Photoactive NO hybrids with pseudo-zero-order release kinetics for antimicrobial applications

Article abstract of DOI:10.1039/d0ob00564a

Bacterial infection is a major threat to the health and life of humans due to the development of drug resistance, which is related to biofilm formation. Nitric oxide (NO) has emerged as an important factor in regulating biofilm formation. In order to harness the potential benefits of NO and develop effective antibacterial agents, we designed and synthesized a new class of NO hybrids in which the active scaffold benzothienoazepine was tagged with a nitroso group and further conjugated with quaternary ammoniums or phosphoniums. The temporal release of NO from these hybrids can be achieved by photoactivation. Interestingly, the NO release follows a pseudo-zero-order kinetics, which is easily determined by measuring the fluorescent benzothienoazepine or NO. Compared to the positive control ciprofloxacin, the NO hybrid with triphenyl phosphonium (TPP) exhibited more effective activity against S. aureus biofilm in darkness. Irradiation of the NO hybrid led to higher inhibition against S. aureus biofilm compared to the parental NO hybrid in darkness or the corresponding NO-released product, indicating the combined effect of NO and the NO-released product. Therefore, this new class of NO hybrids includes very promising antimicrobial agents and this work provides a new way for the design of highly effective antimicrobial agents. This journal is

Full text of DOI:10.1039/d0ob00564a

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. liao, Z. ye, M.
Qian, X. wang, Y. guo, G. Han, Y. Song, J. hou and Y. Liu, Org. Biomol. Chem., 2020, DOI:
1
0.1039/D0OB00564A.
Volume 15
Number 47
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
21
December 2017
Pages 9945-10124
Organic &
Biomolecular
Chemistry
rsc.li/obc
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1477-0520
PAPER
I . J. Dmochowski et al.
Oligonucleotide modifi cations enhance probe stability for
single cell transcriptome in vivo analysis (TIVA)
rsc.li/obc

Please do not adjust margins
Page 1 of 8
Organic & Biomolecular Chemistry
ARTICLE
Journal Name
View Article Online
Photoactive NO hybrids with pseudo-zero-order release kinetics
DOI: 10.1039/D0OB00564A
for antimicrobial applications
1
Received 00th January 20xx,
Yongfang Liao, Zizhen Ye, Meng Qian, Xing Wang, Yuda Guo, Guifang Han, Yuguang Song, Jingli
Hou*, Yangping Liu*
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Bacterial infection is a major threat to health and life of human due to development of drug resistance, which is related to
biofilm formation. Nitric oxide (NO) has emerged as an important factor in regulating the biofilm formation. In order to
harness the potential benefit of NO and develop effective antibacterial agents, we designed and synthesized a new class of
NO hybrids in which active scaffold benzothienoazepine was tagged with a nitroso group and further conjugated with
quaternary ammoniums or phosphoniums. The temporal release of NO from these hybrids can be achieved by
photoactivation. Interestingly, the NO release follows a pseudo-zero-order kinetics, which is easily determined by
measurement of the fluorescent benzothienoazepine or NO. Compared to positive control ciprofloxacin, the NO hybrid
with triphenyl phosphonium (TPP) exhibited more effective activity against S. aureus biofilm in darkness. Irradiation of the
NO hybrid led to higher inhibition against S. aureus biofilm than the parental NO hybrid in darkness or the corresponding
NO-released product, indicating the combined effect of NO and the NO-released product. Therefore, this new class of NO
hybrids are very promising antimicrobial agents and this work provides a new way for design of highly effective
antimicrobial agents.
Furthermore, NO hybrids with combined antibiofilm and
antibacterial activities had been developed to kill
Pseudomonas aeruginosa. Even with the promising results,
Introduction
1
4
Bacterial infection is a major threat to the health and life the therapeutic applications of NO donors are limited since
of human from the onset of human existence. It has been most of them lead to spontaneous generation of NO in
estimated that at least of 60% of bacterial infections currently aqueous solution. In order to harness the potential benefits of
treated by physicians in the developed world are considered to NO, it is extremely desirable to liberate NO at the local site
1
involve biofilm formation. Once established, the biofilms are with precise concentration and specific duration. Photo-
extremely difficult to eradicate by antimicrobial treatment. induced NO donors are potential candidates to meet the needs
Clinically, biofilm infections represent an overwhelming issue, since they allow the release of NO in a precise spatial and
because biofilms usually cause persistent and chronic temporal mode by controlling the site, duration and intensity
infections due to drug resistance. Bacteria with biofilms are of the light.
highly (10-1000 fold) resistant to antimicrobials that are
effective in the treatment of these same bacteria in planktonic developed, such as metal nitrosyls, nitrobenzene and N-
Various types of photo-induced NO donors have been
1
5
16
growth mode.2 However, there is a lack of effective nitroso-amines,
-5
17-19
which enable the NO release in a precise
antibiofilm agents to eradicate biofilm-related infections. spatial and temporal mode by controlling of the site, duration
Therefore, it is of great importance to develop new type of and light intensity. Although significant progress has been
medicines that target bacterial biofilm formation.
