Biocompatible lysine protecting groups for the chemoenzymatic synthesis of K48/K63 heterotypic and branched ubiquitin chains

03 April 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


The elucidation of emerging biological functions of heterotypic and branched ubiquitin (Ub) chains requires new strategies for their preparation with defined length and connectivity. While in vitro enzymatic assembly using expressed E1 activating and E2 conjugating enzymes can deliver homotypic chains, the synthesis of branched chains typically requires extensive mutations of lysines or other sequence modifications. The combination of K48 and K63 biased E2 conjugating enzymes and two new carbamate protecting groups – pyridoxal 5’-phosphate (PLP) cleavable aminobutanamide carbamate (Abac group) and periodate cleavable aminobutanol carbamate (Aboc group) – provides a strategy for the synthesis of heterotypic and branched Ub trimers, tetramers, and pentamers. The Abac- and Aboc-protected lysines are readily prepared and incorporated into synthetic ubiquitin monomers. As these masking groups contain a basic amine, they preserve the overall charge and properties of the Ub structure, facilitating folding and enzymatic conjugations. These protecting groups can be chemoselectively removed from folded Ub chains and monomers with buffered solutions of PLP or NaIO4. Through the incorporation of a cleavable C-terminal His tag on the Ub acceptor, the entire process of chain building, iterative Abac deprotections, and global Aboc cleavage can be conducted on a resin-support, obviating the need for handling and purification of the intermediate oligomers. Simple modulation of the Ub monomers affords various K48/K63 branched chains, including tetramers and pentamers not previously accessible by synthetic or biochemical methods.


protein conjugation
ubiquitin chains
protecting groups

Supplementary materials

Branched Ub Chain Supporting Bode
Supplementary figures, experimental protocols, characterisation data


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