Synthesis of ferrocenylcarbodiimide as a convenient electrochemically active labeling reagent for nucleic acids

Article abstract of DOI:10.1016/j.tet.2005.09.033

Ferrocenylcarbodiimides carrying different redox potentials, 1 and 2, were designed and synthesized as convenient electrochemically active labeling reagents for nucleic acids, which may be used as dually labeling reagents of nucleic acids like Cy3 and Cy5 dyes. These reagents could react with the imino unit of thymine or guanine base on DNA or of uracil base on RNA under a basic buffer condition to yield a labeled product quantitatively in a short period of time. The current responses of the labeled DNAs in square wave voltammetric (SWV) measurement showed a good linear correlation with the amount of the hybridized ones. DNAs labeled with the two different reagents, 1 and 2, could be detected electrochemically at different potentials after hybridization with a DNA probe-immobilized gold electrode.

Full text of DOI:10.1016/j.tet.2005.09.033

Tetrahedron 61 (2005) 11705–11715
Synthesis of ferrocenylcarbodiimide as a convenient
electrochemically active labeling reagent for nucleic acids
Kosuke Mukumoto,b Takahiko Nojimab and Shigeori Takenakaa,
*
aDepartment of Materials Science, Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu-shi 804 8550, Japan
bDepartment of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 812 8581, Japan
Received 13 July 2005; revised 12 September 2005; accepted 13 September 2005
Available online 10 October 2005
Abstract—Ferrocenylcarbodiimides carrying different redox potentials, 1 and 2, were designed and synthesized as convenient
electrochemically active labeling reagents for nucleic acids, which may be used as dually labeling reagents of nucleic acids like Cy3 and
Cy5 dyes. These reagents could react with the imino unit of thymine or guanine base on DNA or of uracil base on RNA under a basic buffer
condition to yield a labeled product quantitatively in a short period of time. The current responses of the labeled DNAs in square wave
voltammetric (SWV) measurement showed a good linear correlation with the amount of the hybridized ones. DNAs labeled with the two
different reagents, 1 and 2, could be detected electrochemically at different potentials after hybridization with a DNA probe-immobilized
gold electrode.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
amide reagent for automated DNA synthesis,5,19,25–27,30–34
ferrocenyl nucleotide triphosphate as substrate of DNA
polymerase,9,35–38 and the reaction of an activated ester of
ferrocene with the amino linked oligonucleotides.2,11,14,39–41
Direct modification of DNA with ferrocene was also
reported by using the Sonogashira reaction of ferrocenyl-
propargylamide with halogenated nucleic base of DNA.42,43
However, all of these methods described here, suffer time-
consuming steps and therefore, simpler and more effecient
ferrocenylation methods for DNA need to be devised.
Recently, conventional DNA detecting systems are required
in the gene diagnosis and many DNA sensing methods
based on electrochemical techniques have been reported,1–19
as they are expected to realize a convenient gene testing
system, which enables direct electronic readout and
miniaturization with good cost performance and high
sensitivity. Furthermore, they are expected as one of the
solutions for the carrier-type gene testing chip and can be
applied to various purposes of gene testing such as the point-
care test.20 A variety of electrochemical DNA detecting
techniques have been reported by using electrochemically
active DNA ligands such as intercalating molecules,4,7–8,21
or DNA labeling with electrochemically active
reagents.2,5,14–15,19,24–28 Direct electrochemical DNA
detection has been achieved under limited conditions
using special electrodes and electrolytes.1,22–23
Ferrocene is often used as an electrochemically active
reagent,2,5,7–9,11,14–15,19,21,24–29 as its reversible redox
potential appears where dissolved and atmospheric oxygen
does not interfere with the measurement. Oligonucleotides
labeled with ferrocene have been used as a DNA probe in
electrochemical DNA sensing. Introduction of a ferrocence
moiety to oligonucleotides was achieved by a ferrocenyl
To achieve a simple labeling method for nucleic acids,
ferrocenylcarbodiimide derivatives, 1 and 2, carrying
different redox potentials were designed and synthesized.
Water-soluble carbodiimide derivatives are known to react
with the imino moiety of thymine and guanine bases on
DNA or of uracil base on RNA reversibly under basic
conditions44–47 with excellent yield and therefore, ferro-
cenylcarbodiimide derivatives should react with DNA and
RNA in the same manner, thereby rapidly labeling natural
single stranded DNA or RNA fragments with ferrocene as
depicted in Scheme 1. Since the redox potential of ferrocene
can be altered readily by changing the nature of its
substituent, ferrocenylcarbodiimide derivatives having
different redox potentials may be prepared by designing a
linker connecting ferrocene with carbodiimide parts. Once
prepared, such compounds will serve as an important tool to
enable competitive analysis of two different samples labeled
differentially with the ferrocenyl groups with a different
redox potential.
Keywords: Ferrocenylcarbodiimide; Electrochemical detection; DNA;
RNA; Dual labeling; Competitive hybridization.
Corresponding author. Tel./fax: C81 93 884 3322;
e-mail: shige@che.kyutech.ac.jp
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.09.033

11706
K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
Scheme 1. Example of a ferrocene-modification reaction for thymine base of nucleic acid with 1.
The principle of this method is illustrated in Figure 1, which
is similar to the expression analysis based on a DNA
2. Results and discussion
microarray coupled with a dual labeling of standard and
sample DNAs with Cy3 and Cy5 dyes, respectively. We
assessed the feasibility of electrochemical gene expression
analysis by using ferrocenyl oligonucleotides, which are
labeled by the activated ester of ferrocenecarboxylic acid
and ferrocenepropionic acid.48 Other electrochemical gene
expression analyses were reported by using 7-deaza guanine
and adenine bases incorporated by PCR49 and nucleoside
triphosphate derivatives carrying ferrocene and anthra-
quinone units.50 These reports underscored the potential
importance of the electrochemical gene expression analysis
and usefulness of ferrocenylation reagents having different
redox potentials.
2.1. Synthesis of ferrocenylcarbodiimides and their
stability in aqueous solution
Ferrocenylcarbodiimides 1 and 2 were synthesized as shown
in Scheme 2. The carbodiimide function was generated by
the method described previously.51 Integrity of 1 and 2 was
1
assessed by H NMR and FT IR measurements, in which
characteristic IR absorption at 2129 cmK1 shown in Figure 2
due to the carbodiimide group was strong evidence for the
structure. Cyclic voltammograms determined in 20 mM
NaH2PO4/Na2HPO4 buffer (pH 7.0) containing 100 mM
NaClO4 revealed a one-electron redox reaction with E1/2
Z
207 mV and DE peakZ63 mV for 1 and E1/2Z443 mV and
DEZ71 mV for 2 (Fig. 3). These half-wave currents were
shifted toward the positive potential side from those of
ferrocenylpropionic acid (E1/2Z171 mV) and ferrocene-
carboxylic acid (E1/2Z328 mV), due presumably to a
difference in their cationic and anionic characters. The
stability of carbodiimides 1 and 2 was tested in several kinds
of buffer by monitoring a change in the intensity of the
absorption at 2130 cmK1 in FT IR. The carbodiimide
function was destroyed within 4 or 12 h in acetic acid buffer
(pH 5.6) or phosphate buffer (pH 7.0), respectively (data not
In this paper, the synthetic methods of 1 and 2 were
established by surveying an effective condition for the
reaction with single stranded DNA or RNA. The stability of
the ferrocenyl oligonucleotides thus obtained was also
evaluated by the melting curve analysis. Finally, we
succeeded in the electrochemical detection of target DNA
by using 1 and 2, to suggest that the electrochemical gene
expression analysis based on DNA or RNA labeled with 1 or
2 is promising for practical use.
