“Xanthene” is a premium bridging group for xanthenoid dyes

Article abstract of DOI:10.1016/j.cclet.2021.04.041

Novel xanthenoid dyes by replacing the central oxygen atom of the xanthene dyes with less electron-rich bridging groups have been intensively sought after primarily for their long spectral wavelengths. However, the new scaffolds are likely prone to nucleophilic attack at their central methane carbon, as the result of the reduced electron density of the fluorochromic scaffolds. We envisage that the bridging group may be harnessed to sterically shield the central methane carbon from incoming nucleophiles and render high stability and synthesized xantheno-xanthene dyes. Additionally, the xantheno-bridging group can be modified via electrophilic aromatic substitution to introduce functionalities, e.g., sulfonate groups.

Full text of DOI:10.1016/j.cclet.2021.04.041

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Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
“Xanthene” is a premium bridging group for xanthenoid dyes
Jin Lia, Mengmeng Zhanga, Lu Yang c, Yubing Hanc, Xiao Luob,∗, Xuhong Qiana,∗
,
Youjun Yang a,∗
a State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
b School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China
c State Key Laboratory of Modern Optical Instrumentation, College of Optical Science and Engineering, Hangzhou 310027, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel xanthenoid dyes by replacing the central oxygen atom of the xanthene dyes with less electron-
rich bridging groups have been intensively sought after primarily for their long spectral wavelengths.
However, the new scaffolds are likely prone to nucleophilic attack at their central methane carbon, as
the result of the reduced electron density of the fluorochromic scaffolds. We envisage that the bridging
group may be harnessed to sterically shield the central methane carbon from incoming nucleophiles and
render high stability and synthesized xantheno-xanthene dyes. Additionally, the xantheno-bridging group
can be modified via electrophilic aromatic substitution to introduce functionalities, e.g., sulfonate groups.
Received 23 February 2021
Revised 11 April 2021
Accepted 22 April 2021
Available online xxx
Keywords:
NIR infrared
Xanthene
© 2021 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia
Medica, Chinese Academy of Medical Sciences.
Carbon rhodamine
Steric protect
Rigidity
Fluorescence is the cornerstone of numerous cut-edge biological
and medical technologies [1–6], e.g., fluorescence in situ hybridiza-
tion (FISH) [7], cell imaging [8], DNA sequencing [9], flow cytom-
etry [10], super-resolution imaging [11], and fluorescence guided-
surgery [12]. Fluorescent materials of different chemical natures
were reported, e.g., organic fluorophores [13], inorganic quan-
tum dots [14], carbon materials [15], lanthanide complexes, up-
converting inorganic materials [16], polymeric emitters [17], and
biological fluorescent proteins [18]. These materials have unique
merits and limitations and mutually complementary. For in vitro
or in vivo applications, organic fluorophores are particularly suit-
able sought after due to their biocompatibility, spectral versatility,
and facile derivatization for functionality [19].
analogs absorbing/emitting beyond 600 nm, by replacing the cen-
tral oxygen atom of the xanthene core into a less electron-donating
moiety than oxygen. The earliest endeavor is the class of dyes com-
monly referred to as carbo-rhodamines [23], which has an sp3 hy-
bridized carbon moiety as the bridging group. They exhibit a re-
duced tendency to aggregate, but an enhanced susceptibility to-
ward nucleophilic attack. While the nucleophilic attack by thiols
or hydride is reversible and can be harnessed for good in localiza-
tion microscopy, the attack by oxidative nucleophiles such as per-
oxynitrite, hypochloride, superoxide, peroxide could lead to perma-
nent destruction of the rhodamine scaffold and place a restriction
over the scope of applicable scenarios. Unfortunately, these above
methods to promote the chemostability of rhodamine dyes are not
applicable to carbo-rhodamines because the steric hindrance of the
Rhodamine is a premium class of organic fluorophores. They
are conveniently synthesized from inexpensive starting materials,
structurally rigid, and therefore very bright. Their spectral prop-
erties can be readily fine-tuned in the range of ca. 490 of Rh110
[20] to 590 nm of Texas Red [21]. Additionally, they can be en-
gineered to exhibit high resistance toward nucleophilic attack or
aggregation by sulfonation at C-4/C-5 of its xanthene core, or in-
ꢀ
ꢀ
central disubstituted methylene unit renders the C4/C5 and C3 /C7
positions inaccessible for chemical modifications.
The use of bulkier chemical groups other than methyl is an
obvious solution. For example, Jacob et al. prepared diethyl and
diphenyl substituted carbo-rhodamines [24]. Recently, Guo et al.
recently reported a new synthesis of carbo-rhodamines allowing
replacement of one of the two methyl groups into a hydroxyl or
methoxyl, or potentially other alkoxyl groups [25]. We prepared
two facilely substituted carbo-rhodamines, with a combination of
carboxyethyl/hydroxyl groups, and a dihydrofuran-2(3H)-one, re-
spectively [26]. Xiao et al. reported carbo-rhodamines with a 1,3-
dioxolane bridge [27]. Yet, these groups are pointing away from the
electrophilic central methine carbon and does not impart sufficient
ꢀ
ꢀ
stallation of bulky groups at C3 /C7 of the bottom benzene ring
(Fig. 1) [22]. Recent years have witnessed a surge for rhodamines
∗
Corresponding authors.
E-mail addresses: xluo@chem.ecnu.edu.cn (X. Luo), xhqian@ecust.edu.cn (X.
Qian), youjunyang@ecust.edu.cn (Y. Yang).
https://doi.org/10.1016/j.cclet.2021.04.041
1001-8417/© 2021 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Please cite this article as: J. Li, M. Zhang, L. Yang et al., “Xanthene” is a premium bridging group for xanthenoid dyes, Chinese Chemical
Letters, https://doi.org/10.1016/j.cclet.2021.04.041

