Synthesis and evaluation of novel chromogenic peptidase substrates based on 9-(4′-aminophenyl)-10-methylacridinium salts as diagnostic tools in clinical bacteriology

Article abstract of DOI:10.1016/j.bmcl.2007.11.018

The synthesis and initial evaluation of novel chromogenic substrates with potential in the detection and differentiation of cultured bacterial colonies are described. The substrates were readily hydrolysed by specific aminopeptidase activity to release th

Full text of DOI:10.1016/j.bmcl.2007.11.018

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 832–835
Synthesis and evaluation of novel chromogenic peptidase
substrates based on 9-(40-aminophenyl)-10-methylacridinium
salts as diagnostic tools in clinical bacteriology
Rosaleen J. Anderson,a,* Paul W. Groundwater,a Yongxue Huang,a Arthur L. James,b
c
b
c
d
´
Sylvain Orenga, Annette Rigby, Celine Roger-Dalbert and John D. Perry
aSunderland Pharmacy School, University of Sunderland, Sunderland SR1 3SD, UK
bSchool of Applied Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, UK
cDepartment of Microbiology, bioMerieux, 38390 La Balme-les-Grottes, France
dDepartment of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK
Received 27 September 2007; revised 5 November 2007; accepted 8 November 2007
Available online 17 November 2007
Abstract—The synthesis and initial evaluation of novel chromogenic substrates with potential in the detection and differentiation of
cultured bacterial colonies are described. The substrates were readily hydrolysed by specific aminopeptidase activity to release the
chromogen, 9-(40-aminophenyl)-10-methylacridinium salt, which provided a clear visual indication of the presence of the corre-
sponding bacteria.
ꢀ 2007 Published by Elsevier Ltd.
The ability to detect the presence of enzymes as diagnos-
tic markers for pathogens using suitable chromogenic
substrates has become a key method for the rapid iden-
tification of microorganisms in clinical1 and food2 sam-
ples. The incorporation of such substrates into a suitable
medium can reduce the need for subculture and further
biochemical tests to establish the identity of certain
microorganisms. In particular, the presence of L-alanine
aminopeptidase in Gram-negative bacteria can be
exploited to distinguish them from Gram-positive bacte-
ria,3,4 which do not express this enzyme, Figure 1. In
addition, chromogenic aminopeptidase substrates can
be used to distinguish between certain bacterial species,
for example, L-pyrrolidonyl aminopeptidase aids in the
identification of enterococci, group A streptococci5,6
and Salmonella spp.7 The identification of b-alanine
aminopeptidase activity in Pseudomonas aeruginosa8 of-
fers the potential for the detection of this important
pathogen, for example, in specimens from patients with
cystic fibrosis.
These bacterial detection systems use a chromogenic
compound covalently linked to a substrate for a specific
bacterial enzyme. The covalent linkage to the substrate
limits the electronic resonance within the chromogen,
resulting in the quenching of the colour. Action of the
target bacterial enzyme(s) results in cleavage of the chro-
mogen-substrate covalent bond, enabling resonance
throughout the conjugated chromogen and the return
of colour. Chromogenic substrates facilitate bacterial
detection and identification when the release of the chro-
mogenic molecule produces an intense colour that can
be visualized by eye, obviating the need for expensive
or specialized equipment.
Unfortunately, most substrates that have been described
for visualization of peptidase activity, including p-nitro-
anilide derivatives, are unsuitable for use in agar media,
due to the widespread diffusion of the chromogen through
agar after hydrolysis from the substrate, and are not suit-
able for the visualization of bacterial colonies.
Keywords: Microorganism detection; Microorganism differentiation;
Clinical bacteriology; Diagnostic reagent; Chromogenic peptidase
substrate; 9-(40-Aminophenyl)-10-methylacridinium salt; Alanine
aminopeptidase; b-Alanine aminopeptidase; Cystic fibrosis.
Recently, the use of acridine-based substrates was re-
ported9; hydrolysis yielded chromogens that were
*
Corresponding author. Tel.: +44 1915152591; fax: +44
1915153405; e-mail: roz.anderson@sunderland.ac.uk
0960-894X/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.11.018