Figure 1. Principle of the electrochemical differential hybridization assay.

K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
11707
Scheme 2. Synthetic route to 1 and 2. The reaction conditions are as follows: (a) S-tert-butoxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine/1,4-dioxane;
(b) ethylisocyanate/dry ether; (c) HCl/1,4-dioxane; (d) ferrocenepropionic acid or ferrocenecarboxylic acid/PyBOP/HOBt/TEA/CHCl3; (e) TsCl/TEA/
dryCH2Cl2, reflux; (f) CH3I/dry ether.
shown). However, it was stable over 1 day in borate buffer
(pH 8.5, 9.0 and 9.5). These data are in good agreement with
the results on carbodiimide derivatives described
previously.47
of retention time was a 1:1 adduct of 1 with D1 as deduced
by MALDI TOF MS measurement shown in Figure 5.
Products of D2–D4 with 1 or 2 could also be assigned as 1:1
adduct analogously (data not shown). No HPLC change was
observed upon reaction of 1 or 2 with D9 as negative control
(Table 1). These results demonstrated that 1 and 2 could
react with thymine and guanine bases on DNA.
2.2. Reactivity for DNA
The reactivity of 1 and 2 was studied with several kinds of
DNAs, namely D1 and D2 carrying one thymine or guanine
base at the 50-terminus of a decamer, respectively, D3
carrying one thymine base in the middle of a decamer, and
D4 carrying a thymine base at the terminus of a 20-mer
(Table 1). The reactivity was assessed from the peak
intensities of the modified and unmodified DNA in HPLC.
Typical HPLC traces before and after reaction of 1 with D1
at pH 8.5 for 12 h are shown in Figure 4. The peak at 23 min
In the next step, the reactivity of 1 and 2 with DNA was
studied at different pH. The time course of the reaction of 1
with D1 or D9 at pH 8.5, 9.0 or 9.5 is shown in Figure 6A.
Quantitative reaction occurred with D1 carrying a thymine
moiety and 1 within 10 h, whereas no reaction was observed
with D9. Since the imino moiety of thymine or guanine base
Figure 3. Cyclic voltammogram of 0.1 mM 1 (solid line) or 2 (dotted line)
in 20 mM NaH2PO4/Na2HPO4 buffer (pH 7.0) containing 100 mM NaClO4.
The scan rate was 100 mV/s.
Figure 2. FT IR spectrum of 1.

11708
K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
Table 1. Synthetic DNAs used in this study
Abbreviation
Sequence
50-TAA AAA AAA A-30
50-GAA AAA AAA A-30
50-AAA ATA AAA A-30
D1
D2
D3
D4
D5
D6
D7
D8
50-TAA AAA AAA AAA AAA AAA AA-30
50-AAA ATA AAA AAA AAA AAA AA-30
50-AAA AAA AAA TAA AAA AAA AA-30
50-AAA ATA AAA TAA AAA AAA AA-30
50-AGG GGT AAG GTT CAT TAG TTG GAA-30
50 HS-(CH2)6-TTC CAA CTA ATG AAC CTT ACC
CCT-30
HS-D8(K)
50-UAA AAA AAA A-30
50-AAA AAA AAA A-30
rUA9
D9
50-TTC CAA CTA ATG AAC CTT ACC CCT-30
50-TTT TTT TTT TAT TTT TTT TT-30
50-TTT TTT TTT TGT TTT TTT TT-30
50-TTT TTT TTT TCT TTT TTT TT-30
50-TTT TTT TTT TTT TTT TTT TT-30
D10
D11
D12
D13
D14
Figure 5. MALDI TOF MS of the HPLC fraction at 23 min in Figure 4.
Matrix, 3-HPA; mode, negative. m/z [MKH]Z3512.6 (theory for
C124H152N54O54P10Fe, 3512.1).
of DNA reacts with carbodiimide, the reactivity depends on
pH.47 The pKa of the imino moiety of thymine or guanine
base is 9.9–10.5 or 9.4–10.0, respectively,52 and therefore,
the reactivity of the imino moieties increased with pH and
the labeling reaction progressed quantitatively in a short
period of time at higher pH. For example, 1 reacted with D1
quantitatively within 4 h at pH 9.5. On the other hand, D2
carrying guanine base reacted with 1 faster than that of
thymine base (Fig. 6B). However, prolongation of the reaction
time resulted in a decrease in the yield. Since the peak of the
starting D1 increased at the expense of the product, the labeled
product of 1 at the guanine base should have hydrolyzed under
alkaline conditions. Similar behavior was reported in the
reaction of carbodiimide with nucleic bases.47
2.3. Reactivity for RNA
The reactivity of 1 with rUA9 as RNA was also studied. The
product was identified by MALDI TOF MS and the
In the third step, the reactivity of 1 was tested at 37 or 50 8C.
The reaction at 50 8C progressed quantitatively in a shorter
period than that at 37 8C as shown in Figure 7. However,
extension of the reaction time brought about a decrease in
yield, because of hydrolysis of the labeled product at the
higher temperature. The same behavior was also observed
for 2. The reactivity of 1 was not influenced by the position
of thymine base in the sequence or the length of DNA
(compare D1 with D3 or D4 in Fig. 9).
Figure 6. (A) pH dependence of reactivity of 0.5 mM D1 with 50 mM 1 in
20 mM borate buffer containing 30% DMSO at pH 8.5 (a), pH 9.0 (b) or
pH 9.5 (c). The reactivity of 0.5 mM D9 with 50 mM 1 in 20 mM borate
buffer at pH 9.0 is also shown by trace (d). (B) pH dependence of the
reactivity of 0.5 mM D2 with 50 mM 1 in 20 mM borate buffer containing
30% DMSO at pH 8.5 (a) pH 9.0 (b) or pH 9.5 (c). All experiments were
conducted at 37 8C.
Figure 4. Reversed phase HPLC before (a) and after (b) reaction of 0.5 mM
D1 with 50 mM 1 in 20 mM borate buffer (pH 8.5) containing 30% DMSO
at 37 8C for 12 h.

K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
11709
Figure 7. Temperature dependence of the reactivity of 0.5 mM D1 with
50 mM 1 in 20 mM borate buffer (pH 9.0) containing 30% DMSO at 37 8C
(a) or 50 8C (b). Temperature dependence for D2 is shown for reaction at
37 8C (c) or 50 8C (d).
