Synthesis and Semi-Synthesis of Alpha-Synuclein: Insight into the Chemical Complexity of Synucleinopathies.

The chemical rules governing protein folding have intrigued generations of researchers for decades. With the advent of artificial intelligence (AI), prediction of protein structure has improved tremendously. However, there is still a level of analysis that is only possible through wet laboratory experiments, especially in respect to the investigation of the pathological effect of mutations and posttranslational modifications (PTMs) on proteins of interest. This requires the availability of pure peptides and proteins in sufficient quantities for biophysical, biochemical, and functional studies. In this context, chemical protein synthesis and semi-synthesis are powerful tools in protein research, which help to enlighten the role of protein modification in the physiology and pathology of proteins. A protein of high interest in the field of biomedicine is alpha-synuclein (aSyn), a protein deeply associated with several devastating neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple systems atrophy (MSA). Here, we describe several methods and pathways to synthesize native or modified aSyn, and discuss how these approaches enable us to address pathological mechanisms that may open novel perspectives for therapeutic intervention.

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.

Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail.

CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.

Two Preparation Methods for Peptide Thioester Containing Tyr(SO3H) Residue(s) without the Use of Protecting Group for Sulfate Moiety.

We report two methods for the preparation of peptide thioesters containing Tyr(SO3H) residue(s), without use of a protecting group for the sulfate moiety. The first was based on direct thioesterification using carbodiimide on a fully protected peptide acid, prepared on a 2-chlorotrityl (Clt) resin with fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc-SPPS). Subsequent deprotection of the protecting groups with trifluoroacetic acid (TFA) (0 °C, 4 h) yielded peptide thioesters containing Tyr(SO3H) residue(s). Peptide thioesters containing one to three Tyr(SO3H) residue(s), prepared by this method, were used as building blocks for the synthesis of the Nα-Fmoc-protected N-terminal part of P-selectin glycoprotein ligand 1 (PSGL-1) (Fmoc-PSGL-1(43-74)) via silver-ion mediated thioester segment condensation. The other method was based on the thioesterification of peptide azide, derived from a peptide hydrazide prepared on a NH2NH-Clt-resin with Fmoc-SPPS. Peptide thioester containing two Tyr(SO3H) residues, prepared via this alternative method, was used as a building block for the one-pot synthesis of the N-terminal extracellular portion of CC-chemokine receptor 5 (CCR5(9-26)) by native chemical ligation (NCL). The two methods for the preparation of peptide thioesters containing Tyr(SO3H) residue(s) described herein are applicable to the synthesis of various types of sulfopeptides.

Accessing Therapeutically-Relevant Multifunctional Antisense Oligonucleotide Conjugates Using Native Chemical Ligation.

Antisense oligonucleotide (ASO) therapies hold significant promise in the realm of molecular medicine. By precisely targeting RNA molecules, ASOs offer an approach to modulate gene expression and protein production, making them valuable tools for treating a wide range of genetic and acquired diseases. As the precise intracellular targeting and delivery of ASOs is challenging, strategies for preparing ASO-ligand conjugates are in exceedingly high demand. This work leverages the utility of native chemical ligation to conjugate ASOs with therapeutically relevant chemical modifications including locked nucleic acids and phosphorothioate backbone modifications to peptides and sugars via a stable amide linkage. A suite of post-ligation functionalizations through modification of the cysteine ligation handle are highlighted, including chemoselective radical desulfurization, lipidation, and alkylation with a range of valuable handles (e.g. alkyne, biotin, and radionuclide chelating ligands), affording multifunctional constructs for further applications in biology and medicine. Application of the methodology to a clinically-relevant triantennary-GalNAc ASO conjugate and validation of its binding and functional activity underpins the applicability of the technique to oligonucleotide-based therapeutics.

Enzymatic Fluoromethylation as a Tool for ATP-Independent Ligation.

S-adenosylmethionine-dependent methyltransferases are involved in countless biological processes, including signal transduction, epigenetics, natural product biosynthesis, and detoxification. Only a handful of carboxylate methyltransferases have evolved to participate in amide bond formation. In this report we show that enzyme-catalyzed F-methylation of carboxylate substrates produces F-methyl esters that readily react with N- or S-nucleophiles under physiological conditions. We demonstrate the applicability of this approach to the synthesis of small amides, hydroxamates, and thioesters, as well as to site-specific protein modification and native chemical ligation.

