A General Strategy to Enhance Donor-Acceptor Molecules Using Solvent-Excluding Substituents

Article abstract of DOI:10.1002/anie.201915744

While organic donor-acceptor (D-A) molecules are widely employed in multiple areas, the application of more D-A molecules could be limited because of an inherent polarity sensitivity that inhibits photochemical processes. Presented here is a facile chemical modification to attenuate solvent-dependent mechanisms of excited-state quenching through addition of a β-carbonyl-based polar substituent. The results reveal a mechanism wherein the β-carbonyl substituent creates a structural buffer between the donor and the surrounding solvent. Through computational and experimental analyses, it is demonstrated that the β-carbonyl simultaneously attenuates two distinct solvent-dependent quenching mechanisms. Using the β-carbonyl substituent, improvements in the photophysical properties of commonly used D-A fluorophores and their enhanced performance in biological imaging are shown.

Full text of DOI:10.1002/anie.201915744

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: A facile strategy to enhance donor-acceptor molecules using
solvent-excluding substituents
Authors: Xin Zhang, Conner A. Hoelzel, Hang Hu, Charles H.
Wolstenholme, Basel A. Karim, Kyle T. Munson, Kwan Ho
Jung, Yu Liu, Hemant P. Yennawar, John B. Asbury, and
Xiaosong Li
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201915744
Angew. Chem. 10.1002/ange.201915744
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10.1002/anie.201915744
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RESEARCH ARTICLE
A general strategy to enhance donor-acceptor molecules using
solvent-excluding substituents
Conner A. Hoelzel,[a] Hang Hu,[c,d] Charles H. Wolstenholme,[a] Basel A. Karim,[a,b] Kyle T. Munson,[a]
Kwan Ho Jung,[a] Prof. Yu Liu,[a,e] Prof. Hemant P. Yennawar,[b] Prof. John B. Asbury,[a] Prof. Xiaosong
Li,*[c] Prof. Xin Zhang*[a,b]
Abstract: While organic donor-acceptor (D–A) molecules are widely
employed in multiple areas, application of a large number of D-A
molecules could be limited due to an inherent polarity sensitivity that
inhibits photochemical processes. Here we present a facile chemical
modification to attenuate solvent-dependent mechanisms of excited
energy, visible light. In organic synthesis, D–A molecules are
employed as photolabile protecting groups for acidic functional
groups[1] and as photoredox catalysts to unlock unique chemical
transformations.[2] In biological research, D–A type fluorophores
have been utilized as labeling reagents to image the behavior,
expression, and localization of biological macromolecules, in
particular via fluorescence microscopy.[3] Yet the application of D–
A type molecules is limited in many regards often due to low
fluorescence quantum yields, short excited state lifetimes, and an
inherent polarity sensitivity that leads to rapid non-radiative decay.
The mechanisms governing the quenching of D–A excited states
include internal conversion to a lower energy, non-emissive
excited state, aggregation,[4] intermolecular hydrogen bonding,[5]
twisted intramolecular charge transfer (TICT),[6] and heat loss to
the solvent upon intramolecular charge transfer, known as
external conversion (EC)[7] (Figure 1b). However, the task of
engineering practical and generalized methods to control these
dynamics remains persistent.
state quenching through addition of
a b-carbonyl-based polar
substituent. Our results reveal a mechanism wherein the b-carbonyl
substituent creates a structural buffer between the donor and the
surrounding solvent. Through computational and experimental
analyses, we demonstrate that b-carbonyl simultaneously attenuates
two distinct solvent-dependent quenching mechanisms. Using the b-
carbonyl substituent, we demonstrate improvements in the
photophysical properties of commonly used D–A fluorophores and
their enhanced performance in biological imaging. In summary, this
work discovers the b-carbonyl substituent as a general method to
enhance the photophysical properties of D-A molecules, thus
potentiating its application in a broad spectrum of D–A molecules and
their excited state chemistry in polar environments.
