Synthesis of hydrazone-modified nucleotides and their polymerase incorporation onto DNA for redox labeling

Article abstract of DOI:10.1002/cplu.201200056

5-(5-Formylthiophen-2-yl)cytosine, 7-(5-formylthiophen-2-yl)-7- deazaadenosine 2′-deoxyribonucleosides, and their 5′-O-triphosphates (dNTPs) were converted into the 2,4-dinitrophenylhydrazone or nitrobenzofurazanyl. The hydrazone-modified dNTPs were enzym

Full text of DOI:10.1002/cplu.201200056

DOI: 10.1002/cplu.201200056
Synthesis of Hydrazone-Modified Nucleotides and Their
Polymerase Incorporation onto DNA for Redox Labeling
[a]
[a]
[a]
[b]
[b]
ˇ
ˇ
ˇ
Veronika Raindlovꢀ, Radek Pohl, Blanka Klepetꢀrovꢀ, Ludek Havran, Eva Simkovꢀ,
Petra Horꢀkovꢀ, Hana Pivonkovꢀ, Miroslav Fojta,*[b, d] and Michal Hocek*[a, c]
[b]
[b]
ˇ
5-(5-Formylthiophen-2-yl)cytosine, 7-(5-formylthiophen-2-yl)-7-
deazaadenosine 2’-deoxyribonucleosides, and their 5’-O-tri-
phosphates (dNTPs) were converted into the 2,4-dinitrophenyl-
hydrazone or nitrobenzofurazanyl. The hydrazone-modified
dNTPs were enzymatically incorporated into DNA by poly-
merase catalyzed primer extension (PEX). This direct incorpora-
tion of hydrazone-linked dNTPs was compared to previously
reported incorporation of aldehyde-modified dNTPs followed
by postsynthetic hydrazone formation on DNA to show that
the direct incorporation can be used for incorporation of more
hydrazone units, however, cleaner PEX products are formed by
incorporation of aldehydes and subsequent reaction with hy-
drazines. Extensive study of electrochemical behavior of the ni-
troarylhydrazone-linked nucleosides and DNA was performed
confirming the potential utility of the hydrazone, nitroaryl, and
benzofurazane groups for redox labeling of DNA.
Introduction
Electrochemical analysis of natural or modified DNA is a promis-
ing tool for applications in diagnostics.[1] Within the framework
of our long-term program on redox-labeling of DNA, we have
designed, synthesized, and enzymatically incorporated diverse
base-modified dNTPs bearing ferrocene,[2] amino- and nitro-
phenyl,[3] [Ru(bpy)3] or [Os(bpy)3] complexes,[4] tetrathiafulva-
lene,[5] alkylsulfanylbenzene,[6] and anthraquinone[7] groups.
Electrochemical behavior of all these markers was thoroughly
studied and the different redox potentials of the different
groups were used for the first generation of multi-potential
redox coding of DNA,[4] where each of the four nucleobases
was bearing a different redox label and square-wave voltam-
metry was used to analyze which particular nucleotide had
been incorporated. However, some of the redox-labeled dNTPs
were of limited stability and some were not incorporated effi-
ciently. Moreover, in DNA bearing two different reducible
labels (nitrophenyl and anthraquinone), the redox potential
partly overlapped.[7] To develop an analytically useful set of
four different labels readable in parallel that can be efficiently
incorporated by polymerase, further search for other types of
redox labels is warranted.
report on the synthesis of hydrazone-modified nucleosides and
dNTPs, their polymerase incorporation into DNA, electrochemi-
cal properties, and potential applications in redox labeling of
DNA.
Results and Discussion
Chemical synthesis
Previous studies[2–7,14–16] on polymerase incorporations of base-
modified dNTPs showed that 5-substituted pyrimidine and
7-substituted 7-deazapurine nucleoside triphosphates are
ˇ
[a] V. Raindlovꢀ, Dr. R. Pohl, Dr. B. Klepetꢀrovꢀ, Prof.Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic, v.v.i.
Gilead Sciences & IOCB Research Center
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
Fax: (+420)220-183-559
E-mail: hocek@uochb.cas.cz
Homepage: http://www.uochb.cas.cz/hocekgroup
ˇ
ˇ
[b] Dr. L. Havran, E. Simkovꢀ, Dr. P. Horꢀkovꢀ, Dr. H. Pivonkovꢀ,
Prof.Dr. M. Fojta
Bioconjugation of DNA with other types of biomolecules or
small molecules is another area of great current interest.[8] Dif-
ferent types of bioorthogonal reactions, that is, 1,3-dipolar cy-
cloaddition reactions (triazole or isoxazoline formation),[9,10]
Staudinger ligation,[11] Diels–Alder reactions,[12] or native pep-
tide ligations[13] have been successfully performed with chemi-
cally modified DNA. Recently, our research group developed[14]
the synthesis of aldehyde-modified dNTPs, their polymerase in-
corporation into DNA, and postsynthetic modification by hy-
drazone formation (applied in staining of DNA). Later, we uti-
lized[15] the aldehyde-modified DNA for bioconjugation with
lysine and peptides through reductive amination. Herein, we
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i.
Kralovopolska 135, 61265 Brno (Czech Republic)
Fax: (+420)541-211-293
E-mail: fojta@ibp.cz
Homepage: http://www.ibp.cz/labs/LBCMO/
[c] Prof.Dr. M. Hocek
Department of Organic and Nuclear Chemistry
Faculty of Science, Charles University in Prague
Hlavova 8, CZ-12843 Prague 2 (Czech Republic)
[d] Prof.Dr. M. Fojta
Central European Institute of Technology, Masaryk University
Kamenice 753/5, CZ-625 00 Brno (Czech Republic)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201200056.
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M. Hocek, M. Fojta et al.
good substrates for the enzymes. Therefore, we focused on
the attachment of aldehyde and hydrazone groups at posi-
tion 5 of dCTP and at position 7 of 7-deaza-dATP. The formyl-
thienyl-modified cytidine dCFT (4a) and adenosine dAFT (5a)
deoxyribonucleosides were prepared by aqueous Suzuki cross-
coupling reaction of iodinated cytidine dCI (1a) and adenosine
dAI (2a) nucleosides with 5-formylthiophene-2-boronic acid 3.
The reactions of dCI (1a; Scheme 1A, Table 1, entries 1 and 2)
and dAI (2a; Scheme 1B, Table 1, entries 6 and 7) with boronic
acid 3 were performed either in DMF/H2O or ACN/H2O in the
presence of Cs2CO3, Pd(OAc)2, and tris(3-sulfonatophenyl)phos-
phine sodium salt (TPPTS). The reactions proceeded smoothly
in DMF/H2O at 1208C and, after separation by reverse-phase
flash chromatography, evaporation of the solvent, and drying
under vacuum the desired dCFT (4a) and dAFT (5a) were both
obtained in very good yields of 85%. In the ACN/H2O mixture
the yields of isolated products for the corresponding alde-
hyde-modified nucleosides dCFT (4a) and dAFT (5a) were lower
(58 and 41%, respectively). Nucleosides dCFT (4a) and dAFT (5a)
were also characterized by X-ray crystal structure analysis.[17]
Unexpectedly, dAFT (5a) was found to be C3’-endo (N-type)
which is typical for ribonucleosides (Figure 1A), whereas dCFT
(4a) adopted the expected C2’-endo configuration (S-type; Fig-
ure 1B).
Figure 1. ORTEP drawings of 5a and 4a with the atom numbering scheme.
Thermal ellipsoids are drawn at the 50% probability level.
Formylthienyl-modified nucleoside monophosphates dCFTMP
(4b) and dAFTMP (5b) were prepared analogously from dCIMP
(1b; Scheme 1A, Table 1, entry 3) and dAIMP (2b; Scheme 1B,
Table 1, entry 8) using ACN/H2O as the solvent. These coupling
reactions proceeded also very smoothly and dCFTMP (4b) and
dAFTMP (5b) were isolated in good yields of 70 and 71%, re-
spectively.
The corresponding FT-modified dNTPs (dCFTTP (4c) and
dAFTTP (5c)) were also prepared by using the Suzuki procedure
with iodinated dCITP (2c; Scheme 1A, Table 1, entries 4 and 5)
and dAITP (2c; Scheme 1B, Table 1, entries 9 and 10) and bor-
onic acid 3. In DMF/H2O the products dCFTTP (4c) and dAFTTP
(5c) were obtained only in 10 and 23% yields, respectively
(owing to the higher decomposition to diphosphate), whereas
in ACN/H2O the yields of isolated product increased up to an
acceptable 65 and 41%, respectively. All formylthienyl deriva-
tives of nucleoside monophosphates and triphosphates were
isolated by semi-preparative HPLC, converted into sodium salts
by ion exchange, and freeze-dried.
Scheme 1. Suzuki cross-coupling reaction of A) dCI (1a), dCIMP (1b), and
dCITP (1c) with boronic acid 3 (6 equiv) ; B) dAI (2a), dAIMP (2b), and
dAITP (2c) with boronic acid 3 (6 equiv) using DMF/H2O or ACN/H2O, Cs2CO3
(5 equiv), Pd(OAc)2 (10 mol%), and TPPTS (5 equiv).
Table 1. Yields of the Suzuki cross-coupling reactions of iodinated nu-
cleosides dCI (1a) and dAI (2a), nucleoside monophosphates dCIMP (1b)
and dAIMP (2b), and triphosphates dCITP (1c) and dAITP (2c) with bor-
onic acid 3.
