PROCESSES FOR THE FORMATION OF FURANDICARBOXYLIC ACID (FDCA) VIA A MULTISTEP BIOCATALYTIC OXIDATION REACTION OF 5-HYDROXYMETHYLFURFURAL (HMF)
20180187224 ยท 2018-07-05
Inventors
- Paul Law (Southampton, GB)
- Paul Mines (Southampton, GB)
- Andrew Carnell (Liverpool, GB)
- Shane Mckenna (Liverpool, GB)
Cpc classification
C12P17/04
CHEMISTRY; METALLURGY
C08G63/199
CHEMISTRY; METALLURGY
C12Y111/01007
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to processes for the formation of furandicarboxylic acid (FDCA), in particular 2,5-furandicarboxylic acid (2,5-FDCA), and mono- and diester derivatives thereof, via a multistep biocatalytic oxidation reaction of 5-hydroxymethylfurfural (HMF) using, for example, an enzyme selected from the group consisting of xanthine oxidoreductase (XOR), galactose oxidase variant M.sub.3-5, aldehyde dehydrogenase, and/or ketoreductase. The invention also relates to copolymers that comprise the furandicarboxylic acid monomers and derivatives thereof, processes for the formation of the copolymers and uses for the copolymers.
Claims
1-47. (canceled)
48. A process for the formation of furandicarboxylic acid (FDCA) from hydroxymethylfurfural (HMF), said process comprising the steps of (i) providing hydroxymethylfurfural, and (ii) (a) adding xanthine oxidoreductase (XOR) and/or galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5), or (b) adding galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5), periplasmic aldehyde oxidase (PaoABC), and horseradish peroxidase (HRP) to the hydroxymethylfurfural, or (c) adding galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5), periplasmic aldehyde oxidase (PaoABC), and a metal complex to the hydroxymethylfurfural, or (d) adding galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5), aldehyde dehydrogenase (ALD), nicotinamide oxidase (NOX) and either nicotinamide adenine dinucleotide phosphate (NADP.sup.+) or nicotinamide adenine dinucleotide (NAD.sup.+) to the hydroxymethylfurfural provided in step (i), or (e) adding aldehyde dehydrogenase (ALD), nicotinamide oxidase (NOX) and either nicotinamide adenine dinucleotide phosphate (NADP.sup.+) or nicotinamide adenine dinucleotide (NAD.sup.+) to the hydroxymethylfurfural provided in step (i), or (f) adding ketoreductase (KRED), and either nicotinamide adenine dinucleotide phosphate (NADP.sup.+) or nicotinamide adenine dinucleotide (NAD.sup.+).
49. The process of claim 48, wherein, in step (ii)(a), the process comprises the addition of xanthine oxidoreductase (XOR) and galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5).
50. The process as claimed in claim 48, wherein, in step (ii)(a), the xanthine oxidoreductase is selected from the group consisting of E. coli XDH, Rhodococcus capsulatus xanthine dehydrogenase (XDH) single variant E232V, and double mutant XDH E232 V/R310, and periplasmic aldehyde oxidase (PaoABC).
51. The process as claimed in claim 48, wherein, in steps (i) and (ii), the process comprises the steps of (i) converting hydroxymethylfurfural (HMF) into formylfurancarboxylic acid (FFCA); and (ii) converting formylfurancarboxylic acid into furandicarboxylic acid (FDCA), wherein steps (i) and (ii) are carried out in the presence of xanthine oxidoreductase (XOR) and galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5).
52. The process as claimed in claim 48, wherein, in step (i) and (ii), the process comprises the steps of (i) providing hydroxymethylfurfural; (ii) adding galactose oxidase variant M.sub.3-5 (GOase M.sub.3-5) to the hydroxymethylfurfural provided in step (i) to convert the hydroxymethylfurfural to diformyl furan (DFF); then (iii) adding periplasmic aldehyde oxidase (PaoABC) to the diformyl furan in step (ii) to convert the diformyl furan to furandicarboxylic acid via formylfurancarboxylic acid (FFCA).
53. A process for the formation of furandicarboxylic acid (FDCA) from diformyl furan (DFF) comprising the steps of (i) providing diformyl furan, and (ii) (a) adding aldehyde dehydrogenase (ALD), nicotinamide oxidase (NOX) and either nicotinamide adenine dinucleotide phosphate (NADP.sup.+) or nicotinamide adenine dinucleotide (NAD.sup.+) to the diformyl furan provided in step (i), or (b) adding periplasmic aldehyde oxidase (PaoABC), catalase and H.sub.2O.sub.2, or (c) adding immobilised periplasmic aldehyde oxidase (PaoABC).
54. A process for the formation of formylfurancarboxylic acid (FFCA) from hydroxymethylfurfural (HMF), said process comprising the steps of (i) providing hydroxymethylfurfural; and (ii) adding ketoreductase (KRED), and either nicotinamide adenine dinucleotide phosphate (NADP.sup.+) or nicotinamide adenine dinucleotide (NAD.sup.+).
55. The process as claimed in claim 54, wherein the process comprises adding nicotinamide oxidase (NOX) in step (ii).
56. The process as claimed in claim 54, wherein the process comprises the step of obtaining the hydroxymethylfurfural from glucose and/or fructose.
57. The process as claimed in claim 56, wherein the process comprises the step of obtaining the glucose and/or fructose from lignocellulose.
58. A process for the formation of a mono- or diester of furandicarboxylic acid from furandicarboxylic acid, comprising the step of (i) providing furandicarboxylic acid; and (ii) adding an alcohol and a catalyst, wherein the furandicarboxylic acid is obtained by a process as defined in claim 48.
59. The process as claimed in claim 58, wherein the mono- or diester of furandicarboxylic acid is selected from the group consisting of ##STR00037## and a combination thereof.