Figure 9. Sequence dependence of the reactivity of 0.5 mM D3 (a) or D4
(b) with 50 mM 1 in 20 mM borate buffer (pH 9.5) containing 30% DMSO
at 37 8C. The reactivity of 0.1 mM rUA9 with 10 mM 1 is also shown by
trace (c) in the same buffer at pH 8.5.
reactivity was evaluated from the peak intensity of HPLC. A
new peak was observed at 23 min with the progress of
reaction in addition to the peak of rUA9 at 9 min. The
former was collected and subjected to MALDI TOF MS
analysis to reveal a 1:1 adduct of rUA9 with 1 as shown in
Figure 8. Ferrocene-labeling reagent 1 gave the labeled
product for rUA9 in 80% yield in 10 h at pH 8.5 and 37 8C
and prolongation of the reaction time resulted in a poorer
yield (Fig. 9). Precipitation was observed during the
reaction of 1 and rUA9 at pH 9.5.
larger destabilization by K14 8C as assessed by Tm
measurement.
To evaluate the effect of the modification with 1 on the
stability of a DNA duplex, D4–D8 modified with 1 and their
adducts were purified by HPLC. After hybridization of these
modified D4–D8 with their complementary DNAs, Tm was
measured in 20 mM KH2PO4/K2HPO4 buffer (pH 7.0)
containing 100 mM KCl (Fig. 10). The Tm values calculated
from the melting curves are summarized in Table 2.
Terminal modification of D4 with 1 did not destabilize its
DNA duplex appreciably (Fig. 10A, entry 1 in Table 2),
whereas middle modification in D5 and D6 caused
considerable destabilization of their DNA duplexes (entries
2 and 3). Modification of D7 with 1 at two sites destabilized
its DNA duplex further (entry 4). Modification with 1 in the
middle destabilized the DNA by K10 8C in Tm values
(entries 2 and 3), which is in agreement with the previous
paper.30 Nevertheless, all of the modified DNAs could still
form a DNA duplex at low temperature and therefore
hybridization was monitored there.
2.4. Stability of double stranded DNA labeled with 1
Since the imino moiety of thymine or guanine is involved in
the hydrogen bonding of a DNA duplex, its modification
with carbodiimide could destabilize the duplex structure of
DNA or RNA. Bucci et al. reported the stability of a
17-meric DNA duplex having thymine modified with a
ferrocenylmethyl group at the imino moiety.30 Terminal
modification with a ferrocenylmethyl moiety exerted only a
small effect on the stability of the duplex, whereas
modification in the middle of the sequence brought about
Compound D8 carrying many thymine and guanine bases in
its sequence was also modified with 1 possibly in more than
one position. In fact, many peaks including three main peaks
were observed in reversed phase HPLC upon reaction of D8
with 1. These three peaks were collected separately and
analyzed by MALDI TOF MS. It turned out that they were
D8 modified by one, two or three molecules of 1. The
melting curves of the DNA duplex of D8 labeled with one to
three molecules of 1 with its complementary DNA are
shown in Figure 10B and entries 5–7 in Table 2. The Tm
curve shown in Figure 10B was broader than that in
Figure 10A. This is reasonable given the fact that the
fraction of D8 modified by one molecule of 1 could still be a
mixture carrying one molecule of 1 in a different position.
As the number of modified 1 increased, the Tm values were
lowered. Nonetheless, all of these DNA duplexes were
stable at 10 8C, as proven by the circular dichroism (CD)
spectra of D8 unlabeled or labeled triply with 1 before
and after hybridization with its complementary DNA
(Fig. 11).
Figure 8. MALDI TOF MS spectrum of rUA9 after reaction with 1. Matrix,
3-HPA; mode, negative. m/z [MKH]Z3658.8 (theory for C123H139N54-
O64P10Fe, 3660.5).

11710
K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
Figure 11. CD spectra of 3 mM D8 unmodified (a) or modified (b) with
three molecules of 1 after hybridization with its complementary DNA in
20 mM KH2PO4/K2HPO4 buffer (pH 7.0) containing 0.1 M KCl at 10 8C.
The CD spectrum of 3 mM D8 before hybridization is also shown (c).
out under uniform conditions, in this case at low
temperature.
2.5. Electrochemical detection of D8 labeled with 1 or 2
Electrochemical DNA detection was carried out by using a
HS-D8(K)-immobilized electrode. Target DNA of D8 was
allowed to react with 1 or 2 and D8 modified with three
molecules of 1 or 2 was used for hybridization reaction
(Fig. 12). The SWV method was used in this experiment
because of its low background current.53 The current peak
based on the ferrocene moieties of D8 modified with 1 was
observed at 0.20 V and its intensity increased with an
increase in the amount of D8 modified with 1, whereas no
peak current was observed for D10 modified with three
molecules of 1 as negative control (Fig. 12A). This result
indicated that D8 modified with 1 hybridized indeed with
the complementary HS-D8(K) on the electrode.
Figure 10. (A) Melting curves of the DNA duplex of 5 mM D4 (B), D5
(,), D6 (6), or D7 (>) modified with 1 with respective complementary
DNA in 20 mM KH2PO4/K2HPO4 buffer (pH 7.0) containing 0.1 M KCl.
(B) Melting curves of DNA duplex of 5 mM D8 modified with one (B), two
(,), or three (6) molecules of 1 with 5 mM its complementary DNA in
20 mM KH2PO4/K2HPO4 buffer (pH 7.0) containing 0.1 M KCl.
The effect of introduction of 1 to the thymine base on the
stability of the DNA duplex was studied (Table 3). Before
the modification, occurrence of a mismatch in the middle of
the sequence lowered Tm by ca. 4.0 8C (entries 1–4). Upon
modification of thymine with 1, the Tm was lowered by ca.
10 8C (entry 5), and the magnitude of this Tm lowering was
barely dependent on the type of mismatch (entries 6–8),
demonstrating that modification in the middle of the
sequence with bulky 1 impairs duplex formation to a larger
extent than ordinary mismatches. Nonetheless, the fact that
the duplex stability is nearly independent of the type of
mismatch is advantageous, as hybridization can be carried
Analogously, the current peak was observed at 0.43 V for
D8 modified with 2 and the peak intensity was proportional
to its concentration (Fig. 12B). Since the peak currents
started to level off above 1 mM D8 (Figs. 12A and B), the
DNA on the electrode seemed to be covered by around
1 mM D8. It was estimated from the peak currents that the
electrode is covered with 4.9!1011–9.9!1012 molecules of
24-meric DNA/cm2 at saturation. This value is in good
agreement with the data previously described by Tarlov and
Georgiadis groups.54–61 In conclusion, the DNA hybridiz-
ation was quantitative at D8 concentrations lower than
1.0 mM.
