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. 2025 Apr 2;10(14):14382-14389.
doi: 10.1021/acsomega.5c00590. eCollection 2025 Apr 15.

Preparative Coupled Enzymatic Synthesis of L-Homophenylalanine and 2-Hydroxy-5-oxoproline with Direct In Situ Product Crystallization and Cyclization

Sven Tiedemann  1 , Annabel Stang  1 , Simon Last  1 , Thierry Gefflaut  2 , Jan von Langermann  1
Affiliations

Preparative Coupled Enzymatic Synthesis of L-Homophenylalanine and 2-Hydroxy-5-oxoproline with Direct In Situ Product Crystallization and Cyclization

Sven Tiedemann et al. ACS Omega. .

Abstract

A continuous in situ crystallization concept is presented for the coupled preparative synthesis of L-homophenylalanine and 2-hydroxy-5-oxoproline (a cyclized form of α-ketoglutarate) using the α-transaminase from Megasphaera elsdenii. The process consists of a spontaneous reactive crystallization step of the enantiopure amino acid itself and a parallel spontaneous cyclization of the deaminated cosubstrate in solution. In parallel, these effects significantly improve the overall productivity of the biocatalytic reaction. Batch, repetitive, and fed-batch processes were investigated, and the fed-batch option proved to be the most viable option. The fed-batch process was subsequently used for a coupled synthesis approach at the gram scale. In total, >18 g of chemically pure L-homophenylalanine and >9 g of 2-hydroxy-5-oxoproline were isolated. This optimized process allows for the design of effective transaminase-catalyzed reactions at a preparative scale utilizing standard (fed-)batch-mode crystallizers.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. General Reaction Concept, Involving the Transaminase from Megaphaera elsdenii (MeTA)-Catalyzed Conversion of OPBA (1a) to L-HPA (2a) with l-Glutamine (1b) Deamination to α-Ketoglutaramat (αKGM, 2b), which Spontaneously Cyclizes to 2-Hydroxy-5-oxoproline (HOP, 2c)
Figure 1
Figure 1
pH-dependency of MeTA; Reaction conditions: 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate-buffer, 30 °C, 24 h, 100 U/mL catalyst.
Figure 2
Figure 2
Investigation of the behavior of MeTA toward higher donor (1b) concentrations with a 1 mL batch reaction of 100 mM 1a, 5 mM PLP, 50 mM phosphate-buffer pH 8, 30 °C, 24 h, 100 U/mL catalyst.
Figure 3
Figure 3
Investigation of substrate (1a, 1b) inhibition for MeTA with a 1 mL batch reaction, C1a:C1b (1:1.2), 5 mM PLP, 50 mM phosphate-buffer pH 8, 30 °C, 24 h, 100 U/mL catalyst.
Figure 4
Figure 4
Temperature dependency of MeTA for a 1 mL batch reaction of 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate-buffer pH 8, 24 h, 75 U/mL catalyst.
Figure 5
Figure 5
Effect of organic solvents on the performance of MeTA, 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate-buffer pH 8, 40 °C with addition of 20 vol % organic, 100 U/mL catalyst.
Figure 6
Figure 6
Time-dependent conversion for a reaction of 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate buffer, pH 8, 40 °C, 100 U/mL catalyst.
Figure 7
Figure 7
Time-dependent conversion for a 10 mL repetitive batch reaction of 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate-buffer pH 8, 40 °C, 100 U/mL catalyst; additional enzyme was added after every 24 h cycle according to legend.
Scheme 2
Scheme 2. Process Concept of the Repetitive Batch Reaction with Re-Use of MeTA and Residual Reactants within the Remaining Mother Liquor for the Subsequent Cycle
Figure 8
Figure 8
Time-dependent conversion for a 10 mL repetitive batch reaction of 100 mM 1a, 120 mM 1b, 5 mM PLP, 50 mM phosphate-buffer pH 8, 40 °C, 100 U/mL catalyst.
Scheme 3
Scheme 3. Process Concept of the MeTA-Catalyzed Fed Batch Reaction Over a 24 h Time Frame with a Continuous Feed of Fresh Substrates OPBA and Gln
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