A dual-functional molecular strategy for in situ suppressing and visualizing of neuraminidase in aqueous solution using iridium(III) complexes

Article abstract of DOI:10.1039/c9cc02189b

We have designed for the first time a dual-functional luminescent probe and inhibitor of neuraminidase (NA), a key influenza target. The lead iridium(iii) complex exhibited enhanced inhibition against NA compared to the FDA-approved antiviral drug, oseltamivir, and could detect NA even in the presence of an autofluorescent background.

Full text of DOI:10.1039/c9cc02189b

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A dual–functional molecular strategy for in–situ suppressing and
visualizing of neuraminidase in aqueous solution using iridium(III)
complexes†
Received 00th January 20xx,
Accepted 00th January 20xx
Chun Wu,‡a Ke-Jia Wu,‡b Jin-Biao Liu,‡a,c Xiao-Ming Zhou,d Chung-Hang Leung*b and Dik-Lung Ma*a
DOI: 10.1039/x0xx00000x
We have designed for the first time a dual–functional
luminescent probe and inhibitor of neuraminidase (NA), a key
influenza target. The lead iridium(III) complex exhibited
enhanced inhibition against NA compared to the FDA–
approved antiviral drug, oseltamivir, and could detect NA even
in the presence of an autofluorescent background.
Scheme 1 Schematic diagram showing the in-situ tracking and
suppressing of NA activity using an iridium(III)-based probe.
Neuraminidase (NA) is a glycoprotein found on the surface of
influenza viruses, and is crucial for enabling the release of newly
generated viruses from infected host cells.1 Hence, NA–
targeting therapeutics are a primary strategy for combating
influenza infection.2 Officially approved NA–targeting drugs
include peramivir, zanamivir and oseltamivir,3 with oseltamivir
showing the greatest protection against influenza among the
three. However, the rapid antigenic mutation rate of influenza
viruses has fostered the emergence of resistance to current
drugs, particularly to oseltamivir,4 creating a critical threat to
influenza treatment. To date, various organic compounds have
been explored for the inhibition of oseltamivir–resistant
influenza viruses.5 However, one drawback of using organic
compounds is their intrinsic fluorescence or quenching effects,
which might lead to false positive or negative results when
screening for NA inhibitors using fluorescence assays.6
Meanwhile, the development of rapid and reliable
diagnostic platforms for influenza infection is urgently needed
for the timely control and management of influenza infection.
For example, the organic dye X-Neu5Ac has been designed as
an imaging reagent for NA.7 The Suzuki group recently
developed a more sensitive fluorogenic and precipitating
imaging reagent, BTP-Neu5Ac, that has been used to visualize
mammalian tissue sialidases, influenza virus NA and sialidase
activity in several other viruses.8 Moreover, the Wong group
developed
a fluorescent difluorosialic acid–based, cell–
permeable probe to label and visualize a variety of human and
influenza NAs.9 Recently, Withers and co-workers have
developed an organic dye as a proximity ligation reagent to
specifically detect influenza virus in mammalian cells.10
However, all organic dye–based probes share a common
weakness of short–lived fluorescence, rapid photobleaching
rates and negligible Stokes shift that can hinder their application
for the real–time sensing of NA in biological situations.11
The development of dual–function therapeutic and imaging
molecules has received recent interest in the literature.12
Combining a drug and a probe into a single molecular entity
could facilitate the treatment of specific diseases while
simultaneously allowing for the real–time and targeted tracking
of the therapeutic efficiency, delivery kinetics, or trafficking
pathways of the drug.13 Moreover, the need for an extraneous
inhibitor or imaging agent is eliminated, which simplifies
administration and eliminates the potential for undesirable
drug–drug interactions.14 However, no example of dual–
function inhibitors and probes for NA have been reported yet.
One great challenge in the design of a dual–functional
inhibitor and probe for NA is how to achieve the desired activity
and selectivity for NA whilst simultaneously maintaining
sufficient photophysical activity in an aqueous environment.
