Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity for Multiplexed Recording of Orchestrated Insulin Secretion

Article abstract of DOI:10.1002/anie.202109510

Zinc biology, featuring intertwining signaling networks and critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red- and far-red-emitting Zn²⁺ probes with K_d

Full text of DOI:10.1002/anie.202109510

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International Edition: doi.org/10.1002/anie.202109510
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Fluorescent Probes
Hot Paper
Red- and Far-Red-Emitting Zinc Probes with Minimal Phototoxicity
for Multiplexed Recording of Orchestrated Insulin Secretion
Junwei Zhang+, Xiaohong Peng+, Yunxiang Wu+, Huixia Ren+, Jingfu Sun, Shiyan Tong,
Tianyan Liu, Yiwen Zhao, Shusen Wang, Chao Tang, Liangyi Chen, and Zhixing Chen*
Abstract: Zinc biology, featuring intertwining signaling net-
works and critical importance to human health, witnesses
exciting opportunities in the big data era of physiology. Here,
we report a class of red- and far-red-emitting Zn2+ probes with
Kd values ranging from 190 nM to 74 mM, which are partic-
ularly suitable for real-time monitoring the high concentration
of Zn2+ co-released with insulin during vesicular secretory
events. Compared to the prototypical rhodamine-based Zn2+
probes, the new class exploits morpholino auxochromes which
eliminates phototoxicity during long-term live recording of
isolated islets. A Si-rhodamine-based Zn2+ probe with high
turn-on ratio (> 100), whose synthesis was enabled by a new
route featuring late-stage N-alkylation, allowed simultaneous
recording of Ca2+ influx, mitochondrial signal, and insulin
secretion in isolated mouse islets. The time-lapse multicolor
fluorescence movies and their analysis, enabled by red-shifted
Zn2+ and other orthogonal physiological probes, highlight the
potential impact of biocompatible fluorophores on the fields of
islet endocrinology and system biology.
the map of dynamic Zn2+ homeostasis. Since the 2000 s,
fluorescence imaging has emerged as an indispensable tool to
study Zn2+ biology, thanks to the coevolution of microscopy
and indicators. To date, a series of fluorescent probes,
including small-molecule probes, genetically encoded indica-
tors and hybrid probes,[8] with different affinities and emission
wavelengths have been developed to detect Zn2+ in living
system, extending the cutting-edge in Zn2+ biology[9] (Fig-
ure 1a). The ever-developing toolbox of Zn2+ probes, how-
ever, can be further upgraded in three ways: (1) In contrast to
numerous bis(pyridylmethyl)amine (DPA) -derived Zn2+
probes with nM affinity, probes for the mM to mM range are
relatively rare. Zn2+ concentrations span around 8 orders of
magnitude, down to 10À10 M in cytoplasm[10] and up to 10À2
M
in some vesicles.[11] Unusually high Zn2+ in insulin secretory
granules[12] (~ 20 mM) and synaptic vesicles[5] (~ 10–30 mM)
suggests its importance in physiology of excitable cells. (2)
The existing toolbox of red- and far-red-emitting Zn2+
probes[9d,f,g,13] need to be further expanded to be used in
combination with the plentiful indicators in green and red
channels (e.g., GCaMP and RCaMP for calcium (Ca2+)
signaling) for studying complex signaling networks. (3). Long-
term, high spatial/temporal resolution fluorescence imaging
probes, bearing superior photostability and minimal photo-
toxicity, are not yet readily available but vitally important.
The core optical motif of the Zn2+ probes often exploit the
classical chromophores for confocal-imaging, but the chro-
mophores are more phototoxic or less biocompatible.[14]
These classic probes cannot meet the challenge of “4D
physiology” in the big data era, where long stretches of high-
resolution video in multiple channels are recorded to
generate the full picture of a molecular signaling network.
Introduction
Zinc (Zn2+) plays a critical role in both physiological and
pathological processes such as signal transduction,[1] apopto-
sis,[2] and gene transcription and expression.[3] Most of Zn2+ in
cells is tightly bound to proteins to serve as structural
components or catalytic centers of enzymes, while another
fraction is free or weakly chelated with small molecules for
signaling purposes in multiple organs.[4] Zn2+ is heterogeneous
in space, yet its dynamics can be fast or even transient, as in
neurophysiological,[5] reproductive,[6] and pancreatic secreto-
ry processes.[7] Investigators of Zn2+ physiology are therefore
challenged to develop bioanalytical methods to fully unveil
[*] J. Zhang,[+] X. Peng,[+] Y. Wu,[+] Y. Zhao, L. Chen, Z. Chen
College of Future Technology, Institute of Molecular Medicine,
National Biomedical Imaging Center, Beijing Key Laboratory of
Cardiometabolic Molecular Medicine, Peking University
Beijing 100871 (China)
J. Sun, Z. Chen
PKU-Nanjing Institute of Translational Medicine
Nanjing 211800 (China)
S. Tong
School of Life Science, Peking University
Beijing 100871 (China)
E-mail: zhixingchen@pku.edu.cn
H. Ren,[+] T. Liu, C. Tang, Z. Chen
Peking-Tsinghua Center for Life Science, Peking University
Beijing 100871 (China)
X. Peng,[+] L. Chen
State Key Laboratory of Membrane Biology, Peking University
Beijing 100871 (China)
H. Ren,[+] C. Tang
Center for Quantitative Biology, Peking University
Beijing 100871 (China)
S. Wang
Organ Transplant Center, Tianjin First Central Hospital, Nankai
University, Tianjin 300192 (China)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under https://doi.org/10.