Introduction
Organic donor-acceptor (D–A) molecules possess
a
common motif of both an electron donating and withdrawing group
separated by a conjugated p system (Figure 1a). Such D–A type
molecules are widely employed in an array of fields within
chemical and biological sciences provided the unique metal-free,
spatiotemporal control over their reactivity and behavior with low-
[a]
C. Hoelzel, C. Wolstenholme, B. Karim, K. Munson, K. Jung, Prof.
Y. Liu, Prof. J. Asbury, Prof. X. Zhang
Department of Chemistry
Pennsylvania State University
University Park, PA 16802, United (United States of America)
E-mail: xuz31@psu.edu
[b]
[c]
B. Karim, Prof. H. Yennawar, Prof. X. Zhang
Department of Biochemistry and Molecular Biology
Pennsylvania State University
University Park, PA 16802 (United States of America)
H. Hu, Prof. X. Li
Department of Chemistry
University of Washington
Seattle, WA 98105 (United States of America)
E-mail: xsli@uw.edu
[d]
[e]
H. Hu
Molecular Engineering and Sciences Institute
University of Washington
Seattle, WA 98105 (United States of America)
Prof. Y. Liu
Dalian Institute of Chemical Physics
Dalian 116023 (China)
Figure 1. Proposed b-carbonyl donor attenuates solvent-mediated excited
state decay. a) Common scaffold of D-A molecules.
b) Established
mechanisms of thermal, non-radiative excited state decay. c) Proposed role of
b-carbonyl based donors to attenuates solvent-mediated excited state decay.
(EWG: electron withdrawing group).
Supporting information for this article is given via a link at the end of
the document.
1
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In recent years, a focus has been placed on achieving the
above goal in order to develop more effective fluorophores with
improved fluorescence quantum yields and photostability for
application in biological and photoorganic chemistry.[8] In
particular, small azacyclic donors have been used to inhibit TICT
in D–A and rhodamine based fluorophores, improving their
application in super-resolution microscopy.[8c, 8d] Additionally, N-
cyclohexanol substituted amino donors have been shown to
inhibit solvent-solute interactions with the excited state,
attenuating the effects of EC in aqueous solutions.[8b] Each of
these works address a single mechanism of excited state
quenching. However, as TICT and other solvent-dependent
quenching mechanisms operate independently of one another, it
is desirable to develop a unique and generally applicable
chemical modification to control solvent interactions with the
excited state that would act cooperatively with these other
established methods to prevent excited state non-radiative decay.
In line with the established theme of perturbing amino
donors to improve the photophysical and photochemical
properties of D–A molecules, our chemical modifications were
aimed at shielding an amino auxochrome from solvent interaction
through appendage of a polar functional group at the b position
relative to the nitrogen donor (Figure 1c). We hypothesized that
such a modification should effectively improve the photophysical
properties of D–A molecules by minimizing the contribution of EC
to the overall rate of excited state quenching. Using D–A
fluorophores as model systems and their fluorescence quantum
yield (Ff) as an evaluation, we demonstrate that inclusion of a b-
carbonyl effectively attenuates the influence of solvent-dependent
quenching mechanisms on the rate of non-radiative decay in D–
A fluorophores. Crystallographic and empirical characterizations
in tandem with computational modeling support a structure-based
argument for attenuation of solvent interaction with the excited
state, thus inhibiting EC. Further, we show that incorporation of
the b-carbonyl effectively inhibits TICT by increasing the excited
state rotational energy barrier of the donating group, allowing us
to propose a mechanism in which the polarity of the b-carbonyl
acts to simultaneously control two independent means of excited
state quenching. In this work, we demonstrate the application of
this b-carbonyl modification to enhance brightness of
fluorophores in live cell imaging. These results promote us to
envision that polar auxiliaries, represented by the b-carbonyl, offer
a general strategy to mitigate multiple mechanisms of non-
radiative quenching and expand the use of D–A type molecules
in a wide range of applications that utilize excited state chemistry.
increases in fluorescence brightness (B) for compounds 2 and 3
in ethanol (B = 2,750 and 3,909, respectively) when compared to
1 (B = 485) (Figure 2b, Table S1 and Figure S1). Furthermore, the
increases in brightness for 2 and 3 persist in a nonpolar solvent
such as a 1,4-dioxane, where solvent-solute interactions are
weak and EC is generally suppressed, albeit the fold change is
reduced (2.5- and 3.7-fold for 2 and 3, respectively). While
brightness is influenced by both fluorescence quantum yield and
molar absorptivity, our photophysical characterization found the
improved brightness was solely due to the increase of
fluorescence quantum yields instead of molar absorptivity (Table
S1). Further, we removed the carbonyl component by
synthesizing S20, an NBD derivative comprised of
a 2-
methylamino-ethyl methyl ether donor with a similar molecular
weight of the b-carbonyl donor. A fluorescence quantum yield of
0.09 in ethanol was determined for S20 (Table S1), compared to
0.18 for 3, indicating the necessity of the b-carbonyl to confer the
observed effect.
