Naphthalimide trifluoroacetyl acetonate: A hydrazine-selective chemodosimetric sensor

Article abstract of DOI:10.1039/c3sc51813b

The trifluoroacetyl acetonate naphthalimide derivative 1 has been synthesized in good yield. In acetonitrile solution, compound 1 reacts selectively with hydrazine (NH₂NH₂) to give a five-membered ring. This leads to OFF-ON fluorescence with a maximum intensity at 501 nm as well as easily discernible color changes. Based on a readily discernible and reproducible 3.9% change in overall fluorescence intensity, the limit of detection for 1 is 3.2 ppb (0.1 μM), which is below the accepted limit for hydrazine set by the U.S. Environmental Protection Agency (EPA). Compound 1 is selective for hydrazine over other amines, including NH ₄OH, NH₂OH, ethylenediamine, methylamine, n-butylamine, piperazine, dimethylamine, triethylamine, pyridine, and is not perturbed by environmentally abundant metal ions. When supported on glass-backed silica gel TLC plates, compound 1 acts as a fluorimetric and colorimetric probe for hydrazine vapor at a partial pressure of 9.0 mm Hg, with selectivity over other potentially interfering volatile analytes, including ammonia, methylamine, n-butylamine, formaldehyde, acetaldehyde, H₂O₂, HCl, and CO₂ being observed. Probe 1 can also be used for the detection of hydrazine in HeLa cells and does so without appreciable interference from other biologically abundant amines and metal ions. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3sc51813b

View Article Online
View Journal
Chemical Science
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: M. H. Lee, B. Yoon, J. S. Kim and J.
Sessler, Chem. Sci., 2013, DOI: 10.1039/C3SC51813B.
This is an Accepted Manuscript, which has been through the RSC Publishing peer
review process and has been accepted for publication.
Chemical Science
Accepted Manuscripts are published online shortly after acceptance, which is prior
www.rsc.org/chemicalscience
Volume 1 | Number 4 | 1 October 2010 | Pages 417–528
to technical editing, formatting and proof reading. This free service from RSC
Publishing allows authors to make their results available to the community, in
citable form, before publication of the edited article. This Accepted Manuscript will
be replaced by the edited and formatted Advance Article as soon as this is available.
To cite this manuscript please use its permanent Digital Object Identifier (DOI®),
which is identical for all formats of publication.
More information about Accepted Manuscripts can be found in the
Information for Authors.
ISSN 2041-6520
PERSPECTIVE
Barry M. Trost et al.
Catalytic asymmetric allylic alkylation
Andrew J. deMello, Joshua B. Edel et al. employing heteroatom nucleophiles:
Please note that technical editing may introduce minor changes to the text and/or
graphics contained in the manuscript submitted by the author(s) which may alter
content, and that the standard Terms & Conditions and the ethical guidelines
that apply to the journal are still applicable. In no event shall the RSC be held
responsible for any errors or omissions in these Accepted Manuscript manuscripts or
any consequences arising from the use of any information contained in them.
EDGE ARTICLE
Rapid cell extraction in aqueous two-
phase microdroplet systems
a powerful method for C–X bond
formation
2041-6520(2010)1:4;1-
X
www.rsc.org/chemicalscience
Registered Charity Number 207890

Dynamic ArticlVeiewLAinrtikclesOn►line
Chemical Science
Page 1 ofJ7ournal Name
DOI: 10.1039/C3SC51813B
Cite this: DOI: 10.1039/c0xx00000x
www.rsc.org/xxxxxx
ARTICLE TYPE
Naphthalimide trifluoroacetyl acetonate: A hydrazine-selective
chemodosimetric sensor
Min Hee Lee,a Byungkwon Yoon,b Jong Seung Kimb,* and Jonathan L. Sesslera,c,*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
The trifluoroacetyl acetonate naphthalimide derivative 1 has been synthesized in good yield. In
acetonitrile solution, compound 1 reacts selectively with hydrazine (NH2NH2) to give a five-membered
ring. This leads to OFF-ON fluorescence with a maximum intensity at 501 nm as well as easily
discernible color changes. Based on a readily discernible and reproducible 3.9% change in overall
10 fluorescence intensity, the limit of detection for 1 is 3.2 ppb (0.1 μM), which is below the accepted limit
for hydrazine set by the U.S. Environmental Protection Agency (EPA). Compound 1 is selective for
hydrazine over other amines, including NH4OH, NH2OH, ethylenediamine, methylamine, n-butylamine,
piperazine, dimethylamine, triethylamine, pyridine, and is not perturbed by environmentally abundant
metal ions. When supported on glass-backed silica gel TLC plates, compound 1 acts as a fluorimetric and
15 colorimetric probe for hydrazine vapor at a partial pressure of 9.0 mm Hg, with selectivity over other
potentially interfering volatile analytes, including ammonia, methylamine, n-butylamine, formaldehyde,
acetaldehyde, H2O2, HCl, and CO2 being observed. Probe 1 can also be used for the detection of
hydrazine in HeLa cells and does so without appreciable interference from other biologically abundant
amines and metal ions.