1002/anie.202109510.
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release is an elaborately regulated tri-
phasic process which lasts for more than
an hour. While modern microscopes
(i.e., spinning disk confocal) are well
equipped for time-lapse imaging, the
recording of such a long process is
eventually limited by the photo-biocom-
patibility of the probes. In our opinion,
these challenges represent general con-
cerns in the ion probe field. Bridging
these technological gaps could provide
a holistic view of orchestrated signaling
in the islet, and at the same time shed
light on a broad community of bioinor-
ganic chemistry.
Herein we present a class of red- and
far-red-emitting rhodamine-based Zn2+
probes with Kd values spanning the mM
range. The new class, named PK Zinc
Red and Far Red (PKZnR and
PKZnFR), is particularly suitable for
imaging the high concentrations of Zn2+
released during vesicular secretory
events in real-time (Figure 1b). Com-
pared to the prototypical rhodamine-
based Zn2+ probes, this new class ex-
ploits hydrophilic morpholino auxo-
chromes, thus eliminating phototoxicity
during live recording from isolated is-
lets. The library of red-emitting Zn2+
probes with different chelating motifs
Figure 1. The history of Zn2+ fluorescent probes and the design of the PK Zinc Family: a) A
map summarizing the state-of-the-art Zn2+ probes with different emission wavelengths and
binding affinities for physiological studies in various tissues. b) Design strategy and chemical
structures of the PK Zinc Family, featuring red-shifted emission and enhanced biocompatibility
for studying dynamic Zn2+ in the mM range.
b-cell endocrinology is a field that would particularly
benefit from a new generation of Zn2+ probes. In the vesicles
of b-cells, Zn2+ co-crystalizes with insulin in the form of 6-
insulin-2-Zn-hexameric complexes, giving rise to a distinctive-
ly high concentration of Zn2+ (~ 20 mM) in secretory gran-
ules.[12] After glucose stimulation, Zn2+/insulin complex is
released from vesicles in a rapid and heterogeneous man-
ner.[15] The malfunction of such process will lead to insulo-
pathic disease.[12] Thanks to modern microscopy that offers
high spatial/temporal resolutions, insulin secretion has been
routinely imaged in cell clusters or intact islets using Zn2+
probes. Prior efforts on probe development include cell-
permeable Zn2+ probes (DA-ZP1,[16] ZIGIR[9e]) for total
granules, plasma membrane-inserted probes (ZIMIR[17]) for
has different binding affinities (Kd values from 190 nM to
74 mM), thus enabling the recording of Zn2+/insulin co-release
in both cell clusters and intact islets. Furthermore, we
developed far-red-emitting Zn2+ probes with a novel synthetic
strategy involving a transient protective group. We demon-
strate four-color simultaneous recording of Ca2+, mitochon-
drial, nuclear, and Zn2+/insulin secretion signals in isolated
islets. These PK Zinc probes revealed the orchestrated and
heterogeneous regulation of secretion, underscoring the
growing importance of photo-biocompatibility, signal multi-
plexing, and molecular tunability in the big data era of 4D
physiology at the tissue level.
secreting cells, and membrane-impermeable extracellular Results and Discussion
probes (FluoZin-3[18] and RhodZin-3[9f,15]) that highlight
opening vesicles as fluorescent puncta which precisely
correlates to individual fusion events of insulin granules
(Figure 2b). The cutting edge of islet biology has moved from
the cell to the tissue level, in order to fully understand the
regulation of b-cell secretion in the context of a whole islet.
