Synthesis and evaluation of novel chromogenic aminopeptidase substrates for microorganism detection and identification

Article abstract of DOI:10.1016/j.bmcl.2006.11.088

The amides 8a-e and 10a-c were prepared as chromogenic aminopeptidase substrates. A range of microorganisms were grown in the presence of these compounds and coloured colonies were produced in several cases after addition of acetic acid-thus giving potent

Full text of DOI:10.1016/j.bmcl.2006.11.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1418–1421
Synthesis and evaluation of novel chromogenic aminopeptidase
substrates for microorganism detection and identification
Arthur L. James,a John D. Perry,b Annette Rigbya and Stephen P. Stanfortha,*
aSchool of Applied Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK
bDepartment of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK
Received 13 September 2006; revised 23 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—The amides 8a–e and 10a–c were prepared as chromogenic aminopeptidase substrates. A range of microorganisms were
grown in the presence of these compounds and coloured colonies were produced in several cases after addition of acetic acid—thus
giving potential methods for the detection of aminopeptidase activity and for microorganism identification.
Ó 2006 Elsevier Ltd. All rights reserved.
Aminopeptidase activity in microorganisms can often be
detected by observing the change in colour when the
amide bond in a weakly coloured amide substrate 1 is
hydrolysed by the aminopeptidase giving a strongly col-
oured amine product 2 (Scheme 1). The strong colour of
the amine is a consequence of delocalisation of the
amine-nitrogen lone-pair throughout the aromatic ring
system. The taxonomic utility of aminopeptidases for
identification and/or discrimination of bacteria has long
been recognised. L-Pyrrolidonyl peptidase has proven to
be a useful test in schemes for the rapid identification of
Salmonella spp.,1 enterococci and Group A streptococ-
ci,2,3 whereas L-c-glutamic acid aminopeptidase can be
used to differentiate Shigella spp.4 Detection of proline
aminopeptidase has been used for the identification of
Neisseria meningitidis5 as well as a diagnostic aid in
the confirmation of bacterial vaginosis.6 There has been
longstanding interest in the detection of L-alanine ami-
nopeptidase for the differentiation of Gram-positive
and Gram-negative bacteria. This is due to the fact that
Gram-negative bacteria readily demonstrate the pres-
ence of this enzyme whereas Gram-positive bacteria do
not. Alanine aminopeptidase has been shown to be a
periplasmic enzyme in Pseudomonas aeruginosa.7 Differ-
entiation is usually achieved using L-alanyl-p-nitroani-
lide as a chromogenic substrate8 although fluorogenic
peptidase substrates have also been applied to this task
in commercially available assays.9 The last 15 yr has
seen an expansion in the use of chromogenic agar media
in diagnostic microbiology for the rapid detection of
pathogenic bacteria in both clinical samples10 and
food.11 Such media exploit chromogenic substrates that
can be hydrolysed to yield chromogens that remain
restricted to bacterial colonies and do not diffuse in
agar. This allows isolation and detection of target
pathogens within polymicrobial cultures. Chromogenic
media have relied on esters of various non-diffusible
chromogens such as glycosides of indoxyl and its halo-
genated derivatives.12 To date, no chromogenic media
have exploited the diagnostic potential of peptidase sub-
strates, largely due to the lack of appropriate substrates
that yield a coloured product that does not diffuse in
agar.
In this paper, the preparation of a selection of novel L-
alanine and b-alanine acridine-based substrates is
reported and their potential application for detecting
and identifying a range of clinically important microor-
ganisms is described. The substrates we have synthesised
and evaluated are the L-alanine 8a–e and b-alanine 10a–
c derivatives of the 9-(40-aminophenyl)acridines 6a–e
(Scheme 2). Upon hydrolysis by aminopeptidases, these
Scheme 1. Aminopeptidases.
Keywords: Aminopeptidase; Microorganism identification; Microor-
ganism detection; Diagnostic reagents; Chromogenic reagents;
9-(40-aminophenyl)acridines.
*
Corresponding author. Tel.: +44 0191 2274784; fax: +44 0191
2273519; e-mail: steven.stanforth@unn.ac.uk
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.088