achieved in the development of photo-induced NO donors,
The signalling molecule nitric oxide (NO) has emerged as some challenges still remain. Particularly, the donors usually
6
an important factor in regulating biofilm formation. It was release NO in a “fast-then-slow” manner, leading to the
reported that nanomolar concentrations of NO prevent initial fluctuation of NO concentrations. Since the therapeutic
biofilm formation and induce a transition from the biofilm to consequence of NO-based drugs depends strongly on the
the planktonic mode of growth via multiple mechanisms, concentrations of NO delivered, effective NO-based therapies
including cyclic-di-GMP signalling, nitric oxide signalling, should only deliver desirable NO doses. In this context, the NO
6
-9
quorum sensing and so on. Moreover, low-dose NO is used donors with constant release rates during the entire release
as targeted adjunctive therapy to disperse Pseudomonas process are very appealing.
1
0
aeruginosa biofilm in cystic fibrosis. Consequently, NO
donors such as sodium nitroprusside and nitrofurazone exhibit bioactive molecules, we focused on N-aryl-nitrosamines to
Considering that aromatic amine is an important moiety in
5
,
11-13
excellent activity to induce biofilm dispersal.
design a novel NO hybrid with high atom economy, which can
undergo homolytic cleavage of N-NO bond upon irradiation to
2
0
give NO and the corresponding aromatic amine derivative.
Tianjin Key Laboratory on Technologies Enabling Development of Similar strategy was used to design near-infrared
ClinicalTherapeutics and Diagnostics, School of Pharmacy, Tianjin Medical
University,Tianjin 300070, P. R. China. E-mail: liuyangping@tmu.edu.cn;
houjingli@tmu.edu.cn
photoactivatable NO donors which enabled integrated
photoacoustic monitoring. In order to develop antimicrobial-
1
8
Electronic Supplementary Information (ESI) available: See DOI:10.1039/x0xx00000x based photoactivatable NO hybrids, an aromatic amine-
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 8
ARTICLE
containing antimicrobial is desirable for the attachment of
Journal Name
To study the antibacterial effect of the cationic groups,
View Article Online
DOI: 10.1039/D0OB00564A
nitroso group. Benzothienoazepine derivatives had been triphenyl phosphonium (TPP), trimethyl ammonium and
2
1
reported as respiratory syncytial virus inhibitors. In the initial pyridinium groups were chosen. The cationic groups TPP and
screening, its derivatives (ester form) displayed weak activity trimethyl ammonium were directly connected to compound 5
against Gram-positive bacteria S. aureus (Fig. S10), thus via amide coupling reaction, followed by N-nitrosation of the
Benzothienoazepine was selected as an antimicrobial (Fig. 1). secondary amine to afford the corresponding NO hybrids (7a
In addition, its fluorescence properties are also appealing and 7b, Scheme 1). The other NO hybrid bearing pyridinium
because NO release can be monitored by fluorescence method. (7c) was obtained by linkage of the pyridine group to the
To enhance the antimicrobial activity, the benzothienoazepine compound 5, followed by sequential N-methylation of the
was conjugated with quaternary ammonium or phosphonium pyridine and N-nitrosation. The target compounds (NO hybrids)
1
13
compounds (QACs or QPCs) that target bacterial cytoplasm were characterized by H NMR, C NMR and HRMS. HPLC
and caused the loss of the structural organization and analysis indicated good purity (>95%) and excellent stability of
integrity.22 These QACs and QPCs have been widely used to these NO hybrids without observable decay in CH
prepare antibacterial materials due to their potent and broad- dark for 24 h (Fig. S3-4).
spectrum activities.2 The final photoactivatable NO hybrids
3
CN in the
3
were then constructed by N-nitrosation on the QACs/QPCs-
O
O
NHR
conjugated benzothienoazepine core (Figure 1). In this work
we demonstrated that this type of NO hybrids could achieve
NO steady release by light at a zero-order rate, evidenced by
the yielded fluorescent product and NO generation. In contrast
to the commonly used antibiotic drug ciprofloxacin, these
hybrids showed potent effects against S. aureus biofilm.
OH
O
NHR
NaNO2, AcOH
HCl, DCM
S
RNH2
S
S
PPh3
R:
HATU, DMAP,
Et3N, DMF
7a
irradiation
N
NO
7
N
H
N
N
H
7b
5
6a, 6b
H2N
HATU, DMAP,
Et3N, DMF
N
Irridiation at 313 nm
Emission at 520 nm
H
H
O
H
O
N
O
N
N
N
N
N
quaternary ammonium or
phosphonium compounds
S
S
NaNO2, AcOH
HCl, DCM
S
CH3I
O
H
Yield: ca. 40%
N
O
H
N
Linker
irradiation
N
ON
7c
N
N
S
S
N
O
H
H
antimicrobial activity
6c
6d
antibiofilm
N
N
Scheme 1. The synthetic routes of the NO hybrids.
