Furfurylamines from biomass: Transaminase catalysed upgrading of furfurals

Article abstract of DOI:10.1039/c6gc02241c

Furfural is recognised as an attractive platform molecule for the production of solvents, plastics, resins and fuel additives. Furfurylamines have many applications as monomers in biopolymer synthesis and for the preparation of pharmacologically active compounds, although preparation via traditional synthetic routes is not straightforward due to by-product formation and sensitivity of the furan ring to reductive conditions. In this work transaminases (TAms) have been investigated as a mild sustainable method for the amination of furfural and derivatives to access furfurylamines. Preliminary screening with a recently reported colorimetric assay highlighted that a range of furfurals were readily accepted by several transaminases and the use of different amine donors was then investigated. Multistep synthetic routes were required to synthesise furfurylamine derivatives for use as analytical standards, highlighting the benefits of using a one step biocatalytic route. To demonstrate the potential of using TAms for the production of furfurals, the amination of selected compounds was then investigated on a preparative scale.

Full text of DOI:10.1039/c6gc02241c

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Furfurylamines from Biomass: Transaminase catalysed upgrading
of furfurals
Alice Dunbabin,a Fabiana Subrizi,a John M. Ward,b Tom D. Shepparda and Helen C. Hailes*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Furfural is recognised as an attractive platform molecule for the production of solvents, plastics, resins and fuel additives.
Furfurylamines have many applications as monomers in biopolymer synthesis and for the preparation of pharmacologically
active compounds, although preparation via traditional synthetic routes is not straightforward due to by-product
formation and sensitivity of the furan ring to reductive conditions. In this work transaminases (TAms) have been
investigated as a mild sustainable method for the amination of furfural and derivatives to access furfurylamines.
Preliminary screening with a recently reported colorimetric assay highlighted that a range of furfurals were readily
accepted by several transaminases and the use of different amine donors was then investigated. Multistep synthetic
routes were required to synthesise furfurylamine derivatives for use as analytical standards, highlighting the benefits of
using a one step biocatalytic route. To demonstrate the potential of using TAms for the production of furfurals, the
amination of selected compounds was then investigated on a preparative scale.
www.rsc.org/
R1
R2
R1
R2
O
NH2
O
O
Introduction
TAm, PLP
R4
R4
Furfurals have attracted significant interest in recent
years as renewable feedstocks for the production of
biofuels and chemicals.1 In 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.3 Indeed, renewable 5-
R3
1a-10a
R3
1b-10b
amine
donor
co-product
Scheme 1 Use of TAms with co-factor pyridoxal-5-phosphate (PLP),
aldehydes and ketone 1a-10a to generate amines 1b-10b.
The 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.7 Moreover, 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
HMF is being manufactured commercially.4 As
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.3,5 Among 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
primary amines.8 These 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.10
Here we describe the use of TAms for the amination of
furfural derivatives 1a-9a and ketone 10a to access amines
pharmaceuticals
such
as
antiseptic
agents, antihypertensives, and diuretics (e.g. Furosemide).6
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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.
However, the less polar 5-methylfurfural 7a showed a 60%
conversion with ArRMut11.
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DOI: 10.1039/C6GC02241C
OH
O
H
O
H
O
H
O
H
O
O
O
O
Results and discussion
Br
4a
2a
Cl
1a
Br
3a
Br
Me
Initial substrate screening
O
H
O
O
H
O
O
O
O
Ten furan-based substrates were screened including
furfural 1a, 5-HMF 2a, halogenated furfurals 3a-6a, 4-
methylfurfural 7a, 5-formyl-2-furancarboxylic acid 8a, 2,5-
furandicarboxaldehyde 9a, and 2-acetylfuran 10a (Figure 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) 11.11-14 The (R)-
selective TAms Arthrobacter sp. variant ArRMut1115 and
Mycobacterium vanbaalenii (Mv-TAm),16 were also selected
due to their complementary enantioselectivities compared
to CV-TAm for use with the ketone 10a. In addition,
ArRMut11 has been used with a wide range of substrates
and also has good tolerance towards IPA 11 as an amine
donor.15-19
H
6a
7a
5a
8a
O
O
O
H
O
O
H
O
O
HO
H
Me
9a
10a
NH2
NH2
11
13
Ph
Figure 1 Furan-based substrates 1a-10a screened against TAms, and
amine donors IPA 11 and MBA 13.
