Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step

Abstract

We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications.

Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides

“…S10†), confirming that these do not arise from further changes in stereochemistry, such as anomerisation. A similar level of diastereoselectivity to our observations described above was also reported for addition of a peptide thiol to Dha in ubiquitin, in either native or denaturing conditions and at two different locations, 28 corroborating the modest stereochemical control observed here.…”

Selenolysine: A New Tool for Traceless Isopeptide Bond Formation

“…[8] Michael addition to the resulting Dha hasb een used to introduce many small-molecule PTMs and entireP TM proteins, as in the case of ubiquitylated a-globin. [9] However,astereoselectivem ethod for addition to Dha has yet to be developed, [9] ad rawback in the chiral world of biology.…”

Activity‐Based Probes Developed by Applying a Sequential Dehydroalanine Formation Strategy to Expressed Proteins Reveal a Potential α‐Globin‐Modulating Deubiquitinase

“…Inspired by our recent study on the ubiquitination of expressed proteins by the use of aprotein DHA precursor, [14] we decided to develop an approach to install aCys residue on preformed DHA, which should enable the formation of af urther DHA moiety at al ater stage. Inspired by our recent study on the ubiquitination of expressed proteins by the use of aprotein DHA precursor, [14] we decided to develop an approach to install aCys residue on preformed DHA, which should enable the formation of af urther DHA moiety at al ater stage.…”

“…Thep robe was purified and characterized by mass spectrometry and circular dichroism for correct folding ( Figure 1A;see also Figure S13 in the Supporting Information). [14] Furthermore,t wo control probes were prepared by total chemical synthesis ( Figure 1B;s ee also Scheme S2 in the Supporting Information): Thef irst probe lacked Ub and was composed of aglobin with aDHA residue,whereas in the second, Ub-DHA was connected to as ingle lysine residue.W ei nitially characterized the activity of the probes in vitro using USP7, aDUB that cleaves Ub and different Ub chains from several ubiquitinated proteins. [14] Furthermore,t wo control probes were prepared by total chemical synthesis ( Figure 1B;s ee also Scheme S2 in the Supporting Information): Thef irst probe lacked Ub and was composed of aglobin with aDHA residue,whereas in the second, Ub-DHA was connected to as ingle lysine residue.W ei nitially characterized the activity of the probes in vitro using USP7, aDUB that cleaves Ub and different Ub chains from several ubiquitinated proteins.…”

“…Our strategy to generate an ABP based on ubiquitinated protein is shown in Scheme 1. Inspired by our recent study on the ubiquitination of expressed proteins by the use of aprotein DHA precursor, [14] we decided to develop an approach to install aCys residue on preformed DHA, which should enable the formation of af urther DHA moiety at al ater stage.…”