Biocatalytic conversion of 5-hydroxymethylfurfural:
synthesis of 2,5-bis(hydroxymethyl)furan and 5-(hydroxymethyl)furfurylamine Antonella Petri,*a Giulia Masiaa and Oreste Piccolo*b
aDipartimento di Chimica e Chimica Industriale, Università di Pisa,
Via Giuseppe Moruzzi 13, 56124 Pisa, Italy
bStudio di Consulenza Scientifica (SCSOP), Via Bornò 5, 23896 Sirtori (LC), Italy
*Corresponding authors. Tel: +39 050 2219279 (A.P.). E-mail addresses: antonella.petri@unipi.it (A. Petri),
orestepiccolo@tin.it (O. Piccolo).
1. Introduction
The growing interest in the preparation of non-oil chemical compounds has led to the development of carbohydrate transformations in valuable chemicals. A considerable range of building blocks derived from renewable resources are already available [1,2]. This trend is highlighted by the production of several di-substituted furan derivatives as useful reagents for the preparation of polymeric materials [3-4]. Furthermore, some pharmacologically active compounds having a substituted furan scaffold present a broad spectrum of biological activity [5-6].
5-hydroxymethyl furfural (HMF) 1, obtained from lignocellulosic biomass, is an important intermediate for the synthesis of di-substituted furan derivatives [7,8]. For example, selective reduction of HMF affords 2,5-hydroxymethyl)furan (BHMF) 2, a versatile building block for the synthesis of relevant target molecules. To date, BHMF is synthesized mainly by chemical reduction of HMF by using different approaches [4, 9-17].
Biocatalysis represents an attractive and promising alternative to the above methodologies offering several advantages such as mild and environment-friendly reaction conditions and high selectivity [18]. However, the use of isolated oxido-reductases may be expensive either for the preparation of the biocatalyst or because a cofactor is needed that must be regenerated in situ [18]. Biocatalytic reduction by microbial resting cells might be cheaper, but HMF is a well-known potent inhibitor to microorganisms [19]. Very recently, reduction of HMF by using resting cells of a new HMF-tolerant yeast strain has been reported [20]. In a greener context, the possibility of using parts of fresh plants as alternative biocatalysts for reduction reactions is well documented [21-25]. In this approach, the involved reagents (oxido-reductase, cofactor and its regeneration system) are all
located in the plant cell. Therefore, the process might be more economically feasible, even if productivity, seasonal variability and supply of plants may be critical issues.
5-(hydroxymethyl)furfurylamine (HMFA) 3 is indeed another useful intermediate not only for the synthesis of pharmaceuticals but also as a curing agent in epoxy resins [26-28]. Due to the sensitivity of the furan ring and the tendency to form mixtures of by-products, synthesis of this compound by using traditional synthetic routes is not straightforward. A method to obtain 3 by reductive amination of HMF with supported ruthenium nanoparticles was recently described [29].Transaminase enzymes (TAs) have found wide applications in the synthesis of amines [30-32]; however, very little attention has been paid to the use of these enzymes with furfural analogues [26, 33-34].
Herein, we targeted sustainable synthetic routes for the synthesis of two industrial relevant building blocks from HMF 1 (Scheme 1). BHMF 2 and HMFA 3 were obtained by green chemistry methodologies using, respectively, plant tissues and immobilized transaminase enzymes as the biocatalysts.
2. Experimental
2.1. General
HMF was purchased from ENCH INDUSTRY CO., LTD. (China), assay 99.5%. Other chemicals were purchased from Sigma-Aldrich, Fluka or Merck and used as received unless otherwise stated. The TA-IMB enzymes were purchased from Purolite; TA enzymes were produced by Codexis and immobilized on a Purolite ECR resin. The enzyme loading is 100 mg protein per gram of wet resin.