R. J. Anderson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 832–835
833
CH3
H
CH3
N
H2N
L-alanine
aminopeptidase
H3N
H3N
CO2
Gram negative
bacteria
O
NO2
NO2
L-alanyl-p-nitroanilide
colourless substrate
L-alanine
p-nitroaniline
yellow
Figure 1. Differentiation of Gram-negative and Gram-positive bacteria through hydrolysis of L-alanyl-p-nitroanilide by L-alanine aminopeptidase.
AA
AA
AA
NH2
NH
NH
NH
X
X
X
X
MeI
AA
DIPCDI,
HOBt, DCM
TFA, DCM
p-xylene, 120oC
N
N
N
N
I
2 CF3CO2
CH3
1a X = H
1b X = OMe
2a-g
CH3
3a-f and 4g
4a-f
Scheme 1. Synthesis of chromogenic substrates 4a–g via precursors 2a–g and 3a–f.
weakly coloured when released from the substrate, but
were intensely red-violet coloured when protonated by
treatment of the plates with acetic acid—due to the for-
mation of the related acridinium salt. Addition of glacial
acetic acid to bacterial colonies is not ideal, not least
because of the loss of viability of bacteria that may be
required for further testing.
the previously reported acridine-based substrates; they
have muted colour when acylated, for example, with
an amino acid, but are intensely red-orange coloured
when released as the methylacridinium salts, without
the requirement for the addition of acid, and they ad-
here to the bacterial colonies making visualization
easier.
We report here the synthesis and evaluation of 9-(40-N-
[aminoacyl]aminophenyl)-10-methylacridinium salts 4a–
g as chromogenic molecules suitable for bacterial detec-
tion and identification. These compounds improve on
The synthesis of 9-(40-aminophenyl)acridine 1a and 9-
(40-amino-30-methoxyphenyl)acridine 1b was achieved
using a slight variation of the method of Acheson and
Birtwistle,10 as previously reported.9
The aminoacyl-10-methylacridinium trifluoroacetate
bacterial enzyme substrates 4a–f were prepared by reac-
tion of 9-(40-aminophenyl)acridine 1a or 1b with the
appropriate tBoc-protected amino acid or peptide, using
established peptide coupling conditions, to form the cor-
Table 1. Yields of synthesized aminoacyl-10-methylacridinium salts
4a–g and related precursors 2a–f and 3a–f
Compound
AA
X
Yield (%)
2a
2b
2c
2d
2e
2f
tBoc-L-Ala
tBoc-b-Ala
tBoc-Gly
tBoc-L-Ala-L-Ala
tBoc-L-Ala-L-Ala-L-Ala
tBoc-L-Ala
L-pyroGlu
tBoc-L-Ala
tBoc-b-Ala
tBoc-Gly
tBoc-L-Ala-L-Ala
tBoc-L-Ala-L-Ala-L-Ala
tBoc-L-Ala
L-Ala
H
78
76
77
79
81
81
80
86
90
80
89
80
92
95
94
95
94
98
98
91
H
t
responding Boc-aminoacyl-acridines 2a–f. Methylation
of the acridines 2a–f with methyl iodide gave the Boc
H
t
H
protected aminoacyl-10-methylacridinium iodide salts
3a–f; Boc removal with TFA gave the expected amino-
H
t
OMe
H
2g
3a
3b
3c
3d
3e
3f
acyl-10-methylacridinium bis-trifluoroacetate salts 4a–f,
Scheme 1 and Table 1.
H
H
The L-pyroglutamyl derivative, 9-(40-N-[L-pyrogluta-
myl]aminophenyl)-10-methylacridinium iodide 4g, was
prepared by a similar route in which Boc protection
H
H
H
t
OMe
H
was not required. The reaction of 1a with L-pyrogluta-
mate under the usual peptide coupling conditions
yielded 9-(40-N-[L-pyroglutamyl]aminophenyl)acridine
2g; methylation of this intermediate, under the condi-
tions indicated, gave the chromogenic substrate 4g. Full
details of the syntheses are provided in the Supplemen-
tary information.
4a
4b
4c
4d
4e
4f
b-Ala
Gly
H
H
L-Ala-L-Ala
L-Ala-L-Ala-L-Ala
L-Ala
H
H
OMe
H
4g
L-pyroGlu

