Miniaturized Traceless Staudinger Ligation Enables Direct-to-Biology Screening of Potent BRD4 PROTACs

Direct-to-biology (D2B) approaches accelerate PROTAC discovery by enabling biological screening of crude reaction mixtures without intermediate purification. However, the range of chemical transformations compatible with these workflows remains limited. Herein, we establish a miniaturized, base-free traceless Staudinger ligation platform diversifying linker chemistry for a preexisting library of E3-ligand-linker adducts bearing azide moieties. As a proof-of-concept target, BRD4 was chosen, yielding amide-linked BRD4-targeting PROTACs. By combining phosphine-functionalized JQ1 derivatives with 72 selected azide building blocks, reactions were performed on a 5 L scale in 384-well plates, achieving a mean LC-MS conversion of approximately 65%. Spiking experiments confirmed that the phosphine starting materials had no significant impact on BRD4 degradation, supporting the direct biological evaluation of crude reaction mixtures in the presence of residual starting material. Cellular screening identified 23 hits at 250 nM screening concentration, reducing BRD4 levels below 40%. Two resynthesized and purified PROTACs were subsequently validated, displaying pDC50 values of 8.2 and 7.0 and a Dmax of 90% and 96%, respectively. This work establishes traceless Staudinger ligation as a complementary D2B strategy for the rapid discovery of amide-linked PROTACs.