 1H, H2 ), 3.02 (q, 6H, (CH3CH2)3N), 3.19 (m, 2H,
0
00
0
H5 and H5 ), 3.78 (s, 6H, O-CH3), 4.69 (m, 1H, H4 ), 4.84
0
(m, 1H, H3 ), 5.30–8.38 (d, 1H, H–P, J = 640 Hz), 6.40
0
(dd, 1H, H1 a, J = 5.9 Hz), 6.80–7.42 (m, 13H, Ar–H), 7.67
c
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(s, 1H, H6), 9.04 (s large, 1H, N–H). RMN 31P (101 MHz,
CDCl3): d (ppm) = 4.46 (P–H). SM (ESI-) m/z: calcd for
C31H32N2O9Pꢀ [M ꢀ H]ꢀ 607.19, found 607.20.
to provide the compound 2a (257 mg, 77%) as a dark
brown oil.
1
RMN H (200 MHz, CDCl3): d (ppm) = 2.40 (t, 2H, H5
0
0
and H5 ), 2.80 (t, 2H, H6 and H6 ), 4.05 (s, 2H, H2), 4.08
(s, 7H, H3 and H4), SM (ESI+) m/z: calcd for C12H15FeN
[M + H]+ 229.06, found 229.10.
50-O-(4,40-Dimethoxytrityl)-4-N-benzoyl-a-20-deoxycytidin-30-yl
hydrogen phosphonate triethylammonium salt (14b). Compound
14b (475 mg, 100%) was obtained as a white powder according
to the general procedure starting from 13b (598 mg, 0.75 mmol)
and diphenylphosphite (1 mL, 5 mmol).
2-Ferrocenylacetic acid (18)49. 9 mL of a solution of com-
pound 17 (4 mmol, 1 eq.) in EtOH was added to 23 mL of a
solution of KOH (2 M) and stirred 5 h at rt. The reaction
mixture was concentrated until a volume of 3 mL and diluted
in 22 mL of water. The aqueous layer was extracted with Et2O
(2 ꢂ 10 mL). 15 mL of a solution of HCl (2.5 M) was added
dropwise with a good stirring. The precipitate was filtered
and washed with cold water (3 ꢂ 12 mL). Compound 18
(724 mg, 74%) was obtained as a yellow powder.
RMN 1H (200 MHz, CDCl3): d (ppm) = 1.31 (t, 9H,
0
(CH3CH2)3N), 2.51 (m, 1H, H2 ), 2.87 (m, 1H, H2 ), 3.02
00
0
(q, 6H, (CH3CH2)3N), 3.19 (m, 2H, H5 and H5 ), 3.74
00
0
(s, 6H, O–CH3), 4.77 (m, 2H, H3 and H4 ), 5.25–8.25
0
0
(d, 1H, P–H, J = 600 Hz); 6.35 (d, 1H, H1 ), 6.76–7.37
(m, 13H, Ar–H), 7.47 (m, 5H, H-benzoyl), 7.87 (d, 1H, H5),
8.12 (d, 1H, H6), 12.01 (s large, 1H, N–H). RMN 31P
(101 MHz, CDCl3): d (ppm) = 4.46 (P–H). SM (ESI-)
m/z: calcd for C37H35N3O9P- [M ꢀ H]ꢀ 696.21, found 696.10.
1
RMN H (200 MHz, CDCl3): d (ppm) = 3.40 (s, 2H, H5
0
and H5 ), 4.08 (s, 7H, H3 and H4), 4.05 (s, 2H, H2), SM
(ESI+) m/z: calcd for C12H12FeO2 [M + H]+ 244.02, found
N,N,N-Trimethyl-1-ferrocenylmethanaminium iodide (16)48.
3.5 mL of methyliodide (51.2 mmol, 5 eq.) was added to
50 mL of solution of N,N-dimethylaminomethylferrocene 15
in MeOH. The reaction mixture was stirred at rt for 1 h, and
then 3.5 mL of methyliodide (51.2 mmol, 5 eq.) was added.
The reaction mixture was stirred for 1 h and evaporated to
dryness. The residue was dissolved in 15 mL of MeOH and
400 mL of Et2O was added. The precipitate was filtered,
washed with cold Et2O (3 ꢂ 30 mL) and dried under reduced
pressure. Compound 16 (3.7 g, 97%) was obtained as a yellow
powder.
244.00.
