Furfurylamines from biomass: transaminase catalysed upgrading of furfurals

Amines derived from furfurals have diverse applications, including use in the synthesis of biologically active compounds and polymers. Here, transaminase enzymes have been used as a mild sustainable method for the amination of furfurals. A range of furfurals was readily accepted by several transaminases, and diff erent amine donors were successfully used to give furfurylamines in good yields. Green Chemistry


Introduction
Furfurals have attracted significant interest in recent years as renewable feedstocks for the production of biofuels and chemicals. 1In particular, furfural 1a and 5-hydroxymethylfurfural (5-HMF) 2a are valuable platform chemicals, prepared via the acid-catalysed dehydration of pentoses and hexoses obtained by the hydrolysis of cellulosic biomass waste including cornstalks, corncobs and rice waste. 2 Due to improved catalytic processes furfurals are becoming more available and their production has been integrated into a biorefinery context. 3Indeed, renewable 5-HMF is being manufactured commercially. 4As a consequence, several processes have been developed for the conversion of furfurals into a number of valuable chemicals and fuels such as furfuryl alcohols, THFs, furfurylamine 1b, 1,5-pentanediol and functionalised aromatics. 3,5Among these, the production of furfurylamines by the selective reductive amination of furfurals has received interest due to many potential applications, including as intermediates in the synthesis of phar-maceuticals such as antiseptic agents, antihypertensives, and diuretics (e.g.Furosemide). 6The 5-HMF 2a derived amine 2b also has potential as a curing agent in epoxy resins.Selective synthesis of these primary amines from carbonyl compounds is still challenging.Indeed, the use of traditional synthetic routes is not straightforward due to the sensitivity of the furan ring to reductive conditions and the tendency to form secondary or tertiary amine by-products. 7oreover, the waste generated from using such reducing agents has to be considered especially within the green chemistry agenda.
Transaminase (TAm) enzymes have been investigated in recent years for the transformation of pro-chiral ketones and aldehydes into the corresponding chiral secondary, or primary amines. 8These transferases can provide a sustainable high yielding, selective route to amines under mild aqueous conditions.The use of TAms with furfural analogues in the literature has surprisingly received very little attention with only two previous reports: a kinetic resolution of racemic 1-(2-furfuryl)ethylamine using commercially available TAms; 9 use of 5-HMF 2a in an enzyme cascade incorporating the Vibrio fluvialis TAm with alanine dehydrogenase to shift the TAm equilibrium. 10ere we describe the use of TAms for the amination of furfural derivatives 1a-9a and ketone 10a to access amines 1b-10b (Scheme 1).High conversions were observed with a range of substrates.The amination of selected compounds was then investigated for the production of value-added chemicals on a preparative scale.

Initial substrate screening
Ten furan-based substrates were screened including furfural 1a, 5-HMF 2a, halogenated furfurals 3a-6a, 5-methylfurfural 7a, 5-formyl-2-furancarboxylic acid 8a, 2,5-furandicarboxaldehyde 9a, and 2-acetylfuran 10a (Fig. 1).TAms selected for screening included the (S)-ω-TAm Chromobacterium violaceum DSM30191 (CV-TAm), which has been used with a range of cyclic and aromatic substrates and demonstrated tolerance towards the low cost amine donor isopropylamine (IPA) 11. [11][12][13][14] The (R)-selective TAms Arthrobacter sp.variant ArRMut11 15 and Mycobacterium vanbaalenii (Mv-TAm), 16 were also selected due to their complementary enantioselectivities compared to CV-TAm for use with the ketone 10a.][17][18][19] Initial screening for the conversion of aldehydes and ketone 1a-10a, was carried out using crude cell extract and our recently reported rapid and sensitive TAm colorimetric assay (Scheme 2). 20It uses an inexpensive amine donor 12 that forms the corresponding aldehyde shown when a TAm reaction occurs: the aldehyde generated can then react with the major amine present (12) to form an imine which tautomerises to give a red precipitate, indicating that the TAm reaction has occurred. 20The results of the colorimetric assay are shown in Fig. 2 against benzaldehyde (+) as a positive control.When using CV-TAm a strong coloration was observed with all aldehyde substrates.Notably, furfural 1a, 5-HMF 2a and acid 8a were readily converted showing a deep red coloration.In addition, aldehydes were accepted by both ArRMut11 and Mv-TAm while ketone 10a showed little coloration with all TAms perhaps due to imine formation with 12.
Notably, the acid 8a was accepted by CV-TAm in approximately 50% conversion, while very low conversions were achieved with ArRMut11.By comparison, 2,5-furan dicarboxaldehyde 9a performed well with all the selected enzymes.Due to complexities arising from the potential formation of monoamine or diamine products when using 9a, product yields were determined using the diamine 9b (see Scheme 3 for synthesis), which was formed in good yield with all the TAms (56-70%).Finally, 2-acetylfuran 10a was accepted by all three TAms, with Mv-TAm giving the highest conversions.Overall, following the initial screening hits from the colorimetric assay, the MBA-screen highlighted that the presence of more polar groups at C-5 reduced conversions for ArRmut11, but this had less effect with CV-TAm and Mv-TAm.

