The synthesis of the required substrates is
The synthesis of the required substrates is shown in (see for experimental details). Treatment of heterocycle with 5,7-Dichlorokynurenic acid of under basic conditions afforded a mixture of the mono-substituted product (22%) and the di-substituted compound (38%) which were separated by column chromatography. Compound (84%) was also prepared from heterocycle using 2 equivalents of the benzylic chloride . De-protection of these individual compounds and with hydrogen chloride produced the hydrochloride salts of the substrates and respectively. In a similar process, Alizarin reacted with compound under basic conditions giving the mono-substituted product from which the required substrate was obtained by removal of the Boc protecting group. Each substrate (100 mgL) was evaluated in Columbia agar media (37°C in air for 18h) on a single plate against 20 clinically important microorganisms including 10 Gram-negative bacteria, 8 Gram-positive bacteria and 2 yeasts (see , top diagram). Also added to the media was either ammonium iron(III) citrate (500mgL) or aluminium potassium sulphate dodecahydrate (500mgL). The substrate concentration and the selection and optimal concentration of metal ions were extrapolated from previous work with glycosides of CHE and Alizarin as documented in previous studies., , The growth of the microorganisms was compared to control plates in which no substrate or metal salt was present. Control plates were also prepared containing metal ions (500mgL) without substrate. All microorganisms grew well on all control plates without inclusion of the two substrates and there was no inhibition caused by the incorporation of metal salts. When substrates and were evaluated as indicators of L-alanylaminopeptidase activity in the presence of ammonium iron(III) citrate, only moderately intense, grey-coloured colonies were observed with most of the panel of Gram-negative microorganisms (data not shown) and these substrates were not examined further. In contrast, with the Alizarin derived-substrate , several species produced intense, purple-coloured colonies in the presence of ammonium iron(III) citrate and intense, pink-coloured colonies in the presence of aluminium potassium sulphate dodecahydrate with all of the selection of Gram-negative microorganisms tested (, ). Less intensely coloured colonies were also produced with two of the Gram-positive microorganisms, and The growth of the other Gram-positive bacteria and also the yeasts was inhibited by the substrate/media combination. Any potential toxicity of these substrates is most likely to arise from the released chromophore or its chelate and these are common to both the previously described glycosides and the peptidase substrates described here. depicts Columbia agar plates inoculated with a representative Gram-negative microorganism, , in the presence of substrate at two concentrations with two metal salts (both salts at 500mgL). A bacterial suspension was prepared with a turbidity equivalent to 0.5 McFarland units. We then cultured 10µL of this suspension and spread the plate to obtain isolated bacterial colonies. The inoculum was approximately 10CFU/plate. With substrate at a concentration of 100mgL, a strong pink colouration is produced by whereas at a lower substrate concentration (50mgL) the depth of colour is diminished but colonies are still clearly visible against the background. When was inoculated onto medium containing substrate at 100mgL in the presence of ammonium iron (III) citrate, highly coloured purple colonies were generated and this substrate remained effective at a concentration of 50mgL. With both metal salts, there was a good contrast between the colour of bacterial colonies and the background colouration. shows four microorganisms (at the same inoculum as the plates depicted in ) in the presence of substrate and aluminium potassium sulphate dodecahydrate (500mgL). The two Gram-negative microorganisms (top row) produced coloured colonies whereas in contrast, the growth of the two Gram-positive organisms was completely inhibited in the presence of the substrate and hence no colouration was produced.