Transition metal complex–based scaffolds enjoy distinct
advantages as bio–sensing and chemo–sensing agents over
organic molecules owing to their unique characteristics,15
including large Stokes shift, high luminescent quantum yield,
long–lived phosphorescent lifetime, high sensitivity of emission
characteristics to subtle variations from chemical environment,
and modular synthesis that enables the efficient production of
analogues with variable photophysical and/or chemical
properties.16 Typically, transition metal complex–based
a. Department of Chemistry, Hong Kong Baptist University, Kowloon, Hong Kong,
China. E-mail address: edmondma@hkbu.edu.hk (Dik-Lung Ma).
b. State Key Laboratory of Quality Research in Chinese Medicine, Institute of
Chinese Medical Sciences, University of Macau, Macao, China. E-mail address:
duncanleung@um.edu.mo (Chung-Hang Leung).
c. School of Metallurgical and Chemical Engineering, Jiangxi University of Science
and Technology, Ganzhou, China.
d. MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science,
College of Biophotonics, South China Normal University, Guangzhou, China.
‡ The authors contribute equally in this work.
* Corresponding authors.
† Electronic Supplementary Information (ESI) available.
This journal is © The Royal Society of Chemistry 20xx
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luminescent probes employ either “always–on”, “switch–on” or
“switch–off” signaling modes after interaction with the target
analyte.17 Always–on or switch–on probes display high target–
to–background ratios by emitting continuous or enhanced
luminescence in the presence of analyte, allowing their signals
to distinguished in the presence of complicated biological
interferents. On the other hand, the switch–off signaling mode
can be susceptible to interference by non–specific quenchers in
the cellular environment. Hence, always–on or switch–on
probes are highly preferred for the real–time monitoring of
targeted analytes in living systems.18 On the basis of our current
research interest in metal complex–based luminescent probes,
we aimed to develop a series of new oseltamivir–conjugated
iridium(III) complexes for the selective inhibition and detection
of NA (Scheme 1). Specifically, the assembled probes consist of
an oseltamivir “binding unit”, which is responsible for the
selectivity and binding affinity of the probe towards NA,
conjugated to an iridium(III) “signaling unit”. Upon selective
binding of the probe to NA, the distinguished luminescence
emission from the iridium(III) signaling moiety allows for the
dynamic tracing of NA. Meanwhile, the introduction of the
oseltamivir moiety is expected to confer NA inhibitory activity,19
allowing the probe to also function as a potential therapeutic
agent for treating influenza infection. Our molecular design of
the complexes also takes into account the emissive behavior of
the cyclometalated metal complex moiety. Most transition
metal complexes display weak emission in aqueous solutions,20
which greatly limits their application for sensing in the biological
environment. Moreover, red–emitting probes are generally
more desirable since they are less susceptible to interference by
optical absorption, light scattering and autofluorescence of
biological issues.21 Hence, our ultimate design goal was to
develop NA–selective iridium(III) complexes with strong red
emission in aqueous condition.
In this study, four kinds of C^N ligands and two kinds of N^N
ligands were conjugated to the iridium(III) metal core. The small
library of iridium(III) complexes have been designed by varying
the auxiliary ligands in order to fine–tune their photophysical
and physicochemical properties, such as emission wavelength,
phosphorescent lifetime, Stokes shift and quantum yield. The
final complexes 1a–1d all contain an oseltamivir moiety grafted
onto the N^N ligand of the parent iridium(III) scaffold.
To synthesize the dual–functional iridium(III) complexes,
oseltamivir phosphate 2 was initially attached to the N^N ligand
through two different strategies, either starting from the
amine–containing 1,10-phenanthrolin-5-amine (Synthesis
Route 1, Scheme S1), or the more reactive carboxyl-containing
1,10-phenanthroline-5-carboxylic acid (Synthesis Route 2,
Scheme S1). Unfortunately, the yield of the targeted oseltamivir
conjugates 5 or 8 was low owing to the poor solubility of
oseltamivir phosphate 2 in organic solvent. To overcome this,
oseltamivir phosphate 2 was first transformed into the organic
solvent-soluble oseltamivir trifluoroacetate 10 (Scheme S2).
