Aminopolyols from Carbohydrates: Amination of Sugars and Sugar‐Derived Tetrahydrofurans with Transaminases

Abstract Carbohydrates are the major component of biomass and have unique potential as a sustainable source of building blocks for chemicals, materials, and biofuels because of their low cost, ready availability, and stereochemical diversity. With a view to upgrading carbohydrates to access valuable nitrogen‐containing sugar‐like compounds such as aminopolyols, biocatalytic aminations using transaminase enzymes (TAms) have been investigated as a sustainable alternative to traditional synthetic strategies. Demonstrated here is the reaction of TAms with sugar‐derived tetrahydrofuran (THF) aldehydes, obtained from the regioselective dehydration of biomass‐derived sugars, to provide access to cyclic aminodiols in high yields. In a preliminary study we have also established the direct transamination of sugars to give acyclic aminopolyols. Notably, the reaction of the ketose d‐fructose proceeds with complete stereoselectivity to yield valuable aminosugars in high purity.


I. General Experimental and analytic methods
The solvents and chemicals were purchased from Sigma-Aldrich or Fluorochem and were used as supplied. Column chromatography was carried out using BDH (40-63 μm) silica gel and analytical thin layer chromatography was carried out using Merck Kieselgel aluminium-backed plates coated with silica gel. Compounds were visualised using combinations of UV (254 nm) or potassium permanganate. 1 H and 13  Transaminase expression and preparation. Overnight cultures (10 mL) of the TAms in E. coli from the UCL TAm library were prepared in 2xTY broth (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) 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 broth at 37 °C and 250 rpm. Transaminases were induced with 1 mM of IPTG when growing in the early exponential phase (OD600 = 0.5 -0.7), and the temperature was dropped to 30 °C until harvesting. Cells were harvested by centrifugation after 5 h of induction, and stored at −20 °C. When needed, the cell pellet after induction was resuspended in the appropriate buffer containing PLP at a 1:25 volume ratio (1 mL of the resuspension buffer per 25 mL of cell suspension) and sonicated (Soniprep 150 sonicator, MSE Sanyo Japan) on ice using 10 cycles of 10 s on, 10 s off at 10 Watts. The sonicated suspension was centrifuged at 12,000 rpm at 4 °C for 45 min to obtain the clarified lysate. The total protein concentration was determined using the Bradford method. TAm concentrations in the crude lysates were determined as 8.8 mg/mL Cv-S3 TAm, 8.0 mg/mL Rh-TAm, 9.6 mg/mL Mv-TAm, 6.4 mg/mL pQR2189, [1] 4.8 mg/mL pQR2191, [1] and 8.0 mg/mL pQR2208. [1] Colorimetric screening. The assay was performed in a 96 well-plate with a total volume of 200 μL containing 2-(4-nitrophenyl)ethan-1-amine hydrochloride 5 (25 mM) as amine donor, amine acceptor (10 mM), PLP (0.5 mM) and potassium phosphate buffer (100 mM, pH 8.0). The reaction was started by the addition of TAm clarified lysate (20 μL) containing the overexpressed TAm and the reaction was incubated at 30 °C and 500 rpm for 24 h. A positive control was performed using benzaldehyde or pyruvate as the amine acceptor as specified. Two negative controls were also performed, one without amine acceptor and another without enzyme. An orange/red coloration indicated that the TAms were active towards the selected aldehydes.
MBA screening. The assay was performed in an Eppendorf tube (500 μL total volume) containing (R)-or (S)-MBA (20 mM), PLP (2 mM), potassium phosphate buffer (100 mM, pH 8.0), amine acceptor (5 mM) and crude cell lysate (30 μL) containing the overexpressed TAm. After incubation at 30 °C and 350 rpm for the time specified, the reaction was stopped by the addition of 0.1% trifluoroacetic acid (TFA) in water (100 μL). Denatured protein was removed by centrifugation (4 °C, 12000 rpm, 10 min) and the supernatant diluted with water and analysed by analytical HPLC using an ACE 5-C18 300 column (150 × 4.6 mm) with UV detection at 250 nm. The concentration of acetophenone produced was determined using a linear gradient 15%-72% over 10 minutes at 1 mL min −1 (A = water with 0.1% of TFA and B = acetonitrile) with subtraction of a negative control without amine acceptor for all substrates. The acetophenone (ACP) produced eluted at a retention time of 8.8 min. Results were verified in duplicate or triplicate.
After incubation at 37 °C and 400 rpm, the reaction was stopped by the addition of 1% TFA in water (100 μL). Denatured protein was removed by centrifugation (4 °C, 13000 rpm, 10 min) and the supernatant analysed by analytical HPLC after derivatization with 9-fluorenylmethyl chloroformate (Fmoc-Cl).

