Chemoselective oxidation of primary alcohols to aldehydes with Gluconobacter oxydans

Article abstract of DOI:10.1016/S0040-4039(02)01221-2

The production of aliphatic and aromatic aldehydes by oxidation of primary alcohols was achieved with Gluconobacter oxydans DSM 2343. The biotransformation was optimised studying the oxidation of 2-phenyl-1-ethanol to 2-phenylacetaldehyde. A high molar conversion (95% chromatographic conversion, 83% of isolated yield) was obtained using cells grown on glycerol as the main carbon source and directly used in the cultural medium after 24 h at 28°C, pH 4.5 and 5 g L^-1 substrate concentration. The conversion of structurally different primary alcohols was performed under these conditions allowing the chemoselective production of aldehydes, sometimes with very good yields.

Full text of DOI:10.1016/S0040-4039(02)01221-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 6059–6061
Chemoselective oxidation of primary alcohols to aldehydes with
Gluconobacter oxydans
Raffaella Villa,a Andrea Romano,a Raffaella Gandolfi,a Jose´ V. Sinisterra Gagob and
Francesco Molinaria,*
aDipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Universita` degli Studi di
Milano, via Celoria 2, 20133 Milan, Italy
bDepartamento de Qu´ımica Orga´nica y Farmace´utica, Facultad de Farmacia, Universidad Complutense de Madrid,
Plaza Ramon y Cajal, 28040 Madrid, Spain
Received 25 January 2002; accepted 20 June 2002
Abstract—The production of aliphatic and aromatic aldehydes by oxidation of primary alcohols was achieved with Gluconobacter
oxydans DSM 2343. The biotransformation was optimised studying the oxidation of 2-phenyl-1-ethanol to 2-phenylacetaldehyde.
A high molar conversion (95% chromatographic conversion, 83% of isolated yield) was obtained using cells grown on glycerol as
the main carbon source and directly used in the cultural medium after 24 h at 28°C, pH 4.5 and 5 g L−1 substrate concentration.
The conversion of structurally different primary alcohols was performed under these conditions allowing the chemoselective
production of aldehydes, sometimes with very good yields. © 2002 Published by Elsevier Science Ltd.
The enzymatic oxidation of primary alcohols for the
production of aldehydes is attractive since it can be
carried out under mild conditions, suited also for labile
products. The use of isolated enzymes is often compli-
cated by the requirements for cofactors and systems for
their regeneration and whole microbial cells have been
used.1 The oxidation of alcohols by acetic acid bacteria
is an old and established microbial method for obtain-
ing production of vinegar and various carboxylic acids,2
sometimes with remarkable chemo- and enantioselectiv-
ity,3 but the accumulation of aldehydes is only seldom
observed. It has been previously reported that a strain
of Gluconobacter oxydans was able to accumulate iso-
valeraldehyde by oxidation of 2-methyl-1-butanol with
good molar conversions, but its substrate specificity
was limited to aliphatic substrates.4 2-Phenyl-1-ethanol
could be also oxidised by Gluconobacter and Acetobac-
ter allowing for maximum accumulation of 50% molar
conversion into the corresponding aldehyde, before its
further oxidation to phenylacetic acid.5 Two-liquid
phase systems proved suitable for the production of
phenylacetaldehyde by oxidation of 2-phenylethanol
using acetic acid bacteria.6
We have carried out further screening experiments
observing that selective oxidation of 2-phenyl-1-ethanol
to 2-phenylacetaldehyde can be obtained using Glu-
conobacter oxydans DSM 2343 and the potential of this
strain is evaluated in this work. The microorganism was
grown in submerged cultures in various media (glucose,
glycerol, mannitol and sorbitol were employed as main
carbon sources) and cells grown for 24 or 48 h were
used for the oxidation of 2-phenyl-1-ethanol (2.5 g
L−1).7 No significant differences in the oxidative activity
were observed if the cells were harvested after 24 or 48
h. The oxidative activity towards 2-phenyl-1-ethanol
was higher with cells grown on glycerol and yeast
extract and gave high molar conversions into phenylac-
etaldehyde (80–85%). No detectable production of
phenylacetic acid was observed even after 24 h. The
optimum temperature and initial pH were 28°C and
4.5, respectively. These results offer a notable simplifi-
cation of the whole procedure, since the temperature
for obtaining optimal growth and biotransformation
coincide and the pH of 24 h submerged cultures of G.
oxydans DSM 2343 (pH 4.3) was already close to the
optimum. Fig. 1 shows the profile of reactions carried
out starting from different substrate concentrations.
The highest rate and product yield was observed at 5 g
L−1 of substrate and no significant further oxidation
could be observed even at prolonged times. Neverthe-
less, molar conversions above 30% were obtained with
higher concentrations (7.5 and 10 g L−1) after 5 h.
Keywords: Gluconobacter oxydans; biotransformation; aldehyde pro-
duction; alcohol oxidation; flavour.
* Corresponding author. Tel.: 0039-0250316695; fax: 0039-
0250316694; e-mail: francesco.molinari@unimi.it
0040-4039/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0040-4039(02)01221-2