2-Ferrocenylethanol (19)49. 15 mL of a solution of LiAlH4
(2,7 mmol, 3,6 eq.) in THF was stirred at reflux for 15 min,
and then 15 mL of a solution of compound 18 (3 mmol, 1 eq.)
in THF was added dropwise. The reaction mixture was stirred
24 h at reflux, cooled at 0 1C and 10 mL of water was added
dropwise. After filtration, the aqueous layer was extracted
with Et2O (3 ꢂ 35 mL). The organic layers were combined,
dried (MgSO4), filtered and evaporated under reduced pressure.
Compound 19 (638 mg, 86%) was obtained as an orange solid.
RMN 1H (200 MHz, CDCl3): d (ppm) = 2.68 (s, 2H, H5 and
1
RMN H (200 MHz, CDCl3): d (ppm) = 3.32 (s, 9H, H6),
4.31 (s, 5H, H4), 4.34 (d, 2H, H2), 4.59 (m, 2H, H3), 4.92
+
0
0
H5 ), 4.05 (s, 9H, H2, H3 and H4), 4.12 (s, 2H, H6 and H6 ),
SM (ESI+) m/z: calcd for C12H14FeO [M + H]+ 230.04,
found 229.00.
0
(s, 2H, H5 and H5 ). SM (ESI+) m/z: calcd for C14H20FeN
[M + H]+ 258.09, found 257.90.
2-Ferrocenylacetonitrile (17)49. Compound 16 (1.7 mmol, 1 eq.)
was added to 7.5 mL of a solution of NaCN (17 mmol, 10 eq.) in
water. The reaction mixture was stirred at reflux for 2 h and
then cooled to rt. The aqueous layer was extracted with Et2O
(3 ꢂ 30 mL). The organic layers were combined, dried
(MgSO4), filtered and evaporated under reduced pressure.
Compound 17 (282 mg, 76%) was obtained as an orange solid.
2-Ferrocenylethan 4-methylbenzenesulfonate (20)50. 600 mL
of pyridine (7.5 mmol, 3 eq.) was added to 3.5 mL of a solution
of compound 19 (2.5 mmol, 1 eq.) in CH2Cl2. The reaction
mixture was cooled to 0 1C and p-TsCl (5.0 mmol, 2 eq.) was
added. The reaction mixture was stirred for 3 h at 0 1C, then
diluted in 15 mL of Et2O and washed with water (2 ꢂ 10 mL),
5 mL of a solution of HCl 0.4 M, 5 mL of saturated aqueous
NaHCO3 and finally 5 mL of saturated aqueous NaCl. The
organic layer was dried (MgSO4), filtered and evaporated
under reduced pressure. The residue was purified by flash
silica gel column chromatography (cyclohexane/triethylamine
99 : 1 then cyclohexane/MeOH 90 : 10) to afford the compound
20 (1.14 g, 80%) as an orange solid.
1
RMN H (200 MHz, CDCl3): d (ppm) = 3.42 (s, 2H, H5
0
and H5 ), 4.18 (d, 2H, H2), 4.24 (s, 5H, H4), 4.26 (d, 2H, H3).
SM (ESI+) m/z: calcd for C14H20FeN+ [M + H]+ 225.02,
found 225.90.
2-Ferrocenylethanamine (2a)49. 12 mL of a solution of
LiAlH4 (10.2 mmol, 28 eq.) in Et2O was stirred at reflux for
30 min, and then 12.5 mL of a solution of compound 17
(1.46 mmol, 1 eq.) in Et2O was added dropwise. The reaction
mixture was stirred 9 h at reflux, cooled at 0 1C and 6 mL of
EtOH/Et2O 1 : 1 v/v was added dropwise. 40 mL of solution
of NaOH (2 M) and 10 mL of saturated aqueous NaCl were
added to the aqueous layer. The aqueous layer was extracted
with Et2O (2 ꢂ 35 mL). The organic layers were washed with
20 mL of a solution of NaOH (1 M) and water (3 ꢂ 15 mL),
dried (MgSO4), filtered and evaporated under reduced pressure.
The residue was purified by flash silica gel column chromato-
graphy (CH2Cl2/triethylamine 99 : 1 then CH2Cl2/MeOH 92 : 8)
RMN 1H (200 MHz, CDCl3): d (ppm) = 2.43 (s, 3H, H11),
0
2.68 (t, 2H, H5 and H5 ), 4.03 (d, 2H, H2), 4.06 (s, 7H, H3
0
and H4), 4.12 (t, 2H, H6 and H6 ), 7.32 (d, 2H, H9), 7.76
(d, 2H, H8), SM (ESI+) m/z: calcd for C19H20FeO3S
[M + H]+ 384.05, found 384.00.
