Several features of Ala B and its derivatives are important to highlight. Ala BMIDA and Ala BPai are convenient reagents in solution-phase and automated solid-phase synthesis of oligopeptides and proteins (Scheme 3). We first demonstrated that boron remains intact under acidic conditions required to cleave peptides from the resin (95% TFA) after solid-phase synthesis (23 and 24). Similarly, the effects of boron substitution on the efficiency of macrocylization are minimal, as demonstrated in total synthesis of boronoalanine analog of pseudostellarin G 24. To further highlight the utility of Ala B in the synthesis of proteins, we prepared modified ubiquitin 28 with Phe4 mutated into Ala B (Scheme 3B). We selected this target because it allows to test the compatibility of Ala BMIDA with denaturating as well as reducing conditions of native chemical ligation (NCL; conversion of 26 into 27) and radical desulfurization (27à28). In a retrosynthetic manner, modified ubiquitin 28 was dissected into two fragments Met1-Phe45 ( ) and Cys46-Gly76 ( ) that were joined by the NCL. Ala MIDA was incorporated into peptide 26 (assembled through Fmoc-SPPS on the methyl Dawson MeDbz linker) that allowed for in situ generation of a thioester intermediate. To our delight, boronoalanine survived several high-temperature (90 o C) couplings and basic Fmoc deprotection conditions with piperidine/DBU. The union of oligopeptides 26 and 29 could be accomplished with a thioester generated from mercaptophenylacetic acid (MPAA), but this step required additional experimentation to achieve optimal yields. We observed that hydrolysis of the peptide backbones well as formation of transient structures where the MIDA group exchanged with intramolecular Lewis basic sites such as amides forming a boraheterocyle (dehydro-27) were the main issues. We hypothesized that the formation of boronic acid anhydrides and the concomitant loss of MIDA group could be controlled by pH. We found that under basic conditions (pH 7.6), substantial cleavage of 27 was observed (72%; conversion into truncated 27) and the intramolecular boronic acid anhydride constituted the remainder of the material. By adjusting pH to 6.4, we were able to improve the yield of 27 to 89% and reduce the fragmentation to ~11%, but we also observed that the MIDA group was removed during HPLC purification resulting a dehydrated boronic acid. Next, in order to complete the chemical synthesis of 28, we subjected 27 to radical desulfurization with glutathione (GSH) as the H-atom donor and reduced Cys46 to Ala46 in 33% isolated yield after HPLC purification. Gratifyingly, these conditions showed no cleavage or truncation of 28. The isolated material shows the formation of dehdro-28 (MS) due to loss of water from boronic acid whereas the HPLC trace indicates a single entity. Collectively, these results demonstrate that Ala B itself can serve as a unique NPAA and is compatible with the current methods for chemical synthesis of proteins.