Design and Synthesis of Near-Infrared Mechanically Interlocked Molecules for Specific Targeting of Mitochondria

Article abstract of DOI:10.1021/acs.orglett.0c01922

The entrapment of squaraine (SQ) within a molecular container to form rotaxane has been shown to improve the dye stability and the fluorescence proficiency inside the mitochondria. The macrocycle provides shelter and protects the near-infrared (NIR) SQ chromophore from nucleophilic attacks made by the exposed thiol of Cys-containing mitochondrial proteins and mitochondrial glutathione. Herein a microwave-assisted template-directed clipping reaction on low-loading 2-chlorotrityl chloride resin is used to develop an NIR unsymmetrical squaraine rotaxane in high quantum yield.

Full text of DOI:10.1021/acs.orglett.0c01922

pubs.acs.org/OrgLett
Letter
Design and Synthesis of Near-Infrared Mechanically Interlocked
Molecules for Specific Targeting of Mitochondria
Rabi Sankar Das, Pranab Chandra Saha, Nayim Sepay, Ayan Mukherjee, Sudipta Chatterjee,
and Samit Guha*
Cite This: https://dx.doi.org/10.1021/acs.orglett.0c01922
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The entrapment of squaraine (SQ) within a molecular
container to form rotaxane has been shown to improve the dye stability
and the fluorescence proficiency inside the mitochondria. The
macrocycle provides shelter and protects the near-infrared (NIR) SQ
chromophore from nucleophilic attacks made by the exposed thiol of
Cys-containing mitochondrial proteins and mitochondrial glutathione.
Herein a microwave-assisted template-directed clipping reaction on low-
loading 2-chlorotrityl chloride resin is used to develop an NIR
unsymmetrical squaraine rotaxane in high quantum yield.
sing smart near-infrared (NIR) fluorescence imaging
be a method of improving the dye performance inside the
Uprobes constructed from organic molecules to target mitochondria.7 Smith and coworkers have made seminal
contributions toward the development of squaraine rotaxane
for various applications.8 However, the synthesis of mitochon-
dria targeting NIR unsymmetrical 1,3,3-trimethylindolin
squaraine rotaxane is a challenging task. The synthesis of
unsymmetrical SQRot can be achieved by the conventional oil-
bath heating method, which takes days and often requires
prolonged and difficult column chromatographic purification
due to unwanted side products. Therefore, a fast, effective
methodology with a high yield and product purity is required
for the synthesis of unsymmetrical SQRot. We propose that
microwave (MW)-assisted solid-phase synthesis on low-
loading resin could be an effective way to construct an
unsymmetrical rotaxane molecule. Moreover, the synthesized
SQRot can be further bioconjugated with various target-
specific functional groups, including organelle-selective target-
ing moieties. To the best of our knowledge, this is the first
report of an NIR unsymmetrical 1,3,3-trimethylindoline
squaraine-rotaxane-based mitochondria targeting and imaging
agent.
specific cellular organelles is an emerging field of contemporary
research.1 NIR fluorescent biomarkers (650−900 nm) for
intracellular locations are indispensable in contrast with visible
and other fluorescent dyes because NIR light can permeate
deep tissues and offers a meaningful bioimaging method with
negligible autofluorescence background.2 Mitochondria are
crucial targets within the numerous cellular organelles.3
Mitochondria are the powerhouse of cells, and they control
cellular functions. The high negative inner mitochondrial
membrane potential (ΔΨm −150 to −180 mV) is distinctive
because it does not exist in other cellular organelles, and this
offers a design opportunity to target mitochondria. So far,
squaraine (SQ) has been overlooked when building probes for
the selective staining of mitochondria.4 We envision that the
electrophilic SQ chromophore could be susceptible to
nucleophilic attack by the reactive thiols (SH) present in
various mitochondrial proteins as well as mitochondrial
glutathione (GSH), and hence NIR fluorescence could be
quenched after the accumulation of SQ inside the mitochon-
dria.5 Moreover, SQ has a propensity to form nonfluorescent
aggregates in an aqueous environment; this leads to substantial
broadening of its absorption as well as emission bands, which is
undesired for cellular organelle imaging.6 These could be
potential downsides for the development of SQ-based
mitochondria tracking agents. We propose that the entrapment
of a mitochondria-targeting SQ probe inside a container
molecule to form mechanically interlocked molecules (rotax-
ane) that are stabilized through noncovalent interactions could
We have designed and constructed an unsymmetrical 1,3,3-
trimethylindoline SQ appended with triphenylphosphonium
Received: June 8, 2020
https://dx.doi.org/10.1021/acs.orglett.0c01922
© XXXX American Chemical Society
Org. Lett. XXXX, XXX, XXX−XXX
A

Organic Letters
pubs.acs.org/OrgLett
Letter
(TPP+) functionality (MSQ) to target mitochondria (Scheme
1).9 A screening inspection is carried out under MW
solution (1:10) of MSQ with GSH. Within a few minutes, the
blue color of MSQ disappeared after the addition of GSH or 2-
mercaptoethanol. UV/vis and fluorescence titration of MSQ in
the presence of GSH and 2-mercaptoethanol showed bleaching
of the λabs as well as λem (Figure 1b and Figures S24 and S25).
