Redox-Controlled Chemical Protein Synthesis: Sundry Shades of Latency.

The last two decades have witnessed the rise in power of chemical protein synthesis to the point where it now constitutes an established corpus of synthetic methods efficiently complementing biological approaches. One factor explaining this spectacular evolution is the emergence of a new class of chemoselective reactions enabling the formation of native peptide bonds between two unprotected peptidic segments, also known as native ligation reactions. In recent years, their application has fueled the production of homogeneous batches of large and highly decorated protein targets with a control of their composition at the atomic level. In doing so, native ligation reactions have provided the means for successful applications in chemical biology, medicinal chemistry, materials science, and nanotechnology research.The native chemical ligation (NCL) reaction has had a major impact on the field by enabling the chemoselective formation of a native peptide bond between a C-terminal peptidyl thioester and an N-terminal cysteinyl peptide. Since its introduction in 1994, the NCL reaction has been made the object of significant improvements and its scope and limitations have been thoroughly investigated. Furthermore, the diversification of peptide segment assembly strategies has been essential to access proteins of increasing complexity and has had to overcome the challenge of controlling the reactivity of ligation partners.One hallmark of NCL is its dependency on thiol reactivity, including for its catalysis. While Nature constantly plays with the redox properties of biological thiols for the regulation of numerous biochemical pathways, such a control of reactivity is challenging to achieve in synthetic organic chemistry and, in particular, for those methods used for assembling peptide segments by chemical ligation. This Account covers the studies conducted by our group in this area. A leading theme of our research has been the conception of controllable acyl donors and cysteine surrogates that place the chemoselective formation of amide bonds by NCL-like reactions under the control of dichalcogenide-based redox systems. The dependency of the redox potential of dichalcogenide bonds on the nature of the chalcogenides involved (S, Se) has appeared as a powerful means for diversifying the systems, while allowing their sequential activation for protein synthesis. Such a control of reactivity mediated by the addition of harmless redox additives has greatly facilitated the modular and efficient preparation of multiple targets of biological relevance. Taken together, these endeavors provide a practical and robust set of methods to address synthetic challenges in chemical protein synthesis.

Phenylthiocarbamate or N-carbothiophenyl group chemistry in peptide synthesis and bioconjugation.

The design of novel chemoselective and site-specific ligation methods provides new tools for obtaining complex scaffolds, peptidomimetics, and peptide conjugates. The chemistry of the N-phenylthiocarbonyl group has led to several developments in peptide ligation chemistry and peptide bioconjugation during the last 10 years. The aim of this review is to provide an overview of this emerging field.

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.

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.

Sequential native peptide ligation strategies for total chemical protein synthesis.

Total chemical synthesis of proteins is usually achieved by assembling unprotected peptide segments using site-specific and chemoselective native peptide ligation methods. Access to large proteins often requires the assembly of at least three segments due to the current limits of solid phase synthesis of individual peptide segments. The aim of this tutorial review is to present the basic concepts and challenges underlying the design of sequential peptide ligation strategies using solution or solid phase chemistry. A special emphasis is given to C-to-N and N-to-C three-segment assembly strategies, which potentially give access to proteins composed of up to 150 amino acid residues.

Electrostatic Assistance of 4-Mercaptophenylacetic Acid-Catalyzed Native Chemical Ligation.

4-Mercaptophenylacetic acid (MPAA) is a popular catalyst of the native chemical ligation (NCL) but has to be used in large excess for achieving practically useful rates (up to 50-100 equiv). We report here that the catalytic potency of MPAA can be boosted by introducing a stretch of arginines in the departing thiol from the thioester. By doing so, the electrostatically assisted NCL reaction proceeds rapidly by using substoichiometric concentrations of MPAA, an advantage that enables useful synthetic applications.

A biomimetic electrostatic assistance for guiding and promoting N-terminal protein chemical modification.

The modification of protein electrostatics by phosphorylation is a mechanism used by cells to promote the association of proteins with other biomolecules. In this work, we show that introducing negatively charged phosphoserines in a reactant is a powerful means for directing and accelerating the chemical modification of proteins equipped with oppositely charged arginines. While the extra charged amino acid residues induce no detectable affinity between the reactants, they bring site-selectivity to a reaction that is otherwise devoid of such a property. They also enable rate accelerations of four orders of magnitude in some cases, thereby permitting chemical processes to proceed at the protein level in the low micromolar range, using reactions that are normally too slow to be useful in such dilute conditions.

Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides.

The design of novel methods giving access to peptide alkylthioesters, the key building blocks for protein synthesis using Native Chemical Ligation, is an important area of research. Bis(2-sulfanylethyl)amido peptides (SEA peptides) 1 equilibrate in aqueous solution with S-2-(2-mercaptoethylamino)ethyl thioester peptides 2 through an N,S-acyl shift mechanism. HPLC was used to study the rate of equilibration for different C-terminal amino acids and the position of equilibrium as a function of pH. We show also that thioester form 2 can participate efficiently in a thiol-thioester exchange reaction with 5% aqueous 3-mercaptopropionic acid. The highest reaction rate was obtained at pH 4. These experimental conditions are significantly less acidic than those reported in the past for related systems. The method was validated with the synthesis of a 24-mer peptide thioester. Consequently, SEA peptides 1 constitute a powerful platform for access to native chemical ligation methodologies.

Synthesis of peptide-protein conjugates using N-succinimidyl carbamate chemistry.

Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate (NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine residue within their sequences, can be converted in good yield into NSC derivatives by reaction with disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification, lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein. To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence. The conjugates were assembled by combining NSC chemistry and alpha-oxo semicarbazone ligation. To this end, a glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely, urea bond formation and alpha-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.

Catalysis of Hydrazone and Oxime Peptide Ligation by Arginine.

Hydrazone and oxime peptide ligations are catalyzed by arginine. The catalysis is assisted intramolecularly by the side-chain guanidinium group. Hydrazone ligation in the presence of arginine proceeds efficiently in phosphate buffer at neutral pH but is particularly powerful in bicarbonate/CO2 buffer. In addition to acting as a catalyst, arginine prevents the aggregation of proteins during ligation. With its dual properties as a nucleophilic catalyst and a protein aggregation inhibitor, arginine hydrochloride is a useful addition to the hydrazone/oxime ligation toolbox.

A cysteine selenosulfide redox switch for protein chemical synthesis.

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.

Silver-catalyzed azaGly ligation. Application to the synthesis of azapeptides and of lipid-peptide conjugates.

Glycine is a amino acid frequently found in peptides. Substitution of a glycine residue by an azaglycine allows the modulation of peptide conformation, bioactivity, or stability. Azapeptides are usually prepared using solid-phase synthetic procedures. We show here that azaGly peptides can be assembled chemoselectively and without racemization using unprotected peptide fragments by silver-catalyzed reaction of C-terminal peptide hydrazides with N-terminal phenylthiocarbonyl peptides. The reaction was performed in a tBuOH/water mixture and the control of apparent pH permitted the clean formation of the azaGly bond in the presence of lysine residues. We show also that this novel ligation method, called azaGly ligation, can be used for the assembly of lipopeptides. For this, lipid hydrazides were reacted with a phenylthiocarbonyl peptide in the presence of silver ions. This ligation method allows incorporation of acid-sensitive lipid moieties that are incompatible with standard solid-phase peptide synthesis procedures, and more generally should be of interest for the modification of peptides by sensitive acyl moieties.

Accelerated microfluidic native chemical ligation at difficult amino acids toward cyclic peptides.

Cyclic peptide-based therapeutics have a promising growth forecast that justifies the development of microfluidic systems dedicated to their production, in phase with the actual transitioning toward continuous flow and microfluidic technologies for pharmaceutical production. The application of the most popular method for peptide cyclization in water, i.e., native chemical ligation, under microfluidic conditions is still unexplored. Herein, we report a general strategy for fast and efficient peptide cyclization using native chemical ligation under homogeneous microfluidic conditions. The strategy relies on a multistep sequence that concatenates the formation of highly reactive S-(2-((2-sulfanylethyl)amino)ethyl) peptidyl thioesters from stable peptide amide precursors with an intramolecular ligation step. With very fast ligation rates (<5 min), even for the most difficult junctions (including threonine, valine, isoleucine, or proline), this technology opens the door toward the scale-independent, expedient preparation of bioactive macrocyclic peptides.

Improved Outcome by Adding Concurrent Chemotherapy to Cetuximab and Radiotherapy for Locally Advanced Head and Neck Carcinomas: Results of the GORTEC 2007-01 Phase III Randomized Trial.