Figure 2. b-carbonyl donors enable enhanced photophysical properties in a
model D–A fluorophore, NBD. a) The amide based b-carbonyl NBD library. b)
Brightness of NBDs 1–12 as determined in ethanol and 1,4-dioxane. (brightness
= Ff • e). Fluorescence quantum yields were measured relative to a fluorescein
standard (Fstd = 0.79 in 0.1 M aqueous NaOH).
Results and Discussion
We next sought to explore whether the effect of the b-
carbonyl can be expanded to small azacyclic donating groups
(Figures 2a-b, Table S1 and Figure S1).[8c, 8d] Of these donors,
azetidine is known to enhance fluorescence quantum yield and
brightness via the inhibition of TICT. In ethanol where the
contribution of EC to the overall rate of non-radiative decay is
pronounced, both b-carbonyl derivatives of azetidino-NBD 5 and
6 reveal notable increases in brightness (B = 5,248 and 6,492,
respectively) and Ff (FEtOH = 0.29 and 0.31, respectively) over the
azetidino control 4 (B = 4568, FEtOH = 0.24). However, in nonpolar
1,4-dioxane, where TICT is the most prominent mechanism of
fluorescence quenching, both 5 and 6 display no increase in
fluorescence quantum yield (Fdioxane = 0.49 and 0.52,
b
-carbonyl enables improved photophysical behavior in a
model D–A fluorophore, NBD.
Our investigations into the designed b-carbonyl donor
began with the synthesis of a library of fluorophores using the
canonical D–A scaffold, 4-dialkylamino-7-nitro-benzoxadiazole
(NBD) (Figure 2a). NBD served as a valuable model system for
initial explorations provided its ease of functionalization and
polarity sensitivity, allowing for rapid and quantifiable observation
of changes in fluorescence properties with derivatization.[9] On the
basis of dimethylamino-NBD 1, the b-carbonyl donor was installed
in the form of amide derivatives of sarcosine to serve as the
donating group (compounds 2 and 3). We observed 6 and 9-fold
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Table 1. b-carbonyl-enabled enhancement of photophysical properties in D–A fluorophores.
respectively) relative to 4 (Fdioxane = 0.55). These results suggest
the b-carbonyl acts to prevent solvent-mediated quenching
mechanisms, in particular EC. For pyrrolidino-based donors 7–9
that are known to undergo TICT,[8c, 8d] increasing fluorescence
donor effectively increases fluorescence quantum yield of NBD
with respect to the corresponding dialkylamino controls. Thus, the
b-carbonyl donor could represent a novel class of simple and
effective modifications to increase fluorescence quantum yield of
D–A fluorophores.
brightness and quantum yield are observed in ethanol (FEtOH
=
0.10 for 7 and 0.28 for 9). In nonpolar 1,4-dioxane, this trend
persists (Fdioxane = 0.31 for 7 and 0.47 for 9), suggesting a
potential role of b-carbonyl to inhibit TICT. b-carbonyl
functionalization fails to improve upon the behavior of the
piperidino derivatives 10–12, likely due to the low-energy, twisted
conformation stably present in the ground state.[8d] In all cases,
Beer’s law plot was used to ensure that all compounds were
soluble at the concentrations and solvents of measurement,
suggesting that the effect of b-carbonyl was not due to the change
of solubility (Figure S16). Importantly, the observable increases in
fluorescence quantum yield and brightness of the b-carbonyl
derivatives of NBD are consistent with the measured fluorescence
lifetimes of these fluorophores, corroborating the presented data
(Table S2). Collectively, these data suggest that the b-carbonyl
b
-carbonyl is generally applicable to enhance fluorescence
quantum yield of D-A fluorophores.
In addition to NBD, we elected to study several
representative D–A scaffolds, including 4-dialkylamino-1,8-
naphthalimide (naphthalimide), 7-dialkylamino-4-methylcoumarin
(coumarin), 2-dialkylamino-6-propionylnaphthalene (PRODAN),
and 4-dialkylamino-(2,2-dicyanovinyl)benzylidene (DCV). The
results of the photophysical characterizations are outlined in
Tables 1 and S3.
