Enzymatic synthesis of 8-vinyl- and 8-styryl-2′-deoxyguanosine modified DNA - Novel fluorescent molecular probes

Article abstract of DOI:10.1016/j.bmcl.2012.03.056

Fluorescent analogs of the natural nucleobases are widely used as molecular probes for investigating DNA hybridization and topology. In this study the guanosine analogs 8-vinyl- and 8-styryl-2′-deoxyguanosine were synthesized and converted into the corres

Full text of DOI:10.1016/j.bmcl.2012.03.056

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3136–3139
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enzymatic synthesis of 8-vinyl- and 8-styryl-20-deoxyguanosine modified
DNA—novel fluorescent molecular probes
Bastian Holzberger a, , Julian Strohmeier b, , Vanessa Siegmund a, Ulf Diederichsen b,à, , Andreas Marx a,
⇑
⇑
a Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany
b Institute of Organic and Biomolecular Chemistry, Georg-August-University Göttingen, 37077 Göttingen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescent analogs of the natural nucleobases are widely used as molecular probes for investigating DNA
hybridization and topology. In this study the guanosine analogs 8-vinyl- and 8-styryl-20-deoxyguanosine
were synthesized and converted into the corresponding 50-triphosphates. These C8 modified nucleotides
were processed by various DNA polymerases to create fluorescent DNA. Whereas the 8-styryl modified
nucleotide somewhat hampers DNA synthesis 8-vinyl-20-deoxyguanosine is processed by DNA polymer-
ases emphasizing the broad applicability as a molecular probe for fluorescence spectroscopy.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 January 2012
Revised 12 March 2012
Accepted 14 March 2012
Available online 20 March 2012
Keywords:
DNA
Modified nucleotide
DNA polymerase
Fluorescent analog
Fluorescence spectroscopy
Fluorescence spectroscopy is a powerful tool for investigating
oligonucleotide conformation and dynamics. The molecular probes
for this technique are fluorescent analogs of the natural nucleo-
bases, for example, for detecting DNA hybridization or to discrimi-
nate between single nucleotide polymorphisms.1 Typically, these
probes are based on fluorophore-base conjugates in which common
fluorophores are tethered to the nucleobases.2 Other methods uti-
lize expanded nucleobases3 or nucleotides bearing a fluorescent
aromatic moiety instead of the natural nucleobase.4 Molecular
probes for investigating oligonucleotide conformation and dynam-
ics should mimic the natural nucleobases to minimize structural
disturbances.5 The most prominent example of this class of mole-
cules is the adenine analog 2-aminopurine.6 Other fluorescent
nucleobase analogs closely resemble their parent nucleobase,
mostly pyrimidine and adenine mimics. There are only a few exam-
ples for guanine analogs that match the requirements for a suitable
fluorescent probe.7
within its microenvironment, the newly introduced fluorescent
20-deoxyguanosine mimic is considered to have a potential compa-
rable to 20-aminopurine. To further expand the scope of applica-
tions for such fluorescent nucleotides by overcoming the
inherent restrictions of chemical synthesis on solid-phase, DNA
polymerases are promising tools to modify DNA in a site-specific
and efficient manner.9 Thereby, researchers have not only utilized
wild-type enzymes but made also use of engineered DNA polymer-
ases to accept modified 20-deoxynucleoside-50-triphosphates
(dNTPs).10 Among 8-substituted purines, especially dATP analogs
have been used to synthesize functionalized DNA, whereby some
of them were rather poor substrates for DNA polymerases.11
In this study, the authors have synthesized the corresponding
triphosphate of 8-vinyl-20-deoxyguanosine 1 for enzymatic synthe-
sis of fluorescent DNA. Additionally, we have investigated the enzy-
matic acceptance of 8-styryl-20-deoxyguanosine-50-triphosphate 2.
This fluorescent analog of 20-deoxyguanosine has already been
introduced into DNA by solid-phase synthesis12 and has been inves-
tigated additionally on nucleoside basis concerning its fluorescent
properties.13 It turned out that despite the increased steric size of
the guanosine analog, the B-form structure of DNA is almost unal-
tered.12 As the styryl modification underlies efficient and reversible
cis–trans photoisomerization13 it has also been shown that photo-
chemical and physical properties can be controlled by external light
stimuli. The conformational switching leads to dramatic changes in
fluorescence intensities,13 the thermal stability of duplex DNA12
and guanosine self-assembling capability14 reflecting the potential
of 8-styryl-20-deoxyguanosine to photochemically control nucleic
acid structures.
Recently, we reported the efficient synthesis of the 8-vinyl-20-
deoxyguanosine phosphoramidite building block for solid-phase
DNA synthesis and the use of 8-vinyl-20-deoxyguanosine as a
fluorescent probe for investigating DNA base pairing and the
formation of different topologies.8 As the emission properties of
8-vinyl-20-deoxyguanosine are very sensitive towards changes
⇑
Corresponding authors. Tel.: +49 551 39 3221; fax: +49 551 39 22944
(U. Diederichsen), tel.: +49 7531 88 5139; fax: +49 7531 88 5140 (A. Marx).
E-mail addresses: udieder@gwdg.de (U. Diederichsen), andreas.marx@
uni-konstanz.de (A. Marx).
These authors contributed equally to this work.
Tel.: +49 551 39 3221; fax: +49 551 39 22944.
à
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.056

