Native Chemical Ligation Method Research Articles

Total Chemical Synthesis of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 and its global spread have created an unprecedented public health crisis. The spike protein of SARS-CoV-2 has gained significant attention due to its crucial role in viral entry into host cells and its potential as both a prophylactic and a target for therapeutic interventions. Herein, we report the first successful total synthesis of the SARS-CoV-2 spike protein receptor binding domain (RBD), highlighting the key challenges and the strategies employed to overcome them. Appropriate utilization of advanced solid phase peptide synthesis and cutting-edge native chemical ligation methods have facilitated the synthesis of this moderately large protein molecule. We discuss the problems encountered during the chemical synthesis and approaches taken to optimize the yield and the purity of the synthetic protein molecule. Furthermore, we demonstrate that the chemically synthesized homogeneous spike RBD efficiently binds to the known mini-protein binder LCB1. The successful chemical synthesis of the spike RBD presented here can be utilized to gain valuable insights into SARS-CoV-2 spike RBD biology, advancing our understanding and aiding the development of intervention strategies to combat future coronavirus outbreaks. The modular synthetic approach described in this study can be effectively implemented in the synthesis of other mutated variants or enantiomer of the spike RBD for mirror-image drug discovery.

Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several “gold-standard” ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0–7.5, resulting in ligation yields of up to 80–95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current “gold-standard” NCL methods.

Homogeneous Sulfopeptides and Sulfoproteins: Synthetic Approaches and Applications To Characterize the Effects of Tyrosine Sulfation on Biochemical Function

Post-translational modification of proteins plays critical roles in regulating structure, stability, localization, and function. Sulfation of the phenolic side chain of tyrosine residues to form sulfotyrosine (sTyr) is a widespread modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection. Tyrosine sulfation commonly occurs in sequences containing clusters of tyrosine residues and is incomplete at each site, resulting in heterogeneous mixtures of sulfoforms. Purification of individual sulfoforms is typically impractical. Therefore, the most promising approach to elucidate the influence of sulfation at each site is to prepare homogeneously sulfated proteins (or peptides) synthetically. This Account describes our recent progress in both development of such synthetic approaches and application of the resulting sulfopeptides and sulfoproteins to characterize the functional consequences of tyrosine sulfation. Initial synthetic studies used a cassette-based solid-phase peptide synthesis (SPPS) approach in which the side chain sulfate ester was protected to enable it to withstand Fmoc-based SPPS conditions. Subsequently, to address the need for efficient access to multiple sulfoforms of the same peptide, we developed a divergent solid-phase synthetic approach utilizing orthogonally side chain protected tyrosine residues. Using this methodology, we have carried out orthogonal deprotection and sulfation of up to three tyrosine residues within a given sequence, allowing access to all eight sulfoforms of a given target from a single solid-phase synthesis. With homogeneously sulfated peptides in hand, we have been able to probe the influence of tyrosine sulfation on biochemical function. Several of these studies focused on sulfated fragments of chemokine receptors, key mediators of leukocyte trafficking and inflammation. For the receptor CCR3, we showed that tyrosine sulfation enhances affinity and selectivity for binding to chemokine ligands, and we determined the structural basis of these affinity enhancements by NMR spectroscopy. Using a library of CCR5 sulfopeptides, we demonstrated the critical importance of sulfation at one specific site for supporting HIV-1 infection. Demonstrating the feasibility of producing homogeneously tyrosine-sulfated proteins, in addition to smaller peptides, we have used SPPS and native chemical ligation methods to synthesize the leech-derived antithrombotic protein hirudin P6, containing both tyrosine sulfation and glycosylation. Sulfation greatly enhanced inhibitory activity against thrombin, whereas addition of glycans to the sulfated protein decreased inhibition, indicating functional interplay between different post-translational modifications. In addition, the success of the ligation approach suggests that larger sulfoproteins could potentially be obtained by ligation of synthetic sulfopeptides to expressed proteins, using intein-based technology.