For naphthalimide, an approximate 10-fold increase in Ff
was observed for the b-carbonyl derivative 14 (FEtOH = 0.11) over
the dimethylamino control 13 in ethanol (FEtOH = 0.01). In water,
an observable Ff of 14 was determined to be 0.05 while such a
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value could not be ascertained for 13 due to an immeasurable
fluorescence intensity (Figure S2). For coumarin, the b-carbonyl
derivative 16 reveals Ff increases in both ethanol (from 0.84 to
0.95) and water (from 0.21 to 0.98), despite the considerably high
efficiency of the dimethylamino control 15 in organic solvents
(Figure S3). Given that coumarin-based probes are commonly
used in microscopy and other analytical techniques with
fluorescence readouts, we sought to explore whether an azetidino
b-carbonyl donor would improve their performance with respect to
the benchmark azetidino donors.[8c] We found that while the fold
change is modest, the β-carbonyl auxiliary increases Ff of
coumarin 18 compared to the alkyl counterpart 17 in both ethanol
(from 0.94 to 0.98) and water (from 0.91 to 0.99). This increase in
Ff is also observed with the incorporation of the b-carbonyl donor
on the PRODAN scaffold (19 and 20), with approximately 3-fold
enhancement in water (from 0.25 to 0.76; Figure S4). However,
an increase in Ff was not observed in both dilute (ethanol) and
viscous solvent (80:20 v/v glycerol:methanol, viscosity= 132
cp[10]) when we applied the b-carbonyl donor to the representative
molecular-rotor DCV scaffold (21 and 22; Figure S5), suggesting
the limited effect of b-carbonyl on molecular-rotor fluorophores.
Taken together, these data along with NBD suggest the b-
carbonyl donor can be generally applicable to increase
fluorescence quantum yield of D–A type fluorophores.
result of reduced 1,3-allylic strain, allowing the donors to attain a
more planar structure with respect to the aromatic p motif.[8c, 8d]
Such a mechanism, however, is not apparent in the case of the b-
carbonyl donors as the calculated geometry of the local excited
state (LE) reveals that the donors of 1 and 3 adopt a roughly
equivalent dihedral angle (Figure 3). Instead, calculation of the
dipole moment of the excited and ground states (µE and µG,
respectively) indicates a lesser overall difference between these
values with b-carbonyl functionalization, supporting a model
wherein intramolecular charge transfer in the excited state is
inhibited (Figure S6c). This result suggests that the increase in
requisite energy for rotation is caused by the inductive electron
withdrawing nature of the carbonyl group, which would give rise
to a destabilization of the amino radical cation of the TICT state.[8e]
This is further supported by the lesser overall difference in the
driving energy between LE and TICT states of 3 (3.50 kcal/mol)
compared to that of 1 (6.72 kcal/mol) (Figure 3).
b
-carbonyl enables
a
solvent exclusion mechanism,
inhibiting EC.
While TICT inhibition sufficiently describes the observed
increases in fluorescence emission of b-carbonyl donors in non-
polar solvents, the increases in fluorescence quantum yield and
brightness persist upon functionalization of azetidino based
donors (6 and 18) where TICT is already mitigated. Particularly,
this effect is most prevalent in polar, hydrogen-bonding solvents
such as ethanol and water. Previous work shows that the
employment of a hydrophobic, 4-(hydroxycyclohexyl)amino donor
acted to exclude solvent interaction with the donating amine in
aqueous environments, yielding improved fluorescence
intensity.[8b] In contrast to this hydrophobic exclusion, we
envisioned a mechanism wherein the carbonyl would shield the
amino donor from solvent interaction, functioning as a sacrificial
hydrogen-bond acceptor.
As transient infrared spectroscopic analysis reveals no
perturbance of the b-carbonyl in the excited state structure of 3
(Figure S7), key to such a hypothesis is the structural orientation
of the b-carbonyl relative to the nitrogen donor in the ground state.
To this end, we solved crystal structures of both 3 for NBD and 14
for naphthalimide (details can be found in Tables S7-S14 for 3
and Tables S15-S22 for 14). Analyses of these structures show
that the carbonyl is oriented toward the nitrogen donor at a
distance of 2.7 Å, placing it in proper spatial position to prevent
solvent from accessing the nitrogen donor (Figures 4a and S8).
