Azetidine-Containing Heterospirocycles Enhance the Performance of Fluorophores

Article abstract of DOI:10.1021/acs.orglett.0c01414

Fluorescent dyes are extensively utilized in various fluorescence imaging techniques. However, many existing modification strategies could not balance the performance (such as brightness, photostability, water solubility, and permeability) of fluorophores. Herein we report a general strategy to enhance the performance of donor-acceptor-type fluorophores by introducing azetidine-containing heterospirocycles to the commonly used fluorophore scaffolds. Such a strategy turned out to be a general way to develop high-quality fluorophores.

Full text of DOI:10.1021/acs.orglett.0c01414

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Letter
Azetidine-Containing Heterospirocycles Enhance the Performance
of Fluorophores
Junliang Zhou,§ Xianfeng Lin,§ Xin Ji, Shuang Xu, Chang Liu, Xiaochun Dong,* and Weili Zhao*
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ABSTRACT: Fluorescent dyes are extensively utilized in various fluorescence imaging techniques. However, many existing
modification strategies could not balance the performance (such as brightness, photostability, water solubility, and permeability) of
fluorophores. Herein we report a general strategy to enhance the performance of donor−acceptor-type fluorophores by introducing
azetidine-containing heterospirocycles to the commonly used fluorophore scaffolds. Such a strategy turned out to be a general way to
develop high-quality fluorophores.
One of the important strategies to develop bright
n recent years, four-membered spirocyclic rings have
Iwitnessed significant prominence in medicinal chemistry fluorophores is to suppress the probable twisted intramolecular
discovery programs.1,2 Since Mu
̈
ller, Carreira, and co-workers
charge transfer (TICT) formation in donor−acceptor (D−A)
type fluorophores.7 There are two main ways to suppress
TICT: modulating steric hindrance8 and electronic effects.9 A
landmark breakthrough reported by Lavis et al. to replace a
common donor of dialkylamino moiety with an azetidine ring
demonstrated much improved brightness of several fluoro-
phores, including rhodamines, rhodols, coumarins, and
naphthalimides.8a−c They termed the nice dyes as Janelia
Fluor dyes (e.g., JF549, JF525).8a,b However, recent observations
by Hell et al. indicated JF549 and JF525 underwent a notable
photobleaching process.10
demonstrated the high potential of azaspirocycles as building
blocks for drug discovery,3 azetidine-containing spirocyclic
synthons have been proven to provide improved physicochem-
ical properties and favorable pharmacokinetic profiles of lead
series, and over 100 patents on the use of 3-azaspiro[3.3]-
heptanes in drug discovery projects have appeared since 2010
as searched via www.drugbank.ca. Herein, we apply four-
membered spirocyclic rings in modification of fluorophores
and report that azetidine-containing spirocycles are eligible to
improve the overall performance of fluorescent dyes.
We intended to install various azetidine-containing hetero-
spirocycles as donors to construct various D−A fluorophores
as shown in Scheme 1. The target molecules could be accessed
by either an aromatic nucleophilic substitution (method A) or
Buchwald−Hartwig coupling (method B). We speculate that
the spiroazetidine moiety as donor may retain the effect of the
azetidine moiety to suppress TICT and afford bright
fluorescence. On the other hand, the second heteroatom in
the spirocycle may address the dilemma of solubility/
permeability by taking advantages of remarkable ability of
Small molecule fluorescent dyes are widely utilized in
fluorescence imaging techniques, such as fluorogenic labels,4
molecular probes,5 and super-resolution microscopy techni-
ques.6 However, the majority of the applications applied
traditional fluorescent dyes which frequently lack sufficient
brightness, water solubility, and photostability. To improve the
performance (such as brightness, photostability, water
solubility, and permeability) of small molecule labels, chemists
introduced different functionalities, such as sulfonates, to
increase water solubility and rigidification motifs to improve
brightness and photostability. The great challenge in improving
existing probes is that these properties cannot be optimized
independently and optimization for one of the photophysical
properties must not sacrifice the others. So far, a general
method to improve brightness without damaging photo-
stability and to improve water solubility without compromising
cell permeability of fluorophore is still lacking.
