Efficient Enzymatic Preparation of ¹³N‐Labelled Amino Acids: Towards Multipurpose Synthetic Systems

Abstract

Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents.

Sustainable and Continuous Synthesis of Enantiopure l‐Amino Acids by Using a Versatile Immobilised Multienzyme System

“…Herein, we have harnessed a recently reported hierarchical architecture to co‐immobilise two well‐known enzymes, l ‐alanine dehydrogenase from Bacillus subtilis ( l ‐AlaDH‐Bs) and formate dehydrogenase from Candida boidinii (FDH‐Cb), with the ultimate goal of synthesising enantiopure l ‐α‐amino acids (Scheme ). This multifunctional and heterogeneous biocatalyst is very versatile for the synthesis of both natural and unnatural α‐amino acids in both batch mode and flow reactors.…”

Biocatalysis in radiochemistry: Enzymatic incorporation of PET radionuclides into molecules of biomedical interest

“…Recycling of the cofactor is an attractive alternative to increase the efficiency of the radiochemical reaction while decreasing the net concentration of the cofactor. Recently, our group has reported a simple and efficient route to synthesize different [ 13 N]amino acids incorporating a NADH recycling system . In this system, L‐ alanine dehydrogenase from Bacillus subtilis was used for the 1‐pot/1‐step noncarrier‐added synthesis of L‐ [ 13 N]alanine, [ 13 N]glycine, and L‐ [ 13 N]serine starting from their corresponding α‐ketoacids and using NADH as the redox cofactor and [ 13 N]NH 3 as the amine source.…”

“…The understanding of the precise mechanisms underlying biocatalytic reactions in radiochemistry opens new avenues for the preparation of radiolabeled compounds in multiple biomedical applications. Thanks to the advances in bioengineering, old approaches are finding new applications (see, for example, recent developments of new enzymatic strategies for the preparation of 13 N‐labeled amino acids reported by Da Silva et al); moreover, new approaches are currently being developed, as exemplified by the most recent work from O'Hagan et al with enzymatic fluorination of sugar and nucleoside derivatives or the elegant strategy from Harada et al for the preparation of 11 C/ 18 F‐labeled proteins using cell‐free translation systems …”

Chemistry for Positron Emission Tomography: Recent Advances in 11C‐, 18F‐, 13N‐, and 15O‐Labeling Reactions

“…[211] [ 13 N]NH 3 could also serve as ab uilding block for 13 N transformations,i ncluding enzymatic reactions,H ofmann rearrangements,a mide or imine reduction, amination of organoboranes,substitutions,and amidations.These reactions were used to prepare 13 N-labeled amino acids,p rimary amines,a mides,u reas,c arbamates,a nd metal complexes (Scheme 37 B). [211,[214][215][216] [ 13 N]NO 2 À is another commonly used synthon for 13 N chemistry,w hich can be prepared by proton irradiation of water, oxidation of [ 13 N]NH 3 using gallium or cobalt oxides,or reduction of [ 13 N]NO 3 À by metals or eukaryotic nitrate reductase (Scheme 37 A). [211,217,218] The 13 N-nitrosation of ureas,s econdary amines,a nd thiols could be realized with [ 13 N]NO 2 À .…”