However, conjugation of oseltamivir trifluoroacetate 10 to the
N^N ligand 1,10-phenanthroline-5-carboxylic acid proceeded
poorly (Synthesis Route 3, Scheme S1).
View Article Online
DOI: 10.1039/C9CC02189B
Fig. 1 (a) Chemical structures of iridium(III) complexes 1a–1d. (b)
Photographs of complexes 1a–1d (2.5 μM) observed under UV
light (365 nm) in PBS buffer containing 0.5% ACN.
Instead, we found that 10 could be directly conjugated to
the cyclometalated iridium(III) complex precursor 11a already
bearing the 1,10-phenanthroline-5-carboxylic acid N^N ligand
under standard EDCI/HOBT coupling conditions, producing the
final oseltamivir–conjugated compound 1a (Fig. 1a) in 62%
yield. The parent iridium(III) complex precursor 11a was
synthesized by the reaction of 1,10-phenanthroline-5-
carboxylic acid with the dichloro–bridged dimer 7, which was in
turn synthesized from the C^N ligand 6 and iridium(III) chloride.
The iridium(III) analogues 1b–1d were prepared in similar
fashion via the intermediate parent complexes 11b–11d.
Complexes 1a–1d were fully characterized by high–resolution
mass spectrometry (HRMS), 1H NMR, 13C NMR and high
performance liquid chromatography (HPLC) (Fig. S1).
Remarkably, complexes 1a–1d displayed strong luminescence
in aqueous PBS buffer containing 0.5% ACN (Fig. 1b), which
endows with the potential to function as dual–functional
inhibitor and probes for NA.
We first studied the NA inhibition activity of the novel
oseltamivir–conjugated complexes 1a–1d using a fluorescence
assay (Fig. 2). Encouragingly, complexes 1a–1d (100 µM)
showed significant inhibition of NA activity compared to the
DMSO control. Notably, complexes 1a–1d all showed superior
inhibition activity over the reference drug oseltamivir under the
same in–vitro experimental conditions. This result suggests that
the combination of the oseltamivir moiety and iridium(III) core
and generate molecules with promising potential for treating
influenza infection. In light of the unique optical properties of
iridium(III) complexes, we next investigated the photophysical
properties of complexes 1a–1d (Table S1). In acetonitrile (ACN),
complexes 1a–1d displayed characteristic absorbance bands at
210–288 nm in the UV–Vis spectra (Fig. S2a). Complexes 1a–1d
also exhibited maximum excitation and emission wavelengths
at 354–381 nm and 585–606 nm, respectively, in PBS buffer
containing 0.5% ACN (Fig. S2b), giving large Stokes shifts of 225–
249 nm. Noticeably, complex 1d exhibited the largest Stokes
shift for 249 nm, which is much larger than the typical small
Stokes shift of organic dyes at around 100 nm, thus giving it a
great advantage in terms of preventing self-quenching.22
Red luminescence is generally more desirable because of its
superior penetration of biological cells and tissues.23 Complexes
1b and 1c exhibited yellow to orange luminescence in PBS
buffer, while complexes 1a and 1d displayed a red–shifted red
luminescence at around 600 nm under the same aqueous
condition. Interestingly, complex 1c showed around 6-fold
higher emission intensity compared to complexes 1a, 1b and 1d.
This might be attributed to the change of the metal–to–ligand–
2 | J. Name., 2012, 00, 1-3
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Fig. 2 Inhibition activity of complexes 1a–1d and oseltamivir
(Ose) (100 μM) against NA (10 μg/mL) as measured by a
fluorescence assay. The fluorescence intensity was detected at
ex/em = 330 nm/450 nm.
