2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the detection of nitroreductase activity in clinically important microorganisms

Article abstract of DOI:10.1016/j.bmcl.2015.10.099

A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential nitroreductase substrates for the purpose of detecting clinically important microorganisms. Several of the substrates produced highly fluorescent colonies with the majority of a panel of 10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.

Full text of DOI:10.1016/j.bmcl.2015.10.099

Accepted Manuscript
2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic sub-
strates for the detection of nitroreductase activity in clinically important micro-
organisms
Marie Cellier, Amandine Gignoux, Arthur L. James, Sylvain Orenga, John D.
Perry, Shaun N. Robinson, Stephen P. Stanforth, Graeme Turnbull
PII:
S0960-894X(15)30209-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.10.099
BMCL 23255
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 September 2015
30 October 2015
31 October 2015
Please cite this article as: Cellier, M., Gignoux, A., James, A.L., Orenga, S., Perry, J.D., Robinson, S.N., Stanforth,
S.P., Turnbull, G., 2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the
detection of nitroreductase activity in clinically important microorganisms, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.10.099
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2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the
detection of nitroreductase activity in clinically important microorganisms
Marie Cellier,a Amandine Gignoux,b,c the late Arthur L. James,b,e Sylvain Orenga,a John D.
Perry,d Shaun N. Robinsonb, Stephen P. Stanforthb* and Graeme Turnbull.b
a Research & Development Microbiology, bioMérieux SA, 3 route de Port Michaud,
38 390 La-Balme-les-Grottes, France
b Department of Applied Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST,
UK
c École Nationale Supérieure de Chimie de Paris, 11 rue Pierre et Marie Curie, F75231 Paris
cedex 05, France
d Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK
e Deceased, May 2014
*Corresponding authors. Tel.: +44-191 227 4784; fax: + 44-191 227 3519; e-mail:
steven.stanforth@northumbria.ac.uk
Abstract
A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-
substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential
nitroreductase substrates for the purpose of detecting clinically important microorganisms.
Several of the substrates produced highly fluorescent colonies with the majority of a panel of
10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.
Keywords Nitroreductase, fluorescence, enzyme substrates, microorganism detection,
bacterial detection
1

Methods that enable the detection, enumeration and identification of harmful bacteria are
important in many sectors of the economy including the health-care sector (e.g. hospitals), the
food industry (e.g. food quality control) and the environment (e.g. monitoring of water
contamination).1-3 The utilisation of synthetic enzyme substrates is an important protocol for
bacterial detection and identification. Relevant to this paper is the reductive transformation of
weakly fluorescent nitro-aromatic substrates 1 by bacterial nitroreductase enzymes into
highly fluorescent aromatic amines 2 (or hydroxylamines) as depicted in Scheme 1. Such a
change in fluorescence intensity can frequently offer a practicable method for analysing
biological and environmental samples because many bacteria are capable of reducing nitro-
aromatic compounds.4 A bioluminescent nitroreductase probe has been reported and its
capacity to detect nitroreductase activity in the Gram-negative Escherichia coli and the
Gram-positive Bacillus subtilis was demonstrated. 5 Chromogenic substrates, including
halogenated nitrophenoxazinones6 and 4-(4-nitrostyryl)quinolines and related compounds,7
have been evaluated for their ability to detect nitroreductase activity across a range of
microorganisms. A substrate derived from 3H-phenoxazin-3-one (resorufin) linked to a
nitrothiophene moiety has been used to detect E. coli.8 In these chromogenic substrates,
weakly coloured nitro-heterocycles are transformed by a nitroreductase enzyme into highly
coloured amine derivatives.
Scheme 1. Reduction of nitro-aromatics by nitroreductase enzymes.
The synthesis of a series of several 7-nitrocoumarin derivatives has been described
previously by one of us (ALJ) and these fluorogenic nitroreductase substrates were evaluated
against 30 bacterial strains which encompassed 24 distinct microbial genera, including a wide
range of clinically important species.9,10 We have also described the synthesis of a series of 2-
(2-nitroaryl)benzothiazoles 3a-d, 2-(2-nitroaryl)benzoxazoles 4a-d and 2-(2-
nitroaryl)benzimidazoles 5a and 5b (Figure 1) and their evaluation against a panel of 20
microorganisms in agar media.11 In general, these heterocycles gave either blue or purple
fluorescent colonies with the majority of Gram-negative bacteria and also with two Gram-
positive strains, methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant
Staphylococcus aureus (MRSA). The benzothiazoles 3a-d and benzoxazoles 4a-d usually
produced highly fluorescent bacterial colonies, whereas in comparison, the fluorescence
intensity of the bacterial colonies in the presence of the benzimidazole derivatives 5a and 5b
was weak. We also demonstrated the benzoxazole derivative 4c produced a fluorescent
solution when incubated with a Escherichia coli BL21 cell-free extract in a Tris buffer
solution (pH 7.4) thus confirming that enzyme activity was responsible for the generation of
fluorescence. Furthermore, the corresponding amine derivative was identified as the product
2