The molecular docking of MSQ with Cys exposed mitochon-
drial protein ATP synthase subunit beta 1 (PDB: 4Q4L), and
other SH-exposed proteins showed interactions between the
SH moieties and MSQ (Figure 1c, Figure S26). The
encapsulation of MSQ dye inside a molecular container has
been shown to greatly improve the MSQ chemical stability and
also alter the fluorescence spectroscopic and microscopic
properties.
Scheme 1. Synthesis of SQ-COOH and MSQ
Herein the SQ-COOH molecule is loaded on a low-loading
2-chlorotrityl chloride (2-CTC) resin through −COOH.11
Efforts to synthesize the rotaxane molecule through a direct
slippage method in solution are ineffective. A template-directed
clipping reaction on 2-CTC resin is used to transform SQ-
COOH to SQRot-COOH under MW conditions (Scheme
2).12 Most strikingly, the unwanted side products of the
conditions to optimize the yield of the hemisquarate
intermediate (5) from diethyl squarate (Table S3, Figures S6
and S7).10 Compound 5 is hydrolyzed with NaOH and treated
with 2 under MW irradiation to obtain unsymmetrical SQ with
one −COOH functionality (SQ-COOH) (Table S4, Figures
S8 and S9). The SQ-COOH is further conjugated with a TPP+
functionality to get MSQ (Figures S10−S12).
The absorption and emission of MSQ are observed in the
NIR region (Figure S22). A cellular uptake and imaging assay
was performed by a confocal laser scanning microscope on an
epithelioid cervix carcinoma HeLa cell line. In the case of
mitochondria targeting MSQ, we observed a weak NIR
fluorescence signal inside the mitochondria (Figure 1a, Figure
Scheme 2. Synthesis of Unsymmetrical SQRot-COOH
Using the MW-Assisted Solid-Phase Protocol on 2-CTC
Resin and the Construction of MSQRot
conventional synthesis, whose formation is the most major
drawback of rotaxane construction, can be easily removed in
the solid phase by following the washing steps with suitable
solvents. Eventually, the desired rotaxane is obtained from the
resin support. The SQRot-COOH is cleaved from the 2-CTC
resin and further conjugated with TPP+ functionality to obtain
MSQRot (Scheme 2). Here MSQRot consists of an unsym-
metrical SQ chromophore as an NIR imaging signaling unit
tethered with a lipophilic cationic TPP+ group to target
mitochondria and a macrocycle to shelter and protect the
chromophore from nucleophilic attack inside the mitochon-
dria. The unsymmetrical MSQRot is acquired in satisfactory
yield and is characterized by numerous spectroscopic
techniques (Figures S13−S15). MSQRot displays one 31P
NMR peak at 24.8 ppm for the TPP+ group. A control
symmetrical rotaxane (SQRot) lacking the TPP+ moiety is also
synthesized (Figures S18−S21).
Figure 1. (a) Confocal images of MSQ colocalized with MitoTracker
Green (MTG) in HeLa cells. (b) A decrease in the fluorescence
intensity of MSQ (2 μM in DMSO/pH 8 buffer solution (1:10)) is
observed on the gradual addition of GSH (2 μM) at mitochondrial
pH 8. (c) Docking poses of MSQ with the exposed Cys (C35)
containing mitochondrial protein ATP synthase subunit beta 1.
S23). We hypothesize that the MSQ is selectively incorporated
inside the mitochondria; however, the chromophore is
vulnerable to nucleophilic attack by the exposed Cys-
containing mitochondrial proteins as well as mitochondrial
GSH. This leads to a discontinuity in conjugation, and thus the
NIR fluorescence of MSQ is dropped. To understand the
fluorescence turn-off of MSQ by the nucleophilic attack of the
−SH group, we used 2-mercaptoethanol with 1 equiv of
Hunig’s base in DMSO as well as GSH in mitochondrial pH
8.0 as model systems.5 We treated a DMSO/pH 8.0 buffer
The changes in 1H NMR chemical shifts caused by MSQRot
formation have been compared with the free macrocycle and
MSQ (Figure 2a). Most notable is the extreme downfield shift
for macrocycle proton “C” and “NH” due to hydrogen bonding
B
https://dx.doi.org/10.1021/acs.orglett.0c01922
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
1
singlet) protons of the MSQ, and hence an upfield shift in H
NMR is observed. The B3LYP/6-31+G** energy-minimized
structure shows that the two 1,3,3-trimethylindolin rings of
MSQ are in the trans configuration in the encapsulated form of
MSQRot (Figure 2d, Figure S27). Tetralactam N−H bonds
and two C−H bonds of the phenyl ring of the macrocycle are
forming hydrogen bonds with two O atoms of the C4O2
residue of MSQ. The central C4O2 core of MSQ, which is
highly electron-deficient and sandwiched between the two
electron-rich anthracene moieties (centroid-to-centroid, an-
thracene residues to C4O2 ring, distance is 3.9 Å), is stabilized
through a D−A−D-type interaction and thus is expected to
block nucleophilic attacks. We are fortunate to obtain the
single-crystal X-ray structure of the control symmetrical SQRot
molecule lacking the TPP+ group, which also exhibited a trans
orientation similar to that of the N-methyl indoline residues
(Figure 2e, Figure S28, Table S5). Four N−H bonds and two
phenyl C−H bonds of the macrocycle are forming hydrogen
bonds with SQ O atoms (N−H···O as well as C−H···O
hydrogen-bond distance is ca. 2.3 Å). The C4O2 core is also
forming D−A−D-type interactions with two anthracene
residues (D−A distance 3.7 Å) in SQRot. Also, the λabs and
λem of MSQRot show a characteristic bathochromic shift ca. 12
nm from the original spectra of MSQ in CHCl3 (Figure 2b,c).
The absorption spectra of unsymmetrical MSQRot revealed an
intense peak (λmax) at 652 nm in CHCl3 with a large molar
extinction coefficient of 1.5 × 105 M−1 cm−1 (Table S6). The
λem of MSQRot is observed to be 670 nm in CHCl3. There is a
3.5-fold augmentation in the MSQRot quantum yield
((Φf)MSQRot = 0.53) in comparison with MSQ ((Φf)MSQ
=
0.15) in DMSO, which is due to the reduced rotational and
vibrational motions of the entrapped MSQ and because the
consequent nonradiative excited-state relaxation pathways are
the least feasible.13 The anthracene fluorescence at λex 365 nm
becomes weaker for MSQRot formation in comparison with
the free macrocycle due to FRET from anthracene to the
trapped MSQ (Figure S29).
Dethreading could be a potential drawback of rotaxane
located inside the subcellular organelles.14 1H NMR studies in
CDCl3 at 37 °C for 2 days showed no dissociation of the MSQ
from MSQRot. The mechanical stability of MSQRot is also
investigated by absorption/emission spectroscopy in CHCl3.
There is no evidence of MSQRot dethreading after 48 h at 37
°C. This confirmed that the gem-dimethyl and N-methyl
groups in 1,3,3-trimethylindoline residues and the TPP+ group
are bulky enough to prevent MSQRot dethreading. The
plausible biological interference from GSH at mitochondrial
pH 8 at 37 °C is inspected over 48 h. No significant emission
changes in MSQRot are perceived in GSH at pH 8 (Figure
S30). Cell viability studies show that the MSQRot is less toxic
to HeLa carcinoma and the noncancerous HEK293 cell line
(Figure S31). The outstanding biocompatibility, insignificant
cytotoxicity, narrow NIR absorption/emission band, and high
quantum yield further make the MSQRot a smart choice for
organelle staining. To confirm the mitochondrial selectivity, a
colocalization test involving MSQRot is executed in HeLa and
A549 carcinoma cells with a mitochondria specific marker,
MitoTracker Green (MTG). The MSQRot displayed decent
colocalization with MTG in the confocal microscopy with a
Pearson’s correlation (PC) coefficient of 0.89 for HeLa and
0.85 for A549 (Figure 3 and Figures S32 and S33). The
accumulation of MSQRot in the mitochondria is anticipated to
be 500−1000 times greater than that in the extracellular region
Figure 2. (a) Partial 1H NMR stacking of the free macrocycle,
MSQRot, and MSQ in CDCl3. (b) Normalized absorption plot of
MSQ (2 μM) and MSQRot (2 μM) in CHCl3. (c) Normalized
fluorescence plot of MSQ (2 μM) and MSQRot (2 μM) in CHCl3.
Absorption and emission plots showing the bathochromic shift for
rotaxane formation. (d) B3LYP/6-31+G** energy-minimized struc-
ture of MSQRot. (e) Single-crystal X-ray structure of control SQRot.
The centroid-to-centroid distance between the anthracene residues
and the C4O2 core is 3.7 Å.
with SQ O atoms. Moreover, an upfield shift of macrocyclic
protons E and F is observed for rotaxane formation. For
MSQRot, the anthracene residues of the macrocycle create
anisotropic shielding zones for the 5,5′ (two well-separated
C
https://dx.doi.org/10.1021/acs.orglett.0c01922
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
orcid.org/0000-0001-6643-9882; Email: samitfsu@
gmail.com, samit.guha@jadavpuruniversity.in
Authors
Rabi Sankar Das − Department of Chemistry, Organic
Chemistry Section, Jadavpur University, Kolkata 700032, India
Pranab Chandra Saha − Department of Chemistry, Organic
Chemistry Section, Jadavpur University, Kolkata 700032,
India; orcid.org/0000-0003-4808-1375
Nayim Sepay − Department of Chemistry, Organic Chemistry
Section, Jadavpur University, Kolkata 700032, India;
orcid.org/0000-0001-7702-3989
Ayan Mukherjee − Department of Chemistry, Organic
Chemistry Section, Jadavpur University, Kolkata 700032, India
Sudipta Chatterjee − Department of Chemistry, Serampore
College, Serampore, West Bengal 712201, India; orcid.org/
0000-0002-3273-1751
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01922
Notes
Figure 3. Confocal images of MSQRot colocalized with MTG in
HeLa cells. The colocalization scatter plot shows PC coefficient: 0.89.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
due to the potential gradient (ΔΨm −150 to −180 mV vs
plasma membrane potential −30 to −60 mV).9 The inner
mitochondrial concentrations of MSQRot in HeLa cells are
determined by fluorescence analysis to be 1.3 0.3 mM and
This work is supported by DST-SERB, Govt. of India (file no.
ECR/2017/001405).
REFERENCES
4.2
0.6 mM, which are greater than the original exterior
■
(1) (a) Zhu, H.; Fan, J.; Du, J.; Peng, X. Acc. Chem. Res. 2016, 49,
2115−2126. (b) Wang, L.; Frei, M. S.; Salim, A.; Johnsson, K. J. Am.
Chem. Soc. 2019, 141, 2770−2781.
concentrations of 2 and 5 μM, respectively (Figure S34). The
mitochondrial target selectivity of MSQRot is compared with a
control symmetrical SQRot lacking the TPP+ functionality.
SQRot stains inside the A549 cells; however, confocal images
show that it is poorly colocalized with mitochondria tracker
MTG (Figure S35).
In summary, the macrocycle gives shelter and protects the
MSQ dye from nucleophilic attack inside the mitochondria.
This rotaxane molecule can be functionalized to target specific
cellular organelles. MSQRot with an appended targeting TPP+
group could be an effective molecular probe for mitochondrial
therapy with NIR imaging diagnostics.
(2) (a) Yan, R.; Hu, Y.; Liu, F.; Wei, S.; Fang, D.; Shuhendler, A. J.;
Liu, H.; Chen, H.-Y.; Ye, D. J. Am. Chem. Soc. 2019, 141, 10331−
10341. (b) Ducharme, G. T.; LaCasse, Z.; Sheth, T.; Nesterova, I. V.;
Nesterov, E. E. Angew. Chem., Int. Ed. 2020, 59, 8440−8444.
(3) Liew, S. S.; Qin, X.; Zhou, J.; Li, L.; Huang, W.; Yao, S. Q.
Angew. Chem., Int. Ed. 2020, DOI: 10.1002/anie.201915826.
(4) Babu, P. S. S.; Manu, P. M.; Dhanya, T. J.; Tapas, P.; Meera, R.
N.; Surendran, A.; Aneesh, K. A.; Vadakkancheril, S. J.; Ramaiah, D.;
Nair, S. A.; Pillai, M. R. Sci. Rep. 2017, 7, 42126.
(5) (a) Hewage, H. S.; Anslyn, E. V. J. Am. Chem. Soc. 2009, 131,
13099−13106. (b) Arunkumar, E.; Forbes, C. C.; Noll, B. C.; Smith,
B. D. J. Am. Chem. Soc. 2005, 127, 3288−3289. (c) Anees, P.; Sreejith,
S.; Ajayaghosh, A. J. Am. Chem. Soc. 2014, 136, 13233−13239.
(6) Chen, H.; Farahat, M. S.; Law, K.-Y.; Whitten, D. G. J. Am.
Chem. Soc. 1996, 118, 2584−2594.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01922.
(7) (a) Stoddart, J. F. Angew. Chem., Int. Ed. 2017, 56, 11094−
11125. (b) Craig, M. R.; Hutchings, M. G.; Claridge, T. D. W.;
Anderson, H. L. Angew. Chem., Int. Ed. 2001, 40, 1071−1074. (c) Lee,
S.; Chen, C.-H.; Flood, A. H. Nat. Chem. 2013, 5, 704−710.
(8) (a) Johnson, J. R.; Fu, N.; Arunkumar, E.; Leevy, W. M.;
Gammon, S. T.; Piwnica-Worms, D.; Smith, B. D. Angew. Chem., Int.
Ed. 2007, 46, 5528−5531. (b) Jarvis, T. S.; Collins, C. G.; Dempsey, J.
M.; Oliver, A. G.; Smith, B. D. J. Org. Chem. 2017, 82, 5819−5825.
(c) Gassensmith, J. J.; Arunkumar, E.; Barr, L.; Baumes, J. M.;
DiVittorio, K. M.; Johnson, J. R.; Noll, B. C.; Smith, B. D. J. Am.
Chem. Soc. 2007, 129, 15054−15059.
Experimental procedures, synthesis and characterization,
crystallographic data, computational results, and con-
focal images (PDF)
Accession Codes
CCDC 1998840 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(9) Zielonka, J.; Joseph, J.; Sikora, A.; Hardy, M.; Ouari, O.;
Vasquez-Vivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B. Chem. Rev.
2017, 117, 10043−10120.
(10) Barbero, N.; Magistris, C.; Park, J.; Saccone, D.; Quagliotto, P.;
Buscaino, R.; Medana, C.; Barolo, C.; Viscardi, G. Org. Lett. 2015, 17,
3306−3309.
AUTHOR INFORMATION
Corresponding Author
■
Samit Guha − Department of Chemistry, Organic Chemistry
Section, Jadavpur University, Kolkata 700032, India;
́
̀
́
(11) Lopez-Macia, A.; Jimenez, J. C.; Royo, M.; Giralt, E.; Albericio,
F. J. Am. Chem. Soc. 2001, 123, 11398−11401.
D
https://dx.doi.org/10.1021/acs.orglett.0c01922
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
pubs.acs.org/OrgLett
Letter
(12) Leigh, D. A.; Murphy, A.; Smart, J. P.; Slawin, A. M. Z. Angew.
Chem., Int. Ed. Engl. 1997, 36, 728−732.
(13) Collins, C. G.; Baumes, J. M.; Smith, B. D. Chem. Commun.
2011, 47, 12352−12354.
(14) Carrasco-Ruiz, A.; Tiburcio, J. Org. Lett. 2015, 17, 1858−1861.
E
https://dx.doi.org/10.1021/acs.orglett.0c01922
Org. Lett. XXXX, XXX, XXX−XXX