Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing

Article abstract of DOI:10.1002/chem.201803653

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tC^O) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tC^O compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.

Full text of DOI:10.1002/chem.201803653

A Journal of
Accepted Article
Title: Electronic modifications of fluorescent cytidine analogues control
photophysics and fluorescent responses to base stacking and
pairing
Authors: Kristine L Teppang, Raymond W Lee, Dillon D Burns, M
Benjamin Turner, Melissa E Lokensgard, Andrew L Cooksy,
and Byron Willis Purse
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To be cited as: Chem. Eur. J. 10.1002/chem.201803653
Link to VoR: http://dx.doi.org/10.1002/chem.201803653
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10.1002/chem.201803653
Chemistry - A European Journal
Electronic modifications of fluorescent cytidine analogues control
photophysics and fluorescent responses to base stacking and
pairing
Kristine L. Teppang, Raymond W. Lee, Dillon D. Burns, M. Benjamin Turner, Melissa E. Lokensgard,
Andrew L. Cooksy, and Byron W. Purse*
Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA, 92182, USA
* To whom correspondence should be addressed. Tel: +1-619-594-6215; Email: bpurse@sdsu.edu. The authors
wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.
ABSTRACT
The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general
method to predict their photophysics, a problem that is especially acute when base pairing and
stacking change fluorescence. To better understand these effects, we have designed and synthesized
a series of tricyclic cytidine (tC and tCO) analogues ranging from electron-rich to electron-deficient,
incorporated them into oligonucleotides, and studied photophysical responses to base pairing and
stacking. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a
fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished
fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential
of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for
the electron-rich compounds, and it enhances a fluorescence decrease for the electron-deficient
compounds. For the tCO compounds, we observe the emergence of vibrational fine structure in the
fluorescence spectra in response to base pairing and stacking, offering a potential new tool for
studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to
rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent
nucleoside analogues for a desired application.
INTRODUCTION
Fluorescent nucleoside analogues are biophysical tools that are providing unique insights into the
structure and dynamics of nucleic acids. Motivated by increased recognition of the importance of
nucleic acid secondary, tertiary and quaternary structure in the maintenance and expression of the
genetic code, chemists have worked to plug gaps in the toolkit for fluorescent labeling of nucleic
acids.[1] Recent advances in the field include Tor’s highly isomorphic RNA alphabet and a growing
number of fluorescent nucleosides that have been designed for use in studying G-quadruplexes, i-
motifs, and lesions.[2–8] The impact of these biophysical tools is clear and exemplified in a number of
recent studies, such as detailed analyses of the mechanisms of riboswitch activation and the origins
1
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of toxicity of HgII through the formation of kinetically stable T-HgII-T base pairs.[9–11] It is equally clear
that serious limitations remain in the capabilities of fluorescent nucleosides. For example, existing
analogues capable of Watson–Crick hydrogen bonding do not emit at wavelengths great than 525 nm
and the brightest known isomorphic analogue, 6-methylisoxanthopterin (6-MI; ε340 = 14000 M−1 cm−1;
Φem,430 = 0.88), has an eight-fold lower extinction coefficient than rhodamine B in water (ε555 = 120000
M−1 cm−1; Φem,576 = 0.31), limiting brightness.[12–14] Moreover, 6-MI is nearly quenched when base-
stacked in duplex DNA (Φem = 0.04), as are most fluorescent nucleobase analogues.[15–18] A small
subset of fluorescent nucleoside analogues retains fluorescence in the base stack, and our lab has
recently reported on a cytidine analogue with the largest known fluorescence turn-on response to
base stacking and pairing.[19] The limited brightness of existing fluorescent nucleoside analogues
explains why they are not widely used in single molecule fluorescence measurements, and the only
existing FRET pairs between nucleobase analogues are nonemissive.[20–22] Ongoing efforts by a
number of groups are focused on developing nucleoside analogues with useful fluorescence changes
in response to base stacking, base pairing, changes in nucleic acid secondary and tertiary structure,
and environmental changes associated with protein binding or changes in nucleic acid solvation.[1,5,23–
31] But probably the greatest impediment to the development of new generations of fluorescent
nucleoside analogues with radically improved properties is that robust methods for predicting
photophysics based on structure are largely unavailable, especially when one is targeting the complex,
anisotropic environment of the base stack.[3]
A number of research groups have studied the relationships between structure and fluorescence
of nucleoside analogues, using a combination of empirical observations and computation. These
studies have generally used substituent effects to tune the photophysical properties of modified
nucleobases and characterized their fluorescence as free nucleosides, sometimes including Stern–
Volmer quenching measurements with natural nucleosides.[12,13,32–35] Our group has focused on
elaborating the tricyclic cytidine scaffold by the introduction of electron-donating groups (EDGs) and
electron-withdrawing groups (EWGs), extending the conjugation, and studying the effects of
substituent placement.[36] Recent works by Wilhelmsson and others have applied time-dependent
density functional theory (TDDFT) calculations to the prediction of absorption and emission spectra of
nucleobase analogues with some success.[32,34,35,37,38] But the application of computational work to
rational design of novel fluorophores is mostly limited to substituent effects.[37] While there are
examples of series of structurally related fluorescent nucleosides that have been studied for their
photophysics as free molecules,[32–34] systematic studies of nucleobase substituent effects on
fluorescence in the base stack have been rarely performed. Wilhelmsson has reported that parent
tricyclic cytidines have fluorescence insensitive to neighboring bases, and neighboring base effects
have been studied for 2-aminopurine quenching in duplex DNA.[39,40]
2
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Figure 1. Tricyclic cytidine derivatives used in this study to probe electronic effects on photophysical properties of free
nucleosides and the effects of base pairing and stacking. X = O in tCO compounds and X = S in tC compounds.
