Protein phosphorylation is by far the most abundant and most studied post-translational modification (PTM). For a long time, phosphate monoesters of serine (pSer), threonine (pThr), and tyrosine (pTyr) have been considered as the only relevant forms of phosphorylation in organisms. Recently, several research groups have dedicated their efforts to the investigation of other, less characterized phosphoamino acids as naturally occurring PTMs. Such apparent peculiar phosphorylations include the phosphoramidates of histidine (pHis), arginine (pArg), and lysine (pLys), the phosphorothioate of cysteine (pCys), and the anhydrides of pyrophosphorylated serine (ppSer) and threonine (ppThr). Almost all of these phosphorylated amino acids show higher lability under physiological conditions than those of phosphate monoesters. Furthermore, they are prone to hydrolysis under acidic and sometimes basic conditions as well as at elevated temperatures, which renders their synthetic accessibility and proteomic analysis particularly challenging. In this Account, we illustrate recent chemical approaches to probe the occurrence and function of these labile phosphorylation events. Within these endeavors, the synthesis of site-selectively phosphorylated peptides, in particular in combination with chemoselective phosphorylation strategies, was crucial. With these well-defined standards in hand, the appropriate proteomic mass spectrometry-based analysis protocols for the characterization of labile phosphosites in biological samples could be developed. Another successful approach in this research field includes the design and synthesis of stable analogues of these labile PTMs, which were used for the generation of pHis- and pArg-specific antibodies for the detection and enrichment of endogenous phosphorylated samples. Finally, other selective enrichment techniques are described, which rely for instance on the unique chemical environment of a pyrophosphate or the selective interaction between a phosphoamino acid and its phosphatase. It is worth noting that many of those studies are still in their early stages, which is also reflected in the small number of identified phosphosites compared to that of phosphate monoesters. Thus, many challenges need to be mastered to fully understand the biological role of these poorly characterized and rather uncommon phosphorylations. Taken together, this overview exemplifies recent efforts in a flourishing field of functional proteomic analysis and furthermore manifests the power of modern peptide synthesis to address unmet questions in the life sciences.

Fretful novelty: We developed two novel doubly labelled fluorescent ceramide analogues that exhibit significant FRET and undergo hydrolysis by ceramidases. We present a fluorescent sphingolipid FRET probe that allows homogeneous ratiometric determination of enzyme activity in real-time.

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A chemical reaction that is triggered by a specific RNA molecule might provide opportunities for the design of artificial feedback loops. We envision a peptidyl transfer reaction in which mRNA encoding an antiapoptotic protein would instruct the synthesis of apoptosis-inducing peptides. In this study, we used the RNA-programmed synthesis of a 16-mer peptide derived from the BH3 domain of the protein Bak, which inhibits the antiapoptotic protein Bcl-x . The reaction involves the transfer of a thioester-linked donor peptide fragment from one PNA conjugate to an acceptor peptide-PNA conjugate. We asked two key questions. What are the chemical requirements that allow RNA-templated synthesis of a 16-mer peptide to proceed at lower (nanomolar) concentrations of RNA, that is, the concentration range found in cancer cells? Will such reactions provide sufficient amounts of peptide product and sufficient affinity to interfere with the targeted protein-protein interaction? Perhaps surprisingly, the lengths of the peptides involved in peptidyl transfer chemistry have little effect on the achievable rate enhancements. However, the nature of the thioester C terminus, the distance between the targeted template annealing sites, and template affinity play important roles. The investigation revealed guidelines for the reaction design for peptidyl transfer with low amounts (1-10 nm) of RNA, yet still provide sufficient product to antagonize a protein-protein interaction.

Invited for the cover of this issue is the group of Christian P. R. Hackenberger at the Leibniz‐Forschungsinstitut für Molekulare Pharmakologie and the Humboldt‐Universität zu Berlin. The image depicts a phospho‐lysine peptide mimic which reflects a phosphorylated lysine but is not identical. Read the full text of the article at 10.1002/chem.202003947.

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The intrinsic lability of the phosphoramidate P−N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post‐translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl‐methyloxycarbonyl‐ (Fmoc‐)based solid‐phase peptide synthesis (SPPS). In order to compare the biophysical properties of natural pLys with our synthetic mimics, the pKa values of pLys and analogues were determined in titration experiments applying nuclear magnetic resonance (NMR) spectroscopy in small model peptides. These results were used to compute electrostatic potential (ESP) surfaces obtained after molecular geometry optimization. These findings indicate the potential of the designed non‐hydrolyzable, phosphonate‐based mimic for pLys in various proteomic approaches.

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