Modified 6-aza uridines: Highly emissive pH-sensitive fluorescent nucleosides

Article abstract of DOI:10.1002/cphc.201200375

Optimized facile syntheses and highly desirable spectroscopic properties of two isomorphic fluorescent pyrimidines, comprising a 1,2,4-triazine motif conjugated to a thiophene (1 a) or a furan (1 b), are described. Although structurally related to their 5-modified uridine counterparts, these modified 6-aza-uridines reveal dramatically improved fluorescence properties and a remarkable sensitivity to polarity and pH changes. The thiophene derivative 1 a has an absorption maximum around 335 nm, which upon excitation yields visible emission with a polarity-sensitive maximum and fluorescence quantum yield ranging from 415 nm (Φ=0.8) to 455 nm (Φ=0.2) in dioxane and water, respectively. Nucleoside 1 a also displays susceptibility to acidity. Correlating emission intensity and solution pH yields a pK_a value of 6.7-6.9, reasonably close to physiological pH values. The results illustrate that highly sought-after fluorescence features (brightness and responsiveness) are not necessarily the trait of large fluorophores alone, but can be observed with probes that meet stringent isomorphic design criteria.

Full text of DOI:10.1002/cphc.201200375

DOI: 10.1002/cphc.201200375
Modified 6-Aza Uridines: Highly Emissive pH-Sensitive
Fluorescent Nucleosides
Renatus W. Sinkeldam, Patrycja A. Hopkins, and Yitzhak Tor*[a]
Optimized facile syntheses and highly desirable spectroscopic
properties of two isomorphic fluorescent pyrimidines, compris-
ing a 1,2,4-triazine motif conjugated to a thiophene (1a) or
a furan (1b), are described. Although structurally related to
their 5-modified uridine counterparts, these modified 6-aza-uri-
dines reveal dramatically improved fluorescence properties
and a remarkable sensitivity to polarity and pH changes. The
thiophene derivative 1a has an absorption maximum around
335 nm, which upon excitation yields visible emission with
a polarity-sensitive maximum and fluorescence quantum yield
ranging from 415 nm (F=0.8) to 455 nm (F=0.2) in dioxane
and water, respectively. Nucleoside 1a also displays susceptibil-
ity to acidity. Correlating emission intensity and solution pH
yields a pKa value of 6.7–6.9, reasonably close to physiological
pH values. The results illustrate that highly sought-after fluo-
rescence features (brightness and responsiveness) are not nec-
essarily the trait of large fluorophores alone, but can be ob-
served with probes that meet stringent isomorphic design cri-
teria.
1. Introduction
The sensitivity and simplicity of fluorescence spectroscopy has
made it one of the most useful tools for the study of biomole-
cules. Despite numerous technological and instrumental ad-
vances, the quality of fluorescence-based analyses ultimately
relies on the fluorescent probes themselves. Countless fluores-
cent tags, labels and probes have been designed over the
years, but only a limited fraction meets the stringent design
criteria of isomorphicity.[1] Useful isomorphic probes are charac-
terized by a high structural resemblance to the molecular
building blocks they mimic, while maintaining distinct spectro-
scopic properties. This imposes structural and application-spe-
cific constraints, where probes capable of innocently replacing
membranes, proteins, or nucleic acid components have to be
fabricated.[1] The design of such probes is further challenged
by the fact that the relationship between structure and photo-
physical properties is hard to deduce, and can typically only be
elucidated experimentally.
benign, with no impact on the anti orientation or sugar pucker
of the nucleosides.[6] The resulting nucleosides emit in the visi-
ble range (390–443 nm), have rather large Stokes shifts (8400–
9700 cmÀ1), but suffer from relatively low emission quantum
efficiency (F=0.01–0.035).[6] These nucleosides behave like
molecular rotors, with the 5-membered heterocycles and the
pyrimidine core acting as the “donor and acceptor”, respective-
ly.[7] Rotation around the biaryl linkage provides an effective
channel for non-radiative torsional relaxation, thus leading to
a low emission quantum yield in non-viscous media.[7,8] We hy-
pothesized that altering the electronics by replacing the pyri-
midine with the corresponding 1,2,4-triazine core (i.e. 6-aza-
pyrimidine)[9] would enhance the charge transfer character, ide-
ally yielding a bathochromic shift as well as a hyperchromic
effect and higher brightness. Herein we report a simplified syn-
thetic pathway to thiophene and furan-modified 6-azauridines,
discuss their most relevant photophysical properties, and
reveal their responsiveness to environmental polarity and pH.
