Aziridinyl Fluorophores Demonstrate Bright Fluorescence and Superior Photostability by Effectively Inhibiting Twisted Intramolecular Charge Transfer

Article abstract of DOI:10.1021/jacs.6b03924

Replacing conventional dialkylamino substituents with a three-membered aziridine ring in naphthalimide leads to significantly enhanced brightness and photostability by effectively suppressing twisted intramolecular charge transfer formation. This replacement is generalizable in other chemical families of fluorophores, such as coumarin, phthalimide, and nitrobenzoxadiazole dyes. In highly polar fluorophores, we show that aziridinyl dyes even outperform their azetidinyl analogues in aqueous solution. We also proposed one simple mechanism that can explain the vulnerability of quantum yield to hydrogen bond interactions in protonic solvents in various fluorophore families. Such knowledge is a critical step toward developing high-performance fluorophores for advanced fluorescence imaging.

Full text of DOI:10.1021/jacs.6b03924

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Communication
Aziridinyl fluorophores demonstrate bright fluorescence
and superior photostability through effectively
inhibiting twisted intramolecular charge transfer
Xiaogang Liu, Qinglong Qiao, Wenming Tian, Wenjuan Liu, Jie Chen, Matthew J Lang, and Zhaochao Xu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03924 • Publication Date (Web): 20 May 2016
Downloaded from http://pubs.acs.org on May 23, 2016
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Aziridinyl fluorophores demonstrate bright fluorescence and supe-
rior photostability through effectively inhibiting twisted intramo-
lecular charge transfer
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,
,§,
Xiaogang Liu, , ¶ * Qinglong Qiao, ¶ Wenming Tian, Wenjuan Liu, Jie Chen, Matthew J. Lang, Zhaochao
⊥
Xu ,*
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,
457 Zhongshan Road, Dalian 116023, China.
Singapore-MIT Alliance for Research and Technology (SMART), 1 CREATE Way, Singapore 138602, Singapore.
§State Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road, Dalian 116012, China.
⊥Department of Chemical and Biomolecular Engineering and Department of Molecular Physiology and Biophysics, Vanderbilt Uni-
versity, Nashville, TN 37235, United States.
Supporting Information Placeholder
ious experimental strategies have been applied to avoid TICT
formation. Commercially available fluorophores, such as
Dylight, CF and Alexa dyes, involve complex structural modifi-
cations via rigidifying amino substituents. Unfortunately, most
molecular structures of these dyes are still not disclosed, con-
straining their further derivatization and functionalization. Song
et al. employed a bulky 7-azabicyclo[2.2.1]heptane to replace
commonly used dimethylamino substituents in rhodamine
dyes.15 This structural modification greatly improves the quan-
tum yields and photostability of rhodamine dyes, but at the cost
of deteriorated water solubility. In a landmark paper, Lavis et al.
changed N,N-dialkylamino substituents to four-membered azet-
idine rings in several chemical families of fluorophores, such as
rhodamines, rhodols and coumarins.2 They demonstrated that
the azetidine ring simultaneously improves dye brightness and
photostability. Moreover, this simple structural modification
retains biochemical properties of the parent compounds (such
as cell-permeability and intracellular labeling), and becomes
accessible even in basic chemical synthesis laboratories. Never-
theless, the degree of improvement with the azetidine ring
based approach varies across different fluorophore families. For
instance, the quantum yield of azetidinyl naphthalimide is
markedly lower than those of coumarins and rhodamines, de-
manding further improvement.2 In this communication, we
show that a three-membered aziridine ring suppresses TICT
formation more effectively over the four-membered azetidine
ring and other dialkylamino substituents; consequently, the
aziridine ring leads to improved brightness and superior photo-
stability in a range of fluorophores, in conjunction with enlarged
Stokes shifts.
