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).