Fuscimiditide: a RiPP with Ω-Ester and Aspartimide Post-translational Modifications

Microviridins and other ω-ester linked peptides (OEPs) are characterized by sidechain-sidechain linkages installed by ATP-grasp enzymes. Here we describe the discovery of a new family of OEPs, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase (PIMT). We produced the first example of this new ribosomally synthesized and post-translationally modified peptide (RiPP), fuscimiditide, via heterologous expression. NMR analysis of fuscimiditide revealed that the peptide contains two ester crosslinks forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We have also fully reconstituted fuscimiditide biosynthesis in vitro establishing that ester formation catalyzed by the ATP-grasp enzyme is an obligate, rate-limiting first biosynthetic step. Aspartimide formation from aspartate is catalyzed by the PIMT homolog in the second step. The aspartimide moiety embedded in fuscimiditide hydrolyzes regioselectively to isoaspartate (isoAsp). Surprisingly, this isoAsp-containing protein is also a substrate for the PIMT homolog, thus driving any hydrolysis products back to the aspartimide form. Whereas aspartimide is often considered a nuisance product in protein formulations, our data here suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.

Synthesis of Multi-Module Low Density Lipoprotein Receptor Class A Domains with Acid Labile Cyanopyridiniumylides (CyPY) as Aspartic Acid Masking Groups

<div><div><div><p>Low-Density Lipoprotein Receptor Class A Domains (LA modules) are common ligand-binding domains of transmembrane receptors of approximately 40 amino acids that are involved in several cellular processes including endocytosis of extracellular targets. Due to their wide-ranging function and distribution among different transmembrane receptors, LA modules are of high interest for therapeutic applications. However, the efficient chemical synthesis of LA modules and derivatives is hindered by complications, many arising from the high abundance of aspartic acid and consequent aspartimide formation. Here, we report a robust, efficient and general applicable chemical synthesis route for accessing such LA modules, demonstrated by the synthesis and folding of the LA3 and LA4 modules of the Low-Density Lipoprotein Receptor, as well as a heterodimeric LA3-LA4 constructed by chemical ligation. The synthesis of the aspartic acid-rich LA domain peptides is made possible by the use of cyanopyridiniumylides (CyPY) – reported here for the first time – as a masking group for carboxylic acids. We show that cyanopyridiniumylide masked aspartic acid monomers are readily available building blocks for solid phase peptide synthesis and successfully suppress aspartimide formation. Unlike previously reported ylide-based carboxylic acid protecting groups, CyPY protected aspartic acids are converted to the free carboxylic acid by acidic hydrolysis and are compatible with all common residues and protecting groups. The chemical synthesis of Cys- and Asp-rich LA modules enable new access to a class of difficult to provide, but promising protein domains.</p></div></div></div>

Synthesis of Multi-Module Low Density Lipoprotein Receptor Class A Domains with Acid Labile Cyanopyridiniumylides (CyPY) as Aspartic Acid Masking Groups

<div><div><div><p>Low-Density Lipoprotein Receptor Class A Domains (LA modules) are common ligand-binding domains of transmembrane receptors of approximately 40 amino acids that are involved in several cellular processes including endocytosis of extracellular targets. Due to their wide-ranging function and distribution among different transmembrane receptors, LA modules are of high interest for therapeutic applications. However, the efficient chemical synthesis of LA modules and derivatives is hindered by complications, many arising from the high abundance of aspartic acid and consequent aspartimide formation. Here, we report a robust, efficient and general applicable chemical synthesis route for accessing such LA modules, demonstrated by the synthesis and folding of the LA3 and LA4 modules of the Low-Density Lipoprotein Receptor, as well as a heterodimeric LA3-LA4 constructed by chemical ligation. The synthesis of the aspartic acid-rich LA domain peptides is made possible by the use of cyanopyridiniumylides (CyPY) – reported here for the first time – as a masking group for carboxylic acids. We show that cyanopyridiniumylide masked aspartic acid monomers are readily available building blocks for solid phase peptide synthesis and successfully suppress aspartimide formation. Unlike previously reported ylide-based carboxylic acid protecting groups, CyPY protected aspartic acids are converted to the free carboxylic acid by acidic hydrolysis and are compatible with all common residues and protecting groups. The chemical synthesis of Cys- and Asp-rich LA modules enable new access to a class of difficult to provide, but promising protein domains.</p></div></div></div>

The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation

Aspartimide formation often complicates the solid phase synthesis of peptides. Much less discussed is the potential occurrence of this side-reaction during the coupling of peptide segments using chemoselective peptide bond forming reactions such as the native chemical ligation and extended methods. Here we describe how to manage this problem using bis(2-sulfenylethyl)amido (SEA)-mediated ligation and SUMO-2/SUMO-3 as protein targets.<br>

The Problem of Aspartimide Formation During Protein Chemical Synthesis Using SEA-Mediated Ligation

Aspartimide formation often complicates the solid phase synthesis of peptides. Much less discussed is the potential occurrence of this side-reaction during the coupling of peptide segments using chemoselective peptide bond forming reactions such as the native chemical ligation and extended methods. Here we describe how to manage this problem using bis(2-sulfenylethyl)amido (SEA)-mediated ligation and SUMO-2/SUMO-3 as protein targets.<br>

Cyanosulfurylides (CSY): Carboxylic Acid Protecting Groups That Prevent Aspartimide Formation During Peptide Synthesis

Peptide chemistry has made great progress in the last decades but the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification steps or even inaccessible peptide sequences, hindering both academic research and industrial applications. Here, we report a new alternative approach to address this longstanding challenge of solid phase peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C–C bond. These functional groups – formally zwitterionic species – are exceptionally stable to all common manipulations and impart improved solubility and processing during peptide synthesis. Deprotection is readily and rapidly achieved under mild, aqueous conditions with electrophilic halogenating agents via a highly selective C–C bond cleavage reaction. This new protecting group was employed for the synthesis a range of peptides and proteins including teduglutide, ubiquitin, and LDLa – a peptide that was not accessible on solid-phase peptide synthesis before due to three aspartimide-prone motifs. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.

Cyanosulfurylides (CSY): Carboxylic Acid Protecting Groups That Prevent Aspartimide Formation During Peptide Synthesis

Peptide chemistry has made great progress in the last decades but the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification steps or even inaccessible peptide sequences, hindering both academic research and industrial applications. Here, we report a new alternative approach to address this longstanding challenge of solid phase peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C–C bond. These functional groups – formally zwitterionic species – are exceptionally stable to all common manipulations and impart improved solubility and processing during peptide synthesis. Deprotection is readily and rapidly achieved under mild, aqueous conditions with electrophilic halogenating agents via a highly selective C–C bond cleavage reaction. This new protecting group was employed for the synthesis a range of peptides and proteins including teduglutide, ubiquitin, and LDLa – a peptide that was not accessible on solid-phase peptide synthesis before due to three aspartimide-prone motifs. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.