The structural consistency between 3 and 14 suggests that the
orientation of the b-carbonyl is relatively independent of the
chromophore employed.
b
-carbonyl increases the excited state energy barrier for
rotation, inhibiting TICT.
Our realization of the broad scope of b-carbonyl donors with
respect to D–A fluorophores warranted an investigation into the
underlying mechanism that governs the improved fluorescence
quantum yields. Given the observed increases in brightness for
the b-carbonyl NBD derivatives in 1,4-dioxane, we first set out to
determine the relative contribution of TICT to the overall rate of
non-radiative decay. In silico calculation of potential energy
surface upon rotation about the C–N bond of the donating amine
in vacuum indicated that incorporation of the β-carbonyl in 3
increased the required activation energy to achieve the TICT state
by 2.5-fold over the dimethylamino control 1 (Ea = 3.77 and 1.50
kcal/mol for 3 and 1, respectively) (Figures 3 and S6). Previous
work of azetidino type donors showed that TICT inhibition was the
With this structural information in mind, we began assessing
the solvent-solute interactions that take place upon excitation of
the naphthalimide derivative 14 in comparison to 13. In silico
analyses of solvent interaction with 13 and 14 were conducted by
calculating the change in electron density (Dr) during excitation
with the inclusion of an explicit ethanol solvent molecule, which
was geometrically optimized near the donating group with both
solvent and dispersion effects taken into account. The results of
these calculations show that electron density shifts about the
ethanol’s hydroxyl upon excitation of 13 (Figure 4b, left),
suggesting that a ground state hydrogen bond between the
solvent and the dimethylamino donor is broken (Figure 4c, left).
However, no electron density shift was observed when the b-
carbonyl derivative 14 was subjected to the same analysis (Figure
Figure 3. TICT inhibition reduces excited-state decay with b-carbonyl
functionalization. Potential energy surface scan for rotation (indicated by the red
arrow) about highlighted bond in 1 and 3. Red markers correspond to the
calculated LE geometry.
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Figure 4. Solvent shielding collectively reduce environmental sensitivity with b-carbonyl functionalization. a) Crystal structures of 3 and 14. Labeled distances (Å)
correspond to yellow dashes. b) Electron density difference during excitation calculated for 13 and 14 with an explicit ethanol solvent molecule geometrically
optimized about the donating group. c) Broken H-bond upon excitation of 13, but not 14. d) Lippert-Mataga plot of 13 and 14 in both aprotic (dimethylformamide,
toluene, 1,4-dioxane, tetrahydrofuran, ethyl acetate, methyl t-butyl ether) and protic (methanol, ethanol, i-propanol, n-butanol, 2,2,2-trifluoroethanol) solvents. e)
Relative emission intensity plotted against solvent ET(30) for the solvents characterized.
4b, right), indicating that the ground state hydrogen-bond
between the ethanol and the carbonyl oxygen persists (Figure
4c, right). Finally, this model shows that the ethanol molecule
migrates away from the nitrogen donor of 13 during emission
(left panel of Figure S9a), indicating a transfer of energy from
the excited state of 13 in the form of heat. By contrast, the
ethanol molecule remained fixed in place during emission of
14 (right panel of Figure S9a), suggesting the inhibition of
thermal non-radiative decay. In addition to naphthalimide,
similar observations were made when NBD derivatives 1 and
3 are subject to the same computational analyses (Figure S9b).
Taken together, these models suggest that the b-carbonyl
effectively shields the nitrogen donor from interacting with the
surrounding solvent, preventing quenching of the charge
transfer state via EC.
obtained structural data (Figure 4a) along with the
computational model of solvent shielding (Figures 4b and S9),
we suggest that this decrease in slope is the result of an
increase in the radius of the solvent shell a, in addition to the
calculated decrease in the difference between µG and µG
(Figure S6c). Furthermore, brightness of 14 is less sensitive to
solvent polarity than 13, based on the relationship between the
relative fluorescence intensity and the solvent ET(30) (Figure
4e).[7] Collectively, these data suggest that the spatial
orientation of the b-carbonyl effectively mitigates solvent-
induced fluorescence quenching by shielding the nitrogen
donor from interaction with its local environment.
Polarity of
yield.
b
-carbonyl controls fluorescence quantum
In support of the computational model, spectroscopic
analyses of napthalimides 13 and 14 were conducted in
several solvents of varying polarity and H-bonding capacity.