electrochemical methods have been exploited for the purpose of
20 Introduction
hydrazine analysis.13 However, there are only a few reports
50 regarding fluorescent probes. Swager et al. reported a fluorescent
polymer that is reduced by hydrazine vapor, giving rise to an
increase in fluorescence intensity.14 Chang et al. developed a
fluorescence turn-on type probe for hydrazine by deprotection of
levulinoyl ester or acetyl groups.15 Fan et al. reported a
55 ratiometric fluorescent probe based on the reaction of hydrazine
with arylidenemalononitrile.16 In addition, Tong et al. reported
that arylaldehydes react with hydrazine to produce a fluorescent
product.17 However, there is still a question concerning the
selective detection of hydrazine over other competitive amines
60 such as ammonia, diamines and alkylamines. A particular
challenge is to develop hydrazine probes that are biocompatible
and which could be used in cellular milieus since this might allow
toxic events to be monitored in real time and the biological
details of hydrazine to be studied in greater detail under
Sensor systems that might allow for the effective and rapid
detection of analytes constitute a major current focus in green
chemistry.1 New analytical tools are needed for real-time
industrial process monitoring and for preventing the formation of
25 toxic materials. Such monitoring allows for the rapid detection of
toxic materials, thus minimizing errors while providing potential
cost reductions relative to traditional laboratory-based analyses.
In this context, fluorescent probes have long been of particular
interest to analytical scientists. Recently, a number of fluorescent
30 probes have been put forward for the detection of anions,2 metal
ions,3 and cellular components,4 etc.
Hydrazine is of particular interest given its toxicity and high
levels of use. It is used as a blowing agent for the production of
plastics, as a gas-forming agent in air bags, in agricultural
35 chemicals and preservatives, and is found in tobacco.5,6 It is also
used as an antioxidant in nuclear and in electrical power plants, as
an anti-corrosion agent, and as a fuel for satellites and rockets.7
Additionally, it was reported that some nitrogen fixing bacteria
may create hydrazine as a by-product.8 Exposure to hydrazine is
40 known to cause critical damage to the liver, kidney, and central
nervous system in humans.9 Hydrazine has been classified as a
probable human carcinogen by the U.S. Environmental Protection
Agency (EPA), which has set an exposure limit of 10 ppb.10 This
has provided an incentive to develop new methods for the
45 detection of hydrazine.
65 controlled in vitro conditions. Herein, we report
a new
fluorescent probe 1 capable of selectively sensing hydrazine over
other amines with fluorescence turn-on manner and show that the
system functions inter alia in HeLa cells.
Results and discussion
70
Probe 1 was prepared by the synthetic route outlined in
Scheme 1. The naphthalimide derivatives
4 and 5 were
synthesized by adapting published procedures.18 Compound 6,
was obtained by reaction intermediate 5 with n-butylamine in
To date,
a variety of analytical techniques, including
chromatography-mass spectrometric,6,11 titrimetric,12 and
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

View Article Online
Chemical Science
DOI: 10.1039/C3SC51P81a3gBe 2 of 7
ethanol. Claisen condensation of 6 with ethyl trifluoroacetate then
the 1) trifluoroacetyl acetonate unit reacts selectively and
gave probe 1 in 88% yield. The chemical structures of 1 and 6
stoichiometrically with hydrazine, but that 2) other amines do not
induce the cyclization reaction that gives rise to the observed
fluorescence enhancement. The contrasting reactions with
were confirmed by 1H NMR and 13C NMR spectroscopy, as well
1
as HR-FAB and ESI mass spectrometry (Fig. S18–S23). The H
5
NMR spectrum of 1 is characterized by a broad -OH peak at 14.7 55 hydrazine and other amines are shown in Scheme 2 and discussed
ppm and a sharp singlet -CH peak at 6.7 ppm, both of which
correspond to the trifluoroacetyl acetonate moiety. Based on
literature precedent, compound 1 is thought to exist as a mixture
of enol forms as depicted in Scheme S1.19
further below.