60. The process as claimed in claim 58, wherein the catalyst is an organic acid or inorganic acid.
61. The process as claimed in claim 58, wherein the catalyst is sulphuric acid.
62. A process for the formation of a copolymer comprising the copolyester of (I) (a) at least one furandicarboxylic acid (FDCA) or a mono- or diester of furandicarboxylic acid, (b) at least one diol, wherein the process comprises reacting together components (a) and (b) (c), and wherein the furandicarboxylic acid is obtained by a process as defined in claim 48; or (II) (a) at least one mono- or diester of furandicarboxylic acid, (b) at least one diol, and (c) at least one aliphatic dicarboxylic acid or a mono- or diester derivative thereof, wherein the process comprises reacting together components (a), (b) and (c), wherein the aliphatic dicarboxylic acid or a mono- or diester derivative thereof is selected from the group consisting of adipic acid, adipic acid monomethyl ester, adipic acid dimethyl ester, adipic acid monoethyl ester, adipic acid diethyl ester, succinic acid, succinic acid monomethyl ester, succinic acid dimethyl ester, succinic acid monoethyl ester, succinic acid diethyl ester, and a combination thereof; or (III) (a) at least one mono- or diester of furandicarboxylic acid, (b) at least one diol, and (c) at least one aliphatic dicarboxylic acid or a mono- or diester derivative thereof, wherein the process comprises reacting together components (a), (b) and (c), and wherein the mono- or diester of furandicarboxylic acid is selected from the group consisting of ##STR00038## and a combination thereof.
63. The process as claimed in claim 62, wherein the copolymer comprises the copolyester of (a) at least one furandicarboxylic acid (FDCA) or a mono- or diester of furandicarboxylic acid; (b) at least one diol; and (c) at least one aliphatic dicarboxylic acid or a mono- or diester derivative thereof.
64. The process as claimed in claim 62(I), wherein the furandicarboxylic acid or a mono- or diester of furandicarboxylic acid is selected from the group consisting of ##STR00039## and a combination thereof.
65. The process as claimed in claim 62, wherein the aliphatic dicarboxylic acid or a mono- or diester derivative thereof is ##STR00040## wherein R.sup.3 is a straight, branched or cyclic, C.sub.1 saturated or C.sub.2 to C.sub.10 saturated or unsaturated alkylene, and wherein each R.sup.4 independently represents H or a straight, branched or cyclic, C.sub.1 to C.sub.6 alkyl group.
66. The process as claimed in claim 62, wherein the aliphatic dicarboxylic acid or a mono- or diester derivative thereof is selected from the group consisting of adipic acid, adipic acid monomethyl ester, adipic acid dimethyl ester, adipic acid monoethyl ester, adipic acid diethyl ester, succinic acid, succinic acid monomethyl ester, succinic acid dimethyl ester, succinic acid monoethyl ester, succinic acid diethyl ester, and a combination thereof.
67. The process as claimed in claim 62, wherein the diol is ##STR00041## wherein R.sup.2 is a straight, branched or cyclic C.sub.2 to C.sub.10 alkylene.
68. The process as claimed in claim 62, wherein the diol is selected from the group consisting of 1,2-ethanediol, 1,4-butanediol, and a combination thereof.
69. The process as claimed in claim 62, wherein the copolymer comprises the copolyester of (1) (A) furandicarboxylic acid diethyl ester; (B) 1,4-butanediol; and (C) adipic acid dimethyl ester or diethyl ester; or (2) (A) furandicarboxylic acid dimethyl ester or diethyl ester; (B) 1,4-butanediol; and (C) adipic acid dimethyl ester or diethyl ester; or (3) (A) furandicarboxylic acid (FDCA) or a mono- or diester of furandicarboxylic acid; and (B) 1,2-ethanediol, 1,4-butanediol, or a combination thereof; or (4) (a) from 1 to 98 mol % of at least one furandicarboxylic acid or a mono- or diester of furandicarboxylic acid; (b) from 1 to 98 mol % of at least one diol; and (c) when present, from 1 to 98 mol % of at least one aliphatic dicarboxylic acid or a mono- or diester derivative thereof.
70. The process as claimed in claim 48, wherein step (ii)(f) comprises adding nicotinamide oxidase (NOX).
71. The process as claimed in claim 48, wherein the process comprises the step of obtaining the hydroxymethylfurfural from glucose and/or fructose.
72. A process for the formation of a mono- or diester of furandicarboxylic acid from furandicarboxylic acid, comprising the step of (i) providing furandicarboxylic acid; and (ii) adding an alcohol and a catalyst, wherein the furandicarboxylic acid is obtained by a process as defined in claim 53.
73. The process as claimed in claim 58, wherein the mono- or diester of furandicarboxylic acid is selected from the group consisting of ##STR00042## and a combination thereof.
74. The process as claimed in claim 58, wherein the catalyst is an organic acid or inorganic acid.
75. The process as claimed in claim 58, wherein the catalyst is sulphuric acid.
76. The process as claimed in claim 62(II), wherein the furandicarboxylic acid or a mono- or diester of furandicarboxylic acid is selected from the group consisting of ##STR00043## and a combination thereof.
77. The process as claimed in claim 62, wherein the diol is a heteroaromatic diol or aromatic diol.
Description
DESCRIPTION OF THE FIGURES
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[0303] The following examples are merely illustrative examples of the invention described herein, and are not intended to be limiting upon the scope of the invention.
[0304] Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or common general knowledge. All references disclosed herein are to be considered to be incorporated herein by reference.
EXAMPLES
[0305] Aldehyde dehydrogenase 3 (ALD-003) is a preferred aldehyde dehydrogenase, and is available from Prozomix Limited, Station Court, Haltwhistle, Northumberland, NE49 9HN, UK (catalogue name: Pro-ALDH (003)).
[0306] Ketoreductase 20 (KRED-020), KRED-089, KRED-141, KRED-143, KRED-163, and KRED-190 are preferred ketoreductases. KRED-089 is a preferred ketoreductase. These KREDs are available from Prozomix Limited, Station Court, Haltwhistle, Northumberland, NE49 9HN, UK, as an Aldo-Keto Reductase Panel (product name kREDy-to-go (AKR/ADH; kit of 96 enzymes); catalogue no. PRO-AKRP(MTP)).
Example 1
[0307] The following is an example of one of ways in which 2,5-furandicarboxylic acids (2,5-FDCA) may be formed from 5-hydroxymethylfurfural (HMF).
Synthesis of 2,5-furandicarboxylic acid (2,5-FDCA)
[0308] ##STR00029##
General Experimental Information and Materials
[0309] The E. coli TP1000 mutant strain used for PaoABC expression is a derivative of MC4100 with a kanamycin cassette inserted in the mobAB gene region. E. coli xanthine dehydrogenase and catalase were sourced from Sigma-Aldrich. Starting materials were purchased from Alfa Aesar and Sigma-Aldrich and used as received. HPLC analysis was performed on an Agilent 1200 system equipped with a G1379A degasser, G1312A binary pump, a G1329 autosampler unit, a G1315B diode array detector and a G1316A temperature controlled column compartment. The columns used were Thermofisher Hypurity C18 (5 m particle size, 4.6 mm diameter250 mm), Thermofisher ODS Hypersil C18 (5 m particle size, 4.6 mm diameter250 mm) and Bio-Rad Aminex HPX-87H, 300 mm7.8 mm pre-packed column. GC analysis was performed on an Agilent 7890A chromatograph using an Alltech SE-30, 30.0 m320 m0.25 m column. Conditions are indicated separately for each compound. .sup.1H NMR spectra were recorded on a Bruker Avance 400 or 500 without additional internal standard.