1H and 13C NMR spectra were recorded at room temperature at the field indicated on a Bruker
Avance II spectrometer. Multiplicities for 1H NMR couplings are shown as s (singlet), d (doublet),
m (multiplet). Chemical shifts (in ppm) are referenced to residual protonated solvent. Thin layer chromatography (TLC) analysis was performed on silica gel 60 F254 plates. Condition were as follow: eluent EtOAc and compounds visualised by exposure to UV light and I2 vapours. The
transamination reactions were performed in a Eppendorf ThermoMixer C. Reactions were performed on a IKA-VIBRAX-VXR or IKA MS 3 Digital. For freeze-drying of the fresh plant material, a laboratory freeze dryer 5 Pascal Lio 5P was used.
The fresh plant materials were purchased from a local market and stored at 4°C. The selected vegetable (Brassica oleracea variety Italica) was used fresh (containing 90 % water by weight) or in lyophilized form. For freeze-drying, the fresh plant material (10 g) was washed with deionized water (20 ml) and disinfected with a diluted (2 % v/v, 20 ml) commercial sodium hypochlorite solution. Then it was washed with deionized water (2 x 20 ml), shredded and suspended in 10 ml of H2O with 10%, 5% or 2 % v/v DMSO. This preparation was frozen at -20 °C or in liquid nitrogen.
After freezing, the samples were dehydrated in a laboratory freeze dryer for 24h. The lyophilized plant material was obtained with a yield of 9-10 % and it was used without any further treatment.
2.3. Chromatographic analysis
Analyses of the reactions were performed a Jasco HPLC system with a Targa C18 column (150 x 4.6 mm). Elution was carried out at 1 ml/min with detection at 220 nm and column temperature of 25°C. The eluents were H2O:CH3CN (90:10, v/v) for bioreduction reactions and H2O:CH3CN:TFA
(97:3:0.1%, v/v) for transamination reactions. Authentic standards were analysed before analysis of the reaction mixtures. Calibration lines are reported as supplementary material.
2.4. General procedure for bioreduction of 1 with fresh plant material
The fresh plant material (100 g) was washed with deionized water, disinfected with a diluted (2 %) commercial sodium hypochlorite solution and washed again with deionized water. Then it was cut into small pieces with a sterilized cutter and suspended in water (250 ml). To these suspension, a solution of 1 (250 mg, 1g/L) in phosphate buffer 0.1M pH7 or in H2O was added. The reaction
mixture was stirred (200 rpm) at room temperature and monitored by TLC and HPLC analysis. At the end of the reaction, the suspension was filtered, treated with activated charcoal and then centrifuged (50 x 100 rpm) for 10 minutes. The supernatant was extracted with EtOAc (110 ml) in a liquid-liquid continuous extractor. The combined organic extracts were dried over Na2SO4 and then
evaporated to yield 2. 1H NMR and 13C NMR were in agreement with those reported in literature
[5,11]. 1H NMR (400 MHz, H
2O-d2, ) 6.27 (2H, s, 2 CH), 4.47 (4H, s, 2 CH2OH); 13C NMR (100
MHz; H2O-d2, ) 153.63, 109.7, 55.83.
2.5. General procedure for bioreduction of 1 with lyophilized plant material
To a previously prepared suspension of lyophilized plant material (0,1g), a solution of 1 (5mg, 1g/L) in H2O was added The reaction mixture was stirred at room temperature and 300 rpm. The
reaction was monitored by TLC and HPLC analysis. At the end of the reaction the suspension was filtered and treated as described for bioreduction with fresh plant material to yield 2.
