A latent green fluorescent styrylcoumarin probe for the selective growth and detection of Gram negative bacteria

Article abstract of DOI:10.1016/j.bmc.2018.08.015

A novel, green fluorescent β-alanylstyrylcoumarin derivative was synthesized and evaluated for its performance as a fluorogenic enzyme substrate on a range of clinically relevant microorganisms. The substrate was selectively hydrolysed by β-alanyl aminopeptidase producing P. aeruginosa resulting in an on-to-off fluorescent signal. Growth inhibitory effect of the substrate was observed on Gram positive bacteria and yeasts. Meanwhile, Gram negative species, despite their extremely protective cell envelope, showed ready uptake and accumulation of the substrate within their healthy growing colonies displaying intense green fluorescence.

Full text of DOI:10.1016/j.bmc.2018.08.015

Accepted Manuscript
A latent green fluorescent styrylcoumarin probe for the selective growth and
detection of Gram negative bacteria
Linda Váradi, Miaoyi Wang, Ramesh R. Mamidi, Jia L. Luo, John D. Perry,
David E. Hibbs, Paul W. Groundwater
PII:
S0968-0896(18)31257-4
https://doi.org/10.1016/j.bmc.2018.08.015
BMC 14502
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 July 2018
9 August 2018
11 August 2018
Please cite this article as: Váradi, L., Wang, M., Mamidi, R.R., Luo, J.L., Perry, J.D., Hibbs, D.E., Groundwater,
P.W., A latent green fluorescent styrylcoumarin probe for the selective growth and detection of Gram negative
bacteria, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.08.015
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A latent green fluorescent styrylcoumarin
probe for the selective growth and detection
of Gram negative bacteria
Leave this area blank for abstract info.
Linda Váradia, Miaoyi Wanga, Ramesh R. Mamidia, Jia L. Luoa, John D. Perryb, David E. Hibbsa, Paul W.
Groundwatera
aFaculty of Medicine and Health, The University of Sydney, Science Road, Camperdown Campus, NSW 2006, Australia
bMicrobiology Department, Freeman Hospital, High Heaton, Newcastle upon Tyne, NE7 7DN, United Kingdom
CONTROL
VIS LIGHT
365 nm
GRAM NEGATIVE
GRAM POSITIVE
P. AERUGINOSA

Bioorganic & Medicinal Chemistry
A latent green fluorescent styrylcoumarin probe for the selective growth and detection
of Gram negative bacteria
Linda Váradia, Miaoyi Wanga, Ramesh R. Mamidia, Jia L. Luoa, John D. Perryb, David E. Hibbsa, Paul W.
Groundwatera
aFaculty of Medicine and Health, The University of Sydney, Science Road, Camperdown Campus, NSW 2006, Australia
bMicrobiology Department, Freeman Hospital, High Heaton, Newcastle upon Tyne, NE7 7DN, United Kingdom
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel, green fluorescent β-alanylstyrylcoumarin derivative was synthesized and evaluated for
its performance as
a fluorogenic enzyme substrate on a range of clinically relevant
Received in revised form
Accepted
Available online
microorganisms. The substrate was selectively hydrolysed by β-alanyl aminopeptidase
producing P. aeruginosa resulting in an on-to-off fluorescent signal. Growth inhibitory effect of
the substrate was observed on Gram positive bacteria and yeasts. Meanwhile, Gram negative
species, despite their extremely protective cell envelope, showed ready uptake and accumulation
of the substrate within their healthy growing colonies displaying intense green fluorescence.
Keywords:
Fluorescent enzyme substrate
Selective growth inhibitor
Gram negative detection
Styrylcoumarin
Dedicated to the memory of our good friend and colleague, Prof. Rosaleen J. Anderson.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
blue fluorescence (λem 445 nm) upon hydrolysis by BAP within 6
hours (Scheme 1b).3 Meanwhile, blue to yellow shift of the
Early-stage, low-cost and reliable detection and identification
of pathogens in clinical settings is crucial in order to facilitate
timely and informed decision-making to initiate appropriate
therapy. Among the range of diagnostic methods which are
available, chromogenic / fluorogenic culture media (exploiting
specific bacterial enzyme activities) are still key components of
clinical practice.1 These techniques are based on the incubation
of bacterial isolates in the presence of chromogenic / fluorogenic
enzyme substrates which can undergo enzymatic hydrolysis,
consequently releasing an optical signal when and only when the
microorganism of interest is present. For example, the incubation
of the multidrug-resistant respiratory pathogens (Pseudomonas
aeruginosa, Burkholderia cepacia, Serratia marcescens) in the
presence of the yellow-coloured β-alanyl-pentylresorufamine (β-
Ala-PRF) 1 (Scheme 1a), produces purple colonies due to the
formation of PRF 2 as a result of the specific β-alanyl
aminopeptidase (BAP) activity they produce.2 Despite such
colorimetric methods often being acknowledged as the gold
standard, due to their excellent sensitivity and specificity, reliable
detection of the colour change against the background requires
24-72 hours. However, application of fluorogenic substrates can
reduce this waiting time to 6-8 hours due to the inherently more
sensitive detection of fluorescence. For example, 7-hydroxy-4-
methylcoumarin 4 was successfully converted into BAP substrate
substrate fluorescence before and after enzymatic hydrolysis was
achieved by 2-(N-β-alanylamino)-10-benzylacridone.4
Scheme 1. Detection of BAP producing P. aeruginosa using a) yellow
coloured β-alanyl PRF 1 resulting in purple colonies upon enzymatic activity;
b) non-fluorescent BAP substrate 3 resulting in blue fluorescent colonies
upon hydrolysis by BAP.