N
H
O
NO release moiety
Nonfluorescence
Fluorescence
Photo-response of NO hybrids
Figure 1. Design of light-induced NO hybrids from benzothienoazepine core and
quaternary ammonium/phosphonium moieties to kill or inhibit bacterial biofilm.
To investigate the photo-response of 7a-c, the UV-vis
absorption spectra of each compound (40 M) in PBS (20 mM,
pH = 7.4, containing 5% DMSO) were recorded intermittently
during irradiation. The maximum absorption of 7a is located at
Results and discussion
13 nm with a molar absorption of 17650 cm- M . Upon
1
-1
3
irradiation 7a with UV light (313  10 nm, 6 W LED light) in
quartz cuvette, the absorption of 7a at 313 nm was weakened,
Synthesis and characterization of NO hybrids
The key intermediate benzothienoazepine (5) was accompanied with the increase in the absorption of 6a at 365
obtained via 4 steps with a total yield of 50% from commercial nm. A linear relationship between the concentration of 6a and
1
,2,3,4-tetrahydro-benzo[b]azepin-5-one (1) according to the time was observed upon irradiation (Fig. 2a), indicating that
2
4
previously reported procedure (see SI). Interestingly, the the photo-induced denitrosation of 7a follows pseudo-zero-
benzothienoazepine (5) has a strong fluorescence emission at order kinetics. The denitrosation rate of 7a was determined to
5
-1
00 nm which has not been revealed since its initial synthesis 0.24 M min . Similarly, the photo-triggered denitrosation of
in 1993 (Fig. S1). The fluorescence quantum yield of 5 was 7b and 7c were also observed with irradiation at 313 nm and
determined to be 0.79 in ethanol (Fig. S2) and a large Stokes followed pseudo-zero-order decomposition kinetics, with the
shift (130 nm) was observed with excitation/emission maxima rates of 0.18 M min- for 7b and 0.23 M min for 7c,
at 370/500 nm in phosphate buffered saline (PBS). Thus, the respectively (Fig. 2b, c). Therefore, it can be concluded that the
compound 5 can be potentially used to develop photo- three NO hybrids have the similar photo-triggered
calibrated NO donors which allow conveniently monitoring of denitrosation rates, and the cationic group has little effect on
1
-1
1
9, 25
NO release in a real-time mode by fluorescence method.
their denitrosation.
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 8
Organic & Biomolecular Chemistry
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/D0OB00564A
Figure 2. (a, b, c): The time course changes of UV−vis absorption of 7a, 7b and 7c solution (40 M) upon irradiation by light 313 nm; Inset: The dynamic relationship
between the concentrations of product (6a-b and 6d) and exposure time to irradiation by fitting the absorption at 365 nm, respectively.
To check if the photo-triggered denitrosation of 7a-c can fluorescent stabilities of the hybrids (7a-b) and their
be monitored by fluorescent spectroscopy, we first measured precursors (6a-b) to ensure the detective reliability. As
the fluorescent spectra of the NO hybrids (7a-c) as well as their indicated in Fig. 3, only negligible changes in fluorescence
precursors (6a-b and 6d). All the NO hybrids are essentially intensity were observed for hybrids (7a-b) and precursors (6a-
non-fluorescent with the fluorescence quantum yields () of b) in the presence of different pH buffer (5.0-8.0), various
+
+
2+
0
.0022, 0.0033 and 0.0013 for 7a, 7b and 7c, respectively (Fig. cations (K , Na and Zn ), and amino acids, such as arginine
S2), while the precursors, except for 6d, show strong (Arg), cystein (Cys) and glutathione (GSH ) and so on. The
fluorescence emission at around 520 nm. The quantum yields results indicated there are no unexpected fluorescence
were estimated to be 0.557 and 0.564 for 6a and 6b in ethanol, changes from hybrids and NO-released products. The above
respectively. The tremendous difference in fluorescent properties ensured the reliability of fluorescence based
intensities of the NO hybrids and precursors suggests that the calibration method to monitor the denitrosation reaction of
denitrosation reaction of the hybrids can be monitored by NO hybrids.
fluorescence method. Subsequently, we investigated the
Figure 3. The fluorescence stability of NO hybrids and their precursors. (a) pH effect on fluorescence intensity; (b) fluorescence interference from amino acids and
cations. The NO hybrids and their precursors were prepared at the concentration of 5 . λex = 375 nm, λem = 520 nm, slit = 5 nm/5 nm.
As shown in Fig. 4a, the fluorescence intensities in the that the photo-triggered generation of 6a from 7a was linearly
solution of 7a (5 M) increased in a time-dependent manner correlated with time and the rate of the pseudo-zero-order
-1
upon irradiation at 313 nm. To confirm the generation of 6a kinetics was calculated to be 0.23 M min (Fig. 4a), in good
from the denitrosation reaction of 7a, the reaction mixture agreement with the value obtained by UV-vis absorption
was subjected to HPLC analysis. In addition to the peak of 7a at method. Moreover, we also plotted the fluorescence intensity
1
0.11 min, a new peak appeared at 6.01 min in the reaction changes for 7b as a function of the irradiation time and the
-1
mixture, which is the same as that of the authentic 6a (Fig. S5). generation rate of 6b was calculated to be 0.21 M min (Fig.