However, the general challenges for probes remain: (1) In the
complex environment of the islet, probes must have high turn-
on ratios to increase sensitivity and appropriate Kd values to
ensure the accuracy of analysis. (2) Red- and far-red Zn2+
probes are ideal, as Ca2+ signals in islets, commonly moni-
tored by genetically-encodable GCaMP, must be recorded
simultaneously for studying signal networks. (3) Insulin
Morpholino auxochromes on a rhodamine scaffold minimize
non-specific staining and phototoxicity during the recording of
Zn2+/insulin co-release in isolated islets
To develop ideal probes meeting the new criteria, we first
evaluated a classic red probe for imaging a whole isolated
islet. RhodZin-1, a prototypical red Zn2+ probe with a binding
affinity of 23 mM, is considered suitable for ex vivo recording
of fusion events.[9d] RhodZin-1 belongs to the impermeable
class of probes, therefore is expected to induce minimal
phototoxicity compared to its cellular-localized counterparts
(DA-ZP1[16] and ZIGIR[9e]). We timed our video recordings
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Figure 2. Morpholino auxochromes on a rhodamine scaffold minimize non-specific staining and phototoxicity during the ex vivo recording of
Zn2+/insulin co-release in mouse islet. a) Protocol to visualize Zn2+/insulin co-release in intact islets. b) Mode of action of PK Zinc for reporting
local Zn2+ elevation in the extracellular space during exocytotic insulin granule fusion. c) Chemical structures of RhodZin-1 and PKZnR-1,
schematic diagram of their interaction with cells. d) Phototoxicity of RhodZin-1 and PKZnR-1 (10 mM) on HeLa cells, measured by cell viability
after green light illumination (568 nm, 4.1 WcmÀ2). e) The maximum-intensity t-projection (540 s) of an islet stained with RhodZin-1 (10 mM).
f) Time-dependent images of ROI 1 depicting an apoptosis event. g) Time-dependent images of ROI 2 highlighting the accumulation of non-
specific staining. h) The maximum-intensity t-projection (540 s) of an islet stained with PKZnR-1 (10 mM). i) Time-dependent images of ROI 3
showing clean cytoplasm with minimal background staining. j) Time-dependent images of ROI 4 showing a typical full-fusion event. For (e) and
(h), scale bar=5 mm; for (f), (g) and (i), scale bar=2 mm; for (j), scale bar=0.2 mm. Imaging conditions for (e) to (j): 561 nm laser illumination,
0.3 WcmÀ2, exposure time 100 ms. k) Time courses of fluorescence of ROI-1’ through ROI-4’ (325 nm ꢁ 325 nm).
from the addition of 18.2 mM glucose and expected to record
Zn2+/insulin co-release spanning long-term imaging. How-
ever, during a half-hour of ex vivo recording, we still observed
a gradual accumulation of RhodZin-1 into the cells (Figure 2e
and Movie 1), giving rise to non-specific staining in cellular
compartments (Figure 2g and k). Consequently, the photo-
toxicity of RhodZin-1 predominated and triggered apoptotic
staining patterns in the nucleus as time-lapse imaging
continued (Figure 2 f and k). The background staining and
apoptotic event were routinely observed in several independ-
ent repeated experiments (Figure S11). We speculated that
this appearance was due to the considerable liposolubility of
RhodZin-1. The hydrophobic upper half of RhodZin-1 and
the ionic chelator rendered the molecule a detergent-like
behavior to interact with membranes. Inspired by a classical
strategy in medicinal chemistry, we proposed to tune the
polarity balance of the rhodamine probe with morpholino
groups (Figure 2c). Compared to the other reported strat-
egies which increase the hydrophilicity of probes by sulfona-
tion,[19] morpholino auxochromes neither change the net
charge nor increase the synthetic difficulty. Therefore, we
synthesized the morpholino auxochrome-based rhodamine
probe, PKZnR-1. The molecule exhibited increased hydro-
philicity (confirmed by the retention time on C18 column of
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liquid chromatography, Figure S10) which reduced its inter-
action with the cell membrane (Figure 2c). Firstly, we
evaluated the phototoxicity of two probes on HeLa cells.
RhodZin-1 caused significant phototoxicity after 30 s of
continuous green light illumination (568 nm, 4.1 WcmÀ2)
while most PKZnR-1 treated cells were still alive (Figure 2d).
We further applied PKZnR-1 to islet imaging. Non-specific
staining in cells was eliminated when using PKZnR-1 for islet
imaging (Figure 2h and Movie 1). The background fluores-
cence intensity in the cytoplasm did not increase during long-
term imaging (Figure 2i and k), nor did we find photo-
induced apoptosis. The minimized non-specific staining and
phototoxicity ensured the recording of physiologically rele-
vant fusion events (exemplified in Figure 2j and k). Further-
more, we incubated the two probes with islets for 1000 s in the
dark. Only non-specific staining signal, but not apoptotic
signal, was observed in islets treated with RhodZin-1. After
laser illumination, apoptotic signal was constantly observed
(Figure S12). Moreover, there was no significant difference in
dark toxicity between the two probes on HeLa cells (Fig-
ure S13). Those evidence further pinpointed the main differ-
ence between the two probes to cell permeability-rendered
phototoxicity, not dark toxicity. Overall, we established the
use of a morpholino auxochrome as a successful strategy for
minimizing the phototoxicity of a Zn2+ probe.
Modular rhodamine chemistry enables the installation of diverse
chelating groups for tunable Zn2+ affinity
The biological pool of Zn2+ features a high heterogeneity
in space, with concentrations spanning the nM to mM ranges.
We sought to elaborate morpholino probes for various
applications by systematically tuning their affinity to Zn2+.
RhodZin-1, featuring a 2-methoxyaniline-N,N-diacetate che-
lating group, was selected as a prototype for further engineer-
ing. Our design retained the main chelating groups (N,N-
diacetic acid) while changing the structure of the side-chain,
so as to tune the affinity on the premise of maintaining the
selectivity of Zn2+. PKZnR-1 was the lead structure of this
morpholino class, with methoxy as the side chain. In PKZnR-
2, we substituted the methoxy group with the more sterically
hindered ethoxy group to reduce affinity. We increased the
chelating sites at the side-chain to devise methoxyethoxy-
substituted PKZnR-3 and o-aminophenol-N,N,O-triacetic
acid (APTRA)[20]-based PKZnR-4. In PKZnR-5, a 2-pyr-
idylmethyl group was installed at the side-chain, as this
pyridine-containing chelator has been reported to be suffi-
ciently selective and have a high affinity.[21] All these
molecules were synthesized via a modular condensation-
oxidation reaction to assemble the rhodamine core, followed
by deprotection to liberate the chelator motif (Scheme S1).
The straightforward chemistry enabled the facile preparation
of PKZnR probes for characterization.
Figure 3. Characterizations of PKZnR 1–5 in vitro: a) Emission spectra
of PKZnR-1 (1 mM) in the presence of various concentrations of free
Zn2+ (0 to 2 mM). (Inset) Absorption spectra of PKZnR-1 (1 mM) in
the presence of 0 and 2 mM free Zn2+. b) Zn2+ titration of PKZnR-
1 (1 mM) as measured from its emission at 580 nm. c) pH dependence
of fluorescence for PKZnR-1 in 0 and 2 mM free Zn2+. Formation of
a white precipitate was observed after Zn2+ addition at pH 9.0,
fluorescence intensity values under these conditions were therefore
not measured. d) Selectivity of PKZnR-1 for Zn2+ compared to other
divalent cations and peptides. Zn2+, Ca2+, Mg2+, Mn2+, Cu2+, Co2+
,
Fe2+, Ni2+, GSH (1 mM) and glucagon (10 mM) were added to 1 mM
PKZnR-1. e) Photophysical properties of PKZnR 1–5. All measurements
were performed in buffers containing 100 mM HEPES (pH 7.4, I
(NaNO3)=0.1) and 0.1% DMSO as a cosolvent at 258C. At free Zn2+
concentrations of <0.1 mM, 10 mM nitrilotriacetic acid (NTA) was
added to chelate the high concentration of total Zn2+. Zero Zn2+
measurements were made in the presence of 10 mM TPEN. The
excitation wavelength was 520 nm. Error bars denote SD; n=3.
The photophysical properties of these probes was tested in
vitro, by measuring Zn2+ -dependent change in fluorescence
(Figure 3a), absorption (Figure 3a (inset)), titration (Fig-
ure 3b), the effect of pH (Figure 3c) and selectivity against
other divalent cations and selected peptides (Figure 3d). The
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PKZnR family generally exhibited excellent Zn2+ selectivity
over other biologically relevant divalent ions or selected
peptides, while offering diverse affinity to Zn2+ spanning
190 nM to 74 mM (Figure 3e, detail in Figure S1–S5, fitting
equation in Supporting Information). It is worth noting that
PKZnR-3 presented a lower affinity than PKZnR-1, suggest-
ing that the steric hindrance effect of this side-chain was
greater than that of the additional methyl ether. The probes in
this family demonstrated a 1:1 metal-to-ligand complex ratio,
which was confirmed by Jobꢀs plots (Figure S1–5e). Because
the chelating group is directly coupled to the fluorophore, the
effective Photoinduced electron Transfer (PeT) quenching
gave considerable “turn-on” responses of the probes to Zn2+
(from 21 to 96, except PKZnR-4). When the pH value
changes in the range of 5.0 to 8.0, the fluorescence of PKZnR
probes in the Zn2+ free and the Zn2+ bound form does not
change significantly. These impressive properties encouraged
us to explore their applications in Zn2+ biology.
yl)-1,2-ethanediamine (TPEN, 50 mM) quenched the fluores-
cence of these puncta (Figure 4a,b and Movie 2), suggesting
the specific and reversible binding of PKZnR-5 to Zn2+. A
similar pattern of insulin granule exocytosis was recorded in
human cell clusters (Figure S15 and Movie 2). Moreover,
PKZnR-5 distinguished different types of fusion events
including full, short-lived and long-lived fusion in mouse b-
cell clusters (Figure S16). Such intricate behaviors, while
consistent with the previous report acquired using RhodZin-
3,[15] can now be monitored over a prolonged window without
severe phototoxicity (Figure S17e). Thus, PKZnR-5 offers
a less photo-damaging solution for cellular studies of insulin
secretion.
We next systematically tested the PKZnR family in ex
vivo recordings of Zn2+/insulin co-release in intact islets
(Figure S17). PKZnR-2 accumulated in cytoplasmic organ-
elles (Figure S17c), while the other four probes gave charac-
teristic punctate patterns. We recommend PKZnR-1 as the
ideal reagent in this application, because its Kd value matched
with the concentration of Zn2+ in the surrounding environ-
ment of islets after stimulation by glucose, therefore ensuring
the accuracy of detection.
PKZnR family covers the detection of Zn2+/insulin co-release
from b-cell clusters to intact mouse and human islets
It is commonly accepted that insulin secretion is defective
in patients with type 2 diabetes.[22] Given that PKZnR-
1 highlighted insulin granules within islets, we tested the
potential of this experiment as an assay for screening drugs.
When stimulated by 11 mM glucose, 326 Æ 171 fusion events
were identified within intact mouse islets (Figure 5a, b and e).
The addition of forskolin, an insulin secretagogue that
elevates the cytosolic cAMP concentration,[23] induced 972 Æ
325 fusion events (Figure 5c and e). Treatment with diazo-
xide, an ATP-sensitive potassium channels activator[24] to
We first screened the PKZnR family to detect insulin
granule exocytosis on pancreatic b-cell clusters. Such cellular-
level imaging differs from islet imaging, as isolated cells lose
their environmental niche and communicational signals, and
secreted contents are extensively diluted into the medium.
Out of the five probes prepared, only PKZnR-5, the strongest
binder with a pyridyl group, efficiently highlighted insulin
granule exocytosis in mouse b-cell clusters (Figure 4a and
S14). The results showed that the concentration of Zn2+ in the
extracellular space of cell clusters was much lower than that of
intact islets. After glucose stimulation, additional fluorescent
puncta appeared, which represented fused insulin granules.
Sequential addition of N, N, N’, N’-tetrakis(2-pyridinylmeth-
Figure 5. PKZnR-1 is capable of ex vivo recording of Zn2+/insulin co-
release in intact mouse islets under chemical stimulations: Represen-
tative examples of islet secretion before stimulation (a, 400 s) or
evoked by 11 mM glucose (b, 1000 s), 11 mM glucose in the presence
of 1 mM forskolin (c, 1000 s), and 11 mM glucose in the presence of
250 mM diazoxide (d, 1000 s). Hot-red puncta represent fusion events
during the recording time. The plasma membrane of individual cells is
highlighted with FM 4–64 dye (10 mM, cyan). Scale bar=10 mm.
e) Numbers of fusion-event under different chemical stimulations.
Error bars denote SEM, n=3 islets. (p*<0.05, t-test).
Figure 4. PKZnR-5 highlights Zn2+/insulin co-release in mouse b-cell
clusters: a) Representative confocal images of PKZnR-5 (10 mM) high-
lighted Zn2+/insulin co-release before (left upper for bright-field image
and right upper for fluorescence image) and after (left lower) glucose
(18.2 mM) stimulation for 10 min, and then chelated by TPEN (50 mM,
right lower) for another 10 min. Scale bar=10 mm. b) The fold change
of numbers of fusion events under glucose stimulation in different cell
clusters. Error bars denote SEM, n=8 cell clusters. (p*<0.05, t-test).
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abolish glucose-stimulated insulin secretion, reduced the
number of fusion events to 3 Æ 1 (Figure 5d and e). These
results suggest that the whole islet imaging assay using
PKZnR-1 is sensitive enough to report the dynamic changes
of insulin secretion, promising a high-throughput method of
screening molecules for treating insulopathic diseases, such as
type 2 diabetes and hyperinsulinemia.
Given that PKZnR-1 is good for detecting the exocytosis
of mouse insulin granules, we also used it to detect the native
insulin granules of human islets, in which genetic manipu-
lation is not practical. Similar to mouse islets, after high
glucose stimulation for ~ 4 min, fluorescent puncta emerged
in a portion of cells (Figure 6a and Movie 3), demonstrating
the native insulin secretion. Moreover, fusion events in
human islets also exhibited all three types of fusion mode
(Figure 6b), which indicates the conservation of insulin
release regulation from mouse to human.[15] Taken together,
these results show that low affinity, biocompatible PKZnR
dyes are suitable for monitoring insulin release within intact
mouse and human islets.
Figure 6. PKZnR-1 reveals three types of Zn2+/insulin fusion mode in
A Strategic late-stage N-alkylation enables practical synthesis of
Si-rhodamine-based Zn2+ probes
a human intact islet: a) The maximum-intensity t-projection (500 s) of
PKZnR-1 (10 mM)- treated islet stimulated with 18.2 mM glucose. Scale
bar=10 mm (b). The time course of PKZnR-1 fluorescence at the
center of ROI-1 through ROI-3 (540 nm ꢁ 540 nm) shows the three
fusion modes. Montages show each consecutive image series at 16 s/
frame.
We next endeavored to expand the morpholino rhod-
amine family to far-red-emitting Zn2+ probes. Far-red channel
is useful as a complementary channel to green and red for
multicolor imaging, especially in phys-
iological studies. While genetically-en-
coded fluorescent indicators have pre-
vailed over the green and red channels,
the far-red window is dominated by
synthetic probes that have advantages
in brightness and flexibility. Conse-
quently, metal probes based on Si-rhod-
amine are rapidly emerging in spite of
the challenges posed by their synthe-
sis.[9g,25]
The first-generation Si-rhodamine-
based cation probes featured a direct
connection between the chelator and the
rhodamine core (Scheme 1a and b).
However, the inevitable strongly-reac-
tive organolithium species were barely
compatible with carbonyl chelators, giv-
ing rise to harsh reaction conditions with
poor yields.[25a] A recent variation on Si-
rhodamine-based Ca2+ probes exploited
an indirect linking strategy that con-
nected the chelator to the fluorophore
through an amide bond (Scheme 1b),
making the synthesis less cumberso-
me.[25b] However, the lone electron pair
on the nitrogen atom of the chelator
could no longer fully quench the fluo-
Scheme 1. Synthesis of PKZnFR 1–3: a) Chemical structure of BAPTA, a Ca2+ chelator which
inspired the Zn2+ chelator, 2-methoxyaniline-N, N-diacetate. b) Structures of BAPTA-based Ca2+
indicators on a Si-rhodamine scaffold. c) Retrosynthetic analyses of PKZnFR 1–3, highlighting
a strategic application of late-stage N-alkylation. d) Synthetic route of PKZnFR 1–3.
rophore through the PeT mechanism,
leading to compromised turn-on ratios.
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In order to solve these problems, we designed a new
synthetic route to create the PKZnFR family (Scheme 1c),
featuring a late-stage N-alkylation to avoid nucleophilic
addition in the presence of ester. A transient protecting
group: 1,2-bis(chlorodimethylsilyl)ethane was used to protect
aniline during the Li-Br exchange reactions.[26] The previously
developed bulky-protection was inherited to block nucleo-
philic attack on the 9 position, ensuring chemical stability of
the products.[27] Practically, compounds 1–3e, bearing tran-
siently protected aniline, were subjected to Li-Br exchange,
followed by nucleophilic addition to the morpholino Si-
xanthone. The reactions were quenched by dilute hydrochlo-
ric acid solution, in which the protecting group was removed
in one-pot. Then the N-alkylation with ethyl bromoacetate
was carried out. Final deprotection of esters gave PKZnFR 1–
3 (Scheme 1d). The new strategy greatly improved the overall
yield, enabling the preparation of final products at the scale of
tens of milligrams, making 13C NMR characterization possi-
ble.
The PKZnFR family had absorption and emission max-
ima at 654 and 676 nm, fitting perfectly with the far-red (Cy5)
channel on fluorescence microscopes (Figure 7a). Notably,
while their affinities for Zn2+ were similar to PKZnR-1 (~
30 mM) (Figure 7b), their fluorescence turn-on ratios were
> 100 (Figure 7d, detail in Figure S6–S8). Bulky groups
successfully enhanced the stability of Si-rhodamine probes
in pH 7.4 HEPES buffer, suppressing their based-induced
degradation (Figure 7c and S9). Both PK ZnFR-2 and 3
exhibited minimal background staining in islet ex vivo
imaging assays (Figure S18). Overall, we recommend PK
ZnFR-3 as the best choice of this family, featuring high serum
stability, minimal non-specific background, and superior
fluorescence turn-on signal.
Figure 7. Characterizations of PKZnFR 1–3 in vitro: a) Emission spec-
tra of PKZnFR-3 (1 mM) in the presence of various concentrations of
free Zn2+ (0 to 2 mM). (Inset) Absorption spectra of PKZnFR-3 (1 mM)
in the presence of 0 and 2 mM free Zn2+. b) Zn2+ titration of PKZnFR-3
(1 mM) as measured from its emission at 675 nm. c) Absorbance
changes of PKZnFR-1 (black squares), PKZnFR-2 (red circles) and
PKZnFR-3 (blue triangles) in HEPES buffer, showing the outstanding
chemical stability of PKZnFR-3. d) Photophysical properties of PKZnFR
1–3. All measurements were performed in buffers containing 100 mM
HEPES (pH 7.4, I(NaNO3)=0.1) and 0.1% DMSO as a cosolvent at
258C. Zero Zn2+ measurements were made in the presence of 10 mM
TPEN. The excitation wavelength was 620 nm. Error bars denote SD;
n=3.
Far-Red PK Zinc probe enables 4D imaging of the signaling
networks that regulate insulin secretion in mouse islets
Insulin secretion is a highly dynamic and multi-tiered
process regulated by complex mechanisms. Briefly, glucose
transported into b-cells is metabolized in mitochondria to
generate ATP. The increased ATP/ADP ratio in cytosol closes
ATP-sensitive potassium channels, which leads to depolari-
zation of the cell membrane and opening of voltage-gated
calcium channels, thereby allowing the flow of calcium into
the cell. Finally, the rise of cytosolic Ca2+ triggers the release
of insulin granules. Research on these pathways has relied
heavily on bioimaging.[23,28] However, integrative studies on
the signal transduction networks of islets are rare, possibly
due to the lack of orthogonal probes for the key components
such as Ca2+, mitochondria, and secreted granules.
with minimal phototoxicity. PKZnFR-3 (red, Ex = 647 nm) to
record insulin release from b-cells; and Hoechst (blue, Ex =
405 nm) to counter-stain nuclei. The mitochondrial signal
recorded with cationic PK Mito Red partly correlates to
mitochondrial membrane potential (Figure S19). The islets
were then recorded under a spinning disk confocal micro-
scope. Four-color imaging showed that, when islets were
stimulated with a high concentration of glucose for ~ 1 mi-
nute, the Ca2+ signal abruptly increased, followed by the
emergence of insulin secretion puncta, meanwhile, the
mitochondrial signal did not change significantly (Figure 8a,
S20 and Movie 4). Furthermore, it revealed an orchestrated
yet complicated relationship between Ca2+, Zn2+ and mito-
chondrial signal. The whole-islet recording revealed that b-
cells were highly heterogeneous in terms of mitochondrial
Using our newly-developed far-red Zn2+ probe, it is now
possible to record time-lapse, four-color and 3D fluorescence
imaging of mitochondrial signal, Ca2+ influx, and insulin
secretion in living mouse islets. We prepared transgenic mice
expressing GCaMP6f (Ex = 488 nm), a genetically-encoded,
green-emitting calcium indicator,[29] under the Ins1 promoter
in b-cells. Islets isolated from these mice were treated with PK
Mito Red[30] (yellow, Ex = 561 nm) to image mitochondria
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Figure 8. PKZnFR-3 enables time-lapse, four-color fluorescence imaging of mouse islets and establishes the relationships between total Ca2+
influx, mitochondrial signal and Zn2+/insulin secretion: a) Four-color confocal image of a mouse islet expressing Ins-GCaMP6f (green,
Ex.=488 nm) stained with 80 mM Hoechst (blue, Ex.=405 nm), 200 nM PK Mito Red (yellow, Ex.=561 nm) and 10 mM PKZnFR-3 (Red,
Ex.=647 nm). (Main Figure) snapshot at 920 s. Scale bar=20 mm. (Right panels) Time-dependent images of the nucleus (blue, Hoechst) and
mitochondria (yellow, PK Mito Red). (Lower panels) Two-color time-lapse images of green and red channels measuring dynamic changes of the
Ca2+ and Zn2+/insulin signals. b) Z-stack of the islet with four-color images at depths of 2–26 mm. c–f) The dynamics of Ca2+ signals (green curve)
and fusion events (red histogram) across a whole islet (f) and representative cells with the highest mitochondrial signal (c), the strongest
secretory activity (d) and the maximum Ca2+ influx (calculated by the area under the curve) (e) after glucose stimulation (18.2 mM).
g) Relationship of cellular Ca2+ influx, mitochondrial signal and fusion events in single b-cells from 7 islets. Dot size represents the number of
fusion events. The color bar shows the mean fluorescence intensity of mitochondria in each cell.
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signal, Ca2+ influx, and levels of insulin secretion. And this
heterogeneity was seen across the entire islet at different
depths (Figure 8b and Movie 5). Thanks to the minimal
phototoxicity of both PKZnFR-3 and PK Mito Red, the four-
color recording lasted for 300 frames (15 min at 0.33 fps)
without perceptible damage.
signaling network. Reminiscent to the neuroimaging field
where time-lapse video data has been routinely processed
using computer programs to map single neuron activities,[33]
Zn2+ biology in islets is now stepping into such digital era. To
a certain extent, the chemical advances in far-red probes may
leverage the integration of artificial intelligence and bioimag-
ing, in which multiplexed biocompatible probes play an
instrumental role.
The PK Zinc family advances the imaging of b-cell
secretion on the islet level and is tailored to fit both mouse
and human islet samples. These integrative imaging ap-
proaches represent a viable alternative to the traditional
screening platform for active compounds. Moreover, 4D and
multi-color imaging provides a system biology perspective on
the cellular functions and communications in islets.
The data generated in 4D recording prompted us to turn
to computer-aided data analysis (see supporting information)
to characterize the Ca2+ influx, mitochondrial signal, and
fusion events in the whole islet (Figure 8 f) and in each b-cell
(Figure 8c–e). We found that the b-cells with the highest
mitochondrial signal released fewer insulin granules (Fig-
ure 8c), while those with the most fusion events had relatively
low mitochondrial signal (Figure 8g). Unexpectedly, those b-
cells with the maximum Ca2+ influx had little-to-no detectable
fusion events (Figure 8e); meanwhile, the Ca2+ influx signal
of the cells with the most fusion events was not significantly
higher than that of other cells (Figure 8d). These results were
verified by imaging experiments on seven individual islets
(Figure 8g, the complete three views in Figure S21). Of note,
the physiological significance of the heterogeneous mito-
chondrial signal, as well as the interplay between mitochon-
drial signal, Ca2+ influx and insulin secretion in islets,
represent appealing topics that are under investigation in
our labs. The multi-color time-lapse imaging approach, made
possible by orthogonal low-phototoxic probes, highlights the
potential impact of red-shifted Zn2+ probes on the field of
islet biology.
Acknowledgements
This work was supported by Beijing Municipal Science &
Technology Commission (Project: Z201100005320017 to
Z.C.), the National Natural Science Foundation of China
(Project 31971375 to Z. C.; 31821091 to Z.C. and L.C.;
92054301, 81925022 to L.C.; 12090053, 32088101 to C.T.,
82070805 to S.W.), Beijing Youth Top-notch Talent Group
(Project: 7350500012 to Z.C. and L.C.), Peking-Tsinghua
Center for Life Sciences (to Z.C. and C.T.), start-up fund from
Peking University (to Z.C.), the National Science and
Technology
Major
Project
Program
Project
(2016YFA0500400 to L.C.), the Beijing Natural Science
Foundation (Z20J00059 to L.C.), National Key Research
and Development Program (2020YFA0803704 to S.W.),
Postdoc fellowship of Peking-Tsinghua Center for Life
Sciences to X.P. and Boya postdoctoral fellowship of Peking
University to H.R. We thank Profs. Chu Wang, Zhen Yang
and Jia-Hua Chen for sharing their chemistry labs, Profs.
Heping Cheng and Xianhua Wang for sharing their spec-
trometers, Prof. Iain Bruce, Dr. Qinsi Zheng and Qi Tang for
helpful discussions. We thank the High-Performance Com-
puting Platform, the Metabolic Mass Spectrometry Platform
of IMM, and the NMR facility of National Center for Protein
Sciences at Peking University for assistance with data
acquisition.
Conclusion
The phototoxicity of fluorescent probes, traditionally
overshadowed by the photobleaching process, has been
increasingly recognized as an independent bottleneck in 4D
and super-resolution imaging.[31] A strategy for alleviating
photodynamic damage to cells was recently developed using
the conjugation of triplet-state quenchers.[30] This work
showcased another parallel strategy for eliminating photo-
toxicity, which focused on improving the specificity of probes
by tuning their polarity. Such morpholino modifications, while
routine in medicinal chemistry, represent an original advance
in the photo-chemical biology of rhodamine. They also
resonate with the development of modern auxochromes for
superior optical properties. We speculate that the next
generation of probes will combine the features of twisted
intramolecular charge transfer (TICT) dyes to compensate
the compromised quantum yield of morpholino rhoda-
mines.[32] This technological trend, in our opinion, exemplifies
the integration of photochemistry with medicinal chemistry to
enable cutting-edge bioimaging applications.
Conflict of Interest
Z.C., J.Z., X.P., and Y.W. have submitted a patent application
based on PK Zinc dyes described in this work.
Keywords: 4D islet physiology · biocompatibility ·
From a chemistry perspective, this work provides a new,
practical synthetic route of far-red Zn2+ probes, the favorite
window to complement genetically-encodable indicators. We
plan to elaborate the transient protection/late-stage alkyla-
tion tactic to generate various probes in the far-red or even
NIR window. It is noteworthy that, adding an orthogonal
channel offers exponentially-expanded information in space
and time, drastically enriching the analysis of an intertwining
fluorescent probe · insulin secretion · zinc
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Accepted manuscript online: August 23, 2021
Version of record online: && &&
,
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Research Articles
Fluorescent Probes
We synthesized a new class of red- and
far-red-emitting Zn2+ probes with mini-
mal phototoxicity, mM affinities and high
turn-on ratios. Tailored for 4D, long-term
J. Zhang, X. Peng, Y. Wu, H. Ren, J. Sun,
S. Tong, T. Liu, Y. Zhao, S. Wang, C. Tang,
L. Chen, Z. Chen*
&&&& — &&&&
and multiple-color recording of Zn2+
/
insulin co-secretion in b-cells and islets,
the new probes promise to unveil the
spatial-temporal regulation of islet endo-
crinology.
Red- and Far-Red-Emitting Zinc Probes
with Minimal Phototoxicity for
Multiplexed Recording of Orchestrated
Insulin Secretion
&&&&
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Angew. Chem. Int. Ed. 2021, 60, 2 – 12
These are not the final page numbers!