A general approach to spirolactonized Si-rhodamines

Article abstract of DOI:10.1039/c4cc06178k

A general approach has been developed for the synthesis of spirolactonized Si-rhodamines (SiRBs). The alternative synthetic route provides a facile opportunity to access various SiRBs bearing ring-expanded scaffolds and substituents for conjugation. By click reaction, a near-infrared (NIR) mitochondrial tracker was prepared, displaying specific mitochondrial staining in cells. This journal is

Full text of DOI:10.1039/c4cc06178k

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A general approach to spirolactonized
Si-rhodamines†
Baogang Wang,a Xiaoyun Chai,a Weiwei Zhu,ab Ting Wang*a and Qiuye Wu*a
Cite this: Chem. Commun., 2014,
50, 14374
Received 12th August 2014,
Accepted 30th September 2014
DOI: 10.1039/c4cc06178k
www.rsc.org/chemcomm
A general approach has been developed for the synthesis of Despite the tunability and utility of SiRBs, the only established
spirolactonized Si-rhodamines (SiRBs). The alternative synthetic synthetic approach to this scaffold is complicated and difficult. In
route provides a facile opportunity to access various SiRBs bearing addition to the long-step synthesis with low overall yield, the
ring-expanded scaffolds and substituents for conjugation. By click synthesis of some derivatives requires cumbersome protection
reaction, a near-infrared (NIR) mitochondrial tracker was prepared, and deprotection of functional groups, which are sensitive to active
displaying specific mitochondrial staining in cells.
lithium reagents,4–6 thus increasing the difficulty of synthesis.
Moreover, some widely-used substituents for covalent conjugation
Recent advances in fluorescence bioimaging have spurred a high (such as –Br) are incompatible with the active lithium reagents,
demand for small-molecule near infrared (NIR) fluorophores,1 further restricting the scope of the reaction. Therefore, the
which are typically active in the spectral region of 650–900 nm. only synthetic approach is far from meeting practical demands
Compared to congeners with shorter excitation and emission and there is a serious need for an alternative synthetic approach
wavelengths, NIR fluorophores are highly desired due to their to SiRBs.
low phototoxicity, low light scattering, deep tissue penetration
Considering the structural similarity of rhodamine B and
and less interference from biological autofluorescence. Although SiRB, we explored the existing synthetic approaches for rhodamine
various synthetic NIR fluorophores exist, due to the lack of to find some clues for preparing SiRB. Recently, Cornish and
biocompatible characteristics such as water solubility, photo- co-workers devised a novel strategy to synthesize rhodamines from
stability, permeability and controllable conjugation, only a small a common diaryl ether intermediate.7 This strategy inspired us to
portion of them can be directly applied in bioimaging in vivo.2 In seek a general route for the direct construction of structurally-
this regard, the development of novel NIR fluorophores suitable similar SiRBs. Herein, we report an alternative approach for the
for bioimaging is still a big concern.
synthesis of various SiRBs from common diaryl silyl ether
Recently, Qian et al. devised a novel class of NIR active (Scheme 1). Due to the avoidance of organometallic addition
fluorophores by perturbing the skeleton of rhodamine with a employed in the prior synthetic route, unprotected condensation
silicon atom.3 The silicon-perturbed rhodamines (SiRs) have reaction is achieved. Thus, the proposed strategy provides a facile
attracted considerable interest on account of their excellent NIR opportunity to access functionalized SiRBs bearing various sub-
photophysical properties as well as biocompatible characteristics.4 stituents for conjugation, such as –Br, –COOH, –NH2, –CRCH
Particularly, spirolactonized Si-rhodamine (SiRB)-based fluores- and –CONH2. Moreover, ring-expanded and asymmetric SiRBs are
cent probes and tags have been proved to be ideally suitable for readily prepared by this method. In bioimaging applications, we
NIR bioimaging of metal ions,5 biomolecules and proteins.6 ‘‘clicked’’ triphenylphosphonium with HC C–SiRB to design the
R
NIR mitochondrial tracker for the specific staining of mitochondria
in cells.
