Protein Synthesis Assisted by Native Chemical Ligation at Leucine

Abstract

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...

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

“…One such strategy is the use of ligation junctions that contain thiolmodified residues that can be desulfurized later to give the native structure. This NCL-desulfurization method, since its invention for an Ala junction, [21] has captured the interest of several laboratories, including ours, and enabled performing NCL at Phe, [22] Val, [23,24] Leu, [25,26] Lys, [27,28] and Thr [29] ligation junctions. Moreover, this concept has been extended to include sugar-assisted ligation [30,31] for glycopeptide synthesis, and lysine-mediated peptide and protein ubiquitination.…”

Synthetic Amino Acids for Applications in Peptide Ligation–Desulfurization Chemistry

“…Madanin-1 (1-27) Chimadanin (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) Chimadanin Chimadanin (1-19)…”

“…This step is typically followed by several protecting group manipulations, including hydrolysis of the thioacetate moiety, to enable protection of the thiol handle as an asymmetric disulfide or the corresponding S-trityl (Trt) derivative, both of which are compatible with standard conditions employed in Fmoc-strategy SPPS. [19] This overall synthetic strategy has been employed for the synthesis of several thiol-derived amino acids, including b-thiol phenylalanine (Phe), [20] g-thiol Val, [21] g-thiol lysine (Lys), [22] d-thiol Lys, [23] g-thiol threonine (Thr), [24] b-thiol leucine (Leu), [25] g-thiol Pro, [26] g-thiol glutamine (Gln), [27] and b-thiol arginine (Arg). [8d] In 2007, Crich and Banerjee prepared b-thiol Phe building block 3 [20a] for use in ligation-desulfurization chemistry in a 9-step synthesis from L-Phe methyl ester 4 (Scheme 3).…”

Application of the logic of cysteine-free native chemical ligation to the synthesis of Human Parathyroid Hormone (hPTH)

“…Such methods are not ideal for the generation of analogs (30)(31)(32)(33). It was recognized that enhancement of the power of chemical synthesis for such objectives, including that of hPTH itself, could be accomplished by extending the reach of the underlying elegant concept of NCL (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). To this end, we report a pleasing and encouraging example wherein these recent findings have been pooled such that the molecule hPTH can be conveniently assembled from small synthetic peptide fragments.…”

“…(2,3). However, given the relative scarcity of cysteine residues in nature, a clear impetus arises for the realization of new NCL capabilities (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”