The tricyclic cytidine (tC) family of compounds was originally developed by Gilead as a part of their
antisense program because these compounds’ enhanced π stacking in the duplex can stabilize
secondary structure.[41] The fluorescence of the two parent compounds tC and tCO (X = O in tCO
compounds and X = S in tC compounds; see Figure 1) has been studied extensively by Wilhelmsson
et al., who noted that they are among the brightest known fluorescent nucleobase analogues that are
not quenched in the base stack.[42] These compounds have been used for biophysical studies of
enzyme mechanisms and Wilhelmsson has more recently developed a FRET pair with tCO as a donor
and nitro-tC as a nonemissive quencher.[20,21] In seeking to add new properties to the fluorescent
nucleoside toolkit, our lab synthesized and studied an extended family of tC compounds that
collectively hint at structure–photophysics relationships.[19,36] That work identified 8-Cl-tCO as the
member of the tC family with the brightest fluorescence and 8-DEA-tC as the least bright nucleoside,
and those results hinted at trends relating analogues’ electronics to photophysical properties such as
Stokes shift.[36] Further studies in duplex DNA showed that 8-DEA-tC exhibits up to a 20-fold increase
in fluorescence when it is base stacked and correctly base paired, making it the most powerful turn-on
fluorescent nucleoside known.[19] Here, we report on the synthesis and properties of 8-CN-tCO, a new,
most electron-deficient tCO analogue, and we assess the fluorescence responses of the family of tC(O)
analogues to base stacking and pairing in double-stranded DNA within the context of a varied set of
nearest neighboring bases (Figure 1; the abbreviation tC(O) is used to encompass both tC and tCO
compounds). The results, supported by density functional theory (DFT) studies, show clear
correlations between the electronic properties of the tC(O) analogues, the electron richness of
neighboring bases, and the fluorescence responses. The observed structure–photophysics
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relationships hint that predicting nucleobase analogue fluorophore responses to base stacking will be
possible when adequately accounting for conformational perturbation of the duplex and the electronic
interactions between nearest neighbors in the base stack.
RESULTS AND DISCUSSION
Design and Synthesis of Cytidine Analogues
The goal of identifying nucleobase analogue electronic effects on photophysics requires a range of
substituents. After problematic initial attempts at late-stage functionalization of tC derivatives through
metal-mediated reactions of 8-Cl-tCO, we adopted a strategy guided by the availability of suitable
starting materials and their reactivity in the tC and tCO syntheses, respectively (Schemes 1 and 2).
Parent compounds tC and tCO were synthesized using previously reported methods.[41,43,44] We
developed synthetic methods to prepare tC(O) derivatives, some of which we have reported previously,
but all are summarized here.[19,36] The amidites of 8-Cl-tCO, and 8-MeO-tC are new compounds, as is
the 8-CN-tCO nucleoside and its precursors. The targeted family of compounds spanned a range of
electronic richness as indicated by Hammett σp from −0.83 to 0.66, comprising the –NEt2, –OMe, –H,
–Cl, and –CN groups (Figure 1). The most electron-rich members, –NEt2 and –OMe, were
synthesized as tC analogues and the most electron-deficient, –Cl and –CN were synthesized as tCO
analogues. We synthesized the –OMe and –H compounds using both tC and tCO frameworks to
provide compound series overlap and to aid in structure–photophysics relationship identification. Past
work by our lab has shown that the photophysical properties of these tC(O) analogue nucleosides are
independent of substituent placement.[36] For example, 7-Cl-tCO (Cl is para to N) has λmax,abs = 357 nm,
λmax,em = 459 nm, ε = 6700 M−1 cm−1 and Φem = 0.35, nearly identical to 8-Cl-tCO (cf. Table 1). For that
reason, and for consistency, we chose to focus on substituents at the synthetically less challenging 8
position of the tC(O) framework.
The synthesis of the tC compounds 8-DEA-tC and 8-MeO-tC was carried out starting with the
corresponding 2-methylbenzothiazoles 1, which were converted to disulfides 2 by hydrazinolysis and
oxidation. This oxidation was necessary because the aminothiophenols were too unstable for
purification and handling. For preparation of the thioethers 3, the disulfides were reduced using
triethylphosphine and then used in a one-pot reaction with bromouracil in the presence of sodium
carbonate. Ring closure to complete the heterocyclic nucleobase analogues 4 was effected by reflux
under acidic conditions. Completion of the tC analogues 8-MeO-tC and 8-DEA-tC was attained by 2′-
deoxyribosylation using silyl Hilbert-Johnson conditions and deprotection by methoxide.
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Scheme 1. Synthesis of tC derivatives.
The synthesis of the tCO analogues follows an alternative route because aminophenols fail to undergo
coupling to bromouracil analogously to aminothiophenols in the synthesis of 3. Beginning with
protected bromouridine 7, Appel chemistry is used in conjunction with cyano-, chloro-, and
methoxyaminophenols to prepare secondary amines 8. Deprotection followed by aryl ether formation
as promoted by fluoride provided 8-CN-tCO, 8-Cl-tCO, and 8-MeO-tCO.
Scheme 2. Synthesis of tCO derivatives.
All of these tC(O) compounds were converted, using standard conditions, to 4,4′-dimethoxytrityl-
protected nucleoside phosphoramidites for solid-phase oligonucleotide synthesis (details in the
Supporting Information).
Photophysical Studies of tC(O) Derivative Nucleosides
With this extended tC(O) family in hand, we sought to measure photophysical properties and to check
for relationships to structure. The goal was to relate the results to empirical structural parameters and
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DFT calculations, seeking to rationalize trends and provide predictive capabilities for future efforts at
nucleoside analogue fluorophore design. These results and trends are also valuable for matching
individual nucleoside analogue fluorophores to biophysical applications.
