The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and their applications in diagnostic microbiology

The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and

their applications in diagnostic microbiology

a

b

c

a

Michael Burton, John D. Perry, Stephen P. Stanforth and Hayley J. Turner*

a

Glycosynth Ltd, 14 Craven Court, Winwick Quay, Warrington, Cheshire, WA2 8QU, UK.

Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, NE7 7DN, UK.

Department of Applied Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK.

b

c

Abstract: The preparation and evaluation of chromogenic substrates for detecting bacterial

glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal

chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene

(6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced

coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A

β-D-riboside of DHN and a β-D-glucuronide derivative of 6,7-dibromo-DHN were particularly

effective for the detection of S. aureus and E. coli respectively.

Key words: enzyme substrates, chromogenic substrates, glycosidase, pathogenic microorganisms,

bacterial detection.

Introduction

Synthetic enzyme substrates are utilised extensively in diagnostic clinical microbiology for the

1

-3

purpose of detecting and identifying pathogenic microorganisms. These substrates are designed to

target microbiological species of interest (or groups of species) based upon their enzyme activity. An

important sub-class of synthetic enzyme substrates are the chromogenic sugar-based enzyme

substrates in which hydrolytic cleavage of the sugar moiety from the aglycone is mediated by an

appropriate enzyme resulting in the liberation of a hydroxyaryl derivative, as shown by the

representative examples in Scheme 1. The hydroxyaryl derivative can be coloured, thus allowing

direct visualisation of the hydrolytic reaction as illustrated by the transformation of the colourless

ortho-nitrophenyl β-D-galactopyranoside 1 into the yellow-coloured ortho-nitrophenol (ONP) (Eqn

4

1

). Alternatively, if the liberated hydroxyaryl derivative is colourless, a subsequent chemical

reaction can be employed to produce a coloured product. Thus, the β-galactoside derivative of 5-

bromo-4-chloro-3-hydroxyindole (‘X-gal’) 2 is hydrolysed to produce 5-bromo-4-chloro-3-

hydroxyindole which then undergoes an oxidative dimerization in air producing the blue-coloured

5

TM

6

indigo derivative 3 (Eqn 2). The indole-derived substrate, ALDOL 455 4, similarly generates a

reactive 3-hydroxyindole intermediate which participates in a subsequent non-oxidative

intramolecular aldol condensation yielding the yellow chromophore 5 (Eqn 3). Sugar-based

chromogenic substrates have also been designed around a glycosidated catechol moiety (Figure 1).

After enzymatic hydrolysis of the substrate, the resulting catechol aglycone undergoes chelation

with metal ions that have been incorporated into the medium, therefore producing coloured metal-

chelates. The hydrolysis of esculin 6 by a β-glucosidase enzyme yields D-glucose and esculetin (6,7-

7

dihydroxycoumarin) which, in the presence of iron salts, produced a brown/black complex.

8

,9

10

Cyclohexenoesculetin-β-D-glucoside 7 (and also its β-D-galactoside derivative) similarly

9

generated a black complex in the presence of iron salts. Alizarin β-D-glucoside 8 (and also its β-D-

1

11

galactoside derivative) yielded a purple-coloured chelate in the presence of iron salts and a pink-

1

2

coloured chelate with aluminium salts. Hydrolysis of 3’,4’-dihydroxyflavone β-D-ribofuranoside

gave black colonies in the presence of iron and yellow colonies in the presence of aluminium. Other

sugar-based substrates, which after enzymatic hydrolysis produce aglycones capable of chelation

with metal ions, have also been prepared from non-catechol cores including glycosides of 8-

1

3

9

hydroxyquinoline and 3-hydroxyflavone.

In this paper, we report the synthesis and microbiological evaluation of a series of novel

chromogenic sugar-based enzyme substrates based upon catechol, 2,3-dihydroxynaphthalene and

1

4,15

6,7-dibromo-2,3-dihydroxynaphthalene 9 cores (Figure 1).

2,3-Dihydroxynaphthalene is

inexpensive and available in large quantities (> 100 g) from several commercial suppliers thus

making this an ideal starting material for the synthesis of enzyme substrates. Additionally, halogen

atoms can be introduced into this ring-system remote from the hydroxyl-groups, i.e. at the 6,7-

positions, whereas the introduction of halogen atoms into known substrates such as compounds 6

and 7 would only be possible adjacent to the hydroxy-groups which may have a detrimental effect

on glycosidase activity. We anticipated that the introduction of halogen atoms would be beneficial

for reducing diffusion of chelates in solid (agar) media. We envisaged that catechol-derived

substrates would have potential applications in liquid media (where the resulting metal chelates

would require appreciable aqueous solubility) and that the increased size of the naphthalene-

derived substrates 9 would potentially generate a more insoluble end-point better suited for use in

solid (agar) media, where diffusion of the chelate must be localised within colonies of

microorganisms. The sugar components of structures 9 have been chosen to target a broad range of

enzymatic activities across a range of clinically important pathogenic microorganisms. The sugar

moieties together with illustrative applications in diagnostic microbiology include: (i) β-D-

glucopyranosides (for the detection of enterococci and Listeria monocytogenes), (ii) β-D-

galactopyranosides (for the detection of coliforms), β-D-glucuronides (for the detection of

Escherichia coli), N-acetylhexosaminides (for the detection of the pathogenic yeast, Candida

albicans) and β-D-ribofuranosides (for the detection of Staphylococcus aureus, including MRSA).

1

6

17

Catechol β-D-ribofuranoside has previously shown efficacy for S. aureus detection in liquid media.

2

Scheme 1. Hydrolysis of chromogenic enzyme substrates by β-galactosidase giving coloured

products.

Figure 1. Chromogenic enzyme substrates possessing a catechol moiety.

Synthesis of substrates

Catechol 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 10¹⁸^-²¹was prepared from catechol in low yield

using a Michael-type glycosidation procedure (Scheme 2). A Zemplén deprotection of compound 10

3

gave the required β-glucosidase substrate 11. The proton-NMR spectral data of compounds 10 and

1

1 were consistent with those reported in the literature with large anomeric coupling constants

2

0, 22

confirming the β-configurations at the anomeric centres.

The direct reaction of glucose and

2

3

catechol has been reported to give a 95:5 ratio of α:β anomers in low (11%) overall yield. 2,3,4,6-

Tetra-O-acetyl-β-D-galactopyranoside 12 was prepared using a Michel-type glycosidation reaction

and after deprotection, the β-galactosidase substrate 13 was obtained. The coupling constant for the

anomeric proton (7.7 Hz, d

6

-DMSO) and the chemical shift of C-1 (104.0 ppm, d -DMSO) in the

6

proton and carbon NMR spectra of compound 13 respectively, confirmed the presence of a β-

glycoside. The tetraacetyl derivative 12 has been described previously in the literature and was

24

reported as comprising a mixture of both α- and β-anomers. Somewhat surprisingly, the substrate

3 appears to be novel although the synthesis of its isomer with the α-configuration has been

1

2

4

claimed but no NMR-spectral data was disclosed to support this assignment. Additionally, the large

negative optical rotation (-33 in DMSO) reported for this proposed α-anomer structure is more

o

aligned to the value expected from a β-D-galactopyranoside. The protected glucuronide derivative

2

5, 26

25

14

was prepared from catechol following a similar procedure to that reported in the literature.

