Highly emissive deoxyguanosine analogue capable of direct visualization of B-Z transition

Article abstract of DOI:10.1039/c3cc48297a

A 2-aminothieno[3,4-d]pyrimidine G-mimic deoxyribonucleoside, ^thdG, was synthesized and incorporated readily into oligonucleotides as a versatile fluorescent guanine analogue. We demonstrate that ^thdG enables the visual detection of Z-DNA successfully based on different π-stacking of B- and Z-DNA. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc48297a

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Highly emissive deoxyguanosine analogue capable
of direct visualization of B–Z transition†‡
Soyoung Park,a Haruka Otomo,a Linjie Zhenga and Hiroshi Sugiyama*abc
Cite this: Chem. Commun., 2014,
50, 1573
Received 30th October 2013,
Accepted 4th December 2013
DOI: 10.1039/c3cc48297a
www.rsc.org/chemcomm
A 2-aminothieno[3,4-d]pyrimidine G-mimic deoxyribonucleoside, synthesis, photophysical properties and DNA incorporation of a
thdG, was synthesized and incorporated readily into oligonucleo- fluorescent base analogue, 2-aminothieno[3,4-d]pyrimidine G-mimic
tides as a versatile fluorescent guanine analogue. We demonstrate deoxyribonucleoside, thdG. In addition, we have achieved successfully
that thdG enables the visual detection of Z-DNA successfully based the direct visualization of B–Z transition using thdG based on different
on different p-stacking of B- and Z-DNA.
p-stacking of B- and Z-DNA.
The synthesis of thdG was performed by following published
Fluorescent probes are powerful and indispensable tools to detect procedures for generating RNA nucleosides (Scheme 1).12 The
biomolecules and monitor their functions. Their development is parent heterocycle with a thiophene group was synthesized from
opening up new fields of research, and many biological phenomena the commercially available methyl 4-aminothiophene-3-carboxylate
have been understood by tracking fluorescent signals in living hydrochloride (1) by reaction with chloroformamidine hydro-
systems.1 The development of fluorescent probes for nucleic acids chloride in dimethylsulfone at 125 1C. The amino group on
is essential because nucleic acids are not fluorescent. In addition, a 2-amino-thieno[3,4-d]pyrimidine-4(3H)-one was protected as the
fluorescent base analogue has great significance for the expansion N,N-dimethylformamidine form. The obtained thienoguanine (2)
of an artificial genetic alphabet with diverse functionality.2–5 Many could be converted into the protected deoxyribonucleoside (3)
artificial fluorescent nucleobase analogues have been developed to through Friedel–Crafts C-glycosylation with an acylated sugar deri-
date and researchers are continuing to design and synthesize new vative. This coupling afforded a mixture of b- and a-anomers with a
forms that fulfill both isomorphicity such as native Watson–Crick
base pairing and practical photophysical properties.6–11 Recently,
Tor and coworkers have developed isomorphic fluorescent RNA
nucleosides derived from thieno[3,4-d]-pyrimidine, which have very
significant photophysical features including visible light emission
and a high quantum yield.12,13 This suggested the potential of using
such nucleobase analogues with a thienopyrimidine heterocycle
and led us to exploit the thieno[3,4-d]-pyrimidine DNA nucleoside.
Here, we focus on guanine as it plays an important role in the
structural dynamics of DNA such as the formation of quadruplexes
and the transition between B-DNA and Z-DNA. We describe the
a Department of Chemistry, Graduate School of Science, Kyoto University,
Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
b Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University,
Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan
c CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku,
Tokyo 102-0075, Japan. E-mail: hs@kuchem.kyoto-u.ac.jp; Fax: +81-75-753-3670;
Tel: +81-75-753-4002
Scheme 1 Synthesis of thdG. Reagents and conditions: (a) (i) chloroform-
amidine hydrochloride, DMSO2, 125 1C; (ii) dimethylformamide dimethyl
acetal, DMF, 98%; (b) b-D-deoxyribofuranose 1-acetate 4,5-dibenzoate, SnCl4,
MeNO2, 0 1C to RT, 54%; (c) NH3–MeOH, 65 1C, 71%; (d) dimethylformamide
dimethyl acetal, DMF–MeOH, 76%; (e) DMTrCl, Py, 51%; (f) 2-cyanoethyl
N,N-diisopropylchloro phosphoramidite, iPr2NEt, DCM–MeCN, 0 1C to RT.