The generation of 6a from the denitrosation reaction of 7a was 4b). Due to the weak fluorescence of 6d (Fig. S7), its
further verified by high resolution mass spectrometry (HRMS) generation rate from 7c could not be obtained using the
studies (Fig. S5). In addition, a good linear relationship was fluorescence method. Taken together, the photo-triggered
observed between the fluorescent intensities of 6a and its denitrosation reactions of 7a and 7b can be also monitored by
concentrations in the range of 0 ~ 5 M (Fig. S6). Based on this fluorescence method, indicating that NO release could be
relationship, the concentration of 6a generated from 7a upon convenient detected by the fluorescence increase.
irradiation was conveniently calculated. The results showed
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 8
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/D0OB00564A
Figure 4. The time course changes of fluorescent spectra of 7a (a) and 7b (b) solutions (5 M) by light irradiation; ex = 375 nm, em = 520 nm, slit = 5 nm/5 nm. Inset:
The linear relationship between the concentrations of the product (6a or 6b) and irradiation time.
NO release from NO hybrids
To confirm NO release from the photo-induced methods due to the different irradiation condition used: 7a
denitrosation of 7a, the EPR spin trapping reagents N-methyl- solution was irradiated in quartz EPR tube (1.0 mm, id) for EPR
2
+
D-glucamine dithiocarbamate-iron (II) Fe -(MGD)
2
was experiments, but in 1-cm quartz cuvette for UV-vis and
employed. MGD-Fe traps NO efficiently to form NO-Fe-MGD fluorescent experiments. The NO release rates for 7b and 7c
complex, which has a typical triplet signal with a nitrogen was similar to that of 7a, with the value as 1.40 and 1.25 M
-1
hyperfine coupling constant (a
N
) between 12.5-13.2 G. All the min respectively (Fig. S8). It was worth mentioning that the
samples were subjected to irradiation in a quartz EPR tube. As zero-order kinetics ensured constant release of NO unlike the
2
6, 27
shown in Fig. 5a, the typical signal was not observed in the previous NO donors with one-order kinetics
. The
solution. accumulative concentration of NO released from 7a (40 M)
= 12.95 G) for NO- was calculated to be about 17.2 M, based on TEMPO
2
+
dark after addition of 7a (40 μM) to the Fe -(MGD)
Upon irradiation, a typical triplet signal (a
2
N
2
8
Fe-MGD complex appeared and increased with the irradiation standard curve. The yield of NO from 7a was about 43%. This
time, indicating that substantial amount of NO was produced result was further confirmed by the Griess method in which
-
29
from 7a. Moreover, the photo-induced generation of NO-Fe- nitrite (NO
2
), the final product of NO in air, was measured.
-
MGD from 7a increased linearly in 0.5 h and then reached a The yield of NO
2
formation was about 38%, 37% and 38% by
plateau. The time course of EPR signal intensities showed a Griess method for 7a, 7b and 7c respectively (Fig. S8). These
pseudo-zero-order kinetics for the NO generation with the rate results support that NO is released from these NO hybrids by
-1
of 1.20 M min . Note: the rate is much higher than the values light irradiation.
-1
(
0.23-0.24 M min ) obtained by UV-vis and fluorescence
2
Figure 5. (a) The EPR spectra of [(MGD) -Fe-NO] in the presence of 7a (40 µM) with or without irradiation at 313 nm; EPR spectra were recorded after photo-irradiation
for 0, 2, 4, 6, 8 and 10 min with a modulation frequency of 100 KHz, a modulation amplitude of 1 G and a microwave power of 10 mW; inset: the relationship between
NO generation and irradiation time; (b) NO-capture curve of 7a under 313 nm light irradiation for different on/off cycles. Samples contained 5 mM of MGD and 1 mM
4 2 4 2
of (NH ) Fe(SO ) .
Next, we examined whether NO release from 7a could be the irradiation was stopped. The re-irradiation by light led to
controlled with temporal precision by light irradiation. A the increase of the EPR signal again. Similar results were also
2
+
buffered solution containing 7a and Fe -(MGD)
to the triggering light (313 nm). As shown in Fig. 5b, the EPR photo-triggered NO generation indicated that temporally
signal for the NO adduct [(MGD) -Fe-NO] increased immediately precise manipulation of NO release from these NO hybrids can
2
was exposed observed in 7b and 7c (Fig. S9). The “on/off” feature of the
2
upon irradiation, while the signal remained unchanged when be achieved.