R1
R1
R2
O
NH2
O
O
TAm, PLP
NH2
R4
R4
R2
H
R3
1a-10a
R3
1b-10b
O
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).20 It 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.20 The results of the colorimetric
assay are shown in Figure 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.
Activity of the TAms towards 1a-10a (with
benzaldehyde as a positive control) was also confirmed
using either (S)- or (R)-α-methylbenzylamine (MBA) 13 as
amine donors, depending on the selectivity of the enzyme
(Figure 3). Notably furfural 1a and 5-HMF 2a were readily
accepted by all three enzymes and in comparable
conversions to benzaldehyde with CV-TAm and Mv-TAm
(see Figure 3 caption). Also, the 3- and 4-bromo derivatives
3a and 4a gave 50-60% conversions with all the selected
transaminases. Within this series, the 5-substituted
furfurals 5a and 7a were particularly well accepted by Mv-
TAm (82% and 79% conversions respectively), while
significantly lower conversions were achieved with 5a and
both CV-TAm and ArRMut11 and 7a with CV-TAm.
12
red precipitate
12
NO2
NO2
Scheme 2 Colorimetric TAm assay using 12.
(+) 1a 2a 3a 4a 5a 6a 7a 8a 9a 10a (-)
CV-TAm
ArRMut11
Mv-TAm
No TAm
Figure 2 Colorimetric assay (in duplicate) using 2-(4-nitrophenyl)ethan-
1-amine 12 as amine donor: 12 (25 mM), amino acceptor (10 mM), PLP (0.2
mM), KPi buffer pH 7.5 (100 mM) and enzyme crude lysate, 24 h, 30 °C, 500
rpm. Benzaldehyde was used as a positive control (+) and water as a
negative control (-).
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
2 | J. Name., 2012, 00, 1-3
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yield with all the TAms (56-70%). Finally, 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.
acid 8b or the corresponding azide with a rangVeieowfArrteicdleuOcnilninge
agents including LiAlH4 and BH3-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.
DOI: 10.1039/C6GC02241C
R
R
R
O
O
O
O
i, ii
iii-v
H
Cl
NH2
CV-TAm
ArRMut11
Mv-TAm
100
80
60
40
20
0
2a R = CH2OH
8a R = CO2H
14 R = CH2Cl
15 R = CO2H
9b R = CH2NH2
8b R = CO2H
Scheme 3 i) NaBH4; ii) HCl; iii) NaN3; iv) PPh3 then Boc2O; v) HCl.
It is worth noting that procedures reported to afford 2b
are typically very low yielding (4% yield) or use
unsustainable metal-based catalysts.21,22 This 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.14 For
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, 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 the
reported stereoselectivity of this TAm). The reaction with
CV-TAm was lower yielding so the ee was not investigated.
1a
2a
3a
4a
5a
6a
7a
8a
9a*
10a
Figure 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).
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 furfurylamines 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
(PPh3). Derivatisation of the amine with tert-
butoxycarbonyl anhydride (Boc2O) 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
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Table 1 Yields for transaminase catalysed reactions producing 1b, 2b, 8b, 9b and 10b using MBA 13 and IPA 11 as amine donorsa,b
OH
NH2
O
Product
NH2
Me
O
O
O
O
HO
O
NH2
NH2
NH2
NH2
1b
MBA 13
80%
2b
8b
9b
10bd
Amine donor
CV-TAm
IPA 11
MBA 13
75%
IPA 11
89%
MBA 13
47%
IPA 11
MBA 13
70%
IPA 11
MBA 13
92%
34%
78%
88%
3%
48%
(60%c)
45%
18% [ee n.d.]
1% [ee n.d.]
ArRMut11
Mv-TAm
59%
69%
44%
4%
57%
(53%c)
46%
75%
53%
66%
32%
59%
56%
54%
[78% ee (R)]
(52%c)
aReactions were performed in triplicate and conversions were determined using HPLC against product standards; bAssays 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; cYields obtained using double the amount of TAm enzyme; dIPA was used as an amine donor except for compound 10b; ee determined using
chiral HPLC; n.d. not determined.
aminoacids which has been incorporated into novel
anticancer peptides.24 The product 8b of the
Preparative scale reaction
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.