Fig. 3 (a) Competition assay of complexes 1a–1d (100 μM) with
oseltamivir (Ose) (100 µM) for NA (10 μg/mL) detection. The
luminescence intensity of the mixture of NA and complex was
utilized as control respectively. (b) Luminescence intensity of
complex 1d in the absence or presence of oseltamivir (Ose) (100
µM) or NA (10 μg/mL). The luminescence intensity was detected
at ex/em = 330 nm/600 nm. (c) Interaction of NA with 1d as
measured by a protein thermal shift assay.
charge–transfer (MLCT) state and increased electron density
induced by the joint interplay of the electron–withdrawing
chloride moiety and the π–π conjugation system located in the
auxiliary C^N and N^N ligands of 1c. Additionally, all four
complexes showed higher quantum yields than Ru(bpy)3 (bpy =
2,2'-bipyridine), a commonly used transition metal complex
standard with quantum yield at 0.062 (Fig. S2c). Finally,
complexes 1a–1d displayed long-lived phosphorescence
lifetimes of ca. 0.277–0.565 μs (Fig. S2d), which is significantly
longer than the typical nanosecond lifetimes of organic
fluorophores. This characteristic could allow the emission of
explored. The data showed that PBS (containing 0.5% ACN) gave
a slightly higher luminescent enhancement in response to NA
compared to the use of Tris-HCl or HEPES buffer systems (Fig.
S4c). Therefore, PBS buffer was used for further experiments.
Under the optimized conditions, the luminescence response of
complex 1d towards different concentrations of NA was tested.
The luminescence of 1d gradually increased with increasing
concentration of NA from 1–20 μg/mL, with a detection limit at
0.68 μg/mL, indicating that 1d allows for NA detection in a
concentration–dependent manner (Fig. S5).
complexes 1a–1d to be discriminated from
auto-fluorescent background.
a highly
Inspired by the promising luminescent characteristics of
complexes 1a–1d, we studied their application as luminescent
probes for NA imaging. Complexes 1a–1d showed negligible
changes in luminescence upon the addition of NA in PBS buffer
containing 0.5% ACN (Fig. S3), suggesting that they could
function as “always–on” probes for NA. To testify whether
complexes 1a–1d can selectively bind to NA, possibly via the
oseltamivir–binding site, we performed a competition assay
with complexes 1a–1d with oseltamivir. Upon the pre-
incubation of NA with oseltamivir (100 µM), complexes 1a–1c
showed negligible luminescence change (Fig. S3), suggesting
that these complexes did not significantly interact with NA.
However, a significant decrease of the luminescence of complex
1d was recorded when NA was pre-incubated with oseltamivir
(Fig. 3a). Complex 1d showed no direct interaction with
oseltamivir alone in the absence of NA (Fig. 3b), suggesting that
the decrease of luminescence intensity observed in the
competition assay was due to competition at the oseltamivir–
binding site of NA rather than through probe–probe
interactions. Moreover, a protein thermal shift assay was
performed to evaluate the binding affinity of complex 1d with
NA (Fig. 3c). Complex 1d showed dose-dependent thermal
stabilization of NA in vitro, with an apparent Kd of 18.95 ± 0.04
µM. To obtain the optimal performance of the probe,
conditions such as pH and the nature of the buffer solution were
optimized. As NA shows the highest activity at pH 5,24 we
explored the effect of pH in the range of pH 4–6 on the NA
sensing activity of 1d. The luminescence intensity of complex 1d
was robust in the absence (Fig. S4a) or presence (Fig. S4b) of NA
in PBS buffer over the pH range from 4–6. Hence, a pH value of
5 was chosen for further investigation to preserve the highest
biological activity of NA. Moreover, the effect of various buffer
systems on the sensing performance of complex 1d was also
To verify that the luminescence of complex 1d could be
maintained in the cellular environment, complex 1d was
challenged with various potentially interfering species,