834
R. J. Anderson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 832–835
The chromogenic substrates 4a–g and 3e were evaluated
for their suitability as chromogenic substrates for the
detection of bacterial aminopeptidase activity, for their
potential to differentiate between Gram-positive and
Gram-negative bacteria, and for their ability to detect
P. aeruginosa.
In general, the substrates were not growth inhibitory to
Gram-negative bacteria and most of the substrates tested
were hydrolysed by these bacteria. In contrast, the Gram-
positive bacteria were either inhibited by the substrates or
were unable to hydrolyse the covalent link between the
amino acid and chromogen, providing a visual means to
differentiate between these different classes of bacteria.
The most promising substrate across the range of bacteria
tested was 9-(40-N-[L-Ala-L-Ala-L-Ala]aminophenyl)-10-
methylacridinium bis-trifluoroacetate 4e, which was read-
ily hydrolysed by Gram-negative bacteria, while being
non-inhibitory to Gram-positive bacteria—allowing the
growth of all bacteria and the visualization of Gram-neg-
ative colonies through hydrolysis of the substrate with a
concomitant release of the coloured chromogen as a
red/orange methylacridinium salt, Figure 2.
An initial evaluation (results not shown) confirmed that
all of the substrates tested could be hydrolysed by bac-
terial aminopeptidases to release the highly coloured
chromogen,
9-(40-aminophenyl)-10-methylacridinium
salt, without the addition of acid. The methoxy deriva-
tive 4f was comparable in activity to the unsubstituted
analogue 4a, but was more difficult to synthesize and
was not evaluated further.
The water soluble substrates 4a–e, 4g and 3e were incor-
porated into Columbia agar at 300 mg/L; 20 mL of this
agar was added to each 90 mm Petri dish to give an agar
depth of 4 mm. The resultant agar plates were each inoc-
ulated with nine Gram-negative and ten Gram-positive
bacteria. Bacterial strains were initially cultivated on a
standard nutrient agar medium. Colonies of each strain
were suspended in sterile deionised water to generate a
suspension with a turbidity equivalent to 0.5 McFarland
units (approximately 1.5 · 108 colony forming units per
ml), as confirmed with a densitometer. One microliter of
this suspension was inoculated onto the agar plates con-
taining the various substrates using an automated mul-
tipoint inoculator. All inoculated media were
incubated at 37 ꢁC for 48 h and examined visually after
24 and 48 h for the presence of growth and coloration of
bacterial colonies, Table 2.
t
The Boc protected analogue, 9-(40-N-[Na-tBoc-L-Ala-L-
Ala-L-Ala]aminophenyl)-10-methylacridinium iodide 3e,
had a similar activity profile, presumably due to facile car-
bamate hydrolysis by unidentified esterase activity to
yield 4e.
The L-pyroglutamyl substrate, 9-(40-N-[L-pyrogluta-
myl]aminophenyl)-10-methylacridinium
showed promise for the differentiation of members of
the enterobacteriaceae family, as it was effectively
hydrolysed by Klebsiella pneumoniae and Enterobacter
cloacae, but not by the strains of Escherichia coli and
Salmonella spp. tested, nor by Shigella sonnei.
iodide
4g,
Two substrates showed potential for the detection of P.
aeruginosa: the L-pyroglutamyl substrate, 9-(40-N-
Table 2. Hydrolysis of substrates 4a–e, 4g and 3e by Gram-negative and Gram-positive bacteria
Bacterial strain
Substratea
4a
4b
4c
4d
4e
4g
3e
Reference
24 hb 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
Gram-negative bacteria
Escherichia coli 0157
Burkholderia cepacia
Pseudomonas aeruginosa
Serratia marcescens
Salmonella poona
c
NCTC 12079
NCTC 10743
NCTC 10662
NCTC 10211
NCTC 4840
NCTC 74
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Salmonella typhimurium
Shigella sonnei
NCTC 9774
NCTC 10896
NCTC 11936
Klebsiella pneumoniae
Enterobacter cloacae
Gram-positive bacteria
Enterococcus faecalis
Enterococcus faecalis
Enterococcus faecium
NCTC 775
NCTC 12697
NCTC 7171
NGd NG
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NG NG
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NG NG
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Enterococcus faecium (VRE) Wild type 141835
ꢀ
Listeria monocytogenes
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
NCTC 11994
NCTC 6571
NCTC 11939
NCTC 4163
ꢀ
NG
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NG NG NG NG NG NG NG
NG NG NG NG
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NG
NG NG NG NG NG
NG
NG NG NG NG NG
NG NG
ꢀ
EMRSA 1729/98 NG
EMRSA 07924/95 NG
ꢀ
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ꢀ
ꢀ
ꢀ
NG NG
a Each substrate was tested at a concentration of 300 mg/L.
b Results after 24 and 48 h of incubation at 37 ꢁC.
c Positive indicates colour observed; negative indicates no colour observed.
d NG, no growth.

