Preparative Enzymatic Desymmetrization of (Acetyl-Leu-Pro-Lys)2-R110 Using Bovine Trypsin Variant D189S

Rhodamine 110 (R110) peptide conjugates are widely used fluorogenic substrates in proteolytic assays; however, their inherent symmetry results in two identical hydrolysis sites, complicating their application as well-defined substrates. Here, we report a preparative enzymatic strategy for the desymmetrization of the symmetric derivative (Acetyl-Leu-Pro-Lys)2-R110 using the bovine trypsin variant D189S. Due to pronounced differences in the rates of the two sequential hydrolysis steps, a mono-substituted intermediate accumulates under controlled reaction conditions. On a preparative scale, Acetyl-Leu-Pro-Lys-R110 was generated by partial hydrolysis and isolated by preparative HPLC in 28.8% yield and 95.8% purity. The structure of the asymmetric product was fully characterized by NMR and high-resolution mass spectrometry. This work demonstrates that selective enzymatic hydrolysis provides a simple and effective preparative route to asymmetric Rhodamine 110 derivatives, offering a practical alternative to conventional multistep synthetic approaches and enabling improved substrate design for kinetic studies.