Tuning macrocycles to design 'turn-on' fluorescence probes for manganese(ii) sensing in live cells

Article abstract of DOI:10.1039/c4cc09542a

We tune the coordination environment of macrocyclic ligands to design two novel fluorescence sensors for Mn²⁺. The BODIPY-based Mn²⁺ sensor M1 affords an excellent, 52 fold, fluorescence 'turn-on' response despite the paramagnetic nature of Mn²⁺. The lipophilic probe is cell-permeable and confocal imaging demonstrates that the sensor distinctly detects Mn²⁺ within live cells. This journal is

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Tuning Macrocycles to Design ‘Turn-on’ Fluorescence
Probes for Manganese (II) Sensing in Live Cells
Cite this: DOI: 10.1039/x0xx00000x
Subha Bakthavatsalam†, Anindita Sarkar†, Ananya Rakshit, Shubhi Jain, Amit
Kumar and Ankona Datta*
Received 00th January 2014,
Accepted 00th January 2014
DOI: 10.1039/x0xx00000x
www.rsc.org/
We tune the coordination environment of macrocyclic have therefore critically assessed the coordination preferences
ligands to design two novel fluorescence sensors for Mn2+. of Mn2+ to design Mn2+ specific ligands and report herein the
The BODIPY-based Mn2+ sensor M1 affords an excellent, synthesis and properties of Mn2+ selective ‘turnꢀon’ fluorescent
52 fold, fluorescence ‘turn-on’ response despite the sensors, manganese sensor 1 (M1) and manganese sensor 2
paramagnetic nature of Mn2+. The lipophilic probe is cell- (M2). The BODIPYꢀbased lipophilic sensors have visible
permeable and confocal imaging demonstrates that the excitation and emission profiles and display 52ꢀfold and 28ꢀfold
sensor distinctly detects Mn2+ within live cells.
fluorescence ‘turnꢀon’ response upon Mn2+ binding, for M1 and
M2 respectively. M1 displayed higher selectivity towards Mn2+
sensing and was successfully applied for in cellulo imaging of
Mn2+ using confocal microscopy.
Mn2+ is essential for biological systems as a cofactor for
enzymes in its bound form and as an antioxidant when labile.1ꢀ3
Mnꢀcontaining compounds also find extensive applications in
industrial processes.4 Pesticides, antiꢀknock agents, and infant
formula contain Mn in different forms.4 Exposure through these
sources however, can lead to Mn accumulation and neuronal
defects in biological systems.4ꢀ6 The subtle balance between
essentiality and toxicity is extremely intriguing, and there are
many open questions related to the cellular effects of Mn2+ and
Mn2+ lies in the bottom of the IrvingꢀWilliams series that
predicts the relative stabilities of complexes formed by divalent
transition metal ions.32 Hence, designing Mn2+ specific ligands
for sensor development proves to be extremely challenging.
Canary and coꢀworkers have reported that ligands containing
mixed oxygen and nitrogen donor atoms might be suitable for
Mn2+ binding.29 A comprehensive body of work on Mn2+
complexes of pentaꢀaza macrocycles acting as superoxide
dismutase mimetics reported by Riley and coꢀworkers and other
7
factors maintaining homeostasis.3, A key requirement for
attempts towards elucidating cellular mechanisms is obtaining
information on the in cellulo localization of Mn2+ both under
physiological and overexposure conditions.
groups indicate that Mn2+ ions prefer pentagonal biꢀpyramidal
34
coordination geometry.33,
We therefore hypothesize that
Optical sensing using fluorescence is ideal for detecting
trace metals because of its high sensitivity.8 Justifiably multiple
groups have reported various elegant fluorescent sensors for
transition metal ions.8ꢀ24 Mn2+, however remains elusive, due to
the lack of Mnꢀselective ligands and the paramagnetic nature of
Mn2+ which leads to fluorescence quenching. Hence, there are
only few reports on fluorescence sensors for Mn2+.25ꢀ31 We
pentaꢀaza macrocyclic motifs with oxygen containing arms can
be optimized for selective Mn2+ sensing over other divalent
metal ions (Scheme1).