R. J. Anderson et al. / Bioorg. Med. Chem. Lett. 18 (2008) 832–835
835
L-pyroglutamyl substrate, 4g showed a similar specificity
and successfully detected 97% of the P. aeruginosa
strains tested.
In conclusion, substrates for bacterial aminopeptidases
based on 9-(40-N-[aminoacyl]aminophenyl)-10-methyl-
acridinium salts were effective at differentiating be-
tween Gram-positive and Gram-negative bacteria on
agar plates, due to the differential expression and
activity of the appropriate aminopeptidases. 9-(40-N-
[L-Ala-L-Ala-L-Ala]amino-phenyl)-10-methylacridinium
t
bis-trifluoroacetate 4e and its Boc protected deriva-
tive, 9-(40-N-[Na-tBoc-L-Ala-L-Ala-L-Ala]aminophenyl)-
10-methylacridinium iodide 3e, showed greatest promise
for this application. 9-(40-N-[L-Pyroglutamyl]amino-
phenyl)-10-methylacridinium iodide 4g was effective
in distinguishing between members of the enterobacte-
riaceae family, being hydrolysed by K. pneumoniae and
E. cloacae, but not by the strains of E. coli and Sal-
monella spp. tested, nor by S. sonnei. Further testing
is still required to evaluate this substrate’s ability to
distinguish Citrobacter species from Salmonella and
E. coli species. 9-(40-N-[b-Alanyl]aminophenyl)-10-
methylacridinium bis-trifluoroacetate 4b and 9-(40-N-
[L-pyroglutamyl]aminophenyl)-10-methylacridinium io-
dide 4g, showed potential for the detection of P. aeru-
ginosa in cultured samples; a reliable detection method
for this bacterium would be valuable in the clinical
management of cystic fibrosis.
Figure 2. Evaluation of 9-(40-N-[L-Ala-L-Ala-L-Ala]aminophenyl)-10-
methylacridinium bis-trifluoroacetate 4e with nine Gram-negative and
ten Gram-positive bacterial strains.
Table 3. Percentage of bacterial strains detected by substrates 4b, 4g
and 3e
Bacterial strain
No. of strains 4b 4g 3e
Burkholderia cepacia
Burkholderia gladioli
Other Burkholderia spp.
Pseudomonas aeruginosa
Other Pseudomonas spp.
Pandoraea spp.
34
6
53
0
9 100
83
0
9
33 56 88
100 97 100
35 24 94
38 38 100
50 50 100
19 38 100
74
17
8
Ralstonia picketti
4
Other Ralstonia spp.
Stenotrophomonas maltophilia
Acinetobacter spp.
16
2
Acknowledgment
0
0
0
0 100
71
0 100
7
0
We thank the EPSRC mass spectrometry service centre
(Swansea, UK) for some high-resolution mass spectra.
Brevindomonas spp.
Moraxella spp.
2
3
67 33 100
Sphingobacterium spiritivorum
Chryseobacterium meningosepticum
Oligella spp.
1
100
0 100
0 100
100 100 100
1
0
Supplementary data
1
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.018.
[L-pyroglutamyl]aminophenyl)-10-methylacridinium io-
dide 4g, and the b-alanyl substrate, 9-(40-N-[b-alanyl]-
aminophenyl)-10-methylacridinium bis-trifluoroacetate
4b. These substrates showed high specificity for P. aeru-
ginosa, along with Burkholderia cepacia and Serratia
marcescens. To evaluate the extent of their specificity,
an extended range of non-fermentative bacteria were
tested with these two substrates, along with 9-(40-
N-[Na-tBoc-L-Ala-L-Ala-L-Ala]aminophenyl)-10-methyl-
acridinium iodide 3e as a control substrate that is hydro-
lysed by most Gram-negative bacteria, Table 3.
References and notes
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2. Manafi, M. Int. J. Food Microbiol. 2000, 60, 205.
3. Cerny, G. Eur. J. Appl. Microbiol. 1976, 3, 223.
4. Cerny, G. Eur. J. Appl. Microbiol. Biotechnol. 1978, 5, 113.
5. Gordon, D. B.; DeGirolami, P. C.; Bolivar, S.; Karafotias,
G.; Eichelberger, K. Am. J. Clin. Pathol. 1988, 90, 210.
6. Wellstood, S. A. J. Clin. Microbiol. 1987, 25, 1805.
7. Inoue, K.; Miki, K.; Tamura, K.; Sakazaki, R. J. Clin.
Microbiol. 1996, 34, 1811.
8. Jensch, T.; Fricke, B. J. Basic Microbiol. 1997, 37, 115.
9. James, A. L.; Perry, J. D.; Rigby, A.; Stanforth, S. P.
Bioorg. Med. Chem. Lett. 2007, 17, 1418.
10. Acheson, R. M.; Birtwistle, D. H. J. Chem. Res. 1986,
3425.
The ability of the b-alanyl substrate, 4b, to detect the
presence of 100% of P. aeruginosa strains was encourag-
ing. This substrate showed promising potential for the
detection of P. aeruginosa in cultured samples and could
be used with complementary substrates and selective
agents to enhance the specificity of detection. The