B. Holzberger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3136–3139
3137
The synthesis of 8-vinyl-20-deoxyguanosine-50-triphosphate
(VdGTP) 1 (Scheme 1) was carried out starting with bromination
at the C8 position of 20-deoxyguanosine 3 with NBS and precipita-
tion in acetone as an efficient purification step.15 Nucleoside 4 was
subjected to Stille coupling with tributyl–vinyltin to give the vinyl-
substituted derivative 5 in good yields.16 The modified triphos-
phate 1 was generated using an established method.17
8-Styryl-20-deoxyguanosine-50-triphosphate (SdGTP) 2 was also
synthesized in three steps from commercially available 20-deoxygua-
nosine (Scheme 2). Bromination with NBS at position C8 followed by
Suzuki–Miyaura cross-coupling with trans-2-phenylvinylboronic
acid yielded compound 6 in good yields.13,18 The corresponding tri-
phosphate 2 was synthesized using a protection-free one-pot syn-
thesis protocol introduced by Huang.19
To investigate the acceptance of VdGTP 1 and SdGTP 2 by DNA
polymerases we carried out primer extension reactions (Fig. 1).
Thereby, we used a radioactively 50-32P labelled DNA primer and
a DNA template containing three cytosines that call for the incorpo-
ration of either dGMP, VdGMP or SdGMP, respectively (Fig. 1a).
After incubation in presence of dNTPs and DNA polymerase reac-
tions were analyzed by denaturing polyacrylamide gelelectropho-
resis (PAGE) and visualized by phosphor imaging. We used the
large fragment of the thermophilic Taq DNA polymerase (KlenTaq)
from family A and the two B-family DNA polymerases KOD (exoꢀ)
and Therminator (A485L mutant of Thermococcus species 9°N-7
DNA polymerase (exoꢀ)).
Scheme 2. Synthesis of 8-styryl-20-deoxyguanosine-50-triphosphate 2. Reagents
and conditions: (a) trans-2-phenylvinylboronic acid, sodium carbonate, tris(3-
sulfophenyl)phosphine trisodium salt, palladium(II) acetate, water/acetonitrile 2:1,
16 h, 80 °C, 72%; (b) 2-chloro-4-H-1,2,3-benzodioxaphosphorin-4-one, bis-tri-n-
butylammonium pyrophosphate, n-tributylamine, N,N-dimethylformamide then
nucleoside 6, iodine solution, 21%.
As expected, if only dATP, dTTP, and dCTP but neither natural
dGTP nor any modified guanosine analog are present (Fig. 1;
ꢀdGTP) the primer strand cannot be elongated to the full length
of 35 nucleotides (nt) by any of the DNA polymerases. Interest-
ingly, KlenTaq DNA polymerase stops primer elongation mainly
by the first cytosine displaying strong termination bands at posi-
tion 24 and 25. As DNA synthesis is also inhibited after incorporat-
ing a mismatched nucleotide opposite the templating cytosine
primer fragments with 28 nt length are prominent as well. In con-
trast, under the applied conditions primer elongation catalyzed by
KOD and Therminator DNA polymerase is mainly paused by reach-
ing the second cytosine at position 30. However, in presence of all
four natural nucleotides (Fig. 1; +dGTP) all DNA polymerases are
able to extend the primer to full-length oligonucleotide. The
Figure 1. Primer extension studies: incorporation of (b) VdGMP and (c) SdGMP by
KlenTaq, KOD, and Therminator (Therm.) DNA polymerase. (a) Partial primer
template sequences employed. (b) and (c) Primer: primer only; ꢀdGTP: primer
extension in the presence of dATP, dTTP, and dCTP; +dGTP: as ꢀdGTP but in the
presence of dGTP; +VdGTP: as ꢀdGTP but in the presence of VdGTP 1; +SdGTP: as
ꢀdGTP but in the presence of SdGTP 2.
incorporation of an additional nucleotide in a non-template direc-
ted manner leading to 36 nt long products has been observed be-
fore using 30–50 exonuclease-deficient DNA polymerases.20
Notably, the use of Therminator DNA polymerase resulted in addi-
tional, slower migrating products probably caused by different
annealing sites and further elongation under the applied reaction
conditions that are too excessive for simple DNA synthesis in pres-
ence of natural dNTPs. By replacing natural dGTP with VdGTP 1 we
observed the formation of full-length products for all tested DNA
polymerases (Fig. 1b; +VdGTP). This clearly demonstrates that
VdGTP is not only processed by DNA polymerases but it also shows
Scheme 1. Synthesis of 8-vinyl-20-deoxyguanosine-50-triphosphate (VdGTP) 1.
Reagents and conditions: (a) N-bromosuccinimide, acetonitrile/water 4:1, 1 h, rt,
91%; (b) tributyl(vinyl)stannane, tetrakis(triphenylphosphine)-palladium(0), N-
methyl-2-pyrrolidone, 2 h, 110 °C, 80%; (c) 1,8-bis-(dimethylamino)naphthalene,
phosphorus oxychloride, trimethyl phosphate then bis-tri-n-butylammonium
pyrophosphate, n-tributylamine, 14%.

