Rémi Desmet

A One-Pot Three-Segment Ligation Strategy for Protein Chemical Synthesis†

Protein chemical synthesis by native peptide ligation of unprotected peptide segments is an interesting complement and potential alternative to the use of living systems for producing proteins. Actually, tremendous efforts are focused on the design of one-pot strategies allowing the assembly of three peptide segments. The goal is to get rapid access to small proteins (less than 150 amino acid residues), while saving intermediate purification steps and obtaining the products in good yield. Such methods are gaining increasing significance for the study of protein function and appear as a potential option for producing various protein-based therapeutics currently under development. To date, proteins were mainly assembled by sequential native chemical ligation (NCL) or extended methodologies in the C-to-N direction (for recent achievements, see Refs. [8, 9]). NCL involves the chemoselective ligation of a Cterminal peptide thioester, usually an alkylthioester, with an N-terminal cysteine (Cys) peptide. The one-pot sequential Cto-N ligation of three peptide segments designed by Kent et al. is increasingly used for synthesizing proteins. Methods that enable the assembly of peptide segments in the reverse N-to-C direction are rare. 11] Fundamentally, the combination of N-to-C and C-to-N assembly techniques is at the basis of the convergent total synthesis of proteins. The general principle of the one-pot assembly of three peptide segments in the N-to-C direction is illustrated in Scheme 1. Ligation of peptide segments A-X and H-Cys-B-Y yields segment A-Cys-B-Y (Scheme 1, ligation 1). Group Y must ideally be inert during ligation 1 or at least be significantly less reactive than group X to avoid oligomerization or cyclization of segment B. Activation of group Y into Y* subsequently allows the ligation with the third segment H-Cys-C (Scheme 1, ligation 2). For designing a one-pot process working in the N-to-C direction, this activation must be carried out in situ after ligation 1 by using reagents compatible with ligation 2. Furthermore, the Y* group must enable an efficient ligation with the Cys segment C. To date only few one-pot strategies have been described that work in the N-to-C direction and enable the coupling of three peptide segments. 5, 12] Fundamentally, these methods, such as kinetically controlled ligation, rely on the differential reactivity of X and Y groups for peptide-bond formation. In other words, the purity of the target polypeptide is highly dependent on the C-terminal residues of A and B segments and more generally on the accessibility of the reactive ends. Clearly, a strategy in which Y is inert during the first ligation step would bypass these limitations and constitute a critical advance. Herein we show that the combination of NCL and SEA ligation (Scheme 1) permitted design of a solution to this important problem. Reaction of a peptide featuring a C-terminal bis(2-sulfanylethyl)amido group, called SEA hereafter (Scheme 1), with a Cys peptide results in the formation of a native peptide bond in water at pH 7. This reaction probably proceeds via Scheme 1. Total protein synthesis by one-pot assembly of three peptide segments in the N-to-C direction. The first step is a native chemical ligation between thioester segment A and Cys segment B, during which the cyclic disulfide SEA acts as a blocked thioester group (SEA = bis(2-sulfanylethyl)amido). Activation of SEA into SEA by reduction with a phosphine and addition of the third Cys segment C triggers the second ligation step.

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.

Highly efficient solid phase synthesis of large polypeptides by iterative ligations of bis(2-sulfanylethyl)amido (SEA) peptide segments

Up to now, the advantages of solid phase protein synthesis have been largely under-utilized due to the difficulty of designing a simple and efficient elongation cycle enabling the concatenation of unprotected peptide segments. The combination of selective N-terminal anchoring (N3-Esoc linker) with the blocked thioester properties of the SEAoff group enabled the solid phase concatenation of unprotected peptide segments by N-to-C sequential formation of native peptide bonds. The strategy was applied to the synthesis of a 60 amino acid-long latent peptide thioester or to the assembly of five peptide segments to give a 15 kDa polypeptide.

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

Total synthesis of biotinylated N domain of human hepatocyte growth factor.

Hepatocyte growth factor/scatter factor (HGF/SF) is the high affinity ligand of MET tyrosine kinase receptor. We report here the total synthesis of a biotinylated analogue of human HGF/SF N domain. Functionally, N domain is part of the HGF/SF high affinity binding site for MET and also the main HGF/SF binding site for heparin. The 97 Aa linear chain featuring a C-terminal biotin group was assembled in high yield using an N-to-C one-pot three segments assembly strategy relying on a sequential Native Chemical Ligation (NCL)/bis(2-sulfanylethyl)amido (SEA) native peptide ligation process. The folded protein displayed the native disulfide bond pattern and showed the ability to bind heparin.

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.

PASE: A Web-Based Platform for Peptide/Protein Microarray Experiments

Peptide microarray technology requires bioinformatics and statistical tools to manage, store, and analyze the large amount of data produced. To address these needs, we developed a system called protein array software environment (PASE) that provides an integrated framework to manage and analyze microarray information from polypeptide chip technologies.

In situ ligation between peptides and silica nanoparticles for making peptide microarrays on polycarbonate.

Bisphenol A polycarbonate (PC) is emerging as an interesting alternative to silicon oxide substrates for making microarrays. We show that the printing of peptide/nanoparticle mixtures allows the creation of complex peptide microarrays. Semicarbazone ligation was used for linking the peptides to the nanoparticles. The reaction occurred probably after printing due to solvent evaporation and to the in situ concentration of the reagents. Peptide microarrays were used successfully for the specific capture of purified antibodies or of antibodies from serum.

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.