by HPLC; the structure of the amine was confirmed by comparison with an authentic sample
which was prepared by chemical reduction of compound 4c.11
Figure 1. Nitroreductase substrates.
In this paper, we report the synthesis of a series of 2-(2-nitroaryl)benzothiazole 6a, 9a-c
and 2-(2-nitroaryl)benzoxazole 7a-d and 10a-c derivatives that all possess a carboxylic acid
group as part of their structure (Figure 1). These substrates have been prepared because the
presence of the carboxy-group (either as the free acid or an appropriate salt) would be
expected to enhance aqueous solubility; an important consideration for applications in the
preparation of microbiological media. Both broth and agar media are frequently prepared by
addition of water to the powdered media (containing the substrate) and hence for commercial
applications the substrate should be able to dissolve quickly and completely in water. In view
of our previous work on fluorogenic nitroreductase substrates,11 it was also of general interest
to investigate and compare the selectivity of these new heterocycles with existing
compounds.
The nitroreductase substrates prepared in this paper have been evaluated in agar media
because multi-point inoculation of plates allows a wide range of microorganisms to be
evaluated concomitantly. Structurally related to the 2-(2-nitroaryl)benzoxazole substrates is
2-(2-nitrophenyl)-[1,3]oxazolo[5,4-b]pyridine (8).12 To our knowledge, this compound has
not been evaluated as a nitroreductase substrate and hence it has been included in this study.
The synthetic routes to the nitroreductase substrates used in this study are shown in
Scheme 2. 3-Mercapto-4-aminobenzoic acid was heated with 2-nitrobenzaldehyde in
nitrobenzene solution at reflux following a literature procedure affording substrate 6a.13
Substrates 7a-d were prepared by condensing 2-aminophenol-3/4-carboxylic acids with an
appropriately substituted 2-nitrobenzaldehyde derivative in ethanol at reflux to give the
Schiff bases 12 which were subjected to an oxidative cyclisation yielding the required
3

substrates 7a-d. Substrate 8 was produced following a method described in the patent
literature;8 3-amino-2-chloropyridine was treated with 2-nitrobenzoyl chloride under basic
conditions giving the amide 13 which was subsequently cyclised by heating in a mixture of
1,2-dichlorobenzene, phosphorus pentoxide and hexamethyldisiloxane. The reaction of the
fluorinated benzothiazole 3c and the fluorinated benzoxazole 4c with an amino acid (D-
proline, L-proline or N-methyl-γ-aminobutyric) under basic conditions furnished the
substrates 9a-c and 10a-c.
Scheme 2. Synthesis of the potential nitroreductase substrates.
A preliminary evaluation of each substrate was carried out in Columbia agar media (37 oC
in air for 18 hours) against 20 clinically important microorganisms, including 10 Gram-
negative bacteria, 8 Gram-positive bacteria and 2 yeasts. Columbia agar media was selected
because this enabled a direct comparison of the substrates described in this paper with those
previously reported by us and, additionally, each substrate could be evaluated simultaneously
against the complete panel of 20 microorganisms on a single plate. The growth of the
microorganisms was compared to control plates in which no substrate was present. The
Gram-negative microorganisms all grew well on the control plates whereas the Gram-positive
microorganisms and the yeasts showed only moderate growth.
Table 1 depicts the results obtained for the evaluation of the substrates 6a, 7a and 8
against the panel of 20 microorganisms. Photographs of agar plates for substrates 6a and 7a
are shown in Figure 2. The majority of the Gram-negative bacteria as well as the two Gram-
positive strains, MSSA and MRSA, produced strongly fluorescent colonies with all of these
4