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Scheme 1. Synthesis of various new carbo-rhodamines (10, 16a, 16b, Sulfo-16a, Sulfo-16b).
steric protection toward nucleophilic attack. Having the two alkyl
groups replaced by two bulkier phenyl groups does not address
this difficulty either because the two phenyl rings are rotatable and
they tend to adopt a geometry, in which the two peripheral hydro-
gen atoms are also twisted away from the central methine carbon
and not provide much steric protection over the central methine
carbon. We envisage that the steric protection of the two phenyl
group could be greatly enhanced if they are tethered together. This
way, the two hydrogen atoms of the phenyl rings, highlighted in
orange colour, are forced to point down toward the central me-
thine carbon (Fig. 2). Following this line of research, our first at-
tempt is a rhodamine with a benzophenone bridge. They exhibit
the desired high chemical stability, and however is still difficult for
further functionalization due to its electron deficient nature.
In this work, it is our goal to design and synthesize new carbo-
rhodamine analogs, which not only exhibit high resistance toward
aggregation or nucleophilic attack, but also allow convenient fur-
ther modification. Herein, we report the design, synthesis, spec-
tral studies and proof-of-concept applications of fluoreno-xanthene
(10) and xantheno-xanthene (16a and 16b), i.e. xanthene dyes with
a “fluorene” or a “xanthene” bridging group, which fulfils the re-
quirements discussed previously (Scheme 1).
2-(4-diethylaminobenzyl)phenyl)diphenylmethanol, which was re-
fluxed with MeSO3H in CH2Cl2 for 2 h and then oxidized to
3,6-bis(diethylamino)-10,10-diphenylanthracen-9(10H)-one (6) by
KMnO4 in an overall 48% yield. Compound 6 reacted with Grig-
nard reagent (7, 2 equiv.) at 45 °C for 5 h to produce 8
in 65% yield. Dye 10 was synthesized similarly with fluo-
a
renone (9) instead of benzophenone (5) in an overall 37% yield.
Surprisingly, the preparation of 16a and 16b from xanthenone
was not successful. So, we proposed an alternative synthesis
ꢀ
starting from 4,4 -methylenebis(3-bromo-N,N-diethylaniline) (11),
whose synthesis was reported during the first synthesis of sil-
icon rhodamine by Xiao and Qian et al. [28,29]. Compound 11
was treated with nBuLi (2.4 equiv.) at −78 °C for 15 min to
prepare the corresponding lithium reagent in situ via halogen-
lithium exchange before methyl 2-phenoxybenzoate 13 (1.0 equiv.)
was added to furnish 2,7-bis(diethylamino)-9-(2-phenoxyphenyl)-
9,10-dihydroanthr-acen-9-ol, which was oxidized by KMnO4 be-
fore refluxing with MeSO3H in CH2Cl2 for 2 h to produce the
ꢀ
desired 2,7-bis(diethylamino)-10H-spiro[anthracene-9,9 -xanthen]-
10-one (14) in a 63% yield. Compound 14 was further treated with
Grignard reagent (15a and 15b, 2 equiv.) at 45 °C for 5 h to pro-
duce 16a and 16b in good yields. The single-crystal structure of
16b was obtained (Fig. S1 in Supporting information). The upper
diphenyl ether plane is perpendicular to the fluorochromic xan-
thene structure.
We devised a new synthesis for carbo-rhodamine dyes, and
the feasibility was first exemplified by synthezing a bis-phenyl
substituted carbo-rhodamine (8). 3-Bromo-N,N-diethylaniline (1)
was prepared via alkylation of 3-bromoaniline with EtI in
We tested the feasibility of 16a and 16b for functionalization,
i.e., sulfonation, which is a valued substitution for dyes because
it improves water solubility, photo/chemo stability, and resistance
toward aggregation simultaneously. Compound 10 was dissolved
in concentrated H2SO4 in an ice bath and stirred for 24 h, and
no desired sulfonated derivatives were observed. Presumably, the
biphenyl moiety is not sufficiently electron-rich. We then tested
a
nearly quantitative yield. Acid-catalysed condensation of
1
with (4-(diethylamino)phenyl)methanol (2) at 80 °C for
3
in
a 96% yield. Compound 3 was treated with nBuLi (1.2 equiv.)
at −78 °C for 15 min to prepare the corresponding lithium
reagent in situ via halogen-lithium exchange before benzophe-
none 5 (1.0 equiv. in THF) was added to furnish (5-diethylamino-
2

