A traceless linker for aliphatic aminesA traceless linker for aliphatic amines

Reduction sensitive linkers (RSLs) have the potential to transform the field of drug delivery due to their ease of use and selective cleavage in intracellular environments. However, despite their compelling attributes, developing reduction sensitive self-immolative linkers for aliphatic amines has been challenging due to their poor leaving group ability and high pK_a values. Here a traceless self-immolative linker composed of a dithiol-ethyl carbonate connected to a benzyl carbamate (DEC) is presented, which can modify aliphatic amines and release them rapidly and quantitatively after disulfide reduction. DEC was able to reversibly modify the lysine residues on CRISPR–Cas9 with either PEG, the cell penetrating peptide Arg₁₀, or donor DNA, and generated Cas9 conjugates with significantly improved biological properties. In particular, Cas9–DEC–PEG was able to diffuse through brain tissue significantly better than unmodified Cas9, making it a more suitable candidate for genome editing in animals. Furthermore, conjugation of Arg₁₀ to Cas9 with DEC was able to generate a self-delivering Cas9 RNP that could edit cells without transfection reagents. Finally, conjugation of donor DNA to Cas9 with DEC increased the homology directed DNA repair (HDR) rate of the Cas9 RNP by 50% in HEK 293T cell line. We anticipate that DEC will have numerous applications in biotechnology, given the ubiquitous presence of aliphatic amines on small molecule and protein therapeutics.

Two major classes of RSLs have been developed to modify aliphatic amines, which are disulfide carbamates (DCBs) and dithiobenzyl carbamates (DTBs). Even though DCBs and DTBs have demonstrated the great potential of RSLs in prodrug design, they have not found widespread applications for protein delivery due to their slow self-immolation rates or incomplete release.

DEC is composed of two rapidly hydrolyzing self-immolative linkers connected in series, and this unique design allows it to overcome the challenges associated with making RSLs for aliphatic amines. The two linkers embedded in DEC are a thiol-ethyl carbonate linker and a 1,6-elimination linker. These two linkers were selected for incorporation in DEC because they individually release alcohols instantaneously and amines after activation. Specifically, the thiol-ethyl carbonate linker rapidly releases a cyclic thiol ether after disulfide reduction, and the 1,6-elimination linker rapidly releases aliphatic amines. In addition, DEC was able to reversibly modify the Cas9 protein via conjugation of its lysine residues with the CPP Arg₁₀. Cas9–CPP was able to successfully edit cells and has great potential as a self-delivering Cas9 RNP formulation. Finally, DEC was also able to increase the HDR rate of the CRISPR system by 50%, by reversibly tethering donor DNA to Cas9 RNP in traceless manner. The DEC linker is a versatile reagent that can be used for multiple applications in protein and small molecule drug delivery.