Synthesis of Nitro-Aryl Functionalised 4-Amino-1,8-Naphthalimides and Their Evaluation as Fluorescent Hypoxia Sensors

Article abstract of DOI:10.1002/chem.202002088

Fluorescent sensors are a vital research tool, enabling the study of intricate cellular processes in a sensitive manner. The design and synthesis of responsive and targeted probes is necessary to allow such processes to be interrogated in the cellular environment. This remains a challenge, and requires methods for functionalisation of fluorophores with multiple appendages for sensing and targeting groups. Methods to synthesise more structurally complex derivatives of fluorophores will expand their potential scope. Most known 4-amino-1,8-naphthalimides are only functionalised at imide and 4-positions, and structural modifications at additional positions will increase the breadth of their utility as responsive sensors. In this work, methods for the incorporation of a hypoxia sensing group to 4-amino-1,8-naphthalimide were evaluated. An intermediate was developed that allowed us to incorporate a sensing group, targeting group, and ICT donor to the naphthalimide core in a modular fashion. Synthetic strategies for attaching the hypoxia sensing group and how they affected the fluorescence of the naphthalimide were evaluated by photophysical characterisation and time-dependent density functional theory. An extracellular hypoxia probe was then rationally designed that could selectively image the hypoxic and necrotic region of tumour spheroids. Our results demonstrate the versatility of the naphthalimide scaffold and expand its utility. This approach to probe design will enable the flexible, efficient generation of selective, targeted fluorescent sensors for various biological purposes.

Full text of DOI:10.1002/chem.202002088

Accepted Article
Accepted Article
Title: Synthesis of nitro-aryl functionalised 4-amino-1,8-naphthalimides
and their evaluation as fluorescent hypoxia sensors
Authors: Liam D Adair, Natalie Trinh, Pauline M. Vérité, Denis
Jacquemin, Katrina A. Jolliffe, and Elizabeth New
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202002088
Link to VoR: https://doi.org/10.1002/chem.202002088
01/2020

10.1002/chem.202002088
Chemistry - A European Journal
FULL PAPER
Synthesis of nitro-aryl functionalised 4-amino-1,8-naphthalimides
and their evaluation as fluorescent hypoxia sensors
Liam D. Adaira, Natalie Trinha, Pauline M. Véritéb, Denis Jacqueminb, Katrina A. Jolliffea,c,d, and
Elizabeth J. Newa,c,d
[a]
[b]
Dr L. D. Adair, N. Trinh, Prof K. A. Jolliffe, A/Prof E. J. New
School of Chemistry
The University of Sydney
NSW 2006, Australia
E-mail: elizabeth.new@sydney.edu.au
P. M. Vérité, Prof D. Jacquemin
CNRS
Université de Nantes
CEISAM UMR 6230
F-44000 Nantes, France
[c]
[d]
Prof K. A. Jolliffe, A/Prof E. J. New
The University of Sydney Nano Institute (Sydney Nano)
The University of Sydney
NSW 2006, Australia
Prof K. A. Jolliffe, A/Prof E. J. New
Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science
The University of Sydney
NSW 2006, Australia
Supporting information for this article is given via a link at the end of the document
Abstract: Fluorescent sensors are a vital research tool, enabling the
study of intricate cellular processes in a sensitive manner. The design
and synthesis of responsive and targeted probes is necessary to allow
such processes to be interrogated in the cellular environment. This
remains a challenge, and requires methods for functionalisation of
fluorophores with multiple appendages for sensing and targeting
groups. Methods to synthesise more structurally complex derivatives
of fluorophores will expand their potential scope. Most known 4-
amino-1,8-naphthalimides are only functionalised at imide and 4-
positions, and structural modifications at additional positions will
increase the breadth of their utility as responsive sensors. Here we
evaluate methods for the incorporation of a hypoxia sensing group to
4-amino-1,8-naphthalimide. We developed an intermediate that
allowed us to incorporate a sensing group, targeting group, and ICT
donor to the naphthalimide core in a modular fashion. Synthetic
strategies for attaching the hypoxia sensing group and how they
affected the fluorescence of the naphthalimide were evaluated by
photophysical characterisation and time-dependent density functional
theory. We then rationally designed an extracellular hypoxia probe
that could selectively image the hypoxic and necrotic region of tumour
spheroids. Our results demonstrate the versatility of the naphthalimide
scaffold and expand its utility. This approach to probe design will
enable the flexible, efficient generation of selective, targeted
fluorescent sensors for various biological purposes.
