Total Synthesis of the TIM-1 IgV Domain via N-to-C Serine/Threonine Ligation Enabled by Knorr Pyrazole Synthesis-Mediated Regeneration of Salicylaldehyde Esters

T-cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) have recently emerged as the B-cell immune checkpoint with the potential to overcome the resistance of traditional immune checkpoint blockade therapy. However, the chemical biology of its post-translational glycosylations remains unexplored due to the absence of synthetic access to its homogeneous isoform. Here we report the total synthesis of the TIM-1 IgV-like domain (21-121) through the N-to-C Ser/Thr ligation (STL) with native chemical ligation (NCL). During the synthesis, we uncovered an unprecedented incompatibility of pyruvic acid-mediated salicylaldehyde (SAL) ester regeneration with tryptophan residues. Mechanistic studies revealed that indole side chains condense with pyruvic acid under acidic conditions, leading to side reactions. Guided by this insight, we developed an acetyl acetone (AcAc)-mediated "off-on" method that harnesses Knorr pyrazole synthesis to regenerate SAL esters with exceptional compatibility toward Trp. By combining this advance with aggregation-disrupting strategies, we achieved the first chemical synthesis of the bioactive TIM-1 IgV domain confirmed by various characterizations including high-resolution mass spectroscopy (HRMS), circular dichroism (CD) spectroscopy, and enzyme-linked immunosorbent assay (ELISA).