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 Km values similar to GABA.23
Moreover, 8b belongs to the class of furanoid sugar
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.4 Three 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-1-amine 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
acetylfuran 10a was more readily converted to the
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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-performance25 and
biodegradable26 properties.
reduced to 30 °C.14 After 5 h cells were harvested by
centrifugation (8000 rpm, 20 min) and the pellet suspended
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DOI: 10.1039/C6GC02241C
in potassium phosphate buffer (100 mM, pH 7.5) containing
pyridoxal-5-phosphate (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 Figure 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 CV-TAm, 4.0
mg/mL ArRMut11 and 2.5 mg/mL Mv-TAm.
Experimental
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: 1H and 13C 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 1H 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
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
coloration indicated that the TAms were active towards the
selected furfurals (Figure 2).
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.
MBA (13) Screening. The assay was performed in an
Eppendorf tube (200 µL total volume) containing (R)- or (S)-
MBA (13) (25 mM), PLP (1 mM), potassium phosphate
buffer (100 mM, pH 7.5), amine acceptor (5 mM) and crude
cell lysate (20 μL). After incubation at 30 ˚C and 300 rpm for
24 h, the reaction was stopped by the addition of 10%
trifluoroacetic acid (TFA) in water (10 μL). Denatured
protein was removed by centrifugation (4 ˚C, 3000 rpm, 4
min) and the supernatant diluted and analysed by analytical
HPLC.
Analytical HPLC. Analyses of the reactions were
performed using an Agilent 1260 Infinity HPLC with an Ace
5 C18 column (150 x 4.6 mm). Elution was carried out at 1
mL/min with
a linear gradient of acetonitrile/H2O
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 DSM3019111 (CV-TAm),
Arthrobacter sp. variant ArRMut1115 and Mycobacterium
vanbaalenii (Mv-TAm)16, from the UCL TAm library were
used to inoculate 2TY broth (5 mL) containing kanamycin
(50 µg/mL) 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 OD600 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
Isopropylamine (11) assay. The assay was performed in
an Eppendorf tube containing isopropylamine (11) (100
mM, pH 8), PLP (1 mM), potassium phosphate buffer (100
mM, pH 8), amine acceptor (10 mM) and crude cell lysate
(20 μL). After incubation at 35 ˚C and 300 rpm for 24 h, the
reaction was stopped by the addition of 10% TFA in water
(10 μL). Denatured protein was removed by centrifugation
(4 ˚C, 3000 rpm, 4 min) and the supernatant diluted and
analysed by analytical HPLC.
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5-Hydroxymethyl-2-furancarboxylic acid. To 5-formyl-
2-furancarboxylic acid 8a (140 mg, 1.00 mmol) in methanol
(5 mL) at 0 °C, NaBH4 (57 mg, 1.5 mmol) was added in
portions and the reaction was stirred for 4 h. The reaction
was quenched with brine (5 mL) and the methanol removed
under reduced pressure. The aqueous residue was acidified
and extracted with ethyl acetate (3 x 30 mL), washed with
brine (2 x 30 mL), dried (Na2SO4) and the solvent removed
under reduced pressure to give 5-hydroxymethyl-2-
furancarboxylic acid27 as a colourless solid (115 mg, 81%).
M.p. 157-158 °C (EtOAc), Lit. 163-164 °C;28 vmax (film) 3236,
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DOI: 10.1039/C6GC02241C
2,5-Bis(hydroxymethyl)furan. NaBH4 (170 mg, 4.49
mmol) was added in portions to 5-HMF 2a (378 mg, 3.00
mmol) in methanol (10 mL) at 0 °C. The reaction was stirred
for 4 h, brine (5 mL) was added and the methanol removed
under reduced pressure. The aqueous residue was
extracted with ethyl acetate (3 x 30 mL), washed with brine
(2 x 30 mL), dried (Na2SO4) and the solvent removed under
reduced pressure to give 2,5-bis(hydroxymethyl)furan29 as a
colourless oil (296 mg, 77%). vmax (film) 3281, 2924, 2866,
1631 cm–1; 1H NMR (CDCl3; 400 MHz) 2.45 (2H, br s, 2 x OH),
4.56 (4H, s, 2 x CH2OH), 6.22 (2H, s, 3-H, 4-H); 13C NMR
(CDCl3; 150 MHz) 57.5, 108.7, 154.2; m/z (CI) 256 ([2M]+,
100%), 191 (38), 173 (15), 146 ([MH+NH3]+ 35), 128 (5).