Amine donor selection and synthesis of 2b, 8b and 9b
Several methods are known to shift the TAm equilibrium towards the amine product, including the use of excess amine donor and enzyme coupled systems.One of the most operationally straightforward and cost-effective is the use of IPA 11 as the amine donor.Since IPA and the co-product acetone are both volatile, the scale-up and isolation of products is also more facile.Furfural 1a, 5-HMF 2a, acid 8a and dialdehyde 9a were taken forward for reaction optimisation using IPA 11 as the amine donor.The reaction with 2-acetylfuran 10a was also explored further to establish stereoselectivities.Since furfuryla-mines 2b, 8b, and 9b were not readily available commercially but required as analytical standards they were synthesised (Scheme 3).As the furan ring is sensitive to reductive conditions, and traditional reductive amination routes resulted in mixtures of di-and trisubstituted amines, 7 a multi-step route for the preparation of 9b and 8b was established through chlorination to 14 and 15 and azide formation followed by reduction with triphenylphosphine (PPh 3 ).Derivatisation of the amine with tert-butoxycarbonyl anhydride (Boc 2 O) was necessary to enable more straightforward product isolation and purification from side-products.The amine standards were then used as chemical standards to determine TAm reaction yields by HPLC.
Efforts to prepare 5-hydroxymethylfurfurylamine 2b starting from 5-HMF 2a following a similar strategy were unsuccessful.Moreover, attempts to reduce the carboxylic acid 8b or the corresponding azide with a range of reducing agents including LiAlH 4 and BH 3 -THF resulted in complex mixtures of products that could not be purified.Instead, an enzymatic reaction using CV-TAm and IPA as amine donor was the most efficient method of preparing 2b, which was isolated after scale-up (see below) and used as a standard.
It is worth noting that procedures reported to afford 2b are typically very low yielding (4% yield) or use unsustainable metal-based catalysts. 21,22This example, together with the generally low yields of the multi-step synthesis, emphasises problems with such traditional synthetic approaches and the benefits of a one step biocatalytic route to compounds such as the amino acid 8b.
Reaction product yields when using amine donors 11 and 13 are shown in Table 1 using typical reaction conditions previously utilised in such TAm reactions. 14For IPA 11 (10 equiv.)higher yields were generally observed than when using MBA 13 (5 equiv.),highlighting the benefits of using excess amine donor and possibly the slightly higher reaction temperature.Yields of up to 92% were obtained for furfurylamine 1b with CV-TAm, but again lower yields (34%) were observed when using ArRMut11.The same trend was observed using 2a, which was readily accepted by CV-TAm and Mv-TAm, but showed a lower product yield with ArRMut11.Notably, with CV-TAm the amino acid 8b was produced in 88% yield with IPA, compared to 47% with MBA, and again ArRMut11 gave 8b in very low yield.For the dialdehyde 9a, when using IPA, yields of the diamine 9b were slightly lower than for MBA.However, since a double transamination was required the use of twice as much enzyme was explored with IPA 11.Yields of the diamine increased up to 60% for CV-TAm (Table 1).It was not possible to determine the yield of the mono-aminated product since it was unstable during synthetic investigations, forming complex polymerised mixtures via imine formation: this may also account for why the TAm yields observed did not increase markedly when using twice as much enzyme with 9a.The product yields for 2-acetylfuran 10a with MBA were confirmed using a commercial sample of 10b and HPLC, again highlighting Mv-TAm (54%) as the most productive transaminase, giving (R)-10b in 78% ee (absolute stereochemistry based on Fig. 3 Screening results for substrates 1a-10a (5 mM) using 25 mM (S)-13 (CV-TAm) or 25 mM (R)-13 (ArRMut11 and Mv-TAm) as amine donors, 24 h, 30 °C.The product acetophenone was detected by HPLC analysis (at 254 nm) and used to determine % conversions.Background levels of acetophenone production were subtracted from the assay results in control reactions, and all reactions were performed in triplicate (standard deviations were less than 10%).Benzaldehyde (conversion of 68% with CV-TAm, 34% with ArRMut11, and 64% with Mv-TAm) was used as a positive control.*For 9a product formation was determined using a synthesised diamine standard 9b (see below). the reported stereoselectivity of this TAm).The reaction with CV-TAm was lower yielding so the ee was not investigated.