A. L. James et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1418–1421
1419
Scheme 2. Synthesis of the substrates 8 and 10. Reagents and conditions: (i) S8, heat; (ii) BOC-L-alanine, NMM, IBCF, ꢀ10 to ꢀ15 °C, then add 6;
(iii) BOC-b-alanine, NMM, IBCF, ꢀ10 to ꢀ15 °C, then add 6; (iv) EtOAc, HCI. BOC, tert-butyloxycarbonyl; NMM, N-methylmorpholine; IBCF,
iso-butylchloroformate.
Scheme 3. Hydrolysis of the substrates 8 and 10.
substrates afford the 9-(40-aminophenyl)acridines 6a–e
which are pale yellow at physiological pH but give red
colours when protonated by acetic acid (Scheme 3).13
The aminopeptidase substrates 8a–e and 10a–c were
evaluated against the range of clinically important
microorganisms shown in Table 1. In general, the
L-alanyl substrates 8a–d did not inhibit growth of the
Gram-negative organisms and cream or yellow colonies
were produced in the presence of the substrate. Addition
of acetic acid resulted in the production of various
shades of red-coloured colonies (see Scheme 3) due to
protonation of the amino-acridines 6a–d which are pro-
duced by hydrolysis of the substrates. The L-alanyl sub-
strate 8e inhibited the growth of the Gram-negative
organisms Escherichia coli and Klebsiella pneumoniae.
Figure 1 shows hydrolysis of substrate 8c by colonies
of E. coli (NCTC 10418). Yellow colonies become red-
violet within seconds on addition of glacial acetic acid.
The 9-(40-aminophenyl)acridine derivatives 6 that pro-
vide the aromatic amine fragment of the substrates were
prepared by heating the appropriately substituted ani-
lines 4 with acridine hydrochloride 5 in the presence of
elemental sulfur. This method of preparing 9-(40-amino-
phenyl)acridine derivatives 6, which has been used previ-
ously by other workers, afforded the known compounds
6a14,15 and 6b16 and the novel products 6c–e in 42–65%
yield. Reaction of the amines 6a–e with BOC-L-alanine
afforded the protected amides 7a–e, respectively (76–
83%), from which the substrates 8a–e (65–76%) were ob-
tained as their dihydrochloride salts by deprotection
with hydrogen chloride. The dihydrochloride salts of
the b-alanyl substrates 10a–c (67–71%) were similarly
prepared from the corresponding protected amides 9a–
c which in turn were synthesised from amines 6a–c
and BOC-b-alanine (75–80%). Full details of the syn-
thetic work are given in the Supplementary information.
In order to demonstrate that the development of colour
was due to specific enzyme activity, substrates 8a–d were
incubated with purified L-alanine aminopeptidase. Sub-
strates 8a–d all generated an immediate pink-orange col-
ouration on addition of acetic acid only in the tubes
containing enzyme. No change of colour was observed

