Direct Conversion of Hydrazones to Amines using Transaminases

Abstract Transaminase enzymes (TAms) have been widely used for the amination of aldehydes and ketones, often resulting in optically pure products. In this work, transaminases were directly reacted with hydrazones in a novel approach to form amine products. Several substrates were investigated, including those with furan and phenyl moieties. It was determined that the amine yields increased when an additional electrophile was added to the reaction mixture, suggesting that they can sequester the hydrazine released in the reaction. Pyridoxal 5’‐phosphate (PLP), a cofactor for transaminases, and polyethylene glycol (PEG)‐aldehydes were both found to increase the yield of amine formed. Notably, the amination of (S)‐(−)‐1‐amino‐2‐(methoxymethyl)pyrrolidine (SAMP) hydrazones gave promising results as a method to form chiral β‐substituted amines in good yield.

To a solution of benzaldehyde (0.45 mL, 4.4 mmol) in anhydrous ethanol (40 mL) under argon was added N,N-dimethylhydrazine (0.50 mL, 6.6 mmol) and the resulting mixture stirred at 50 °C for 3 h.
The solution was diluted with water (50 mL) and extracted with dichloromethane (3 × 50 mL). The combined organic layers were dried with MgSO4, filtered, and concentrated under reduced pressure to yield the product as a yellow oil (490 mg, 75%  To a solution of hydroxymethylfurfural (630 mg, 5.0 mmol) in water (30 mL) was added N,N-dimethylhydrazine (0.49 mL, 6.5 mmol) and the resulting mixture was stirred at 50 °C for 2 h.
The solution was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried with MgSO4, filtered, and concentrated under reduced pressure to yield the product as an orange oil (840 mg, 100%
The combined organic layers were dried with MgSO4, filtered, and concentrated under reduced pressure to yield the product as a yellow oil (680 mg, 91%

PEG-2000 supported aldehyde 13
To a solution of PEG-2000 (5.0 g, 2.5 mmol) in dichloromethane (50 mL) under argon was added 4formylbenzoic acid (1.5 g, 10 mmol, 4 equiv.), DCC (1.0 g, 5.0 mmol, 2 equiv.) and DMAP (60 mg, 0.50 mmol, 0.2 equiv.) and the solution stirred overnight at room temperature. Diethyl ether (200 mL) was added to precipitate the polymer and the solution cooled to 0 °C. The resulting crystals were filtered, washing with diethyl ether, and the filtrate was concentrated under reduced pressure to yield the crude product as a white solid (4.50 g, 90%

PEG-1000 supported aldehyde 14
To a solution of PEG-1000 ( After incubation, the reactions were visualised by NMR, with reference to the solvent peak at 4.79 ppm. There appears to be no reaction between IPA and the PEG-aldehyde, however, there does appear to be some imine formation between the amine product and PEG-aldehyde. As this is a reversible reaction, and the yield increases with the addition of this PEG-aldehyde 13, it can be assumed that the binding of the hydrazine is favourable enough that the resin is overall beneficial to this reaction. PEG-aldehyde 13 + furfurylamine Terrific broth (TB) 47.6 g of TB and 4 mL glycerol in 1 L distilled water then autoclaved.

Protein expression
Overnight cultures (10 mL) of the TAms in E. coli from the UCL TAm library were prepared in terrific broth supplemented with kanamycin (50 µg/mL) and incubated overnight at 37 °C. Cells were subcultured using 1% v/v inoculum in 2 L shake flasks containing 500 mL of the same supplemented   10 , Rhodobacter sphaeroides (Rh-TAm) 11 , Mycobacterium vanbaalenii (Mv-TAm) 12 , Arthrobacter sp. variant ArRMut11 (As-TAm) 13 and three metagenomic enzymes from a domestic drain 14 94-TAm pQR2189, 553-TAm pQR2191 and 3588-TAm pQR2208. Negative controls (no enzyme) were carried out with hydrazone in buffer; hydrazone and IPA in buffer;

Assays
hydrazone and PLP in buffer; hydrazone, IPA and PLP in buffer. In all reactions there was no amine product detected.
When measuring the enantiomeric excess of the amine product, an acetylation reaction was performed.
To 100 µL of the supernatant, pyridine (4.5 µL, 55 equiv.) and acetic anhydride (5.2 µL, 55 equiv.) were added, and the reaction was shaken at 20 °C for 2 h. The reaction was diluted with dichloromethane (100 µL) and extracted with water (2 × 100 µL). The organic layer was concentrated then dissolved in the HPLC mobile phase and analysed by analytical HPLC.
The total protein concentration of the Cv-TAm 10 lysate was measured by the Bradford method 9 and found to be 40.0 mg/mL. Analysis of the gel using ImageJ showed that the proportion of Cv-TAm was 31% (12.4 mg/mL).

Furfurylamine 11
The concentration of furfurylamine was determined with UV detection at 210 nm using a linear

3-Phenylpropylamine 19
The concentration of 3-phenylpropylamine was determined with UV detection at 210 nm using a linear