Developing deprotectase biocatalysts for synthesis.

Area of Science:

• Biocatalysis and Green Chemistry
• Organic Synthesis and Protecting Group Chemistry
• Enzymology and Structural Biology

Background:

• Organic synthesis frequently employs protecting groups (PGs) to mask functional groups (FGs), necessitating selective removal steps.
• Traditional PG removal often involves harsh conditions, excess reagents, long reaction times, and low yields.
• Biocatalysis offers an underutilized approach using deprotectase enzymes for selective PG removal, aligning with green chemistry principles.

Purpose of the Study:

• To construct and demonstrate a biocatalytic cascade for the deprotection of doubly-protected amino acids.
• To explore the use of specific enzymes, Bacillus BS2 esterase and Sphingomonas Cbz-ase, for sequential PG removal.
• To provide structural insights into the enzymes involved in substrate recognition and catalysis.

Main Methods:

• Screening of Sphingomonas Cbz-ase with various N-carbobenzyloxy (Cbz)-protected amino acids.
• Utilizing Bacillus BS2 esterase for O-tert-butyloxycarbonyl (OBu) deprotection.
• Combining both enzymes in a one-pot, two-step cascade to deprotect CBz-L-Phe OBu to L-Phe.
• Employing molecular docking and structural modeling to analyze enzyme active sites and substrate interactions.

Main Results:

• Successful deprotection of doubly-protected amino acids using a sequential enzymatic cascade.
• Demonstrated the efficacy of Bacillus BS2 esterase and Sphingomonas Cbz-ase in a combined system.
• Identified key residues for substrate recognition and catalysis in BS2 esterase and Cbz-ase through structural analysis.

Conclusions:

• A novel biocatalytic cascade for efficient and selective deprotection of protected amino acids has been developed.
• This enzymatic approach offers a greener and potentially more efficient alternative to traditional chemical deprotection methods.
• The findings support further exploration of this biocatalytic cascade for broader applications in chemical synthesis.