To explore nucleic acids, the non-emissive canonical nucleo-
sides need to be structurally modified to endow them with
useful fluorescent properties. Such alterations cannot impede
native Watson–Crick base-pairing and subsequent helix forma-
tion.[2,3] Successful nucleoside probes reconcile these structural
and photophysical demands with an exclusively excitable
wavelength, red-shifted from the ~260 nm absorption band of
the native nucleobases. Responsive emissive nucleosides,
unlike fluorescent tags or labels, are characterized by spectro-
scopic properties, which are sensitive to changes in the local
environment. Several groups, including ours, have reported
a rich variety of isomorphic fluorescent nucleoside analogs.[1,4,5]
The basic design principle of our first-generation emissive nu-
cleobase analogs has relied on conjugating five-membered ar-
omatic heterocycles to the pyrimidine core at the 5-position.[2]
The modification at this position appears to be structurally
2. Chromophore Design
As articulated above, replacing the uridine core with 6-azaura-
cil, a more electron-deficient heterocycle, while keeping the
electron-rich thiophene and furan moieties in the 5-position,
was expected to maintain an extended p system while aug-
[a] Dr. R. W. Sinkeldam, P. A. Hopkins, Prof. Dr. Y. Tor
Department of Chemistry and Biochemistry
University of California, San Diego
La Jolla, California 92093 (USA)
Fax: (+1)858-534-0202
E-mail: ytor@ucsd.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cphc.201200375.
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Only a few fluorescent nucleosides have been shown to be
pH sensitive.[8,16,17] 8-Aza-guanosine, an isomorphic fluorescent
purine analog,[18] shows its pH-dependent emission characteris-
tics as a nucleoside[19] and after enzymatic incorporation into
RNA.[20] This feature has been exploited in the investigation of
the catalytic function of RNA.[21] An application reveals interest-
ing opportunities for fluorescent nucleosides that display spec-
tral sensitivity toward (de)protonation. No emissive pyrimidines
with pKa values near physiological pHs have, however, been
documented. Importantly, the presence of the nitrogen atom
at position 6 has a profound influence on the acidity of the
NH, lowering its pKa to 6.8 vs a pKa value of 9.3 for the parent
uridine.[22] Like uridine, 6-aza-uridine has no appreciable fluo-
rescence properties hampering visualization of (de)protonation
events with fluorescence spectroscopy. Assuming the emissive
features discovered for the parent 5-modified uridines translate
to (or are enhanced in) the 6-aza motif, deprotonation and
protonation events are likely to be revealed by changes in the
emission profile. This new motif would therefore constitute the
first example of a pH-responsive emissive pyrimidine with
a pKa value near physiological pHs.
3. Results
3.1. Synthesis
Condensation of commercially available thiophene glyoxylic
acid (2a) with semicarbazide (3) gave 4a which, in situ, cy-
clized upon heating under basic conditions to furnish 5a in
good yields. The same approach, starting from 2b, provided
5b. Glycosylation of 5a and 5b was performed in a two-step
one-pot procedure. First, the nucleobase was activated by re-
action with bis(trimethylsilyl)acetamide (BSA) in acetonitrile for
~30 min at room temperature (rt), turning the initial suspen-
sion into a clear solution. Secondly, the reaction mixture was
brought to 858C after which b-d-ribofuranose 1-acetate 2,4,5-
tribenzoate (6) was added, immediately followed by addition
of trimethylsilyltriflate (TMSOTf). The reaction was allowed to
run for 30 min after which TLC analysis indicated full conver-
sion of the nucleobase to the desired glycosylated product. An
aqueous work-up followed by purification with column chro-
matography yielded 7a and 7b in good yields. Deprotection
with methanolic ammonia at 608C, followed by column chro-
matographic purification gave the desired 1a and 1b.
Scheme 1. a) Syntheses of 1a and 1b, reagents and conditions: i) semicarba-
zide hydrochloride (3) (1.1 equiv), H2O, 608C, 17 h, ii) add. 1m NaOH aq.
