Tropomyosin receptor kinases (TRKs, i.e., TRKA, TRKB, and TRKC) of the surface receptor tyrosine kinase family are of great interest in recent years as they are recognized as potential oncogenic factors. These transmembrane proteins, which serve as the high affinity receptors for neurotrophins, are predominantly expressed in the neuronal tissues and play important roles in regulating cell proliferation, differentiation, and apoptosis, and survival of neurons and other cells [1]. TRKA, TRKB, and TRKC are encoded by the NTRK1, NTRK2, and NTRK3 genes, respectively. The abnormal expression of TRKs caused by gene fusion, copy number aberration, and/or single nucleotide variation of the NTRK genes have been found to implicate a wide variety of adult and pediatric tumors [2]. Since the first identification of NTRK gene alternation in cancer [3], a lot of efforts have been made in understanding their carcinogenic mechanisms and the development of novel anti-tumor drugs targeting TRKs [4], [5], [6].

In the development of novel TRK inhibitors, the chiral amine (R)-2-(1-aminoethyl)-4-fluorophenol serves as an important intermediate for the synthesis of several candidate drug entities, such as repotrectinib [7]. The chemical synthesis of (R)-2-(1-aminoethyl)-4-fluorophenol is generally conducted according to the following procedure [8], [9] (Fig. 1a). The condensation of 5-fluoro-2-hydroxybenzaldehyde and (R)- or (S)-2-methylpropane-2-sulfinamide generates an imine intermediate. The target quaternary carbon stereocenter is established by the nucleophilic attack of methylmagnesium bromide or chloride against imine under −65 ℃. After removal of the protecting group (i.e., tert-butyl sulfonamide), (R)-2-(1-aminoethyl)-4-fluorophenol is generated. Due to the strict conditions (e.g., −65 ℃ low temperature), limited stereospecificity, and use of toxic agents such as 1,4-dioxane, it is challenging to scale up this method for industrial production. Recently, a novel chemical route synthesizing (R)-2-(1-aminoethyl)-4-fluorophenol in one step was reported [10] (Fig. 1b). In the presence of H2, ammonium salt (such as ammonium acetate), and Ru-based catalyst (such as Ru[OAc]2), the ketone substrate 5′-fluoro-2′-hydroxyacetophenon was efficiently converted to the target amine. Although this method greatly simplified the process of chemical synthesis, the cost, metal pollution, and safety issues associated with the use of Ru-based catalysts and high pressure of H2 atmosphere (40 atm) need to be carefully considered in scaled-up production.

ω-transaminases (hereafter ωTAs) are a group of pyridoxal 5′-phosphate (PLP) dependent enzymes that catalyze the reversible transfer of amino groups between diverse amino (such as amino acids, alkyl amines, aromatic amines) and carbonyl (such as aldehydes, ketones, ketoacids) compounds. Due to several advantages over conventional chemical synthesis, such as high stereoselectivity, broad substrate scope, and environmental friendliness, ωTA-based asymmetric catalysis has emerged as a promising strategy for chiral amine biosynthesis in the pharmaceutical and fine chemical industries. A number of chiral amine intermediates, which are of pharmaceutical and agronomic importance, have been synthesized employing ω-TAs on small or industrial scales [11], [12], [13], [14]. A classic example is the successful biosynthesis of sitagliptin, one of the bestselling antidiabetic drugs [15]. Employing an engineered (R)-selective ωTA of Arthrobacter sp., 200 g prositagliptin ketone l−1 was successfully converted to sitagliptin via one-step-biocatalysis with a 92% yield and excellent optical purity (up to >99.95% enantiomeric excess [e.e.]). This allows a simpler and greener production of sitagliptin in industrial scale. Since then, an increasing number of novel ωTAs have been identified and their applications in chiral amine biosynthesis were intensively investigated [16], [17], [18]. As a type of enzyme, ωTAs exhibit a wide scope of substrates. Ketone compounds of diverse sizes and structures can be accepted by ωTAs for the production of different kinds of amines. However, in a defined case of synthesis, it is important to employ suitable enzymes and develop proper reaction conditions for efficient catalysis. In the present study, 21 (R)-selective ωTAs were screened to catalyze the synthesis of (R)-2-(1-aminoethyl)-4-fluorophenol from its precursor ketone 5′-fluoro-2′-hydroxyacetophenon (Fig. 1c). Using a potent enzyme AbTA, which was the first identified (R)-ωTA from Arthrobacter sp. KNK168 [19], catalytic conditions were optimized. A scaled-up production in 1-litre reaction was then performed, generating the target product with high enantiopurity at gram scale. An overall process for (R)-2-(1-aminoethyl)-4-fluorophenol bioproduction was therefore developed.