We began by performing photophysical measurements on all these members of the tC(O) family as
free nucleosides in 1× PBS buffer (pH 7.4) and 1,4-dioxane (Table 1), including the recording of
absorption and emission spectra and the measurement of quantum yields of fluorescence emission,
Φem. Visual inspection of the plotted absorption and emission spectra of the tC(O) compounds shows a
number of readily apparent trends (Figures 2 and 3). First, all of these compounds have a low-energy
excitation at λ > 350 nm in addition to much stronger absorbance at shorter wavelengths (< 300 nm;
data shown in the Supporting Information). The absorption of these fluorophores at λ > 300 nm is
useful for their selective excitation when they are present in nucleic acids, which absorb at shorter
wavelengths. 8-CN-tCO is unique in that it has two distinct absorption bands at λ > 300 nm, the lower-
energy band being a shoulder at 355 nm, which is at nearly the same λmax as the other tCO
compounds. In the tCO series, considering only 8-CN-tCO’s longer wavelength absorption (its HOMO–
LUMO transition; see next paragraph), λmax,abs varies little, indicating that the substituents have little
effect on excitation energy. No trend is apparent in the extinction coefficients of the tCO compounds.
In contrast, the absorption spectra of the tC compounds show a different trend. Extinction coefficients
ε show a strongly correlated decreasing trend with more electron-donating substituents (Figure S1),
but patterns in λmax,abs are not apparent.
To better understand the electronic transitions and transition probabilities associated with
absorption and emission spectra, we performed DFT and TDDFT calculations on free nucleosides
and short, trimer oligonucleotides (single- and double-stranded with the B conformation; for details,
see Supporting Information). Briefly, a small series of benchmark calculations was carried out on
parent tCO using every combination of B3LYP,[45,46] BH&HLYP,[46,47] PBE0,[48] and M06[49] methods
with SVP,[50] TZVP,[51] cc-pVDZ,[52] and pc-2[53] basis sets. The best agreement with experimental
excitation and emission spectra was found for B3LYP-D2/cc-pVDZ and this methodology was applied
to the remaining calculations described in this paper. For free nucleosides, solvation was modeled by
optimizing the geometry with explicit water molecules hydrogen-bonding to the two oxygen atoms,
and then applying the IEFPCM continuum solvation model[54] to the entire system. Computational
modeling of the > 350 nm transitions described in the preceding paragraph indicates that they arise
from π to π* transitions between orbitals highly delocalized across the aryl system. Our calculations
ascribe the 8-CN-tCO band at 323 nm to a HOMO–LUMO+1 transition, still predominantly π to π* in
character, but somewhat stronger than the HOMO–LUMO transition. This observation is consistent
with a predicted trend towards increasing HOMO–LUMO+1 transition strength with increasing
electron-withdrawing character of the substituent.
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Table 1. Photophysical properties of the tC and tCO nucleosides and their derivatives.a
Compound
8-CN-tCO
8-Cl-tCO
tCO
Substituent
σp
0.66
Solvent
1× PBS
λmax,abs
λmax,em
Stokes shift ε at λmax,abs
Φem
Brightness
(ε·Φem)
1600
n.d.
2000
3500
1600
3400
400
1500
200
1800
40
323, 355
429
n.d.
458
453
473
445
513
472
499
474
550
500
493
524
106, 74
n.d.
97
8000, 5100
n.d.
0.32
n.d.
1,4-dioxane n.d.
1× PBS 361
1,4-dioxane 366
1× PBS 355
1,4-dioxane 357
1× PBS 377
1,4-dioxane 369
1× PBS 361
1,4-dioxane 360
1× PBS 379
1,4-dioxane 372
1× PBS 395
1,4-dioxane 389
0.23
0
5600
7200
6800
7400
4700
4300
4000
4800
3800
3800
2700
n.d.
0.35
0.49
0.24
0.46
0.09
0.34
0.05
0.38
0.01
0.28
0.006
0.06
87
118
88
tC
0
136
103
138
114
171
128
98
8-MeO-tCO
8-MeO-tC
8-DEA-tC
−0.27
−0.27
−0.83
1100
16
n.d.
135
a Measurements performed in 1× PBS buffer, pH 7.4, 296 K. n.d. = not determined
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Figure 2. Absorption (solid lines) and corrected emission (dashed lines; normalized at λmax to Φem) spectra of tCO derivatives in
1× PBS buffer (pH 7.4) recorded at 296 K.
Figure 3. Absorption (solid lines) and corrected emission (dashed lines and inset; normalized at λmax to Φem) spectra of tC
derivatives in 1× PBS buffer (pH 7.4) recorded at 296 K.
A plot of the Stokes shifts of the tC and tCO compounds in 1× PBS buffer (pH 7.4) against Hammett σp
values shows a clear correlation in the tCO series, but the values for the tC compounds are scattered
(Figure 4). Because wavelength is reciprocally dependent on energy, we plotted the absorption and
emission energies of each of these nucleoside analogues at λmax,abs and λmax,em in electron volts
against the Hammett σp values (Figure S2). Stokes shift decreases with increasingly electron-
withdrawing substituents on tCO, and this trend derives from the substituents’ stronger influence on
emission energy as compared with absorption energy. The more electron-deficient compounds emit
at higher energy. The strong correlation between absorption and emission energies of the tCO
compounds and σp indicates that the inductive and resonance effects of the substituents are the
major determinants of changes in Stokes shift.
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Figure 4. Linear correlation of Stokes shift to Hammett σp for tCO compounds (red) and plot for tC compounds (blue).
