Vitamin C for peptide ligation: Phosphine and thiol additives maintain reducing environments and increase the reactivities of peptide thioesters in native chemical ligation. Thiol additives such as thiophenol also act as radical scavengers that inhibit phosphine‐induced desulfurization of cysteine. This role can be assumed by the odourless, nontoxic, highly water‐soluble and inexpensive ascorbate, which can replace the usually added thiols.

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.

Total chemical synthesis of proteins offers exceptional opportunities for preparing targets with exquisite control over the covalent structure, high purity and large quantities for functional and structural analysis. [1] In this regard, native chemical ligation (NCL) continues to be the method of choice for joining two unprotected peptides through thiol capture followed by S-N acyl transfer to form the amide bond at the ligation site.[2] The desulfurization reaction introduced by Yan and Dawson [3] as a post-NCL step greatly expanded the scope of ligation chemistry beyond Xaa-Cys (Xaa is any amino acid) by making ligation at Xaa-Ala sites accessible in the synthesis of functional proteins.[4] This seminal work has prompted several groups to extend this strategy to other b-and g-mercapto amino acid derivatives (e.g., Phe and Val) as part of the ongoing efforts to extend NCL to essentially any ligation junction. [5] In addition, by positioning a thiol handle at the Lys side chain and at the 2-acetamido group of the glycan moiety in a glycopeptide allowed NCL to be used for peptide ubiquitylation [6] and glycopeptide ligation [7] respectively, and after a desulfurization step, the native structures could be achieved.The above-described examples testify to the power of desulfurization when combined with NCL to assist the synthesis of naturally occurring proteins. In addition, recent reports on the mild and highly versatile free-radical Cys reduction protocol, [5a,b, 8] as well as the compatibility of these methods in the presence of other thiol functionalities [9] permit the use of this two-step approach in the synthesis of a variety of protein targets. Despite the introduction of mercaptoPhe and mercaptoVal to assist efficient peptide ligation, [5] their utility in protein synthesis has not yet been demonstrated. The preparation of proteins by using ligation methods is often challenging because decreases in rate and chemoselectivity only come to light in more complex systems. To date, only ligation at XaaCys followed by desulfurization has been demonstrated in the synthesis of full-length proteins, [4] this diminishes the generality of the desulfurization approach in the total chemical synthesis of proteins. Here we report an innovative strategy for ligation at Xaa-Leu sites by using b-mercaptoleucine combined with desulfurization (Scheme 1) and its application in the total chemical synthesis of HIV-1 Tat protein.To implement the approach outlined in Scheme 1, we first had to design a synthetic strategy for b-mercaptoleucine, the key residue in this strategy. We envisioned two routes to achieve the synthesis of this building block by starting from the commercially available threo-b-hydroxy-l-leucine. In the first route, the b-hydroxy group could be converted to a good leaving group followed by nucleophilic substitution with a thiol nucleophile, whereas in the second one an aziridine intermediate could be employed to introduce the thiol functionality through a regioselective ring opening. Applying the first s...

This work presents the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step.

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