Evaluation of fluorogenic aminonaphthalenesulfonamides and 6-hydrazinobenz[de]isoquinoline-1,3-diones for the detection of bacteria

Article abstract of DOI:10.1016/j.dyepig.2015.09.031

New fluorogenic enzyme substrates were synthesized by the coupling of aminonaphthalenesulfonamides or 6-hydrazinobenz[de]isoquinoline-1,3-diones with β-alanine. The 6-hydrazinobenz[de]isoquinoline-1,3-diones were also condensed with a range of aryl aldehydes to give the corresponding hydrazones. The photophysical properties of the synthesized amines and hydrazines and their amide, hydrazide and hydrazone derivatives, were examined and they were also incorporated into Columbia agar in order to determine their potential for the detection of pathogenic bacteria.

Full text of DOI:10.1016/j.dyepig.2015.09.031

Accepted Manuscript
Evaluation of fluorogenic aminonaphthalenesulfonamides and 6-
hydrazinobenz[de]isoquinoline-1,3-diones for the detection of bacteria
Jia Lin Luo, Terry Jin, Linda Váradi, John D. Perry, David E. Hibbs, Paul W.
Groundwater
PII:
S0143-7208(15)00378-2
10.1016/j.dyepig.2015.09.031
DYPI 4942
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 19 June 2015
Revised Date: 19 September 2015
Accepted Date: 22 September 2015
Please cite this article as: Luo JL, Jin T, Váradi L, Perry JD, Hibbs DE, Groundwater PW, Evaluation
of fluorogenic aminonaphthalenesulfonamides and 6-hydrazinobenz[de]isoquinoline-1,3-diones for the
detection of bacteria, Dyes and Pigments (2015), doi: 10.1016/j.dyepig.2015.09.031.
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ACCEPTED MANUSCRIPT
Evaluation of
fluorogenic aminonaphthalenesulfonamides
and
6-
hydrazinobenz[de]isoquinoline-1,3-diones for the detection of bacteria
¶
a
¶
a
a
b
a
Jia Lin Luo, Terry Jin, Linda Váradi, John D. Perry, David E. Hibbs, Paul
1
a
W. Groundwater,
a
Faculty of Pharmacy, University of Sydney, Science Road, Camperdown Campus, NSW
2
006, Australia
b
Microbiology Department, Freeman Hospital, High Heaton, Newcastle upon Tyne, NE7
7
DN, United Kingdom
Abstract
New fluorogenic enzyme substrates were synthesized by the coupling of
aminonaphthalenesulfonamides or 6-hydrazinobenz[de]isoquinoline-1,3-diones with β-
alanine. The 6-hydrazinobenz[de]isoquinoline-1,3-diones were also condensed with a range
of aryl aldehydes to give the corresponding hydrazones. The photophysical properties of the
synthesized amines and hydrazines and their amide, hydrazide and hydrazone derivatives,
were examined and they were also incorporated into Columbia agar in order to determine
their potential for the detection of pathogenic bacteria.
Keywords
Aminonaphthalenesulfonamides, 6-hydrazinobenz[de]isoquinoline-1,3-diones, hydrazones,
fluorescence, pathogenic bacteria.
1
.
Introduction
Antimicrobial resistance has been a major obstacle to the effective treatment of bacterial
infections for several decades and has recently become a serious threat to global public
health, contributing to over 720,000 potentially preventable healthcare-associated infections
(HAIs) in the US p.a.[1] and more than 177,000 p.a. in Australia.[2] Although a natural
phenomenon, antimicrobial resistance has been accelerated by the misuse of antimicrobial
agents, allied to a lack of new agents to replace those which have become ineffective (only
1
Corresponding author; Tel.: +61 2 9114 1232; fax: +61 2 9351 4391.
e-mail: paul.groundwater@sydney.edu.au
¶
These authors contributed equally to this work
1

ACCEPTED MANUSCRIPT
two truly novel antibacterial agents have been approved in the last 30 years). Antibiotic
stewardship programs have been introduced worldwide in attempts to improve patient
outcomes, reduce adverse sequelae associated with antimicrobial use, and to preserve the ever
more limited array of antibacterials which infectious disease clinicians have at their
disposal.[3, 4] In addition, effective prevention and control of infection can help to reduce the
spread of antibiotic resistance, and this can be informed by universal screening protocols,[5]
which are ideally based upon rapid, simple, and cost-effective techniques for the detection
and identification of pathogenic bacteria.
One of the most commonly employed techniques for the detection and identification of
bacteria involves the use of chromogenic culture media,[6] which employ an initially non-
coloured substrate, consisting of an enzyme-targeting moiety covalently attached to a
chromogen. In the presence of an enzyme which is specific to a particular bacterium, the
covalent bond is cleaved, releasing the chromogen, Figure 1, and thus allowing the detection
and identification of this bacterium.
For example, chromID™ P. aeruginosa, exploits β-alanyl-1-pentyl resorufamine 1, which is
cleaved by β-alanyl aminopeptidase to give the chromogen 1-pentylresorufamine 2, Figure 1,
to enable the rapid identification of P. aeruginosa in patients with cystic fibrosis before
conversion into a difficult-to-treat mucoid phenotype.[7] Other chromogenic media have
been employed for the detection of multi-resistant organisms, such as methicillin-resistant
Staphylococcus aureus (e.g. CHROMagar™ MRSA),[9] vancomycin-rsistant enterococci
(e.g. VRE-BMX),[10] and NDM-1 (carbapenemase) producing Enterobacteriaceae.[11]
Although sufficiently specific to allow presumptive species identification, a limiting factor in
the use of chromogenic media is the time taken (typically 24-48 hours) for the development
of sufficient quantities of the indicative colour of the chromogen, which is essential for
reliable detection against the usually lightly coloured background. This limitation can be
addressed through the development of fluorogenic media, since the detection of fluorescence
is inherently more sensitive than the perception of colour change — media containing
fluorogenic substrates should lead to a reduction in the time required for bacterial
detection.[12]
In order to develop fluorogenic media for the detection of bacteria, the reliable detection of
the enzymatically-released fluorophores is necessary, and this can be facilitated by two
methods; (i) either an off-to-on mechanism analogous to chromogenic media or (ii) as the
2

ACCEPTED MANUSCRIPT
result of a significant change in the observed fluorescent emission wavelengths between the
substrate and subsequent fluorogen . In addition to displaying intense fluorescence, the ideal
fluorogen should be non-growth inhibitory and retained within bacterial cells / colonies,
while the substrate should be water soluble and readily taken up by bacterial cells.
Based on their reported fluorescent properties, we identified aminonaphthalenesulfonamide
(ANS) and 6-hydrazinobenz[de]isoquinoline-1,3-dione (6-HBI) cores as potential fluorogens
for the detection of bacterial enzymes, Figure 2. Aminonaphthalenesulfonamides (ANS) have
previously been reported as useful fluorogenic substrates for proteolytic enzymes involved in
fibrinolysis and blood coagulation, and the quantitation of Lys-plasmin.[13] Similiarly,
benz[de]isoquinoline-1,3-diones have been used as enzymatic markers for dipeptidyl
peptidase IV and tripeptidyl peptidase I activity in rodents,[14] as a probe for nitroreductase
activity for the detection of hypoxic cells,[15, 16] and as a marker for the detection of
Mycobacterium tuberculosis.[17]
In order to evaluate their potential for the detection of bacteria in clinical samples, various
derivatives of both sets of fluorescent cores were designed, synthesised and coupled to a
suitable amino acid. β-Alanine was initially chosen as the amino acid moiety in order to
demonstrate the utility of these fluorogens in bacterial systems by targeting the β-alanyl
aminopeptidase activity of P. aeruginosa. The hydrolysis of the amide bond in the presence
of this bacterium would result in the release of the fluorogen. In the case of the 6-HBI core, a
secondary reaction, with a range of para-substituted benzaldehydes and cinnamaldehyde, was
also envisaged in order to enhance the fluorescence observed as a result of the extension of
the conjugation. Both fluorescent core molecules would be modified through the introduction
of substituents with different alkyl (R) chain lengths in order to ascertain the optimum
uptake, adherence, and lack of growth inhibition.
Synthesis of the desired fluorophores was first achieved using the methods described below
and their subsequent coupling to Boc-β-alanine was achieved with the same symmetrical
anhydride method for both core molecules.
2
.
Experimental
General
2
.1
All reagents and solvents were purchased from Sigma-Aldrich, Alfa Aesar, and ChemSupply,
and used without any further purification or treatment. Thin layer chromatography was
1
13
performed on Grace Reveleris® Silica Aluminum-backed TLC Plates (UV254). H and C
3

ACCEPTED MANUSCRIPT
NMR spectra were acquired on a Varian 400MR at 400 MHz and 100 MHz, respectively.
Coupling constants (J) are in Hertz (Hz), chemical shifts (δ) are expressed in parts per million
(ppm) and reported relative to residual solvent peaks. Melting points were obtained on a
Stuart Scientific SMP 10. Low resolution mass spectra were obtained on TSQ Quantum
Access Max (Triple Quadrupole) LCMS/MS in positive ion mode. High resolution mass
spectra were obtained on a Bruker 7T Fourier Transform Ion Cyclotron Resonance Mass
Spectrometer (FTICR) in positive ion mode. Infrared spectra were recorded on a Shimadzu
FTIR-8400S and Shimadzu IRTracer-100. Elemental analyses were obtained from the
Campbell Microanalytical Laboratory in the University of Otago, NZ.
2
.2
General procedure for the preparation of acetamides 6a-e
Imide 4 was prepared by the method of Xu et al.[18] and sulfonyl chloride 5 by the method
of Guan et al..[19]
To a solution of 5 (1 equiv.) in DCM, triethylamine (2 equiv.) and the appropriate amine (1.5
equiv.) were added. The resulting solution was then left to stir at room temperature overnight.
Upon completion, the organic layer was washed with 1M HCl (3 × 20 mL), water (3 × 20
mL) and brine (3 × 20 mL), then dried over Na SO . The solvent was removed in vacuo and
2
4
the crude product was purified by column chromatography, eluting with ethyl acetate: hexane
25:75 to 75:25) to give acetamides 6a-e.
(
2
.3
General procedure for the deacetylation of acetamides 6a-e to produce
sulfonamides 7a-e
The appropriate acetamide 6a-e was dissolved in methanol / 5M aqueous NaOH mixture (3:2
v/v) and kept at 85 °C. Upon completion of the reaction, the methanol was removed in vacuo
and the aqueous residue was extracted with DCM (6 × 25mL). After drying over Na SO , the
2
4
organic solvent was removed and the crude product was purified by column chromatography
on silica, eluting with ethyl acetate: hexane (50:50 to 60:40) to yield the desired sulfonamides
7
a-e.
2
.4
General method for preparation of 6-bromo-1H-benz[de]isoquinoline-
1
,3(2H)-diones 9
4
-Bromo-1,8-naphthalic anhydride 8 (1 equiv.) and the corresponding amine (1 equiv.) were
refluxed in ethanol (25 mL) for 2 hours. The solution was then allowed to cool to room
temperature and the precipitate which formed was collected by filtration to give the desired
product 9a-c.
4

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2
.5
General
method
for
the
preparation
of
6-hydrazino-1H-
benz[de]isoquinoline-1,3(2H)-diones 10
The 2-alkyl-6-bromobenz[de]isoquinolin-1,3(2H)-dione 9a-c (1 equiv.) was reacted with
hydrazine hydrate (2 equiv.) in 2-butanol (25 mL) under reflux. After 2 hours, additional
hydrazine hydrate (2 equiv.) was added to the solution and after an additional 4 hours, the
solution was allowed to cool to room temperature. The precipitate which formed was washed
with diethyl ether to give the desired product 10a-c.
2
.6
General procedure for the coupling with Boc-β-alanine to give
sulfonamides 11a-e and hydrazides 13a-c
Boc-β-alanine (8 equiv.) was dissolved in DCM and cooled to 0 °C. To this solution, DCCI
(4 equiv.) was added and the mixture was allowed to warm to room temperature and left to
stir for 1 hour. This mixture was then filtered into a flask containing the appropriate amine 7
or hydrazine 10 (1 equiv.), EDIPA (1.2 equiv.) and DMAP (0.6 equiv.) in DCM. The
resulting solution was then heated to 50 °C and left to stir. Upon completion of the reaction,
the organic layer was washed with saturated NaHCO (3 × 20 mL), 1M aqueous HCl (3 × 20
3
mL) and water (3 × 20 mL) then dried over Na SO . The solvent was removed in vacuo and
2
4
the crude products 11a-e were purified by column chromatography on silica, eluting with
ethyl acetate: hexane (20:80 to 50:50).
For the coupling to the hydrazinobenz[de]isoquinolin-1,3(2H)diones 10a-c, after 3 hours,
additional Boc-β-Alanine anhydride (4 equiv.) was added to the solution. After a further 20
hours, the solvent was removed in vacuo and the residue was purified via column
chromatography on silica gel. Recrystallization from ethanol gave the Boc-protected
substrates 13a-c.
2.6.1 tert-Butyl [3-oxo-3-{5-(N-propylsulfamoyl)naphthalen-1-
yl}aminopropyl)]carbamate (11a)
-1
Yield of 11a: 20%; White solid; mp 177-179 °C; FTIR (neat, v/cm ): 3291 (NH), 3271 (NH),
1
1
684 (C=O), 1653 (C=O), 1597 (C=C, aromatic), 1290 (S=O), 1153 (S=O); H NMR (400
MHz, CD OD) δ: 0.76 (3H, t, J = 7.4 Hz, CH -3′), 1.36 (2H, sextet, J = 7.2 Hz, CH -2′), 1.46
3
3
2
(
9H, s, C(CH ) ), 2.75 (2H, t, J = 6.7 Hz, CH -α), 2.79 (2H, t, J = 7.0 Hz, CH -1′), 3.48 (2H,
3 3 2 2
t, J = 6.7 Hz, CH -β), 7.62-7.73 (3H, m), 8.24 (1H, d, J = 6.4 Hz), 8.31 (1H, d, J = 8.4 Hz),
2
1
3
8
.65 (1H, d, J = 8.4 Hz); C NMR (100 MHz, CD OD) δ: 11.45 (CH , C-3′), 24.0 (CH , C-
3 3 2
2
′), 28.8 (3 × CH , C(CH ) ), 37.8 (CH , C-α), 38.1 (CH , C-β), 45.8 (CH , C-1′), 80.2 (quat.,
3
3 3
2
2
2
5

ACCEPTED MANUSCRIPT
C(CH ) ), 124.6 (CH), 125.4 (CH), 125.5 (CH), 128.5 (CH), 129.5 (CH), 130.3 (quat.), 130.4
3
3
(CH), 131.3 (quat.), 135.1 (quat.), 137.5 (quat.), 158.5 (C=O, carbamate CO), 173.6 (C=O,
amide CO); ESI-MS = 436 (M + H). Anal. Calcd. for C H N O S (435): C, 57.91; H, 6.71;
21
29
3
5
N, 9.65. Found: C, 57.99; H, 6.92; N, 9.72%.
2
.6.2 tert-Butyl [3-oxo-3-{(5-(N-butylsulfamoyl)naphthalen-1-
yl}aminopropyl]carbamate (11b)
-1
Yield of 11b: 51%; White solid; mp 175-178 °C; FTIR (neat, v/cm ): 3308 (NH), 3265
1
(
NH), 1680 (C=O), 1651 (C=O), 1620 (C=C, aromatic), 1290 (S=O), 1150 (S=O); H NMR
(
400 MHz, CD OD) δ: 0.74 (3H, t, J = 7.4 Hz, CH -4′), 1.18 (2H, sextet, J = 7.4 Hz, CH -3′),
3
3
2
1
.32 (2H, quintet, J = 7.2 Hz, CH -2′), 1.46 (9H, s, C(CH ) ), 2.745 (2H, t, J = 6.7 Hz, CH -
2 3 3 2
α), 2.82 (2H, t, J = 6.8 Hz, CH -1′), 3.48 (2H, t, J = 6.7 Hz, CH -β), 7.62-7.73 (3H, m), 8.24
2
2
1
3
(
1H, d, J = 6.8 Hz), 8.31 (1H, d, J = 8.4 Hz), 8.64 (1H, d, J = 8.4 Hz); C NMR (100 MHz,
CD OD) δ: 13.8 (CH , C-4′), 20.65 (CH , C-3′), 28.8 (3 × CH ), 32.7 (CH , C-2′), 37.8 (CH ,
3
3
2
3
2
2
C-α), 38.05 (CH , C-β), 43.6 (CH , C-1′), 80.2 (quat., C(CH ) ), 124.55 (CH), 125.4 (CH),
2
2
3 3
1
1
25.5 (CH), 128.5 (CH), 129.5 (CH), 130.3 (quat.), 130.4 (CH), 131.3 (quat.), 135.1 (quat.),
37.4 (quat.), 158.5 (C=O, carbamate CO), 173.6 (C=O, amide CO); ESI-MS = 450 (M + H).
Anal. Calcd. for C H N O S (449): C, 58.78; H, 6.95; N, 9.35. Found: C, 58.80; H, 7.13; N,
22
31
3
5
9
.40%.
2
.6.3 tert-Butyl [3-oxo-3-{(5-(N-pentylsulfamoyl)naphthalen-1-
yl}aminopropyl]carbamate (11c)
-1
Yield of 11c: 27%; White solid; mp 166-170 °C; FTIR (neat, v/cm ): 3320 (NH), 3269 (NH),
1
1
686 (C=O), 1653 (C=O), 1622 (C=C, aromatic), 1287 (S=O), 1153 (S=O); H NMR (400
MHz, CD OD) δ: 0.74 (3H, t, J = 6.8 Hz, CH ), 1.09-1.14 (4H, m, 2 × CH , 2 × H-3′ plus 2 ×
3
3
2
H-4′), 1.29-1.36 (2H, m, CH -2′), 1.46 (9H, s, C(CH ) ), 2.75 (2H, t, J = 6.8 Hz, CH -α), 2.82
2
3 3
2
(
2H, t, J = 7.0 Hz, CH -1′), 3.48 (2H, t, J = 6.6 Hz, CH -β), 7.62-7.73 (3H, m), 8.24 (1H, d, J
2
2
1
3
=
7.2 Hz), 8.31 (1H, d, J = 8.4 Hz), 8.64 (1H, d, J = 8.4 Hz); C NMR (100 MHz, CD OD)
3
δ: 14.2 (CH , C-5′), 23.1 (2 × CH , C-3′ and C-4′), 28.8 (3 × CH ), 30.3 (CH , C-2′), 37.8
3
2
3
2
(
(
(
(
CH , C-α), 38.1 (CH , C-β), 43.9 (CH , C-1′), 80.2 (quat., C(CH ) ), 124.6 (CH), 125.4
2 2 2 3 3
CH), 125.5 (CH), 128.5 (CH), 129.5 (CH), 130.3 (quat.), 130.4 (CH), 131.3 (quat.), 135.2
quat.), 137.5 (quat.), 158.5 (C=O, carbamate CO), 173.6 (C=O, amide CO); ESI-MS = 486
M + Na). HRMS Calcd. for C H N O SNa: 486.2033. Found: 486.2038.
23
33
3
5
6

ACCEPTED MANUSCRIPT
2
.6.4 tert-Butyl [3-oxo-3-{5-(N-octylsulfamoyl)naphthalen-1-yl}aminopropyl]carbamate
(11d)
-1
Yield of 11d: 37%; White solid; mp 175-177 °C; FTIR (neat, v/cm ): 3275 (NH), 1676
1
(
C=O), 1653 (C=O), 1595 (C=C, aromatic), 1321 (S=O), 1150 (S=O); H NMR (400 MHz,
CD OD) δ: 0.84 (3H, t, J = 7.2 Hz, CH ), 1.07-1.15 (8H, m, 4 × CH ), 1.20-1.32 (4H, m, 2 ×
3
3
2
CH ), 1.44 (9H, s, C(CH ) ), 2.72 (2H, t, J = 6.7 Hz, CH -α), 2.80 (2H, t, J = 7.0 Hz, CH -1′),
2
3 3
2
2
3
8
.46 (2H, t, J = 6.7 Hz, CH -β), 7.59-7.72 (3H, m), 8.22 (1H, d, J = 6.8 Hz), 8.29 (1H, d, J =
2
1
3
.4 Hz), 8.61 (1H, d, J = 8.4 Hz); C NMR (100 MHz, CD OD) δ: 14.4 (CH , C-8′), 23.7
3
3
(
CH ), 27.55 (3 × CH ), 28.8 (CH ), 30.1 (CH ), 30.2 (CH ), 30.6 (CH ), 32.9 (CH ), 37.8
2 3 2 2 2 2 2
(
CH , C-2′′), 36.6 (CH , C-3′′), 42.4 (CH , C-1′), 80.23 (quat., C(CH ) ), 124.5 (CH), 125.3
2 2 2 3 3
(
CH), 125.5 (CH), 128.5 (CH), 129.5 (CH), 130.3 (quat.), 130.4 (CH), 131.2 (quat.), 135.15
quat.), 137.5 (quat.), 158.5 (C=O, carbamate CO), 173.55 (C=O, amide CO); ESI-MS = 506
M + H); Anal. Calcd. for C H N O S (505): C, 61.76; H, 7.77; N, 8.31. Found: C, 61.76;
(
(
2
6
39
3
5
H, 8.00; N, 8.34%.
2
.6.5 tert-Butyl [3-{2-(1,3-dioxo-2-propyl-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)hydrazinyl}-3-oxopropyl]carbamate (13a)
-1
Yield of 13a: 21%; Light yellow solid; mp 193-195 °C; FTIR (neat, v/cm ): 3336 (NH),
3
325 (NH), 3279 (NH), 1684 (C=O), 1655 (amide I), 1647 (C=O), 1585 (amide II), 1528
1
(
C=C); H NMR (400 MHz, (CD ) CO) δ: 0.97 (3H, t, J = 7.6 Hz, H-3′), 1.45 (9H, s,
3
2
C(CH ) ), 1.72 (2H, sextet, J = 7.6 Hz, H-2′), 2.66 (2H, t, J = 6.8 Hz, CH -α), 3.44 (2H, q, J
3
3
2
=
=
6.8 Hz, CH -β), 4.04-4.08 (2H, m, H-1′), 6.08 (1H, broad s, carbamate NH), 7.11 (1H, d, J
2
8.4 Hz, H-5), 7.60 (1H, dd, J = 8.4, 7.6 Hz, H-8), 8.34 (1H, d, J = 8.4 Hz, H-4), 8.43 (1H,
dd, J = 7.6, 1.2 Hz, H-9), 8.54 (1H, dd, J = 8.4, 1.2 Hz, H-7), 9.16 (1H, broad s, amide NH);
1
3
C NMR (100 MHz, (CD ) CO) δ: 12.8 (CH , C-3′), 23.1 (CH , C-2′), 29.7 (3 × CH ,
3
2
3
2
3
C(CH ) ), 35.9 (CH , CH -α), 38.7 (CH , CH -β), 43.0 (CH , C-1′), 79.8 (quat., C(CH ) ),
3
3
2
2
2
2
2
3 3
107.6 (CH, C-5), 114.6 (quat., C-3a), 121.5 (quat., C-6a), 124.7 (quat., C-9a), 127.0 (CH, C-
8), 129.0 (CH, C-7), 131.1 (quat., C-3b), 132.3 (CH, C-9), 134.9 (CH, C-4), 152.1 (quat., C-
6), 157.6 (C=O, carbamate CO), 165.1 (C=O, C-3), 165.6 (C=O, C-1), 172.9 (C=O, amide
CO); ESI-MS = 442 (M + H).
2
.6.6 tert-Butyl [3-{2-(1,3-dioxo-2-pentyl-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)hydrazinyl}-3-oxopropyl]carbamate (13b)
7

ACCEPTED MANUSCRIPT
-1
Yield of 13b: 19%; Yellow-brown solid; mp 150-154 °C; FTIR (neat, v/cm ): 3357 (NH),
3
312 (NH), 3221 (NH), 1688 (C=O), 1674 (amide I), 1639 (C=O), 1614 (amide II), 1587
1
(
C=C), 1519 (C=C); H NMR (400 MHz, (CD ) CO) δ: 0.93 (3H, t, J = 7.2 Hz, H-5′), 1.38-
3
2
1
.41 (4H, m, H-3′, H-4′), 1.46 (9H, s, C(CH ) ), 1.72 (2H, quintet, J = 7.6 Hz, H-2′), 2.70
3 3
(
2H, t, J = 6.4 Hz, CH -α), 3.47 (2H, q, J = 6.4 Hz, CH -β), 4.07 (2H, t, J = 7.6 Hz, H-1′),
2
2
6
.13 (1H, broad s, carbamate NH), 7.06 (1H, d, J = 8.0 Hz, H-5), 7.39 (1H, dd, J = 8.4, 7.6
Hz, H-8), 8.26-8.29 (2H, m, H-4, H-9), 8.38 (1H, dd, J = 8.4, 0.8 Hz, H-7), 9.08 (1H, broad s,
13
NH), 9.49 (1H, broad s, NH); C NMR (100 MHz, (CD ) CO) δ: 15.3 (CH , C-5′), 24.1
3
2
3
(
CH , C-4′), 29.5 (CH , C-2′), 29.7 (3 × CH , C(CH ) ), 31.1 (CH , C-3′), 36.0 (CH , CH -α),
2 2 3 3 3 2 2 2
3
8.7 (CH , CH -β), 41.4 (CH , C-1′), 80.0 (quat., C(CH ) ), 107.3 (CH, C-5), 114.6 (quat., C-
2 2 2 3 3
3
a), 121.2 (quat., C-6a), 124.4 (quat., C-9a), 126.8 (CH, C-8), 128.8 (CH, C-7), 130.8 (quat.,
C-3b), 131.9 (CH, C-9), 134.8 (CH, C-4), 151.9 (quat., C-6), 157.7 (C=O, carbamate CO),
65.0 (C=O, C-3), 165.5 (C=O, C-1), 173.4 (C=O, amide CO); ESI-MS = 469 (M + H).
1
2
.6.7 tert-Butyl [3-{2-(1,3-dioxo-2-benzyl-2,3-dihydro-1H-benzo[de]isoquinolin-6-
yl)hydrazinyl}-3-oxopropyl]carbamate (13c)
-1
Yield of 13c: 34%; Yellow solid; mp 175-177 °C; FTIR (neat, v/cm ): 3387 (NH), 3318
(NH), 3298 (NH), 1684 (C=O), 1653 (amide I), 1647 (C=O), 1638 (amide II), 1578 (C=C),
1
1
541 (C=C), 1521 (C=C), 1508 (C=C); H NMR (400 MHz, (CD ) CO) δ: 1.45 (9H, s,
3
2
C(CH ) ), 2.67 (2H, t, J = 6.8 Hz, CH -α), 3.44 (2H, t, J = 6.8 Hz, CH -β), 4.56 (1H, s,
3
3
2
2
carbamate NH), 5.32 (2H, s, CH ), 7.11 (1H, d, J = 8.4 Hz, H-5), 7.19-7.23 (1H, m, H-4′),
2
7
.27-7.31 (2H, m, H-3′, 5′), 7.44-7.47 (2H, m, H-2′, 6′), 7.58 (1H, dd, J = 8.4, 7.2 Hz, H-8),
.35 (1H, d, J = 8.4 Hz, H-4), 8.43 (1H, dd, J = 7.2, 0.8 Hz, H-9), 8.54 (1H, dd, J = 8.4, 0.8
8
1
3
Hz, H-7), 8.89 (1H, broad s, amide NH), 9.54 (1H, broad s, amide NH); C NMR (100 MHz,
(
CD ) CO) δ: 29.7 (3 × CH , C(CH ) ), 35.9 (CH , CH -α), 38.5 (CH , CH -β), 44.7 (CH ),
3 2 3 3 3 2 2 2 2 2
7
9.9 (quat., C(CH ) ), 107.7 (CH, C-5), 114.4 (quat., C-3a), 121.4 (quat., C-6a), 124.5 (quat.,
3 3
C-9a), 127.0 (CH, C-8), 128.8 (CH, C-4′), 129.3 (CH, C-7), 130.01 (2 × CH, C-2′, 6′), 130.04
2 × CH, C-3′, 5′), 131.1 (quat., C-3b), 132.6 (CH, C-9), 135.2 (CH, C-4), 140.2 (quat., C-1′),
(
1
5
52.3 (quat., C-6), 165.1 (C=O, C-3), 165.7 (C=O, C-1), 174.1 (C=O, amide CO); ESI-MS =
11 (M + Na).
2
.7
General procedure for the removal of Boc protecting group to give salts
1
2a-e and 14a-c
8

ACCEPTED MANUSCRIPT
Boc-protected substrates 11a-e and 13a-c were dissolved in DCM (5 mL) and the solution
cooled to 0 °C in an ice bath. DCM / TFA (5 drops of 1:1 mixture) was added to this solution
which was allowed to stir for 2 hours while warming to room temperature. The solvent and
excess TFA were removed in vacuo after the addition of methanol (50 mL). The residue was
then triturated with diethyl ether and the solid formed was filtered to give the products 12a-e,
or the reaction vessel was cooled at -80 °C overnight to give the final substrate 14a-c as a
TFA salt.
2
.7.1 3-Amino-N-{5-(N′-propylsulfamoyl)naphthalen-1-yl}propanamide
trifluoroacetate salt (12a)
-1
Yield of 12a: 87%; Off-white solid; mp 155-158 °C; FTIR (neat, v/cm ): 3265 (NH), 3163
1
(
NH), 1668 (C=O, amide), 1597 (C=C, aromatic), 1305 (S=O), 1134 (S=O); H NMR (400
MHz, D O) δ: 0.68 (3H, t, J = 7.4 Hz, CH ), 1.32 (2H, sextet, J = 7.2 Hz, CH -2′), 2.86 (2H,
2
3
2
t, J = 6.8 Hz, CH -1′), 3.09 (2H, t, J = 6.6 Hz, CH -α), 3.41 (2H, t, J = 6.0 Hz, CH -β), 7.65-
2
2
2
13
7
.71 (2H, m), 7.78 (1H, t, J = 8.2 Hz), 8.26-8.30 (2H, m), 8.58 (1H, d, J = 8.8 Hz); C NMR
(
(
100 MHz, D O) δ: 10.2 (CH -3′), 22.0 (CH , C-2′), 31.9 (CH , C-α), 35.4 (CH , C-β), 44.45
2
3
2
2
2
CH , C-1′), 123.4 (CH), 125.0 (CH), 125.4 (CH), 128.1 (CH), 128.2 (quat.), 128.8 (CH),
2
1
3
29.9 (CH), 130.0 (quat.), 132.5 (quat.), 133.8 (quat.), 172.3 (C=O, amide CO); ESI-MS =
36 (M + H); HRMS Calcd. for C H N O S: 336.1376. Found: 336.1379.
16
22
3
3
2
.7.2 3-Amino-N-{5-(N′-butylsulfamoyl)naphthalen-1-yl}propanamide trifluoroacetate salt
(12b)
-1
Yield of 12b: 53%; Off-white solid; mp 152-155 °C; FTIR (neat, v/cm ): 3267 (NH), 1670
1
(
C=O, amide), 1597 (C=C, aromatic), 1319 (S=O), 1134 (S=O); H NMR (400 MHz, D O) δ:
2
0
.60 (3H, t, J = 7.4 Hz, CH ), 1.06 (2H, sextet, J = 7.4 Hz, CH -3′), 1.235 (2H, quintet, J =
3 2
7
.2 Hz, CH -2′), 2.88 (2H, t, J = 6.8 Hz, CH -1′), 3.08 (2H, t, J = 6.5 Hz, CH -α), 3.40 (2H, t,
2
2
2
J = 6.5 Hz, CH -β), 7.63-7.68 (2H, m), 7.76 (1H, t, J = 8.2 Hz), 8.24-8.27 (2H, m), 8.56 (1H,
2
13
d, J = 8.8 Hz); C NMR (100MHz, D O) δ: 12.4 (CH ), 18.8 (CH , C-3′), 30.4 (CH , C-2′),
2
3
2
2
3
1.9 (CH , C-α), 35.4 (CH , C-β), 42.2 (CH , C-1′), 123.3 (CH), 125.0 (CH), 125.3 (CH),
2 2 2
1
28.1 (CH), 128.2 (quat.), 128.8 (CH), 130.0 (CH and quat.), 132.5 (quat.), 133.7 (quat.),
1
72.3 (C=O, amide CO); ESI-MS = 350 (M + H); HRMS Calcd. for C H N O S: 350.1533.
1
7
24
3
3
Found: 350.1536.
2
.7.3 3-Amino-N-{5-(N′-pentylsulfamoyl)naphthalen-1-yl}propanamide
trifluoroacetate salt (12c)
9

ACCEPTED MANUSCRIPT
-1
Yield of 12c: 95%; White solid; mp 141-144 °C; FTIR (neat, v/cm ): 3294 (NH), 3265 (NH),
1
1
670 (C=O, amide), 1624 (C=C, aromatic), 1290 (S=O), 1125 (S=O); H NMR (400 MHz,
D O) δ: 0.53 (3H, t, J = 6.4 Hz, CH ), 0.84-0.97 (4H, m, 2 × CH , CH -3′,4′), 1.19 (2H, t, J =
2
3
2
2
6
.6 Hz, CH -2′), 2.86 (2H, t, J = 6.6 Hz, CH -1′), 3.07 (2H, t, J = 6.6 Hz, CH -α), 3.40 (2H, t,
2 2 2
J = 6.4 Hz, CH -β), 7.60-7.64 (2H, m), 7.74 (1H, t, J = 7.6 Hz), 8.20-8.245 (2H, m), 8.54
2
1
3
(
1H, d, J = 8.8 Hz); C NMR (100 MHz, D O) δ: 12.8 (CH ), 21.05 (CH , C-4′), 27.6 (CH ,
2 3 2 2
C-3′), 27.85 (CH , C-2′), 32.0 (CH , C-α), 35.4 (CH , C-β), 42.4 (CH , C-1′), 123.3 (CH),
2
2
2
2
1
1
24.95 (CH), 125.2 (CH), 128.0 (CH), 128.15 (quat.), 128.7 (CH), 129.8 (quat.), 129.9 (CH),
32.5 (quat.), 133.8 (quat.), 172.2 (C=O, amide CO); ESI-MS = 364 (M + H); HRMS Calcd.
for C H N O S: 364.1689. Found: 364.1692.
18
26
3
3
2
.7.4 3-Amino-N-{5-(N′-octylsulfamoyl)naphthalen-1-yl}propanamide trifluoroacetate salt
(12d)
-1
Yield of 12d: 58%; Off-white solid; 175-177 °C; FTIR (neat, v/cm ): 3250 (NH), 1668
1
(
C=O, amide), 1599 (C=C, aromatic), 1314 (S=O), 1132 (S=O); H NMR (400 MHz, D O) δ:
2
0
.67 (3H, t, J = 7.2 Hz, CH ), 0.80-1.11 (10H, m, 5 × CH ), 1.20 (2H, br s, CH -2′), 2.70 (2H,
3 2 2
br s, CH -1′), 2.91 (2H, t, J = 6.4 Hz, CH -β), 3.24 (2H, t, J = 6.4 Hz, CH -α), 7.33-7.44 (3H,
2
2
2
1
3
m), 7.94 (1H, d, J = 6.8 Hz), 8.09 (1H, d, J = 8.4 Hz), 8.40 (1H, d, J = 8.0 Hz); C NMR
(
100 MHz, D O) δ: 13.5 (CH ), 22.2 (CH ), 26.0 (CH ), 28.5 (CH ), 28.7 (CH ), 29.1 (CH -
2 3 2 2 2 2 2
2
1
′), 31.3 (CH ), 32.0 (CH -β), 35.3 (CH -α), 42.6 (CH -1′), 123.0 (CH), 124.5 (2 × CH),
2 2 2 2
27.75 (CH), 128.1 (quat.), 128.5 (CH), 129.0 (CH), 129.45 (quat.), 132.5 (quat.), 134.4
(
quat.), 171.6 (C=O, amide CO); ESI-MS = 406 (M + H); HRMS Calcd. for C H N O S:
21 32 3 3
4
06.2159. Found: 406.2161.
2
.7.5 3-Amino-N-{5-(N′-benzylsulfamoyl)naphthalen-1-yl}propanamide
trifluoroacetate salt (12e)
The reaction of 5-amino-N-propylnaphthalene-1-sulfonamide 7e (0.55 g, 1.76 mmol) with
Boc-β-alanine (2.665 g, 14.08 mmol), DCCI (1.451 g, 7.04 mmol), EDIPA (0.273 g, 2.11
mmol) and DMAP (0.129 g, 1.06 mmol), as described above, gave a white solid 11e (1.038
g) which was used without further purification.
Carbamate 11e (0.9g) was deprotected using the general method to give a beige solid which
was taken up in water, then the pH was adjusted to 14 with 1M aq. NaOH (aq). The aqueous
layer was then extracted with ethyl acetate (5 × 30 mL), and the combined organic layers
were dried over Na SO and reduced in vacuo to give the free amine of amide 12e (0.49g) as
2
4
1
0

ACCEPTED MANUSCRIPT
a white solid. This free base was then converted into the trifluoroacetate salt with TFA (0.15
mL) in DCM (10 mL) at 0°C. The white solid product 11e was obtained after trituration with
-1
diethyl ether (0.438g, 50% over 2 steps); mp 100-102 °C; FTIR (neat, v/cm ): 3273 (NH),
1
1
655 (C=O, amide), 1599 (C=C, aromatic), 1311 (S=O), 1138 (S=O); H NMR (400 MHz,
D O) δ: 3.06 (2H, t, J = 6.6 Hz, CH -α), 3.395 (2H, t, J = 6.6 Hz, CH -β), 4.08 (2H, s, NCH ),
2
2
2
2
6
.83-6.98 (5H, m, CH ), 7.49-7.58 (2H, m), 7.67 (1H, t, J = 8.0 Hz), 8.06-8.12 (2H, m), 8.41
Ph
13
(
1H, d, J = 8.8 Hz); C NMR (100 MHz, D O) δ: 31.9 (CH , CH -α), 35.4 (CH , CH -β),
2 2 2 2 2
4
6.2 (NCH ), 123.1 (CH), 124.9 (CH), 125.0 (CH), 127.2 (C-4′), 127.4 (C-2′ and C-6′), 127.8
2
(
C-3′ and C-5′), 127.9 (CH), 128.0 (quat.), 128.7 (CH), 129.6 (quat.), 130.2 (CH), 132.3
quat.), 133.8 (quat.), 135.5 (C-1′), 172.1 (C=O, amide CO); ESI-MS (ESI) = 384 (M + H);
(
HRMS Calcd. for C H N O S: 384.1376. Found: 384.1379.
20
22
3
3
2
.7.6 3-Amino-N′-(2-propyl-1,3-dioxo-2,3-dihydro-1H-benz[de]isoquinolin-6-
yl)propanehydrazide trifluoroacetate (14a)
-1
Yield of 14a: 89%; Light orange crystals; mp 134-136 °C; FTIR (nujol, v/cm ): 3482 (NH),
3
252 (NH), 1687 (C=O), 1672 (amide I), 1646 (C=O), 1614 (amide II), 1582 (C=C), 1533
1
(
C=C); H NMR (400 MHz, CD OD) δ: 0.99 (3H, t, J = 7.6 Hz, C-3′), 1.73 (2H, sextet, J =
3
7
.6 Hz, C-2′), 2.86 (2H, t, J = 6.8 Hz, CH -α), 3.31-3.34 (2H, m, CH -β), 4.07-4.11 (2H, m,
2 2
H-1′), 7.03 (1H, d, J = 8.4 Hz, H-5), 7.70-7.74 (1H, m, H-8), 8.40 (1H, d, J = 8.4 Hz, H-4),
1
3
8
1
1
8
6
.51 (1H, d, J = 8.8 Hz, H-9), 8.55 (1H, d, J = 7.6 Hz, H-7); C NMR (100 MHz, CD OD) δ:
3
1.8 (CH , C-3′), 22.5 (CH , C-2′), 31.3 (CH , CH -α), 36.8 (CH , CH -β), 42.7 (CH , C-1′),
3
2
2
2
2
2
2
06.5 (CH, C-5), 113.4 (quat., C-3a), 120.9 (quat., C-6a), 123.7 (quat., C-9a), 126.4 (CH, C-
), 129.1 (CH, C-7), 130.7 (quat., C-3b), 132.4 (CH, C-9), 135.0 (CH, C-4), 151.7 (quat., C-
), 165.7 (C=O, C-3), 166.0 (C=O, C-1), 172.3 (C=O); ESI-MS = 341 (M + H); HRMS
Calcd. for C H N O : 341.1608. Found: 341.1605.
18
21
4
3
2
.7.7 3-Amino-N′-(2-pentyl-1,3-dioxo-2,3-dihydro-1H-benz[de]isoquinolin-6-
yl)propanehydrazide trifluoroacetate (14b)
-1
Yield of 14b: 94%; Bright yellow solid; mp 122-127 °C; FTIR (nujol, v/cm ): 3392 (NH),
3
336 (NH), 3197 (NH), 1707 (C=O), 1674 (amide I), 1638 (C=O), 1615 (amide II), 1585
1
(
(
(
C=C), 1532 (C=C); H NMR (400 MHz, CD OD) δ: 0.92 (3H, t, J = 6.8 Hz, H-5′), 1.35-1.40
3
4H, m, H-3′, H-4′), 1.68 (2H, quintet, J = 7.6 Hz, H-2′), 2.85 (2H, t, J = 6.4 Hz, CH -α), 3.29
2
2H, m, CH -β), 4.08 (2H, t, J = 7.6 Hz, H-1′), 7.00 (1H, d, J = 8.4 Hz, H-5), 7.66 (1H, dd, J
2
=
8.4, 7.2 Hz, H-8), 8.36 (1H, d, J = 8.4 Hz, H-4), 8.46 (1H, dd, J = 8.4, 0.8 Hz, H-9), 8.49
1
1

ACCEPTED MANUSCRIPT
1
3
(
1H, dd, J = 7.2, 0.8 Hz, H-7); C NMR (100 MHz, CD OD) δ: 14.4 (CH , C-5′), 23.5 (CH ,
3 3 2
C-4′), 28.9 (CH , C-2′), 30.4 (CH , C-3′), 31.2 (CH , CH -α), 36.8 (CH , CH -β), 41.2 (CH ,
2
2
2
2
2
2
2
C-1′), 106.5 (CH, C-5), 113.4 (quat., C-3a), 120.9 (quat., C-6a), 123.7 (quat., C-9a), 126.4
(
CH, C-8), 129.1 (CH, C-7), 130.7 (quat., C-3b), 132.4 (CH, C-9), 134.9 (CH, C-4), 151.7
quat., C-6), 165.6 (C=O, C-3), 166.0 (C=O, C-1), 172.3 (C=O); ESI-MS = 369 (M + H);
(
HRMS Calcd. for C H N O : 369.1921. Found: 369.1918.
20
25
4
3
2
.7.8 3-Amino-N′-(2-benzyl-1,3-dioxo-2,3-dihydro-1H-benz[de]isoquinolin-6-
yl)propanehydrazide trifluoroacetate (14c)
-1
Yield of 14c: 96%; Orange solid; mp 131-133 °C; FTIR (nujol, v/cm ): 3462 (NH), 3219
1
(
NH), 1701 (C=O), 1684 (amide I), 1640 (C=O), 1614 (amide II), 1577 (C=C); H NMR (400
MHz, CD OD) δ: 2.86 (2H, t, J = 6.8 Hz, CH -α), 3.30-3.35 (2H, m, CH -β), 5.32 (2H, s,
3
2
2
CH ), 7.03 (1H, d, J = 8.4 Hz, H-5), 7.19-7.23 (1H, m, H-4′), 7.26-7.30 (2H, m, H-3′, 5′),
2
7
.40-7.42 (2H, m, H-2′, 6′), 7.72 (1H, dd, J = 8.4, 7.6 Hz, H-8), 8.41 (1H, d, J = 8.4 Hz, H-4),
1
3
8
.52 (1H, dd, J = 8.4, 0.8 Hz, H-9), 8.56 (1H, dd, J = 7.6, 0.8 Hz, H-7); C NMR (100 MHz,
(CD OD) δ: 31.2 (CH , CH -α), 36.8 (CH , CH -β), 44.3 (CH ), 106.6 (CH, C-5), 113.2
3 2 2 2 2 2
(
quat., C-3a), 120.9 (quat., C-6a), 123.6 (quat., C-9a), 126.5 (CH, C-8), 128.3 (CH, C-4′),
29.2 (2 × CH, C-2′, 6′), 129.3 (CH, C-7), 129.4 (2 × CH, C-3′, 5′), 130.8 (quat., C-3b), 132.6
1
(CH, C-9), 135.2 (CH, C-4), 139.1 (quat., C-1′), 151.8 (quat., C-6), 165.6 (C=O, C-3), 166.0
(
C=O, C-1), 172.3 (C=O); ESI-MS = 389 (M + H); HRMS Calcd. for C H N O : 389.1608.
22
21
4
3
Found: 389.1606.
2
.8
General method for the coupling of hydrazines 10 to aryl aldehydes
The hydrazines 10a-c (1 equiv.) and arylaldehydes (1.1 equiv.) were dissolved in ethanol (25
mL) and stirred at 80 °C for 2 hours with five drops of glacial acetic acid as a catalyst. The
solution was then allowed to cool and the formed precipitate was taken to obtain final
compounds 17-21.
2
.9
Fluorescence properties
-
4
-4
-5
In vitro fluorescent assessment of fluorophores (7a-e: 5 × 10 M; 10a-c: 1 × 10 M),
-4
-4
substrates (12a-e: 5 × 10 M; 14a-c: 1 × 10 M), and hydrazones 17-21 (17,18: 1 × 10 M;
-4
1
9-21: 1 × 10 M) in ethanol was carried out on a Shimadzu RF-5301PC spectrophotometer
using identical scan parameters, utilising a 5 mm slit width, if not stated otherwise.
2
.10 Microbiological testing
1
2

ACCEPTED MANUSCRIPT
2
.10.1 Preparation of culture media containing substrates 12a-e and 14a-c
Columbia agar was prepared as follows; 41 g of Columbia agar (Oxoid Basingstoke, UK)
was added to deionised water and the volume was made up to 1 L. The medium was sterilised
by autoclaving at 116 °C for 20 minutes and left to cool at 50 °C. 2 mg of each substrate 12a-
e and 14a-c 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 incorporating an
equivalent concentration of N-methylpyrrolidone was used as a growth control.
2
.10.2 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.
2
.10.3 Multipoint inoculation
Colonies of each microbial strain were harvested using a loop from overnight cultures on
Columbia agar. These were suspended in sterile deionised 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
5
bacterial suspension, giving 1.5 × 10 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.
2
.10.4 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.
3
Results and discussion
3
.1
Preparation of 5-amino-1-naphthalenesulfonamides (ANS)
Starting from 5-amino-1-naphthalenesulfonic acid 3, the amino group was protected via N-
acetylation as described by Xu et al.[18] to give imide 4 in excellent yield, Scheme 1. In
order to synthesise the fluorophores 7a-e, the sulfonic acid group was converted to a sulfonyl
chloride 5 [19] which was subsequently reacted with a series of amines, to form the
1
3

ACCEPTED MANUSCRIPT
acetamides 6a-e in moderate yields via a nucleophilic substitution. Removal of the N-acetyl
group of the acetamides 6a-e gave the sulfonamide fluorophores 7a-e.
3
.2
Preparation of 6-hydrazinobenz[de]isoquinoline-1,3-diones (6-HBI) 10a-c
Synthesis of the desired 6-hydrazinobenz[de]isoquinoline-1,3-diones (6-HBI) 10a-c was
achieved in two steps; a range of 6-bromobenz[de]isoquinoline-1,3-diones 9a-c was
prepared, in high yields, Scheme 2, from 4-bromo-1,8-naphthalic anhydride 8 by using an
adaptation of the method of Wang et al..[20] The subsequent substitution of the bromine was
achieved by reaction with hydrazine hydrate in ethanol, under reflux, to give the fluorophores
1
0a-c,[14] Scheme 3. This step was optimised via the addition of hydrazine in portions over a
period of time, resulting in an approximately 20 % increase in reaction yield, and the
products were then isolated by recrystallization of the crude residues from 2-butanol.
3
.3
Amide 12 and hydrazide 14 formation
Each of the fluorophores 7a-e and 10a-c were initially coupled to Boc-β-Ala-OH using the
activated pentafluorophenyl ester, as described by Nakatani et al..[21] However, the
presumed instability of the ester resulted in low yields of the desired products 11a-e and 13a-
c. Alternatively, the coupling was carried out by the formation of the symmetrical anhydride
of Boc-β-Ala-OH (using DCCI as a dehydrating agent), Scheme 3, which was then added to a
mixture of 7a-e or 10a-c and DMAP, EDIPA in DCM.[22] The subsequent deprotection of
1
1a-e and 13a-c in TFA / DCM (1:6 v/v) gave the trifluoroacetate salts 12a-e and 14a-c, in
moderate to good yields, for microbiological evaluation.
3
.4
Secondary reaction of 6-hydrazinobenz[de]isoquinoline-1,3-diones (6-HBI) 10a-c
with aldehydes
A range of aryl aldehydes 15 and 16 and the appropriate 6-hydrazinobenz[de]isoquinoline-
1
,3-dione 10a-c were stirred under reflux in ethanol for two hours, in the presence of catalytic
amounts of glacial acetic acid, Scheme 4. The isolation of products 17-21, as a range of
orange to dark purple solids, was achieved in good yields by the filtration of the precipitate
which formed upon cooling.
3
.5
Fluorescence properties
As discussed earlier, the fluorescence properties of a substrate compared to those of a
fluorophore are sufficient for their application in bacterial diagnostics when a significant
change in fluorescence intensity or a significant shift in the emission wavelength is observed.
1
4

ACCEPTED MANUSCRIPT
In order to fully assess the suitability of our core molecules, in vitro fluorescent assessment of
fluorophores 7a-e, 10a-c, substrates 12a-e, 14a-c, and hydrazones 17-21 was carried out in
ethanol.
3
.5.1 Fluorescence properties of ANS derivatives
The ANS substrates 12a-e displayed excitation maxima of 339 nm across the five compounds
while the emission maxima ranged from 421-426 nm, resulting in Stokes shifts of 82-87 nm,
Table 1. Changes in the N-substituent had little or no effect on the fluorescence properties, as
shown by the identical excitation and narrow range of emission maxima. The ANS
fluorophores 7a-e, however, exhibited excitation and emission maxima of 391-408 nm and
5
14-519 nm, respectively, with Stokes shifts of 111-125 nm as shown in Table 1. Similarly to
the substrates, the alkyl chain length of the N-substituents had a negligible effect on the
observed fluorescence properties, while the benzyl substituent had only a slight effect. When
comparing the emission maxima between the substrates 12 and fluorophores 7, e.g. Figure 3,
an average bathochromic shift of 93 nm was observed, suggesting that these would be ideal
fluorogenic substrates for the detection of bacteria.
3
.5.2 Fluorescent properties of 6-HBI derivatives
Evaluation of the fluorescence properties of all synthesized hydrazines 10a-c, Boc-protected
hydrazides 13a-c, intended enzyme substrates 14a-c and hydrazones 17-21, was performed at
-4
a concentration of 1 × 10 M in ethanol. The hydrazones 17a-c and 18a-c displayed
significantly enhanced fluorescence, surpassing the detection limit of the spectrophotometer,
-5
so that the fluorescence of these compounds was studied at a concentration of 1 × 10 M.
Similarly to the ANS derivatives, differences in the N-substituents on the 6-HBI core resulted
in relatively little effect on the fluorescence properties observed (Figures 4 and 5, Table 2)
with the exception of 10c, where a markedly increased emission intensity was observed
compared to derivatives 10a-b. Interestingly, this increase in fluorescence disappeared once
1
0c was coupled to Boc-β-alanine (13,14) or when reacted with an aldehyde to form
hydrazones 17-21. This decrease in intensity is presumably due, in part, to the engagement of
the lone pair of the 4-hydrazinyl moiety in further resonance structures of 13-14 and 17-21.
The 6-HBI substrates 14a-c exhibited excitation maxima of 442-444 nm, with corresponding
emission maxima of 509-511 nm, with a Stokes shift of 67 nm. In comparision, the
fluorophores 10a-c displayed excitation maxima of 465-476 nm and emission maxima of
5
34-536 nm. This would correspond to a slight bathochromic shift (23-27 nm) if the
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substrates 14a-c were enzymatically cleaved to give the fluorophores 10a-c. Unfortunately,
such a small shift would be likely produce little or no effect on the observed colour of the
fluorescence and a secondary reaction wold need to be utilized to further enhance the
differentiation between the fluorophore and the substrate.
Reaction of the fluorophores 10a-c with aryl aldehydes 15 and 16 resulted in the formation of
hydrazones 17-21 with greatly increased fluorescence intensities (2-fold to > 10-fold) in most
cases (except for 20a-c), Figure 5 and Table 3, and a bathochromic shift (compounds 19-21)
of the maximum emission wavelength. The extent of this bathochromic effect on the emission
maximum can be related to the electron withdrawing / donating characteristics of the aryl
aldehydes used (4-N(CH3)2 > 4-OCH3 > benzaldehyde > 4-Cl). Electron-donating
substituents 19,20 displayed greater bathochromic effects (∆ 23-52 nm), while electron-
withdrawing substituents 17,16 had insignificant effects on the emission wavelengths. Further
extension of the conjugation, with an additional double bond, as seen in compounds 21a-c,
resulted in a similar bathochromic effect to that of compounds 19a-c, containing a weakly
electron-donating group.
Hydrazones 20a-c, containing a dimethylamino group, produced the greatest recorded
bacthochromic shift, but were distinguished by relatively weak fluorescence at the studied
-4
concentrations (10 M), presumably due to the facilitated rotational decay to the ground state
offered by the dimethylamino group disrupting the electronic properties, and resulting in
quenching of the fluorescence.[23] The greatest intensification of fluorescence emission was
observed for the benzaldehyde (17a-c) and 4-chlorobenzaldehyde (18a-c) derived
hydrazones.
3
.6
Microbiological testing of β-alanylaminonaphthalenesulfonamides (ANS) 12 and
-(β-alanyl)hydrazinobenz[de]isoquinoline-1,3-diones 14
6
The substrates 12a-e and 14a-c were incorporated into a commercially available Columbia
medium and subjected to microbiological evaluation against a range of clinically relevant
microorganisms. All of the substrates were tested on ten Gram negative and eight Gram
positive bacteria and two yeasts.
When observed under long-wave UV light, the colonies of strains hydrolysing the substrates
should display a significant change in fluorescence in comparison to those which do not
hydrolyse the substrate and the dark background of the medium, Figures 7 and 8. However,
none of the visualized plates exhibited the fluorescence associated with the expected released
1
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of fluorophores 7a-e, Figure 8. Upon further investigation, it was discovered that the medium
used for bacterial growth quenches the fluorescence of the released fluorophores 7a-e, Figure
6
.
Furthermore, while substrates 12a-c and 12e did not inhibit bacterial growth, in the presence
of substrate 12d only Providencia rettgeri, Serratia marcescens and Bukholderia cepacia
were able to grow. The greater toxicity of 12d compared to the other substrates is presumably
due to its longer octyl chain disrupting the integrity of the bacterial cytoplasmic membrane
and this is supported by the fact that all three of the surviving microorganisms are known to
be resistant to antibacterial agents which target the cell membrane.[24]
Incorporation of substrates 14a-c into Columbia agar produced a yellow agar with a slightly
yellow fluorescent background, Figures 7 and 8. Uptake of the substrates 14a-c into the
bacterial cell greatly increased and localized the fluorescence, which was shown to be
suitable for the detection of all bacteria, but it was impossible to differentiate between β-
alanyl aminopeptidase producing (spots 5, 7, and 9, Table 4) and non-β-alanyl
aminopeptidase producing bacteria. Incorporation of an aryl aldehyde, as previously
discussed, would thus be necessary to shift the emission wavelengths to one which is distinct
from that of the substrates 10a-c.
Once again, the substrates 10b and 10c containing longer chain lengths inhibited bacterial
growth, presumably again due to the disruption of the cell wall. Gram positive and yeast
species appeared to be affected more than Gram negative species.
Preliminary data for the inclusion of 4-chlorobenzaldehyde (4-CBA) into growth media
showed little to no effect on the observed fluorescence of substrate 14a, Figure 9. This may
be due to a number of factors, such as the lack of enzymatic cleavage of the hydrazide bond
to release the fluorophore, lack of reaction between aldehyde and the released hydrazide,
failure of the 4-CBA to be taken up into the cell, a reduction in fluorescence emission due to
a suboptimal wavelength of 365 nm being used to excite the compounds (λ of 465 nm and
ex
4
67 nm for fluorophore and hydrazone respectively), or a cleavage of the formed hydrazone
bond by enzymes expressed by P. aeruginosa, similar to results seen by Taniyama et al..[25]
4
Conclusions
A series of novel ANS and 6-HBI fluorophores 7a-e and 10a-c were synthesized and were
then coupled to β-alanine to form substrates 12a-e and 14a-c for β-alanyl aminopeptidase.
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Substrates 12d, 14b and 14c, with longer N-substituent chains, were associated with growth
inhibition of all Gram-positive and a majority of the Gram-negative organisms.
Although preliminary fluorescence data suggested that the ANS fluorophores 9a-e would
display favourable fluorescence characteristics, with a bathochromic shift of ~93 nm from the
substrates 12a-e, as a result of quenching by the medium, Columbia agar incorporating these
susbstrates did not appear to selectively differentiate between β-alanyl aminopeptidase
producing and non-β-alanyl aminopeptidase producing bacteria. The use of other media or
detection formats may, however, alleviate this issue and thus allow for their use in bacterial
detection.
The increased fluorescence intensity upon localization of substrate 14a into all the Gram
negative and 7/8 Gram positive bacteria (with the exception of S. pyogenes) does, however
suggest a use for this substrate when the detection of any bacterial presence is required. For
example, as one of the leading causes of sepsis is bacteraemia, a rapid method for the
detection of viable bacteria in blood would enable the timely administration of antibacterial
agents. This would help to reduce the prevalence of mortality associated with sepsis (10-20 %
fatality rate), its progression to severe sepsis (20-50 % fatality rate), and, ultimately, septic
shock (40-80 % fatality rate). In the US in the period 1999-2008, the leading bacterial causes
of severe sepsis were Gram negative (51.5 %) organisms,[26] with the most common being
E. coli (20 %), and Pseudomonas species (9 %). Gram-positive organisms were responsible
for 45.6 % of severe sepsis cases, with methicillin-sensitive S. aureus (28 %) being the most
common. Of the cases in which E. coli was present, 16 % resulted in mortality, while
methicillin-sensitive S. aureus (MSSA) bacteraemia had a mortality rate of 31 %.[27]
The fluorophore 10a-c was also reacted with a series of para-substituted benzaldehydes and
cinnamaldehyde to alter the fluorescence of the fluorophore. Addition of electron-donating
para-substituted benzaldehydes appeared to form hydrazones 20a-c with the desired emission
wavelengths, although the intensities of emission were severely reduced. Preliminary data
with the inclusion of substrate 14a with 4-chlorobenzaldehyde in Columbia agar showed little
to no effect on the observed intensity of fluorescence, and further work must be undetaken to
ascertain the mechanism behind these observations. Although the synthesized hydrazones 17-
2
1 did not produce the desired characteristics for incorporation into media, future work
should also examine the effects of more strongly electron-donating aldehydes with less
rotatable bonds.
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Acknowledgements
PWG, DEH and JDP would like to thank the National Health and Medical Research Council
NHMRC) for funding.
(
Supplementary data
Supplementary data relating to this article can be found at http://
1
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References
1]
[
Scott RD. The Direct Medical Costs of Healthcare-Associated Infections in U.S.
Hospitals and the Benefits of Prevention. http://stackscdcgov/view/cdc/11550. 2009.
2] Ferguson JK. Preventing healthcare-associated infection: risks, healthcare systems
and behaviour. Internal Med J. 2009;39:574-81.
3] Huttner A, Harbarth S, Carlet J, Cosgrove S, Goossens H, Holmes A, et al.
[
[
Antimicrobial resistance: a global view from the 2013 World Healthcare-Associated
Infections Forum. Antimicrob Res Infect Control. 2013;2:31.
[
4]
hospitals: the value of antimicrobial usage data. Med J Australia. 2010;193.
5] The evolving threat of antimicrobial resistance: options for action. World Health
Organization Rep No 9789241503181: WHO Press; 2012. p. 120.
6] Van Belkum A, Durand G, Peyret M, Chatellier S, Zambardi G, Schrenzel J, et al.
Rapid clinical bacteriology and its future impact. Ann Lab Med. 2013;33:14-27.
7] Zaytsev AV, Anderson RJ, Bedernjak A, Groundwater PW, Huang YX, Perry JD,
McNeil V, Cruickshank M, Duguid M, Journal M. Safer use of antimicrobials in
[
[
[
et al. Synthesis and testing of chromogenic phenoxazinone substrates for beta-alanyl
aminopeptidase. Org Biomol Chem. 2008;6(4):682-92.
[
8]
Anderson RJ, Groundwater PW, James AL, Monget D, Zaytsev AV. Novel
Enzymatic Substrates Derived from Phenoxazinone and their Use as Developer in
Detection of Micro-organism with Peptidase Activity. EP1786828 B1 (11 Jan 2012).
[
9]
Perry JD, Davies A, Butterworth La, Hopley ALJ, Nicholson A, Kate Gould F.
Development and evaluation of a chromogenic agar medium for methicillin-resistant
Staphylococcus aureus. J Clin Microbiol. 2004;42:4519-23.
[
10] Ledeboer Na, Das K, Eveland M, Roger-Dalbert C, Mailler S, Chatellier S, et al.
Evaluation of a novel chromogenic agar medium for isolation and differentiation of
vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates. J Clin
Microbiol. 2007;45:1556-60.
[
11] Perry JD, Naqvi SH, Mirza IA, Alizai SA, Hussain A, Ghirardi S, et al.
Prevalence of faecal carriage of Enterobacteriaceae with NDM-1 carbapenemase at
military hospitals in Pakistan, and evaluation of two chromogenic media. J Antimicrob
Chemother. 2011;66:2288-94.
[
12] Váradi L, Gray M, Groundwater PW, Hall AJ, James AL, Orenga S, et al.
Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the
detection of bacteria. Org Biomol Chem. 2012;10:2578.
[
13] Butenas S, Orfeo T, Lawson JH, Mann KG. Aminonaphthalenesulfonamides, a
new class of modifiable fluorescent detecting groups and their use in substrates for serine
protease enzymes. Biochem. 1992;31(23):5399-411.
[
14] Ivanov I, Tasheva D, Todorova R, Dimitrova M. Synthesis and use of 4-
peptidylhydrazido-N-hexyl-1,8-naphthalimides as fluorogenic histochemical substrates
for dipeptidyl peptidase IV and tripeptidyl peptidase I. Eur J Med Chem. 2009;44:384-92.
[
15] Hodgkiss RJ, Jones GW, Long A, Middleton RW, Parrick J, Stratford MR, et al.
Fluorescent markers for hypoxic cells: a study of nitroaromatic compounds, with
fluorescent heterocyclic side chains, that undergo bioreductive binding. J Med Chem.
1
991;34:2268-74.
20

ACCEPTED MANUSCRIPT
[
16] Cui L, Zhong Y, Zhu W, Xu Y, Du Q, Wang X, et al. A new prodrug-derived
ratiometric fluorescent probe for hypoxia: High selectivity of nitroreductase and imaging
in tumor cell. Org Lett. 2011;13:928-31.
[
17] Anthony R, den Hertog A, Ingham CJ, Ter Maat J. Method for detecting bacteria.
EP2764113 A1 (13 Aug 2014).
18] Xu YW, Zhao GS, Shin CG, Zang HC, Lee CK, Lee YS. Caffeoyl
naphthalenesulfonamide derivatives as HIV integrase inhibitors. Bioorg Med Chem.
003;11:3589-93.
19] Guan B, Minor C, Dai M, Ghosh S, Jenkins T, J., Li G, et al. CCR8 Inhibitors.
WO/2004/058709 (15 July 2004).
20] Wang Y, Zhang Y, Yi X, Qin W. Design and Synthesis of Some 1,8-
Naphthalimides as Fluorescence Probes for Transition Metal Ions. Asian J Chem.
012;24:323-6.
21] Nakatani K, Horie S, Murase T, Hagihara S, Saito I. Assessment of the sequence
[
2
[
[
2
[
dependency for the binding of 2-aminonaphthyridine to the guanine bulge. Bioorg Med
Chem. 2003;11:2347-53.
[
22] Kadirvel M, Fairclough M, Cawthorne C, Rowling EJ, Babur M, McMahon A, et
al. Detection of apoptosis by PET/CT with the diethyl ester of [¹⁸F]ML-10 and
fluorescence imaging with a dansyl analogue. Bioorg Med Chem. 2014;22:341-9.
[
23] Gan J-A, Song QL, Hou XY, Chen K, Tian H. 1,8-Naphthalimides for non-doping
OLEDs: the tunable emission color from blue, green to red. J Photochem Photobiol A.
004;162:399-406.
24] Ball AP, McGhie D, Geddes AM. Serratia marcescens in a general hospital. Quart
J Med. 1977;46:63-71.
25] Taniyama K, Itoh H, Takuwa A, Sasaki Y, Yajima S, Toyofuku M, et al. Group X
2
[
[
Aldehyde dehydrogenases of pseudomonas aeruginosa PAO1 degrade hydrazones. J
Bacteriol. 2012;194:1447-56.
[
26] Ani C, Farshidpanah S, Bellinghausen Stewart A, Nguyen HB. Variations in
organism-specific severe sepsis mortality in the United States: 1999-2008. Critical care
medicine. 2015;43(1):65-77.
[
27] Martin GS. Sepsis, severe sepsis and septic shock: changes in incidence,
pathogens and outcomes. Exp Rev Anti-infect Ther. 2012;10(6):701-6.
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Figure Legends
Figure 1.
Figure 2.
Figure 3.
Illustration of the principle for the chromogenic / fluorogenic detection of bacteria
and the basis of the colour change in chromID P. aeruginosa.[7, 8]
Chemical structures and reported excitation and emission wavelengths for core
heterocycles, ANS and 6-HBI.
Emission spectra for substrate 12c (slit width = 3 mm) (dashed blue line) and
-4
fluorophore 7c (slit width = 5 mm) (solid green line) (both 5 × 10 M in EtOH)
intensities are shown in arbitrary units).
(
-4
Figure 4.
a) Emission spectra (at λex = 443 nm) of substrate 14a in EtOH (1 × 10 M)
(black line), water (blue line), brine (red line), Mueller-Hinton broth (green line)
(100 mg/L in water, brine and Mueller-Hinton broth); b) Emission spectra (at λex
-4
=
465 nm) of fluorophore 10a in EtOH (1 × 10 M) (black line), water (blue line),
brine (red line), Mueller-Hinton broth (green line) (100 mg/L in water, brine and
Mueller-Hinton broth). The dashed green line in both figures represents the
spectrum of Mueller-Hinton broth alone. All iintensities are shown in arbitrary
units).
Figure 5.
Figure 6.
Emission spectra of fluorophore 10a (dashed blue line) and hydrazones 17a (solid
red line), 18a (solid green line), 19a (solid purple line), 20a (solid black line), and
-5
2
1a (solid orange line on baseline) (17a, 18a: 1 × 10 M in EtOH, 10a, 19a-21a:
-
4
1
× 10 M in EtOH) (slit width = 5 mm) (intensities are shown in arbitrary units).
-4
a) Emission spectra (at λ = 339 nm) of substrate 12b in EtOH (5 × 10 M)
ex
(black line), water (blue line), brine (red line), Mueller-Hinton broth (green line)
(100 mg/L in water, brine and Mueller-Hinton broth); b) Emission spectra (at λex
-
4
=
394 nm) of fluorophore 7b in EtOH (5 × 10 M) (black line), water (blue line),
brine (red line), Mueller-Hinton broth (green line) (100 mg/L in water, brine and
Mueller-Hinton broth) The dashed green line in both figures represents the
spectrum of Mueller-Hinton broth alone. All iintensities are shown in arbitrary
units); c) Observed fluorescence (λ = 365 nm) of sulfonamide 7b in Mueller-
ex
Hinton broth (left) and ethanol (right).
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Figure 7.
Figure 8.
Culture of various bacterial species with substrates 12a-e and 14a-c and control
on Columbia agar; inoculated colonies correspond to numbered plate.
Observed fluorescence of various bacterial species with substrates 12a-e and 14a-
c and control on Columbia agar (λ = 365 nm); inoculated colonies correspond to
ex
numbered plate.
Figure 9.
Observed fluorescence of Pseudomonas aeruginosa colonies incubated on
Columbia agar in the presence of substrate 14a (100 mg/L) (left) and substrate
1
4a (100 mg/L) along with 4-chlorobenzaldehyde (100 mg/L) (right).
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Figure 1
24

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Figure 2
25

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Figure 3
2
50
00
50
00
2
1
1
50
0
350
400
450
500
550
600
Wavelength (nm)
2
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Figure 4
50
40
30
20
10
0
500
550
600
Wavelength (nm)
a)
b)
27

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Figure 5
28

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Figure 6
a)
b)
29