Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates: Synthesis, polymerase incorporation to DNA and electrochemical study

Article abstract of DOI:10.1002/chem.201003496

Aqueous Suzuki-Miyaura cross-coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5-iodocytosine and 7-iodo-7-deazaadenine with methyl-, benzyl- and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo-) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl- or tritylsulfanylphenyl moieties produced signals in [Co(NH₃) ₆]³⁺ ammonium buffer, attributed to the Brdicka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdicka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions. Copyright

Full text of DOI:10.1002/chem.201003496

FULL PAPER
DOI: 10.1002/chem.201003496
Alkylsulfanylphenyl Derivatives of Cytosine and 7-Deazaadenine
Nucleosides, Nucleotides and Nucleoside Triphosphates: Synthesis,
Polymerase Incorporation to DNA and Electrochemical Study
Hana Macꢀcˇkovꢁ-Cahovꢁ,[a] Radek Pohl,[a] Petra Horꢁkovꢁ,[b] Ludeˇk Havran,[b]
[b]
Jan Spacˇek, Miroslav Fojta,*[b] and Michal Hocek*[a]
ˇ
Abstract: Aqueous Suzuki–Miyaura
cross-coupling reactions of halogenated
nucleosides, nucleotides and nucleoside
triphosphates derived from 5-iodocyto-
sine and 7-iodo-7-deazaadenine with
methyl-, benzyl- and tritylsufanylphe-
nylboronic acids gave the correspond-
ing alkylsulfanylphenyl derivatives of
nucleosides and nucleotides. The modi-
fied nucleoside triphosphates were in-
corporated into DNA by primer exten-
sion by using Vent
G
tylsulfanylphenyl moieties produced
signals in [Co
(NH3)6]3+ ammonium
The electrochemical behaviour of the
alkylsulfanylphenyl nucleosides indicat-
ed formation of compact layers on the
electrode. Modified nucleotides and
DNA with incorporated benzyl- or tri-
ACHTUNGTRENNUNG
ˇ
buffer, attributed to the Brdicka cata-
lytic response, depending on the nega-
tive potential applied. Repeated con-
stant current chronopotentiometric
scans in this medium showed increased
ˇ
Brdicka catalytic response, which sug-
Keywords: DNA polymerases
electrochemistry · nucleosides · nu-
cleotides · organosulfur compounds
gests the deprotection of the alkylsul-
fanyl derivatives to free thiols under
the conditions.
Introduction
herently decreases the sensitivity of the electrochemical sen-
sors. In many applications, the free thiols can be replaced by
dialkylsulfides (in particular methylsulfanyl derivatives),[5]
which also bind efficiently to the gold surface. We envisaged
that preparation of ON probes with an alkylsulfanyl group
attached to a nucleobase through a conjugated system may
lead to the construction of more sensitive and accurate elec-
trochemical DNA sensors and may also potentially be used
for multiple attachment(s) of an ON to a metal surface to
form stable loops, hairpins and other structures.
Polymerase incorporations of base-modified 2’-deoxynu-
cleoside triphosphates (dNTPs) is now a very popular ap-
proach for the construction of functionalised oligonucleo-
tides and DNA.[6] It has been used for attachment of diverse
fluorescent,[7] redox[8] or spin[9] labels, and reactive function-
al groups.[10] Within our project of “multi-colour” redox la-
belling of DNA,[11] we are interested in the development of
other alternative electroreducible or -oxidisable labels (both
reversible and irreversible) with different redox potentials.
Alkylsulfanyl groups, apart from being anchors for binding
to metal surfaces,[5] are potentially interesting both as redox
labels and as precursors of free thiols by chemical deprotec-
tion[12] or electroreduction.[13]
Attachment of thiol-end-labelled oligonucleotides (HS-
ONs) at gold surfaces to facilitate the formation of a DNA
self-assembled monolayer (SAM)[1] is one of the most fre-
quently used methods of DNA immobilisation on solid sur-
faces. This approach is widely applied in the construction of
diverse sensors of DNA hybridisation, DNA chips and other
important technologies.[2] In addition, formation of a SAM
of HS-ONs at mercury electrodes[3] was used to generate
electrochemical sensors of DNA hybridisation. In all of
these applications, the HS-ONs used are end-labelled with a
thiol group tethered by a longer alkyl linker. The conforma-
tional behaviour of such HS-ON probes is quite complex
and not yet fully understood,[4] and the separation of the
ON probe from the thiol group by the saturated tether in-
ˇ
[a] Dr. H. Macꢀckovꢁ-Cahovꢁ, 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)220183559
The goals of our present study were the synthesis of nu-
cleosides, nucleotides and dNTPs bearing either a free sul-
fanyl- or alkylsulfanylphenyl group attached directly to a
nucleobase and the development of polymerase incorpora-
tions of the sulfur-containing dNTPs into DNA. Additional-
ly, we studied the electrochemical behaviour of the modified
nucleotides and DNA and their assembly onto the metal
electrode surfaces.
E-mail: hocek@uochb.cas.cz
Homepage: http://www.uochb.cas.cz/hocekgroup
ˇ
ˇ
[b] Dr. P. Horꢁkovꢁ, Dr. L. Havran, J. Spacek, Prof. Dr. M. Fojta
Institute of Biophysics, v.v.i.
Academy of Sciences of the Czech Republic
Kralovopolska 135, 61265 Brno, Czech Republic
Fax : (+420)541211293
E-mail: fojta@ibp.cz
Homepage: http://www.ibp.cz/labs/LBCMO/
Chem. Eur. J. 2011, 17, 5833 – 5841
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5833

Results and Discussion
Chemistry: Previously, we discovered that the best position
for attachment of a modification at pyrimidine nucleobases
is the 5-position, whereas purines should be replaced by 7-
deazapurines modified at the 7-position.[14] Such modifica-
tions are accommodated into the major groove of DNA in
which they do not destabilise duplexes (in some cases they
even stabilise them). Moreover, dNTPs modified at these
positions are good substrates for polymerases and can be en-
zymatically incorporated into DNA. Therefore, our target
compounds were nucleotides functionalised with alkylsulfa-
nylphenyl groups at the 5- (pyrimidine) or 7-position (deaza-
purine), respectively.