(3 equiv), 608C, 4 h, iii) BSA (3 equiv), CH3CN, rt, 30’, iv) b-d-ribofuranose 1-
acetate 2,4,5-tribenzoate (6) (1.1 equiv), TMSOTf (1.1 equiv), CH3CN, 858C,
30’, v) NH3/MeOH, 608C, 24 h; and b) structures of reported 5-(thiophene-2-
yl)-2’-deoxyuridine (8a) and 5-(furan-2-yl)-2’-deoxyuridine (8b).
menting the donor–acceptor nature of this motif (Scheme 1).
This enhanced “push–pull” interaction is expected to affect
a red-shift of the absorption maxima and likely a concomitant
red-shifted emission maxima. Such a donor–acceptor interplay
is also frequently associated with enhanced sensitivity toward
environmental polarity.[10–13] The single-bond linkage between
the 5-membered heterocycle and the 6-membered nucleobase
introduces a molecular rotor element likely to render 1a and
1b sensitive toward viscosity (molecular crowding) as demon-
strated for other 5-aryl modified pyrimidines.[7,8] Chromophores
possessing such a feature typically reveal a much higher emis-
sion intensity in viscous compared to non-viscous media. Vis-
cosity (molecular crowding) hampers formation of a twisted
excited state, thereby limiting the rotational relaxation, a non-
emissive decay pathway, to the ground state.[14,15]
3.2. Photophysics
3.2.1. Basic photophysics in water
In unbuffered deionized water, 1a is characterized by a red-
shifted absorption maximum at 332 nm, which upon excitation
gives a visible fluorescence signal, peaking at 455 nm, with a re-
spectable quantum yield (F=0.20) and a fluorescence lifetime
of 4.9 ns (Table 1). In contrast, a solution in dioxane yields an
absorption maximum at 335 nm, an emission maximum at
415 nm (F=0.80) and a slightly longer fluorescence lifetime of
5.4 ns. The small change in absorption maximum, but 40 nm
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Highly Emissive pH-Sensitive Fluorescent Nucleosides
value of 6.7 was calculated (Fig-
ure 2b). Using the same ap-
proach a pKa value of 6.6 for
probe 1b was determined.[25]
The difference between the
quantum yield under acidic and
basic conditions was also reflect-
ed by the difference in fluores-
cence lifetimes, 3.0 and 6.6 ns,
respectively (Table 1).
Table 1. Selected spectroscopic properties of 1a and 1b.[a]
Solvent
Absorption
Fluorescence
Brightness
Fꢁe
Stokes Shift
lmax
e
lmax
F
t
nabsÀnem
[nm]
[ꢁ104 MÀ1 cmÀ1
]
[nm]
[ns]
ꢁ103
[cmÀ1
]
Dioxane
Water
pH 2.55
pH 10.13
MeOH
Glycerol
Dioxane
Water
pH 2.55
pH 10.13
MeOH
335
332
336
325
334
339
327
320
329
319
326
331
1.3
1.1
1.2
1.2
1.1
1.0
1.2
1.0
1.1
1.1
1.1
1.1
415
455
462
426
433
444
414
443
485
446
442
434
0.80
0.20
0.13
0.39
0.50
0.66
0.60
0.05
<0.01
0.11
0.04
0.46
5.4
4.9
3.0[b]
6.6[b]
6.6
7.9
5.6
3.1
<1[b]
2.8[b]
1.6
10.4
2.2
1.6
4.7
5.5
6.6
7.2
0.5
<0.1
1.2
0.4
5.1
6025
8492
8485
7588
7332
7372
6824
9107
9861
9273
8585
7574
1a
1b
3.2.4. Sensitivity to Viscosity
Absorption and emission spectra
of 1a in samples of methanol,
glycerol and mixtures thereof
Glycerol
2.7
[a] The Stokes shifts are calculated with corrected emission maxima obtained using: Intensity [n]=l2 ꢁIntensity
[l].[10] [b] Values for pH 2.55 and pH 10.13 (aq. 10 mm phosphate, 0.1m NaCl buffers) represent the lower- and
upper plateau values of the sigmoidal fit (Figure 2). All values represent averages, see the Supporting Informa-
tion for error analysis.
reveal virtually no responsive-
ness toward viscosity for 1a
(Figure 3), but indicate a signifi-
cant sensitivity to viscosity for
1b.[25] A double-log plot of the
difference between the emission maxima in the two solvents
prompted us to study the sensitivity to polarity in greater
detail.
fluorescence intensity as a function of sample viscosity for 1b,
however, reveals poor linearity.[25] The high fluorescence quan-
tum yields for 1a in methanol (0.50) and glycerol (0.66), are ac-
companied by the longest lifetimes observed in this study, 6.6
and 7.9 ns, respectively.
