Emmanuelle Boll

One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis (2-sulfanylethyl)amido peptide latent thioester surrogates

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO–peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO–peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO–peptide conjugates on a milligram scale takes 20 working days.

Access to cyclic or branched peptides using bis(2-sulfanylethyl)amido side-chain derivatives of Asp and Glu.

Bis(2-sulfanylethyl)amido (SEA) side-chain derivatives of aspartic and glutamic acids enable the synthesis of tail-to-side chain cyclic or branched peptides using standard Fmoc-SPPS followed by SEA native peptide ligation.

Access to Large Cyclic Peptides by a One-Pot Two-Peptide Segment Ligation/Cyclization Process

The use of the N-acetoacetyl protecting group for N-terminal cysteine residue enabled creation of an efficient and mild one-pot native chemical ligation/SEA ligation sequence giving access to large cyclic peptides.

A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate

A bis(2-sulfanylethyl)amino PEG-based resin enabled the synthesis of large (∼50 Aa) SEA or thioester peptides using Fmoc-SPPS. These peptide segments permitted the first total synthesis of a 97 amino-acid long SUMO-1-SEA peptide thioester surrogate and of a functional and reversible SUMO-1 peptide conjugate.

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters†

Protein total chemical synthesis enables the atom‐by‐atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C‐terminal peptide thioesters with N‐terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc‐SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2‐sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C‐terminal bis(2‐sulfanylethyl)amido peptides and of peptide thioesters derived from 3‐mercaptopropionic acid. Copyright

Synthesis of peptide thioacids at neutral pH using bis(2-sulfanylethyl)amido peptide precursors.

Reaction of bis(2-sulfanylethyl)amido (SEA) peptides with triisopropylsilylthiol in water at neutral pH yields peptide thiocarboxylates. An alkylthioester derived from β-alanine was used to trap the released bis(2-sulfanylethyl)amine and displace the equilibrium toward the peptide thiocarboxylate.

Selenopeptide transamidation and metathesis.

Selenopeptides can be transamidated by cysteinyl peptides in water using mild conditions (pH 5.5, 37 °C) in the presence of an arylthiol catalyst. Similar conditions also catalyze the metathesis of selenopeptides. The usefulness of the selenophosphine derived from TCEP (TCEP=Se) for inhibiting the TCEP-induced deselenization of selenocysteine residue is also reported.

Exploration of an imide capture/N,N-acyl shift sequence for asparagine native peptide bond formation.

Imide capture of a C-terminal peptidylazide with a side-chain thioacid derivative of an N-terminally protected aspartyl peptide leads to the formation of an imide bond bringing the two peptide ends into close proximity. Unmasking of the N(α) protecting group and intramolecular acyl migration results in the formation of a native peptide bond to asparagine.

A Central Cysteine Residue Is Essential for the Thermal Stability and Function of SUMO-1 Protein and SUMO-1 Peptide–Protein Conjugates

SUMOylation constitutes a major post-translational modification (PTM) used by the eukaryote cellular machinery to modulate protein interactions of the targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a central and conserved cysteine residue (Cys52) that is located in the hydrophobic core of the protein and in tight contact with Phe65, suggesting the occurrence of an S/π interaction. To investigate the importance of Cys52 on SUMO-1 thermal stability and biochemical properties, we produced by total chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide-protein conjugates featuring a native isopeptidic bond between SUMO-1 and a peptide derived from p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1 secondary structure and resulted in a dramatic loss of protein thermal stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating enzyme Upl1 was significantly less efficient than for the wild-type conjugate. Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was significantly less efficient with the SUMO-1 C52A analog compared to wild-type SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1 conformation and function and the importance of keeping this cysteine intact for the study of SUMO-1 protein conjugates.

Synthesis of Unprotected Linear or Cyclic O-Acyl Isopeptides in Water Using Bis(2-sulfanylethyl)amido Peptide Ligation

SEA ligation proceeds chemoselectively at pH 3, i.e., at a pH where the O-acyl isopeptides are protected by protonation. This property was used for synthesizing unprotected O-acyl isopeptides in water, starting from peptide segments which are easily accessible by the Fmoc SPPS.