Table 2. Melting temperature of several combinations of DNA duplexes between 1-modified DNAs and their complementary DNAs
Entry
Sequencea
Tm (8C)
DTm (8C)b
50-TFcAA AAA AAA AAA AAA AAA AA-30 30-A TT TTT TTT TTT TTT TTT TT-50
50-AAA ATFcA AAA AAA AAA AAA AA-30 30-TTT TA T TTT TTT TTT TTT TT-50
50-AAA AAA AAA TFcAA AAA AAA AA-30 30-TTT TTT TTT A TT TTT TTT TT-50
50-AAA ATFcA AAA TFcAA AAA AAA AA-30 30-TTT TA T TTT A TT TTT TTT TT-50
50-AGG GGT AAG GTT CAT TAG TTG GAA-30(1Fc) 30-TCC CCA TTC CAA GTA ATC AAC CTT-50
50-AGG GGT AAG GTT CAT TAG TTG GAA-30(2Fc) 30-TCC CCA TTC CAA GTA ATC AAC CTT-50
50-AGG GGT AAG GTT CAT TAG TTG GAA-30(3Fc) 30-TCC CCA TTC CAA GTA ATC AAC CTT-50
1
2
3
4
5
6
7
44.2
34.2
34.1
22.1
55.3
47.8
39.1
K0.9
K10.5
K10.3
K22.2
K4.5
K12.0
K20.7
a Fc represents ferrocene of 1 modifying the site(s) marked in the sequence.
b DTmZTm(modified with 1)KTm(unmodified).

K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
Table 3. Effect of the type of mismatch on the DNA duplex stability
11711
Entry
Sequence
X
Tm (8C)
DTm (8C)a
50-AAA AAA AAA TAA AAA AAA AA-30 30-TTT TTT TTT XTT TTT TTT TT-50
1
2
3
4
5
6
7
8
A
G
C
T
A
G
C
T
44.4
42.0
40.0
39.8
34.1
35.2
34.9
36.3
—
K2.4
K4.4
K4.6
K10.3
K9.2
K9.5
K8.1
50-AAA AAA AAA TFcAA AAA AAA AA-30 30-TTT TTT TTT X TT TTT TTT TT-50
a DTmZTm(T/X or TFc/X)KTm(T/A).
2.6. Electrochemical gene expression analysis
Figure 13B shows a plot of the observed ratio of peak
current of D8 modified with 1 or 2 against the mixed ratio of
D8 modified with 1 or 2. A good correlation obtained
suggested the feasibility of electrochemical expression
analysis by using D8 modified with 1 and 2 coupled with
a HS-D8(K)-immobilized electrode.
As a model gene expression experiment was successful with
D8 modified with 1 or 2, competitive hybridization was
attempted with a HS-D8(K)-immobilized electrode. D8
modified with 1 or 2 was mixed at various ratios and
allowed to hybridize with the HS-D8(K)-immobilized
electrode. As shown in Figure 13A, two current peaks
were obtained at 0.20 and 0.43 V in SWV measured in
20 mM NaH2PO4/Na2HPO4 buffer (pH 7.0) containing
100 mM NaClO4. The intensity of these peaks was
proportional to the amount of D8 modified with 1 or 2 and
the total peak current was ca. 40 nA, which correspond to
ca. 4.0!1012 molecules/cm2. This result shows that D8
modified with 1 or 2 can hybridize on the electrode
competitively.
3. Conclusion
Ferrocenylcarbodiimide derivatives 1 and 2 having a
different redox potential were designed and synthesized.
They could react with DNA or RNA quantitatively under
basic conditions. Although the labeling of DNA and RNA
with 1 and 2 destabilized their DNA duplex, a DNA duplex
could still form at low temperature. The ferrocene-labeled
Figure 12. (A) Square wave voltammogram of HS-D8(K) immobilized on
the electrode after hybridization with different concentrations of D8
modified with three molecules of 1 (,) or D10 (B) in 20 mM NaH2PO4/
Na2HPO4 buffer (pH 7.0) containing 100 mM NaClO4 at 10 8C. DEpZ
50 mV, DEsZ10 mV, fZ10 Hz. A standard line for the current peak at
0.20 V was plotted against the concentration of D8. (B) Square wave
voltammogram of HS-D8(K) immobilized on the electrode after
hybridization with different concentrations of D8 modified with three
molecules of 2 (,) or D10 (B) in 20 mM NaH2PO4/Na2HPO4 buffer (pH
7.0) containing 100 mM NaClO4 at 10 8C. DEpZ50 mV, DEsZ10 mV, fZ
10 Hz. A standard line for the current peak at 0.43 V was plotted against the
concentration of D8.
Figure 13. (A) Square wave voltammogram of a HS-D8(K)-immobilized
electrode after hybridization with a mixture of D8 modified with 1 or 2
(1.5:0.5, 1.2:0.8, 0.8:1.2, 0.5:1.5 mM/mM) in 20 mM NaH2PO4/Na2HPO4
buffer (pH 7.0) containing 100 mM NaClO4 at 10 8C. DEpZ50 mV, DEsZ
10 mV, fZ10 Hz. (B) Plot of the observed ratio of the peak current of D8
modified with 1 to that with 2 against the mixed ratio of D8 modified with 1
to that with 2.

11712
K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
DNA could hybridize with its complementary DNA probe-
immobilized electrode and the peak current was pro-
portional to the amount of the target DNA. When DNA
was labeled differentially with 1 or 2, the resulting DNA
gave rise to signals competitively on the electrode, making
electrochemical gene expression analysis promising with
the detection limit being ca. 0.05 mM DNA sample in 1 ml
(ca. 50 fmol).
Hitachi C-7300 column oven, L-7450H diode array
detector, L-7100 pump and D-7000 interface chromato-
graph. Reversed phase HPLC was run using a Lichrospher
RP-18 (Cica-Merck, Kanto Chemicals Co., Tokyo, Japan)
column with the gradient condition where the acetonitrile
content in 100 mM TEAA buffer (pH 7.0) was linearly
changed from 0 to 40% over 30 min at a flow rate of 1.0 ml/
min with detection at 260 nm. The reactivity of 1 and 2 with
single stranded DNAs or RNA was assessed from the ratio
of peak heights for unmodified and modified DNAs or RNA.
4. Experimental
4.1. Chemicals
4.2.3. MALDI TOF MS analysis. DNAs or RNA modified
with 1 or 2 were characterized by matrix-assisted laser
desorption ionization time-of-flight mode mass spec-
trometry (MALDI TOF MS, Voyagere Linear-SA,
PerSeptive Biosystems Inc., Fostercity, CA) measurement
of the products separated by HPLC. They were desalted by
Dowex 50WX8 cation exchange resin and dissolved in a
solution of 50 mg/ml 3HPA (3-hydroxypicolinic acid) in
0.1% TFA/50% CH3CN and dried. Mass spectra were
measured by the negative mode.
N,N-bis(3-aminopropyl)methylamine, S-tert-butoxycarbon-
yl-4,6-dimethyl-2-mercaptopyrimidine, ethylisocyanate,
ferrocenylcarboxylic acid, benzotriazol-1-yloxytris(pyrro-
lidino)phosphonium hexafluorophosphate (PyBOP),
1-hydroxybenzotriazole (HOBt), p-toluenesulfonyl chloride
(TsCl), and iodomethane were purchased from Tokyo Kasei
Co., (Tokyo, Japan). HCl/1,4-dioxane was purchased from
Watanabe Chemical Inc. (Hiroshima, Japan). Solvents used
in this paper, were purchased from Wako Chemicals Inc.