a Department of Organic Chemistry, College of Pharmacy, Second Military Medical
To pursue our objective, we initially investigated the synthesis of
University, Guohe Road 325, Shanghai 200433, P.R. China.
critical diaryl silyl ether (DASE) intermediates. DASE-1 and DASE-2
with identical monomers were prepared under mild conditions
with high yield. To extend the scaffold, asymmetrical DASE-3 with
two different monomers was readily synthesized for the prepara-
tion of asymmetrical SiRB. Having established a robust method in
synthesizing the critical DASE intermediates, we next explored the
direct transformation of DASEs into SiRBs (Scheme 2).
E-mail: wangting1983927@gmail.com, wuqy6439@sohu.com
b College of Pharmacy, Yantai University, Qingquan Road 32, Yantai,
Shandong 264005, P.R. China
† Electronic supplementary information (ESI) available: Synthetic and experi-
mental details, 1H NMR, 13C NMR and HRMS spectra, absorption and fluores-
cence spectra, crystallographic data and fluorescence images. CCDC 1011227. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4cc06178k
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tube in the presence of 5.0 equiv. of 2-formylbenzoic acid
(Table S1, ESI†). Of note, the solvent-free system was adopted
for the advantages of no solvent pollution. Furthermore, the
desired product was easy to purify using standard silica-based
flash chromatography after washing off the unreacted acid
materials. Over one gram of SiRB was prepared in one pot by
this method, thus demonstrating the scalability of our approach.
Plenty of products also provided an opportunity for the investi-
gation of crystals of SiRB in which a silicon-perturbed ring and a
unique spirolactone were found (Fig. S1, ESI†). The reliable
synthesis of SiRB will significantly contribute to the development
of NIR probes based on the spiroring-opening mechanism.
With the conditions for synthesizing SiRB in hand, we
proceeded to explore the substrate scope of this transformation
by varying the 2-formylbenzoic acid bearing substituents, such
as –Br, –CN, –NO2, –COOH and –CRCH. These functional
substituents were selected for following reasons. Firstly, some
of them are widely used in covalent conjugation through cross-
coupling (–Br), esterification (–COOH) and click reaction
(–CRCH). Secondly, by a prior method, some substituents
require protection or even are difficult to conduct protection
because they are sensitive to or incompatible with the active
lithium reagents. Finally, these substituted derivatives are
commercially available or could be readily prepared in high
yield. Extraordinarily, most studied functional groups were
tolerated the present reaction conditions. The substrates sub-
stituted with –Br, –CN, –NO2 and –CRCH yielded corres-
ponding substituted SiRBs in 16–40% isolated yields
(Scheme 3). Although O2N–SiRB and NC–SiRB are difficult to
directly apply in conjugation, they could be efficiently converted to
H2N–SiRB and NH2CO–SiRB separately by reduction and hydro-
lyzation with high yield. However, the synthesis of HOOC–SiRB
under similar conditions experienced relatively low yield, probably
due to the high melting point of 2-formylterephthalic acid. To
improve the reaction system, a modified condensation reaction
Scheme 1 Synthetic routes to prepare SiRBs.
Scheme 2 Synthesis of DASEs. (a) n-BuLi, 0 1C, Et2O, then 0.6 equiv. of
SiMeCl2; (b) n-BuLi, 0 1C, Et2O, then 5.0 equiv. of SiMeCl2; (c) n-BuLi, 0 1C, Et2O.
A typical synthesis of rhodamine B derivatives involves the performed at 140 1C in a sealed tube with p-TsOH as a promoting
condensation between a phthalic anhydride and aminophenol, solvent, affording the final product HOOC–SiRB in 11% yields.