Antibacterial activities of the NO hybrids
Please do not adjust margins

Please do not adjust margins
Page 5 of 8
Organic & Biomolecular Chemistry
ARTICLE
Journal Name
Subsequently, we tested the antibacterial activities of the activities than the hybrids with quaternary ammoniums (6b, 6d,
View Article Online
DOI: 10.1039/D0OB00564A
NO hybrids and their precursors against planktonic bacteria. and 7b-c), evidenced by the remarkable reduced OD600nm and
GFP
Positive-gram bacteria S. aureus ATCC12600
was selected the lower MIC (ca. 30 g/mL) and MBC (ca. 60 g/mL) value
which is sensitive to cationic disinfectants. Minimum inhibitory (Fig.6 and Table S1). It was consistent with the previous report
MIC) and bactericidal (MBC) concentrations were detected to that phosphonium-bearing polymers showed better
evaluate the antibacterial activity of the NO hybrids and their antibacterial activity than the polymers containing the
(
2
2, 30, 31
precursors. The results indicated that the TPP-bearing hybrids quaternary ammonium salt.
a and its NO precursor 6a exhibited superior antibacterial
7
Figure 6. (a, c) Optical density measurements for S .aureus ATCC12600GFP in the presence of different concentrations of 6a and 7a (7.5-120 g/mL). The error bars
represent the standard deviations of three independent measurements. (b, d) Pictures of Agar plated solutions (30, 45, 60 and 120 g/mL) which were used to
determine the minimum bactericidal concentrations (MBC) after 24-h treatment in the dark. MBC was determined by agar dilution method as being the lowest
concentration of drug to kill 99.9% of bacteria which produce no colonies on the plate.
The biofilm formation of S. aureus protects the bacterium treatment with 7a or 6a at the concentration of 60 g/ mL for
3
2, 33
against antibiotic assault and enhances its recalcitrance.
8 h, while only 2-log reduction in the CFUs count was observed
Therefore, we tested the antimicrobial efficacy of the most for ciprofloxacin at the concentration of 100 g/mL after
potent compounds 7a and 6a against S. aureus bioflim. After incubation for 8 h (Fig. S11). Although only a slight reduction in
4
8-h growth, S. aureus biofilm was exposed to 7a, 6a or CFUs count was detected at lower concentration (30 and 45
antibiotic drug ciprofloxacin at 37C. Fig. 7 showed the viable g/mL), increasing the concentration (120 or 180 g/mL) led
cell counts in the biofilm after treatment with 6a, 7a and to further reduction of the CFUs count (more than 6-log). At 24
ciprofloxacin for different times. Clearly, both 7a and 6a were h, the effect became weaker, and 2-log reduction in CFUs was
very effective against biofilm cells and showed the similar detected at the concentration of 60 g/mL.
efficacy. 4-log colony forming units (CFUs) were reduced after
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 8
ARTICLE
Journal Name
Figure 7. The antimicrobial efficacy of 6a and 7a at various concentrations against 48-h-old S. aureus biofilm after incubation at 37 C in the dark for different times (2,
View Article Online
DOI: 10.1039/D0OB00564A
4
, 8, 24 h). Data are expressed as means  S.D. over three independent experiments; ***P <0.0001.
NO is identified as a key mediator of biofilm dispersal clear that 7a at a low concentration of 45 g/mL still gave a
3
4
conserved across microbial species . Subsequently, we carried significantly larger reduction in biofilm CFUs (ca. 3-log) at 8 h
the experiment with light-irradiation to test the effect of NO compared with 6a (ca. 0.3-log) under light irradiation or 7a
on biofilm. UV irradiation could damage the mammalian cells without irradiation (ca. 0.3-log) (Fig. 8). At the concentration of
3
5
due to the formed reactive oxygen and nitrogen species , 60 g/mL, 6-log reduction in CFUs was observed for 7a at 8 h
although UV irradiation has been used as a therapeutic agent under irradiation, which is about 2-log reduction than 6a or
3
6
for various skin diseases . It was reported that UVA (315-400 non-irradiated 7a. Taking into account the possibility of the NO
nm) induces erythema after 24 h, whereas the erythema formation from 7a under irradiation, as well as known anti-
3
6
develops 4-6 h after the treatment of UVB (280-315 nm) . biofilm activity of a number of NO donors, we can conclude
Therefore, we set the irradiation time as 4 h to maximize NO that the enhanced anti-biofilm activity of 7a under irradiation
release but minimize the potential toxic effect. To rule out any is due to the combined effect from the photo-generated NO.
effect of the light irradiation itself on bacterial biofilm, a set of However, the efficacy difference between the irradiation and
control experiments (PBS) with up to 4 h exposure to light (313 non-irradiation group disappeared after 24 h (Fig. 8d). Taken
nm) were conducted. Our results showed no light-induced together, 7a is a “two-in-one” hybrid with good antimicrobial
reduction in bacterial biofilm. In the experimental groups, it is efficacy on S. aureus biofilm cells.
Figure 8. The antibiofilm activities of 7a (45 and 60 g/mL) against 48-h-old S. aureus biofilm. The biofilm was incubated with 7a and then exposed to light irradiation
(
313 nm) for 4 h to allow the NO release. Subsequently, the antibiofilm effect was evaluated at different times (2, 4, 8, 24 h). Data are expressed as means  S.D. over
three independent experiments;*** p<0.0001.