Entry SM[a]
R1
Solvent
T
t
Product
Yield
[%]
All the aldehyde-modified cytidine and adenosine deoxyri-
bonucleosides (dCFT (4a), dAFT (5a)), nucleoside monophos-
phates (dCFTMP (4b), dAFTMP (5b)), and triphosphates (dCFTTP
(4c), dAFTTP (5c)) were then used as substrates for the reaction
with two different aryl hydrazines: 4-(1-methylhydrazino)-7-ni-
trobenzofurazane (6, MHNBF)[18] and 2,4-dinitrophenylhydra-
zine (7, DNPH)[19] to form the corresponding hydrazone deriva-
tives (Scheme 2). The nitrobenzofurazane (dCNBF (8a), dANBF
(10a); Table 2, entries 1 and 7) and dinitrophenyl (dCNPh (9a),
dANPh (11 a); Table 2, entries 4 and 10) nucleoside derivatives
were prepared by precipitation from a mixture of methanol/
sulfuric acid/water and after filtration and washing the precipi-
[8C] [h]
1
2
3
4
5
6
7
8
9
10
dCI (1a)
dCI (1a)
H
H
DMF/H2O (10:1) 120 1 dCFT (4a)
ACN/H2O (1:2) 120 1 dCFT (4a)
85
58
dCIMP (1b) PO3H2 ACN/H2O (1:2) 100 1 dCFTMP (4b) 70
dCITP (1c) P3O9H4 DMF/H2O (10:1) 120 0.5 dCFTTP (4c) 10
dCITP (1c) P3O9H4 ACN/H2O (1:2) 100 1 dCFTTP (4c) 65
dAI (2a)
dAI (2a)
H
H
DMF/H2O (10:1) 120 1 dAFT (5a)
ACN/H2O (1:2) 120 1 dAFT (5a)
85
41
dAIMP (2b) PO3H2 ACN/H2O (1:2) 100 1 dAFTMP (5b) 71
dAITP (2c) P3O9H4 DMF/H2O (10:1) 120 0.5 dAFTTP (5c) 23
dAITP (2c) P3O9H4 ACN/H2O (1:2) 100 1 dAFTTP (5c) 41
[a] SM=starting material. ACN=acetonitrile, DMF=N,N-dimethylforma-
mide.
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Nucleotides
Scheme 2. Hydrazone formation between aldehyde-modified cytidine (dCFT (4a), dCFTMP (4b), dCFTTP (4c)) or aldehyde-modified adenosine (dAFT (5a),
dAFTMP (5b), dAFTTP (5c)) and two arylhydrazines 6 (MHNBF) and 7 (DNPH).
tate with 5% K2CO3 and water the desired nucleosides were
obtained in good yields of 70–76%.
for monophosphates and ranged between 42 and 66%
(Table 2, entries 3, 6, 9, and 12).
In contrast, for the facilitation of condensation under aque-
ous conditions,[20] the preparation of nitrobenzofurazane and
dinitrophenyl monophosphates (dCNBFMP (8b), dCNPhMP (9b),
dANBFMP (10b), dANPhMP (11 b)) or triphosphates (dCNBFTP
(8c), dCNPhTP (9c), dANBFTP (10c), dANPhTP (11 c)) was per-
formed in a mixture of aniline/ammonium acetate/water ac-
cording to Dawson’s protocol[21] for the aqueous conjugation
of peptides. After purification by HPLC, conversion into the
sodium salt, and freeze drying the modified dCRMPs (8b, 9b)
were obtained in acceptable 53 and 70% yields, respectively
(Table 2, entries 2 and 5), and modified dARMPs (10b, 11b)
were prepared in 42 and 59% yields (Table 2, entries 8 and 11),
respectively. The reaction yields of modified dCRTPs (8c, 9c)
and dARTPs (10c, 11c) were consistent with yields obtained
Enzymatic incorporation
There are basically two approaches for the enzymatic construc-
tion of hydrazone-modified DNA. The first approach reported
in our preliminary Communication[14] consists of polymerase in-
corporation of aldehyde-modified dNTPs (dCFTTP (4c), dAFTTP
(5c)) and subsequent postsynthetic treatment with an arylhy-
drazine 6 or 7 to form the corresponding hydrazones. The
second pathway, which will be also discussed in this work, is
based on the direct polymerase incorporation of hydrazone-
modified triphosphates (dCNBFTP (8c), dCNPhTP (9c), dANBFTP
(10c), or dANPhTP (11 c)).
To investigate the feasibility of this second approach, the
four hydrazone-modified dCRTPs
(8c, 9c) and dARTPs (10c, 11c)
Table 2. Yields of hydrazone formation from aldehyde-modified nucleos(t)ides dCFT (4a), dCFTMP (4b), dCFTTP
(4c), dAFT (5a), dAFTMP (5b), and dAFTTP (5c).
were studied as substrates for
enzymatic incorporation into
DNA by primer extension (PEX)
using four different polymerases:
DeepVent(exo-), Pwo, Vent(exo-),
and KOD XL. The PEX reactions
were tested on a 31-mer tem-
plate with four modifications
(either A or C) in separate posi-
tions.
Entry SM[a]
R1
Reagent Solvent
T [8C] t [h]
Product
Yield [%]
1
2
3
4
5
6
7
8
dCFT (4a)
H
6
6
6
7
7
7
6
6
6
7
7
7
MeOH/H2SO4/H2O
RT
aniline/NH4OAc/H2O RT
aniline/NH4OAc/H2O 30
5 min dCNBF (8a)
71
53
60
70
70
66
75
dCFTMP (4b) PO3H2
24
20
dCNBFMP (8b)
dCNBFTP (8c)
dCFTTP (4c)
dCFT (4a)
P3O9H4
H
MeOH/H2SO4/H2O
aniline/NH4OAc/H2O RT
aniline/NH4OAc/H2O RT
MeOH/H2SO4/H2O
aniline/NH4OAc/H2O RT
aniline/NH4OAc/H2O 30
MeOH/H2SO4/H2O
aniline/NH4OAc/H2O RT
aniline/NH4OAc/H2O RT
RT
5 min dCNPh (9a)
dCFTMP (4b) PO3H2
24
24
dCNPhMP (9b)
dCNPhTP (9c)
dCFTTP (4c)
dAFT (5a)
P3O9H4
H
RT
5 min dANBF (10a)
dAFTMP (5b) PO3H2
24
20
dANBFMP (10b) 42
9
dAFTTP (5c)
dAFT (5a)
P3O9H4
H
dANBFTP (10c)
42
76
In general, the modified
dCRTPs (8c, 9c) were better sub-
strates for polymerases than
dARTPs (10c, 11c) and were effi-
ciently incorporated into DNA by
10
11
12
RT
5 min dANPh (11 a)
dAFTMP (5b) PO3H2
24
24
dANPhMP (11 b) 59
dAFTTP (5c)
P3O9H4
dANPhTP (11 c)
55
[a] SM=starting material.
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M. Hocek, M. Fojta et al.
DeepVent(exo-) DNA polymerase and gave 31-mer products
(Figure 2). The dCRTPs (8c, 9c) were also efficiently incorporat-
ed by Vent(exo-) and KOD XL DNA polymerases (but the full-
Figure 2. Polyacrylamide gels of PEX experiments of dCNBFTP (8c), dCNPhTP
(9c), dANBFTP (10c), and dANPhTP (11 c). A) Vent(exo-) DNA polymerase,
(B) KOD XL DNA polymerase, (C) Deep Vent(exo-) DNA Polymerase; P: Primer
(5’-32P-end labeled primer-template); +: natural dNTPs; C-: dTTP, dATP,
dGTP; A-: dTTP, dCTP, dGTP; CNBF: dCNBFTP (8c), dTTP, dATP, dGTP; CNPh
:
dCNPhTP (9c), dTTP, dATP, dGTP; ANBF: dANBFTP (10c), dTTP, dCTP, dGTP; ANPh
dANPhTP (11 c), dTTP, dCTP, dGTP.
:
length products were accompanied by 1-nt shorter deletion
products (Figure 2). Pwo DNA polymerase gave only mixtures
of ON products of various lengths (data not shown). Moreover,
the thermal denaturation study showed significant destabiliza-
tion of the duplex by nitrobenzofurazane or dinitrophenyl
groups (À3.008C or À2.758C per modification; Table S1 in the
Supporting Information). On the other hand, dARTPs (10c,
11 c) were found to be worse substrates for the PEX experi-
ments. In all experiments using any of the studied poly-
merases, only mixtures of shorter (by 2–6-nts) PEX products
were obtained (Figure 2).
Figure 3. A) Direct incorporation of hydrazone-modified dNTPs and subse-
quent magnetoseparation. B) PEX synthesis of aldehyde-modified ssON and
subsequent hydrazone formation.
was directly performed with hydrazone-modified dNTPs
(dCNBFTP (8c) or dCNPhTP (9c)) followed by magnetoseparation
to isolate ssON (the yields were 8–12% based on the biotiny-
lated template). Because some of the hydrazone units could
have been hydrolyzed during the PEX and magnetoseparation
steps, in an additional experiment, the DNA after PEX was
treated with either hydrazine 6 or 7 for 24 hours followed by
magnetoseparation. In approach B, the PEX was performed
with aldehyde-modified dCFTTP (4c) and the resulting alde-
hyde-modified DNA was treated with hydrazine 6 or 7 for
either 24 or 48 hours followed by magnetoseparation.
Therefore, we have chosen the modified cytidine derivatives
for the comparative study of DNA–hydrazone modification by
the direct incorporation of hydrazone derivatives and by post-
synthetic hydrazone formation from aldehyde-modified DNA.
Because the hydrazone-formation reaction in water is reversi-
ble, the hydrazone modification on DNA (prepared by any of
the two methods mentioned above) is inherently unstable in
water and could be hydrolyzed to aldehyde and/or re-formed
from an aldehyde and hydrazine.[20] In our previous study,[14]
we reported an approximate 50% efficiency for the DNA stain-
ing by postsynthetic hydrazone formation. Now we have opti-
mized both approaches and compared which one is more effi-
cient at attaching a higher number of hydrazone units.