giving an intractable mixture of regioisomers.8 To avoid iso-
A further extension of this synthetic methodology was
merization, 2-formylbenzoic acid and its derivatives were studied by varying the DASE intermediates. As expected, the
selected to directly condense with DASE under acidic conditions. condensation of DASE-2 and DASE-3 provided target products
Our first attempt was carried out in propionic acid with catalytic SiRB-2 and SiRB-3 respectively, with 16% and 31% isolated
p-toluenesulfonic acid (p-TsOH) from 2-formylbenzoic acid.9 yields. The results suggest that this synthetic approach can be
Unfortunately, the formation of the desired product was not applied to the synthesis of SiRBs with advantages as follows:
observed, probably due to the relatively low reaction activity of (1) a straightforward approach to prepare SiRBs with a long
DASE. Realizing the low melting point of 2-formylbenzoic acid emission wavelength, which are hard to achieve by the prior
(95 1C), we envisioned that it could serve as an acid solvent under method; (2) synthesis of asymmetrical SiRBs (such as SiRB-3)
melting conditions. Pleasingly, the improved condensation reac- for the first time, which facilitates the design of potentially
tion performed at 140 1C in a sealed tube afforded a modest yield useful NIR fluorescent probes and tags.
of SiRB (40%, Table S1, ESI†). To further understand the role of
Fig. S2 (ESI†) shows the normalized absorption and fluores-
2-formylbenzoic acid, control reactions with 3-formylbenzoic acid cence spectra of SiRBs in EtOH solution (containing 0.1 mM HCl
(m.p. 175 1C) and 4-formylbenzoic acid (m.p. 247 1C) were carried to ensure the ring opening of SiRBs). All SiRBs display similar
out. Trace amounts of corresponding products were obtained. absorption and emission maxima in the NIR region. Expansion
These results further confirmed the unique role of 2-formylbenzoic of the ring of SiRB-2 and SiRB-3 results in significant red-shifted
acid. We found that the yield obviously improved to B50% in the absorption and fluorescence. H2N–SiRB exhibits low fluores-
presence of catalytic amounts of CuBr2. Optimization revealed that cence quantum yield due to the PET of the amino group. Spectral
the condensation reaction performed best at 140 1C in a sealed properties of SiRBs are summarized in Table S3 (ESI†).
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Fig. 1 Fluorescence images of bEnd3 (left) and HepG2 (right) cells
co-stained with 10.0 mM Mito-SiRB and 50 nM rhodamine 123.
(a) Fluorescence microscopic images from Mito-SiRB. (b) Fluorescence
microscopic images from rhodamine 123. (c) Microscopic images.
(d) Merged fluorescence microscopic images.
with that of rhodamine 123 (Fig. S4, ESI†), proving the specific
mitochondrial staining of Mito-SiRB in both normal and
cancer cells.
In conclusion, we have delivered a general approach to
prepare SiRBs from common diaryl silyl ether intermediates
based on a condensation process, which is compatible with
various functional groups without protection. Thus, SiRBs with
various substituents, including –Br, –COOH, –NO2, –CRCH
and –CN, which are difficult or unpractical to synthesize by the
prior method were readily prepared. Further extension of this
synthetic methodology can be applied to the synthesis of ring-
expanded and asymmetric SiRBs. The scope for synthesizing
SiRBs is extensively enlarged by the reported strategy on two
different aspects: the variety of functional groups and the variety
of scaffolds. Versatility of the present synthetic methodology
enabled its adoption for a broad range of applications in NIR
bioimaging. By click reaction, Mito-SiRB was prepared, which
displayed specific mitochondrial staining in cells, confirming
the controllable conjugation of functionalized SiRBs for targeted
imaging. Further development of NIR fluorescence probes based
on the synthetic methodology is in progress.
Scheme 3 Synthesis of SiRBs. a Isolated yields. b The reaction was performed
with 1.0 equiv. of p-TsOH.
To test the application of functionalized SiRBs, a controllable
conjugation was performed to develop an NIR fluorescent tag for
targeted bioimaging. In agreement with reported results, SiRB
is cell permeable and tends to homogeneously spread in
the cytoplasm in both bEnd3 and HepG2 cells, confirming the
nonspecific binding of SiRB in cells (Fig. S3, ESI†). In the
intracellular environment, mitochondria play a crucial role in
the regulation of cell functions. Thus, the development of a
mitochondrion-specific NIR fluorescent tracker will benefit the
research of chemical biology.10 To achieve the targeted NIR
bioimaging, Mito-SiRB was readily synthesized through click
11
R
reaction from HC C–SiRB (Scheme 4). Co-staining with
This research was supported by National Natural Science
Foundation of China (No. 21205135). We also sincerely appreciate
Xiaoyan Cui for her useful discussion and suggestion.