including 20 common amino acids, glutathione (GSH), glutathiol


2
(GSSG), homocysteine, homocysteine, S2 , HCO3 , CO32, SO3
,



Br, Cl, SO42, F, ClO4 , NO3 , HPO42, H2PO4 , I, CH3COO,
Mg2+, Fe3+, Cd2+, Ag+, Ni+, Pb2+, Ba2+, Cu2+, Al3+, Na+, K+, Ca2+, Zn2+,
Fe2+ and Mn2+ (Fig. S6). The results revealed that complex 1d
could maintain robust luminescence in the presence of the
various interfering species. Next, we measured the emission of
1d using time–resolved emission spectroscopy (TRES). The
fluorescent organic dyes thioflavin S (THS) and coumarin 460
(Cm460) were used as model matrix interferences. In steady–
state emission mode, the emission of complex 1d was
perturbed by the strong emission peak of THS and Cm460 at 428
nm and 460 nm respectively (Fig. S7a), leading to reduced
accuracy in the measurement of NA concentration. However,
when the time gate was defined as after the completion of the
fluorescence decay of THS and Cm460, the interfering signals
from THS and Cm460 were almost completely lost and only the
emission of complex 1d was observed (Fig. S7b). These results
demonstrate the potential utilization of the long–lived 1d in
highly auto–fluorescent environments using TRES.
As the complex with the highest binding affinity to NA,
complex 1d was therefore chosen as the most promising dual–
function agent. The biological potency of 1d was subsequently
assessed using a dose–response experiment with various
concentrations of complex 1d. With increasing concentration of
complex 1d (10–200 μM) as inhibitor, the activity of NA was
gradually reduced with IC50 at around 20 μM, indicating that
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7. M. Saito, H. Hagita, Y. Iwabuchi, I. Fujii, K. IkedVaiewanAdrticMle O. nIltinoe,
complex 1d inhibited the NA activity in a dose–dependent
manner (Fig. S8).
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In conclusion, using a structure–based molecular design
strategy, we have for the first time successfully developed a
dual–functional Iridium(III) complex–based platform for in–situ
suppressing and visualizing of NA under aqueous conditions. In
terms of the inhibition properties, complexes 1a–1d showed
superior inhibition efficiency compared to the FDA–approved
NA inhibitor oseltamivir under the same experimental
conditions. Notably, complex 1d also displayed promising
potential as an “always–on” luminescent probe for NA due to
its long–lived lifetime, large Stokes shift, high quantum yield
and strong red emission in aqueous PBS buffer containing 0.5%
ACN. Moreover, complex 1d also enables the dose–dependent
detection of NA with high selectivity at 0–20 μg/mL. In
summary, complex 1d is the first highly promising transition
metal–based dual–functional candidate for the in–situ
monitoring and inhibition of NA. We envision that complex 1d
can facilitate a new route for developing real–time and in–field
assessment tools against targeted biomarkers for guided
therapy for influenza viruses.
This work is supported by Hong Kong Baptist University
(FRG2/16-17/007 and FRG2/17-18/003), the Health and
Medical Research Fund (HMRF/14150561), the Research Grants
Council (HKBU/12301115), the National Natural Science
Foundation of China (21575121, 21775131, 21628502 and
21762018), the Hong Kong Baptist University Century Club
Sponsorship Scheme 2018, the Interdisciplinary Research
Matching Scheme (RC-IRMS/16-17/03), Interdisciplinary
Research Clusters Matching Scheme (RC-IRCs/17-18/03),
Matching Proof of Concept Fund (MPCF-001-2017/18),
Collaborative Research Fund (C5026-16G), SKLEBA and HKBU
Strategic Development Fund (SKLP_1718_P04), the Science and
Technology Development Fund, Macao SAR (077/2016/A2), and
the University of Macau (MYRG2016-00151-ICMS-QRCM and
MYRG2018-00187-ICMS).
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Conflicts of interest
There are no conflicts of interest to declare.
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