In order to solve the problems during total chemical synthesis, such as the waste of N-glycans and low efficiency of long peptide synthesis, semi-synthesis of glycoprotein combining cell expression (usually E. coli expression) and chemical synthesis (mainly SPPS) are developed. E. coli expression can produce peptide without glycan up to over 200 residues, while SPPS can produce short glycopeptide with homogeneous glycan structure. Subsequent ligation between peptide prepared in E. coli and glycopeptide from SPPS can give full-length homogeneous glycopeptide in only few conversion steps.During semi-synthesis of (glyco)protein, the main challenge is the thioesterification of peptide obtained from E. coli expression, namely expressed peptide thioesterification.[35-36] Expressed peptide thioesterification requires selective C-terminal activation in the absence of any side chain protection. (Fig14) The thioesterification methods using such as Intein system [35] or specific proteases for the recombinant polypeptides [37-39], however these enzymatic methods required specific amino acid sequences. On the other hand, Okamoto and coworkers developed reactions for chemical thioesterification of recombinant peptides by using the unique both of C-terminal and internal Cys modification strategies. In 2012 they achieved internal Cys modification, by S-thiocarbonylation (Fig14, A).[40] They designed a new thioester synthesis consisting of three steps: the selective S-thiocarbonylation of a β-thiol group of cysteine residue to provide a phenyl xanthate group by using O-phenylchlorothionoformate. Treatment of the resultant peptide 115 with N-acetylguanidine to give peptide thioester surrogate 116. Finally, conversion of N-acetylguanidine derivative 116 into desired thioester 117. They also found that N-Boc protection of both amino groups of the Lys side chains, and the N terminus are necessary to avoid side reaction by the N-acetylguanidine treatment. In addition to this efficient reaction, peptidyl N-acetylguanidine is much less reactive for NCL than a corresponding thioester, they could be directly used for one-pot sequential NCL. Using their thioesterification approach, they succeeded to synthesize semi-synthesis of interleukin 13, which is introduced in next web page (Fig15).

Two years later, Kajihara and coworkers developed another thioesterification methods by using cyanylation of Cys residue.41 Reaction mechanism is shown in the following figure (Fig14, B). Firstly, the thiol group of the cysteine residue is cyanylated by cyanylation reagents. This intermediate 119 is comparably stable, and in the presence of nucleophiles such as hydrazine or amines, the amide nitrogen of the cysteine attacks the sp carbon of cyanyl groups nucleophilically, subsequently forming a five-membered ring intermediate, which gave the corresponding peptide hydrazide as thioester surrogate efficiently. In their reports, they demonstrated the preparation of polypeptide thioester consists of 94 amino acids by E. coli expression followed by cyanylation, hydrazinolysis, and thiolysis. Their attempts to synthesize expressed peptides thioester utilizing internal Cys residue succeeded to synthesize several homogeneous glycoproteins. However, these thioesterification protocols by Cys residue required multiple reaction steps. The selective modification of the C-terminal Cys was also difficult on peptides containing internal Cys residues at that time. To solve these problems, the Okamoto and Kajihara group found two types of highly chemo-selective peptides thioesterification reactions in 2022.[42] These new thioesterification reactions progressed without any protecting groups, applying an intramolecular N-S acyl shift reaction at a C-terminal Cys residue (Fig14, C,D). They made a well improved thioesterification based of the Aimoto [43-44] and Macmillan [45] groups. The first method is chemical transformation of peptide-Cys 122 using bis(2-sulfanylethyl)amine (SEA) efficiently yielded peptide-SEA 125, which can be utilized as a peptide thioester surrogate (Figure 4-1, c).[42] Reaction starts from conversion of peptide-Cys 122 to peptide-S-Cys-thioester 123 by an intramolecular N-S acyl shift at the C-terminal Cys and the trans-thioesterification of the peptide-S-Cys-thioester 123 with an external thiol such as SEA to afford peptide-SEA 124 (peptide thioester surrogate). Fortunately, the equilibrium state is biased toward peptide-SEA 125 because peptide-SEA 124 was oxidized in air to form C-terminal disulfide bonds, which cannot be converted to peptide-S-Cys-thioester 123 . Alternatively, they also found a peptide having cysteinyl glycyl cysteine 127 (peptide-CGC) can be used for the synthesis of peptide thioesters from unprotected polypeptides (Fig 14, D).[42] Peptides-CGC 127 could be transformed to peptide-thioesters 130 through peptides with CG-thioester units 128 at the C terminus via an N-S acyl shift at Cys. Then peptide-CG-thioester 128 can be converted to peptide thioesters 130 via diketopiperazine (DKP) formation and a subsequent N-S acyl shift at Cys. Both methods were successfully applied to recombinant polypeptides and utilized for NCL. In the next paragraph, several examples of the semi-synthesis applying Cys-based thioesterification protocols developed by the Okamoto and Kajihara group will be introduced.

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