One-pot chemoenzymatic synthesis of aldoximes from primary alcohols in water

Article abstract of DOI:10.1039/c2gc35764j

A new synthetic method for the one-pot preparation of aldoximes in water was developed; the method is based on the combination of the enzymatic oxidation of primary alcohols to aldehydes using different acetic acid bacteria and in situ condensation of the aldehydes with hydroxylamine.

Full text of DOI:10.1039/c2gc35764j

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COMMUNICATION
One-pot chemoenzymatic synthesis of aldoximes from primary alcohols in
water
Paolo Zambelli,a Andrea Pinto,b Diego Romano,a Elena Crotti,a Paola Conti,b Lucia Tamborini,b
Raffaella Villac and Francesco Molinaria
Received 18th May 2012, Accepted 18th June 2012
DOI: 10.1039/c2gc35764j
A new synthetic method for the one-pot preparation of aldo-
ximes in water was developed; the method is based on the
combination of the enzymatic oxidation of primary alcohols
to aldehydes using different acetic acid bacteria and in situ
condensation of the aldehydes with hydroxylamine.
Enzymatic oxidation of alcohols can be catalytically efficient
and may offer distinct advantages over classical chemical
synthetic methods, since it can be carried out under mild and
ecologically compatible conditions, being often chemo-, regio-
and enantioselective.1 Oxidation of primary alcohols catalyzed
by membrane-bound alcohol and aldehyde dehydrogenases of
acetic acid bacteria generally leads to carboxylic acids or
ketones.2,3 Acetic acid bacteria, which are generally regarded as
safe (GRAS) microorganisms, can also be employed to perform
the partial oxidation of primary alcohols to aldehydes, using
strains lacking aldehyde dehydrogenase activity or when systems
for the in situ extraction of the aldehydes are applied.4 Other-
wise, enzymatic oxidation of primary alcohols to aldehydes can
be obtained by using isolated enzymes.3,5
In this work, aldoximes were prepared using a one-pot chemo-
enzymatic reaction which combines the oxidation of primary
alcohols by acetic acid bacteria with the subsequent conden-
sation of the aldehyde with hydroxylamine; this protocol allows
the preparation of the corresponding aldoximes (Scheme 1).
Aldoximes have wide application in medicine, industry and
analytical chemistry.6 Moreover, they are very useful and versa-
tile intermediates in synthetic organic chemistry. Notably, they
can be reduced to amines7 or oxidized to nitrile oxides,8 which
are in turn precursors for the synthesis of isoxazoles. In addition,
they can undergo Beckmann rearrangement leading to the corre-
sponding amides.9 Aldoximes can be chemically10 or enzymati-
cally11 converted into nitriles through a dehydration reaction.
Finally, it has also been reported that dehydrogenases can cata-
lyse the reduction of phenylacetaldoxime to the corresponding
alcohol.12 One-pot preparation of aldoximes from primary
Scheme 1 One-pot chemoenzymatic synthesis of aldoximes from
primary alcohols.
alcohols deserves special interest since often aldehydes have low
stability, making it difficult to prepare them in high yields.13
Thus, a method where aldehydes are not isolated, but in situ
directly converted into aldoximes seems advantageous.
Three strains of acetic acid bacteria (belonging to different
genera) have been used in this study: Acetobacter sp.
MIM 2000/61, Gluconobacter oxydans DSM 2343 and Asaia
bogorensis SF2.1. G. oxydans DSM 2343 had been previously
used for the preparation of aliphatic and aromatic aldehydes with
isolated yields ranging from 43% to 82%.4c Acetobacter sp.
MIM 2000/61 was recently isolated from vinegar and demon-
strated good oxidative capacities. Asaia bogorensis SF2.1
showed some unusual properties for acetic acid bacteria such as
rapid growth and low production of acetic acid and had never
been used before for biotransformation of primary alcohols.14
Our initial studies were aimed at finding the most suitable con-
ditions for the one-pot chemoenzymatic reaction; 2-phenyl-
1-ethanol (1a) was initially chosen as a substrate, since it can be
easily converted into phenylacetic acid (3a) by different strains
of acetic acid bacteria2e,15 and into phenylacetaldehyde (2a) by
G. oxydans DSM 2343, as reported in previous work from our
group.4c Biotransformations were performed starting from
10 mM of substrate with and without NH2OH·HCl, using the
same amount of cells (dry weight = 10 g L−1) at 28 °C in 0.1 M
phosphate buffer, pH 7.5 (Table 1).
aDepartment of Food, Environmental and Nutritional Sciences
(DeFENS), University of Milan, Milan, Italy.