Deprotection of compound 14 under basic conditions, followed by acidification using an ion-

exchange resin, afforded the required β-glucuronic acid derivative which was conveniently isolated

as the cyclohexylamine salt 15. The impure glucuronic acid has been proposed as a catechol

2

7

metabolite produced from rabbits but this compound was not characterised directly. Methylation,

per-acylation and finally hydrolysis of the metabolite gave 2-methoxyphenol (guaiacol) which

suggested the rabbit metabolite was a mono-glucuronide.

Scheme 2. Preparation of catechol-derived substrates. Reagents and conditions: (i) α-D-

acetobromoglucopyranoside, acetone, aq. NaOH, rt, overnight; (ii) MeOH, NaOMe, rt, overnight; (iii)

α-D-acetobromogalactopyranoside, acetone, aq. NaOH, rt, overnight; (iv) 1,2,3,4-tetra-O-acetyl α-D-

2

5

o

glucuronic acid methyl ester, 4-toluenesulfonic acid (cat.), reduced pressure, 70-75 C, 50 min; (v)

(

+

a) aq. NaOH, acetone, rt, 2 h, (b) Amberlite IR 120 H ion exchange resin, (c) cyclohexylamine.

4

The synthetic routes to the substrates based on the 2,3-dihydroxynaphthalene and 6,7-dibromo-2,3-

dihydroxynaphthalene cores are depicted in Scheme 3. A Michael-type glycosylation of 2,3-

dihydroxynaphthalene 16a gave the acetylated sugar 17a which was deprotected giving the required

β-glucosidase substrate 18a.²⁸^-³⁰A photoluminescence approach for the detection of β-D-glucosidase

3

1

activity (but not in microorganisms) using compound 18a has recently been reported. The physical

2

8

and spectral data of compounds 17a and 18a were in good accord with published data. Similarly

prepared were the novel dibrominated analogues 17b and 18b, both of which were associated with

the large anomeric proton coupling constants and negative optical rotations confirming the presence

of the β-isomers. The novel β-galactosidase substrates 20a and 20b were prepared from compounds

19a and 19b respectively by analogous procedures. The β-glucosaminidase substrate 22a was

synthesised from the reaction of 2,3-dihydroxynaphthalene 16a and α-acetochloroglucosamine

under basic conditions (giving the intermediate 21a) followed by deprotection. The β-glucuronidase

substrate 24a was synthesised using a similar procedure to that shown in Scheme 2 for the

preparation of substrate 15. The intermediate 23b was produced from the reaction of naphthalene

3

2

1

6b with a glucuronide trichloroacetimidate in the presence of boron trifluoride etherate.

Deprotection of compound 23b, followed by treatment with cyclohexylamine then afforded the

3

substrate 24b. The reaction of either α-D-ribofuranosyl trichloroacetimidate or 1,2,3,5-tetra-O-

acetyl-β-D-ribofuranose with naphthalenes 16a and 16b in the presence of boron trifluoride

etherate produced the β-anomers of the acetylated intermediates 25a and 25b. Deprotection of

these two compounds afforded the required ribofuranoside substrates 26a and 26b respectively.

5

Scheme 3. Preparation of 2,3-dihydroxynaphthalene and 6,7-dibromo-2,3-dihydroxynaphthalene

derived substrates. Reagents and conditions: (i) α-D-acetobromoglucopyranoside or α-D-

o

acetobromogalactopyranoside, acetone, aq. NaOH, rt, overnight; (ii) MeOH, NaOMe, 4 C, overnight;

(iii) α-acetochloroglucosamine, acetone, K

2

CO , heat on water bath, 15 min.; (iv) 1,2,3,4-tetra-O-

3

acetyl-α-D-glucuronic acid methyl ester, 4-toluenesulfonic acid (cat.), AcOH, Ac

2

O, reduced pressure,

o

1

20 C, 1 h (compound 23a) or α-D-glucuronide trichloroacetimidate, BF

3

•Et

2

O, CH Cl , rt, 30 min.

2

+

(compound 23b); (v) (a) aq. NaOH, acetone, rt, 2 h, (b) Amberlite IR 120 H ion exchange resin, (c)

cyclohexylamine; (vi) α-D-ribofuranosyl trichloroacetimidate, BF

5a); (vii) 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose, BF •Et O, 3 Å mol. sieves, CH

compounds 25b).

3

•Et

2

O, CH

2

Cl

2

, 5 min., rt (compound

2

(

3

2

Cl , 15 min., rt

2

Evaluation of substrates

In order to simplify the microbiological evaluation of the substrates, each assay comprised a

representative panel of 20 clinically important microorganisms which were inoculated

simultaneously onto a single Columbia agar plate. A standardised inoculum of approximately 10

8

colony forming units (CFU)/mL was prepared using a densitometer and 1 µL was delivered onto the

6

agar surface using a multipoint inoculation device (final inoculum: approximately 10 CFU/spot). All

o

assays were conducted at 37 C in air for 18 hours. For each assay, the panel of microorganisms

comprised 10 Gram-negative bacteria, 8 Gram-positive bacteria and 2 yeasts. Each substrate was

-

1

incorporated into the agar medium at a concentration of 300 mgL in the presence of ammonium

-

1

iron(III) citrate (500 mgL ). The substrates were added to molten agar at 50°C, after the agar had

been sterilised by autoclaving.

Microorganism growth was compared to control plates in which no substrate or metal salt was

-

1

present. Control plates were also prepared containing metal ions (500 mgL ) in the absence of

substrates. All of the collection of microorganisms exhibited good growth on the substrate-free

control plates and no growth inhibition was apparent in the presence of the metal salt.

For illustrative purposes, selected substrates were subjected to additional testing in a broth-based

medium. For this purpose, we selected tryptone soya broth (Oxoid) that was supplemented with

-

1

ammonium iron(III) citrate (500 mgL ) before sterilization by autoclaving. The broth was then

-

1

6

supplemented aseptically with 300 mgL of substrate and inoculated with 10 CFU of test organism

before incubation for 18 h at 37°C.