Chemical Synthesis of Peptides and Proteins Bearing Base-Labile Post-Translational Modifications: Evolution of the Methods in Four Decades.

The S-palmitoylation on Cys residue and O-acetylation on Ser/Thr residues are two types of base-labile post-translational modifications (PTMs) in cells. The lability of these PTMs to bases and nucleophiles makes the peptides/proteins bearing S-palmitoyl or O-acetyl groups challenging synthetic targets, which cannot be prepared via the standard Fmoc-SPPS and native chemical ligation. In this review, we summarized the efforts towards their preparation in the past 40 years, with the focus on the evolution of synthetic methods.

D-Peptide and D-Protein Technology: Recent Advances, Challenges, and Opportunities.

Total chemical protein synthesis provides access to entire D-protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D-enantiomeric forms facilitating the development of mirror-image life. Crystallization of a racemic mixture of L- and D-protein enantiomers provides access to high-resolution X-ray structures of polypeptides. Additionally, D-enantiomers of protein drug targets can be used in mirror-image phage display allowing discovery of non-proteolytic D-peptide ligands as lead candidates. This review discusses the unique applications of D-proteins including the synthetic challenges and opportunities.

Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation.

Alpha-Synuclein (α-Synuclein) is a 140 amino acid protein implicated in neurodegenerative disorders known as synucleinopathies, where it accumulates in proteinaceous inclusions in the brain. The normal physiological function of α-Synuclein remains obscure, as it exists in several non-neuronal cells in which its function has not been studied. Given the tremendous interest in studying α-Synuclein, and the existing limitations in the production of modified forms of the protein, we developed a method for the chemical synthesis of α-Synuclein by combining peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis and ligation strategies. Our synthetic pathway enables the synthesis of protein variants of interest, carrying either mutations or posttranslational modifications, for further investigations of the effects on the structure and aggregation behavior of the protein. Ultimately, our study forms the foundation for future syntheses and studies of other custom-made α-Synuclein variants with a single or several modifications, as necessary.

Selenium in Peptide Chemistry.

In recent years, researchers have been exploring the potential of incorporating selenium into peptides, as this element possesses unique properties that can enhance the reactivity of these compounds. Selenium is a non-metallic element that has a similar electronic configuration to sulfur. However, due to its larger atomic size and lower electronegativity, it is more nucleophilic than sulfur. This property makes selenium more reactive toward electrophiles. One of the most significant differences between selenium and sulfur is the dissociation of the Se-H bond. The Se-H bond is more easily dissociated than the S-H bond, leading to higher acidity of selenocysteine (Sec) compared to cysteine (Cys). This difference in acidity can be exploited to selectively modify the reactivity of peptides containing Sec. Furthermore, Se-H bonds in selenium-containing peptides are more susceptible to oxidation than their sulfur analogs. This property can be used to selectively modify the peptides by introducing new functional groups, such as disulfide bonds, which are important for protein folding and stability. These unique properties of selenium-containing peptides have found numerous applications in the field of chemical biology. For instance, selenium-containing peptides have been used in native chemical ligation (NCL). In addition, the reactivity of Sec can be harnessed to create cyclic and stapled peptides. Other chemical modifications, such as oxidation, reduction, and photochemical reactions, have also been applied to selenium-containing peptides to create novel molecules with unique biological properties.

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization.

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

Backbone-Installed Split Intein-Assisted Ligation for the Chemical Synthesis of Mirror-Image Proteins.

Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone-Installed Split Intein-Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L-CfaN and L-CfaC ) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (µM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any "ligation scar" on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets.

Pedal to the Metal: The Homogeneous Catalysis of the Native Chemical Ligation Reaction.

The native chemical ligation reaction of peptide thioesters with cysteinyl peptides is a pivotal chemical process in the production of native or modified peptides and proteins, and well beyond in the preparation of various biomolecule analogs and materials. To benefit from this reaction at its fullest and to access all the possible applications, the experimentalist needs to know the factors affecting its rate and how to control it. This concept article presents the fundamental principles underlying the rate of the native chemical ligation and its homogeneous catalysis by nucleophiles. It has been prepared to serve as a quick guide in the search for an appropriate catalyst.