Received: April 23, 2020
https://dx.doi.org/10.1021/acs.orglett.0c01414
© XXXX American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

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Scheme 1. Synthesis of Azetidine-Containing
Heterospirocycle-Substituted Fluorophores: (A)
Nucleophile Substitution and (B) Palladium-Catalyzed
Table 1. Photophysical and Physicochemical Data of
Naphthalimides in Water
a
Cross-Coupling Approach
a
Nap dye
λmax/λem/ε
ΦF
ε × ΦF
LYSA
Peff
Nap-NMe2
Nap-Azet
Nap-01
Nap-02
Nap-03
Nap-04
Nap-05
Nap-06
Nap-07
Nap-08
Nap-09
Nap-10
435/550/0.89
463/553/1.47
456/550/1.63
460/546/1.72
449/549/1.01
449/546/1.24
449/549/1.89
448/540/1.32
449/545/1.20
447/546/1.06
457/545/1.43
446/552/1.19
<0.01
0.19
0.35
0.42
0.14
0.38
0.27
0.18
0.24
0.15
0.28
0.17
<89
2793
5705
7224
1414
4712
5103
2376
2880
1590
4004
2023
<1
<1
<1
<1
<1
<1
27
0.03
0.65
0.45
0.20
b
nd
0.27
0.11
0.30
0.03
0.11
10
14
138
170
nd
0.14
b
b
nd
a
ΦF, fluorescence quantum yield in water using fluorescein as
b
reference (ΦF = 0.95 in 0.1 M NaOH solution); nd = not
determined; λmax, absorption maxima (nm); λem, emission maxima
(nm); ε, molar extinction coefficient (×104 M−1 cm−1); LYSA,
lyophilization solubility assay (μg/mL) with <1 μg/mL to be low and
>100 μg/mL to be high; PAMPA, parallel artificial membrane
permeation assay (Peff, 10−6 cm/s) with <0.2 to be low.
a
Reaction conditions: Method A: HNR2, Et3N, DMSO. For Nap: X =
Br, yields 22−47%. For NBD: X = Cl, yields 64%, 37%. Method B:
HNR2, Pd2(dba)3, X-Phos, Cs2CO3, 1,4-dioxane. For RhO: 29%, 21%
after hydrolysis with NaOH. For RhN: 19−59%. For Cou: 47%, 39%.
For DAN: 56%, 68%. For Oxa: 57%, 42%. All yields for
diazaspirocycles were combined yields after deprotection of Boc
with TFA in CH2Cl2.
of Nap-NMe2, the replacement of the dimethylamino group
with spirooxetane or spirothietane species did not reveal
improvement in water solubility. In contrast, the diazaspir-
ocyclic naphthalimides (Nap-05−Nap-09) generally greatly
improved water solubility and have diverse effects on
membrane permeability. Both Nap-05 and Nap-06 possessed
a reasonable balance of membrane permeability and water
solubility. Nap-07−Nap-09 presented slightly diminished
membrane permeability while maintaining favorably water
solubility.