1
2414 br, 1650, 1595 cm–1; H NMR (MeOH-d4; 500 MHz)
4.56 (2H, s, CH2OH), 6.45 (1H, d, J 3.4 Hz, 4-H), 7.15 (1H, d, J
3.4 Hz, 3-H); 13C NMR (MeOH-d4; 125 MHz) 57.5, 110.2,
120.0, 145.7, 160.7, 161.8; m/z (EI) 142 ([M]+, 28%), 123
(22), 97 (100), 69 (50).
2,5-Bis(aminomethyl)furan hydrochloric salt (9b). To
2,5-bis(hydroxymethyl)furan (256 mg, 2.00 mmol) in CH2Cl2
(6 mL), conc. HCl (37%; 2 mL) was added. The reaction was
stirred at room temperature for 16 h. Water (10 mL) was
added and the product was extracted with CH2Cl2 (3 x 15
mL), washed with brine (2 x 15 mL), dried (Na2SO4) and the
solvent removed under reduced pressure to give 1430 as a
brown oil (82 mg, 25%) which was taken directly through to
the next step. 1H NMR (CDCl3; 600 MHz) 4.58 (4H, s, CH2Cl),
6.34 (2H, s, 3-H, 4-H); 13C NMR (CDCl3; 150 MHz) 37.4,
110.9, 151.1.
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). PPh3 (525 mg, 2.00 mmol)
was added and the reaction was stirred at room
temperature. After 2 h Boc2O (436 mg, 2.00 mmol) was
added and the reaction was stirred for 16 h. The methanol
was removed under reduced pressure, sat. NaHCO3 added
(30 mL) and the product was extracted with ethyl acetate (3
x 30 mL), dried (Na2SO4) and the solvent removed under
reduced pressure. 2,5-Bis(Boc-aminomethyl)furan was
5-Chloromethyl-2-furancarboxylic acid (15). To 5-
hydroxymethyl-2-furancarboxylic acid (91 mg, 0.64 mmol)
in CH2Cl2 (6 mL), conc. HCl (37%, 2 mL) was added. The
reaction was stirred at room temperature for 24 hours.
Water (10 mL) was added and the reaction was extracted
with CH2Cl2 (3 x 15 mL), washed with brine (2 x 15 mL),
dried (Na2SO4) and the solvent removed under reduced
pressure to give 15 as a colourless solid (45 mg, 44%). M.p.
123-124 °C (CH2Cl2); vmax (film) 3121, 2929, 2800 br, 1675,
1590 cm–1; 1H NMR (MeOH-d4; 600 MHz) 4.70 (2H, s,
CH2Cl), 6.59 (1H, d, J 3.5 Hz, 3-H), 7.15 (1H, d, J 3.5 Hz, 4-H);
13C NMR (CDCl3; 125 MHz) 36.6, 111.8, 121.1, 144.0, 155.5,
163.1; m/z MS (EI) 162 ([M37Cl]+, 6%), 160 ([M35Cl]+, 16),
125 (100), 79 (33); HRMS (FTMS) found [M-H]– 158.9858;
C6H435ClO3 requires 158.9854.
5-Aminomethyl-2-furancarboxylic acid (8b). Sodium
azide (53 mg, 0.82 mmol) was added to 15 (44 mg, 0.27
mmol) in dry DMF (5 mL) under argon, and the reaction was
heated at 65 °C for 16 h. The DMF was removed under
reduced pressure and the residue dissolved in methanol (8
mL). PPh3 (212 mg, 0.809 mmol) was then added and the
reaction was stirred at room temperature. After 3 h Boc2O
(118 mg, 0.541 mmol) was added and the reaction was
stirred for 16 h. The methanol was removed under reduced
pressure, sat. NaHCO3 was added (30 mL) and side-products
removed with ethyl acetate (3 x 30 mL). The aqueous layer
was then acidified (HCl) and extracted with ethyl acetate (3
x 30 mL). The combined organic extracts were dried
(Na2SO4) and the solvent removed under reduced pressure.
