br Conclusions Enzyme prodrug therapy mediated by implantabl
Conclusions Enzyme prodrug therapy mediated by implantable biomaterials combines the benefits offered by the site specific drug delivery techniques and the systemic administration of drugs. From the former, SMEPT inherits the localized mode of drug delivery with lower systemic distribution of the drug and hence lower side effects. From the latter, SMEPT enjoys the flexibility of changing treatment in terms of drug dosage, duration of treatment, sequential or combination treatments. In vitro and in vivo validation of this technology offers this platform for translational research – which is the next necessary step for a broader acceptance of this methodology, academically and clinically.
Acknowledgements We gratefully acknowledge financial support to this work from the European Research Council Consolidator Grant (ERC-2013-CoG 617336 BTVI).
Introduction Molecules that contain self-immolative spacers have found widespread applications in areas such as drug delivery, prodrug systems, chemical sensors and enzyme sensors. In one aspect of self-immolative spacer design, fluorescent substrates of general structure 1 have been constructed around a para-aminobenzyl alcohol (PABA) core for the purpose of enabling the detection of protease enzyme activity as outlined in Scheme 1. This system is designed so that the presence of a protease enzyme results in the release of a fluorescent phenolic derivative 4. Thus, the weakly fluorescent substrates 1 are hydrolytically cleaved by an appropriate enzyme producing the O-arylated-4-aminobenzyl alcohol intermediate 2 which subsequently undergoes fragmentation with concomitant liberation of the imine 3 and the fluorescent phenolic derivative 4. Renard, Romieu and co-workers described the preparation of two fluorescent substrates based on the general structure 1; a sensor 5 for the detection of JNJ 5207852 dihydrochloride receptor G acylase2, 3 and a probe 6 for determining Caspase-3 activity (Fig. 1). Structurally related chemiluminescent substrates for detecting penicillin G acylase and Caspase-3 activities have also been described. A fluorogenic assay based on structure 7 for monitoring the activity of the autophagy-initiating enzyme ATG4B has recently been reported (Fig. 1). The identification of specific types of enzyme activity in microorganisms has proved tremendously useful in diagnostic microbiology. Of particular relevance to this paper is the detection of l-alanylaminopeptidase activity which has enabled the differentiation between Gram-positive and Gram-negative microorganisms.7, 8 This enzyme is widely distributed in Gram-negative microorganisms whereas, in contrast, it is generally absent or less abundant in most Gram-positive microorganisms. Fluorogenic substrates that have been used for the detection of l-alanylaminopeptidase activity include the commercially available coumarin derivative 8 which liberates the highly fluorescent 7-amino-4-methylcoumarin 9 in the presence of an l-alanylaminopeptidase enzyme (Scheme 2). We have previously described the synthesis and evaluation of a series of fluorogenic substrates 10 (X=S, O) that produced the corresponding fluorescent 2-(2-aminophenyl)benzoxazole 11 (X=O) and 2-(2-aminophenyl)benzothiazoles 11 (X=S) in the presence of l-alanylaminopeptidase. Fluorogenic l-alanylaminopeptidase substrates derived from 2-(4-aminophenyl)benzothiazoles were also prepared and evaluated.
Synthesis of substrates The substrates depicted in Scheme 2 all liberate fluorescent heterocyclic amine derivatives. In view of the availability and structural diversity of fluorescent phenols, we wished to develop substrates that would enable the detection of l-alanylaminopeptidase activity in microorganisms such that a fluorescent phenolic derivative is produced. A potential benefit of these proposed substrates would be that fluorogenic phenols with specific properties, e.g., tailored excitation/emission wavelengths, could be selected from an extensive pool of known molecules hence extending the availability of fluorophores that might be incorporated into aminopeptidase substrates. Thus, in this paper we describe the synthesis and evaluation of a series of novel self-immolative spacer substrates 18 (Table 1) for the purpose of detecting l-alanylaminopeptidase activity in microorganisms.