In contrast, our calculations at present only predict that λmax,abs should decrease with increasing
electron-withdrawing character, for the tC and tCO species alike. This effect appears to arise from
unequal shifts in the HOMO and LUMO energies as the electron density in the aryl π system changes.
EWGs reduce the electron density in the π system, dropping the HOMO energy by slightly more than
the LUMO energy. There is a greater density of MO energies at the higher excitation level of the
LUMO, and therefore the HOMO energy tends to respond more freely to changes in electron density
than the LUMO.
Trends are also apparent in the fluorescence quantum yields of the tC(O) analogues as a function
of their substituents. While no clearly linear correlations were observed, it is clear that the quantum
yield of fluorescence emission is greatly impacted by EDGs and EWGs (Figure 5). For both tC and
tCO derivatives, EDGs are associated with attenuated Φem, whereas EWGs increase Φem with respect
to the parent tC(O). 8-CN-tCO is an outlier with Φem = 0.32, a value similar to that of 8-Cl-tCO (Φem
=
0.35). We wondered whether our measurements of Φem for 8-CN-tCO were underreporting the true
value because the absorption at 355 nm might include a significant contribution from the HOMO–
LUMO+1 band at λmax = 323 nm. Curve fitting the 8-CN-tCO absorption spectrum to two Gaussian
functions using Origin 8.1 (OriginLab, Northampton, MA) shows that only around 10% of the
absorbance at 355 nm is derived from the tailing end of absorption centered at 323 nm (Figure S3).
The extent of overlap of the absorption centered at 323 nm is insufficient to explain why 8-CN-tCO
falls from the trend line of the other tCO derivatives. One possible explanation is that 8-CN-tCO may be
more prone to quenching by excited-state proton transfer because the cyano group increases this
compound’s acidity. We measured the acidity of 8-CN-tCO by recording the pH dependence of its Φem,
and obtained an inflection point indicative of pKa = 10.3 (see Supporting Information). We performed
TDDFT calculations to try to rationalize 8-CN-tCO’s relatively weak fluorescence, but the origin of this
intensity is not apparent from a visual analysis of the relevant orbitals or from the MO energies.
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Figure 5. Correlation of fluorescence quantum yield Φem to Hammett σp for tC (blue) and tCO compounds (red).
Figure 6. Normalized fluorescence emission spectra of the 8-Cl-tCO nucleoside in mixtures of 1× PBS buffer (pH 7.4) and 1,4-
dioxane. Vibrational fine structure appears only under nonaqueous conditions.
To better understand fluorescence responses to solvation, we measured absorption and emission
spectra of the tC(O) compounds in 1,4-dioxane as compared with 1× PBS buffer at pH 7.4 (Table 1).
Excitation energies (λmax,abs) were generally similar between solvents, but emission is blue-shifted in
1,4-dioxane, indicating that this solvent has less ability than water to stabilize the polar excited state
of the fluorophores. 8-DEA-tC is a notable exception, with a red-shifted λmax,em in dioxane. Quantum
yields of fluorescence were universally higher in dioxane. We also observed that distinct shoulders
indicative of vibrational fine structure appeared in the emission spectra of tCO, 8-MeO-tCO, and 8-Cl-
tCO as solvent mixtures approached 100% dioxane (Figure 6; shoulders were not observed for tC
derivatives and 8-CN-tCO was not measured). These shoulders are separated from λmax,em by
approximately 29 nm, corresponding to vibrational stretching wavenumbers near 1400 cm−1 and the
vibrational energy level transitions for arene C–C and C–N bonds. Interestingly, a similar appearance
of vibrational fine structure was observed in several cases when tCO analogues were base stacked in
double-stranded oligonucleotides, and this was especially true with 8-Cl-tCO (vide infra).
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Photophysical Studies of tC(O) Derivatives in Single- and Double-Stranded Oligonucleotides
We chose a representative set of 10-mer oligonucleotides with varied 5′ and 3′ neighboring bases for
these studies (Table 2). Attempts to prepare oligonucleotides containing 8-CN-tCO failed, seemingly
because this analogue is sensitive to degradation under the standard conditions of solid-phase
synthesis. Only one oligonucleotide incorporating tC was prepared because the photophysics of tC-
and tCO-containing oligonucleotides have been thoroughly studied by Wilhelmsson.[42] The
oligonucleotides were prepared using standard solid-phase synthesis conditions from the appropriate
nucleoside phosphoramidites, purified by HPLC, and confirmed by mass spectrometry. Absorption
and fluorescence spectra were recorded for these oligonucleotides in 1× PBS buffer (pH 7.4) at 296 K
and the measurements were repeated after duplex formation with matched, complementary DNA
oligonucleotides. The quantum yields of fluorescence emission Φem we measured using the
comparative method of Williams et al. and a fluorescence standard of quinine sulfate in 0.1 M
H2SO4.[55] Temperature-dependent circular dichroism spectra were recorded and used to measure
melting temperatures Tm and to assess conformational perturbations relative to the natural duplexes.
Molecular modeling shows how tC(O) compounds are likely to be base paired and stacked in
duplex oligonucleotides (Figure 7). This model was prepared beginning with an idealized duplex
trimer in the native B form conformation, modifying a cytidine, and minimizing with the MMFF force
field (Spartan ’08) with all natural atoms held at fixed positions. CD spectra show that the B form is
maintain when all of the tC(O) compounds substitute for cytidine (see Supporting Information). The
model shows extensive π system overlap in the form of base stacking with the central heterocyclic
ring of the 8-DEA-tC nucleobase, but most of the diethylaminobenzene group protrudes into the major
groove, where the diethylamino group is exposed to solvent.
Figure 7. Molecular model (Spartan ’08) showing 8-DEA-tC base paired and stacked in idealized B form DNA.