The number of modifications was calculated based on the
known extinction coefficients of the hydrazone-modified nucle-
otides (at 412 nm for NPh or 542 nm for NBF) and concentra-
tion of the modified ONs. Because the chromophores are ex-
posed to the major groove of DNA through the linker and we
study ssONs, we assumed that the sequence should not signifi-
cantly influence the extinction coefficient of the label (in con-
trast to the chromophores that participate on the p–p stacking
of DNAduplex where a strong influence of the sequence was
reported[22]). The results (Table 3) show that, on average, be-
tween two and four (out of four) hydrazone modifications
could be attached by these approaches. The nitrobenzofuraza-
nehydrazone is apparently more stable than the dinitrophenyl-
Because both approaches inherently gave mixtures of DNA
products (with varying number of modifications) and MALDI
analysis of the hydrazone-modified oligonucleotides (ONs)
failed (presumably owing to low stability of the hydrazones),
the comparison was based on UV/Vis spectroscopy of isolated
modified ssONs. The study was performed on 5’-biotinylated
31-mer template suitable for magnetoseparation on streptavi-
dine-coated magnetic beads (Figure 3). In approach A, the PEX
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Nucleotides
Table 3. Efficiency of hydrazone incorporation by different approaches.
Sample
Modif Approach
Number of
hydrazone
modifs
B1
B2
B3
B4
D1
D2
D3
D4
CNBF[a]
CNBF[b]
CFT[c]
CFT[d]
CNPh[e]
CNPh[f]
CFT[g]
CFT[h]
dCNBFTP direct incorporation
2.3Æ0.2
2.6Æ0.2
3.3Æ0.2
3.1Æ0.5
4.0Æ0.2
dCNBFTP direct incorporation + 6 (24 h)
dCFTTP incorporation + 6 (24 h)
dCFTTP incorporation + 6 (48 h)
dCNPhTP direct incorporation
dCNPhTP direct incorporation + 7 (24 h) 2.8Æ0.3
dCFTTP incorporation + 7 (24 h)
dCFTTP incorporation + 7 (48 h)
3.1Æ0.3
3.7Æ0.5
[a] CNBF: dCNBFTP (8c), dTTP, dATP, dGTP. [b] CNBF: dCNBFTP (8c), dTTP, dATP,
dGTP, 6, 24 h/308C. [c] CFT: dCFTTP (4c), dTTP, dATP, dGTP, 6, 24 h/308C.
[d] CFT: dCFTTP (4c), dTTP, dATP, dGTP, 6, 48 h/308C. [e] CNPh: dCNPhTP (9c),
dTTP, dATP, dGTP, 24 h/308C. [f] CNPh: dCNPhTP (9c), dTTP, dATP, dGTP, 7,
24 h/308C. [g] CFT: dCFTTP (4c), dTTP, dATP, dGTP, 7, 24 h/308C. [h] CFT:
dCFTTP (4c), dTTP, dATP, dGTP, 7, 48 h/308C. Modif=modification.
hydrazone. For the CNPh-modified ON, the postsynthetic ap-
proach is more efficient, whereas for the CNBF-modified ON,
both approaches are comparably efficient. Additional treat-
ment of hydrazone-modified DNA with hydrazines 6 or 7 does
not increase the efficiency (in some cases it even decreases the
number of modifications). Because the dCFTTP (4c) is a more
efficient substrate for DNA polymerases (it works very well
both for PEX and PCR)[14] compared with dCNBFTP (8c) or
dCNPhTP (9c), the indirect approach based on incorporation of
aldehyde-modified nucleotides and subsequent hydrazone for-
mation is a more general and efficient procedure for the syn-
thesis of hydrazone-modified DNA.
Figure 4. Cyclic voltammograms of modified nucleoside monophosphates
(A), separate building blocks (B), and modified dNXTPs (C; see legend in the
panels) measured at HMDE in 0.2m sodium acetate pH 5, with initial poten-
tial 0.0 V, switching potential À1.6 V, and scan rate 1 VsÀ1. All compounds at
a concentration of 40 mm.
Electrochemistry
Electrochemical properties of the NPh- and NBF-hydrazone
modified nucleotides and dNXTPs were studied by means of
cyclic voltammetry (CV) at the surface of a hanging mercury
drop electrode (HMDE; Figure 4) and a basal plane pyrolytic
graphite electrode (PGE; Figure 5). Depending on the function-
al groups present in individual compounds, specific peaks cor-
responding to their electrochemical reduction were observed
at both electrodes. Results obtained with the HMDE for a set
of modified cytosine nucleoside monophosphates, dCFTMP
(4b), dCNPhMP (9b), and dCNBFMP (8b) are shown in Figure 4A.
All dCXMPs gave a cathodic peak Cred close to À1.25 V resulting
from irreversible reduction of the cytosine moiety[1] (see
Table 4 for potential values obtained with individual com-
pounds). For dCFTMP (4b), another well-developed current
signal (peak C=Ored) was detected around À0.91 V, which has
been ascribed to two-electron/two-proton reduction of the
formyl C=O bond. Both hydrazone derivatives yielded a reduc-
tion peak CH=Nred at about the same potential, corresponding
to reduction of the hydrazone C=N bond. In dCNBFMP (8b) the
latter signal partly overlaps with a slightly less negative peak
BFred corresponding to six-electron/six-proton reduction of the
furazane heterocycle[23] at À0.88 V.
Nitro groups present in both hydrazone conjugates were re-
red
duced between À0.25 and À0.42 V, giving a single peak NO2
at À0.33 V for the mononitro derivative dCNBFMP (8b) or to
two separate peaks resulting from consecutive reduction of
two nitro groups[24] in dCNPhMP (9b). Attribution of the above
reduction signals to individual moieties has been verified by
measurements carried out with separate building blocks not
attached to the nucleoside (Figure 4B). Voltammograms of
these compounds naturally did not show the peak Cred but
other signals corresponded to the present moieties, as pro-
posed above. 5-formylthiophene boronic acid (3) yielded
a single reduction peak C=Ored around À1.0 V. The MHNBF (6)
red
produced a single peak NO2 and a symmetrical peak BFred
,
meanwhile the overlapping peak CH=Nred was absent in
agreement with absence of the hydrazone C=N bond. Similarly,
DNPH (7) yielded the double peak corresponding to the dini-
trophenyl group and no other signal. For model thiophen-2-
carbaldehyde-dinitrophenylhydrazone (13), the double peak
corresponding to dinitrophenyl and the hydrazone peak CH=
Nred were detected (Figure 4B). In Figure 4C, CVs of triphos-
phates dCNBFTP (8c), dCFTTP (4c), dANBFTP (10c), and dAFTTP
(5c) are compared. Reduction peaks displayed on the voltam-
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M. Hocek, M. Fojta et al.
responding to adenine reduction was observed at a more neg-
ative potential around À1.38 V; Table 4) and the conjugate
group (dNFTTPs gave peak C=Ored whereas dNNBFTPs gave peak
red
NO2 and overlapping peaks BFred and CH=Nred). All observed
electrode processes were irreversible, as evidenced by absence
of any counter peak in the anodic parts of the CVs (it should
be noted that no re-oxidation peaks were detected regardless
of the switching potential value i.e. even when the potential
scan was reversed just after the given cathodic signal to pre-
vent further reduction and breakdown of the primary reduc-
tion product). Alterations in the peak intensities observed for
individual derivatives (Figure 4), albeit measured for the same
solution concentration, are naturally caused by their different
absorbability/solubility.
Analogous measurements as above were performed with
the pyrolytic graphite electrode (PGE; Figure 5). The nucleo-
base reduction signals could not be observed owing to limited
negative potential window of the carbon electrode given by
considerably lower hydrogen overvoltage.[25] On the other
hand, concerning reduction processes of the extrinsic conju-
gate groups, similar results were obtained. In contrast to
HMDE, the PGE in general provided worse resolution of signals
occurring at close potentials (e.g. it was not possible to resolve
peak BFred and CH=Nred in the NBF hydrazone derivatives, and
the dinitrophenyl moiety in dCNPhMP (9b) gave one developed
peak with a shoulder; Figure 5A), which may be related to less
facile electron transfer at the PGE compared with HMDE. Nev-
ertheless, it is evident that the carbon electrodes are suited for
the analysis of the nucleic acids components modified with or-
ganic electroactive moieties as well.
Figure 5. Cyclic voltammograms of cytosine modified nucleoside monophos-
phates (A), separate building blocks (B), and modified dNXTPs (C; see legend
in the panels) measured at PGE in 0.2m sodium acetate pH 5, with initial po-
tential 0.0 V, switching potential À1.4 V, and scan rate 1 VsÀ1. All compounds
at a concentration of 40 mm.
For the next experiments with modified oligonucleotides we
chose the NBF hydrazone as a new type of electroactive label.
