Aspartic Acid Forming α-Ketoacid–Hydroxylamine (KAHA) Ligations with (S)-4,4-Difluoro-5-oxaproline

Abstract

The α-ketoacid-hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments. Currently, the most applied hydroxylamine is the 5-membered cyclic hydroxylamine (S)-5-oxaproline, which forms a homoserine ester as the primary ligation product. In order to access native aspartic acid residues at the ligation site, we synthesized a 4,4-difluoro version of this monomer. Upon KAHA ligation, the resulting difluoro alcohol hydrolyzes to an aspartic acid residue with little or no formation of aspartamide. We applied this monomer for the synthesis of the hormone peptides glucagon and an insulin variant, and as well for segment ligation of the peptides UbcH5a and SUMO3.

Aza‐Heterocyclic Building Blocks with In‐Ring CF2‐Fragment

“…First, the pyrrolidine 268 was built via [4 + 1] addition method; second, introduction of gem-di-F moiety in lactam 269 was achieved stepwise with electrophilic N- ). [118] The synthetic pathway started from coupling D-mannose derived auxiliary with hydroxylamine. The formed 273 was one-pot converted to the corresponding nitrone with tert-butyl glyoxylate and coupled with 1,1-di-F ethene, thus establishing gem-di-F isoxazolidine motif 275 (also regarded as 4,4difluoro-5-oxaproline).…”

“…(Scheme 45). [118] The synthetic pathway started from coupling D ‐mannose derived auxiliary with hydroxylamine. The formed 273 was one‐pot converted to the corresponding nitrone with tert ‐butyl glyoxylate and coupled with 1,1‐ di ‐F ethene, thus establishing gem ‐ di ‐F isoxazolidine motif 275 (also regarded as 4,4‐difluoro‐5‐oxaproline).…”

Investigation of Peptidic Templates for Bioorthogonal Ligations

“…The ligation strategy was further optimized by the synthesis of SPPS-compatible resins that form enantiopure α-ketoacids upon cleavage, [43][44][45] the synthesis of oxaproline and oxazetidine derivatives that lead to different amino acid side chains at the ligation site [44,[46][47][48] and the introduction of (photocleavable) protecting groups for the hydroxylamine and ketoacid functionalities. [49,50] Despite being limited to a lower pH and possessing a slower reaction rate than NCL, the KAHA ligation is broadly applicable in the synthesis of proteins by utilizing aqueous acidic NMP or DMSO, allowing various ligation sites that are more common than cysteine (the ligation site of NCL), utilizing easily protected functional groups, while being tolerant to all canonical amino acids.…”