E-mail: francesco.molinari@unimi.it; Fax: +39 0250319191;
Tel: +39 0250319148
bDepartment of Pharmaceutical Sciences, University of Milan, Milan,
Italy
Biotransformations carried out without NH2OH gave mostly
the corresponding aldehyde 2a with G. oxydans, as previously
cSchool of Applied Sciences, Cranfield University, Bedford, UK
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Table 1 Oxidation of 2-phenyl-1-ethanol (1a), 3-phenyl-1-propanol (1b), p-nitro-2-phenyl-1-ethanol (1c) and 2-phenyl-1-propanol (1d) with acetic
acid bacteria with or without addition of hydroxylamine. Starting concentration of the alcohols was 10 mM
Entry
Strain
Substrate
Equivalents of NH2OH
2 (%)
3 (%)
ee (%)
4 (%)
ee (%)
Time (h)
1
2
3
4
5
6
7
8
Acetobacter sp.
Acetobacter sp.
Gluconobacter oxydans
Gluconobacter oxydans
Asaia bogorensis
Asaia bogorensis
Acetobacter sp.
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1b
1c
1c
1c
1c
1c
1c
1d
1d
1d
1d
1d
1d
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
0
75
0
0
0
0
0
52
0
0
0
0
0
0
0
0
0
0
0
8
0
0
0
100
0
21
0
48
0
100
0
37
0
38
0
93
0
17
0
0
94
0
96
0
68
0
97
0
86
0
80
0
65
0
32
0
12
0
45
0
34
0
1
48
5
48
48
48
1
5
5
24
48
24
5
48
48
48
24
24
24
48
48
48
24
48
Acetobacter sp.
9
Gluconobacter oxydans
Gluconobacter oxydans
Asaia bogorensis
Asaia bogorensis
Acetobacter sp.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Acetobacter sp.
Gluconobacter oxydans
Gluconobacter oxydans
Asaia bogorensis
Asaia bogorensis
Acetobacter sp.
91
0
50
0
0
0
92 (R)
Acetobacter sp.
92 (R)
38 (R)
<5
Gluconobacter oxydans
Gluconobacter oxydans
Asaia bogorensis
Asaia bogorensis
94
0
<5
59
reported, while Ac. aceti and As. bogorensis gave the carboxylic
acid 3a as the only detectable product. All reactions in the pres-
ence of NH2OH gave the aldoxime 4a as product, with yields
ranging from 87 to 96%, depending on the strain used. The high
conversions into aldoxime suggest that condensation of inter-
mediate 2a with hydroxylamine is faster than further oxidation
to acid. It is noteworthy that no acid or aldehyde was detected in
the biotransformations performed in the presence of NH2OH. In
addition, for Gluconobacter and Asaia, with both substrates
(entries 4 and 6), the yield of the aldoxime was higher than the
yields of aldehyde or acid in the absence of hydroxylamine,
showing that removal of the formed aldehyde is beneficial for
the overall yield of the biotransformation.
The good results obtained in the preparation of 4a were
confirmed in the case of the preparation of 3-phenylpropionalde-
hyde oxime 4b (entries 7–12, Table 1), which was obtained with
satisfactory yields (80–97%). When p-nitro-2-phenylethanol 1c
was used as a substrate, higher yields were observed in the
oxidation without NH2OH; indeed, Acetobacter and Asaia, in
the absence of NH2OH, furnished the carboxylic acid 3c with 93
and 91% yield, respectively, while working in the presence of
NH2OH, 4c was prepared with moderate yields (12–65%) from
1c (Table 1, entries 14, 16, and 18).