Cyclic voltammograms measured at the HMDE with products
of PEX performed with dCNBFTP (8c) complemented with three
natural dNTPs (Figure 6, red curve) and with an unlabeled PEX
product (black curve) show a clear discrimination between the
modified and unmodified ON. The latter produced a cathodic
peak CAred around À1.5 V corresponding to reduction of cyto-
sine and adenine (it is known that in random-sequence DNA
both bases produce a single reduction peak[1]) and an anodic
peak around À0.25 V specific for guanine,[1,7] whereas the la-
beled ON gave additional cathodic peaks corresponding to re-
Table 4. Potentials of reduction of modified nucleotides and building
blocks (measured at HMDE).[a]
Compound
Peak type (see Figure 4)
red
NO2
BFred
–
CH=Nred C=Ored
Cred
Ared
–
dCNPh
À0.260
À0.415
À0.910
–
–
À1.235
dCNBF
dCFT
MHNBF
DNPH
À0.330 À0.880 À0.920
À1.235
–
–
–
–
red
–
–
–
–
–
À0.910 À1.255
duction of the nitro group (peak NO2 around À0.45 V) and
À0.400 À0.895
–
–
–
–
reduction of the furazane heterocycle (peak BFred at around
À1.0 V). In contrast to measurements with monomeric nucleo-
tides (Figure 4A) and dNNBFTPs (Figure 4C), here we did not
observe a separate signal of the hydrazone C=N group. On the
other hand, comparison of responses of the CNBF-modified (red
curve in Figure 6) and CFT-modified (blue curve in Figure 6) PEX
products revealed a good separation of the peak BFred from
peak C=Ored (at À1.16 V) produced by the formyl moiety, thus
allowing us to discriminate between both labeled ONs and to
follow postsynthetic modification of the FT-functionalized DNA
with the MHNBF. This is demonstrated by the green curve in
Figure 6, representing a CV obtained for CFT-modified PEX
product incubated with MHNBF under conditions favoring for-
mation of the corresponding hydrazone.[14] Such treatment re-
À0.270
À0.430
–
–
3
13
–
–
–
À1.040
–
–
–
–
À0.260
À0.430
À0.950
–
dCNBFTP
dANBFTP
dAFTTP
dCFTTP
À0.355 À0.885 À0.935
À0.310 À0.890 À0.930
–
–
À1.235
–
–
–
À1.375
À1.385
–
–
–
–
–
–
–
À0.940
À0.930 À1.245
[a] Cyclic voltammograms measured at HMDE under conditions given in
Figure 4. Potentials measured against the Ag/AgCl/3m KCl reference elec-
trode
mograms reflected both types of nucleobases (dCXTPs pro-
duced peak Cred around À1.24 V whereas dAXTPs peak Ared cor-
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Nucleotides
tion conditions. Because the polymerase incorporation of more
bulky and relatively unstable (in water) hydrazone-dNTPs is
less efficient than the incorporation of small formylthienyl-
dNTPs, the indirect approach based on postsynthetic hydra-
zone formation is more suitable, especially for larger DNA se-
quences.
Apart from DNA staining reported in our previous study,[14]
the hydrazone-modifications in nucleos(t)ides and DNA can be
used for redox labeling. Electrochemical experiments revealed
an easy detection of various oxygenous or nitrogenous func-
tional groups attached onto DNA or nucleic acids components,
including formyl (through reduction of the C=O bond), hydra-
zone (C=N), nitro derivatives, and the furazane heterocycle by
their electroreduction at mercury or carbon electrodes. The al-
dehyde and hydrazone groups are rather reactive and/or un-
stable to serve, by themselves, as electroactive labels for bio-
sensing applications. Their specific electrochemical properties
can nevertheless be used for monitoring of the preparation of
functionalized DNA to attach further electroactive, optical, or
affinity tags as well as of the subsequent reactions. On the
other hand, the stable nitro derivatives and the benzofurazane
moiety appeared to be suitable reducible labels. Our results
suggest that various aromatic nitro derivatives differing in the
position and number of nitro groups can be used for differen-
tial DNA coding based on specific patterns of signals in the
region between À0.25 and À0.45 V. Reduction of the furazane
heterocycle offers an intense electrochemical signal in the po-
tential region reasonably separated from the region of nucleo-
base reduction (in DNA around À1.5 V) as well as from redox
potentials of previously applied reducible labels based on an-
thraquinone[7] or the nitro compounds[3,7,25] (less negative than
À0.5 V) allowing its application as a new independent reduci-
ble marker. DNA labeling with nucleobase–benzofurazane con-
jugates is a subject of our ongoing separate study.
Figure 6. A) Ex situ CVs of CFT- or CNBF-modified 31-mer PEX products (see
legend in the panels). The CVs were measured at HMDE in 0.3m ammonium
formate, 0.05m sodium phosphate pH 6.9 with initial potential 0.0 V, switch-
ing potential À1.85 V, and scan rate 1 VsÀ1. B) Detail of cathodic branches of
the CV showing potential region around peaks COred and BFred. For other de-
tails see text.
sulted in decrease of the peak C=Ored and appearance of peak
BFred (together with peak NO2red). Intensities of the NBF signals
resulting from this treatment were by about 40–50 % lower
compared with intensities of the same signals resulting from
direct PEX incorporation of CNBF (while intensities of intrinsic
DNA peaks CAred and G were practically identical, thus suggest-
ing comparable yields of the ONs), thereby reflecting partial
conversion and the dynamic equilibrium discussed above. Im-
portantly, MHNBF treatment of the unmodified DNA (magenta
curve in Figure 6B) did not result in appearance of the NBF sig-
nals, thus confirming that in the case of MHNBF treatment of
the CFT-modified DNA the observed responses were a result of
postmodification of the formyl groups in the functionalized
DNA and not did not correspond to unremoved hydrazine
units.
Experimental Section
General chemistry
The NMR spectra were measured with Bruker Avance 500 at
500 MHz for 1H and 125.7 MHz for 13C, or with Bruker Avance at
1
600 MHz for H and 150.9 MHz for 13C, instruments in D2O (refer-
ence to dioxane as internal standard, dH =3.75 ppm, dC =
67.19 ppm), [D4]Methanol (reference to TMS as internal standard),
or in [D6]DMSO referenced to the residual solvent signal (1H NMR
dH =2.50 ppm, 13C NMR dC =39.7 ppm). Chemical shifts are given in
ppm (d scale) and coupling constants (J) in Hz. Complete assign-
ment of all NMR signals was achieved by using a combination of
H,H COSY, H,C HSQC, and H,C HMBC experiments. Semi-preparative
separation of nucleoside triphosphates and monophosphates was
performed by HPLC on a reversed-phase column packed with
10 mm of reversed-phase silica gel (Phenomenex, Luna C18 (2)).
Semi-preparative separation of nucleosides was performed by flash
chromatography on a column packed with 40–63 mm of reversed-
phase silica gel (C18, Biotage). The IR spectra were measured by
using KBr micro-tablets. Mass spectra were measured by ESI mass
spectrometry. High-resolution mass spectra were measured on
a LTQ Orbitrap XL (Hermo Fischer Scientific) spectrometer using ESI
ionization technique. The UV/Vis spectra were measured on
Conclusion
In conclusion, nitroarylhydrazone modification can be easily in-
troduced into DNA either 1) by polymerase incorporation of
formylthienyl-modified dNTPs and subsequent treatment of
the resulting aldehyde-modified DNA with hydrazine com-
pounds or 2) by direct polymerase incorporation of hydrazone-
modified dNTPs. Owing to the reversibility of hydrazone-forma-
tion in water, the efficiency of both methods is only between
50–100% depending on the nature of the hydrazone and reac-
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M. Hocek, M. Fojta et al.
a Varian CARY 100 Bio spectrophotometer at RT. Known starting
compounds were either purchased and used without any further
treatment (5-formylthiophene-2-boronic acid 3 (Frontier Scientific),
4-(1-methylhydrazino)-7-nitrobenzofurazane (6; Sigma–Aldrich),
2,4-dinitrophenylhydrazine (7; Sigma–Aldrich)) or prepared by liter-
ature procedures (compounds dCITP[3] (1c), dAITP[27] (2c), dAIMP[28]
(2b), dCIMP[14] (1b), dCFTTP[14] (4c), dCFTMP[14] (4b), dAFTTP[14] (5c)).
5’), 72.87 (CH-3’), 86.48 (CH-1’), 89.17 (CH-4’), 102.17 (C-4a), 110.20
(C-5), 124.95 (CH-6), 128.60 (CH-3 thienyl), 139.77 (CH-4 thienyl),
144.06 (C-5 thienyl), 147.50 (C-2 thienyl), 151.54 (C-7a), 152.93 (CH-
2), 158.78 (C-4), 184.94 ppm (CHO); IR (KBr): u=3460, 3104, 2914,
1645, 1590, 1544, 1439, 1274, 1177, 1097, 1043, 905, 815 cmÀ1; UV/
Vis (DMSO, 100 mm): lmax1 (e)=361 (11298), lmax2 (e)=273 nm
(12345 molÀ1 dm3 cmÀ1); MS (ESI+): m/z (%): 742.7 (100) [2M+Na]+,
361.0 (72) [M+H]+, 383.1 (38) [M+Na]+; HRMS (ESI+): m/z calcd
for C16H17O4N4S: 361.0965 [M+H]+; found 361.0965; m.p.=192–
1938C.
General procedure A for the preparation of dCFT (4a) or dAFT
(5a) by the Suzuki cross-coupling reaction
Procedure for the preparation of dAFTMP (5b)
A mixture of DMF/H2O (10:1, 2 mL) was added through a septum
into an argon-purged vial containing 5-iodo-2’-deoxycytidine dCI
(1a) or 7-iodo-7-deaza-2’-deoxyadenosine dAI (2a), 5-formylthio-
phene-2-boronic acid (3), and Cs2CO3. Then a solution of Pd(OAc)2
and TPPTS in DMF/H2O (10:1, 0.5 mL) was injected into the mixture
and the resulting mixture was stirred at 1208C for 1h. The crude
reaction mixture was pre-evaporated and then loaded onto silica
gel by evaporation and subsequently separated by flash chroma-
tography on reverse-phase silica gel (C18) and eluted with a linear
gradient of H2O to MeOH. The relevant fractions were evaporated
and dried under vacuum to give purified products.
This compound was prepared by Suzuki cross-coupling reaction
under aqueous conditions. An mixture of ACN/H2O (1:2, 1 mL) was
added through a septum into an argon-purged vial containing 7-
iodo-7-deaza-2’-deoxyadenosine monophosphate dAIMP (2b;
0.040 g, 0.084 mmol), 5-formylthiophene-2-boronic acid 3 (0.078 g,
6 equiv, 0.500 mmol), and Cs2CO3 (0.136 g, 5 equiv, 0.417 mmol).
