AMPylation, a critical protein post-modification process, is gaining significant research interest. Yet, a major roadblock existed – the lack of a readily available probe molecule for studying this process. This case study explores how Sigut Labs tackled this challenge by making N6-propargyl adenosine phosphoramidate (pro-N6pA) commercially accessible, paving the way for more efficient AMPylation research.

Background information

Researchers from the Technical University of Munich and Max Planck Institute of Psychiatry, Munich, Germany recently reported (Nat Commun 11, 517 (2020)) novel N6-propargyl adenosine phosphoramidate (pro-N6pA). This innovative compound offers a simplified approach to identifying proteins undergoing AMPylation, a crucial protein post-translational modification (PTM) involved in the modulation of neurodevelopment and neurodegeneration (Cell Chemical Biology, 27(7), 773-779).

Pro-N6pA’s effectiveness lies in its ability to enter cells readily and undergo metabolic activation. Once inside, it transforms into N6pATP, a molecule accepted by AMP-transferases, the enzymes responsible for the AMPylation of proteins.

Identifying the issue

Sigut Labs was contacted by Dr. Pavel Kielkowski, one of the inventors of the molecule, to inquire about our ability to prepare it on a larger scale. At their lab at the TU Munich Munich and later LMU Munich, pro-N6pA was typically prepared on a milligram (mg) scale solely for their internal research. This meant that any researcher needing pro-N6pA for their application had to synthesize it themselves.

While the synthesis is well-described with several preparation options available, it remains time-consuming and requires a skilled synthetic chemist along with appropriate laboratory equipment for handling sensitive compounds.

Additionally, many scientists from various academic institutions worldwide were contacting Dr. Kielkowski to request pro-N6pA for their studies.

Methodology insight

Equipped with the necessary skills and advanced equipment, Sigut Labs’ team of experienced chemists tackled the challenge of large-scale pro-N6pA production. Here’s how we achieved it:

• Pathway evaluation: We scrutinized existing and novel synthetic pathways for pro-N6pA.
• Optimization and scale-up: The most promising route was selected and optimized, enabling multigram quantities within just two weeks.
• Exceptional purity: The synthesized pro-N6pA exceeded 99% purity, verified through advanced analytical techniques including 1H-NMR, 31P-NMR, and LC-MS.

Positive outcome

Sigut Labs’ collaboration with the Kielkowski Research Group has resulted in the successful large-scale production of pro-N6pA. This accomplishment offers significant benefits to the scientific community:

• Increased availability: Researchers can now access pro-N6pA readily, eliminating the need for in-house synthesis and saving valuable time and resources.
• Simplified research: Ready-made pro-N6pA facilitates a quicker start to AMPylation research projects.
• Consistent quality: Sigut Labs’ expertise ensures consistent quality and purity of pro-N6pA, leading to reliable research data.

Need high-purity pro-N6pA for your next AMPylation project? Visit our e-shop.