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Fig. 1. The generic structure of rhodamine dyes, their limitations, and remedies.
Fig. 2. The generic structures of existing carbo-rhodamines and the main idea of this work to develop novel highly stable xanthenoid dyes.
Table 1
16a and 16b. Under the same condition, the diphenyl ether moi-
The spectral properties of 16b, Sulfo-16b and Sulfo-Cy5 in different solvents.a
ety of 16a was smoothly bis-sulfonated to Sulfo-19a in a 64% yield.
Sulfonation of 16b produced a tris-sulfonated product (Sulfo-16b)
in a 64% yield.
Dyes
Solvent
λabs (nm)
ε (L mol−1 cm−1
)
λem (nm)
ꢀfb
16b
DCM
DMSO
MeOH
PBS
640
648
638
642
650
651
644
650
650
130,000
127,000
120,000
103,000
89,700
659
672
659
663
673
672
666
670
667
0.91
0.66
0.69
0.46
0.70
0.70
0.58
0.38
0.27
Dyes 16b and Sulfo-16b were selected for further spectral and
imaging studies. Dye 16b has an absorption band typical of a xan-
thene dye, including a major peak with a maximum at ca. 640
nm in such solvents as CH2Cl2/DMSO/MeOH/phosphate buffer with
their molar absorptivities ranging of (1.0–1.3) × 105 L mol−1 cm−1
and a shoulder peak. The emission band of 16b is a good mirror
image of their absorption band, including a major peak at ca. 660
nm in different solvents and a tailing shoulder band. It is partic-
ularly noteworthy that the fluorescence quantum yields in these
solvents are very high, ranging from 0.91 in CH2Cl2, 0.66 in DMSO,
0.69 in MeOH, to 0.46 in phosphate buffer. The maximum ab-
sorption and emission wavelengths of Sulfo-16b exhibit a slight
bathchromic shift of ca. 5–10 nm compared to 16b in the same sol-
vents. Comparatively the molar absorptivities and the fluorescence
quantum yields witnessed a drop. Nevertheless, Sulfo-16b is still a
bright dye with a molar absorptivity of 81,000 L mol−1 cm−1 and
quantum yield of 0.38 in phosphate buffer. We note that the spec-
tral properties of Sulfo-16b are essentially the same to those of a
classic Cy5 dye (Sulfo-Cy5). The molar absorptivity of Sulfo-Cy5 is
high at 234,000 L mol−1 cm−1 and the fluorescence yield is lower
at 0.27. (Table 1 and Fig. S2 in Supporting information).
Sulfo-
DCM
DMSO
MeOH
PBS
16b
84,500
92,300
81,000
Sulfo-Cy5
PBS
234,000
a
Measured in solution containing 0.2% DMSO.
b
Quantum yields determined with Sulfo-Cy5 as the reference (ꢀf = 0.27 in
PBS [30]).
stabilities of Sulfo-16b were studied with Sulfo-Cy5 as a reference.
An addition of hypochlorite (up to 100 equiv., Fig. 3A) or peroxyni-
trite (up to 5 equiv., Fig. 3B) caused a dose-dependent decrease of
the absorbance of Sulfo-Cy5 at 648 nm, verifying it poor chemo-
stabilities toward these reactive oxygen species. Under the same
condition, Sulfo-16b remained unchanged, suggesting that Sulfo-
16b is chemically stable even in a large excess of these oxidative
species. The stabilities of Sulfo-Cy5 and Sulfo-16b toward GSH (up
to 300 equiv., Fig. 3C) were checked and both remained stable.
Lastly, the photo-stability of Sulfo-16b and Sulfo-Cy5 were com-
pared by irradiation of their solution in neutral phosphate buffer
with a halogen lamp (Fig. 3D). In 30 min, the absorbance of Sulfo-
Cy5 decreased by 80%, while the absorbance of Sulfo-16b again
remains unchanged. Absorption/emission maxima in the deep-red
Therefore, 16b and Sulfo-16b can potentially replace Cy5 dyes
for practical applications. Cy5 type fluorophores are known for
their poor stabilities. Their floppy polymethine backbone is prone
to nucleophilic attack by thiolates or reactive oxygen species, in-
cluding hypochlorite, peroxynitrite, etc. Therefore, chemo-/photo-
3