3.2.2. Sensitivity to Polarity
Influence of polarity on spectroscopic parameters of fluores-
cent probes was studied using samples in dioxane [ET(30)=
36.0 kcalmolÀ1], water [ET(30)=63.1 kcalmolÀ1] and mixtures
thereof.[23] Water–dioxane samples of 1a and 1b were pre-
pared from a concentrated DMSO stock solution and subjected
to an absorption and steady-state emission spectroscopy study
(Figure 1 and Table 1). The small amount of DMSO has been
established to have negligible impact on polarity.[24]
The Stokes shifts (nabsÀnem) were calculated for the samples
in dioxane, water, and mixtures thereof and plotted, after con-
version from cmÀ1 to kcalmolÀ1, as a function of the experi-
mentally determined samples’ ET(30) values (Figure 3). The data
points clearly line up linearly, as was established by a good
linear fit with a slope value of 0.268, quantifying the sensitivity
to polarity (Table 2). In addition to a poorer linear fit, the sensi-
tivity to polarity for 1b was lower (Table 2 and Supporting In-
formation).
3.2.3. Sensitivity to pH
The sensitivity of the emissive 6-aza-U chromophores to pH
was studied in aqueous phosphate buffers with pH values
ranging from 0.5 to 12. Absorption spectra only showed small
differences in maxima (lmax =326 nm), while the corresponding
emission spectra revealed a strong influence of pH on the po-
sition as well as intensity of the emission maximum (Figure 2a,
Table 1). The emission maximum shifted from ~426 nm (F=
0.39) to ~462 nm (F=0.13), going from pH 10.13 to 2.55, re-
spectively. Curve-fitting the normalized fluorescence intensity
as a function of pH gave a sigmoidal curve from which a pKa,
Figure 1. a) Absorption (dotted line) and emission (solid line) spectra of 1a
in dioxane (bold black line, solid circles), water (bold black line, open circles),
and binary mixtures of 10, 30, and 70 v% water in dioxane (black lines).
Samples’ concentration is 7.9ꢁ10À6 m, emission is recorded after excitation
at 335 nm. b) Stokes shift [kcalmolÀ1] vs ET(30) values [kcalmolÀ1] obtained
from dioxane–water mixtures (data points with error bars) and linearization
(grey line). Absorption and emission spectra of each sample are recorded in
duplicate; only one set is shown.
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necessitating a third step to convert the thiocarbonyl into a car-
bonyl moiety.[27] To shorten the synthesis of 5a and 5b we
started from semicarbazide and converted the original two-pot
two-step synthesis[26] to a simplified one-pot two-step proce-
dure.[28] Importantly, this streamlined approach works well for
the synthesis of 5a and, albeit with lower yield, also for 5b,
while the three-step three-pot synthesis by Walker et al. works
for the synthesis of 5a, but proved challenging for the synthe-
sis of 5b.[27,29] Glycosylation of 5-substituted 6-aza uracils has
been reported.[27,30,31] We used a slightly modified procedure
involving activation of the nucleobase with BSA in CH3CN in-
stead of TMSCl and Et3N in benzene. We performed the glyco-
sylation directly in situ after BSA treatment, activating the
ribose (6) with TMSOTf, instead of changing the solvent from
benzene to dichloroethane and using stannic chloride to acti-
vate the ribose (6). Our procedure proved facile, quick, and
high-yielding for the synthesis of 7a, and 7b. Deprotection of
the benzoyl groups to give 1a and 1b was performed using
methanolic ammonia for 24 h at 608C in a pressure vessel. De-
protection also succeeded with aqueous ammonia in methanol
in 17 h at 408C, albeit in lower yield. Both 1a and 1b were
synthesized in three steps from commercially available starting
materials in 41% and 20% overall yield, respectively.