† This communication is dedicated to Professor Andrew D. Hamilton on the
occasion of his 60th birthday.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cc48297a
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Table 1 Photophysical data of thdG
stability and base pairing selectivity indicate that thdG could
replace a G base in the strand without causing structural
disruption. Furthermore, the fluorescent properties of ODN1
were evaluated upon hybridization with complementary strands
containing matched or mismatched bases (Fig. S6, ESI‡).
Interestingly, significant changes in the fluorescence intensity
(match o T o G o A) were observed, indicating that the
fluorescence of thdG is sensitive to the local environment of DNA.14
The isomorphicity and the photophysical properties of thdG
prompted us to exploit this new G-mimic deoxyribonucleoside. We
intended to monitor the conformational changes of DNA through
changes in fluorescence intensity of thdG.15 DNA has a remarkable
conformational flexibility. Among them, the most typical example is
the structural transition between right-handed B-DNA and left-handed
Z-DNA.16 In particular, the dynamic structural change from the
continuous p-stacks in B-DNA to the discrete four-base p-stacks in
Z-DNA is a very attractive option for constructing DNA-based nano-
devices.17,18 We have demonstrated that the B–Z transition could be
detected by measuring the fluorescence intensity of 2-aminopurine.18
Based on previous studies, we expected that thdG fluorescence could
be applied to monitor the change of electronic properties between
B- and Z-DNA. To test this possibility thdG-containing the duplex
decamer, 50–d(CGCGCXCGCG)–30 (ODN8), where X = thdG, was
prepared and investigated. However CD spectroscopy indicated that
the B–Z transition became more difficult when thdG was incorporated
as a replacement for a G nucleotide. Indeed, the duplex ODN8
produced an almost B-conformation even in 5 M NaCl. This might
be explained because thdG favors the anti-conformation and stabilizes
B-form DNA when it is located in DNA oligonucleotide strands. The
theoretical calculations for the syn–anti conformation of thdG were
performed at the ground state with the density functional B3LYP and
the 6-31* basis set. This optimized the lowest energy conformations,
suggesting that the anti-conformation is more stable than the syn
conformation (Fig. S10, ESI‡). We therefore decided on the introduc-
tion of a Z-stabilizing unit into DNA sequences and 8-methylguanine
(m8G) was chosen corresponding to this objective. Previously, we
demonstrated that the incorporation of a methyl group at the guanine
C8 position (m8G) markedly stabilizes the Z conformation at low salt
conditions. The self-complementary dodecamer d(CGCXCYCGCG)2
(ODN9), where X = thdG and Y = m8G, was synthesized and
fluorescence intensity was observed at various salt concentrations.
To our delight, ODN9 exhibited a dramatic change of fluorescence
intensity in response to the B–Z transition. ODN9 showed the typical
CD spectrum of Z-DNA, a negative cotton effect around 295 nm and a
positive cotton effect around 260 nm, at high salt concentrations. As
shown in Fig. 2, a very strong fluorescence enhancement was
observed in Z-DNA, whereas the fluorescence was weak in B-DNA.
The intense fluorescence in Z-DNA can be explained by disruption of
charge transfer attributable to its four-base p-stacks (Fig. S11, ESI‡).18
The fluorescence of ODN9 increased proportionally when we
increased the ratio of the Z-conformation by adjusting the NaClO4
concentration. Furthermore, as shown in Fig. 2c, robust brightness
of ODN9 in the visible region enabled visualization of the B–Z
transition. This inspired us with visual detection of DNA–protein
interactions.19 The DNA-binding domain of double-stranded RNA
adenosine deaminase (ADAR1), called Za domain, specifically binds
l
abs/nm
Stokes
Solvent
Water
(e/103 Mꢀ1 cmꢀ1
)
l
em/nm (f) fe
t/ns shift/cmꢀ1
319 (4.26)
Dioxane 329 (5.37)
MeOH 327 (3.80)
455 (0.58)
429 (0.85)
460 (0.47)
2471 20.5 9370
4565 14.0 7085
1786 13.7 8842
52% yield at a b:a proportion of 3 : 1. The benzoyl protecting groups
were removed in a methanolic ammonia and the N,N-dimethyl-
formamidine group was reintroduced for protection of the purine
amino group. Subsequently, the 50-hydroxyl group was protected as
the dimethoxytrityl ether (DMTr) and the desired b-isomer could be
isolated at a yield of 51%. The configuration at the C-1 carbons of
1
anomers was confirmed by 1D and 2D (NOESY) H NMR experi-
ments (see ESI‡).
The fundamental photophysical properties of the thdG monomer
were investigated and are shown in Table 1. The fluorescence of the
thdG deoxyribonucleoside (4) shows absorption at 319 nm and visible
emission at 455 nm with a high quantum yield under neutral
conditions in water, dioxane, and MeOH. These results are similar
to those of thG RNA nucleoside reported by the Tor group.12 In
addition, the thdG deoxyribonucleoside displays a relatively high
quantum yield under dioxane (0.85) and a longer excited state lifetime
in water (20.5 ns) compared with a RNA nucleoside thG. To evaluate
oligonucleotides including the isomorphic dG surrogate, the phos-
phoramidite of thdG was synthesized and incorporated into the center
of 18-mer DNA oligonucleotides of 50–d(CGTCCGTCXTACGCACGC)–30,
where X = thdG, by automated solid-phase synthesis and phosphor-
amidite chemistry. The complementary strands of ODN1 containing
matched or mismatched bases and the corresponding natural DNA
duplexes with guanine were also prepared. As shown in Fig. 1, the
thdG–C base pair afforded almost identical thermal stability
(Tm = 72.3 1C) compared with natural duplex DNA with a G–C base
pair (Tm = 72.1 1C). A very similar tendency to decrease Tm was observed
for thdG (ODN1) and G (ODN2) using the complementary strands
including mismatched bases (ODN3BODN7). The thermodynamic
Fig. 1 Thermal stability and selectivity of base pairing. (a) Thermal melts of
thG containing DNA. ODN1:50-CGTCCGTCthdGTACGCACGC-30 paired
with ODN3-7:50-GCGTGCGTAYGACGGACG-30 Y = C, T, A, G, or dSpacer.
(b) Comparison of Tm values. ODN1,2:50 CGTCCGTCX TACGCACGC-30
X = thdG or G paired with ODN3-7. All samples contained 5 mM of each
oligonucleotide strand, 20 mM Na cacodylate (pH 7.0) and 100 mM NaCl.
50-O-Dimethoxytrityl-10,20-dideoxyribose-30-[(2-cyanoethyl)-(N,N-diisopropyl)]-
phosphoramidite was used as dSpacer (dS).
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our hope that the present fluorescent nucleobase, thdG, could
be superior to 2-Ap which is quenched in DNA and emits in the
UV region.6,22 We believe that thdG will expand the repertoire of
fluorescent base analogues. We are currently exploring the
application of thdG further, including its charge transfer pro-
perties and incorporation into living cells.
We express our sincere thanks for the CREST grant from the
Japan Science and Technology Corporation (JST), grants from the
WPI program (iCeMS, Kyoto University), and for the global COE
program from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT), Japan.
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Fig. 2 Observation of the conformational changes from B-DNA to Z-DNA
through changes in fluorescence intensity (a) observation of the B–Z
transition by CD spectroscopy. (b) Change of fluorescence of NaClO4 at
5 1C. (c) Visual detection of B–Z transition. Samples contained 5 mM of
ODN9 in 20 mM sodium cacodylate buffer (pH 7.0). 5 M (left) or 50 mM
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This journal is ©The Royal Society of Chemistry 2014
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