Then
a solution of Pd(OAc)2 (1.8 mg, 10 mol%.) and TPPTS
(0.023 g, 5 equiv, 0.040 mmol) in ACN/H2O (1:2, 0.5 mL) was inject-
ed into the mixture and the resulting mixture was stirred at 1008C
for 1h. The crude reaction mixture was extracted with CHCl3 (2ꢂ
4 mL). The H2O layer that contained product was concentrated by
evaporation and the product was isolated by semi-preparative
HPLC on a reversed-phase column packed with C18 and eluted
with a linear gradient of 0.1m TEAB (triethylammonium bicarbon-
ate) in H2O to 0.1m TEAB in H2O/MeOH (1:1). Several co-distillations
with H2O, conversion into the sodium salt form (Dowex 50WX8 in
Na+ cycle), and subsequent freeze drying from H2O gave the prod-
uct dAFTMP (5b) as a bright-yellow powder (0.0276 g, 71%).
1H NMR (499.8 MHz, D2O, ref(dioxane)=3.75 ppm): d=2.48 (ddd,
1H, Jgem =14.0, J2’b,1’ =6.1, J2’b,3’ =3.1 Hz, H-2’b), 2.715 (ddd, 1H,
dCFT (4a): This compound was prepared according to general pro-
cedure A using 5-iodo-2’-deoxycytidine dCI (1a; 0.050 g,
0.142 mmol), 5-formylthiophene-2-boronic acid 3 (6 equiv, 0.133 g,
0.852 mmol), Cs2CO3 (5 equiv, 0.231 g, 0.709 mmol), Pd(OAc)2
(3.1 mg, 10 mol%.), and TPPTS (5 equiv, 0.040 g, 0.704 mmol). The
product dCFT (4a) was obtained as a light-yellow solid (0.041 g,
85%). Note: When using a mixture of ACN/H2O (1:2) the reaction
yield was lower (58%). 1H NMR (600.1 MHz, CD3OD): d=2.23 (dt,
1H, Jgem =13.6, J2’b,1’ =J2’b,3’ =6.2 Hz, H-2’b), 2.43 (ddd, 1H, Jgem
13.6, J2’a,1’ =6.2, J2’a,3’ =4.6 Hz, H-2’a), 3.72 (dd, 1H, Jgem =12.0,
5’b,4’ =3.3 Hz, H-5’b), 3.82 (dd, 1H, Jgem =12.0, J5’a,4’ =3.0 Hz, H-5’a),
3.96 (ddd, 1H, J4’,3’ =4.1, J4’,5’ =3.3, 3.0 Hz, H-4’), 4.39 (ddd, 1H,
3’,2’ =6.4, 4.6, J3’,4’ =4.1 Hz, H-3’), 6.25 (t, 1H, J1’2’ =6.2 Hz, H-1’), 7.32
=
J
J
gem =14.0, J2’a,1’ =8.1, J2’a,3’ =6.2 Hz, H-2’a), 3.85, 3.87 (2 ꢂ dt, 2ꢂ1H,
gem =10.9, JH,P =J5’,4’ =5.3 Hz, H-5’), 4.17 (td, 1H, J4’,5’ =5.3, J4’,3’
J
=
3.1 Hz, H-4’), 4.66 (dt, 1H, J3’,2’ =6.2, 3.1, J3’,4’ =3.1 Hz, H-3’), 6.57 (dd,
1H, J1’,2’ =8.1, 6.1 Hz, H-1’), 7.27 (d, 1H, J3,4 =3.9 Hz, H-3-thienyl),
7.71 (s, 1H, H-6), 7.955 (d, 1H, J4,3 =3.9 Hz, H-4-thienyl), 8.13 (s, 1H,
H-2), 9.73 ppm (s, 1H, CHO); 13C NMR (125.7 MHz, D2O, ref-
(dioxane)=69.3 ppm): d=40.82 (CH2-2’), 66.72 (d, JC,P =4.5 Hz, CH2-
5’), 74.34 (CH-3’), 85.50 (CH-1’), 88.42 (d, JC,P =8.4 Hz, CH-4’), 103.17
(C-4a), 112.47 (C-5), 125.37 (CH-6), 130.87 (CH-3 thienyl), 143.22
(CH-4 thienyl), 144.24 (C-5 thienyl), 149.64 (C-2 thienyl), 152.88 (C-
7a), 154.60 (CH-2), 159.68 (C-4), 189.17 ppm (CHO); 31P {1H} NMR
(202.3 MHz, D2O, ref(H3PO4)=0 ppm): d=4.13 ppm; IR (KBr): u=
3438, 2928, 1653, 1588, 1546, 1513, 1473, 1310, 1231, 1173, 1095,
976, 932 cmÀ1; UV/Vis (H2O, 100 mm): lmax1 (e)=347 (9539), lmax2
(e)=270 nm (10739 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 439.0
(100) [MÀH]À, 440.0 (22) [M]À, 461.0 (5) [MÀH+Na]À; HRMS (ESIÀ):
m/z calcd for C16H16O7N4PS: 439.04828 [MÀH]À; found 439.04793.
J
(d, 1H, J3,4 =3.9 Hz, H-3-thienyl), 7.93 (d, 1H, J4,3 =3.9 Hz, H-4-thien-
yl), 8.44 (s, 1H, H-6), 9.88 ppm (s, 1H, CHO); 13C NMR (150.9 MHz,
CD3OD): d=42.52 (CH2-2’), 62.13 (CH2-5’), 71.42 (CH-3’), 87.97 (CH-
1’), 89.03 (CH-4’), 102.63 (C-5), 129.87 (CH-3 thienyl), 139.23 (CH-4
thienyl), 143.49 (CH-6), 144.98 (C-5 thienyl), 145.90 (C-2 thienyl),
157.22 (C-2), 164.82 (C-4), 185.03 ppm (CHO); IR (KBr): u=3390,
3105, 2921, 2852, 1625, 1453, 1410, 1355, 1224, 1100, 1049, 938,
816 cmÀ1
;
UV/Vis
(DMSO,
100 mm):
lmax1
(e)=341 nm
(3534 molÀ1 dm3 cmÀ1); MS (ESI+): m/z (%): 360.0 (100) [M+Na]+,
337.8 (13) [M]+, 338.9 (5) [M+H]+; HRMS (ESI+): m/z calcd for
C14H16O5N3S: 338.0805 [M+H]+; found 338.0805; m.p.=222–2268C
dAFT (5a): This compound was prepared according to general pro-
cedure A using 7-iodo-7-deaza-2’-deoxyadenosine dAI (2a; 0.050 g,
0.133 mmol), 5-formylthiophene-2-boronic acid 3 (6 equiv, 0.124 g,
0.795 mmol), Cs2CO3 (5 equiv, 0.216 g, 0.663 mmol), Pd(OAc)2
(3.0 mg, 10 mol%.), and TPPTS (5 equiv, 0.038 g, 0.669 mmol). The
product dAFT (5a) was obtained as a bright-yellow solid (0.041 g,
85%). Note: When using a mixture acetonitrile/water (1:2) the reac-
tion yield was lower (41%). 1H NMR (600.1 MHz, CD3OD): d=2.37
(ddd, 1H, Jgem =13.5, J2’b,1’ =6.0, J2’b,3’ =2.9 Hz, H-2’b), 2.67 (ddd, 1H,
General procedure B for the preparation of dCNBF (8a) or
dANBF (10a) and dCNPh (9a) or dANPh (11a)
Formylthiophene-2’-deoxycytidine dCFT (4a) or formylthiophene-2’-
deoxyadenosine dAFT (5a) was weight out to flask and dissolved in
hot MeOH. Then H2O, conc. H2SO4, and MeOH were added through
a septum into a vial containing 4-(1-methylhydrazino)-7-nitroben-
zofurazane (6) or 2,4-dinitrophenylhydrazine (7). The dissolved hy-
drazine 6 or 7 was subsequently added to a flask containing previ-
ously dissolved formylthiophene-2’-deoxycytidine dCFT (4a) or for-
mylthiophene-2’-deoxyadenosine dAFT (5a). The product started to
precipitate immediately and was left stirring for a further 5 min.
J
J
gem =13.5, J2’a,1’ =8.0, J2’a,3’ =6.0 Hz, H-2’a), 3.74 (dd, 1H, Jgem =12.2,
5’b,4’ =3.7 Hz, H-5’b), 3.82 (dd, 1H, Jgem =12.2, J5’a,4’ =3.2 Hz, H-5’a),
4.02 (ddd, 1H, J4’,5’ =3.7, 3.2, J4’,3’ =2.6 Hz, H-4’), 4.54 (dddd, 1H,
3’,2’ =6.0, 2.9, J3’,4’ =2.6, J3’,1’ =0.4 Hz, H-3’), 6.56 (dd, 1H, J1’2’ =8.0,
J
6.0 Hz, H-1’), 7.25 (bd, 1H, J3,4 =3.7 Hz, H-3-thienyl), 7.71 (bs, 1H, H-
6), 7.89 (bs, 1H, H-4-thienyl), 8.14 (s, 1H, H-2), 9.82 ppm (bs, 1H,
CHO); 13C NMR (150.9 MHz, CD3OD): d=41.59 (CH2-2’), 63.47 (CH2-
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Nucleotides
General procedure C for the preparation of dCNBFMP (8b),
dCNBFTP (8c), dANBFMP (10b), dANBFTP (10c), dCNPhMP (9b),
dCNPhTP (9c), dANPhMP (11b), and dANPhTP (11c)
The precipitated product was then filtrated, washed with 5%
K2CO3 and H2O, and dried under vacuum.
dCNBF (8a): This compound was prepared according to general pro-
cedure B using formylthiophene-2’-deoxycytidine dCFT (4a, 0.020 g,
0.059 mmol), hot MeOH (3 mL), 4-(1-methylhydrazino)-7-nitroben-
zofurazane (6; 0.025 g, 2 equiv, 0.120 mmol), H2O (0.17 mL), conc.
