Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization

Article abstract of DOI:10.1039/d1sc00542a

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namelyNIMCsby attaching a morpholine moiety at theperiposition of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesizedNIMCswere explored by¹H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications.NIMC6displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, allNIMCswere employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probeNIMC6was also employed in BHK-21 cells for 3-D spheroid imaging and inCaenorhabditis elegans(C. elegans) forin vivoimaging, to evaluate its efficacy for imaging animal models.

Full text of DOI:10.1039/d1sc00542a

Showcasing research from Professor Apurba Lal Koner’s
and Professor Deepak Chopra’s laboratories, Department
of Chemistry, Indian Institute of Science Education and
Research Bhopal (IISERB), Bhopal, Madhya Pradesh, India.
As featured in:
Strategic engineering of alkyl spacer length for a pH-
tolerant lysosome marker and dual organelle localization
Dual organelle localization has been achieved by a
1
,8-naphthalimide probe tethered with a long alkyl spacer
terminated by a morpholine moiety. The long alkyl chain
brings suitable lipophilicity to the probe effectuating the
localization into the endoplasmic reticulum. Concurrently,
lysosomal localization was observed owing to the attached
morpholine moiety. Besides, this article also introduces an
alkyl spacer length engineering strategy that helped us to
understand the genesis of the pH tolerance. These findings
are foreseen as building blocks to study lysosome-related
dysfunctions and inter-organelle communication.
See Deepak Chopra,
Apurba L. Koner et al.,
Chem. Sci., 2021, 12, 9630.
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Strategic engineering of alkyl spacer length for
a pH-tolerant lysosome marker and dual organelle
localization†
Cite this: Chem. Sci., 2021, 12, 9630
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
a
a
b
Suprakash Biswas,‡ Tanoy Dutta, ‡ Akshay Silswal, Rohit Bhowal,
b
a
Deepak Chopra * and Apurba L. Koner
*
Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their
dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes
precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-
sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome
these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of
lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri
position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-
1
naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by H-
NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical
spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and
highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-
tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast
cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal
micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER)
was also achieved by controlling the linker length and this phenomenon was subsequently applied in
determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21
cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate
its efficacy for imaging animal models.
Received 28th January 2021
Accepted 13th June 2021
DOI: 10.1039/d1sc00542a
rsc.li/chemical-science
into their constituent building blocks to regenerate nutrients,
catalyzed by a group of proteases, lipases, nucleases, and other
Introduction
Lysosomes are the most important membrane-bound cellular hydrolytic enzymes. The constituents are then transported to
3
–8
organelles in eukaryotic cells, frequently regarded as the the cytoplasm via dedicated lysosomal membrane permeases.
cellular degradative compartment of the endocytic pathway.
1,2
To execute such catalytic propensity the hydrolases demand an
The metabolic function of lysosomes proceeds through the acidic pH gradient of 4.5–5.5 and to maintain such a pH range,
disruption of various complex extracellular macromolecules protons are pumped to the lysosomal interior from membrane-
9,10
bound ATPase through ion-channels.
Besides, the acidic
environment (pH 4.5–5.5) plays a crucial role in a variety of
a
Bionanotechnology Lab, Department of Chemistry, Indian Institute of Science
physiological processes, such as cellular homeostasis and
Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal, Madhya
11,12
Pradesh, India. E-mail: akoner@iiserb.ac.in
b
plasma membrane repair.
Additionally, malfunction of
Crystallography and Crystal Chemistry Laboratory, Department of Chemistry, Indian various lysosomal hydrolases, proteases, and transporter
Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri,
enzymes caused by mutation of genes results in a gradual
Bhopal, Madhya Pradesh, India. E-mail: dchopra@iiserb.ac.in
accumulation of undesired cargo inside lysosomes (lysosomal
†
Electronic supplementary information (ESI) available: The synthetic procedure
13
storage). Over-accumulation of undesired substances inside
the lysosomes can commence a cascade of secondary disorders
1
13
and characterization ( H,
C-NMR, HRMS) of NIMCs, pH- and
1
solvent-dependent optical properties, variable temperature H-NMR, cell
viability assay for NIMCs, live-cell imaging for co-localization analysis, which ultimately affects the pathway of calcium homeostasis,
l-scanning experiments for micropolarity determination, live-cell 3D spheroid oxidative stress management, inammatory immune response,
imaging, crystal renement parameters, and
a
table showing inter and
14
and cell death. As per example, the central nervous system
intra-molecular interactions. CCDC 2053600–2053604. For ESI and
expresses variable amounts of glycosphingolipid, and for this
reason, some neuronal lysosomes allow this molecule to be
stored and effectively released at a particular time. However, the
crystallographic data in CIF or other electronic format see DOI:
1
0.1039/d1sc00542a
‡
These authors contributed equally to this work.
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storage of these macromolecules by other neuronal lysosomes numerous synthetic organic lysotrackers have been reported by
13
38–41
leads to neuronal damage and cell death. Further, autopha- various research groups.
The majority of such reported
gosomes engulf intracellular constituents and consequently lysosome targeting probes and commercially available lyso-
fuse with lysosomes promoting the formation of autolysosomes trackers such as Neutral Red (NR), DND-189, and DND-99 suffer
15–20
during autophagy.
Lysosomes and late endosomes from pH-dependent uorescence, interference from non-
frequently knot with other intracellular organelles such as specic binding, background uorescence, insufficient photo-
26,42,43
mitochondria and endoplasmic reticulum, without fusing with stability, and cytotoxicity.
them, and form functional membrane contact sites, which To target the most acidic organelle, that is, the lysosome,
allow transfer of different lipid and protein composites between recently reported lysotrackers are designed to operate via the
21
the organelles. Hence, the transfer of such substances makes protonation of tertiary amine groups inside the lysosomal
3
8,44
the lysosomal compartment dynamic in terms of its micro- compartment.
environment, morphology, and spatial distribution.
For many such reported probes, the dynam-
13,22
Any icity and acidity of the lysosomal compartment impose limita-
disruption of such a dynamic balance may be a cause for tions such as quenching of uorescence intensity governed by
diseases such as cardiovascular diseases, inammation, and the well-known phenomenon of photo-induced electron trans-
23–29
even cancer.