Purpose To investigate the effect of adding concurrent chemotherapy (CT) to cetuximab plus radiotherapy (RT; CT-cetux-RT) compared with cetuximab plus RT (cetux-RT) in locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). Patients and Methods In this phase III randomized trial, patients with N0-2b, nonoperated, stage III or IV (nonmetastatic) LA-SCCHN were enrolled. Patients received once-daily RT up to 70 Gy with weekly cetuximab or with weekly cetuximab and concurrent carboplatin and fluorouracil (three cycles). To detect a hazard ratio (HR) of 0.64 for progression-free survival (PFS) with 85% power at a two-sided significance level of P = .05, 203 patients needed to be included in each arm. Results Four hundred six patients were randomly assigned to either CT-cetux-RT or cetux-RT. Patient and tumor characteristics were well balanced between arms, including p16 status. With a median follow-up of 4.4 years, the HR for PFS favored the CT-cetux-RT arm (HR, 0.73; 95% CI, 0.57 to 0.94; P = .015), with 3-year PFS rates of 52.3% and 40.5% and median PFS times of 37.9 and 22.4 months in the CT-cetux-RT and cetux-RT arms, respectively. The HR for locoregional control was 0.54 (95% CI, 0.38 to 0.76; P < .001) in favor of CT-cetux-RT. These benefits were observed regardless of p16 status for oropharynx carcinomas. Overall survival (HR, 0.80; P = .11) and distant metastases rates (HR, 1.19; P = .50) were not significantly different between the two arms. The CT-cetux-RT arm, compared with cetux-RT, had a higher incidence of grade 3 or 4 mucositis (73% v 61%, respectively; P = .014) and of hospitalizations for toxicity (42% v 22%, respectively; P < .001). Conclusion The addition of concurrent carboplatin and fluorouracil to cetux-RT improved PFS and locoregional control, with a nonsignificant gain in survival. To our knowledge, this is the first evidence of a clinical benefit for treatment intensification using cetux-RT as a backbone in LA-SCCHN.

Ti-Cp functionalization by deposition of organic/inorganic silica nanoparticles.

In orthopaedics and cardiovascular surgery, titanium has become the metal of choice, due to its excellent mechanical properties and biocompatibility. In many surgical operations, chemicals and/or biomolecules (such as antibiotics or growth factors) are used in conjunction with prostheses, so as to avoid or stimulate targeted biological events. Often, immobilization instead of release of such molecules is preferred to optimize their effects, thus avoiding ectopic transformations. A versatile method for the functionalization of pure Ti is shown here, which allows the covalent immobilization of polypeptides. In order to avoid the hydrolysable Ti-O-Si bond found in directly silanized Ti, we use organic/inorganic silica colloids, derived from commercially available 25 nm Ludox silica nanoparticles. Prior to deposition onto Ti-Cp, the silica nanoparticles are functionalized by a propylsemicarbazide moiety by silanization. After spin-coating onto the Ti substrates, the colloids were shown by SEM to form a uniform layer, and to be very strongly adsorbed; the reactivity of the supported semicarbazide (Sc) functionalities being maintained. Chemoselective reaction of semicarbazide groups on the surface with aldehyde moieties present on the polypeptide of interest was chosen in this work due to its efficiency, to its compatibility with the proteinogenic amino acids and in particular cystein and to the use of mild experimental conditions. Aldehyde groups are also easily introduced onto polypeptides by synthesis, oxidation of N-terminal Ser residue or polysaccharide moieties of glycoproteins. Biological assays with MC3T3-E1 osteoblasts revealed an excellent cytocompatibility as shown by the assessment of cell viability, vitality and morphology.

Accelerating chemoselective peptide bond formation using bis(2-selenylethyl)amido peptide selenoester surrogates.

Given the potential of peptide selenoesters for protein total synthesis and the paucity of methods for the synthesis of these sensitive peptide derivatives, we sought to explore the usefulness of the bis(2-selenylethyl)amido (SeEA) group, i.e. the selenium analog of the bis(2-sulfanylethyl)amido (SEA) group, for accelerating peptide bond formation. A chemoselective exchange process operating in water was devised for converting SEA peptides into the SeEA ones. Kinetic studies show that SeEA ligation, which relies on an initial N,Se-acyl shift process, proceeds significantly faster than SEA ligation. This property enabled the design of a kinetically controlled three peptide segment assembly process based on the sequential use of SeEA and SEA ligation reactions. The method was validated by the total synthesis of hepatocyte growth factor K1 (85 AA) and biotinylated NK1 (180 AA) domains.

Fmoc solid-phase synthesis of peptide thioesters using an intramolecularn,S-acyl shift.

[reaction: see text] We describe the Fmoc solid-phase synthesis of peptide thioesters based on the alkylation of the safety-catch sulfonamide linker with a protected 2-mercaptoethanol derivative. The thioester is generated on the solid phase after the peptide chain assembly as a consequence of an intramolecular N,S-acyl shift. Depending on the stability of the spacer separating the sulfonamide linker from the resin toward TFA, treatment of the peptidyl resin with TFA led to a soluble or supported deprotected thioester.

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.

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.