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A bar chart plotting Φem for 8-DEA-tC, 8-MeO-tC, tC, and 8-Cl-tCO as free nucleosides and in
single- and double-stranded oligonucleotides (AXA sequence; Table 2) immediately reveals a trend in
photophysical responses to base stacking and pairing (Figure 8). The electron-rich analogues, while
less emitting as free nucleosides, exhibit a large fluorescence increase when incorporated into duplex
oligonucleotides. In contrast, the electron-deficient 8-Cl-tCO shows a fluorescence decrease in the
duplex as compared with the free nucleoside analogue. The unsubstituted parent tC, in contrast again,
has relatively little fluorescence change in response to base stacking and Watson–Crick hydrogen
bonding. To further study this relationship, we plotted the fluorescence change upon duplex
incorporation, as expressed by Φem,ds/Φem,nuc on a logarithmic scale, against Hammett σp for each
substituent (Figure 9). The clear trend indicates that the electronic nature of the cytidine analogue is a
major determinant of its fluorescence response to base stacking and pairing. While similar data were
collected for the single-stranded oligonucleotides containing the tC(O) analogues, we hesitate to
ascribe meaningful trends to their photophysics. The well-characterized conformational variability of
short, single-stranded DNA oligonucleotides makes it unlikely that the cytidine analogue fluorophores
experience consistent local environments.
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Table 2. Photophysical properties single- and double-stranded oligonucleotides containing tC(O) compounds.a
Analogue
8-Cl-tCO
Sequen
ce Name
Sequence
ss Φem
ds
Φem
ds λmax,abs
/ nm (ss)
ds λmax,em
nm (ss)
/
Tm / °C
ΔTm
/ °C b
AXA
GXC
TXA
TXT
AXA
AXA
GXC
TXA
TXT
AXA
GXC
TXA
TXT
GXA
CXT
CXA
GXG
CXC
AXA
GXC
TXA
TXT
5′-CGC-AAX-ATC-G-3′
5′-CGC-AGX-CTC-G-3′
5′-CGC-ATX-ATC-G-3′
5′-CGC-ATX-TTC-G-3′
5′-CGC-AAX-ATC-G-3′
5′-CGC-AAX-ATC-G-3′
5′-CGC-AGX-CTC-G-3′
5′-CGC-ATX-ATC-G-3′
5′-CGC-ATX-TTC-G-3′
5′-CGC-AAX-ATC-G-3′
5′-CGC-AGX-CTC-G-3′
5′-CGC-ATX-ATC-G-3′
5′-CGC-ATX-TTC-G-3′
5′-CGC-AGX-ATC-G-3′
5′-CGC-ACX-TTC-G-3′
5′-CGC-ACX-ATC-G-3′
5′-CGC-AGX-GTC-G-3′
5′-CGC-ACX-CTC-G-3′
5′-CGC-AAX-ATC-G-3′
5′-CGC-AGX-CTC-G-3′
5′-CGC-ATX-ATC-G-3′
5′-CGC-ATX-TTC-G-3′
0.40
0.20
367 (363)
367 (366)
368 (368)
366 (363)
391 (391)
399 (397)
399 (399)
398 (398)
403 (392)
410 (415)
398 (425)
420 (417)
416 (413)
422 (425)
414 (413)
410 (420)
400 (415)
413 (421)
392 (391)
391 (394)
395 (390)
399 (390)
447 (446)
451 (453)
445 (445)
447 (444)
502 (502)
529 (531)
532 (536)
530 (532)
530 (526)
498 (497)
500 (499)
495 (495)
495 (495)
499 (499)
492 (495)
494 (493)
499 (498)
496 (496)
507 (510)
507 (509)
503 (509)
503 (507)
51.4
29.6
55.3
n.s.‡
n.d.
50.8
59.2
55.5
n.s.‡
48.4
49.1
48.7
48.5
50.7
54.7
57.4
58.2
60.2
42
+0.4
−34
+5.0
n.s.‡
n.d.
−0.2
−4.4
+5.2
n.s.‡
−2.6
−14.5
−1.6
+0.2
−5.1
+2.0
+2.1
−7.0
+6.7
+1
0.15
0.45
0.32
0.11
0.14
0.18
0.22
0.11
tC
8-MeO-tC
0.023
0.024
0.029
0.036
0.008
0.032
0.014
0.025
0.020
0.020
0.014
0.027
0.013
0.21
0.024
0.023
0.029
0.029
0.042
0.12
0.026
0.041
0.050
0.025
0.017
0.056
0.024
0.18
8-DEA-tC
tC†
0.19
0.21
0.22
0.16
0.20
0.20
46
45
44
−3
+4
+5
a Measurements performed in 1× PBS buffer, pH 7.4, 296 K. tC† = data from Wilhelmsson for tC-containing oligonucleotides of the same sequences,
measured in 50 mM sodium phosphate buffer, pH 7.5.[39] ‡ The melting curve was not sigmoidal (n.s.) and therefore no distinct Tm is indicated. b ΔTm =
Tm(analogue-containing duplex) − Tm(corresponding duplex with natural cytidine)
13
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Chemistry - A European Journal
Figure 8. Changes in fluorescence quantum yield of four cytidine analogues up incorporation into single- and double-stranded
DNA oligonucleotides, measured in 1× PBS buffer (pH 7.4). nuc = free nucleoside, ss = single-stranded oligonucleotide with the
AXA sequence 5′-CGC-AAX-ATC-G-3′ (Table 2), where X = the fluorescent cytidine analogue, ds = matched, double-stranded
oligonucleotide.