targeted, responsive fluorescent sensors and their development
remains challenging.[1] Responsive fluorescent sensors typically
comprise a sensing moiety tethered to a fluorophore, often
through a conjugated linker. A particular challenge in the rapid
development of new fluorescent sensors is the identification of
fluorophore precursors to which sensing groups can be readily
conjugated. To achieve this, it is important to identify fluorophore-
linker scaffolds that enable facile conjugation of sensing groups,
and for which the analyte induces a measurable fluorescence
response.
4-Amino-1,8-naphthalimides are donor-acceptor molecules with
an internal charge-transfer (ICT) excited state, and typically
display strong fluorescence emission.[2] They are commonly used
in bioimaging due to their chemical stability, photostability, high
quantum yields, and large Stokes shifts.[3] Responsive 4-amino-
1,8-naphthalimide based sensors have been reported for several
analytes including metal ions,[4] anions,[5] and reactive small
molecules.[6] Functionalisation at the imide or the 4-position
predominates the literature (Figure 1), with additional
functionalisation uncommon. Because of the limited scope for
derivatisation to date, naphthalimides remain an underexplored
class of fluorophore, and in order to maximise the potential of
naphthalimides as responsive fluorescent sensors, it is essential
to develop methods for functionalisation at other positions.
Importantly, we identified the need to determine robust synthetic
methods for introducing a broad range of sensor groups. This will
allow rapid access to more structurally complex derivatives and
enable more efficient development of a set of fluorescent sensors.
We were particularly interested in a third point of attachment
which could be functionalised orthogonally to the imide and 4-
position.
Introduction
Fluorescent sensing, particularly when applied to bioimaging, is a
vital tool for studying chemical processes in biological systems.
Many unanswered biochemical questions require selective,
1
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hypoxic regions can aid in tumorigenicity and promote changes
that lead to disease progression, with acute hypoxia being
associated with more aggressive tumour phenotypes and
resistance to treatment.[12] Fluorescence imaging is a useful
technique for imaging hypoxic regions of tumours as it is non-
invasive, highly sensitive, and can provide spatiotemporal
resolution. Other commonly-used methods for measuring hypoxia,
such as microelectrodes, are invasive techniques.[13] To
investigate the ease with which the nitro-aryl hypoxia sensing
groups could be introduced onto the 4-amino-1,8-naphthalimide
scaffold, together with the impact of different linkers on the
photophysical properties of these systems we prepared a small
series of 6-substituted-4-amino-1,8-naphthalimides and report
here our investigations of these as fluorescent sensors of hypoxic
microenvironments..
R1
N
O
O
1
4
8
7
2
6
3
5
N
R3 R2
Figure 1. 4-Amino-1,8-naphthalimide structure.
We have previously reported several substituted 4-amino-1,8-
naphthalimide derivatives with various simple structural
modifications and found that the addition of substituents,
particularly at the 6-position, expanded the range of emission
wavelengths available.[7] An additional electron donating group at
this position was shown to have a large impact as a secondary
charge transfer (CT) donor, making it a promising position for
incorporation of sensing groups. Among the compounds
previously synthesised was a 6-bromo analogue, which had the
potential to be used as a synthetic handle. This work aims to
assess the synthetic strategies that are most promising for
attaching sensing groups at this position, both in terms of optimal
photophysical properties, and synthetic considerations.
