Carbon Isotope Labeling of Amino Acids by CO₂ Carboxylic Acid Exchange

α-amino acid is one of the cornerstones of the building of life, and is the basic unit of polypeptide drugs and biological agents. At the same time, α-amino acid is also the key structure of many small molecule drugs. In recent years, isotope-labeled α-amino acids and their derivatives have been widely used in structural and mechanistic biology, quantitative proteomics, pharmacokinetic analysis, positron emission tomography (PET) and other fields. Since hydrogen isotope labeling is easy to eliminate or undergo metabolic transformation, α-amino acids and their derivatives are usually labeled with carbon isotopes. In the past 70 years, scientists have also made some progress in the synthesis of carbon isotope-labeled α-amino acids. The general strategy is to introduce the *C-labeled body into the precursor molecule, and then undergo several steps of transformation to synthesize the target molecule. However, the construction of isotope-labeled α-amino acids is still a major challenge due to the constraints of synthetic steps, radiochemical yield (RCY), and separation and purification.

In contrast, CO₂ is the major source of all isotopically labeled carbon reagents. In recent years, scientists have developed several methods for carbon labeling of biologically relevant compounds using the principle of dynamic isotope exchange, including: 1) Pd-catalyzed decarbonylation of acid chlorides to construct isotope-labeled carboxylic acids; 2) nickel-catalyzed active esters to generate isotope ¹³C- and ¹⁴C-labeled carboxylic acids; 3) thermal or photochemical conditions to achieve reversible decarboxylation-carboxylation of electrically stable carboxylic acids to construct isotopic carbon-labeled carboxylic acids. On this basis, recent researchers used aromatic aldehyde catalysts and isotope-labeled *CO₂ to realize the carboxylic acid exchange reaction of α-amino acids (Figure 1), and constructed a series of ¹¹C, ¹³C or ¹⁴C isotope-labeled α-amino acids.

Figure 1. Isotopic labeling of α-amino acids

In the system of adding [¹³C]CO₂, the researchers used (±)-phenylalanine as the template substrate to optimize the reaction conditions, and the results showed that 4-anisaldehyde (20 mol%) was used as the catalyst, Cs₂CO₃ (40%) as the base, [¹³C]CO₂ (~8 equiv.), and DMSO as the solvent, the reaction was carried out at 70°C, and the target product was obtained with a ¹³C incorporation rate of 75% and a yield of 84%. It should be pointed out that 4-anisaldehyde is the best catalyst because of its electrophilicity. On the other hand, after an in-depth study of the electrophilicity of the catalyst, it was found that 4-anisaldehyde had a slower reaction rate but a longer service life, while a more electrophilic catalyst had a faster initial rate but was easily decomposed, thereby reducing ¹³C incorporation rate. In addition, this reaction can also be used to introduce ¹⁴C labeling, and the target product can be prepared with 53% ¹⁴C incorporation and 51% yield.

Under optimal conditions, the researchers investigated the substrate scope of various α-amino acids, and the results showed that aliphatic/aromatic α-protein amino acids (such as: (±)-phenylalanine, (±)-alanine, (±)-leucine, (±)-glycine, (±)-tyrosine, (±)-tryptophan, (±)-methionine), β-branched substituted α-protein amino acids (eg: (±)-isoleucine, (±)-valine, (±)-proline) and even α-protein amino acids with acidic/basic side chains are compatible with the reaction, the corresponding ¹³C-labeled products were obtained with moderate to good yields. In addition, phenylalanine substituted by different groups and other aryl/alkyl substituted α-amino acids can also be isotopically labeled.

In general, this study used aldehyde catalysts and isotope-labeled *CO₂ to realize the carboxylic acid exchange reaction of α-amino acids under mild conditions, and constructed a series of ¹¹C, ¹³C or ¹⁴C isotope-labeled α-amino acids. This method not only has mild conditions and good substrate tolerance, but also has important significance for the construction of isotope-labeled α-amino acids, drug development and medical imaging.

Reference

1. Aldehyde-catalysed carboxylate exchange in α-amino acids with isotopically labelled CO₂.
   Odey Bsharat, Michael G. J. Doyle, Maxime Munch, Braeden A. Mair, Christopher J. C. Cooze, Volker Derdau, Armin Bauer, Duanyang Kong, Benjamin H. Rotstein, Rylan J. Lundgren