title: 4-Hydroxy-N-propyl-1,8-naphthalimide esters: New fluorescence-based assay for analysing lipase and esterase activity.
abstract: Research using 1,8-naphthalimide derivatives has expanded rapidly in recent years owing to their cell-permeable nature, ability to target certain cellular locations and fluorescent properties. Here we describe the synthesis of three new esters of 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) and the development of a simple and sensitive assay protocol to measure the activity of carboxylester hydrolases. The NAP fluorophore was esterified with short (butyrate), medium (octanoate) and long (palmitate) chain fatty acids. The esters were spectroscopically characterised and their properties investigated for their suitability as assay substrates. The esters were found to be relatively stable under the conditions of the assay and levels of spontaneous hydrolysis were negligible. Non-specific hydrolysis by proteins such as bovine serum albumin was also minimal. A simple and rapid assay methodology was developed and used to analyse a range of commercially available enzymes that included enzymes defined as lipases, esterases and phospholipases. Clear differences were observed between the enzyme classes with respect to the hydrolysis of the various chain length esters, with lipases preferentially hydrolysing the medium chain ester, whereas esterases reacted more favourably with the short ester. The assay was found to be highly sensitive with the fluorophore detectable to the low nM range. These esters provide alternate substrates from established coumarin-based fluorophores, possessing distinctly different excitation (447 nm) and emission (555 nm) optima. Absorbing at 440-450 nm also offers the flexibility of analysis by UV-visible spectrophotometry. This represents the first instance of a naphthalimide-derived compound being used to analyse these enzymes.
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Victoria, 3216, Australia; Marine Products Group, The New Zealand Institute for Plant & Food Research Limited, 300 Wakefield Quay, Nelson, New Zealand. Electronic address: tim.nalder@deakin.edu.au.
authors.forename: Tim D
authors.initials: TD
authors.lastname: Nalder
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Victoria, 3216, Australia.
authors.forename: Trent D
authors.initials: TD
authors.lastname: Ashton
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Victoria, 3216, Australia.
authors.forename: Frederick M
authors.initials: FM
authors.lastname: Pfeffer
authors.affiliation: Marine Products Group, The New Zealand Institute for Plant & Food Research Limited, 300 Wakefield Quay, Nelson, New Zealand.
authors.forename: Susan N
authors.initials: SN
authors.lastname: Marshall
authors.affiliation: Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Deakin University, Waurn Ponds, Victoria, 3216, Australia. Electronic address: colin.barrow@deakin.edu.au.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Barrow
mesh_terms: D001203:Ascomycota / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D002175:Candida / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D053644:Naphthalimides / Q000737:chemistry*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013053:Spectrophotometry
chemical_list: D001426:Bacterial Proteins; D004952:Esters; D005227:Fatty Acids; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D053644:Naphthalimides; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; D008049:Lipase
keywords: 1,8-Naphthalimide; Carboxylester hydrolase; Enzyme assay; Fluorescence; Lipase