substrates. These nitroreductase profiles are similar to those previously reported by us for the
substrates 3a-d and 4a-d, and illustrate that the presence of the carboxy-groups in substrates
6a and 7a are not detrimental to the substrates’ effectiveness. The results obtained for the
evaluation of the substrates 7b-7d (data not shown) were broadly similar to those of substrate
7a.
6a
7a
8
Growthb
Fluorescencec
Growthb
Fluorescencec
Growthb
Fluorescencec
Microorganism / Referencea
Gram-negative microorganisms
Escherichia coli NCTC 10418
Klebsiella pneumoniae NCTC 9528
Providencia rettgeri NCTC 7475
Enterobacter cloacae NCTC 11936
Serratia marcescens NCTC 10211
Salmonella typhimurium NCTC 74
Pseudomonas aeruginosa NCTC
10662
1
2
3
4
5
6
7
++
++
++
++
++
++
++ green
++ green
++ green
++ green
++ green
++ green
++
++
++
++
++
++
++
++ blue
++ blue
++ blue
++ blue
++ blue
++ blue
++
++
++
++
++
++
++ blue
++ blue
++ blue
++ blue
++ blue
++ blue
++
+
++
+/- green
+/- green
++ green
-
-
++
++
++
++ blue
+ blue
+ blue
8
9
Yersinia enterocolitica NCTC 11176
Burkholderia cepacia NCTC 10743
++
++
++
++ blue
10 Acinetobacter baumannii NCTC
12156
++
++ green
++ blue
++
++ blue
Gram-positive microorganisms
11 Streptococcus pyogenes NCTC 8306
12 Staphylococcus aureus (MRSA)
NCTC 11939
13 Staphylococcus aureus (MSSA)
NCTC 6571
+
+
+
-
+
+
+
-
+
+
+
-
++ green
++ green
++ blue
++ blue
++ blue
++ blue
14 Staphylococcus epidermidis NCTC
11047
15 Listeria monocytogenes NCTC 11994
16 Enterococcus faecium NCTC 7171
17 Enterococcus faecalis NCTC 775
18 Bacillus subtilis NCTC 9372
+
+
+
+
+
Tr. green
Tr. green
Tr. green
Tr. green
+ green
+
+
+
+
+
-
-
-
-
+
+
+
+
+
-
-
-
-
+ blue
+ blue
Yeasts
19 Candida albicans ATCC 90028
20 Candida glabrata NCPF 3943
+
+
Tr. green
-
+
+
-
-
+/-
+
-
-
aNCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National
Collection of Pathogenic Fungi.
b ++ strong growth, + moderate growth, +/- weak growth, Tr. trace of growth.
c ++ strong fluorescence, + moderate fluorescence, +/- weak fluorescence, Tr. trace of fluorescence.
Table 1. Evaluation of substrates 6a, 7a and 8. Substrate concentration = 100 mg L-1;
inoculum = 100 000 colony-forming units (cfu)/spot.
5

