A highly selective fluorogenic probe for the detection and: In vivo imaging of Cu/Zn superoxide dismutase

Article abstract of DOI:10.1039/c6cc00095a

Copper/zinc superoxide dismutase (Cu/Zn SOD) is an essential enzyme that protects tissue from oxidative damage. Herein we report the first fluorogenic probe (SODO) for the detection and in vivo imaging of Cu/Zn SOD. SODO represents a unique chemical probe for translational imaging studies of Cu/Zn SOD in inflammatory disorders.

Full text of DOI:10.1039/c6cc00095a

View Article Online
View Journal
ChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. Zhang, J. C. Er,
H. Jiang, X. Li, Z. Luo, T. Ramezani, Y. Feng, M. K. Tang, Y. Chang and M. Vendrell, Chem. Commun.,
2016, DOI: 10.1039/C6CC00095A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/chemcomm

Please do not adjust margins
ChemComm
Page 1 of 4
View Article Online
DOI: 10.1039/C6CC00095A
Journal Name
COMMUNICATION
A Highly Selective Fluorogenic Probe for the Detection and in vivo
Imaging of Cu/Zn Superoxide Dismutase
Liyun Zhang,†,a,* Jun Cheng Er,†,b,c Hao Jiang,d Xin Li,a Zhaofeng Luo,d Thomas Ramezani,e Yi Feng,e
Mui Kee Tang,b Young-Tae Changb and Marc Vendrelle,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Copper/zinc superoxide dismutase (Cu/Zn SOD) is an essential injury).5-7 On the contrary, decreased levels of Cu/Zn SOD have
enzyme that protects tissue from oxidative damage. Herein we been associated with an inhibition of the immune response
report the first fluorogenic probe (SODO) for the detection and in and the promotion of oxidative stress in age-related
vivo imaging of Cu/Zn SOD. SODO represents a unique chemical disorders.8,9
probe for translational imaging studies of Cu/Zn SOD in Despite the importance of Cu/Zn SOD in regulating the balance
inflammatory disorders.
between healthy and disease states, the exact mechanism that
correlates Cu/Zn SOD to the progression of different
pathologies remains largely unknown.5 Current probes to
visualize SODs mainly rely on the intrinsic fluorescence of Tyr
or Trp residues10,11 or the use of non-specific metal chelators,
such as bathocuproine.12 These methods have very limited
practical use in vivo, due to spectral shortcomings (e.g. short
excitation/emission wavelengths) and their poor selectivity
between SODs and other ROS-related enzymes.
Superoxide dismutases (SOD, EC 1.15.1.1) are metalloenzymes
that protect tissue from the oxidative stress caused by reactive
oxygen species (ROS).1 The main function of SODs is to catalyse
the dismutation of superoxide radicals (O2•-) to hydrogen
peroxide (H2O2) and oxygen. There are several isoforms of
SODs, which can be distinguished by their metal cofactors and
their distribution in cells.2 Among the different isoforms of
SODs, copper/zinc superoxide dismutase (Cu/Zn SOD or SOD1)
is widely distributed and comprises around 90% of the total
SODs. Alterations in the expression and activity of Cu/Zn SOD
have been associated with the onset of a number of diseases.
Mutations in human Cu/Zn SOD are implicated in the
development of neurological disorders, such as familial
amyotrophic lateral sclerosis (fALS), Alzheimer’s disease and
Parkinson’s disease.3-5 Furthermore, elevated activities of
Cu/Zn SOD have been reported in cancer (e.g. acute
myelogenous leukaemia, Hodgkin’s lymphoma) and chronic
inflammatory diseases (e.g. rheumatoid arthritis, ischemic
Fluorogenic probes are advantageous for in vivo imaging since
they provide high signal-to-noise ratios without the need for
washing steps.13,14 Our group and others have reported the
preparation of fluorogenic probes based on the 4,4-difluoro-4-
bora-3a,4a-diaza-s-indacene (BODIPY) scaffold,15,16 one of the
most exploited fluorophores for cell imaging due to its
photostability and permeability properties.17,18 BODIPY
fluorogens can be synthesized by direct conjugation of
electron-rich groups (e.g. substituted benzene rings) to the
BODIPY core, leading to photoinduced electron transfer (PeT)
quenching and subsequent turn-on fluorescence emission in
hydrophobic environments. In order to enhance the
fluorogenic response of probes binding to Cu/Zn SOD, we
designed a new class of BODIPY fluorogens combining PeT-
quenching substituents and chemical groups restricting the
rotational flexibility of the BODIPY core. The restriction of
torsional motion has proven an effective strategy to generate
turn-on fluorescent probes,19,20 and previous studies have
shown that “–NH” groups directly linked to the position C3 of
BODIPY can form intramolecular hydrogen bonds with the
fluorine atoms.21 We synthesized MK fluorogens by modifying
a 3,5-dichloro-BODIPY scaffold (
forming intramolecular hydrogen bonds with the fluorine
atoms, and triazole groups ( ) as PeT-quenchers (Scheme 1).