To reveal the unique advantages of our strategy, we
developed fluorescent probes (P1−P3) for detection of
biothiols with maleimide as recognizing moiety (Figure 1).14
These probes were expected to exhibit significant fluorescence
enhancement upon treatment with biothiols (GSH, Cys, and
Hcy) via Michael addition to restrict the PET process (Figure
1A).15 As shown in Figure 1B, introduction of a polar
maleimide moiety improved water solubility and membrane
permeability (P1). In contrast, azetidinyl-modified P2
possessed disappointing poor water solubility, although
membrane permeability was reasonable. Even though P2
with an azetidinyl moiety would potentially provide better
fluorescence turn-on over P1, the poor solubility of P2 limited
the application for biothiol detection. Therefore, we performed
the necessary measurements with P1 and P3. To our pleasure,
spirooxetane-spiroazetidinyl probe P3 illustrated significant
improvement on water solubility while keeping fine membrane
permeability. In aqueous media, P1 (10 μM) reacted quickly
with GSH (100 μM) with 6.4-fold fluorescence enhancement
(Figure 1C,D). In stark contrast, P3 showed much stronger
fluorescence emission (53-fold of intensity of that produced by
P1) in the presence of GSH with 19.5-fold fluorescence turn-
on. As for intracellular imaging of biothiols in HeLa cells
spirooxetane/spiroazetidine moieties in the enhancement of
solubility and permeability.3 The steric congestion may also be
beneficial for photostability. The azetidine-containing diazas-
pirocycle may provide a linking site for further modification.
The implementation of our design concept was initially
tested in naphthalimide, which suffers from weak fluorescence
in water (Tables 1 and S1 and Figures S1 and S2).11 Various
spirocyclic azetidine-substituted naphthalimides (Nap) were
obtained in reasonable yields (Scheme 1) with a dimethyla-
mino derivative (Nap-NMe2) and an azetidino derivative
(Nap-Azet) as reference dyes. Computational studies showed
that azetidine-derived spirocyclic compounds possessed flat
conformations (Figure S1) similar to that of the azetidine
moiety and disliked dimethylamino (up−down).8d In CHCl3,
all naphthalimides emitted bright fluorescence (0.6−0.84).
However, Nap-NMe2 showed a very weak fluorescence
quantum yield in EtOH (ΦF = 0.02) and in H2O (<0.01).
In comparison, Nap-Azet revealed a high fluorescence
quantum yield of 0.64 in EtOH (0.19 in water), which
confirmed the suppression TICT.8a Gratifyingly, all spirocyclic
naphthalimides (Nap-01−Nap-10) exhibited bright emission
both in EtOH (ΦF = 0.36−0.78) and in water (0.14−0.42). As
an example, the brightness for Nap-02 improved more than
80-fold over that of Nap-NMe2.
We then studied the critical water solubility (LYSA12) and
membrane permeability (PAMPA13). Nap-NMe2 showed poor
water solubility and membrane permeability (Table 1).
Azetidinyl modification (Nap-Azet) and spirooxetane species
(Nap-01, Nap-02)/spirothietaneoxide Nap-04 displayed im-
proved membrane permeability. Due to the very poor solubility
B
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Table 2. Photophysical and Physicochemical Data of
Rhodamines in Water
a
b
c
Data reported in the literature (ref 8a). nd = not determined. Data
taken from the literature (ref 8b); λmax, absorption maxima (nm); λem,
emission maxima (nm); ε, molar extinction coefficient (×104 M−1
cm−1); LYSA, lyophilization solubility assay (μg/mL); PAMPA,
parallel artificial membrane permeation assay (Peff, 10−6 cm/s).
Figure 1. (A) Structures and detection mechanism of the probes. (B)
Physicochemical data. (C) Fluorescence spectra of P1, and P3 (10
μM) with GSH (100 μM) in PBS buffer (10 mM, pH 7.4, containing
1% DMSO) at 25 °C for 1 min. (D) Emission intensities of P1, and
P3 with addition of GSH at 545 nm. (E) Confocal fluorescence
images of P1, and P3 to biothiols in HeLa cells: (E1) cells without
treatment of any probes; (E2) cells with treatment of 10 μM P1; (E3)
cells with treatment of 10 μM P3. (F) Relative intensity shown in (E).
Scale bar = 50 μm.
exhibited much better antiphotobleaching performance than
JF549 and maintained photostability at the same level of TMR.9
shown in Figure 1E,F, P3 resulted in a much brighter green
image and exhibited 4.5-fold greater fluorescence intensity than
those treated with P1.