Preparation of Biocatalysts
[0310] Galactose Oxidase Variant M.sub.3-5 (GOase M.sub.3-5)
[0311] GOase mutant M.sub.3-5 (Escalettes; Turner J. ChemBioChem 2008, 9, 857-860) was transformed into E. coli BL21 Star (DE3) cells (Invitrogen) according to manufacturer's specifications. A single colony was picked from an overnight LB plate containing 1 L of kanamycin of a 30 mg/mL stock solution per mL of agar and used to inoculate 5 mL LB medium supplemented with 5 L kanamycin and grown overnight at 37 C. and 250 rpm. 500 L of the overnight culture was used to inoculate 250 mL of an autoinduction medium (8ZY-4LAC) as described by Deacon and McPherson (Deacon; McPherson J. ChemBioChem. 2011, 12, 593-601) and supplemented with 250 L of kanamycin in a 2-L-baffled Erlenmeyer flask. The cells were grown at 26 C. and 250 rpm for 60 hour. Cells were harvested by centrifugation at 6,000 rpm and 40 C. for 20 min and subsequently prepared for protein purification.
Purification of GOase M.SUB.3-5
[0312] The cell pellet from a 250-mL-culture was resuspended in 30 mL lysis buffer containing 50 mM piperazine-N,N-bis(2-ethanesulfonic acid) (PIPES), 25% sucrose (w/v), 1 mg mL.sup.1 lysozyme, 5 mM MnCl.sub.2 and 1% Triton X-100 (v/v). The suspension was gently shaken at 4 C. for 20 min. Afterwards, cells were mechanically disrupted via ultrasonication (30 sec. on, 30 sec. off; 20 cycles) followed by ultracentrifugation (20,000g, 30 min, 4 C.). The cleared crude extract was transferred into a flexible tubing (30 kDa cut-off), dialysed into buffer C (50 mM NaPi buffer, 300 mM NaCl, pH 8.0) for 12 hours at 4 C. and subsequently passed through a syringe filter with a 0.22 m pore size. Protein purification was accomplished with a peristaltic tubing pump (Thermo Scientific) equipped with a 5-mL-Strep-Tag-II column (GE Healthcare) pre-equilibrated with buffer C. After loading with crude extract, the column was washed with 5 column volumes of buffer C followed by protein elution with 70 mL of buffer D (50 mM NaPi buffer, 300 mM NaCl, 5 mM desthiobiotin, pH 8.0).
[0313] For copper-loading, GOase M.sub.m-containing fractions were pooled and subsequently transferred into flexible dialysis tubing (30 kDa cut-off) and dialysed twice for 12 hours into buffer E (50 mM NaPi buffer saturated with CuSO.sub.4, pH 7.4) at 4 C. Removal of excess CuSO.sub.4 was attained by two cycles of dialysis into buffer E (without CuSO.sub.4) for 12 hours at 4 C. and protein samples concentrated to approximately 3 mg/mL using a Sartorius Vivaspin 6 spin column (30 kDa mass cut-off). The protein samples were aliquoted and the aliquots were frozen in liquid nitrogen prior to storage at 80 C.
E. coli Perisplasmic Aldehyde Oxidase (PaoABC)
[0314] For PaoABC expression (Neumann et al. FEBS Journal 2009, 276, 2762-2774), the plasmid pMN100 derived from pTrcHisA (Invitrogen), containing the PaoABC genes with a His6 tag fused to the N-terminus of PaoA, was used. For heterologous expression in E. coli, pMN100 was transformed into E. coli TP1000 cells, containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. One litre of LB supplemented with 1 mM sodium molybdate and 10 M isopropyl thio--D-galactoside was inoculated with 2 mL of an overnight culture and incubated for 24 hours at 22 C. and 100 rpm. The cells were harvested by centrifugation at 4,000g for 15 min.
Purification of PaoABC
[0315] The cell pellet was resuspended in 8 volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, 10 mM imidazole and cell lysis was achieved by sonication (MSE Soniprep) with cooling on ice (20 bursts of 20 s on/off at 14 u). After addition of DNase I, the lysate was incubated for 30 min. After centrifugation at 17,000g for 25 min the supernatant was filtered through 0.45 and 0.2 M membranes before loading onto Ni.sub.2-nitrilotriacetic agarose (HiTrap 1 mL column (GE Healthcare)). The column was washed with 2 column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with 10 column volumes of the same buffer with 20 mM imidazole. His-tagged PaoABC was eluted with 20 mL of 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing PaoABC were buffer exchanged into 50 mM Tris, 1 mM EDTA, pH 7.5. The yield of protein was about 13 mg/L of E. coli culture.
Xanthine Dehydrogenase Variants E232V and E232VR310 (XDH E232V, XDH E232VR310)
[0316] For expression of XDH mutants, the plasmid pSL207 derived from pTrcHisA (Invitrogen), containing the xdh genes with a His6 tag fused to the N-terminus of XDHA, was used. For heterologous expression in E. coli, pSL207 was transformed into E. coli TP1000 cells, containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. The enzyme was expressed in 500-mL-cultures of TP1000 cells carrying plasmid pSL207 grown at 30 C. in LB medium supplemented with 150 g/mL ampicillin, 1 mM molybdate, and 0.02 mM isopropyl-D-thiogalactopyranoside until the OD.sub.600nm=1. This culture was then transferred to a bottle containing 8 L of supplemented LB medium and subsequently grown at 30 C. for 18 to 20 hours. Cells were harvested by centrifugation at 5000g at 4 C. and subsequently prepared for protein purification.