(Osaka, Japan). Ferrocenepropionic acid was synthesized
according to the method reported previously.35 Synthetic
DNAs, rUA9 of RNA, and thiolated DNA were custom-
synthesized by Genenet Co., (Fukuoka, Japan). The
sequences of these DNAs are listed in Table 1. D8(K)
carried a sequence complementary to that of mRNA coding
for the cytochrome c gene of Xenopus. MilliQ water was
used throughout (Millipore, Billerica, MA). Buffers were
prepared from the following chemicals: boric acid,
disodium hydrogenphosphate dodecahydrate, sodium
dihydrogenphosphate dihydrate, dipotassium hydrogen-
phosphate, potassium dihydrogenphosphate, sodium
hydroxide, triethylamine, and hydrochloric acid were
purchased from Wako. Tris(hydroxymethyl)aminomethane
was purchased from Nacalai Tesque (Kyoto, Japan). Buffers
20!SSC (0.3 M sodium citrate containing 3 M NaCl) and
2!SSC for hybridization were purchased from Wako.
Buffer for reaction of 1 and 2 with DNA and RNA was
prepared as 50 mM NaH2BO3/NaOH buffers (pH 8.5, 9.0 or
9.5) and used after dilution. Two hundred mM KH2PO4/
K2HPO4 buffer (pH 7.0) containing 1 M NaClO4 or 1 M
KCl was prepared and used after dilution in electrochemical
or spectrophotometric measurements, respectively, and
100 mM triethylammonium acetate (TEAA) buffer
(pH 7.0) was used as an eluent in high performance liquid
chromatography (HPLC).
4.2.4. Melting curve measurement. Melting curves of
DNA duplexes were measured on a Hitachi 3300
spectrometer equipped with an SPR 10 temperature
controller. The concentration of DNAs unmodified or
modified with 1 or 2 was estimated from the molar
absorptivity at 260 nm, 6229 or 6201 cmK1 MK1 for 1 or
2, respectively. Melting temperature was measured in
20 mM KH2PO4/K2HPO4 buffer (pH 7.0) containing
100 mM KCl. A mixture of 5 mM DNAs modified with 1
or 2 and 5 mM their complementary DNA was placed in the
cell of 1 cm in light path length (total 200 ml) and
absorbance change at 260 nm was monitored with raising
to 85 8C at a rate of 0.5 8C/min.
4.2.5. Electrochemical measurement. Electrochemical
measurement was made on an ALS Electrochemical
Analyzer Model 900 (CH Instrument Inc., Austin, TX).
The redox behavior of 100 mM 1 or 2 was monitored by
cyclic voltammetric (CV) measurement over a scan range of
0–0.7 V at a scan rate of 100 mV/s. The Osteryoung square
wave voltammetry (SWV) method was used in the
experiments for DNA-immobilized electrodes before and
after hybridization with an amplitude of 50 mV, applied
potential of 10 mV, and frequency of 10 Hz. The electrolyte
used was 20 mM NaH2PO4/Na2HPO4 buffer (pH 7.0)
containing 100 mM NaClO4, as it is known that the stable
redox reaction of ferrocence occurs in this buffer.62 The cell
was furnished with three electrodes of Ag/AgCl as reference
electrode, Pt wire as counter electrode, and DNA-
immobilized electrode as working electrode. All measure-
ments were conducted at 10 8C where the double stranded
structure of DNA used is stable on the working electrode.
4.2. Instruments
4.2.1. Identification of product. Compounds 1 and 2 were
characterized mainly by H NMR (250 MHz spectrometer,
1
Bruker, Rheinstetten, Germany) and Fourier transform
infrared (FT IR, Spectrum One FT IR, Perkin Elmer Co.,
Wellesley, MA). Tetramethylsilane (TMS) was used as a
standard in H NMR measurement. IR was measured with
4 cmK1 resolution after sandwiching the sample between
CaF2 single crystal plates.
4.3. Synthesis of 1 and 2
1
Ferrocenylcarbodiimides 1 and 2 were synthesized accord-
ing to the route shown in Scheme 2.
4.3.1. [3-[(3-Aminopropyl)methylamino]propyl]carba-
mic acid tert-butyl ester (3). N,N-bis(3-aminopropyl)-
methylamine (10 ml, 60 mmol) was dissolved in 20 ml of
4.2.2. HPLC analysis. The HPLC system used in this
experiment, was composed of the following components:

K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
11713
1,4-dioxane and a solution of S-tert-butoxycarbonyl-4,6-
dimethyl-2-mercaptopyrimidine (7.7 g, 30 mmol) in 50 ml
of 1,4-dioxane was dropped to the solution over 10 h at
room temperature and the mixture was stirred for 20 h. After
filtration of the yellow solid, the solvent was removed under
reduced pressure and 50 ml of water were added. The white
precipitates formed were removed by filtration and NaCl
was added to the filtrate. Further precipitates and remaining
solid NaCl were removed by filtration and mono Boc
derivative 3 was extracted with ethyl acetate (30 ml!4).
The extract was dried over magnesium sulfate and 3 was
obtained as a yellow oil (4.7 g, 32% yield) after evaporation
3.02–3.35 (8H, m, FcCH2CH2, CH2NHCO), and 4.12 (9H,
m, C5H5FeC5H4CH2) ppm.
4.3.5. N-[3-[[3-(3-Ethylureido)-propyl]-methylamino]-
propyl]-3-ferrocenylamide (7). Compound 5 (2.5 g,
8.8 mmol) was dissolved in TEA (5.1 ml, 36 mmol) and
chloroform (30 ml), ferrocenecarboxylic acid (2.3 g,
10 mmol), PyBOP (5.2 g, 10 mmol), and HOBt (1.3 g,
10 mmol) were added to the solution and stirred for 18 h at
room temperature. The progress of reaction was monitored
by the spot of RfZ0.20 on TLC (CHCl3/CH3OH/TEAZ
95:5:1). The reaction mixture was washed with saturated
NaHCO3 aqueous solution (30 ml!2) and the solvent was
removed under reduced pressure. The yellow fraction of
RfZ0.20 (CHCl3/CH3OH/TEAZ95:5:1) was collected
from silica gel chromatography. After removing the solvent
and drying under reduced pressure 7 was obtained as a pale
yellow oil (1.9 g, 51% yield). 1H NMR (250 MHz, CDCl3)
dZ1.11 (3H, t, NHCONHCH2CH3), 1.80 (4H, q, CH2CH2-
CH2), 2.14 (3H, s, NCH3) 2.38 (4H, m, CH2NCH3), 3.05–
3.43 (6H, m, CH2NHCO), 4.15 (5H, s, (C5H5)Fe(C5H4)-
CONH), 4.33 (2H, m, (C5H5)Fe(C2H2C2H2C)CONH), and
4.63 (2H, m, (C5H5)Fe(C2H2C2H2C)CONH) ppm.
1
and drying under reduced pressure. H NMR (250 MHz,
CDCl3) dZ1.44 (9H, s, C(CH3)3), 1.66 (4H, m, CH2CH2-
CH2), 2.19 (3H, s, NCH3), 2.36 (4H, m, NCH2CH2), 2.75
(2H, m, CH2CH2NH2), and 3.16 (2H, m,
CH2CH2NHCO) ppm.
