A Modular Nucleoside Kinase Cascade for the Synthesis of Ribonucleoside Triphosphates.

Area of Science:

• Biotechnology
• Enzymatic Synthesis
• Nucleotide Chemistry

Background:

• High demand for affordable nucleoside 5 -triphosphates (NTPs) requires efficient and scalable synthesis methods.
• Current synthesis methods may lack efficiency or scalability for widespread application.

Purpose of the Study:

• To develop standardized one-pot enzymatic cascades for the production of all canonical NTPs (ATP, CTP, GTP, UTP).
• To optimize these cascades for high conversion rates and yields.
• To demonstrate the scalability and broad applicability of the developed enzymatic platform.

Main Methods:

• Utilized Saccharomyces cerevisiae UMP kinase (ScURA6) and GMP kinase (ScGMPK) for initial phosphorylation.
• Employed Escherichia coli acetate kinase (EcAcK) with acetyl phosphate for NDP phosphorylation.
• Optimized reaction conditions for flux and full conversion of substrates.
• Developed an alternative cascade using NTP as a phosphate shuttle.
• Performed preparative-scale syntheses.

Main Results:

• Achieved high conversion rates (≥93%) for substrates within 60-120 minutes.
• Demonstrated NTP conversion of ≥84% using the modified phosphate shuttle cascade.
• Reached up to 89% isolated yield (≥150 mg product) in preparative-scale syntheses.
• Confirmed the absence of NMP and NDP intermediates in the optimized cascades.

Conclusions:

• Developed a robust and scalable enzymatic platform for the production of canonical NTPs.
• The enzymatic cascades offer flexibility for synthesizing noncanonical nucleosides and analogs.
• The method provides a cost-effective and efficient approach to meet the growing demand for NTPs.