H2SO4 (0.12 mL), and MeOH (0.63 mL). The product dCNBF (8a) was
obtained as dark-violet solid (0.023 g, 71%). 1H NMR (499.8 MHz,
[D6]DMSO, T=508C): d=2.09 (ddd, 1H, Jgem =13.4, J2’b,1’ =6.9,
An aqueous solution of aniline (20 mm) and ammonium acetate
(0.1m) adjusted to pH 4.5 using acetic acid, was added to an
argon-purged vial containing cytidine monophosphate dCFTMP
(4b) or adenosine monophosphate dAFTMP (5b) and 4-(1-methyl-
hydrazino)-7-nitrobenzofurazane (6; 1–1.2 equiv) or 2,4-dinitrophe-
nylhydrazine (7; 2 equiv). dNXMPs were stirred at RT for 24 h,
dNXTPs at 308C for 20 h. The product was isolated from the crude
reaction mixture by semi-preparative HPLC on a reversed-phase
column packed with C18 and eluted with a linear gradient of 0.1m
TEAB in H2O to 0.1m TEAB in H2O/MeOH (1:1).The product was co-
distillated several times with H2O, converted into the sodium salt
form (Dowex 50WX8 in Na+ cycle), and subsequently freeze dried
from H2O.
J
2’b,3’ =6.0 Hz, H-2’b), 2.23 (ddd, 1H, Jgem =13.4, J2’a,1’ =6.1, J2’a,3’
=
3.7 Hz, H-2’a), 3.57, 3.63 (2 ꢂ dt, 2ꢂ1H, Jgem =12.0, J5’,4’ =J5’,OH
=
3.5 Hz, H-5’), 3.84 (q, 1H, J4’,5’ =J4’,3’ =3.5 Hz, H-4’), 4.09 (s, 3H,
CH3N), 4.25 (m, 1H, J3’,2’ =6.0, 3.7, J3’,OH =3.6, J3’,4’ =3.5 Hz, H-3’), 4.92
(bt, 1H, JOH,5’ =3.5 Hz, OH-5’), 5.12 (bd, 1H, JOH,3’ =3.6 Hz, OH-5’),
6.18 (dd, 1H, J1’2’ =6.9, 6.0 Hz, H-1’), 7.19 (d, 1H, J3,4 =3.8 Hz, H-3
thienyl), 7.27 (bd, 1H, J5,6 =9.0 Hz, H-5 oxadiazole), 7.57 (d, 1H,
J
4,3 =3.8 Hz, H-4 thienyl), 8.14 (s, 1H, H-6), 8.63 (d, 1H, J6,5 =9.0 Hz,
dCNBFMP (8b): This compound was prepared according to general
procedure C using aniline (4 mL, 20 mm, 7 equiv, 0.797 mmol), am-
monium acetate (0.1m, 0.614 g, 7.966 mmol), cytidine monophos-
phate dCFTMP (4b; 0.050 g, 0.114 mmol), and 4-(1-methylhydrazi-
no)-7-nitrobenzofurazane (6; 0.029 g, 1.2 equiv, 0.139 mmol). The
H-6 oxadiazole), 8.65 ppm (s, 1H, CHN); 13C NMR (125.7 MHz,
[D6]DMSO, T=508C): d=36.76 (CH3N), 40.91 (CH2-2’), 61.10 (CH2-
5’), 70.20 (CH-3’), 85.60 (CH-1’), 87.58 (CH-4’), 100.44 (C-5), 106.14
(CH-5 oxadiazole), 124.22 (C-7 oxadiazole), 127.99 (CH-3 thienyl),
132.70 (CH-4 thienyl), 135.44 (CH-6 oxadiazole), 138.70, 138.71 (C-
2,5 thienyl), 140.44 (CHN), 141.27 (CH-6), 142.59 (C-4 oxadiazole),
144.35 (C-3a oxadiazole), 144.74 (C-7a oxadiazole), 153.95 (C-2),
162.88 ppm (C-4); IR (KBr): u=3468, 3436, 2930, 2726, 1697, 1650,
1541, 1414, 1374, 1339, 1295, 1061, 1009, 983 cmÀ1; UV/Vis (DMSO,
product dCNBFMP (8b) was isolated as violet powder (0.039 g,
1
53%). H NMR (499.8 MHz, D2O, T=508C): d=2.25 (ddd, 1H, Jgem
=
14.1, J2’b,1’ =7.1, J2’b,3’ =6.6 Hz, H-2’b), 2.35 (ddd, 1H, Jgem =14.1,
J
J
J
2’a,1’ =6.4, J2’a,3’ =3.9 Hz, H-2’a), 3.81 (s, 3H, CH3N), 3.82 (dt, 1H,
gem =11.2, JH,P =J5’b,4’ =6.0 Hz, H-5’b), 3.88 (ddd, 1H, Jgem =11.2,
100 mm):
lmax1
(e)=538
(26786),
lmax2
(e)=355 nm
(14962 molÀ1 dm3 cmÀ1); MS (ESI+): m/z (%): 551.0 (100) [M+Na]+,
1079.4 (15) [2M+Na]+; HRMS (ESI+): m/z calcd for C21H20O7N8NaS:
551.10679 [M+Na]+; found 551.10669; m.p.=240–2428C
H,P =6.3, J5’a,4’ =5.1 Hz, H-5’a), 4.07 (ddd, 1H, J4’,5’ =6.0, 5.1, J4’,3’
=
3.9 Hz, H-4’), 4.46 (dt, 1H, J3’,2’ =6.6, 3.9, J3’,4’ =3.9 Hz, H-3’), 6.02 (dd,
1H, J1’2’ =7.1, 6.4 Hz, H-1’), 7.00 (bd, 1H, J5,6 =9.2 Hz, H-5 oxadia-
zole), 7.13 (d, 1H, J3,4 =3.7 Hz, H-3 thienyl), 7.38 (d, 1H, J4,3 =3.7 Hz,
H-4 thienyl), 7.71 (s, 1H, H-6), 8.20 (s, 1H, CHN), 8.49 ppm (d, 1H,
dCNPh (9a): This compound was prepared according to general pro-
cedure B using formylthiophene-2’-deoxycytidine dCFT (4a; 0.035 g,
0.104 mmol), hot MeOH (10 mL), 2,4-dinitrophenylhydrazine (7;
0.125 g, contains 50% H2O as stabilizer, 3 equiv, 0.631 mmol), H2O
(0.8 mL), conc. H2SO4 (0.6 mL), and MeOH (3.1 mL). The product
dCNPh (9a) was obtained as dark-brown solid (0.038 g, 70%).
J
6,5 =9.2 Hz, H-6 oxadiazole); 13C NMR (125.7 MHz, D2O, T=508C):
d=38.26 (CH3N), 41.41 (CH2-2’), 66.67 (d, JC,P =4.1 Hz, CH2-5’), 73.98
(CH-3’), 88.59 (d, JC,P =7.4 Hz, CH-4’), 89.01 (CH-1’), 105.40 (C-5),
108.64 (CH-5 oxadiazole), 126.13 (C-7 oxadiazole), 132.43 (CH-3
thienyl), 135.56 (CH-4 thienyl), 138.84 (CH-6 oxadiazole), 139.40 (C-
2 thienyl), 142.68 (CHN), 142.74 (C-5 thienyl), 143.78 (CH-6), 145.61
(C-4 oxadiazole), 146.72 (C-3a oxadiazole), 147.13 (C-7a oxadiazole),
158.81 (C-2), 166.48 ppm (C-4); 31P{1H} NMR (202.3 MHz, D2O, T=
508C): d=4.21 ppm. IR (KBr): u=3436, 1647, 1614, 1586, 1541,
1498, 1442, 1423, 1367, 1291, 1212, 1077, 1000, 976, 923 cmÀ1; UV/
Vis (H2O, 100 mm): lmax1 (e)=542 (20995), lmax2 (e)=341 nm
(10491 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 607.1 (100) [MÀH]À,
608.1 (25) [M]À; HRMS (ESIÀ): m/z calcd for C21H20O10N8PS:
607.07662 [MÀH]À; found 607.07623.
1H NMR (499.8 MHz, [D6]DMSO, T=508C): d=2.09 (ddd, 1H, Jgem
13.4, J2’b,1’ =6.8, J2’b,3’ =6.1 Hz, H-2’b), 2.21 (ddd, 1H, Jgem =13.4,
=
J
J
2’a,1’ =6.2, J2’a,3’ =3.8 Hz, H-2’a), 3.56 (2 ꢂ ddd, 2ꢂ1H, Jgem =11.8,
5’,OH =5.0, J5’,4’ =3.6 Hz, H-5’), 3.83 (q, 1H, J4’,5’ =J4’,3’ =3.6 Hz, H-4’),
4.25 (m, 1H, J3’,2’ =6.1, 3.8, J3’,OH =4.4, J3’,2’ =3.6 Hz, H-3’), 4.94 (t, 1H,
OH,5’ =5.0 Hz, OH-5’), 5.12 (d, 1H, JOH,3’ =4.4 Hz, OH-3’), 6.18 (dd,
1H, J1’,2’ =6.8, 6.2 Hz, H-1’), 6.87 (bs, 2H, NH2), 7.16 (d, 1H, J3,4
J
=
3.7 Hz, H-3-thienyl), 7.45 (d, 1H, J4,3 =3.7 Hz, H-4-thienyl), 7.90 (d,
1H, J6,5 =9.6 Hz, H-6-C6H3(NO2)2), 8.13 (s, 1H, H-6), 8.36 (bd, 1H,
J
5,6 =9.6 Hz, H-5-C6H3(NO2)2), 8.838 (s, 1H, CHN), 8.845 (d, 1H, J3,5 =
dCNBFTP (8c): This compound was prepared according to general
procedure C using aniline (2.5 mL, 20 mm, 7 equiv, 0.354 mmol),
ammonium acetate (0.1m, 0.27 g, 3.503 mmol), cytidine triphos-
phate dCFTTP (4c; 0.044 g, 0.068 mmol), and 4-(1-methylhydrazi-
no)-7-nitrobenzofurazane (6; 0.014 g, 1 equiv, 0.067 mmol). The
product dCNBFTP (8c) was isolated as violet powder (0.034 g, 60%).