4.3.2. [3-[[3-(3-Ethylureido)propyl]methylamino]pro-
pyl]carbamic acid tert-butyl ester (4). A solution of
ethylisocyanate (1.5 g, 20 mmol) in 13 ml of diethyl ether
was added slowly to a solution of 3 (4.7 g, 9.0 mmol) in
7 ml of diethyl ether at 0 8C with stirring. The reaction
mixture was stirred for 3 h at room temperature after
standing for 10 min at 0 8C. The mixture became negative to
ninhydrin in 3 h and the solvent was removed and the
residue dried under reduced pressure to give 4 as a yellow
oil (5.5 g, 95% yield). 1H NMR (250 MHz, CDCl3) dZ1.11
(3H, t, NHCONHCH2CH3), 1.45 (9H, s, C(CH3)3), 1.64
(4H, m, CH2CH2CH2), 2.18 (3H, s, NCH3), 2.37 (4H, m,
NCH2CH2), and 3.18 (6H, m, CH2CH2NHCO, CH2CH2-
NHCOO(tBu), CONHCH2CH3) ppm.
4.3.6.
N-[3-[(3-Ethyliminomethyleneaminopropyl)
methylamino]propyl]-3-ferrocenylpropionamide (8).
The reaction was carried out under the nitrogen atmosphere.
Compound 6 (2.0 g, 4.4 mmol) was dissolved in TEA
(2.4 ml, 18 mmol) and dry dichloromethane (20 ml) and
stirred for 15 min. The solution was kept for 15 min at
K20 8C. A dry dichloromethane solution (15 ml) of
p-toluenesulfonyl chloride (1.7 g, 8.8 mmol) was added to
the solution slowly. After standing at room temperature, the
reaction mixture was refluxed for 4 h and the progress of
reaction was monitored by the spot of RfZ0.48 on TLC
(CHCl3/TEAZ100:0.5). The reaction mixture was washed
with 40% potassium carbonate aqueous solution (20 ml!4)
and the solvent was removed. The solid obtained was
dissolved in 30 ml of diethyl ether and insoluble material
was removed by filtration. The solvent was evaporated
under reduced pressure and the residue was dried to give 8
as an orange viscous oil (0.81 g, 41% yield). 1H NMR
(250 MHz, CDCl3) dZ1.23 (3H, t, N]C]NCH2CH3),
1.68 (4H, m, CH2CH2CH2), 2.16 (3H, s, NCH3) 2.38 (4H,
m, CH2NCH3), 2.68 (2H, t, FcCH2CH2), 3.32–3.49 (8H, m,
FcCH2CH2, CH2N]C]N, CH2NHCO), and 4.10 (9H, m,
C5H5FeC5H4CH2) ppm.
4.3.3. 1-[3-[(3-Aminopropyl)methylamino]propyl]-3-
ethylurea (5). Compound 4 (5.5 g, 17 mmol) was dissolved
in 4 N HCl/1,4-dioxane (13 ml, 52 mmol) and stirred for
3 h. The precipitates formed were collected and dried under
reduced pressure to give 5 as a yellow gum-like solid (5.2 g,
95% yield). 1H NMR (250 MHz, DMSO-d6) dZ1.00 (3H, t,
NHCONHCH2CH3), 1.80 (2H, m, CH2CH2NHCO), 2.03
(2H, m, CH2CH2NCH3) 2.70 (3H, s, NCH3), and 3.03 (10H,
m, NCHCH2CH2, CH2CH2NCH3, CH2CH2NHCO,
CONHCH2CH3) ppm.
4.3.4. N-[3-[[3-(3-Ethylureido)-propyl]-methylamino]-
propyl]-3-ferrocenylpropionamide (6). Compound
5 (2.5 g, 8.8 mmol) was dissolved in TEA (5.1 ml,
36 mmol) and chloroform (30 ml). Ferrocenepropionic
acid (2.6 g, 10 mmol), PyBOP (5.2 g, 10 mmol), and
HOBt (1.3 g, 10 mmol) were added to the solution and
stirred for 10 h at room temperature. The progress of
reaction was monitored by the spot of RfZ0.22 on thin-
layer chromatography (TLC) (CHCl3/CH3OH/TEAZ
95:5:1) on silica gel. The solution was washed with
saturated NaHCO3 aqueous solution (30 ml!2) and the
solvent was removed under reduced pressure. After
collection of the RfZ0.22 fraction on silica gel chromato-
graphy with the eluent (CHCl3/CH3OH/TEAZ95:5:1),
compound 6 was obtained as a pale yellow oil (2.8 g, 70%
yield). 1H NMR (250 MHz, CDCl3) dZ1.11 (3H, t,
NHCONHCH2CH3), 1.80 (4H, q, CH2CH2CH2), 2.14 (3H,
s, NCH3) 2.38 (4H, m, CH2NCH3), 2.68 (2H, t, FcCH2CH2),
4.3.7.
N-[3-[(3-Ethyliminomethyleneaminopropyl)
methylamino]propyl]-3-ferrocenylamide (9). The
reaction was carried out under the nitrogen atmosphere.
Compound 7 (1.5 g, 3.5 mmol) was dissolved in TEA
(1.9 ml, 14 mmol) and dry dichloromethane (15 ml) and
stirred for 15 min. The solution was kept for 15 min at
K20 8C. A dry dichloromethane solution (10 ml) of
p-toluenesulfonyl chloride (1.3 g, 7.0 mmol) was added to
the solution slowly. After standing at room temperature, the
reaction mixture was refluxed for 4 h. The progress of
reaction was monitored by the spot of RfZ0.42 on TLC
(CHCl3/TEAZ100:0.5). The reaction mixture was washed
with 40% potassium carbonate aqueous solution (20 ml!4)
and the solvent was removed. The solid left was dissolved in
30 ml of diethyl ether and insoluble material was removed

11714
K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
by filtration. The solvent was evaporated under reduced
pressure and the residue was dried to give 9 as an orange
viscous oil (0.55 g, 38% yield). 1H NMR (250 MHz,
CDCl3) dZ1.11 (3H, t, N]C]NCH2CH3), 1.70 (4H, q,
CH2CH2CH2), 2.14 (3H, s, NCH3) 2.38 (4H, m, CH2NCH3),
3.02–3.35 (6H, m, CH2N]C]N, CH2NHCO), 4.15 (5H, s,
(C5H5)Fe(C5H4)CONH), 4.33 (2H, m, (C5H5)Fe(C2H2C2-
H2C)CONH), and 4.63 (2H, m, (C5H5)Fe(C2H2C2H2C)-
CONH) ppm.
1 ml of the flow-through, RNA was eluted with 1.5 ml of
0.1 M TEAA buffer and lyophilized. After addition of
100 ml of water, 20 ml of this solution were subjected to
reversed phase HPLC to assess the composition.
