TPZ, a bright centrosymmetric two-photon scaffold for bioimaging

Article abstract of DOI:10.1039/c7cc06031a

The development of biocompatible two-photon fluorophores with a large absorption cross-section is challenging, despite the presence of theoretical guidelines. By rendering asymmetric PRODAN dye centrosymmetric, we designed and synthesized a novel class of two-photon fluorophores (TPZ). Their photophysical properties were investigated and their imaging potentials in cells, tissues and zebrafish were showcased.

Full text of DOI:10.1039/c7cc06031a

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Wang, X. Li, Y. Li, H. He, X. Luo, X. Meng, J. Chen, X. H. Qian and Y. C. Yang, Chem. Commun., 2017, DOI:
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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
first methanofullerene derivatives of Sc
3
N@C2n (2n
=
68 and 80). Synthesis of the
3
N@D5h-C80
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TPZ, a bright centrosymmetric two-photon scaffold for bioimaging
b,
c,
d
b
b
b
b
c,
Zuhai Lei †, Ping Yue †, Xueli Wang , Xinran Li , Yi Li , Haihong He , Xiao Luo , Xiangming Meng *,
d,
a,b
a,b,
Received 00th January 20xx,
Accepted 00th January 20xx
Jinquan Chen *, Xuhong Qian , Youjun Yang
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
5
6
7
8
Development of biocompatible two-photon fluorophores with plaques , and probes for pH , metal ions , biological thiols ,
9
10
11
large absorption cross-section is challenging, despite the presence oxidative species , saccharides , and enzymes , etc. were
of therotical guidelines. By rendering assymetric PRODAN dye reported. However, PRODAN is not a strong two-photon
3
centrosymmetric, we designed and synthesized a novel class of absorber with a two-photon cross-section of ~70 GM.
two-photon fluorophores (TPZ). Their photophysical properties Therefore, optimizing the two-photon properties of PRODAN
1
were investigated and their imaging potentials in cells, tissues and has attracted attention. For example, PRODAN analogs were
2
1
zebrafish were showcased.
developed through p-extension and rigidification (Figure 1).
We report herein the rational design, synthesis, spectral
studies and bioimagings of a novel PRODAN congener (TPZ)
with improved two-photon absorption cross-section.
Two-photon absorption refers to a non-linear photophysical
process, during which a molecule simultaneously absorbs two-
photons and gets promoted to the excited state, first
O
O
1
theoretically proposed by Göppert-Mayer in 1930s. Over the
R
past three decades, fluorescence based microscopic methods
has become routine in basic biological studies and biomedical
R
2
N
2
R N
2
theranostics. Two-photon (TP) microscopy has become a
PRODAN
π-extended PRODAN
2
c
prominent imaging method for the following advantages.
1. π-extension
O
First, photobleaching is minimized by restricting the photo-
excitation to dye molecules within the minute volume of the
focal point. Second, higher penetration depth may be achieved
for a fluorophore with two-photon excitation than one-photon
excitation. Two-photon molecular tools for biomedical studies
2. regidification
OH
O
R2N
O
R
2
N
O
O
π-extended and rigidified PRODAN
O
3
have been actively sought after and recently flourished.
Fluorophores exhibiting high two-photon absorption cross-
section are the cornerstones for construction of probes or
imaging agents. PRODAN type fluorophores, i.e. 2-amino-7-
carbonyl substituted naphthalenes have been frequently
employed. For example, PRODAN derivatives for tracking
R
2
N
NR
2
TPZ, R = alkyls
O
Centrosymmetrically π-extended, rigidified, and stacking-inhibited PRODAN
4
4
4
plasma membrane , lysosome , mitochondria , and amyloid-b
Figure 1. A) PRODAN and its asymmetric derivatives. B) The structure of
centrosymmetric TPZs.
It has been theoretically rationalized that centrosymmetry can
promote two-photon absorption cross-section, via enhancing
the transition dipole moment between the ground state and
1
,13
the intermediate state.