According to the Lippert-Mataga equation (Equation 1),
solvatochromism can be quantified by linear fit of Stokes’ shift
(nabs–nem, cm-1) as a function of the empirical solvent polarity
parameter, Df.[11]
Because the polarity of b-carbonyl is the key towards
inhibiting both the EC and TICT pathways, we aimed to finely
tune this property to control the fluorescence quantum yields
and brightness of fluorophores. As the b-carbonyl is engaged
in polar interactions with the solvent, we hypothesized that
modulation of the electron density of the carbonyl oxygen
would allow for control over the strength of these interactions.
As such, NBD derivatives 23–26 were synthesized and the
photophysical properties were characterized in ethanol (Figure
5a and S11, Tables S4 and S6). We predicted that carbonyls
with lesser polarity such as the aldehyde (23) and ketone (24)
would offer weaker H-bond acceptors, conferring lower
fluorescence quantum yields compared to the more polar
carbonyls represented by the amide (3), ester (25), and
thioester (26) derivatives. As expected, the quantum yield
values of 23 (FEtOH = 0.05) and 24 (FEtOH = 0.14) are lower
(Equation 1)
(nabs–nem) = ∆f • (µG–µE)2 / a3 + const.
In this equation, the slope of the line (m) is directly related to
the square of the difference in dipole moment between µG and
µG and indirectly related to the cube of the radius of the
Onsager cavity (a), or the solvent shell. Our data show that this
slope is decreased for the b-carbonyl derivative 14 with respect
to the dimethylamino control 13 in both protic (m = 7675.2 and
12,593.0, respectively) and aprotic (m = 37.3 and 1,784.3,
respectively) solvents (Figures 4d and S10). Based on the
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relative to that of 3 (FEtOH = 0.18), 25 (FEtOH = 0.26), and 26
(FEtOH = 0.28). Consistent to these measurements, brightness
of these fluorophores shows a similar trend (Figure 5a). In
compound 23 and 24, the aldehyde and ketone functional
groups have been used as potent d-PET quenchers when
attached to fluorophores.[3b, 12] We found that the fluorescence
quantum yield of 23 and 24 were higher than that of the control
compound 1 (FEtOH = 0.02), suggesting the lower fluorescence
quantum yield of 23 and 24 were largely due to the lower
polarity of b-carbonyl.
Figure 6. Improved brightness of a bis-b-amide coumarinyl probe in cellular
imaging. a) Structures of P1 and P2. b) In vitro characterization of P1 and
P2 (5 µM) fluorescence intensity in 50 mM tris•HCl buffer (100 mM NaCl, pH
7.5) and in conjugation with purified HaloTag protein (25 µM) in tris buffer
(standard error, n = 3). c) Excitation (dashed lines) and emission spectra
(solid lines) of P1 (grey) and P2 (black) upon conjugation with purified
HaloTag protein in tris•HCl buffer. d) Normalization strategy to account for
expression level in cell imaging analysis of P1 and P2 by ratio of mean pixel
intensity of probe (Iprobe) to that of the mCherry (ImCherry) domain in a
transiently expressed N-HaloTag–mCherry-C fusion protein. e) Live-cell
confocal microscopy of HeLa cells transiently expressing an N-HaloTag–
mCherry-C fusion protein for 24 h in the presence of P1 or P2 (0.5 µM) after
a single wash. f) Quantified mean pixel intensity fold-change of probe signal
relative to that of mCherry (standard error, n = 3). g) Intensity profiles of
mCherry (red) and probe (blue) signal corresponding to the marked cross
sections (white dotted lines) in panel f.
Figure 5. Control of quantum efficiency though b-carbonyl derivation. a)
Efficiency of b-carbonyl NBD derivative 25-28 quantified in terms of
brightness. b) Brightness of bis-b-carbonyl derivatives 27 and 28 compared
to the corresponding mono counterparts. Fluorescence quantum yields were
measured relative to a fluorescein standard (Fstd = 0.79 in 0.1 M aqueous
NaOH).
Further, we rationalized that an alternative approach to
amplify the solvent-shielding effect of b-carbonyl is to install an
additional b-carbonyl. To this end, we synthesized and
characterized the bis-b-carbonyl derivatives of NBD, 27 and 28.
