Fluorescent nitric oxide donor for the detection and killing of: Pseudomonas aeruginosa

Article abstract of DOI:10.1039/c8tb02552e

The epidemic of multidrug-resistant bacteria calls for the improvement of both detection methods for bacterial infections and methods of treatment. Nitric oxide is a known potent antibacterial agent, but due to its gaseous and highly reactive nature, it is difficult to incorporate into a stable antibacterial compound. In this paper, we synthesize a nitric oxide donor attached to a fluorescent compound, creating a material that can both detect and kill the deadly multi-drug resistant bacteria strain Pseudomonas aeruginosa. Detection occurs through a bacterial enzyme-activated color change, showing a clear and obvious change from blue to yellow under UV light. The synthesized compound spontaneously releases 853 μmol of nitric oxide/g from a 10 mM initial concentration. Antibacterial efficacy studies after exposing Pseudomonas aeruginosa to a 10 mM dose of the synthesized compound show a 55-75% reduction in bacteria after 24 hours. This work is the first instance of a small molecule dual-function material that can both detect and kill bacteria.

Full text of DOI:10.1039/c8tb02552e

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PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
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Journal of Materials Chemistry B
ARTICLE
Fluorescent nitric oxide donor for the detection and killing of
Pseudomonas aeruginosa†
Received 00th January 20xx,
Accepted 00th January 20xx
Hailey A. J. Hibbarda and Melissa M. Reynolds*a b
DOI: 10.1039/x0xx00000x
The epidemic of multidrug-resistant bacteria calls for the improvement of both detection methods for bacterial infections
and methods of treatment. Nitric oxide is a known potent antibacterial agent, but due to its gaseous and highly reactive
nature, it is difficult to incorporate into a stable antibacterial compound. In this paper, we synthesize a nitric oxide donor
attached to a fluorescent compound, creating a material that can both detect and kill the deadly multi-drug resistant bacteria
strain, Pseudomonas aeruginosa. Detection occurs through a bacterial enzyme-activated color change, showing a clear and
obvious change from blue to yellow under UV light. The synthesized compound spontaneously releases 853 µmol of nitric
oxide/g from a 10 mM initial concentration. Antibacterial efficacy studies after exposing Pseudomonas aeruginosa to a 10
mM dose of the synthesized compound show a 55-75% reduction in bacteria after 24 hours. This work is the first instance
of a small molecule dual-function material that can both detect and kill bacteria.
www.rsc.org/
studied class of fluorescent molecules, aminoacridones, is
attached to the amino acid proline to form an indicator for the
bacterial enzyme prolyl aminopeptidase, which is known to
cleave N-terminal proline from peptides.8 Next, a nitric oxide
(NO) donor, a diazeniumdiolate similar to PROLI/NO, shown in
Figure 2, is added to impart antibacterial activity.
1. Introduction
The Review on Antimicrobial Resistance predicts that by 2050,
one person will die every three seconds from a bacterial
infection,1 surpassing both heart disease and cancer as the most
common cause of death.2 This epidemic of antibiotic-resistant
bacteria creates a critical need for researchers to develop better
methods for detection and treatment of bacterial infections.
Correctly detecting a bacterial infection is critical to preventing
the over-use of antibiotics.3 A multifunctional material that
could both detect and eradicate bacterial infections would be
ideal.
The fluorescent compound 2-aminoacridone is
a
commercially available, well-characterized fluorescent
reporter.9 It is commonly used to derivatize oligosaccharides for
fluorescent analysis.10 Previous reports have also shown that
attaching amino acids to derivatives of 2-aminoacridones allows
for enzymatic detection of bacteria.11 For these reasons, a 2-
aminoacridone derivative is chosen as the fluorescent reporter
to detect bacteria in this work.
Multifunctional materials are a promising new area of
research, offering improved efficiency and versatility, while
reducing costs. Unfortunately, few examples exist of materials
that can both detect and kill bacteria, and these examples are
exclusively larger molecules, including polymers and
nanoparticles.4–6 Small molecules, on the other hand, tend to
release active drug compounds much faster.7 Additionally, the
small molecule could be incorporated into a polymer support to
make biomedical devices.
By attaching an amino acid to the fluorescent
aminoacridone compound, the wavelength the emitted light
changes, and a color change can be relied upon for bacterial
detection. In this work, proline is attached to an aminoacridone,
offering several advantages. Proline is cleaved by prolyl
aminopeptidase, an enzyme found in Pseudomonas aeruginosa,
a deadly bacteria species that is growing increasingly resistant
to antibiotics and causes a significant amount of hospital-
acquired infections.8,12 In addition, a known NO donor,
PROLI/NO (Figure 2), is formed from the secondary amine on
proline, conveying antibacterial activity. A few recent reports
have shown NO donors with fluorescent reporters, however,
fluorescent changes in this case are used to quantify the
amount of NO released from the compound, not to detect an
external stimulus.13–16 The compound synthesized in this work
uses fluorescence to detect bacteria and NO release to kill the
bacteria.
In this work, a dual-function small molecule is specifically
designed, synthesized, and tested to detect the presence of and
kill bacteria, shown in Figure 1. Briefly, a derivative of a well-
a. Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA.
E-mail: Melissa.Reynolds@colostate.edu
b. School of Biomedical Engineering, Colorado State University, Fort Collins, CO
80523, USA
† Electronic Supplementary Information (ESI) available: Details of syntheses, 1H
NMR, 13C NMR, additional bacterial studies images, and nitric oxide analyzer data.