We envisaged that the incorporation of nitro-aryl substituents onto
the 4-amino-1,8-naphthalimide scaffold would provide a suitable
model system in which to examine the above properties,
anticipating that the resulting molecules would be useful probes
for sensing hypoxic environments, since aromatic nitro
compounds are known to be reduced to the corresponding aniline
in hypoxic conditions.[8] Nitro groups generally quench
fluorescence,[9] whereas in amine-containing fluorophores, strong
fluorescence is common. This difference in fluorescence
properties, coupled with the ease of reduction of the nitro group
makes this an attractive strategy for the design of fluorogenic
hypoxia probes and an ideal model system for our studies.[10]
Hypoxia develops in advanced solid tumours due to uncontrolled
proliferation of cancer cells, resulting in regions that are distant
from blood vessels, or possess inadequate tumoral vasculature,
and therefore are starved of oxygen.[11] The development of these
Results and Discussion
Design/Synthesis
We chose to incorporate the aryl-nitro functionality via four robust
methodologies that are commonly used in synthetic chemistry,
with readily available substrates: directly conjugated by a Suzuki-
coupling, with an alkyne linker using a Sonogashira coupling, a
triazole linker using Cu-catalysed azide-alkyne cycloaddition
(CuAAC) click reaction, and by Buchwald-Hartwig amination. We
designed four nitro-compounds (1NO2 - 4NO2), and their reduced
aniline analogues (1NH2 - 4NH2) (Figure 2), to investigate how the
aryl-nitro functionality is incorporated impacts the emission.
The reported synthesis of 6-bromonaphthalimide 7 contains a
non-selective nitration and is low yielding for the desired
regioisomer.[7] We therefore developed an alternative route to 7
with selective bromination of 4-nitro-1,8-naphthalic anhydride as
the initial step. Commercially available 4-nitronaphthalic
anhydride 5 was brominated regioselectively at the 6-position
using NBS in strongly acidic conditions, giving the desired product
6 with no need for chromatographic purification. This substituted
anhydride 6 is a key intermediate and allows for facile and
O
N
O
O
N
O
O
N
O
N
O
O
N
O2
N
O2
N
N
N
N
HN
H
HN
HN
HN
N
O2
N
O2
3NO2
1NO2
2NO2
4NO2
O
N
O
O
N
O
O
N
O
O
N
O
H2N
H2N
N
N
N
HN
H
HN
H2N
N
HN
HN
H2N
1NH2
2NH2
3NH2
4NH2
Figure 2. Structure of 6-aryl-nitro naphthalimides 1 – 4NO2 and their reduced aniline derivatives 1 – 4NH2.
2
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O
N
O
HN
O
N
O
R
1NO
1NH
R = NO2
R = NH2
,
,
2
2
HN
(iii)
H2N
2NH2
(iv)
O
N
O
O
O
O
O
O
O
O
N
O
O
N
O
(v)
(vi)
(i)
(ii)
Br
Br
HN
2NO2
NO2
NO2
HN
HN
5
6
O2N
7
8
(vii)
(viii)
O
N
O
O
N
O
O
N
O
H2N
(ix) O2N
R
N
N
H
N
H
N
HN
N
HN
4NH2
HN
4NO2
R = NO2 3NO
,
2
R = NH2 3NH
,
2
Scheme 1. Synthesis of 1NO2 – 4NO2 and 1NH2 – 4NH2. Reagents and conditions: (i) NBS, H2SO4, 60 °C, 93%; (ii) nBuNH2, EtOH, 80 °C, 55%; (iii) 4-
nitrophenyl boronic acid or 4-aminophenyl boronic acid, Pd(PPh3)4, K2CO3, THF:H2O, 70 °C, 41% (1NO2), 98% (1NH2); (iv) 4-ethynylaniline, Pd(PPh3)2Cl2,
CuI, THF:Et3N, 80 °C, 76%; (v) 1. TMS-acetylene, Pd(PPh3)2Cl2, CuI, THF:Et3N, 80 °C, 2. TBAF, THF, 69% (over 2 steps); (vi) 4-bromo-1-nitrobenzene,
Pd(PPh3)2Cl2, CuI, THF:Et3N, 80 °C, 62%; (vii) 1-azido-4-nitrobenzene or 4-azidoaniline, CuSO4.5H2O, NaAsc, Na2CO3, THF:H2O, 66% (3NO2), 98%
(3NH2); (viii) Pd(OAc)2, RuPhos, Cs2CO3, toluene, 110 °C, 72%; (ix) Pd/C, NH4CO2H, EtOAc:MeOH, 98%.
selective functionalisation at each of the 4-, 6- and anhydride
positions of the scaffold. Treatment with excess n-butylamine in
EtOH gave bromo-naphthalimide 7, in good overall yield leaving
the 6-bromide handle for further functionalisation. Suzuki coupling
of 7 with 4-nitrophenylboronic gave the desired product 1NO2.
The aniline derivative 1NH2 was similarly synthesised by Suzuki
coupling, using 4-aminophenylboronic acid. Sonogashira
coupling of 7 directly with 4-nitrophenylacetylene proved to be
very low yielding. However, using TMS-acetylene as the coupling
partner, followed by desilylation with TBAF, gave alkyne 8 in good
yield over two steps. Subsequent coupling with 4-bromo-1-
nitrobenzene gave the desired compound 2NO2. In contrast, 4-
ethynylaniline was successfully coupled directly to the 6-
bromonaphthalimide to generate the corresponding aniline
analogue 2NH2 in good yield. Acetylene 8 pleasingly also gave an
intermediate to allow incorporation of the nitro-aryl functionality
using CuAAC. 1-Azido-4-nitrobenzene and 4-azidoaniline were
each prepared from the corresponding boronic acids via copper
mediated conditions.[14] CuAAC reaction of acetylene 8 with 1-
azido-4-nitrobenzene and 4-azidoaniline, respectively using
CuSO4 in THF:H2O gave the desired triazoles 3NO2 and 3NH2 in
excellent yield for both products. Buchwald-Hartwig amination of
7 using 4-nitroaniline gave the desired product 4NO2. Subsequent
reduction of the nitro group using Pd-catalysed transfer
hydrogenation with ammonium formate as the hydrogen source,
gave the aniline derivative 4NH2 (Scheme 1).
Photophysical studies
The photophysical properties of 1NO2 - 4NO2 and 1NH2 - 4NH2
were next measured in CH2Cl2 to determine both how these
changed upon reduction of the nitro group and how these varied
with the nature of the linker (Figure 3, Table 1). For the NO2 series,
compounds 3NO2, and to a lesser extent compound 1NO2, show
appreciable fluorescence, whereas 2NO2 and 4NO2 are almost
completely quenched. In all four cases, reduction to the aniline
resulted in a turn-on in fluorescence intensity rather than a change
in emission wavelength. 4NH2 has the weakest emission of the
NH2 series, but also shows a significant bathochromic shift (Δ 30
nm).
Table 1. Comparison of excitation and emission maxima (in CH2Cl2) of 1 – 4
and unsubstituted N-butyl-4-butylamino-1,8-naphthalimide.
λex (nm)
λem (nm)
Unsubstituted
421
503
NO2
442
445
444
456
NH2
440
450
446
459
NO2
507
520
511
497
NH2
509
509
512
527
1
2
3
4
3
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Figure 4: Density difference plots for the lowest transition in all compounds. The
blue and red lobes represent regions of decrease and increase of density upon
photon absorption. Contour threshold 8 × 10-4 au.
Figure 1: Excitation and emission spectra of 1 (1 µM in CH2Cl2, λex 440 nm, λem
510 nm), 2 (1 µM in CH2Cl2, λex 440 nm, λem 510 nm), 3 (1 µM in CH2Cl2, λex
440 nm, λem 510 nm), 4 (5 µM in CH2Cl2, λex 450 nm, λem 520 nm).
Next, we examined the emission behaviour. In the NH2 series, the
S1 optimisations led to small structural changes in 1NH2 – 3NH2
with computed emission wavelengths reasonably fitting the
values that were observed experimentally. Again, 4NH2 was an
exception to this general rule, as the optimisation of the lowest
state led to a very low-lying “theoretical fluorescence” at 1.64 eV
with a large ICT effect involving the amine at the 6-position (as in
the absorption figure 4). Such a low-lying transition will be dark
experimentally, due to the well-known energy gap law, and hence
the very low residual emission observed experimentally is likely
coming from a side effect, e.g., an emission from S2 or a fast
emission from S1 before its full relaxation. In that sense, the CT in
4NH2 is too large and it consequently becomes an ineffective
emitter. On the other hand, the three other NH2 compounds show
the more classical behaviour of effective emitters with a more
moderate CT, which fits with the experimental observations. As
stated in the introduction, it is well known that nitro groups quench
fluorescence, hence it is not surprising to see that all the NO2
compounds are less bright than their corresponding NH2 systems.