Table 2. Sensitivity to polarity.[a]
Slope
cmÀ1/kcalmolÀ1
Y-int.
cmÀ1
Slope
–
Y-int.
R2
kcalmolÀ1
1a
8a
1b
8b
93.7
63.8
75.4
79.5
2548
5188
4290
4437
0.268
0.182
0.216
0.227
7.29
0.99510
0.86159
0.84211
0.96922
14.83
12.27
12.69
[a] Polarity sensitivity equals the slope of the linearized Stokes shift vs
ET(30) plot. Values for 5-(thiophen-2-yl)-2dU (8a) and 5-(fur-2-yl)-2dU (8b)
in cmÀ1 kcalmolÀ1 have been reported.[7,25] A unitless value for polarity
sensitivity is obtained by conversion of the Stokes shift value from cmÀ1
to kcalmolÀ1 and replotting it as a function of the sample ET(30) value
[kcalmolÀ1]. The adjusted R2 represents the goodness of the linear fit.
4.2. Fluorescence Characteristics and their Sensitivity to
Polarity
Diverse events can impact the structure of nucleic acids [e.g.,
(un)folding, binding of low MW ligand, lesions], which often
result in changes in the local polarity. Nucleic acid probes able
to detect such changes in environmental polarity have proven
to be useful tools.[1,24,32–39] We therefore thoroughly evaluate
the influence of polarity on a probe’s photophysical properties.
We regularly use water–dioxane mixtures, as they not only
vary in polarity, but also present changes in hydrogen-bonding
donor and acceptor composition prone to exert specific sol-
vent–solute interactions.[23] Moreover, the polarity window ob-
tainable with such water–dioxane mixtures likely reflects most
biological relevant environmental polarities. Using a microenvir-
onmental polarity parameter such as the ET(30) scale with
values ranging from 36.0 to 63.1 kcalmolÀ1 for pure dioxane to
water, respectively,[11] typically provides good correlation with
spectral features like Stokes shifts.[23]
Figure 2. a) Absorption (dashed lines) and emission plots (solid lines), cor-
rected for small differences in the optical density at the lex(335 nm) of 1a at
pH 0.61 (open circles), pH 10.99 (solid circles), pH 6.55 (open triangles) and
intermediate pH values (black lines). b) Normalized emission intensity as
function of sample pH (solid circles) fitted to a sigmoidal curve (grey line).
The dashed lines illustrate a graphical determination of the pKa value.
While the quantum yield for 1a in water is modest (F=
0.20) and low for 1b (F=0.05), the quantum yield displayed
by 1a in dioxane (F=0.80) is among the highest values re-
ported for an isomorphic nucleoside (Table 1).[1] Both the quan-
tum yield in water and dioxane represent a huge improvement
compared to the values reported for the corresponding 2’-de-
oxyuridine modified with either a thiophene (8a) (Fwater =0.01)
or furan (8b) (Fwater =0.03) in the five position (Scheme 1).[2,6]
Interestingly, uridine analogs 8a and 8b display the opposite
behavior with even lower quantum yields in apolar organic
media.[2,6,24] Incidentally, this remarkable difference in fluores-
cence quantum yields between these isoelectronic nucleoside
analogs shows that photophysical properties are difficult to
predict from the molecular structure alone.
4. Discussion
4.1. Synthesis
The earliest scheme described for the synthesis of 5b, is
a two-pot two-step procedure starting from furanglyoxylic acid
(2b), which was reacted with semicarbazide hydrochloride (3)
to give the corresponding formic acid semicarbazone (4b).
This, in turn, yielded 5b upon refluxing in a mixture of propyl-
ene glycol–ethanol in the presence of sodium ethoxide.[26]
Later, Walker et al. reported a procedure for the synthesis of
2a, but used thio-semicarbazide instead of semicarbazide (3),
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Highly Emissive pH-Sensitive Fluorescent Nucleosides
It is worth noting that we previously reported the sensitivity
to polarity for 8a and 8b in cmÀ1 kcalmolÀ1 (Table 2). To obtain
unitless values, the values for the Stokes shift in cmÀ1 were
converted to kcalmolÀ1, and replotted as a function of the
samples’ ET(30) value. The slope for the linear relationship, the
polarity sensitivity, for modified uridines 8a and 8b was deter-
mined to be 0.182 and 0.227,[25] respectively, both significantly
less sensitive than 1a (Table 2). These substantial differences il-
lustrate how the enhanced push-pull motif present in 1a and
1b, as compared to 8a and 8b indeed results in the expected
enhanced susceptibility to polarity.