See DOI: 10.1039/x0xx00000x
NO is an excellent small molecule candidate for
incorporation into a new antibiotic, due to its broad-spectrum
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Journal Name
View Article Online
DOI: 10.1039/C8TB02552E
O
O
O
H
H
N
N
N
N
N
NH2
H
spontaneous
enzyme
O
O
O
N
O
N
N
N
Me
Me
Na
Me
NO
NO
NO
NO
Fig. 1 Schematic showing nitric oxide release and color change of indicator. Nitric oxide is released from the diazeniumdiolate functional group spontaneously, killing
Pseudomonas aeruginosa bacteria cells. A bacterial enzyme then cleaves proline from the fluorescent aminoacridone/proline compound, changing the fluorescent
color from blue to yellow.
antimicrobial activity, killing both gram-positive and gram- infection is caused by P. aeruginosa. To our knowledge, this is
negative strains of bacteria.17–20 Researchers have shown that the first example of a multifunctional small molecule that can
NO uses multiple mechanisms of action to kill bacteria, meaning both detect and kill P. aeruginosa.
that bacterial resistance is much more difficult to develop,
which is critical in stemming the spread of antibiotic-resistant
bacterial infections.21,22 However, NO is gaseous at room
temperature and highly reactive, leading to difficulties in safely
delivering it as a drug.23 As a result, NO is almost always
delivered as a prodrug, as an NO donor, that is metabolized into
free radical NO via a chemical or enzymatic transformation.
Some common NO donors include S-nitrosothiols, organic
nitrates and nitrites, metal nitrosyls, and diazeniumdiolates.24–
28 Diazeniumdiolates have the advantage of being very stable as
solids and in basic media, releasing two equivalents of NO per
one equivalent of diazeniumdiolate, and having predictable
half-lives for spontaneous NO release.29 One disadvantage of
diazeniumdiolates is that they are known to decompose into
nitrosamines, which are typically carcinogenic.30 However, the
nitrosamine formed from proline, N-nitrosoproline, is one of
the few non-carcinogenic nitrosamines.31,32 This could mean
that if the nitrosamine forms from our synthesized proline
aminoacridone compound, it would also be non-carcinogenic,
although future studies would be necessary to confirm its non-
toxicity. Because of these advantages, a derivative of the
diazeniumdiolate PROLI/NO (Figure 2) is chosen as the NO
donor in this work.
2. Results and Discussion
2.1 Synthesis of Pro/Fluoro
The steps we developed to synthesize the enzyme-activated
bacterial indicator Pro/Fluoro (6) are shown in Scheme 1. First,
we nitrated acridone (1) with nitric acid in the presence of acetic
acid to form 2a, 2-nitroacridone. Based on the 1H-NMR
spectrum of the product, we found that this reaction forms a
4:1 ratio of the desired product 2a with the nitro group in the 2-
position to an undesired side product 2b with the nitro group in
the 4-position, shown in Figure 3. We found that boiling the
mixture of isomers in acetic acid dissolves the 4-isomer 2b,
leaving the 2-isomer 2a as a solid, however, we carry on the
mixture of isomers to the next step, as the isomers are
separated in a later step.
In next step of the synthesis, we methylate 2 to form 3 in
Scheme 1. We modified a previously reported synthesis using
sodium hydride and methyl iodide to methylate the amine.34
We reduce the nitro group, 3a and 3b to 4 in Scheme 1, using
tin(II) chloride dihydrate and hydrochloric acid, following a
previously published procedure.35 The crude product showed
evidence of the undesirable 4-isomer as well as the desired 2-
isomer, similar to Figure 3. Fortunately, we developed column
chromatography conditions on silica gel to separate the
isomers, giving an isolated yield of 4 of 42% over three steps.
This report details the design and synthesis of a compound
for its application as a dual-function bacterial indicator and
antibacterial agent. The proposed process is shown in Figure 1,
where the proline-based diazeniumdiolate releases NO
spontaneously to kill bacteria. The bacterial enzyme prolyl
aminopeptidase found in P. aeruginosa then cleaves proline
from the fluorescent aminoacridone compound, causing a
fluorescent color change from blue to yellow that indicates the
presence of bacteria. P. aeruginosa commonly causes infections
in burn wounds33 and our compound could be useful in this
case, releasing NO as site-specific broad-spectrum antibacterial
agent to kill bacteria in the wound, and determining if the
O
N
Na
O
N
N
Scheme 1 Overall reaction scheme developed for the synthesis of fluorescent indicator
Pro/Fluoro 6, for an overall yield of 8% over 5 steps. HNO3 = nitric acid, AcOH = acetic
acid, NaH = sodium hydride, DMF = N,N-dimethylformamide, MeI = methyl iodide,
SnCl2·2H2O = tin (II) chloride dihydrate, HCl = hydrochloric acid, iBuCF = isobutyl
chloroformate, Et3N = triethylamine, TFA = trifluoroacetic acid, DCM = dichloromethane,
Boc = tert-butyloxycarbonyl
O
O
Na
Fig.
2
Structure of proline-based diazeniumdiolate, PROLI/NO, a previously
synthesized diazeniumdiolate formed by reacting nitric oxide with proline
2 | J. Name., 2012, 00, 1-3
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To perform the bacterial sensing experiments, P. aeruginosa
View Article Online
DOI: 10.1039/C8TB02552E
is plated on agar mixed with Pro/Fluoro 6, then a color change
is determined visually under UV light. Yellow fluorescence
indicates that enzymatic cleavage occurred, releasing proline
and compound 4, with an emission wavelength of 546 nm, to
fluoresce yellow. Blue fluorescence indicates that no enzymatic
cleavage occurred; it is the color of fluorescence of intact
Pro/Fluoro/H+ 7, with an emission wavelength of 489 nm.
The results of these experiments can be seen in Figure 4,
showing that there is a clear color change due to the presence
of P. aeruginosa. The positive control, P. aeruginosa without
Pro/Fluoro/H+ 7, shows no fluorescence under UV light (Figure
S9), indicating that fluorescence is due entirely to the
synthesized compound. The negative control, Pro/Fluoro/H+ 7
without P. aeruginosa, left in Figure 4, clearly fluoresces blue,
indicating that cleavage of Pro/Fluoro/H+ 7 does not occur when
P. aeruginosa is not present. The experimental plate,
Pro/Fluoro/H+ 7 with P. aeruginosa, right in Figure 4, fluoresces
yellow under UV light, indicating that proline is cleaved from
Pro/Fluoro/H+ 7 by P. aeruginosa, leaving compound 4 to
Fig. 3 The 2-isomer 2a and 4-isomer 2b products resulting from the reaction of
acridone 1 to nitroacridone. The 2-isomer 2a is the desired product, which was
separated from the undesired 4-isomer 2b in a later step.