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B. Holzberger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3136–3139
that incorporated VdG units can be further elongated in primer
extension reactions. Next, we studied the action of the even more
Considering the incorporation opposite matching cytosine, KOD
and Therminator DNA polymerase were able to process VdGTP as
efficiently as natural dGTP whereas KlenTaq DNA polymerase
showed a drastically decreased incorporation efficiency in pres-
ence of VdGTP (Fig. 2b–d; N@C). Notably, higher concentrations
of VdGTP inhibited the incorporation step catalyzed by KOD and
Therminator DNA polymerase yielding less elongated primer prod-
ucts. Interestingly, we observed only opposite the mismatching
thymidine (N@T) some incorporation of VdGMP. In contrast, natu-
ral dGMP was apparently better incorporated opposite all mis-
matching nucleotides (Fig. 2b–d; N@G, A, T). In case of KlenTaq
DNA polymerase lacking VdGMP incorporation opposite the mis-
matching nucleotides might be due to a generally diminished
acceptance of VdGTP. However, KOD and Therminator DNA poly-
merase process VdGTP as efficiently as dGTP in the match case
(N@C) but show less incorporation opposite mismatching nucleo-
tides compared to reactions in presence of natural dGTP. Thus,
VdGTP is apparently more selectively processed by these DNA
polymerases than dGTP. Nevertheless, VdGTP showed only poor
efficiencies in PCR experiments using the above mentioned DNA
polymerases (data not shown).
size-demanding dGTP analog SdGTP
2 on DNA polymerases
(Fig. 1c). Thereby, primer extension experiments in presence of
dATP, dTTP, dCTP and SdGTP (+SdGTP) yielded DNA products sim-
ilar to those that are formed when only the three natural dNTPs but
neither dGTP nor SdGTP are present (ꢀdGTP). This suggests that
SdGTP is not accepted by the tested DNA polymerases.
To investigate the efficiency and selectivity of VdGMP incorpo-
ration in more detail, we carried out single incorporation experi-
ments in presence of different concentrations of VdGTP and
dGTP, respectively (Fig. 2). Therefore, we used four different primer
template combinations displaying all four natural nucleotides in
the template at the first position after the primer binding site
(Fig. 2a). In case of correct nucleotide incorporation (N@C), reac-
tions were incubated for 5 min (KlenTaq) and 10 min (KOD and
Therminator). To induce mismatch incorporation (N@G, A, T) reac-
tions were performed for 60 min. Doing so, two primer bands can
be detected after PAGE analysis reflecting the given 23 nt long
primer and the elongated one (24 nt).
To study multiple incorporation of the fluorescent nucleotides
in a row we performed primer extension experiments using a
DNA template that bears only cytosine residues after the primer
binding site (Fig. 3). Thus, we were able to investigate whether
multiple VdG or SdG nucleotides can be incorporated in a row. If
only natural dGTP is present the primer strand is extended effi-
ciently to DNA products that migrate markedly slower (Fig. 3;
dGTP). As up to 46 guanidine moieties can be incorporated in a