ABSTRACT: Replacing conventional dialkylamino substituents with
a three-membered aziridine ring in naphthalimide leads to significantly
enhanced brightness and photostability, via effectively suppressing
twisted intramolecular charge transfer (TICT) formation. This re-
placement is generalizable in other chemical families of fluorophores,
such as coumarin, phthalimide and nitrobenzoxadiazole dyes. In highly
polar fluorophores, we show that aziridinyl dyes even outperform their
azetidinyl analogues in aqueous solution. We also proposed one simple
mechanism that can explain the vulnerability of quantum yield to hy-
drogen bond interactions in protonic solvents in various fluorophore
families. Such knowledge is a critical step towards developing high-
performance fluorophores for advanced fluorescence imaging.
Rapid evolution of fluorescence imaging techniques in recent
years demands fluorophores with enhanced brightness and pho-
tostability.1-4 This evolution, particularly in biomolecular label-
ling5,6 and super-resolution imaging techniques,7-10 has facilitat-
ed fluorescence imaging with single-molecule precision in nu-
merous biological and biomedical studies.11-13 However, many
existing fluorophores lack sufficient brightness and photostabil-
ity for single-molecule and live-cell imaging. Rational molecular
engineering of fluorophores, based on a deep understanding of
their working mechanism, is thus crucial and imperative to yield
novel fluorophores with superior brightness and photostability.
On the theoretical side, it is proposed that twisted intramo-
lecular charge transfer (TICT) acts as one of the major non-
radiative de-excitation pathways in fluorophores.14 In the TICT
state, a dialkylamino donor twists out the fluorophore scaffold
by approximately 90° upon photoexcitation, forming a non-
emissive and highly reactive chemical species. Accordingly, var-
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Figure 1. The three-membered aziridine ring enhances the brightness and photostability of naphthalimide fluorophores. (a) Illustration of steric
hindrance between donor and acceptor moieties of a fluorophore. (b) Three types of alignment conformations between the t
lighted in yellow) and the main fluorophore scaffold (green) in the ground state (S0). Arrows indicate potential rotations to enter the twisted intra-
molecular charge transfer (TICT) state in the first excited state (S1). (c) The top and side views of the theoretically optimized molecular structures of
1
7 in ethanol in the ground state. (d) Calculated potential energy surfaces (PES) of the ground (S0, in blue) and the first excited singlet (S1, in red)
states of 1 3 in ethanol, as a function of the rotational angle ( ) between the amino substituent and the main fluorophore scaffold. is the average
The LE state has a more planar conformation and less charge transfer
compared to the TICT state. (e) Experimental spectroscopic data for 1 7 in ethanol abs, peak UV
and , the Stokes shift; , quantum yield. (f) Experimental quantum yields of 1 3 in various sol-
em
vents. (g) Relative intensity changes of 1, 2, and Rhodamine B in DMSO/ water mixture (volume ratio, 30:70) during 12 hours of strong white light
exposure. (h) Relative intensity changes of 8 and 9 in water during 500 seconds of strong laser radiation (416 nm) in microfluidic channels. The in-
sets show the molecular structures of 8 and 9.