Preparative scale reaction
Furfurylamines are valuable biomass-derived products and direct syntheses were then investigated on a preparative scale using CV-TAm, due to its high yields using IPA 11 as the amine donor (Table 1), together with aldehydes 1a, 2a, and 8a.
The reactions were performed on a 20 mM scale (in 50 mL) for 24 h after which no starting materials remained.HPLC analysis established product yields of 83%, 58%, and 40% for 1b, 2b, and 8b respectively.The lower yields for 2b and 8b may have been due to some product inhibition at higher substrate concentrations.Furfurylamine 1b was not isolated as it is commercially available.As mentioned above, 2b was required as a product standard for analytical purposes in this work and was readily generated using this one-step enzymatic route from 5-HMF 2a.The product was isolated in 54% yield, highlighting the potential and benefits of this synthetic strategy compared to traditional chemical approaches.The preparative scale reaction using acid 8a was also of particular interest as amino acid 8b is a cyclic analogue of γ-aminobutyric acid (GABA) and has activity as an inhibitor of GABA aminotransferase, with K m values similar to GABA. 23Moreover, 8b belongs to the class of furanoid sugar amino acids which has been incorporated into novel anticancer peptides. 24The product 8b of the biotransformation reaction was successfully isolated as Boc-8b for ease of isolation purposes, and directly deprotected to give 8b in 31% overall isolated yield and high purity.

Conclusions
Amines derived from furfural have diverse applications, including the synthesis of furan containing polymers from the amine products of 5-HMF and 2,5-furandicarboxaldehyde, which are a renewable alternative to conventional polymers. 4Three TAms have been investigated for the transamination of a range of furan-based aldehydes and a ketone.The initial colorimetric assay highlighted that all the furfurals 1a-9a were readily accepted by the selected TAms using 2-(4-nitrophenyl)ethan-1amine hydrochloride 12 as the donor, and this was validated using an MBA screen.The use of IPA was then investigated as an alternative amine donor with 1a, 2a, 8a, and 9a.In most cases higher yields were obtained with some yields in excess of 90%, while 2-acetylfuran 10a was more readily converted to the corresponding amine when MBA was used as the amine donor.Procedures using IPA allowed reactions to be performed on a preparative scale in one-step and good yields.This approach uses a sustainable feedstock for the preparation of known and novel fine chemicals.The biocatalytic process for the production of furfurylamine and derivatives has significant potential in applications for manufacturing drug synthons and monomers for use in polymer production with high-performance 25 and biodegradable 26 properties.

General
All chemicals were obtained from commercial suppliers and used as received unless otherwise stated.Thin layer chromatography (TLC) analysis was performed on Merck Kieselgel precoated aluminium-backed silica gel plates and compounds visualised by exposure to UV light, potassium permanganate or ninhydrin stains.Flash column chromatography was carried out using silica gel ( particle size 40-60 μm).NMR: 1 H and 13 C NMR spectra were recorded at 298 K at the field indicated using Bruker spectrometers AMX400, Avance 500, and Bruker Avance III 600.Coupling constants ( J) are measured in Hertz (Hz) and multiplicities for 1 H NMR couplings are shown as s (singlet), d (doublet), and m (multiplet).Chemical shifts (in ppm) are given relative to tetramethylsilane and referenced to residual protonated solvent.Infrared spectra were recorded on a Perkin Elmer Spectrum 100 FTIR spectrometer.Mass spectrometry analyses were performed at the UCL Chemistry Mass Spectrometry Facility using a Finnigan MAT 900 XP mass spectrometer and the EPSRC UK National Mass Spectrometry  Facility at Swansea University using a Thermo scientific LTQ Orbitaltrap XL.Melting points were recorded on an Electrothermal IA9000 Series melting point apparatus and are uncorrected.

Analytical HPLC
Analyses of the reactions were performed using an Agilent 1260 Infinity HPLC with an Ace 5 C18 column (150 × 4.6 mm).Elution was carried out at 1 mL min −1 with a linear gradient of acetonitrile/H 2 O containing 0.1% TFA, with detection at 250 or 210 nm, injection volume of 10 µL and column temperature of 30 °C.