4.6. Preparation of a DNA-immobilized electrode and its
hybridization with complementary DNA
A gold electrode having 2.0 mm2 in area was polished with
6 mm, 1 mm of diamond slurry, and 0.05 mm of alumina
slurry in this order and sonicated in MilliQ water for 10 min.
This electrode was electrochemically polished by scanning
40 times from K0.2 to 1.5 V at a scan rate of 100 mV/s in
1 M H2SO4 aqueous solution and sonicated in MilliQ water
for 15 min. One microliter of 1 M NaCl solution containing
2 mM thiolated DNA (see Table 1) was placed on the gold
electrode held upside down and kept in a closed container
under high humidity for 24 h at room temperature. After
washing with MilliQ water, 1 ml of 1 mM 6-mercapto-
hexanol was placed on the electrode for 1 h at 45 8C.
4.3.8. (3-Ethyliminomethyleneaminopropyl)dimethyl[3-
(3-ferrocenylpropionylamino)propyl]ammonium iodide
(1). Compound 8 (0.81 g, 1.8 mmol) was dissolved in
diethyl ether (3 ml) and iodomethane (1.1 ml, 3.6 mmol)
was added and stirred for 18 h. The precipitates formed
were collected by filtration and dried under reduced pressure
1
to give 1 as a yellow solid (0.76 g, 72% yield). H NMR
(CDCl3) dZ1.23 (3H, t, N]C]NCH2CH3), 1.96–2.16
(4H, m, CH2CH2CH2), 2.68 (2H, m, FcCH2CH2), 3.21 (6H,
s, NC(CH3)2), 3.24–3.48 (12H, m, CH2NC(CH3)2, FcCH2-
CH2, CH2N]C]N, CH2NHCO), and 4.10 (9H, m,
C5H5FeC5H4CH2) ppm, FT IR (CaF2) 2129 cmK1
(–N]C]N–), 1643 cmK1 (–NH–CO–), 1559 cmK1
(–NH–CO–).
One microliter of 2!SSC containing 0.1, 0.2, 0.5, 0.8, 1 or
2 mM DNA modified with 1 or 2 was placed on the electrode
for 6 h at 10 8C to allow hybridization to proceed. The
electrode was kept in 20 mM NaH2PO4/Na2HPO4 buffer
(pH 7.0) containing 100 mM NaClO4 and SWV was
measured with an ALS model 900 Electrochemical
Analyzer. Competitive hybridization of two different DNA
samples was carried out as follows. One microliter of a
mixture of DNAs modified with 1 or 2 (1.8:0.2, 1.5:0.5,
1.2:0.8, 1.0:1.0, 0.8:1.2, 0.5:1.5, or 0.2:1.8 mM/mM) was
placed on the DNA-probe immobilized electrode for 8 h at
10 8C to allow hybridization to proceed. These electrodes
were dipped in the same electrolyte as above for 1 min and
SWV was measured at 10 8C.
4.3.9. (3-Ethyliminomethyleneaminopropyl)dimethyl[3-
(3-ferrocenylamino)propyl]ammonium iodide (2). Com-
pound 9 (0.55 g, 1.3 mmol) was dissolved in diethyl ether
(3 ml) and iodomethane (0.80 ml, 2.6 mmol) was added and
stirred for 18 h. The precipitates formed were collected by
filtration and dried under reduced pressure to give 2 as a
yellow solid (0.49 g, 68% yield). 1H NMR (CDCl3) dZ1.24
(3H, t, N]C]NCH2CH3), 1.93–2.14 (4H, m, 2!CH2-
CH2CH2), 3.19 (6H, s, NC(CH3)2), 3.20–3.49 (10H, m,
CH2NC(CH3)2, CH2N]C]N, CH2NHCO), 4.19 (5H, s,
(C5H5)Fe(C5H4)CONH), 4.42 (2H, m, (C5H5)Fe(C2H2C2-
H2C)CONH), and 4.75 (2H, m, (C5H5)Fe(C2H2C2H2-
C)CONH) ppm, FT IR (CaF2) 2127 cmK1 (–N]C]N–),
1642 cmK1 (–NH–CO–), 1557 cmK1 (–NH–CO–).
Acknowledgements
Special thanks are due to Professor Hiroki Kondo of Kyushu
Institute of Technology for reading the manuscript. This
work was supported in part by a Japan Health Sciences
Foundation (KH51045).
4.4. Labeling reaction of DNAs with 1 or 2
Ten microliters of a solution of 1 mM DNA in 20 mM
borate buffer (pH 8.5, 9.0 or 9.5) were mixed with 10 ml of a
solution of 100 mM 1 or 2 in 20 mM borate buffer
containing 60% DMSO at proper temperature for a specified
period of time. The mixture was diluted to 1 ml with 0.1 M
TEAA buffer (pH 7.0) and then loaded on a NAP-10 column
(Pharmacia Sephadex G-25, Amersham Biosciences Co.,
Uppasala, Sweden). After discarding the first 1 ml of the
flow-through, DNA was eluted with 1.5 ml of 0.1 M TEAA
buffer and lyophilized. After addition of 100 ml of water,
20 ml of this solution were subjected to reversed phase
HPLC to evaluate the composition.
References and notes
1. Wang, J.; Cai, X.; Wang, J.; Jonsson, C. Anal. Chem. 1995, 67,
4065–4070.
2. Ihara, T.; Maruo, Y.; Takenaka, S.; Takagi, M. Nucleic Acids
Res. 1996, 24, 4273–4280.
3. Korri-Youssoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.;
Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388–7389.
4. Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.;
Barton, J. K. Nat. Biotechnol. 2000, 18, 1096–1100.
5. Yu, C. J.; Wan, Y.; Yowanto, H.; Li, J.; Tao, C.; James, M. D.;
Tan, C. L.; Blackburn, G. F.; Meade, T. J. J. Am. Chem. Soc.
2001, 123, 11155–11161.
4.5. Labeling reaction of RNA with 1 or 2
Ten microliters of a solution of 0.2 mM rUA9 in 20 mM
borate buffer (pH 8.5) were mixed with 10 ml of a solution of
20 mM 1 or 2 in 20 mM borate buffer containing 60%
DMSO at 37 8C for a specified period of time. The mixture
was diluted to 1 ml with 0.1 M TEAA buffer (pH 7.0) and
then loaded on a NAP-10 column. After discarding the first
6. Jung, S.; Taton, T. A.; Mirkin, C. A. Science 2002, 295,
1503–1506.

K. Mukumoto et al. / Tetrahedron 61 (2005) 11705–11715
11715
7. Yamashita, K.; Takagi, M.; Kondo, H.; Takenaka, S. Anal.
Biochem. 2002, 306, 188–196.
34. Navarro, A. E.; Spinelli, N.; Chaix, C.; Moustrou, C.;
Mandrandb, B.; Brisseta, H. Bioorg. Med. Chem. Lett. 2004,
14, 2439–2441.
8. Yamashita, K.; Takagi, A.; Takagi, M.; Kondo, H.; Ikeda, Y.;
Takenaka, S. Bioconjugate Chem. 2002, 13, 1193–1199.
9. Patolsky, F.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc.
2002, 124, 770–772.