It has been reported that QACs and QPCs are able to kill investigated the combined effect of NO and cationic groups on
2
2
bacteria by damaging cell walls and membrane.
To the morphology of the bacteria. No difference in cell walls was
investigate structural modifications of S. aureus biofilm- found for the bacteria only exposed to light or in the dark
detached cells after treatment with 7a and 6a in the dark, the (control and 6a groups), indicating that the light irradiation has
morphology of the S. aureus bacteria was analyzed by scanning no contribution to bacterial biofilm. In contrast, exposure to
electron microscopy (SEM). As shown in Fig. 9, S. aureus light in the presence of 7a led to more significant damage of
biofilm cells looked round in the PBS-treated group and the cell walls as compared to the treatment in the dark,
exhibited an undamaged normal smooth lining. After supporting that NO and cationic groups have the combined
treatment with 7a or 6a (60 g/mL) for 24 h in the dark, the effect on the bacterial biofilm. The change in the morphology
morphology of the bacteria underwent a transformation from of the cell walls was considered to be detrimental to the
their initial round and smooth surfaces to irregular structures. bacteria, accounting for the antibacterial activity of 7a.
Since NO release from 7a can be induced by light, we
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 7 of 8
Organic & Biomolecular Chemistry
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/D0OB00564A
Figure 9. Scanning electron microscope photographs of S. aureus biofilm treated with 6a or 7a (60 g/mL) for 24 h after exposure to light for 4 h or in the dark. The
control group was treated with sterile PBS in the same condition. Scan bar = 3 m
In addition, we also assessed the selectivity of the NO Natural Science Foundation of China (No. 81973269, 81603064
,
hybrids for bacterial over mammalian cell membranes by 21871210 and 21603163), Science & Technology Projects of
conducting hemolytic assay on sheep red blood cells. As shown Tianjin (18JCYBJC95300 and 18JCQNJC76100), The Open
in Fig. S12, very low haemolysis (less than 1%) was observed Project Program of Tianjin Key Laboratory on Technologies
for all the hybrids (7a-c) and their corresponding NO-released
Enabling Development of Clinical Therapeutics and Diagnostics
products (6a-b, 6d) at the concentration range from 300 to
(
(
Theranostics) and Tianjin Municipal 13th five year plan
Tianjin Medical University Talent Project).
6
00 g/mL. Comparatively, QPCs 7a and 6a showed slightly
higher haemolysis compared to QACs 7b and 7c possibly due
to the high hydrophobicity of the TPP group, which facilitated
their insertion into the red blood cell membrane. These results
indicated that these compounds have high hemocompatibility.
Notes and references
1
.
C. A. Fux, J. W. Costerton, P. S. Stewart and P. Stoodley, Survival
strategies of infectious biofilms, Trends Microbiol, 2005, 13, 34-40.
D. Davies, Understanding biofilm resistance to antibacterial agents, Nat
Rev Drug Discov, 2003, 2, 114-122.
U. Romling and C. Balsalobre, Biofilm infections, their resilience to
therapy and innovative treatment strategies, J Intern Med, 2012, 272, 541-
2
.
Conclusions
We have designed and synthesized a new type of
multifunctional NO hybrids based on benzothienoazepine
scaffold and quaternary phosphoniumor ammonium. NO
release from the NO hybrids is controlled with temporal
precision by light irradiation and calibrated by the generated
fluorescence from the NO-released product. In addition, light
irradiation of the NO hybrids leads to the generation of NO at
nearly zero-order release rate and ensures constant release of
NO, unlike the previously reported NO donors which have one-
order release kinetics. Upon photoactivation, such “two-in-
one” hybrids release active gas molecule NO and the
conjugates of benzothienoazepine with quaternary
phosphonium or ammonium, enabling potent activity against
gram-positive bacteria S. aureus biofilm. Our work provides a
new strategy for design of controllable NO hybrids and new
antibacterial compounds.
3
.
5
4
61.
.
P. S. Stewart and J. William Costerton, Antibiotic resistance of bacteria in
biofilms, The Lancet, 2001, 358, 135-138.
N. Barraud, B. G. Kardak, N. R. Yepuri, R. P. Howlin, J. S. Webb, S. N.
5
.
Faust, S. Kjelleberg, S. A. Rice and M. J. Kelso, Cephalosporin-3'-
diazeniumdiolates: targeted NO-donor prodrugs for dispersing bacterial
biofilms, Angew Chem Int Ed Engl, 2012, 51, 9057-9060.
6
.
D. P. Arora, S. Hossain, Y. Xu and E. M. Boon, Nitric Oxide Regulation of
Bacterial Biofilms, Biochemistry, 2015, 54, 3717-3728.
J. Zaitseva, V. Granik, A. Belik, O. Koksharova and I. Khmel, Effect of
7
.
nitrofurans and NO generators on biofilm formation by Pseudomonas
aeruginosa PAO1 and Burkholderia cenocepacia 370, Res Microbiol, 2009,
1
60, 353-357.