Research on lysosomal activity indicates that fer (PET) or delocalization of the probe from lysosomes to
lysosomal polarity plays a pivotal role in their functionality and elsewhere, resulting in non-specic targeting for long-term
45
any unusual change in the lysosomal micro-polarity triggers monitoring. Recently, a few dextran conjugated lysosome-
signicant disorder in apoptosis enabling uncontrolled cell specic probes have been reported for understanding phago-
27,30–32
death.
Recent experimental ndings also indicate higher cytosis. However, these probes are less suitable for long-term
lysosomal micro-polarity in cancerous cells than in normal imaging, as dextran itself interrupts the natural physiological
33,34
46
cells.
However, the dynamicity of lysosomal pH prohibits the activity of lysosomes. Besides, the development of polarity
use of various techniques for long-term visualization of their sensitive uoroprobes for monitoring the micro-environment of
behavior. Therefore, convenient techniques are required to be subcellular compartments is extremely essential for early-stage
47,48
developed for real-time monitoring of lysosomal activity, as well disease diagnosis.
Although very few lysosomal micro-
as their micro-polarity, to get an explicit vision of lysosome polarity sensitive uorescent probes have already been re-
35,36
dysfunction-related diseases.
ported, their applicability in aspects such as differentiating
The development of synthetic organic luminophores to stain between cancer cells/tissue and normal cells/tissue is less
34,49
biological samples for imaging applications is considered to be explored.
an extraordinary avenue in modern research trends. Organic nonspecic background uorescence outside the region of
uoroprobe-based imaging techniques are oen convenient to interest (ROI) of many probes are major limitations for imaging
Additionally, vulnerable photostability and

visualize specic regions of cells, model organisms, and bio- in xed cells or static snapshots, without the possibility of
logical processes, even with a few nanometer resolution, continuous data acquisition for long-term tracking under
5
0,51
because of their excellent sensitivity and operational a high-power LASER scanning confocal microscope (LSCM).
simplicity.
37,38
Due to the dynamicity of the lysosomal Therefore, the predicament demands a pH-tolerant, polarity
compartment, imaging lysosomal properties to comprehend sensitive, extremely photostable, non-toxic, and synthetically
their activities is extremely fascinating, and to pursue this,
Scheme 1 Schematic representation of the synthesis of NIMCs, and the corresponding general conditions are as follows: (a) K
2
CO
3
, dry MeCN,
ꢀ
reflux, 8 h, 78–83%; (b) morpholine, K
2
CO
3
, dry DMF, 100 C, overnight, 84–91%; (c) N
H $H
2 4 2
O, EtOH, reflux, 2 h, quantitative yield; (d) n-
ꢀ
butylamine, EtOH, reflux, 2 h, 77%; (e) N-morpholinoalkane-1-amine, dry DMSO, 105 C, overnight, 51–57%.
©
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simple lysosome targeting uorescent marker for routinely NIMC6 encouraged us to employ it for 3-D spheroid imaging
monitoring lysosomal polarity.
and in vivo imaging in C. elegans.
Herein, we have addressed the limitation of uorescence
quenching with increasing pH due to PET and obtained
a promising pH-tolerant lysotracker, afforded by a simple yet
Results and discussion
Probe design, synthesis, and characterization
52
convenient synthetic approach.
Consequently, we have
designed a series of uoroprobes namely NIMCs (Scheme 1) by
a simple alkyl chain length variation strategy and synthesized
them by attaching a morpholine moiety at the peri position of
the 1,8-naphthalimide (NI) ring through various alkyl spacers.
The design strategy of NIMCs lies in the suppression of PET to
52
avoid the quenching of uorescence. Here, we followed
a reporter–spacer–targeting (R–S–T) strategy and synthesized
a library of eight uorescent molecules namely NIMCn (n ¼ 2–6,
1
13
The synthesized molecules were characterized by H, C-NMR
spectroscopy, mass spectrometry, and single-crystal X-ray
analysis. Aerward, a detailed spectroscopic investigation was
conducted to comprehend the photophysical properties of all
NIMCs in an aqueous medium. Further, the NIMCs were
screened to evaluate their pH-tolerant uorescence properties
where NIMC6 exhibited superior pH-tolerant uorescence
compared to other NIMCs. Moreover, the solvatochromic
properties of all the NIMCs were probed, which demonstrates
a conducive agreement between solvent polarity and sol-
vatochromism. The NIMCs were subjected to live-cell imaging
in BHK-21 cells where NIMC2–6 were found to be localized
exclusively in the lysosomal compartment. Interestingly, NIMCs
with longer alkyl spacers, NIMC8 and NIMC10, exhibit dual
organelle localization in lysosomes and the endoplasmic retic-
ulum (ER) wherein the localization shis more towards the ER
with increasing alkyl spacer length. The photostability of all
synthesized NIMCs was assessed, and the results display
excellent photostability, well suited for long-term imaging
application. Consequently, NIMC6 was selected and applied for
determining lysosomal micro-polarity in various cell lines
through live-cell imaging. Additionally, the micro-polarity of the
ER was also estimated using NIMC10. Finally, the potential of
8, 10) by choosing 1,8-naphthalimide as a uorescent reporter
and morpholine as the lysosome targeting unit. The afore-
mentioned units are connected via exible alkyl spacers of
varying lengths through a secondary amine at the peri-position
of the naphthalimide core (Scheme 1). It is well-known that
upon increasing the pH of the medium, PET is facilitated from
donor morpholine (D) to electron acceptor 1,8-naphthalimide
53
(
A), leading to uorescence quenching. To overcome this
quenching and to develop a pH-tolerant uoroprobe we
modulated the distance between the D–A pair by introducing
a exible alkyl spacer of increasing length from two to ten
carbons. Such an elongation of the distance between the D–A
pair causes a reduction in the extent of electron transfer. A
distance greater than 0.7 nm, similar to the alkyl spacer length
of six-carbon chains, is known to drastically reduce the electron
54
transfer rate between the D–A pair. Such a suppression in
electron transfer rate results in an unaltered uorescence
intensity throughout the pH scale. Additionally, the lone pair on
the nitrogen atom of the secondary amine participates in
intramolecular charge transfer (ICT) with the 1,8-naph-
thalimide core making NIMCs excellent non-exible ICT
55
probes. Such an ICT character introduces an efficient sol-
vatochromism in NIMCs, making them potent probes for
determining organelle micro-polarity.