Figure 9. The fluorescence change upon incorporation of a tC(O) compound into a double-stranded oligonucleotide of the AXA
sequence (Table 2), as expressed by Φem,ds/Φem,nuc on a logarithmic scale, is strongly correlated to the Hammett σp for the tC(O)
compounds’ substituents.
As the data for the AXA sequences indicates a clear relationship between the electronics of the
cytidine analogue and its fluorescence response to base stacking and pairing, we next examined the
effects of neighboring bases on this response (Figure 10 and Table 2). The data show that 8-DEA-tC
has the greatest sensitivity to neighboring bases of all analogues tested. The least bright sequence,
CXA, shows approximately triple the Φem as compared with the 8-DEA-tC nucleoside, whereas the
brightest sequence, GXC, exhibits a 20-fold greater Φem, the greatest known fluorescence turn-on
response to base pairing and stacking. Considering only sequences with A as the 3′ neighbor, Φem
increases from C, T, A, to G, following the order of decreasing oxidational potential of these canonical
nucleobases.[56] A plot of Φem for these sequences against the oxidation potentials of the 5′-
neighboring nucleobases gives a strong linear correlation (Figure 11). In contrast, when the 5′
neighbor is fixed as C, Φem increases slightly from 3′ neighbors of A to C (CXG was not measured),
14
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10.1002/chem.201803653
Chemistry - A European Journal
an opposite but much diminished trend (c.f. Figure 12). While it is not at this time entirely clear how
these trends can be rationalized by structure, it is apparent that the different π stacked overlap on the
5′ and 3′ sides gives rise to very different electronic interactions between bases.
Figure 10. Fluorescence quantum yield of tC(O) derivatives as free nucleosides (black) and in duplex oligonucleotides with
varied neighboring bases (full sequences given in Table 2), measured in 1× PBS buffer, pH 7.4 (columns plotted against left y-
axis). Excitation and emission energy, derived from λmax,abs and λmax,em respectively, are plotted as points against the right y-axis.
tC† = data from Wilhelmsson for tC-containing duplex oligonucleotides of the same sequences, measured in 50 mM sodium
phosphate buffer, pH 7.5.[39]
Figure 11. Dependence of the Φem of 8-DEA-tC-containing duplex oligonucleotides (for sequences, see Table 2) on the
oxidation potential of the 5′-neighboring nucleobase.[56]
The other analogues 8-MeO-tC, tC, and 8-Cl-tCO have fluorescence quantum yields that are much
less sensitive to neighboring base effects. Clear trends in these smaller changes in Φem are not
apparent, but it is notable that the least bright sequence including 8-Cl-tCO, GXC, is the brightest
sequence for 8-DEA-tC. Here, the relatively electron-deficient 8-Cl-tCO has the most electronic rich
canonical nucleobase, G, as its 5′ neighbor, and there is an associated decrease in fluorescence. The
opposite was observed for electron-rich 8-DEA-tC. Again, tunable electronic interactions between
neighboring bases are critical determinants of fluorescence changes. In contrast to this observation,
15
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we found that duplex stability as measured by ΔTm was lowest in the GXC sequence for all of the
analogues, irrespective how Φem changes in response to base stacking (Table 2). Similarly, CD
spectra for all analogue-containing duplexes indicate the B form conformation, but the spectra are
most perturbed in the GXC sequences. Specifically, there is a decrease in the local minima centered
at 255 nm (see Supporting Information). These data suggest an intriguing local structural perturbation
that may be very important for understanding changes in fluorescence, but that will likely require NMR
or x-ray structural determination to fully understand.
Figure 12. Fluorescence quantum yield of tC(O) derivatives as free nucleosides (black) and in duplex oligonucleotides with
varied neighboring bases (full sequences given in Table 2), measured in 1× PBS buffer, pH 7.4. tC† = data from Wilhelmsson
for tC-containing duplex oligonucleotides of the same sequences, measured in 50 mM sodium phosphate buffer, pH 7.5.[39]
Figure 13. Fluorescence emission spectra of 8-Cl-tCO as a free nucleoside (black), in ssDNA (blue; AXA sequence from Table
2), and dsDNA (red; annealed to matched complement), in mismatched dsDNA (green; 8-Cl-tCO:A mismatch), and in dsDNA
opposite the 1,2-dideoxy-D-ribose surrogate for an abasic site (orange; 8-Cl-tCO:AP) all in 1× PBS buffer (pH 7.4), and the
emission spectrum of 8-Cl-tCO nucleoside in 1,4-dioxane (purple dashed line).
16
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The vibrational fine structure of the fluorescence emission spectra of some analogues is also
affected by the duplex. Wilhelmsson et al. reported previously that parent tCO exhibits vibrational fine
structure in its fluorescence emission spectrum when incorporated into a duplex DNA oligonucleotide,
irrespective of the identity of the neighboring bases.[44] We find the same with 8-Cl-tCO in the present
study (Figure 13 and additional spectra in the Supporting Information). We also observed that
fluorescence emission intensity and the appearance of vibrational structure are further enhanced
when 8-Cl-tCO is mispaired with adenosine (see also next paragraph). Wilhelmsson has previously
proposed that the appearance of this vibrational fine structure can be attributed to the fact that “tCO is
firmly stacked and has a very well-defined position and geometry at least on the time scale of
fluorescence.”[44] In light of our observation that very similar vibrational fine structure is observed in
the emission spectra when the tCO nucleosides are simply dissolved in 1,4-dioxane—conditions under
which they are surely not base stacked—we propose an alternative explanation. Solvation of the tCO
compounds in aqueous buffer in all likelihood involves an ensemble of multiple types of hydrogen
bonding arrangements between water and analogue, each with slightly perturbed vibrational energy
levels. Collectively, such solvation states would broaden the emission spectra. The tCO analogues are
engaged in Watson–Crick hydrogen bonds in duplex DNA, but this is a highly ordered arrangement of
hydrogen bonds that stands in contrast with the ensemble of arrangements possible in solution. For
this reason, solvation-induced perturbation of the vibrational energy levels is greatly diminished, and
the vibrational fine structure emerges in the emission spectra. This model is further supported by the
observation that vibrational fine structure is not seen when 8-Cl-tCO (and parent tCO) is present
opposite an abasic site, and therefore is not engaged in ordered hydrogen bonding. The tC
compounds, particularly 8-DEA-tC, do show hints of a similar emergence of vibrational fine structure
upon base stacking, although this effect is much diminished (see spectra in the Supporting
Information).