The product was then stirred in a 1:1 mixture of methanol
and 4 M HCl (6 mL) for 2 h, and the solvent removed under
reduced pressure to give 5-aminomethyl-2-furancarboxylic
acid hydrochloride salt 8b·HCl23 (29 mg, 60%) as a
colourless solid. M.p. 250 °C (decomp.; H2O); vmax (film)
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·2HCl31 as a brown solid (34 mg, 34% from 14). M.p. 240
°C (decomp.; H2O); vmax (film) 3090, 2851 br, 1596 cm–1; 1H
NMR (MeOH-d4; 600 MHz) 4.21 (4H, s, 2 x CH2NH2), 6.61
(2H, s, 3-H, 4-H); 13C NMR (MeOH-d4; 150 MHz) 36.8, 113.2,
149.6; m/z (EI) 126 ([M]+, 30%), 96 (100); HRMS (EI) found
[M]+ 126.0788; C6H10N2O requires 126.0788.
1-Furan-2-ethylamine ee determination for Mv-TAm.
The assay was performed (800 µL total volume) containing
(R)-(13) (25 mM), PLP (1 mM), potassium phosphate buffer
(100 mM, pH 7.5), amine acceptor (5 mM) and crude cell
lysate (80 μL). After incubation at 30 ˚C and 300 rpm for 24
h, the reaction was stopped by the addition of 10%
trifluoroacetic acid (TFA) in water (40 μL). Denatured
1
3150, 3011, 2791 br, 1685, 1586 cm–1; H NMR (MeOH-d4;
600 MHz) 4.26 (2H, s, CH2NH2), 6.71 (1H, d, J 3.5 Hz, 4-H),
7.22 (1H, d, J 3.5 Hz, 3-H); 13C NMR (MeOH-d4; 600 MHz)
36.8, 113.8, 119.9, 147.4, 152.1, 161.2; HRMS (CI) found
[M+H]+ 142.0499; C6H7NO3 requires 142.0499.
6 | J. Name., 2012, 00, 1-3
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ARTICLE
protein was removed by centrifugation (4 ˚C, 3000 rpm, 4
min), the supernatant extracted with diethyl ether (1 mL)
and the solvent evaporated. The residue was dissolved in
THF (1 mL), 2 M NaOH (50 µL), benzyl chloroformate (100
µL) added and the reaction shaken at room temperature for
16 h. Solvents were removed under reduced pressure and
the residue dissolved in water (500 µL), extracted with
diethyl ether (1 mL) and the solvent evaporated. The
product was dissolved in EtOH (100 µL) and analysed by
chiral HPLC to give the ee (78% (R)-assigned on the basis of
the reported selectivity for Mv-TAm). Retention times: (R)-
isomer 27.5 min, (S)-isomer 35.1 min.
deprotected in a 1:1 mixture of methanol and 4 M HCl (6
mL) for 2 h, and the solvent removed under reduced
pressure to give 5-aminomethyl-2-furancarboxylic acid
hydrochloride salt 8b·HCl23 (55 mg, 31%). The
characterisation data was identical to 8b·HCl synthesised
above.
View Article Online
DOI: 10.1039/C6GC02241C
Acknowledgements
The authors would like to thank the UK Engineering and
Physical Sciences Research Council (EPSRC) for financial
support of this work to A. D. and for F. S. (EP/K014897/1),
as part of their Sustainable Chemical Feedstocks
programme. Also the Department of Chemistry UCL for
part-funding A. D. Furthermore, we gratefully acknowledge
the UCL Mass Spectrometry and NMR Facilities in the
Department of Chemistry UCL and the EPSRC UK National
Mass Spectrometry Facility at Swansea University.
Preparative scale biocatalytic reactions. The TAm
reaction was scaled up (50 mL) with substrate (20 mM),
isopropylamine (200 mM, pH 8), potassium phosphate
buffer (100 mM, pH 8), PLP (1 mM) and CV-TAm crude cell
lysate (10 mL). The reaction was incubated at 37 °C and 200
rpm for 24 h.
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 yield in 83%.
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View Article Online
DOI: 10.1039/C6GC02241C
TOC and text
A biocatalytic approach using transaminases has been used for the generation of a range
of furfurylamines in good yields.