Chiral amine synthesis using ω-transaminases: an amine donor that displaces equilibria and enables high-throughput screening

Abstract

The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity.

Keywords: asymmetric catalysis; biocatalysis; chiral amines; high-throughput screening; transaminases.

Figure 1

Selected examples of widely utilized conditions for small/medium-scale (a) and large-scale (b) processes that employ ω-TAs.

Scheme 1

Conversion of 2 (100 mm) into (S)-3 using diamine donor 1 (1.1 equiv). [a] Yield of isolated product. HEPES=4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid.

Figure 2

a) Conversion of 10 (5 mm) into the corresponding amine using commercially available ω-TAs and diamine 1 (5 mm). L1, L3, and L5 A–F contain the (R)-selective Codexis enzymes ATA 025, 303, 013, 301, 415, and 117, respectively. L2, L4, and L6 contain the (S)-selective Codexis enzymes ATA 254, G 05, 260, 256, 234, and 113, respectively. L1/L2: diamine 1 only, 3 h; L3/L4: 15 min after addition of substrate 10; L5/L6: 24 h after addition of substrate 10; L7: A–F=Almac TAm 106, 107, 115, 121, 125, and 140, respectively, substrate 10, diamine 1, 24 h. b) Colony-based screen with ortho-xylylenediamine (1). Cells expressing the pf-ATA gene turn dark in color after 30 min (right). Cells lacking the pf-ATA gene remain colorless (left).