1H NMR (499.8 MHz, D2O, ref(dioxane)=3.75 ppm, pD=7.1, phos-
phate buffer, T=508C): d=2.29 (ddd, 1H, Jgem =14.4, J2’b,1’ =7.2,
2.6 Hz, H-3-C6H3(NO2)2), 11.64 ppm (bs, 1H, NH); 13C NMR
(125.7 MHz, [D6]DMSO, T=508C): d=40.87 (CH2-2’), 61.05 (CH2-5’),
70.14 (CH-3’), 85.52 (CH-1’), 87.52 (CH-4’), 100.39 (C-5), 116.47 (CH-
6-C6H3(NO2)2), 123.02 (CH-3-C6H3(NO2)2), 127.83 (CH-3 thienyl),
129.68 (C-2-C6H3(NO2)2), 129.79 (CH-5-C6H3(NO2)2), 132.24 (CH-4
thienyl), 136.98 (C-4-C6H3(NO2)2), 138.01 (C-5 thienyl), 138.39 (C-2
thienyl), 141.26 (CH-6), 144.27 (CHN), 144.27 (C-1-C6H3(NO2)2),
153.92 (C-2), 162.86 ppm (C-4); IR (KBr): u=3454, 3283, 3091, 2923,
2085, 1646, 1615, 1599, 1507, 1422, 1334, 1112, 983 cmÀ1; UV/Vis
(DMSO, 100 mm): lmax1 (e)=508 (3308), lmax2 (e)=331 nm
(1559 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 516.3 (100) [MÀH]À,
517.3 (30) [M]À, 518.3 (10) [M+H]À; HRMS (ESIÀ): m/z calcd for
C20H18O8N7S: 516.09430 [MÀH]À; found 516.09396; m.p.=3008C.
J
2’b,3’ =6.4 Hz, H-2’b), 2.41 (ddd, 1H, Jgem =14.4, J2’a,1’ =6.3, J2’a,3’ =
3.9 Hz, H-2’a), 3.85 (s, 3H, CH3N), 4.12 (m, 2H, H-5’), 4.17 (m, 1H, H-
4’), 4.56 (m, 1H, H-3’), 6.10 (dd, 1H, J1’2’ =7.2, 6.3 Hz, H-1’), 7.01 (d,
1H, J5,6 =9.1 Hz, H-5-oxadiazole), 7.17 (d, 1H, J3,4 =3.8 Hz, H-3-
thienyl), 7.42 (d, 1H, J4,3 =3.8 Hz, H-4-thienyl), 7.80 (s, 1H, H-6), 8.27
(s, 1H, CHN), 8.49 ppm (d, 1H, J6,5 =9.1, H-6-oxadiazole); 13C NMR
(125.7 MHz, D2O, ref(dioxane)=69.3 ppm, pD=7.1, phosphate
buffer, T=508C): d=38.36 (CH3N), 41.78 (CH2-2’), 68.11 (d, HzC,P
=
6.0 Hz, CH2-5’), 73.45 (CH-3’), 88.27 (d, JC,P =8.2 Hz, CH-4’), 89.21
ChemPlusChem 2012, 00, 1 – 12
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chempluschem.org
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M. Hocek, M. Fojta et al.
(CH-1’), 105.50 (C-5), 108.68 (CH-5 oxadiazole), 126.06 (C-7 oxadia-
zole), 132.31 (CH-3 thienyl), 135.69 (CH-4 thienyl), 138.92 (CH-6 oxa-
diazole), 139.50 (C-2 thienyl), 142.58 (C-5 thienyl), 142.98 (CHN),
143.75 (CH-6), 145.74 (C-4 oxadiazole), 146.79 (C-7a oxadiazole),
147.19 (C-3a oxadiazole), 158.82 (C-2), 166.45 ppm (C-4);
31P{1H} NMR (202.3 MHz, D2O, ref(H3PO4)=0 ppm, pD=7.1, phos-
phate buffer, T=508C): d=À21.32 (bdd, J=18.7 Hz, 17.8, Pb),
À10.50 ppm (bd, J=17.8 Hz, Pa); À7.34 (bd, J=18.7 Hz, Pg); IR
(KBr): u=3436, 2924, 2855, 1647, 1616, 1541, 1501, 1443, 1423,
1367, 1317, 1290, 1258, 1130, 1080, 1000, 915 cmÀ1; UV/Vis (H2O,
(CH-6-C6H3(NO2)2),
126.41
(CH-3-C6H3(NO2)2),
131.83
(C-2-
C6H3(NO2)2), 132.19 (CH-3-thienyl), 132.76 (CH-5-C6H3(NO2)2), 135.38
(CH-4 thienyl), 139.19 (C-2 thienyl), 140.12 (C-4-C6H3(NO2)2), 141.82
(C-5 thienyl), 143.80 (CH-6), 146.43 (CH=N), 147.07 (C-1-
C6H3(NO2)2), 159.00 (C-2), 166.59 ppm (C-4); 31P{1H} NMR
(202.3 MHz, D2O, T=508C, ref(H3PO4)=2.35 ppm, pD=7.1, phos-
phate buffer): d=À21.45 (t, J=19.3 Hz, Pb), À10.88 (d, J=19.3 Hz,
Pa), À6.63 ppm (d, J=19.3 Hz, Pg); IR (KBr): u=3479, 3401, 3118,
2919, 1647, 1618, 1600, 1506, 1468, 1422, 1341, 1239, 1128, 1092,
1052, 1008, 912 cmÀ1; UV/Vis (H2O, 100 mm): lmax1 (e)=410 (24353),
lmax2 (e)=312 nm (8539 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 756.0
(3) [MÀH]À, 676.0 (45) [MÀH2PO3]À, 596.2 (100) [MÀH3P2O6]À;
HRMS (ESIÀ): m/z calcd for C20H21O17N7P3S: 755.99330 [MÀH]À;
found 755.99381.
100 mm):
lmax1
(e)=545
(27186),
lmax2
(e)=341 nm
(13311 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 687.0 (100) [MÀHPO3]À,
767.0 (15) [MÀH]À, 814.3 (10) [M+2Na]À, HRMS (ESIÀ): m/z calcd
for C21H22O16N8P3S: 767.0093 [MÀH]À; found 767.0078.
dCNPhMP (9b): This compound was prepared according to general
procedure C using aniline (4 mL, 20 mm, 7 equiv, 0.797 mmol), am-
monium acetate (0.1m, 0.614 g, 7.966 mmol), cytidine monophos-
phate dCFTMP (4b; 0.050 g, 0.114 mmol), and 2,4-dinitrophenylhy-
drazine (7; 0.090 g, contains 50% H2O as stabilizer, 2 equiv,
0.454 mmol). The product dCNPhMP (9b) was isolated as light-
General procedure D for the preparation of ssDNA contain-
ing nitrobenzofurazane or dinitrophenyl moiety for the UV/
Vis measurement
1
orange powder (0.049 g, 70%). H NMR (499.8 MHz, D2O, T=508C):
d=2.30 (dt, 1H, Jgem =14.1, J2’b,1’ =J2’b,3’ =7.0 Hz, H-2’b), 2.44 (ddd,
Reaction mixtures +, B1–B4 and D1–D4 (250 mL each, Table 3)
contained Deep VentR(exo-) DNA Polymerase (New England Bio-
Labs, 2 UmLÀ1, 30 mL), natural dNTPs (10 mm, 8 mL), functionalized
dCFTTP (4c; 4 mm, 20 mL), or dCNBFTP (8c; 4 mm, 40 mL) or dCNPhTP
(9c; 4 mm, 40 mL), primer (10 mm, 60 mL, Prim248 short: 3’-
GGGTACGGCGGGTAC-5’), and 31-mer biotinylated template (10 mm,
90 mL, Prb4basII-bio (5’-biotine): 5’-CTAGCATGAGCTCAGTCC-
CATGCCGCCCATG-3‘) in Deep VentR(exo-) reaction buffer (25 mL)
supplied by the manufacturer. Reaction mixtures were incubated
for 30 min at 608C in a thermal cycler, then heated for 5 min at
958C. For the separation on magnetic beads (Novagen) it was used
240 mL of beads for each 250 mL reaction mixture and was carried
out according to standard techniques (washing with 1xPBS, 0.3m
NaCl in 10 mm Tris, H2O). Samples + and B1 or D1 were immedi-
ately separated on magnetic beads and re-purified by NucAway
Spin Columns and stored in freezer while working with other sam-
ples. Samples B2, D2, B3, D3, B4, and D4 were still held on mag-
netic beads and treated with hydrazine 6 (MHNBF, 320 mL of
120 mm suspension in aniline/ammonium acetate/water, pH 4.5) or
hydrazine 7 (DNPH, 38 mL of 1m suspension in aniline/ammonium
acetate/water, pH 4.5). Samples B2, D2 and B3, D3 were incubated
for 24 h at 308C, samples B4, D4 for 48 h at 308C. After the reac-
tion with hydrazine 6 or 7, samples B2, D2, B3, D3 and B4, D4
were re-purified by NucAway Spin Columns and then all prepared
samples +, B1-B4 and D1-D4 were precipitated (3m acetate
buffer, iPrOH) and again re-purified by NucAway Spin Columns.
UV/Vis spectra of all samples were then measured on a Varian
CARY 100 Bio spectrophotometer at RT (Figure S9, S10).
1H, Jgem =14.1, J2’a,1’ =6.3, J2’a,3’ =3.6 Hz, H-2’a), 3.88 (dt, 1H, Jgem
11.3, JH,P =J5’b,4’ =5.6 Hz, H-5’b), 3.94 (ddd, 1H, Jgem =11.3, JH,P =6.6,
5’a,4’ =5.6 Hz, H-5’a), 4.14 (td, 1H, J4’,5’ =5.6, J4’,3’ =3.6 Hz, H-4’), 4.50
=
J
(m, 1H, overlapped with HDO signal, H-3’), 6.16 (dd, 1H, J1’2’ =7.0,
6.3 Hz, H-1’), 7.19 (d, 1H, J3,4 =3.7 Hz, H-3-thienyl), 7.37 (dd, 1H,
J
4,3 =3.7 Hz, H-4-thienyl), 7.80 (s, 1H, H-6), 7.84 (d, 1H, J6,5 =9.6 Hz,
H-6-C6H3(NO2)2), 8.25 (dd, 1H, J5,6 =9.6, J5,3 =2.6 Hz, H-5-C6H3(NO2)2),
8.35 (s, 1H, CH=N), 8.93 ppm (d, 1H, J3,5 =2.6 Hz, H-3-C6H3(NO2)2);
13C NMR (125.7 MHz, D2O, T=508C): d=41.66 (CH2-2’), 66.72 (d,
J
C,P =4.6 Hz, CH2-5’), 74.03 (CH-3’), 88.67 (d, JC,P =7.8 Hz, CH-4’),
89.15 (CH-1’), 105.39 (C-5), 119.19 (CH-6-C6H3(NO2)2), 126.38 (CH-3-
C6H3(NO2)2), 130.83 (C-2-C6H3(NO2)2), 132.17 (CH-3 thienyl), 132.72
(CH-5-C6H3(NO2)2), 135.31 (CH-4 thienyl), 139.16 (C-2 thienyl),
140.12 (C-4-C6H3(NO2)2), 141.86 (C-5 thienyl), 143.80 (CH-6), 146.22
(CH=N), 147.00 (C-1-C6H3(NO2)2), 158.96 (C-2), 166.56 ppm (C-4);
31P{1H} NMR (202.3 MHz, D2O, T=508C): d=4.21 ppm; IR (KBr): u=
3436, 2925, 2855, 1646, 1617, 1599, 1505, 1468, 1422, 1338, 1276,
1235, 1086, 976 cmÀ1; UV/Vis (H2O, 100 mm): lmax1 (e)=412 (18428),
lmax2 (e)=314 nm (6447 molÀ1 dm3 cmÀ1); MS (ESIÀ): m/z (%): 596.0
(100) [MÀH]À, 597.1 (30) [M]À, 1193.1 (5) [2MÀH]À; HRMS (ESIÀ): m/
z calcd for C20H19O11N7PS: 596.06063 [MÀH]À; found 596.06010.
dCNPhTP (9c): This compound was prepared according to general
procedure C using aniline (3.5 mL, 20 mm, 7 equiv, 0.797 mmol),
ammonium acetate (0.1m, 0.614 g, 7.966 mmol), cytidine triphos-
phate dCFTTP (4c; 0.021 g, 0.033 mmol), and 2,4-dinitrophenylhy-
drazine (7; 0.027 g, contains 50% H2O as stabilizer, 2 equiv,
0.136 mmol). The product dCNPhMP (9c) was obtained as brown
powder (0.018 g, 66%). 1H NMR (499.8 MHz, D2O, T=508C, ref-
(dioxane)=3.75 ppm, pD=7.1, phosphate buffer): d=2.31 (ddd,
1H, Jgem =14.1, J2’b,1’ =7.1, J2’b,3’ =6.7 Hz, H-2’b), 2.44 (ddd, 1H,
J
gem =14.1, J2’a,1’ =6.1, J2’a,3’ =3.8 Hz, H-2’a), 4.15 (m, 2H, H-5’), 4.21
(q, 1H, J4’,5’ =J4’,3’ =3.8 Hz, H-4’), 4.58 (dt, 1H, J3’,3’ =6.7, 3.8, J3’,4’
3.8 Hz, H-3’), 6.16 (dd, 1H, J1’2’ =7.1, 6.1 Hz, H-1’), 7.19 (d, 1H, J3,4
3.8 Hz, H-3 thienyl), 7.39 (dd, 1H, J4,3 =3.8 Hz, H-4 thienyl), 7.84 (s,
1H, H-6), 7.88 (d, 1H, J6,5 =9.6 Hz, H-6-C6H3(NO2)2), 8.26 (dd, 1H,
=
=
Aldehyde-hydrazine coupling on DNA for electrochemical
experiments (see Figure 6)
FT-labeled PEX products were purified using Qiagen Nucleotide Re-
moval Kit. The samples were eluted from the spin columns with
deionized H2O. Then, 30 mL of the aldehyde-functionalized ON was
mixed with 40 mL of 4 mm MHNBF and 5 mL of ammonium acetate/
aniline (2:1; pH adjusted at 4.5). After overnight incubation at 308C
the modified ONs were purified by the Nucleotide Removal Kit
again and analyzed by ex situ voltammetry.
J
5,6 =9.6, J5,3 =2.6 Hz, H-5-C6H3(NO2)2), 8.38 (s, 1H, CH=N),
8.96 ppm (d, 1H, J3,5 =2.6 Hz, H-3-C6H3(NO2)2); 13C NMR (125.7 MHz,
D2O, T=508C, ref(dioxane)=69.30 ppm, pD=7.1, phosphate
buffer): d=41.88 (CH2-2’), 68.03 (d, JC,P =5.8 Hz, CH2-5’), 73.37 (CH-
3’), 88.35 (d, JC,P =8.1 Hz, CH-4’), 89.25 (CH-1’), 105.49 (C-5), 119.25
&10
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2012, 00, 1 – 12
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These are not the final page numbers!

Nucleotides
Schmidt, G. A. Burley, T. Carell, Chem. Eur. J. 2007, 13, 9486–9494;
c) P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell, Angew. Chem. 2008,
120, 3491–3493; Angew. Chem. Int. Ed. 2008, 47, 3442–3444; d) F. Seela,
V. R. Sirivolu, P. Chittepu, Bioconjugate Chem. 2008, 19, 211–224; e) F.
Seela, V. R. Sirivolu, Org. Biomol. Chem. 2008, 6, 1674–1687; f) G. A.
Burley, J. Gierlich, M. R. Mofid, H. Nir, S. Tal, Y. Eichen, T. Carell, J. Am.
Chem. Soc. 2006, 128, 1398–1399.
Electrochemical analysis
Nucleosides, dNMPs and other building blocks were analyzed by
conventional in situ cyclic voltammetry (CV). Modified ONs, purified
from reaction mixtures using the Nucleotide Removal Kit (Qiagen),
were analyzed by ex situ (adsorptive transfer stripping) CV. The
PEX products were accumulated for 60 s from 5 mL aliquots con-
taining 0.2m NaCl at the surface of the working electrode (hanging
mercury drop electrode (HMDE) or basal-plane pyrolytic graphite
electrode (PGE)). The electrode was then rinsed with deionized
H2O and placed in the electrochemical cell. CV settings: scan rate
was 1.0 VsÀ1, initial potential was 0.0 V, for switching potentials see
Figure legends. Background electrolyte: 0.5m ammonium formate,
0.05m sodium phosphate, pH 6.6 (for analysis of modified DNA) or
0.2m sodium acetate pH 5.0 (for measurements of nucleosides and
separate building blocks at both HMDE and PGE) All measure-
ments were performed at RT using an Autolab analyzer (Eco
Chemie, The Netherlands) in connection with VA-stand 663 (Met-
rohm, Herisau, Switzerland). The three-electrode system was used
with Ag/AgCl/3m KCl electrode as a reference and platinum wire
as an auxiliary electrode. All measurements were done after deaer-
ation of the solution by argon purging.
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Acknowledgements
This study was supported by the Academy of Sciences of the
Czech Republic, Grant Agency of the Academy of Sciences of the
Czech Republic (IAA400040901) to M.F., the Czech Science Foun-
dation (P206/12/G151) to M.H. and V.R., and by Gilead Sciences,
´
Inc. (Foster City, CA, USA). We thank to Dr. Jan Sykora and Dr.
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Ivana Cꢁsarovꢀ for X-ray diffraction measurements.
Keywords: cross-coupling
hydrazones · nucleotides · polymerases
·
DNA
·
electrochemistry
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Received: March 9, 2012
Published online on && &&, 0000
ChemPlusChem 2012, 00, 1 – 12
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chempluschem.org
&
11
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
ˇ
V. Raindlovꢀ, R. Pohl, B. Klepetꢀrovꢀ,
Redox labeling of DNA through nitroar-
ylhydrazone modification has been ach-
ieved either by polymerase incorpora-
tion of hydrazone-modified dNTPs or by
incorporation of aldehydes and subse-
quent hydrazone formation (see figure).
Electrochemical studies revealed the po-
tential utility of different redox-active
groups for DNA labeling for bioanalysis.
ˇ
L. Havran, E. Simkovꢀ, P. Horꢀkovꢀ,
ˇ
H. Pivonkovꢀ, M. Fojta,* M. Hocek*
&& – &&
Synthesis of Hydrazone-Modified
Nucleotides and Their Polymerase
Incorporation onto DNA for Redox
Labeling
&12
&
www.chempluschem.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2012, 00, 1 – 12
ÝÝ
These are not the final page numbers!