Fig. 1 Representation of the solid-state geometry of the synthesized NIMCs, (a) NIMC2, (b) NIMC3, (c) NIMC5, (d) NIMC4 and (e) NIMC10
obtained from the single-crystal X-ray diffraction. Crystal packing reveals that the molecules are stacked via p/p interactions and the shorter
alkyl spacers adopt a bent chain conformation, while a straight chain geometry is adopted by intermediate and longer alkyl spacers.
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Taking into account the above design strategy, we synthe- the orthorhombic Pbcn space group, with constituent molecules
sized NIMC2–6, 8, and 10, according to Scheme 1. The detailed stacked in a herringbone fashion, where the alkyl spacer bends
synthetic procedure of NIMCs is provided in the ESI.† In brief, to a greater extent, allowing the morpholine moiety to come
NIMCs were obtained by substituting the bromine atoms of 4- closer to the aromatic core (Fig. 1b). NIMC4 crystallizes in the
ꢀ
bromo-1,8-naphthalimide with n-morpholinoalkane-1-amines. triclinic P1 space group and structural analysis conrms the
Wherein, n-morpholinoalkane-1-amines were synthesized by molecular assembly through alternate p/p and C–H/p
substituting one bromine of 1,n-dibromoalkanes with phthali- interactions along the a axis. Interestingly, in this case, the alkyl
mide followed by the substitution of other bromine atoms with spacer has been found to bend signicantly, mimicking a scor-
morpholine and nally deprotecting the phthalimide moiety pion tail-like conformation that brings the morpholine moiety
using N H $H O.
in very close proximity to the aromatic core (Fig. 1d). NIMC5
2
4
2
1
13
ꢀ
Aer the synthesis, NIMCs were characterized by H, C- crystallizes in the triclinic P1 space group and the constituent
NMR spectroscopy and mass spectrometry. Single crystals of molecular layers are assembled via p/p interactions between
NIMC2, NIMC3, NIMC4, NIMC5, and NIMC10 were grown by the aromatic cores along the b axis (Fig. 1c). In this case, the
solvent evaporation of suitable solvent mixtures (see the ESI† alkyl spacer has been found to adopt a straight-chain confor-
for details) for several days and were characterized by single- mation, perhaps to favor hydrophobic interactions for attaining
56
crystal X-ray diffraction analysis. The details of the entire crys- greater thermodynamic stability. Furthermore, NIMC10 crys-
ꢀ
tallographic data are tabulated in Table S8.† Crystal structure tallizes in the triclinic P1 space group, with constituent mole-
analysis reveals that NIMC2 crystallizes in the orthorhombic cules being uniformly stacked via p/p interactions along the
Pbca space group, with constituent molecules being stacked a axis. Here also the alkyl spacer embraces a straight-chain
along the crystallographic a axis via p/p interactions between conformation very similar to that of NIMC5, to promote
naphthalene monoimide rings. The short length of the alkyl hydrophobic interactions. Hence, structural analysis elucidates
spacer in NIMC2 allows the morpholine moiety to be in close the stark difference in the structure of NIMCs, where the shorter
proximity to the aromatic core (Fig. 1a). NIMC3 crystallizes in alkyl spacer adopts a bent chain conformation bringing the
1
Fig. 2 (a) Comparative H-NMR spectra of all NIMCs in CDCl
3
at RT, and an unexpected downfield shift of the secondary amine proton in NIMC3
. QTAIM analysis of (c) NIMC2, (d) NIMC3, and
e) NIMC4, respectively. The bond critical points are shown as brown spheres and ring critical points as green spheres.
was observed which prompted us to carry out (b) a variable temperature-dependent NMR in CDCl
3
(
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Fig. 3 Representation of photophysical properties: (a) absorption, (b) emission (lex ¼ 450 nm), and (c) emission lifetime of the synthesized
NIMCs in water (pH ¼ 6.0), where the fluorescence decays were recorded using a 440 nm excitation LASER.
morpholine moiety in close proximity to the NI core. In bond donors in NIMC2 (Fig. 2c), NIMC3 (Fig. 2d) and NIMC4
57–59
contrast, intermediate and long alkyl spacers direct the mor- (Fig. 2e) using QTAIM.
Interestingly, it has been observed
pholine moiety far away from the NI core. Fascinatingly, the that the molecular conformation of NIMC3 shows the existence
crystal structure of NIMC10 reveals that the long alkyl spacer of a stable six-membered ring, formed via an intramolecular
enables the morpholine moiety of the other molecule to be in N–H/N hydrogen bond (involving H2N and N3), corroborated
the adjacency of neighboring naphthalimide (Fig. 1e).
by the presence of a bond path and a bond critical point (BCP)
With the aim of understanding the behavior of NIMCs in (3, ꢁ1) (shown by the brown colored circle in Fig. 2d). The
1
solution, a comparative H-NMR of all the synthesized NIMCs formation of such a six-membered ring through the directional
was performed in CDCl
3
at room temperature (RT), where the N–H/N (H-bond) interaction makes the secondary amine
comparative proton NMR plot displays an unexpected down- proton of NIMC3 highly deshielded and hence exhibits the
60

eld shi (d ¼ 7.62) of the secondary amine proton in the case of maximum downeld shi. Similarly in the case of NIMC2,
NIMC3 (Fig. 2a). The systematic comparison of chemical shis Fig. 2c shows that intramolecular N–H/H–C interaction
d) reveals that the introduction of different alkyl spacers does (involving H2N and H14) locks the molecular conformation
(
not produce any signicant shi of the aromatic protons in resulting in a relatively reduced magnitude of the downeld
NIMCs (Fig. 2a). However, the downeld shi for NIMC3 might shi. The presence of intermolecular N–H/O and the absence
be attributed to the formation of intramolecular hydrogen of intramolecular N–H/N interactions (Fig. 2e and S1†) in
bonding between the secondary amine proton (N2H) and the higher analogs due to the straight chain conformation adopted
nitrogen lone pair of the morpholine moiety. To obtain further by the alkyl spacer contribute to almost similar shis of
1
insight, we also performed a variable temperature H-NMR secondary amine protons as observed in the NMR spectra
experiment for NIMC2, C3, and C4 (Fig. 2b, S2 and S3†), in (Fig. 2a).