When dissolved in ethanol, we observe that compound 4
appears yellow under a 365 nm UV light.
In the next step, we couple Boc-protected proline (5) to the
2-aminoacridone, 4, using isobutyl chloroformate and
triethylamine. After an aqueous work-up, we remove the Boc
protecting group using trifluoroacetic acid to give compound 6,
Pro/Fluoro. We purify the deprotected product by column
chromatography, giving a 29% yield. We use mass spectrometry
1
and H- and 13C-NMR spectra to confirm the synthesis of
Pro/Fluoro 6 (Fig. S1-S2). We see that Pro/Fluoro 6 appears blue
under a 365 nm UV light when dissolved in ethanol.
fluoresce yellow. By serial dilution of the initial bacteria solution
and plating on agar, then counting colony forming units (CFUs),
we have determined that 3.7 ± 0.1 × 107 CFU/mL is enough to
cause a visible color change. Further experiments are necessary
to determine the limit of detection of our synthesized indicator.
Based on the above experimental observations, we
conclude that Pro/Fluoro/H+ 7 can be used as an indicator to
detect if P. aeruginosa is present using UV light. We observe a
clear and obvious color change from blue to yellow indicating
the presence of P. aeruginosa. Our hypothesis is that the
enzyme prolyl aminopeptidase, produced by P. aeruginosa,8
cleaves proline, causing the color change. If our hypothesis is
correct, this is the first report of a prolyl aminopeptidase-
specific indicator. Based on our hypothesis, any bacteria species
containing this enzyme would be detectable via this sensing
mechanism, such as Clostridium difficile, which can cause life-
threatening gastrointestinal infections.37
The synthesis of Pro/Fluoro 6 we developed gives an overall
yield of 8% over five steps. This synthetic scheme shows the
development of an indicator specially designed to detect the
presence of the bacterial enzyme prolyl aminopeptidase.
2.2 Bacterial Detection Studies
With the synthesis of Pro/Fluoro 6 complete, we wanted to test
the ability of Pro/Fluoro 6 to detect the presence of P.
aeruginosa as a proof of concept, to ensure that a color change
would be possible once NO is released (Figure 1). We selected
P. aeruginosa for the bacterial studies because it produces a
prolyl aminopeptidase enzyme, which we hypothesized would
cleave N-terminal proline from Pro/Fluoro 6, causing a color
change.36 P. aeruginosa is also a deadly gram-negative bacteria
strain, resistant to many common antibiotics and is the cause of
death in many immunocompromised hospital patients.12 The
goal of this experiment is to show that Pro/Fluoro 6 displays a
color change when P. aeruginosa is present. Future studies will
determine the exact number of bacteria needed to cause a
detectable color change.
2.3 Synthesis of Pro/Fluoro/NO (8)
We attempted bacterial detection experiments with
Pro/Fluoro 6, but it has low solubility in the aqueous-based agar
and precipitated out before the agar set, preventing sensing. To
overcome this problem, Pro/Fluoro 6 was protonated using
trifluoroacetic acid to form 7 (Pro/Fluoro/H+), shown in Scheme
2. Protonation solubilized Pro/Fluoro/H+ 7 in the agar, forming
a homogenous solution.
Fig. 4 Representative agar plates showing color change from blue to yellow under a 365
nm UV lamp in the presence of Pseudomonas aeruginosa, as expected. Left: negative
control- not inoculated with Pseudomonas aeruginosa, Pro/Fluoro/H+ 7, appears blue;
right- inoculated with Pseudomonas aeruginosa, Pro/Fluoro/H+ 7, appears yellow
Scheme 1 Synthesis of fluorescent indicator Pro/Fluoro/H+ 7, used for Pseudomonas
aeruginosa detection experiments. TFA = trifluoroacetic acid, DCM = dichloromethane
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Once we demonstrated the detection function, the last step in 2.4 NO Release Measurements
the synthesis is to attach a diazeniumdiolate group to
View Article Online
DOI: 10.1039/C8TB02552E
Once we synthesized Pro/Fluoro/NO 8, we measured the
amount of NO released at different concentrations using a nitric
oxide analyzer (NOA). An NOA uses chemiluminescence to
directly quantify the amount of NO released by oxidizing NO
with ozone, forming nitrogen dioxide in an excited state. Upon
relaxation, it emits a photon, which is measured and related to
the moles of NO released via a previously determined
calibration constant. We performed NOA experiments to
determine the NO release profile of Pro/Fluoro/NO 8 and to
quantify the amount of NO release at different concentrations.
During the NOA experiments, we observed that NO release
from Pro/Fluoro/NO 8 is light sensitive. We observe a visible
decrease in NO release when the overhead lights are turned off,
and a return to previous NO release levels when the lights are
turned on again, although there is still above-baseline NO
release when the lights are off. This light sensitivity could be due
to the photosensitivity of acridone compounds, which has been
previously reported.41 Light could be exciting the acridone
portion of Pro/Fluoro/NO 8, leading to an increase in the release
of NO.
First, we determine the total amount of possible NO release
from Pro/Fluoro/NO 8. Previous reports show that NO release
from diazeniumdiolates displays first order kinetics, and occurs
when the amine on which the diazeniumdiolate is protonated,
causing up to two molecules of NO to be released from the
compound.30 We chose experimental conditions to release all
the NO in Pro/Fluoro/NO 8. Most diazeniumdiolates are stable
as solids when stored at -20 ˚C or below, or in solutions at pH
12 or above.42 Diazeniumdiolates are unstable in acidic and
neutral conditions, so exposure to these conditions forces the
release of NO. To determine the total amount of NO released,
we forced NO off the diazeniumdiolate by injecting a basic
solution containing the diazeniumdiolate into an acidic buffer
(pH = 3), as well as shining a UV light on the solution to address
the light sensitivity of the diazeniumdiolate. By comparing the
actual total moles of NO release from Pro/Fluoro/NO 8 in these
experiments to the theoretical amount of NO release possible,
the amount of NO released from the diazeniumdiolate can be
calculated. In these calculations, we assume two moles of NO
Pro/Fluoro 6. Adding the diazeniumdiolate group to the
compound imparts the ability to release NO to kill bacteria and
develops a dual-function material.