The transformation was also evaluated on the chiral substrate
1d. Oxidations performed without NH2OH showed that Aceto-
bacter and Asaia (Table 1, entries 19 and 23) oxidized 1d to 3d
as the only product; Acetobacter gave 3d with good enantio-
selectivity (92%, R-enantiomer at 50% molar conversion), while
no kinetic resolution could be observed with Asaia (Table 1,
entry 23). Gluconobacter (Table 1, entry 21) gave only traces of
aldehyde 3d. When working in the presence of NH2OH, aldox-
ime 4d was obtained with 34–59% yields (Table 1, entries 20,
22, and 24); good enantioselectivity (92%, R-enantiomer,
Table 2 Optimization of the preparation of p-nitro-1-phenylethanol
oxime 4c with Acetobacter sp. 2000/61 MIM
[Biocatalyst]
gdry weight L−1
1c
(mM)
Equivalents of
NH2OH
4c
(%)
Time
(h)
Entry
1
2
3
4
5
6
7
8
10
10
10
10
10
10
15
15
15
15
15
15
5
5
10
10
20
20
5
1.0
1.5
1.0
1.5
1.0
1.5
1.0
1.5
1.0
1.5
1.0
1.5
85
24
65
6
11
<5
85
95
75
90
24
30
24
24
48
48
48
48
24
24
24
24
48
48
5
9
10
10
20
20
10
11
12
45% molar conversion) was observed only with Acetobacter
(Table 1, entry 20).
The preparation of p-nitro-2-phenylethanol oxime 4c, which
gave the lowest yields under the conditions employed for the
experiments described in Table 1, was optimized using Aceto-
bacter sp. 2000/61 MIM by changing substrate and biocatalyst
concentration (Table 2). Under optimized conditions (12 g per
cell of biocatalyst, 10 mM of both substrates, entry 10), a 90%
yield was obtained after 24 h. Higher concentrations of substrates
resulted in lower yields; this is likely due to the known toxic
effect of hydroxylamine towards many enzymatic systems.16
Finally, the one-pot transformation was applied to perillyl
alcohol [1e, (4-isopropenyl-cyclohexen-1-enyl)-methanol]. Two
products of the oxidation of perillyl alcohol have great interest in
the food industry: perillyl aldehyde 2e (or perillaldehyde
used as a flavor component)17 and perillyl aldehyde oxime 4e
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Table 3 Oxidation of perillyl alcohol 1e (15 mM) with acetic acid
bacteria with or without addition of hydroxylamine
into 500 mL Erlenmeyer flasks containing 50 mL of the
liquid medium containing yeast extract (10 g L−1) and glycerol
(25 g L−1) at pH 5 in distilled water and incubated on a
reciprocal shaker (100 spm). The dry weights were determined
after centrifugation of 100 mL of cultures; cells were washed
with distilled water and dried at 110 °C for 24 h.
Equivalents of 2e
3e
4e
Time
Entry Strain
NH2OH
(%) (%) (%) (h)
1
2
3
4
5
6
Acetobacter
0
1.5
0
1.5
0
25
0
16
0
12
0
70
0
0
0
0
0
96
0
25
0
70
2
Acetobacter
G. oxydans
G. oxydans
As. bogorensis
48
24
48
24
48
Biotransformations
As. bogorensis 1.5
0
Biotransformations were carried out with cells centrifuged and
suspended in phosphate buffer (0.1 M, pH 7.5). Neat substrates
were directly added to the suspensions and flasks were shaken
on a reciprocal shaker (100 spm). The results were expressed as
molar conversion, defined as the number of converted moles per
number of starting moles. Products were recovered after centrifu-
gation and extraction with EtOAc; the organic extracts were
dried over Na2SO4, the solvent evaporated under reduced
pressure and the products purified by flash chromatography.
1H NMR spectra of oximes (4a–e) were recorded with a
Varian Mercury 300 (300 MHz) spectrometer. Chemical shifts
(δ) are expressed in parts per million and coupling constants (J)
in hertz.
(or perillartine used as a sweetener).18 Table 3 summarizes the
results obtained using perillyl alcohol as a substrate. The reaction
conditions were those optimized for the conversion of 1d into
4d. For the first time, aldehyde 2e was accumulated (16–25%) as
the only product at the end of the reaction in the absence of
NH2OH with Gluconobacter and Asaia. In the presence of
NH2OH, Acetobacter was able to give 96% yield of 4e after
48 h. Here again, it should be stressed that generally much
higher yields were obtained for the preparation of 4e, than
for 2e.
The one-pot chemoenzymatic synthesis of perillartine 4e with
Acetobacter sp. MIM 2000/61 was also carried out on a prepara-
tive scale (2 L of total volume, under the conditions of entry 2).
The oxime 4e was recovered with 94% yield and no detectable
formation of by-products was observed.