MK fluorogens were prepared by loading onto 2-
1) with benzylamines (M)
K
1
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
View Article Online
DOI: 10.1039/C6CC00095A
COMMUNICATION
Journal Name
Scheme 1 Solid-phase synthesis of MK fluorogens.
NH2
NH2
NH2
HN
aniline
meso-
a
b - e
N
N+
B-
NH
O
N
N
F
F
N
N
N+
N
N+
N
N+
B-
B-
B-
3
amine
'arm'
triazole
'arm'
R2
Cl
Cl
Cl
Cl
N
N
N
F
F
F
F
F F
R1
N
H
MK103-48
(SODO)
1
2
MK fluorogens
abs. 474 nm, λem.: 564 nm
F (DMSO): 0.014
λ
Φ
Reaction conditions: a) CTC-PS, DIPEA, CH2Cl2:DMF (1:1), r.t.; b) NaN3, DMF, r.t; c)
R1NH2, DIPEA:DMF (1:4), r.t.; d) R2-C≡CH, CuI, L-ascorbic acid, r.t.; e) TFA:CH2Cl2
(0.5:99.5), r.t.
Fig. 1 Chemical structure and fluorescence spectra of SODO (10 ꢀM) after incubation
with serial concentrations of hCu/Zn-SOD from 0.01 to 5 mg mL-1 in 20 mM Tris-HCl
buffer (pH = 7.4). λexc.: 460 nm. ФF in hCu/Zn-SOD: 0.45.
chlorotrityl chloride polystyrene (CTC-PS) resin, followed by
nucleophilic substitution and copper-catalysed azide-alkyne
cycloaddition. A total of 40 MK compounds with diverse amine
and alkyne groups were isolated in moderate to high yields
with very high purities using mild acidic cleavage conditions22
(for detailed chemical structures and characterisation data,
see Electronic Supporting Information (ESI)).
We examined the binding of SODO at Cu/Zn SODs from
different species. As with hCu/Zn-SOD, SODO displayed a
concentration- dependent response in Cu/Zn SODs from
bovine blood (bCu/Zn SOD) and from Arabidopsis thaliana
(aCu/Zn SOD) (Fig. 2). These results suggest that the binding of
SODO is species-independent and occurs at a conserved
hydrophobic region of Cu/Zn SOD. While Cu/Zn SOD stands for
the majority of SOD in tissue, high selectivity for the Cu/Zn
SOD isoform is essential for imaging studies. We assessed the
fluorescence response of SODO in the other two SOD isoforms
(i.e. Mn-SOD and Fe-SOD) and observed high selectivity for
Cu/Zn SOD over Mn-SOD and Fe-SOD, where minimal binding
was detected (Fig. 2). To the best of our knowledge, SODO is
the first small fluorophore able to detect Cu/Zn SOD with high
specificity over other SODs. We also assessed the fluorescence
emission of SODO in other ROS-related enzymes (e.g. catalase,
The spectral characterisation of the MK derivatives confirmed
their strong fluorogenic behaviour with minimal fluorescence
in aqueous media, long Stokes shifts (i.e. around 90 nm) and
red-shifted emission wavelengths when compared to the
BODIPY core (Table S1 in ESI). As expected, the incorporation
of benzylamines at the position C3 of the BODIPY scaffold
restricted the torsional motion of the fluorophore, leading to
an increase in the quantum yields in non-polar solvents. We
also observed that the fluorescence emission of MK fluorogens
correlated with solvent viscosity as a result of the decreased
rotation of both triazole and aniline substituents (Fig. S1 in
ESI). Moreover, the fluorogenic response of MK derivatives
was stronger in non-polar solvents due to the reduced PeT
quenching effect from the meso-aniline group in non-polar
environments (Figs. S2-S3 and Table S2 in ESI). Altogether,
these results assert MK derivatives as BODIPY fluorogens with
excellent spectral properties to detect polarity changes
associated to the binding at large macromolecules.
peroxidase) (Fig. 2) and metabolites (e.g. H2O2, O2• •)
−, 1O2, OH
(Fig. 2 inset). SODO exhibited very high specificity for Cu/Zn-
SOD, showing minimal fluorescence in other enzymes and
metabolites involved in oxidative damage and inflammatory
processes.