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Fig. 3. Normalized absorption spectra over time for the solutions of Sulfo-16b and Sulfo-Cy5 in PBS (pH 7.4) with the addition of different equivalents of NaClO (A), ONOO−
(B), GSH (C), and upon irradiation with a halogen lamp (300 W) (D).
Fig. 4. (A) Wide-field and reconstructed SIM images of cell mitochondria in U2OS cells with 16b. (B) Further magnified region of interest (ROI). (C) The intensity profiles
of mitochondria in super-resolution SIM image and wide-field image. Exc: 640 nm. Scale bar: 5 μm. (D) STED imaging of U2OS cells incubated with 16b and the magnified
image of the region of interest (ROI). Exc: 640 nm; Depletion lasers: 775 nm; Scale bar: 10 μm.
region, high chemo-/photo-stabilities, and bright fluorescence ren-
der 16b and its hydrophilic Sulfo-16b value fluorophores for bio-
imaging. Their potentials for imaging were showcased with 16b in
live U2OS cells via three different microscopic methods, i.e., epi-
fluorescence, structured illumination (SIM), and stimulated emis-
sion depletion (STED). Cells were incubated with compound 16b
(1.2 μmol/L) for 10 min, rinsed with PBS three times, and cell im-
ages were acquired. Bright images of filamentous structures, pre-
sumably mitochondria, were observed by all three methods (Fig. 4
for wide-field/SIM and STED). Cells did not exhibit structural ab-
normalities, and the fluorescence did not exhibit noticeable bleach-
ing during the course of imaging. Though preliminary, these imag-
ing results are sufficient to verify that 16b and Sulfo-16b are feasi-
ble for cell-based imaging applications.
Often, xanthene dyes are installed with various functionalities,
e.g., benzyl bromide, amino, carboxyl, or activated carboxyl. We
4

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showcase that this chemistry is also applicable to the novel carbo-
rhodamines reported herein. The benzephenone 14 was reacted
with 2-methoxymethylphenyl lithium to prepare dye 17, which
was further treated with BBr3 to furnish the dye (18) with a ben-
zyl bromide, which is a good reaction partner for various biolog-
ical thiols. Then, by reacting 19 with aminothiol ethanol or 3-
mercaptopropanoic acid, respectively, dyes with a primary amino
group or a carboxyl group were prepared in excellent yields. If
necessary, the carboxyl group of 20 can be conveniently activated
into a succinimidyl ester (21) for conjugation with an amino group
(Scheme S1 in Supporting information).
pality for the Shanghai International Cooperation Program (No.
18430711000), the China Postdoctoral Science Foundation (Nos.
2019M651427, 2020T130197).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.cclet.2021.04.041.
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Declaration of competing interest
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The work is financially supported by the National Natural Sci-
ence Foundation of China (Nos. 21822805, 21908065, 22078098),
the Science and Technology Commission of Shanghai Munici-
5