1
4a: a 1 : 1 mixture of geometric isomers a and b. H NMR
(300 MHz, CDCl3): δ = 7.57 (t, J = 6.3, 1H, isomer b),
7.45–7.15 (m, 5H, isomer a and 5H, isomer b), 6.93 (t, J =
5.3 Hz, 1H, isomer a), 3.75 (d, J = 5.3 Hz, 2H, isomer a), 3.57
(d, J = 6.3, 2H, isomer b).
1
4b: a 3 : 2 mixture of geometric isomers a and b. H NMR
(300 MHz, CDCl3): δ = 7.45 (t, J = 5.8 Hz, 1H, isomer b),
7.35–7.18 (m, 5H, isomer a and 5H, isomer b), 6.78 (t, J =
5.8 Hz, 1H, isomer a), 2.90–2–80 (m, 2H, isomer a and 2H,
isomer b), 2.78–2.68 (m, 2H, isomer a), 2.58–2.50 (m, 2H,
isomer b).
Conclusions
In conclusion, we have established an easy and efficient alterna-
tive to heavy metal-based solutions for the one-pot preparation
of aldoximes directly from alcohols. Satisfactory yields can be
obtained for different substrates by choosing suitable microor-
ganisms and reaction conditions. Oxidation of alcohols to alde-
hydes is catalyzed by acetic acid bacteria dehydrogenases: a high
turnover of dehydrogenases under medium–high dissolved
oxygen tension is observed with consequent shift of the equili-
brium towards oxidation of primary alcohols. The produced
aldehydes are immediately converted into aldoximes by nearly
stoichiometric amounts of hydroxylamine, preventing further
oxidation to carboxylic acids. The yields and productivity of the
batch transformations described in this communication can be
increased by using continuous systems in membrane reactors, as
previously described for oxidations catalyzed by acetic acid
bacteria.19
1
4c: a 1 : 1 mixture of geometric isomers a and b. H NMR
(300 MHz, CDCl3): δ = 8.19 (m, 2H, isomer a and 2H, isomer
b), 7.55 (t, J = 6.1, 1H, isomer b), 7.42–7.38 (m, 2H, isomer a
and 2H, isomer b), 6.90 (t, J = 5.5 Hz, 1H, isomer a), 3.84 (d,
J = 5.5 Hz, 2H, isomer a), 3.65 (d, J = 6.1, 2H, isomer b).
1
4d: a 2 : 1 mixture of geometric isomers a and b. H NMR
(300 MHz, CDCl3): δ = 7.55 (d, J = 7.0 Hz, 1H, isomer a),
7.25–7.42 (m, 5H, isomer a and 5H, isomer b), 6.82 (d, J =
7.2 Hz, 1H, isomer b), 4.42 (dq, J = 7.2, 7.2 Hz, 1H, isomer b),
3.68 (dq, J = 7.0, 7.0 Hz, 1H, isomer a), 1.45 (d, J = 7.0 Hz,
3H, isomer a), 1.43 (d, J = 7.2 Hz, 3H, isomer b).
1
4e: H NMR (300 MHz, CDCl3): δ = 7.70 (s, 1H), 6.05–6.00
(m, 1H), 4.80–4.65 (m, 2H), 2.45–2.00 (m, 4H), 1.95–1.80
(m, 2H), 1.70 (s, 3H), 1.60–1.40 (m, 1H).
Experimental
Analytical methods
Microorganisms and growth conditions
Samples (0.5 mL) were taken at intervals, brought to pH 1 by
addition of 0.5 M HCl, extracted with an equal volume of
EtOAc and the organic extracts were dried over Na2SO4. The
biotransformations of 2-phenyl-1-ethanol (1a), 3-phenyl-1-pro-
panol (1b), p-nitro-2-phenyl-1-ethanol (1c) and 2-phenyl-1-pro-
panol (1d) were routinely analyzed by HPLC. Analysis was
performed on an HPLC Merck Hitachi 655A, with a UV detector
(254 nm) Merck Hitachi L-4000 using a Purospher® STAR
Strains from an official collection (Gluconobacter oxydans DSM
2343; DSM, Deutsche Sammlung von Mikroorganismen) and
from our collection (Acetobacter sp. MIM 2000/61 and Asaia
bogorensis) were used. Acetic acid bacteria were routinely main-
tained on GYC slants (glucose 50 g L−1, yeast extract 10 g L−1
,
CaCO3 30 g L−1, agar 15 g L−1, pH 6.3) at 28 °C. The strains,
grown on GYC slants for 24 h at 28 °C, were inoculated
2160 | Green Chem., 2012, 14, 2158–2161
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