This post-SPPS thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester. N-alkyl cysteine derivatives were also used for the preparation of peptide thioesters [ 69 , 70 ]. Despite an inconvenience in loading the first amino acid to the CPE linker and the need for a relatively reactive glycolic ester at the C-terminus, this method is appealing for its clever design. Recently, Dr. Liu group [ 73 ] and Dr.
Although formed transiently, the thioester possesses a lifetime that is sufficient to allow its capture for direct reaction with a cysteinyl peptide at weakly acidic pH.
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Remarkably, low-power microwave irradiation has been shown to be a viable means to assist BMEA-mediated ligation. The reaction system is applicable to a wide range of C-terminal amino acid residues, pointing to its broad utility.
The successful demonstration of the synthesis of histone H3 protein validates the practical value of this BMEA methodology in synthetic protein chemistry. Compared with most existing methods, this system does not require a preactivation step or an intrinsic driving device for the thioester formation reaction, making it one of the simplest and most straightforward Fmoc-based systems for peptide thioester synthesis. The scope of ligation chemistry can be expanded by the use of removable thiol-based auxiliaries. The auxiliaries are linked synthetically to the N-terminal amine of the second peptide.
Once the two peptides are ligated together, the removal of the extraneous atoms of the auxiliary gives the final ligation product as shown in Fig. A new method for the synthesis of cysteine-free glycopeptides using auxiliary-assisted ligation was developed by Brik et al. In this ligation see Fig. The final product can be obtained by desulfurization [ 81 ]. This method can be applied for the total synthesis of the antibacterial glycoprotein diptericin. Both the thioester and cysteinyl segments can be acquired by recombinant protein expression.
A cysteinyl protein can be expressed directly from prokaryotic systems after the removal of Met via endogenous methionyl aminopeptidases MetAPs [ 82 , 83 ]. However, the expression of thioester protein is difficult. The idea of thioester protein expression was inspired by protein splicing, a posttranslational process that was first described by Anraku and coworkers in [ 84 ]. The mechanism is now relatively well understood see Fig. Conserved residues are found at both the splice junctions in standard inteins. Amino acids at the N-terminus of the intein and C-extein bear a nucleophilic side chain such as cysteine, serine, or threonine, which can provide a nucleophilic attack on an amide or ester bond.
At the C-terminus of intein, asparagine is conserved, which is engaged in spontaneous cyclization reactions that result in deamidation or peptide bond cleavage. The process of protein splicing was elucidated well [ 85 ]. This branched intermediate is cleaved by the attack of the amido group of the asparagine side chain to the carbonyl carbon.
Mutation of the C-terminal asparagine residue within the intein to an alanine residue blocks the final step in protein splicing by preventing succinimide formation [ 86 ] see Fig. The addition of a thiol reagent such as MESNa can induce the thioester exchange, resulting in the formation of a reactive thioester protein, which is the building block for subsequent EPL Fig. In this system, a recombinant intein fusion protein is fused to a chitin binding domain CBD from Bacillus circulans. When the target protein-intein-CBD passes through a chitin resin, it is separated from other cell proteins by binding to the resin.
Using the same principle, a thioacid protein can be prepared similarly when the nucleophile is HS— [ 88 ]. Recently, this strategy has been successfully used in the synthesis of H3 histone proteins by thioacid-capture ligation [ 89 ]. Ras protein was divided into three unprotected peptide segments. The central peptide thioester segment was S -Acm acetamidomethyl -protected at its N-terminal cysteine residue to prevent cyclization or self-ligation. The S -protecting group Acm was removed by mercury acetate or silver acetate , and the reaction was quenched by mercaptoethanol or dithiothreitol.
Ras protein was synthesized successfully after two NCLs. However, the procedure for the removal of Acm led to the formation of a metal-thiol precipitate, which resulted in a significant loss of material. They used the 1,3-thiazolidinecarboxo Thz group in place of Acm to protect the N-terminal Cys of the central peptide segment. The protein was divided into six segments. The first three left half part segments were ligated from the C-to-N direction using Thz protecting group for the N-terminal Cys residue of the central segment as described in the last section, and the other three right half part segments were ligated from the N-to-C direction utilizing the different reactivities of aryl and alkyl thioesters in NCL.
Alkyl thioesters were sufficiently unreactive and did not participate in ligation reactions when competing aryl thioesters were present. Furthermore, these alkyl thioesters could be activated by adding excess aryl thiols in NCL. The sequential ligation could be controlled by the CPE unit see Fig. When the thiol group in the CPE unit was protected, the peptide A could not be converted to thioester.
Thus, this method represented a promising alternative for achieving ligation in the N-to-C direction. Our lab also developed a ligation method in the N-to-C direction, which combined enzymatic peptide ligation with thioacid-capture ligation [ 98 ]. A model peptide was demonstrated see Fig. Thioacid group was negatively charged and unlikely to be recognized by subtiligase. As a result, a thioacid peptide with a suitable sequence could be ligated to a peptide-ester by the enzymatic catalyst of subtiligase.
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Several years ago, successful application of thioacids as the central segments to sequential NCL in the N-to-C direction was achieved. Our research group also developed an alternative method, which allowed the sequential peptide ligation of several segments from the N-to-C direction. The BMEA peptide reacts with cysteinyl peptide slowly at room temperature.
Given that the reaction rate of BMEA-mediated ligation is much slower than that of NCL, this kinetic difference can be exploited in a sequential ligation strategy. Based on this difference, we hypothesized that thioester may be ligated with a cysteinyl BMEA peptide at room temperature, with minimal risk of self-ligation or cyclization of the cysteinyl BMEA peptide.
The ligated BMEA peptide can then react with another cysteinyl peptide under microwave irradiation.
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Quantity Our equipment allows us to produce down to 1 milligram and up to several grams of peptide. Chemical Modifications Thanks to our expertise in peptide chemistry, we can also bring very precise chemical modifications such as: addition of one or several functional groups acetylation, phosphorylation… labelling with a probe coupling to a carrier protein alteration of structure cyclisation, disulphide bonds… For more details, please consult our complete list of available modifications.
Recommendations Solubility All peptides are shipped lyophilised. Increase the concentration of acid stepwise if the peptide does not dissolve. Please note that these solvents may have a damaging effect on your experiments. For peptides with secondary structures, it may be necessary to add chaotropic agents such as urea or guanidium-HCl.
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Jump to Figure 2. H-IA-OR 1. Supporting Information. Supporting Information File 1: Experimental procedures and characterization data of peptides. Format: PDF Size: 1. News , 89, 21— Nature , , — News , 95, 27— Amino Acids and Peptides in Ball Milling. Carbon—Nitrogen Bond-Formation Reactions. B , , — Fundamentals of Modern Peptide Synthesis. Nature , , 20— London , — News , 93, Return to citation in text: [ 1 ]. Reference Go to reference References References 1,2.
Reference 8. Go to reference 8. References 4,7. Go to references Reference 3. References 25, References 21, Reference 9. All issues All volumes Article is part of the issue Mechanochemistry.
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