1420
A. L. James et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1418–1421
Table 1. Colour formation as a result of aminopeptidase activity in a range of microorganisms
Substrate
Organism
Organism growth:
substrate absenta
Organism growth:
substrate presenta
Colony colour in
presence of substrate
Colony colour
after acidification
8a
Escherichia colib
++
++
++
++
++
Cream
Cream
Cream
Pink-orange
Pink-orange
Diffuse orange
Klebsiella pneumoniaeb
Pseudomonas aeruginosab
Burkholderia cepaciab
Staphylococcus aureusc
Enterococcus faecalisc
++
Not evaluated
++
++
++
++
Pale yellow
Yellow
Yellow
Yellow
8b–d
Escherichia coli
++
++
++
++
++
++
++
++
Yellow
Yellow
Yellow
Yellow
—
Red-violet
Red-brown
Orange-brown
Orange-brown
—
Klebsiella pneumoniae
Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus
Enterococcus faecalis
++
++
No growth
No growthd
—
—
8e
Escherichia coli
++
++
++
++
++
++
Trace
+
++
None
None
None
None
—
Red-violet
Red-brown
Pale orange
Orange-brown
—
Klebsiella pneumoniae
Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus
Enterococcus faecalis
++
No growth
No growth
—
—
10a
10b
10c
Escherichia coli
++
++
++
++
++
++
No growth
++
++
—
—
None
Orange-brown
Klebsiella pneumoniae
Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus
Enterococcus faecalis
None
None
None
—
++
No growth
No growth
None
—
—
—
Escherichia coli
++
++
++
++
++
++
No growth
+
++
—
—
Klebsiella pneumoniae
Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus
Enterococcus faecalis
None
Pale yellow
Yellow
—
None
Red-brown
Orange
—
++
No growth
No growth
—
—
Escherichia coli
++
++
++
++
++
++
No growth
+
++
—
—
Klebsiella pneumoniae
Pseudomonas aeruginosa
Burkholderia cepacia
Staphylococcus aureus
Enterococcus faecalis
Pale yellow
Yellow
Yellow
—
Orange
Red-brown
Red-brown
—
++
No growth
No growth
—
—
a ++, Good growth ; +, moderate growth; +/ꢀ, poor growth.
b Gram-negative organism.
c Gram-positive organism.
d Moderate growth with substrate 8d.
in any tubes without the enzyme demonstrating that col-
our generation was associated with hydrolysis by a spe-
cific enzyme. Figure 2 shows the colour resulting from
hydrolysis of substrate 8c by purified L-alanine amino-
peptidase following acidification.
The b-alanyl substrates 10a–c inhibited the growth of
the Gram-negative organism E. coli and with K. pneu-
moniae inhibition was also noted with substrates 10b
and 10c. No significant colour change in the colonies
produced by these organisms was observed on exposure
to acetic acid suggesting that hydrolysis of the substrates
had not occurred. In contrast, P. aeruginosa and Burk-
holderia cepacia produced coloured colonies with sub-
strates 10b and 10c in the presence of acetic acid.
Substrates 10a–c did not produce any colour change in
the presence of purified L-alanine aminopeptidase.
Figure 1. Hydrolysis of substrate 8c by colonies of E. coli (NCTC
10418) after addition of glacial acetic acid.

A. L. James et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1418–1421
1421
of Gram-negative ones and also as a method of distin-
guishing between these two types of organisms.
Acknowledgments
We thank Drs Sylvain Orenga and Celine Roger-Dal-
bert for their input to this project and for useful discus-
´
sions. We thank BioMerieux SA for funding this project
and the EPSRC mass spectrometry service centre
(Swansea, UK) for high-resolution mass spectra.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.11.088.
Figure 2. Colour resulting from hydrolysis of substrate 8c by L-alanine
aminopeptidase following acidification (left, substrate with enzyme;
right, substrate without enzyme).
References and notes
In contrast to the Gram-negative organisms described
above, and with the exception of substrate 8a, the other
substrates described in this paper inhibited the growth
of the Gram-positive organisms Staphylococcus aureus
and Enterococcus faecalis. In cases where growth had
occurred with substrate 8a, no significant change in col-
ony colour was observed on addition of acetic acid. This
suggests that the substrates had not been hydrolysed by
aminopeptidases.
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13. James, A.; Rigby, A. 2004, Patent WO 2004/069804 A1.
14. Trofimov, V. A.; Chupakhin, O. N.; Pushkareva, Z. V.;
Rusinov, V. L. Khim. Geterotsikl. Soedin. 1971, 112.
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For the bacterial strains that generated coloured colo-
nies on addition of acetic acid, it was of particular value
to note that the colour remained tightly localised on the
colony and was not seen in the surrounding agar. This
may be due to either the relatively large molecular size
and lipophilic nature of the chromogen or localisation
of the chromogen on or within the microorganism. This
indicates that such substrates have the potential to dis-
criminate aminopeptidase-producing pathogens within
heavily mixed polymicrobial cultures.
In conclusion, a series of novel aminopeptidase sensitive
substrates have been prepared, characterised and evalu-
ated. Many of the substrates proved useful for the detec-
tion of Gram-negative microorganisms. Particularly
interesting was the inhibitory effect of most of the sub-
strates on the growth of Gram-positive organisms and
this could have practical applications in suppressing
the growth of Gram-positive organisms in the presence