A fluorogenic H2S-triggered prodrug based on thiolysis of the NBD amine

Article abstract of DOI:10.1016/j.bmcl.2019.126627

Based on thiolysis of the NBD amine, a H₂S-triggered prodrug has been designed and synthesized for localized production of ciprofloxacin under micromolar H₂S. Activation of the prodrug can be monitored through fluorescence in real-time. We envision that thiolysis of the NBD amine could be readily used for development of other H₂S-triggered prodrugs in the future.

Full text of DOI:10.1016/j.bmcl.2019.126627

Journal Pre-proofs
A fluorogenic H2S-triggered prodrug based on thiolysis of the NBD amine†
Yonghui Xie, Haojie Huang, Ismail Ismail, Hongyan Sun, Long Yi, Zhen Xi
PII:
S0960-894X(19)30569-4
DOI:
https://doi.org/10.1016/j.bmcl.2019.126627
BMCL 126627
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
22 March 2019
8 July 2019
16 August 2019
Please cite this article as: Xie, Y., Huang, H., Ismail, I., Sun, H., Yi, L., Xi, Z., A fluorogenic H2S-triggered prodrug
based on thiolysis of the NBD amine†, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.126627
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Bioorganic & Medicinal Chemistry Letters
A fluorogenic H2S-triggered prodrug based on thiolysis of the NBD amine†
a
b
Yonghui Xie,‡ Haojie Huang,‡ Ismail Ismail,a Hongyan Sun,c,d Long Yi*b,e and Zhen Xi*a,e
aState Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, National Engineering Research Center of Pesticide (Tianjin),
College of Chemistry, Nankai University, Tianjin 300071, China. Email: zhenxi@nankai.edu.cn
bState Key Laboratory of Organic-Inorganic Composites and Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing
100029, China. Email: yilong@mail.buct.edu.cn
cDepartment of Chemistry and Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong
Kong, P. R. China.
dKey Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen, 518057, P. R.
China
eCollaborative Innovation Center of Chemical Science and Engineering (Tianjin)
†Electronic Supplementary Information (ESI) available: experimental procedures and additional figures. See DOI: 10.1039/b000000x/
‡These authors contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Based on thiolysis of the NBD amine, a H2S-triggered prodrug has been designed and
synthesized for localized production of ciprofloxacin under micromolar H2S. Activation of the
prodrug can be monitored through fluorescence in real-time. We envision that thiolysis of the
NBD amine could be readily used for development of other H2S-triggered prodrugs in the future.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
prodrug
H2S-triggered
ciprofloxacin
NBD amine
fluorescence probes since 2013.12 The fluorescence of the
fluorophore can be quenched by the NBD moiety in aqueous
Introduction
Recent studies deomonstrated that endogenously produced
H2S possess important physiological functions, and H2S is named
as the third gasotransmitter after NO and CO.1,2 Accumulating
solution, whereas the fluorescence will be restored upon the
thiolysis of the NBD moiety. To this end, a series of NBD-based
H2S probes with emissions from blue to near-infrared range have
been developed by our groups.13 In this work, we continue to
investigate this field and develop a fluorogenic H2S-triggered
prodrug (Fig. 1) based on thiolysis of the NBD amine for the first
time.
evidence suggests that H2S influences
a wide range of
physiological and pathological processes, including modulation
of blood vessel and cardioprotection,3 endogenous stimulator of
angiogenesis,4 mitochondrial bioenergetics,5 and crosstalk with
MicroRNA.6 H2S also plays important roles in tumour biology,
and it is suggested that both inhibition of H2S biosynthesis and
elevation of H2S concentration beyond a certain threshold could
exert anticancer effects.7 In plants, H2S participates in seed
germination, plant growth and development, as well as the
acquisition of stress tolerance including cross-adaptation and
cadmium tolerance.8,9 In bacteria, H2S is a key player in
maintaining intracellular redox balance to counteract a lethal
degree of oxidative stress induced by ampicillin.10 Due to its
widespread existence and functions, we spectulate that H2S might
Fig. 1 Schematic illustration of H2S-triggered release of the
prodrug 1 to produce ciprofloxacin 2 and NBD-SH accompanying
with fluorescence increase.
be employed to develop H2S-triggered prodrugs
improved properties.
with
One major challenge in developing H2S-triggered prodrugs
and fluorescence probes is the development of a chemical
reaction to effectively differentiate the reactivity of high
concentration of biothiols (millimolar) versus low concentration
of H2S (micromolar).11 To address this issue, we and Pluth et al.
A fluorogenic prodrug can be used to in situ produce active
drug accompanying with significant fluorescence increase.14
A
modification of prodrug is commonly designed by conjugating a
parent drug with a promoiety, which could be eliminated by a
specific reaction under a certain condition.15 Therefore, selective
delivery of the active drug can be achieved with a specific
have
developed
thiolysis
of
the
NBD
(7-nitro-1,2,3-benzoxadiazole) amine for design of H2S-specific