(2-Azidoethyl)ferrocene (6). Compound 20 (0.6 mmol, 1 eq.)
diluted in 3 mL of acetonitrile was added to 2 mL of a solution
of NaN3 (5.5 mmol, 9 eq.) in water. The reaction mixture was
stirred at reflux for 15 h and then cooled to rt. 1 mL of water
was added and the filtrate was extracted with Et2O (3 ꢂ 5 mL).
The organic layers were combined, dried (MgSO4), filtered and
c
898 New J. Chem., 2011, 35, 893–901
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evaporated under reduced pressure. The residue was purified
by flash silica gel column chromatography (CH2Cl2/triethyl-
amine 99 : 1 then CH2Cl2) to afford the compound 6
(48 mg, 29%) as a brown solid.
(3 ꢂ 0.1 mL), and then dried by flushing with nitrogen and in a
desiccator over P2O5 for 2 h under vacuum.
General procedure for Cu(I)-catalyzed 1,3-dipolar cyclo-
addition (7). To the CPG-supported oligonucleotide penta-
propargyl 5 (0.5 mmol) was added the azide 6 (20 eq., 10 mmol,
50 mL of a 200 mM solution in MeOH), CuSO4 (1.6 eq.,
0.8 mmol, 20 mL of a 40 mM solution in water), freshly prepared
sodium ascorbate (8 eq., 4 mmol, 80 mL of a 50 mM solution
in water) and MeOH (50 mL). The resulting preparation
was placed in a sealed tube then treated with a microwave
synthesizer Initiatort from Biotage set at 35 W for 30 min at
60 1C with a 30 s premixing time to afford 7. Temperature was
monitored with an internal infra-red probe. CPG was washed
with H2O/MeOH (2 mL, 1 : 1, v/v) and MeOH (1 mL) then
dried in a desiccator over P2O5 for 2 h under vacuum.
1
RMN H (200 MHz, CDCl3): d (ppm) = 2.62 (t, 2H, H5
0
0
and H5 ), 3.36 (t, 2H, H6 and H6 ), 4.10 (s, 4H, H2 and H3),
4.11 (s, 5H, H4), SM (ESI+) m/z: calcd for C12H13FeN3
[M + H]+ 255.05, found 254.00.
3-Ferrocenyl-propanenitrile (21). Compound 20 (2.5 mmol, 1 eq.)
diluted in 12 mL of acetonitrile was added to 3 mL of a
solution of NaCN (25 mmol, 10 eq.) in water. The reaction
mixture was stirred at reflux for 15 h and then cooled to rt.
Then, 20 mL of water was added and the solution was filtered.
The filtrate was extracted with Et2O (2 ꢂ 35 mL). The organic
layers were combined, dried (MgSO4), filtered and evaporated
under reduced pressure. The residue was purified by flash silica
gel column chromatography (cyclohexane/triethylamine 99 : 1
then cyclohexane) to afford the compound 21 (664 mg, 99%)
as an orange solid.
General procedure for deprotection, purification and charac-
terization (4a, 4b, 8). The beads were placed into a sealed vial
and treated with concentrated aqueous ammonia (1 mL) for
5 h at 55 1C. The beads were eliminated by filtration and the
solution was evaporated. The residue was dissolved in water
and purified by HPLC on a column Delta Pak (15 mm, C18,
100 A, 7.8 ꢂ 300 mm) from Waters using a linear gradient of
acetonitrile from 12% to 64% in 30 min and 64% to 80% in
5 min at 2 mL minꢀ1 in TEAAc 50 mM pH 7 buffer affording
pure Fc5-a-DNAs 4a, 4b and 8. They were characterized
by HPLC on a column X-terra (2.5 mm, MS C18 2.5 mm,
4.6 ꢂ 50 mm) from Waters at 60 1C using a linear gradient of
acetonitrile from 12% to 64% in 30 min, then 64% for 5 min
and finally 64% to 80% in 5 min at 1 mL minꢀ1 in TEAAc
50 mM pH 7 (Fig. S1, ESIw), and by MALDI-TOF mass spectro-
metry on a Voyager DE Perseptive Biosystem spectrometer
(Table 1 and Fig. S2, ESIw).
1
RMN H (200 MHz, CDCl3): d (ppm) = 2.45 (t, 2H, H5
0
0
and H5 ), 3.63 (t, 2H, H6 and H6 ), 4.09 (s, 9H, H2, H3 and H4),
SM (ESI+) m/z: calcd for C13H13FeN [M + H]+ 239.04,
found 237.80.
3-Ferrocenylpropan-1-amine (2b). 12 mL of a solution of
LiAlH4 in Et2O was stirred at reflux for 30 min, and then 12.5 mL
of a solution of compound 21 in Et2O was added dropwise.