CDCl . Eventually, we found a gradual upeld shi of the amine
3
proton of NIMC3 with increasing temperature, whereas the
other congeners display a considerably less shi (Fig. 2b, S2 and Photophysical investigation of NIMCs
S3†), also signifying the formation of intramolecular H-bonding
in the case of NIMC3. To comprehend such H-bonding and
other non-covalent interactions present, we carried out an
analysis of the topological parameters (Table S10†) for a few
To obtain a deeper insight into the photophysical properties of
NIMCs, we recorded their absorption and emission spectra, as
well as uorescence lifetime decays in an aqueous medium at
pH 6.0. The absorption and emission proles of NIMCs reveal
selected motifs highlighting the involvement of N–H hydrogen
that their absorption maximum lies at around 450 nm whereas
Table 1 Photophysical parameters of all NIMCs in an aqueous medium (pH ¼ 6)
abs
max/nm
a
1
ꢁ1
em
max/nm
b
F
ꢂ 3)/Mꢁ1 cmꢁ1
c
Probes
l
3 /M cm
l
F
Brightness (F
F
savg /ns
NIMC2
NIMC3
NIMC4
NIMC5
NIMC6
NIMC8
NIMC10
441
446
449
452
454
453
452
16 000
10 800
12 000
11 600
12 200
11 000
11 400
538
548
554
553
556
559
558
0.22
0.47
0.34
0.27
0.32
0.23
0.08
3520
5076
4080
3132
3904
2530
912
6.9
5.4
4.6
4.3
4.2
4.1
4.2
a
b
61
c
3
is the molar extinction coefficient. F is the uorescence quantum yield, calculated with respect to uorescein in 0.1 N NaOH. s is the average

uorescence lifetime.
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their emissions fall in the range of 490–720 nm (Fig. 3a and b) alkyl spacer length which might arise from the self-assembling
photophysical values are tabulated in Table 1). NIMC2 shows effect in the case of higher congeners. The outcome of all
absorption and emission maxima centered at 441 nm and spectroscopic measurements of NIMCs established that they
38 nm, whereas the absorption and emission maxima are are visible light excitable with a large Stokes shi, along with
(
5
shied to 454 nm, 452 nm, and 556 nm, 559 nm for NIMC6 and high uorescence quantum yield and brightness, evincing their
NIMC10, respectively (Fig. 3a and b). A comparative analysis of suitability towards uorescence-based imaging applications.
the spectral behavior indicates that NIMCs with intermediate
and longer alkyl spacers exhibit almost similar absorption and
emission maxima, where NIMC2 and C3 show a signicant Fluorescence intensity variation with the pH of the medium
hypsochromic shi in absorption and emission maxima
compared to their higher congeners. This hypsochromic shi
To elucidate the applicability of NIMCs towards imaging lyso-
somal activity, we inspected their emission behavior with the
most possibly could be attributed to the hydrogen bonding
variation of pH to screen their pH tolerance. Hence, we recorded
interaction between the lone pair of the secondary amine
nitrogen and the protonated morpholine hydrogen at pH 6
+
the emission spectra of all NIMCs at pH 2.50 (NIMCH ) and 9.25
NIMC), by exciting at 450 nm (Fig. 4a and S9†). The emission
(
(Fig. S4†).
proles indicate a decrease in uorescence intensity with
a small bathochromic shi in the emission maximum with
increasing alkyl spacer length at both the pH values (Fig. 4a and
Further, the comparative absorption spectra also indicate
a signicant tailing effect in absorption at a higher wavelength
in the case of NIMC8 and C10 (Fig. 3a) which perhaps arises due
to lower solubility of the long alkyl chains in water. The uo-
rescence quantum yield of the synthesized NIMCs in the
aqueous medium (pH ¼ 6) was also determined using uores-
S9†). Additionally, we plotted the difference in uorescence
em
intensities DFlu. Int. ¼ Int.(NIMH ꢁ Int.(NIM) at lmax against the
+
)
alkyl spacer length for all NIMCs (Fig. 4b). The plot indicates
a gradual decrease in DFlu. Int. with increasing alkyl spacer
length from C2 to C6. Interestingly, the difference starts
increasing again with increasing alkyl spacer length from C8 to
C10 showing a minimal effect in the case of C6 (Fig. 4b).
Further, we examined the variation of uorescence intensity not
only at highly acidic or basic regions but also for the whole
range of pH. The uorescence titration with pH showed
61
cein as a reference. NIMCs show uorescence quantum yields
ranging from 0.47–0.08 and the average uorescence lifetime
ranges from 6.9–4.2 ns, which is quite desirable for live-cell

uorescence imaging and uorescence lifetime imaging
microscopy (FLIM) (Fig. 3c and Table 1). The uorescence life-
times were also found to decrease gradually with increasing
Fig. 4 The pH-dependency of the emission profile of NIMCs, where (a) presents the emission profiles of all NIMCs at pH 2.5 and (b) presents the
difference in fluorescence intensity (DFlu. Int.) at the emission maxima of individual NIMCs between pH 2.5 and 9.2, plotted against alkyl spacer
0
00
length of the alkyl spacer where the inset (b and b ) presents the change in the emission profiles of NIMC2 and NIMC6 at these two pH values.
The variation of fluorescence intensity at 540 nm and 560 nm is plotted against pH for NIMC2 and NIMC6, respectively (c and d), and the
corresponding inset shows the change in the emission profiles.
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a quenching of uorescence intensity with increasing pH where to lower values of DFlu. Int. than those of NIMC2 (Fig. 4b).
the obtained values yield a sigmoidal tting with a pK around Therefore, NIMC6 emerges as a potent pH-tolerant uorescent
a
6.8 to 5.0 for NIMCs except for NIMC6, which exhibits a negli- probe for lysosomal imaging.