stimulus. To this end, bioorthogonal approaches are employed for
"on-demand" activation of prodrugs.14a In this work, we report a
NBD-capped ciprofloxacin 1 as a fluorogenic prodrug (Fig. 1),
which can be activated by micromolar H2S.
fast and selectively activated via H2S and be used as a
fluorescence probe for detection of H2S in aqueous buffer.
The synthesis of the prodrug 1 can be prepared from direct
coupling of NBD-Cl and 2.16 The facile and economic synthesis
is important for the wide use of the prodrug and probe. The
chemical structure of 1 was confirmed by 1H NMR, and
high-resolution mass spectra (HRMS). It is noted that the signals
for 13C NMR were quite weak due to the poor solubility of 1 in
common deuterated solvents.
With the prodrug in hand, we firstly tested the absorbance
spectra of 1 in the presence of H2S (using Na2S as an equivalent)
in PBS buffer (20 mM, pH 7.4). 1 showed relative low solubility
in buffer (~ 17 µM, Fig. S1). As shown in Fig. 2a, 1 exhibited
absorbance peaks at 350 nm and 490 nm, which should be
assigned to the ciprofloxacin and NBD fluorophores, respectively.
After reaction with H2S, new absorbance peak appeared at 530
nm, which might be due to the production of NBD-SH.12b The
intensities at 560 nm can be used to monitor the reaction kinetics
to give the reaction rate of 12.9 M-1s-1 (Fig. S2). Such thiolysis
reaction was further characterized by HPLC analysis (Fig. 2b).
The reaction was nearly complete within 5 min, and the peak for
1 was completely disappeared at 40 min. These studies indicated
the prodrug 1 could be activated by H2S efficiently.
Fig. 3 (a) Time-dependent fluorescence spectra of 1 (5 μM) and
Na2S (100 μM) in PBS buffer (Ex. = 280 nm). Fluorescence intensity
at 415 nm versus time is indicated inset. The red line represents the
best fitting to give kobs of 0.08 min−1. (b) The linear relationship
between concentrations of Na2S and kobs. The slope of the linear
fitting gives k2
Encouraging with the above results, we next employed 1 for
antibacterial tests under different conditions. The concentration
of H2S in E. coli strains of urinary tract infections is between
300-600 µM approximately.[17] E. coli was chosen as a model
bacterium in our study. After incubation of 30 nM 1 and E. coli
in the LB medium for 12 h, the OD600 value was recorded for
each sample to show the bacterial concentration (Fig. 4). The
prodrug 1 or H2S, L-Cys or 1 + GSH (Fig. S7) did not show
obvious inhibition on the bacterial growth. However, the
combined usage of 30 nM 1 and 50 µM H2S resulted in
significant inhibition activity against E. coli, and such inhibition
effect could be directly visualized by naked eye (Fig. 4). L-Cys
can induce E. coli to produce endogenous micromolar H2S (Fig.
S8), which can be also employed to activate the prodrug.
However, a mixture of NBD-diethanolamine and H2S (that can
produce NBD-SH) has no inhibition on bacterial growth. The
results implied that 1) the prodrug can be activated by exogenous
or endogenous H2S; 2) the release of ciprofloxacin produced a
bactericidal effect, not the by-product NBD-SH.
Fig. 2 (a) Time-dependent absorption spectra of 1 (10 µM) and
Na2S (200 μM) in PBS buffer (50 mM, pH 7.4). (b) Time-dependent
HPLC traces of the reaction of 1 with Na2S to give ciprofloxacin 2.
The probe 1 showed very weak fluorescence upon excitation
at 280 nm, indicating that the fluorescence from ciprofloxacin
may be majorly quenched by the FRET effect (Fig. 1). After
reaction with H2S, large fluorescence turn-on was observed, with
about 62-fold increase at 415 nm (Fig. 3a). The reaction kinetics
of the prodrug during the activation is an important parameter to
examine its biological applicability on account of the rapid
catabolism of H2S under physiological conditions. To this end,
time-dependent fluorescence intensities at 415 nm for the probe
in the presence of H2S were acquired for data analysis (Fig. S3).
The pseudo-first-order rate, kobs, was found by fitting the data
with a single exponential function. The reaction rate k2 (12.0
M-1s-1) was obtained by linear fitting of the kobs versus H2S
concentrations (Fig. 3b). Moreover, the fluorescence of the
prodrug could be efficiently released by low-micromolar H2S
(Fig. S4). The prodrug cannot be activated by millimolar
biothiols (10 mM GSH, 5 mM Cys, Fig. S5), which is consistent
with the highly H2S-specific reactivity of our thiolysis of the
NBD amine.13 In addition, we examined the reactivity of the
prodrug with different concentrations of H2O2 (Fig. S6), the
prodrug cannot be activated, even if the concentration of H2O2 is
as high as 200 μM. These results imply that the prodrug can be
Fig. 4 OD600 values of E. coli in the different cultured conditions.
Experimental conditions: 30 nM 1, 30 nM NBD-SH, 50 μM Na2S, 5
mM L-Cys, 50 μM Na2S + 30 nM 1, or 5 mM L-Cys + 30 nM 1, 30
nM 2 was added into the LB medium as indicated below each bar.
Inset: photographs of
E coli in tubes under the conditions
corresponding to each column. Images of 1 (15 μM) with or without
H2S (400 μM) in PBS buffer under white light or 254 nm UV light is
indicated inset.

Take
a
step further, we attempted to study the
concentration-dependent inhibition of bacterial growth. Various
concentrations of 1 in the absence and presence of H2S were
added into the culture medium. After incubation at 37 ºC for
another 12 h, the OD600 of the medium was measured for each
sample (Fig. 5). As expected, the prodrug 1 at low concentration
showed weak inhibition effect on the bacterial growth, while the
addition of H2S or L-Cys significantly increased the inhibition
effect of 1. However, the prodrug could also inhibit bacterial
growth at high concentrations (> 0.2 μM), which may be
explained by the inhibitory mechanism of ciprofloxacin. Firstly,
the X-ray crystallography of ciprofloxacin in complex with DNA
gyrase18 showed that piperazinyl ring of ciprofloxacin protruding
toward the protein surface had little interaction with its target
protein. Secondly, the substituted piperazinyl ring of drug
variants19 (levofloxacin, gatifloxacin, moxifloxacin and
C8-Me-moxifloxacin) were all effective antibiotics. Therefore,
the inhibitory activity of prodrug 1 was reduced rather than
completely lost. The MIC of the combination of prodrug 1 and
H2S (50 μM Na2S or 5 mM L-Cys) was about 30 nM, and in this
condition E. coli cell were almost killed (Fig. 5) within 12 hours.
The NBD moiety even at micromolar concentrations did not have
obvious effect on bacterial growth.
Acknowledgments
This work was supported by the National Key R&D Program
of China (2017YFD0200500, 16JCYBJC20200), NSFC
(21572190, 21877008).
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In conclusion, we have designed and synthesized the first
H2S-triggered prodrug based on thiolysis of the NBD amine. The
prodrug can be used for localized production of ciprofloxacin in
the presence of micromolar H2S. Activation of the prodrug can be
indicated via significant turn-on (62-fold) fluorescence in
real-time. We also envision that thiolysis of NBD amine could be
generally used for development of other H2S-triggered prodrugs,
including that for sparfloxacin, grepafloxacin, pipemidic acid,
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Graphical Abstract
This work reports a fluorogenic H2S-triggered prodrug for in
situ production of ciprofloxacin under micromolar H2S.
A fluorogenic H2S-triggered prodrug based
Leave this area blank for abstract info.
on thiolysis of the NBD amine†
a
b
Yonghui Xie,‡ Haojie Huang,‡ Ismail Ismail, a Hongyan Sun,c,d Long Yi*b,e and Zhen Xi*a,e