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.     

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     

Proximity‐Based Sortase‐Mediated Ligation

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site‐specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity‐based sortase‐mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag–SpyCatcher peptide–protein pair. By expressing the target protein with SpyTag C‐terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher–SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.     

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.     

Chemical Protein Synthesis by Native Chemical Ligation and Variations Thereof

For a long time, the total synthesis of proteins was considered as a “mission impossible” because of the tedious and complex synthetic steps and demanding purification processes. However, with the development of modern synthetic methodologies, many protein syntheses have now been reported. More importantly, through chemical synthesis, desired modifications can be installed to target proteins precisely, which is a major advantage over traditional bio‐synthesis approaches. This review summarizes the techniques developed for protein assembly, including native chemical ligation, Se‐mediated ligation, and a range of other ligation methods. A few synthetic examples, whereby synthetic proteins with desired modifications have been utilized for related biological research, are also included. We believe that chemical synthesis can provide alternative pathways to solve problems that have hitherto proved insurmountable by traditional biological approaches.     

In Situ Vesicle Formation by Native Chemical Ligation

Phospholipid vesicles are of intense fundamental and practical interest, yet methods for their de novo generation from reactive precursors are limited. A non‐enzymatic and chemoselective method to spontaneously generate phospholipid membranes from water‐soluble starting materials would be a powerful tool for generating vesicles and studying lipid membranes. Here we describe the use of native chemical ligation (NCL) to rapidly prepare phospholipids spontaneously from thioesters. While NCL is one of the most popular tools for synthesizing proteins and nucleic acids, to our knowledge this is the first example of using NCL to generate phospholipids de novo. The lipids are capable of in situ synthesis and self‐assembly into vesicles that can grow to several microns in diameter. The selectivity of the NCL reaction makes in situ membrane formation compatible with biological materials such as proteins. This work expands the application of NCL to the formation of phospholipid membranes.     

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.     

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.     

脯氨酸硫酯自然化学连接反应中的轨道相互作用

采用密度泛函理论(DFT)方法(M06//B3LYP)对脯氨酸硫酯在自然化学连接(NCL)反应中低反应活性的现象进行了详细的理论研究.通过对脯氨酸和丙氨酸硫酯NCL反应的具体反应路径(Path-Pro和Path-Ala)分别进行计算发现,两个分子的反应路径均先后经历外源硫醇-硫酯交换、硫酯-硫酯交换、分子内S→N酰基化重排三个主要步骤.两者的决速步骤均为第一步骤.其中Path-Pro的总能垒较高,即脯氨酸硫酯的反应活性较低,这与实验结果一致.对两路径的决速步骤进一步考察,发现脯氨酸中αN上的羰基对反应中心羰基的n→π*相互作用使得脯氨酸硫酯的LUMO轨道能量升高,相应羰基C与S(芳基硫醇)原子之间的相互作用较小,从而使得反应能垒升高.     

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.     

Imidazole‐Aided Native Chemical Ligation: Imidazole as a One‐Pot Desulfurization‐Amenable Non‐Thiol‐Type Alternative to 4‐Mercaptophenylacetic Acid

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4‐mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co‐elution of MPAA with the ligation product during preparative high‐performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide‐alkylthioester. The efficiency of the imidazole‐aided NCL (Im‐NCL) is similar to that of traditional MPAA‐aided NCL. As model cases, we successfully synthesized adiponectin(19‐107) and [Ser(PO₃H₂)⁶⁵]‐ubiquitin using Im‐NCL with a one‐pot desulfurization.     

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.     

Enhancing Auxiliary-Mediated Native Chemical Ligation at Challenging Junctions with Pyridine Scaffolds

To expand the scope of native chemical ligation (NCL) beyond reactions at cysteine, ligation auxiliaries are appended to the peptide N-terminus. After the introduction of a pyridine-containing auxiliary, which provided access to challenging junctions (proline or β-branched amino acids), we herein probe the role of the pyridine-ring nitrogen. We observed side reactions leading to preliminary auxiliary loss. We describe a new easy to attach β-mercapto-β-(4-methoxy-2-pyridinyl)-ethyl (MMPyE) auxiliary, which 1) has increased stability; 2) enables NCL at sterically encumbered junctions (e. g., Leu-Val); and 3) allows removal under mildly basic (pH 8.5) conditions was introduced. The synthesis of a 120 aa long peptide containing eight MUC5AC tandem repeats via ligation of two 60mers demonstrates the usefulness. Making use of hitherto unexplored NCL to tyrosine, the MMPyE auxiliary provided access to a head-to-tail-cyclized 21-mer peptide and a His₆-tagged hexaphosphorylated peptide comprising 6 heptapeptide repeats of the RNA polymerase II C-terminal domain.     

Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein

The inhibition of the G protein‐coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41‐residue and three‐disulfide cross‐bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N^α‐deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca²⁺ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.