Internal Activation of Peptidyl Prolyl Thioesters in Native Chemical Ligation

Abstract

Prolyl thioesters have shown significantly lower reactivities in native chemical ligation (NCL) in comparison to that of the alanyl thioester. This report describes a mild and efficient internal activation protocol of peptidyl prolyl thioesters in NCL without using any thiol-based additives, where the introduction of a 4-mercaptan substituent on the C-terminal proline significantly improves the reactivity of prolyl thioesters via the formation of a bicyclic thiolactone intermediate. The kinetic data indicate that the reaction rate is comparable to that of the reported data of alanyl thioesters, and the mechanistic studies suggest that the ligation of two peptide segments proceeds through an NCL-like pathway instead of a direct aminolysis, which ensures the chemoselectivity and compatibility of various amino acid side chains. This 4-mercaptoprolyl thioester-based protocol also allows an efficient one-pot ligation-desulfurization procedure. The utility of this method has been further demonstrated in the synthesis of a proline-rich region of Wilms tumor protein 1.

Photoprotected Peptide α‐Ketoacids and Hydroxylamines for Iterative and One‐Pot KAHA Ligations: Synthesis of NEDD8

“…Fmoc-SPPS using photoprotected a-ketoacid building blocks (a) and analytical HPLC traces (220 nm) of crude (c and e) and purified (b and d) peptide segments with photoprotected a-ketoacids. In NEDD8 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) 17 (b and c), the two diastereoisomeric peptides resulting from the racemic protecting group rac-2 elute as separate peaks.…”

“…All peptides were prepared by standard Fmoc-SPPS. NEDD8 (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) 25 was prepared from protected isoleucine a-ketoacid and assembled until Thr7 after which Fmoc deprotection appeared to be difficult. From this point on, the deprotection was achieved using 20% piperidine in DMF with 3% of DBU, preceded by a LiCl (0.8M in DMF) wash. Two variants of the NEDD8 segments were prepared, one (18) with the photoprotected tyrosine a-ketoacid and (S)-Boc-5-oxaproline, and the other one (30) with a tyrosine a-ketoacid and photoprotected (S)-5-oxaproline.…”

“…[11] Dong and collaborators also recently described a ligation forming, after desulfurization, a native proline at the ligation site using an internal activation protocol of a prolyl thioester. [12] Despite these advances, NCL is not ideal for the synthesis of some proteins due to necessity for either correctly placed Cys residues or additional desulfurization steps.…”

Synthesis of Thiol‐Containing Oligopeptides via Tandem Activation of γ‐Thiolactones by Silver‐DABCO Pair

“…[17] Dong et al have also reported NCL via a bicyclic thiolactone intermediate generated from prolyl thioesters. [18] However, the use of γ-thiolactone in peptide synthesis had been limited to those early aminolysis examples by glycine and glycylglycine. [10] The fact that large excess of amine was required to achieve moderate yields is not practical for peptide synthesis since one segment of a peptide could be valuable or only available in small amount.…”

Traceless β-mercaptan-assisted activation of valinyl benzimidazolinones in peptide ligations

“…Besides the search for more effective and practical additives, 15 studies have also been conducted exploiting the presence of –SH functionalities within the peptide segments to activate the C-termini under thiol additive-free conditions, 16 including a recent report from our group on a 4-thioproline-based ligation at the Pro-Xaa sites. 17 In contrast to the internal activation originating from the cysteine residues within the sequence, a constrained thiolactone structure was suggested to be crucial in accounting for the drastically increased reactivity in comparison to the regular prolyl thioesters. 18 Despite these improvements, methods that can improve efficiencies at other sterically demanding sites under mild reaction conditions are still of great importance, and call for further investigations.…”