Transaminase expression and cell crude extract preparation
Selected TAm glycerol stocks of Chromobacterium violaceum DSM30191 11 (CV-TAm), Arthrobacter sp.variant ArRMut11 15 and Mycobacterium vanbaalenii (Mv-TAm), 16 from the UCL TAm library were used to inoculate 2TY broth (5 mL) containing kanamycin (50 μg mL −1 ) and incubated at 37 °C for 16-18 h.This pre-inoculum was then used to inoculate a larger culture (500 mL) containing the same antibiotic which was incubated at 37 °C for 3 h until an OD 600 of 0.5-0.7 was reached.Enzyme expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (1 mM), and the temperature was reduced to 30 °C. 14After 5 h cells were harvested by centrifugation (8000 rpm, 20 min) and the pellet suspended in potassium phosphate buffer (100 mM, pH 7.5) containing pyridoxal-5phosphate (PLP) (1 mM) and freeze dried.The freeze dried cells were used fresh or stored at −20 °C for up to six months.
To prepare crude cell extract, freeze dried cells (25 mg) were suspended in potassium phosphate buffer (1 mL, 100 mM, pH 7.5), lysed by sonication on ice (10 s on and 10 s off for 5 cycles) and used as a crude lysate (10% v/v).TAm concentrations were determined by SDS-PAGE densitometry: densitometry of samples electrophoresed on a NuPAGE 10% Bis-Tris gel (Novex) was used.The Coomassie stained gel was imaged using LabWork image software to calculate the TAm band density.A range of concentrations of commercial BSA was run in each SDS-PAGE gel (ESI Fig. 1 †), and used to calculate a standard curve based on integrated optical density for calibration of the enzyme concentration.Total protein was determined using a standard Bradford assay.TAm concentrations in the crude lysates were determined as 5.7 mg mL −1 CV-TAm, 4.0 mg mL −1 ArRMut11 and 2.5 mg mL −1 Mv-TAm.

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 12 (25 mM) as amine donor, amine acceptor (10 mM), PLP (0.2 mM) and potassium phosphate buffer (100 mM, pH 7.5).The reaction was started by the addition of E. coli crude cell extract (20 μL) containing the overexpressed TAm and the reaction was incubated at 30 °C and 500 rpm for 24 h.Two negative controls were also performed, one without amine acceptor and another without enzyme.An orange/red color-ation indicated that the TAms were active towards the selected furfurals (Fig. 2).
Sodium azide (130 mg, 2.00 mmol) was added to 14 (83 mg, 0.50 mmol) in dry DMF (8 mL) under argon, and the reaction was heated at 65 °C for 16 h.The DMF was removed under reduced pressure and the residue was dissolved in methanol (8 mL).PPh 3 (525 mg, 2.00 mmol) was added and the reaction was stirred at room temperature.After 2 h Boc 2 O (436 mg, 2.00 mmol) was added and the reaction was stirred for 16 h.The methanol was removed under reduced pressure, sat.NaHCO 3 added (30 mL) and the product was extracted with ethyl acetate (3 × 30 mL), dried (Na 2 SO 4 ) and the solvent removed under reduced pressure.2,5-Bis(Boc-aminomethyl) furan was purified by column chromatography (ethyl acetate/ petroleum ether (40-60), 1 : 4) to give a brown solid (63 mg, 39%).It was then directly deprotected by stirring in a 1 : 1 mixture of methanol and 4 M HCl (6 mL) for 1 h, and the solvent removed under reduced pressure to give 9b•2HCl 31

Furfurylamine (1b)
Furfural 1a (83 µL, 1.0 mmol) was subjected to the preparative scale reaction conditions, and after removal of the denatured protein by centrifugation (4000 rpm, 4 °C, 20 min) was analysed by HPLC, to give the product in 83% yield.

a
Reactions were performed in triplicate and conversions were determined using HPLC against product standards.b Assays were performed on a 200 µL total reaction volume containing MBA 13 (25 mM) or IPA 11 (100 mM), PLP (1 mM), potassium phosphate buffer (100 mM, pH 7.5 for MBA and pH 8 for IPA), amine acceptor (5 mM when MBA was used or 10 mM when IPA was used) and crude cell lysate (20 μL) at 30 °C with MBA and 35 °C with IPA, for 24 h.(S)-13 or (R)-13 was used depending on enzyme selectivity.c Yields obtained using double the amount of TAm enzyme.d IPA was used as an amine donor except for compound 10b; ee determined using chiral HPLC; n.d.not determined.