gible variation of uorescence intensity with pH (Fig. 4c, d and
S10–S13†) in the selected pH window. The inverse relationship
of uorescence intensity with pH is attributed to the well-known
phenomenon of photo-induced electron transfer (PET) from the
lone-pair of morpholine nitrogen (D) to the electron-decient
Polarity-dependent photophysics
Specically, the selected pH-invariant uorophore NIMC6 and
other synthesized NIMCs were subjected to the measurement of
absorption, emission, and uorescence lifetimes (using time-
correlated single-photon counting) in solvents of varying
polarity, to validate their applicability for probing lysosomal
micro-polarity. NIMC6 is pH invariant, and hence, to get further
insight we checked its optical purity in water (pH 6.0). A suitable
overlap between the absorption and excitation spectra was
observed, validating its optical purity and its suitability towards
live-cell imaging (Fig. S5e†). Further, the optical purity of all
other NIMCs in water was also checked where the absorption
and excitation spectra of NIMC2 and C3 show slight deviation,
which might be due to hydrogen bonding interaction between
the protonated morpholine and the lone pair of the secondary
amine nitrogen at that pH (Fig. S5 and S4†). NIMCs contain
a secondary amine group at the peri-position of the 1,8-naph-
thalimide ring and the nitrogen lone-pair is capable of conju-
gating with the imide group through the naphthalene p-
conjugated ring. Fluorescent molecules containing both
electron-donating and accepting groups connected via
62
naphthylamide ring (A). The straight-chain conformation
adopted by the alkyl spacers of NIMC5 and NIMC6 places the
D–A units of constituent molecules far away from each other
causing the suppression in intramolecular PET, and as a result,
they enjoy a minimal change in uorescence intensities (Fig. 4d
and 1c). However, the long alkyl spacers in NIMC8 and C10 help
the D–A units of neighboring molecules to come in close prox-
imity to each other, facilitating intermolecular PET and hence
higher DFlu. Int. (Fig. 1e, 4b, and S13†). The short distance
between the D–A pair in NIMC2, along with the absence of any
inter or intramolecular interactions involving morpholine as
the donor (Fig. 1a and 2c), makes PET a very facile process,
52
affording the highest DFlu. Int. (Fig. 4b and c). However, the
presence of intramolecular N–H/N and C–H/N interactions
in NIMC3 (Fig. 2d) and NIMC4 (Fig. 2e), respectively, conrmed
by the presence of bond paths and BCPs (details of interactions
shown in Table S10†) reduces their intramolecular PET, leading
Fig. 5 Solvent-dependent emission properties of NIMC6. (a) Bathochromic shift of emission maxima with increasing solvent polarity. (b)
2
Emission maximum varies linearly (R ¼ 0.93) with the solvent polarity scale E
T
(30), and the inset shows its ICT character further confirmed by the
visible color change. (c) Solvent-dependent fluorescence lifetimes of NIMC6 in various solvents. (d) Photostability of NIMC6, where the fluo-
rescence intensity remains unaltered under continuous illumination with 450 nm light and the inset shows the corresponding fluorescence
kinetics.
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a conjugated p-linker are known as intramolecular charge (Fig. 5c). The excited-state lifetime of NIMCs increases with
transfer (ICT) dyes, and possess a high dipole moment in the increasing solvent polarity, signifying their applicability in
63,64
ground and in the excited state.
The photophysical investi- uorescence lifetime imaging. In a nutshell, polarity-dependent
gations indicate that all NIMCs exhibit typical characteristics of measurement conrms their applicability for determining
ICT dyes because they show a small red shi in absorption lysosomal micro-polarity.
maxima and a huge bathochromic shi in emission maxima
with increasing solvent polarity (Fig. 5a, S6 and S7, for values
see Tables S1–S7†). Eventually, the absorption and emission
maxima of NIMC6 in toluene are 426 nm and 502 nm, respec-
tively, but in a highly polar aqueous medium, they shi to
Photostability of NIMC6
The photobleaching effect is a well-known phenomenon in uo-
rescence quenching which imposes limitations on their utility in
uorescence-based live-cell imaging. Several uoroprobes get
453 nm and 556 nm (Fig. 5a and Table S5†). This larger Stokes
photo-bleached due to their low photostability when excited
using high LASER power in confocal microscopy. Hence, these
probes are less suitable for prolonged imaging purposes. To test
their applicability for long-term imaging, the photostability of
NIMC6 was measured by exciting with 450 nm light (450 W xenon
lamp, 100 lx) and the uorescence kinetics was monitored for an
hour (Fig. 5d). No change in uorescence intensity was observed
in the experimental time frame indicating its high photostability.
Additionally, the photostability of NIMC6 was also compared with
that of commercially available LysoTracker Green by exciting both
with 480 nm light. The result displays similar photo-stability of
NIMC6 to LysoTracker Green further evidencing its potential
towards long-term lysosomal imaging (Fig. S14†). The photo-
stability of NIMC6 and LysoTracker Red was compared under live-
cell imaging conditions using confocal laser excitation
shi of ICT probes in various dielectric media can be rational-
ized by the Lippert–Mataga equation as follows:
ꢀ
ꢁ
2
2
Df À
Á
2
3 ꢁ 1
n ꢁ 1
!
ƒ!
Dn ¼
m ꢁ m þb; Df ¼
ꢁ
e
g
3
2
hca
23 þ 1 2n þ 1
where Dn ¼ nabs ꢁ nem stands for the Stokes shi, nabs and nem
are absorption and emission frequencies, respectively,
ꢁ
1
in cm , h is the Planck's constant, c is the velocity of light in
a vacuum, a is the Onsager radius and b is a constant. Df is the
orientation polarizability, 3 is the dielectric constant of the
medium, n is the refractive index, and m and m are the
dipole moments of the emissive and ground states, respectively.
ƒ
!
ƒ!
e
g
ƒ
!
ƒ! 2
ð m ꢁ m Þ is proportional to the slope of the Lippert–Mataga
e
g
6
5
plot. From this equation, it can be inferred that the emission
maximum varies linearly with the solvent polarity scale E (30),
T
(Fig. S15†), vide infra, which establishes its prospects as a long-
as observed for NIMC6 and other analogs (Fig. 5b and S7†).
Such linear polarity-dependent emission properties with
a large Stokes shi corroborate a non-twistable ICT character
for NIMCs. To acquire further knowledge of their photophysical
properties, we recorded the uorescence lifetime decay in
term live-cell imaging agent.
Live-cell imaging
Sub-cellular localization. Following the detailed spectro-
different solvents which exhibits single-exponential decay scopic investigations, live-cell imaging experiments were
Fig. 6 CLSM images of BHK-21 cells stained with (a) 0.2 mM NIMC2 and (b) 0.3 mM LysoTracker Deep Red; (c) merged image of (a) and (b); (d)
scatter plot showing Pearson's correlation coefficient of 0.89 ꢃ 0.04; (e) 0.2 mM NIMC6; (f) 0.3 mM LysoTracker Deep Red; (g) merged image of (e)
and (f); (h) scatter plot showing Pearson's correlation coefficient of 0.88 ꢃ 0.02 (scale bar: 20 mm) [lex/lem ¼ 488 nm/500–540 nm, lex/lem
¼
6
40 nm/650–680 nm, dwell time: 8 ms per pixel, 1 airy disk, laser power: 0.2%].
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Fig. 7 CLSM images of BHK-21 cells stained with (a) 0.2 mM NIMC8, (b) 0.3 mM ER Tracker Red, and (c) 0.3 mM LysoTracker Deep Red; (d) merged
image (Pearson's correlation coefficient of 0.78 ꢃ 0.03) of (a) and (b); (e) merged image (Pearson's correlation coefficient of 0.88 ꢃ 0.02) of (a)
and (c); staining with (f) 0.2 mM NIMC10, (g) 0.3 mM ER Tracker Red, and (h) 0.3 mM LysoTracker Deep Red; (i) merged image (Pearson's correlation
coefficient of 0.91 ꢃ 0.02) of (f) and (g); (j) merged image (Pearson's correlation coefficient of 0.87 ꢃ 0.04) of (f) and (h) (scale bar: 20 mm) [lex/lem
¼
488 nm/500–540 nm, lex/lem ¼ 561 nm/575–620, lex/lem ¼ 640 nm/650–680 nm, dwell time: 8 ms per pixel, 1 airy disk, laser power: 0.2%].
performed to visualize the intracellular localization of the efficient localization of all the NIMCs in the lysosomal
NIMCs. Prior to performing extensive live-cell microscopy, we compartment.

rst monitored the cytotoxic effects of all NIMCs by the stan-
However, to conrm the accumulation of NIMCs exclusively
dard
MTT
[3-(4,5-dimethylthiazole-2-yl)-2,5- in the lysosomes, they were co-stained with two other
diphenyltetrazolium bromide] assay in normal BHK-21 (baby commercially available organelle-specic dyes, namely
hamster kidney) cell lines. Following a 24 h incubation of BHK-
21 cells, cytotoxicity assays were performed and the results
indicated that the half-maximal inhibitory concentrations, i.e.,
IC50 values, range from 5 mM to 10 mM for NIMCs (Fig. S16†).
Hence, considering this inhibitory concentration range, we
used 0.2 mM probe, as ꢄ90% of cells were viable in each case, to
execute live-cell uorescence imaging. Furthermore, cellular
uptake phenomena of NIMCs were studied to understand their
internalization process through the plasma membrane. At two
ꢀ
ꢀ
different incubation temperatures, 4 C and 37 C, comparable
uorescence signals were observed from the lysosomal

compartments suggesting a pH-driven simple diffusion mech-
anism for the internalization process. To optimize the incuba-
tion time of the NIMCs, the cells were incubated for different
periods. Much to our delight, it was observed that a 10 min
incubation period is sufficient for confocal imaging, indicating
fast and efficient internalization of NIMCs. Besides, to conrm
the colocalization of the synthesized NIMCs inside the lyso-
somal compartments, BHK-21 cells were stained with all the
NIMCs at 0.2 mM concentration along with 0.3 mM commercially
available LysoTracker Deep Red for 10 min. Upon excitation
with 488 nm and 640 nm laser, uorescence signals coming
from the cells were collected, from which the shapes of lyso-
somes were deciphered with low background conrming their Fig. 8 CLSM images of BHK-21 cells stained with (a) 0.2 mM NIMC6
and stimulated with 20 mM chloroquine for (b) 10 min and (c) 30 min;
suitability for lysosomal imaging. Further, sequential imaging
was performed to compute the colocalization inside the lyso-
somal compartment, which revealed high Pearson's coefficients
(d) the average fluorescence intensity measured in different ROIs from
the same field of view (FOV) at the aforementioned time-points of the
experiment (see the ESI† for complete information) (scale bar: 10 mm)
around 0.9 (Fig. 6, and see the ESI† for others), indicating an [lex ¼ 488 nm, l ¼ 500–540 nm, dwell time: 8 ms per pixel, 1 airy
em
disk, laser power: 0.2%].
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MitoTracker Red FM and ER-Tracker Red. The colocalization hold a prominent place due to their efficient localization and
analysis divulged that signals from the NIM (C –C ) lightened unaltered emission signal even if lysosomal pH varies. Chloro-
2
6
up only the lysosomal compartments with bright green uo- quine causes lysosomal stress by inducing lysosomal proton
26,69
rescence (Fig. 6, S17 and S18†), whereas the colocalization leakage resulting in a gradual increase of lysosomal pH.
studies for NIMC8 and NIMC10 provided an intriguing result. Here, the effect of lysosomal pH variation on the emission
In this case, uorescent signals were observed from both lyso- intensity of NIMC6, in the presence of lysosomal stressor
somes and the endoplasmic reticulum (ER), which was further chloroquine, is delved into. For this purpose, live BHK-21 cells
veried through colocalization studies by co-incubation with were co-incubated with 20 mM chloroquine and 0.2 mM NIMC6
NIMC8 or NIMC10, ER-Tracker Red, and LysoTracker Deep Red, for 10, 20, and 30 min. The average uorescence intensity was
and through sequential imaging (Fig. 7). Possibly, the longer measured over different ROIs in both same eld of view (FOV)
alkyl chain of NIMC8 and NIMC10 accounted for greater and different FOVs of the stimulated and untreated cells at
hydrophobicity, driving them to localize in the ER. As the different time points (Fig. 8a, d, S19a and b†). The uorescence
endoplasmic reticulum (ER) is a major site for lipid synthesis intensity of NIMC6 remains invariant for both stimulated and
and metabolism, it poses a hydrophobic environment, which untreated cells throughout the entire period of incubation.
induces a hydrophobicity driven accumulation inside the ER of Such a fascinating result encouraged us to visualize the lyso-
these two derivatives. Therefore, dual organelle localization was somal activity with the aid of pH-tolerant probe NIMC6.
achieved by NIMC8 and NIMC10. Notably, the result implies
that a long alkyl spacer plays a decisive role even if a pH-
directive moiety is present for localization in the lysosomes.
Besides, our observation creates another opportunity to get
more insights into ER–lysosome contact through this dual-
mode targeting. Therefore, pH-dependent spectroscopic
scopic results, we speculated that NIMC6 would be superior to
measurements and live-cell imaging indicate that NIMC6 is
a superior choice among other congeners for imaging lysosomal
activity.
Fluorescence recovery aer photobleaching (FRAP)
The synthesized NIMCs are quite excellent in terms of low
cytotoxicity, cell permeability, and brightness. Nonetheless,
based on the comprehensive spectroscopic and initial micro-
other analogs. We veried this apprehension by understanding
the binding properties of NIMCs in the lysosomal compart-
ments. Fluorescence recovery aer photobleaching (FRAP) is
a widely used approach that provides information regarding the
Effect of stimulated pH change in live cells
dynamics of binding interactions, spatial distribution, and
70,71
Lysosomes are acidic organelles crucial for the digestion of diffusibility of a constituent of interest.
The FRAP experi-
proteins, lipids, and enzymes and thereby maintain cellular ment is a useful technique to judge the suitability of a probe to
66
homeostasis. Lysosomal pH is subject to change during image in living cells for a longer period. Hence, we performed
autophagy due to the accumulation of undigested and insoluble the FRAP experiment to explicate the diffusional properties of
67,68
metabolites.
pH-independent lysosome targeting probes the synthesized NIMCs to acquire further acumen into the local
Fig. 9 Fluorescence recovery after photobleaching (FRAP) measurement was performed in the BHK-21 cell line stained with 0.2 mM NIMC6
probe. The results show the bleaching and post-bleaching (recovery) phenomena. The circular region (1S) represents the bleached region: (a)
pre-bleach cycle; (b) bleaching; (c), (d), (e) and (f) show post-bleaching recovery of fluorescence, respectively (scale bar: 10 mm); (g) FRAP
recovery kinetics of NIMC2, C4, C6, C8 and C10 derivatives. The fluorescence emission was collected from 500 to 550 nm [lex/lem ¼ 488 nm/
5
00–540 nm, photobleach dwell time: 100 ms per pixel, 1 airy disk, bleaching duration: 5 s, bleaching laser power: 20%].
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organization exhibited by them. To perform the FRAP experi- where r
n
is the radius of a uniformly bleached spot and s1/2 is
ment the scanning parameters, i.e., scanning speed, laser the half-time for recovery. In this case, the effective bleach
power, and bleaching duration, were optimized to avoid radius (r ) and bleaching spot radius were considered equal
e
consequential uorescence photobleaching due to repetitive because of instantaneous bleaching, small bleaching radius,
imaging. Aer optimization, the FRAP experiment was per- and small pixel size so that the Gaussian prole can be
formed and the results showed an increase in half-time for neglected. The FRAP analysis indicates NIMC2 to be the
recovery with increasing alkyl spacer length. Interestingly, preferred one for bioimaging applications compared to all other
NIMC2 and NIMC10 showed the highest and lowest diffusion NIMCs due to its faster dynamics and intensity recovery.
rate, respectively, whereas NIMC6 maintained an intermediate However, NIMC2 shows the highest pH-sensitive uorescence
diffusional rate (Fig. 9). The smaller size of NIMC2 enables it to compared to all other analogs making it the least favorite due to
diffuse and bind rapidly, evident from the fastest half-time of dynamic lysosomal pH. Additionally, NIMC10 shows a fair
recovery where the diffusion coefficients were calculated by enough pH sensitivity and dual organelle targeting making it
72,73

tting the raw FRAP data using the Soumpasis equation:
less preferable for imaging lysosomal dynamics. Therefore,
these two competing parameters establish NIMC6 as the supe-
rior one for imaging lysosomal properties which is also sup-
ported by its intermediate diffusion rate.
2
r
n
Diffusion coefficient; Drn ¼ 0:224 s
1=2
Fig. 10 (a–h) Series of individual images at different wavelengths recorded using the confocal lambda scanning technique; (i) emission spectra of
NIMC6 inside lysosomes (lmax ¼ 524 nm) showing a 32 nm blue shift compared to that of water (lmax ¼ 556 nm). However, the same technique
reveals a 37 nm blue shift (lmax ¼ 519 nm) of NIMC6 inside the lysosomal compartment of HEK-293 cells compared to that of water (i) [lex
¼
4
88 nm, lem ¼ 500–700 nm, dwell time: 8 ms per pixel, 1 airy disk, laser power: 1%].
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Micro-polarity in live cells
Additionally, we measured the lysosomal micro-polarity in HEK-
2
93 cells by employing the same probe where we found that the
micro-polarity was similar to that of dichloromethane (DCM)
Fig. S20 and Table S5†), indicating the potential of NIMC6 for
Inspired by the pH-tolerance, lysosomal colocalization, and
suitable diffusion rate of NIMC6, it is considered as a potential
candidate to explore the lysosomal micro-environment in living
cells. Lysosomal polarity is an important parameter to monitor
as any alteration of such a parameter causes internal environ-
(
differentiating the lysosomal micro-environment polarity in
various living cells. Further, the accumulation of NIMC10 inside
the endoplasmic reticulum (ER) demonstrates its applicability
for monitoring the micro-environment polarity inside the ER.
Hence, we performed a spectral scanning experiment to deter-
mine ER-micro-polarity by employing NIMC10 in BHK-21 and
33,49,74–76
ment disorganization under pathological conditions.
Hence, monitoring micro-polarity inside the lysosomal
compartment by employing the synthesized NIMC6 would
surely be an extra advantage. To perform such measurements,
BHK-21 cells were incubated with 0.2 mM NIMC6 followed by the
spectral scanning under a confocal laser scanning microscope.
The lysosome rich area gave rise to an emission spectrum
HEK-293 cells. NIMC10 exhibits an emission maximum at
around l
max
¼ 536 nm (Fig. S21 and S22†) in both the cell
em(ER)
types compared with the solvent polarity dependent emission
prole of NIMC10 (Table S7†), signifying that the micro-
environment polarity inside the ER is similar to that of
dimethyl sulfoxide (DMSO) for both the cell types. Therefore,
the experimental results evidence captivating applicability of
NIMC6 and NIMC10 towards the determination of lysosomal
and endoplasmic reticulum micro-environment polarity in
various live cells.
having completely different emission maxima of NIMC6
max
(l
em(lyso) ¼ 524 nm) compared to its emission maxima in bulk
water (Fig. 10). Upon comparing the emission maxima, with
solvent polarity-dependent emission spectra (Table S5†),
surprisingly we found that the micro-environmental polarity in
lysosomes was similar to that of acetonitrile in BHK-21 cells.