Strong growth was observed for the whole panel of microorganisms in the presence of the catechol-

derived substrates 11, 13 and 15 exemplifying the non-inhibitory nature of these substrates (Table

1). Where hydrolysis of the substrates had occurred and catechol was liberated, the brown colour of

the resulting iron chelate was extensively dispersed around the border of the colonies and this was

attributed to diffusion of the catechol into the surrounding medium. The microbial strains showed

mostly expected activity with the glucopyranoside substrate 11 with the exception of an unexpected

weak positive reaction with Salmonella typhimurium, which is not known to produce β-glucosidase.

The weak reaction observed with E. coli is consistent with the low level of inducible enzyme known

to be produced by this species.⁹^,³⁴For substrate 13, coloration was only generated by known

producers of β-galactosidase and hydrolysis of the glucuronide substrate 15 only occurred with E.

coli as expected.

6

Substrate 11

Substrate 13

Substrate 15

Microorganism / Reference^a

Gram-negative microorganisms

Escherichia coli NCTC 10418

Serratia marcescens NCTC

Growth^b

Colour^c

Growth^b

Colour^c

Growth^b

Colour^c

1

2

++

+/- brown

++

+ brown

++

+ brown

1

0211

Pseudomonas aeruginosa NCTC

0662

Burkholderia cepacia ATCC

5416

Yersinia enterocolitica NCTC

1176

Salmonella typhimurium NCTC

+ brown

-

+/- brown

-

3

4

5

6

++

1

-

2

+ brown

+/- brown

1

-

7

4

++

-

7

8

Citrobacter freundii 46262 (wild)

Morganella morganii 462403

+ brown

-

+ brown

++

(

wild)

Enterobacter cloacae NCTC

1936

++

-

9

1

+ brown

++

1

++

+ brown

-

0 Providencia rettgeri NCTC 7475

Gram-positive microorganisms

1 Bacillus subtilis NCTC 9372

2 Enterococcus faecalis NCTC 775

3 Enterococcus faecium NCTC

1

++

-

++

-

++

-

+ brown

7171

++

+ brown

-

++

+/- brown

++

-

1

4 Staphylococcus epidermidis

NCTC 11047

5 Staphylococcus aureus NCTC

-

6571

+ brown

6 Staphylococcus aureus (MRSA)

NCTC 11939

7 Streptococcus pyogenes NCTC

8

306

8 Listeria monocytogenes NCTC

1994

1

Yeasts

1

9 Candida albicans ATCC 90028

++

-

++

-

++

-

20 Candida glabrata NCPF 3943

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

+

+ strong growth, + moderate growth, +/- weak growth.

c

++ strong colour, + moderate colour, +/- weak colour, - no noticeable colour. Diffusion of the colour

into the medium was noted in all cases.

Table 1. Evaluation of the catechol substrates.

7

The 2,3-dihydroxynaphthalene-derived substrates 18a, 20a and 22a (Table 2) allowed only moderate

growth of all members of the panel of microorganisms suggesting that these substrates were

inhibitory to some extent. Strong growth of the Gram-negative microorganisms and moderate

growth of the Gram-positive microorganisms and the yeasts were seen with the glucuronide

substrate 24a. In contrast, strong growth of all 20 microorganisms was observed in the presence of

the ribofuranoside substrate 26a. Purple coloured colonies were produced with this series of

substrates when appropriate enzymatic activity was present. As with the catechol-derived

substrates, diffusion of colour into the surrounding medium occurred and hence these substrates

would be better suited for use in liquid media. For substrate 18a, coloration was generated only by

known producers of β-glucosidase including enterococci and Listeria monocytogenes, suggesting this

substrate could be exploited for detection of these species. Similarly, for substrate 20a activity was

only observed for known producers of β-galactosidase. Substrate 22a is expected to detect β-

hexosaminidase activity and several bacteria demonstrated hydrolysis of this substrate including

35

known producers of this enzyme e.g. Serratia marcescens. Disappointingly, no coloration was

generated by Candida albicans within the 18 h incubation period and this species is known to

3

6

produce β-hexosaminidase. The glucuronide substrate 24a was only hydrolysed by E. coli, as

expected, producing strongly coloured colonies. Substrate 26a effectively detected β-ribosidase

activity in a number of Gram-negative species and activity was consistent with that previously

1

2, 37

reported for other β-ribosidase substrates.

Among Gram-positive bacteria, reactivity was

restricted to S. aureus strains and there was clear differentiation from other Gram-positive species,

suggesting that this may be a useful substrate for detection of S. aureus. Figure 2 illustrates the

differentiation of S. aureus from other Gram-positive microorganisms in liquid media. The usefulness

1

6

of β-ribosidase detection for the identification of S. aureus has previously been observed.

Substrate Substrate Substrate Substrate Substrate

b

c

d

1

8a

20a

Colour^e

22a

24a

Colour^e

26a

Colour^e

e

Colour^e

Microorganism /

Colour

a

Reference

Gram-negative

microorganisms

1

2

3

4

5

6

7

8

9

1

Escherichia coli NCTC

Tr.

purple

+

+ purple

-

++ purple ++ purple

10418

Serratia marcescens

NCTC 10211

Pseudomonas

aeruginosa NCTC 10662

Burkholderia cepacia

ATCC 25416

Yersinia enterocolitica

NCTC 11176

Salmonella typhimurium

NCTC 74

Citrobacter freundii

NCTC 9750

+

+ purple

++ purple

-

++ purple

-

+ purple

-

++ purple

++ purple^f

++ purple

NT

Morganella morganii

-

462403 (wild)

Enterobacter cloacae

NCTC 11936

Providencia rettgeri

NCTC 7475

+/-

purple

+

purple ++ purple

+ purple

0

+

-

8

1

2

Klebsiella pnueumoniae

NCTC 10896

Acinetobacter

+

+ purple ++ purple

NT

1

-

baumannii ATCC 19606

Gram-positive

microorganisms

13

14

15

16

17

18

19

20

Bacillus subtilis NCTC

+

purple

-

9372

Enterococcus faecalis

NCTC 775

Enterococcus faecium

NCTC 7171

+

purple

-

++ purple

-

Tr.

purple

++ purple

-

Staphylococcus

-

epidermidis NCTC 11047

Staphylococcus aureus

NCTC 6571

-

+ purple

Staphylococcus aureus

-

+ purple

(MRSA) NCTC 11939

Streptococcus pyogenes

NCTC 8306

Listeria monocytogenes

NCTC 11994

-

++ purple

-

+

purple

Yeasts

2

1

2

Candida albicans ATCC

-

9

0028

Candida glabrata NCPF

943

2

3

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

all microorganisms exhibited moderate growth in the presence of these substrates.

the Gram-negative microorganisms exhibited strong growth and the Gram-positive microorganisms

c

and yeasts showed moderate growth in the presence of this substrate.

d

all microorganisms exhibited strong growth in the presence of this substrate.

e

++ strong colour, + moderate colour, +/- weak colour, - no noticeable colour. NT, not tested.

Diffusion of the colour into the medium was noted in all cases.

f

Citrobacter freundii 4626 (wild).

