Design and Synthesis of Minimalist Terminal Alkyne‐Containing Diazirine Photo‐Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell‐ and Tissue‐Based Proteome Profiling

Abstract

Klein und zackig: Nach der Einführung eines minimalen Photovernetzers mit Eignung für Klick‐Reaktionen (siehe Schema) in zahlreiche niedermolekulare Kinase‐Inhibitoren wurden die resultierenden Sonden zum Proteom‐Profiling in vitro (in Zelllysaten) und in situ (in lebenden Zellen) eingesetzt. Auch bei der Identifizierung ihrer potenziellen Zielkinasen im großen Maßstab waren sie bekannten Sonden überlegen.

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

“…P2,o n the other hand, was designed by conveniently attaching our previously developed "minimalist"l inker to FED1.W ep reviously showed such ap hoto-cross-linker having both an alkyl diazirine and at erminal alkyne could effectively minimize potential interference of the probe upon binding to the target proteins. [19] This strategy has thus far been applied to taggingavariety of bioactive compounds with reasonables uccess. [19,23,24] Since both P1 and P2 were carefullyd esigned to resemble FED1's core structure with minimal modifications, they should be suitable for accurate interrogation of FED1 in live cellular environments.…”

“…[19] This strategy has thus far been applied to taggingavariety of bioactive compounds with reasonables uccess. [19,23,24] Since both P1 and P2 were carefullyd esigned to resemble FED1's core structure with minimal modifications, they should be suitable for accurate interrogation of FED1 in live cellular environments. With both probe designs,u pon photo-irradiation, the diazirine moiety in each case would generate ah ighly reactive carbene, resulting in an effective covalentl inkage with nearbyr esidues from the probe-bound target protein.…”

“…P2 was prepared through anothers ynthetic route using some of the intermediates described above. The minimalist linker L7 wass ynthesized as previousr eported, [19] and was used to alkylate the amino group in compound 16 under basic conditions. After alkylation, the azide group was reduced with PPh 3 to afford compound 18.F inally,c ompounds 8 a and 18 reactedt hrough reductivea minationf ollowed by subsequent removal of the acetal protecting group, generating probe P2.…”

“…[12][13][14][15][16] By using the socalled activity-based probes (ABPs) or affinity-based probes (AfBPs) that were derived from bioactive compounds, this strategyh as recently becomeapowerful tool for large-scale identification of potential "on" and "off" cellular targets of drug candidates. [13] Up to now,w ea nd others have successfully applied such "in situ drug profiling" strategies to an umber of biologically important drugs( and candidates), including the FDA-approved anti-obesity drug Orlistat, [17] severala nti-cancer small-molecule kinase inhibitors, [18][19][20][21][22] and the newly developed epigenetic protein-protein interaction (PPI) inhibitor JQ-1. [23] Furthermore, a" clickable" affinity-based probe derived from 3-Deazaneplanocin A( DzNep), which is ag lobal histone methylation inhibitor that also targetsm any other SAM-binding proteins, was recently reported.…”

Affinity purification in target identification: the specificity challenge

“…These ''all-in-one'' probes have high potential to reduce the level of non-specific binding in affinity purification. In 2013, the Yao laboratory designed a type of ''minimalist probes'' containing a terminal alkyne, a diazirine crosslinker, and a functional group for conjugation with the small molecule (Li et al 2013c;Su et al 2013). Without the ligand, these probes are only seven atoms long so that they have minimal perturbation on the binding property of the ligand.…”