Our prior studies of 8-DEA-tC’s fluorescence turn-on responses to base stacking and Watson–
Crick hydrogen bonding prompted an investigation of how tC(O) analogue substituents influence
fluorescence responses to mispairing and the presence of abasic sites. Choosing the AXA sequence
and the analogues 8-DEA-tC, 8-MeO-tC, tC, and 8-Cl-tCO, we measured oligonucleotide fluorescence
and the change in Φem upon duplex formation with a matched complement and a tC(O) derivative :
adenosine mismatch (Table 3 and Figure 14). We also measured 8-DEA-tC and 8-Cl-tCO
fluorescence when opposite a 1,2-dideoxy-D-ribose (AP) surrogate for an abasic site. While 8-DEA-
tC’s fluorescence turn-on response to base stacking and base pairing is greatly diminished when
opposite adenosine or an abasic site, the opposite trend is observed for 8-Cl-tCO. 8-MeO-tC and
parent tC are in the middle, exhibiting slightly increased fluorescence when opposite adenosine as
compared with when they are base paired with guanosine. Again, electronic modification of these
tricyclic cytidines flips their responses to DNA duplex formation.
17
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Figure 14. Fluorescence quantum yield of tC(O) derivatives in single-stranded oligonucleotides (AXA sequence; see Table 2), in
matched duplexes X:G, with adenosine mismatches X:A, and opposite the abasic site mimic 1,2-dideoxy-D-ribose.
Measurements were performed in 1× PBS buffer, pH 7.4.
Looking at all of these data in aggregate, it is also apparent that there are distinct trends in
excitation and emission energies, influenced by the electronic nature of the tC(O) analogues and the
effects of base stacking and pairing (Figures 10, 12, and 14). For all analogues studied, base stacking
lowers the excitation energy. Base pairing, in contrast, has little influence on excitation energy. The
excitation energy for all analogues in all sequence contexts is little changed by base pairing with
guanine, adenine, or the AP abasic site mimic. In contrast, when any tC(O) analogue is paired with the
AP site, fluorescence emission occurs at greater energy (Figure 14). This observation suggests that
hydrogen bonding at the Watson–Crick interface stabilizes the excited state of the tC(O) analogue. For
all of the analogues except 8-DEA-tC, base stacking raises emission energy. 8-DEA-tC’s emission
energy is slightly lowered by base stacking, especially with the neighboring bases associated with the
strongest fluorescence turn-on. We note the parallel to 8-DEA-tC’s blue-shifted emission in buffer as
compared with dioxane, unique among these tC(O) analogues.
Computational modeling of these effects remains challenging. Because 8-DEA-tC exhibits some of
the strongest variation in quantum yield with complexation, its excitation spectrum was predicted by
TDDFT as a function of its separation distance from the base pair partner guanosine and from C, G,
and A π-stacking neighbor nucleosides. The base-pairing (with no π-stacking) redshifts the predicted
absorbance spectrum by roughly 15 nm at an H-bonding distance of 1.6 Å, and subsequent π-
complexation with C and G cause additional redshifts of similar magnitude. This is qualitatively
consistent with the drop in excitation energies shown in Figure 10 from monomer to oligonucleotide.
The delocalization of MOs across adjacent nucleosides upon complexation is one promising line of
inquiry, as previously suggested by Hardman and Thompson.[40] Molecular orbitals were calculated for
three of the 8-DEA-tC duplex trimers appearing in Fig. 10 (stacked as CXA, GXA, and GXC). Table 4
reports the distribution of electron density of the (nominal) 8-DEA-tC HOMO and LUMO for each of
these three complexes, relative to the density localized on the analogue itself. The results show that
orbital delocalization onto the neighboring nucleoside is consistently and significantly greater upon
complexation with G than with A or C. Although this demonstrates a base-specific response to the
18
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stacking, additional calculations are planned to elucidate the origin of the effect and its relationship to
the relatively high fluorescence response of 8-DEA-tC when stacked with guanine.
19
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Table 3. Photophysical properties matched and mismatched oligonucleotides of the sequence 5′-CGC-AAX-ATC-G-3′, where X = a tC(O) compound.a
Analogue
8-Cl-tCO
Experiment
ss
Complement
Φem
0.40
λmax,abs / nm
363
λmax,em / nm
446
X:G match
X:A mismatch
X:AP
5′-CGA-GGC-TGC-G-3′
5′-CGA-GAC-TGC-G-3′
5′-CGA-GAPC-TGC-G-3′
0.20
0.41
0.49
367
370
369
447
449
444
tC
ss
0.11
391
502
X:G match
X:A mismatch
ss
X:G match
X:A mismatch
ss
X:G match
X:A mismatch
X:AP
5′-CGA-GGC-TGC-G-3′
5′-CGA-GAC-TGC-G-3′
0.11
0.13
391
394
397
399
401
415
410
417
502
501
529
532
526
497
498
501
8-MeO-tC
8-DEA-tC
0.023
0.024
0.041
0.008
0.042
0.016
0.007
5′-CGA-GGC-TGC-G-3′
5′-CGA-GAC-TGC-G-3′
5′-CGA-GGC-TGC-G-3′
5′-CGA-GAC-TGC-G-3′
5′-CGA-GAPC-TGC-G-3′
421
466
a Measurements performed in 1× PBS buffer, pH 7.4, 296 K. ss = single-strand including tC(O) analogue.