Table 2. Evaluation of the 2,3-dihydroxynaphthalene-based substrates.

9

Figure 2. Detection of β-ribosidase activity using substrate 26a in Staphylococcus aureus NCTC 6571

left) and lack of reaction shown by Staphylococcus epidermidis NCTC 11047 (middle) and

(

Enterococcus faecalis NCTC 775 (right).

In order to produce non-diffusible chelates that might be suited for use in solid (agar) media, the

series of 6,7-dibromo-2,3-dihydroxynaphthalene substrates 18b, 20b, 24b and 26b were prepared

(Table 3) with the expectation that the incorporation of the bromine substituents would reduce the

solubility of the resulting chelates in this medium, thus limiting their diffusion out of bacterial

colonies. Microorganism growth was generally moderate in the presence of substrates 18b and 20b

and strong in the presence of substrate 24b whereas substrate 26b was inhibitory to Gram-positive

bacteria. In agreement with expectation, the brown colours generated by enzymatic hydrolysis of

these substrates were largely localised within the boundaries of the microorganism colonies and

diffusion of colour into the surrounding medium was minimal.

A β-D-ribofuranoside derived from 1,4-dibromo-2,3-dihydroxynaphthalene was also prepared (i.e.

the isomer of compound 26b) but this substrate produced a strong background coloration in agar

media and no discernible enzyme activity could be observed against the background. This suggests

that the location of bromine atoms adjacent to hydroxy-groups would not produce practical

substrates as noted in the introduction.

10

Substrate 18b

Substrate 20b

Substrate

4b

Substrate 26b

2

Microorganism /

Gro_bColour^c

wth

Gro Colour Gro Colour Gro Colour

a

b

c

b

c

Reference

wth

wt

wth

b

h

Gram-negative

microorganisms

Escherichia coli

NCTC 10418

Serratia

marcescens NCTC

1

2

Tr.

brown

++

brown

++

brown

+

++

-

+

++

brown

+

-

+

brown

10211

3

4

5

6

7

8

9

1

Pseudomonas

aeruginosa NCTC

+

-

+

-

++

+

brown

10662

Burkholderia

cepacia ATCC

+

+/-

brown

+/-

brown

25416

Yersinia

enterocolitica

NCTC 11176

Salmonella

typhimurium

NCTC 74

-

+

-

+/-

+

brown

Citrobacter

freundii NCTC

+

++

brown

+ brown

brown

9750

Morganella.

morganii 462403

+

-

+

brown

(wild)

Enterobacter

cloacae NCTC

+

+/-

+

brown

11936

0

Providencia

rettgeri NCTC

+

-

brown

7475

Gram-positive

microorganisms

Bacillus subtilis

NCTC 9372

Enterococcus

faecalis NCTC 775

Enterococcus

faecium NCTC

1

2

3

+

+ brown

+

-

++

-

Tr.

brown

Tr.

brown

+

/-

+/-

brown

+

+/-

+

++

-

brown

7171

14

Staphylococcus

epidermidis NCTC

-

11047

11

15

16

17

18

Staphylococcus

aureus NCTC

Tr.

brown

+

-

++

-

6571

Staphylococcus

aureus (MRSA)

NCTC 11939

Streptococcus

pyogenes NCTC

Tr.

brown

-

+/-

+

-

8306

Listeria

monocytogenes

NCTC 11994

+

/-

+/-

brown

Yeasts

1

9

0

Candida albicans

ATCC 90028

Candida glabrata

NCPF 3943

+

-

+

-

++

+

-

+/-

-

2

-

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

+

+ strong growth, + moderate growth, +/- weak growth, no noticeable growth.

c

++ strong colour, + moderate colour, +/- weak colour, Tr. trace of colour, - no noticeable colour.

Table 3. Evaluation of brominated substrates.

Substrate 18b did not show potential as a suitable substrate for detection of β-glucosidase

producing pathogens as weak reactions were observed for enterococci and Listeria monocytogenes.

Some unexpected (albeit weak) positive reactions were also observed for other species such as S.

aureus. β-Galactosidase is most often targeted as a useful marker for coliforms and moderate to

strong positive reactions were obtained as expected for E. coli, S. marcescens and C. freundii with

substrate 20b. However, E. cloacae (a β-galactosidase producing coliform) did not produce

coloration. It is likely that activity of this and other species could be improved by inclusion of an

inducer of β-galactosidase such as isopropyl-β-D-thiogalactoside (IPTG). As anticipated, the

glucuronide substrate 24b was only hydrolysed by E. coli producing strongly coloured colonies and

illustrative agar plates are depicted in Figure 3. In addition to the 20 microorganism plate which

clearly shows a positive response for E. coli, four single microorganism plates which included two E.

coli strains (expected to give positive responses) and S. marcescens and S. typhimurium (both

expected to produce negative responses) were prepared. These four plates also showed a clear

difference between positive and negative results and a good contrast between the colour of the

colonies and the background. Growth inhibition was even more pronounced with substrate 26b with

inhibition observed for E. coli, P. rettgeri and all of the eight Gram-positive bacteria tested. This

precluded its potential application for the detection of S. aureus.

12

Figure 3. Agar plates produced using substrate 24b. Left frame: arrangement of 20 microorganisms

on a single agar plate (blue spots represent Gram-negative bacteria, red spots represent Gram-

positive bacteria and the two yellow spots are the yeast species). Numbers correspond to

microorganisms in Table 3. Right frame: left, 20 selected microorganisms on a single agar plate;

middle top, E. coli NCTC 10418; top right, E. coli NCTC 12241; middle bottom, S. marcescens NCTC

10211; bottom right, S. typhimurium NCTC 74.

Conclusion

We have reported the synthesis and evaluation of a novel series of chromogenic glycosidase

substrates, some of which show potential for the detection of pathogenic bacteria. By introducing

two bromine atoms at the 6,7-positions in the naphthalene-based substrates, diffusion of the

resulting chromogens into agar media is minimised. The chromogens produced by the non-

brominated substrates diffused from the microorganism colonies thus precluding their use in agar

media, but these substrates have potential for use in liquid media.

Acknowledgements

We thank the EPSRC UK National Mass Spectrometry Facility at Swansea University, UK, for high

resolution mass spectra. We also thank Dr Karen Haggerty for performing the HPLC work.

Experimental

1

NMR spectra were recorded on a Jeol spectrometer at either 270 or 400 MHz for H spectra and

1

3

either 68 or 100 MHz for C spectra. All chemical shifts are quoted in ppm relative to

tetramethylsilane. In the assignment of signals the abbreviation DHN is used for 2,3-

dihydroxynaphthalene and CHA for cyclohexylamine. Optical rotations were measured on an Optical

13

Activity AA10 polarimeter. High resolution mass spectra (HRMS) were obtained from the EPSRC

Mass Spectrometry Facility (Swansea). Flash chromatography was performed using Fluorochem Ltd

silica gel (60 Å). Mixed solvents are recorded as volumetric ratios. EtOH is methylated spirit. Water is

deionised water. HPLC was performed using an Agilent 1260 modular HPLC instrument fitted with a

multiple wavelength detector and a Phenomenex Kinetex 2.6 μ C18, 150 x 2.1 mm column. Data was

acquired at 280 nm with a flow rate of 0.4 mL per minute at ambient temperature. Mobile phase A

was water containing 0.1% v/v formic acid and mobile phase B was acetonitrile containing 0.1% v/v

formic acid. Substrates were prepared in a mixture 95% A/ 5% B. The gradient used was: 0-10 mins

95% A/ 5% B; 10-13.5 mins 5% A/ 95% B; 13.5-18 mins 95% A/ 5% B. The HPLC traces of key

substrates are shown in the supplementary information.

3

8

6

,7-Dibromo-2,3-dihydroxynaphthalene 16b was prepared following a literature procedure.

2

0

24

28

The known acylated sugar derivatives 10, 12 and 17a were prepared by a Michael-type

glycosidation procedure (420-503 mmol scale) in a solution of acetone and aqueous sodium

hydroxide. The crude products were purified by column chromatography (eluent toluene: acetone

10:1) followed by trituration with EtOH (compounds 10 and 12) or trituration (MeOH) followed by

recrystallization from MeOH (compound 17a). A Zemplén deprotection of compounds 10 and 17a

1

8,22,39

28

afforded the known glycosides 11

similar method to that described in the literature, m.p. 128-130 C (lit. 136-137 C). H NMR:

DMSO-d ) 9.28 (1H, s, OH), 6.99 (1H, d, J 7.9 Hz, Ar-H), 6.93-6.83 (2H, m, Ar-H), 6.73 (1H, m, Ar-H),

.44 (2H, m), 5.09-5.02 (2H, m), 4.62 (1H, d, J4,5 9.9 Hz, H-5), 3.35 (1H, s, OCH ), 2.01 (3H, s, OAc),

) 170.1 (Ac), 169.9 (Ac), 169.6 (Ac), 167.8

and 18a respectively. The ester 14 was prepared by a

o

25 1

(

5

2

6

3

1

3

.00 (3H, s, OAc), 1.99 (3H, s, OAc); C NMR: (DMSO-d

6

(C=O), 148.4, 145.0, 124.7, 119.7, 119.3, 117.2 (catechol), 99.4 (C-1), 71.7, 71.5, 71.3, 69.6 (C-

2

/3/4/5), 53.1 (OCH ), 21.0 (Ac), 20.9 (Ac), 20.8 (Ac).

3

The synthesis of the β-D-glucuronic acid derivatives and the β-D-ribofuranosides are described

below. The preparation of all other compounds is described in the supplementary information.

Catechol-β-D-glucuronic acid, cyclohexylamine salt 15.

Compound 14 (5.46 g, 12.8 mmol) was dissolved in acetone (75 mL) and a solution of NaOH (2.81 g,

7

0.3 mmol) in H O (37.5 mL) was added. The reaction mixture was stirred at room temperature for 2

2

+

hours and then passed down a column of Amberlite IR 120 H ion exchange resin (50 g). Fractions

containing the product were combined and basified with cyclohexylamine (5 mL). The solution was

kept at 4 C overnight and then evaporated, giving an amber oil which was triturated with EtOH (100

mL). A white precipitate formed and the mixture was left at 4 C overnight. The precipitate was then

collected affording compound 15 (3.23 g, 65%) as a white solid, m.p. 208-210 C, [α]

o

22

o

D

-51 (c 0.504

1

in H

2

O). H NMR: (DMSO-d

6

) 7.05, (1H, dd, J 1.5 Hz, J 7.9 Hz, Ar-H), 6.90-6.69 (3H, m, Ar-H), 4.60 (1H,

d, J1,2 7.9 Hz, H-1), 3.40 (1H, d, J4,5 10.0 Hz, H-5), 3.26-3.13 (2H, m), 2.85 (1H, m), 1.81 (2H, m, CHA),

1

3

1

.64 (2H, m, CHA), 1.51 (1H, m, CHA), 1.25-1.18 (5H, m, CHA). C NMR: (DMSO-d

6

) 172.8 (C=O),

1

47.8, 146.1, 123.7, 119.8, 118.3, 116.7, 103.2 (C-1), 76.4, 74.4, 73.7, 72.7 (C-2,3,4,5), 49.7 (C-NH

2

,

-

CHA), 31.1 (CHA), 25.2 (CHA), 24.3 (CHA). HRMS (ESI) for C12

H

13

8

O : m/z calcd 285.0616; measured:

285.0616. The HPLC of this substrate showed a single compound (see supplementary data).

6′-Methyl (3-hydroxynaphthalen-2-yl)-2′,3′,4′-tri-O-acetyl-β-D-glucuronate 23a.

A mixture of compound 16a (34.4 g, 215 mmol) and 1,2,3,4-tetra-O-acetyl-α-D-glucuronide 6-methyl

o

ester (MTAG) (40 g, 106 mmol) were heated in an oil bath to 120 C on a rotary evaporator under

reduced pressure until a homogeneous melt was obtained. 4-Toluenesulphonic acid (PTSA) (150 mg,

o

0

.8 mmol) in acetic acid/acetic anhydride (1 mL) was added and the mixture was stirred at 120 C on

14

a rotary evaporator under reduced pressure for 1 hour. A TLC of the reaction mixture showed some

remaining MTAG and a further quantity of PTSA (150 mg, 0.8 mmol) in acetic acid/acetic anhydride

o

(

1 mL) was added and the mixture stirred at 120 C under reduced pressure for a further 30 min. The

dark oil was allowed to cool to room temperature and after standing overnight was dissolved in

CH Cl (300 mL). The solution was washed sequentially with sat. aq. NaHCO (4 x 50 mL), H O (500

mL), brine (500 mL) and the organic fraction was then separated, dried (MgSO ) and evaporated

2

3

2

4

giving a brown foaming oil (59.1 g). The foam was purified by flash chromatography over silica gel (1

Kg) (eluent; toluene: acetone 10:1) and fractions of approximately 200 mL were collected. Fractions

1

9-26 were combined and evaporated giving a red solid (29.66 g). The red solid was triturated with

o

EtOH (150 mL) and kept at 4 C overnight. The resulting pale yellow, fluffy solid was collected

o

23

o

1

affording compound 23a (12.6 g, 25%), m.p. 191-192 C, [α]

): 9.80 (1H, s, OH), 9.67 (2H, dd, J 8.0 and 12 Hz, Ar-H), 7.45 (1H, s, Ar-H), 7.28 (2H, m, Ar-H), 7.18

