Methoxyphenol and dihydrobenzofuran as oxidizable labels for electrochemical detection of DNA

Article abstract of DOI:10.1002/cplu.201402194

4-Hydroxy-3-methoxyphenyl (MOP) and 2,3-dihydrobenzofuran- 5-yl (DHB) groups were tested as potential new oxidizable labels for electrochemical detection of DNA. The corresponding 5-aryl-cytosine and 7-aryl-7-deazaadenine 2′-deoxyribonucleoside triphospha

Full text of DOI:10.1002/cplu.201402194

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DOI: 10.1002/cplu.201402194
Methoxyphenol and Dihydrobenzofuran as Oxidizable
Labels for Electrochemical Detection of DNA
[a]
[a]
[a]
[b]
[b, c]
ˇ
Anna Simonova, Jana Balintovꢀ, Radek Pohl, Ludek Havran, Miroslav Fojta,*
Michal Hocek*[a, d]
and
4-Hydroxy-3-methoxyphenyl (MOP) and 2,3-dihydrobenzofur-
an-5-yl (DHB) groups were tested as potential new oxidizable
labels for electrochemical detection of DNA. The correspond-
ing 5-aryl-cytosine and 7-aryl-7-deazaadenine 2’-deoxyribonu-
cleoside triphosphates (dNTPs) were prepared by the Suzuki
coupling of halogenated dNTPs or by triphosphorylation of
modified nucleosides, and were incorporated enzymatically
into DNA by polymerase. In parallel, the electrochemical be-
havior of the labeled nucleosides, nucleotides, and DNA was
studied to show that most of them give analytically useful sig-
nals of oxidation on the carbon electrode. Particularly, MOP ap-
pears to be a useful oxidizable DNA label since it offers inde-
pendent detection of the modified DNA.
Introduction
Electrochemical DNA biosensors[1] are increasingly applied in
the life sciences and diagnostics and have become a viable al-
ternative to the current fluorescence and microarray tech-
niques.[2] Apart from inherent electrochemical detection of un-
labeled nucleic acids, additional DNA labels have been devel-
oped extensively for diverse bioanalytical applications.[3] We
have previously reported the labeling of nucleos(t)ides and
DNA by several new redox labels, including ferrocene,[4] amino-
and nitrophenyl,[5] Os(bpy)3 (bpy=2,2’-bipyridine),[6] tetrathia-
fulvalene,[7] anthraquinone,[8] alkylsulfanylphenyl,[9] hydra-
zones,[10] and benzofurazane,[11] and several more detailed elec-
trochemical studies[12] of these labels. Some of these labels
were combined in a new concept of multipotential redox
coding of DNA nucleobases (each base was labeled by a differ-
ent label with a different redox potential) but the first genera-
tion[6] suffered from weak and overlapping signals, limited sta-
bility, and difficult incorporation of some labels into DNA. Re-
cently, we reported[11] the first orthogonal and ratiometric set
of two reducible labels (nitrophenyl and benzofurazane) useful
for electrochemical minisequencing of short DNA stretch-
es.[5,6,11] However, for any practical bioanalytical applications,
we need a fully orthogonal set of four labels that can be readi-
ly incorporated into DNA by polymerase and are then inde-
pendently readable in the presence of all the other labels. This
could then be used for simultaneous detection of multiple nu-
cleobase mutations in short sequences (1–3 codons) in one ex-
periment (as opposed to current sequencing techniques,[2]
which always use detection of one nucleotide at a time).
[a] A. Simonova, J. Balintovꢀ, Dr. R. Pohl, Prof. Dr. M. Hocek
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Gilead Sciences & IOCB Research Center
Flemingovo nam. 2, 16610 Prague 6 (Czech Republic)
Fax: (+420)220-183-559
Herein, we report the development of two new potential ox-
idizable labels, 4-hydroxy-3-methoxyphenyl (MOP) and 2,3-di-
hydrobenzofuran-5-yl (DHB) groups, and their electrochemical
studies. 2-Methoxyphenol is known to undergo 2eÀ electrooxi-
dation to o-benzoquinone.[12] The electrochemistry of 2,3-dihy-
drobenzofurans has been less investigated[13] but they are
known to be easily oxidized to aromatic benzofurans.[14]
E-mail: hocek@uochb.cas.cz
Homepage: http://www.uochb.cas.cz/hocekgroup
[b] L. Havran, Prof. Dr. M. Fojta
Institute of Biophysics
Academy of Sciences of the Czech Republic
Kralovopolska 135, 61265 Brno (Czech Republic)
Fax: (+420)541-211-293
Results and Discussion
E-mail: fojta@ibp.cz
Homepage: http://www.ibp.cz/labs/LBCMO/
Synthesis
[c] Prof. Dr. M. Fojta
The synthesis of model labeled nucleosides (2’-deoxycytidine
and 2’-deoxy-7-deazaadenosine) and 2’-deoxyribonucleoside
triphosphates (dNTPs) was based on aqueous-phase Suzuki–
Miyaura cross-coupling reactions[15] of the corresponding halo-
genated nucleosides or dNTPs with commercially available
MOP- or DHB-substituted boronic acids or pinacolatoboronates
(MOP-B(pin) and DHB-B(OH)2, Scheme 1). At first, the reactions
were performed with nucleosides. Thus the reactions of either
Central European Institute of Technology
Masaryk University
Kamenice 753/5, 625 00 Brno (Czech Republic)
[d] Prof. Dr. M. Hocek
Department of Organic Chemistry
Faculty of Science, Charles University in Prague
Hlavova 8, 12843 Prague 2 (Czech Republic)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201402194.
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isolated by semipreparative HPLC in approximately 40% yields
(Table 1). Only in the case of dCMOPTP was the product not iso-
lated in pure form owing to a similar retention time to TPPTS.
Therefore, this compound was prepared by an alternative ap-
proach using chemical triphosphorylation of the corresponding
modified nucleoside dCMOP with POCl3 in P(O)(OMe)3 followed
by (NHBu3)2H2P2O7, Bu3N in dimethylformamide (DMF), and trie-
thylammonium bicarbonate (TEAB). The desired dCMOPTP was
isolated in pure form in 28% yield.
Polymerase incorporation of dNXTPs
The four new dNTPs (dAMOPTP, dCMOPTP, dADHBTP, and dCDHBTP)
were then tested as substrates for DNA polymerases[16] in
primer extension experiments (PEX) using KOD XL and Pwo
polymerases (for sequences of primers and templates, see
Table 2). At first, single nucleotide incorporation of each of the
dNXTPs into a 15-nucleotide (nt) primer was followed by three
natural dG using tempA or tempC templates (Figure 1). In all
Scheme 1. Reagents and conditions: i) MOP-B(pin) or DHB-B(OH)2, Pd(OAc)2,
TPPTS, Cs2CO3, MeCN/H2O (2:1), 1 h, 758C. ii) 1) PO(OMe)3, POCl3, 08C;
2) (NHBu3)2H2P2O7, Bu3N, DMF, 08C; 3) 2m TEAB (phosphorylation of modi-
fied nucleosides to triphosphates was applied only in the case of dCMOP).
iii) MOP-B(pin) or DHB-B(OH)2, Pd(OAc)2, TPPTS, Cs2CO3, MeCN/H2O (2:1),
40 min, 508C.
Figure 1. Primer extension with a) KOD XL and b) Pwo polymerases using
primrnd, tempA, and tempC: (Pr) primer (5’-32P-end labeled); (A+) dATP, dGTP;
(AÀ) dGTP; (ADHB) dADHBTP, dGTP; (AMOP) dAMOPTP, dGTP; (C+) dCTP, dGTP;
(CÀ) dGTP; (CDHB) dCDHBTP, dGTP; (CMOP) dCMOPTP, dGTP.
dAI or dCI with MOP-B(pin) and DHB-B(OH)2 were performed in
the presence of Pd(OAc)2, 3,3,’3“-tris(sulfonato)-triphenylphos-
phane (TPPTS), and Cs2CO3 in acetonitrile (MeCN)/H2O (2:1) at
758C for 1 hour. The desired aryl-substituted nucleosides
dAMOP, dCMOP, dADHB, and dCDHB were obtained in acceptable
yields of 42–78% after chromatographic separation (Table 1).
The same reactions were then performed with iodinated
dNTPs (dAITP and dCITP) for 40 minutes at 508C (to avoid hy-
drolysis of the triphosphates). The desired labeled dNTPs
(dAMOPTP, dCMOPTP, dADHBTP, and dCDHBTP) were formed and
cases, fully extended product was observed on polyacrylamide
gel electrophoresis (PAGE). All the products were also charac-
terized by MALDI-TOF mass spectrometry (see Table 3). Kinetic
experiments (see Figures S1–S12 in the Supporting Informa-
tion) showed that the modified dNXTPs were incorporated
with only slightly slower rate than dATP or dCTP.
Then, the dNXTPs were incorporated individually into
a longer oligonucleotide (ON) by using temprnd16 designed to
encode for four modifications at each nucleobase. Also here,
the PEX gave full-length products in the presence of KOD XL
polymerase (Figure 2). Also, a combination of a modified A and
modified C (ADHB +CMOP or AMOP +CDHB) was successfully incor-
porated to give full-length ONs bearing two different modifica-
tions at A and C bases. These ONs were characterized by
MALDI-TOF mass spectrometry (Table 3).
Table 1. Synthesis of MOP- and DHB-modified nucleosides and dNTPs
Entry Starting
compound
Reagent
Product Yield [%][a]
1
2
3
4
5
6
7
8
9
dAI
MOP-B(pin)
MOP-B(pin)
DHB-B(OH)2
DHB-B(OH)2
MOP-B(pin)
MOP-B(pin)
DHB-B(OH)2
DHB-B(OH)2
dAMOP
dCMOP
dADHB
dCDHB
dAMOPTP 42
dCMOPTP 35[b]
dADHBTP 42
dCDHBTP 41
dCMOPTP 28
77
42
78
69
dCI
dAI
dCI
dAITP
dCITP
dAITP
dCITP
dCMOP
Then we tried to challenge the polymerases by incorpora-
tion of four modifications in a row by using temp4C or temp4A.
Figure 3 shows that KOD XL gave full-length products in all
cases (also confirmed by MALDI-TOF analysis), whereas Pwo
gave mixtures of full-length and n+1 products in the case of
incorporation of ADHB and AMOP (Figure 3a) and nÀ1 products
with CMOP (Figure 3b).
To study the compatibility of the MOP label with previously
reported aminophenyl[5] and benzofurazane[11] groups, we pre-
pared PEX products containing combinations of CMOP with 7-
1) PO(OMe)3,POCl3;
2) (NHBu3)2H2P2O7, Bu3N, DMF;
3) 2m TEAB
[a] Yield of isolated product. [b] Product was not separable from TPPTS.
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temprnd16. Figure 4 shows the in-
corporation of either dCMOPTP or
dANH2TP separately and in com-
bination. In all cases, full-length
ONs bearing either one or two
Table 2. List of sequences of templates, primer, and products.[a]
Oligonucleotide
primerrnd
Sequence
5’-CATGGGCGGCATGGG-3’
temprnd16
temp4A
5’-CTAGCATGAGCTCAGTCCCATGCCGCCCATG-3’
5’-TTTTCCCATGCCGCCCATG-3’
5’-GGGGCCCATGCCGCCCATG-3’
5’-CCCTCCCATGCCGCCCATG-3’
5’-CCCGCCCATGCCGCCCATG-3’
5’-TCCCATGCCGCCCATG-3’
different labels at
A and/or
C bases were obtained.
temp4C
tempA
tempC
temptempA
temptempC
Similarly, PEX incorporations
of dCMOPTP in combination with
dABFTP
were
performed
5’-GCCCATGCCGCCCATG-3’
(Figure 5). KOD XL polymerase
gave clean full-length products
for each modified DNA, whereas
when using Pwo, some shorter
ON side-products were also ob-
served on PAGE. Therefore, la-
beled ONs prepared by KOD XL
polymerase incorporations were
further used in electrochemical
studies. These products were
also characterized by MALDI-TOF
mass spectrometry (Table 3).
ONrnd16 ADHB
ONrnd16 AMOP
ONrnd16 CDHB
ONrnd16 CMOP
ONrnd16 ADHB CMOP
ONrnd16 AMOP CDHB
ONrnd16 ANH2 CMOP
ONrnd16 ABF CMOP
ON4A ADHB
5’-CATGGGCGGCATGGGADHBCTGADHBGCTCADHBTGCTADHBG-3’
5’-CATGGGCGGCATGGGAMOPCTGAMOPGCTCAMOPTGCTAMOPG-3’
5’-CATGGGCGGCATGGGACDHBTGAGCDHBCDHBATGCDHBTAG-3’
5’-CATGGGCGGCATGGGACMOPTGAGCMOPTCMOPATGCMOPTAG-3’
5’-CATGGGCGGCATGGGADHBCMOPTGADHBGCMOPTCMOPADHBTGCMOPTADHBG-3’
5’-CATGGGCGGCATGGGAMOPCDHBTGAMOPGCDHBTCDHBAMOPTGCDHBTAMOPG-3’
5’-CATGGGCGGCATGGGANH2CMOPTGANH2GCMOPTCMOPANH2TGCMOPTANH2G-3’
5’-CATGGGCGGCATGGGABFCMOPTGABFGCMOPTCMOPABFTGCMOPTABF-3’
5’-CATGGGCGGCATGGG ADHB ADHB ADHB ADHB-3’
5’-CATGGGCGGCATGGG AMOP AMOP AMOP AMOP-3’
5’-CATGGGCGGCATGGG ADHB ADHB ADHB ADHB-3’
5’-CATGGGCGGCATGGG AMOP A MOP AMOP AMOP-3’
5’-CATGGGCGGCATGGG CDHB CDHB CDHB CDHB-3’
5’-CATGGGCGGCATGGG CMOP CMOP CMOP CMOP-3’
5’-CATGGGCGGCATGGG ADHB GGG-3’
ON4A AMOP
ON4A1C ADHB
ON4A1C AMOP
ON4C CDHB
ON4C CMOP
Next, we tried incorporation
of a large number of modifica-
tions into double-stranded DNA
by the polymerase chain reac-
tion (PCR). The experiments
using 98-mer template and each
of the modified dNXTPs in the
presence of KOD XL polymerase
proceeded well to give good
amplification of the modified
DNA. Also, both combinations of
ONA ADHB
ONA AMOP
5’-CATGGGCGGCA GGG AMOP GGG-3’
ONC CDHB
5’-CATGGGCGGCATGGG CDHB GGG-3’
ONC CMOP
5’-CATGGGCGGCATGGG CMOP GGG-3’
ONtempA ADHB
ONtempA AMOP
ONtempC CDHB
ONtempC CMOP
5’-CATGGGCGGCATGGG ADHB-3’
5’-CATGGGCGGCATGGG AMOP-3’
5’-CATGGGCGGCATGGG CDHB-3’
5’-CATGGGCGGCATGGG CMOP-3’
[a] For magnetic separation of the extended primer strands, the templates were 5’-end biotinylated; acronyms
used in the text for primer extension products correspond to those used for the templates.
the modified A and C (ADHB
+
(3-aminophenyl)-7-deazaadenine (ANH2) or with 7-(benzofura-
zan-5-yl)-7-deazaadenine (ABF). The PEX experiments were per-
formed using KOD XL or Pwo polymerases and template
CMOP or AMOP +CDHB) gave the correct product (Figure 6). This
result confirms that all four modified dNXTPs are very good
substrates, at least for KOD XL polymerase.
Table 3. List of MALDI-TOF mass spectrometry data of ONs bearing MOP
and DHB derivatives.
Oligonucleotide
M calcd [Da]
M found [Da]
ONrnd16 ADHB
ONrnd16 AMOP
ONrnd16 CDHB
ONrnd16 CMOP
ONrnd16 ADHB CMOP
ONrnd16 AMOP CDHB
ONrnd16 ANH2 CMOP
ONrnd16 ABF CMOP
ON4A ADHB
10085.90
10101.86
10089.90
10105.86
10574.46
10574.46
10466.34
10574.26
6395.50
6411.50
6303.40
6319.36
6092.05
6096.04
6069.05
6073.04
10085.9
10101.3
10090.6
10107.0
10575.2
10575.3
10467.8
10575.8
6396.7
ON4A AMOP
6433.3[a]
6304.9
Figure 2. Denaturing PAGE analysis of a) ADHBTP and CMOPTP, and b) AMOPTP
and CDHBTP by using KOD XL polymerase, primrnd, and temprnd16. Composi-
tions of the dNTP mixes and nucleotide labeling are as follows: (Pr) primer
(5’-32P-end labeled); (+) natural dNTPs; (AÀ) dCTP, dTTP, dGTP; (CÀ) dATP,
dTTP, dGTP; (ADHBÀ) dCMOPTP, dTTP, dGTP; (AMOPÀ) dCDHBTP, dTTP, dGTP;
(CMOPÀ) dADHBTP, dTTP, dGTP; (CDHBÀ) dAMOPTP, dTTP, dGTP; (ADHB) dADHBTP,
dCTP, dTTP, dGTP; (AMOP) dAMOPTP, dCTP, dTTP, dGTP; (CMOP) dCMOPTP, dATP,
dTTP, dGTP; (CDHB) dCDHBTP, dATP, dTTP, dGTP; (ADHBCMOP) dADHBTP, dCMOPTP,
dTTP, dGTP; (AMOPCDHB) dAMOPTP, dCDHBTP, dTTP, dGTP.
ON4C CDHB
ON4C CMOP
6320.7
6093.4
6097.2
6070.1
ONA ADHB
ONA AMOP
ONC CDHB
ONC CMOP
6074.0
[a] Found mass corresponds to [M+Na]+.
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Figure 3. Primer extension with a) temp4A and b) temp4C templates using
primrnd, KOD XL, and Pwo polymerases: (Pr) primer (5’-32P-end labeled); (A+)
dATP; (AÀ) no dNTPs; (ADHB) dADHBTP; (AMOP) dAMOPTP; (C+) dCTP; (CÀ) no
dNTPs; (CDHB) dCDHBTP; (CMOP) dCMOPTP.
Figure 6. PCR synthesis of 98-mer by KOD XL polymerase: (L) DNA ladder;
(+) dATP, dCTP, dTTP, dGTP; (AÀ) dCTP, dTTP, dGTP; (CÀ) dATP, dTTP, dGTP;
(ADHB) dADHBTP, dCTP, dTTP, dGTP; (AMOP) AMOPTP, dCTP, dTTP, dGTP; (CDHB
)
dCDHBTP, dATP, dTTP, dGTP; (CMOP) CMOPTP, dATP, dTTP, dGTP; (ADHBCMOP
dADHBTP, dCMOPTP, dTTP, dGTP, (AMOPCDHB) dAMOPTP, dCDHBTP, dTTP, dGTP.
)
Figure 4. Denaturing PAGE analysis of ANH2TP and CMOPTP by using a) KOD
XL or b) Pwo polymerases, primrnd, and temprnd16. Compositions of the dNTP
mixes and nucleotide labeling are as follows: (Pr) primer (5’-fluorescein ami-
dite (FAM)&&OK?&& labeled); (+) natural dNTPs; (AÀ) dCTP, dTTP, dGTP;
(CÀ) dATP, dTTP, dGTP; (ANH2À) dCMOPTP, dTTP, dGTP; (CMOPÀ) dANH2TP, dTTP,
dGTP; (ANH2) dANH2TP, dCTP, dTTP, dGTP; (CMOP) dCMOPTP, dATP, dTTP, dGTP;
(ANH2CMOP) dANH2TP, dCMOPTP, dTTP, dGTP.
Figure 5. Denaturing PAGE analysis of ABFTP and CMOPTP by using a) KOD XL
or b) Pwo polymerases, primrnd, and temprnd16. Compositions of the dNTP
mixes and nucleotide labeling are as follows: (Pr) primer (5’-FAM labeled);
(+) natural dNTPs; (AÀ) dCTP, dTTP, dGTP; (CÀ) dATP, dTTP, dGTP; (ABFÀ)
dCMOPTP, dTTP, dGTP; (CMOPÀ) dABFTP, dTTP, dGTP; (ABF) dABFTP, dCTP, dTTP,
dGTP; (CMOP) dCMOPTP, dATP, dTTP, dGTP; (ABFCMOP) dABFTP, dCMOPTP, dTTP,
dGTP.
Figure 7. Square-wave voltammograms of boronic acids (a), modified nucle-
osides (b), and dNXTPs (c). All experiments were done at a pyrolytic graphite
electrode in 0.2m acetate buffer (pH 5.0) with 40 mm concentration of sam-
ples. The peak around 200 mV is related to oxygen-containing groups at the
graphite electrode surface.
Electrochemistry
The electrochemical behavior was first studied on model nu-
basal-plane pyrolytic graphite electrode (Figure 7). The MOP-
B(pin) yielded under these conditions a single anodic (oxida-
tion) peak around 600 mV (peak MOPox, Figure 7a, Table 4),
whereas DHB-B(OH)2 produced two distinct signals at 785 and
950 mV (peaks marked DHBox, Figure 7a). All electrode process-
cleosides (dAMOP dCMOP dADHB and dCDHB and dNXTPs
, , , )
(dAMOPTP, dCMOPTP, dADHBTP, and dCDHBTP) and compared to
that of the starting boronic acids/boronates MOP-B(pin) and
DHB-B(OH)2 by using square-wave voltammetry (SWV) at the
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Table 4. Potential peaks produced by boronic acids, nucleosides, dNxTPs,
and PEX products measured at a pyrolytic graphite electrode using SWV
(values in mV against Ag/AgCl/3m KCl reference electrode).
Sample
Peaks
A*ox
MOPox
DHBox
Gox
Aox
MOP-B(pin)
DHB-B(OH)2
dCMOP
600
–
510
450
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
785, 950
–
–
1150
dAMOP
1045
–
–
dCDHB
dADHB
845
dCMOPTP
dAMOPTP
dCDHBTP
dADHBTP
PEX CMOP
PEX AMOP
PEX APh
PEX CDHB
PEX ADHB
unlabeled PEX
330, 500
435
–
–
–
–
–
–
–
–
–
–
–
–
1040
1070
820
–
–
–
–
–
–
–
–
–
–
–
590
510
–
–
–
1100
1095
1100
1100[a]
1090
1100
1360
1370
1380
1360
1360
1380
–
1100?[a]
870
–
–
[a] Overlapping signals.
Figure 8. AdTS square-wave voltammograms of single-stranded PEX prod-
ucts bearing four MOP (a) or four DHB (b) labels in sequences synthesized
on the temprnd16 template.
es giving rise to these signals took place in the adsorbed state
and were irreversible (data not shown).
Oxidation signals of the MOP moiety were retained in both
nucleoside conjugates (Figure 7b) and appeared at less posi-
tive potentials than observed for the corresponding boronic
beled with CMOP, an additional anodic signal appeared around
590 mV (Figure 8a) whereas ON bearing the AMOP conjugate
gave two signals at potentials of 510 and 900 mV. For the
ADHB-labeled ON (Figure 8b), an extra peak was detected at
870 mV. For ON bearing the CDHB conjugates, no signal outside
the region of natural purine oxidation was detected but a re-
markable increase of the peak occurring at 1100 V was ob-
served, which suggests that in the ON, the peak of DHB oxida-
tion in CDHB overlapped with the peak Gox (Figure 8b).
acid (around 450 mV for dAMOP and around 510 mV for dCMOP
,
Figure 7b). In addition to these well-developed peaks, both
MOP-labeled nucleosides produced poorly developed double
peaks around 750 and 880 mV for dAMOP and dCMOP, respective-
ly. Another distinct anodic signal was produced by dAMOP at
1045 mV (peak A*ox), which can be attributed to oxidation of
the 7-deazaadenine moiety[17] in the nucleobase conjugate.[6]
The DHB-labeled nucleosides gave single peaks around
1070 mV (peak DHBox(C) of dCDHB, Figure 7b) or 820 mV (peak
DHBox(A) of dADHB; interestingly, no separate signal correspond-
ing to the 7-deazaadenine oxidation was detected in the DHB
conjugate). The nucleoside triphosphates (Figure 7c) displayed
qualitatively the same electrochemical behavior as the corre-
sponding nucleosides (Figure 7b) with the exception of
dCMOPTP, which yielded two well-developed anodic signals in
the potential region around 300 and 500 mV. Differences in
the peak heights produced by corresponding species (boronic
acid versus nucleosides versus triphosphates) may be a result
of effects of the nucleobase type and/or of the negatively
charged triphosphate group on the adsorbability of the given
substance at the positively charged electrode surface and ac-
cessibility of the oxidizable moieties for the electrode reaction.
Then, the single-stranded DNA PEX products prepared by
the magnetoseparation procedure, each bearing four modifica-
tions, were studied by adsorptive transfer stripping (AdTS)
SWV (Figure 8). Regardless of the presence or absence of any
of the two labels, all PEX products gave peaks Gox (around
1100 mV) and Aox (around 1370 mV) owing to oxidation of nat-
ural guanine and adenine nucleobases, respectively. For ON la-
Taken together, the MOP moiety appears to be a potentially
useful label for both cytosine and adenine nucleobases, as the
corresponding conjugates produce signals detectable in a po-
tential region in which natural DNA components (as well as
unlabeled 7-deazapurines[17]) are electrochemically silent. Dif-
ference in peak potentials makes it possible to distinguish be-
tween DNA bearing CMOP and that containing AMOP. Moreover,
CMOP producing a single oxidation signal can easily be used for
multipotential DNA coding in combination with other oxidiza-
ble labels differing in the potentials of their electrooxidation.
For example, a PEX product labeled simultaneously with CMOP
and ANH2 produced two independently readable peaks MOPox
ox
and NH2 (the latter a result of oxidation of the aminophenyl
moiety[5] around 830 mV, Figure 9a). An example of combina-
tion of MOP with an irreversibly reducible label, benzofurazane
(BF),[11] is demonstrated in Figure 9b and c. In this case the oxi-
dizable label MOP produces a specific signal only in the anodic
potential scan and BF only in the cathodic one (peak BFred
around À900 mV, Figure 9c), thereby making their differentia-
tion even more obvious.
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Conclusion
4-Hydroxy-3-methoxyphenyl (MOP) and 2,3-dihydrobenzofur-
an-5-yl (DHB) groups were attached to nucleosides and 2’-de-
oxyribonucleoside triphosphates (dNTPs) and tested as new
oxidizable redox labels for DNA. The synthesis of the modified
nucleosides and dNTPs by the aqueous-phase Suzuki cross-
coupling reactions is straightforward and efficient. The corre-
sponding modified dNXTPs were good substrates for DNA
polymerases and were easily incorporated into DNA by primer
extension experiments or polymerase chain reaction. The la-
beled nucleosides, dNTPs, and DNA were studied systematical-
ly by several electrochemical techniques. In nucleosides and
dNTPs, both MOP and DHB labels show distinct signals result-
ing from oxidation of the label. However, after incorporation
into DNA, only the MOP label appears to be useful since its ox-
idation signals occur at about 500–600 mV, at which they do
not interfere either with any signals of inherent oxidation of
DNA or with those of some other earlier designed oxidizable
labels. Particularly, the CMOP conjugate appears to be a candi-
date for application in multipotential DNA labeling in combina-
tion with other oxidizable or reducible labels. The use of DHB
label would be limited only for modified adenine, which gives
an oxidation signal at 870 mV, whereas the oxidation of the
corresponding cytosine conjugate (CDHB) interferes with oxida-
tion of guanine.
Experimental Section
General
NMR spectra were measured on a Bruker Avance 600 spectrometer
1
(600 MHz for H and 151 MHz for 13C nuclei) and a Bruker 500 spec-
1
trometer (500 MHz for H, 125.7 MHz for 13C, and 202.3 for 31P) in
D2O (referenced to dioxane as internal standard, dH=3.75 ppm,
31
dC=67.19 ppm, standard for P NMR was external H3PO4). Chemi-
cal shifts are given in ppm (d scale), and coupling constants (J) in
Hertz. Complete assignment of all NMR signals was performed
using a combination of H,H-COSY, H,C-HSQC, and H,C-HMBC ex-
periments. Mass spectra were measured on an LCQ classic
(Thermo-Finnigan) spectrometer using electrospray ionization (ESI)
or Q-Tof Micro (Waters, ESI source, internal calibration with lock-
spray). Preparative HPLC separations were performed on a column
packed with 10 mm C18 reversed phase (Phenomenex, Luna
C18(2)). IR spectra were measured by using the attenuated total re-
flectance technique or by using KBr tablets. High-resolution mass
spectra were measured by using the ESI technique. Mass spectra
of functionalized DNA were measured by MALDI-TOF, Reflex IV
(Bruker) with a nitrogen laser. UV/Vis spectra were measured on
a Varian CARY 100 Bio spectrophotometer at room temperature.
Melting points were determined on a Kofler block. Known starting
compounds dAITP,[5] dCITP,[15b] dAPhTP,[18] and dABFTP[11] were pre-
pared by literature procedures.
Figure 9. Sections of baseline-corrected AdTS square-wave voltammograms
of PEX products labeled with CMOP in combination with ABF (a, b) or with
ANH2 (c); in (a,c) electrochemical oxidation and in (b) electrochemical reduc-
tion are shown. The temprnd16 template was used for the PEX.
On the other hand, utilization of DHB as a DNA label is limit-
ed to ADHB owing to coincidence of CDHB oxidation with gua-
nine oxidation. Moreover, DHB cannot be used for independ-
ent DNA labeling in combination with AMOP because of coinci-
dence of their oxidation peaks around 900 mV. Compared with
CMOP, the applicability of both ADHB and AMOP in the multipoten-
tial labeling scheme in combination with earlier introduced ox-
idizable labels is also limited as a result of interference of oxi-
dation signals close to 900 mV, at which the oxidation signals
of aminophenyl (Figure 9a) and 7-deazaguanine[17] also appear.
General procedure for the synthesis of modified nucleo-
sides: Suzuki–Miyaura cross-coupling
Method A: A 2:1 mixture of H2O/CH3CN (2 mL) was added through
a septum to an argon-purged flask containing halogenated nucleo-
sides dNI (1 equiv), boronic acid/boronate (2 equiv), and Cs2CO3
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(3 equiv). In a separate flask, Pd(OAc)2 (10 mol%) and TPPTS
(2.5 equiv with respect to Pd) were combined, the flask was evacu-
ated and purged with argon, and then a 2:1 mixture of H2O/CH3CN
(1 mL) was added. This catalyst solution was injected into the reac-
tion mixture, which was then stirred at 758C for 1 h until complete
consumption of the starting material and then evaporated in va-
cuo. The products were purified by silica gel column chromatogra-
phy using chloroform/methanol (0 to 10%) as eluent.
147.96 (C-3-C6H3OHOMe), 150.34 (C-7a), 151.77 (CH-2), 157.48 ppm
(C-4); IR (KBr): n˜ =3497, 3385, 2968, 2865, 1735, 1630, 1470, 1291,
1097, 1052 cmÀ1; MS (ESI+): m/z (%): 373.2 (100) [M++H], 395.2
(54) [M++Na]; HRMS (ESI+): m/z calcd for C18H21N4O5: 373.15060;
found: 373.15065; calcd for C18H20N4O5Na: 395.13254; found:
395.3259.
dCMOP: Compound dCMOP was prepared from dCI according to the
general procedure (Method A). The product was isolated as
a yellow solid (48 mg, 83%). M.p. 2048C; 1H NMR (499.8 MHz,
[D6]DMSO): d=2.12 (m, 2H; H-2’), 3.50, 3.57 (2ꢂddd, Jgem =11.7,
General procedure for the synthesis of modified nucleotide
triphosphates: Suzuki–Miyaura cross-coupling
J
5’,OH =5.1, J5’,4’ =3.4 Hz, 2ꢂ1H; H-5’), 3.77 (q, J4’,3’ =J4’,5’ =3.4 Hz,
1H; H-4’), 3.78 (s, 3H; CH3O), 4.22 (m, 1H; H-3’), 4.96 (t, JOH,5’
5.1 Hz, 1H; OH-5’), 5.19 (d, JOH,3’ =4.2 Hz, 1H; OH-3’), 6.20 (dd,
=
Method B: A 2:1 mixture of H2O/CH3CN (1 mL) was added through
a septum to an argon-purged flask containing halogenated nucleo-
tides dNITP (1 equiv), boronic acid/boronate (2 equiv), and Cs2CO3
(3 equiv). In a separate flask, Pd(OAc)2 (10 mol%) and TPPTS
(2.5 equiv with respect to Pd) were combined, the flask was evacu-
ated and purged with argon, and then a 2:1 mixture of H2O/CH3CN
(0.5 mL) was added. This catalyst solution was injected into the re-
action mixture, which was then stirred at 508C for 40 min until
complete consumption of the starting material and then evaporat-
ed in vacuo. The product was isolated from the crude reaction mix-
ture by HPLC on a C18 column with the use of a linear gradient of
0.1m triethylammonium bicarbonate (TEAB) in H2O to 0.1m TEAB
in H2O/MeOH (1:1) as eluent. Several co-distillations with water and
conversion to sodium salt form (Dowex 50WX8 in Na+ cycle) fol-
lowed by freeze-drying from water gave a solid product.
J
J
1’,2’ =7.1, 6.1 Hz, 1H; H-1’), 6.35 (bs, 1H; NHaHb), 6.70 (dd, J6,5 =8.0,
6,2 =2.0 Hz, 1H; H-6-C6H3OHOMe), 6.81 (d, J5,6 =8.0 Hz, 1H; H-5-
C6H3OHOMe), 6.83 (d, J2,6 =2.0 Hz, 1H; H-2-C6H3OHOMe), 7.35 (bs,
1H; NHaHb), 7.79 (s, 1H; H-6), 9.10 ppm (bs, 1H; OH-4-
C6H3OHOMe); 13C NMR (125.7 MHz, [D6]DMSO): d=40.75 (CH2-2’),
55.60 (CH3O), 61.25 (CH2-5’), 70.37 (CH-3’), 85.20 (CH-1’), 87.41 (CH-
4’), 108.11 (C-5), 113.00 (CH-2-C6H3OHOMe), 115.98 (CH-5-
C6H3OHOMe), 121.40 (CH-6-C6H3OHOMe), 124.75 (C-1-C6H3OHOMe),
139.64 (CH-6), 146.41 (C-4-C6H3OHOMe), 147.86 (C-3-C6H3OHOMe),
154.52 (C-2), 163.73 ppm (C-4); IR (KBr): n˜ =3305, 2928, 2859, 1744,
1642,.1601, 1473, 1280, 1092, 1059 cmÀ1; MS (ESI+): m/z (%): 350.2
(20) [M++H]; HRMS (ESI+): m/z calcd for C16H20N3O6: 350.13466;
found: 350.13466; calcd for C18H20N4O5Na: 395.13254; found:
395.3259.
dCDHB: Compound dCDHB was prepared from dCI according to the
general procedure (Method A). The product was isolated as a dark
purple solid (20 mg, 69%). M.p. 1788C; 1H NMR (500.0 MHz,
[D6]DMSO): d=2.05 (ddd, Jgem =13.1, J2’b,1’ =7.1, J2’b,3’ =6.1 Hz 1H;,
H-2’b), 2.13 (ddd, Jgem =13.1, J2’a,1’ =6.1, J2’a,3’ =3.5 Hz, 1H; H-2’a),
3.19 (t, J3,2 =8.7 Hz, 2H; H-3-dihydrobenzofuryl), 3.50, 3.55 (2ꢂddd,
Synthesis of modified nucleoside triphosphates: triphos-
phorylation
Method C: POCl3 (1.2 equiv) in PO(OMe)3 was added through
a septum to an argon-purged flask containing modified nucleo-
sides dNX (1 equiv). The reaction mixture was then stirred at 08C
for 3 h until complete consumption of the starting material. Then
an ice-cooled solution of (NHBu3)2H2P2O7 (5 equiv) and Bu3N
(4.2 equiv) in dry DMF (2 mL) was added and the mixture was
stirred at 08C for another 1.5 h. The reaction was quenched by ad-
dition of 2m aqueous TEAB (2 mL), the solvents were evaporated
in vacuo, and the residue was co-distilled with water three times.
The product was isolated by HPLC on a C18 column with the use
of a linear gradient of 0.1m TEAB in H2O to 0.1m TEAB in H2O/
MeOH (1:1) as eluent. Several co-distillations with water and con-
version to sodium salt form (Dowex 50WX8 in Na+ cycle) followed
by freeze-drying from water gave a solid product.
J
gem =11.8, J5’,OH =5.0, J5’,4’ =3.5 Hz, 2ꢂ1H; H-5’), 3.76 (q, J4’,3’ =J4’,5’
3.5 Hz, 1H; H-4’), 4.21 (m, 1H; H-3’), 4.54 (t, J2,3 =8.7 Hz, 2H; H-2-di-
hydrobenzofuryl), 4.94 (t, JOH,5’ =5.0 Hz, 1H; OH-5’), 5.19 (d, JOH,3’
=
=
4.2 Hz, 1H; OH-3’), 6.20 (dd, J1’,2’ =7.1, 6.1 Hz, 1H; H-1’), 6.24 (bs,
1H; NHaHb), 6.79 (d, J7,6 =8.2 Hz, 1H; H-7-dihydrobenzofuryl), 7.01
(dd, J6,7 =8.2, J6,4 =2.0 Hz, 1H; H-6-dihydrobenzofuryl), 7.16 (d, J4,6
=
2.0 Hz, 1H; H-4-dihydrobenzofuryl), 7.34 (bs, 1H; NHaHb), 7.75 ppm
(s, 1H; H-6); 13C NMR (125.7 MHz, [D6]DMSO): d=29.31 (CH2-3-dihy-
drobenzofuryl), 40.70 (CH2-2’), 61.27 (CH2-5’), 70.41 (CH-3’), 71.30
(CH2-2-dihydrobenzofuryl), 85.12 (CH-1’), 87.36 (CH-4’), 108.04 (C-5),
109.43 (CH-7-dihydrobenzofuryl), 125.95 (C-5-dihydrobenzofuryl),
126.08 (CH-4-dihydrobenzofuryl), 128.24 (C-3a-dihydrobenzofuryl),
128.86 (CH-6-dihydrobenzofuryl), 139.58 (CH-6), 154.72 (C-2),
159.53 (C-7a-dihydrobenzofuryl), 163.94 ppm (C-4); IR (KBr): n˜ =
dAMOP: Compound dAMOP was prepared from dAI according to the
general procedure (Method A). The product was isolated as
a yellow solid (38 mg, 77%). M.p. 908C; 1H NMR (500.0 MHz,
[D6]DMSO): d=2.18 (ddd, Jgem =13.1, J2’b,1’ =6.0, J2’b,3’ =2.6 Hz, 1H;
H-2’b), 2.56 (ddd, Jgem =13.1, J2’a,1’ =8.3, J2’a,3’ =5.7 Hz, 1H; H-2’a),
3.50, 3.57 (2ꢂddd, Jgem =11.7, J5’,OH =5.3, J5’,4’ =4.4 Hz, 2ꢂ1H; H-5’),
3.81 (s, 3H; CH3O), 3.82 (td, J4’,5’ =4.4, J4’,3’ =2.5 Hz, 1H; H-4’), 4.35
3410, 2929, 2860, 1649, 1601, 1475, 1233,1192, 1094, 1053 cmÀ1
;
MS (ESI+): m/z (%): 346.1 (35) [M++H]; 368.1 (100) [M++Na];
HRMS (ESI+): m/z calcd for C17H20O5N3: 346.13976; found:
346.13975; calcd for C17H19O5N3Na: 368.12157; found: 368.12169.
dADHB: Compound dADHB was prepared from dAI according to the
general procedure (Method A). The product was isolated as a weak
yellow solid (38 mg, 78%). M.p. 1078C; 1H NMR (499.8 MHz,
[D6]DMSO): d=2.18 (ddd, Jgem =13.1, J2’b,1’ =6.0, J2’b,3’ =2.6 Hz, 1H;
H-2’b), 2.55 (ddd, Jgem =13.1, J2’a,1’ =8.4, J2’a,3’ =5.8 Hz, 1H; H-2’a),
3.23 (t, J3,2 =8.7 Hz, 2H; H-3-dihydrobenzofuryl), 3.50, 3.57 (2ꢂddd,
(bm, 1H; H-3’), 5.07 (bt, JOH,5’ =5.3 Hz, 1H; OH-5’), 5.27 (bd, JOH,3’
=
2.9 Hz, 1H; OH-3’), 6.12 (bs, 2H; NH2), 6.57 (dd, J1’,2’ =8.3, 6.0 Hz,
1H; H-1’), 6.84 (dd, J6,5 =8.0, J6,2 =1.9 Hz, 1H; H-6-C6H3OHOMe),
6.87 (d, J5,6 =8.0 Hz, 1H; H-5-C6H3OHOMe), 6.98 (d, J2,6 =1.9 Hz, 1H;
H-2-C6H3OHOMe), 7.41 (s, 1H; H-6), 8.12 (s, 1H; H-2), 9.14 ppm (bs,
1H; OH-4-C6H3OHOMe); 13C NMR (125.7 MHz, [D6]DMSO): d=39.53
(CH2-2’), 55.76 (CH3O), 62.20 (CH2-5’), 71.25 (CH-3’), 83.06 (CH-1’),
87.49 (CH-4’), 100.79 (C-4a), 113.02 (CH-2-C6H3OHOMe), 116.04 (CH-
5-C6H3OHOMe), 116.86 (C-5), 120.03 (CH-6), 121.08 (CH-6-
C6H3OHOMe), 125.57 (C-1-C6H3OHOMe), 146.03 (C-4-C6H3OHOMe),
J
J
gem =11.7, J5’,OH =5.5, J5’,4’ =4.5 Hz, 2ꢂ1H; H-5’), 3.82 (td, J4’,5’ =4.5,
4’,3’ =2.4 Hz, 1H; H-4’), 4.35 (bm, 1H; H-3’), 4.57 (t, J2,3 =8.7 Hz, 2H;
H-2-dihydrobenzofuryl), 5.05 (t, JOH,5’ =5.5 Hz, 1H; OH-5’), 5.25 (d,
OH,3’ =4.1 Hz, 1H; OH-3’), 6.10 (bs, 2H; NH2), 6.57 (dd, J1’,2’ =8.4 Hz,
1H; 6.0, H-1’), 6.86 (dd, J7,6 =8.1, J7,4 =0.5 Hz, 1H; H-7-dihydroben-
J
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zofuryl), 7.16 (ddt, J6,7 =8.1, J6,4 =2.0, J6,3 =0.7 Hz, 1H; H-6-dihydro-
benzofuryl), 7.31 (dtd, J4,6 =2.0, J4,3 =1.1, J4,7 =0.5 Hz, 1H; H-4-dihy-
drobenzofuryl), 7.40 (s, 1H; H-6), 8.12 ppm (s, 1H; H-2); 13C NMR
(125.7 MHz, [D6]DMSO): d=29.33 (CH2-3-dihydrobenzofuryl), 39.82
(CH2-2’), 62.21 (CH2-5’), 71.27 (CH2-2-dihydrobenzofuryl), 71.28 (CH-
3’), 83.06 (CH-1’), 87.50 (CH-4’), 100.77 (C-4a), 109.35 (CH-7-dihydro-
benzofuryl), 116.70 (C-5), 120.11 (CH-6), 125.64 (CH-4-dihydroben-
zofuryl), 126.65 (C-5-dihydrobenzofuryl), 128.34 (C-3a-dihydroben-
zofuryl), 128.39 (CH-6-dihydrobenzofuryl), 150.35 (C-7a), 151.73
(CH-2), 157.44 (C-4), 159.17 ppm (C-7a-dihydrobenzofuryl); IR (KBr):
n˜ =3332, 3191, 2929, 2860,1493, 1300, 1223, 1093, 1050 cmÀ1; MS
(ESI+): m/z (%): 369.2 (100) [M++H]; HRMS (ESI+): m/z calcd for
C19H21N4O4: 369.15573; found: 369.15575.
19.2 Hz; Pa), À6.65 ppm (d, J=19.8 Hz; Pg); MS (ESI+): m/z (%):
611.1 (8) [M++H]; 633.0 (20) [M++Na]; HRMS (ESI+): m/z calcd for
C18H22O14N4P3:
611.03508;
found: 611.03483;
calcd
for
C18H21O14N4NaP3: 633.01703; found: 633.01664.
dCDHBTP: Compound dCDHBTP was prepared from dCITP according
to the general procedure (Method B). The product was isolated as
a white solid (17 mg, 41%). 1H NMR (500.0 MHz, D2O, pD=7.1,
phosphate buffer, ref(dioxane)=3.75 ppm): d=2.35 (ddd, Jgem
14.1, J2’b,1’ =7.5, J2’b,3’ =6.4 Hz, 1H; H-2’b), 2.42 (ddd, Jgem =14.1,
2’a,1’ =6.3, J2’a,3’ =3.6 Hz, 1H; H-2’a), 3.28 (t, J3,2 =8.7 Hz, 2H; H-3-di-
hydrobenzofuryl), 4.15 (m, 2H; H-5’), 4.21 (m, 1H; H-4’), 4.61 (dt,
3’,2’ =6.4,. 3.6, J3’,4’ =3.6 Hz, 1H; H-3’), 4.64 (t, J2,3 =8.7 Hz, 2H; H-2-
dihydrobenzofuryl), 6.35 (dd, J1’,2’ =7.5, 6.3 Hz, 1H; H-1’), 6.91 (d,
7,6 =8.2 Hz, 1H; H-7-dihydrobenzofuryl), 7.18 (dd, J6,7 =8.2, J6,4
=
J
J
dADHBTP: Compound dADHBTP was prepared from dAITP according
to the general procedure (Method B). The product was isolated as
a purple solid (14 mg, 42%). 1H NMR (499.8 MHz, D2O, pD=7.1,
J
=
2.0 Hz, 1H; H-6-dihydrobenzofuryl), 7.31 (d, J4,6 =2.0 Hz, 1H; H-4-di-
hydrobenzofuryl), 7.70 ppm (s, 1H; H-6); 13C NMR (125.7 MHz, D2O,
pD=7.1, phosphate buffer, ref(dioxane)=69.3 ppm): d=31.62
(CH2-3-dihydrobenzofuryl), 41.59 (CH2-2’), 68.03 (d, JC,P =5.4 Hz;
CH2-5’), 73.37 (CH-3’), 74.76 (CH2-2-dihydrobenzofuryl), 88.16 (d,
phosphate buffer, ref(dioxane)=3.75 ppm): d=2.46 (ddd, Jgem
14.0, J2’b,1’ =6.2, J2’b,3’ =3.2 Hz, 1H; H-2’b), 2.72 (ddd, Jgem =14.0,
2’a,1’ =7.8, J2’a,3’ =6.6 Hz, 1H; H-2’a), 3.76 (t, J3,2 =8.7 Hz, 2H; H-3-di-
=
J
hydrobenzofuryl), 4.11 (ddd, Jgem =11.0, JH,P =5.0, J5’b,4’ =4.4 Hz, 1H;
H-5’b), 4.17 (ddd, Jgem =11.0, JH,P =6.4, J5’a,4’ =4.4 Hz, 1H; H-5’a),
4.24 (bq, J4’,5’ =J4’,3’ =4.4 Hz, 1H; H-4’), 4.63 (t, J2,3 =8.7 Hz, 2H; H-2-
dihydrobenzofuryl), 4.80 (m, 1H; H-3’ overlapped with HDO signal),
6.66 (dd, J1’,2’ =7.8, 6.2 Hz, 1H; H-1’), 6.90 (d, J7,6 =8.2 Hz, 1H; H-7-
dihydrobenzofuryl), 7.24 (dd, J6,7 =8.2, J6,4 =1.7 Hz, 1H; H-6-dihy-
drobenzofuryl), 7.35 (d, J4,6 =2.0 Hz, 1H; H-4-dihydrobenzofuryl),
7.42 (s, 1H; H-6), 8.15 ppm (s, 1H; H-2); 13C NMR (125.7 MHz, D2O,
pD=7.1, phosphate buffer, ref(dioxane)=69.3 ppm): d=31.67
(CH2-3-dihydrobenzofuryl), 40.92 (CH2-2’), 68.27 (d, JC,P =5.5 Hz;
CH2-5’), 73.88 (CH-3’), 74.69 (CH2-2-dihydrobenzofuryl), 85.42 (CH-
1’), 87.76 (d, JC,P =8.7 Hz; CH-4’), 103.88 (C-4a), 112.16 (CH-7-dihy-
drobenzofuryl), 121.05 (C-5), 122.29 (CH-6), 128.57 (CH-4-dihydro-
benzofuryl), 128.96 (C-5-dihydrobenzofuryl), 131.27 (CH-6-dihydro-
benzofuryl), 131.45 (C-3a-dihydrobenzofuryl), 152.37 (C-7a), 153.90
(CH-2), 159.88 (C-4), 161.24 ppm (CH-7a-dihydrobenzofuryl);
31P NMR (202.3 MHz, D2O, pD=7.1, phosphate buffer, ref(phos-
phate buffer)=2.35 ppm): d=À21.53 (t, J=19.6 Hz; Pb), À10.55 (d,
J=19.6 Hz; Pa), À6.48 ppm (d, J=19.6 Hz; Pg); MS (ESI+): m/z (%):
629.0 (8) [M++Na]; 651.0 (20) [M++2Na]; HRMS (ESI+): m/z calcd
for C19H22O13N4P3: 607.04017; found: 607.04022; calcd for
C19H21O13N4NaP3: 629.02211; found: 629.02208.
JC,P =8.8 Hz; CH-4’), 88.62 (CH-1’), 112.38 (CH-7-dihydrobenzofuryl),
113.56 (C-5), 127.38 (C-5-dihydrobenzofuryl), 129.13 (CH-4-dihydro-
benzofuryl), 131.68 (C-3a-dihydrobenzofuryl), 131.96 (CH-6-dihydro-
benzofuryl), 142.03 (CH-6), 159.88 (C-2), 161.99 (CH-7a-dihydroben-
zofuryl), 167.79 ppm (C-4); 31P NMR (202.3 MHz, D2O, pD=7.1, re-
f(phosphate buffer)=2.35 ppm): d=À21.60 (t, J=19.7 Hz; Pb),
À10.74 (d, J=19.7 Hz; Pa), À6.63 ppm (d, J=19.7 Hz; Pg); MS
(ESI+): m/z (%): 628.0 (15) [M++2Na]; HRMS (ESI+): m/z calcd for
C17H21O14N3P3: 584.02418; found: 584.02419; calcd for
C17H20O14N3NaP3: 606.00613; found: 606.00623.
dCMOPTP: Compound dCMOPTP was prepared from dCI according to
the general procedure (Method C). The product was isolated as
a white solid (11 mg, 28%). 1H NMR (500.0 MHz, D2O, pD=7.1,
phosphate buffer, ref(dioxane)=3.75 ppm): d=2.36 (ddd, Jgem
14.0, J2’b,1’ =7.4, J2’b,3’ =6.4 Hz, 1H; H-2’b), 2.42 (ddd, Jgem =14.0,
2’a,1’ =6.4, J2’a,3’ =3.5 Hz, 1H; H-2’a), 3.89 (s, 3H; CH3O), 4.15 (m, 2H;
=
J
H-5’), 4.22 (td, J4’,5’ =4.4, J4’,3’ =3.5 Hz, 1H; H-4’), 4.60 (dt, J3’,2’ =6.4,.
3.5, J3’,4’ =3.5 Hz, 1H; H-3’), 6.35 (dd, J1’,2’ =7.4, 6.4 Hz, 1H; H-1’),
6.93 (dd, J6,5 =8.1, J6,2 =2.0 Hz, 1H; H-6-C6H3OHOMe), 7.02 (d, J5,6
=
8.1 Hz, 1H; H-5-C6H3OHOMe), 7.03 (d, 2,6 =2.0 Hz, 1H; H-2-
J
C6H3OHOMe), 7.71 ppm (s, 1H; H-6); 13C NMR (125.7 MHz, D2O,
pD=7.1, phosphate buffer, ref(dioxane)=69.3 ppm): d=41.59
(CH2-2’), 58.72 (CH3O), 68.05 (d, JC,P =5.5 Hz; CH2-5’), 73.41 (CH-3’),
88.18 (d, JC,P =8.7 Hz; CH-4’), 88.70 (CH-1’), 113.36 (C-5), 116.11 (CH-
2-C6H3OHOMe), 118.90 (CH-5-C6H3OHOMe), 125.22 (CH-6-
C6H3OHOMe), 127.33 (C-1-C6H3OHOMe), 142.12 (CH-6), 147.97 (C-4-
C6H3OHOMe), 150.42 (C-3-C6H3OHOMe), 159.88 (C-2), 167.73 ppm
(C-4); 31P NMR (202.3 MHz, D2O, pD=7.1, ref(phosphate buffer)=
2.35 ppm): d=À21.57 (t, J=19.6 Hz; Pb), À10.72 (d, J=19.6 Hz;
Pa), À6.60 ppm (d, J=19.6 Hz; Pg); MS (ESI+): m/z (%): 610.0 (15)
[M++H]; 653.9 (10) [M++Na]; HRMS (ESI+): m/z calcd for
C16H20O15N3P3: 610.00104; found: 610.00062; calcd for
C16H18O15N3Na3P3: 653.96493; found: 653.96465.
dAMOPTP: Compound dAMOPTP was prepared from dAITP according
to the general procedure (Method B). The product was isolated as
a purple solid (21 mg, 42%). 1H NMR (500.0 MHz, D2O, pD=7.1,
phosphate buffer, ref(dioxane)=3.75 ppm): d=2.46 (ddd, Jgem
=
14.0, J2’b,1’ =6.2, J2’b,3’ =3.0 Hz, 1H; H-2’b), 2.73 (ddd, Jgem =14.0,
J
J
J
2’a,1’ =8.1, J2’a,3’ =6.4 Hz, 1H; H-2’a), 3.88 (s, 3H; CH3O), 4.11 (ddd,
gem =10.7, JH,P =5.6, J5’b,4’ =4.4 Hz, 1H; H-5’b), 4.17 (ddd, Jgem =10.7,
H,P =6.3, J5’a,4’ =4.4 Hz, 1H; H-5’a), 4.24 (td, J4’,5’ =4.4, J4’,3’ =3.0 Hz,
1H; H-4’), 4.76 (dt, J3’,2’ =6.4, 3.0, J3’,4’ =3.0 Hz, 1H; H-3’), 6.67 (dd,
1’,2’ =8.1, 6.2 Hz, 1H; H-1’), 6.98 (dd, J6,5 =8.0, J6,2 =1.7 Hz, 1H; H-6-
C6H3OHOMe), 7.01 (d, J5,6 =8.0 Hz, 1H; H-5-C6H3OHOMe), 7.10 (d,
J
J
2,6 =1.7 Hz, 1H; H-2-C6H3OHOMe), 7.45 (s, 1H; H-6), 8.16 ppm (s,
1H; H-2); 13C NMR (125.7 MHz, D2O, pD=7.1, phosphate buffer, ref-
(dioxane)=69.3 ppm): d=40.93 (CH2-2’), 58.73 (CH3O), 68.31 (d,
Materials for biochemistry
J
C,P =5.5 Hz; CH2-5’), 73.95 (CH-3’), 85.46 (CH-1’), 87.79 (d, JC,P =
Synthetic oligonucleotides (ONs) and unmodified nucleoside tri-
phosphates (dATP, dTTP, dCTP, and dGTP) were purchased from
Sigma, Dynabeads M-270 Streptavidin (DBStv) were obtained from
Dynal A.S. (Norway), Pwo polymerase was from New England Bio-
labs (Great Britain), KOD XL DNA from Novagen, and g-32P-ATP
from MP Empowered Discovery (USA). Other chemicals were of an-
alytical grade.
8.9 Hz; CH-4’), 103.82 (C-4a), 115.81 (CH-2-C6H3OHOMe), 118.68
(CH-5-C6H3OHOMe), 120.89 (C-5), 122.36 (CH-6), 124.55 (CH-6-
C6H3OHOMe), 128.93 (C-1-C6H3OHOMe), 147.14 (C-4-C6H3OHOMe),
150.38 (C-3-C6H3OHOMe), 152.40 (C-7a), 153.70 (CH-2), 159.86 ppm
(C-4); 31P NMR (202.3 MHz, D2O, pD=7.1, ref(phosphate buffer)=
2.35 ppm): d=À21.49 (dd, J=19.8, 19.2 Hz; Pb), À10.44 (d, J=
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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tolab analyzer (Eco Chemie, The Netherlands) in connection with
VA-stand 663 apparatus (Metrohm, Herisau, Switzerland). The
three-electrode system was used with an Ag/AgCl/3m KCl elec-
trode as a reference and platinum wire as an auxiliary electrode.
PEX experiment
The reaction mixture (20 mL) contained template (for sequences,
see Table 2, 3 mm), primer (5’-32P- or 5’-FAM-labeled, for sequences
see Table 2, 3 mm), dNTPs (all natural, three natural and one modi-
fied, or two natural and two modified; 1, 2, or 4 mm depending on
the experiment), DNA polymerase (0.125–0.5 U KOD XL or 0.25–
0.5 U Pwo), and buffer. Primer was labeled at its 5’-end by use of
[g-32P]-ATP according to standard techniques. The reaction mixture
was incubated for 10 min (20 min for temprnd16) at 608C. The PEX
reaction was stopped by the addition of PAGE stop solution [40 mL,
formamide (80%, v/v), ethylenediaminetetraacetic acid (EDTA,
20 mm), bromophenol blue (0.025%, w/v), xylene cyanol
(0.025%,w/v)] and heating for 2 min at 958C. Samples were ana-
lyzed by use of a 12.5% denaturing polyacrylamide gel electropho-
resis (PAGE; 1 h, 508C) and visualized by phosphorimager (Ty-
phoon 9410, Amersham Biosciences).
Acknowledgements
This study was supported by the Academy of Sciences of the
Czech Republic (RVO 61388963 and RVO 68081707), the Czech
Science Foundation (P206/12/G151), and Gilead Sciences, Inc.
Keywords: DNA · electrochemistry · nucleotides · polymerase
chain reactions · redox labeling
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Kinetics of PEX: The PEX reaction mixtures using KOD XL and Pwo
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mediate heating (see Figures S1–S12).
Towards Electrochemical Sensors for Genomics and Proteomics (Eds.: E.
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MALDI-TOF experiment
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The MALDI-TOF spectra were measured on an UltrafleXtreme
MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany)
with 1 kHz smartbeam II laser technology. The measurements were
done in reflectron mode by the droplet technique, with the mass
range up to 30 kDa. The matrix consisted of 3-hydroxypicolinic
acid/picolinic acid/ammonium tartrate in the ratio 9:1:1. The matrix
(1 mL) was applied on the target (ground steel) and dried down at
room temperature. The sample (1 mL) and matrix (1 mL) were mixed
and added on top of the dried matrix preparation spot and dried
at room temperature.
Preparation of ONs for MALDI-TOF analysis: Streptavidin magnetic
particles stock solution (Roche, 50 mL) was washed with binding
buffer (3ꢂ200 mL, 10 mm Tris, 1 mm EDTA, 100 mm NaCl, pH 7.5).
The PEX solution (prepared as described above) and binding buffer
(50 mL) were added. The suspension was shaken (1200 rpm) for
30 min at 158C. The magnetic beads were collected on a magnet
(DynaMag-2, Invitrogen) and washed with wash buffer (3ꢂ200 mL,
10 mm Tris, 1 mm EDTA, 500 mm NaCl, pH 7.5) and water (4ꢂ
200 mL). Then water (50 mL) was added and the sample was dena-
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a magnet and the solution was transferred into a clean vial. The
product was analyzed by MALDI-TOF mass spectrometry (the re-
sults are summarized in Table 3; for copies of mass spectra, see
Figures S18–S33).
Electrochemical analysis
Nucleosides, dNXTPs, and boronic acids were analyzed by conven-
tional in situ square-wave voltammetry (SWV). Purified PEX prod-
ucts were analyzed by ex situ (adsorptive transfer stripping) SWV.
The PEX products were accumulated for 60 s from aliquots (5 mL)
containing 0.2m NaCl at the surface of the working electrode
(basal-plane pyrolytic graphite). The electrode was then rinsed
with deionized water and placed in the electrochemical cell. SWV
settings: initial potential 0.0 V, end potential +1.5 V (for oxidation)
or À1.5 V (for reduction, Figure 9c), frequency 200 Hz, amplitude
50 mV. Background electrolyte: 0.2m sodium acetate pH 5.0. All
measurements were performed at room temperature using an Au-
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Received: June 27, 2014
Revised: August 12, 2014
Published online on && &&, 0000
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Simonova, J. Balintovꢀ, R. Pohl,
L. Havran, M. Fojta,* M. Hocek*
&& – &&
Methoxyphenol and
Dihydrobenzofuran as Oxidizable
Labels for Electrochemical Detection
of DNA
Clearly labeled: 4-Hydroxy-3-methoxy-
phenyl (MOP) and 2,3-dihydrobenzofur-
an-5-yl (DHB) groups have been devel-
oped as new oxidizable labels for the
electrochemical detection of DNA and
for redox coding of nucleotides (see
figure). MOP offers independent detec-
tion of the modified DNA.
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 0000, 00, 1 – 11
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