20
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Table 4. B3LYP/cc-pVDZ HOMO and LUMO electron densities of nucleosides stacked with X=8-DEA-tC, relative to the density
localized on the central 8-DEA-tC.
Complex
HOMO relative density
LUMO relative density
A/X
0.008
0.068
-
C/X
0.003
-
G/X
-
A/X
0.045
0.108
-
C/X
0.002
-
G/X
-
CXA
GXA
GXC
0.456
0.621
0.453
0.539
0.002
0.003
CONCLUSION
In this project, we report on the synthesis and photophysical characterization of an extended series of
tC(O) compounds as free nucleosides and in single- and double-stranded oligonucleotides. The
compounds range from electron-deficient (8-CN-tCO) to electron-rich (8-DEA-tC). Photophysical
studies of the free nucleosides show that EWGs shorten the Stokes shift of tCO compounds and raise
Φem, although 8-CN-tCO’s Φem is slightly depressed relative to 8-Cl-tCO. There is no clear pattern of
substituent effects on molar extinction coefficient ε. Electron-richness is also associated with low Φem
for tC compounds and there is a clear correlation between Hammett σp and ε, but patterns of Stokes
shift are less apparent. Substituents clearly have the ability to alter photophysics in ways that are
strongly correlated with σp, but the nature of these correlations—positive or negative, and exactly
which photophysical parameters are correlated—depends on the heterocyclic scaffold of the
fluorophore.
Electronic substituent effects play a large role in determining the fluorescent responses to base
pairing and stacking and, in some cases such as predicting neighboring base effects on 8-DEA-tC
fluorescence, are critical parameters. In general, whereas parent tC is insensitive to base stacking,
electron-deficient 8-Cl-tCO is partially quenched by base stacking, while electron-rich 8-DEA-tC
exhibits a fluorescence turn-on. 8-DEA-tC’s fluorescence turn-on is greatest when base-stacked
against the most electron-rich natural neighbor, guanine, a trend that appears to be inverted for
electron-deficient 8-Cl-tCO. Computation correctly predicts that emission energy is lowered (i.e. λmax,em
is red-shifted) by base pairing and stacking and it is clear that MOs mix between neighboring bases,
providing a general rationale for the strong effects on fluorescence and its dependence on the
electronic nature of the fluorescent base analogue and its nearest neighbors. Electronic modifications
to tC(O) compounds also invert the fluorescent responses to base pairing. 8-DEA-tC’s fluorescence
turn-on is greatly diminished by mispairing with adenine or an abasic site, whereas 8-Cl-tCO is
brightest in these contexts.
21
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A striking observation in these studies is that the tCO compounds, especially 8-Cl-tCO, show
enhanced vibrational fine structure in their emission spectra when in duplex oligonucleotides and
base paired with G or A, and when dissolved in 1,4-dioxane. This vibrational fine structure is absent in
aqueous buffer and when the analogue is present in the duplex opposite an abasic site. The data
strongly suggest that the appearance of this vibrational fine structure is the result of highly organized
hydrogen bonding as one would expect at the Watson–Crick interface or in a tautomeric base pair
with adenine. Accordingly, this spectral feature may offer special applications for probing nucleic acid
structure and dynamics at fast timescales.
Computational modeling predicts the orbital mixing between stacked bases that, along with
electron richness or deficiency, is essential for determining fluorescence changes. Spectral
predictions can, to some extent, rationalize changes in absorption and emission wavelengths and how
they change in response to the duplex. But changes in CD spectra and duplex melting temperature
upon analogue incorporation, particularly in the GXC sequences, indicate some local structural
perturbation. For that reason, we expect that NMR or x-ray structure determination will be needed to
determine the exact nature of base stacking of these analogues, information that will be invaluable for
further computational studies.
Research on fluorescent nucleoside analogues continues to be inspired by the need for powerful,
accurate, and nonperturbing probes for nucleic acid biophysics. 8-DEA-tC has a uniquely powerful
ability to report on matched duplex formation by fluorescence turn-on, and the fluorescence spectra of
8-Cl-tCO have vibrational fine structure with exciting potential for use as a reporter on local structure
and dynamics. The aggregate results of this study show that electronic tuning of fluorescent
nucleosides modulates fluorescent responses to base pairing and stacking that are clearly determined
by the electronic nature of neighboring bases and the hydrogen bonding at the Watson–Crick
interface. Moreover, the diversity of fluorescent responses observed, including turn-on, turn-off, and
changes in vibrational fine structure, invites a variety of applications in nucleic acid biophysics. We
believe that further studies of structure–photophysics relationships will lead to a deeper understanding
of nucleoside analogue fluorescence responses to the anisotropic environment of duplex nucleic
acids, eventually allowing for useful fluorescent properties to be planned in rational design.
EXPERIMENTAL SECTION
General experimental information, synthetic procedures, compound characterization, photophysical
experimental methods, and computational methods are described in the Supporting Information.
SUPPORTING INFORMATION
Supporting Information is available online.
22
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FUNDING
This work was supported by the National Science Foundation [CHE-1709796 to B.W.P]; the National
Institutes of Health [GM058906 to K.L.T.]; the California State University Program for Education &
Research in Biotechnology; and San Diego State University.
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TOC Graphic and Caption
Base stacking and pairing of fluorescent nucleosides such as DEA-tC changes their photophysical
properties in ways that are strongly correlated with the electronic nature of neighboring bases.
25
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