We initially choose naphthalimide-derived 1 7 as model
compounds to investigate the effect of azacyclic substituents on
minimizing TICT (Figure 1). Our computational studies show
1
7 can align in three different conformations, due to competing
contributions from azacyclic strain energy, steric hindrance, and
resonance effect (Figure 1a, b). The resonance effect depends
on the electron-donating/ withdrawing strength of both the
azacyclic rings and the fluorophore scaffolds. We find that com-
pounds 1, 2 and 3 7 possess the up-up, flat and up-down con-
formations, respectively (Figure 1c). Intuitively, the up-up con-
formation should possess the highest TICT resistance, because
one of its amino arms faces strong steric repulsion to enter the
TICT state. Similarly, the up-down conformation should exhibit
the lowest TICT resistance, because steric hindrance facilitates
its TICT rotation after photoexcitation.16 Indeed, our theoreti-
cally calculations show that in a polar environment (such as
ethanol), the TICT state of 1 (the up-up conformation) is ener-
getically unfavorable, and a significant energy barrier (0.393 eV)
exists between the local excited (LE) and TICT states (Figure
1d). The TICT state of 2 (the flat conformation), however,
becomes slightly more stable than the LE state, in conjunction
with a reduced energy barrier (0.246 eV). In contrast, a strong
tendency to the TICT state is most evident in 3 (the up-down
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Figure 2. (a) Molecular structures of 10 17. (b) Experimental spectroscopic data for 10 17 in aqueous solution abs, peak UV vis absorption
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and , the Stokes shift; , quantum yield. (c). The atomic contribu-
em
tions to the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) electron densities of 1, 10, 14 and 16,
calculated based on the optimized molecular structures in the S0 state. The blue/ pink circle size represents the atomic contribution; only contribu-
tions greater than 0.02 are shown. Hydrogen bond formation sites in naphthalimide, coumarin, phthalimide, and NBD fluorophore scaffolds are indi-
cated by arrows. Molecular sites that experience significant partial charge increase upon photoexcitation are highlighted by red arrows in 14 and 16.
conformation; Figure 1d). Nevertheless, in a non-polar envi-
ronment, our calculations show that 1 3 all favor LE emission,
but not TICT rotation (Supporting Information Section 1).
dine ring in 1 upon photoexcitation results in a large geometry
relaxation in the excited state, thus leading to a favorable large
Stokes shift ( = 127 nm for 1 versus 88 nm for 2).17 The large
Stokes shift of 1
-colour super-
Moreover, owing to the larger quan-
Experimental results are in excellent agreement with our the-
oretical calculations. In non-polar solvents, 1 7 all emit bright
18
tum yield of 1, its brightness is ~27% higher than that of 2 in
aqueous solution. The same patterns were observed between 8
and 9 (Supporting Information Section 2).
fluorescence (quantum yield,
. In polar solvents, only
1 and 2 demonstrate high quantum yields. We notice that 1
exhibits higher quantum yields than 2 in all tested solvents. This
difference is most apparent in water ( = 0.432 for 1 and 0.199
for 2; Figure 1e, f; Supporting Information Section 2). It is thus
clear that the aziridine ring possesses higher TICT resistance
than the azetidine ring and other dialkylamino substituents.
Inspired by the improved brightness, enhanced stability and
enlarged Stokes shifts in naphthalimide dyes, we then employed
the aziridine ring in coumarin, rhodamine, phthalimide and
nitrobenzoxadiazole (NBD) dyes, and compared the fluoro-
phore performance against their azetidinyl and dimethylamino
analogues (Figure 2a, b). Our quantum chemical calculations
show that aziridinyl fluorophores demonstrate the highest
TICT resistance, followed by azetidinyl dyes. Experimental data
also show that both aziridinyl and azetidinyl fluorophores exhib-
it higher brightness than conventional dialkylamino fluoro-
phores ( = 0.058 for Coumarin 1; < 0.01 for dimethylamino
NBD and phthalimide in water). Between aziridinyl and azet-
idinyl fluorophores, coumarins 10 and 11 display comparably
We then tested the photostability of 1 and 2 (under white
light radiation; Figure 1g), and their analogues with enhanced
water solubility, 8 and 9 (under 416 nm laser radiation; Figure
1h). Our tests show that aziridinyl 1 and 8 exhibit considerably
higher photostability than azetidinyl 2 and 9. The chemical
stability of 1 was also evaluated in solutions of different pH. We
found that the fluorescence intensities of 1 remained stable
from pH 4.2 to 10.8. We also treated the solution of 1 with vari-
ous metal ions, oxidants and nucleophiles including H2O2,
NaHS, cysteine and glutathione. The unperturbed emission
indicates that 1 has good chemical stability (Supporting Infor-
mation Section 2).