2.6. General procedure for transaminationof 1 with immobilized enzymes
To 5 ml of phosphate buffer 0.1 M pH 7.5 containing IPA 1.1 M, TA-IMB enzyme (200 mg) and PLP (1.4 mM) were added. The mixture was stirred at 35°C and 600 rpm for 5 minutes and then a preheated solution of 1 (50 mg, 0.4 mmol, 70 mM) in phosphate buffer 0.1 M pH 7.5 was added. The reaction was stirred (600 rpm) at 35°C for 10 minutes and then the temperature was changed to 50°C. The reaction was monitored by TLC and HPLC analysis. At the end of the reaction the enzyme was filtered under vacuum and washed with phosphate buffer 0.1 M pH 7.5 (3 x 2 ml). The recovered enzyme was suspended in buffer and stored at 4°C. KOH 5M was then added to the reaction mixture to obtain pH 10. The solution was diluted to 10 ml with H2O, saturated with NaCl
and extracted with EtOAc (110 ml) in a liquid-liquid continuous extractor. The combined organic extracts were dried over Na2SO4 and then evaporated to yield 3 as a yellow oil. 1H NMR and 13C
NMR were in agreement with those reported in literature [35]. 1H NMR (400 MHz; MeOH-d 4, )
6.23 (1H, d, 4-H), 6.18 (1H, d, 3-H), 4.49 (2H, s, CH2OH), 3.76 (2H, s, CH2NH2); 13C NMR (100
MHz; MeOH-d4, ) 156.2, 155.3, 109.2, 107.7, 57.4, 39.2.
3. Results and discussion
3.1.Bioreduction of HMF 1 with plant tissues
In order to evaluate the catalytic potential of different plants as reducing agents, bioreduction of 1 by using various types of fresh vegetables was investigated (Table 1). Complete conversion of the substrate was obtained in all entries at room temperature in 2-4 days. These results showed that the most efficient plants for this reaction are carrot (Daucus carota) and broccoli (Brassica oleracea) . In view of the valorisation of less edible parts of vegetables (possible waste), we focused our attention on the use of broccoli stems.
Table 1
Bioreduction of HMF 1 by using plant tissues
Entrya Plant tissue Time (h) Conv.(%) b
1 Brassica oleracea var. botrytis (leaves) 96 >99
2 Brassica oleracea var. botrytis (flowers) 96 >99
3 Daucus carota 48 >99
4 Brassica oleracea var. italica (stems) 48 >99
5 Brassica oleracea var. italica (florets) 24 >99
a Reaction conditions: substrate = 100 mg, 2 g/L, plant tissue = 20 g, phosphate buffer = 0.1 M pH
7, 20°C, 200 rpm.
b Conversion determined by HPLC.
3.2.Optimization of bioreduction of HMF 1
Initially the use of buffer-free water as solvent was evaluated (Table 2). The reaction was equally efficient, . as already reported for bioreduction of other compounds with plant tissues [20-21]. The use of double quantity of substrate yielded similar results (entries 4,5).
Table 2
Bioreduction of HMF 1 by using stems of B. oleracea
Entrya Solvent Time (h) Conv. [Yield] (%)b,c
1 Buffer 43 >99 [52]
2 H2O 42 >99 [(71]
3 H2O 48 >99 [91]d
4 Buffer 48 >99 [76]e
5 H2O 48 >99 [70]e
aReaction conditions: substrate = 250 mg, 1 g/L, plant tissue = 100 g, phosphate buffer =0.1 M pH
7 or deionized water, 20°C, 200 rpm.
b Conversion and yield determined by HPLC. c Plant from a local market.
d Plant freshly picked. e Substrate = 500 mg.
In an attempt to avoid potential irreproducibility of bioreduction reaction due to variations in origin, age and seasonality of the vegetable, we decided to use B. oleracea stems in lyophilised form (Table 3).
In a preliminary test (entry 1) no product formation was observed even after 144 h, This negative result, probably due to degradation of the biocatalyst during the freeze-drying, was the same obtained with commercial frozen B. oleracea florets.
Bioreduction of HMF 1 by using lyophilised stems of B. oleracea
Entrya Lyophilisation methodb Time (h) Conv.[ Yield] (%)c
1 A >144 0 2 B 144 97[79] 3 C 75 99 [99] 4 D >144 0 5 E 55 99 [95] 6 F 48 99 [87] 7 F 48 >99 [93]d 8 G 48 >99 [91]d
a Reaction conditions: 5 mL total reaction volume, 0.1 g lyophilised material and 1 g/L
of substrate.
b Lyophilisation method A: without cryoprotectant; B or C or D: with 10% or 5% or 2
% DMSO respectively; E: with 10% DMSO, frozen at -20°C; F: with 10% DMSO, frozen in liquid N2; G: with 5% DMSO, frozen in liquid N2.
c Conversion and yield determined by HPLC. d T=30 °C.
