Proteins are subject to numerous post-translational modifications (PTMs) that can alter the chemical structure, and hence function, of the molecule.[1] The astonishing diversity of PTMs possible on proteins is exemplified by histones, nuclear proteins that form the protein core of the nucleosome particle.[2] Histones can be modified in a variety of ways including acetylation, phosphorylation, methylation, ADP-ribosylation and ubiquitylation.[3] Moreover, many, if not all, of these modifications can occur in combination.[4, 5] Indeed, there is growing evidence that functional cross-talk between histone PTMs is essential for the regulation of gene expression[3] and ultimately cell fate and identity.[6] Biochemical studies into the role of histone PTMs are often confounded by the difficulty associated with obtaining large quantities of homogeneously modified proteins. For this reason chemical approaches to obtaining post-translationally modified histones have received considerable attention in recent years.[7–9] Among the available strategies, the protein ligation approach, expressed protein ligation (EPL), offers the most flexibility in terms of the number and type of PTMs that can be incorporated.[10] To date, EPL has been used to generate phosphorylated,[8] acetylated, and methylated forms of histone H3,[7] acetylated H4,[11] and ubiquitylated H2B.[12] Nonetheless, many modified histones have yet to be accessed using semi-synthesis. A notable case in point is the N-terminal region of H2B, which has been described to possess several PTMs, including (poly)lysine acetylation and serine 14 phosphorylation, which have been implicated in transcription[13] and apoptotic chromatin compaction,[14] respectively. Differentially modified semi-synthetic H2B analogs would be useful to assess the affect of acetylation on both antibody recognition as well as on the efficiency of phosphorylation. In this report, we describe a general semi-synthetic route to H2B that allows the installation of PTMs into an otherwise native polypeptide background.