row we suggest that the smearing of the full-length band is caused
by unspecific primer binding during catalysis at the applied tem-
perature of 72 °C. Additionally, dG and dC rich DNA sequences
are known to fold into stable secondary structures that may affect
DNA mobility in PAGE as well.21 In presence of VdGTP the incorpo-
ration of multiple fluorescent nucleotides can be observed for all
studied DNA polymerases (Fig. 3b; VdGTP). Whereas in case of
KlenTaq and KOD DNA polymerase (Fig. 3b; left and middle panel)
Figure 2. Single incorporation experiments in presence of differing nucleotide
concentrations employing natural dGTP and VdGTP. P: primer only (23 nt).
Reactions were carried out for 10 min (KlenTaq 5 min) in case of N@C or for
Figure 3. Multiple incorporation of the modified nucleotides (b) VdGMP and (c)
SdGMP by KlenTaq, KOD, Therminator (Therm.), and Therminator L408Q (L408Q)
DNA polymerase, respectively. (a) Partial primer template sequences employed. (b)
and (c) Primer: primer only; no dNTP: incubation without any dNTP; dGTP: primer
extension in the presence of dGTP only; VdGTP: primer extension in the presence of
VdGTP 1 only; SdGTP: primer extension in the presence of SdGTP 2 only.
60 min for N@G, A, and T in presence of 10, 50, and 200 lM dGTP and VdGTP,
respectively. (a) Partial primer template sequences employed. (b) Incorporation
catalyzed by KlenTaq DNA polymerase. (c) Incorporation catalyzed by KOD DNA
polymerase. (d) Incorporation catalyzed by Therminator (Therm.) DNA polymerase.

B. Holzberger et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3136–3139
3139
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In summary, we report the synthesis of 8-vinyl- and 8-styryl-20-
deoxyguanosine-50-triphosphates and attempts to enzymatically
introduce the fluorescent nucleotides into DNA by various DNA
polymerases. It clearly turned out that the vinyl-modified nucleo-
tide VdGTP is suitable for the enzymatic synthesis of fluorescent
DNA. It can be easily and specifically incorporated within a grow-
ing primer strand at single and even at multiple adjacent positions.
In contrast, we observed that the enzymatic conversion of the sty-
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caused by the rather large modification at position C8 of the guan-
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8-styryl guanosines can be successfully attached to the 30-end of
a DNA primer strand. Interestingly, DNA polymerases behave dif-
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catalysis if B-family DNA polymerases are used. Taken together,
at least the fluorescent analog VdGTP is suitable for the enzymatic
introduction into DNA emphasizing the broad applicability and po-
tential of 8-vinyl-guanosine.
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Financial Support through the Konstanz Research School Chem-
ical Biology and the DFG is gratefully acknowledged. B.H. thanks
the Carl-Zeiss-Foundation for a Ph.D. scholarship. We also thank
Nadine Staiger for the expression and purification of Therminator
DNA polymerases.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.056.
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