Purification of XDH E232V and XDH E232VR310
[0317] The cell pellet was resuspended in eight volumes of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, and cell lysis was achieved by several passages through a French press. After addition of DNase I, the lysate was incubated for 30 min. After centrifugation at 17,000g for 25 min, imidazole was added to the supernatant to a final concentration of 10 mM. The supernatant was mixed with 2 mL of Ni.sub.2-nitrilotriacetic agarose (Qiagen) per litre of cell growth, and the slurry was equilibrated with gentle stirring at 4 C. for 30 min. The slurry was poured into a column, and the resin was washed with two column volumes of 10 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, pH 8.0, followed by a wash with ten column volumes of the same buffer with 20 mM imidazole. His-tagged XDH was eluted with 100 mM imidazole in 50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Fractions containing XDH were combined and dialyzed against 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, pH 7.5. The dialyzed sample was applied to a Q-Sepharose fast protein liquid chromatography column and eluted with a linear gradient of 0-250 mM NaCl. To the pool of fractions containing XDH, 15% ammonium sulphate was added, and the protein was then applied to a phenyl-Sepharose column equilibrated with 50 mM Tris, 1 mM EDTA, 2.5 mM dithiothreitol, 15% ammonium sulphate, pH 7.5. XDH E232V was eluted from the column with a linear gradient of from 15 to 0% ammonium sulphate. During purification, fractions were monitored using SDS-PAGE, whereas enzyme activity was measured spectrophotometrically as described above.
Screening of Xanthine Oxidoreductases for Oxidation of HMF, DFF and FFCA
[0318] XORs were screened using potassium phosphate buffer (50 mM, pH 7.6), 3 L 0.1 M HMF (in MeCN), 30 L 0.01 M DCPIP (aq.) final volume 300 L, 36 C. When DCPIP was the oxidant, activity was determined by the colour change from blue to colourless; when O.sub.2 was the oxidant, activity was detected using NBT assay (Agarwal; Banerjee Open Biotech J. 2009, 3, 46-49). The results can be found in Table 1.
TABLE-US-00001 TABLE 1 Screening of xanthine oxidoreductases Enzyme HMF DFF FFCA Oxidant E. coli XDH.sup.a Active O.sub.2 XDH E232V.sup.b Active Active Active DCPIP XDH E232V R310.sup.c Active Active Active DCPIP PaoABC.sup.d Active Active Active O.sub.2 .sup.aE. coli XDH (1.1 mg/mL); .sup.bXDH E232V (25.4 mg/mL); .sup.cXDH E232V/R310M (23 mg/mL); .sup.dPaoABC (13.3 mg/mL); .sup.ePaoABC (13.3 mg/mL).
Optimisation of the HMF 2-Step Oxidation Cascade
[0319] ##STR00030##
[0320] GOase M.sub.3-5 (3.3 mg/mL; 103 L), catalase (3.3 mg/mL; 33 L), HMF (3 L of a solution defined in Table 2 in MeCN), potassium phosphate buffer (concentration as per Table 2, pH 7.6) were combined and made up to 300 L. After full conversion of HMF to DFF, PaoABC (13.3 mg/mL; 5 L) was added. Formation of DFF was monitored via RP-HPLC using a Thermofisher Hypurity C18 column with flow rate 0.6 mL/min using 85% water+0.1% Acetic acid and 15% MeCN. Formation of 2,5-FDCA was monitored via RP-HPLC using a Thermofisher Hypurity C18 column with flow rate 1 mL/min using a 98% 10 mM phosphate buffer (pH 6.5) and 2% MeCN mobile phase. The results can be found in Table 2.
TABLE-US-00002 TABLE 2 Optimisation of the HMF multistep oxidation cascade HMF Buffer Entry mM pH mM DFF % 2,5-FDCA % 1.sup.a 10 7.5 50 97 2.sup.a 20 7.5 50 55 3 20 7.5 50 >99 >99 4 30 7.5 50 >99 >99 5 50 7.5 50 >99 0 6 50 7.5 100 >99 >99 7 70 7 300 >99 >99 (80.sup.c) 8 100 7 400 >99 0 9.sup.b 100 7 400 >99 >99 10.sup.b 100 7 400 0 >99 (74.sup.c) .sup.aOne-pot reactions with all enzymes present. .sup.bAdditional catalase was added with PaoABC. .sup.cReactions on a preparative scale.
Optimisation of Conversion of DFF to 2,5-FDCA
[0321] ##STR00031##
[0322] To a solution of potassium phosphate buffer was added DFF (2 M in MeCN; 33 L), catalase (3.3 mg/mL) and PaoABC (13.3 mg/mL). The final volume was 300 L. The reaction was vigorously shaken and placed in a shaking incubator at 37 C. Aliquots of the reaction mixture were removed, acidified with 2 M HCl and analysed by RP HPLC using a Thermo Fisher Hypurity C18 column, 98% 10 mM phosphate buffer pH 6.5, 2% MeCN with a flow rate of 1 mL/min. The results can be found in Table 3.
TABLE-US-00003 TABLE 3 Optimisation of conversion of DFF to 2,5-FDCA 2,5- 2,5- En- DFF Buffer PaoABC Time FFCA FDCA.sup.b try mM pH.sup.c mM L hours % % 1 50 6 200 5 2 100 0 2 50 7 200 5 2 0 >99 3 50 8 200 5 2 0 >99 4 50 9 200 5 2 18 0 5 80 7 200 5 4 0 >99 6 80 8 200 5 4 0 66 7.sup.a 100 7 200 5 16 40 60 8 100 7 400 5 8 0 >99 .sup.apH 4.5 after 16 hours. .sup.bConversion adjusted by analysing a 1:1 standard of the aldehyde:acid by .sup.1H NMR and comparing the HPLC trace of the same sample and adjusting the absorbance accordingly. .sup.cInitial pH.
Preparative Scale Oxidation of DFF with PaoABC
##STR00032##
[0323] To a solution of phosphate buffer (400 mM; pH 7) was added DFF (37 mg, 0.29 mmol), catalase (3.3 mg/mL; 330 L), MeCN (150 L) and PaoABC (13.3 mg/mL; 50 L). The final volume was 3 mL. The reaction was vigorously shaken and placed in a shaking incubator at 37 C. The pH was maintained at pH 7 by the addition of 1 M NaOH. The reaction was heated to 80 C. for 5 minutes and left cool to ambient temperature. The solution containing denatured protein was centrifuged and the supernatant removed. The supernatant was then cooled to 0 C. and concentrated HCl was added until a precipitate formed. The solution was then centrifuged, the supernatant removed and the resulting pellet washed with 1 M HCl. The pellet was dissolved in acetone and then concentrated in vacuo (3) to form 2,5-FDCA as a slightly yellow solid (41 mg, 0.26 mmol, 90%). .sup.1H NMR (500 MHz, DMSO-d6) ppm: 13.63 (b s, 2H), 7.29 (s, 2H); .sup.13C NMR (125 MHz, DMSO-d6) ppm: 159.4, 147.5, 118.86.