high quantum yields (
in water. Since these 4-methyl-
coumarin dyes have a low polarity and weak TICT tendency,
the azetidine ring seems sufficient to suppress TICT formation
and afford high quantum yields. The aziridine substituent in 12,
however, leads to the colorless and non-fluorescent lactone
form of rhodamine dyes, while azetidinyl 13 are highly emissive
= 0.459). These results are consistent with Lavis and
observations on similar rhodamine dyes, where both
dialkylamino groups are replaced by aziridine or azetidine
rings.2 In contrast, for phthalimide and NBD dyes 14 17, the
Several other differences between 1 and 2 are of note. The up-
up conformation of 1 yields a lower molar extinction coefficient
and a blue shift in its UV vis absorption peak ( = 9,200 M-1
cm-1 and abs = 382 nm) with respect to the planar 2 ( = 15,900
M-1 cm-1 and abs = 445 nm; Figure 1e). This is due to a poorer
resonance effect in 1. However, a slight flattening of the aziri-
(
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Synthesis, characterization, and computational details. This material is
available free of charge via the Internet at http:/ / pubs.acs.org.
quantum yields of aziridinyl fluorophores are approximately
twice as large as those of azetidinyl analogues in aqueous solu-
tion, in conjunction with enlarged Stokes shifts (Figure 2b).
These results are remarkable, considering that dimethylamino
phthalimide and NBD dyes are non-emissive in water.19,20 Un-
fortunately, the absolute quantum yields of 14 17 ( = 0.011
to 0.084) are low, largely due to intensive hydrogen bond inter-
actions around the fluorophore scaffolds.19,21
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Corresponding Author
*Email: xiaogang@smart.mit.edu; Tel: +65-6516-1462.
*Email: zcxu@dicp.ac.cn; Tel: +86-411-84379648.
9
We have found one plausible mechanism to explain the vul-
nerabilities of quantum yield to hydrogen bond interactions
among different fluorophores. This vulnerability is closely relat-
ed to the partial charge increase upon photoexcitation, at hy-
drogen bond formation sites in the fluorophore scaffolds (Fig-
ure 2c, highlighted by arrows). We denote the total charge
change at these sites during HOMO
Author Contributions
¶X.L. and Q.Q. contributed equally to this work.
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Notes
The authors declare no competing financial interests.
X.L. and M.J.L. thank National Research Foundation of Singapore
(through BioSyM Interdisciplinary Research Group at Singapore-MIT
Alliance for Research and Technology) for supporting this project.
Z.X. acknowledges the National Natural Science Foundation of China
(21276251), the 100 Talents Program funded by Chinese Academy of
Sciences, Dalian Cultivation Fund for Distinguished Young Scholars
(2014J11JH130 and 2015J12JH205) and the National Science Fund
for Excellent Young Scholars (21422606). The authors thank Dr. Jie
Pan for her assistance on photostability tests.
are observed. Coincidentally, the quantum yields of 1 and 10
are high ( = 0.432 for 1 and 0.899 for 10). In contrast, we no-
14
and 16 (Figure 2c, highlight by red arrows), indicating intensive
hydrogen bond interactions after photoexcitation. Accordingly,
their quantum yields are low ( = 0.020 for 14 and 0.084 for
16). Interestingly, this mechanism also works perfectly in ex-
plaining the quantum yield differences of several other fluoro-
phore families in protonic solvents, such as rhodol and oxazine
dyes (Supporting Information Section 1). Additional quantum
chemical calculations including explicit solvent molecules are
required to further understand such hydrogen bond interactions,
and are the subject of future work.
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In conclusion, we have demonstrated that the up-up con-
formed aziridine ring is highly effective in suppressing TICT
rotation. Aziridinyl fluorophores thus demonstrate considerably
enhanced brightness and superior photostability, in comparison
to conventional dialkylamino substituted dyes. In highly polar
fluorophores with a strong TICT tendency (such as naph-
thalimide dyes), aziridinyl fluorophores even outperform their
azetidinyl analogues in aqueous solution. As another favorable
feature, a slight flattering of the aziridine ring upon photoexcita-
tion affords large Stokes shifts in aziridinyl fluorophores. More-
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