Fluorogenic/chromogenic enzyme substrates constructed around the 2-amino-7-nitrofluorene core structure for the detection of aminopeptidase activities in clinically important microorganisms

Chromogenic/fluorogenic enzyme substrates have been widely employed for the detection and identification of pathogenic microorganisms. In this paper we describe the synthesis and evaluation in solid (agar) media of a series of novel enzyme substrates for the detection of aminopeptidase activities in clinically important microorganisms. Amino acid derivatives of 2-amino-7-nitrofluorene and its 9,9-dimethyl analogue were prepared and these substrates produced yellow coloured/fluorescent colonies when aminopeptidase activity was present. The L-alanylaminopeptidase substrates 2-(L-alanyl)-amino-7-nitrofluorene (18a) and 2-(L-alanyl)-amino-7-nitro-9,9-dimethylfluorene (18b) could differentiate between Gram-negative and Gram-positive bacteria and the β-alanylaminopeptidase substrates 2-(β-alanyl)-amino-7-nitrofluorene (19a) and 2-(β-alanyl)-amino-7-nitro-9,9-dimethylfluorene (19b) were effective in detecting Pseudomonas aeruginosa, a microorganism commonly encountered in people with cystic fibrosis. A homologue of substrate 19b, 2-(γ-aminobutyryl)-amino-7-nitro-9,9-dimethylfluorene (20b), was also prepared and evaluated for the detection of Burkholderia cepacia (also associated with people with cystic fibrosis).