Preparative Scale Synthesis of FDCA
[0324] ##STR00033##
[0325] To a solution of potassium phosphate buffer (400 mM; pH 7; 1.09 mL), MeCN (0.03 mL) and catalase (0.33 mL of a 3.3 mg/mL solution) was added HMF (38 mg, 0.3 mmol; final concentration 100 mM). GOase M.sub.3-5 (1.5 mL of a 3.3 mg/mL solution) was added and the reaction shaken at 37 C. in an incubated shaker for 10 hours. Another portion of catalase (0.33 mL of a 3.3 mg/mL solution) was then added, together with PaoABC (0.05 mL of a 13.2 mg/mL solution). The reaction was left for another 5 hours in the shaking incubator. The pH was carefully monitored and adjusted to pH 7 with 1 M NaOH. The reaction was heated to 80 C. for 5 minutes and left to cool to ambient temperature. The solution containing denatured protein was centrifuged and the supernatant removed. The supernatant was then cooled to 0 C. and concentrated HCl was added until a precipitate formed. The solution was then centrifuged and the supernatant removed and the pellet washed with 1M HCl. The pellet was dissolved in acetone and then concentrated in vacuo (3) to form 2,5-FDCA as a slight yellow solid (35 mg, 0.22 mmol, 74% yield). .sup.1H NMR (500 MHz, DMSO-d6) ppm: 13.63 (b s, 2H), 7.29 (s, 2H); .sup.13C NMR (125 MHz, DMSO-d6) ppm: 159.4, 147.5, 118.86.
Example 2
[0326] HMF (1.9 mg, 0.015 mmol, final concentration=50 mM) and catalase (0.033 mL of a 3.3 mg mL.sup.1 solution) were added to KPi buffer (400 mM pH 7) (0.109 mL) and MeCN (0.003 mL). GOase M.sub.3-5 (0.15 mL of a 3.3 mg mL.sup.1 solution) was then added and the reaction shaken at 37 C. for 5 hours in a shaking incubator. PaoABC (0.005 mL of a 13.2 mg mL.sup.1 solution) was added and the reaction shaken for a further 3 hours in the incubator. The pH was monitored and adjusted to pH 7 with 1 M NaOH. The reaction was heated to 80 C. for 5 minutes and left to cool. The solution containing denatured protein was centrifuged and the supernatant removed and analysed by RP-HPLC (see
[0327] HMF is converted into DFF during the first five hours of the reaction. The addition of PaoABC to the reaction rapidly converts DFF into 2,5-FDCA.
Example 3
[0328] HMF (final concentration 50 mM), GOase M.sub.3-5 (103 L of 3 mg/mL), PaoABC (1 L of 28.9 mg/mL), catalase (33 L of 3.3 mg/mL) and horseradish peroxidase (HRP) (70 L of 1 mg/mL) were combined in KPi buffer (500 mM) at 37 C. and the pH continuously adjusted with 2M NaOH to give a conversion of 93-100% of 2,5-FDCA. The reaction was heated to 80 C. for 5 minutes and left to cool. The solution containing denatured protein was centrifuged and the supernatant removed and analysed by RP-HPLC (see
[0329] HMF may be converted into 2,5-FDCA via DFF or HMFCA under the reaction conditions. A peak of about 65% HMFCA after 60 minutes may indicate that the conversion of HMFCA into FFCA may be the rate limiting step.
Example 4
[0330] ##STR00034##
[0331] HMF (100 mM) was added to KPi buffer (500 mM pH 7.0). GOase M.sub.3-5 (103 l of 3.3 mg/mL), PaoABC (1 l of 28.9 mg/mL) and a metal complex (see Table 4) were added at 37 C. and the pH was continuously adjusted with NaHCO.sub.3 for a period of 16 hours. The reaction was heated to 80 C. for 5 minutes and left to cool. The solution containing denatured protein was centrifuged and the supernatant removed and analysed by RP-HPLC.
TABLE-US-00004 TABLE 4 Metal complex 2,5- (mol %.sup.1) HMF DFF HMFCA FFCA FDCA Entry (electron acceptor) (%.sup.2) (%.sup.2) (%.sup.2) (%.sup.2) (%.sup.2) 1 V(II)acac (400%) 0 0 90 7 3 2 V(II)acac (100%) 0 0 60 0 33 3 V(II)acac (50%) 0 0 56 0 36 4 V(II)acac (10%) 0 0 6.6 86 7.3 5 Mn(III)acac(50%) 0 0 86 14 0 6 Fe(II)phthalocy- 23 9 7 60 1 anine (50%) 7 Fe(III)acac (50%) 0 0 56 0 44 8 Fe(III)EDTA (50%) 0 0 0 0 0 9 V(V)OEt.sub.3 (50%) 0 0 13 58 28 10 V(V)OEt.sub.3 (10%) 0 0 3 52 45 11 V(V)Oxide (50%) 0 0 26 0 73 12 Co(II,III)oxide 0 49 3 46 1 (50%) 13 Fe(III)oxide (50%) 3.4 44.6 5.8 45 0.7 14 Vanadyl acac (50%) 0 0 77.8 0 22.2 15 VO(IV)sulphate 0 0 22.3 0 77 (50%) 16 Hematin (50%) 0 43 0 56 0 17 Hemin (50%) 0 60 0 40 0 18 Mn(II)sulphate 0 39 0 61 0 (50%) .sup.1mol % of metal complex based on the amount of HMF at the start of the reaction. .sup.2percentage based upon calibration by NMR of equimolar mixtures.