Department of Chemical Sciences, Tata Institute of Fundamental
Research, 1 Homi Bhabha Road, Colaba, Mumbaiꢀ 400005, India.
†
Equal Contribution
Electronic Supplementary Information (ESI) available: Synthetic procꢀ
edures, characterization, and spectra. See DOI: 10.1039/c000000x/
Scheme 1 Design strategy for ‘turn-on’ Mn2+ sensing.
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Scheme 2 Synthesis of ‘turn-on’ Mn2+ sensors.
In order to achieve efficient ‘turnꢀon’ Mn2+ sensing a
M1 and M2 have ester arms which increase their
dimethylaniline moiety is strategically fused with the ligand. lipophilicity. Hence, in vitro absorbance and fluorescence
The dimethylaniline moiety and the additional Nꢀdonors in the experiments to examine the ‘turnꢀon’ response of the probes
ligand will quench the fluorescence of the covalently attached were conducted in acetonitrile. Metalꢀunbound aka ‘apo’ M1
BODIPY via photoꢀinduced electron transfer (PET) mechanism and M2 both display a single absorption maxima at 496 nm (
in the absence of Mn2+. Upon Mn2+ binding to the macrocycle, 6.9 x 104 Mꢀ1 cmꢀ1 for M1 and M2, Figure S3). The emission
the fluorophore will afford an increase in fluorescence intensity maxima for apo M1 and M2 were at 508 and 507 nm ( ex = 480
(Scheme 1).
nm), respectively (Figures 1 and S2). Upon Mn2+ addition
An important requirement for Mn2+ sensors is the selectivity neither M1 nor M2 showed any significant shifts in either the
for Mn2+ over Ca2+ due to the high abundance of Ca2+ in absorption ( = 7.1 x 104 Mꢀ1 cmꢀ1 for M1 and
abs=497 nm,
cellulo 35 Specifically, anionic oxygen donors will increase Ca2+ M2, Figure S3) or emission maxima (λem = 508 nm for M1 and
ε =
λ
λ
ε
.
binding. Hence, we include methyl ester groups as oxygen M2, Figures 1 and S2).
donors in the ligand design. The ratio of nitrogen to oxygen
donor atoms is varied to design M1 and M2 with the aim of
tuning Mn2+ affinity and selectivity.
The synthesis of M1 and M2 is outlined in Scheme 2.
RichmanꢀAtkins cyclization is used to synthesize the two novel
differentially substituted
3b in 29 % and 47 % yields respectively, starting from
compounds and . The synthesis of the macrocycle
precursors, compounds and are described in the
Nꢀtosylꢀprotected macrocycles 3a and
1
2
1
2
supplementary information. The macrocycles are deprotected in
the presence of sodium naphthalenide following which methyl
acetate arms are attached on the free secondary amines with
yields of 45 % and 21 % for compounds 4a and 4b
,
Figure 1 Fluorescence response of M1 (2.5
acetonitrile. ex = 480 nm. (inset) Image depicting quartz cuvettes containing M1
(10 µM, left) and M1 (10 µM) in presence of Mn2+ (20 µM, right). Both cuvettes
were illuminated equally under long-wave UV radiation ( ex = 365 nm).
ꢀ - 48 ꢀM) in
M) to Mn2+ (0
λ
respectively. Vilsmeier–Haack reaction is used to generate
aldehydes 5a in 33 % and 5b in 31 % yields. Condensation
reactions of the aldehydeꢀcontaining macrocycles 5a and 5b
with 2,4ꢀdimethylpyrrole, followed by treatment with BF3·Et2O
affords M1 in 29 % yield and M2 in 14 % yield following
purification.
λ
Both M1 and M2 displayed excellent fluorescence ‘turnꢀon’
response in the presence of Mn2+. The apo forms of M1 and M2
had very low quantum yields (
φ
= 0.0038 for M1 and 0.0055
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for M2) due to effective quenching of the BODIPY fluorophore paramagnetic metal ion Fe2+ did not afford any increase in
via the dimethylaniline moiety fused to the pentaꢀaza emission in the presence of either sensor (Figures 2a and 2b).
macrocycle (Figures 1 and S2). Upon addition of Mn2+, M1
afforded a 52 fold emission enhancement (
and 1 inset), while M2 afforded a 28 fold emission ions that afforded a response with M1 were Zn2+ and Cu2+
M1 afforded better selectivity towards Mn2+ when
φ
= 0.197, Figure 1 compared to M2. The only other biologically relevant metal
enhancement ((
φ
= 0.156, Figure S2).
(Figure 2a). Since, intracellular Cu is mostly present as Cu+ in
the reducing intracellular environment, interference from Cu2+
during in cellulo Mn2+ imaging is not expected.21 Also,
intracellular labile Zn2+ is in the pM level, while the putative
labile Mn2+ is predicted to be in the sub µM level.35ꢀ37
Competition experiments performed by adding Mn2+ following
additions of Zn2+ or Cu2+ to either probe indicated further
increase in fluorescence emission (Figure S5). Hence, M1 and
M2 respond to Mn2+ even in the presence of these interfering
ions, proving higher Mn2+ specificity. Another metal ion that
responded to the sensors was Cd2+ (Figure S6). However Cd2+
is highly toxic to cells and is not generally present in labile
form.35 Hence, we do not expect any interference due to this ion
in the cellular milieu.
Figure 2 Observed fluorescence intensity (F) over initial fluorescence intensity in
the absence of any metal ion (F0) for (a) M1 (2.5 M) and (b) M2 (2.5 M) in the
presence of various biologically relevant metal ions ([Mn+]= 20
M) at 508 nm, in
ꢀ
ꢀ
ꢀ
acetonitrile. λex = 480 nm. Insets: F/F0 at 508 nm for (a) M1 and (b) M2 in the
presence of Mn2+ (blue squares, fitted data red line), Ca2+ (orange circles), and
Fe2+ (green triangles) over a [Mn+] range (0-48
ꢀM).
The fluorescence binding plots (Figures 2a inset and 2b
inset) were fitted to obtain apparent dissociation constants for
Mn2+ binding to M1 and M2 in acetonitrile, which were 4.0 ±
0.9 µM and 8.3 ± 0.4 µM respectively. The plot for M1 could
be best fitted to a 1:1 binding model (Equation S1) with no
cooperativity as indicated by the corresponding Hill plot
(Figure S4a). On the other hand, the binding plot for M2 was
best fitted to a 1:2 (M2:Mn2+) binding model (Equation S2). A
Hill plot in this case displayed cooperative binding (Figure
S4b), hinting at a possible second Mn2+ binding site due to the
presence of additional methyl acetate arms.
We probed the selectivity of M1 and M2 towards other
biologically relevant metal ions by performing fluorescence
titration experiments. F/F0 values for the probes at the emission
maxima, 508 nm, have been plotted for various metal ions in
Figures 2a and 2b. Amongst the ions tested, both M1 and M2
afforded maximum fluorescence enhancement in the presence
of Mn2+ and the fluorescence response saturated above 20 µM
Mn2+ concentration (Figures 2a inset and 2b inset).
Importantly, Ca2+, a major competitor for Mn2+ binding ligands,
did not display any fluorescence enhancement with
M1(Figures 2a and 2a inset) and only a modest 2.6 times
increase with M2 (Figures 2b and 2b inset). Similarly, another
Figure 3 Live-cell imaging of intracellular Mn2+ by confocal microscopy in HEK
293T cells. Overlay of confocal and transmission images of cells treated with (a)
M1 (b) MnCl2 (25 µM) in growth media followed by M1 (c) M1 followed by TPEN
(2mM) (d) MnCl2 followed by M1 and TPEN. λex 488 nm (e-g) Confocal images
depicting co-localization of M1 (green channel, λex 488 nm) with Nile Red (100
nM, red channel, λex 543 nm) in Mn-treated cells. M1 concentration 5 µM. Scale:
25 µm (a-d), 10 µm (e-g).
The probes are lipophilic and hence would be applicable for
in cellulo imaging of Mn2+ in lipidꢀrich bodies and organelles.
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DOI: 10.1039/C4CC09542A
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