Rhodamines are among the most useful fluorophores due to
their favorable brightness and the photostability of this
fluorophore scaffold.16 Though Lavis and co-workers demon-
strated that azetidine modifications improved the brightness of
rhodamine dyes over tetramethylrhodamine (TMR),8a as a
recent report identified,10 these azetidine derivatives encoun-
tered greater photobleaching. We prepared various azaspir-
ocyclic rhodamines and evaluated their properties (Table 2
and Figure S3). Pleasantly, in aqueous media, our azetidine-
derived heterospirocyclic rhodamines (RhN-01−RhN-05)
exhibited higher fluorescence quantum yields (ΦF = 0.88−
0.93) than that of TMR (ΦF = 0.41) as well as JF549 (ΦF =
0.85). These azaspirocyclic rhodamines also showed a higher
molar extinction coefficient (ε = 0.9−1.2 × 105 M−1·cm−1)
compared to that of TMR (ε = 0.8 × 105 M−1·cm−1). JF549 also
did not alter the water solubility and membrane permeability
too much. In contrast, RhN-02 offered markedly improved
water solubility. Although the Peff of RhN-02 was lower than
JF549 and TMR, RhN-02 still maintained good membrane
permeability. On the other hand, introduction of diazaspiro-
cycles renders the dyes highly water soluble, which is very
important for labeling (e.g., RhN-05). It may allow further
chemical modification on the second nitrogen atom. We
performed the photostability studies of TMR, JF549, RhB,
RhN-02, and RhN-05 under white light radiation (Figures 2
and S4). To our pleasure, our azaspirocyclic rhodamines
Figure 2. Time-dependent absorbance of fluorophore (RhN-02,
RhN-05, TMR, RhB, JF549; A = 0.28) during light irradiation (Xe
lamp, 190 mW cm−2) for 1 h monitored at absroption maximum
(548, 548, 552, 558, 553 nm, respectively) in water (containing 10%
EtOH, 0.1% TFA).
These results revealed important improvements on the
properties of naphthalimides and rhodamines using azetidin-
derived heterospirocyclic modifications, especially with spi-
rooxtane-spiroazetidine and 2,7-diazaspiro[3,5]nonane. We
then tested the effects of 2-oxa-6-azaspiro[3,3]heptane and
2,7-diazaspiro [3,5]nonane on other D−A fluorophores.
For rhodol dyes, the dimethylamino rhodol (RhO-NMe2)
exhibited modest quantum yields (ΦF = 0.21) (Table S2 and
Figure S5). Azetidinyl rhodol (RhO-Azet) presented high
fluorescence quantum yield (ΦF = 0.85).8a Our azetidine-
containing heterospirocyclic derivatives (RhO-01, RhO-02)
illustrated excellent illumination properties with ΦF = 0.89,
0.90, respectively.
Dimethylamino-substituted coumarin Cou-NMe2 has a
modest quantum yield in aqueous media (ΦF = 0.20).
Replacement of the dimethylamino group with azetidine
(Cou-Azet) was reported to dramatically enhance fluorescence
C
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emission (ΦF = 0.96).8a A potential disadvantage of azetidine
modification (Cou-Azet) is likely that diminished solubility
though membrane permeability is well retained (Table S3,
Figure S6). The oxetane-containing azaspirocyclic coumarin
Cou-01 and diazaspirocyclic coumarin Cou-02 maintained
excellent fluorescence emission and significantly improved
both water solubility and membrane permeability.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01414.
■
sı
Experimental procedures, additional photophysical data,
DFT calculations, and copies of NMR spectra (PDF)
Next, NBD modifications were performed similarly (Table
S3 and Figure S7). NBD-NMe2 showed very weak
fluorescence (ΦF = 0.014), good permeability, and low water
solubility. NBD-Azet only improved the fluorescence quantum
yield (ΦF = 0.034). Oxetane-derived NBD-01 and diazaspir-
ocyclic NBD-02 possessed stronger fluorescence emission (ΦF
= 0.050, 0.054) and balanced water solubility/membrane
permeability. Among these examples, we have clearly
demonstrated that spirooxetane−spiroazetidine modifications
maintained balanced water solubility and membrane perme-
ability. On the other hand, diazaspirocyclic dyes improved
water solubility remarkably while the impaired membrane
permeability.
AUTHOR INFORMATION
Corresponding Authors
■
Xiaochun Dong − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China;
Email: xcdong@fudan.edu.cn
Weili Zhao − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China; Key
Laboratory for Special Functional Materials of the Ministry of
Education, School of Materials Science and Engineering, Henan
University, Kaifeng 475004, P.R. China; orcid.org/0000-
0001-9403-1382; Email: zhaoweili@fudan.edu.cn
For oxazone dyes (Table S4 and Figure S8), Oxa-NMe2 had
fluorescence emission in the red region with λmax/λem = 601/
632 nm but with poor quantum yield in water (ΦF = 0.03).
Oxa-Azet improved ΦF to 0.08 in water. To our delight,
spirocyclic dyes Oxa-01 and Oxa-02 exhibited significantly
enhanced quantum yields (ΦF = 0.16, 0.17 respectively).
Acedan dye DAN-NMe2 is an important two-photon excitable
fluorophore with ΦF of 0.2.17 Our spiroazetidine modifications
resulted in improved quantum yields (ΦF = 0.45 for both
DAN-01 and DAN-02) over the azetidinyl derivative DAN-
Azet (ΦF = 0.39, Table S4 and Figure S9).
Authors
Junliang Zhou − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China
Xianfeng Lin − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China
Xin Ji − School of Pharmacy, Institutes of Integrative Medicine,
Fudan University, Shanghai 201203, P.R. China
Shuang Xu − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China
Chang Liu − School of Pharmacy, Institutes of Integrative
Medicine, Fudan University, Shanghai 201203, P.R. China
To further inspect the generality and limitation, we prepared
and tested other D−A dyes with extended conjugation systems
(Figure S10). As shown in Table S5 and Figure S11,
dimethylamino-substituted dicyanoisophorone (DCI-NMe2)
afforded low quantum yields in EtOH (ΦF = 0.07). Though
azetidine- and oxetane-derived spiroazetidine analogues
provided similar photophysical properties, they did not
improve the fluorescence emission (ΦF = 0.08, for both
DCI-Azet and DCI-01). Among BODIPY dyes (Table S5 and
Figure S12), all BODIPYs (BDP-NMe2, BDP-Azet, BDP-01)
displayed equal efficiency within experimental error. Therefore,
it is much more likely that the molecular rotation of aromatic
rings plays a dominant role in TICT formation (Figure
S10).7b,18
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01414
Author Contributions
§J.Z. and X.L. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Natural Science
Foundation of China (21372063) and the Development
Project of Shanghai Peak Disciplines-Integrative Medicine
(20180101).
In summary, through inspections of various DA fluoro-
phores, we have come to a general method to improve
brightness/photostability and modulate water solubility/
membrane permeability with azetidine-containing heterospir-
ocyclic species in various D−A systems. The modifications
were only effective in the direct linkage of the azetidine/
spiroazetidine to the chromophore acceptor without rotating
the fragment in D−A fluorophores. Moreover, the secondary
amines of diazaspirocyclic fluorophores could also serve as
attachment sites for further derivatization and functionaliza-
tion, for instance: (1) linking to various targeted ligand;19 (2)
conjugation of trolox or cyclooctatetraene to improve photo-
stability of fluorophore;20 (3) modulating solubility (such as
methylation, acetylation, etc.);3b (4) fluorogenic labeling (such
as Halo-Tag, Snap-Tag, etc.);4a and (5) click chemistry.21 Our
efforts for these potential applications are undergoing and will
be reported in the near future.
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