Example 5DFF OxidationStandard Process (No Shaking)
[0332] To 490 L of 0.2M KPi buffer (pH 7.0) was added DFF (final concentration 100 mM) and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L of water and quenched with 15 L 1M HCl. The aliquots were analysed by RP HPLC (see
Example 6DFF OxidationInitial Shaking
[0333] To 490 L of 0.2M KPi buffer (pH 7.0) was added DFF (final concentration 100 mM) and 1 mg catalase. The Eppendorf was vigorously shaken. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1M HCl. The aliquots were analysed by RP HPLC (see
Example 7DFF OxidationOxygen Sparged Buffer
[0334] To 490 L of 0.2M KPi (pH 7.0), sparged with compressed air (HPLC filter) for 5 hours, was added DFF (final concentration 100 mM) and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 5 L of the reaction mixture was extracted, diluted with 80 L water and quenched with 15 L 1M HCl. The aliquots were analysed by RP HPLC (see
Example 8Periodic Hydrogen Peroxide Addition
[0335] To 490 L of 0.2M KPi buffer (pH 7.0) was added DFF (final concentration 100 mM) and 1 mg catalase. 10 L of a 100 M PaoABC was then added and the reaction was left in a shaking incubator. 1 l of a 1% H.sub.2O.sub.2 was added every 15 minutes. 5 L of the reaction mixture was extracted, diluted with 80 L of water and quenched with 15 L 1M HCl. The aliquots were analysed by RP HPLC (see
Example 9A
[0336] ALD-003 (5 mg), NOX-009 or NOX-001 (5 mg) and NAD.sup.+ or NADP.sup.+ (20 mol % based upon the amount of ALD-003) was added to 0.5 mL 0.25M KPi (pH 8.5). The pH was adjusted to pH 8.5 with 1M NaOH. 10 mM DFF or HMF was added and the reaction was left in a shaking incubator at 35 C. After a specified time the reaction was quenched with 1M HCl, centrifuged and analysed by RP-HPLC. The results are found in Tables 5, 6, and 7A.
TABLE-US-00005 TABLE 5 Entry Enzyme Substrate Co-Factor NOX Conv Product 1 ALD-003 HMF NAD+ NOX-009 100% HMFCA 2 HMF NADP+ NOX-001 100% HMFCA 3 DFF NAD+ NOX-009 100% FDCA 4 DFF NADP+ NOX-001 100% FDCA Reaction Conditions: 0.5 mL KPi 0.25M pH 8.5, 5 mg CFE, 20 mol % cofactor, 5 mg NOX, 10 mM Substrate, 35 C., reaction time 30 minutes.
TABLE-US-00006 TABLE 6 Sub- Entry Enzyme [DFF] strate Nox Time FFCA FDCA 1 ALD-003 50 DFF NOX-009 3 hr 20% 80% 2 100 NOX-009 80% 20% Reaction Conditions: 0.5 mL KPi 0.25M pH 8.5, 5 mg ALD-003 CFE, 30 l catalase (3.3 mg/mL) 20 mol % cofactor, 5 mg NOX, 35 C.
TABLE-US-00007 TABLE 7A NOX-009 CFE NAD.sup.+ Yield Entry [DFF] pH (mg) (mg) (mol %) DFF:FFCA:FDCA 1 100 8.5 5 5 20% 0:80:20 2 30% 0:44:56 3 40% 0:50:50 Reaction Conditions: 0.5 mL KPi 0.25M pH 8.5, 5 mg ALD-003 CFE, 30 L catalase (3.3 mg/mL) Xmol % cofactor (NAD.sup.+), 5 mg NOX, 35 C., reaction time 3 hr.
Example 9B
[0337] HMF (10 mM), KPi Phosphate buffer (1 mL, 100 mM), KRED CFE (15 mg), were combined with NADP.sup.+ (30 mM). Aliquots were removed every hour and quenched with 1M HCl, centrifuged and analysed by RP-HPLC. The results from the reaction can be seen in
Example 9C
[0338] Reaction Conditions: HMF (10 mM), KRED (089) (7.5 mg), 0.5 mL KPi Buffer (pH x), and NOX-1 and NADP.sup.+ as defined in Table 7B were reacted at 37 C. for 2 hours. A sample was quenched with 1M HCl, centrifuged and analysed by RP-HPLC. The results from the reaction can be seen in Table 7B.
[0339] Using 10 mol % of NADP.sup.+ (relative to the amount of HMF used) and 5 mg NOX-1 provided the highest conversion of HMF to 2,5-FDCA. Reducing the amount of NADP.sup.+ and NOX-1 lead to a lower conversion of HMF to HMFCA.
TABLE-US-00008 TABLE 7B NADP.sup.+ NOX-1 (mol Conversion Entry pH (mg) %).sup.[a] HMF:DFF:HMFCA:FFCA:2,5-FDCA 1 7 5 50 0:0:23:53:23 2 7 7.5 0:0:28:54:17 3 7 10 0:0:33:52:15 4 7 5 10 8:0:12:57:29 5 8 13:0:7.5:49:30 6 9 10 20:0:5:46:30 .sup.[a]mol % relative to amount of HMF used.
Example 10AEntrapment of PaoABC in SiO.SUB.2 .Hydrogel
[0340] Tetramethyl orthosilicate (TMOS) (0.450 g) was placed in a small vial, cooled in an ice bath, and stirred at about 600 rpm. HCl (108 L, 2.44 mM) was added, and the solution was stirred for 10 min. The solution was adjusted to pH 5.1 by adding 60 L of 20 mM sodium phosphate buffer (pH 7.4). In a separate small vial, 1 mg of PaoABC, 540 L of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (sodium salt) buffer solution (pH 7.5), and, when polymer was added, 60 L of 20 mg/mL aqueous PVI or PEI were mixed. The PaoABC-containing solution was added to the TMOS-containing solution, and the resulting mixture was stirred for 1 min. A vacuum was applied to the stirred mixture until a gel formed. The vacuum was released, and the gel was rinsed with 2 mL of distilled water three times. The gel was then soaked in 2 mL of distilled water overnight at 4 C. The water was removed from the vial, and the gel was allowed to dry at room temperature overnight in a vial. The dried gel was first collected and then ground to a powder with a mortar and pestle. 750 mg of hydrogel was produced with 1 mg of PaoABC, as determined by Bradford assay of the supernatant.
Example 10BEntrapment of PaoABC in Ni-Sepharose
[0341] 30 mL binding buffer (400 mM NaCl, 20 mM Imidazole, 50 mM KPi pH 7.0), 3 g of PaoABC CFE and 5 g Ni.sup.+2 5 g of resin was stirred for 40 minutes. The slurry was centrifuged at 500 RPM and the supernatant removed. This was repeated twice with washing with binding buffer.
