Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

P−B Desulfurization: An Enabling Method for Protein Chemical Synthesis and Site‐Specific Deuteration

Cysteine‐mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH₄ or TCEP/LiBEt₃H; TCEP=tris(2‐carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation–desulfurization approach. This method, termed P−B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D₂O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post‐translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotomin C and E and synthetic proteins, including ubiquitin, γ‐synuclein, and histone H2A.     

Synthesis of Histidine‐Containing Oligopeptides via Histidine‐Promoted Peptide Ligation

Histidine‐containing peptides are valuable therapeutic agents for a treatment of neurodegenerative diseases. However, the synthesis of histidine‐containing peptides is not trivial due to the potential of imidazole sidechain of histidine to act as a nucleophile if unprotected. A peptide ligation method utilizing the imidazole sidechain of histidine has been developed. The key imidazolate intermediate that acts as an internal acyl transfer catalyst during ligation is generated by deprotonation. Transesterification with amino acids or peptides tethered with C‐terminal thioester followed by N→N acyl shifts led to the final ligated products. A range of histidine‐containing dipeptides could be synthesized in moderate to good yields via this method without protecting the imidazole sidechain. The protocol was further extended to tripeptide synthesis via a long‐range N→N acyl transfer, and tetrapeptide synthesis.     

Parallel Chemical Protein Synthesis on a Surface Enables the Rapid Analysis of the Phosphoregulation of SH3 Domains

Analysis of postranslationally modified protein domains is complicated by an availability problem, as recombinant methods rarely allow site‐specificity at will. Although total synthesis enables full control over posttranslational and other modifications, chemical approaches are limited to shorter peptides. To solve this problem, we herein describe a method that combines a) immobilization of N‐terminally thiolated peptide hydrazides by hydrazone ligation, b) on‐surface native chemical ligation with self‐purified peptide thioesters, c) radical‐induced desulfurization, and d) a surface‐based fluorescence binding assay for functional characterization. We used the method to rapidly investigate 20 SH3 domains, with a focus on their phosphoregulation. The analysis suggests that tyrosine phosphorylation of SH3 domains found in Abl kinases act as a switch that can induce both the loss and, unexpectedly, gain of affinity for proline‐rich ligands.     

Efficient Palladium‐Assisted One‐Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis

The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd^II complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one‐pot fashion by native chemical ligation where the Acm moiety was used to protect the N‐terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin‐like protein UBL‐5, which contains two native Cys residues, was accomplished through the one‐pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Selenocysteine‐Mediated Native Chemical Ligation

C‐Terminal peptide thioesters are shown to react efficiently with peptide fragments containing an N‐terminal selenocysteine to give selenoproteins. In analogy to the native chemical ligation of thioesters and peptides containing N‐terminal cysteines, the selenol presumably attacks the thioester nucleophilically to give a selenoester intermediate that subsequently rearranges to give a native chemical bond. The utility of this procedure was demonstrated by the synthesis of a selenium‐containing derivative of bovine pancreatic trypsin inhibitor (BPTI) in which Cys³⁸ is replaced by selenocysteine. The artificial selenoprotein folds into a conformation similar to that of wild‐type BPTI and inhibits trypsin and chymotrypsin with unaltered affinity.     

Synthesis of l‐ and d‐Ubiquitin by One‐Pot Ligation and Metal‐Free Desulfurization

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one‐pot native chemical ligation and metal‐free desulfurization for chemical protein synthesis. This one‐pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X‐ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis coupled with a one‐pot ligation–desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.     

Controlling Peptide Self‐Assembly through a Native Chemical Ligation/Desulfurization Strategy

Self‐assembled peptides were synthesized by using a native chemical ligation (NCL)/desulfurization strategy that maintained the chemical diversity of the self‐assembled peptides. Herein, we employed oxo‐ester‐mediated NCL reactions to incorporate cysteine, a cysteine‐based dipeptide, and a sterically hindered unnatural amino acid (penicillamine) into peptides. Self‐assembly of the peptides resulted in the formation of self‐supporting gels. Microscopy analysis indicated the formation of helical nanofibers, which were responsible for the formation of gel matrices. The self‐assembly of the ligated peptides was governed by covalent and non‐covalent interactions, as confirmed by FTIR, CD, fluorescence spectroscopy, and MS (ESI) analyses. Peptide disassembly was induced by desulfurization reactions with tris(2‐carboxyethyl)phosphine (TCEP) and glutathione at 80 °C. Desulfurization reactions of the ligated peptides converted the Cys and penicillamine functionalities into Ala and Val moieties, respectively. The self‐supporting gels showed significant shear‐thinning and thixotropic properties.     

Long‐Range Chemical Ligation from N→N Acyl Migrations in Tryptophan Peptides via Cyclic Transition States of 10‐ to 18‐Members

Chemical ligations to form native peptides from N→N acyl migrations in Trp‐containing peptides via 10‐ to 18‐membered cyclic transition states are described. In this study, a statistical, predictive model that uses an extensive synthetic and computational approach to rationalize the chemical ligation is reported. N→N acyl migrations that form longer native peptides without the use of Cys/Ser/Tyr residues or an auxiliary group at the ligation site were achieved. The feasibility of these traceless chemical ligations is supported by the N?C bond distance in N‐acyl isopeptides. The intramolecular nature of the chemical ligations is justified by using competitive experiments and theoretical calculations.     

Leveraging the Knorr Pyrazole Synthesis for the Facile Generation of Thioester Surrogates for use in Native Chemical Ligation

Facile synthesis of C‐terminal thioesters is integral to native chemical ligation (NCL) strategies for chemical protein synthesis. We introduce a new method of mild peptide activation, which leverages solid‐phase peptide synthesis (SPPS) on an established resin linker and classical heterocyclic chemistry to convert C‐terminal peptide hydrazides into their corresponding thioesters via an acyl pyrazole intermediate. Peptide hydrazides, synthesized on established trityl chloride resins, can be activated in solution with stoichiometric acetyl acetone (acac), readily proceed to the peptide acyl pyrazoles. Acyl pyrazoles are mild acylating agents and are efficiently exchanged with an aryl thiol, which can then be directly utilized in NCL. The mild, chemoselective, and stoichiometric activating conditions allow this method to be utilized through multiple sequential ligations without intermediate purification steps.     

N‐Linked Glycosyl Auxiliary‐Mediated Native Chemical Ligation on Aspartic Acid: Application towards N‐Glycopeptide Synthesis

A practical approach towards N‐glycopeptide synthesis using an auxiliary‐mediated dual native chemical ligation (NCL) has been developed. The first NCL connects an N‐linked glycosyl auxiliary to the thioester side chain of an N‐terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C‐terminal thioester oligopeptide. Mild cleavage provides the desired N‐glycopeptide.     

Theoretical Analysis of the Detailed Mechanism of Native Chemical Ligation Reactions

The method of native chemical ligation between an unprotected peptide α‐thioester and an N‐terminal cysteine–peptide to give a native peptide in aqueous solution is one of the most effective peptide ligation methods. In this work, a systematic theoretical study was carried out to fully understand the detailed mechanism of ligation. It was found that for the conventional native chemical ligation reaction between a peptide thioalkyl ester and a cysteine in combination with an added aryl thiol as catalyst, both the thiol‐thioester exchange step and the transthioesterification step proceed by an anionic concerted S_N2 displacement mechanism, whereas the intramolecular rearrangement proceeds by an addition–elimination mechanism, and the rate‐limiting step is the thiol‐thioester exchange step. The theoretical method was then extended to study the detailed mechanism of the auxiliary‐mediated peptide ligation between a peptide thiophenyl ester and an N‐2‐mercaptobenzyl peptide in which both the thiol‐thioester exchange step and intramolecular acyl‐transfer step proceed by a concerted S_N2‐type displacement mechanism. The energy barrier of the thiol‐thioester exchange step depends on the side‐chain steric hindrance of the C‐terminal amino acid, whereas that of the acyl‐transfer step depends on the side‐chain steric hindrance of the N‐terminal amino acid.     

Chemical Synthesis of Proteins with Non‐Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125‐residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C‐terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N‐terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild‐type PHPT1 and an analogue in which the active‐site histidine was substituted with the unnatural and isosteric amino acid β‐thienyl‐l ‐alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.     

Chemical Synthesis of Native S‐Palmitoylated Membrane Proteins through Removable‐Backbone‐Modification‐Assisted Ser/Thr Ligation

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBM^GABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBM^GABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBM^GABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis.     

Traceless Purification and Desulfurization of Tau Protein Ligation Products

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin–protein conjugate on streptavidin‐coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On‐bead desulfurization is followed by delivery of the final tag‐free protein product. The strategy is demonstrated in the isolation of a tag‐free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C‐terminus of tau.     

Total Chemical Synthesis of the Enzyme Sortase AΔN59 with Full Catalytic Activity

The enzyme sortase A is a ligase which catalyzes transpeptidation reactions. 1 , 2 Surface proteins, including virulence factors, that have a C terminal recognition sequence are attached to Gly₅ on the peptidoglycan of bacterial cell walls by sortase A. 1 The enzyme is an important anti‐virulence and anti‐infective drug target for resistant strains of Gram‐positive bacteria. 2 In addition, because sortase A enables the splicing of polypeptide chains, the transpeptidation reaction catalyzed by sortase A is a potentially valuable tool for protein science. 3 Here we describe the total chemical synthesis of enzymatically active sortase A. The target 148 residue polypeptide chain of sortase A_ΔN59 was synthesized by the convergent chemical ligation of four unprotected synthetic peptide segments. The folded protein molecule was isolated by size‐exclusion chromatography and had full enzymatic activity in a transpeptidation assay. Total synthesis of sortase A will enable more sophisticated engineering of this important enzyme molecule.     

Aziridine‐Mediated Ligation at Phenylalanine and Tryptophan Sites

An efficient approach towards peptide synthesis that allows easy access to variety of small peptides via one‐pot aziridine‐mediated ligation/desulfurization strategy has been described. The protocol afforded a library of phenylalanine‐ and tryptophan‐containing α‐peptides in good yields by regioselective ring‐opening of aziridine‐3‐aryl‐2‐carboxylates with peptide thioacids, followed by desulfurization.     

Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation

Increased versatility for the synthesis of proteins and peptides by native chemical ligation requires the ability to ligate at positions other than Cys. Here, we report that Raney nickel can be used under standard conditions for the selective desulfurization of Cys in the presence of Cys(Acm). This simple and practical tactic enables the more common Xaa-Ala junctions to be used as ligation sites for the chemical synthesis of Cys-containing peptides and proteins. [reaction: see text].     

Semisynthesis of Biologically Active Glycoforms of the Human Cytokine Interleukin 6

Human interleukin 6 (IL‐6) is a potent cytokine with immunomodulatory properties. As the influence of N‐glycosylation on the in vivo activities of IL‐6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL‐6 was established by sequential native chemical ligation. The four cysteines of IL‐6 are convenient for ligations and require only the short synthetic glycopeptide 43–48. The Cys‐peptide 49–183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1–42 was accessible by the simultaneous cleavage of two inteins, leading to the 1–42 thioester with the native N‐terminus. Ligation and refolding studies showed that the inherently labile Asp? Pro bond 139–140 was detrimental for the sequential C‐ to N‐terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full‐length IL‐6 glycoproteins, which showed full bioactivity after efficient refolding and purification.