Coumarins are often used as fluorogenic core molecules for the
detection of enzymatic processes due to their low toxicity and
good cell-permeability as substrates, and their reasonable cell
6
retention, allowing for localized signal release.5, Moreover,
sufficiently substituted coumarin-based substrates allow free
enzymatic access to the targeting moiety (here, the β-alanyl
fragment), which is essential for the hydrolysis and release of the
signalling metabolic product.
———
3 (by the incorporation of a self-immolative linker) displaying
 Corresponding author. Tel.: +61395457861; e-mail: linda.varadi@csiro.au; Current address, CSIRO Manufacturing, Normanby Road,
Clayton, VIC 3169, Australia

Scheme 2. Synthetic route for the preparation of substrate 10. Reagents and conditions: i) methyl (triphenylphosphoranylidene)acetate, 180 °C, 1 hr,
inert atm.; ii) DMF, POCl3, 60 °C, 12 hrs, inert atmosphere; iii) 4-nitrobenzyltriphenylphosphonium bromide, CH3OH, CH3ONa, rt, 12 hrs; iv)
iodine, chloroform, rt, 12 hrs; v) SnCl2·2H2O, EtOAc, 85 °C, 12 hrs. vi) Boc-β-alanine anhydride, DCM, DIPEA, 35 °C, 72 hrs. vii) TFA, DCM, 0
°C, 3 hrs
Designing substrates with hydrolysis products that emit
outside of the blueish autofluorescence region of some naturally
occurring peptide moieties remains a desired outcome. To enable
this desirable red-shift (away from the autofluorescence range)
we have designed a coumarin-based fluorophore 9 containing an
electron donor diethylamino group, to enhance intramolecular
charge transfer (ICT) and a styryl moiety, in order to extend
conjugation associated with coumarins. Herein, we report the
synthesis (Scheme 2) and evaluation of novel, green fluorescent
β-alanylstyrylcoumarin derivative 10 with a green emission
wavelength of 519 nm targeting BAP.
mixtures fluorescence emission of both nitrostyrylcoumarin 8
and amino derivative 9 were quenched, while substrate 10
displayed an intense green fluorescence, with an emission
maximum of 519 nm (Figure 1). In general, the bright
fluorescence of 7-dialkylamino-coumarins is a result of excited
state ICT enhanced by the strongly electron donating
dialkylamino group.15 However, especially in polar solvents,
when a strongly electron withdrawing group (such as nitro) is
conjugated via position 3 of the coumarin ring, the enhanced
push-pull effect of the EDG-EWG substituents stabilizes this
charge separation in the excited state, thus opening up the
possibility of non-radiative twisted intramolecular charge transfer
(TICT) decay and consequently resulting in the weakening of the
fluorescence emission intensity, as is observed in the case of the
nitro derivative 8.16 The strength of the push-pull effect across
the coumarin ring has been reported to narrow the HOMO-
LUMO gap, thus resulting in a red shift of the emission
wavelength. Although, amine 9 and substrate 10 both have
electron donating substituents, amides are much less electron
donating than amines, hence they displayed rather different
fluorescence properties. The above ICT process was described
between the 7-diethylamino and coumarin carboxyl moieties.
When the styryl-amino lone pair is occupied in the amide
resonance structures of 10, bright green fluorescence is
displayed. However, in amine 9, the freely available amino lone
pair is presumed to reduce the probability of ICT, resulting in an
‘isolated’ diethylamino rotor, and allowing for non-radiative
decay pathways (Scheme 3).6 Due to these observed properties,
instead of the often-exploited fluorescence quenching by the
amide bond-formation,17 substrate 10 would provide ‘on-to-off’
signalling of BAP activity.
2. Synthesis and characterization
The synthetic route to obtain substrate 10 combined a series of
well-established methods (Scheme 2); for the synthesis of 7-
diethylaminocoumarin 6, a Wittig reaction7 between methyl
(triphenylphosphoranylidene)acetate
and
4-diethyl-
aminosalicylaldehyde 5 resulted in a significantly higher yield in
comparison to the conventional Knoevenagel condensation8 (see
Section 5.1.1.). Introduction of the formyl functionality onto
position 3 of the coumarin ring (7) involved a Vilsmeier-Haack
formylation via a previously reported procedure.8 A para-
nitrostyryl moiety was then introduced into coumarin 7 for the
dual purpose of extending the conjugation and facilitating the
covalent attachment of the β-alanine enzyme targeting moiety.
To achieve this,
a
Wittig reaction9 using p-nitrobenzyl
triphenylphosphonium ylide gave higher yield of 8 than the
condensation between p-nitrophenylacetic acid and coumarin 7
(see Section 5.1.2.).5, 10 The approx. 1:0.8 mixture of (E)- and
(Z)-7-diethylamino-3-(4-nitrostyryl)coumarin
8 obtained was
converted into the sole (E)-isomer product via isomerization in
the presence of iodine.11 To enable attachment of β-alanine (the
N-terminal recognition site for BAP), reduction of the nitro group
was carried out (using stannous chloride dihydrate) to give amine
9. This was followed by amide bond formation12 and consequent
Boc-deprotection to obtain BAP substrate 10 as a TFA salt.13
This cationic form of the substrate is proven to facilitate water
solubility and cell permeability.14 In vitro fluorescence of the
nitro 8, amine 9, and amide 10 derivatives were firstly recorded
in THF at a concentration of 1 x 10-4 M (Figure 1 and Figure
S1). A change in the substituent at the para-styryl position, from
the strongly electron withdrawing nitro group in 8 to the electron
donating amine in 9 and amide in 10, resulted in the blue shift of
the emission wavelength from 574 to 510 nm and 497 nm,
respectively. A significant increase in emission intensity was also
observed from -NO2 8 < -NH2 9 < amide 10. In aqueous THF 1:1
λex
(nm)
λem
(nm)
THF
574
510
497
THF/H2O
na
519
Int
(a.u.)
8
9
10
522
497
473
26
70
192
9
10
na
482
0
200
Figure 1. Emission of amine
9 (purple) in THF (dotted line) (λex: 497
nm), and in THF:water 1:1 (solid line); Emission of substrate 10 (green)
in THF (dotted line) (λex: 473 nm), and THF:water 1:1 (solid line) (λex:
482 nm)

in aqueous solution (Figure 1), BAP producing P. aeruginosa
displayed non-fluorescent colonies due to the hydrolysis of
substrate 10 to non-fluorescent metabolic product 9 (Figure 2b).
This change provides a ready visual differentiation between BAP
and non-BAP producing Gram negative species.
Preliminary results in liquid growth media confirmed these
results described above; the pathogens were the Gram negative
non-BAP producing E. coli, BAP producing S. marcescens, B.
cepacia, and P. aeruginosa, as well as Gram positive S. aureus.
The pathogens were incubated, in the absence and presence, of
substrate 10 in a well plate for 20 hours before determining
optical densities (OD) by measuring the increase in absorbance
intensity at 690 nm, and the fluorescence intensity increase (λex
365 nm, λem 460 nm) (Table S2, Figure S3). OD values
confirmed the growth inhibitory effect of the substrate on S.
aureus, along with weak suppression of OD of Gram negative S.
marcescens and P. aeruginosa (Figure S3a). Substrate 10
exhibited a background fluorescence as measured in the blank
control wells containing no microorganisms (Figure S3b). Non-
BAP producing E. coli, and BAP producing S. marcescens
displayed increased green fluorescence within the time frame of
the test. Although S. marcescens and B. cepacia express BAP, its
activity is significantly lower than that of P. aeruginosa.18
Substrate 10 is thus virtually unhydrolysed by S. marcescens, and
partly hydrolysed by B. cepacia within the time frame of this
study. The expression of BAP by P. aeruginosa is apparent
through the quenched fluorescence of substrate 10 via its
hydrolysis to non-fluorescent metabolic product 9. Wells
inoculated with S. aureus displayed equal fluorescence intensity
to that of the blank wells, confirming that there was no growth of
these Gram positive microorganisms.
Scheme 3. Proposed resonance structures resulting in ICT in substrate 10,
and inhibition of ICT in amine 9
3. Biological evaluation
Substrate 10 was incorporated into both solid agar-based and
liquid growth media for their incubation with microorganisms.
The agar plates were subjected to in vivo evaluation on both
multispot inoculation and individually streaked clinical isolates
of commonly encountered pathogens. For the multispot
inoculation, 10 Gram negative (spots no. 1-10), 8 Gram positive
bacteria (spots no. 11-18), and 2 yeasts (spots no. 19-20) (Figure
2a) were selected as representative cell lines for the clinical
evaluation. Colonies of Gram negative E. coli, S. enteritidis, P.
aeruginosa, and Gram positive S. aureus, and E. faecalis were
streaked on individual plates (Figure 2b, Figure S2). There are
two apparent effects of the incorporation of substrate 10 into the
growth media; the growth of all Gram positive bacteria and yeast
species was inhibited, while the Gram negative species formed
colonies (Figure 2a-iii, Table S1), and secondly, Gram negative
cell lines displayed intense green fluorescence as a result of
accumulating substrate 10 within the colonies (Figure 2a-iv).
Moreover, as was suggested by the in vitro fluorescence studies
a) Multipoint Innoculation
b) Streaked Colonies of Isolates
Gram negative organisms
Accumulation induced fluorescence
of substrate 10 in non-BAP
producers
Substrate 10
hydrolysed by
BAP-producer(s)
i
ii
iii
iv
Organism
Organism
E. coli NCTC 10418
K. pneumoniae NCTC 9528
P. rettgeri NCTC7475
S. pyogenes NCTC 8306
S. aureus (MRSA) NCTC 11939
S. aureus NCTC 6571
S. epidermidis NCTC 11047
L. monocytogenes NCTC 11994
E. faecium NCTC 7171
1
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
19
20
E. coli
S. enteritidis
P. aeruginosa
Gram positive organisms
E. cloacae NCTC11936
Growth inhibitory effect of substrate 10 on Gram positive
S. marcescens NCTC 10211
S. typhimurium NCTC 74
P. aeruginosa NCTC 10662
Y. enterocolitica NCTC 11176
B. cepacia ATCC 25416
A. baumannii ATCC 19606
organisms
E. faecalis NCTC 775
B. subtilis NCTC 9372
C. albicans ATCC90028
C. glabrata NCPF 3943
10
S. aureus
E. faecalis
Figure 2. a) Multispot inoculation Columbia agar plates with isolates after 20 hours of incubation. i) Numbered spots correlating to species named in
the table and ii) corresponding microorganisms in the absence of substrate 10; iii) in the presence of substrate 10 (50 mg/L) under VIS light, and iv)
under 365 nm light source; b) Streaked Columbia agar plates containing substrate 10 (50 mg/L) under UV light (365 nm) after 20 hours incubation in
the presence of (from top left to bottom right): Gram negative non-BAP producing E. coli and S. enteritidis, BAP producing P. aeruginosa, and Gram
positive S. aureus (inhibited) and E. faecalis (inhibited).

Although substrate 10 was metabolized by the targeted
enzyme, BAP, it displayed on-to-off rather than the more
desirable off-to-on fluorescence. However, the combination of
selective growth (enrichment) of Gram negative organisms, and
the display of intense green fluorescence upon accumulation by
viable colonies provides substrate 10 with a highly sought after
dual role. These attributes exhibited by the same enzyme
substrate can assist the every-day clinical task of isolating and
identifying multi-drug resistant Gram negative pathogens.
Furthermore, Gram negative bacteria exhibit greater resistance to
most known antibiotics due to a combination of the effect of their
complex outer membrane providing low permeability, and their
multidrug efflux pumps deterring the intracellular accumulation
of drugs.19 Thus, the combination of cellular uptake and
accumulation of substrate 10 by Gram negative species can be
exploited as a lead towards identifying molecular structural
requirements for both intracellular uptake and retention by Gram
negative species.
5.1.1. Synthesis of 7-diethylaminocoumarin 6
Method A: 4-Diethylaminosalicylaldehyde 5 (0.773 g, 4
mmol) was dissolved in ethanol (30 mL), then 1,8-
diazabicycloundec-7-ene (DBU) (1.5 mL) and diethylmalonate
(1.5 mL) were added. The mixture was stirred for 6 hours at 80
°C. The ethanol was removed under vacuum and glacial acetic
acid (20 mL) and concentrated HCl (37%) (20 mL) were added to
the reaction, which was stirred overnight at 75 °C. The reaction
mixture was cooled to room temperature and poured into ice
water (50 mL). Aq. NaOH solution (5 mol/L) was added
dropwise to the mixture to adjust the pH of the solution to 5. The
precipitate formed was filtered, and washed with water to give a
1
7-diethylaminocoumarin-3-carboxylic acid (0.314 g, 36 %). H
NMR (400 MHz, CDCl3) δ: 1.25 (6H, t, J = 7.2 Hz, 2 × CH3),
3.48 (4H, q, J = 7.2 Hz, 2 × CH2), 6.51 (1H, d, J = 2.4 Hz, Ar-
H), 6.70 (1H, dd, J = 8.8, 2.4 Hz, Ar-H), 7.44 (1H, d, J = 8.8 Hz,
Ar-H), 8.64 (1H, s, Ar-H); 13C NMR (100 MHz, CDCl3) δ: 12.4,
45.3, 96.8, 105.6, 108.6, 110.9, 131.9, 150.3, 153.8, 158.0, 164.5,
165.5; LRMS (ESI) m/z 262 (MH+). 7-Diethylaminocoumarin-3-
carboxylic acid (0.314 g, 1.45 mmol) was then further reacted
with glacial acetic acid (5 mL) and HCl (5 mL) at 120 °C
overnight. The reaction was cooled to room temperature and
poured over ice water (50 mL). Aq. NaOH solution (5 mol/L)
was added to the mixture dropwise to adjust pH of the solution to
5. The precipitate formed was filtered, and washed with water to
give product 6 as a dark brown solid (0.158 g, 18 %).8
4. Conclusion
In conclusion, a novel β-alanylstyrylcoumarin aminopeptidase
substrate 10 was synthesized and characterized. Substrate 10
displayed intense green fluorescence (outside the inherent
autofluorescence region) upon localization within Gram negative
bacterial colonies, while inhibited the growth of all tested Gram
positive bacteria and yeasts. Thus, this compound has the
potential to be exploited as a selective enrichment agent and a
detection tool at the same time. Selective detection of BAP
activity (specific to P. aeruginosa) was achieved via fluorescence
quenching, instead of the more desired off-to-on signal
generation. More significantly, the substrate was taken up and
retained by Gram negative colonies. This is an essential
requirement for both diagnostic enzyme substrates for the
localization of the detection signal, and for therapeutic agents to
reach and act on their intracellular targets. Additionally,
exchange of the β-alanine targeting moiety for other amino acids,
or the modification of the fluorescent coumarin allow for the
design of customized agents for the detection, selective growth,
or advanced cellular uptake of other microorganisms.
Method B: A mixture of 4-diethylaminosalicylaldehyde 5 (1 g,
5.17 mmol) and methyl (triphenylphosphoranylidene)acetate (2
g, 5.96 mmol) was heated at 180 °C under nitrogen for 1 hour.
The mixture was cooled to room temperature. On the obtained
solid mixture column chromatography was performed using
hexane : ethyl acetate (10 to 15% ethyl acetate ) to give product 6
as a light orange solid (0.749 g, 67 %).7 Mp 87-88 °C; H NMR
1
(400 MHz, CDCl3) δ: 1.20 (6H, t, J=7.2 Hz, 2 × CH3), 3.40 (4H,
q, J=7.2 Hz, 2 × CH2), 6.02 (1H, d, J=9.6 Hz, Ar-H3), 6.48 (1H,
d, J=2.4 Hz, Ar-H8), 6.56 (1H, dd, J=8.8, 2.4 Hz, Ar-H6), 7.23
(1H, d, J=8.8 Hz, Ar-H5), 7.52 (1H, d, J=9.6 Hz, Ar-H4); 13C
NMR (100 MHz, CDCl3) δ: 12.4 (2 × CH3), 44.8 (2 × CH2), 97.6
(CH), 108.3 (quat.), 108.6 (CH), 109.2 (CH), 128.7 (CH), 143.7
(CH), 150.7 (quat.), 156.7 (quat.), 162.3 (C=O); IR (υmax/cm-1):
1701 (C=O); LRMS (ESI) m/z 218 (MH+); HRMS (ESI) m/z
found: MNa+, 240.0997; Calcd. for C13H15NO2Na+ , 240.0995.
5. Experimental
5.1. Synthetic chemistry
5.1.2. Synthesis of 7-diethylaminocoumarin-3-carbaldehyde 7
All solvents and reagents were obtained from Sigma-Aldrich
(Castle Hill, Sydney, Australia) and used without any further
purification or treatment. The THF for the fluorescence
measurements specifically contained no fluorescent stabilizers.
Thin layer chromatography was performed on silica gel plates
from Grace Davison Discovery Sciences (US). Column
chromatography was performed on a Grace Reveleris X2. 1H and
13C NMR spectra were obtained on a Varian 400MR at 400 MHz
and 100 MHz, respectively. Coupling constants (J) are in Hertz
(Hz), and chemical shifts (δ) are expressed in parts per million
(ppm) and reported relative to residual solvent peaks. Melting
points were recorded on a Stuart Scientific SMP 10. Infrared
spectra were obtained on Shimadzu FTIR IR Tracer-100 and
Shimadzu FTIR-8400S spectrophotometers. Excitation and
emission spectra were obtained on a Shimadzu RF-5301PC
spectrophotometer. Microwave reactions were performed on
CEM Discover. Low resolution mass spectra were obtained on
Thermo Scientific TSQ Quantum Access Max LCMS/MS &
TLX1 Turboflow Chromatography System in positive ion mode.
High resolution mass spectra were generated on Bruker 7T
Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
(FTICR) in positive ion mode.
Anhydrous DMF (6 mL) was added dropwise to POCl3 (6 mL)
in an ice water bath and the mixture was stirred for 30 minutes
under nitrogen. 7-Diethylaminocoumarin 6 (1.5 g, 6.91 mmol)
was dissolved in DMF (15 mL) and the solution was then added
dropwise to the above solution. The mixture was stirred at 60 °C
overnight under nitrogen, and poured into ice water(50 mL). Aq.
NaOH solution (5 mol/L) was added dropwise to adjust the pH of
the mixture to about 6, resulting in a precipitate which was
filtered to give product 7 as an orange solid (0.993 g, 59 %).8 Mp
1
162-164 °C; H NMR (400 MHz, CDCl3) δ: 1.25 (6H, t, J=7.2
Hz, 2 × CH3), 3.47 (4H, q, J=7.2 Hz, 2 × CH2), 6.48 (1H, d,
J=2.4 Hz, Ar-H8), 6.63 (1H, dd, J=8.8, 2.4 Hz, Ar-H6), 7.40 (1H,
d, J=8.8 Hz, Ar-H5), 8.24 (1H, s, Ar-H4), 10.12 (1H, s, CHO);
13C NMR (100 MHz, CDCl3) δ: 12.4 (2 × CH3), 45.3 (2 × CH2),
97.2 (CH), 108.3 (quat.), 110.2 (CH), 114.4 (quat.), 132.5 (CH),
145.3 (CH), 153.4 (quat.), 158.9 (CH), 161.9 (C=O), 187.9
(CHO); IR (υmax/cm-1): 1701 (C=O), 1670 (CHO); LRMS (ESI)
m/z 246 (MH+); HRMS (ESI) m/z found: MNa+, 268.0946; Calcd.
for C14H15NO3Na+, 268.0944.

5.1.3. Synthesis of (E)-7-diethylamino-3-(4-nitrostyryl)coumarin
8
found: M+, 364.1421 and MH+, 365.1500; Calcd. for C21H20N2O4,
364.1418 and for C21H21N2O4 , 365.1451.
+
Method A: A mixture of 7-diethylaminocoumarin-3-aldehyde
7 (245 mg, 1 mmol), 2-(4ʹ-nitrophenyl)acetic acid (207 mg, 1.15
mmol) and piperidine (196 mg, 2.3 mmol) was stirred for 1.5
hours at 140 °C under reflux. The reaction mixture was cooled to
room temperature then ethanol (15 mL) was added and sonicated
for 10 minutes. The mixture was then stirred overnight and the
precipitate was filtered under vacuum, washed with ethanol (3
mL) and hexane (3 mL) resulting in coumarin 8 as a dark red
solid (7 mg, 2 %).5
Method B: A mixture of 7-diethylaminocoumarin-3-aldehyde
7 (50 mg, 0.204 mmol), 2-(4-nitrophenyl)acetic acid (40.9 mg,
0.226 mmol), imidazole (21 mg, 0.308 mmol), piperidine (26.2
mg, 0.308 mmol) and ethylene glycol (0.6 mL) was stirred under
microwave irradiation at i) 150W, 160 °C) or ii) 300W, 160 °C
for 30 minutes. Both reaction mixtures were cooled to room
temperature and acidified with HCl (1 mol/L) to adjust the pH to
5. The aqueous solution was then extracted with ethyl acetate (2-
mL), which was separated and dried over sodium sulfate. The
organic layer was evaporated, ethanol (10 mL) was added and
sonicated for 90 minutes to form a precipitate, which was then
filtered under vacuum, washed with ethanol (3 mL) and hexane
(3 mL) resulting in a dark red solid 8 (5 mg, 7 %).10
5.1.4. Synthesis of (E)-3-(4-aminostyryl)-7-(diethylamino)
coumarin 9
(E)-7-Diethylamino-3-(4-nitrostyryl)coumarin
8 (200 mg,
0.549 mmol) and SnCl2·2H2O (1.23 g, 5.49 mmol) in ethyl
acetate (10 mL) was refluxed overnight. Saturated Na2CO3 was
added to adjust the pH to 10. Ethyl acetate (20 mL) was added to
the mixture which was then filtered through celite, and washed
with ethyl acetate (60 mL). The organic layer was dried over
sodium sulfate and evaporated under vacuum resulting in the
product 9 as a dark orange solid (182 mg, 99 %). 5 Mp 188-192
°C (lit. 166-168 °C); 1H NMR (400 MHz, DMSO-d6) δ: 1.13 (6H,
t, J=7.2 Hz, 2 × CH3), 3.44 (4H, q, J=7.2 Hz, 2 × CH2), 5.33 (2H,
broad s, NH2), 6.54 (1H, d, J=2.4 Hz, Ar-H8), 6.57 (2H, d, J=8.8
Hz, Ar-H3ʹ,5ʹ), 6.71 (1H, dd, J=8.8, 2.4 Hz, Ar-H6), 6.79 (1H, d,
J=16.4 Hz, CHA or CHB), 7.23 (2H, d, J=8.4 Hz, Ar-H2ʹ,6ʹ), 7.29
(1H, d, J=16.4 Hz, CHA or CHB), 7.43 (1H, d, J=9.2 Hz, Ar-H5),
7.93 (1H, s, Ar-H4); 13C NMR (100 MHz, DMSO-d6) δ: 13.0,
44.7, 97.0, 109.3, 109.9, 114.6, 117.9, 118.0, 125.6, 128.1, 129.6,
130.8, 137.3, 149.5, 150.5, 155.4, 161.2; IR (υmax/cm-1): 3456
(NH2), 3360 (NH2), 1686 (C=O), 1605 (C=C); LRMS (ESI) m/z
335 (M+); HRMS (ESI) m/z found: M+, 334.1677 and MH+,
+
335.1756; Calcd. for C21H22N2O2, 334.1676 and for C21H23N2O2 ,
335.1754.
Method C: Synthesis of 4-nitrobenzyltriphenylphosphonium
bromide: 4-Nitrobenzyl bromide (3 g, 13.89 mmol) was
dissolved in toluene (50 mL), then triphenylphosphine (3.643 g,
13.89 mmol) was added and the mixture was heated at 120 °C
overnight. The product was filtered under vacuum, washed with
hexane (50 mL) to give a fine white powdered solid (6.333 g,
5.1.5. Synthesis of (E)-3-(4-β-alanylstyryl)-7-(diethylamino)
coumarin trifluoroacetate 10
Boc-β-alanine (508.97 mg, 2.69 mmol) was added to
dicyclohexylcarbodiimide (610.53 mg, 2.959 mmol) dissolved in
DCM (10 mL) and stirred in an ice bath for 3 hours. The reaction
mixture was filtered and the filtrate was added into a mixture of
1
13.18 mmol, 95 %).20 Mp 273-274 °C; H NMR (400 MHz,
CDCl3) δ: 5.99 (2H, d, J=15.6 Hz, Ar-H), 7.45 (2H, dd, J=7.2,
2.8 Hz, Ar-H), 7.56-7.84 (17H, m, Ar-H); LRMS (ESI) m/z 398
(M+); HRMS (ESI) m/z found: M+, 398.1306; Calcd. for
C25H21NO2P+, 398.1304. Synthesis of (E)-7-diethylamino-3-(4-
nitrostyryl)coumarin 8: In a round bottom flask fitted with drying
tube, 7-diethylaminocoumarin-3-aldehyde 7 (0.8 g, 3.2 mmol)
and 4-nitrobenzyltriphenylphosphonium bromide (1.872 g, 3.92
mmol) were dissolved in dry methanol (80 mL) and then
CH3ONa (0.96 g, 17.6 mmol) was added. The mixture was stirred
at room temperature overnight and was then poured into ice
water (160 mL), and stirred for 2 hours at room temperature. The
solid was filtered under vacuum and triturated with ethanol (3 ×
100 mL) resulting in a mixture of the (E) and (Z) isomers of 8
(0.743 g, 62%). This mixture of (E) and (Z)-8 (743 mg, 2.036
mmol) was dissolved in chloroform (60 mL) in a round bottom
flask fitted with drying tube then iodine (103.43 mg, 0.204
mmol) was added and the reaction mixture was stirred at room
temperature overnight. The solution was washed thoroughly with
saturated sodium metabisulfite solution (5 × 20 mL). The organic
layer was dried over sodium sulfate and evaporated under
reduced pressure to give the product (E)-8 as a dark red solid
(743 mg, 100 %).9, 11 Mp 259-260 °C (lit. 253-255 °C); 1H NMR
(400 MHz, CDCl3) δ: 1.23 (6H, t, J=7.2 Hz, 2 × CH3), 3.44 (4H,
q, J=7.2 Hz, 2 × CH2), 6.51 (1H, d, J=2.4 Hz, Ar-H8), 6.62 (1H,
dd, J=8.8, 2.4 Hz, Ar-H6), 7.19 (1H, d, J=16 Hz, CHA or CHB),
7.31 (1H, d, J=8.8 Hz, Ar-H5), 7.59 (1H, d, J=16 Hz, CHA or
CHB), 7.61 (2H, d, J=8.8 Hz, Ar-H2ʹ,6ʹ), 7.71 (1H, s, Ar-H4),
8.19 (2H, d, J=8.8 Hz, Ar-H3ʹ,5ʹ); 13C NMR (100 MHz, CDCl3)
δ: 12.5 (2 × CH3), 45.0 (2 × CH2), 97.2 (CH), 108.9 (quat.), 109.4
(CH), 116.4 (quat.), 124.1 (CH-2ʹ,6ʹ or CH-3ʹ,5ʹ), 126.8 (CH-2ʹ,6ʹ
or CH-3ʹ,5ʹ), 127.6 (CH), 128.0 (CH), 129.3 (CH), 140.5 (CH),
144.4 (CH), 146.5 (quat.), 151.0 (quat.), 156.0 (quat.), 161.0
(C=O); IR (υmax/cm-1): 1705 (C=O), 1616 (C=C), 1519 (NO2),
1331 (NO2); LRMS (ESI) m/z 365 (MH+); HRMS (ESI) m/z
(E)-3-(4-aminostyryl)-7-(diethylamino)coumarin
9 (300 mg,
0.897 mmol) and diisopropyl ethyl amine (DIPEA) (197.08 mg,
1.525 mmol) in DCM (20 mL). The mixture was heated at 35 °C
over 72 hours. Aqueous HCl (1 mol/L, 10 mL) was added and the
organic layer was washed with saturated NaHCO3 solution (10
mL) then dried over sodium sulfate and filtered under reduced
pressure to give a yellow solid (214 mg, 47 %).12 Mp 216-218
°C; 1H NMR (400 MHz, DMSO-d6) δ: 1.14 (6H, t, J= 7.2 Hz, 2 ×
CH3), 1.39 (9H, s, 3 × CH3), 2.47-2.52 (2H, m, CH2-C or CH2-D),
3.23 (2H, m, J=6.8, 6.0 Hz, CH2-C or CH2-D), 3.45 (4H, q, J=7.2
Hz, 2 × CH2), 6.56 (1H, d, J=2.4 Hz, Ar-H8), 6.73 (1H, dd,
J=6.8, 2.4 Hz, Ar-H6), 6.86 (1H, t, J=5.2 Hz, NH), 7.02 (1H, d,
J=16.4 Hz, CHA or CHB), 7.42 (1H, d, J=16.4 Hz, CHA or CHB),
7.45-7.48 (3H, m, 3 × Ar-H), 7.61 (2H, d, J=8.8 Hz, Ar-H3ʹ,5ʹ),
8.03 (1H, s, Ar-H4), 10.01 (1H, s, NH); 13C NMR (100 MHz,
DMSO-d6) δ: 13.0, 28.9, 37.1, 37.4, 44.7, 78.2, 96.9, 109.1,
110.0, 117.0, 119.9, 122.5, 127.3, 129.5, 129.9, 132.8, 139.4,
150.9, 155.8, 156.2, 161.0, 170.0; IR (υmax/cm-1): 1709 (C=O),
1667 (C=O), 1616 (C=C), 1593 (amide I), 1508 (amide II);
LRMS (ESI) m/z 506 (MH+); HRMS (ESI) m/z found: M+,
505.2573 and MH+, 506.2607; Calcd. for C29H35N3O5, 505.2571
+
and for C29H36N3O5 , 506.2605.
The
obtained
(E)-3-(4-tert-butoxycarbonylamino-
propanamidostyryl)-7-(diethylamino)coumarin (150 mg, 0.297
mmol) was dissolved in DCM (10 mL) on an ice bath. A mixture
of trifluoroacetic acid (2 mL) and DCM (2 mL) was added
dropwise. The reaction was then allowed to warm to room
temperature and stirred for 3 hours. The reaction mixture was
concentrated in vacuo then methanol (2 × 10 mL) and the
mixture was concentrated in vacuo (for the removal of residual
TFA). The reaction mixture was triturated with diethyl ether
twice and the solid was filtered and washed with diethyl ether (20
mL) resulting in product 10 as an orange solid (132 mg, 86 %).13

1
Mp 222-226 °C; H NMR (400 MHz, DMSO-d6) δ: 1.14 (6H, t,
used. Microplate reader TECAN Infinite M-200 was used to
record fluorescence (365/460 setting) and absorption (at 690 nm
for organism density). After a 20 hour period of incubation of the
assessed strains (Table S2 and S3) on GREINER 96 well plate
(ref. 655 090) sealed with transparent GREINER viewseal (ref.
676 070). For 8, 9 and 10, a 10 µL solution of substrate (at 50g/L
in NMP) was suspended in 5 mL of TSB (final concentration 100
mg/L). Every well was filled with 100 µL of specified solution of
substrate and 100 µL of bacterial suspension at 0.5 McFarland
for a final concentration of 0.25 McFarland (about 7.5 × 107
bacteria/mL). For control, in the absence of enzyme substrates
the wells were filled with 100 μL TSB and 100 µL of bacterial
suspension at 0.5 McFarland. For negative control (blank), the
wells were filled with 100 µL TSB in the absence or presence of
any of the substrates and 100 µL Suspension Medium. The
absorption (at 690 nm for microbial growth) and relative
fluorescent intensities (at λex= 365 nm / λem= 460 nm) for
enzymatic activity were recorded after a period of 20 hours.
J= 7.2 Hz, 2 × CH3), 2.72 (2H, t, J= 6.8 Hz, CH2-C or CH2-D),
3.11 (2H, t, J=6.8 Hz, CH2-C or CH2-D), 3.45 (4H, q, J=7.2 Hz, 2
× CH2), 6.56 (1H, d, J=2.4 Hz, Ar-H8), 6.74 (1H, dd, J=8.8, 2.4
Hz, Ar-H6), 7.03 (1H, d, J=16.4 Hz, CHA or CHB), 7.41-7.51
(4H, m, CHA or CHB and 3 × Ar-H), 7.62 (2H, d, J=8.8 Hz, Ar-
H3ʹ,5ʹ), 7.76 (3H, broad s, NH3+), 8.03 (1H, s, Ar-H4), 10.23
(1H, s, NH); 13C NMR (100 MHz, DMSO-d6) δ: 12.8, 33.8, 35.4,
44.6, 96.8, 108.9, 109.9, 116.8, 119.8, 122.6, 127.3, 129.2, 129.8,
133.0, 138.8, 139.3, 150.8, 155.6, 160.8, 168.8; IR (υmax/cm-1):
3325 (NH3+), 1697 (C=O), 1670 (C=C), 1585 (amide I), 1508
(amide II); LRMS (ESI) m/z 406 (M+); HRMS (ESI) m/z found:
M+, 406.2126; Calcd. for C24H28N3O3 , 406.2125.
+
5.2. In vitro fluorescence properties
The concentration of stock solution for (E)-3-(4ʹ-nitrostyryl)-
7-(diethylamino)coumarin
8,
(E)-3-(4ʹ-β-alanylstyryl)-7-
(diethylamino)coumarin trifluoroacetate 10 and (E)-3-(4ʹ-
aminostyryl)-7-(diethylamino)coumarin 9 was 2 × 10-4 mol/L in
THF. 2 mL of THF, or 2 mL water was added to 2 mL of the
stock solution of each compound, respectively. The slit width of
fluorescence spectrophotometer was set at 3 mm.
Acknowledgments
PWG, DEH, and JDP would like to thank the National Health
and Medical Research Council (NHMRC) for funding.
5.3. Biological evaluation
References and notes
5.3.1. Solid agar media
5.3.1.1. Preparation of culture media containing substrate.
Columbia agar was prepared as follows; 41 g of Columbia agar
(Oxoid Basingstoke, UK) was added to deionized water and the
volume was made up to 1 L. The medium was sterilized by
autoclaving at 116 °C for 20 minutes and left to cool at 50 °C. 2
mg of the substrate to be tested was initially dissolved in 100 μL
of N-methylpyrrolidone and this was added to Columbia agar
(made up to 20 mL), then poured into sterile Petri dishes to give a
final concentration of 100 mg/L for the substrates. Columbia agar
1
2
Varadi, L.; Luo, J. L.; Hibbs, D. E.; Perry, J. D.; Anderson, R. J.;
Orenga, S.; Groundwater, P. W. Chem. Soc. Rev. 2017, 46, 4818-
4832.
Zaytsev, A. V.; Anderson, R. J.; Bedernjak, A.; Groundwater, P.
W.; Huang, Y.; Perry, J. D.; Orenga, S.; Roger-Dalbert, C.;
James, A. Org. Biomol. Chem. 2008, 6, 682-692.
Varadi, L.; Hibbs, D. E.; Orenga, S.; Babolat, M.; Perry, J. D.;
Groundwater, P. W. RSC Adv. 2016, 6, 58884-58889.
Cellier, M.; James, A. L.; Orenga, S.; Perry, J. D.; Turnbull, G.;
Stanforth, S. P. PLOS ONE 2016, 11, e0158378.
Herner, A.; Estrada Girona, G.; Nikić, I.; Kállay, M.; Lemke, E.
A.; Kele, P. Bioconjugate Chem. 2014, 25, 1370-1374.
Anila, A. H.; Ali, F.; Kushwaha, S.; Taye, N.; Chattopadhyay,
S.; Das, A. Anal. Chem. 2016, 88, 12161-12168.
Flašík, R.; Stankovičová, H.; Gáplovský, A.; Donovalová, J.
Molecules 2009, 14, 4838.
Jiang, N.; Fan, J.; Xu, F.;Peng, X.; Mu, H.; Wang, J.; Xiong, X.
Angew. Chem. Int. Edit. 2015, 54, 2510-2514.
Wang, L.; Long, L.; Zhou, L.; Wu, Y.; Zhang, C.; Han, Z.;
Wang, J.; Da, Z. RSC Adv. 2014, 4, 59535-59540.
3
4
5
6
7
8
9
incorporating
an
equivalent
concentration
of
N-
methylpyrrolidone was used as a growth control.
5.3.1.2. Microbial suspension preparation. Microbial reference
strains were obtained from either the National Collection of Type
Cultures (NCTC) or the National Collection of Pathogenic Fungi
(NCPF) which are both located at the Central Public Health
England Laboratory, Colindale, UK or the American Type
Culture Collection (ATCC), Manassas, USA. The 20 test
microorganisms were maintained on Columbia agar.
10 Sinha, A. K.; Kumar, V.; Sharma, A.; Sharma, A.; Kumar, R.
Tetrahedron 2007, 63, 11070-11077.
11 Gaukroger, K.; Hadfield, J. A.; Hepworth, L. A.; Lawrence, N.
J.; McGown, A. T. J. Org. Chem. 2001, 66, 8135-8138.
12 Montalbetti, C. A. G. N.; Falque, V. Tetrahedron 2005, 61,
10827-10852.
13 Luo, J. L.; Jin, T.; Váradi, L.; Perry, J. D.; Hibbs, D. E.;
Groundwater, P. W. Dyes Pigments 2016, 125, 15-26.
14 Chevalier, A.; Mercier, C.; Saurel, L.; Orenga, S.; Renard, P.-Y.;
Romieu, A. Chem. Commun. 2013, 49, 8815-8817.
15 Avhad, K.; Jadhav, A.; Sekar, N. J. Chem. Sci. 2017, 129, 1829-
1841.
16 Eisold, U.; Sellrie, F.; Memczak, H.; Andersson, A.; Schenk, J.
A.; Kumke, M. U. Bioconjugate Chem. 2018, 29, 203-214.
17 Varadi, L.; Gray, M.; Groundwater, P. W.; Hall, A. J.; James, A.
L.; Orenga, S.; Perry, J. D.; Anderson, R. J. Org. Biomol. Chem.
2012, 10, 2578-2589.
5.3.1.3. Multipoint inoculation. Colonies of each microbial strain
were harvested using a loop from overnight cultures on Columbia
agar. These were suspended in sterile deionized water to a
suspension equivalent to 0.5 McFarland units using
a
densitometer. 100 μL of this suspension was pipetted into the
corresponding wells of a multipoint inoculation device. Each set
of plates received 1 μL of bacterial suspension, giving 1.5 × 105
organisms per spot on each inoculation. Twenty strains were
inoculated per plate and the plates were incubated for 18 hours in
air at 37 °C.
5.3.1.4. Activity determination. After incubation, the activity of
the microorganisms with the test substrates was determined by
observing the plates under UV irradiation at 365 nm and
comparing with the substrate-free control.
5.3.2. Liquid media
18 Bedernjak, A. F.; Zaytsev, A. V.; Babolat, M.; Cellier, M.;
James, A. L.; Orenga, S.; Perry, J. D.; Groundwater, P. W.;
Anderson, R. J. J. Med. Chem. 2016, 59, 4476-4487.
19 Zgurskaya, H. I.; Rybenkov, V. V.; Krishnamoorthy, G.; Leus, I.
V. Res. Microbiol. 2018.
For the evaluation of substrates 8, 9, and 10 in liquid media,
N-methyl-2-pyrrolidinone (NMP) was purchased from ACROS
ORGANICS. Trypcase Soy Broth (TSB BIOMERIEUX ref. 42
100) and Suspension Medium BIOMERIEUX (ref. 70 640) were

20 Shu, X.; Lu, Z.; Zhu, J. Chem. Mater. 2010, 22, 3310-3312.
A. Supplementary Material
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.xxxx