Arrangement of microorganisms on platesa
Substrate 6a
Substrate 7a
Substrate 9b
Figure 2. Columbia agar plates depicting fluorescence generated by substrates 6a, 7a and 9b
with various microorganisms. a Microorganisms are numbered in the sequence shown in the
Tables. Pink spots represent Gram-negative bacteria, blue spots represent Gram-positive
bacteria and the yeast species
9a
9b
9c
Growthb
Fluorescencec
Growthb
Fluorescencec
Growthb
Fluorescencec
Microorganism / Referencea
Gram-negative microorganisms
Escherichia coli NCTC 10418
Klebsiella pneumoniae NCTC 9528
Providencia rettgeri NCTC 7475
Enterobacter cloacae NCTC 11936
1
2
3
4
++
++
++
++
+ yellow
+ yellow
+ yellow
+ yellow
++
++
++
++
++ blue
++ blue
++ blue
++ blue
++
++
++
++
+ yellow
+ yellow
+ yellow
+ yellow
6

5
6
7
Serratia marcescens NCTC 10211
Salmonella typhimurium NCTC 74
Pseudomonas aeruginosa NCTC
10662
Yersinia enterocolitica NCTC 11176
Burkholderia cepacia NCTC 10743
Acinetobacter baumannii NCTC
12156
++
++
+ yellow
+ yellow
++
++
++
++ blue
++ blue
++
++
+ yellow
+ yellow
++
++
++
Tr. yellow
Tr. yellow
+ yellow
++
++ blue
+ blue
++ blue
++
++
++
+ yellow
Tr. yellow
+ yellow
++
8
9
10
++
++
++
++
yellow
++ blue
++
yellow
Gram-positive microorganisms
11
12
13
14
15
16
Tr.
yellow
Streptococcus pyogenes NCTC 8306
Staphylococcus aureus (MRSA)
NCTC 11939
Staphylococcus aureus (MSSA)
NCTC 6571
Staphylococcus epidermidis NCTC
11047
Listeria monocytogenes NCTC
11994
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+ blue
+ blue
+ yellow
-
+ yellow
+ yellow
++ blue
++ blue
+ blue
-
+/- yellow
Tr.
yellow
Tr.
Enterococcus faecium NCTC 7171
Enterococcus faecalis NCTC 775
Bacillus subtilis NCTC 9372
+
+
+
yellow
Tr. yellow
-
+
+
+
-
-
-
+
+
+
-
-
-
17
18
Yeasts
19
20
Candida albicans ATCC 90028
Candida glabrata NCPF 3943
+
+
+ yellow
-
+
+
-
-
+
+
-
-
aNCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National
Collection of Pathogenic Fungi.
b ++ strong growth, + moderate growth, +/- weak growth, Tr. trace of growth.
c ++ strong fluorescence, + moderate fluorescence, +/- weak fluorescence, Tr. trace of fluorescence.
Table 2. Evaluation of substrates 9a-c. Substrate concentration = 100 mg L-1; inoculum =
100 000 colony-forming units (cfu)/spot.
Of the three substrates 9a-c, only the L-prolyl derivative 9b produced strongly fluorescent
colonies (Table 2, Figure 2). This substrate gave the expected pattern of nitroreductase
activity with the panel of microorganisms, i.e. fluorescent colonies were formed with all of
the Gram-negative bacteria and also with the MSSA and MRSA strains. Substrate 9c gave a
similar nitroreductase profile to substrate 9b but the colonies were yellow fluorescent and the
fluorescence intensity was only moderate. There was an interesting and unexpected contrast
between the prolyl substrates 9a and 9b. The substrate 9a produced yellow fluorescent
colonies with most microorganisms although the fluorescence intensity was very weak in
many cases, with the exception of Acinetobacter baumannii which gave strongly fluorescent
colonies. The observed yellow fluorescence of substrate 9a is in contrast to the strong blue
fluorescence produced by substrate 9b and the reason for this is as yet unclear.
Unexpected evaluation results were also obtained for the benzoxazole substrates 10a-10c.
These substrates only produced weakly fluorescent colonies (blue) with three of the panel of
20 microorganisms [Pseudomonas aeruginosa, A. baumannii and MRSA (data not shown)].
7

Substrate 10a also gave weakly fluorescent colonies with Burkholderia cepacia. This
suggests that reduction of these substrates is not occurring with the majority of
microorganisms. This observation is surprising in view of our previous work on
benzothiazole and benzoxazole nitroreductase substrates in which the nitroreductase activity
profiles of both of these heterocyclic systems with the majority of microorganisms has
generally been comparable.11
The potassium salt of substrate 7a was prepared by addition of an equimolar quantity of
methanolic potassium hydroxide solution to the substrate followed by evaporation of the
solvent. The potassium salt (30 mmol) was added to 100 mL of a potassium phosphate buffer
(0.1 M, pH 7.4) in order to simulate the pH of typical culture media. The potassium salt was
completely soluble in the buffer, whereas in contrast, the corresponding substrate 4a, lacking
the carboxy-group, was visibly insoluble at the same concentration.
We have evaluated substrate 7a in brain heart infusion broth over a range of substrate
concentrations against the Gram-negative E. cloacae (NCTC 11936) and the Gram-positive
S. aureus (NCTC 6571) (Figure 3). The broths were incubated at 37 oC for 18 hours and
bacterial activity could be detected at concentrations as low as 0.02 mmol/L-1.
2500
2000
1500
S. aureus / 7a
1000
E. cloacae / 7a
No bacteria / 7a
500
0
0
0.02
0.04
0.08
0.17
0.33
Concentration (mmol/L)
-500
Figure 3. Fluorescence generated from substrate 7a by two pathogenic bacteria at a range of
substrate concentrations.
In conclusion, the nitroreductase substrates 6a and 7a-d, which bear a carboxy-group in
the benzothiazole/benzoxazole ring, all exhibit a pattern of activity against a selection of
8

microorganisms in agar media which is broadly similar to that previously observed for the
substrates 3a-d and 4a-d. All of these new substrates 6a and 7a-d produce highly fluorescent
colonies with the majority of Gram-negative bacteria in most cases. The pyridine-ring
containing substrate 8 also conforms to this pattern of activity. In contrast, the heterocycles
9a-c and 10a-c are less effective substrates, generally showing diminished fluorescent
intensities and in the cases of substrates 10a-c a greatly reduced nitroreductase activity
profile. The presence of the carboxy-groups in these substrates has been shown to be
beneficial to their aqueous solubility, thus allowing potential applications of these substrates
in culture media.
Supplementary information
Synthetic and microbiological experimental procedures are given in the supplementary
information.
Acknowlegements
We thank bioMérieux SA for generous financial support and the EPSRC Mass Spectrometry
Centre, Swansea, for high resolution mass spectra.
References and notes
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2009, 79, 139.
2. Manafi, M. Int. J. Food Microbiol. 2000, 60, 205.
3. Manafi, M.; Kneifel, W.; Bascomb, S. Microbiol. Rev. 1991, 55, 335 and refs therein.
4. Roldán, M. D.; Pérez-Reinado, E.; Castillo, F.; Moreno-Vivián, C. FEMS Microbiol. Rev.,
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5. Wong, R. H. F.; Kwong, T.; Yau, K.-H.; Au-Yeung, H. Y. Chem. Commun., 2015, 51,
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6. Bedernjak, A. F.; Groundwater, P. W.; Gray, M.; James, A. L.; Orenga, S.; Perry, J. D.;
Anderson, R. J. Tetrahedron, 2013, 69, 8456.
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WO/2011/012824, 2011.
8. Li, Zhao, gao, X.; Shi, W.; Li, X.; Ma, H. Chem. Commun., 2013, 49, 5859.
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Microbiol., 2001, 33, 403.
10. James, A.; Monget, D. European Patent EP 1124986, 2001.
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Salwatura, V. L.; Stanforth, S. P. Bioorg. Med. Chem., 2011, 19, 2903.
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