Mito-SiRB and the commercial mitochondrial tracker rhodamine
123 was performed in bEnd3 and HepG2 cells (Fig. 1). The yellow
merged images indicate that the staining of Mito-SiRB fits well
Notes and references
1 (a) Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43, 16;
(b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev.,
2013, 42, 622; (c) S. A. Hilderbrand and R. Weissleder, Curr. Opin.
Chem. Biol., 2010, 14, 71.
2 Y.-Q. Sun, J. Liu, X. Lv, Y. Liu, Y. Zhao and W. Guo, Angew. Chem.,
Int. Ed., 2012, 51, 7634.
3 M. Fu, Y. Xiao, X. Qian, D. Zhao and Y. Xu, Chem. Commun., 2008, 1780.
4 (a) Y. Koide, Y. Urano, K. Hanaoka, W. Piao, M. Kusakabe, N. Saito,
T. Terai, T. Okabe and T. Nagano, J. Am. Chem. Soc., 2012, 134, 5029;
(b) Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, ACS
Chem. Biol., 2011, 6, 600; (c) T. E. McCann, N. Kosaka, Y. Koide,
M. Mitsunaga, P. L. Choyke, T. Nagano, Y. Urano and H. Kobayashi,
ˇ
Bioconjugate Chem., 2011, 22, 2531; (d) T. Pastierik, P. Sebej,
ˇ
´
´
J. Medalova, P. Stacko and P. Klan, J. Org. Chem., 2014, 79, 3374;
(e) P. Shieh, M. S. Siegrist, A. J. Cullen and C. R. Bertozzi, Proc. Natl.
Acad. Sci. U. S. A., 2014, 111, 5456.
Scheme 4 Synthesis of Mito-SiRB. (a) CuSO4Á5H2O, sodium ascorbate,
(3-azidopropyl)triphenylphosphonium bromide, THF, RT.
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5 T. Wang, Q.-J. Zhao, H.-G. Hu, S.-C. Yu, X. Liu, L. Liu and Q.-Y. Wu,
Chem. Commun., 2012, 48, 8781.
8 (a) H. Yu, Y. Xiao and H. Guo, Org. Lett., 2012, 14, 2014;
(b) J. B. Grimm and L. D. Lavis, Org. Lett., 2011, 13, 6354;
(c) L. D. Lavis and R. T. Raines, ACS Chem. Biol., 2014, 9, 855.
ˇ
6 (a) G. Lukinavicius, K. Umezawa, N. Olivier, A. Honigmann, G. Yang,
ˆ
´
T. Plass, V. Mueller, L. Reymond, I. R. Correa Jr, Z.-G. Luo,
9 I. C. S. Cardoso, A. L. Amorim, C. Queiros, S. C. Lopes, P. Gameiro, B. de
C. Schultz, E. A. Lemke, P. Heppenstall, C. Eggeling, S. Manley
Castro, M. Rangel and A. M. G. Silva, Eur. J. Org. Chem., 2012, 2012, 5810.
and K. Johnsson, Nat. Chem., 2013, 5, 132; (b) E. Kim, K. S. Yang, 10 A. R. Sarkar, C. H. Heo, H. W. Lee, K. H. Park, Y. H. Suh and
R. J. Giedt and R. Weissleder, Chem. Commun., 2014, 50, 4504. H. M. Kim, Anal. Chem., 2014, 86, 5638.
7 A. V. Anzalone, T. Y. Wang, Z. Chen and V. W. Cornish, Angew. 11 H. Yu, X. Zhang, Y. Xiao, W. Zou, L. Wang and L. Jin, Anal. Chem.,
Chem., Int. Ed., 2013, 52, 650.
2013, 85, 7076.
This journal is ©The Royal Society of Chemistry 2014
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