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  • br Conclusion The past few years have witnessed substantial

    2019-07-11


    Conclusion The past few years have witnessed substantial progress in understanding the structural mechanisms of substrate recognition and the reactions catalyzed by the O-GlcNAc-cycling enzymes, but more work remains. In particular, future research will be needed to establish how OGT and OGA interact with protein substrates and to advance understanding of substrate specificity. Structural work that reveals new binding modes and guides the design of cellular experiments to deconvolute OGT and OGA’s functions will continue to play a crucial role in linking in vitro PF-04449913 molecular to cellular function. In addition, regulatory mechanisms of OGT and OGA that limit futile O-GlcNAc cycling remain to be uncovered.
    Conflict of interest statement
    References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:
    Molecules that possess a self-immolative spacer unit have been studied and utilised in several areas of chemical science including drug delivery systems, prodrug design, chemical sensors and enzyme sensors. In one theme of self-immolative spacer chemistry, molecules of general structure have been constructed around a -aminobenzyl alcohol (PABA) core for the purpose of enabling the detection of protease enzyme activity as outlined in . This system is designed so that protease-induced fragmentation of compounds results in the release of a fluorescent phenolic derivative and hence the presence of protease activity results in an increase in fluorescence. Thus, the protease substrates are hydrolytically cleaved resulting in the formation of the intermediates which subsequently fragment with concomitant liberation of the imine and the fluorescent phenolic derivative . A sensor for the detection of penicillin G acylase,, a probe for determining Caspase-3 activity, an assay for monitoring the activity of the autophagy-initiating enzyme ATG4B and a Resorufin-based fluorogenic/chromogenic substrate for the detection of proteases in biological matrices are recent examples of the application of self-immolative spacers of general structure . We have recently described the synthesis and evaluation of a series of novel fluorogenic self-immolative enzyme substrates based on the general structure (AA=L-alanyl) for the detection of L-alanylaminopeptidase activity in a panel clinically important Gram-negative and Gram-positive microorganisms, an area of importance in diagnostic microbiology. Several of these substrates enabled the detection of L-alanylaminopeptidase activity in Gram-negative microorganisms in Columbia agar media because L-alanylaminopeptidase activity is frequently abundant in Gram-negative microorganisms whereas it is generally less pronounced in the Gram-positive microorganisms., The growth of many of the panel of Gram-positive microorganisms in Columbia agar was frequently inhibited by these fluorogenic substrates, and hence fluorescent microorganism colonies were not observed except in a few cases where some growth had occurred. A fluorogenic, self-immolative substrate designed for the detection of β-alanylaminopeptidase activity has recently been reported. In this Letter, we describe the synthesis and evaluation of some novel chromogenic self-immolative enzyme substrates for the detection of L-alanylaminopeptidase activity in microorganisms. The core molecules (equivalent to compound ) chosen for study were cyclohexenoesculetin (CHE) and Alizarin () because both of these molecules are annulated catechol derivatives and as such, they are known to form coloured chelates in the presence of selected metal ions. For example, the 7-β-D-glucoside derivative of compound in the presence of ammonium iron(III) citrate produced black colonies with microorganisms that exhibited β-D-glucosidase activity. The 2-β-galactoside derivative of Alizarin produced either violet or pink coloured colonies in the presence of ammonium iron(III) citrate or potassium aluminium sulphate respectively with microorganisms that displayed β-galactosidase activity. The benefit of the work described in this Letter would be that L-alanylaminopeptidase activity might be detected through the formation of highly coloured metal chelates of catechol-type molecules.