With compound 9 in hand, microscale thermophoresis (MST) was used to determine if the PI5P diazirine probe 9 could bind hUHRF1 (Figure ). While binding of the diazirine probe 9 to recombinant hUHRF1 was observed at concentra,ons above 50 µM, a Kd value could not be determined due to limita,ons in the ,tra,on scheme. The Kd of PI5P (1) for hUHRF1 was approximately 8.6 µM. Modest altera,on a single C16 acyl chains, by introduc,on of a diazirine photo crosslinking group and terminal alkyne, resulted in a Kd of over 50 µM for the PI5P diazirine (9), highligh,ng the selec,vity of UHRF1 for the di-C16 acyl chains of PI5P. To confirm the probe would s,ll be func,onal given this lower level of affinity, compound 9 was combined with either purified recombinant hUHRF1, or purified recombinant hTAF3 [human TATA box binding protein associated factor 3], another known protein binder of PI5P, and irradiated with light at a wavelength of 365 nm for 30 minutes to induce photo cross-linking. A copper-catalysed click reac,on was then performed to conjugate a TAMRA-PEG-azide fluorophore to the covalently bound PI5P (Figure ). Using SDS-PAGE gel electrophoresis, fluorescent bands were observed at the running posi,ons corresponding to the molecular weights of either hUHRF1 or hTAF3 (Figure ), confirming that the diazirine probe bound to recombinant hUHRF1 and hTAF3, and could undergo the photo-crosslinking with either protein. Next, to determine that the diazirine probe 9 was selec,vely binding its targets, and not non-specifically labelling hydrophobic proteins, the diazirine probe 9 was incubated with bovine serum albumin (BSA), and subject to the photo-crosslinking/click chemistry reac,on. Following SDS-PAGE analysis, Figure , shows that there was no substan,al labelling of BSA, confirming the selec,vity of the diazirine probe 9.