Design and Synthesis of Glycosylated Cholera Toxin B Subunit as a Tracer of Glycoprotein Trafficking in Organelles of Living Cells.

Glycosylation of proteins is known to be essential for changing biological activity and stability of glycoproteins on the cell surfaces and in body fluids. Delivering of homogeneous glycoproteins into the endoplasmic reticulum (ER) and the Golgi apparatus would enable us to investigate the function of asparagine-linked (N-) glycans in the organelles. In this work, we designed and synthesized an intentionally glycosylated cholera toxin B-subunit (CTB) to be transported to the organelles of mammalian cells. The heptasaccharide, the intermediate structure of various complex-type N-glycans, was introduced to the CTB. The synthesized monomeric glycosyl-CTB successfully entered mammalian cells and was transported to the Golgi and the ER, suggesting the potential use of synthetic CTB to deliver and investigate the functions of homogeneous N-glycans in specific organelles of living cells.

RNA-templated chemical synthesis of proapoptotic L- and d-peptides.

Nucleic acid-programmed reactions find application in drug screening and nucleic acid diagnosis, and offer prospects for a RNA-sensitive prodrug approach. We aim for the development of a nucleic acid-templated reaction providing nucleic acid-linked molecules that can act on intracellular protein targets. Such reactions would be useful for in situ drug synthesis and activity-based DNA-encoded library screening. In this report, we show native chemical ligation-like chemical peptidyl transfer reactions between peptide-PNA conjugates. The reaction proceeds on RNA templates. As a chemical alternative to ribosomal peptide synthesis access to both L- and d-peptides is provided. In reactions affording 9 to 14 amino acid long pro-apoptotic L- and d-peptides, we found that certain PNA sequence motifs and combinations of cell penetrating peptides (CPPs) cause surprisingly high reactivity in absence of a template. Viability measurements demonstrate that the products of templated peptidyl transfer act on HeLa cells and HEK293 cells. Of note, the presence of cysteine, which is required for NCL chemistry, can enhance the bioactivity. The study provides guidelines for the application of peptide-PNA conjugates in templated synthesis and is of interest for in situ drug synthesis and activity-based DNA-encoded library screening.

Comparison of different strategies towards the chemical synthesis of long-chain scorpion toxin AaH-II.

Long-chain scorpion toxin AaH-II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage-gated sodium ion channel Nav 1.7 responsible for pain sensation. Efficient chemical synthesis of AaH-II and its derivatives is beneficial to the study of the function and mechanism of Nav 1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid-phase peptide synthesis, hydrazide-based two-segment native chemical ligation, and hydrazide-based three-segment native chemical ligation for the synthesis of AaH-II. The hydrazide-based two-segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH-II to study the biological functions and modulation of Nav 1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.

Development of Naturally Inspired Peptide and Protein Chemistry.

Diverse naturally occurring events relevant to proteins, including processing and post-translational modification, provide significant clues enabling advances in peptide/protein chemistry. Our motivation to synthesize large proteins prompted us to seek scientific bases utilized in synthetic experiments on the intein-mediated protein processing system. This account describes peptide/protein thioester-producing protocols whose design is based on acyl transfer reactions observed in the intein system, and the development of the stimulus-responsive amide cleavage and its application to the modulation of peptide function. Finally, several findings derived from nature-inspired research efforts, including peptide mimetic synthesis and S-protected cysteine chemistry, are described.

Advances in Preparation of Peptide and Protein Thioesters Aiming to Use in Medicinal Sciences.

The growing interest in artificial proteins modified by synthetic functional units has fueled the demand for their facile preparation. Native chemical ligation (NCL) enables the chemoselective condensation of peptide thioesters with a cysteine-installed synthetic partner and has enjoyed great success in the production of artificial proteins with up to 100-150 residues. A practical method for converting expressed proteins to the corresponding thioesters should lead to significant progress in the NCL-mediated technology. This account describes our recent contributions to the conversion of naturally occurring peptides to the corresponding thioesters by chemical or chemoenzymatic protocols aiming at their future prevalent use in the preparation of sophisticated protein biologics.

Repetitive Thiazolidine Deprotection Using a Thioester-Compatible Aldehyde Scavenger for One-Pot Multiple Peptide Ligation.

Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N',N'-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.