Example 10CEntrapment of PaoABC in Eupergit EC
[0342] 35 l of PaoABC was dissolved in 1 mL 1M KPi buffer with a pH as specified in Table 8. The concentration of PaoABC before immobilisation was recorded. 10 mg of Eupergit EC was added and the mixture was left in a shaking incubator at 150 rpm and 25 C. for 24 hours. The amount of enzyme absorbed onto the resin was determined by UV spectroscopy (428 nm). Blocking buffer (0.2M ethanolamine in 0.1 M KPi pH 7) was added and the mixture was left to shake for 30 minutes. The immobilised enzyme was filtered through a sinter funnel and washed with KPi buffer pH 7 and then KPi buffer pH 7 with 1M NaCl. The immobilised beads were tested for activity.
TABLE-US-00009 TABLE 8 Immobilisation Enzyme Enzyme Conversion.sup.[a] Entry pH (mg/mL) Immobilized (mg) (%).sup.[b] 1 6 0.85 0.7 44 2 7 1 0.97 48 3 8 0.94 0.8 60 .sup.[a]Reaction conditions: 0.1 mg PaoABC (on resin), 2.5 l benzaldehyde, 500 l 100 mM KPi pH 7.6, 37 C., 5 Hr .sup.[b]Conversion calculated by RP-HPLC
Example 10DEntrapment of PaoABC in Eupergit CM
[0343] 70 L (2 mg) of paoABC was dissolved in 1 mL 1M KPi buffer pH 7. The concentration before immobilisation was recorded. 200 mg of Eupergit EC was added and left in a shaking incubator on 150 rpm at 25 C. for 24 Hr. The amount of enzyme absorbed onto the resin was determined by UV spectroscopy (428 nm). After this time the pH was increased to pH 8.5 to facilitate multipoint attachment to the resin (5 hr). Blocking buffer (3M Glycine pH 8.5) was added and left shake for 5 hours. The immobilised enzyme was filtered through a filter paper and washed with 100 mM KPi Buffer pH 7 and, 100 mM KPi buffer pH 7 with 1M NaCl. The immobilised beads were tested for activity.
TABLE-US-00010 Conversion Entry Resin Amount Run (%) 1 Eupergit CM 50 mg 1 100 2 2 100 Reaction conditions: 100 mM KPi phosphate buffer pH 7.0, 100 mM DFF, 5 hr, 37 C.
Example 11Formation of 2,5-FDCA from DFF Using Immobilised PaoABC
[0344] To 300 L of 0.3M TrisHCl pH 7.0, was added 5 mg of catalase-CLEA (catalase cross-linked enzyme aggregates) and 0.065 mg of immobilized PaoABC (50 mg of hydrogel, 10 mg for Eupergit-immobilised PaoABC, 2.2 L of soluble PaoABC (29.2 mg/mL)) in a 1.5 mL Eppendorf. The reaction was vigorously shaken and the pH adjusted to pH 7.0. DFF (1.9 mg; 50 mM) was added and the reaction was placed in a shaking incubator at 25 C. 5 L of the reaction mixture was extracted and diluted with 80 L of water and 15 L of 1M HCl before being centrifuged for 5 mins. The aliquots were analysed by reverse phase HPLC.
[0345] The hydrogel could be used 14 times with no loss in activity.
Example 12
Synthesis of 2,5-diethyl-2,5-furandicarboxylate
[0346] ##STR00035##
[0347] 2,5-furandicarboxylic acid (25.18 g; 160 mmol) was added to ethanol (1,800 mL). Aqueous sulfuric acid (1.32 mL) was added. The mixture was heated at reflux (about 78 C.) for 67 hours, during which time water was removed from the reaction by the use of a Dean-Stark apparatus. The reaction progress was monitored using NMR spectroscopy. After the 2,5-diethyl-2,5-furandicarboxylate had been formed in >97% purity by NMR, the reaction mixture was allowed to cool to ambient temperature and was extracted with 2-methyltetrahydrofuran. The combined organic layers were washed with a saturated aqueous brine solution and deionised water, and dried (MgSO.sub.4). The organics were filtered and the volatiles were removed in vacuo to afford the title compound (26.77 g; 130 mmol; >98% conversion).
General Methodology for the Formation of Copolymers
[0348] A 250 mL flange flask with 5 quick-fit ports was used in connection with a Dean-Stark apparatus. Stirring was achieved via a magnetic stirrer using a large precious metal stirrer bar. The rates of stirring were gradually decreased from the initial 400 rpm down to 200 rpm to avoid issues as a result of the increasing viscosity of the reaction mixture. All reagents were added to the reactor and warmed to 110 to 130 C. as described below to allow total melting and achieve miscibility. A flow of N.sub.2 gas was applied for 20 minutes to purge the reagents and reactor of oxygen. The temperature was then increased to the desired point as stated below. After a further four hours of very low N.sub.2 flow the gas line was removed, the Dean-Stark drained and a vacuum pump turned on, initially at a low vacuum (200 mbar) but slowly increased as stated below.
Example 13
Synthesis of 2,5-polybutyrate adipate furandicarboxylate (2,5-PBAF)
[0349] ##STR00036##
[0350] 2,5-Diethyl-2,5-furandicarboxylate (21.20 g; 100 mmol), diethyl adipate (20.23 g; 100 mmol), 1,4-butane diol (22.53, 250 mmol) and T titanium(IV) tert-butoxide (0.77 mL; cat.) were combined. The reaction mixture was heated at 110 C. for 4 hours at atmospheric pressure with stirring at 400 rpm, 180 C. for 17 hours at 200 mbar and 350 rpm, and at 180 C. for 3 hours at 25 mbar and 250 rpm. The polymer was formed (37.20 g). The .sup.1H NMR spectra for 2,5-PBAF can be found at
[0351] The molar ratio of 2,5-furandicarboxylate:adipate was determined by .sup.1H NMR to be 0.90:1. The molecular weight of the 2,5-PBAF was estimated using end-group analysis, wherein the ratio of end groups to those of the bulk polymer were calculated using .sup.1H NMR to give the number of constitutional repeating units (CRU), which was estimated to be 20.71. One ideal CRU is 410.43 gmol.sup.1. Therefore, the molecular weight of the 2,5-PBAF was estimated to be 8,497.5 gmol.sup.1.