Preparation of truncated orf virus entry fusion complex proteins by chemical synthesis

Members of the Chordopoxvirinae subfamily possess an unusual 11 protein entry-fusion complex (EFC) that is highly conserved and present in all species. The mode of action of this EFC is unknown, and the interactions of the constituent proteins are uncharacterised. Here, we present the chemical synthesis of membrane domain truncated linear constructs of two EFC proteins in orf virus, ORFV036 and 049. By using Boc solid phase peptide synthesis and native chemical ligation methods, these truncated proteins have been readily prepared in milligram quantities. These robust synthetic protocols allow ready access to these polypeptides to facilitate biological studies.

Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (1–3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

Nucleosome-Nucleosome Stacking: A Major Element of Chromatin Structure

Interactions between nucleosome core particles (NCP) are key features of chromatin folding and are inherently related to regulation of DNA replication, transcription and repair. However, little is known about structures of NCP-NCP contacts as well as about the nature and scale of the forces involved. The NCP and chromatin are polyelectrolytes which show a sensitivity of folding and self-association both to ionic environment and to position, sequence and modifications of histone N-termini (tails). Combining experimental and computer modeling approaches, we study nucleosome-nucleosome interaction in both general polyelectrolyte and specific structural context. using native chemical ligation methods, large quantities of H4 histones containing acetylated and methylated lysines were synthesized. The influence of these posttranslational modifications on folding and intermolecular association of the NCPs and model chromatin arrays was studied as a function of the nature and concentration of monovalent and multivalent cations (K+, Na+, Mg2+, Co(NH3)63+, spermine4+). Additionally, computational modelling was used to investigate NCP-NCP interaction. A coarse-grained model of the NCP takes into account the details of its structure, based on the single crystal structure at atomic resolution and adequately describes the strong electrostatic forces acting in chromatin by explicit modelling of ions. Most experimental data is well-described by this electrostatic model. However, NCP-NCP close contact (stacking) shows sensitivity to the presence of the H4 histone tail and to acetylation of lysine 16 and other modifications in this histone which is beyond electrostatics. We suggest a structural model explaining the mechanism of these combined modification factors.

At last: erythropoietin as a single glycoform.

At last: erythropoietin as a single glycoform.

Influence of Histone Tails and H4 Tail Acetylations on Nucleosome–Nucleosome Interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg2+, Ca2+, cobalt(III)hexammine3+, spermidine3+ and spermine4+] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K→Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.

Hexafluoro-2-propanol as a potent cosolvent for chemical ligation of membrane proteins

The study on membrane proteins is an important challenge mainly because of their very poor solubility in various solvents. The traditional recombinant expression strategy and the native chemical ligation method both have difficulty in generating sufficient amounts of desired proteins with high efficiency. Previous studies have shown that multiply fluorinated alcohols exhibit good ability to dissolve difficult peptide sequences, especially hexafluoro-2-propanol (HFIP). In the present study we systematically studied the capability of solvents containing different percentage of HFIP in dissolving transmembrane peptides. Through both HPLC and UV analyses we concluded that 60% HFIP/8 M urea constituted a good solvent system. In this solvent system we also optimized conditions to perform native chemical ligation (NCL). Under the optimized conditions we successfully achieved NCL’s for both dipeptide formation and the synthesis of a model protein (Trifolitoxin). These results suggested that HFIP was a potential cosolvent that could be used in the ligation of poorly soluble peptides for the generation of membrane proteins.

Development of highly pure α-helical lipoglycopeptides as self-adjuvanting vaccines

The incorporation of lipid moieties into synthetic peptide vaccines has been demonstrated to self-adjuvant otherwise poorly immunogenic peptides, whereas carbohydrates have emerged to be advantageous carriers for assembling these peptides. With the advent of an efficient native chemical ligation method, which is compatible with both peptides and carbohydrates, we have developed highly pure self-adjuvanting tetravalent group A streptococcal vaccine candidates assembled on carbohydrate templates. The utility of chemoselective ligation has overcome difficulties in the synthesis and purification of branched high molecular weight peptides. Circular dichroism measurements provided the evidence of α-helix formation of the assembled peptide epitopes, which may have impact on their immunogenicity.

