A novel flavin derivative reveals the impact of glucose on oxidative stress in adipocytes

Article abstract of DOI:10.1039/c4cc03464c

We present the synthesis and characterisation of naphthalimide-flavin redox sensor 1 (NpFR1), a novel flavin molecule that is almost non-fluorescent in reduced form, but oxidation gives rise to a 125-fold increase in fluorescence. Confocal microscopy experiments confirm that NpFR1 is sensitive to redox changes within living cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03464c

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A novel flavin derivative reveals the impact of
glucose on oxidative stress in adipocytes†
Cite this: Chem. Commun., 2014,
50, 8181
Jonathan Yeow, Amandeep Kaur, Matthew D. Anscomb and Elizabeth J. New*
Received 8th May 2014,
Accepted 5th June 2014
DOI: 10.1039/c4cc03464c
www.rsc.org/chemcomm
We present the synthesis and characterisation of naphthalimide-flavin
redox sensor 1 (NpFR1), a novel flavin molecule that is almost non-
fluorescent in reduced form, but oxidation gives rise to a 125-fold
increase in fluorescence. Confocal microscopy experiments confirm
that NpFR1 is sensitive to redox changes within living cells.
Oxidative stress – a sustained elevation in the oxidative capacity of
cell – arises from a build-up of oxidising cellular components,
such as reactive oxygen species (ROS) and redox-active metals.1,2 It
is a natural consequence of aging, but is also implicated in many
diseases such as cardiovascular disease,3 arthritis,4 Alzheimer’s
disease,5 diabetes1,6 and obesity.7,8 Oxidative stress is commonly
studied by monitoring the causes of increased oxidative capacity
(such as levels of an individual ROS) or analysing the downstream
effects (for example, expression of antioxidant genes). A much
Fig. 1 Generic structure of flavin, showing alloxazine and isoalloxazine forms.
clearer picture will be gained, however, from the cell’s oxidative make flavins ideal for incorporation into a fluorescent redox
capacity itself. This can be best achieved by measurement of the sensor. Since flavins are naturally found within cells, and con-
redox state of a cell, or sub-cellular compartment. To date, a tribute to cellular autofluorescence,15 we aimed to design a probe
limited number of fluorescent redox sensors have been developed, with fluorescence distinct from naturally-occurring flavins. This
based on both fluorescent proteins9 and small organic mole- was achieved by incorporating a naphthalimide fluorophore into
cules.10–12 The challenge in the design of such probes is in tuning the final probe. The probe design also included a non-hydrogen
the reduction potential of the probe such that its dynamic range is atom at the N-10 position of the flavin, as this is required to
appropriate to measurement of cellular processes. We report here stabilise the isoalloxazine form over the alloxazine form, and to
a novel fluorescent redox sensor, naphthalimide-flavin redox maintain reversibility of reduction.16
sensor 1 (NpFR1), and its application to the study of oxidative
stress in adipocytes.
NpFR1 was synthesised by the alloxan monohydrate method
first reported by Kuhn and Reinemund (Scheme 1).17 Alkylation of
NpFR1 was designed based on the flavin moiety (Fig. 1), a 4-bromo-1,8-naphthalic anhydride (1) gave 4-bromo-N-butyl-1,8-
group which plays important roles in sub-cellular redox processes, naphthalimide (2), followed by nitration of 2 to afford 3-nitro-4-
in the redox cofactors flavin adenine dehydrogenase (FAD) and bromo-N-butyl-1,8-naphthalimide (3). Alkylation with propylamine
flavin mononucleotide (FMN), and in the vitamin riboflavin.13 in acetonitrile gave the corresponding propylamino compound (4),
Flavin reductions are therefore well tuned to biologically-relevant which was reduced with stannous chloride to give a naphthalimide
potentials. In addition, flavins are typically fluorescent in oxidised derivative bearing adjacent primary and secondary amines (5), a
form and non-fluorescent upon reduction.14 These properties suitable substrate for the alloxan monohydrate reaction. 5 was
reacted with alloxan monohydrate in the presence of boric acid to
give NpFR1.
School of Chemistry, The University of Sydney, New South Wales, 2006, Australia.
Photophysical characterisation of NpFR1 was performed in
E-mail: elizabeth.new@sydney.edu.au
HEPES buffer (100 mM, pH 7.4). NpFR1 showed maximum
absorption at 395 and 463 nm (log e = 6.2 and 4 respectively),
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4cc03464c
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Scheme 1 Synthesis of naphthalimide-flavin redox sensor 1 (NpFR1).
and maximum emission at 545 nm (F = 0.186). This represented a
27 and 14 nm red-shift in absorption and emission respectively
compared to riboflavin (Fig. S1, ESI†). While we had originally
expected a larger shift in the fluorescence upon addition of the
naphthalimide group, the new molecule fulfilled our aims of a
distinct emission spectrum that could be resolved by spectral
imaging confocal microscopy. Treatment of NpFR1 with various
mild reductants, including sodium thiosulfate, sodium cyano-
borohydride, dithiothreitol (DTT) and glutathione (GSH) gave the
reduced form of the probe, which exhibited 110-fold lower
absorbance and 125-fold lower emission (Fig. 2a). NpFR1 could
be re-oxidised by air or by hydrogen peroxide, restoring its original
fluorescence, and this oxidation–reduction cycle could be
repeated multiple times without loss of response (Fig. 2b). Cyclic
voltammetry confirms the reversibility of the process, with a
reduction potential of À336 mV vs. SHE (Fig. S2, ESI†). Interest-
ingly, upon higher energy excitation (380 nm), an additional
emission peak could be observed at 440 nm which was unaffected
by reduction and oxidation, suggesting potential application
as a ratiometric probe (Fig. S3, ESI†). Re-oxidation occurred
very rapidly, with 95% of the initial fluorescence regained after
4 minutes (Fig. S4, ESI†). The magnitude of the fluorescence
change upon reduction and the kinetics of re-oxidation for NpFR1
are very similar to those of riboflavin,18 suggesting a similar
sensing mechanism, involving fluorescence from the planar
oxidised flavin and loss of the fluorescence in the non-planar,
reduced form. Control experiments confirmed that the emissions Fig. 2 Photophysical behaviour of NpFR1. (a) Fluorescence emission of NpFR1
(5 mM, lex = 405 nm) with the incremental addition of sodium dithionite.
of NpFR1 in oxidised and reduced form are constant over the pH
(b) Fluorescence response of NpFR1 to cycles of oxidation and reduction. Reduction
range 2 to 9 (Fig. S5, ESI†) and were unaffected by the presence
was achieved with sodium dithionite (100 mM) followed by re-oxidation with 250 mM
of common metal ions (Fig. S6, ESI†). Fluorescence intensity
H2O2. Spectra were recorded 5 min after the addition of reducing and oxidising
increases were observed for a range of diverse oxidants (Fig. S7,
ESI†), demonstrating the utility of NpFR1 as a probe of the general
redox environment. Furthermore, the probe shows sensitivity
for small changes in reduced:oxidised glutathione ratios in a
biologically-relevant range (Fig. S8, ESI†).19
agents. All spectra were acquired in HEPES buffer (100 mM, pH 7.4).
Having demonstrated the redox sensitivity and reversibility of
NpFR1, we next sought to test its ability to respond to changes in
8182 | Chem. Commun., 2014, 50, 8181--8184
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Fig. 4 Changes in oxidative capacity of 3T3-L1 pre-adipocytes and
mature adipocytes incubated under different glucose conditions. (a), (b),
(d), and (e) are confocal microscopy images of 3T3-L1 cells treated
incubated with NpFR1 (50 mM, 2 hours). (a) Pre-adipocytes cultured in
low glucose (10 mM, 7 d), (b) pre-adipocytes cultured in high glucose
(25 mM, 7 d), (d) mature adipocytes cultured in low glucose (10 mM, 7 d), (e)
mature adipocytes cultured in high glucose (25 mM, 7 d). Scale bars
represent 50 mm. Graphs show relative integrated emission from 510 nm
to 610 nm from (c) 3T3-L1 preadipocytes and (f) mature adipocytes. Values
are the mean ratio generated from the intensity from ten fields of cells.
Error bars represent standard error measurement (s.e.m.).
mature adipocytes, we found that fluorescence intensity corre-
lated with lipid accumulation, indicating that oxidative capacity
increases as adipocytes mature (Fig. S9, ESI†). We cultured
preadipocytes and mature adipocytes in differing concentra-
tions of glucose, and measured their oxidative capacity by
microscopy using NpFR1. For both cell types, cells cultured in
higher glucose concentrations exhibited higher fluorescence,
indicative of an increased oxidative capacity (Fig. 4). These
results are consistent with higher levels of metabolism under
conditions of greater glucose consumption, which leads to
greater superoxide formation due to increased electron leakage
from the electron transport chain. They are also consistent with
findings that obesity, in which cells experience constitutively
Fig. 3 Imaging of NpFR1 in 3T3-L1 preadipocytes treated with (a) vehicle
control (b) H2O2 (100 mM, 2 min), (c) NpFR1 (50 mM, 2 h) and (d) NpFR1
(50 mM, 2 h) followed by H2O2 (100 mM, 2 min). Scale bar represents 50 mm,
l
ex = 405 nm. (e) Integrated emission from 510 nm to 610 nm. Values are
the mean ratio generated from the intensity from five fields of cells. Error
bars represent standard error measurement (s.e.m.).
redox state in cultured cells. 3T3-L1 preadipocytes that had been higher levels of glucose, can lead to oxidative stress.7,20
incubated with 50 mM NpFR1 showed noticeable fluorescence In summary, we have presented here NpFR1, a new fluorescent
(Fig. 3c), whilst untreated, control cells exhibited negligible sensor for redox state in biological systems. NpFR1 exhibits a
fluorescence (Fig. 3a). Cells which had been treated with H2O2 greater than 100-fold increase in fluorescence upon oxidation, and
for 2 minutes after incubation with NpFR1 showed a significant oxidation–reduction events are highly reversible. NpFR1 is not
fluorescence enhancement (Fig. 3d), consistent with oxidation selective for any individual oxidising or reducing species, but
of the cellular environment. Importantly, cells treated with H2O2 rather the ratio of oxidised to reduced NpFR1 reflects the redox
but no NpFR1 showed no fluorescence, confirming that any status of its environment. We have been able to demonstrate the
fluorescence observed in this experiment is due to the probe, and utility of NpFR1 in the observation of glucose-induced oxidative
not to the oxidation of endogenous flavins. This provides impor- stress in cultured adipocytes. This probe is a valuable contribution
tant validation that there is no interference from intracellular to the limited number of reversible probes for redox state. We have
flavins, in either oxidised or reduced form.
been able to ensure that we are operating at biologically-relevant
With these microscopy studies showing that NpFR1 is able to reduction potentials by utilising a biologically-relevant sensing
report on changes in oxidative capacity within cells, we aimed to group. We continue to explore the utility of NpFR1 in various
use the probe to detect induced changes in redox state, and biological systems, and we are developing improved versions for
specifically to study the effect of glucose in adipocytes. For these the study of intracellular oxidation events.
studies, both 3T3-L1 preadipocytes and mature 3T3-L1 adipo-
This work was supported by a Discovery Early Career
cytes were studied. By imaging cells as they differentiated into Researcher Award from the Australian Research Council and
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