S4
Derivatization with Fmoc-Cl and analytical HPLC. 25 μL of reaction sample was diluted with 5 μL of milliQ water or quenched with 1% TFA and mixed with 70 μL of sodium borate buffer solution (7 mM, pH 8) and 70 μL of Fmoc-Cl (40 mM in acetonitrile), and then incubated at r.t.
for 5 min. After this time the reaction was stopped by adding 80 μL of L-alanine (100 mM in water:acetonitrile 2:1). After 1 min the reaction mixture was centrifuged (25 °C, 13000 rpm, 10 min) and the supernatant was analysed by HPLC using an ACE 5-C18 column (150 × 4.6 mm).
The mobile phase A consisted of trifluoroacetic acid 0.1% in MilliQ water and the mobile phase B was trifluoroacetic acid 0.1% in acetonitrile 80% and MilliQ water 20%. The gradient elution was performed from 0 to 55% of phase B and run at 0.8 mL/min for 10 min at 30 °C for 3a and from 40 to 60% of phase B and run at 0.8 mL/min for 20 min at 30 °C 3b and for 28 min for 3c and 3d. The eluted products were detected at 360 nm and quantitative analysis was performed by the integration of peak areas using the external standard method (standard concentrations from 5 mM to 25 mM).
[a] Control reaction without DMSO. It is noteworthy that the selected TAm pQR2191 performs well in 25% DMSO at 30 °C, as shown from the positive control with pyruvate (+), while the yield slightly decreased at 45 °C. Conversely, when sugars were used as substrates an increase in the conversion yields was observed at the higher temperature. Interestingly, the control reaction for D-ribose without DMSO showed lower conversion yields at both 30 °C or 45 °C. The conversion yields of D-ribose with pQR2191 also dropped when MeOH was used instead of DMSO. Amberlyst 15 (0.5 g/mmol) was added to a solution of hydrazone in water (C = 0.2 mol/L). The reaction was stirred at room temperature for 10 min before filtration on a sinter funnel (porosity 3). The resulting mixture was concentrated under vacuum and the resulting thick oil was lyophilised to afford the hydrate as a white foam.

yl)methyl)carbamate (syn-4d)
The protected amine was obtained by following General procedure 2 using the THF-hydrazone Amines 3a-d synthesis. Compounds 3a-d were prepared following the General procedure 3.

General procedure 3
The protected amine was dissolved in a solution of HCl in MeOH (0.5 M) and heated at 40 °C for 4 hours. The reaction mixture was then evaporated to dryness. The residue was washed with Et2O and dried under high vacuum to give the ammonium salt.

(2S,3R,4R)-2-(Aminomethyl)tetrahydrofuran-3,4-diol hydrochloride (3b)
The amine hydrochloride salt was obtained by following the General procedure 3 using the Boc- The aldehyde 2a (300 mg, 2.00 mmol) was subjected to the General procedure 4 using Rh-TAm The aldehyde 2d (328 mg, 2.00 mmol) was subjected to the General procedure 4 using Rh-TAm supernatant was basified to pH 12 with NaOH and concentrated to remove excess IPA. The yellow residue was redissolved in water (50 mL, pH adjusted to 8.0), loaded on to a Dowex 50WX8 ionexchange resin, and washed several times with water (100 mL) to remove excess salt. The amine product was then eluted with 28% aq NH3 (60 mL) and the eluent was concentrated under reduced pressure. The off-white residue obtained was resuspended in 1 M HCl and concentrated to dryness to give the amine salt as a single isomer.