Conjugation of Au11 cluster with Cys-rich peptides containing the α-domain of metallothionein

The stoichiometrically controlled complexation of abiological inorganic clusters with proteins is a challenging task that enables us to combine the functions of inorganic clusters with those of biological systems on the basis of chemical characterisation. In this study, we synthesised a 1:1 conjugate between Au11(PPh3)8Cl3 (Au11) and CGMTIIalpha, where Au11 is the smallest cluster, which is situated on the boundary of molecules and nanoparticles, having discrete electronic states and exhibiting Coulomb blockade; on the other hand, CGMTIIalpha is a twelve-cysteine (Cys)-containing peptide consisting of the alpha-domain of rat liver metallothionein II (MTIIalpha)--a heavy metal-binding protein--and an addendum, cysteine-glycine (CG). The Cys residue of the N-terminus is used as a site for elongation by the native chemical ligation (NCL) method. Titration experiments monitored by circular dichroism spectroscopy indicate that CGMTIIalpha reacts with Au11 in a ratio of 1:1. The product exhibits a negative Cotton effect at 222 nm, indicating the formation of a short a-helix. The composition of this hybrid module is quantified as Au11(PPh3)4(S-Cys)6 by transmission electron microscopy (core size observation), energy-dispersive X-ray spectroscopy (elemental analysis), Ellman test (uncoordinated Cys residues), and high-performance liquid chromatography (quantification of eliminated PPh3). These results, as well as those obtained from the simulations of Au 4f X-ray photoelectron spectra, suggest that the Au cluster of this product is Au11(PPh3)4(S-Cys)6. We also prepared F3F3-CGMTIIalpha, where F3F3 is a zinc finger peptide with two domains, in order to demonstrate the applicability of the NCL method to the elongation of CGMTIIalpha. F3F3-CGMTIIalpha also reacts with Au11 to yield a 1:1 conjugate without interfering in the folding of the zinc finger.

Use of thiosulfonate for the protection of thiol groups in peptide ligation by the thioester method

Use of thiosulfonate for protecting thiol (-SH) groups in peptide ligation by the thioester method was examined. Thiosulfonate was introduced and was stable in the presence of silver ion, 4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and diisopropylethylamine. Based on these results, a strategy for using the thioester method and the native chemical ligation method in the synthesis of a single polypeptide is described.

A photoremovable ligation auxiliary for use in polypeptide synthesis

A photoremovable auxiliary for use in peptide synthesis via the native chemical ligation method is described. A 2-mercapto-1-(2-nitrophenyl)ethylamine (Mnpe-amine) moiety was attached to the N-terminus of a peptide via the periodate oxidation of a seryl peptide. The resulting peptide was then ligated to a peptide thioester, and UV (365 nm) irradiation resulted in the removal of the auxiliary from the peptide.

Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro.

Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.

Fmoc-Based Synthesis of Peptide-αThioesters: Application to the Total Chemical Synthesis of a Glycoprotein by Native Chemical Ligation

The technique of native chemical ligation has enabled the total chemical synthesis of proteins with molecular weights far in excess of those achievable by conventional stepwise solid-phase peptide synthesis. The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal αthioester and the other an N-terminal cysteine residue, to afford a protein with a native amide linkage at the site of ligation. Here we report an extension of the native chemical ligation method to the total synthesis of a glycosylated protein, the antimicrobial O-linked glycoprotein diptericin. The major challenge in our synthesis was preparation of a 24-residue glycopeptide-αthioester segment, which was complicated by the incompatibility of glycosidic linkages with Boc chemistry and by the incompatibility of thioesters with Fmoc chemistry. The use of an alkanesulfonamide “safety-catch” linker circumvented this problem and permitted the solid-phase synthesis of the glycopeptide-αthioester using standa...

Solid Phase Synthesis of Peptide C-Terminal Thioesters by Fmoc/t-Bu Chemistry

Peptide C-terminal thioesters are key intermediates in a variety of applications, most notably the recently developed native chemical ligation methods for the total chemical synthesis of proteins. So far they have been prepared only by the use of the least prevalent Boc/benzyl solid-phase method on ad hoc prepared resin supports. We describe here a novel method for the solid phase synthesis of thioesters by the most prevalent Fmoc/t-Bu method. The method is based on the use of a 3-carboxypropanesulfonamide safety-catch linker, which is fully stable to repetitive exposure to the basic conditions needed for Fmoc cleavage. Activation with diazomethane or iodoacetonitrile followed by displacement with a suitable thiol produces the thioester in good to excellent yields. The method is also compatible with Boc/benzyl chemistry. Moreover, all the necessary reagents are commercially available.