Selective and diagnostic labelling of serine hydrolases with reactive phosphonate inhibitors.
Reactive phosphonates are important probes to target the active site of serine hydrolases, one of the largest and most diverse family of enzymes. Developing such inhibitory probes is of special importance in activity based protein profiling, a strategy that is increasingly used to gain information about a certain class of enzymes in complex proteosomes. Therefore, gaining detailed information about these inhibition events on the individual protein level is important since it affords information that can be used to fine-tune the probe for a specific task. Here, we report a novel and versatile synthesis protocol to access a variety of functionalised p-nitrophenyl phosphonate (PNPP) inhibitors from a common azide functionalised precursor using click chemistry. The obtained PNPPs were successfully used to covalently label serine hydrolases in their active sites with molecular tags. Furthermore, a model study is described in which we developed straightforward protocols that can be used to study protein inhibition events. Kinetic studies using UV-Vis and fluorescence spectroscopy techniques revealed that these PNPPs possess different inhibition rates for various proteins and were shown to be suitable probes to discriminate between various lipases. Additionally, we demonstrate that PNPPs are highly selective for serine hydrolases, making these probes very interesting as diagnostic or affinity probes for studying proteins in complex proteosomes.
Dijkstra
Harmen P
HP
Department of Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands. h.p.dijkstra@uu.nl
Sprong
Hein
H
Aerts
Bas N H
BN
Kruithof
Cornelis A
CA
Egmond
Maarten R
MR
Klein Gebbink
Robertus J M
RJ
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism*; D001665:Binding Sites; D003619:Dansyl Compounds / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009596:Nitrophenols; D063065:Organophosphonates / Q000138:chemical synthesis* / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000345:Affinity Labels; D003619:Dansyl Compounds; D009596:Nitrophenols; D063065:Organophosphonates; D020543:Proteome; C001026:5-dimethylaminonaphthalene-1-sulfonamide; C085778:4-nitrophenyl phosphonate; D012694:Serine; D006867:Hydrolases