1H, s, Ar-H), 5.69 (1H, d, J1,2 8 Hz, H-1), 5.48 (1H, t, J3,4=3,2 10 Hz, H-3), 5.18-5.04 (2H, m, H-2/4), 4.71

D

-26 (c 0.5 in CHCl ). H NMR (DMSO-

3

d

6

(

1

3

1H, d, J 10 Hz, H-5), 3.63 (3H, s, CO

DMSO-d ): 170.1 (Ac), 169.9 (Ac), 169.6 (Ac), 167.8 (C-6) 148.2, 146.4, 131.2, 128.2, 127.3, 126.2,

25.2, 123.9, 114.1, 111.0 (10 x DHN), 98.7 (C-1), 71.8, 71.6, 71.3, 69.6 (C-2/3/4/5), 53.2 (CH ) 21.0

] : m/z calcd 494.1657; measured:

2

CH ), 2.01 (3H, s, Ac), 2.00 (3H, s, Ac), 1.99 (3H, s, Ac); C NMR

3

6

1

3

+

(

Ac), 20.9 (Ac), 20.8, (Ac). HRMS (ESI) for C23

H

28NO11 [M+NH

4

494.1646.

6′-Methyl (6,7-dibromo-3-hydroxynaphthalen-2-yl)-2′,3′,4′-tri-O-acetyl-β-D-glucuronate 23b.

To a stirred solution of α-D-glucuronide trichloroacetimidate (10 g, 21 mmol) and compound 16b (6

g, 18.9 mmol) in CH Cl (100 mL) was added BF •OEt (10 drops, ~ 20 μL). The reaction mixture was

stirred at room temperature for 30 min. and then poured into a mixture of CH Cl (500 mL) and sat.

aq. NaHCO (500 mL). The organic layer was separated and washed with sat. NaHCO (4 x 500 mL),

O (500 mL) and then dried (MgSO ) and evaporated. The residue was triturated with EtOH (100

mL) and the mixture kept at 4 C overnight. The solid was collected giving compound 23b (2.89 g,

2

3

2

3

H

2

4

o

25

o

1

2

4%) as a white solid, m.p. 176-177 C, [α]

D

-16 (c 0.25 in acetone). H NMR (DMSO-d ): 10.22 (1H,

6

broad s, OH), 8.19 (1H, s, Ar-H), 8.17 (1H, s, Ar-H), 7.51 (1H, s, Ar-H), 7.20 (1H, s, Ar-H), 5.66 (1H, d,

1,2 8 Hz, H-1), 5.51 (1H, t, J3,4=3,2 9 Hz, H-3), 5.19-5.05 (2H, m, H-2/4), 4.70 (1H, d, J4,5 9 Hz, H-5), 3.64

1

3

2

CH

3

), 2.03 (3H, s, Ac), 2.01 (3H, s, Ac), 2.00 (3H, s, Ac); C NMR (DMSO-d

6

): 170.1 (Ac),

1

70.0 (Ac), 169.6 (Ac), 167.7 (C-6) 149.5, 147.6, 131.6, 131.3, 130.6, 128.3, 120.1, 118.1, 112.9, 110.0

(

10 x DHN), 98.5 (C-1), 71.7, 71.7, 71.2, 69.7 (C-2/3/4/5), 53.1 (CH

3

) 21.0 (Ac), 20.9 (Ac), 20.8, (Ac).

] : m/z calcd 649.9867; measured: 649.9868.

+

HRMS (ESI) for C23

H

26Br

2

NO11 [M+NH

4

(

3-Hydroxynaphthalen-2-yl)-β-D-glucuronic acid, cyclohexylamine salt 24a.

Compound 23a (6.1 g, 12.8 mmol) was dissolved in acetone (75 mL) and a solution of NaOH (2.81 g,

7

0.25 mmol) in H O (37.5 mL) was added. The mixture was stirred at room temperature for 2 hours

2

+

and then passed down an Amberlite IR120 H ion exchange resin column (50 g). The eluent

containing the product was basified using cyclohexylamine (CHA) (5 mL) producing a white

o

precipitate. The mixture was kept at 4 C overnight and the white fluffy solid was collected, washed

o

19

o

with H

2

O and then acetone affording compound 24a (2.8 g, 53%), m.p. 223-224 C, [α]

D

-96 (c 0.5

1

in H

2

O). H NMR (DMSO-d

6

): 7.64 (2H, m, Ar-H), 7.46 (1H, s, Ar-H), 7.28 (2H, m, J 6.93 Hz, Ar-H), 7.20

(

1H, s, Ar-H), 4.95 (1H, d, J1,2 7 Hz, H-1), 3.58 (1H, d, J3,4=4,5 9 Hz, H-4), 3.35 (2H, m, H-2/3), 3.24 (1H,

1

3

m, H-5), 2.83 (1H, m, CHA), 1.92-1.48 (5H, m, CHA), 1.23-1.05 (5H, m, CHA); C NMR (DMSO-d

1

C

6

):

72.7 (C-6), 147.8, 147.1, 130.7, 128.7, 127.3, 126.3, 124.9, 123.7, 112.1, 110.7 (10 x DHN), 100.0 (C-

), 76.6, 74.8, 73.6, 72.8 (C-2/3/4/5), 49.6 (CHA), 31.0 (CHA), 25.1 (CHA), 24.3 (CHA). HRMS (ESI) for

+

16

H16NO

8

[M+H] : m/z calcd 335.0772; measured: 335.0767. The HPLC of this substrate showed a

single compound (see supplementary data).

(6,7-Dibromo-3-hydroxynaphthalen-2-yl)-β-D-glucuronic acid, cyclohexylamine salt 24b.

15

Following the procedure described above for the preparation of compound 24a, compound 23b (2.5

g, 3.9 mmol) gave compound 24b (1.32 g) and a second crop (840 mg) from concentration of the

o

25

o

filtrate. Compound 24b (2.16 g, 95%) was obtained as a white solid, m.p. 221-224 C, [α]

D

-159 (c

1

0.25 in H

2

O). H NMR (DMSO-d

6

): 8.12 (1H, s, Ar-H), 8.09 (1H, s, Ar-H), 7.47 (1H, s, Ar-H), 7.20 (1H, s,

2

O), 2.86

1

3

1

48.3, 131.4, 130.6, 130.5, 128.7, 119.2, 117.2, 110.6, 109.7 (10 x DHN), 101.4 (C-1), 76.6, 74.7, 73.5,

+

72.7 (C-2/3/4/5), 49.7 (CHA), 31.0 (CHA), 25.1 (CHA), 24.3 (CHA). HRMS (ESI) for C16

H

13Br

2

8

O [M+H] :

m/z calcd 490.8983; measured: 490.8976. The HPLC of this substrate showed a single compound

(see supplementary data).