Quantitative assessments of the fluorescence spectral changes
were made by treating probe 1 with increasing concentrations of
hydrazine (0–20.0 μM) in CH3CN (Fig. 1b). The fluorescence
60 intensity at 445 nm decreases in a monotonic fashion whereas
that of the feature at 501 nm increases (33-fold, Фf = 0.43)20 with
an isosbestic point being observed at 430 nm. The response
produced at any given hydrazine concentration was not
instantaneous. However, at all concentrations the response
65 occurred within one hour, with no further appreciable spectral
changes taking place (see Fig. S5). The fluorescence intensity
reached a plateau upon the addition of 1.0 equiv (10.0 μM) of
hydrazine. The UV/Vis spectra of 1 with various hydrazine
concentrations (0–20.0 μM) were also recorded; they are shown
70 in Fig. S6 and reveal a pattern of ratiometric changes that
10
O
O
Na2Cr2O7
acetic acid
n-butylamine
O
EtOH
O
O
4
5
O
H
O
O
O
O
CF3
O
O
CF3
NaOEt, DCM
N
N
O
O
6
1
matches the fluorescence behavior. A linear relationship (R2
0.9931) was found between the emission intensity at 501 nm and
=
Scheme 1 Synthesis of probe 1.
Initial spectroscopic studies of
1
were undertaken by
the hydrazine concentration over the 0–5.0 μM range. From this
15 monitoring the UV/Vis absorption and fluorescence spectral
changes observed upon the addition of hydrazine and other
amines in CH3CN solution at room temperature. In the absence of
hydrazine, probe 1 displays two absorption bands centered at 295
(ε = 7.4 X 103 M-1cm-1) and 348 nm (ε = 1.7 X 104 M-1cm-1),
20 respectively, as well as a weak emission feature at 445 nm (Фf =
0.004)20, as shown in Fig. 1 and S1. On the basis of comparisons
with 6, the absorption band at 348 nm is thought to originate with
the naphthalimide moiety.
linear relationship, and using
a reproducible and readily
75 discernible 3.9% change in overall fluorescence intensity as the
standard, the detection limit of probe 1 for hydrazine is 0.1 μM
(3.2 ppb) (cf. inset to Fig. 1b). On this basis we conclude that
compound 1 may be used as an Off-On fluorescent signaling
agent with a limit of detection that is below the 10 ppb limit for
80 hydrazine exposure set by the U.S. EPA.10
As shown in Fig. 1a, addition of hydrazine (1.0 mM) to a
25 CH3CN solution of 1 leads to an increase of fluorescence
intensity along with a red-shift from 445 to 501 nm. In contrast,
only a fluorescence quenching at 445 nm or an insignificant
change was observed upon the addition of a variety of other test
amines, including ammonium hydroxide, hydroxylamine,
30 ethylenediamine, methylamine, n-butylamine, piperazine,
dimethylamine, triethylamine, and pyridine (1.0 mM,
respectively). However, when exposed to most amines other than
pyridine the absorbance feature at 348 nm was observed to
decrease in intensity, whereas that at 295 nm was found to
35 increase (Fig. S2). Nevertheless it is important to appreciate that
even when added at a 30-fold excess relative to hydrazine, no
inhibition of the hydrazine-induced fluorescence response was
seen (vide infra). The effect of environmentally abundant metal
ions, such as Cs+, Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Mn2+, Fe2+,
40 Fe3+, Hg2+, Pb2+, Cd2+, Ni2+, and Ba2+ (as the chloride salts), was
also tested. However, no appreciable spectroscopic changes were
seen (Fig. S3). Taken in concert, these findings lead us to suggest
that 1 could be used to detect hydrazine selectively even in
milieus that contain potential interferants.
Fig. 1 (a) Fluorescence spectra of 1 (10.0 μM) recorded in the presence of
various amines, including hydrazine, ammonium hydroxide (NH4OH),
85 hydroxylamine, ethylenediamine, methylamine, n-butylamine, piperazine,
dimethylamine, triethylamine, and pyridine (1.0 mM, respectively). (b)
Fluorescence increase observed for 1 (10.0 μM) in the presence of
increasing concentrations of hydrazine (0, 0.1, 0.2, 0.4, 1.0, 1.8, 3.0, 4.0,
5.0, 6.0, 10.0, 20.0 μM). Inset: plot showing the linear relationship
90 between the fluorescence intensity at 501 nm and the hydrazine
concentration (0, 0.1, 0.2, 0.4, 1.0, 1.8, 3.0, 4.0, 5.0 μM). Fluorescence
quenching observed for 1 (10.0 μM) in the presence of increasing
concentrations of NH4OH (c) and n-butylamine (d), respectively. All
spectra were acquired 1 h after addition of the amine at room temperature
95 in CH3CN, with excitation effected at 348 nm.
45
The reaction of 1 with hydrazine is thought to produce a
cyclized product (2) resulting in an increase in the intensity of the
emission feature at 501 nm (Scheme 2). In contrast to what
proved true for 1, neither the absorption nor fluorescence spectral
features undergo appreciable changes when 6 is exposed to
50 hydrazine in CH3CN (Fig. S4). On this basis, we conclude that
2
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

View Article Online
DOI: 10.1039/C3SC51813B
Page 3 of 7
Chemical Science
To gain insight into the selectivity of probe 1 for hydrazine
over other amines, the fluorescence changes of 1 in the presence
of increasing concentrations of other amines were recorded in
CH3CN under conditions identical to those used for hydrazine.
