Design and synthesis of fluorescent 7-deazaadenosine nucleosides containing π-extended diarylacetylene motifs

Article abstract of DOI:10.1039/c4ob02081b

C-modified 7-deazaadenosines containing a diphenylacetylene moiety have been synthesised using cross-coupling approaches. The C-modified nucleosides exhibit remarkable fluorescence properties, including high quantum yields. Solvatochromic studies show a n

Full text of DOI:10.1039/c4ob02081b

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Design and synthesis of fluorescent
7-deazaadenosine nucleosides containing
Cite this: Org. Biomol. Chem., 2015,
13, 68
π-extended diarylacetylene motifs†
Sara De Ornellas,a,b John M. Slattery,a Robert M. Edkins,c Andrew Beeby,c
Christoph G. Baumann*b,d and Ian J. S. Fairlamb*a,d
Received 30th September 2014,
Accepted 30th October 2014
DOI: 10.1039/c4ob02081b
www.rsc.org/obc
C-modified 7-deazaadenosines containing
a diphenylacetylene
moiety have been synthesised using cross-coupling approaches.
The C-modified nucleosides exhibit remarkable fluorescence pro-
perties, including high quantum yields. Solvatochromic studies
show a near linear correlation between the Stokes shift and solvent
polarity which is indicative of intramolecular charge transfer.
DFT calculations have allowed us to correlate the experimentally
observed photophysical properties with the calculated HOMO–
LUMO energy gaps within a series of real and model compounds.
Introduction
Fluorescently-labelled nucleosides and nucleotides are invalu-
able tools in the study of biological systems.1 The addition of
compact substituents at C8 in adenosine, e.g. phenyl-(1)
or phenylethynyl-(2), induces significant fluorescence.2 This
property cannot be exploited fully however, as C8-modified
analogues are incompatible with base-pair formation (due to
an unfavourable conformational preference).3
On the other hand, 7-deazaadenosine nucleosides adopt
conformations commensurate with adenosine, allowing the
C7-position to be utilised for functionalisation.4 C7-groups
orientate into the major groove of double helices, resulting in
minimal duplex destabilisation.5 There are a limited number
Fig. 1 Fluorescent nucleosides, including C7 and C8 modifications.
The series of compounds 4a–e are the target molecules in this paper.
rescent C7-modified analogues, which is the principal aim of
the current study.
In this paper, we report the synthesis and photophysical
properties of novel 7-deazaadenosine nucleosides containing
compact π-extended fluorophores. An extrinsic fluorophore, i.e.
a diarylacetylene group (tolan), has been introduced at the
C7-position (as illustrated for generic compound 4 in Fig. 1). The
design of the series of compounds 4a–e was guided by compu-
tational studies, which predict that such molecules should
function as classical ‘push–pull’ diarylacetylene systems,
which are known to exhibit strong fluorescence.7 The theore-
tical predictions go some way to explain the experimentally
observed photophysical properties of compounds within the
series 4a–e. The fluorescence lifetimes and quantum yields
make these artificial C-modified nucleosides potentially useful
as probes in biological systems.
of
7-deaza-2′-deoxyadenosine
analogues
incorporating
compact fluorophores (e.g. 3), but they typically exhibit weak
fluorescence.6 It is therefore necessary to develop highly fluo-
aDepartment of Chemistry, University of York, York, YO10 5DD, UK.
E-mail: ian.fairlamb@york.ac.uk; Tel: +44 (0)1904 324091
bDepartment of Biology, University of York, York, YO10 5DD, UK.
E-mail: christoph.baumann@york.ac.uk; Tel: +44 (0)1904 328828
cDepartment of Chemistry, University of Durham, South Road, Durham, DH1 3LE,
UK. E-mail: andrew.beeby@durham.ac.uk
dBiological Physical Sciences Institute (BPSI), University of York, York, YO10 5DD,
Results and discussion
UK
†Electronic supplementary information (ESI) available: Characterisation data of
new compounds and detailed flurorescence measurements and spectra. See
DOI: 10.1039/c4ob02081b
The following section is divided into theoretical (DFT
calculations) and experimental (synthesis and photophysical
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properties) parts. The theoretical work establishes the credi- Synthesis of target compounds (4a–4e)
bility of 4 as a potentially useful new class of fluorescent
We set about synthesising the core structure of 4a {and deriva-
tives (4b–e)} using 7-iodo-7-deazaadenosine 11 as a convenient
starting material (Scheme 1). The synthesis of 11 involves reac-
tion of 6-chloro-7-deazapurine 7 with iodine in DMF to give 8.
nucleosides.
Density functional theory (DFT) calculations
We studied structures containing both N-ribose and N-methyl An alternative iodination method using N-iodosuccinimide
groups, i.e. 3 and 4a, and 5 and 6a, respectively. Several confor- required recrystallisation of 8 from MeOH, which leads to the
mations within the ribose sugar and the π-system within each undesired formation of 7-iodo-6-methoxy-7-deazapurine.
molecule were considered in the calculations. In these deriva- Compound 8 was coupled to protected ribose sugar 9 using
tives, the ribose with a C3′-endo conformation was selected. Vorbrüggen glycosylation conditions to give 10.8 Optimal yields
The methyl substituent was viewed as an electronically of 10 were achieved using sub-stoichiometric quantities of tri-
similar group to the more conformationally-flexible ribose. De- methylsilyl triflate and N,O-bis(trimethylsilyl)acetamide (BSA).
coupling the potential complexity of the latter was necessary, Sugar deprotection of 10 and installation of the exocyclic
especially when studying a larger series of compounds (4a–e), amine was carried out with aqueous NH3 in dioxane (in a
vide infra. All of the calculated structures were optimised at the sealed tube at 60 °C), which avoids methanolic ammonia and
(RI-)PBE0/def2-TZVPP level and their frontier molecular orbi- an autoclave, affording 11 in good overall yield.
tals (MOs) and vertical excitation energies (from TDDFT
For the diarylacetylene arm, an organoboron species was
studies at the same level of theory) were used to probe the required for coupling to 11. Diarylacetylene boronate esters
effect of chemical structure on their photophysical properties.
Interestingly, a qualitative analysis of the MOs of 3 and 5
(Fig. 2) goes some way to explaining a general preference for
non-radiative decay and the weak fluorescence observed6
experimentally. The HOMO/LUMO in both 3 and 5 are charac-
teristic of a weaker push–pull system and suggests limited
intramolecular charge transfer upon excitation.
12a–e were synthesised by chemoselective Pd-catalysed cross-
By contrast, the MOs for ribose derivative 4a and N-methyl
derivative 6a (shown in Fig. 2) show localisation of the HOMO
primarily on the 7-deazaadenine unit and the LUMO on the
diarylacetylene. This observation suggests that there is poten-
tial for intramolecular charge transfer upon excitation in this
type of structure, i.e. as expected in a ‘push–pull’-type system,
and that the ribose and methyl groups do not appreciably
affect the distribution of electron density within the π-system.
Scheme 1 Synthesis of key starting material 11. Reaction conditions:
i. I2 (1.1 eq.), KOH (2.5 eq.), DMF, rt, 30 min. ii. 9 (1.1 eq.), BSA (0.5 eq.),
TMSOTf (0.5 eq.), MeCN, 80 °C, 1.5 h. iii. NH3 (aq, 25%)/dioxane (1 : 1),
60 °C, 3 days.
Fig. 2 (RI-)PBE0/def2-TZVPP optimised structures for N-substituted-7-phenylethynyl-7-deazaadenosines 3 and 5 (left) and N-substituted-7-
[4-(phenylethynyl)-phenyl]-7-deazaadenosines 4a and 6a (right). In all cases, the frontier MOs (HOMO/LUMO) are illustrated (isosurface at 0.05 a.u.).
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Scheme 2 Synthesis of compounds 12a–e. Reaction conditions:
i. PdCl2(PPh3)2 (5 mol%), CuI (5 mol%), PPh3 (20 mol%), Et2NH, DMF,
120 °C (MW), 20 min. ii. TBAF, THF. iii. As for i, without DMF, 25 min.
Scheme 3 Synthesis of target compounds 4a–e.
coupling of 4-bromophenyl boronic acid neopentyl glycol ester 13.9
Two approaches were used. Firstly, 13 was cross-coupled
with three terminal arylacetylenes to give 12a, 12b and 12d,
respectively (Scheme 2).
in biological systems. The 7-modified-7-deazaadenosines 4a–e
possess high molar absorbance coefficients (ca. 2 × 104 M−1 cm−1),
suggesting the diphenylacetylene motif connected to the
7-deaza-adenine unit is an effective chromophore.
For access to 12c and 12e, 13 was cross-coupled with tri-
methylsilylacetylene to give 14, which was then deprotected
quantitatively with TBAF to reveal terminal acetylene 15. The
latter compound was then cross-coupled with the respective
aryl halide to give boronate esters 12c and 12e (Scheme 2).
With the diarylacetylene boronate esters 12a–e in hand,
they were cross-coupled with 7-iodo-7-deazaadenosine 11
under reported aqueous conditions,10,4a with the water-soluble
phosphine ligand TPPTS (Scheme 3). Pure C7-diarylacetylene
functionalised nucleosides 4a–4e were all isolated by column
chromatography on silica gel or by preparative thin-layer chrom-
atography in good yields (see ESI† for characterisation details).
The 7-modified-7-deazaadenosines 4a–e exhibit promising
fluorescence properties (Table 1). High emission quantum
yields were recorded for all compounds in DMSO, but of par-
ticular note are compounds 4a, 4b and 4c, which have
quantum yields >0.70. The thiophene-containing analogue 4d
has a somewhat lower quantum yield (Φ = 0.32), as does the
trifluoromethyl-compound 4e (Φ = 0.41). The variation in wave-
length of the emission maxima for these compounds is much
larger than the variation in their absorption maxima. This
gives rise to large differences in their Stokes shifts, e.g. trifluoro-
methyl compound 4e, has a remarkably large Stokes shift of
1 × 104 cm−1
.
In order to further explore the substituent effects seen in
4a–e, the model structures (6a–e, R = Me rather than ribose)
were optimised computationally at the (RI-)PBE0/def2-TZVPP
level, allowing their UV-visible absorption spectra to be calcu-
lated in the gas phase (TDDFT at the same level of theory,
50 singlet excitations considered). Vertical excitation energies
were used to probe the effects of various substituents on the
photophysical properties of these molecules. The frontier MOs
and their energies are shown in Fig. 3. The calculated λmax
values for 6a–e are given in Table 1, where there are differences
with the experimental data. Crucially, the trend within the
Photophysical studies
We turned our attention to studying the photophysical pro-
perties of 7-modified-7-deazaadenosines 4a–e. UV-visible absorp-
tion spectra of the modified nucleosides exhibit maxima at
wavelengths >310 nm, with the lowest λmax observed for the
thiophene-containing compound 4d (Table 1). The absorb-
ance maxima of 4a–c (at ca. 320 nm) are shifted away
from the intrinsic absorbance of nucleic acids and proteins
(ca. >280 nm), which is important when considering the potential
applications of these novel nucleosides as fluorescent probes
Table 1 UV-visible absorption and fluorescence data for compounds 4a–4e in dilute DMSO solution
λmax/nm
(exp.)
λmax/nm
Stokes
Cpd
(calc.)a
ε/104 M−1 cm−1
λem/nm
shift/ cm−1
Φ
τ/ns
4a
4b
4c
4d
4e
321
321
331
313
328
345
341
353
349
363
2.2
2.3
2.5
2.1
1.5
428
405
431
404
491
7790
6460
7010
7200
10 000
0.74
0.78
0.76
0.32
0.41
2.2 (92%), 0.5 (8%)
1.6 (90%), 0.5 (10%)
2.0 (92%), 0.5 (8%)
1.5 (88%), 0.5 (12%)
2.1 (87%), 0.8 (12%)
a Calculated λmax are for model complexes 6a–6e.
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Fig. 3 HOMO and LUMO surfaces (isosurface at 0.05 a.u.) and energies for compounds 6a–6e, calculated by DFT at the (RI-)PBE0/def2-TZVPP)
level.
Table 2 Effect of solvent on the fluorescence properties of 4a
Solvent
λmax
nm
/
ε/104 M−1 λem
/
Stokes
dielectric
Solvent
cm−1
nm
shift/cm−1
Φ
constantb
H2Oa
302
321
308
306
304
304
312
311
318
2.0
2.2
2.4
2.5
2.6
3.1
2.5
2.4
2.7
429
428
409
407
393
396
394
389
420
9800
7790
8020
8110
7450
7640
6670
6450
7640
0.15 80.1
0.74 47.2
0.62 36.6
0.46 33.0
0.31 25.3
0.29 20.2
DMSO
MeCN
MeOH
EtOH
iPrOH
CH2Cl2
EtOAca
DMF
a
0.61
0.33
9.0
6.1
0.78 38.3
a Solutions made from stocks of 4a in DMSO (2.5 × 10−3 M), due to
poor compound solubility in non-polar solvents, which were diluted to
1–100 µM, so that A = 0.1 for each sample. The % (v/v) DMSO in the
final solutions was typically <1%. b Measured at 20 °C.
component (ca. 10%) that was consistently ca. 0.5 ns. The fluo-
rescence lifetime values recorded are in keeping with measure-
ments on related tolan derivatives made by Marder and co-
workers.12
Solvatochromism of fluorophores can be informative about
the nature of the excited state and the electronic transitions
giving rise to the absorption and emission spectra. For
example, solvatochromic fluorescent probes can be used to
gain insight into the polarity of the local environment.11 The
variation in the absorption and emission spectra of 7-[4-(phenyl-
ethynyl)-phenyl]-7-deazaadenosine 4a was, therefore, investi-
gated in a variety of solvents (Table 2).
Fig. 4 Correlation between calculated and experimental λmax (nm) for
all optimised structures and conformational isomers (see ESI† for
details). The structures that are plotted against each other are indicated
as their compound numbers (e.g. 4d/6d). TDDFT refers to Time-Depen-
dent Density Functional Theory; calculated λmax values for structures 5,
6a–e are shown on the y-axis. The experimental λmax values for com-
pounds 3 and 4a–e are shown on the x-axis.
series can be considered. For example, when all calculated
(which includes 4a, 5 and 6a–e) and experimental λmax data are
plotted against each other, there is a good correlation between
the experimentally and theoretically derived data (Fig. 4). Even
in the case of the 3-thienyl-system (4d and 6d), a correlation is
seen within the compound series. Interestingly, the 3-thienyl
group in 6d contributes to the LUMO, but not appreciably to
the HOMO (see Fig. 3), which is perhaps surprising given the
electron-rich nature of the thienyl group.
Fluorescence lifetimes were measured using time-correlated
single photon counting (TCSPC), see Table 1.11 All of the
7-modified-7-deazaadenosines 4a–e were found to exhibit bi-
exponential decays, with a major component that varied
according to the substituent at the 7-position, and a minor
Both the absorption and emission maxima vary slightly
when the solvent is changed. Whilst no distinct correlation
was observed between wavelength and solvent polarity, the
data shows a reasonable correlation between (ν –ν ) and the
¯
A
¯
F
orientation polarizability (Δf ) in
a Lippert–Mataga plot
(Fig. 5), indicating the sensitivity of the fluorophore to its local
chemical environment. The linear correlation is improved if
one omits water (R2 = 0.89), which could suggest that 4a is
behaving quite differently in this solvent. It is of particular
note that the fluorescence quantum yield of 4a in water was
found to be 0.15, which is significantly higher than 7-phenyl-
ethynyl-7-deaza-2′-deoxyadenosine (Φ = 0.02)6 and, indeed,
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computational equipment used in this study (ref. EP/H011455/1).
This work was supported in-part by EPSRC ‘ENERGY’ grant,
ref. no. EP/K031589/1.
Notes and references
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the majority of other bespoke fluorescent C-modified nucleo-
sides reported to date.
In conclusion, a novel class of 7-modified-7-deazaadenosine
nucleosides 4a–e have been designed and synthesised from
the corresponding 7-iodo nucleoside 11, using multi-step
Pd-catalysed cross-coupling approaches. The nucleosides exhibit
promising UV-visible absorption and fluorescence properties,
with large quantum yields and absorption maxima shifted
away from the intrinsic absorption associated with amino acid
and nucleic acid residues. The changes in the absorption
and emission wavelengths in different solvents indicate that
the fluorescence properties of 4a are quite complex, whereas
the Stokes shift exhibits a reasonable linear correlation with the
orientation polarizability of the solvent. DFT calculations pro-
vided valuable insight into the fluorescent properties observed
experimentally for reported compound 3, and the herein
described novel compounds 4a–e.
We were particularly pleased with the quantum yield for 4a
in water (Φ = 0.15). The incorporation of a compact fluoro-
phore means that the chemical modification should not have a
pronounced effect on either: (i) the biological compatibility of
the nucleoside, or (ii) the stability of nucleic acids containing
these C7-modified nucleotides. Applications for this class of
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Acknowledgements
We thank BBSRC (S. De O./ref. BB/D527034/1), University of
York and the Royal Society (I. J. S. F.) for funding this
work. R. M. E. thanks Durham University for a Durham
Doctoral Fellowship. We are grateful to EPSRC for funding the
72 | Org. Biomol. Chem., 2015, 13, 68–72
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