. D. McDougald, S. A. Rice, N. Barraud, P. D. Steinberg and S. Kjelleberg,
8
Should we stay or should we go: mechanisms and ecological consequences for
biofilm dispersal, Nat Rev Microbiol, 2011, 10, 39-50.
Conflicts of interest
9
.
S. Hossain, L. M. Nisbett and E. M. Boon, Discovery of Two Bacterial Nitric
There are no conflicts to declare.
Oxide-Responsive Proteins and Their Roles in Bacterial Biofilm Regulation, Acc
Chem Res, 2017, 50, 1633-1639.
1
0. R. P. Howlin, K. Cathie, L. Hall-Stoodley, V. Cornelius, C. Duignan, R. N.
Acknowledgements
Allan, B. O. Fernandez, N. Barraud, K. D. Bruce, J. Jefferies, M. Kelso, S.
This work was financially supported by the National Kjelleberg, S. A. Rice, G. B. Rogers, S. Pink, C. Smith, P. S. Sukhtankar, R. Salib,
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 8 of 8
ARTICLE
Journal Name
J. Legg, M. Carroll, T. Daniels, M. Feelisch, P. Stoodley, S. C. Clarke, G. and Biological Evaluation of Anilide Derivatives with a QuaternVaierwy AArmticmleoOnniluinme
DOI: 10.1039/D0OB00564A
Connett, S. N. Faust and J. S. Webb, Low-Dose Nitric Oxide as Targeted Anti- Moiety, J Med Chem, 2000, 43, 2049-2063.
biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa 24. V. Peesapati and N. Lingaiah, Thiopheno[3,2][1]benzazepine,
Infection in Cystic Fibrosis, Mol Ther, 2017, 25, 2104-2116.
1. S. K. Kutty, N. Barraud, A. Pham, G. Iskander, S. A. Rice, D. S. Black and N. benzo[3,4]cyclohepta[2,1-d]thiazoles, Org Prep Proced Int, 1993, 25, 602-606.
Kumar, Design, synthesis, and evaluation of fimbrolide-nitric oxide donor 25. Z. Zhang, J. Wu, Z. Shang, C. Wang, J. Cheng, X. Qian, Y. Xiao, Z. Xu and Y.
hybrids as antimicrobial agents, J Med Chem, 2013, 56, 9517-9529. Yang, Photocalibrated NO Release from N-Nitrosated Napthalimides upon
2. N. R. Yepuri, N. Barraud, N. S. Mohammadi, B. G. Kardak, S. Kjelleberg, S. One-Photon or Two-Photon Irradiation, Anal Chem, 2016, 88, 7274-7280.
benzo[3,4]cyclohepta[2,1-b]thiophenes, thiazolo[5,4-d][1]benzazepine and
1
1
A. Rice and M. J. Kelso, Synthesis of cephalosporin-3'-diazeniumdiolates: 26. Y. Duan, Y. Wang, X. Li, G. Zhang, G. Zhang and J. Hu, Light-triggered
biofilm dispersing NO-donor prodrugs activated by beta-lactamase, Chem nitric oxide (NO) release from photoresponsive polymersomes for corneal
Commun 2013, 49, 4791-4793.
wound healing, Chem Sci, 2020, 11, 186-194.
1
3. Z. Sadrearhami, T.-K. Nguyen, R. Namivandi-Zangeneh, K. Jung, E. H. H. 27. A. Ramamurthi and R. S. Lewis, Measurement and modeling of nitric
Wong and C. Boyer, Recent advances in nitric oxide delivery for antimicrobial oxide release rates for nitric oxide donors, Chem Res Toxicol, 1997, 10, 408-
applications using polymer-based systems, J Mater Chem B, 2018, 6, 2945- 413.
2
959.
28. Y. Kotake, T. Tanigawa, M. Tanigawa, I. Ueno, D. R. Allen and C. S. Lai,
1
4. A. Rineh, O. Soren, T. McEwan, V. Ravikumar, W. H. Roh, F. Azamifa, M. R. Continuous monitoring of cellular nitric oxide generation by spin trapping with
Naimi-Jamal, C.-Y. Cheung, A. G. Elliott, J. Zuegg, M. A. T. Blaskovich, M. A. an iron-dithiocarbamate complex, Biochim Biophys Acta, 1996, 1289, 362-
Copper, V. Dolange, M. Christodoulides, G. M. Cook, S. A. Rice, S. N. Faust, J. S. 368.
Webb M. J. Kelso, Discovery of Cephalosporin-3′-Diazeniumdiolates That Show 29. J. S. Wishnok, J. A. Glogowski and S. R. Tannenhaum, Quantitation of
Dual Antibacterial and Antibiofilm Effects against Pseudomonas Nitrate, Nitrite, and Nitrosating Agents, Methods Enzymol., 1996, 268, 130-
aeruginosa Clinical Cystic Fibrosis Isolates and Efficacy in a Murine Respiratory 141.