Fig. 1c and 1d are indicative of fluorescence changes seen when
probe 1 is exposed to NH4OH and n-butylamine, respectively. In
the presence of high concentrations of these bases, the
fluorescence intensity of probe 1 at 445 nm is somewhat
quenched while a slight blue shift in the emission maximum is
5
10 seen. This stands in sharp contrast to what is seen with hydrazine
(vide infra).
Competition experiments were also carried out. Upon the
addition of various amines (0.3 mM, respectively) to a solution of
1
(10.0 μM), fluorescence quenching was observed as
Scheme 2 Proposed reactions of 1 with hydrazine and other amines.
15 demonstrated in Fig. S7. However, when 0.3 mM of hydrazine
was then added to the resulting mixtures containing 1 and the
amine in question, a strong fluorescent emission at 501 nm was
seen. On this basis, we conclude that probe 1 undergoes a
hydrazine-induced cyclization to give
a
fluorescence
20 enhancement even in the presence of other potentially
competitive amines.
In Scheme 2 proposed reactions of 1 with hydrazine and other
amines are shown. Proton NMR spectroscopic and ESI mass
spectrometric data consistent with the structure proposed for
25 product 2 were obtained. Fig. 2 shows the 1H NMR spectrum of 1
recorded in the absence and presence of 2.0 equiv of hydrazine,
n-butylamine,
triethylamine,
dimethylamine,
ammonium
hydroxide, and pyridine, respectively, in CD3CN. Upon the
addition of hydrazine, the –CH (a) peak of the trifluoroacetyl
30 acetonate moiety at δ 6.7 (1H) disappears. Concurrently, two pair
of doublets at δ 3.8 (1H) and 3.4 (1H), corresponding to the –CH2
resonances (b) of the pyrazole subunit are observed. Moreover,
two broad peaks at δ 7.3 (1H) and 5.3 (1H), corresponding to the
–NH and –OH signals of 2, respectively, were observed. In
35 addition, changes in the chemical shifts for the aromatic protons
1
of the naphthalimide core were found. On the basis of the H
NMR spectra recorded in the presence of varying concentrations
of hydrazine it is concluded that only 1.0 equiv of hydrazine is
required to convert probe 1 completely into 2 (Fig. S8). ESI-MS
40 analyses of 2 revealed the expected fragmentation patterns and
primary peaks ([2+H]+ and [2+Na]+, m/z 406.138 and 428.119,
respectively) (Fig. S9).
The proposed daughter product 2 was also isolated and its
structure confirmed by 1H NMR and ESI-MS spectroscopic
45 analyses, as shown in Fig. S10 and S11. Based on time dependent
analyses, product 2 is found to be quite stable in CD3CN over the
course of several days (Fig. S12). Interestingly, in CDCl3, as
opposed to CD3CN, compound 2 proved unstable; it undergoes
dehydration to give a crystalline product 3 whose identity was
50 confirmed by 1H NMR, ESI-MS, and X-ray crystallographic
analyses (Fig. S13, S24, and Tables S1-S7). This solvent
dependent behavior is ascribed to the fact that CHCl3 can
decompose, especially under UV light, to produce H+, which can
trigger the proposed dehydration reaction that serves to produce 3
55 from 2.21
60 Fig. 2 Partial 1H NMR spectra of 1 recorded upon the addition of 2.0
equiv of n-butylamine, triethylamine, dimethylamine, ammonium
hydroxide (NH4OH), hydrazine, and pyridine, respectively, in CD3CN.
In contrast to what is seen with hydrazine, the addition of other
amines, including n-butylamine, triethylamine, dimethylamine,
65 and ammonium hydroxide, to probe 1 causes the –CH (a) to shift
1
to higher field (from δ 6.7 to 5.7). The H NMR spectra of n-
butylamine, dimethylamine, and triethylamine were also recorded
in the absence and presence of 1 in CD3CN. Upon the addition of
1, the alkyl protons of the amines were found to shift to lower
70 field as shown in Fig. S14–S16. However, in the case of pyridine,
a negligible change was observed probably due to its weak
basicity. These results lead us to propose that the amines
deprotonate the acidic –OH proton within the trifluoroacetyl
acetonate moiety of 1 leading to a quenching of the fluorescence
75 signal at 445 nm as depicted in Scheme 2.
Next, we investigated whether 1 displays a hydrazine-
dependent change in its fluorescence signature when used in
aqueous environments. Toward this end, probe 1 was exposed to
a 1.0 mM solution of hydrazine in a mixture of H2O/CH3CN (v/v,
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

View Article Online
Chemical Science
DOI: 10.1039/C3SC51P81a3gBe 4 of 7
1:9). Under these conditions, a new emission band, presumably
abundant vapors, such as formaldehyde, acetaldehyde, H2O2, HCl,
induced by the cyclization reaction of 1 with hydrazine, was
observed at 534 nm as shown in Fig. 3. We also monitored the
changes in the fluorescence intensity at 534 nm when probe 1
and CO2. Again, these findings provide support for the contention
that 1 can selectively detect hydrazine vapor as the result of a
specific cyclization reaction that gives rise to fluorescent and
5
was treated with increasing concentrations of hydrazine. The 45 visual color changes that are detectable both in solution and when
fluorescence intensity increases by approximately 3-fold with
good linearity with respect to [hydrazine] being seen over a wide
concentration range (0–10.0 μM) (R2 = 0.99, Fig. 3b). On this
basis we conclude that probe 1 is capable of detecting hydrazine
the probe is present on the surface of a TLC plate.
10 both in mixed aqueous-organic environments.
The pH dependence of the hydrazine-induced cyclization
reaction was also investigated. As seen in Fig. S17, probe 1 is
stable over a pH range of 2 to 12. On the other hand, it readily
reacts with hydrazine within a pH range of 2 to 5. Such findings
15 led us to consider that 1 may be used to detect hydrazine
effectively in acidic environments.
Fig. 4 (a) Fluorescent and (b) visual color changes of probe 1 (5.0 mM)-
50 coated TLC plates after exposure to an excess quantity of various vapors,
including
hydrazine,
ammonia,
methylamine,
n-butylamine,
formaldehyde, acetaldehyde, H2O2, HCl, and CO2 for 1.0 min,
respectively. The fluorescent color changes were observed using a hand-
held UV lamp producing an excitation at 365 nm. In the specific case of
55 hydrazine a partial pressure of 9.0 mm Hg was used by exposing the TLC
plate directly to the equilibrated head space of a 20.7 M (65%) solution of
hydrazine hydrate.
We also examined whether 1 can be used for the detection of
hydrazine in cells. In this study, HeLa cells were incubated with
60 probe 1 and then separately treated with various concentrations of
hydrazine (0, 0.1, 0.5 and 1.0 mM). Fluorescence images were
then acquired via confocal microscopy. As shown in Fig. 5, upon
two-photon excitation at 740 nm, probe 1 displays a strong
fluorescence signature in the presence of hydrazine. Moreover,
Fig. 3 (a) Fluorescence spectra of 1 (10.0 μM) recorded without and with
20 hydrazine (1.0 mM) in an aqueous mixture (H2O/CH3CN = v/v, 1:9). (b)
The changes in the fluorescence intensity (FI) at 534 nm observed for 1
(10.0 μM in H2O/CH3CN = v/v, 1:9) as a function of hydrazine 65 the fluorescence intensity of 1 in HeLa cells was seen to depend
concentration (0, 0.8, 1.2, 2.0, 3.0, 5.0, 6.0, 8.0, 10.0 μM). All spectra
were acquired 1 h after hydrazine addition and obtained using excitation
results lend further support to the inference that the hydrazine-
25 at 348 nm.
on the concentration of hydrazine in the cellular medium. These
induced cyclization of 1 to produce the fluorescent species 2 is
very selective and is not subject to appreciable interference from
70 other biologically abundant amines and metal ions.
We also tested whether probe 1 would react with hydrazine gas
or other vaporized organic materials (i.e., formaldehyde,
acetaldehyde, H2O2, HCl, CO2, ammonia, methylamine, and n-
butylamine). For these tests, glass TLC plates were soaked in a
30 CH3CN solution of 1 (5.0 mM) and dried. The TLC plates coated
with 1 were then exposed to vapors of each sample for 1.0 min,
respectively. This was done under conditions where the vapors
were in considerable excess relative to 1. As shown in Fig. 4,
upon exposure of hydrazine vapor at a partial pressure of 9.0 mm
35 Hg, distinctive changes were observed in the fluorescence (from
blue to green) and color (from white gray to yellow) of the TLC
plates. While in the case of other amines a visual color change
from white gray to yellow is seen, the change in the fluorescent is
negligible. Moreover, no detectable changes or trace differences
40 were observed upon exposure of probe 1 to other environmentally
Fig. 5 Confocal microscopic images of HeLa cells treated with 1. Cells
75 were incubated with 1 (10.0 μM) and separately incubated with media
containing hydrazine (0, 0.1, 0.5 and 1.0 mM) for 2 h at 37 °C.
Fluorescence images were obtained using
a two-photon excitation
wavelength of 740 nm and emission wavelengths of 400−600 nm.
4
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

View Article Online
DOI: 10.1039/C3SC51813B
Page 5 of 7
Chemical Science
removed and replaced with DMEM without FBS. The cells were
treated and incubated with 10 μM of 1 at 37 °C under 5% CO2 for
2 h. The cells were washed three times with phosphate buffered
saline (PBS, Gibco) and then cell images were obtained using a
60 confocal microscope from Leica (Leica TCS SP2 model). Other
information is available in the Figure captions.
Conclusions
Compound 1, containing
a naphthalimide and bearing a
trifluoroacetyl acetonate moiety for hydrazine sensing, was
synthesized. This probe undergoes a selective hydrazine-induced
cyclization to give a fluorescent species 2. This results in a turn-
on of the fluorescence emission signal at 501 nm and color
changes within the time course of 60 min. The detection limit of 1
was found to be 3.2 ppb, which falls below the 10 ppb limit for
hydrazine exposure set by the U.S. EPA. Competition
10 experiments were carried out and served to confirm that probe 1
selectively reacts with hydrazine, even in the presence of other
test amines. When coated on silica gel TLC plates, probe 1 can be
used to detect vaporized hydrazine selectively as inferred from
the easy-to-visualize fluorescent and color changes. Finally, it
15 was found that probe 1 may be used to detect hydrazine in HeLa
cells without appreciable interference from other biologically
abundant amines or metal ions. We thus propose that probe 1 and
structurally related species that exploit a selective ring-forming
reaction could be used to sense hydrazine in a range of
20 environments.
5
4. Two-photon fluorescence microscopy
Two-photon fluorescence microscopy images of HeLa cells
65 incubated with 1 were obtained using spectral confocal and
multiphoton microscopes (Leica TCS SP2 model) with a ×10
(NA = 0.30 DRY) and ×100 (NA = 1.30 OIL) objective lens. The
two-photon fluorescence microscopic images were obtained using
a DM IRE2 Microscope (Leica) by exciting probe 1 with a mode-
70 locked titanium sapphire laser source (Coherent Chameleon, 90
MHz, 200 fs) set at wavelength 740 nm and using an output
power of 1580 mW, which corresponded to approximately 10
mW average power in the focal plane. To obtain images, internal
photomultiplier tubes (PMTs) were used to collect the signals in
75 an 8 bit unsigned 512 × 512 pixels at 400 Hz scan speed.
5. Synthesis
Experimental section
Compounds 4 and 5 were prepared by adapting published
procedures.18
1. Synthetic materials and methods
All reagents, including amines, metal ions, and other chemicals
for synthesis, were purchased from Aldrich, TCI and used as
25 received. All solvents were HPLC reagent grade, and triple-
deionized water was used throughout the analytical experiments.
Silica gel 60 (Sorbent, 40–63 mm) was used for column
chromatography. Analytical thin layer chromatography was
performed using Silicycle 60 F254 silica gel (precoated sheets,
30 0.25 mm thick). 1H and 13C NMR spectra were recorded in
CDCl3 (Cambridge Isotope Laboratories, Cambridge, MA) on
Varian 400 MHz spectrometers.
80 Synthesis of 6. Compound 5 (2.0 g, 8.3 mmol) and n-butylamine
(1.2 mL, 16.6 mmol) were dissolved in ethanol (100 mL). The
reaction mixture was then stirred and heated at reflux for 3 h.
After removal of solvent under reduced pressure, the crude
product was purified over silica gel using ethyl acetate/hexanes
85 (v/v, 1:2) as the eluent to yield 6 as a white solid (2.0 g, 81%).
1
HRESI-MS m/z ([M+Na]+) calc 318.11006, obs 318.11015. H
NMR (CDCl3, 400 MHz): δ 8.91 (d, 1 H, J = 8.8 Hz); 8.59 (d, 2
H, J = 8.6 Hz); 8.08 (d, 1 H, J = 8.1 Hz); 7.78 (t, 1 H, J = 7.8 Hz);
4.12 (t, 2 H, J = 4.2 Hz); 2.76 (s, 3 H); 1.70–1.63 (m, 2 H); 1.42–
90 1.37 (m, 2 H); 0.92 (t, 3 H, J = 0.9 Hz). 13C NMR (CDCl3, 100
MHz): 200.6, 163.8, 163.3, 139.7, 132.3, 131.5, 129.6, 128.8,
128.3, 125.6, 122.3, 40.4, 30.3, 20.4, 14.2 ppm.
2. UV/Vis absorption and fluorescence spectroscopy
35 Stock solutions of compounds 1, 6, and amines were prepared in
CH3CN. The chloride salts of the test metal ions Cs+, Na+, K+,
Ca2+, Mg2+, Zn2+, Cu2+, Mn2+, Fe2+, Fe3+, Hg2+, Pb2+, Cd2+, Ni2+,
and Ba2+ were prepared in triple-distilled water. The fluorescence
quantum yields (Φf) were measured relative to quinine sulfate (Φf
40 = 0.54 in 0.5 M H2SO4).20 Absorption spectra were recorded on
Varian-5000 UV/Vis-NIR spectrophotometer, and fluorescence
spectra were recorded using a FL3-11T spectrofluorometer
(Nanolog) equipped with a xenon lamp (FL 1039). Samples for
absorption and emission measurements were contained in quartz
45 cuvettes (3 mL volume). Excitation was provided at 348 nm with
excitation and emission slit widths both set at 5 nm.
Synthesis of 1. To a solution of 6 (1.3 g, 4.4 mmol) in
dichloromethane was added ethyl trifluoroacetate (10 mL). The
95 mixture was stirred for 5 min before an ethanolic 21% NaOEt
solution (20 mL) was added. The reaction mixture was then
heated at reflux for 2 h under a nitrogen atmosphere in the dark.
After allowing the vessel to cool to room temperature, the
reaction was quenched by adding dilute HCl(aq.). The solution was
100 then extracted with dichloromethane several times. The combined
organic phases were dried over Na2SO4, and filtered. After
removal of solvent under reduced pressure, the crude product was
purified over silica gel using ethyl acetate/hexanes (v/v, 4:1) as
the eluent to yield 1 as a brownish solid (1.2 g, 88%). HRESI-MS
105 m/z ([M+Na]+) calc 414.09236, obs 414.09219. 1H NMR (CDCl3,
400 MHz): δ 14.8 (s, 1 H); 8.79 (d, 1 H, J = 8.8 Hz); 8.70–8.65
(m, 2 H); 8.06 (d, 1 H, J = 8.1 Hz); 7.88 (t, 1 H, J = 7.8 Hz); 6.54
(s, 1 H); 4.19 (t, 2 H, J = 4.2 Hz); 1.77–1.71 (m, 2 H); 1.50–1.41
(m, 2 H); 0.99 (t, 3 H, J = 0.9 Hz). 13C NMR (CDCl3, 100 MHz):
110 188.5, 163.4, 163.0, 136.6, 131.7, 131.2, 129.7, 128.5, 128.1,
125.7, 122.8, 97.7, 41.0, 30.5, 20.1, 13.1 ppm.
3. Cell culture and imaging
A human cervical cancer cell line (HeLa) was cultured in
50 Dulbecco's Modified Eagle’s Medium (DMEM) supplemented
with 10% FBS (WelGene), penicillin (100 units/mL), and
streptomycin (100 μg/mL). Two days before imaging, the cells
were passed and plated on glass-bottomed dishes (MatTek). All
the cells were maintained in a humidified atmosphere of 5/95 (v/v)
55 of CO2/air at 37 °C. For labelling, the growth medium was
6. X-ray crystallography for 3
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

View Article Online
Chemical Science
DOI: 10.1039/C3SC51P81a3gBe 6 of 7
11 J.-A. Oh, J.-H. Park and H.-S. Shin, Anal. Chim. Acta, 2013, 769, 79.
65 12 Z. K. He, B. Fuhrmann and U. Spohn, Anal. Chim. Acta, 2000, 409,
83.
Crystals were grown as yellow prisms by slow evaporation from
chloroform-d3 (CCDC number: 942177). The data were collected
at -120 C on a Nonius Kappa CCD diffractometer using a
Bruker AXS Apex II detector and a graphite monochromator with
MoKα radiation (λ = 0.71075 Å ). Reduced temperatures were
maintained by use of an Oxford Cryosystems 600 low-
temperature device. A total of 2194 frames of data were collected
using ɷ-scans with a scan range of 0.9 and a counting time of 62
seconds per frame. Details of crystal data, data collection and
13 (a) A. Umar, M. M. Rahman, S. H. Kim and Y.-B. Hahn, Chem.
Commun., 2008, 166. (b) J. Liu, Y. Li, J. Jiang and X. Huang, Dalton
Trans., 2010, 39, 8693.
70 14 S. W. Thomas and T. M. Swager, Adv. Mater., 2006, 18, 1047.
15 (a) M. G. Choi, J. Hwang, J. O. Moon, J. Sung and S.-K. Chang, Org.
Lett., 2011, 13, 5260. (b) M. G. Choi, J. O. Moon, J. Bae, J. W. Lee
and S.-K. Chang, Org. Biomol. Chem., 2013, 11, 2961.
16 J. Fan, W. Sun, M. Hu, J. Cao, G. Cheng, H. Dong, K. Song, Y. Liu,
5
75
80
85
S. Sun and X. Peng, Chem. Commun., 2012, 48, 8117.
17 X. Chen, Y. Xiang, Z. Li and A. Tong, Anal. Chim. Acta, 2008, 625,
41.
18 W. Zhu, R. Yao and H. Tian, Dyes and Pigments, 2002, 54, 147.
19 (a) Y. Kinoshita, M. Kunieda, Y. Mikata and H. Tamiaki,
Tetrahedron Lett., 2013, 54, 1243; (b) J. C. Sloop, C. L.
Bumgardner, G. Washington, W. D. Loehle, S. S. Sankar and A. B.
Lewis, J. Fluorine Chem., 2006, 127, 780.
10 structure refinement are listed in Table S1.
Acknowledgment
This work was supported by the U.S. National Science
Foundation (CHE-1057904; J.L.S.), the Robert A. Welch
Foundation (F-1018 to J.L.S.) and by a CRI project grant from
15 National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIP) (No. 2009-0081566; J.S.K.).
20 G. A. Crosby and J. N. Demas, J. Phys. Chem., 1971, 75, 991.
21 Z. Muňoz, A. S. Cohen, L. M. Nguyen, T. A. McIntosh and P. E.
Hoggard, Photochem. Photobiol. Sci., 2008, 7, 337.
Notes and references
a Department of Chemistry and Biochemistry, The University of Texas at
Austin, Austin, TX 78712-1224, USA. c Department of Chemistry, Yonsei
20 University, Seoul 120-749, Korea. Fax: 512 471-7550; E-mail:
sessler@cm.utexas.edu
b Department of Chemistry, Korea University, Seoul 136-701, Korea. Fax:
+82-2-3290-3121; E-mail: jongskim@korea.ac.kr
25 † Electronic Supplementary Information (ESI) available: Experimental
details, Mass and NMR spectra; X-ray crystallographic details
corresponding to CCDC number 942177. See DOI: 10.1039/b000000x/
1
(a) P. T. Anastas, Crit. Rev. Anal. Chem., 1999, 29, 167; (b) P. T.
Anastas and J. C. Warner, Green Chemistry: Theory and Practice,
Oxford University Press, New York, 1998; (c) J. Buffle and G.
Horvai, In Situ Monitoring of Aquatic Systems: Chemical Analysis
and Speciation, Wiley: New York, 2001.
30
35
40
45
50
55
2
3
(a) P. D. Beer and P. A. Gale, Angew. Chem. Int. Ed., 2001, 40, 486;
(b) S. K. Kim and J. L. Sessler, Chem. Soc. Rev., 2010, 39, 3784.
(a) H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev.,
2012, 41, 3210; (b) D. T. Quang and J. S. Kim, Chem. Rev., 2010, 110,
6280; (c) D.-G. Cho and J. L. Sessler, Chem. Soc. Rev., 2009, 38,
1647.
(a) X. Chen, G. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2012,
41, 4610; (b) Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52; (c)
Y. Zhou, Z. Xu and J. Yoon, Chem. Soc. Rev., 2011, 40, 2222.
(a) E. W. Schmidt, Hydrazine and its Derivatives: Preparation,
Properties, Applications, Wiley, New York, 1984; (b) H. W. Schiessl,
Encyclopedia of Chemical Technology, ed. K. Othmer, Wiley, New
York, 3rd edn., 1980, 12, 734; (c) K. Yamada, K. Yasuda, N.
Fujiwara, Z. Siroma, H. Tanaka, Y. Miyazaki and T. Kobayashi,
Electrochem. Commun., 2003, 5, 892.
Y.-Y. Liu, I. Schmeltz and D. Hoffmann, Anal. Chem., 1974, 46, 885.
(a) J.-W. Mo, B. Ogorevc, X. Zhang and B. Pihlar, Electroanalysis,
2000, 12, 48; (b) S. D. Zelnick, D. R. Mattie and P. C. Stepaniak,
Aviat. Space Environ. Med., 2003, 74, 1285; (c) Ragnarsson, U.
Chem. Soc. Rev., 2001, 30, 205; (d) V. Andries and D. Couturier,
Materials Performance, 2000, 39, 58.
M. Strous and M. S. M. Jetten, Ann. Rev. Microbiol., 2004, 58, 99.
(a) B. Toth, Cancer Res., 1975, 35, 3693; (b) S. Garrod, M. E.
Bollard, A. W. Nicholls, S. C. Connor, J. Connelly, J. K. Nicholson
and E. Holmes, Chem. Res. Toxicol., 2005, 18, 115; (c) C. A. Reilly
and S. D. Aust, Chem. Res. Toxicol., 1997, 10, 328.
4
5
6
7
8
9
60 10 U.S. Environmental Protection Agency (EPA). Integrated Risk
Information System (IRIS) on Hydrazine/Hydrazine Sulfate. National
Center for Environmental Assessment, Office of Research and
Development, Washington, DC. 1999.
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

View Article Online
DOI: 10.1039/C3SC51813B
Page 7 of 7
Chemical Science
TOC
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7