In view of these properties, we assessed our MK fluorogens in
vitro to bind at the macromolecular dimeric structure of Cu/Zn
SOD. The screening of diversity-oriented fluorescence libraries
has become an effective strategy to identify highly selective
molecular probes,23 and we observed that compounds with 2-
ethoxybenzylamine (M103) as the amine group displayed high
fluorescence emission after incubation with human Cu/Zn SOD
(hCu/Zn SOD) (Fig. S4 in ESI). MK103-48 showed the strongest
response among all compounds and was selected for further
studies (hereinafter named as SODO (SOD Orange), Fig. 1).
SODO displayed up to 150-fold increase in fluorescence
emission after binding at hCu/Zn SOD, with a limit of detection
ꢀ
g mL-1 (Figure S5 in ESI). Notably, SODO reached
Fig. 2 Fluorogenic response of SODO upon binding to different proteins (at 0.1, 0.2, 0.3
and 0.4 mg mL-1) and metabolites (inset, ROS & RNS: 100 ꢀM) in 20 mM Tris-HCl buffer
(pH = 7.4). λexc.: 460 nm, λem.: 560 nm. Values are represented as means and error bars
as standard deviations (n = 3), *** for p < 0.001.
of 10
quantum yields around 45% and displayed a remarkable 60 nm
hypsochromic shift in the fluorescence spectrum after binding
(Fig. 1 and Figs. S5-S6 in ESI).
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 3 of 4
View Article Online
DOI: 10.1039/C6CC00095A
Journal Name
COMMUNICATION
Finally we studied whether the binding of SODO affected the Cell viability assays in fibroblasts also corroborated the
catalytic activity of Cu/Zn SOD (Fig. S7 in ESI). SODO did not marginal cytotoxicity of SODO within the working
significantly perturb enzymatic function; hence being an concentration range (Fig. S10 in ESI). In order to determine the
excellent reporter of Cu/Zn SOD without altering the normal binding mode of SODO in Cu/Zn SOD, we performed docking
physiology of cells. Altogether, these results confirm SODO as calculations to analyse the interaction between SODO at
the first fluorogenic probe to detect Cu/Zn SOD without cross- hCu/Zn SOD. Cu/Zn-SOD is found in all eukaryotic species as a
reacting with other SOD isoforms, enzymes or ROS.
homodimeric enzyme of ~32 kDa containing one Cu and one
In view of the high selectivity and fluorogenic properties of Zn ion in each of the subunits, which are stabilized by an intra-
SODO, we employed it to visualise changes in the expression chain disulfide bond.26 Our model predicted the interaction of
of Cu/Zn SOD in vivo. We used SODO to image Cu/Zn SOD SODO at the interface of the two subunits of Cu/Zn SOD (Fig.
during the onset of inflammatory processes in zebrafish 4a). The binding at this conserved hydrophobic pocket, which
embryos.9 We employed a zebrafish tail fin injury model of is away from the catalytic site of the enzyme, is consistent with
inflammation by amputating the tail fin of embryos at 3 days the previously observed species-independent response of
post fertilization (dpf),24 which allowed us to examine the in SODO (Fig. 2) and the fact that the enzymatic activity of Cu/Zn
vivo fluorogenic response of SODO in the inflammatory milieu. SOD remained unaffected by SODO (Fig. S7 in ESI). A closer
As shown in Fig. 3b, zebrafish undergoing inflammation examination of the binding revealed four hydrogen bonds
displayed bright fluorescence in the wound margins (white between SODO and hCu/Zn SOD: one hydrogen bond between
arrows), which correspond to inflamed areas where Cu/Zn SOD the oxygen atom of the ethoxy group and Val148, two
is highly expressed. High magnification images corroborated hydrogen bonds between the nitrogen atoms of the triazole
the expression of Cu/Zn SOD in the cytoplasm of epithelial cells ring and the residues Lys9 and Asn53, and a final hydrogen
(Fig. 3c). We further confirmed these results by measuring the bond between the meso-aniline group and Asp11 (Fig. 4b). The
levels of the sod1 gene before and after wounding using semi- binding analysis suggests that the fluorogenic response of
quantitative reverse transcription-polymerase chain reaction SODO is the result of combining the restriction in the rotation
(RT-PCR). As shown in Fig. 3d, the sod1 gene was highly of the fluorophore by forming four hydrogen bonds and the
upregulated 5 h after wounding, in agreement with the deactivation of the quenching PeT due to the migration to a
fluorescence emission profile of SODO in vivo (Fig. S8 in ESI). hydrophobic environment, as observed in our results from the
We also observed that SODO brightly stained oxidatively- in vitro characterisation assays.
stressed fibroblasts (Fig. S9 in ESI), containing high levels of In order to corroborate this hypothesis, we prepared two
Cu/Zn SOD.25
derivatives of SODO lacking the chemical groups involved in
the interaction with hCu/Zn SOD (Fig. 5). We synthesized
SODO1 as the derivative without the ethoxy group in the
amine ‘arm’ and SODO2 as the derivative lacking the triazole
nitrogen atoms (ESI for synthetic details and characterisation),
and compared their fluorogenic response to SODO after
binding to hCu/Zn SOD. SODO1 and SODO2 showed
remarkably lower fluorescence emission than SODO
,
confirming the relevance of both ethoxy
Fig. 3 In vivo imaging of Cu/Zn SOD in inflamed zebrafish after treatment with SODO
(10 ꢀM). a) Unwounded tail fin of a zebrafish embryo (3 dpf). b) Tail fin of a zebrafish
embryo (3 dpf) 5 h after wounding (5 hpw). Strong fluorescence emission is observed
towards the wound margin (white arrows). Bright spots in a) and b) away from the
wound edge correspond to auto-fluorescence signals from pigment cells. c) High
magnification images showing bright fluorescence from SODO at the wound margin (i)
compared to non-fluorescent unwounded areas (ii). d) Semi-quantitative RT-PCR of
Fig. 4 Molecular docking for the binding of SODO at hCu/Zn-SOD. a) Illustration of the
binding site of SODO (yellow) at the interface between the two monomeric subunits
(blue and pink) of hCu/Zn-SOD (Cu and Zn are shown as green and magenta spheres,
respectively). b) Suggested hydrogen bonding interactions between SODO and
different residues of hCu/Zn SOD.
sod1 and α-actin genes at 0 and 5 hpw with corresponding ladders. Scale bars (a, b): 40
μm; (c): 20 μm.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
View Article Online
DOI: 10.1039/C6CC00095A
COMMUNICATION
Journal Name
Foundation (2013T60613). J.C.E. acknowledges
a
NGS
scholarship. Y.F. is a Wellcome Trust Sir Henry Dale Fellow (WT
100104/Z/12/Z). Y.-T. C. acknowledges funding from the
National Medical Research Council (NMRC/CBRG/0015/2012).
M. V. acknowledges funding from Medical Research Council,
Marie Curie Career Integration Grant (333487) and the WT
Institutional Strategic Support Fund.
References
1
2
I. Fridovich, Annu. Rev. Biochem., 1995, 64, 97.
I. N. Zelko, T. J. Mariani and R. J. Folz, Free Radical Biol. Med.,
2002, 33, 337.
3
4
H. J. Lee, K. J. Korshavn, A. Kochi, J. S. Derrick and M. H. Lim,
Chem. Soc. Rev., 2014, 43, 6672.
M. A. Hough, J. G. Grossmann, S. V. Antonyuk, R. W. Strange,
P. A. Doucette, J. A. Rodriguez, L. J. Whitson, P. J. Hart, L. J.
Hayward, J. S. Valentine and S. S. Hasnain, Proc. Natl. Acad.
Sci. U. S. A., 2004, 101, 5976.
5
6
R. Noor, S. Mittal and J. Iqbal, Med. Sci. Monit., 2002, 8, 210.
R. Gonzales, C. Auclair, E. Voisin, H. Gautero, D. Dhermy and
P. Boivin, Cancer Res., 1984, 44, 4137.
L. Papa, G. Manfredi and D. Germain, Genes and Cancer,
2014, 5, 15.
A. Vasilaki and M. J. Jackson, Free Radical Biol. Med., 2013,
65, 317.
Fig.
5 Fluorogenic response of SODO derivatives upon incubation with serial
7
8
9
concentrations of hCu/Zn SOD in 20 mM Tris-HCl buffer (pH = 7.4). λexc.: 460 nm for
SODO and SODO1, 510 nm for SODO2. ФF in hCu/Zn SOD: SODO: 0.45, SODO1: 0.11,
SODO2: 0.22. Values are represented as means and error bars as standard deviations (n
= 3).
M. Marikovsky, V. Ziv, N. Nevo, C. Harris-Cerruti and O.
Mahler, J. Immunol., 2003, 170, 2993.
10 S. T. Ferreira, L. Stella and E. Gratton, Biophys. J., 1994, 66
1185.
11 E. Luchinat, A. Gianoncelli, T. Mello, A. Galli and L. Banci,
Chem. Commun., 2015, 51, 584.