Nevertheless, there are notable differences in the nitro series. For
1NO2 – 4NO2 the theoretical calculations predict fluorescence in
the 2.52 – 2.62 eV range, which again fits the measurements
(2.38 – 2.46 eV), with similar excited state topologies for all four
compounds. Therefore, the experimental differences, and
specifically the fact that (relative) quenching is much stronger in
1NO2 and 2NO2 than in 3NO2, should be explained by another
phenomena. We reasoned that intersystem crossing (ISC) might
be taking place and we have therefore computed S-T gaps on the
lowest S1 geometry for 1NO2 – 4NO2. In all cases, the gap
between S1 and T1 is very large (>0.6 eV) and therefore ISC is
unlikely with the lowest triplet. In contrast, the S1-T2 gaps are very
low in 4NO2 (0.00 eV) and 2NO2 (0.07 eV) and strong quenching
would be expected, as is indeed found experimentally. In 3NO2,
T2 lies 0.49 eV higher than S1 making ISC very unlikely, consistent
with the relatively bright fluorescence observed. 1NO2 is
intermediate with a S1-T2 gap of 0.18 eV. These trends are clearly
consistent with the experimental measurements.
Computational studies
To shed more light on the photophysical properties of the
compounds, we have used first principle calculations using a
technique detailed in the SI and successfully applied to many
dyes before.[15] Table 2 reports the computed positions of the
lowest dipole-allowed transitions. The agreement between the
theoretical and experimentally observed values is satisfactory: i)
the mean absolute error is 0.20 eV, typical of such an
approach;[15a] ii) theory overshoots the experimental value, which
is the expected error sign when neglecting vibronic couplings;[15b]
iii) the experimental fact that only 4NH2 undergoes a significant
bathochromic shift is reasonably reproduced.
Table 2. Comparison between theoretical (CC2, see the SI) and experimental
excitation wavelengths given in eV.
1NO2
2NO2
3NO2
4NO2
1NH2
2NH2
3NH2
4NH2
Theoretical
2.94
2.70
2.89
2.72
2.93
2.71
2.93
2.78
2.99
2.75
2.95
2.72
2.94
2.71
2.69
2.58
Experimental
Figure 4 represents the density difference plots for all compounds.
In all cases, except for 4NH2, there is a significant ICT from the
N-butylamino group (mostly in blue, donor) at position 4 to the
core of the compound (mostly in red, acceptor). The side groups
added at the 6-position play a minor role. 4NH2 is a notable
exception, with a strong ICT from the amine at the 6-position to
the core of the molecule, an effect well in line with the large
redshift measured for that compound. In 4NO2, there is likely a
counterbalancing effect between the nitro and amino groups, and
hence, a small overall effect is evident.
4
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With these results we then evaluated which scaffold would be
optimal for a useful hypoxia sensor. As compound 3 had
significant fluorescence in the 3NO2 form (Φ 0.06 in EtOH, Table
S1), reducing the turn on response and compound 4NH2 was not
bright enough to be a promising candidate, these scaffolds were
ruled out. The turn-on response for compounds 1 and 2 made
them the most promising candidates for a hypoxia probe. Non-
radiative decay in 4-amino-1,8-naphthalimides is generally
increased in aqueous solvents,[16] but interestingly, the
fluorescence of 2NH2 was significantly quenched in aqueous
buffer, whereas 1NH2 remained bright (Figure 5). 1NH2 was also
the scaffold with the highest quantum yield (Φ 0.42 in EtOH, Table
S1).
butylamine in MeCN gave the bromide 10 in excellent yield. The
bromide was then subjected to Suzuki coupling with 4-
nitrophenylboronic acid to give di-ester 11, followed by basic
hydrolysis of the methyl esters to give the final extracellular
hypoxia probe Asp-1NO2 (Scheme 2).
CO2Me
CO2Me
O
MeO2C
CO2Me
MeO2C
O
MeO2C
O
NH2
O
O
O
N
O
N
nBuNH2
MeCN, 82 °C
97%
1,4-dioxane
100 °C
87%
Br
Br
Br
NO2
NO2
HN
6
9
10
B(OH)2
CO2Me
O
CO2H
O2N
MeO2C
O
HO
O
2C
N
N
O
, Cs2CO3
2O, 75 °C
Pd(PPh3)4
THF:H
NaOH (aq), THF
93%
88%
HN
HN
Asp-1NO2
O2N
O2N
11
Scheme 2. Synthesis of extracellular hypoxia sensor Asp-1NO2.
With probe Asp-1NO2 in hand, we first investigated the
fluorescence response by chemical reduction with sodium
dithionite in aqueous solution. Asp-1NO2 showed a strong
fluorescence emission increase at 550 nm upon reduction (Figure
6). The cytotoxicity to DLD-1 cells was assessed, by Alamar blue
assay, and Asp-1NO2 was shown to be non-toxic to cells after 24
h incubation, indicating its suitability for biological studies (Figure
S3).
Figure 5. Excitation and Emission spectra of 1 and 2 (50 µM in HEPES buffer
100 mM pH 7.4)
Preliminary biological studies
1NO2 was therefore the most promising hypoxia sensor amongst
those synthesised, and its potential utility was therefore evaluated
in preliminary biological studies. Firstly, the cytoxicity of 1NO2 was
assessed by Alamar blue assay, which confirmed that the
compound was not toxic to DLD-1 cells (Figure S3). Next, we
carried out a preliminary imaging experiment with a tumour
spheroid model. DLD-1 cells were allowed to grow into three-
dimensional spheroids of varying size. Such systems develop a
hypoxic region which is far from the oxygenated cell culture media,
mimicking the environment of a solid tumour. Imaging of
spheroids incubated with 1NO2 showed high fluorescence
intensity at the periphery of the spheroid, consistent with rapid
uptake of probe into the outer cells, preventing diffusion into the
hypoxic region of the spheroid (Figure S4). Furthermore, the
bright fluorescence intensity observed indicates that 1NO2 is too
easily reduced in the highly reducing intracellular environment,
consistent with a previous work.[8] We therefore sought to modify
this system to enable imaging of the hypoxic region by developing
an extracellular version of 1NO2, in which cellular uptake is
inhibited through the incorporation of an aspartic acid group that
would exist in the dicarboxylate form at biologically relevant pH.
Figure 6. Fluorescence titration of Asp-1NO2 with increasing concentration of
sodium dithionite (10 µM in PBS pH 7.4, λex 445 nm).
Asp-1NO2 was then used in an analogous imaging experiment to
that performed with 1NO2. Spheroids of varying size were
incubated with probe Asp-1NO2 then imaged and a turn-on in
fluorescence was observed approximately 100 µm from the
spheroid surface. This distance corresponds to the limit of where
oxygen can diffuse, and the beginning of the hypoxic region.[17] In
the larger spheroids, fluorescence was also observed at the very
centre of the spheroid, this corresponds to the necrotic region. In
the smallest spheroid, which is lacking a hypoxic region, no
fluorescence was observed demonstrating the selectivity for
hypoxic microenvironments (Figure 7). The clear differences in
fluorescence of 1NO2 and Asp-1NO2 within spheroids (Figures
S4, 7) confirm the role of the aspartic acid group in enabling the
probe to travel between cells into the centre of the spheroid.
Synthesis of extracellular analogue
We envisaged that naphthalic anhydride 6 could be functionalised
selectively at the anhydride, 4-, and 6- position. This would allow
for modular, orthogonal incorporation of a targeting group,
sensing group, and ICT donor. We first formed naphthalimide 9
through selective condensation of anhydride 6 with aspartic acid
dimethyl ester. The use of 1,4-dioxane as solvent allowed for
selective reaction of the amine at the imide position, giving 9 as
the sole product in good yield, with no chromatographic
purification. Substitution of the nitro group upon treatment with N-
5
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Experimental Section
Full experimental details are included in the ESI
Acknowledgements
The authors would like to acknowledge the Australian Research
Council (DP180101353) for funding, the Westpac Scholars Trust
for a Research Fellowship (EJN), the University of Sydney for a
SOAR Fellowship (EJN) and the Australian Government for a
Research Training Program Scholarship (NT). We acknowledge
the scientific and technical assistance of Sydney Analytical, and
the Australian Microscopy and Microanalysis Research Facility
at the Australian Centre for Microscopy and Microanalysis
(ACMM).
Figure 7. Confocal microscopy images of DLD-1 spheroids of varying sizes
treated with 50 µM of probe Asp-1NO2 (scale bar represents 100 µm).
Conclusion
We have demonstrated a versatile approach to the synthesis of
4-amino-1,8-napthhalimide based probes and a flexible approach
to incorporation of sensing groups to the naphthalimide core using
simple, modular chemistry. All linkers gave rise to a change in
fluorescence intensity, and from these results we expect various
sensing groups could be incorporated using an aryl-linker at the
6-position and make effective, responsive sensors. Theoretical
calculations allowed us to rationalise the weak fluorescence of
4NH2 as due to a too strong excited-state relaxation and also
evidenced differences in the singlet-triplet gaps that nicely
correlate with experimental fluorescence quenching for the nitro
series.
Keywords: fluorescent sensor • hypoxia • 4-amino-1,8-
naphthalimide
[1]
[2]
E. J. New, ACS Sensors 2016, 1, 328-333.
a) S. Saha, A. Samanta, The Journal of Physical Chemistry
A 2002, 106, 4763-4771; b) P. Kucheryavy, G. Li, S. Vyas,
C. Hadad, K. D. Glusac, The Journal of Physical Chemistry
A 2009, 113, 6453-6461.
[3]
[4]
R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger, T.
Gunnlaugsson, Chemical Society Reviews 2010, 39, 3936-
3953.
a) K. Hanaoka, Y. Muramatsu, Y. Urano, T. Terai, T.
Nagano, Chemistry – A European Journal 2010, 16, 568-
572; b) Z. Xu, K.-H. Baek, H. N. Kim, J. Cui, X. Qian, D. R.
Spring, I. Shin, J. Yoon, Journal of the American Chemical
Society 2010, 132, 601-610; c) R. K. Jackson, Y. Shi, X.
Yao, S. C. Burdette, Dalton Transactions 2010, 39, 4155-
4161; d) Z. Xu, J. Yoon, D. R. Spring, Chemical
Communications 2010, 46, 2563-2565; e) Z. Xu, Y. Xiao, X.
Qian, J. Cui, D. Cui, Organic Letters 2005, 7, 889-892; f) R.
Parkesh, T. Clive Lee, T. Gunnlaugsson, Org Biomol Chem
2007, 5, 310-317.
For use in sensing hypoxia, the Suzuki coupled product 1NO2 was
determined to be the optimal structure, as this had the highest
quantum yield, a large turn-on response upon reduction to 1NH2,
and was the most intensely fluorescent in aqueous media. Using
a modular approach, we have selectively functionalised the
naphthalimide core with a targeting group at the imide position,
amino donor at the 4-position, and a sensing group at the 6-
position to synthesise the rationally designed extracellular
hypoxia naphthalimide based probe Asp-1NO2. Asp-1NO2 was
used to successfully image the hypoxic and necrotic regions of
tumour cell spheroids. One challenge for the use of green emitting
fluorophores for tumour imaging, however, is their limited tissue
penetration. Two-photon microscopy has been employed to
address this issue for several naphthalimide based sensors, and
could allow for Asp-1NO2 to be used for imaging in tissues.[6c, 18]
Our ongoing research also involves the development of more red-
shifted probes.
[5]
a) Z. Xu, Y.-Y. Ren, X. Fan, S. Cheng, Q. Xu, L. Xu,
Tetrahedron 2015, 71, 5055-5058; b) S. N. Berry, V. Soto-
Cerrato, E. N. W. Howe, H. J. Clarke, I. Mistry, A.
Tavassoli, Y.-T. Chang, R. Pérez-Tomás, P. A. Gale,
Chemical Science 2016, 7, 5069-5077; c) X. Sun, S. D.
Dahlhauser, E. V. Anslyn, Journal of the American
Chemical Society 2017, 139, 4635-4638; d) F. M. Pfeffer, A.
M. Buschgens, N. W. Barnett, T. Gunnlaugsson, P. E.
Kruger, Tetrahedron Letters 2005, 46, 6579-6584; e) J. F.
Zhang, C. S. Lim, S. Bhuniya, B. R. Cho, J. S. Kim,
Organic Letters 2011, 13, 1190-1193.
[6]
a) D. Srikun, E. W. Miller, D. W. Domaille, C. J. Chang,
Journal of the American Chemical Society 2008, 130, 4596-
4597; b) X. Sun, Q. Xu, G. Kim, S. E. Flower, J. P. Lowe, J.
Yoon, J. S. Fossey, X. Qian, S. D. Bull, T. D. James,
Chemical Science 2014, 5, 3368-3373; c) J. Fan, Z. Han, Y.
Kang, X. Peng, Scientific Reports 2016, 6, 19562; d) L.
Zhang, W.-q. Meng, L. Lu, Y.-S. Xue, C. Li, F. Zou, Y. Liu,
J. Zhao, Scientific Reports 2014, 4, 5870; e) X.-L. Liu, X.-J.
Du, C.-G. Dai, Q.-H. Song, The Journal of Organic
Chemistry 2014, 79, 9481-9489.
This work further illustrates the versatility of the naphthalimide
scaffold: we could readily substitute the group at the imide
position to target any cell type or sub-cellular organelle, and easily
append
a variety of sensing groups. This approach to
naphthalimide based probe design and synthesis is flexible
allowing for the efficient generation of targeted, selective
fluorescent probes and work is underway in applying this
approach to the synthesis of various probes for biological
purposes.
[7]
[8]
K. G. Leslie, D. Jacquemin, E. J. New, K. A. Jolliffe, Chem-
Eur J 2018, 24, 5569-5573.
K. Yang, K. G. Leslie, S. Y. Kim, B. Kalionis, W.
Chrzanowski, K. A. Jolliffe, E. J. New, Org Biomol Chem
2018, 16, 619-624.
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Chemistry - A European Journal
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[9]
E. Collado-Fregoso, J. S. Zugazagoitia, E. F. Plaza-Medina,
J. Peon, J Phys Chem A 2009, 113, 13498-13508.
R. B. P. Elmes, Chemical Communications 2016, 52, 8935-
8956.
K. L. Eales, K. E. R. Hollinshead, D. A. Tennant,
Oncogenesis 2016, 5, e190-e190.
aJ. Pouysségur, F. Dayan, N. M. Mazure, Nature 2006, 441,
437-443; bM. Höckel, P. Vaupel, JNCI: Journal of the
National Cancer Institute 2001, 93, 266-276.
X. Sun, G. Niu, N. Chan, B. Shen, X. Chen, Molecular
Imaging and Biology 2011, 13, 399-410.
[10]
[11]
[12]
[13]
[14]
[15]
K. D. Grimes, A. Gupte, C. C. Aldrich, Synthesis-Stuttgart
2010, 1441-1448.
a) D. Jacquemin, I. Duchemin, X. Blase, Journal of
Chemical Theory and Computation 2015, 11, 5340-5359; b)
A. D. Laurent, C. Adamo, D. Jacquemin, Physical
Chemistry Chemical Physics 2014, 16, 14334-14356.
D. Yuan, R. G. Brown, The Journal of Physical Chemistry A
1997, 101, 3461-3466.
[16]
[17]
[18]
A. J. Franko, R. M. Sutherland, Radiation Research 1979,
79, 439-453.
a) B. Zhu, P. Li, W. Shu, X. Wang, C. Liu, Y. Wang, Z.
Wang, Y. Wang, B. Tang, Analytical Chemistry 2016, 88,
12532-12538; b) Z.-R. Dai, G.-B. Ge, L. Feng, J. Ning, L.-
H. Hu, Q. Jin, D.-D. Wang, X. Lv, T.-Y. Dou, J.-N. Cui, L.
Yang, Journal of the American Chemical Society 2015, 137,
14488-14495.
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Entry for the Table of Contents
Synthetically-versatile fluorophore cores will enable the rapid preparation of novel responsive fluorescent sensors. We have
developed methods for incorporating sensing groups at the 6-position of 4-amino-1,8-naphthalimide, and have used this to prepare a
set of hypoxia-responsive fluorophores. We further demonstrate the versatility of this scaffold by preparing an extracellular analogue
that can image the hypoxic region of a tumour spheroid.
Institute and/or researcher Twitter usernames: @liamthechemist @kate_jolliffe @lizjnew @newgroupchem @sydneychemistry
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