4.4. Sensitivity to Viscosity
The nucleobase moiety in 1a and 1b can be described as
a molecular rotor, being comprised of a single bond linking
two p systems.[7] Such molecules can lose their excitation
energy through a non-emissive rotational decay from a twisted
excited state. Molecular crowding effects such as that induced
by viscous solvents can hinder formation of a twisted excited
state, thereby limiting the non-emissive rotational decay pro-
cess, resulting in an increased emission intensity.[43] The contri-
bution of this non-emissive decay pathway can be studied by
plotting the emission intensity as a function of sample viscosi-
ty.[44] A practical approach to controlling sample viscosity is the
use of binary mixtures of solvents with very low and very high
viscosities that differ minimally in polarity.[7,8,45] Suitable non-
4.3. Sensitivity to pH
Sensitivity to pH as a characteristic of fluorescent nucleosides
is relatively uncommon, although a few examples have been
reported.[8,17,19] In these cases the absorption spectra show
a two-state transition with an isosbestic point. The influence of
pH on the emission properties is reflected by a strong increase
or decrease of the fluorescence intensity not necessarily ac-
companied by significant shifts of the emission maxima.
viscous and highly viscous solvents are methanol (h208C
=
0.583 cp), and glycerol (h208C =1317 cp),[7,46] respectively. They
can be mixed in all ratios to control sample viscosity, which
can be calculated based on the weight fraction and viscosity
values of its pure components.[47]
Clearly, 1a shows no fluorescence intensification upon in-
creasing viscosity, suggesting that 1a does not adopt a twisted
excited state upon excitation (Figure 3a). This contrasts the
Nucleoside 1a reveals its pH sensitivity by small changes in
absorption, but large changes in emission intensity and
maxima (Figure 2a). Changing from basic to acidic conditions,
the fluorescence curve of 1a shifts from a maximum located at
426 nm to 462 nm with a concurrent drop in the fluorescence
quantum yield from 0.39 to 0.13, respectively (Figure 2a,
Table 1). Fluorescence decay analysis reveals that the fluores-
cence lifetime in basic conditions is more than doubled com-
pared to the value in acidic conditions. The drop in normalized
fluorescence intensity as a function of pH gives, after a Boltz-
mann fit of the data points, an S-curve indicative of a two-
state (de)protonation process with a pKa of 6.7 and 6.6 for 1a,
and 1b respectively (Figure 2band the Supporting Informa-
tion). These values are in agreement with a reported value for
6-aza-uridine (pKa =6.8).[22] It further shows that the nature of
the modification in the 5-position of the 6-membered hetero-
cycle has little effect on the pKa of the NH as its electronic in-
fluence is limited. The observed pKa values significantly deviate
from the reported values for the parent uridine (9.3–9.5),[22,40,41]
demonstrating that the additional nitrogen renders the NH
~1000-fold more acidic. This finding is corroborated by others
who have reported that substitution of H-6 in native uracil
with an electron-withdrawing chloride has a spectacular influ-
ence on the pKa value, causing a drop from 9.5 to 5.8.[42] Closer
inspection of Figure 2ashows a downward trend of the fluores-
cent intensity at very low pHs. Although speculative at this
stage, this might suggest that the 6-aza nitrogen gets proton-
ated at pHs far below 2. A normalized plot excluding these
low pH data points (pH<2.5) gives a slightly higher pKa value
of 6.85 for the heterocycle’s NH (Figure S4.1, Supporting Infor-
mation).[25] Regardless of this putative second transition, 1a
shows fluorescence intensity as well as emission maximum
sensitivity with a pKa close to physiological pH. This makes 1a
an attractive probe for studies involving (de)protonation
events on RNA, as there are no known isomorphic pyrimidine
analogs with pKa values at this range.[21]
Figure 3. Absorption (dashed lines) and emission (solid lines) curves for 1a,
corrected for small differences in the O.D. at the lex(340 nm), in methanol
(solid circles), glycerol (open circles) and mixtures thereof (black lines).
significant sensitivity for viscosity observed for 1b.[25] The dif-
ference can perhaps be explained by their respective quantum
yields in solvents of low viscosity (Table 1). Samples in non-vis-
cous, protic solvents such as water and methanol display
quantum yields of 0.20 and 0.50 for 1a, but 0.05 and 0.04 for
1b, respectively. The already much higher quantum yields for
1a suggest that rotational relaxation might not be a major
non-radiative pathway for this chromophore’s electronic decay.