Infection Model, ACS Infect. Dis., 2020, doi: 10.1021/acsinfecdis.0c00070
5. M. Guo, H. J. Xiang, Y. Wang, Q. L. Zhang, L. An, S. P. Yang, Y. Ma, Y. Wang
30. S. Garcia-Arguelles, M. C. Serrano, M. C. Gutierrez, M. L. Ferrer, L. Yuste,
F. Rojo and F. del Monte, Deep eutectic solvent-assisted synthesis of
biodegradable polyesters with antibacterial properties, Langmuir, 2013, 29,
1
and J. G. Liu, Ruthenium nitrosyl functionalized graphene quantum dots as an
efficient nanoplatform for NIR-light-controlled and mitochondria-targeted
delivery of nitric oxide combined with photothermal therapy, Chem Commun,
9
3
525-9534.
1. T. J. Cuthbert, B. Hisey, T. D. Harrison, J. F. Trant, E. R. Gillies and P. J.
Ragogna, Surprising Antibacterial Activity and Selectivity of Hydrophilic
Polyphosphoniums Featuring Sugar and Hydroxyl Substituents.pdf, Angew
Chem Int Ed Engl, 57, 12707-12710.
2
1
017, 53, 3253-3256.
6. K. Hishikawa, H. Nakagawa, T. Furuta, K. Fukuhara, H. Tsumoto, T. Suzuki
and N. Miyata, Photoinduced Nitric Oxide Release from
a Hindered
3
2. M. R. Parsek and P. K. Singh, Bacterial biofilms: an emerging link to
disease pathogenesis, Annu Rev Microbiol, 2003, 57, 677-701.
3. S. Kumar Shukla and T. S. Rao, Dispersal of Bap-mediated Staphylococcus
aureus biofilm by proteinase K, J Antibiot (Tokyo), 2013, 66, 55-60.
4. N. Barraud, M. J. Kelso, S. A. Rice and S. Kjelleberg, Nitric oxide: a key
Nitrobenzene Derivative by Two-Photon Excitation, J Am Chem Soc, 2009, 131,
7
1
488-7489.
3
7. S. Namiki, T. Arai and K. Fujimori, High-Performance Caged Nitric Oxide: A
New Molecular Design, Synthesis, and Photochemical Reaction, J Am Chem
Soc, 1997, 119, 3840-3841.
3
mediator of biofilm dispersal with applications in infectious diseases, Curr
Pharm Des, 2015, 21, 31-42.
1
8. E. Y. Zhou, H. J. Knox, C. J. Reinhardt, G. Partipilo, M. J. Nilges and J. Chan,
Near-Infrared Photoactivatable Nitric Oxide Donors with Integrated
3
5. T. J. McMillan, E. Leatherman, A. Ridley, J. Shorrocks, S. E. Tobi, J. R.
Photoacoustic Monitoring, J Am Chem Soc, 2018, 140, 11686-11697.
Whiteside, Cellular Effects of Long Wavelength UV Light (UVA) in Mammalian
Cells, J Pharm Pharmacol, 2008, 60, 969-976.
1
9. H. He, Y. Liu, Z. Zhou, C. Guo, H. Y. Wang, Z. Wang, X. Wang, Z. Zhang, F.
G. Wu, H. Wang, D. Chen, D. Yang, X. Liang, J. Chen, S. Zhou, X. Liang, X. Qian
and Y. Yang, A Photo-triggered and photo-calibrated nitric oxide donor:
Rational design, spectral characterizations, and biological applications, Free
Radic Biol Med, 2018, 123, 1-7.
3
6. Y. Matsumura, H. N. Anathaswamy, Toxic Effects of Ultraviolet Radiation on
the Skin, Toxicol Appl Pharmacol, 2004, 195, 298-308
2
0. J. P. Cheng, M. Xian, K. Wang, X. Zhu, Z. Yin and P. G. Wang, Heterolytic
and Homolytic Y-NO Bond Energy Scales of Nitroso-Containing Compounds:
Chemical Origin of NO Release and NO Capture, J Am Chem Soc, 1998, 120,
1
2
0266-10267.
1. E. A. F. Fordyce, D. W. Brookes, C. Lise-Ciana, M. S. Coates, S. F. Hunt, K.
Ito, J. King-Underwood, S. T. Onions, G. F. Parra, G. Rapeport, V. Sherbukhin,
J. A. Stockwell, P. Strong, J. C. Thomas and J. Murray, Discovery of novel
benzothienoazepine derivatives as potent inhibitors of respiratory syncytial
virus, Bioorg Med Chem Lett, 2017, 27, 2201-2206.
2
2. A. Muñoz-Bonilla and M. Fernández-García, Polymeric materials with
antimicrobial activity, Prog Polym Sci, 2012, 37, 281-339.
3. A. Y. Shiraishi M, Seto M, Imoto H, Nishikawa Y, Kanzaki N, Okamoto M,
2
Sawada H, Nishimura O, Baba M, Fujino M, Discovery of Novel, Potent, and
Selective Small-Molecule CCR5 Antagonists as Anti-HIV-1 Agents: Synthesis
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins