A bodipy based hydroxylamine sensor

Article abstract of DOI:10.1039/c7cc05872a

With this research, we have developed a bodipy based system as the first "turn-on" fluorescence system for the detection hydroxylamine.

Full text of DOI:10.1039/c7cc05872a

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2017, DOI: 10.1039/C7CC05872A.
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DOI: 10.1039/C7CC05872A
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A bodipy based hydroxylamine sensor
Cite this: DOI: 10.1039/x0xx00000x
Adam C. Sedgwick,a Robert S. L. Chapman,a Jordan E. Gardiner,a Lucy R. Peacock,a
Gyoungmi Kim,b Juyoung Yoon,b* Steven D. Bulla* and Tony D. James a*
Received 00th January 2016,
Accepted 00th January 2016
DOI: 10.1039/x0xx00000x
www.rsc.org/
With this research we have developed a bodipy based system of HA. Luckily, an appropriate system probe 1 had previously been
as the first “turn-on” fluorescence system for the detection prepared for the detection of hydrogen sulphide (H2S).21, 22 Scheme
hydroxylamine.
1.
Hydroxylamine, (HA) NH2OH, is an oxygenated form of ammonia,
which is widely used in industrial and pharmaceutical processes1.
HA is widely known as a nitric oxide (NO) donor and participates in
a wide range of biological processes.2-4 In cellular metabolism, HA is
an intermediate in the conversion L-arginine to nitric oxide (NO).5, 6
This process involves the hydrolysis of oxime arginine to L-citrulline
and HA. HA is then converted by catalase to NO and superoxide (-O2
) in the presence of hydrogen peroxide.7 It is also believed that HA
can be converted to NO by the non-enzymatic attack by -O2.
-
Therefore, in inflammatory cells a reaction between HA and O2
could take place to generate NO.3, 8 For example, HA has shown to
be a vasodilator in a dose-dependent manner in the blood vessels
of rats’ kidneys.9. HA has also shown to be an inhibitor for the
release of insulin and HA has been shown to activate K+ channels.3,
10
Despite HA being a product of metabolism and a NO donor, it is
moderately toxic to humans, animals and plants. HA toxicity occurs
at concentrations that are substantially greater than the normal
physiological concentrations. Exposure to HA has been shown to be
hemotoxic, mutagenic whilst also being an enzyme and virus
inhibitor.11
Scheme 1. Nitrone synthesis and sensor for hydrogen sulphide.
The synthesis of probe 1, is given in the electronic supplementary
information (ESI)
We also confirmed that probe 1 reacts as predicted with hydroxyl
amine as shown by the 1HNMR, 13CNMR and MS. (ESI). More
importantly when HA is added to a 0.5 µM solution of probe 1 in a
1% DMSO PBS Buffer solution at pH 7.4 the fluorescence is
significantly enhanced as the concentration of HA is increased. (Fig
1)
Given these important biological aspects of HA we set out to
develop a fluorescence probe for the detection of HA. To our
knowledge, there are currently no HA fluorescence probes. HA is
currently detected using: HPLC, GC, potentiometric, polarographic,
biamperometric and electrochemical methods.12-19
Previously we have developed a synthetic route to nitrones by
reacting an aromatic aldehyde containing an ortho α,β-unsaturated
ester with NH2OH (50 % wt in H2O) in THF at – 20 OC to produce
isoindole nitrones with high yields,20 Scheme 1. Therefore, we set
out to integrate this reaction with a fluorophore for the detection
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DOI: 10.1039/C7CC05872A
We have carried out a more extensive screen amongst
relavant biological species (ESI), and as expected only
hydrogen sulphide (50 µM) produced a fluorescence response.
We then attempted to detect hydroxylamine in a cellular
environment using probe 1. HeLa cells were incubated HA (0 -
150 μM) for 30 min and washed with DPBS then 1 μM of probe
300
6
5
4
3
2
1
NH2OH
250
200
150
100
50
1
was added and left for 30 minutes, fluorescence images were
0
5
10
15
20
then acquired by confocal microscopy. From these
fluorescence images it is clear that probe 1 was fluorescent in
HeLa cells even without added HA (ESI Fig 3). The strong
background fluorescence is attributed to cellular thiols
including endogenous H2S present in the HeLa cells.
Concentration (HA)/ micromolar
0
480
500
520
540
560
580
600
620
Wavelength/ nm
Fig 1. Fluorescence changes observed for probe 1 (0.5 µM) during the addition of HA (0
– 20 µM), in 1% DMSO PBS Buffer at pH 7.4. λex = 465 nm, with a 5 min delay between
additions of HA. Insert: Relative fluorescence changes (I/IO) as
a function of HA
concentration (0 – 20 µM)
Having shown that probe
1 works with HA we set out to
investigate the selectivity amongst several hydroxyl amines: N-
(Benzyl)hydroxylamine, N-(Propargyl)Hydroxylamine, N-(tert-
Butyl)Hydroxylamine, O-(Benzyl)Hydroxylamine, and N-
(Phenyl)Hydroxylamine. (Fig 2)
10
Fig 3. Mechanism for the reaction of probe
20).
1 with HA (adapted from reference
8
In conclusion we have developed a fluorescence turn-on probe for
the detection of hydroxylamine. The system works well in vitro but
with the in vivo cellular experiments endogenous thiols react to
turn on probe 1. We are currently working to develop a system with
enhanced selectivity for hydroxyl amines over thiols. However, it is
interesting to note that the system using N-(Propargyl)
Hydroxylamine could be used as fluorescent “click” tag.
6
4
2
1
2
3
4
5
6
7
8
0
Acknowledgements
Fig 2. Selectivity of probe
Hydroxylamine, (2) N-(methyl)hydroxylamine, (3) N-(Benzyl)hydroxylamine, (4)
N-(Propargyl)Hydroxylamine, (5) N-(tert-Butyl)Hydroxylamine, (6) O-
(Benzyl)Hydroxylamine, (7) N-(Phenyl)Hydroxylamine, (8) Blank. 15 min
1 (0.5 µM) towards various hydroxylamines (1)
We would like to thank the EPSRC and the University of Bath for
funding. ACS, JEG, and LRP thank the EPSRC for studentships. RSLC
incubation with each analyte (50 µM) in PBS Buffer, 1% DMSO pH = 7.4. slit thanks the EPSRC Doctoral Training Centre in Sustainable Chemical
width ex = 5 nm, em = 2.5 nm. λexc = 465 nm, λem = 510 nm.
Technologies: EP/G03768X/1 for a studentship. JY thanks to the
support from the National Research Foundation of Korea (NRF),
From these results it is clear that the system works for simple
which was funded by the Korea government (MSIP) (No.
primary hydroxyl amines (1) Hydroxylamine, (2) N-
2012R1A3A2048814). Catherine Lyall is thanked for help in
(methyl)hydroxylamine (3) N-(Benzyl)hydroxylamine, (4) N-
obtaining NMR spectra using NMR characterisation facilities
(Propargyl)Hydroxylamine,) However, larger alkyl groups or
provided through the Chemical Characterisation and Analysis
aromatic hydroxyl amines such as (5) N-(tert-
Facility (CCAF) at the University of Bath (www.bath.ac.uk/ccaf). The
Butyl)Hydroxylamine and (7) N-(Phenyl)Hydroxylamine do not
EPSRC UK National Mass Spectrometry Facility at Swansea
work. It is evident that the system also requires the hydroxyl of
University is thanked for analyses.
the hydroxyl amine (6) O-(Benzyl)Hydroxylamine. These
observations are consistent with the proposed mechanism of
the reaction (Fig 3). Where, a nucleophillic nitrogen23 and
Notes and references
exchangeable OH are required to facilitate condensation of HA
a Department of Chemistry, University of Bath, Bath, BA2 7AY,
with probe 1.
UK. Email: t.d.james@bath.ac.uk; s.d.bull@bath.ac.uk
b
Department of Chemistry and Nano Science, Ewha Womans
University, Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr.
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Electronic Supplementary Information (ESI) available: [details of
any supplementary information available should be included
here]. See DOI: 10.1039/x0xx00000x
All data created during this research are openly available from
the University of Bath data archive at http://doi.org/XXXXXXX
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