(

3-Hydroxynaphthalen-2-yl)-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside 25a.

To a stirred mixture of compound 16a (14.9 g, 93 mmol) in CH

mL) followed by a solution of α-D-ribofuranosyl trichloroacetimidate (13 g, 30.9 mmol) in CH

2

Cl

2

(200 mL) was added BF

3

•OEt

Cl

2

(3

(100

2

mL). After approximately 5 min. the reaction mixture was poured into a mixture of sat. aq. NaHCO

300 mL) and CH Cl (200 mL). The organic layer was separated and washed with sat. aq. NaHCO

solution (4 x 500 mL). A TLC showed the organic fraction still contained a significant amount of

unreacted compound 16a, therefore it was washed with sat. aq. Na CO solution (2 x 500 mL) (no

remaining compound 16a by TLC) and then with H O (500 mL), dried (MgSO ) and evaporated giving

an amber foam. The foam was triturated with MeOH (50 mL) and the resulting white solid collected

3

(

2

3

2

3

2

4

o

22

o

1

yielding compound 25a (6.24 g, 54%), m.p. 142-144 C. [α]

DMSO-d ) 9.61 (1H, s, OH), 7.64 (2H, t, J 9 Hz, Ar-H), 7.39 (1H, s, Ar-H), 7.26 (2H, m, Ar-H), 7.17 (1H,

s, Ar-H), 5.94 (1H, s, H-1), 5.53 (2H, m, H-2/3), 4.35 (2H, m, H-5a/5b), 4.00 (1H, dd, J 11 and 5 Hz, H-

D

-69 (c 0.99 in acetone). H NMR:

(

6

1

3

4

), 2.12 (3H, s, Ac), 2.06 (3H, s, Ac), 1.86 (3H, s, Ac); C NMR: (DMSO-d

6

) 170.6 (Ac), 170.2 (Ac), 170.1

(

7

Ac) 147.7, 145.3, 130.7, 128.4, 127.2, 125.0, 126.2, 123.8, 111.6, 110.7 (10 x DHN), 103.3 (C-1), 79.9,

+

4.8, 71.2, 63.9 (C-2/3/4/5), 21.0 (Ac), 20.9 (Ac), 20.8 (Ac). HRMS (ESI) for C21

H

26NO

9

[M+NH ] : m/z

4

calcd 436.1602; measured: 436.1609.

(6,7-Dibromo-3-hydroxynaphthalen-2-yl)-2′,3′,5′-tri-O-acetyl-β-D-ribofuranoside 25b.

Compound 16b (3 g, 9.4 mmol), 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3.3 g, 10.3 mmol) and 3Å

molecular sieves (1 g) were stirred in CH Cl (30 mL) for 10 min. and then BF •OEt (3 mL, 24 mmol)

2

3

2

was added. The reaction mixture gradually formed a clear amber solution after approximately 5 min.

of stirring at room temperature. After 15 min., a thick white precipitate had formed and the reaction

mixture became difficult to stir. The reaction mixture was poured into a mixture of CH

and sat. NaHCO (200 mL). The organic layer was separated, washed sequentially with sat. aq.

NaHCO solution (2 x 200 mL), H O (200 mL) and then dried (MgSO ) and evaporated giving a white

solid. The solid was triturated with EtOH (50 mL) and then collected affording compound 25b (4.42 g,

2

Cl

2

(200 mL)

3

2

4

o

23

o

1

8

(

1%), m.p. 185-186 C, [α]

1H, s, Ar-H), 8.14 (1H, s, Ar-H), 7.42 (1H, s, Ar-H), 7.20 (2H, m, Ar-H), 5.95 (1H, s, H-1), 5.53 (2H, m,

H-2/3), 4.42-4.31 (2H, m, H-4/5a), 4.00 (1H, dd, J4,5b 5.0 and J5a,5b 11 Hz, H-5b), 2.14 (3H, s, Ac), 2.07

D

-51 (c 1 in CHCl

3

). H NMR (DMSO-d

6

): 10.01 (1H, broad s, OH), 8.15

1

3

(

3H, s, Ac), 1.85 (3H, s, Ac); C NMR (DMSO-d

6

): 170.5 (Ac), 170.1 (Ac), 170.0 (Ac), 149.1, 146.3,

1

31.4, 130.8, 130.6, 128.5, 119.5, 118.1, 110.3, 109.8 (10 x DHN), 103.1 (C-1), 80.0, 74.8, 71.1, 63.6

+

(

C-2/3/4/5), 21.0 (Ac), 20.9 (Ac), 20.8 (Ac). HRMS (ESI) for C21

H

24Br

2

NO

9

[M+NH ] : m/z calcd

4

591.9812; measured: 591.9805.

(

3-Hydroxynaphthalen-2-yl)-β-D-ribofuranoside 26a.

Deprotection of compound 25a (1.0 g, 1.7 mmol) by a similar procedure to that described above for

the preparation of compound 18b afforded compound 26a (542 mg, 39%) as a white solid, m.p. 181-

o

22

o

1

7

3

82 C, [α]

D

-143 (c 0.55 in acetone/H

2

O). H NMR: (DMSO-d ) 7.63 (2H, m, Ar-H), 7.39 (1H, s, Ar-H),

6

.25 (2H, m, Ar-H), 7.14 (1H, s, Ar-H), 5.53 (1H, s, H-1), 4.14 (2H, broad m, H-2/3), 3.92 (1H, m, H-4),

.59 (1H, dd, J5a,4 4 Hz and J5a,5b 11 Hz, H-5a), 3.41 (1H, m, H-5b and H

1

3

2

O); C NMR: (DMSO-d ) 148.2,

6

16

1

46.5, 130.5, 128.7, 127.1, 126.1, 124.8, 123.7, 112.4, 110.5 (10 x DHN), 106.9 (C-1), 85.1, 75.1, 70.9,

+

63.0 (C-2/3/4/5). HRMS (ESI) for C15

H

16NaO [M+Na] : m/z calcd 315.0839; measured: 315.0844. The

6

HPLC of this substrate showed a single compound (see supplementary data).

(6,7-Dibromo-3-hydroxynaphthalen-2-yl)-β-D-ribofuranoside 26b.

Deprotection of compound 25b (1.0 g, 1.7 mmol) by a similar procedure to that described above for

the preparation of compound 18b afforded compound 26b (697 mg, 89%) as a white solid, m.p. >240

o

23

o

1

C (decomp.), [α]

.04 (1H, s, Ar-H), 6.30 (1H, s, Ar-H), 5.25 (1H, d, J1,2 2.0 Hz, H-1), 4.32 (1H, t, J3,4=2,3 5.0 Hz, H-3), 4.05

1H, dd, J1,2 2.0 Hz and J2,3 5 Hz H-2), 3.79 (1H, m, H-4), 3.52 (1H, dd, J4,5a 3.0 Hz and J5a,5b 12 Hz, H-5a),

D

-90 (c 0.5 in MeOH). H NMR (DMSO-d

6

): 7.73 (1H, s, Ar-H), 7.53 (1H, s, Ar-H),

7

(

3

1

3

.41 (1H, H-5b and H

2

O); C NMR (DMSO-d

6

): 163.3, 153.4, 134.3, 130.7, 127.1, 124.3, 117.7, 111.8,

NO

1

10.6, 108.6 (10 x DHN), 108.4 (C-1), 85.5, 75.0, 70.2, 61.7 (C-2/3/4/5). HRMS (ESI) for C15

H

18Br

2

6

+

[

M+H] : m/z calcd 465.9495; measured: 465.9495. The HPLC of this substrate showed a single

compound (see supplementary data).

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1

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18

Substrate 11

Substrate 13

Microorganism / Reference^a

Gram-negative microorganisms

Escherichia coli NCTC 10418

Growth^b

Colour^c

Growth^b

Colour^c

Grow

1

2

3

4

5

6

7

8

9

1

++

+/- brown

+ brown

-

++

+ brown

+/- brown

+

Serratia marcescens NCTC 10211

Pseudomonas aeruginosa NCTC 10662

Burkholderia cepacia ATCC 25416

Yersinia enterocolitica NCTC 11176

Salmonella typhimurium NCTC 74

Citrobacter freundii 46262 (wild)

Morganella morganii 462403 (wild)

Enterobacter cloacae NCTC 11936

Providencia rettgeri NCTC 7475

-

+ brown

+/- brown

+ brown

-

+ brown

-

+ brown

-

0

Gram-positive microorganisms

11

12

13

14

15

16

17

18

Bacillus subtilis NCTC 9372

++

-

++

-

+

Enterococcus faecalis NCTC 775

Enterococcus faecium NCTC 7171

Staphylococcus epidermidis NCTC 11047

Staphylococcus aureus NCTC 6571

Staphylococcus aureus (MRSA) NCTC 11939

Streptococcus pyogenes NCTC 8306

Listeria monocytogenes NCTC 11994

+ brown

-

+ brown

+/- brown

-

Yeasts

1

2

9

0

Candida albicans ATCC 90028

Candida glabrata NCPF 3943

++

-

++

-

+

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

+

+ strong growth, + moderate growth, +/- weak growth.

c

++ strong colour, + moderate colour, +/- weak colour, - no noticeable colour. Diffusion of the colour

into the medium was noted in all cases.

Substrate

b

c

18a

20a

22a

24a

Microorganism / Reference^a

Gram-negative microorganisms

Colour

e

Colour^e

1

2

3

4

5

6

7

Escherichia coli NCTC 10418

Tr. purple

++ purple

+ purple

-

++ purple

Serratia marcescens NCTC 10211

Pseudomonas aeruginosa NCTC 10662

Burkholderia cepacia ATCC 25416

Yersinia enterocolitica NCTC 11176

Salmonella typhimurium NCTC 74

Citrobacter freundii NCTC 9750

++ purple

-

NT

-

NT

-

+ purple

-

19

8

9

1

Morganella morganii 462403 (wild)

Enterobacter cloacae NCTC 11936

Providencia rettgeri NCTC 7475

Klebsiella pnueumoniae NCTC 10896

Acinetobacter baumannii ATCC 19606

NT

++ purple

-

NT

+ purple

++ purple

-

+/- purple

0

1

2

-

NT

++ purple

-

Gram-positive microorganisms

13

14

15

16

17

18

19

20

Bacillus subtilis NCTC 9372

+ purple

-

Enterococcus faecalis NCTC 775

Enterococcus faecium NCTC 7171

Staphylococcus epidermidis NCTC 11047

Staphylococcus aureus NCTC 6571

Staphylococcus aureus (MRSA) NCTC 11939

Streptococcus pyogenes NCTC 8306

Listeria monocytogenes NCTC 11994

++ purple

Tr. purple

-

++ purple

+ purple

Yeasts

2

1

2

Candida albicans ATCC 90028

Candida glabrata NCPF 3943

-

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

all microorganisms exhibited moderate growth in the presence of these substrates.

the Gram-negative microorganisms exhibited strong growth and the Gram-positive microorganisms

c

and yeasts showed moderate growth in the presence of this substrate.

d

all microorganisms exhibited strong growth in the presence of this substrate.

e

++ strong colour, + moderate colour, +/- weak colour, - no noticeable colour. NT, not tested.

Diffusion of the colour into the medium was noted in all cases.

f

Citrobacter freundii 4626 (wild).

Substrate 18b

Substrate 20b

Substr

Microorganism / Reference^a

Gram-negative microorganisms

Escherichia coli NCTC 10418

Growth^b

Colour

c

Growth^b

Colour

c

Growth^b

1

2

3

4

5

6

7

8

9

1

+

Tr. brown

++ brown

+

++ brown

+ brown

++

Serratia marcescens NCTC 10211

Pseudomonas aeruginosa NCTC 10662

Burkholderia cepacia ATCC 25416

Yersinia enterocolitica NCTC 11176

Salmonella typhimurium NCTC 74

Citrobacter freundii NCTC 9750

Morganella. morganii 462403 (wild)

Enterobacter cloacae NCTC 11936

Providencia rettgeri NCTC 7475

-

+ brown

+/- brown

-

+ brown

-

+/-

+

0

++ brown

Gram-positive microorganisms

20

1

2

3

4

Bacillus subtilis NCTC 9372

+

+ brown

Tr. brown

+/- brown

+

+/-

-

++

Enterococcus faecalis NCTC 775

Enterococcus faecium NCTC 7171

Staphylococcus epidermidis NCTC

Tr. brown

+/- brown

11047

+

-

+

-

15

6

Staphylococcus aureus NCTC 6571

Staphylococcus aureus (MRSA) NCTC

Tr. brown

++

1

1939

+

Tr. brown

-

+/- brown

+

+/-

+

-

1

7

8

Streptococcus pyogenes NCTC 8306

Listeria monocytogenes NCTC 11994

++

+/- brown

Yeasts

1

2

9

0

Candida albicans ATCC 90028

Candida glabrata NCPF 3943

+

-

+

-

++

+

a

NCTC: National Collection of Type Cultures; ATCC: American Type Culture Collection; NCPF: National

Collection of Pathogenic Fungi.

b

+

+ strong growth, + moderate growth, +/- weak growth, no noticeable growth.

+ strong colour, + moderate colour, +/- weak colour, Tr. trace of colour, - no noticeable colour.

c

21

Article Doi

The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and their applications in diagnostic microbiology

DOI: 10.1016/j.bmc.2018.08.023

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Authors:

Article abstract of DOI:10.1016/j.bmc.2018.08.023

Full text of DOI:10.1016/j.bmc.2018.08.023

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