Abstract
Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50–90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60–80% isolated yield (n = 3, μ = 71, σ = 8.3) with an isotopic incorporation of 70–88% (n = 18, μ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield.
Key points
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Amino acids can be labeled with carbon isotopes via carboxylate exchange using [*C]CO2 where *C is either 13C, 11C or 14C. Three procedures are described using phenylalanine as the example starting material; the main differences relate to the sources of [*C]CO2 and how it is delivered.
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The products can be used immediately after a straightforward HPLC separation.
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Data availability
All experimental data supporting the findings of this study can be found within the paper and its Supplementary Information. Additional data related to the protocol can be found in the key reference supporting this paper27.
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Acknowledgements
Support was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) (RGPIN-2019-06050 and RGPAS-2019-00051 to R.J.L., RGPIN-2017-06167 to B.H.R., Canada Graduate Scholarship-Doctoral (CGS-D) fellowship to M.G.J.D., Postgraduate Scholarship-Doctoral (PGS-D) fellowship to B.A.M.), the Canadian Foundation for Innovation (IOF 32691 to R.J.L., JELF 36848 to B.H.R.), the Province of Alberta (AGES fellowship to M.G.J.D., AGES fellowship to O.B.), the Province of Ontario (ER17-13-119 to B.H.R.), the University of Ottawa Heart Institute (Endowed fellowship to M.M.) and the Killam trusts (Izaak Walton Killam (IWK) fellowship to M.G.J.D.). We thank M. Muñoz for HPLC recommendations as well as B. Reiz and A. Morales-Izquierdo for assistance with MS analyses.
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M.G.J.D. optimized the described procedure involving 13C. M.G.J.D., A.S. and V. D. carried out experiments and analyzed data involving 14C. B.A.M. optimized the described procedure involving 11C. O.B. optimized the initial reaction involving 13C. M.G.J.D. and O.B. carried out experiments and analyzed data related to studies involving the 13C procedure. B.A.M. and M.M. carried out experiments and analyzed data related to studies involving the 11C procedure. R.J.L. and B.H.R. directed the research. R.J.L. conceived the project. M.G.J.D. wrote the manuscript and supplementary information with contributions from all authors.
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A.S. and V.D. are employees of Sanofi and may hold shares or options of the company. The other authors declare no competing interests.
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Nature Protocols thanks Sukwon Hong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Bsharat, O. et al. Nat. Chem. 14, 1367–1374 (2022): https://doi.org/10.1038/s41557-022-01074-0
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Supplementary calculations, compound characterization data and discussion.
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Doyle, M.G.J., Mair, B.A., Sib, A. et al. A practical guide for the preparation of C1-labeled α-amino acids using aldehyde catalysis with isotopically labeled CO2. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00974-4
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DOI: https://doi.org/10.1038/s41596-024-00974-4
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