Comparative Example 14
Synthesis of polybutyrate adipate terephthalate (PBAT)
[0352] Diethyl terephthalate (22.22 g; 100 mmol), diethyl adipate (20.23 g; 100 mmol), 1,4-butane diol (22.73, 230 mmol) and titanium(IV) tert-butoxide (0.77 mL; cat.) were combined. The reaction mixture was heated at 130 C. for 2 hours at atmospheric pressure with stirring at 400 rpm, 180 C. for 2 hours at atmospheric pressure and 400 rpm, 180 C. for 17 hours at 200 mbar and 350 rpm, and at 180 C. for 3 hours at 25 mbar and 250 rpm. The copolymer was formed (40.51 g). The .sup.1H NMR spectra for PBAT can be found at
[0353] The molecular weight of the PBAT was estimated by .sup.1H NMR using end-group analysis as described for 2,4-PBAP. The molar ratio of terephthalate:adipate was determined to be 1.047:1. The number of CRUs was estimated to be 16.4. One ideal CRU is 420.45 gmol.sup.1. Therefore, the molecular weight of the PBAT was estimated to be 6,893 gmol.sup.1.
[0354] PBAT is available commercially under a range of trade names. The molecular weight of one particular commercial PBAT was estimated by .sup.1H NMR using end-group analysis as described for 2,4-PBAP. The molar ratio of terephthalate:adipate was determined to be 0.93:1. The number of CRUs was estimated to be 25.7. One ideal CRU is 420.45 gmol.sup.1. Therefore, the molecular weight of the commercial PBAT was estimated to be 10,809 gmol.sup.1. The .sup.1H NMR spectra for commercial PBAT can be found at
Example 15
[0355] Thermal analysis of polymers using (STA and DSC)
[0356] The thermal stability of cured copolymer was analysed using Simultaneous Thermal Analysis (STA) using a Stanton Redcroft STA 625. Approximately 10-20 mg of copolymer was heated from ambient temperature to 625 C. at a heating rate of 10 C. min.sup.1 under nitrogen. The decomposition may be that of the copolymer backbone. The results can be found in Table 9.
TABLE-US-00011 TABLE 9 STA analysis of polymers Temperature of Temperature of 5 wt % loss decomp. Copolymer C. C. STA trace 2,5-PBAF 315.0 391.7 FIG. 4 Comparative Example 14 289.5 406.0 FIG. 5 Commercial PBAT 341.5 409.5 FIG. 6
[0357] The glass transition temperature (T.sub.g) and melting point (T.sub.m) of the copolymers were obtained by Differential Scanning calorimetry (DSC) analysis using a TA Instruments Q2000 DSC. Indium was used as the standard to calibrate the temperature and heat capacity. Copolymer samples (7-10 mg) were sealed in Tzero aluminum hermetic DSC pans. The method was carried out under a constant flow of dry nitrogen of 50 mL/min, at 10 C./min over a temperature range of 80 C. to 250 C. The results can be found in Table 10. The DSC traces can be found at
TABLE-US-00012 TABLE 10 DSC analysis of copolymers T.sub.g1 T.sub.g2 T.sub.m Copolymer C. C. C. 2,5-PBAF 30.4 40.5 87.2 Comparative Example 14 39.5 42.4 134.6 Commercial PBAT 30.1 45.4 122.2
Example 16
[0358] The molecular weight (M.sub.n and M.sub.w) and polydispersity (Pd.sub.i) data as generated by GPC can be found in Table 11. GPC was conducted on an Agilent SECurity GPC System 1260 Infinity using THF as the solvent, a polystyrene standard, and a light scattering detector.
TABLE-US-00013 TABLE 11 GPC analysis of copolymers GPC Copolymer M.sub.n M.sub.w Pd.sub.i chromatogram 2,5-PBAF 862.5 6,121 7.097 FIG. 8 Comparative Example 14 5,582 8,615 1.544 FIG. 9 Commercial PBAT 42,190 113,100 2.680 FIG. 10
Example 17
Tensile Strength Measurement
[0359] Mechanical properties including tensile strength, elongation at break and Young's modulus of samples are summarised in Table 12. Film samples were prepared by heating about 8 g of copolymer in a fan-assisted oven at 160 C. for 15 min (180 C. for PBAT). The resulting films were cut into standard dumb-bell shapes (60 mm10 mm). Film thickness was in the region of 1.5-2.0 mm. Tensile studies were conducted in triplicate using an Instron 3367 universal testing machine fitted with 1000 N capacity load cell. The initial grip separation was set at 35 mm and the crosshead speed was 20 mm/min. The results reported were the average of the three measurements (the elongation at break was obtained automatically from the software). Commercially PBAT is a typical elastomer with elongation over 293%. It has the highest tensile strength over 19.5 MPa and good Young's modulus of 100.8 MPa.
TABLE-US-00014 TABLE 12 Tensile strength measurement of copolymers Young's Tensile strength Elongation Modulus Copolymer MPa at break % MPa 2,5-PBAF 2.2 0.4 4.7 0.8 75.3 2.0 Comparative Example 14 4.8 0.5 2.3 0.2 269.8 0.2 Commercial PBAT >19.5 >293.1 100.8
[0360] The 2,5-PBAF copolymer produced is soft like that of the commercial PBAT. The expected ratio of FDCA to adipate of about 1:1 has been incorporated into the copolymer. The observed molecular weight of 2,5-PBAF and comparative example 14 (PBAT) are significantly lower than that of commercial PBAT. This is expected given the relatively small scale on which the copolymerisations were conducted and will be higher in a full scale production process. The NMR data provides an indication of the relative number of constitutional repeating units (CRU) and hence an indication of molecule weight, though the GPC provides more accurate values.
[0361] The differences in the data obtained for the copolymers of the invention and the commercial BPAT may be attributed to a lack of branching in 2,5-PBAF.
Example 18
[0362] Stabilised green waste compost is matured in a composting bin under controlled aeration conditions. Before use, the mature compost is sieved on a screen of 5 mm. The fine fraction forms the inoculum with a total solids content of approximately 50-55% and the volatile content of the total solids is more than 30%.
[0363] The standard and control materials are mixed with the inoculum in a ratio of approximately 1 to 1.5 parts of total solids to 6 parts of total solids and introduced into a reactor. These reactors are closed and put into an incubator. The temperature of the reactors is maintained at 58 C.+1-2 C. Pressurised air is pumped through a gas flow controller and blown into the composting vessel at the bottom through a porous plate. During biodegradation, solid carbon of the test sample is converted into CO.sub.2.
[0364] The gas leaving each individual reactor is analysed at regular intervals for CO.sub.2 and C.sub.2 concentrations. As the flow rate is continually measured, the cumulative CO.sub.2 production can be determined. The percentage of biodegradation is determined as the percentage of solid carbon of the test compound that is converted into CO.sub.2.
[0365] Those skilled in the art will recognise or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.