This has inspired us to extend the
conjugation of PRODAN in a centrosymmetric fashion. Also, a
xanthene moiety was incorporated for the following two
considerations. First, it rigidifies the scaffold of TPZ and
eliminates rotational deactivation of the excited state. Also, it
is orthogonal to the large D-
π-A-π-D backbone of TPZ and
effectively inhibits aggregation via stacking.
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O
O
O
F
F
F
Br
OH
F
Br
O
F
CHO
HO
b
O
4
O
O
O
O
O
O
a,
c
O
O
O
O
1
2
3
5
OPh
O
O
OPh
Li
OH
O
F
F
F
F
F
NMe2
e
f
d
g
O
O
O
O
O
O
O
6
7
TPZ1
O
O
O
O
R
R
TfO
OTf
Et
2
N
NEt
2
h
i
7
O
O
8
9
, R = -OMe
, R = -OH
10
TPZ2
o
o
o
Scheme 1. Synthesis of TPZ dyes. a) pTSA, toluene, 80 C, 98%; b) (i) nBuLi, THF, -78 oC; (ii) DMF, THF, -78 C, 82%; c) NaOH, EtOH/H
2
O, rt, 92%.; d) THF, -78 C; e) MeSO
3
H, rt, 32%;
o
o
f) NHMe
2
, Na
2
o
CO
3
, DMSO, rt, 65%; g) (i) MeONa, MeOH/DMSO, 80 C, 82%; (ii) BBr
3
2
, ClCH CH
2
Cl, rt, 85%; h) Tf
2
O, pyridine, CH
2
Cl
2
, 0 C, 95%; i) Pd(OAc)
2
, BINAP, Cs CO , 1,4-
2
3
dioxane, 100 C, 80%.
Synthesis of TPZ dyes was from acid catalysed condensation of Table 1. Spectroscopic Properties of Dyes TPZ1 and TPZ2.
2
-bromo-4-fluoro-benzaldehyde (
give in a 98% yield. Halogen-lithium exchange followed by
quenching with DMF furnished in a 82% yield. Base catalysed
double Aldol condensation yields in a 92% yield. Nucleophilic
addition of 2-lithium diphenylether to followed by an acid
treatment gave compound in a 32% combined yield. It was
1) with ethylene glycol to
2
λ
abs
λ
em
lgε
φ
δ
max
(λTP nm)
Dyes Solvent
3
(nm) (nm)
(
GM)
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
5
356
354
358
356
363
440
436
461
489
456
435
478
490
4.50 0.19
4.58 0.02
4.49 0.25
4.35 0.32
4.28 0.39
4.32 0.05
4.28 0.42
4.28 0.37
CHCl
THF
3
5
7
8
9
DMSO
EtOH
our initial design to substitute the two fluorine atoms by alkyl
amines. However, the reaction did not proceed further upon
completion of the first substitution, and TPZ1 was obtained in
case dimethylamine was used as the nucleophile. Alternative,
CHCl
THF
3
360
we substituted these two fluorine atoms of
yield , which was deprotected by BBr to furnish
hydroxyl groups were converted to triflate, which can cross-
7
by methoxide to
DMSO 363
8
3
9. The two
362
435
432
445
440
441
456
446
465
465
457
EtOH
CHCl
THF
3
526 4.35 0.95 204.4 (890)
530 4.31 1.00 174.9 (850)
600 4.34 0.50 196.5 (920)
600 4.31 0.26 232.5 (850)
14
couple with various amines under Buchwald conditions to
give the desired centrosymmetric TPZ dyes, e.g. TPZ2
.
TPZ1
DMSO
1000
A
TPZ1 in CHCl
C
3
EtOH
a
TPZ2
7
50
00
50
H
2
O
617 4.36 0.07
94.4 (850)
In DMSO
CHCl3
THF
510 4.65 0.53 1018.4 (810)
479 4.70 0.54 1082.2 (810)
552 4.69 0.52 738.6 (810)
573 4.64 0.51 1226.9 (810)
600 4.55 0.22 222.1 (790)
5
TPZ1
B
TPZ2 in CHCl
3
TPZ2
2
DMSO
EtOH
0
300
400
500
600
700
700
750
800
850
900
950
a
H2O
Wavelength (nm)
Wavelength (nm)
Figure 2. One-photon absorption, fluorescence emission and two-photon absorption
a. with 10% DMSO as co-solvent.
spectra of TPZ1 (A, C) and TPZ2 (B, C).
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The UV-Vis absorption and fluorescence emission spectra 30 mins and readily permeable to the plasma membrane. The
TPZ1 and TPZ2 were collected in different solvents, e.g. two-photon fluorescence images were then acquired. For TPZ1
, THF, DMSO, EtOH and H O (with 10% DMSO as a co- an excitation at 850 nm was applied and the emission in the
solvent) (Table 1). The maximal absorption and fluorescence spectral channel of 570-630 nm was collected. Cell cytoplasm
emission wavelengths of and in organic solvents are rather was lit up by the strong green fluorescence. For TPZ2, an
of
CHCl
8
,
9
,
,
3
2
8
9
short at ~350 nm and ~430 nm, respectively. For this reason, excitation at 920 was used and the emission channel was
they are not optimal for imaging of biological specimens, to chosen to be 530-580 nm. Similarly, cell cytoplasm was
which UV-light at ~350 nm could be harmful. A second result strongly fluorescent. These results have clearly delineated the
of the short excitation wavelength is the lack of two-photon potentials of TPZ1-2 for in vitro cell imaging.
absorption of the light in the region of 700-950 nm. The
a 2
phenolic OH group of 9 exhibits a pK of 11.7 in H O containing
10% DMSO, higher than that of a typical phenol. This is likely
attributed to the steric bulkiness of the diphenyl ether moiety,
which has affected the stabilization of the resulting phenoxide
anion via solvation. TPZ1 exhibits an absorption and emission
maximum at 435 nm and 526 nm, respectively (Figure 2A). It
-1
-1
has a modest molar absorptivity of 22,500 M cm and a
fluorescence quantum yield of 0.95. While its absorption
maximum and molar absorptivity was not strongly influenced
by solvent polarity, the fluorescence emission red-shifted by ca.
70 nm with a large decrease of fluorescence quantum yield.
The two-photon absorption cross-section of TPZ1 was
determined to be ca. 200 GM in organic solvents, and 94.4 in
H O with 10% DMSO (Figure 2C). TTPZ2 exhibited a longer-
2
wavelength absorption, and a shorter-wavelength emission
than TPZ1 (Figure 2B). The molar absorptivity of TPZ2 in
different solvents are roughly two-times higher than TPZ1. It is
very interesting to note that the fluorescence quantum yields Figure 5. Two-photon confocal images of a fresh rat liver slice treated with TPZ1-2 (10
μM). TPZ1: (a, b) λex = 850 nm, λem = 570-630 nm; TPZ2: (c, d) λex = 920 nm, λem = 530-
of TPZ2 in different organic solvents are essentially the same
at ca. 0.5. Also, the two-photon absorption cross-section of
TPZ2 is much improved to 738.6-1226.9 GM in various organic
5
80 nm. magnification of 10×.
TPZ1-2 were further employed in the 3D fluorescence
imaging of rat liver tissue slices with a thickness of 180
solvents and 222.1 GM in H
C). This is in good agreement with the theoretical guideline
that centrosymmetry promotes two-photon cross-section.
2
O containing 10% DMSO (Figure
ꢀM.
The liver slices were incubated with TPZ1-2 (10 M) in PBS
2
ꢀ
buffer for 1 h before rinsed off. The fluorescence images at
varying depth were collected. As shown in figure 5., the
penetration depth of TPZ1 and TPZ2 was ca. 150 μm and 170
μm, respectively, upon two-photon fluorescence excitation.
These results suggested that TPZ1-2 have excellent properties
for depth imaging (<170 μm) in living tissues with two-photon
fluorescence microscopy.
(
a)
(b)
(e)
(c)
(f)
2
0 μm
(
d)
20 μm
Figure 4. Two-photon confocal image (a), the bright field (b), and the merged image (c)
of Mcf-7 cells incubated with TPZ1 (10 μM) for 30 min. Two-photon confocal image (d),
the bright field (e), and the merged image (f) of Mcf-7 cells incubated with TPZ2 (10
μM) for 30 min. TPZ1: λex = 850 nm, λem = 570-630 nm; TPZ2: λex = 920 nm, λem = 530-
Figure 6. Two-photon confocal images of 5-day-old zebrafish incubated with
TPZ1-2 (10 μM) for 60 min. (a, d) Blue channel; (b, e) Bright field; (c, f)
Merge. TPZ1: λex = 850 nm, λem = 570-630 nm; TPZ2: λex = 920 nm, λem = 530-
5
80 nm. Magnification is 20 ×, scale bars: 400 μm.
5
80 nm. Scale bars: 20 μm.
We investigated the in vivo imaging capability of TPZ1-2 in
Cytotoxicity of TPZ1-2 was studied prior to any in vitro
studies by MTT assay. Mcf-7 cells were incubated with TPZ1-2
zebrafish. Both dyes were readily uptaken by the zebrafish.
Upon two-photon excitation, fluorescence images were
acquired for both TPZ1 and TPZ2 stained zebrafish. Generally
speaking, two dyes has a similar staining pattern. A strong
fluorescence was observed at intestines and liver (Figure 6).
Comparatively weaker fluorescence intensity was observed at
other parts of the zebrafish.
o
(up to 25 ꢀM) for 24 hrs at 37 C and the cell viability was
found to be higher than 85%, suggesting both has minimal
cytotoxicity and compatible for cellular applications (SI). Then,
the potentials of TPZ1 in in vitro imaging were examined
-
2
(
Figure 4). TPZ1 (10 ꢀM) was incubated with Mcf-7 cells for
-
2
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In conclusion, through rational design, we developed a 10 Y. S. Tian, H. Y. Lee, C. S. Lim, J. Park, H. M. Kim, Y. N. Shin, E.
S. Kim, H. J. Jeon, B. R. Cho. Angew. Chem. Int. Ed. 2009, 48
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011, 133, 12009-12020. (b) Z. Na, L. Li, M. Uttamchandani
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,
class of novel two-photon fluorophores, TPZ. Compared to the
classic PRODAN type fluorophores, TPZs exhibit advantageous
such as longer wavelength excitation and emission, higher
two-photon absorption cross-section, and lower degree of
solvatochromism. Our experimental results resonate with the
theoretical guideline that centrosymmetry promotes two-
photon absorption cross-section.
8
1
2
The work is financially supported by the National Natural
Science Foundation of China (Nos. 21372080, 21572061,
21236002 and 11674101), and the Fundamental Research
Zhu, K. L. Lim, S. Q. Yao. Nat. Commun. 2014,
2 T. Parasassi, E. K. Krasnowska, L. Bagatolli, E.Gratton, J.
Fluores. 1998, , 365-373.
3 (a) M. Tasior, D. Kim, S. Singha, M. Krzeszewski, K. H. Ahn, D.
T. Gryko, J. Mater. Chem. C 2015, , 1421-1446. (b) A. Purc,
K. Sobczyk, Y. Sakagami, A. Ando, K. Kamada, D. T. Gryko, J.
Mater. Chem. C. 2015, , 742-749.
5, 3276.
Funds for the Central Universities (Nos. WY1514053 and
WY1516017).
1
1
8
Author contributions: Z. Lei, X. Li, and Y. Li synthesized TPZ1
and 2. Z. Lei, H, He, and X. Luo performed the spectral studies.
P. Yue and X. Meng carried out the biological imaging studies.
X. Wang and J. Chen carried out the transient spectroscopy. X.
Qian and Y. Yang conceived the project. All have contributed to
writing and proof-reading.
3
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