The very low initial quantum yields of 1b, however, benefit
greatly from curtailment of this non-emissive decay pathway.
Explaining the distinct differences between the two related
chromophores is challenging and must be sought in searching
for differences in the relative ground state orientation of the
thiophene (or furan) with respect to the nitrogenous nucleo-
base. In the reported crystal structure of the deoxy-ribose form
of 1a, the sulfur atom of the thiophene moiety, which is copla-
nar with the nucleobase, points away (anti) from the 4-carbon-
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Acknowledgements
We thank the National Institutes of Healthfor their generous sup-
port (grant number GM 069773), and the National Science Foun-
dation(instrumentation grant CHE-0741968).
Keywords: acidity
· donor-acceptor systems · fluorescent
probes · nucleosides · photophysics
Figure 4. Crystal structures of 8a,[6] 8b,[6] and the 2‘-deoxy-ribose form of
1a.[48] Note: For clarity, the heteroatoms of the nucleobases are labeled.
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[2] N. J. Greco, Y. Tor, J. Am. Chem. Soc. 2005, 127, 10784–10785.
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entation is opposite to that observed in our reported crystal
structure of 8a, in which the thiophene moiety, also coplanar
with the nucleobase, is oriented with its sulfur atom pointing
toward the 4-carbonyl.[2,6] The crystal structure of 8b shows
again the opposite configuration, with the furan oxygen facing
away from the 4-carbonyl (Figure 4).[2] In lieu of a crystal struc-
ture of 1b, we can only speculate on the orientation of its
furan oxygen. While differences between solid and solution
states could exist, the distinct orientational preferences of the
thiophene and furan units in the crystal structures might also
reflect their ground-state orientation in solution. The electronic
nature of the atom in the 6-position, CH (8a and 8b) or N (1a
and 1b) seems, therefore, to influence the orientation of the 5-
membered heterocycle. This, in turn, could have a profound in-
fluence on the excited-state geometry and hence sensitivity
toward viscosity.
5. Summary
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The 6-aza-uridine motif, decorated at the 5-position with either
a thiophene (1a) or furan (1b), is a synthetically accessible pyr-
imidine analog with highly desirable photophysical properties.
The Stokes shift of 1a is very sensitive to polarity. The pH-sen-
sitive fluorescence intensity starkly increases upon deprotona-
tion with a pKa value of 6.7–6.9, close to physiological pHs.
Most importantly, the fluorescence quantum yield remains suf-
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conditions and is thereby amongst the highest reported for
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and Fglycerol =0.66) suggest minimal population of non-radia-
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rescence quantum yield with increasing medium viscosity. In-
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a weaker sensitivity to polarity. Despite their differences, both
1a and 1b exhibit more desirable photophysical properties
than their reported 2‘-deoxyuridine analogs (8a and 8b),
making the 6-aza motif one of the most attractive and promis-
ing emissive pyrimidines.
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Received: May 4, 2012
Revised: June 19, 2012
Published online on && &&, 2012
ChemPhysChem 0000, 00, 1 – 8
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemphyschem.org
&
7
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These are not the final page numbers!

ARTICLES
R. W. Sinkeldam, P. A. Hopkins, Y. Tor*
&& – &&
Modified 6-Aza Uridines: Highly
Emissive pH-Sensitive Fluorescent
Nucleosides
Twinkle, twinkle: Modified 6-azauri-
dines are promising highly emissive iso-
morphic nucleoside analogs that display
a remarkable sensitivity to polarity and
pH (see graphic).
&8
&
www.chemphyschem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPhysChem 0000, 00, 1 – 8
ÝÝ
These are not the final page numbers!