Comprehensive synthesis of K48/K63 ubiquitin pentamers by graph-empowered automation

Linear, branched, and heterotypic ubiquitin (Ub) chains regulate essential cellular processes, yet their structural and functional analysis remains limited by the difficulty of preparing defined, homogeneous samples by chemical synthesis or biochemical assembly. We established a fully automated platform for the comprehensive synthesis of all 56 K48/K63-linked Ub tetramers and pentamers. This is achieved by iterative cycles of K48 or K63-specific chemoenzymatic conjugations of chemically synthesized Ub monomers bearing a new, fast-cleaving lysine protecting group compatible with folded proteins. A graph-based digital encoding of the Ub chain topologies enabled systematic enumeration and filtering of reaction pathways, guiding the selection of viable synthetic routes. This computational system generates reagent maps and executable code to direct iterative cycles of deprotection and conjugation on a standard, laboratory liquid handling robot. All 14 Ub tetramers and 42 Ub pentamers were obtained with high fidelity, as validated by SDS-PAGE and mass spectrometry. This work establishes a general framework for automated protein conjugate synthesis and provides on-demand access to the complete space of K48/K63 branched ubiquitin chains for biological and structural studies.