Diazeniumdiolates are typically formed by reacting
secondary amines with gaseous NO at elevated pressures,30
which in our case is at the proline amine location, seen in
Scheme 3. Our diazeniumdiolate reaction is based on the
previously reported procedure to synthesize the proline-based
diazeniumdiolate, PROLI/NO (Figure 2).38 As can be seen in
Scheme 3, we react Pro/Fluoro 6 with NO at high pressure in the
presence of sodium methoxide in methanol. Over the course of
the reaction, we observe the formation of a yellow precipitate,
evidence of the formation of 8, Pro/Fluoro/NO, in 88% yield.
Diazeniumdiolates are typically white powders, but in our case,
the fluorescent 2-aminoacridone compound imparts a yellow
color to the product, and is not indicative of diazeniumdiolate
decomposition to the nitrosamine.29
The formation of diazeniumdiolates is typically confirmed by
UV-Vis spectroscopy due to their distinctive peak at 250 nm.29
However, an absorbance peak due to the fluorescent acridone
component occurs at 260 nm (Figure S8), interfering with the
diazeniumdiolate absorbance peak. To confirm the formation of
the diazeniumdiolate Pro/Fluoro/NO 8, we used several
techniques. We observe the formation of a precipitate in the
course of the reaction shown in Scheme 3, which is typical of
diazeniumdiolate-forming reactions.38 The physical properties
of the product of the reaction are different than the starting
material; the product is no longer soluble in organic solvents like
Pro/Fluoro 6, but is soluble in water and basic solutions, which
is indicative of the formation of a diazeniumdiolate salt.39 The
1H-NMR spectrum of Pro/Fluoro/NO 8 (Figure S5) shows the
disappearance of the proline N-H peak and a shift in the other
1
peaks compared to the H-NMR spectrum of Pro/Fluoro 6
(Figure S1), which provides indirect evidence that the desired
reaction took place. Electrospray ionization time-of-flight mass
spectrometry (ESI-TOF MS) was performed in a basic solvent
mixture, and the accurate mass of Pro/Fluoro/NO 8 was found
at 381 m/z (Figure S7), further leading us to conclude that the
reaction was successful.
per mole of Pro/Fluoro/NO 8, as is typical in
a
Taken together, this evidence shows that we successfully
synthesized the diazeniumdiolate, Pro/Fluoro/NO 8. The overall
yield of the synthesis of Pro/Fluoro/NO 8 over six steps is 7%,
requiring significantly fewer steps with a higher overall yield
compared to total syntheses of other common antibiotics and
their analogs.40 To the best of our knowledge, our synthesized
compound Pro/Fluoro/NO 8 is the first example of a
diazeniumdiolate with a fluorescent marker to detect bacteria.
diazeniumdiolate.30 Based on our calculations, Pro/Fluoro/NO 8
releases 75 ± 15% of the theoretical 200% of NO possible.
Previous literature reports have shown that based on their
structure, some diazeniumdiolates do not release the entirety
of the theoretical 200% of NO, due to decomposition or other
factors.43–47
Next, we measure the amount of NO release at initial
concentrations of 0.1, 1, and 10 mM. We chose the conditions
of the NO release measurements to most closely mimic the cell
viability assay conditions. A representative NO release profile
can be seen in Figure 5, showing an initial spike of high NO
release in the inset, then dropping off to a steady release after
about one hour. This release profile matches the release profile
of other diazeniumdiolates.48 The results of these experiments
can be found in Table 1. In column 2 of Table 1, the amount of
Scheme 3 Reaction developed to form the fluorescent diazeniumdiolate Pro/Fluoro/NO
8. NO = nitric oxide, NaOMe = sodium methoxide, MeOH = methanol
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Fig. 6 Cell viability after 24-hour exposure of Pseudomonas aeruginosa to
Pro/Fluoro/NO 8 at initial concentrations of 0.1, 1, and 10 mM as determined by
CellTiter Blue assay. Average and 95% confidence interval displayed, n = 3.
Statistically significant differences between cellular viabilities are indicated (*) as
determined by a student’s t test. PC=positive control
Fig. 5 Representative real-time NO release profile of Pro/Fluoro/NO 8 at an initial
concentration of 1 mM in nutrient broth at 37 °C, showing steady release of NO
from 1-24 hours. Inset shows an initial spike of NO release in the first hour.
NO release is normalized to the mass of Pro/Fluoro/NO 8 used compared to a positive control (PC).49 The positive control in
in each experiment. At 10 mM, 853 µmol of NO/ g of this case is P. aeruginosa culture that is not exposed to
Pro/Fluoro/NO 8 is released, compared to only 322 µmol of NO/ Pro/Fluoro/NO 8. For these experiments, we mix P. aeruginosa
g of Pro/Fluoro/NO 8 at a concentration of 0.1 mM. As can be culture in nutrient broth media with Pro/Fluoro/NO 8 to initial
seen in column 3 of Table 1, this corresponds to 46% of the total concentrations of 0.1, 1, and 10 mM, and then incubate for 24
possible NO released at 10 mM and 17% at 0.1 mM, assuming hours at 37 °C.
75% of the theoretical NO is released based on previous
The results of the CTB assay of P. aeruginosa after exposure
experiments. The data in Table 1 show that less NO is released to Pro/Fluoro/NO 8 (Figure 6) indicate that it has antibacterial
at the 0.1 mM concentration. This could be due to less activity at certain doses. At concentrations of 0.1 mM, we find
availability of NO to be released at lower concentrations or that the antibacterial effect of Pro/Fluoro/NO 8 is not significant
because the level of NO release approaches the limit of compared to the positive control, with only a 0-14% reduction
detection of the NOA instrument. An increased amount of NO in viable bacteria observed. The 1 mM dose is effective, as we
release observed at initial concentrations of 10 mM should lead observe a 14-32% reduction, a statistically significant reduction
to an increase in antibacterial activity, which we observe in our in bacteria compared to the positive control. The 10 mM dose
cell viability studies.
is the most effective, with a 55-75% reduction observed after
exposure to Pro/Fluoro/NO 8. This reduction is statistically
significant compared to the positive control, as well as
compared to the reduction from the 1 mM dose. We also
performed a study to count CFUs and determined that the
positive control contains 1.8 ± 0.5 × 109 CFU/mL. The percent
reduction determined by the CellTiter Blue assay is relative to
this value. For example, the 10 mM dose would correspond to
a reduction to 1.0-1.4 × 109 CFU/mL. While typically a more
significant reduction in the number of bacteria remaining is
necessary for antibacterial materials,50 we demonstrate that
this compound has antibacterial effects as a proof of concept.
We show that Pro/Fluoro/NO 8 has antibacterial effects
statistically significant compared to the positive control at
concentrations of 1 and 10 mM.
Wink and Mitchell, as well as Schairer and co-workers, show
that concentrations of NO in the low micromolar range have
antibacterial effects.22,51 At an initial concentration of 0.1 mM
Pro/Fluoro/NO 8, at the peak of the instantaneous NO release
measurements represented by the spike of NO release in Figure
5, the concentration of NO in the solution ranges from 0.6-4.1
nM. Because the concentration of NO does not reach
micromolar ranges, this could explain why at an initial
2.5 Cell Viability Studies
With the amount of NO release measured, we determined the
antibacterial efficacy of Pro/Fluoro/NO 8. To study this, we
expose P. aeruginosa to Pro/Fluoro/NO 8, and then we perform
a spectroscopic assay [CellTiter Blue (CTB)] to determine the
percent of viable bacteria cells left. The CTB assay relies on the
transformation of blue resazurin (λmax = 600 nm) to pink
resorufin (λmax = 570 nm) accomplished by healthy bacteria,
where the absorbance is measured to determine an increase or
decrease in the metabolic activity of viable bacteria cells
Table 1 NO release data showing the normalized amount and the percent
of total NO released at different concentrations
Initial
concentration
Percentage of
total possible NO
released (%)
17 ± 2
µmol NO/g
Pro/Fluoro/NO 8
(mM)
0.1
1
322 ± 45
731 ± 87
853 ± 35
40 ± 5
46 ± 2
10
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concentration of 0.1 mM Pro/Fluoro/NO 8, there is not a infections.37 In the long term, we can envision incorporating
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significant reduction in the number of bacteria cells. The NO Pro/Fluoro/NO 8 into a polymer support, which could be
concentration does not reach levels high enough to have made into a bandage that would have antibacterial properties,
antibacterial activity, based on literature values.22,51 The as well as visualizing when a bacterial infection is present. While
experiments at an initial concentration of 1 mM Pro/Fluoro/NO there are complications to overcome in the practical application
8 reach concentrations of NO ranging from 167-196 nM. The of the synthesized compound, this is an exciting proof of
experiments at an initial concentration of 10 mM concept of a dual function compound to detect and kill bacteria.
Pro/Fluoro/NO 8 reach concentrations of NO ranging from 149-
576 nM. At concentrations of 1 and 10 mM, the concentration
of NO is higher than at 0.1 mM. This could explain why bacteria
4. Experimental Methods
cell viability is significantly lower at 1 and 10 mM
concentrations, as NO concentrations are closer to the deadly
micromolar range.
4.1 Materials and Methods
Acridone (98%) was purchased from Oxchem Corp (Wood Dale,
IL, USA). Nitric acid (68-70%), iodomethane (98%), PBS tablets,
and triethylamine (99.5%) were purchased from EMD Chemicals
(Gibbstown, NJ, USA). Acetic acid (glacial) was purchased from
BDH Chemicals (Radnor, PA, USA). Sodium hydride (57-63% oil
dispersion), tin(II) chloride dihydrate, isobutyl chloroformate
(98%), and anhydrous methanol (99.9%) were purchased from
Alfa Aesar (Ward Hill, MA, USA). N,N-dimethylformamide
(99.9%), hydrochloric acid (1 N), sodium hydroxide (98.8%),
tetrahydrofuran, Oxoid nutrient broth (OXCM0001B), and
Oxoid nutrient agar (OXCM0003B) were purchased from Fisher
Scientific (Hampton, NH, USA). Boc anhydride (99.5%) was
purchased from Chem Impex (Bensenville, IL, USA).
Dichloromethane (99.5%) and trifluoroacetic acid (99%) were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Nitric oxide
(99%) was purchased from Matheson Tri-gas (Montgomeryville,
PA). Sodium methoxide (30% in methanol) was purchased from
Acros Organics (Geel, Belgium). CellTiter Blue was purchased
from Promega (Madison, WI, USA). 24-well and 96-well tissue
culture nontreated plates were obtained from Corning
(Corning, NY, USA). Argon (ultrahigh purity), nitrogen (ultrahigh
purity), and oxygen were purchased from Airgas (Denver, CO,
USA). Deionized water (18.2 MΩ·cm) was obtained from a
Millipore Direct-Q water purification system (EMD Millipore,
Billerica, MA, USA). Pseudomonas aeruginosa (PAO1) was
provided by Dr. Brad Borlee at Colorado State University.
Commercially available reagents were purchased and used
without further purification unless otherwise indicated.
All nuclear magnetic resonance (NMR) spectra were
obtained on Varian Inova 400 (400 MHz for 1H; 101 MHz for 13C)
and/or Bruker US400 (400 MHz for 1H; 101 MHz for 13C) at room
temperature unless noted otherwise. Chemicals shifts were
reported in parts per million (δ scale), and referenced according
the following standards: chloroform residual signal (δ 7.16),
water residual signal (δ 4.79), or dimethyl sulfoxide residual
signal (δ 2.50) for 1H signals; deuterated chloroform or
deuterium dimethyl sulfoxide carbon resonances (middle peak
is δ 77.1, or δ 39.5, respectively) or tetramethylsilane for
samples in deuterated water for 13C signals. Coupling constants
were reported in Hertz (Hz) and multiplicities were reported as
follows: singlet (s), doublet (d), doublet of doublets (dd), triplet
(t), quartet (q), and multiplet (m). IR spectra were obtained on
After exposure, we observe a color change from blue to
yellow in the presence of P. aeruginosa (Figure S13) similar to
the previous detection experiments. The solutions of P.
aeruginosa in nutrient broth media that are exposed to
Pro/Fluoro/NO 8 at all concentrations appear yellow under a UV
light at 365 nm. Solutions of Pro/Fluoro/NO 8 at varying
concentrations in nutrient broth media without P. aeruginosa
are blue under UV light as expected. This demonstrates that the
detection function also works post-NO release.
Together, the results of these experiments demonstrate the
dual-function of Pro/Fluoro/NO 8 to both detect and kill
Pseudomonas aeruginosa.
3. Conclusion
To the best of our knowledge, this paper is the first example of
a small molecule multifunctional material that can both detect
and kill bacteria. A synthetic procedure is developed to attach
proline to a fluorescent aminoacridone compound, Pro/Fluoro
6. Experiments show a clear and distinct color change from blue
to yellow under UV light in the presence of Pseudomonas
aeruginosa, a novel prolyl aminopeptidase-specific indicator. A
reaction is developed to react Pro/Fluoro 6 with NO to form the
new diazeniumdiolate Pro/Fluoro/NO 8. NO release
measurements show that Pro/Fluoro/NO 8 releases 853 µmol
of NO/g at an initial concentration of 10 mM. P. aeruginosa is
exposed to Pro/Fluoro/NO 8, and bacteria cell viability is
reduced by 55-75% at a concentration of 10 mM after 24 hours
of exposure. After exposure, a color change from blue to yellow
is observed under UV light in the presence of P. aeruginosa.
These results demonstrate the dual function of Pro/Fluoro/NO
8 to fluorescently detect P. aeruginosa and kill P. aeruginosa by
releasing NO, creating a very versatile molecule.
In the future, we want to increase the amount of NO
released from Pro/Fluoro/NO 8, which we hypothesize would
lead to an increase in antibacterial efficacy. This could allow a
lower dose of the diazeniumdiolate to be applied with the same
or better results. More controlled release of NO could be
achieved
with a
protecting group on O2 of the
diazeniumdiolate.30 Incorporation into a polymer could also
help to control NO release. We want to test Pro/Fluoro/NO 8
with other bacteria species that produce the prolyl
aminopeptidase enzyme, such as Clostridium difficile, a gram-
positive bacteria strain that causes severe gastrointestinal
a
Thermo Scientific Nicolet 6700 FT-IR spectrometer.
Electrospray ionization mass spectrometry data were obtained
on an Agilent 6224 TOF mass spectrometer equipped with a
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dual electrospray ion source operated in positive and negative 1423s, 1275s, 1179s, 1066s, 879w, 806s, 749s, 684s. λEX = 419
View Article Online
DOI: 10.1039/C8TB02552E
mode. Excitation and emission spectra were obtained on a nm, λEM = 499 nm (EtOH).
Horiba Jobin-Yvon FluoroLog-3 Spectrofluorometer. Melting
points were obtained with an Electrothermal Mel-Temp
apparatus. UV-Vis measurements were obtained with a Thermo
Scientific Nicolet Evolution 300 spectrometer. Well plate
absorbance measurements were obtained with a Biotek
Synergy 2 Multi-Mode Reader. Reactions were analyzed by thin
layer chromatography (TLC) on aluminum sheets that were pre-
coated with silica gel 60 F254, and the reactions were purified by
column chromatography using Alfa Aesar silica gel 60 (0.06-0.2
mm).
2-((10-methyl-9-oxo-9,10-dihydroacridin-2-
yl)carbamoyl)pyrrolidin-1-ium (7, Pro/Fluoro/H+). Crude 6
(200 mg, 0.47 mmol, post work-up, prior to column
chromatography) was dissolved in DCM (2 mL, 0.235 M) and
trifluoroacetic acid (1 mL, 13.1 mmol) was added to the
reaction. The reaction stirred at rt for 2 h, and the solvent was
then removed in vacuo. The product was precipitated out in
diethyl ether and filtered, affording the title compound as a
yellow powder (145 mg, 96%). 1H NMR (400 MHz, d6-DMSO) δ
10.76 (s, 1H, H15), 9.33 (s, 1H, H21), 8.73 (s, 1H, H21), 8.63 (d, J
= 2.6 Hz, 1H, H6), 8.34 (d, J = 8.3 Hz, 1H, H14), 8.04 (dd, J = 9.3,
2.6 Hz, 1H, H2), 7.96 – 7.77 (m, 3H, H1, H3, H11), 7.34 (t, J = 7.8
Hz, 1H, H12), 4.37 (d, J = 7.9 Hz, 1H, H20), 3.96 (s, 3H, H19), 3.31
(m, 2H, H22), 2.40 (m, 1H, H24), 2.02 – 1.93 (m, 2H, H23).13C
NMR (101 MHz, d6-DMSO) δ 176.62 (C10), 167.22 (C16), 142.50
(C4), 139.48 (C13), 134.43 (C8), 132.41 (C2), 126.92 (C6), 126.78
(C9), 122.08 (C5), 121.56 (C1), 117.56 (C12), 116.53 (C11),
116.41 (C14), 116.39 (C5), 60.20 (C20), 46.28 (C22), 34.12 (C19),
29.99 (C24), 24.05 (C23). HRMS (+ESI) Found 322.1563 (Calcd.
4.2 Synthetic Procedures
Compounds 2-5 were synthesized according to previously
reported procedures; see SI for details.
N-(10-methyl-9-oxo-9,10-dihydroacridin-2-yl)pyrrolidine-2-
carboxamide (6, Pro/Fluoro). To a solution of 5 (430.5 mg, 2
mmol) in distilled DCM dried over 3 Å molecular sieves (20 mL,
0.1 M) at 0 °C, isobutyl chloroformate (285 µL, 2.2 mmol) and
triethylamine (307 µL, 2.2 mmol) were added. After stirring for
30 min at 0 °C, 4 (493.4 mg, 2.2 mmol) was added and the
reaction was warmed to rt and stirred for 18 h. The reaction was
washed sequentially with aq. HCl (1 M, 20 mL), sat. aq. NaHCO3
(20 mL) and brine (20 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo to afford an oily orange
crude solid. The crude product was dissolved in DCM (6.7 mL,
0.3 M) and trifluoroacetic acid (3.1 mL, 40 mmol) was added.
The reaction was stirred at rt for 3 h, and the solvent was then
removed in vacuo. The concentrate was dissolved in 15 mL of
DCM, then washed with sat. aq. NaHCO3 (10 mL) and extracted
with DCM (3 × 10 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo to afford an orange oily solid. The
crude product was purified by flash column chromatography
(SiO2, 100% EtOAc, 5% MeOH/DCM, 10% MeOH/DCM gradient),
affording the title compound as an orange solid (188 mg, 29%).
1H NMR (400 MHz, CDCl3) δ 10.02 (s, 1H, H15), 8.64 (dd, J = 9.3,
2.7 Hz, 1H, H6), 8.56 (dd, J = 8.0, 1.5 Hz, 1H, H14), 8.20 (d, J =
2.6 Hz, 1H, H2), 7.73 (t, J = 8.7 Hz, 1H, H1), 7.54 (dd, J = 8.9, 6.8
Hz, 2H, H3, H11), 7.29 (t, J = 7.5 Hz, 1H, H12), 3.92 (s, 3H, H19),
3.91 – 3.81 (m, 1H, H20), 3.08 (dq, J = 16.4, 6.5 Hz, 2H, H22),
2.23 (m, 1H, H24), 2.08 (m, 1H, H24), 1.84 – 1.73 (m, 2H, H23).53
13C NMR (101 MHz, CDCl3) δ 177.68 (C10), 173.60 (C16), 142.34
(C4), 139.11 (C13), 133.76 (C8), 132.35 (C2), 127.72 (C6), 126.30
(C9), 122.47 (C5), 121.88 (C1), 121.06 (C12), 116.31 (C11),
115.66 (C14), 114.70 (C3), 60.95 (C20), 47.35 (C22), 33.68 (C19),
30.80 (C24), 26.29 (C23). HRMS (+ESI) Found 322.1556 (Calcd.
322.1556 for C19H20N3O2; [M]+). m.p. 210.5-211.5 C. νmax/cm-1
o
3084br, 1665s, 1637w, 1617w, 1586s, 1550w, 1507s, 1466w,
1430w, 1351w, 1292w, 1280w, 1200s, 1181s, 1128s, 1051w,
1039w, 1002w. λEX = 421 nm, λEM = 489 nm (H2O).
Sodium
1-(
N-(10-methyl-9-oxo-9,10-dihydroacridin-2-
(8,
yl)pyrrolidine-2-carboxamide)diazen-1-ium-1,2-diolate
Pro/Fluoro/NO). A solution of 7 (100 mg, 0.31 mmol) in
anhydrous MeOH (3.1 mL, 0.1 M) was mixed with 30% sodium
methoxide in methanol (133 µL, 0.62 mmol) that had previously
been dried over activated 3 Å molecular sieves for at least 24 h.
The resulting solution was flushed with argon, then charged
with 5 atm of NO and stirred at rt. A thick yellow precipitate
began to form within 3 h of exposure to NO. After 24 h, the
pressure was released, and the product was collected by
filtration, washed with ether, and dried under vacuum to give
the title compound as a yellow powder (110 mg, 88%). 1H NMR
(400 MHz, 0.5 mM NaOD in D2O) δ 7.64 (d, J = 7.7 Hz, 1H, H6),
7.57 (d, J = 2.3 Hz, 1H, H14), 7.32 (dd, J = 9.2, 2.4 Hz, 1H, H2),
7.28 – 7.13 (m, 2H, H1, H3), 6.98 (d, J = 8.9 Hz, 1H, H11), 6.78 (t,
J = 7.5 Hz, 1H, H12), 3.71 (t, J = 7.2 Hz, 2H, H22), 3.22 (s, 3H,
H19), 3.21 – 3.09 (m, 2H, H24), 2.81 – 2.66 (m, 1H, H20), 2.07
(m, 1H, H23), 1.93 (m, 1H, H23). 13C NMR (101 MHz, 0.5 mM
NaOD in D2O) δ 177.60 (C10), 165.99 (C16), 143.53 (C4), 140.28
(C13), 137.49 (C8), 133.44 (C2), 132.13 (C6), 125.00 (C9), 120.78
o
322.1556 for C19H20N3O2; [M+H]+). m.p. 54-56 C. νmax/cm-1
3676br, 3255br, 2989w, 2972w, 2901w, 1675w, 1589s, 1504s,
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J. Name., 2013, 00, 1-3 | 7
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Journal Name
(C5), 120.55 (C1), 119.07 (C12) , 117.63 (C11), 115.89 (C14), nitrogen gas for 5-10 min to collect a baseline release. A 5 mM
View Article Online
114.67 (C3), 93.98 (C20), 47.46 (C22), 34.36 (C24), 32.84(C19), solution of Pro/Fluoro/NO 8 in 0.01 M NaDOOHI:(110..71013m9/Cg8inTB8042855µ2EL
18.89 (C23). HRMS (+ESI) Found 381.1427 (Calcd. 381.1437 for of 0.01 M NaOH) was made. After a baseline was collected, a
o
C19H19N5O4; [M+H]+). m.p. 174 C decomp. νmax/cm-1 3678br, 100 µL aliquot of the 5 mM Pro/Fluoro/NO 8 solution was
2989s, 2901w, 1668s, 1581s, 1506s, 1466s, 1406s, 1279w, injected into the NOA cell. The overhead lights were kept on for
1250w, 1241w, 1177s, 1125s, 1066s. λEX = 397 nm, λEM = 462 nm the duration of the experiment, and a Blak-Ray B-100AP High
(0.01 M NaOH).
Intensity UV lamp was shined on the solution. Data was
collected until the baseline returned to zero, and the
experiment was repeated three times. For the experiments to
determine NO release at different concentrations,
Pro/Fluoro/NO 8 powder was added into the NOA sample cell
in the dark at rt. The powder was purged continuously with
ultrapure nitrogen gas for 5-10 min to collect a baseline release.
An aliquot of deoxygenated nutrient broth (2.6 g in 200 mL of
Millipore water, pH 7.4) was added through the side injection
port of the NOA vessel to give the specified initial
concentration. The lights were turned on and the vessel was
placed in a 37 °C water bath. The nutrient broth solution was
purged continuously with ultrapure nitrogen gas for the
duration of the experiment. NO release was recorded in 1 s
intervals as parts per billion with a gas sampling rate of 200
mL/min. The solution was maintained in a nitrogen atmosphere
with bubbling rate of 48-54 mL/min N2 directly into the solution
and flow gas introduced into the remaining headspace.
Cell Viability Assay Solutions of Pro/Fluoro/NO (0.1 mM, 40
mg/L; 1 mM, 400 mg/L; and 10 mM, 4,000 mg/L) were made in
Pseudomonas aeruginosa culture solution in NBM. To a 24 well
plate was added the Pro/Fluoro/NO solutions (1 mL; 0.1 mM, 1
mM, or 10 mM) in triplicate. The positive control, Pseudomonas
aeruginosa culture only, and the negative control,
Pro/Fluoro/NO in sterile nutrient broth, were plated in the same
manner. The plates were incubated at 37 °C for 24 h before
analysis. The plates were illuminated by an Aldrich Spectroline
F-series hand-held UV lamp at a wavelength of 365 nm. A
standard Cell Titer Blue cell viability assay was performed to
determine antibacterial efficacy of Pro/Fluoro/NO. Three
aliquots (100 µL) of each solution in the 24 well plate were
transferred to a 96 well plate. To each well containing solution,
20 µL of Cell Titer Blue stock solution (1:5 Cell Titer Blue
reagent:nutrient broth), warmed to 37 °C, was added. The plate
was incubated at 37 °C for 2 h, then the absorbance was
measured at 570 and 600 nm using a plate reader. The number
of CFUs was also determined by the procedure described above
(Bacteria Detection Study) (n = 9).
4.3 Bacteria Studies
Pseudomonas aeruginosa Bacteria Culture Initial stock
culture of Pseudomonas aeruginosa (PAO1) was made by
streaking with a sterile inoculating loop onto agar plates and
incubating overnight at 37 °C. A colony was inoculated in
sterilized nutrient broth media (NBM, 13 g nutrient broth/1 L
Millipore water) and grown overnight at 37 °C until an optical
density at 600 nm (OD600nm) ≈0.7 was reached. This bacterial
solution was combined with glycerol (30% v/v) in a 1:1 fashion
to obtain a final glycerol concentration of 15% (v/v). These
solutions were stored at −80 °C until use. Prior to each bacterial
experiment, a frozen culture was thawed at rt and then
centrifuged at 4700 rpm for 10 min. The supernatant was
discarded, and the pellet was resuspended using 5 mL NBM.
This was transferred to an additional NBM (15 mL) and allowed
to grow overnight with shaking at 120 rpm until the OD600nm
≈1.0. The culture was diluted to an OD600nm ≈0.35 using warmed
NBM prior to beginning experiments.
Bacterial Detection Study Pro/Fluoro/H+ 7 (5 mg) was
dissolved in dimethyl sulfoxide (DSMO, 200 μL) and added to
sterilized nutrient agar [powder base (2.8 g) in Millipore water
(100 mL)] at 50 °C to a final concentration of 50 mg/L. The
mixture was divided among five sterile Petri dishes and allowed
to set overnight before inoculating each plate with 20 µL of a P.
aeruginosa suspension (OD600=0.033-0.038) using a sterile
plastic spreader. The positive control was made using the same
procedure by mixing DMSO (200 μL) with sterile nutrient agar
[powder base (2.8 g) in Millipore water (100 mL)] before
inoculation. The plates were incubated at 37 °C for 24 h before
analysis. The plates were illuminated by an Aldrich Spectroline
F-series hand-held UV lamp at a wavelength of 365 nm. In
addition, the number of colony-forming units (CFUs) was
determined using a plating method. A 1 mL aliquot of the
bacteria solution was serial diluted and 20 µL was plated on
agar. The agar plates were placed in a static 37 ˚C incubator
overnight and CFUs were counted after 24 h (n = 9). CFUs were
calculated using the equation:
Statistical Analysis All biological assays were performed
using at least three samples and assayed in replicate form.
Viability assays are reported as the mean and 95% confidence
interval. All data were evaluated for potential outliers using the
Grubbs Test. The statistical differences in data were evaluated
using the Student’s t test (p < 0.05) at the 95% confidence level.
퐶퐹푈 (# 퐶퐹푈 푐표푢푛푡푒푑)(퐷푖푙푢푡푖표푛 푓푎푐푡표푟)
=
푚퐿
푉표푙푢푚푒 푝푙푎푡푒푑 (푚퐿)
Nitric Oxide Release Measurements Nitric oxide generation
was recorded in real time using a chemiluminescence-based GE
Nitric Oxide Analyzer (NOA). Before each use, the NOA was
calibrated using a 39.6 ppm NO calibration gas cylinder. For the
experiments to force NO release, deoxygenated 0.1 M PBS
buffer was acidified to pH 3 with 1 M hydrochloric acid. The
acidified buffer (4.9 mL) was placed in the NOA cell and warmed
in a 37 ˚C water bath, then purged continuously with ultrapure
5. Conflicts of interest
There are no conflicts to declare.
6. Acknowledgements
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26 L. K. Keefer, ACS Chem. Biol., 2011, 6, 1147–1155.
This work was supported by a grant from the Monfort
Foundation. The authors would like to thank Jesus B. Tapia for
performing LC-MS experiments. The authors thank Dr. Joe
Saavedra (NIH) for helpful discussions and advice. The authors
would like to thank Dr. Brad Borlee (Colorado State University)
for providing the bacteria strain, Pseudomonas aeruginosa
PAO1.
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DOI: 10.1039/C8TB02552E
This paper reports a novel fluorescent nitric oxide donor for the simultaneous detection and
killing of Pseudomonas aeruginosa.