Suzuki–Miyaura cross-coupling reactions are considered
to be the superior method for introduction of functionalised
aryl groups to unprotected nucleosides or nucleotides in
aqueous solutions.[15] Therefore, our project started with a
model study of cross-couplings of halogenated nucleosides
with alkylsulfanylphenylboronic acids (PBARS). Because the
free-sulfanyl phenylboronic acid PBAHS was presumed to be
potentially unreactive because of Pd-catalyst poisoning, we
selected stable methylsulfanyl derivatives, as well as more
labile benzyl- and tritylsulfanyl derivatives with potentially
cleavable protecting groups at sulfur. Sulfanyl-, methylsul-
fanyl- and benzylsulfanylphenylboronic acids were commer-
cially available, whereas novel S-trityl-protected sulfanyl-
phenylboronic acid was prepared in excellent yield by the
reaction of triphenylmethanol with PBAHS in the presence
of trifluoroacetic acid (TFA) in dichloromethane
(Scheme 1).[16]
Scheme 2. Synthesis of sulfur-containing nucleosides.
[(alkylsulfanyl)phenyl]-7-deaza-2’-deoxyadenosines dAMeS
dABnS and dATrS in excellent yields (76–92%, Scheme 2).
Similarly, the same boronic acids reacted with dCI to give
,
the corresponding 5-substituted 2’-deoxycytidines dCMeS
,
dCBnS and dCTrS in good yields (78–82%, Scheme 2).
Apparently, the free HS-containing nucleosides could not
be prepared by direct cross-couplings, therefore, possible de-
protection methods were considered for the cleavage of the
benzyl or trityl protecting groups. Unfortunately, the portfo-
À
lio of R S cleavage methods operable under mild condi-
tions, and thus compatible with fragile nucleosides or nucle-
otides, is very limited. Therefore, we focused on the S-detri-
tylation.
Several methodologies for cleavage of this group are
known, but most of them were excluded due to the require-
ment for harsh reaction conditions or extremely toxic re-
agents (e.g. HCl in AcOH (aq);[17] Hg
ACTHNUTRGNE(UNG OAc)2, reflux, then
H2S;[17] (SCN)2;[18] electrolysis;[19] PhHgOAc, then H2S[20]).
Two methods were selected as potentially useful for our pur-
poses. Treatment of dATrS with triethylsilane in dichlorome-
thane and TFA[21] gave an inseparable mixture of products.
On the other hand, the reaction of dATrS with AgNO3[22] pro-
ceeded smoothly (TLC analysis showed complete conver-
sion of the starting compound to an insoluble polar product)
to give a silver salt of the desired thiolate dAHS. The proce-
dure was followed by trapping of Ag+ by dithioerythritol
(DTE) under mild conditions[22] (Scheme 3). Unfortunately,
the crude product obtained after the treatment with DTE
was unstable and decomposed during workup and chromato-
graphic purification. The only product isolated after a com-
bination of silica gel and reversed-phase flash chromatogra-
phy was the disulfide dinucleoside dASSdA, in a low yield of
13%. Apparently, even if an efficient method for the syn-
thesis of free sulfanylphenyl nucleosides was developed,
Scheme 1. Phenylboronic acids used in this study. i) TrOH, TFA, CH2Cl2,
RT, 1 h (95%).
Iodinated nucleosides, 7-iodo-7-deaza-2’-deoxyadenosine
(dAI) and 5-iodo-2’-deoxycytidine (dCI) were selected as
model starting compounds for the development of the aque-
ous Suzuki–Miyaura cross-coupling methodology. Previously
reported conditions (PdACTHNUTGRNE(UNG OAc)2, P(m-C6H4SO3Na)3 (TPPTS)
ligand, Cs2CO3, 2:1 water/acetonitrile)[4] were applied in re-
actions with PBAHS and PBARS. The reactions were per-
formed at 1008C for 30 min, for compatibility with the syn-
thesis of labile dNTPs. The attempted reactions of dAI or
dCI with PBAHS did not proceed, which confirmed the ex-
pected outcome of catalyst poisoning. On the other hand, all
of the 4-(alkylsulfanyl)phenylboronic acids (PBAMeS
PBABnS and PBATrS) reacted with dAI to give the desired 7-
,
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Chem. Eur. J. 2011, 17, 5833 – 5841

Alkylsulfanylphenyl Derivatives of Cytosine and 7-Deazaadenine Nucleosides
FULL PAPER
Scheme 3. Attempted trityl deprotection of dATrS
.
Scheme 5. Synthesis of sulfur-containing dNTPs. i) 1. PdACHTNUTRGNEG(UN OAc)2, Cs2CO3,
these compounds would be very unstable and prone to oxi-
dation to disulfides. Therefore, no further attempts on the
thiol nucleosides were pursued and we focused on the syn-
thesis and use of the more stable alkylsulfanyl nucleotides.
To act as simplified models of DNA for electrochemical
studies, a small series of 5-(alkylsulfanylphenyl)-2’-deoxycy-
tidine monophosphates (dCRSMP) was prepared. Thus, the
TPPTS, CH3CN, H2O, 1008C, 30 min; 2. Dowex 50WX8 (Na+ cycle) con-
version to sodium salts.
dAITP and dCITP with the series of alkylsulfanylphenylbor-
onic acids PBARS proceeded under the same conditions
within 30 min. (Scheme 5), with similar efficiency as for the
monophosphates (Scheme 4). However, the cross-coupling
reactions were accompanied by unwanted partial hydrolysis
of the dNTPs (both starting compounds and products) to di-
phosphates. The hydrolytic reactions dramatically decreased
the isolated yields of the dNTPs. In all cases, the desired tri-
phosphates dNRSTP were isolated by semi-preparative
HPLC and were accompanied by substantial amounts (15–
25%) of the corresponding diphosphates. The isolated yields
of the methyl- and benzylsulfanylphenyl derivatives
(dAMeSTP, dABnSTP, dCMeSTP and dCBnSTP) were acceptable
(26–50%), whereas the tritylsulfanylphenyl derivatives
(dATrSTP and dCTrSTP) were only obtained in moderate
yields of 20 and 10%, respectively. An alternative synthesis
of dCTrSTP by triphosphorylation of 5-tritylsulfanyl-2’-de-
reactions
of
5-iodo-2’-deoxycytidine
monophosphate
(dCIMP) with boronic acids PBARS under the conditions de-
scribed above proceeded well to give the methylsulfanyl and
benzylsulfanyl derivatives dCMeSMP and dCBnSMP in good
yields (Scheme 4). The reaction with PBATrS gave a much
lower conversion and the desired nucleotide dCTrSMP was
isolated in only 14% yield.
Finally, we applied the cross-coupling reactions for the
synthesis of modified dNTPs, substrates for polymerase in-
corporation to DNA. The reactions of iodinated dNTPs
AHCTUNGTRENNUNG
oxycytidine under standard conditions[8c] was attempted but
the yield was also low (approximately 15%).
Enzymatic incorporations: The sulfur-containing dNTPs
were then tested as substrates of DNA polymerases, to
serve as building blocks for enzymatic DNA synthesis.
Primer extension (PEX) experiments with dAMeSTP,
dABnSTP, dATrSTP, dCMeSTP, dCBnSTP and dCTrSTP were per-
formed with VentACTHNUTRGNEUNG
(exo-) DNA polymerase and temprnd16 tem-
plate (see the Experimental Section). The PEX reaction ex-
tended the primer by 16 nucleotides, which included four A
Scheme 4. Synthesis of sulfur-containing 2’-deoxycytidines. i) 1. PdACTHNUTRGNE(UNG OAc)2,
Cs2CO3, TPPTS, CH3CN, H2O, 1008C, 30 min; 2. Dowex 50WX8 (Na+
cycle) conversion to sodium salts.
and four C nucleotides. The VentACTHUGNETRNNU(G exo-) polymerase showed
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5835

M. Hocek, M. Fojta et al.
good tolerance towards the presence of the sulfur modifica-
tions on the dNTPs. Namely, the enzymatic incorporation of
dAMeSTP, dABnSTP, dCMeSTP and dCBnSTP gave the desired
full-length products with four modifications within one
DNA molecule in every case (Figure 1, lines 4, 5, 9 and 10).
presence of the sulfur groups and/or strongly adsorbing aro-
matic moieties coupled to the nucleobases. The 2’-deoxynu-
cleosides and 2’-deoxynucleoside monophosphates showed
cathodic peaks due to the nucleobase reduction in acetate
buffer, pH 5.0. This is illustrated in Figure 2 for cytosine de-
rivatives, which produced peak Cred. This peak was shifted
towards less negative potentials by 50 mV in dCBnS relative
to unmodified 2’-deoxycytidine (dC) and by a further 40 mV
in dCBnSMP. The dCRS compounds produced two sharp
Figure 1. PEX with temprnd16 in the presence of: dCTP, dGTP, dTTP and
dATP (lines 2, 7); dCTP, dGTP and dTTP (line 3); dATP, dGTP and
dTTP (line 8); dCTP, dGTP, dTTP and dARSTP (lines 4, 5, 6); dATP,
dGTP, dTTP and dCRSTP (lines 9, 10, 11). Line 1: 32P radiolabelled
primer.
The incorporation of dATrSTP and dCTrSTP was more com-
plicated (Figure 1, lines 6 and 11); mixtures of products of
various lengths were observed in both cases (Figure 1).
Other DNA polymerases (DyNAzyme and Klenow frag-
ment) were tested with similar results (not shown). The
methyl- and benzylsulfanyl dNTPs (dAMeSTP, dABnSTP,
dCMeSTP and dCBnSTP) are excellent substrates, with good
potential for the construction of sulfur-containing DNA,
whereas the tritylsulfanylphenyl group is probably too bulky
to be incorporated by the polymerase at multiple positions
in random sequences. Nevertheless, even the bulky dATrSTP
and dCTrSTP are substrates for polymerases and can be in-
corporated into DNA.
Electrochemistry: Electrochemical and interfacial properties
of the title compounds, their respective building blocks and
modified ONs were studied by means of cyclic voltammetry
(CV), AC voltammetry (ACV) or constant current chrono-
potentiometric stripping (CPS) at basal plane pyrolytic
graphite (PGE) or hanging mercury drop (HMDE) electro-
des. At the PGE we only observed the oxidation signal of
the 7-deazaadenine residue[11,23] in the dARS compounds and
their respective monophosphates and triphosphates (not
shown). The behaviour of the alkylsulfanylphenyl nucleo-
sides at the HMDE was more complex, which reflected the
Figure 2. A) CV in sodium acetate buffer, pH 5.0. dCBnS (c); dCBnSMP
(c); dC (c); PBABnS (c); background electrolyte (g). Insets:
details of CV measured in i) sodium acetate buffer; ii) borax buffer.
B) ACV of dCRS: R=methyl (c), benzyl (c), trityl (c); dCBnSMP
(c); background electrolyte (g). Inset: detail. C) ACV of PEXrnd16
products. Unmodified (c) and modified with 5-(RS)cytidine: R=
methyl (c), benzyl (c), trityl (c); background electrolyte (g).
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Alkylsulfanylphenyl Derivatives of Cytosine and 7-Deazaadenine Nucleosides
FULL PAPER
cathodic peaks (spikes) around À0.6 and À0.8 V in the ace-
tate buffer. These spikes were best developed in dCBnS (Fig-
ure 2A; peaks Sb1 and Sb2), followed by dCMeS, then dCTrS
(not shown). In borax buffer (pH 9.3), the spikes were shift-
ed to more negative potentials and the more negative Sb2
was much better developed than at the weakly acidic pH
(Figure 2A, inset i). The spikes were only observed with the
alkylsulfanylphenyl-modified nucleosides; neither of the
building blocks (unmodified nucleosides, PBARS) nor the
dNRSMP (N=A or C) compounds produced such effects,
which suggests the involvement of both the nucleobase and
the RS moiety. Occurrence of sharp spikes in voltammo-
grams has been shown to be connected with phase transi-
tions in compact, two-dimensionally condensed layers of
various surface-active substances (including nucleic acid
components) at electrically charged surfaces.[24] The 2D con-
densation processes are usually connected with a strong de-
crease of differential capacity of the electrode bilayer in AC
voltammetric modes, reflected in the decrease of capacitive
currents well below values that correspond to a clean elec-
trode surface in the background electrolyte. Formation of
capacitance pits with flat bottoms and distinct edges that
correspond to the above mentioned spikes are also ob-
served, at potentials where the phase transitions (condensa-
tion/reorientation/disruption of the compact layers) occur.
ACV of the modified nucleosides revealed such behaviour
in the borax buffer for all three dCRS compounds (Fig-
ure 2B), but not for unmodified dC, dCRSMP or dCRSTP.
The tendency towards compact-layer formation and its sta-
bility at the negatively charged HMDE surface decreased in
the order dCBnS >dCMeS >dCTrS. Hence, attachment of the al-
kylsulfanylphenyl groups to dC contributed significantly to
adsorption of the nucleoside at the HMDE surface to form
the compact layers. In dCBnS, the third sterically uncon-
strained planar aromatic moiety further enhanced these ef-
fects, whereas the presence of the bulky, non-planar trityl
group in dCTrS made formation of a regular surface structure
less feasible. In the dNRSMP and dNRSTP series, electrostatic
repulsion between the molecules and between a molecule
and the negatively charged electrode surface prevented the
condensation processes. More detailed analysis of the prop-
erties of adsorption and interactions at the electrode surfa-
ces is out of the scope of this paper and will be published at
a later date.
(with the exception of dCTrS, which produced an extra signal
close to À0.9 V, Figure 2C). Similarly, CV analysis in neutral
ammonium formate background electrolyte[25] showed redox
processes of C, A and G nucleobases but no significant con-
tribution from the conjugate groups (not shown).
Further, we studied the behaviour of the modified dNMPs
and ONs in ammonium buffer that contained [CoACTHNUTRGNEUNG
(NH3)6]3+
ions by CPS. In solutions that contain cobalt ions, thiol and
disulfide compounds are known to give characteristic cata-
ˇ
lytic currents (namely, the Brdicka catalytic response, BCR)
at mercury[26] and silver amalgam electrodes,[27] whereas
thioACTHUNRTGNEUNGethers have been reported to be inactive. Notably, our
results revealed the BCR appearance with dNMPs and ONs
that possessed trityl- or benzylsulfanylphenyl groups
(Figure 3), suggestive of deprotection of the thiol directly at
ACV traces of the oligonucleotide PEX products with in-
corporated sulfanyl groups did not significantly differ from
those of unmodified ONs. For dCRS (Figure 2C), two capaci-
tive peaks were produced at potentials À1.20 and À1.34 V
due to reorientation/desorption of the polyanionic ON
chains at the negatively charged electrode surface.[25] Such
behaviour was in agreement with the strong adsorption of
single-stranded nucleic acids at mercury and amalgam elec-
trodes through hydrophobic nucleobase residues. The excess
of unmodified nucleotides in flat-lying ON chains at the
HMDE[3] dictated the overall behaviour of the ONs that
contained the alkylsulfanylphenyl conjugates, the contribu-
tions of which were insignificant under the given conditions
Figure 3. A) BCR of dCTrSMP after pre-polarisation to negative poten-
tials. À0.25 V (c), À0.4 V (c), À0.5 V (c), À0.6 V (c), À0.75 V
(c), background electrolyte (a). Peaks Co and Co’ correspond to re-
duction of CoII and its complexes; peaks a, b and c have been attributed
to catalytic hydrogen evolution in the presence of Co and thiol species.
B) BCR of PEXRS products modified with: CMeS (c), CBnS (c), CTrS
(c); negative PEX control (no modified base incorporated, c).
Dashed and solid curves correspond to first and second scans, respective-
ly, of the same DNA adsorbed at the HMDE. The peak denoted as
“DNA” is due to adsorption/desorption processes of ON chains. Inset:
detailed section. Peaks Co and Co’ correspond to reduction of CoII and
its complexes; peaks a, b and c have been attributed to catalytic hydro-
gen evolution in the presence of Co and thiol species.
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5837

M. Hocek, M. Fojta et al.
the electrode in the cobalt-containing medium, depending
on the negative potential applied. Compound dCTrSMP was
exposed to various negative potentials (by applying poten-
tial cycles between 0.0 V and the given more negative poten-
tial) prior to measurement of the BCR. As shown in Fig-
ure 3A, there was practically no signal in the region of the
BCR (i.e. more negative than the cobalt reduction peak)
after pre-polarisation to À0.25 V. Application of potentials
of À0.4 or À0.5 V resulted in the appearance of a small
peak a close to À1.3 V (Figure 3A, peak a). After pre-polar-
isation to À0.6 V, a peak around À0.5 V appeared (Fig-
ure 3A, peak b). Pre-polarisation to À0.75 V resulted in a
significant increase of the intensity of peak b and appear-
ance of another signal at À1.62 V (Figure 3A, peak c). In
the absence of cobalt, there were no such effects, which
strongly suggests that the appearance of peaks b and c was
due to reductive deprotection of the thioether groups to
free thiols.
to DNA (for example, nucleobases would be reduced under
these conditions). Our results indicate that in the aqueous
ammonium buffer, the complex redox electrochemistry of
[CoACHTNUTGRNEUNG
(NH3)6]3+ coupled with catalytic hydrogen gas evolution
À
might facilitate the C S cleavage under less drastic condi-
tions (Scheme 6).
Figure 3B shows the results from CPS of PEXrnd16 prod-
ucts generated from dCRSTP conjugates. It has been shown
previously that unmodified DNA did not give the BCR,
whereas end-alkylthiolated ONs produced specific catalytic
currents in the presence of cobalt ions.[28] In accordance with
the former observation, unmodified PEXrnd16 did not pro-
duce the BCR. The only distinct signal (around À1.5 V),
produced by the unmodified PEXrnd16 at potentials more
negative than the cobalt reduction peak, was dependent on
the presence of neither cobalt ions (not shown), nor the
modified nucleotides (compare curves in Figure 3B). Most
likely the signal corresponded to the tensammetric DNA
signal (see ACV traces in Figure 2C). The PEX product ob-
tained from dCMeSTP also did not show any significant ef-
fects. On the other hand, ONs that contained 5-benzylsul-
fanyl- or 5-tritylsulfanylphenyl cytidine gave distinct signals
in the presence, but not absence, of cobalt ions. These sig-
nals were attributed to the BCR and suggest the presence of
unprotected thiol groups. PEXrnd16 derived from dCBnSTP
produced only a weak BCR peak at À1.7 V (Figure 3B,
peak c) in the first CPS scan. The signal increased signifi-
cantly in the second CPS scan, which suggested deprotection
of more thiol groups due to repeated negative polarisation
of the HMDE with adsorbed modified DNA and/or reorien-
tation of the DNA layer to facilitate communication of the
sulfur groups with the electrode surface. For the PEX prod-
uct containing CTrS, stronger BCR responses were observed
and the effect of repeated potential scanning on the BCR
intensity was observed as well. Notably, the ON containing
CMeS did not give the BCR signal, even in the second CPS
scan. Such behaviour accorded well with a more difficult re-
Scheme 6. Debenzylation of benzylsulfanylphenyl-modified DNA by
ˇ
electroreduction followed by the Brdicka reaction.
Conclusion
Methyl-, benzyl- and tritylsulfanylphenyl-substituted 7-de-
AHCTUNGTREGaNNUN zaadenosine and cytosine nucleosides, nucleotides and nu-
cleoside triphosphates were prepared by Suzuki–Miyaura
cross-coupling reactions of halogenated nucleosides and nu-
cleotides with the appropriate alkylsulfanylphenylboronic
acids. The attempted deprotection of the tritylsulfanylphenyl
derivatives was unsuccessful. The methyl- and benzylsulfanyl
dNTPs (dAMeSTP, dABnSTP, dCMeSTP and dCBnSTP) were
good substrates for DNA polymerases and were successfully
incorporated into DNA by primer extension. The trityl de-
rivatives (dATrSTP and dCTrSTP) were less suitable sub-
strates; they were incorporated but did not lead to fully ex-
tended products.
Electrochemical deprotection of the alkylsulfanylphenyl
nucleosides and DNA by using literature conditions[29] was
not considered due to the incompatibility of highly negative
potentials with the preparation of functional DNA probes.
However, a remarkable tendency to condensation of the al-
kylsulfanyl nucleosides into layers at the electrode was ob-
served, which suggests that they might bind to the metal sur-
face. Moreover, behaviour of the benzyl- or trityl-protected
DNA in [CoACTHNUTRGNEUNG
(NH3)6]3+-containing ammonium buffer sug-
À
À
ductive cleavage of the Me S bond relative to Bn S and, es-
gests that electrochemical deprotection may be achieved at
potentials less negative than the potentials of nucleobase re-
duction.[25] Repeated potential scans in this medium showed
increased formation of characteristic peaks of the BCR,
which indicates the presence of free (deprotected) SH
groups. The most promising building block for the applica-
tions is dCBnSTP, which is perfectly incorporated by DNA
polymerases to give full-length DNA products and appears
À
pecially, Tr S bonds.
It has been reported that benzyl or trityl protective
groups were cleaved electrochemically from aryl thiols, cys-
teine and peptides at platinum or mercury electrodes in am-
monia, methanol or DMF solutions only at highly negative
potentials (approximately À2.5 V).[29] Such cleavages are not
useful for analytical or preparative applications with regards
5838
www.chemeurj.org
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 5833 – 5841

Alkylsulfanylphenyl Derivatives of Cytosine and 7-Deazaadenine Nucleosides
FULL PAPER
2H; H-5’), 4.19 (td, J=4.0, 3.2 Hz, 1H; H-4’), 4.51 (dt, J=6.3, 3.2 Hz,
1H; H-3’), 6.33 (dd, J=7.5, 6.2 Hz, 1H; H-1’), 7.37 (m, 2H; H-o-phenyl-
ene), 7.41 (m, 2H; H-m-phenylene), 7.75 ppm (s, 1H; H-6); 13C NMR
to be efficiently deprotected by electrochemical reduction at
moderate potentials in the presence of cobalt ions. Exploita-
tion of these outcomes would enable immobilisation of ON
probes at the electrode surfaces by using protected building
blocks and enzymatically constructed probes to avoid oxida-
tive dimerisation or coupling to other thiol species (such as
proteins), followed by deprotection directly at the electrode
to which the probe is to be attached. Recently, it has been
shown that not only traditional gold electrodes, but also
mercury electrodes, are suitable for the preparation of thiol-
terminated ON monolayers.[3] Moreover, formation of such
monolayers at mercury-based electrodes (including silver
amalgam) has been reported to be more facile. Above and
beyond the prospective utilisation of the sulfur moieties as
anchors, we have shown that the BCR in the presence of
cobalt ions can be exploited analytically to monitor the
modified ON synthesis.
(125.7 MHz, D2O, ref
(CH3S), 42.06 (CH2-2’), 49.39 (CH3CH2N), 67.34 (d, J
CH2-5’), 73.91 (CH-3’), 88.40 (d, J(C,P)=8.5 Hz; CH-4’), 89.03 (CH-1’),
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
112.84 (C-5), 129.37 (CH-m-phenylene), 131.55 (C-i-phenylene), 132.62
(CH-o-phenylene), 141.34 (C-p-phenylene), 142.41 (CH-6), 159.49 (C-2),
167.19 ppm (C-4); 31P{1H} NMR (202.3 MHz, D2O): d=1.26 ppm; MS
(ESIÀ): m/z (%): 428 (100) [M]+; HRMS: m/z calcd for C16H19O7N3PS:
428.0687; found: 428.0687.
7-[(4-Methylsulfanyl)phenyl]-7-deaza-2’-deoxyadenosine
phate (dAMeSTP): Yield: 38%; 1H NMR (500.0 MHz, D2O, pD =7.1,
phosphate buffer, ref(dioxane)=3.75 ppm): d=2.44 (ddd, J=14.0, 6.0,
5’-O-triphos-
AHCTUNGTRENNUNG
3.0 Hz, 1H; H-2’b), 2.52 (s, 3H; CH3S), 2.69 (ddd, J=14.0, 7.5, 6.0 Hz,
1H; H-2’a), 4.11 (brm, 1H; H-5’), 4.15 (brm, 1H; H-5’), 4.23 (brm, 1H;
H-4’), 4.74 (dt, J=6.0, 3.0 Hz, 1H; H-3’), 6.62 (dd, J=7.3, 6.4 Hz, 1H; H-
1’), 7.33 (m, 2H; H-m-phenylene), 7.36 (m, 2H; H-o-phenylene), 7.45 (s,
1H; H-8), 8.13 ppm (s, 1H; H-2); 13C NMR (125.7 MHz, D2O, pD =7.1,
phosphate buffer, ref
(CH2-2’), 68.30 (d, J(C,P)=5.6 Hz; CH2-5’), 73.90 (CH-3’), 85.48 (CH-1’),
87.85 (d, J(C,P)=8.5 Hz; CH-4’), 103.58 (C-5), 120.50 (C-7), 122.75 (CH-
ACHTUNGRTENN(UNG dioxane)=69.3 ppm): d=17.31 (CH3S), 40.98
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
8), 129.73 (CH-m-phenylene), 131.92 (CH-o-phenylene), 133.01 (C-i-
phenylene), 139.65 (C-p-phenylene), 152.48 (C-4), 153.69 (CH-2),
159.61 ppm (C-6); 31P{1H} NMR (202.3 MHz, D2O, pD =7.1, phosphate
buffer, ref(phosphate buffer)=2.35 ppm): À21.09 (brt, J=19.0 Hz; Pb),
À10.24 (d, J=19.0 Hz; Pa), À6.37 ppm (d, J=19.0 Hz; Pg); MS (ESIÀ):
m/z (%): 611 (100) [MÀH]+, 633 (40) [M+NaÀH]+; HRMS: m/z calcd
for C11H22O12N4P3S: 611.0173; found: 611.0160.
Experimental Section
General procedure for Suzuki–Miyaura cross-coupling reactions of halo-
genated deoxynucleosides with alkylsulfanylphenylboronic acids: Water/
acetonitrile mixture (2:1, 1.5 mL) was added through septum to an
argon-purged vial that contained halogenated dNTP (0.14 mmol), boron-
ic acid (0.28 mmol) and Cs2CO3 (228 mg, 0.7 mmol). A solution of Pd-
7-[(4-Benzylsulfanyl)phenyl]-7-deaza-2’-deoxyadenosine
phate (dABnSTP): Yield: 26%; 1H NMR (499.8 MHz, D2O, pD =7.1, ref-
(dioxane)=3.75 ppm): d=2.41 (ddd, J=13.9, 6.0, 3.2 Hz, 1H; H-2’b),
5’-O-triphos-
ACHTUNGTRENNUNG(OAc)2 (3.1 mg, 0.014 mmol) and TPPTS (40 mg, 0.07 mmol) in water/
AHCTUNGTRENNUNG
acetonitrile (2:1, 1.2 mL) was added and the mixture was stirred and
heated to 1008C for 30 min. Products were isolated from crude reaction
mixture by silica gel column chromatography with gradient elution
(CHCl3/0–10% MeOH) and evaporation of the solvents, then dried
under vacuum.
2.65 (ddd, J=13.9, 7.1, 6.2 Hz, 1H; H-2’a), 4.10 (dt, J=10.6, 4.5 Hz, 1H;
H-5’b), 4.12 (s, 2H; CH2S), 4.14 (dt, J=10.6, 4.5 Hz, 1H; H-5’a), 4.23 (td,
J=4.5, 3.2 Hz, 1H; H-4’), 4.71 (dt, J’ =6.2, 3.2 Hz, 1H; H-3’), 6.58 (dd,
J=7.1, 6.0 Hz, 1H; H-1’), 7.20–7.28 (m, 7H; H-o,m,p-Ph, H-o-phenyl-
ene), 7.30 (m, 2H; H-m-phenylene), 7.41 (s, 1H; H-8), 8.13 ppm (brs,
7-[(4-Benzylsulfanyl)phenyl]-7-deaza-2’-deoxyadenosine (dABnS): Yellow-
ish foam, 88%; m.p. 76–818C; 1H NMR (499.8 MHz, CD3OD): d=2.32
(ddd, J=13.4, 6.0, 2.7 Hz, 1H; H-2’b), 2.68 (ddd, J=13.4, 8.3, 6.0 Hz,
1H; H-2’a), 3.72 (dd, J=12.1, 3.6 Hz, 1H; H-5’b), 3.80 (dd, J=12.1,
3.2 Hz, 1H; H-5’b), 4.01 (ddd, J=3.6, 3.2, 2.7 Hz, 1H; H-4’), 4.13 (s, 2H;
CH2S), 4.53 (dt, J=6.0, 2.7 Hz, 1H; H-3’), 6.55 (dd, J=8.3, 6.0 Hz, 1H;
H-1’), 7.18 (m, 1H; H-p-Ph), 7.23 (m, 2H; H-m-Ph), 7.28 (m, 2H; H-o-
Ph), 7.30 (m, 2H; H-o-phenylene), 7.33 (m, 2H; H-m-phenylene), 7.36 (s,
1H; H-8), 8.12 ppm (brs, 1H; H-2); 13C NMR (125.7 MHz, CD3OD): d=
39.30 (CH2S), 41.42 (CH2-2’), 63.65 (CH2-5’), 73.03 (CH-3’), 86.48 (CH-
1’), 89.05 (CH-4’), 102.73 (C-5), 118.02 (C-7), 122.65 (CH-8), 128.15 (CH-
p-Ph), 129.41 (CH-m-Ph), 129.96 (CH-o-Ph), 130.21 (CH-o-phenylene),
131.28 (CH-m-phenylene), 133.50 (C-i-phenylene), 136.96 (C-p-phenyl-
ene), 138.92 (C-i-Ph), 151.07 (C-4), 152.22 (CH-2), 158.79 ppm (C-6); IR
(KBr): n˜ =3474, 3388, 3351, 3063, 1658, 1620, 1558, 1548, 1536, 1493,
1466, 1454, 1300, 1216, 1093, 1050, 700 cmÀ1; MS (ESI+): m/z (%): 449
(100) [M+H]+, 471 (80) [M+Na+H]+; HRMS: m/z calcd for
C24H24O3N4S: 449.1642; found: 449.1642.
1H; H-2); 13C NMR (125.7 MHz, D2O, pD =7.1, ref
69.3 ppm): d=40.45 (CH2S), 40.99 (CH2-2’), 68.35 (d, J(C,P)=5.6 Hz;
CH2-5’), 73.93 (CH-3’), 85.54 (CH-1’), 87.74 (d, J(C,P)=8.6 Hz; CH-4’),
ACHTUNGTRENUN(NG dioxane)=
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
103.44 (C-5), 120.41 (C-7), 123.03 (CH-8), 130.08 (CH-p-Ph), 131.35
(CH-m-Ph), 131.56 (CH-o-Ph), 131.80 (CH-o-phenylene), 133.12 (CH-m-
phenylene), 134.43 (C-i-phenylene), 136.78 (C-p-phenylene), 140.30 (C-i-
Ph), 152.34 (C-4), 153.13 (CH-2), 159.12 ppm (C-6); 31P{1H} NMR
(202.3 MHz, D2O, pD =7.1, ref(phosphate buffer)=2.35 ppm): d=
À21.04 (brdd, J=19.4, 18.4 Hz; Pb), À9.76 (d, J=19.4 Hz; Pa),
À7.32 ppm (d, J=18.4 Hz; Pg); MS (ESIÀ): m/z (%): 687 (100) [MÀH]+,
709 (20) [M+NaÀH]+; HRMS: m/z calcd for C24H25O12N4P3S/2:
343.0207; found: 343.0196.
7-[(4-Triphenylmethylsulfanyl)phenyl]-7-deaza-2’-deoxyadenosine 5’-O-
triphosphate (dATrSTP): Yield: 20%; 1H NMR (499.8 MHz, CD3OD):
d=1.29 (brt, J=6.9 Hz, 18H; CH3CH2N), 2.25 (ddd, J=13.1, 5.6, 2.9 Hz,
1H; H-2’b), 2.43 (ddd, J=13.1, 8.1, 5.6 Hz, 1H; H-2’a), 3.18 (brq, J=
6.9 Hz, 12H; CH3CH2N), 4.14 (m, 1H; H-4’), 4.22 (brt, J=5.0 Hz, 2H;
H-5’), 4.57 (brdt, J=5.6, 2.9 Hz, 1H; H-3’), 6.57 (dd, J=8.1, 5.6 Hz, 1H;
H-1’), 6.99 (m, 4H; H-o,m-phenylene), 7.22 (m, 9H; H-m,p-trityl), 7.41
(m, 6H; H-o-trityl), 7.54 (s, 1H; H-8), 8.23 ppm (brs, 1H; H-2);
13C NMR (150.9 MHz, CD3OD): d=9.12 (CH3CH2N), 41.16 (CH2-2’),
General procedure for Suzuki–Miyaura cross-coupling reactions of 5-
iodo-dCMP or halogenated dNTPs with alkylsulfanylphenylboronic
acids: The reactions were performed as described above for sulfur-con-
taining deoxynucleosides. Products were isolated from the crude reaction
mixture by HPLC (C18 column, linear gradient elution: 0.1m triethylam-
monium bicarbonate (TEAB) in H2O to 0.1m TEAB in 1:1 H2O/
MeOH). Several co-distillations with water and conversion to the sodium
salt form (Dowex 50WX8 in Na+ cycle) followed by freeze-drying from
water gave white solid products.
47.47 (CH3CH2N), 66.91 (d, J
ACHTUNGTRENNUNG
(C-trityl), 84.44 (CH-1’), 87.28 (d, JACHTUNGTRENNNUG
119.40 (C-7), 123.04 (CH-8), 127.99 (CH-p-trityl), 128.83 (CH-m-trityl),
129.32 (CH-o-phenylene), 131.18 (CH-o-trityl), 133.90 (C-i-phenylene),
135.25 (C-p-phenylene), 136.45 (CH-m-phenylene), 145.90 (C-i-trityl),
147.30 (CH-2), 150.21 ppm (C-4), C-6 not detected; 31P{1H} NMR
(202.3 MHz, CD3OD): d=À22.46 (br; Pb), À10.14 (d, J=19.8 Hz; Pa),
À9.44 ppm (d, J=18.6 Hz; Pg); MS (ESIÀ): m/z (%): 839 (20) [MÀH]+,
759 (50) [MÀPO3HÀH]+, 516 (100) [MÀPO3HÀTrÀH]+; HRMS: m/z
calcd for C36H33O12N4P3S/2: 419.0520; found: 419.0516.
5-[(4-Methylsulfanyl)phenyl]-2’-deoxycytidine
5’-O-monophosphate
(dioxane)=
(dCMeSMP): Yield: 73%; 1H NMR (499.8 MHz, D2O, ref
AHCTUNGTRENNUNG
3.75 ppm): d=1.27 (t, J=7.3 Hz, 9H; CH3CH2N), 2.33 (ddd, J=14.2, 7.5,
6.3 Hz, 1H; H-2’b), 2.45 (ddd, J=14.2, 6.2, 3.2 Hz, 1H; H-2’a), 2.53 (s,
3H; CH3S), 3.19 (q, J=7.3 Hz, 6H; CH3CH2N), 4.00 (dd, J=5.5, 4.0 Hz,
Chem. Eur. J. 2011, 17, 5833 – 5841
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5839

M. Hocek, M. Fojta et al.
5-[(4-Methylsulfanyl)phenyl]-2’-deoxycytidine
(dCMeSTP): Yield: 50%; 1H NMR (600.1 MHz, D2O, pD =7.1, phosphate
buffer, ref(dioxane)=3.75 ppm): d=2.36 (ddd, J=14.3, 7.3, 6.6 Hz, 1H;
H-2’b), 2.42 (ddd, J=14.3, 6.4, 3.3 Hz, 1H; H-2’a), 2.53 (s, 3H; CH3S),
4.15 (brm, 2H; H-5’), 4.22 (brm, 1H; H-4’), 4.60 (dt, J=6.6, 3.3 Hz, 1H;
H-3’), 6.34 (dd, J=7.3, 6.4 Hz, 1H; H-1’), 7.38 (m, 2H; H-o-phenylene),
7.42 (m, 2H; H-m-phenylene), 7.73 ppm (s, 1H; H-6); 13C NMR
5’-O-triphosphate
Electrochemical analysis: Nucleosides, dNMPs and other building blocks
were analysed in situ by conventional CV, ACV and CPS. The PEX prod-
ucts were analysed by ex situ (adsorptive transfer stripping) CV, ACV
and CPS. The PEX products were accumulated over 60 s from 5 mL ali-
quots that contained 0.2m NaCl at the surface of the working electrode
(HMDE). The electrode was then rinsed with deionised water and placed
in the electrochemical cell. CV settings: scan rate 0.5 VsÀ1, initial poten-
tial 0.15 V, switching potential À1.6 V (electrolyte: 0.2m acetate buffer,
pH 5.0); À1.9 V (electrolyte: 0.05m sodium tetraborate, pH 9.3), end po-
tential 0.15 V. ACV settings: frequency 230 Hz, amplitude 10 mV, initial
potential 0.15 V, end potential À1.9 V (electrolyte: 0.05m sodium tetrabo-
rate, pH 9.3). CPS settings: stripping current À10 mA, initial potential 0 V
ACHTUNGTRENNUNG
(150.9 MHz, D2O, pD =7.1, phosphate buffer, ref
d=17.07 (CH3S), 41.63 (CH2-2’), 68.04 (d, J(C,P)=5.4 Hz; CH2-5’), 73.36
(CH-3’), 88.16 (d, J(C,P)=8.7 Hz; CH-4’), 88.75 (CH-1’), 112.97 (C-5),
ACHTUNGTREN(NUNG dioxane)=69.3 ppm):
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG
129.42 (CH-m-phenylene), 131.62 (C-i-phenylene), 132.63 (CH-o-phenyl-
ene), 141.27 (C-p-phenylene), 142.35 (CH-6), 159.83 (C-2), 167.42 ppm
(C-4); 31P{1H} NMR (202.3 MHz, D2O, pD =7.1, phosphate buffer, re-
f(phosphate buffer)=2.35 ppm): d=À21.16 (brt, J=19.3 Hz; Pb), À10.52
(d, J=19.3 Hz; Pa), À6.31 ppm (d, J=19.3 Hz; Pg); MS (ESIÀ): m/z (%):
588 (100) [MÀH]+, 610 (40) [M+NaÀH]+; HRMS: m/z calcd for
C16H21O13N3P3S: 588.0013; found: 587.9992.
(electrolyte: 0.1m ammonium buffer+1 mm [CoACTHNUTRGNEUNG
(NH3)6]3+, pH 9.5). All
electrochemical measurements were performed with an Autolab analyzer
(Eco Chemie, The Netherlands) in connection with VA-stand 663 (Met-
rohm, Herisau, Switzerland). The three-electrode system was used with a
Ag/AgCl/3m KCl electrode as a reference and platinum wire as an auxili-
ary electrode. As a working electrode, HMDE with an area of 0.4 mm2
was used. CV measurements were performed after the solution was
purged with argon. All electrochemical measurements were performed at
room temperature.