Indeed, both the bis-b-amide 27 and the bis-b-ester 28 display
a significant improvement relative to the mono analogs 3 and
25 in both fluorescence quantum yield and brightness (Figures
5b and S12, Tables S5-S6). It is also noted that the bis-b-
carbonyl mimics metal binding domain, we added EDTA to
chelate possible metal ion that could be bound to bis-b-
carbonyl and found no change in brightness, suggesting the
bis-b-carbonyl group barely binds with metal ions (Figure S13).
Taken together, these data indicate that photophysical
behavior of b-carbonyl functionalized D–A fluorophores can be
controlled by tuning the solvent-shielding effect of the b-
carbonyl.
Enhance brightness for live cell imaging using the bis-
amide modification.
b
-
We envision a wide range of applications can be enabled
by the b-carbonyl modification on D–A molecules. As an
example, we aimed to develop b-carbonyl functionalized
fluorophores for general labeling of proteins in live cell imaging
using fluorescence microscopy. Due to higher fluorescence
quantum yield and brightness, we expect such fluorophores
would require less laser power to obtain desired signal-to-
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noise ratios. To this end, coumarinyl probes for HaloTag[13] P1
and P2 were synthesized with a dimethylamino control and bis-
b-amide donor, respectively (Figure 6a). The bis-b-amide
derivative was chosen over the ester and thioester
counterparts given the known instability of the latter in vivo. In
vitro characterization of P1 and P2 indicates a greater than 5-
fold increase in fluorescence intensity upon conjugation to
purified HaloTag protein (Figure 6b-c). HeLa cells transiently
expressing a HaloTag-mCherry fusion protein were incubated
with P1 or P2 prior to imaging. Brightness of each image was
quantified by the fold-change of mean pixel intensity over that
of mCherry to account for expression level differences (Figure
6d). The results reveal an approximate 3-fold change in
emission intensity of P2 over P1 (Figures 6e-g and S14).
Similarly, 4-dialkylamino-7-sulfonylbenzoxadiazole derivatives
SP1 and SP2 reveal an approximate 1.5-fold change in
emission intensity with inclusion of the b-carbonyl donor when
subjected to the same experimental conditions and analysis as
described above (Figure S15). These results show that b-
carbonyl functionalized fluorophores exhibit enhanced
brightness in biological imaging applications.
the b-carbonyl additive to other modifications such as small
azacyclic donating groups. We show that these functional
groups improve upon the brightness of several classes of D–A
scaffolds, and further that these properties can be finely tuned
by perturbation of the carbonyl itself. Our demonstration of the
improved brightness of these dyes in a cellular setting offers a
single application of the b-carbonyl in practice, but still many
opportunities remain. We suggest this modification can be
used broadly to improve signal-to-noise ratios in analytical
techniques that employ a D–A chromophore for fluorescence
read-out. Further, we propose that these donors can be used
to improve the kinetics of D–A based photolabile protecting
groups or the turnover and longevity of photoredox catalysts in
polar environment.
Acknowledgements
We thank A. Winter, R. Giri, and S. Benkovic for their
contributed insight and helpful discussions as well as J.
Cotruvo for access to his UV/Vis instrumentation. We thank
support from the Burroughs Wellcome Fund Career Award at
the Scientific Interface (X.Z.), Paul Berg Early Career
Professorship (X.Z.), Lloyd and Dottie Huck Early Career
Award (X.Z.), Sloan Research Fellowship (X.Z.), PEW
Biomedical Scholars Program (X.Z.), NSF CHE-1856210
(X.L.). We thank Dr. Gang Ning and Ms. Missy Hazen of the
Penn State Microscopy Core Facility and Dr. Tatiana Laremore
of the Penn State Proteomics and Mass Spectrometry Core
Facility for technical assistance.
Keywords: excited state • fluorescence • twisted
intramolecular charge transfer • external conversion • donor-
acceptor molecule
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RESEARCH ARTICLE
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RESEARCH ARTICLE
Entry for the Table of Contents
The presented work outlines the characterization and application of a novel means to improve the photophysical behavior of donor-
acceptor molecules through installation of a polar, solvent-shielding auxiliary to an amino donating group. This b-carbonyl substitute
generally improves fluorescence quantum yield of donor-acceptor fluorophores, enabling brighter labeling probes for live-cell imaging.
Institute and/or researcher Twitter usernames: @psu_chemistry, @kfzhangxin
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