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PreprintCC BYChemRxiv

Expanding Non-Natural Prenyl Diphosphate Chemical Space through Promiscuous yet Regioselective C2-Methylation

  • Vikas Kumar1,2
  • Prashant S. Mandal1,2
  • Anshi Pandey1,2
  • Gavin J. Williams3,4
  • Shanteri Singh1,2
4 affiliations
  1. 1Department of Chemistry & Biochemistry
  2. 2Oklahoma
  3. 3Department of Chemistry
  4. 4NC State University
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Abstract

Prenyl diphosphate methyltransferases are emerging as powerful biocatalysts that bypass the classic isoprene rule, installing methyl appendages directly onto standard (iso)prenyl diphosphate substrates and transforming conventional terpene architectures into non-canonical terpenoids. Although their activity toward natural precursors is well documented, their biocatalytic tolerance toward non-natural analogs remains largely underexplored. Here, we systematically probe the substrate scope of the C2-geranyl diphosphate methyltransferase from Streptomyces coelicolor A3(2) (ScGPP-MT) using a library of 40 structurally diverse synthetic allylic, benzylic and heterocyclic diphosphate (alkyl-PP) analogs spanning 4–15 carbons. Direct screening revealed that 10 analogs serve as substrates, including the natural substrate geranyl diphosphate (GPP), establishing a minimum linear chain length of seven carbons for acceptance. To expand this scope, we developed a coupled enzymatic cascade with Lithospermum erythrorhizon GPP synthase (LeGPPS), in which non-natural GPP derivatives are generated by condensation of synthetic alkyl-PP analogs with isopentenyl diphosphate (IPP) and subsequently methylated by ScGPP-MT. This cascade successfully processed 25 additional analogs, including the natural substrate. Collectively, ScGPP-MT accommodates allylic substrates spanning 7–11 linear carbons from diphosphate headgroup, enabling modification of 33 distinct non-natural analogs. To investigate the regiochemistry of ScGPP-MT-catalyzed methylation, which cannot be directly determined because methylated prenyl diphosphates and their alcohol derivatives are difficult to isolate, we coupled the methylation reaction with an engineered aromatic prenyltransferase, NphB(M31S), affording an array of structurally unique, non-canonical cannabinoid precursors. Scale-up and subsequent structural characterization of selected cannabinoid precursors revealed that ScGPP-MT is strictly regio-specific, performing C2-methylation of non-natural substrates just as it does with GPP. Together, these results establish a versatile biocatalytic platform that circumvents traditional biosynthetic constraints, offering a powerful toolkit for the de novo synthesis of diverse unnatural terpenoids with potential pharmaceutical value.

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Cite

BibTeXRIS
BibTeX
@misc{kumar2026expanding,
  title = {{Expanding Non-Natural Prenyl Diphosphate Chemical Space through Promiscuous yet Regioselective C2-Methylation}},
  author = {Vikas Kumar and Prashant S. Mandal and Anshi Pandey and Gavin J. Williams and Shanteri Singh},
  howpublished = {ChemRxiv},
  year = {2026},
  month = oct,
  doi = {10.26434/chemrxiv.15010712/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010712/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Expanding Non-Natural Prenyl Diphosphate Chemical Space through Promiscuous yet Regioselective C2-Methylation
AU  - Vikas Kumar
AU  - Prashant S. Mandal
AU  - Anshi Pandey
AU  - Gavin J. Williams
AU  - Shanteri Singh
PY  - 2026
DA  - 2026/10/20
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15010712/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010712/v1
AB  - Prenyl diphosphate methyltransferases are emerging as powerful biocatalysts that bypass the classic isoprene rule, installing methyl appendages directly onto standard (iso)prenyl diphosphate substrates and transforming conventional terpene architectures into non-canonical terpenoids. Although their activity toward natural precursors is well documented, their biocatalytic tolerance toward non-natural analogs remains largely underexplored. Here, we systematically probe the substrate scope of the C2-geranyl diphosphate methyltransferase from Streptomyces coelicolor A3(2) (ScGPP-MT) using a library of 40 structurally diverse synthetic allylic, benzylic and heterocyclic diphosphate (alkyl-PP) analogs spanning 4–15 carbons. Direct screening revealed that 10 analogs serve as substrates, including the natural substrate geranyl diphosphate (GPP), establishing a minimum linear chain length of seven carbons for acceptance. To expand this scope, we developed a coupled enzymatic cascade with Lithospermum erythrorhizon GPP synthase (LeGPPS), in which non-natural GPP derivatives are generated by condensation of synthetic alkyl-PP analogs with isopentenyl diphosphate (IPP) and subsequently methylated by ScGPP-MT. This cascade successfully processed 25 additional analogs, including the natural substrate. Collectively, ScGPP-MT accommodates allylic substrates spanning 7–11 linear carbons from diphosphate headgroup, enabling modification of 33 distinct non-natural analogs. To investigate the regiochemistry of ScGPP-MT-catalyzed methylation, which cannot be directly determined because methylated prenyl diphosphates and their alcohol derivatives are difficult to isolate, we coupled the methylation reaction with an engineered aromatic prenyltransferase, NphB(M31S), affording an array of structurally unique, non-canonical cannabinoid precursors. Scale-up and subsequent structural characterization of selected cannabinoid precursors revealed that ScGPP-MT is strictly regio-specific, performing C2-methylation of non-natural substrates just as it does with GPP. Together, these results establish a versatile biocatalytic platform that circumvents traditional biosynthetic constraints, offering a powerful toolkit for the de novo synthesis of diverse unnatural terpenoids with potential pharmaceutical value.
ER  -