,
and triazole groups for binding at hCu/Zn SOD (Fig. 5). These
results confirmed the need of four hydrogen bonds, which are
missing in the analogues SODO1 and SODO2 (Fig. S11 in ESI),
to restrict the torsional motion of SODO and induce its 12 D. V. Martyshkin, S. B. Mirov, Y. X. Zhuang, J. P. Crow, V.
Ermilov and J. S. Beckman, Spectrochim. Acta A Mol. Biomol.
Spectrosc., 2003, 59, 3165.
maximal fluorogenic response.
In summary, we have designed a new class of BODIPY
fluorogens with enhanced spectral properties by incorporating
both rotational restriction and PeT-quenching groups. These
new BODIPY fluorogens show excellent properties as polarity
probes with minimal background emission in aqueous media
and long Stokes shifts upon fluorescence activation. In vitro
studies identified one derivative (SODO) as a highly selective
fluorogenic probe for Cu/Zn SOD. SODO shows remarkable
fluorescence emission only after binding to Cu/Zn SOD with
very high selectivity over ROS-related enzymes and
metabolites as well as the other SOD isoforms (i.e. Mn-SOD
and Fe-SOD). The high selectivity of SODO enabled its use for
imaging Cu/Zn SOD in vivo during the onset of an inflammatory
13 P. Shieh, V. T. Dien, B. J. Beahm, J. M. Castellano, T. Wyss-
Coray and C. R. Bertozzi, J. Am. Chem. Soc., 2015, 137, 7145.
14 Y. Hori, T. Norinobu, M. Sato, K. Arita, M. Shirakawa and K.
Kikuchi, J. Am. Chem. Soc., 2013, 135, 12360.
15 J. C. Er, M. K. Tang, C. G. Chia, H. Liew, M. Vendrell and Y.-T.
Chang, Chem. Sci., 2013, 4, 2168.
16 H. Sunahara, Y. Urano, H. Kojima and T. Nagano, J. Am.
Chem. Soc., 2007, 129, 5597.
17 T. Kowada, H. Maeda and K. Kikuchi, Chem. Soc. Rev., 2015,
44, 4953-4972.
18 A. Vázquez-Romero, N. Kielland, M. J. Arévalo, S. Preciado, R.
J. Mellanby, Y. Feng, R. Lavilla and M. Vendrell, J. Am. Chem.
Soc., 2013, 135, 16018.
19 Y.-H. Ahn, J.-S. Lee and Y.-T. Chang, J. Comb. Chem., 2008, 10
376.
,
response in a zebrafish tail fin injury model. Furthermore, we 20 Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose and T.
Nagano, J. Am. Chem. Soc., 2005, 127, 4888.
performed computational modelling to analyse the binding of
SODO at Cu/Zn SOD. Structure-activity studies suggest that the
binding occurs at the interface of the two enzymatic subunits
and involves four residues to restrict the torsional motion of
21 W. Qin, V. Leen, T. Rohand, W. Dehaen, P. Dedecker, M. van
der Auweraer, K. Robeyns, L. van Meervelt, D. Beljonne, B.
van Averbeke, J. N. Clifford, K. Driesen, K. Binnemans and N.
l. Boens, J. Phys. Chem. A, 2008, 113, 439.
the BODIPY fluorophore and deactivate its PeT-quenching 22 M. Vendrell, G. G. Krishna, K. K. Ghosh, D. Zhai, J.-S. Lee, Q.
Zhu, Y. H. Yau, S. G. Shochat, H. Kim, J. Chung and Y.-T.
Chang, Chem. Commun., 2011, 47, 8424.
23 J.-S. Lee, M. Vendrell and Y.-T. Chang, Curr. Opin. Chem. Biol.
2011, 15, 760.
24 L. Li, B. Yan, Y. Q. Shi, W. Q. Zhang and Z. L. Wen, J. Biol.
Chem. 2012, 287, 25353.
groups. SODO is the first fluorogenic probe for Cu/Zn SOD and
represents a unique probe for the detection and in vivo
imaging of Cu/Zn SOD during the progression of inflammatory
disorders.
25 J. H. Chen, K. Stoeber, S. Kingsbury, S. E. Ozanne, G. H.
Williams and C. N. Hales, J. Biol. Chem., 2004, 279, 49439.
26 J. A. Tainer, E. D. Getzoff, K. M. Beem, J. S. Richardson and D.
C. Richardson, J. Mol. Biol., 1982, 160, 181.
L.Z. acknowledges the ‘973’ program (2014CB932002), the
Natural Science Foundation of China (11105150) and the
Special Financial Grant from China Postdoctoral Science
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins