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     

An Efficient One‐Pot Four‐Segment Condensation Method for Protein Chemical Synthesis

Successive peptide ligation using a one‐pot method can improve the efficiency of protein chemical synthesis. Although one‐pot three‐segment ligation has enjoyed widespread application, a robust method for one‐pot four‐segment ligation had to date remained undeveloped. Herein we report a new one‐pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one‐pot four‐segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.     

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.     

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.     

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.     

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.     

Total Chemical Synthesis and Biological Activities of Glycosylated and Non‐Glycosylated Forms of the Chemokines CCL1 and Ser‐CCL1

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T‐cells and acts as a chemoattractant for monocytes. 1 Originally, CCL1 was identified as a 73 amino acid protein having one N‐glycosylation site, 1 and a variant 74 residue non‐glycosylated form, Ser‐CCL1, has also been described. 2 There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser‐CCL1. Here we report the total chemical syntheses of both N‐glycosylated and non‐glycosylated forms of (Ser‐)CCL1, by convergent native chemical ligation. We used an N‐glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide‐^αthioester building block. 3 Chemotaxis assays of these glycoproteins and the corresponding non‐glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser‐)CCL1 using homogeneous N‐glycosylated protein molecules of defined covalent structure.     

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.     

A Type of Auxiliary for Native Chemical Peptide Ligation beyond Cysteine and Glycine Junctions

Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol‐containing auxiliary groups have been used to extend the reaction scope beyond N‐terminal cysteine residues. However, the N‐benzyl‐type auxiliaries used so far result in rather low reaction rates. Herein, a new N^α‐auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2‐mercapto‐2‐phenethyl auxiliary is not limited to Gly‐containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on‐resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD‐1L and opistoporin‐2.     

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.     

One‐Pot N2C/C2C/N2N Ligation To Trap Weak Protein–Protein Interactions

Weak transient protein–protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity‐induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N‐terminus to N‐terminus) and C2C (C‐terminus to C‐terminus) ligation approaches. By using N2C (N‐terminus to C‐terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t_1/2 of 4.8 min and near quantitative conversion. The Ypt1‐GDI conjugate revealed that binding of Ypt1 G‐domain causes opening of the lipid‐binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.     

Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation

The chemical synthesis of the 184‐residue ferric heme‐binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α‐ketoacid‐hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild‐type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well‐suited for the preparation of hydrophobic protein targets.     

Traceless Preparation of C‐Terminal α‐Ketoacids for Chemical Protein Synthesis by α‐Ketoacid–Hydroxylamine Ligation: Synthesis of SUMO2/3

A novel protecting group for enantiopure α‐ketoacids delivers C‐terminal peptide α‐ketoacids directly upon resin cleavage and allows the inclusion of all canonical amino acids, including cysteine and methionine. By using this approach, SUMO2 and SUMO3 proteins were prepared by KAHA ligation with 5‐oxaproline. The synthetic proteins containing homoserine residues were recognized by and conjugated to RanGAP1 by SUMOylation enzymes.     

SnCl4/SiO2: An Efficient Heterogeneous Alternative for One‐Pot Synthesis of β‐Acetamidoketones

Enolizable ketones have been reacted in a one‐pot method with aromatic aldehydes, acetyl chloride and acetonitrile at room temperature in the presence of SnCl₄/SiO₂ to furnish the corresponding β‐acet‐amidoketones in improved yields. Acetylation of an aromatic hydroxyl group was observed while using 4‐hydroxybenzaldehyde or vanillin and the corresponding β‐acetamidoketones were isolated in an excellent yield.