Fig. 11 CLSM images (a–f) of 3D spheroids in different Z-planes obtained from BHK-21 and incubated with 0.2 mM NIMC6 (scale bar 10 mm);
images of wild-type N2 worms: (g) bright field, (h) incubated with 5 mM NIMC6, (i) merged image showing the in vivo distribution of NIMCs (scale
bar: 100 mm) [lex ¼ 488 nm, lem ¼ 500–540 nm, dwell time: 8 ms per pixel, 1 airy disk, laser power: 0.2%].
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Imaging of 3D-spheroids and lysosome-related organelles in
C. elegans
conditions. Further, NIMC6 is also applicable for specic lyso-
somal imaging under normal and stress conditions, which is
illustrated by colocalization with LysoTracker, Mito-tracker, ER-
tracker, and intensity monitoring in the presence of the lyso-
somal stressor chloroquine. Surprisingly, the higher congeners,
i.e., NIMC8 and C10, exhibit dual organelle localization in
lysosomes and the endoplasmic reticulum due to augmented
lipophilicity. Moreover, specically selected pH-tolerant NIMC6
Three-dimensional (3D)-spheroidal cell models have become
a mainstay in life science research due to their ability to mimic
77
in vivo environments. Cells in monolayer culture lose many of
their in vivo characteristics. As a result, this platform provides
limited predictive value in determining the clinical outcome of
the compounds of interest. The use of a technique known as 3D-
spheroid culture may be the answer to this conundrum. 3D-cell
culture models are better able to reproduce not only the in vivo
morphology, but also the cell connectivity, polarity, gene
expression, and tissue architecture, making them more physi-
ologically relevant and predictive than 2D-models, thereby
ꢁ
3
diffuses nicely, with a diffusion coefficient value of 2.7 ꢂ 10
2
ꢁ1
mm
s , into lysosomes and allows real-time monitoring of
lysosome-polarity which is similar to the polarity of acetonitrile.
Eventually, the employment of NIMC6 for 3D-spheroid imaging
in BHK-21 cells and in vivo imaging in C. elegans ascertains its
potential as a marker for staining lysosome-related organelles
78
bridging the gap between in vitro and in vivo models. As the
live-cell microscopy experiments came up with some intriguing
results we further wanted to verify the applicability of NIMC6 in
(LRO). Therefore, we strongly believe that pH-tolerant NIMC6
will emerge as a competent candidate for monitoring lysosome-
related dysfunctions and the higher congeners (NIMC8 & C10)
play a commendable role in elucidating organelle communi-
cation through uorescence imaging in live cells as well as in
animal models.
3D-spheroid imaging. Hence, we cultured spheroids of BHK-21
cells and treated them with NIMC6 (Fig. 11a–f and S23†).
Signicant cellular uptake was observed with only 10 min
incubation suggesting that the probes possess the potential for
imaging in tissue samples.
Caenorhabditis elegans is one of the simplest animal models Data availability
79
used for lysosome-related studies. They have gut granules
which are acid hydrolase containing membrane-bound cellular
compartments oen called lysosome-related organelles
Electronic supplementary information including the synthetic
1
13
procedure and characterization ( H,
C-NMR, HRMS) of
80,81
NIMCs, pH and solvent-dependent optical properties, variable
(LRO).
These gut granules mostly ll the intestinal cells of C.
1
temperature H-NMR, cell viability of NIMCs, live-cell imaging
elegans. Hence, to further explore the probe's versatility for
bioimaging applications, C. elegans were imaged by NIMC6
staining. The hermaphrodite worms were synchronized and
for co-localization analysis, lamda-scanning experiments for
micropolarity determination, live-cell 3D spheroid imaging,
crystal renements parameters, inter and intra-molecular
interaction table.
grown to the young adult stage in nematode growth media
ꢀ
(
NGM) treated with 5 mM NIMC6 for 60 h at 20 C. They were
transferred to an agar pad on a glass slide and paralyzed with
5
mM levamisole and imaged under a confocal microscope. The Author contributions
intestine and the gut granules are visible as evident from the
green channel image (Fig. 11g–i). Therefore, NIMC6 can be used
as a potential marker for staining the LROs in C. elegans.
SB designed the synthetic procedures and performed the
synthesis, characterization, and spectroscopic experiments. TD
performed all the live-cell imaging experiments. AS assisted
with the synthesis and spectroscopic measurements. RB
collected and analyzed single-crystal X-ray data. The manuscript
was written from the inputs from all authors. All authors have
Conclusions
In summary, we have demonstrated an alkyl spacer length participated in analyzing the data presented in the manuscript.
variation strategy by simple chemical modication to improve All the experiments were performed and the project was
pH-dependent uorescence quenching and synthesized a series completed under the guidance of DC and ALK.
of uorescent derivatives namely NIMCs. Structural analysis
reveals that while a shorter alkyl spacer adopts a bent-chain
conformation, the intermediate and longer ones adopt
Conflicts of interest
a straight-chain conformation. NIMCs exhibit similar behavior There are no conicts to declare.
1
in the H-NMR analysis except NIMC3 which shows an unex-
pected downeld shi of the secondary amine proton attributed
to the formation of intramolecular H-bonding between the
Acknowledgements
amine proton and the nitrogen lone pair of morpholine. The We wish to acknowledge the nancial support from the host
staining character of most reported PET-lysotrackers is usually institute IISER Bhopal. SB acknowledges fellowship support
driven out of lysosomes and quenched signicantly upon the from UGC, India. TD and RB acknowledge doctoral fellowship
increase in local pH, whereas NIMC6 retains its uorescence support from IISERB. AS thanks the CSIR for his doctoral
intensity throughout the pH range. The rationally designed fellowship. We also thank NMR, the mass facility in the central
photostable and solvatouorochromic NIMCs showed potential instrumentation facility (CIF), and the departmental instru-
for determining lysosomal micro-polarity under pathological mentation facility at IISERB. We acknowledge the Department
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of Science and Technology (DST), India, for the FIST support to 25 B. Cabukusta and J. Neees, Traffic, 2018, 19, 761–769.
our Biological Sciences Department at the IISERB for live-cell 26 X. Zhang, C. Wang, Z. Han and Y. Xiao, ACS Appl. Mater.
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