35. Anne, A.; Blanc, B.; Moiroux, J. Bioconjugate Chem. 2001,
12, 396–405.
36. Wlassoff, W. A.; King, G. C. Nucleic Acids Res. 2002, 30, e58.
37. Brazill, S.; Hebert, N. E.; Kuhr, W. C. Electrophoresis 2003,
24, 2749–2757.
10. Fritz, J.; Cooper, E. B.; Gaudet, S.; Sorger, P. K.; Manalis,
S. R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 14142–14146.
11. Brazill, S. A.; Kuhr, W. G. Anal. Chem. 2002, 74, 3421–3428.
12. Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125,
3214–3215.
38. Di Giusto, D. A.; Wlassoff, W. A.; Giesebrecht, S.; Gooding,
J. J.; King, G. C. J. Am. Chem. Soc. 2004, 126, 4120–4121.
39. Takenaka, S.; Uto, Y.; Kondo, H.; Ihara, T.; Takagi, M. Anal.
Biochem. 1994, 218, 436–443.
13. Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am.
Chem. Soc. 2003, 125, 324–325.
40. Long, Y. T.; Li, C. Z.; Sutherland, T. C.; Chahma, M.; Lee,
J. S.; Kraatz, H. B. J. Am. Chem. Soc. 2003, 125, 8724–8725.
41. Chahma, M.; Lee, J. S.; Kraatz, H. B. J. Electroanal. Chem.
2004, 567, 283–287.
14. Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 9134–9137.
15. Wang, J.; Li, J.; Baca, A. J.; Hu, J.; Zhou, F.; Yan, W.; Pang,
D. W. Anal. Chem. 2003, 75, 3941–3945.
42. Beilstein, A. E.; Grinstaff, M. W. Chem. Commun. 2000,
509–510.
16. Xie, H.; Yu, Y. H.; Xie, F.; Lao, Y. Z.; Gao, Z. Anal. Chem.
2004, 76, 4023–4029.
43. Beilstein, A. E.; Grinstaff, M. W. J. Organomet. Chem. 2001,
637–639 pp 398–406.
17. Fojta, M.; Havran, L.; Vojtiskova, M.; Palecek, E. J. Am.
Chem. Soc. 2004, 126, 6532–6533.
44. Gilham, P. T. J. Am. Chem. Soc. 1962, 84, 687–688.
45. Naylor, R.; Ho, N. W. Y.; Gilham, P. T. J. Am. Chem. Soc.
1965, 87, 4209–4210.
18. Long, Y. T.; Li, C. Z.; Sutherland, T. C.; Kraatz, H. B.; Lee,
J. S. Anal. Chem. 2004, 76, 4059–4065.
19. Immoos, C. E.; Lee, S. J.; Grinstaff, M. W. ChemBiochem
2004, 5, 1100–1103.
46. Ho, N. W. Y.; Gilham, P. T. Biochemistry 1967, 6, 3632–3639.
47. Mertz, D. H.; Brown, G. L. Biochemistry 1969, 8, 2312–2328.
48. Mukumoto, K.; Nojima, T.; Furuno, N.; Takenaka, S. Nucleic
Acids Res. Suppl. 2003, 43–44.
20. Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol.
2003, 21, 1192–1199.
21. Wakai, J.; Takagi, A.; Nakayama, M.; Miya, T.; Miyahara, T.;
Iwanaga, T.; Takenaka, S.; Ikeda, Y.; Amano, M. Nucleic
Acids Res. 2004, 32, e141.
49. Yang, I. V.; Ropp, P. A.; Thorp, H. H. Anal. Chem. 2002, 74,
347–354.
50. Di Giusto, D. A.; Wlassoff, W. A.; Giesebrecht, S.; Gooding,
J. J.; King, G. C. Angew. Chem., Int. Ed. 2004, 43, 2809–2812.
51. Sheehan, J. C.; Cruickshank, P. A.; Boshart, G. L. J. Org.
Chem. 1961, 26, 2525–2528.
22. Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem.
Soc. 1995, 117, 8933–8938.
23. Wang, J.; Kawde, A. N.; Erdem, A.; Salazar, M. Analyst 2001,
126, 2020–2024.
24. Thorp, H. H. Trends Biotechnol. 2003, 21, 522–524.
25. Anne, A.; Bouchardon, A.; Moiroux, J. J. Am. Chem. Soc.
2003, 125, 1112–1113.
52. Saenger, W. Principles of nucleic acid structure; Springer:
New York, 1984.
53. Bard, A. J.; Faulkner, L. R. Electrochemical methods
fundamentals and applications; Wiley: Hoboken, 2001.
54. Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J.
J. Am. Chem. Soc. 1997, 119, 3401–3402.
26. Immoos, C. E.; Lee, S. J.; Grinstaff, M. W. J. Am. Chem. Soc.
2004, 126, 10814–10815.
27. Kim, K.; Yang, H.; Park, S. H.; Lee, D. S.; Kim, S. J.; Lim,
Y. T.; Kim, Y. T. Chem. Commun. 2004, 1466–1467.
28. Gibbs, J. M.; Park, S. J.; Anderson, D. R.; Watson, K. J.;
Mirkin, C. A.; Nguyen, S. T. J. Am. Chem. Soc. 2005, 127,
1170–1178.
55. Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119,
8916–8920.
56. Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70,
4670–4677.
29. Staveren, D. R.; Metzler-Nolte, N. Chem. Rev. 2004, 104,
5931–5985.
57. Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. J. Am. Chem.
Soc. 2000, 122, 7837–7838.
30. Bucci, E.; De Napoli, L.; Di Fabio, G.; Messere, A.;
Montesarchio, D.; Romanelli, A.; Piccialli, G.; Varra, M.
Tetrahedron 1999, 55, 14435–14450.
58. Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J.
Biophys. J. 2000, 79, 975–981.
59. Peterson, A. W.; Heaton, R. H.; Georgiadis, R. M. Nucleic
Acids Res. 2001, 29, 5163–5168.
31. Yu, C. J.; Yowanto, H.; Wan, Y.; Meade, T. J.; Chong, Y.;
Strong, M.; Donilon, L. H.; Kayyem, J. F.; Gozin, M.;
Blackburn, G. F. J. Am. Chem. Soc. 2000, 122, 6767–6768.
32. Yu, C. J.; Wang, H.; Wan, Y.; Yowanto, H.; Kim, J. C.;
Donilon, L. H.; Tao, C.; Strong, M.; Chong, Y. J. Org. Chem.
2001, 66, 2937–2942.
60. Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem.
Soc. 2002, 124, 14601–14607.
61. Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov,
M. J. J. Am. Chem. Soc. 2003, 125, 5219–5226.
62. Valincius, G.; Niaura, G.; Kazakeviciene, B.; Talaikyte, Z.;
Kazemekaite, M.; Butkus, E.; Razumas, V. Langmuir 2004,
20, 6631–6638.
33. Pike, A. R.; Ryder, L. C.; Horrocks, B. R.; Clegg, W.;
Elsegood, M. R. J.; Connolly, B. A.; Houlton, A. Chem. Eur. J.
2002, 8, 2891–2899.