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

Installation of electrophiles onto the C-terminus of recombinant ubiquitin and ubiquitin-like proteins

Ubiquitin and related ubiquitin-like proteins (Ubls) influence a variety of cellular pathways including protein degradation and response to viral infections. The chemical interrogation of these complex enzymatic cascades relies on the use of tailored activity-based probes (ABPs). Herein, we report the preparation of ABPs for ubiquitin, NEDD8, SUMO2 and ISG15 by selective acyl hydrazide modification. Acyl hydrazides of Ubls are readily accessible by direct hydrazinolysis of Ubl-intein fusions. The suppressed pK(a) and superior nucleophilicity of the acyl hydrazides enables their selective modification at acidic pH with carboxylic acid anhydrides. The modification proceeds rapidly and efficiently, and does not require chromatographic purification or refolding of the probes. We modified Ubl-NHNH2 with various thiol-reactive electrophiles that couple selectively with E2s and DUBs. The ease of modification enables the rapid generation and screening of ubiquitin probes with various C-terminal truncations and warheads for the selection of the most suitable combination for a given E2 or DUB.ISSN:2041-6520ISSN:2041-653

Prevention of aspartimide formation during peptide synthesis using cyanosulfurylides as carboxylic acid-protecting groups

Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challenge of 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 during synthesis. Deprotection is readily and rapidly achieved under aqueous conditions with electrophilic halogenating agents via a highly selective C–C bond cleavage reaction. This protecting group is employed for the synthesis of a range of peptides and proteins including teduglutide, ubiquitin, and the low-density lipoprotein class A. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.ISSN:2041-172

Anisotropic 3D texture synthesis with application to volume rendering

We present a novel approach to improving volume rendering by using synthesized textures in combination with a custom transfer function. First, we use existing knowledge to synthesize anisotropic solid textures to fit our volumetric data. As input to the synthesis method, we acquire high quality images using a 12.1 megapixel camera. Next, we extend the volume rendering pipeline by creating a transfer function which yields not only color and opacity from the input intensity, but also texture coordinates for our synthesized 3D texture. Thus, we add texture to the volume rendered images. This method is applied to a high quality visualization of a pig carcass, where samples of meat, bone, and fat have been used to produce the anisotropic 3D textures

Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1

Aberrations in protein modification with ubiquitin-fold modifier (UFM1) are associated with a range of diseases, but the biological function and regulation of this post-translational modification, known as UFMylation, remain enigmatic. To provide activity-based probes for UFMylation, we have developed a new method for the installation of electrophilic warheads at the C-terminus of recombinant UFM1. A C-terminal UFM1 acyl hydrazide was readily produced by selective intein cleavage and chemoselectively acylated by a variety of carboxylic acid anhydrides at pH 3, without detriment to the folded protein or reactions at unprotected amino acid side chains. The resulting UFM1 activity-based probes show a range of tunable reactivity and high selectivity for proteins involved in UFMylation processes; structurally related E1s, E2s, and proteases associated with Ub or other Ubls were unreactive. The UFM1 probes were active both in cell lysates and in living cells. A previously inaccessible alpha-chloroacetyl probe was remarkably selective for covalent modification of the active-site cysteine of de-UFMylase UFSP2 in cellulo.ISSN:2374-795

Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon beta-sheet structure binding to the backside of LC3. We show that the beta-sheet conformation is crucial for its interaction with LC3 and used this insight to design synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabled in cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3 similar to LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3.ISSN:2374-795

Widespread microbial utilization of ribosomal β-amino acid-containing peptides and proteins

β-Amino residues are regarded as extremely rare features among ribosomal products. They can be installed by a remarkable non-canonical enzymatic splicing process occurring in some Nif11-type ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways from select cyanobacteria. The functions of the final pathway products remained unknown. Here, a global bioinformatic analysis suggested an unexpectedly broad distribution of ribosomal β-amino acid products in diverse bacterial lineages as well as archaea. Characterization of 27 bacterial splicease-substrate pairs confirmed the modification in all cases. The “spliceotide” products include many previously unrecognized RiPP types as well as proteins, contain 35 to >600 residues, and feature single to multiple α-keto-β-amino acid moieties, with 15 different naturally occurring β units characterized and 20 predicted. Of three tested spliceotides, all exhibited exceptionally potent protease inhibitory activity, providing a potential rationale for the widespread splicease chemistry in prokaryotes and highlighting substantial potential for drug discovery and gene-based biomolecule diversification.ISSN:2451-9294ISSN:2451-930