The reaction mixture was stirred 9 h at reflux, cooled at 0 1C
and 6 mL of EtOH/Et2O 1 : 1 v/v was added dropwise. 40 mL
of solution of NaOH (2 M) and 10 mL of saturated aqueous
NaCl were added to the aqueous layer. The aqueous layer was
extracted with Et2O (2 ꢂ 35 mL). The organic layers were
washed with 20 mL of a solution of NaOH (1 M) and water
(3 ꢂ 15 mL), dried (MgSO4), filtered and evaporated under
reduced pressure. The residue was purified by flash silica gel
column chromatography (CH2Cl2/triethylamine 99 : 1 then
CH2Cl2/MeOH 85 : 15) to afford the compound 2b (261 mg,
43%) as a dark brown oil.
Thermodynamic characterization
Molar extinction coefficient calculation. The molar extinc-
tion coefficient (e) of 2a was found to be 2176 L molꢀ1 cmꢀ1, in
methanol, at 260 nm. This value was evaluated from solutions
of 2a at different concentrations. The e of the related Fc5-a-DNA
4a was calculated by adding 5 fold the e value of 2a to the e
of the unmodified strand. The resulting e value of 4a was
105 080 L molꢀ1 cmꢀ1. For the calculation of the e of Fc5-a-DNAs
4b and 8, we hypothesized that the e of the other ferrocene
residues (propyl ferrocene, triazol ferrocene) were close to
the one of 2a. Consequently, we used the same e for the
Fc5-a-DNAs 4b and 8. After HPLC purification, quantities
of Fc5-a-DNAs were evaluated from the UV measurement
according to the Beer–Lambert equation and characterized by
MALDI TOF MS (Table 1).
1
RMN H (200 MHz, CDCl3): d (ppm) = 1.64 (q, 2H, H6
0
0
0
and H6 ), 2.36 (t, 2H, H5 and H5 ), 2.70 (t, 2H, H7 and H7 ),
4.04 (s, 9H, H2 and H3), 4.08 (s, 5H, H4), SM (ESI+) m/z:
calcd for C13H17FeN [M + H]+ 243.07, found 242.90.
General procedure for automated synthesis of Fc5-a-DNAs
4a, 4b and 8. a-DNA 1 was designed with a parallel orientation
with respect to the target. Modified a-DNA 1 was first
synthesized (1 mmol scale) with an ABI model 381 A DNA
synthesizer (Applied Biosystems) via the phosphoramidite
chemistry, using a-nucleoside 30 phosphoramidites prepared
according to a published procedure.32 The elongation of chimeric
a-DNA was performed on an ABI Model 394 DNA synthesizer
(Applied Biosystems) using the H-phosphonate chemistry with
a-nucleoside 30 H-phosphonates 14a and 14b.
UV melting temperature measurement
Optical measurements were achieved on a Uvikon UV
spectrophotometer XL from Secomam (Ales, France). UV
cell temperature was controlled by a Peltier thermosystem
(Bio-Tek Instruments, Colmar, France). 1 mM solutions of
the two complementary strands were prepared in sodium
phosphate buffer (25 mM)/NaCl (250 mM)/pH 6.4. Minimum
General procedure for amidative oxidation (3a, 3b and 5). The
solid support a-DNA 1 (0.5 mmol) was treated with a 1.5 M
solution of 2a, 2b or propargylamine in CCl4/pyridine 50 : 50 v/v
(1 mL) for 30 min to afford 3a, 3b and 5 respectively. The CPG
beads were washed with pyridine (2 ꢂ 0.1 mL), acetonitrile
c
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of five successive increases and decreases of the temperature
Acknowledgements
from 15 to 80 1C were recorded.
The authors thank bioMe
´
rieux for the financial support of
´
Gregory Chatelain’s PhD thesis and for providing the gold
Thermodynamic parameter calculation (Tm, DS1, DH1 and DG1298
)
electrode systems. This research is supported by the ANR
(French national research agency) through the project VirProbe
(PIRIBio program), the Lyonbiopole and Eurobiomed clusters.
Dissociation constants (K(T)) were calculated for Fc5-a-DNA/
target duplexes as described by Breslauer and Marky51 and
Durand et al.52 A van’t Hoff plot of Ln(K(T)) versus 1/T was
generated for duplex formation. The slope and intercept of the
calculated line yielded to the enthalpy (DH1) and the entropy
(DS1), respectively, as described in the ESI.w Then, the Gibbs
free energy at 298 K (DG1298) was calculated according to the
following equation:
Notes and references
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;
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c
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