6060
R. Villa et al. / Tetrahedron Letters 43 (2002) 6059–6061
Figure 1. Phenylacetaldehyde production by G. oxydans DSM 2343 using different substrate concentrations.
G. oxydans DSM 2343 was utilised for testing the
oxidation of different primary alcohols under the con-
ditions optimised for the production of phenylacetalde-
hyde (Scheme 1).8
alcohol producing 3-hydroxy butanal. The racemic sub-
strates employed were generally transformed with low
enantioselectivity (E in the range of 2–5).
The low conversions observed in the case of benzyl
alcohols can be explained by the stability of the carbo-
cation (generated by the cofactor-assisted removal of
the hydride) which is stabilised by conjugation with the
aromatic ring. The good activity observed with 2-
hydroxy-benzyl alcohol can be due to the steric hin-
drance of the hydroxy group in o-position which
disturbs the conjugation, giving a less stable and more
reactive carbocation. Therefore, a higher yield is
obtained in the case of 10 than with benzyl alcohol.
Substrates (5 g L−1) were directly added to submerged
24 h cultures and biotransformations performed at
28°C. Gluconobacter o. DSM 2343 was able to produce
several aldehydes, sometimes in very good yields (Table
1).
The production of carboxylic acids (not reported in the
table) was observed at prolonged times only with
aliphatic substrates. Maximum production of aromatic
aldehydes was achieved in the first h of the reaction and
they were not transformed even after 24 h.
In conclusion, the use of G. oxydans DSM 2343 appears
to be a very promising method for the production of
aldehydes. The procedure is very simple as the alcohols
are added directly to 24 h submerged cultures without
The biotransformation of 1,3-butanediol proceeded
with complete chemoselective oxidation of the primary
Scheme 1.

R. Villa et al. / Tetrahedron Letters 43 (2002) 6059–6061
Table 1. Maximum production of aldehydes with G. oxydans DSM 2343
6061
Entry
Substrate
Molar conversion (%)
Isolated yield (%)
Time (h)
1
2
3
4
5
6
7
8
Ethanol
Propanol
Butanol
1,3-Butanediol
Pentanol
Hexanol
2-Methyl-1-butanol
3-Methyl-1-butanol
Benzyl alcohol
2-Hydroxy-benzyl alcohol
4-Hydroxy-3-methoxy-benzyl alcohol
3,4-Dimethoxy-benzyl alcohol
2-Phenyl-1-ethanol
2-(4-Hydroxy)-phenylethanol
2-Phenyl-1-thioethanol
2-Phenyl-1-propanol
3-Phenyl-1-propanol
3-Phenyl-1-butanol
65
79
84
40
60
84
74
45
15
51
B5
B5
95
5
51
62
71
35
50
72
65
38
12
43
–
–
83
–
64
25
82
43
5
3
2
4
3
3
6
6
24
5
9
10
11
12
13
14
15
16
17
18
8
24
24
24
24
4
70
30
90
50
3
8
any modification of conditions (pH, temperature) and
the aldehydes are often produced quickly under mild
conditions.
Microbiol. Biotechnol. 1995, 43, 989–994.
5. Manzoni, M.; Molinari, F.; Tirelli, A.; Aragozzini, F.
Biotechnol. Lett. 1993, 15, 341–345.
6. (a) Molinari, F.; Gandolfi, R.; Aragozzini, F.; Le`on, R.;
Prazeres, D. M. F. Enzyme Microb. Technol. 1999, 25,
729–735; (b) Gandolfi, R.; Ferrara, N.; Molinari, F. Tetra-
hedron Lett. 2001, 42, 513–514.
Acknowledgements
This work was partially supported by the C.N.R.
Target Project on Biotechnology (n 97.01019. PF
115.08601).
7. G. oxydans DSM 2343 (Deutsche Sammlung von Mikroor-
ganismen) was routinely maintained on GYC solid
medium (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. Submerged
cultures were carried out in 1 L Erlenmeyer flasks contain-
ing 150 mL of different media with various carbon sources
and incubated at 28°C on a reciprocal shaker (100 spm).
8. Neat substrates were directly added to the submerged
cultures or to cells centrifuged and resuspended in phos-
phate buffers (pH in the range 6.0–8.0) and flasks were
shaken on a reciprocal shaker (100 spm). All compounds
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1
were characterised by a combination of H NMR, GC/MS
and molar conversions determined by GC or reversed
phase HPLC analysis. The oxidation of 2-phenyl-1-ethanol
was also carried out in 5 L reactor with 1 L working
volume with cells grown directly inside the reactor vessel,
agitation speed 250 rpm, air flow rate 1 v v−1 m−1. The
reaction was started with 5 g of the alcohol. The work-up
of this biotransformation is reported as an example. After
5 h the reaction mixture was centrifuged (15 000 g, 10 min)
to remove the bacterial cells and the supernatant was
extracted with ethyl acetate. The organic extracts were
dried over anhydrous Na2SO4 and the solvent removed.
The crude product was purified by flash chromatography
(hexane/ethyl acetate, 7/3) to give 4.05 g of phenylac-
etaldehyde.
4. Molinari, F.; Villa, R.; Manzoni, M.; Aragozzini, F. Appl.