Therefore, the use of DMSO as a cryoprotectant was investigated. By using 5-10% DMSO, the product 2 was obtained with high conversion and yield. No product formation was observed in entry 4, in which the biocatalyst was obtained by using 2% of DMSO. It has to be noted that performing the reaction at 30°C had no relevant effect on the reaction time (entries 7 and 8). When bioreduction was performed on 0.1 g of 1, pure 2 was obtained after 72 h with 95% yield as determined by HPLC and recovered with an isolated yield of 60%.
3.3.Transaminatyion of HMF 1 with immobilized transaminase enzymes
Transaminase enzymes are important biocatalysts for the selective and efficient transformation of ketones and aldehydes into the corresponding amines. Immobilized TAs may offer several advantages over the free form such as: i) increased stability to temperature; ii) recovery and reuse of the catalyst with a potential decreased cost; iii) simpler and easier work-up and purification of the product; iv) possible use of isopropylamine (IPA) as an amine donor . Owing to its ability to shift the reaction equilibrium to the side of the product and the ease with which byproducts can be removed, the use of IPA represents a significant advance in improvement of the method [31]. Preliminary screening was carried out by using commercially available immobilized TAs and IPA as amine donor at 50°C (Table 4).
Table 4.
Entrya Enzyme Conv.[Yield](%)b 1 ATA-254-IMB >99 [92] 2 ATA-260-IMB >99 [95] 3 ATA-256-IMB 99 [93] 4 ATA-303-IMB 99 [91] 5 ATA-P1-G05 68 [37] 6 ATA-234-IMB 63 [35] 7 ATA-025-IMB 40 [26]
a Reaction conditions: substrate (50 mg, 0.4 mmol, 9 g/L), TA-IMB enzyme
200 mg, phosphate buffer 0.1 M, pH 7.5 containing IPA 1.1 and PLP 1.4 mM, 600 rpm, 50° C, 24 hours.
b Conversion and yield determined by HPLC.
The product was obtained with high conversion and yield in 24 hours with four enzymes (entries1-4). An important issue is the recovery of 3 from the reaction mixture. Very recently the preparation of HMFA has been reported [34], in which by using crude cell lysates containing overexpressed TAs, 3 was obtained with 58% yield and recovered with an isolated yield of 54%. However the described isolation procedure was not successful in our hands and we had to find alternative conditions (see experimental part).
3.4.Re-use of enzyme in transamination of HMF 1
Enzyme recycling was carried out with ATA-260-IMB under the same experimental conditions used in the screening. At the end of each reaction, the enzyme was washed with phosphate buffer, suspended and re-used for five reaction cycles, obtaining in all entries, the product 3 with high conversion and yield (Figure 1).
4. Conclusions
The synthesis of two di-substituted furan derivatives was obtained by biocatalytic conversion of HMF. Bioreduction by using lyophilised stems of Brassica oleracea was optimized overcoming the typical problems found with plant tissue biocatalysis Several immobilized transaminase enzymes were investigated for the transamination reaction and enzyme recycling was performed. The results showed the potential of the described protocols for the valorisation of HMF allowing the preparation of valuable derivatives in a more sustainable and green chemistry perspective
Conflicts of interest
The authors declare no conflict of interest. Acknowledgements
This study was financially supported by research funding from University of Pisa, Italy (Fondi di Ateneo; PRA-2017-25) and by Studio di Consulenza Scientifica (SCSOP), Sirtori (LC), Italy. The authors gratefully acknowledge Prof. Federica Chiellini and the BIOLab Research Group (University of Pisa) for their support in lyophilisation experiments.
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Scheme 1. Biotransformation of HMF 1 with (i) Plant tissue; (ii) immobilized TAs, PLP and amine donor