5-[(4-Benzylsulfanyl)phenyl]-2’-deoxycytidine-5’-O-triphosphate
(dCBnSTP): Yield: 34%; 1H NMR (499.8 MHz, D2O, pD =7.1, ref-
ACHTUNGTRENNUNG(dioxane)=3.75 ppm): d=2.35 (ddd, J=14.1, 7.5, 6.3 Hz, 1H; H-2’b),
2.43 (ddd, J=14.1, 6.3, 3.4 Hz, 1H; H-2’a), 4.13 (ddd, J=11.3, 5.8, 4.3 Hz,
1H; H-5’), 4.16 (ddd, J=11.3, 5.8, 4.3 Hz, 1H; H-5’), 4.22 (td, J=4.3,
3.4 Hz, 1H; H-4’), 4.28 (s, 2H; CH2S), 4.59 (dt, J=6.3, 3.4 Hz, 1H; H-3’),
6.32 (dd, J=7.5, 6.3 Hz, 1H; H-1’), 7.30 (m, 1H; H-p-Ph), 7.34 (m, 2H;
H-o-phenylene), 7.35 (m, 2H; H-m-Ph), 7.42 (m, 2H; H-o-Ph), 7.47 (m,
2H; H-m-phenylene), 7.72 ppm (s, 1H; H-6); 13C NMR (125.7 MHz,
Acknowledgements
D2O, pD =7.1, ref
ACHTUNGTRENNUNG
This work was supported by the Academy of Sciences of the Czech Re-
public (Z4 055 0506, Z5 004 0507 and Z5 004 0702), the Ministry of Edu-
cation (LC06035, LC512), Grant Agency of the Academy of Sciences of
the Czech Republic (IAA400040901), Czech Science Foundation (203/09/
0317) and by Gilead Sciences, Inc. (Foster City).
2’), 68.08 (d, J(C,P)=5.7 Hz; CH2-5’), 73.44 (CH-3’), 88.17 (d, JACHTUGNTRENNNUG
G
8.7 Hz; CH-4’), 88.81 (CH-1’), 112.79 (C-5), 130.16 (CH-p-Ph), 131.49
(CH-m-Ph), 131.59 (CH-o-Ph), 132.63, 132.66 (CH-o,m-phenylene),
133.08 (C-i-phenylene), 138.45 (C-p-phenylene), 140.43 (C-i-Ph), 142.49
(CH-6), 159.77 (C-2), 167.28 ppm (C-4); 31P{1H} NMR (202.3 MHz, D2O,
pD=7.1, ref(phosphate buffer)=2.35 ppm): d=À21.38 (t, J=19.4 Hz;
Pb), À10.35 (d, J=19.4 Hz; Pa), À7.07 ppm (d, J=19.4 Hz; Pg); MS
(ESIÀ): m/z (%): 664 (50) [MÀH]+, 584 (100) [MÀPO3HÀH]+; HRMS:
m/z calcd for C22H24O13N3P3S/2: 331.5127; found: 331.5125.
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5-[(4-Triphenylmethylsulfanyl)phenyl]-2’-deoxycytidine 5’-O-triphosphate
(dCTrSTP): Yield: 10%; 1H NMR (499.8 MHz, CD3OD): d=1.29 (t, J=
7.0 Hz, 36H; CH3CH2N), 2.19 (ddd, J=13.7, 7.6, 6.7 Hz, 1H; H-2’b), 2.36
(ddd, J=13.7, 5.9, 3.1 Hz, 1H; H-2’a), 3.18 (q, J=7.0 Hz, 24H;
CH3CH2N), 4.11 (m, 2H; H-5’), 4.23 (m, 1H; H-4’), 4.55 (dt, J=6.7,
3.1 Hz, 1H; H-3’), 6.26 (dd, J=7.6, 5.9 Hz, 1H; H-1’), 7.08 (m, 4H; H-
o,m-phenylene), 7.21 (m, 3H; H-p-trityl), 7.26 (m, 6H; H-m-trityl), 7.41
(m, 6H; H-o-trityl), 7.69 ppm (s, 1H; H-6); 13C NMR (125.7 MHz,
CD3OD): d=9.11 (CH3CH2N), 41.14 (CH2-2’), 47.39 (CH3CH2N), 66.72
(d, J
ACHTUNGTRENNUNG
1’), 87.71 (d, JACHTUNGTRENNUNG
128.83 (CH-m-trityl), 130.01 (CH-o-phenylene), 131.18 (CH-o-trityl),
133.65 (C-i-phenylene), 135.89 (CH-m-phenylene), 136.51 (C-p-phenyl-
ene), 140.93 (CH-6), 145.84 (C-i-trityl), 157.54 (C-2), 165.33 ppm (C-4);
31P{1H} NMR (202.3 MHz, CD3OD): d=À22.54 (br; Pb), À10.51 (d, J=
20.6 Hz; Pa), À9.43 (d, J=18.8 Hz; Pg); MS (ESIÀ): m/z (%): 816 (40)
[MÀH]+, 736 (30) [MÀPO3HÀH]+, 493 ppm (100) [MÀPO3HÀTrÀH]+;
HRMS: m/z calcd for C34H33O13N3P3S: 816.0952; found: 816.0936.
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first crystal structure of DNA polymerase with a modified dNTP:
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Primer extension experiment: The reaction mixture (20 mL) contained
VentACHTUNGTRENNUNG(exo-) DNA polymerase (New England Biolabs, 0.1 unit), natural
dNTPs (Fermentas, 0.2 mm), modified surrogates dARSTP or dCRSTP
(0.2 mm), 3’-GGGTACGGCGGGTAC-5’ primer (Sigma–Aldrich oligoes,
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0.15 mm),
temprnd16
template
5’-CTAGCATGAGCTCAGTCC-
CATGCCGCCCATG-3’ (Sigma–Aldrich oligoes, 0.225 mm) in 1ꢃTher-
moPol reaction buffer. The primer was labelled with of [g32P]-ATP by
using standard techniques. Reaction mixtures were incubated for 30 min
at 608C in a thermal cycler and were stopped by addition of the stop so-
lution (40 mL, 80%v/v, formamide, 20 mm ethylenediaminetetraacetic
acid, 0.025%w/v, bromophenol blue, 0.025%w/v, xylene cyanol). Reac-
tion mixtures were separated by use of 12.5% denaturing polyacrylamide
gel electrophoresis. Visualisation was performed by phosphoimaging.
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Received: December 3, 2010
Published online: April 6, 2011
Chem. Eur. J. 2011, 17, 5833 – 5841
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5841