Reductive aminations by imine reductases: from milligrams to tons

Abstract

The synthesis of secondary and tertiary amines through the reductive amination of carbonyl compounds is one of the most significant reactions in synthetic chemistry. Asymmetric reductive amination for the formation of chiral amines, which are required for the synthesis of pharmaceuticals and other bioactive molecules, is often achieved through transition metal catalysis, but biocatalytic methods of chiral amine production have also been a focus of interest owing to their selectivity and sustainability. The discovery of asymmetric reductive amination by imine reductase (IRED) and reductive aminase (RedAm) enzymes has served as the starting point for a new industrial approach to the production of chiral amines, leading from laboratory-scale milligram transformations to ton-scale reactions that are now described in the public domain. In this perspective we trace the development of the IRED-catalyzed reductive amination reaction from its discovery to its industrial application on kg to ton scale. In addition to surveying examples of the synthetic chemistry that has been achieved with the enzymes, the contribution of structure and protein engineering to the understanding of IRED-catalyzed reductive amination is described, and the consequent benefits for activity, selectivity and stability in the design of process suitable catalysts.

Scheme 1. The asymmetric synthesis of chiral secondary amines via hemiaminal and imine formation by reductive amination (adapted from ref. 3).

Scheme 2. Examples of asymmetric reductive aminations catalyzed by chiral transition metal complexes. (A) Synthesis of (S)-metolachlor precursor; (B) synthesis of precursor of tofacitinib.

Scheme 3. Biocatalytic amination of ketones by various enzymes. (A) ω-Transaminases (ω-TAs); (B) amine dehydrogenases (AmDHs); (C) opine dehydrogenases (OpDHs); (D) imine reductases (IREDs); (E) reductive aminases (RedAms).

Fig. 1. (A) Structure of dimer of (R)-selective IRED Q1EQE0 from S. kanamyceticus (PDB 3ZHB) with monomers (A) and (B) shown in brown and blue respectively; (B) detail of active site clefts in 3ZHB and (S)-selective IRED from Bacillus cereus (4D3F) showing cofactor NADP⁺ and residues D187 and Y188 from the respective enzymes.

Fig. 2. (A) Reduction of 1-methyl-3,4-dihydroisoquinoline 12 to (R)-MTQ 13 by AoIRED (B) detail of active site of AoIRED in complex with NADP⁺ (5A9S) and (R)-MTQ (5FWN). Residues are from monomer (A) unless marked as (B). The trajectory for delivery/acceptance of hydride from/to the C4 atom of NADP⁺ is shown with a dashed black line.

Scheme 4. Early examples of intramolecular asymmetric reductive amination reactions enabled by IREDs at high amine: ketone ratios. (m.e. = molar equivalents). ‘NADPH’ in this and other schemes signifies the requirement of this reduced cofactor for the reaction, and is usually generated with the assistance of a cofactor recycling system comprising e.g. glucose dehydrogenase (GDH) and glucose, omitted from this and other schemes.

Scheme 5. Screening of an IRED library by Roche identified IR_20 as a candidate enzyme for enabling reductive aminations of 16 and (R)-17 to form 16b and (1S,3R)-17d respectively.

Scheme 6. IREDs from the Roche library catalyzed the synthesis of both enantiomers of rasagiline 18e and tertiary amine 19f.

Scheme 7. IR-Sip catalyzed amination of α-racemic aldehydes.

Scheme 8. Reductive amination reactions catalyzed by the reductive aminase from Aspergillus oryzae (AspRedAm).

Fig. 3. (A) Structure of dimer of AspRedAm (5 G6S) with monomers (A) and (B) in brown and blue respectively; (B) active site of AspRedAm with NADP⁺ and (R)-rasagiline (R)-18e. Residues are from monomer (A) unless marked as (B). The interaction between the nitrogen atom of 18e and the side chain of Y177 is indicated by a black dashed line.

Fig. 4. Active site of AtRedAm in complex with redox-inactive cofactor NADPH₄, cyclohexanone 19 and allylamine j. Residues are from monomer (A) unless marked as (B). Interactions between j and the side chains of D175 and N98 are shown as black dashed lines.

Scheme 9. Proposed mechanism for a fungal RedAm-catalyzed reductive amination using residue numbering from AtRedAm. (A) The active site of AtRedAm binds NADPH, (B) cyclohexanone, and (C) the amine sequentially, to form a quaternary complex; (D) the amine is activated by D175 for attack at the electrophilic carbon of the ketone to give a hemiaminal intermediate; (E) the hemiaminal intermediate loses water to give the iminium intermediate; (F) this is reduced by NADPH to give the amine product and NADP⁺; (G) the amine product and (H) NADP⁺ are sequentially expelled from the active site to regenerate the enzyme for another catalytic cycle.

Scheme 10. Resolution of racemic amine 24 by AspRedAm.

Scheme 11. Reductive aminations catalyzed by RedAms from Neosartorya spp.

Scheme 12. The formation of melanin-concentrating hormone receptor agonist 19k using GSK IR-01.

Scheme 13. (A) Chemical basis of IRED-y-to-go screen. The oxidative activity of an IRED, acting on a secondary amine substrate, generates NADPH, which is used by diaphorase to reduce tetrazolium salt 28 to red dye 29. (B) The IRED-y-to-go screen was used to identify IREDs that enabled aminations of acetophenones 30 and the production of tertiary amines. (C) The IRED-y-to-go screen enabled also identified enzymes for the amination of esters.

Scheme 14. Synthesis of anti-Parkinson's agent (S)-rotigotine 36.

Scheme 15. Intramolecular reductive amination for the preparation of piperazines.

Scheme 16. Intramolecular reductive aminations for the formation of azepanes.

Scheme 17. Enzyme cascade for the amination of cyclohexane. FDH = formate dehydrogenase.

Scheme 18. Flow system for the oxidation-reductive amination cascade towards the synthesis of 4-O-methyl norbelladine 47.

Scheme 19. Conjugate Reduction (CR) – Reductive Amination (RA) catalyzed by EneIRED.

Scheme 20. Synthesis of lysine-specific demethylase-1 (LSD) inhibitor GSK2879552 intermediate 53o by reductive amination with IR46.

Fig. 5. (A) Model of structure of ‘IR-46’ derived using AoIRED (5FWN). The model shows the location of amino acid residue sites that were mutated in variant ‘M3,’ applied in the kg-scale synthesis of 53n in Scheme 20; (B) structure of IRED88 (ref. 81) (PDB 7OG3) showing locations of residues mutated in variant Q194L/S220T/H230Y, used in the gram-scale synthesis of 54b in Scheme 21; (C) model of structure of SpRedAm showing locations of residues mutated in variant ‘R3-V6’ used in the ton-scale synthesis of 58b (Scheme 22).

Scheme 21. Synthesis of ZPL389 54b using enzyme IRED88 Q194L/S220T/H230Y evolved by Novartis.

Scheme 22. Reductive amination reactions investigated by Pfizer.